US010138517B2

(12) United States Patent ( 10 ) Patent No.: US 10 , 138,517 B2

Mehta et al. (45) Date of Patent: Nov . 27 , 2018
(54 ) MANIPULATION OF MICROPARTICLES IN C120 1/6869 (2018.01)
MICROFLUIDIC SYSTEMS BOIF 13/00 ( 2006 .01)
C40B 60 / 14 ( 2006 .01)
(71) Applicant: Caliper Life Sciences , Inc., Hopkinton , GOIN 27/447 (2006 .01)
MA (US ) GOIN 35 /00 (2006 .01 )
(72 ) Inventors: Tammy Burd Mehta , San Jose, CA (52) CPC
U .S. .CI.......... C120 1 /6874 ( 2013 . 01 ) ; BOIL 3 /5027
(US); Anne R . Kopf-Sill, Portola ( 2013 .01) ; BOIL 3 /502761 ( 2013 .01) ; CO7H
Valley , CA (US ) ; J . Wallace Parce , 21/02 (2013.01); CO7H 21/04 (2013.01 );
Palo Alto , CA (US ); Andrea W . Chow , C12Q 1/6869 ( 2013.01); BOIF 13/0059
Los Altos , CA (US ); Luc J . Bousse , ( 2013 .01) ; BOIJ 2219 /00317 (2013 .01); B01J
Los Altos, CA (US ); Michael R . 2219 / 00459 ( 2013.01); B01J 2219 /00468
Knapp , Palo Alto , CA (US); Theo T. (2013.01 ); BOIL 3/ 502715 (2013 .01); BOIL
Nikiforov , San Jose , CA (US ); Steve 2200 / 0668 ( 2013 .01) ; BOIL 2400 /043
Gallagher , Palo Alto , CA (US ) (2013 .01); BOIL 2400 /0409 (2013 . 01 ); BOIL
2400/ 0415 ( 2013.01); BOIL 2400 /0487
(73 ) Assignee: CALIPER LIFE SCIENCES, INC ., (2013 .01); C40B 60 / 14 ( 2013 . 01) ; GOIN
Mountain View , CA (US ) 27/ 44756 (2013 .01); GOIN 35/0098 (2013 .01 );
( * ) Notice : Subject to any disclaimer, the term of this GOIN 2035 /00574 (2013.01); Y1OT
patent is extended or adjusted under 35 436 / 143333 ( 2015 .01)
U .S .C . 154 (b ) by 0 days . 8 ) Field of Classification Search
None
(21) Appl. No.: 15 /615 ,469 See application file for complete search history .
(22 ) Filed : Jun. 6, 2017 (56 ) References Cited
(65 ) Prior Publication Data U .S. PATENT DOCUMENTS
US 2017 /0356040 A1 Dec. 14 , 2017 5 ,028,545 A * 7 /1991 Soini .................. GOIN 21/6408
435 / 7 . 1
Related U .S. Application Data 6,051,380 A * 4/2000 Sosnowski .......... B01J43519//0046
6 . 11
(60 ) Continuation of application No. 14 /823,571, filed on * cited by examiner
Aug. 11 , 2015 , now Pat. No. 9 ,670 , 541, which is a
division of application No. 13 /015 ,242, filed on Jan . Primary Examiner — Christopher M Gross
27 , 2011, now Pat. No . 9 , 101, 928 , which is a (74 ) Attorney, Agent, or Firm — Day Pitney LLP
continuation of application No. 11/ 928,808 , filed on
Oct . 30 , 2007, now abandoned , which is a division of (57 ) ABSTRACT
application No. 10 /606 ,201, filed on Jun . 25 , 2003, An array of transportable particle sets is used in a micro
now abandoned , which is a continuation of fluidic device for performing chemical reactions in the
application No. 09/510 ,626 , filed on Feb . 22, 2000 , microfluidic device . The microfluidic device comprises a
now Pat. No. 6,632,655 . main channel and intersecting side channels , the main chan
(60) Provisional application No. 60 / 128,643 , filed on Apr. nel and side channels forming a plurality of intersections.
9 , 1999, provisional application No. 60 / 127 ,825 , filed The array of particle sets is disposed in the main channel,
on Apr. 5, 1999, provisional application No. and the side channels are coupled to reagents . As the particle
60/ 121 ,223, filed on Feb . 23, 1999 . sets are transported through the intersections of the main
channel and the side channels , reagents are flowed through
(51 ) Int. Cl. the side channels into contact with each array member (or
BOIL 3/00 ( 2006 .01) selected array members ), thereby providing a plurality of
C12Q 1/6874 ( 2018 .01) chemical reactions in the microfluidic system .
COFH 21/02 ( 2006 .01)
CO7H 21/04 (2006 .01) 14 Claims, 19 Drawing Sheets
U .S. Patent Nov.27, 2018
Nov . 27, 2018 Sheet 1 of 19 US 10 , 138,517 B2

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 US 10 ,138 ,517 B2
MANIPULATION OF MICROPARTICLES IN able. For example , Paree et al. “High Throughput Screening
MICROFLUIDIC SYSTEMS Assay Systems in Microscale Fluidic Devices ” WO
98 / 00231 and Knapp et al. " Closed Loop Biochemical
CROSS -REFERENCE TO RELATED Analyzers ” (WO 98 / 45481; PCT/US98 / 06723 ) provide pio
APPLICATIONS 5 neering technology for the integration of microfluidics and
sample selection and manipulation . For example , in WO
This application is a continuation of U . S . patent applica - 98 /45481, microfluidic apparatus, methods and integrated
tion Ser. No . 14 / 823 ,571. filed Aug. 11 . 2015 which is a systems are provided for performing a large number of
divisional of U .S . patent application Ser. No. 13 /015 . 242. iterative , successive , or parallel fluid manipulations . For
filed Jan . 27 , 2011 , which is a continuation of U . S . patent 10 example , integrated sequencing systems, apparatus and
application Ser. No . 11/ 928 , 808, filed Oct. 30 , 2007 , which methods are provided for sequencing nucleic acids (as well
is a divisional of U . S . patent application Ser. No . 10 /606 . as for many other fluidic operations, e . g ., those benefiting
201, filed Jun . 25 , 2003, which is a continuation of U .S . from automation of iterative fluid manipulation ). This ability
patent application Ser. No . 09/510 ,626 , filed Feb. 22, 2000 , to iteratively sequence a large nucleic acid (or a large
now U . S . Pat. No . 6 ,632,655 , which claims benefit of and 15 number of nucleic acids) provides for increased rates of
priority to U . S . Ser. No . 60 / 121,223 , filed Feb . 23 , 1999 by sequencing, as well as lower sequencing reagent costs .
Mehta et al. ; U . S . Ser. No . 60 / 127 ,825 , filed Apr. 5 , 1999 , by Applications to compound screening , enzyme kinetic deter
Mehta et al.; U .S . Ser. No.60 /128 ,643 , filed Apr. 9 , 1999 , by mination , nucleic acid hybridization kinetics andmany other
Mehta et al.; co - filed PCT application PCT/US00 /04522 , processes are also described by Knapp et al.
filed Feb . 22, 2000; co - filed U .S . application U . S . Ser. No. 20 As an alternative to microfluidic approaches, small scale
09/510 .205 , filed Feb . 22. 2000 : and co - filed PCT applica - array based technologies can also increase throughput of
tion PCT /US00 / 04486 , filed Feb . 22 . 2000. Each of these screening, sequencing , and other chemical and biological
applications is incorporated herein by reference in its methods, providing robust chemistries for a variety of
entirety for all purposes. screening, sequencing and other applications. Fixed solid
25 phase arrays of nucleic acids , proteins, and other chemicals
STATEMENT REGARDING FEDERALLY have been developed by a number of investigators. For
SPONSORED RESEARCH OR DEVELOPMENT example , U .S . Pat. No. 5 ,202,231, to Drmanac et al. and ,
e . g ., in Drmanac et al. (1989 ) Genomics 4 : 114 - 128 describe
The present invention was made with government funding sequencing by hybridization to arrays of oligonucleotides .
from the United States National Institute of Standards and 30 Many other applications of array -based technologies are
Technology (NIST), through the Advanced Technology Pro commercially available from e.g., Affymetrix, Inc . (Santa
gram (ATP ) under Grant No. 70NANB8H4000 , and the Clara , Calif.), Hyseq Technologies , Inc . (Sunnyvale , Calif.)
United States government may have certain rights in the and others . Example applications of array technologies are
invention . described e.g ., in Fodor ( 1997 ) “Genes, Chips and the
35 Human Genome” FASEB Journal. 11: 121- 121; Fodor
FIELD OF THE INVENTION ( 1997) “Massively Parallel Genomics” Science . 277 :393
395 ; Chee et al. (1996 ) “ Accessing Genetic Information
The invention is in the field of microfluidics and microar - with High -Density DNA Arrays” Science 274 :610 -614 ; and
ray technology. Apparatus, methods and integrated systems Drmanac et al. ( 1998 ) “ Accurate sequencing by hybridiza
for detecting molecular interactions are provided . The appa - 40 tion for DNA diagnostics and individual genomics ." Nature
ratus include microscale arrays which are movable or fixed Biotechnology 16 : 54 - 58 .
in a microfluidic system . The systems are capable of per The present invention is a pioneering invention in the field
forming integrated manipulation and analysis in a variety of ofmicrofluidics and mobile array technologies , coupling the
biological, biochemical and chemical experiments, includ - fluid handling capabilities of microfluidic systems with the
ing , e . g ., DNA sequencing. Applications to chemical and 45 robust chemistries available through array technologies
biological systems, e . g., nucleic acid sequencing , enzyme ( e .g ., solid phase chemistries ) to facilitate laboratory and
kinetics , diagnostics, compound screening, and the like are industrial processes. Many applications and variations will
provided . be apparent upon complete review of this disclosure .
BACKGROUND OF THE INVENTION 50 SUMMARY OF THE INVENTION
The development of microfluidic technologies by the The present invention provides microfluidic arrays. The
inventors and their co -workers has provided a fundamental arrays include particle sets (or “ packets” ) which can be
paradigm shift in how artificial biological and chemical mobile or fixed in position , e . g ., within a microfluidic
processes are performed . In particular , the inventors and 55 system . The particle sets can include fixed chemical com
their co -workers have provided microfluidic systems which ponents or can be modifiable . The arrays are used in a wide
dramatically increase throughput for biological and chemi- variety of assays, as chemical synthesis machines, as nucleic
calmethods , as well as greatly reducing reagent costs for the acid or polypeptide sequencing devices , as affinity purifica
methods. In these microfluidic systems, small volumes of tion devices, as calibration and marker devices, asmolecular
fluid ( e .g ., on the order of a few nanoliters to a few 60 capture devices, as molecular switches and in a wide variety
microliters ) are moved through microchannels (e .g., in glass of other applications which will be apparent upon further
or polymer microfluidic devices ) by electrokinetic or pres - review .
sure -based mechanisms. Fluids can bemixed , and the results in one implementation , the invention provides microflu
of the mixing experiments determined by monitoring a idic devices comprising one or more array( s ) of particles .
detectable signal from products of the mixing experiments. 65 The device includes a body structure having a microscale
Complete integrated systems with fluid handling, signal cavity (e . g ., microchannel, microchannel network , microw
detection , sample storage and sample accessing are avail - ell, microreservoir or combination thereof) disposed within
 US 10 ,138 ,517 B2
the body structure . Within the microscale cavity , an ordered results in contacting the at least one set of nucleic acid
array of a plurality of sets of particles (each particle set is templates with the plurality of sequencing reagents. Signals
constituted of similar or identical particle “ members ” or resulting from exposure of the first set of particles to the
" types ” ) constitute the array . The array is optionally mobile reagent train are selected , thereby providing a portion of
( e . g ., flowable in a microfluidic system , with flow being in 5 sequence of the nucleic acid template . For example , the
either the same or in a different direction relative to fluid reagent train can include a polymerase , a sufurylase , an
flow ) or can be fixed (e . g ., having flowable reagents flowed apyrase , an inorganic phosphate , ATP , a thermostable poly
across the system ) . merase , luciferin , luciferase , an endonuclease , an exonu
The arrays of the invention include a plurality of particle clease , Mg + + , a molecular crowding agent, a buffer , a dNTP ,
sets . The precise location of the particle sets within the 10 a dNTP analog, a fluorescentnucleotide , a chain terminating
arrays is not critical, and can take many configurations. In nucleotide , a reversible chain terminating nucleotide , a
one simple embodiment, particle sets abut in channels . For p hosphatase , a reducing agent, an intercalator, a salt, DTT,
example , 2, 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 100 , 1,000, 10 ,000 , 100 ,000 BSA , a detergent ( e.g ., Triton® or Tween® ), chemicals to
or more particle sets can abut in a single channel. Alterna inhibit or enhance EO flow ( e.g ., polyacrylamide ), or other
tively , non -abutting sets of particles dispersed within a 15 sequencing reagent . One preferred use for the arrays of the
microfluidic device can also be used , e . g ., where the spatial invention is sequencing by " synthesis ” or “ incorporation , "
location of each set of particles is known or can be deter - e.g., pyrosequencing . For example , the reagent train or array
mined . Fluidic reagents and particles are optionally flowed optionally include reagents for sequencing nucleic acid
through the same or through different microchannels (or templates by pyrosequencing . A variety of other sequencing
other microfluidic structures such as wells or chambers ). For 20 approaches are described herein .
example , fluidic reagents are optionally flowed from a first Steps in the methods herein can be performed repeatedly
channel into a second channel which includes particle sets of or reiteratively for chemistries such as sequencing that
the array . involve repetitive synthesis and/or analysis steps . As
Particles (alternatively “ microparticles ” ) of the arrays of reagents are depleted e. g., in the reagent train noted above ,
the invention can be essentially any discrete material which 25 the method further optionally includes flowing a second
can be flowed through a microscale system . Example par - train of reagents comprising a plurality of sequencing
ticles include beads and biological cells . For example , reagents across the first set of particles, or flowing the first
polymer beads, silica beads, ceramic beads, clay beads, glass set of particles through the second reagent. Alternatively,
beads, magnetic beads , metallic beads , inorganic beads, and reagents are flowed in excess for a period of time, after
organic beads can be used . The particles can have essentially 30 which the channel (s ) are rinsed , e . g ., with a buffer before
any shape , e . g ., spherical, helical , irregular , spheroid , rod - flowing a second reagent.
shaped , cone- shaped , disk shaped , cubic , polyhedral or a To further avoid contamination between repetitions or
combination thereof. Particles are optionally coupled to steps, methods are provided for loading and unloading
reagents, affinity matrix materials, or the like, e. g., nucleic reagents from a microfluidic device using a pair of split
acid synthesis reagents , peptide synthesis reagents, polymer 35 reagent wells and apair of split waste wells .
synthesis reagents , nucleic acids, nucleotides , nucleobases , Integrated systems and methods for performing fluidic
nucleosides, peptides, amino acids, monomers , cells , bio - analysis of sample materials in a microfluidic system having
logical samples , synthetic molecules , or combinations a particle array are also provided . For example , an integrated
thereof. Particles optionally serve many purposes within the microfluidic system is provided which has a microfluidic
arrays, including acting as blank particles, dummy particles , 40 device with the particle array, a material transport system ,
calibration particles , sample particles, reagent particles, test and a fluidic interface in fluid communication with the
particles , and molecular capture particles , e . g ., to capture a particle array. The interface samples a plurality of materials
sample at low concentration . Additionally the particles are from one or a plurality of sources ofmaterials and introduces
used to provide particle retention elements. Particles are the materials into contact with the particle array .
sized to pass through selected microchannel regions ( or 45 In the integrated methods , a first material from the plu
other microscale elements ). Accordingly , particles will rality of materials is sampled with the fluidic interface . The
range in size, depending on the application , e.g ., from about first material is introduced into contact with at least one
0 .1 to about 500 microns in at least one cross -sectional member of the particle array , whereupon the first sample
dimension . material and at least a first member of the particle array
In one aspect, the microfluidic system comprises an 50 react. A reaction product of the first sample material and the
intersection of at least two microchannels. At least one particle array is then analyzed and a second material (which
member of the particle array is transported within a first of may be the same as or different than the first material) is
the at least two channels to a point proximal to or within the selected , based upon the reaction product . The second mate
channel intersection . At least one of the reagents is trans - rial is contacted with the particle array , where the second
ported through a second of the at least two intersecting 55 material and at least a second member of the particle array
microchannels to a point proximal to or within the channel react. A second reaction product of the second material and
intersection . The at least one member of the particle array the particle array is analyzed , thereby providing a fluidic
and the at least one reagent are contacted proximal to or analysis of the first and second materials .
within the channel intersection . For example, in sequencing applications, the first material
Methods of sequencing nucleic acids are provided . In the 60 can include a first DNA sequencing template, a first sequenc
methods , a first set of particles comprising at least one set of ing primer , or a first sequencing reagent while the second
nucleic acid templates is provided , e . g., in a first microflu - material can include a second DNA template, a second
idic channel. A train of reagents (i. e., an ordered or semi- sequencing primer, or a second sequencing reagent. The
ordered arrangement of fluidic reagents in a channel) com - array, in this example , includes a first mixture of reagents
prising a plurality of sequencing reagents is flowed across 65 having DNA sequencing reagents or DNA templates. The
the first set of particles, or the first set of particles is flowed first reaction product includes products of a DNA sequenc
through the reagent train , depending on the application . This ing reaction (e. g., primer extension , sequencing by incorpo
 US 10 ,138,517 B2
ration , e. g., by the pyrosequencing reaction or the like). A changing the array members .New components can be added
second sequencing primer is selected for inclusion in the to the array members following such washing. For example
second mixture of reagents based upon the products of the one or more particle modification reagents can be removed
DNA sequencing reaction . Optionally, a third material is from one or more of the particle sets following washing, to
selected based upon the results of the analysis of the second 5 provide one or more stripped particle sets . At least one
reaction product. The third material is optionally introduced additional particle modification reagent can be flowed across
into proximity with the array, whereupon the third material the one or more stripped particle set, thereby producing an
and the array react. As above, the third reaction product is additional particle set.
analyzed . This process is optionally reiterated several times Many additional aspects of the invention will be apparent
( e . g ., easily 10 times or more , often 100 - 1 ,000 times or 10 upon review including uses of the devices and systems of the
more ). Indeed , the process can be repeated thousands of invention , methods of manufacture of the devices and sys
times in a single experiment, e . g ., to sequence a long stretch
of DNA, or the like. Ordinarily , the integrated system tems of the invention , kits for practicing the methods of the
includes a computer for performing or assisting in selection invention and the like. For example , kits comprising any of
of the second material. The integrated system can also 15 the devices or systems set forth above , or elements thereof,
in conjunction with packaging materials (e . g ., containers,
include fluid handling elements, e.g., electrokinetic or pres sealable
sure flow controllers . plastic bags etc .) and instructions for using the
Systems for optimizing or performing a desired chemical devices , e. g ., to practice the methods herein , are also con
reaction are provided . The system includes a microfluidic templated . Methods of Manufacture and manufactured
device which includes a microscale cavity having a particle 20 devices comprising arrays or array members are set forth in
array disposed therein . The particle array includes a plurality detail herein .
of particle sets. The system includes an electrokinetic or
pressure based fluid direction system for transporting a BRIEF DESCRIPTION OF THE FIGURES
selected volume of a first reactant to the array, or for
reconfiguring the position of the array or for reconfiguring 25 FIGS. 1A & 1B are a schematic top view and expanded
the arrangement of array members , or for loading array v iew , respectively , of an example microfluidic system com
members (e . g ., constituting a plurality of array sets ) into the prising array elements .
microscale cavity . The system also includes a control sys - FIGS. 2A & 2B are side view schematics of a microchan
tem , e . g., including a computer, which instructs the fluid nel and particle retention region arrangement.
direction system to deliver a first selected volume of first 30 FIGS. 3A & 3B are a top view and expanded view
reactant to the array, or formoving members of the array into schematic , respectively, of an example microfluidic system
proximity with the first reactant,where contacting or mixing comprising array elements.
of the first reactant and at least one member of the array FIGS. 4A -4C are top view and expanded view schematics
produces a first chemical reaction product. The control of an example microfluidic system comprising array ele
system optionally directs a plurality of mixings of the first 35 ments .
reactant and the array ( e.g ., by electrokinetic and/or pressure FIG . 5 is a side- view schematic of a main channel with
based manipulation of reagent or array member as described reagent introduction channels for sequencing nucleic acids.
herein ), wherein a reaction condition selected from : tem - FIG . 6 is a schematic of a sipper for reagent/array train
perature , pH , and time is systematically varied in separate introduction into microfluidic devices .
mixings reactions. The system typically includes a detection 40 FIG . 7 is a schematic of a multiplexing arrangement
system for detecting the first chemical reaction product e . g ., useful for DNA sequencing.
as set forth above and supra . Other optional elements include FIG . 8 is a schematic showing a device with pipettor
a temperature control element for controlling temperature of access to a particle library .
reaction of the first and second element, a source of acid , a FIGS . 9A & 9B are a schematic of a device with particle
source of base and a source of reactants , reagents , array 45 packets in channels having reagent flow across the top of the
members, or the like . particles. FIG . 9A is a cross section . FIG . 9B is a top view .
In one aspect, the system instructs the fluid direction FIG . 10 is a schematic illustration of a top view of a
system to contact a second selected volume of the first or a device showing loading of bead packets .
second reactant with the array. This contact produces a FIGS. 11A - 11C are schematic illustrations of alternate
second chemical reaction product . 50 bead packet loading embodiments . FIG . 11A is a top view .
The particles of the arrays optionally include a tag and one FIG . 11B is a top view . FIG . 11C is a top view .
or more of the particle modification reagents comprising an FIG . 12 is a schematic of a system comprising a computer,
anti- tag ligand . A “ tag ” is a component that can be detected , detector, array , sampling system , and particle library .
directly or indirectly ( e .g ., by binding to a detectable ele - FIG . 13 is a schematic of a reversible terminator labeling
ment ). Exemplar tag and anti -tag ligands include nucleic 55 strategy .
acids ; nucleic acid bindingmolecules , amidin , biotin , avidin , FIG . 14 is a side view of a capillary or microchannel
streptavidin , antibodies , antibody ligands, carbohydrate containing a porous barrier formed from a plurality of
molecules, carbohydrate molecule binding reagents , pro - particles which captures or retains a solid phase reaction
teins, protein binding molecules, organic molecules, organic element, e .g ., another set of particles .
molecule binding reagents , receptors , receptor ligands, etc . 60 FIG . 15 is a side view of a capillary or microchannel in
The particle modification reagent can also include a func - a housing, which housing comprises a porous barrier.
tional domain , e . g ., independently selected from those noted FIG . 16 is a side view of a capillary or microchannel
for the tag and tag ligand . For example, in one embodiment, comprising an integral or formed porous barrier made from
the one or more particle modification reagent has a nucleic a set of particles . The porous barrier is used , e. g ., to capture
acid having a biotin or avidin attached thereto . 65 multiple packets , i. e ., sets of particles .
Wash buffers, heat application , or an electric pulse are FIG . 17 is a schematic illustrating sequencing by synthe
optionally used to strip components from arrays , thereby sis in a high throughput system .
 US 10 ,138 ,517 B2
FIGS. 18A - 18C are schematic views of example micro - A " serial ” stream of components , e .g., reagents is a train
fluidic devices useful in split well loading and unloading, of reagents , is an ordered linear arrangement of the compo
e.g., of reagents and particles , e.g., to avoid contamination . nents
A “ parallel” set of components is an array of components
DEFINITIONS 5 which are in a plurality of linear arrangements, e .g ., separate
linear arrangements, of the components .
Unless indicated to the contrary, the following definitions A “ particle modification reagent” is a reagent thatbinds to
supplement those in the art . or chemically alters one or more component that is physi
“ Microfluidic,” as used herein , refers to a system or cally associated with the particle .
device having fluidic conduits or chambers that are generally 10
fabricated at the micron to submicron scale , e.g., typically DETAILED DESCRIPTION
having at least one cross -sectional dimension in the range of
from about 0 . 1 pm to about 500 pm . The microfluidic system The present invention provides microfluidic arrays . The
of the invention is fabricated from materials that are com - array components can be mobile or fixed . They can also be
patible with the conditions present in the particular experi- 15 of a selected type or type switchable , and can incorporate
ment of interest. Such conditions include, but are not limited any of a wide variety of chemical or biochemical compo
to , pH , temperature , ionic concentration , pressure , and appli- nents . The arrays are broadly useful as tools for screening
cation of electrical fields. The materials of the device are assay components , biopolymer sequencing, drug screening,
also chosen for their inertness to components of the experi- assay normalization , as miniaturized chemical and bio
ment to be carried out in the device . Such materials include , 20 chemical synthesis machines, as molecular switches, as
but are not limited to , glass , quartz , silicon , and polymeric fluidic logic circuits and a variety of other applications that
substrates, e . g ., plastics , depending on the intended appli- will become apparent upon complete review of this disclo
cation . sure . The arrays can be components of integrated systems.
A “microscale cavity ” is a conduit or chamber having at Methods of performing a plurality of chemical reactions
least one dimension between about 0 . 1 and 500 microns. 25 in a microscale device are an aspect of the invention . In the
A "microchannel” is a channel having at least one methods , an array within a microfluidic system ( e. g ., having
microscale dimension , as noted above . A microchannel a body structure with a microscale interior cavity, etc . as
optionally connects one or more additional structure for described above) is provided . One or more liquid reagent is
moving or containing fluidic or semi-fluidic ( e. g ., gel- or flowed into the interior cavity and into contact with particle
polymer solution - entrapped ) components . 30 sets of the array . The liquid reagent chemically reacts with
An " ordered array of a plurality of sets of particles” is an one or more of the plurality of particles, thereby providing
array of particle sets ( each particle set is constituted of a chemical reaction in the microscale device .
similar or identical particle “ members" or " types" ) having a Optionally , one or more of the plurality of sets of particles
spatial arrangement. The spatial arrangement of particle sets of the array ( or the entire array or a substantial portion of )
can be selected or random . In a preferred embodiment, the 35 is moved into or through an intersection of at least two
spatial arrangement is selected . The arrangement can be channels present in a microfluidic system .Mixing can occur
known or unknown. In a preferred embodiment, the spatial in the intersections of channels , or within chambers , chan
arrangement of particle sets is known . nels, wells , reservoirs, or the like. Thus, in the methods of
A “ set” of particles is a group or “ packet” of particles the invention , at least one of the plurality of sets of particles
having similar or identical constituents . 40 can be moved through at least one of the at least two
A “ particle movement region ” is a region of a microscale channels into an intersection of the at least two channels ,
element in which particles are moved . A " fluid movement while (separately or simultaneously ) flowing the liquid
region ” is a region of a microscale element in which fluidic reagent through a second of the at least two channels into the
components are moved . As discussed supra , fluidic and channel intersection , where the liquid reagent flows into
particulate elements are moved by any of a variety of forces, 45 contact with at least one set of particles of the array. In one
including capillary, pressure , electrokinetic and the like . aspect, the cavity comprises a main channel having a plu
A “ particle retention region ” is a region of a microscale rality of intersecting side channels, forming a plurality of
element in which particles can be localized , e . g ., by placing channel intersections between the main channel and each of
a physical barrier or porous matrix within or proximal to the the intersecting side channels . The methods optionally
retention region , by application of magnetic or electric 50 include transporting at least one of the plurality of sets of
fields , by application of pressure , or the like . For example , particles in themain channel into at least two of the plurality
a porousmatrix optionally comprises a fixed set of particles , of channel intersections .
e . g., particles about 100 um to about 200 um in cross Similarly , in one aspect, the method includes transporting
sectional dimension , within a microchannel. at least one fluidic reagent through at least one of the side
A “microwell plate ” is a substrate comprising a plurality 55 channels into at least one of the plurality of channel inter
of regions which retain one or more fluidic components . sections , where the reagent flows into at least one of the
A “ pipettor channel” is a channel in which components plurality of sets of particles in the main channel. Alterna
can be moved from a source to a microscale element such as tively , one or more of the particle sets of the array is flowed
a second channel or reservoir. The source can be internal or through a single microchannel and various reagents, e. g.,
external to a microfluidic device comprising the pipettor 60 liquid reagents, are optionally flowed through the particles
channel. or the particles are flowed through the reagent. The liquid
Two components are “ physically associated ” when they reagent is optionally flowed through a capillary fluidly
are in direct or indirect contact. coupled to the single microchannel, e .g ., a capillary that sips
A particle array is “ mobile ” when a plurality of sets of the fluid from a microwell plate into a microfluidic device , e. g.,
array can be moved in a selected or selectable manner, e . g ., 65 a single channel or multi- channel device .
by electrokinetic , pressure - based or capillary fluid move - Methods also optionally include moving particles ( or
ment systems, or a combination of movement systems. reagents ) into the interior cavity . For example, in one
 US 10 ,138 ,517 B2
10
embodiment, the interior cavity has a broad channel with fluidic pressure, atmospheric pressure, capillary force , grav
narrow channels within the broad channel. The narrow ity, electrokinesis , electric and magnetic fields, and centrip
channels are deeper in at least one dimension than the broad etal or centrifugal force.
channel. A plurality of sets of particles are transported into The inventors and their co -workers have provided a
one or more of the narrow channels to form the array . 5 variety of microfluidic systems in which the arrays of the
Optionally , a liquid reagent is also ( subsequently or previ- invention can be constructed . For example , Ramsey WO
ously ) transported through the broad channel and the narrow 96 / 04547 provides a variety of microfluidic systems. See
channel and into contact with the plurality of sets of par also , Ramsey et al. ( 1995 ), Nature Med . 1 (10 ): 1093 - 1096 ;
ticles. For example , a liquid reagent is optionally sipped Kopf - Sill et al. ( 1997 ) “ Complexity and performance of
from a microtiter plate by a capillary fluidly coupled to a 10 on -chip biochemical assays," SPIE 2978: 172 - 179 February
microfluidic device , in which device the liquid reagent 10 - 11 ; Bousse et al. ( 1998 ) “ Parallelism in integrated fluidic
contacts one or more particle set. Microparticles are also circuits,” SPIE 3259 : 179 - 186 ; Chow et al. U . S . Pat. No .
optionally brought into a microfluidic device through a 5 ,800 ,690 ; Kopf-Sill et al. U . S . Pat. No. 5 ,842 ,787; Paree et
sipper capillary attached to a microfluidic device . Exemplar al., U .S . Pat. No. 5 ,779 , 868; Paree, U . S . Pat. No . 5 ,699, 157;
liquid reagents include those described above such as 15 U .S . Pat. No. 5 ,852 ,495 (J. Wallace Paree ) issued Dec . 22,
nucleic acid sequencing reagents. 1998 ; U .S . Pat. No . 5 ,869,004 (J.Wallace Paree et al.) issued
One preferred use for the arrays ofthe invention is nucleic Feb . 9 , 1999 , U . S . Pat . No. 5 ,876 ,675 (Colin B . Kennedy )
acid or protein sequencing (or sequencing monomer ele - issued Mar. 2 , 1999; U . S. Pat. No . 5 ,880,071 (J. Wallace
ments of any polymer). For example the methods of the Paree et al.) issued Mar. 9 , 1999 ; U . S . Pat. No. 5,882 ,465
invention optionally include sequencing by hybridization , 20 (Richard J. McReynolds ) issued Mar . 16 , 1999 ; U . S . Pat.
sequencing by synthesis or incorporation , sequencing by No. 5 ,885,470 (J. Wallace Paree et al.) issued Mar. 23, 1999;
photobleaching, sequencing by intercalation , specific detec - U .S . Pat . No. 5,942,443 (J. Wallace Paree et al.) issued Aug .
tion of nucleic acid polymorphisms, specific detection of a 24 , 1999 ; U . S . Pat. No. 5,948,227 (Robert S . Dubrow )
nucleic acid , diagnosing or predicting prognosis of a disease issued Sep. 7 , 1999; U .S . Pat. No. 5, 955 ,028 ( Calvin Y. H .
or infectious condition associated with presence or absence 25 Chow ) issued Sep . 21, 99 ; U .S . Pat. No. 5 ,957,579 (Anne R .
of a nucleic acid in a biological sample, diagnosing or Kopf-Sill et al.) issued Sep . 28 , 1999 ; U . S . Pat. No. 5 , 958 ,
predicting prognosis of a disease or infectious condition 203 ( J. Wallace Paree et al.) issued Sep . 28, 1999 ; U . S . Pat.
associated with presence or absence of a protein in a No . 5 , 958 ,694 ( Theo T .Nikiforov ) issued Sep . 28 , 1999 ; and
biological sample , serial or parallel hybridization between U .S . Pat. No. 5 ,959,291 Morten
( J . Jensen ) issued Sep . 28
multiple liquid reagents and members of the array, and serial 30 1999 ; Paree et al. WO 98 /00231; Paree et al . WO 98 /00705 ;
or parallel detection of results of multiple hybridization Chow et al. WO 98 /00707 ; Paree et al. WO 98 /02728; Chow
reactions between liquid reagents and members of the array . WO 98 /05424 ; Paree WO 98 /22811; Knapp et al., WO
Methods of contacting samples and reagents in a micro - 98 /45481; Nikiforov et al. WO 98 /45929 ; Paree et al. WO
fluidic system are also provided . In these methods, a plu - 98 / 46438 ; Dubrow et al., WO 98 /49548 ; Manz , WO
rality of members of a particle array which includes a 35 98 /55852; WO 98 / 56505 ; WO 98 /56956 ; WO 99 / 00649;
plurality of samples is transported to a selected location WO 99 /10735 ; WO 99 / 12016 ; WO 99 / 16162 ; WO
within the microfluidic system . Simultaneously, separately 99/ 19056 ; WO 99 / 19516 ; WO 99 /29497; WO 99/31495 ;
or sequentially , the reagents are also transported within the WO 99/ 34205; WO 99/43432 ; and WO 99 /44217 ; U . S . Pat.
microfluidic system such that members of the particle array No. 5 , 296 , 114 ; and e .g ., EP O 620 432 Al; Seiler et al.
and the reagents are contacted .Members of the particle array 40 (1994 ) Mitt Gebiete Lebensm . Hyg . 85:59-68; Seiler et al.
and the reagents are optionally repeatedly transported (1994 ) Anal. Chem . 66 :3485 - 3491 ; Jacobson et al. (1994 )
sequentially or simultaneously within the microfluidic sys “ Effects of Injection Schemes and Column Geometry on the
tem . In microfluidic systems having one or more emission Performance of Microchip Electrophoresis Devices” Anal.
detectors, the method optionally includes transporting the Chem . 66 : 66 . 1107 - 1113 ; Jacobsen et al. ( 1994 ) " Open
plurality of particle members past the one ormore emission 45 Channel Electrochromatography on a Microchip ’ Anal.
detectors, before, during , or after contacting the plurality of Chem . 66 : 2369- 2373 ; Jacobsen et al. ( 1994 ) “ Precolumn
particle members with one or more of the reagents . Reactions with Electrophoretic Analysis Integrated on
Array Configurations Microchip ’ Anal. Chem . 66 :4127 -4132 ; Jacobsen et al.
As noted , the devices and systems of the invention ( 1994 ) " Effects of Injection Schemes and Column Geometry
typically include a body structure having a microscale cavity 50 on the Performance ofMicrochip Electrophoresis Devices .”
( e .g ., microchannel, microchannel network , microwell, Anal. Chem . 66 : 1107 - 1113 ; Jacobsen et al. ( 1994 ) “ High
microreservoir or combination thereof) disposed within the Speed Separations on a Microchip .” Anal. Chem . 66 : 1114
body structure . Within the microscale cavity , an ordered 1118 ; Jacobsen and Ramsey ( 1995 ) “ Microchip electropho
array of a plurality of sets of particles constitute the array. resis with sample stacking" Electrophoresis 16 : 481-486 ;
The array is optionally mobile ( e.g., flowable in a micro - 55 Jacobsen et al . ( 1995 ) “ Fused Quartz Substrates for Micro
fluidic system ) or can be fixed (e.g., having flowable chip Electrophoresis” Anal. Chem . 67 : 2059 - 2063 ; Harrison
reagents flowed across the system ). A fixed array of particles et al. ( 1992 ) “ Capillary Electrophoresis and Sample Injec
optionally comprises a porous barrier to capture or retain tion Systems Integrated on a Planar Glass Chip .” Anal.
other solid phase components, e.g ., another set of particles . Chem . 64 : 1926 - 1932; Harrison et al. ( 1993 ) “ Micromachin
Mobile Arrays 60 ing a Miniaturized Capillary Electrophoresis -Based Chemi
The arrays of the invention are optionally mobile . Arrays cal Analysis System on a Chip .” Science 261: 895 -897 ;
comprise particle sets such as beads , microspheres, cells , Harrison and Glavania ( 1993 ) " Towards Miniaturized Elec
fluidly transportable substrates, or the like . The particles are trophoresis and Chemical System Analysis Systems on
optionally moved by flowing fluids comprising the particles Silicon : An Alternative to Chemical Sensors .” Sensors and
through or to a desired location , e.g ., within a microfluidic 65 Actuators 10 : 107 - 116 ; Fan and Harrison ( 1994 ) “Microma
system . Particles or reagents can be moved within micro - chining of Capillary Electrophoresis Injectors and Separa
fluidic arrays by any of a variety of techniques, including tors on Glass Chips and Evaluation of Flow at Capillary
 US 10 ,138 ,517 B2
12
Intersections. Anal. Chem . 66 : 177 -184; Effenhauser et al. phoretic mobility of charged species within the electric field
( 1993) “Glass Chips for High -Speed Capillary Electropho applied to the structure . Such systems are more particularly
resis Separations with Submicrometer Plate Heights ” Anal. referred to as electrophoretic material transport systems. For
Chem . 65:2637 -2642 ; Effenhauser et al. (1994 ) “ High electrophoretic applications, the walls ofinterior channels of
Speed Separation of Antisense Oligonucleotides on a Micro - 5 the electrokinetic transport system are optionally charged or
machined Capillary Electrophoresis Device .” Anal. Chem . uncharged . Typical electrokinetic transport systems are
66 :2949 -2953 ; and Kovacs EP 0376611 A2 . made of glass, charged polymers, and uncharged polymers .
In general, these microfluidic systems can be adapted for The interior channels are optionally coated with a material
use in the present invention , i.e ., by introducing arrays into which alters the surface charge of the channel.
the microfluidic systems, e .g ., for the assays set forth herein . 10 A variety of electrokinetic controllers are described , e . g .,
A variety of array component introduction approaches for in Ramsey WO 96 /04547, Paree et al. WO 98 /46438 and
introducing array elements into microfluidic systems are
noted herein . Dubrow et al., WO 98 /49548, as well as a variety of other
Movement of Particles within Microfluidic Systems references noted herein .
The microfluidic devices which include arrays also can 15 allyToincludes
provide appropriate electric fields, the system gener
a voltage controller that is capable of applying
include other features of microscale systems, such as fluid
transport systems which direct particle movement within the selectable voltage levels , simultaneously , to each of the
array channel, incorporating any movementmechanism set reservoirs, including the ground . Such a voltage controller is
forth herein (e. g., fluid pressure sources for modulating fluid optionally implemented using multiple voltage dividers and
pressure in the array channel, electrokinetic controllers for 20 multiple relays to obtain the selectable voltage levels . Alter
modulating voltage or current in the array channel, gravity natively ,multiple independent voltage sources are used . The
flow modulators , magnetic control elements for modulating voltage controller is electrically connected to each of the
a magnetic field within the array channel, or combinations reservoirs via an electrode positioned or fabricated within
thereof). The microscale cavity can also include fluid each of the plurality of reservoirs. In one embodiment,
manipulation elements such as a parallel stream fluidic 25 multiple electrodes are positioned to provide for switching
converter, i.e ., a converter which facilitates conversion of at of the electric field direction in a microchannel, thereby
least one serial stream of reagents into parallel streams of
reagents for parallel delivery of reagents to a reaction site
causing the analytes to travel a longer distance than the
within the microscale cavity . For example , a capillary is physical length of the microchannel. Use of electrokinetic
transport to control material movement in interconnected
optionally used to sip a sample or samples from a microtiter 30 channel
|
plate and to deliver it to one of a plurality of channels, e.g., Ramsey . structures
An
was described , e.g., in WO 96 /04547 to
exemplary controller is described in U .S . Pat.
parallel reaction or assay channels . In addition , particle sets No. 5, 800 ,690 . Modulating voltages are concomitantly
are optionally loaded into one or more channels of a micro
fluidic device through one sipper fluidly coupled to each of applied to the various reservoirs to affect a desired fluid flow
the one or more channels and to a sample or particle source , 25 35 characteristic, e .g., continuous or discontinuous (e .g., a
such as a microwell plate . Indeed , one advantage of the regularly pulsed field causing the sample to oscillate direc
present system is the ability to provide parallel streams of tion of travel) flow of labeled components toward a waste
reagents to samples fixed on arrays. This is particularly the reservoir. Particularly ,modulation of the voltages applied at
advantageous, because the volumetric accuracy require various reservoirs can move and direct fluid flow through
ments for delivery of reagents is often less than the volu - 40 the interconnected channel structure of the device .
metric requirements for samples . A wide variety of inte Other Particle and Fluid Movement Approaches
grated microfluidic systems comprising serial to parallel Other methods of transport are also available for situa
fluid manipulation are described in Bousse et al., “ Parallel- tions in which electrokinetic methods are not desirable . For
ism in integrated fluidic circuits ,” SPIE 3259: 179 -186 example, sample introduction and reaction are optionally
( 1998 ) and in CLOSED LOOP BIOCHEMICAL ANALYZ - 45 carried out in a pressure- based system to avoid electroki
ERS ; WO 98 / 45481 and the references therein . netic biasing during sample mixing and high throughput
Electrokinetic Controllers systems typically use pressure induced sample introduction .
One method of achieving transport or movement of Pressure based flow is also desirable in systems in which
particles through microfluidic channels is by electrokinetic electrokinetic transport is also used . For example , pressure
material transport. “ Electrokinetic material transport sys - 50 based flow is optionally used for introducing and reacting
tems," as used herein , includes systems that transport and reagents in a system in which the products are electropho
direct materials within a microchannel and / or chamber con retically separated . In the present invention particle arrays
taining structure , through the application of electrical fields are optionally loaded using electrokinetic fluid control and
to the materials, thereby causing materialmovement through other reagents are flowed through the particle arrays under
and among the channel and/ or chambers , i.e ., cations will 55 pressure .
move toward a negative electrode, while anions will move Pressure is optionally applied to microscale elements ,
toward a positive electrode . For example , movement of e . g ., to a channel, region , or reservoir, to achieve fluid
fluids toward or away from a cathode or anode can cause movement using any of a variety of techniques . Fluid flow
movement of particles suspended within the fluid ( or even and flow of materials suspended or solubilized within the
particles over which the fluid flows). Similarly , the particles 60 fluid , including cells or particle sets, is optionally regulated
can be charged , in which case they will move toward an by pressure based mechanisms such as those based upon
oppositely charged electrode (indeed , in this case, it is fluid displacement, e. g ., using a piston, pressure diaphragm ,
possible to achieve fluid flow in one direction while achiev - vacuum pump , probe or the like to displace liquid and raise
ing particle flow in the opposite direction ). In this embodi- or lower the pressure at a site in the microfluidic system . The
ment, the fluid can be immobile or flowing. 65 pressure is optionally pneumatic , e. g ., a pressurized gas, or
In general, electrokinetic material transport and direction uses hydraulic forces, e. g ., pressurized liquid , or alterna
systems include those systems that rely upon the electro tively, uses a positive displacement mechanism , i.e ., a
 US 10 ,138,517 B2
13 14
plunger fitted into a material reservoir, for forcing material Particle Retention Regions and Other Specific Configu
through a channel or other conduit, or is a combination of rations
such forces . As noted , the particle sets of the arrays of the invention
In some embodiments, a vacuum source is applied to a can be fixed in place, or mobile . For example , the microscale
reservoir or well at one end of a channel to draw a fluidic 5 cavity can have a first microchannel which includes a
material through the channel. For example, a vacuum source particle movement region and a particle retention region .
The particle retention region typically includes at least one
is optionally placed at a reservoir in the present devices for set
drawing fluid into or through a channel, e. g., through a particle of the plurality of sets of particles disposed within the
porous particle retention element, e.g., a particle set . Pres 10 one set. retention
The
region , although it can include more than
particle “ retention ” area or region optionally
sure or vacuum sources are optionally supplied external to includes a region of increased or decreased microchannel
the device or system , e . g ., external vacuum or pressure depth or width or other physical barrier ( groove , mesh , net,
pumps sealably fitted to the inlet or outlet of the channel, or matrix , etc .), an electromagnetic field , a porous matrix ( e . g .,
they are internal to the device, e . g ., microfabricated pumps sieving matrices are optional components of microfluidic
integrated into the device and operably linked to the channel. 15 systems , as described below ), or other means of inhibiting
Examples of microfabricated pumps have been widely particle movement in or adjacent to the region .
described in the art. See, e . g ., published International Appli- For example , in embodiments where the microfluidic
cation No . WO 97 /02357 . cavity comprising the array has a first microchannel, the
Hydrostatic , wicking and capillary forces are also option - microchannel is optionally configured to include at least one
ally used to provide fluid pressure for continuous fluid flow 20 reagent flow region and at least one particle capture region .
of materials such as enzymes , substrates, modulators , or The at least one particle capture region optionally has an
protein mixtures . See , e . g ., "METHOD AND APPARATUS increased or decreased depth or width relative to the at least
FOR CONTINUOUS LIQUID FLOW IN MICROSCALE one reagent flow region , or is bounded by a region of
CHANNELS USING PRESSURE INJECTION , WICKING increased or decreased depth or width . For example , in one
AND ELECTROKINETIC INJECTION ,” by Alajoki et al. 25 embodiment, the devices include regions of width (or depth )
in U .S . Pat. No. 6 ,416 ,642. In these methods, an adsorbent sufficient to permit passage of particles, where the particle
material or branched capillary structure is placed in fluidic capture region is insufficient in dimension to permit free
contact with a region where pressure is applied , thereby passage of the particles. For example , where particles larger
causing fluid to move towards the adsorbent material or than 3 -4 microns are used , a channel region having a cross
branched capillary structure . The capillary forces are option - 30 sectional dimension smaller than 3 - 4 microns blocks flow of
ally used in conjunction with the electrokinetic or pressure the particles , while still permitting flow of fluids. Preferred
based flow in the present invention . The capillary action particle size ranges from about 0 . 1 microns to about 50
pulls material through a channel. For example a wick is microns; accordingly , in this embodiment, a physical barrier
optionally added to draw fluid through a porous matrix fixed which does not permit passage of a selected particle within
in a microscale channel or capillary . 35 this range of particle sizes can be used as a particle capture
Mechanisms forreducing adsorption of materials during element. In one aspect, a " particle capture region ” is
fluid -based flow are described in “ PREVENTION OF SUR - bounded on at least one side by such particle capture
FACE ADSORPTION IN MICROCHANNELS BY APPLI- elements . In another example , the reagent flow region com
CATION OF ELECTRIC CURRENT DURING PRES prises a cross -sectional dimension comprising passage of
SURE -INDUCED FLOW ” filed May 11 , 1999 by Paree et 40 selected particles and the particle retention or capture region
al., application Ser. No. 09/310 ,027 (now U .S . Pat. No. abuts a narrow channel region comprising a dimension
6 ,458 , 259) . In brief, adsorption of components, proteins, sufficiently small to inhibit movement of a selected particle
enzymes , markers and other materials to channel walls or through the narrow channel. Such narrow channel regions
other microscale components during pressure -based flow are typically less than about 10 um , more typically less than
can be reduced by applying an electric field such as an 45 5 um . Preferable narrow channel dimensions are about 5 um
alternating current to thematerial during flow . Alternatively, or smaller or about 3 um or smaller .
flow rate changes due to adsorption are detected and the flow In other embodiments , the particle capture region , e.g ., in
rate is adjusted by a change in pressure or voltage . a capillary or microchannel, comprises a device to capture a
Mechanisms for focusing labeling reagents, enzymes , set of particles , e . g ., chemically coated microspheres or
modulators, and other components into the center of 50 other chemically coated solid phase objects , e . g ., for a
microscale flow paths , which is useful in increasing assay sequential chemistry assay . The device typically comprises
throughput by regularizing flow velocity , e . g ., in pressure a physical barrier, e . g ., a porous barrier with a defined pore
based flow , is described in “ FOCUSING OF MICROPAR - size , that captures solid phase objects having a larger mean
TICLES IN MICROFLUIDIC SYSTEMS” by H . Garrett diameter than the pore size. The upstream end of the
Wada et al . filed May 17, 1999 , application Ser. No . 60 / 134 , 55 capillary or microchannel is typically inserted in a reagent,
472. In brief, sample materials are focused into the center of e .g ., a liquid reagent, and the downstream end is typically
a channel by forcing fluid flow from opposing side channels coupled to a vacuum source or electrokinetic controller
into the main channel comprising the cells, or by other fluid which transports the reagents across the bed of the captured
manipulation . solid phase . The first reagent brought into the channel is
In an alternate embodiment, microfluidic systems can be 60 typically the solid phase in a suitable buffer and subsequent
incorporated into centrifuge rotor devices , which are spun in reagents follow sequentially, e. g ., sequencing regents. For
a centrifuge . Fluids and particles travel through the device example , DNA coated microspheres are optionally captured
due to gravitational and centripetal/ centrifugal pressure in a particle retention area for sequencing by synthesis ,
forces . wherein subsequent reagents comprise dNTPs brought in
Fluid flow or particle flow in the present devices and 65 one at a time, e. g., with sufficient rinsing between nucleo
methods is optionally achieved using any one of the above tides . Other applications include DNA probes and the like .
techniques , alone or in combination . The captured solid phase is typically captured in the particle
 US 10 ,138 ,517 B2
15 16
retention area , e.g., by a physical barrier. Such barriers are ods for making the arrays, microfluidic devices, integrated
optionally formed by a fixed particle set, by a sintered glass systems and the like are also provided .
frit, e. g., a preformed frit that has been inserted into the In addition to the aspects set forth above , in one specific
channel or a frit that has been fabricated integral to the set ofmethods, a plurality of particle sets is flowed through
capillary or channel. Alternative barriers , include a frit, e . g ., 5 a first microscale channel having a particle flow region
a fixed set of particles, formed from epoxy coated micro - having a height to width aspect ratio greater than 1.
spheres, e. g ., providing an appropriate pore size . The fixed In another set of manufacturing embodiments, a first
set of particles, e. g., epoxy coated microspheres, form a particle set is flowed through the particle flow region and the
porous barrier which captures other particle sets or packets. 10 resulting in a setfirstis fixed
first particle fixed in a first location in the channel,
For example, a set of particles is optionally flowed through retention area to capture particle set, e. g., forming a particle
a capillary , e.g., with glue or epoxy . The capillary is centri second particle set is flowed throughother
or retain sets of particles. A
the particle flow region
fuged to flow the glue or epoxy resin over the particles. to a region abutting the first fixed particle set, where the first
Excess glue is then removed , leaving a fixed particle matrix fixed particle set blocks further flow of the second particle
which is optionally used to capture or retain flowing or 15
tain Towing of 15 set in the direction of flow , resulting in a second fixed
mobile beads, e. g., chemically coated beads . Othermaterials particle set. In some embodiments , a third particle set
used to form the barrier include, but are not limited to , glass , through the particle flow region to a region abutting the
plastic , and other materials comprising an appropriate pore second fixed particle set , wherein the second fixed particle
size . The barrier or frit is optionally contained in a housing , set blocks further flow of the third particle set in the
which housing is optionally a capillary or a machined or 20 direction of flow , resulting in a third fixed particle set. In
molded plastic or glass unit. some embodiments , the first particle set is fixed in a particle
The flow region can be in the same plane or transverse to retention region , which inhibits movement of selected par
the particle capture region . In typical embodiments , the ticles from the particle retention region e .g., by a physical
microfluidic device comprising the microscale cavity barrier to movement of the particles that is proximal to or
includes a plurality of microchannels , which are optionally 25 within the particle retention region , a magnetic field proxi
intersecting or non intersecting and which optionally fluidly mal to or within the particle retention region , a chemical
connect with reservoirs , wells or the like. The microfluidic particle capture moiety that is proximal to or within the
device can include , or be coupled to ( e . g ., through a channel, particle retention region or the like .
microchannel, sipper capillary , pipettor, pressure pipettor, In one aspect, the first particle set is fixed in a particle
electropipettor, etc . ), external sources of reagents or par- 30 retention region , which abuts the particle flow region . The
ticles , permitting loading of the particles , or reagents which particle retention region has a decreased or increased height
interact with the particles . Thus, in one embodiment, the to width aspect ratio as compared to the particle flow region ,
device comprises a loading channel region (or particle which decreased or increased height to width aspect ratio , in
loading channel region ) coupled to a source of at least a first combination with the dimensions of the particle flow region ,
selected particle type or reagent coupled to the microscale 35 creates a physical barrier at the point where the dimensions
cavity . For example , the source of particle type or reagent vary and inhibits movement of the first particle set from the
can be a microwell plate external to the body structure particle retention region . Alterations in the aspect ratio can
having at least one well with the selected particle type or also be used to provide regions of faster or slower particle
reagent, a well disposed on the surface of the body structure flow , depending on the shape of the particles. In another
comprising the selected particle type or reagent, a reservoir 40 aspect, the particle retention area is formed by a set of
disposed within the body structure comprising the selected particles , e .g ., epoxy coated particles, which has been fixed
particle type or reagent ; a container external to the body in position in the channel, e . g ., in a housing or by glue or
structure comprising at least one compartment comprising epoxy. Alternatively, a sintered glass frit or other porous
the selected particle type or reagent, or a solid phase matrix is placed in the channel or fabricated within the
structure comprising the selected particle type or reagent. 45 channel to form a barrier .
The particle loading channel region is optionally fluidly In another set of manufacturing embodiments, methods of
coupled to one or more of: a pipettor channel with a port making a microfluidic particle array are provided . In the
external to the body structure , an electropipettor channel methods, a microfluidic device comprising a microscale
with a port external to the body structure , a sipper capillary, cavity is provided . One or more particle sets are flowed into
e . g ., external to the body structure , fluidly coupled to one or 50 the microscale cavity . One or more particle modification
more microwell plates, a pipettor channel with a port inter - reagents are flowed into contact with the one or more
nal to the body structure , an internal channelwithin the body particle sets, thereby producing a plurality of particle sets
structure fluidly coupled to a well on the surface of thebody comprising the microfluidic particle array .
structure , an internal channel within the body structure Further understanding of specific configurations is devel
fluidly coupled to a well within the body structure, or the 55 oped by consideration of the following specific embodi
like. ments.
The ordered array optionally includes a plurality of FIG . 1 provides microfluidic device 1 comprising reagent
sample sets associated with the plurality of particle sets , wells 10 -30 , additionalwells 40 -70 and waste well 80. The
where each sample set is physically associated with one or w ells are fluidly connected to microchannel 90 comprising
more ofthe plurality of particle sets . In addition , the ordered 60 intersections 100 and 120 . Additionalwells 40 - 70 are fluidly
array optionally includes a plurality of reagent sets associ- coupled to intersections 100 - 130 . Channel constriction 140
ated with the plurality of particle sets, where each reagent set provides a bead capture area . As shown in detail , constric
is physically associated with one or more of the plurality of tion 140 traps beads/particles 150 in particle retention region
particle sets . These reagent and particle sets are optionally 160 . In one example of this embodiment, the particles are
associated with each other, typically such that each reagent 65 between about 10 and about 4 microns, while particle
set and each particle set is physically associated with one or retention region 160 is about 15 microns and constriction
more of the plurality of particle sets . Manufacturing meth 140 , e.g., a narrow channel region , is about 3 microns in
 US 10 ,138 ,517 B2
17 18
diameter. Alternatively , the particles are larger than about 12 in a microfluidic device ) comprises particle retention ele
microns and particle retention region 160 is about 20 ment 1410 and particle set 1415 . Particle retention element
microns to about 50 microns and constriction 140 is about 3 1410 comprises a particle set that is fixed or immobilized in
microns to about 12 microns. For particles larger than about capillary 1405 , thus creating particle retention region 1420
15 microns, constriction region 140 is less than about 15 5 in capillary 1405 . In a sequencing reaction , a set of particles,
microns . Various particle sizes are used with various con - e .g ., coated with a DNA template , is optionally flowed
striction measurements . Typically , the constriction is smaller through capillary 1405 from inlet region 1425 . For example ,
than the particles of interest. An additional embodiment inlet region 1425 is optionally fluidly coupled to a sipper
utilizing only one channel is provided in FIG . 14 . Channel capillary, which during operation is fluidly coupled to a
or capillary 1405 is optionally coupled to a sipper capillary 10 microwell plate . The set of DNA coated particles flows
that draws reagents , e.g ., from a microwell plate , and through capillary 1405 and is held or retained against
through the capillary, e. g., through a pressure or electroki particle retention element 1410 , causing the set of DNA
netic controller. particles to become fixed in capillary 1405 , such as particle
FIG . 2 , Panel A , provides microfluidic device 201 com - set 1415 . Sequencing reagents are then optionally flowed
prising channel 210 . In a region ( e.g ., a bottom region ) of 15 across particle set 1415 and optionally rinsed from the
channel 210 several particle retention regions 220 - 250 retain channel between introductions of different reagents . Particle
particle sets 260- 290 . In this simple embodiment, particle retention element 1410 forms a porous barrier, which stops
sets 260 - 290 constitute an array , or a portion of an array . particle set 1415 , thus allowing, e .g ., a series of reagents to
Particle sets 260 - 290 optionally comprise particle members be flowed across the DNA template and then rinsed away if
which are of smaller dimension than channel 210 . In this 20 desired , e. g., four different nucleotides are serially flowed
configuration , particle sets 260 - 290 are optionally magnetic across the template coated particle set 1415 and unincorpo
and held in place by application of an appropriate magnetic rated nucleotides are optionally rinsed away between steps .
field . In other embodiments , channel 210 is of sufficiently At the same time, the pore size of particle retention element
narrow dimension that members of particle sets 260 - 290 1410 has a pore size allowing, e . g ., liquid reagents through
cannot exit retention regions 220 - 250 . This embodiment is 25 so they are optionally rinsed out of capillary 1405 , e . g ., into
depicted in FIG . 2B . Effective channel dimensions can be a waste reservoir proximal to downstream region 1430 .
altered during operation by addition of a matrix or size An alternate version of a particle retention element is
exclusion gel to the channel. shown in FIG . 15 . Particle retention element 1510 is sealed
FIG . 3 , panels A and B provide an additional aspect of the within housing 1540. Particle sets and/or liquid reagents are
invention . The channels and particles used are typically 30 flowed through capillary 1505 . Particle sets with a mean
smaller than the ones shown . An expanded view is used for diameter greater than the pore size of the particle retention
ease of illustration .Microfluidic device 301 comprises wells element are stopped in capillary retention region 1515 by
310 - 340 . The wells are fluidly connected through a network particle retention element 1510 and liquid reagents and
of channels comprising channels 350 - 390 and intersections particle sets with a smaller mean diameter than the pore size
400 -410 . Channel region 415 comprises particle sets 420 - 35 of particle retention area are flowed past particle retention
440. These particle sets can be immobile or mobile . For area 1515 , e .g ., by a vacuum fluidly coupled to downstream
example , the particles are optionally held in place by a capillary region 1530 . In another embodiment, centrifugal
particle retention element, e. g ., a porous barrier. Particle sets force is used to draw reagents through capillary 1505 .
420- 440 are optionally the same size and/ or shape or dif- Alternatively,multiple solid phase components are flowed
ferent sizes and /or shapes . In addition , each particle set 40 through and retained by a particle retention element. FIG . 16
optionally comprises a different number of particles . shows particle retention element 1610 serving as a barrier to
FIG . 4 , panels A , Band C show an additional embodiment particle set 1615 , which serves as a barrier to particle set
(the channel dimensions are shown larger than typical chan 1617, which serves as a barrier to particle set 1619 . Three
nel dimensions for ease of illustration ). Microfluidic device particle sets are thus stacked in capillary 1605 . Particle sets
4001 comprises wells 4005 -4025 . The wells are fluidly 45 1615 , 1617, and 1619 optionally have the same mean
connected through channels 4030 -4040 and through double - diameter or different mean diameters. Particle retention
depth channels 4045 -4050 and single depth wide channel element 1610 comprises a set of particles which when fixed
regions 4060 -4070 . Double - depth channel regions 4045 - within capillary 1605 forms a porous barrier to the move
4050 act as particle retention regions by trapping particles ment of other particle sets. Liquid reagents are optionally
sets 4055 -4057. Reagents can be passed across the trapped 50 flowed through capillary 1605 to contact particle sets 1615 ,
particle sets by flowing the reagents through single - depth 1617 , and 1619 . The liquid reagents are optionally intro
wide channel regions 4060 -4070 . In one aspect , packets of duced from a microwell plate via a sipper capillary fluidly
particles are aligned to form trains of particles in double coupled to upstream region 1625 . The liquid reagents flow
depth channels 4045 - 4050 . across and through particle sets 1615 , 1617 , and 1619 and
FIG . 5 shows an embodiment adapted to sequencing . 55 react, e. g ., with a DNA template and primer on the particles,
Microfluidic device 500 comprises main channel 510 and and flow through particle retention element 1610 , e. g.,
reagent introduction channels 515 -530 (as depicted , these pulled by a vacuum fluidly coupled to downstream capillary
are coupled to reagents for separate sequencing reactions, region 1630 .
e . g ., comprising A , G , C , or T nucleotides. Sample train 531 FIG . 6 shows an embodiment adapted to high -throughput
comprising a plurality of samples , e . g ., particle sets 535 - 60 methods. In particular, a “ sipper” (e . g ., an electropipettor ),
550 , is passed back and forth through intersections 560 -590 . e .g ., sipper 610 , draws one or more sets of beads or reagents
Reagent from channels 515 -530 is flowed across each ABCD and dispenses it into channel 615 or into multiple
sample (or selected samples ) in train 531 as the train passes channels, e. g ., parallel assay channels, where the sets are
the corresponding coupled intersection . Particle retention sent as packet 620 across target beads or reagent sites, e. g .,
element 595 is an optional element. 65 regions 625 or 630 . This is faster than sipping beads or
FIG . 14 shows an additional device embodiment adapted reagents individually and passing them across the relevant
to sequencing . Capillary 1405 (which is optionally a channel reaction site .
 US 10 ,138 ,517 B2
19 20
FIG . 7 shows a multiplexing arrangement useful for DNA barrier that has a pore size smaller than the mean diameter
sequencing. The bold straight lines represent fluid channels of the particle sets of interest, e . g ., particle sets 1005 - 1020 .
connected to a sipper channel. The dotted lines represent FIG . 11 , panel A , shows a parallel particle array in which
electrical wires or pressure conduits connecting each of the sample particle sets 1105 are separated by blank particle sets
reagent lines to a single controller. In this example, 3 sipper 5 1110 in channels 1115 - 1125 . Panel B shows particle sets
channels and 12 electrodes or pressure ports require only 4 1130 - 1145 in particle retention region 1150 abutting narrow
channel region 1160 . Panel C depicts an alternate embodi
controllers, instead of the more typical 12 . An example of an ment
assay using this arrangement is DNA sequencing , in which retention in which particle sets 1130 - 1145 are held in particle
DNA samples are on beads, one per sipper channel. The side 10 FIG . 12region 1150 which abuts physical barrier 1155 .
shows a schematic integrated system of the
channels have different nucleotides that are passed over the invention . A computer or other microprocessing device
beads sequentially . All of the bead samples will have the directs materials from a sample system to an array for
same reagent stream pass across them . For example , mul analysis . If appropriate to the assay , array members are
tiple DNA samples , e . g ., three different DNA templates , are directed the
from a particle library to the array to comprise or
simultaneously sequenced using only four controllers, one one 1515 modify
for each reagent line and one for the main line.Alternatively , optionally in array
modify the . The selection of particle members is
response to an assay signal from the array. In
introduction of reagents from a sipper capillary fluidly general, assay signals are directed from the array to a
coupled to a microwell plate and to each of themain reaction detector which detects the signals. The signal information is
channels in FIG . 7 allows the DNA samples to be sequenced converted to a digital format and sent to the computer, which
using only one controller coupled to each of the reaction 20 reads and /or stores the information . Optionally , the infor
channels. mation is used to select additional samples by or from the
FIG . 8 shows access to a particle library using one ormore sampling system and/or additional particle sets from the
sippers. In particular, body 801 comprises particle channels particle library .
810 -825 connected to sipper channel 830 which accesses a For example , during operation of the integrated system
particle library (e . g ., particle sets on a microwell plate ). 25 illustrated in FIG . 12 , an array is formed by accessing a
Packets of particles ( e . g ., comprising nucleic acid templates particle library , following an instruction set provided by the
for sequencing) are directed (e .g ., by electrokinesis or pres computer. The computer directs flow of fluidic ( or particu
sure -based mechanisms) into channels 810 - 825 to provide late ) reagents to the array through the sampling system (e . g .,
an array of particle sets . For example , an electrical potential a system comprising a sipper channel which contacts and
or a vacuum is optionally applied at one or more of wells 30 sips fluid from a relevant source of material, e . g ., a micro
835 - 850 . Similarly , reagents (e . g ., sequencing reagents ) are titer plate ). The fluidic reagents interact with the array
flowed from any of wells 860 - 875 through fine channel members , providing a detectable signal. The signal is
network 880 and into broad channel 885 , across the particle detected by the detector and converted into digital informa
sets within channels 810 -825 , where the reagents interact tion which is stored and /or manipulated by the computer.
with the particles in the channels . Alternatively, an indi- 35 Optionally, the digital information is used to provide the
vidual channel is used in place of fine channel network 880 . logical basis for selecting additional array or fluidic or
The particles are first flowed within fine channel network particulate reagents from the particle library and /or through
880 to provide more even flow of reagents into broad the sampling system . In these cases , the computer selects the
channel 885 . Waste reagents are flowed into waste well 890 . additional array or fluidic or particulate reagent(s ) and
Flow is provided by applying electrical or pressure poten - 40 directs flow to the array and or modification or movement of
tials at one or more of wells 860 -875 and 890 . Although the array .
illustrated for clarity as a single sipper channel arrangement, Particle Stacking
multiple sipper channels interconnected to microfluidic As noted above , the particle retention region can take any
structures can also be used , e .g ., for simultaneous and /or of a number of forms in the present invention. For example ,
parallel access to samples or fluidic reagents . Alternatively , 45 in one embodiment, a first set of particles is flowed into a
a train of reagents is flowed from a microwell plate into microfluidic region ( e . g ., channel region , chamber, etc . )
sipper 830 and directly into each of channels 810 -825 . having a region with sufficiently small dimension to inhibit
FIG . 9A shows an expanded cross-sectional view of movementof the first particle set. The first particle set stacks
particle packets within deep channels having an intersecting against the small dimensioned region of the channel. Sub
broad shallow channel. In particular, particle sets 900 -910 50 sequently , a second , third , fourth . . . nth set of particles can
are shown in channels 815 - 825 , i. e., the view is a cross be moved into the channel, where they will stack against,
section of a portion of FIG . 8 comprising the channels. Flow e. g ., the first particle set. Even though the second . . . nth
of reagents is through broad channel 915 , across particle sets particle sets can be small enough to pass through the small
900 - 910 . As depicted , body 801 comprises upper layer 930 dimensioned region of the channel, they are retained by
and lower layer 940 . FIG . 9B shows essentially the same 55 stacking against the first particle set. Thus, the first particle
elements in an expanded top view , additionally depicting set acts as a matrix preventing passage of subsequent
particle packets 950 - 975 . particle sets. In this embodiment, each set of stacked par
FIG . 10 shows sequential loading of particle sets or ticles is larger in diameter than the typical voids between the
packets into a channel. Particle set 1005 is flowed through particles of the adjacent set. Thus, large retention particles
channel 1000 until blocked by particle retention element 60 can be trapped in a region of a microfluidic device and
1040 . Over time, particle sets 1010 - 1020 are accessed from medium sized particles can then be stacked next to the large
a particle library, with each particle set abutting the previ- particles, where the medium sized particles are larger in
ously loaded particle set(s ). Particle sets 1005 - 1020 are dimension than the voids between the large particles . Sub
optionally the same or different sizes . Particle retention sequently , small particles can be stacked next to the medium
element 1040 is optionally a sintered glass frit , glass, plastic, 65 sized particles , where the small particles are larger in
a fixed set of particles , e . g ., epoxy coated particles , or the diameter than the voids between the medium sized particles .
like . Typically , particle retention element 1040 is a porous Optionally, the small particles can be smaller than the voids
 US 10 , 138 ,517 B2
21 22
between the large particles, as they will be blocked from into the particle retention region where it is retained by
downstream flow by the medium sized particles. Even magnetic or affinity forces (e . g ., covalent or non - covalent
smaller particles can be stacked next to the small particles , chemical bonding between the particle or molecules dis
etc . Of course , large particles can also be stacked next to posed on the particle and a region of the relevant microflu
fixed or otherwise retained small particles, as long as the 5 idic system ). Second , third . . . nth particle sets are flowed
large particles cannot pass between the voids of the stacked into contact with the fixed particle , where the fixed particle
or fixed small particles . acts as a retention element to block flow of the fixed
For example , beads having a cross - section of about 100 particles . As above , small particles are typically stacked next
um to about 200 um are optionally used to form a fixed to fixed or otherwise retained larger particles , e . g ., where the
porous matrix or barrier in a microchannel. The porous 10 small particles cannot pass between the voids of the stacked
matrix is optionally used to capture or retain smaller par - or fixed larger particles (of course , the small particles can be
ticles , e.g ., particles comprising a cross -section of about 30 magnetic or affinity particles, with larger particles stacking
um to about 80 um , e .g ., a 40 um particle set. Even smaller against the fixed smaller particles as well ) .
particles, e .g., particles having a cross-section less than 30 As described supra , particles can beessentially any size or
um , e . g ., about 5 um to about 11 um , are then captured and 15 shape. In embodiments where arrays are made by stacking
retained, e . g ., by the second particle set, i. e., the 40 um of adjacent sets of particles, it is desirable for sets to be of
particle set. sufficient diameter that they cannot flow between adjacent
There are several advantages to embodiments where particle sets . Thus, in one typical aspect, the smallest dimen
particle sets are retained by other particle sets , rather than sion of a set of particles is larger than the voids between the
simply by e . g ., physical dimensions of the microfluidic 20 adjacent downstream particle set.
system in which the particles are flowed . For example , the Number and Types of Array Members
particle retention region is switchable in these configura - The number of ordered sets constituting the array depends
tions , providing for dynamic construction and removal of on the selected application . For example, as discussed in
the arrays and of the particle retention region (advantages of more detail herein , one exemplar array for sequencing
switchable arrays are set forth supra , including, e . g ., creation 25 nucleic acids comprises about 2 , 3 , or 4 sets of particles ( e. g .,
of “ smart” and “ programmable ” particle arrays ) . Another beads, cells , microspheres, etc .). In other implementations ,
advantage to this configuration is that particles of extremely 5 , 10 , 50 , 100 , 500 , 1000 , 5 ,000 , 10 , 000 , 50 , 000 or even
small dimensions can be used in the second . . . nth position, 100,00 or more different sets of particles can be present in
which increases the diversity of particle types which are the arrays. The precise number of particles in an array
accessible by this approach . 30 depends on the intended use of the array . For example , larger
Small particles also have properties which are , them - arrays are especially useful, e .g ., in screening molecular
selves, advantageous for some embodiments . For example , libraries against one or more targets bound to the member
several small particles have significantly more surface area particles of the particle sets . Smaller arrays can be used as
than a single large particle that occupies the same volume as to screen a smaller number of targets, as is common , e .g ., in
the smaller particles . This increase in surface area allows 35 diagnostic applications, where one or a few targets ( e . g .,
attachment/ association of a greater number of molecules to nucleic acids corresponding to various disease states, such
the particle, increasing the density of the molecules of as altered levels or altered types of oncogene products , p53 ,
interest in the array. This is useful, e . g ., for detecting assay presence of infectious organisms (HIV and other viruses,
signals which result from interaction with the molecules of bacteria , etc .) are desirably screened .
interest, i.e ., for increasing the signal-to -noise ratio of assay 40 The array components ( i. e ., particles ) of the arrays of the
signals in the assay . invention can be essentially any discreet material which can
For example, in one embodiment, a linear array of beads be flowed through a microscale system . Example particles
is made in a microfluidic channel by stacking beads of a include beads and biological cells . For example, polymer
diameter larger than the depth of the downstream channel beads (e. g ., polystyrene , polypropylene , latex , nylon and
created by a shelf, raised area, narrowed area , or other 45 many others ), silica or silicon beads, clay or clay beads,
constriction within the channel (or other microfluidic struc - ceramic beads, glass beads , magnetic beads, metallic beads ,
tural element such as a chamber, cavity or the like). The size inorganic compound beads, and organic compound beads
of the beads determines the surface area and binding capac - can be used . An enormous variety of particles are commer
ity of the beads. The available binding capacity of the array cially available , e. g ., those typically used for chromatogra
is increased by stacking beads of a smaller diameter adjacent 50 phy ( see, e . g ., the 1999 Sigma “ Biochemicals and Reagents
to the larger diameter beads, such that the diameter of the for Life Sciences Research ” Catalog from Sigma ( Saint
smaller diameter beads is larger than the voids between the Louis, Mo.), e . g ., pp . 1921- 2007 ; The 1999 Suppleco “ Chro
larger diameter beads. For example , in one embodiment, a matography Products ” Catalogue , and others ), as well as
selected set of 4 . 4 micron beads have surface area of, those commonly used for affinity purification ( e. g ., Dyna
roughly , 1.286 * 1012 um´/g and the spaces between the beads 55 beadsTM from Dynal, as well asmany derivatized beads, e.g.,
are approximately 1. 8 microns. Packing beads of 2.5 various derivatized DynabeadsTM (e.g ., the various magnetic
microns next to the 4 . 4 micron beads provides about 1 . 7 DynabeadsTM , which commonly include coupled reagents )
times as much surface area per unit volume as the 4 .4 micron supplied e . g ., by Promega , the Baxter Immunotherapy
beads. This procedure is optionally repeated with increasing Group , and many other sources ).
surface area as the adjacent bead packages become smaller 60 A wide variety of particles useful in the present invention
and smaller. include those used as components of sieving and molecular
In a similar aspect, magnetic particles or affinity particles partition matrixes in the art . Many such matrixes are avail
can be used to create particle retention regions for non - able, and can be used to constitute particle arrays in the
magnetic / non affinity particles . In these embodiments , the apparatus of the invention . For example , a variety of sieving
first particle set (which is, e .g., a magnetic particle (i.e ., a 65 matrixes , partition matrixes and the like are available from
particle which generates a magnetic field or which is Supelco , Inc. (Bellefonte , Pa ., see , e . g ., the 1997 (or later )
attracted to a magnetic field ), or an affinity particle ) is flowed Suppleco catalogue). Common matrixes which are useful in
 US 10 ,138,517 B2
23 24
the present invention include those generally used in low thione resins , hydrophobic resins , immunochemical resins,
pressure liquid chromatography, gel electrophoresis and lectin resins , nucleotide/ coenzyme resins, nucleic acid res
other liquid phase separations. Matrix materials designed ins , and specialty resins are available , e . g ., from Suppleco ,
primarily for non - liquid phase chromatography are also SIGMA , Aldrich or the like . See also , Hermanson et al .
useful in certain contexts, as the materials often retain 5 ( 1992 ) Immobilized Affinity Ligand Techniques Academic
particulate characteristics when suspended in fluids. For a Press.
discussion of electrophoresis matrixes see , e . g., Weiss Other particulate media commonly used , e.g., in chroma
( 1995 ) Ion Chromatography VCH Publishers Inc .; Baker tography are also adaptable to the present invention , includ
( 1995 ) Capillary Electrophoresis John Wiley and Sons ; ing activated aluminas, carbopacks , carbosieves, carbo
Kuhn (1993 ) Capillary Electrophoresis : Principles and Prac - 10 waxes, chromosils, DEGS, Dexsil , Durapak , Molecular
tice Springer Verlag ; Righetti ( 1996 ) Capillary Electropho Sieve , OV phases, porous silica, chromosorb series packs ,
resis in Analytical Biotechnology CRC Press ; Hill ( 1992 )
Detectors for Capillary Chromatography John Wiley and HayeSep series, Porapak series , SE - 30 , Silica Gel, SP -1000 ,
Sons; Gel Filtration : Principles and Methods (5th Edition ) SP - 1200, SP - 2100 , SP -2250 , SP - 2300 , SP2401, Tenax ,
TCEP, supelcosil LC - 18 -S and LC - 18 - T , Methacrylate /
Pharmacia ; Gooding and Regnier ( 1990 ) HPLC of Biologi - 15 DVBm
cal Macromolecules: Methods and Applications (Chrom . , polyvinylalcohols, napthylureas, non -polar methyl
Sci. Series, volume 51) Marcel Dekker and Scott ( 1995 ) silicone , methylpolysiloxane , poly ( ethylene glycol) biscya
Techniques and Practices of Chromatography Marcel nopropyl polysiloxane and the like .
Dekker, Inc. Ion exchange chromatography resins comprising particu
Commercially available low pressure liquid chromatog - 20 late material are commercially available , including from EM
raphy media suitable as particulate material (i.e .,material for Separations (Gibbstown, N .J.), BioSepra (Marlborough ,
making particle sets) in a variety of applications include , Mass.), Polymer Laboratories (Amherst, Mass.), Perseptive
e .g .,non -ionic macroreticular and macroporous resinswhich Biosystems (Cambridge , Mass.), Toso Haas (Montgomery
adsorb and release components based upon hydrophilic or ville, Pa.) and Pharmacia (Uppsala , Sweden ).
hydrophobic interactions, such as Amberchrom resins 25 As noted herein , the definition for particles as intended
(highly cross - linked styrene /divinylbenzene copolymers herein includes both biological and non - biological particle
suitable for separation of peptides , proteins, nucleic acids, material. Thus , cells are included within the definition of
antibiotics, phytopharmacologicals, and vitamins ); the particles for purposes of the present invention .
related Amberlite XAD series resins (polyaromatics and Cell based microfluidic assays are described in a variety
acrylic esters ) and amberchroms (polyaromatic and 30 of publications by the inventors and their co -workers ,
polymethacrylates ) (manufactured by Rohm and Haas, including, Paree et al. " High Throughput Screening Assay
available through Suppleco ); Diaion (polyaromatic or Systems in Microscale Fluidic Devices” WO 98 /00231 and
polymethacrylic beads ); Dowex (polyaromatics or substi - Knapp et al. “ Closed Loop Biochemical Analyzers ” (WO
tuted hydrophilic functionalized polyaromatics) (manufac 98 / 45481 ; PCT/US98 /06723 ). It is expected that one of skill
tured by Dow Chemical, available through Suppleco ); Duo - 35 is fully able to culture cells and introduce them into micro
lite ( phenol-formaldehyde with methanolic functionality ), fluidic systems. In addition to Paree et al. and Knapp et al.,
MCI GEL sephabeads, supelite DAX -8 (acrylic ester ) and many references are available for the culture and production
Supplepak ( polyaromatic ) ( all of the preceding materials are of many cells , including cells of bacterial, plant, animal
available from Suppleco ). For a description of uses for (especially mammalian ) and archebacterial origin . See e .g .,
Amberlite and Duolite resins , see , Amberlite /Duolite Anion 40 Sambrook , Ausubel, and Berger (all supra ), as well as
Exchange Resins ( Available from Suppleco , 1997 Cat No . Freshney (1994 ) Culture of Animal Cells , a Manual of Basic
T412141) . Gel filtration chromatography matrixes are also Technique , third edition Wiley -Liss , New York and the
suitable , including sephacryl, sephadex , sepharose, super - references cited therein , Humason (1979 ) Animal Tissue
dex , superose, toyopearl, agarose , cellulose, dextrans, mixed Techniques , fourth edition W .H . Freeman and Company ;
bead resins, polystyrene , nuclear resins, DEAE cellulose , 45 Ricciardelli , et al., ( 1989 ) In Vitro Cell Dev. Biol . 25 : 1016
Benzyl DEA cellulose , TEAE cellulose, and the like 1024 ; Payne et al. (1992 ) Plant Cell and Tissue Culture in
(Suppleco ). Liquid Systems John Wiley & Sons, Inc. New York , N . Y .
Gel electrophoresismedia comprising particulate material (Payne ); Gamborg and Phillips (eds ) ( 1995 ) Plant Cell ,
useful in making particle sets include silica gels such as Tissue and Organ Culture; Fundamental Methods Springer
Davisil Silica , E . Merck Silica Gel, Sigma -Aldrich Silica 50 Lab Manual, Springer- Verlag (Berlin Heidelberg New York )
Gel (all available from Suppleco ) in addition to a wide range (Gamborg ); and Atlas and Parks ( eds ) The Handbook of
of silica gels available for various purposes as described in MicrobiologicalMedia ( 1993 ) CRC Press, Boca Raton , Fla .
the Aldrich catalogue/handbook (Aldrich Chemical Com (Atlas ). Additional information for plant cell culture is found
pany (Milwaukee , Wis .)) . Preferred gel materials include in available commercial literature such as the Life Science
agarose based gels , various forms of acrylamide based gels 55 Research Cell Culture Catalogue ( 1998 ) from Sigma-Al
(reagents available from , e .g ., Suppleco , SIGMA , Aldrich , drich , Inc (St Louis , Mo.) (Sigma -LSRCCC ) and, e. g., the
SIGMA -Aldrich and many other sources ) colloidal solutions Plant Culture Catalogue and supplement ( 1997 ) also from
such as protein colloids ( gelatins ) and hydrated starches. Sigma-Aldrich , Inc (St Louis, Mo.) (Sigma- PCCS ). One
A variety of affinity media for purification and separation particularly preferred use for cell-based microfluidic assays
of molecular components are also available , including a 60 is to screen binding and /or internalization of cell ligands ,
variety of modified silica gels available from SIGMA , e.g., cell receptor ligands, drugs, co - factors, etc . This screen
Aldrich and SIGMA- Aldrich , as well as Suppleco , such as ing is considerably facilitated by arraying different cell sets
acrylic beads , agarose beads, Mono beads, cellulose , sep into arrays of cells, which can then have reagent trains
harose , sepharose CL , toyopearl or the like chemically comprising any factor to be tested for in vitro cellular
linked to an affinity ligand such as a biological molecule . A 65 activity flowed across the cell sets. Of course , cells can also
wide variety of activated matrixes, amino acid resins, avidin be present in reagent trains and passed into contact with
and biotin resins, carbohydrate resins, dye resins, gluta other array members .
 US 10 ,138,517 B2
25 26
Cells can exist as sets of particles in a variety of formats example, Silicon Compounds Registry and Review , United
in the present invention . For example , cells can be fixed to Chemical Technologies, Bristol, Pa. Additionally , the art in
solid supports such as beads or other microparticles. Thus, this area is very well developed and those of skill will be
arrays of the invention can include heterogeneous particles able to choose an appropriate molecule for a given purpose .
comprising solid supports and cells or other components of 5 Appropriate molecules can be purchased commercially , syn
interest. Cells can also be trapped using strategies similar to thesized de novo , or it can be formed by modifying an
those described herein for particles generally, i.e ., by physi- available molecule to produce one having the desired struc
cal trapping mechanisms. In addition , as cells comprise ture and / or characteristics.
surface proteins and other molecules, it is convenient to fix The substrate linker attaches to the solid substrate through
cell binding molecules (cell receptor ligands, cell wall 10 any of a variety of chemical bonds. For example , the linker
binding molecules , antibodies, etc .) either to regions of the is optionally attached to the solid substrate using carbon
channels of the microfluidic device, or to particles which are carbon bonds , for example via substrates having (poly )
then fixed or localized in position by the methods described trifluorochloroethylene surfaces , or siloxane bonds (using,
herein . for example , glass or silicon oxide as the solid substrate ).
The array particles can have essentially any shape, e .g ., 15 Siloxane bonds with the surface of the substrate are formed
spherical, helical, spheroid , rod - shaped , cone- shaped , cubic, in one embodiment via reactions of derivatization reagents
polyhedral, or a combination thereof (of course they can also bearing trichlorosilyl or trialkoxysilyl groups. The particular
be irregular, as is the case for cell -based particles ). In linking group is selected based upon, e .g., its hydrophilic /
addition , the particles can be a variety of sizes. Typically, the hydrophobic properties where presentation of an attached
particles are about 0 .1 um to about 500 um . Alternatively , 20 polymer in solution is desirable . Groups which are suitable
the particles are about 0 .5 um to about 50 um or about 1 um for attachment to a linking group include amine, hydroxyl,
to about 20 um . Particles are optionally coupled to reagents, thiol, carboxylic acid , ester, amide , isocyanate and isothio
affinity matrix materials , or the like , e. g ., nucleic acid cyanate . Preferred derivatizing groups include aminoalkyl
synthesis reagents, peptide synthesis reagents, polymer syn trialkoxysilanes , hydroxyalkyltrialkoxysilanes , polyethyl
thesis reagents , nucleic acids, nucleotides, nucleobases, 25 eneglycols , polyethyleneimine, polyacrylamide,
nucleosides , peptides , amino acids, monomers , cells, bio - polyvinylalcohol and combinations thereof.
logical samples, synthetic molecules, or combinations By way of example, the reactive groups on a number of
thereof. Particles optionally serve many purposes within the siloxane functionalizing reagents can be converted to other
arrays, including acting as blank particles , dummy particles , useful functional groups :
calibration or marker particles , capture devices for low 30 1. Hydroxyalkyl siloxanes (Silylate surface, functionalize
concentration reagents , sample particles, reagent particles with diborane, and H2O2 to oxidize the alcohol);
and test particles . a . allyl trichlorosilane 3 -hydroxypropyl
Linking Chemistries b . 7 -oct- 1- enyl trichlorchlorosilane 8 -hydroxyoctyl
The particles within the arrays of the invention can 2 . Diol (dihydroxyalkyl) siloxanes (silylate surface and
present a solid or semi-solid surface for any of a variety of 35 hydrolyze to diol)
linking chemistries , allowing the incorporation of biological a . (glycidyl trimethoxysilane (2,3 -dihydroxypropy
and chemical components of interest into the particle mem loxy)propyl
bers of the arrays. A wide variety of organic and inorganic 3 . Aminoalkyl siloxanes (amines requiring no intermedi
polymers, both natural and synthetic may be employed as ate functionalizing step )
the material for the solid surface . Illustrative polymers 40 a. 3 - aminopropyl trimethoxysilane aminopropyl
include polyethylene, polypropylene, poly (4 -methylbutene ), 4 . Dimeric secondary aminoalkyl siloxanes
polystyrene, polymethacrylate , poly ( ethylene terephthalate ), a . bis (3 -trimethoxysilylpropyl) amine bis (silyloxylpro
rayon , nylon , poly ( vinyl butyrate ), polyvinylidene difluoride pyl)amine . See , for example, Leyden et al., Sympo
( PVDF ) , silicones , polyformaldehyde, cellulose , cellulose sium on Silylated Surfaces , Gordon & Breach 1980 ;
acetate , nitrocellulose, and the like . Other materials thatmay 45 Arkles , Chemtech 7 , 766 ( 1977); and Plueddemann ,
be employed include papers , ceramics , such as glass , metals , Silane Coupling Reagents , Plenum , N . Y., 1982 .
metalloids, semiconductive materials , cements , or the like . These examples are illustrative and do not limit the
In addition , substances that form gels, such as proteins ( e .g ., types of reactive group interconversions which are
gelatins), lipopolysaccharides, silicates , agarose and are also useful in conjunction with the present invention .
optionally used . 50 Additional starting materials and reaction schemes
A wide variety of linking chemistries are available for will be apparent to those of skill in the art.
linking molecules to a wide variety of solid or semi-solid The components which can be attached to a derivatized
particle support elements . These chemistries can be per- particle surface include peptides, nucleic acids , mimetics ,
formed in situ (i.e., in the microfluidic system , by flowing large and small organic molecules, polymers and the like .
appropriate reagents , e . g ., nucleic acids, proteins, and 55 For example , moieties bearing a permanent charge or a pH
samples present in low concentrations, into contact with the dependent charge are useful in practicing the present inven
particles, or vice -versa ), or outside of a microfluidic envi- tion . For example, the charged group can be a carboxylate ,
ronment , e . g ., prior to introduction of the particles into the quaternary amine or protonated amine that is a component of
microfluidic system . It is impractical and unnecessary to an amino acid that has a charged or potentially charged side
describe all of the possible known linking chemistries for 60 chain . The amino acids can be either those having a structure
linking molecules to a solid support. It is expected that one which occurs naturally or they can be of unnatural structure
of skill can easily select appropriate chemistries , depending (i. e., synthetic ). Useful naturally occurring amino acids
on the intended application . include , arginine, lysine, aspartic acid and glutamic acid .
In one preferred embodiment, the particles of the inven - Surfaces utilizing a combination of these amino acids are
tion comprise silicate elements ( e . g ., glass or silicate beads). 65 also of use in the present invention . Further, peptides com
An array of silicon -based molecules appropriate for func prising one or more residues having a charged or potentially
tionalizing surfaces are commercially available . See , for charged side chain are useful coating components and they
 US 10 ,138 ,517 B2
27 28
can be synthesized utilizing arginine, lysine, aspartic acid , multiple polymorphic nucleotides. Typically , a nucleic acid
glutamic acid and combinations thereof. Useful unnatural probe is chemically linked to a solid support and a target
amino acids are commercially available or can be synthe nucleic acid (e.g ., an RNA or corresponding amplified
sized utilizing art- recognized methodologies. In those DNA ) is hybridized to the probe . Either the probe, or the
embodiments in which an amino acid moiety having an 5 target , or both , can be labeled , typically with a fluorophore.
acidic or basic side chain is used , these moieties can be Where the target is labeled , hybridization is detected by
attached to a surface bearing a reactive group through detecting bound fluorescence . Where the probe is labeled ,
standard peptide synthesis methodologies or easily acces - hybridization is typically detected by quenching of the label
sible variations thereof. See, for example , Jones , Amino by the bound nucleic acid . Where both the probe and the
Acid and Peptide Synthesis , Oxford University Press , 10 target are labeled, detection of hybridization is typically
Oxford , 1992 . In addition , nucleic acids attached to a performed by monitoring a signal shift such as a change in
particle surface are optionally sequenced or used as a color , fluorescent quenching , or the like, resulting from
calibration particle or marker. proximity of the two bound labels . In some assay formats ,
Linking groups can also be placed on the particles of the the above format is inverted , with expression products being
invention . Linking groups of use in the present invention can 15 fixed to array members and probes being moved into contact
have a range of structures , substituents and substitution with the array .
patterns. They can , for example be derivatized with nitrogen , In another variation , solid - phase arrays are used to detect
oxygen and / or sulfur containing groups which are pendent samples even at very low concentrations. Particles and
from , or integral to , the linker group backbone . Examples particle arrays are very efficient at capturingmolecules in a
include , polyethers, polyacids (polyacrylic acid , polylactic 20 fluid stream . Therefore, they provide suitable molecule
acid ), polyols (e .g., glycerol,), polyamines (e . g., spermine, capture devices for studying systems in which a sample or
spermidine ) and molecules having more than one nitrogen , reagent is in very low concentration , e. g., in single cell RNA
oxygen and /or sulfur moiety ( e.g ., 1 ,3 -diamino - 2 - propanol, analysis . Typicalmethods for studying such systems involve
taurine ). See , for example , Sandler et al. Organic Functional DNA arrays having the non -precious reagents , e . g ., those
Group Preparations 2nd Ed ., Academic Press , Inc. San 25 available in large or adequate quantities and concentrations ,
Diego 1983 . A wide range of mono -, di- and bis - function spread out in defined locations across a substrate . The
alized poly ( ethyleneglycol) molecules are commercially precious or low concentration sample , e. g ., cell contents or
available and will prove generally useful in this aspect of the amplification product, is typically diffused across the array ,
invention . See , for example , 1997 - 1998 Catalog, Shearwater e .g ., to find a hybridization partner. By attaching the pre
Polymers, Inc., Huntsville , Ala . Additionally , those of skill 30 cious or low concentration sample to a particle or particle
in the art have available a great number of easily practiced , array, it can be placed in a defined location and then
usefulmodification strategies within their synthetic arsenal. non -precious samples are optionally flowed across the array ,
See , for example , Harris, Rev . Macromol. Chem . Phys ., allowing detection of the sample at very low concentrations .
C25 ( 3 ), 325 - 373 (1985) ; Zalipsky et al., Eur. Polym . J., For example, a few cells are optionally flowed through a
19 ( 12 ), 1177 - 1183 ( 1983 ); U .S . Pat. No . 5 , 122 ,614 , issued 35 capillary comprising a particle array , e. g ., beads , e. g .,
Jun . 16 , 1992 to Zalipsky ; U .S . Pat. No. 5,650 ,234 , issued to hybridized with a capture reagent. The cells are lysed and the
Dolence et al. Jul. 22 , 1997 , and references therein . mRNA from the cells is captured on the particles . The
In a preferred embodiment of the invention , the coupling capillary is then optionally used to flow reagents across the
chemistries for coupling materials to the particles of the particles , e.g., hybridization reagents that bind , e.g., specifi
invention are light-controllable, i.e., utilize photo - reactive 40 cally , to the mRNA of interest, if it is present. The capillary
chemistries . The use of photo -reactive chemistries and is rinsed after each exposure and any resulting hybridization
masking strategies to activate coupling of molecules to is detected , e . g ., by fluorescence detection . Alternatively , the
substrates , as well as other photo -reactive chemistries is captured molecule is amplified and the product captured ,
generally known (e .g ., for semi- conductor chip fabrication e .g ., immediately or in about the same vicinity to enhance
and for coupling bio - polymers to solid phase materials ). The 45 the signal before bringing hybridization or probe reagents
use of photo - cleavable protecting groups and photo -masking across the capture area . In other aspects, different probe
permits type switching of both mobile and fixed array reagents comprising different fluorophores are mixed to
members , i. e ., by altering the presence of substrates present detect several components at once . Alternatively , capillary
on the array members ( i.e ., in response to light) . Among a or channel surfaces are used to capture the low concentration
wide variety of protecting groups which are useful are 50 molecule instead of particles.
nitroveratryl (NVOC )-methylnitroveratryl (Menvoc ), ally In other aspects , the particles of the present invention are
loxycarbonyl (ALLOC ), fluorenylmethoxycarbonyl used as marker particles or calibration particles. For
(FMOC ), -methylnitro -piperonyloxycarbonyl (MeNPOC ), example , charged beads, e.g ., to which a dye is attached , are
- NH -FMOC groups, t-butyl esters, t-butyl ethers , and the optionally used as markers in capillary electrophoresis ,
like. Various exemplary protecting groups ( including both 55 especially when very low mobility is required . For example,
photo - cleavable and non - photo -cleavable groups ) are a marker that flows slower than any sample is optionally
described in , for example , Atherton et al., ( 1989) Solid used . Alternatively, a neutral particle or bead is used with an
Phase Peptide Synthesis, IRL Press , and Greene, et al. attachment, functional group , or linker that is charged .
( 1991) Protective Groups In Organic Chemistry, 2nd Ed ., Typical molecules for use on the particles include, but are
John Wiley & Sons , New York , N . Y ., as well as, e. g ., Fodor 60 not limited to , molecules that are similar to the sample of
et al. (1991 ) Science , 251: 767 -777, Wang (1976 ) J. Org . interest. For example, a nucleic acid is preferably used in
Chem . 41 : 3258 ; and Rich , et al. ( 1975 ) J. Am . Chem . Soc . electrophoresis of nucleic acids . A typical DNA or RNA
97 : 1575 - 1579. The use of these and other photo -cleavable molecule attached to such a marker or calibration bead
linking groups for nucleic acid and peptide synthesis on comprises about 10 thousand base - pairs to about 20 thou
solid supports is a well -established methodology . 65 sand base pairs , e. g ., a 17 thousand base pair nucleic acid .
In one useful variation of these methods , solid phase The marker particle or bead is used , for example , to deter
arrays are adapted for the rapid and specific detection of mine the position of one or more member of an array . For
 US 10 ,138,517 B2
29 30
example , the marker is optionally used to determine the Although light- directed coupling chemistries are pre
position of an array member that has captured a low con - ferred , and described above in some detail for exemplary
centration sample as described above. The particle or bead purposes , these are not the only feasible routes to producing
typically comprises a charged moiety or particle and a label arrays with a variety of different particle members . The use
moiety , e . g ., a fluorescent dye or a charged labelmoiety . The 5 of microfluidic fluid movement to move reagents into con
labelmoiety is optionally detected , e .g., by fluorescence , to tact with array members can also be used . In particular, solid
determine and calibrate positions of array members. surfaces are derivatized as noted above in preparation for
In one embodiment of this concept, an array of probes are addition of components such as nucleotide synthesis
synthesized on solid support particles constituting array reagents , peptides , or the like . Between coupling steps,
members . Using typical array masking technologies and 10 photoprotective
protective groups can be used as noted above. Instead of
photoprotective chemistry, it is possible to generate ordered depending on thecleavage , other cleavage agents can be used ,
arrays of nucleic acid probes with large numbers of probes . the like. Indeed , because itthe
nature of reaction , e. g ., acids, bases , or
In the embodiments of the present invention , even photo reagents into contact with selected arrayto members
is possible flow only desired
, it is not
masking is unnecessary , making photoprotective chemistry istry 15 necessary to use blocking groups at all. The elimination of
particularly useful. In particular, the array members can be blocking groups is one of themany advantages of the present
flowed past a light source in a selected order in the presence invention over standard solid phase synthesis techniques.
of selected reagents, permitting selective addition of com Array Templates
ponents to the array , without actually performing chip mask The array or reagents contacting the array can involve
ing . Ofcourse , however, chip masking strategies can also be 20 template hybridization reagents or the like, including a first
used , i.e., array members can be fixed in place and selec nucleic acid which is fully or partially complementary to a
tively exposed to light. Either method is used , for example , second nucleic acid complexed with a particle set of the
to place a precious sample , e. g ., a sample that is only array, a first protein which specifically hybridizes to one or
available in small amounts , in a defined location and detect more component with a particle set of the array , a first
it at very low concentrations. 25 antibody which specifically hybridizes to one or more com
In brief, a combinatorial strategy allows for the synthesis ponent with a particle set of the array, a hybridization buffer,
of arrays containing a large number of different elements a blocking reagent, and a labeled probe nucleic acid . The
such as fixed nucleic acids, using a minimal number of methods optionally comprise flowing liquid reagents into
synthetic steps . For instance , in general in solid - phase contact with one or more array member and detecting the
masking technologies, it is possible to synthesize and attach 30 resulting hybridization of the liquid reagent to the array
all possible DNA 8 -mer oligonucleotides (48 , or 65,536 member .
possible combinations ) using only 32 chemical synthetic Sequencing and PCR in Microfluidic Systems
steps. In general, these procedures provide a method of In a preferred embodiment of the invention , the microar
producing 4n different oligonucleotide probes on an array rays of the invention are used for sequencing nucleic acids .
using only 4n synthetic steps. Alternatively , due to the 35 The devices of the invention optionally include reagents
high -throughput reaction speeds of microfluidic systems, it (which may be part of the array or flowed into contact with
is possible to perform large numbers of reactions with linear the array , e .g . in a reagent train ) for performing a biological
or parallel fluidic manipulations in feasibly short periods of or chemical assay. The liquid reagent or array can include a
time. nucleic acid sequencing reagent such as a liquid solution
As noted , light-directed combinatorial synthesis of oligo - 40 comprising a nucleotide, a liquid solution comprising a
nucleotide arrays on glass array members is performed with polymerase , a liquid solution comprising a dNTP , a ddNTP ,
automated phosphoramidite chemistry and, optionally, chip a dNTP analog , or a fluorescent dNTP , a liquid solution
masking techniques similar to photoresist technologies in comprising a sufurylase , a liquid solution comprising an
the computer chip industry . Typically, a glass surface is apyrase, a liquid solution comprising inorganic phosphate, a
derivatized with a silane reagent containing a functional 45 liquid solution comprising ATP, a liquid solution comprising
group , e . g ., a hydroxyl ( for nucleic acid arrays ) or amine a thermostable polymerase , a liquid solution comprising an
group (for peptide or peptide nucleic acid arrays ) blocked by endonuclease , a liquid solution comprising an exonuclease ,
a photolabile protecting group . Photolysis through a photo - a liquid solution comprising a phosphatase , a liquid solution
lithographic mask , or by selective flow , e .g ., in a microflu - comprising an intercalator, a liquid solution comprising a
idic system past a light source , is used selectively to expose 50 reducing agent, a liquid solution comprising Mg+ + , a liquid
functional groups which are then ready to react with incom - solution comprising a molecular crowding agent, e. g ., PEG ,
ing photoprotected elements (e .g ., for nucleic acids, 5 '- pho - a liquid solution comprising a buffer, a liquid solution
toprotected nucleoside phosphoramidites ). The photopro - comprising a salt, a salt, DTT, BSA , a detergent ( e. g ., triton
tected elements react with those sites which are illuminated or tween ), chemicals to inhibit or enhance electroosmotic
(and thus exposed by removal of the photolabile blocking 55 flow (e .g., polyacrylamide ) or the like .
group ). Thus , e. g ., phosphoramidites only add to those areas Standard Chain Termination Sequencing
selectively exposed from the preceding step . These steps are Most DNA sequencing today is still carried out by chain
repeated until the desired array of sequences has been termination methods of DNA sequencing . The most popular
synthesized on the array particle . Combinatorial synthesis of chain termination methods of DNA sequencing are variants
different molecules at different locations on the array is 60 of the dideoxynucleotide mediated chain termination
determined by the pattern of illumination during synthesis , method ofSanger. See, Sanger et al. ( 1977) Proc . Nat. Acad .
relative to the array ( again , the array can be mobile or fixed Sci., USA 74 :5463 -5467. For a simple introduction to dide
in the present invention ) and the order of addition of oxy sequencing, see , Current Protocols in Molecular Biol
coupling reagents. Monitoring of hybridization of target ogy , F . M . Ausubel et al., eds., Current Protocols , a joint
elements to the array is typically performed with fluores - 65 venture between Greene Publishing Associates , Inc. and
cence microscopes, laser scanningmicroscopes, CCD arrays John Wiley & Sons, Inc ., ( Supplement 38 , current through
or the like. 1998 ) (Ausubel), Chapter 7 . Thousands of laboratories
 US 10 ,138 ,517 B2
31 32
employ dideoxynucleotide chain termination techniques . BRL Life Technologies , Inc . (Gaithersberg, Md.); Fluka
Commercialkits containing the reagents most typically used Chemica -Biochemika Analytika (Fluka Chemie AG , Buchs ,
for these methods of DNA sequencing are available and Switzerland ); Invitrogen (San Diego , Calif.); Perkin Elmer
widely used . These methods of DNA sequencing are adapt- (Foster City , Calif.); and Strategene ; as well as many other
able to the arrays of the invention . In particular, array 5 commercial sources known to one of skill.
members can comprise e .g ., template nucleic acids , e .g ., In one aspect, the generation of large nucleic acids is
chemically coupled or hybridized to particle surfaces . useful in practicing the invention , e. g ., as templates fixed to
Reagent trains comprising sequencing reagents are passed array members, e. g ., for sequencing long regions of nucleic
across the template nucleic acids ( e . g ., using electrophoresis , acids, or for monitoring expression products by hybridiza
or electroosmotic or pressure -based reagent flow ) where 10 tion of biologicalmaterials to the fixed templates . It will be
they contact the templates . Reaction products can be ana appreciated that such templates are particularly useful in
lyzed directly, or following dissociation and electrophoresis some aspects where the methods and devices of the inven
within the microfluidic system . tion are used to sequence large regions of DNA , e . g ., for
In addition to the Sanger methods of chain termination ,
new PCR exonuclease digestion methods have also been 15 genomics types of applications. An introduction to large
developed for DNA sequencing. Direct sequencing of PCR clones such as YACs, BACs, PACs and MACs as artificial
generated amplicons by selectively incorporating boronated chromosomes is provided by Monaco and Larin ( 1994 )
nuclease resistant nucleotides into the amplicons during Trends Biotechnol 12 (7 ): 280 -286 .
PCR and digestion of the amplicons with a nuclease to The construction of nucleic acid libraries of template
produce sized template fragments has been performed (Por- 20 nucleic acids is described in the above references . YACs and
ter et al. ( 1997 ) Nucleic Acids Research 25 (8 ): 1611- 1617 ). YAC libraries are further described in Burke et al. (1987 )
In the methods, 4 PCR reactions on a template are per - Science 236 :806 -812 . Gridded libraries of YACs are
formed , in each ofwhich one of the nucleotide triphosphates described in Anand et al. ( 1989) Nucleic Acids Res . 17 ,
in the PCR reaction mixture is partially substituted with 2a 3 425 - 3433 , and Anand et al. ( 1990 ) Nucleic Acids Res .
deoxynucleoside 5 - \ ffP -boranol-triphosphate . The boro - 25 Riley (1990 ) 18 : 1951 - 1956 Nucleic Acids Res . 18 ( 10 ) :
nated nucleotide is stochastically incorporated into PCR 2887 - 2890 and the references therein describe cloning of
products at varying positions along the PCR amplicon in a YACs and the use of vectorettes in conjunction with YACs.
nested set of PCR fragments of the template. An exonuclease See also , Ausubel, chapter 13 . Cosmid cloning is also well
which is blocked by incorporated boronated nucleotides is known . See , e . g ., Ausubel , chapter 1 . 10 .11 (supplement 13 )
used to cleave the PCR amplicons . The cleaved amplicons 30 and the references therein . See also , !sh -Horowitz and Burke
are then separated by size using polyacrylamide gel elec- ( 1981) Nucleic Acids Res . 9 : 2989-2998 ; Murray (1983 )
trophoresis , providing the sequence of the amplicon. An Phage Lambda and Molecular Cloning in Lambda II (Hen
advantage of this method is that it requires fewer biochemi drix et al., eds) 395 -432 Cold Spring Harbor Laboratory ,
cal manipulations than performing standard Sanger- style NY ; Frischauf et al. ( 1983 ) J. Mol. Biol. 170 :827 - 842; and ,
sequencing of PCR amplicons. These methods are similarly 35 Dunn and Blattner (1987 ) Nucleic Acids Res. 15 :2677 -2698 ,
adaptable to the arrays and microfluidic systems of the and the references cited therein . Construction of BAC and
invention . In particular, PCR can be performed by heating P1 libraries is well known; see , e.g ., Ashworth et al. ( 1995 )
and cooling all or part of a microfluidic system . Anal Biochem 224 ( 2 ): 564 - 571; Wang et al. (1994 ) Genom
It is expected that one of skill is familiarwith fundamental ics 24 (3 ): 527 -534 ; Kim et al. ( 1994 ) Genomics 22 ( 2 ):
sequencing methodologies applicable to the present inven - 40 336 - 9; Rouquier et al . (1994 ) Anal Biochem 217 (2 ): 205 -9 ;
tion . Examples of techniques for making and sequencing Shizuya et al. (1992 ) Proc Natl Acad Sci USA 89 ( 18 ):
nucleic acids , and instructions sufficient to direct persons of 8794 - 7 ; Kim et al. ( 1994 ) Genomics 22 (2 ): 336 - 9 ; Woo et
skill through most standard cloning and other template al. ( 1994 ) Nucleic Acids Res 22 (23 ) : 4922 -31 ; Wang et al.
preparation exercises are found in Berger and Kimmel, ( 1995 ) Plant (3 ): 525 - 33 ; Cai (1995 ) Genomics 29 ( 2 ):
Guide to Molecular Cloning Techniques , Methods in Enzy - 45 413 - 25 ; Schmitt et al. ( 1996 ) Genomics 1996 33 ( 1 ) : 9 -20 ;
mology volume 152 Academic Press, Inc ., San Diego , Calif. Kim et al. ( 1996 ) Genomics 34 (2 ): 213 -8 ; Kim et al. (1996 )
(Berger ); Sambrook et al. (1989 ) Molecular Cloning — A Proc Natl Acad SciUSA ( 13 ): 6297 - 301; Pusch et al. ( 1996 )
Laboratory Manual (2nd ed .) Vol. 1- 3, Cold Spring Harbor Gene 183 ( 1 - 2 ): 29-33; and, Wang et al. ( 1996 ) Genome Res
Laboratory, Cold Spring Harbor Press , N . Y ., (Sambrook ) ; 6 ( 7 ): 612 - 9 .
and Current Protocols in Molecular Biology, F . M . Ausubel 50 In general, where the desired goal of a sequencing project
et al., eds., Current Protocols, a joint venture between is the sequencing of a genome or expression profile of an
Greene Publishing Associates, Inc . and John Wiley & Sons, organism , a library of the organism ' s cDNA or genomic
Inc., ( 1997, supplement 37 ) (Ausubel). Basic procedures for DNA is made according to standard procedures described ,
cloning and other aspects of molecular biology and under- e. g., in the references above. Individual clones are isolated
lying theoretical considerations are also found in Lewin 55 and sequenced , and overlapping sequence information is
( 1995 ) Genes V Oxford University Press Inc., NY (Lewin ); ordered to provide the sequence of the organism . See also ,
and Watson et al. (1992 ) Recombinant DNA Second Edition Tomb et al. (1997) Nature 539 - 547 describing the whole
Scientific American Books, NY. Product information from genome random sequencing and assembly of the complete
manufacturers of biological reagents and experimental genomic sequence ofHelicobacter pylori; Fleischmann et al.
equipment also provide information useful in known bio - 60 ( 1995 ) Science 269 :496 -512 describing whole genome ran
logical methods. Such manufacturers include the Sigma dom sequencing and assembly of the complete Haemophilus
Chemical Company (Saint Louis , Mo.); New England Bio - influenzae genome; Fraser et al. ( 1995 ) Science 270 :397
labs (Beverly, Mass.); R & D systems (Minneapolis, Minn .); 403 describing whole genome random sequencing and
Pharmacia LKB Biotechnology ( Piscataway, N .J.); CLON - assembly of the complete Mycoplasma genitalium genome
TECH Laboratories, Inc . (Palo Alto , Calif.); ChemGenes 65 and Bult et al. ( 1996 ) Science 273: 1058- 1073 describing
Corp ., (Waltham Mass .) Aldrich Chemical Company (Mil- whole genome random sequencing and assembly of the
waukee , Wis.); Glen Research , Inc . (Sterling, Va .); GIBCO complete Methanococcus jannaschii genome.
 US 10 , 138 ,517 B2
33 34
Recently, Hagiwara and Curtis ( 1996 ) Nucleic Acids between cycles of label addition . This can be accomplished ,
Research 24 ( 12 ): 2460 - 2461 developed a " long distance e .g ., by interspersing cycles of photobleaching between
sequencer” PCR protocol for generating overlapping nucleic labeling steps to reduce fluorescence of previously labeled
acids from very large clones to facilitate sequencing, and components . For example , nucleic acid template molecules
methods of amplifying and tagging the overlapping nucleic 5 are optionally attached to the surface of a microfluidic
acids into suitable sequencing templates . The methods can
be used in conjunction with shotgun sequencing techniques channelA
or to an array member, e. g., a bead or set of beads.
primer is bound to the template , e.g., by flowing the
to improve the efficiency of shotgun methods typically used
in whole organism sequencing projects . As applied to the reagents, e .g., athepolymerase
primer across nucleic acid template . Other sequencing
present invention , the techniques are useful for identifying 10 flowed across the template , e.and a series of nucleotides are
g ., incorporating a nucleotide .
and sequencing genomic nucleic acids using the arrays of For example , a solution comprising at least one of the four
the present invention . In particular, one or more component
of the PCR reactions , YACs, vectorettes, or the like used in standard nucleotides, at least a portion of which nucleotides
are labeled nucleotides, is flowed across the template . The
the long distance sequencer method are fixed to an array, and10 15 channel is then washed , removing any unincorporated
the array performed in a microfluidic system by flowing 15 cha
other components of the long distance sequencer method nucleotides . If a nucleotide was incorporated , a fluorescence
into contact with the fixed component on the array. This signal associated with the incorporated nucleotide is
method particularly benefits from the use of arrays due to the detected , thus determining the identity of the added nucleo
need to organize several reactions simultaneously for the tide and providing a portion of the sequence. The template
long distance sequencer method. Products can be assayed in 20 nucleic acid is then photobleached to reduce the background
parallel or sequentially , facilitating the selection of subse - level of fluorescence before repeating the procedure with
quent reaction components. another nucleotide.
Sequencing by Incorporation /Synthesis In one preferred sequencing by incorporation method ,
In a preferred embodiment, the present invention provides label is produced indirectly, i.e ., incorporation of nucleotide
for sequencing by synthesis or incorporation . A number of 25
basic sequencing by incorporation methods are known , e . g ., a coupledis signal
reagents measured by production of a detectable label in
reaction , e.g ., when sequencing by pyro
as set forth in Hyman U . S . Pat. No . 4 , 971, 903 ; Malemede
63 . 840 . Cheeseman ís Pat ' No 5300 phosphate
U .S . Pat . No. 4 ,863 ,849; Cheeseman U .S . Pat. No. 5, 302, PI methods .
509 , and Canard U . S . Pat. No. 5,798 ,210 . Generally , any Polymerase reactions sometimes show incorporation of
detectable event associated with incorporation of a nucleo - 30 non - complementary bases at a low frequency , particularly
tide can be used to monitor sequencing reactions. In when the bases are present in excess, or when complemen
sequencing by incorporation methods, incorporation of tary bases are not present. In one aspect , non - incorporatable
nucleotide reagents into nucleic acids (typically by a using nucleotides ( e.g., analogues which lack moieties which
a polymerase to extend a primer hybridized to a comple - provide for coupling to the phosphate backbone) are added
mentary template nucleic acid ) is monitored to provide an 35 to increase the fidelity of a polymerase reaction , by com
indication of the sequence of a template nucleic acid . This peting with the non -complementary bases . In addition , the
can be performed by selectively adding reagents comprising non - incorporatable nucleotides reduce polymerase proces
labels such as bases comprising fluorescent moieties, e . g ., sivity , which is desirable , e .g ., in reactions where a comple
four detectably different fluorescent moieties, to e . g ., a mentary residue is not present in the reaction mixture .
member of an array set and monitoring incorporation of the 40
label into the nucleic acid . In one aspect, chain termination is reversible . See also ,
A variety of nucleotides which have fluorescent labels can Cheeseman U .S . Pat. No. 5 ,302,509 and Canard U . S . Pat.
be added in a base specific fashion by a polymerase . For No . 5 ,798 ,210 . In one method of sequencing by synthesis, a
example , Hawkins et al. U . S . Pat. No. 5 , 525 ,711 describe " reversible ” label is used . In particular, a terminating base
pteridine nucleotide analogs for use in fluorescent DNA 45 comprising a label is added by a polymerase as in standard
probes. These analogs can be incorporated by, e .g., Taq chain termination methods. The label is cleavable , e .g., by
polymerase , sequenase , DNA polymerase Klenow fragment, photolysis , or by exposure to heat or to one or more
or the like. chemicals , e. g ., a reducing agent and /or a phosphatase .
In both sequencing methodologies and elsewhere herein , Base -specific incorporation of the label is first detected and
it will be recognized that fluorescent labels are not to be 50 then the label is cleaved and washed from the array. Alter
limited to single species organic molecules , but include natively , the label; is cleaved , washed from the array and
inorganic molecules, multi -molecular mixtures of organic then detected
then detected .. The
The ararray is then exposed to nucleotides
and / or inorganic molecules , crystals , heteropolymers , and comprising a label and the process is repeated . Sequence
the like . Thus, for example , CdSeCdS core - shell nanoc information is provided by assessing which nucleotides
rystals enclosed in a silica shell can be easily derivatized for 55 comprising a label are incorporated and compiling the
coupling to a biological molecule (Bruchez et al. ( 1998 ) information . The typical four nucleotides
Science, 281 : 2013 -2016 ). Similarly , highly fluorescent added in series or they are all added together inareoneoptionally
solution.
quantum dots (e .g ., zinc sulfide-capped cadmium selenide ) In the latter case , the four nucleotides each have a detectably
have been covalently coupled to biomolecules for use in
ultrasensitive biological detection (Warren and Nie (1998 ) 60 different label , which is used to identify the nucleotide
Science, 281: 2016 -2018 ). The use of quantum dots as labels incorporated .
in the present invention is particularly useful, both in direct Examples of reversible chain terminating nucleotides
sequencing application and more generally as a labeling include , but are not limited to , nucleotides with 3'-phosphate
methodology for any system set forth herein . blocking groups, e.g ., comprising a disulfide , and nucleo
In certain aspects, it is useful to reduce the fluorescence 65 tides with 3 '- carbamate blocking groups. Example of such
of labeled nucleic acids before adding additional labels to compounds include , but are not limited to compounds hav
the nucleic acids, e .g ., to reduce background fluorescence ing the following formulas:
 US 10 , 138 ,517 B2
35 36
appropriate for incorporation into a nucleic acid oligomer

H117
0 - P - O - P - 0 - P - 04
Ó
-

O= P- 0
B
synthesis is also optionally utilized to derivatize a nucleic
acid monomer.
At least two methods can be utilized to prepare nucleic
acids with a disulfide linker having a detectable moiety at
one terminus of the linker. In the first method , the disulfide
moiety , preferably supplied by an agent such as 2 -hydroxy
ethyl disulfide is mono - functionalized at one hydroxy ter
VOR4 and 10
minus with a detectable group . The remaining hydroxy
group is converted to a phosphoramidite . This linker arm
detectable agent conjugate can then be incorporated into a
10
=

0— P- O - P- 0 - P normal nucleic acid synthetic cycle .

A second method , exemplified below , allows a trityl
protected linker to be tethered to a growing nucleic acid
chain . The trityl group is subsequently removed in a normal
OO nucleic acid deprotection cycle and the liberated hydroxy
group is conjugated to another base or to a detectable
moiety .
20
CH3
HO
wherein B comprises a nitrogenous base and R4 typically
sYCOH TÚ Trtci

comprises a label moiety , e .g ., a fluorescent label moiety. . ?? ,

OTrt
These nucleotides are added to a growing nucleotide chain
by a polymerase , e.g., taq polymerase , and then the blocking
group is removed to provide a 3'-OH group , to which another CIP (OCNE)N (iPr)2)
nucleotide is optionally added as sequencing continues.
Removal of the phosphate blocking group comprises 30 ( iPr)(Et)2N
reducing the disulfide linkage with a suitable reducing agent, (i-Pr)2NP — 0
e . g ., including, diborane and other boron - containing reduc OTrt
tants, dithiothreitol, and enzymes such as reductases specific OCNE
for the disulfide group . The cleavage of the disulfide results 3
in a molecule having the following formula : 35
OOO O Synthesis of monotrityl-2 -hydroxyethyl disulfide (2 )
0 -5 -0 -5 -0 -" — oyoys O = P - 0 2 - hydroxyethyl disulfide ( 1, Aldrich Chemical Co.) is
00 40 monotritylated by the action of tritychloride and a base such
OS - R as triethylamine . The reaction is weighted towards produc
tion of the monotrityl derivative by using a substantial
which is typically unstable and spontaneously degrades, e .g ., stoichiometric excess (3 -5 fold ) of 2 -hydroxyethyl disulfide
through nucleophilic attack , providing a 3'- phosphate group . relative to tritylchloride. The reaction is typically carried out
The phosphate group is then cleaved , e. g ., using an alkaline 45 in ethyl acetate of a chlorinated hydrocarbon at room
phosphatase . Cleavage of the 3'-phosphate group leaves an temperature . The monotritylated product will exhibit mark
extendable 3'-OH group. edly different chromatographic behavior than both unreacted
Removal of the carbamate typically comprises reduction tritylchloride and the ditritylated derivative . Thus, the mono
of the carbamate linkage, thus producing an unblocked tritylated derivative can be easily purified by, for example ,
3 – OH group , which is optionally extended , e .g ., by addi- 50 silica gel chromatography , optionally in a microfluidic sys
tion of another dNTP . tem .
Ribonucleic acid base monomers comprising removable
labels having a disulfide linker group at the 3'-hydroxyl Synthesis of monotrityl- 2 -hydroxyethyl
group are optionally prepared by normal solid or solution disulfide -2 -cyanoethyldiisopropyl-phosphoramidite
phase phosphoramidite chemistry , optionally in a microflu - 55
idic system . As the 3 -hydroxyl is effectively blocked by the The purified monotrityl 2 -hydroxyethyl disulfide is con
conjugated linker arm , these monomers can then be used as verted to a phosphoramidite appropriate for inclusion into
a terminating residue in standard dideoxy chain termination , solid phase nucleic acid synthesis as follows. The montrityl
or can be used as reversible terminators in sequencing by compound is contacted with 2 - cyanoethyldiisopropylchlo
synthesis protocols as noted above . The free hydroxy ter - 60 rophosphoramidite in the presence of an organic base such
minus of the linker arm is preferably derivatized with a as diisopropylethylamine. As the reaction proceeds, amine
detectable label such as a fluorescent or chemiluminescent hydrochloride is produced and precipitates from the reaction
dye or a radioactive isotope . Methods for preparing linker medium . When the reaction is complete , the solid is
arms that can be incorporated into monomers or nucleic acid removed by filtration and the organic layer is washed with
oligomers are discussed below in the context of nucleic acid 65 water at approximately neutral pH . The solvent is removed
oligomers . It is, however, understood that this is by way of by evaporation and the crude product is purified by silica gel
example. One of skill will recognize that a linker that is chromatography to provide the desired product.
 US 10 , 138 ,517 B2
37 38
In one embodiment, the phosphoramidite derivative is NADPH +6 phosphoglutamate . The ADP is converted into
brought into solution in , for example , acetonitrile and placed ATP by addition of phosphoenoyl pyruvate (PEP ) and a
into a reactant vessel on an art -recognized nucleic acid phosphokinase enzyme. ATP formation can be monitored
synthesis apparatus. The disulfide linker arm is added to the with luciferase . Preferably , NADPH formation can be moni
growing chain at any desired point in the synthesis . The use 5 tored by monitoring fluorescence . In essence , as long as PEP
of the phosphoramidite derivative allows the disulfide linker is present in the reaction , this set of reactions provides a
to be added in a manner identical to any other nucleic acid signal amplification cycle by producing additional ATPs.
base phosphoramidite. Following the addition of the disul- This is helpful in luciferase mediated signals, because
fide linker, the trityl group is removed using a standard luciferase produces a relatively low level of detectable
deprotection cycle and , if desired , chain elongation can then 10 signal, e . g ., as compared to fluorescence .
proceed . Maxam Gilbert Sequencing
In any of the embodiments herein which comprise fixation In addition to chain termination and coupled enzymatic
of targets to array members, the targets can be related ( e. g., methods, chemical nucleic acid degradation methods have
sequence fragments of a single clone ) or unrelated . In been in use for specialized applications; see , Maxam and
embodiments where the sequences are related , the system 15 Gilbert ( 1980 ) in Grossman and Moldave ( eds .) Academic
optionally includes a microprocessor for compiling overlap - Press, New York , Methods in Enzymology 65 :499 -560 . As
ping sequence information . with the sequencing methodologies noted above , the Maxam
Direct Sequencing — Real Time Pyrophosphate Gilbert method is entirely amenable to use with the arrays of
One recent approach to sequencing by synthesis is set the invention. In particular, the template can be fixed to an
forth in Ronaghi et al. ( 1998 ) “ A Sequencing Method Based 20 array ( covalently or non -covalently ) and selectively
on Real Time Pyrophosphate ” Science 281: 363 - 364 (See degraded by the Maxam Gilbert degradation method . Reac
also , Nyren and Uhlen ( 1996 ) Anal. Biochem . 242 : 84 -89 tion products can be viewed concurrentwith degradation , or
and ( 1993 ) 208 : 171 - 175 and Canard U .S . Pat . No. 5 ,798 , following degradation (i.e., downstream of the reaction ).
509) . In this method , four nucleotides are added stepwise to Indeed , one advantage of the present invention over standard
a template nucleic acid hybridized to a primer. In the 25 Maxam Gilbertmethods is that extremely small quantities of
applications of the present invention , templates are option - reagent can be used . One of skill will appreciate that some
ally fixed to one or more particle members of the arrays. A of the reagents in the Maxam Gilbert method are highly
polymerase adds a nucleotide to the primer based upon toxic and /or explosive , making working with large quanti
standard base -pairing rules and standard polymerase activ - ties of these reagents (i.e ., as in standard methods ) somewhat
ity . The addition of a nucleotide to the primer results in the 30 problematic .
release of an inorganic pyrophosphate from the nucleotide . Sequencing by Hybridization
An ATP sulfurylase enzyme is used to convert the inorganic Sequencing by hybridization to complementary oligo
pyrophosphate into ATP or an ATP analogue ( e. g ., compris - nucleotides has also been developed e .g ., in U .S . Pat. No .
ing a sulfur atom ). A luciferase enzyme releases light in the 5 , 202,231 , to Drmanac et al., Drmanac et al. ( 1989 ) Genom
presence of the ATP , providing an indication as to when a 35 ics 4 : 114 -128 and , e . g ., Drmanac et al. ( 1998 ) “ Accurate
nucleotide is added to the primer. To remove excess ATP sequencing by hybridization for DNA diagnostics and indi
from the system , an apyrase is added to degrade the ATP into vidual genomics” Nature Biotechnology 16 : 54 - 58 . Methods
AMP + 2PPibetween dNTP addition cycles. The apyrase also of detecting genetic differences by hybridization are
degrades any nucleotide from the system which is not added described e. g ., in Fodor (1997 ) “Genes, Chips and the
to the primer. Any or all of these reagents can be present in 40 Human Genome” FASEB Journal. 11: 121- 121; Fodor
a reagent train which passes over particle members com - (1997 ) “Massively Parallel Genomics” Science. 277 :393
prising template , or, alternatively can be themselves fixed to 395 ; Chee et al. (1996 ) “ Accessing Genetic Information
array members, where a reagent train comprising the tem - with High -Density DNA Arrays ” Science 274 :610 -614 , and
plate is passed across the array members . As described in a variety of other publications.
above , chemistries for fixing either nucleic acids or proteins 45 The arrays of the present invention are particularly well
( or both ) to any of a variety of array members is well known . suited to sequencing and/ or detection of differences at the
Optionally , the dATP for incorporation into growing nucleic nucleic acid level, by hybridization methods. In particular,
acids is a - thio dATP which can be incorporated by poly - either probe or target nucleic acids (or both in multiplexed
merase , but not by luciferase , thus reducing background assays ) are fixed to array members . Either probes , or target
signal production in the assay. 50 members, or both , are flowed sequentially, simultaneously
Indeed , one advantage of the present invention is that it or in parallel into contact. Typically , either the probe or
makes the reaction outlined by Ronaghi et al. much more target member are labeled to facilitate detection of any
practical. In the Ronaghi et al. reference , due to the use of hybridization event . Alternatively, nucleic acids can simply
relatively crude enzyme fractions and fluidic inefficiencies, be hybridized to and denatured from the array , with detec
signal to noise ratios gradually decreased as the reaction 55 tion occurring downstream from the hybridization event
proceeded (due in part to incomplete washing of reactants (e . g ., where the detection and hybridization are timed to
and products between steps), making it difficult to read provide meaningful information regarding the hybridiza
longer nucleic acid templates. In contrast, using the present tion ). Downstream detection can be performed with a
invention , it is possible to isolate completely the relevant labeled probe (which is optionally a component of the array,
reagents , and to wash them from the templates using micro - 60 or of a second fluidly connected array ), or by simple
fluidic fluid movement. detection of the physical presence of the nucleic acid ( e . g.,
One additional aspect of the present invention is a new detection downstream can be performed by mass spectros
pyrophosphate sequencing reaction . In this new sequencing copy ).
reaction , PPi is converted to a thio analogue form of ATP It is expected that one of skill is thoroughly familiar with
with a sulfurylase . The ATP is combined with glucose and 65 the theory and practice of nucleic acid hybridization and
converted to glucose -6 - phosphate +NADP and ADP. Glu - probe selection . Gait , ed . Oligonucleotide Synthesis: A
cose -6 -phosphate dehydrogenase is used to produce Practical Approach , IRL Press, Oxford ( 1984); W . H . A .
 US 10 ,138 ,517 B2
39 40
Kuijpers Nucleic Acids Research 18 ( 17 ), 5197 ( 1994 ); K . L . at least one additional probe. Specific sequences are gener
Dueholm J. Org . Chem . 59 , 5767-5773 (1994 ); S . Agrawal ated by compiling the sequences of probes bound to the
( ed .) Methods in Molecular Biology, volume 20 ; and Tijssen targets . Examples of devices for storing and accessing large
( 1993) Laboratory Techniques in biochemistry and molecu sets of small probes in conjunction with a microfluidic
lar biology -hybridization with nucleic acid probes, e . g ., part 5 system are described , e . g ., in " Closed Loop Biochemical
I chapter 2 " overview of principles of hybridization and the Analyzers” WO 98 / 45481 .
strategy of nucleic acid probe assays” , Elsevier , New York Other investigators have also reported immunological
provide a basic guide to nucleic acid hybridization . Hybrid detection of DNA :RNA hybrids, including Bogulayski et al .
ization of nucleic acids to nucleic acids fixed to solid ( 1986 ) J. Immunol. Methods 89: 123 - 130 ; Prooijen -Knegt
substrates is described , e .g ., in U . S . Pat. No . 5 , 202, 231, to 10 ( 1982 ) Exp . Cell Res. 141: 397 -407; Rudkin ( 1976 ) Nature
Drmanac et al., Drmanac et al. ( 1989) Genomics 4 : 114 - 128 265:472 -473 , and Stollar (1970) PNAS 65: 993- 1000. Simi
and , e .g ., Drmanac et al. ( 1998 ) “ Accurate sequencing by larly, detection of DNA :DNA hybrids and RNA :RNA
hybridization for DNA diagnostics and individual genom - hybrids has also been described . See, Ballard ( 1982 ) Mol.
ics ” Nature Biotechnology 16 : 54 - 58. Methods of detecting Immunol. 19 :793 -799; Pisetsky and Caster ( 1982 ) Mol.
genetic differences by hybridization are described e .g ., in 15 Immunol. 19 :645 -650 , and Stollar ( 1970 ) PNAS 65 : 993
Fodor (1997 ) “Genes , Chips and the Human Genome” 1000 . These methods are similarly adapted to the present
FASEB Journal. 11: 121- 121 ; Fodor (1997 ) “Massively Par invention by fixing one or more component of the assay to
allel Genomics” Science. 277 :393 - 395 ; Chee et al. (1996 ) one or more array members , flowing other components of
“ Accessing Genetic Information with High -Density DNA the assay into contact with the one or more array members
Arrays ” Science 274 :610 -614 . 20 and detecting any resulting signal.
These methods of sequencing and assessing genetic dif- An alternative sequencing method useful in the present
ference are adapted to the arrays of the invention by per - invention , e . g ., using particle arrays and microfluidic
forming hybridization assays in a microfluidic format. In devices, involves the use of an intercalator. A template is
particular, nucleic acids ( e .g . either probes or targets ) are sequenced in the presence of an intercalator, e. g ., an inter
fixed to an array member e . g ., by covalent synthesis strat- 25 calating nucleic acid dye . Nucleotides are flowed across the
egies as noted supra ( e. g . by light directed synthesis and template or the template is flowed across a nucleotide
photomasking procedures ), or by hybridization to a mol solution as described above. Upon addition of a nucleotide
ecule which captures the nucleic acid , such as a comple - to the primer, the intercalator , e . g ., a fluorescent or chemi
mentary nucleic acid , or antibody specific for DNA or RNA . luminescent intercalator, intercalates into the new double
In addition to the references noted above regarding nucleic 30 stranded region produced by the addition of a nucleotide to
acid hybridization , antibodies to , e. g ., DNA , RNA and the primer. The intercalator is then detected , e. g ., by an
DNA -RNA duplexes are known, as are immunological increase in signal, i.e ., a fluorescent signal. The signal is
methods of screening for and differentiating between ) optionally photobleached after detection , as described
DNA , RNA and RNA -DNA . For example , Coutlee et al. above , to decrease the background signal before addition of
( 1989 ) Analytical Biochemistry 181 :153 -162 describe non - 35 more nucleotides .
isotopic detection of RNA in an enzyme immunoassay using The above sequencing methods are optionally performed
a monoclonal antibody which binds DNA : RNA hybrids. In using the microfluidic devices and particle arrays of the
these assays, hybridization of an RNA target with a bioti invention to provide , e.g ., a high throughput system of
nylated DNA probe is performed , followed by incubation of sequencing. A schematic of such a system is provided in
the hybridized target-probe duplex on an anti-biotin plate , 40 FIG . 17 . FIG . 17 shows three 384 -well microtiter plates,
reaction of the resulting bound duplex with a beta - galacto - plates 1705, 1710 , and 1715 . Each well contains a set of
sidase labeled monoclonal antibody specific for RNA - DNA particles comprising a nucleic acid template . Therefore , the
hybrids , and addition of a fluorescent substrate . In another system shown optionally comprises 1152 different nucleic
example, a “ sandwich ” hybridization method is described acid templates that are optionally sequenced in a high
for non - isotopic detection of e . g ., RNA using oligonucle - 45 throughput manner. Additional microwell plates and chan
otides (Ishii & Ghosh ( 1993 ) Bioconjugate Chem . 4 :34 -41). nels are optionally used to provide a greater number of
In these assays , the RNA target is hybridized to a first templates . A plate of blank particle sets is also optionally
complementary oligonucleotide , which is linked to a bead . included , e .g ., plate 1720 . The particle sets are loaded into
The RNA target is then hybridized to a second complemen - a set of capillaries or channels as shown by capillary set
tary oligonucleotide conjugated to alkaline phosphatase . The 50 1725 . For example, 96 particle sets are optionally loaded
RNA target is detected by providing a chemiluminescent into each of 12 channels using 12 sipper capillaries or one
alkaline phosphatase substrate. These methods are readily sipper capillary fluidly coupled to each of the 12 channels .
adapted to the present invention , e. g ., by providing arrays of The particle sets are typically retained in the capillary or
such beads in a microfluidic format, as well as downstream , microchannel by a porous particle retention element, e .g ., a
sequential, or simultaneous detection of the alkaline phos - 55 sintered glass frit , a set of epoxy coated particles, or the like.
phatase reaction . Alternative particle retention devices are described above ,
For example , in one aspect, targets for sequencing are e .g ., narrowed channel dimensions . The particle retention
fixed to particles (for example, unsequenced clones can be element fixes or retains the particle sets, e. g., particle sets
fixed to particle sets, or specific fragments of such clones comprising nucleic acid templates , in the channel. The
can be fixed to particular particle sets . The particle sets are 60 particle sets , e . g ., templates, are then optionally exposed to
then flowed into e . g ., selected portions of a particle retention a series or train of reagents . The reagents are typically added
region . Small labeled probes ( e . g., 6 -15 mers , typically 6 - 12 through each capillary , e . g ., from another set of microwell
mers ) are then flowed into contact with the fixed targets for plates, to perform various assays , e . g ., sequencing. A single
sequencing under selected hybridization conditions ( typi- controller, e . g ., controller 1730 is optionally used to control
cally stringent hybridization conditions), and the arrays are 65 fluid flow through the sipper and channels. One or more
monitored for specific binding by the probes. The probes are detector is used to monitor the particle packets in the
then washed free of the targets and the process repeated with channels as various nucleotides are added . Alternatively,
 US 10 , 138,517 B2
detectors are positioned downstream of the channels to suited to use in methods and devices of the present invention
monitor the waste products , e.g ., to detect a fluorescent label (see also , U . S . Pat . Nos. 5,498 ,392 and 5 ,587 ,128 to Will
that has since been washed from the channels . For example ingham et al.).
detection optionally occurs in detection region 1735 . Using Thermocycling for PCR and other thermocyclic applica
a system such as that shown in FIG . 17 , one particle set is 5 tions ( e.g ., the ligase chain reaction , or LCR ) can be con
optionally loaded in about one minute . Therefore 96 tem ducted in microfluidic systems in at least two ways . First, a
plates are optionally analyzed , e . g ., sequenced , in 1 .6 hours. heat source ( external or internal ) can be used to thermocycle
Alternatively, particles with different chemistries are arrayed all or part of a device, thereby heating and cooling the array
sequentially in a single capillary and a template is flowed within the microfluidic system . In a second approach , joule
across the array, e .g., for sequencing . 10
heating is used . Thermocycling in microscale devices, e.g.,
In addition to sequencing by hybridization , essentially using joule heating, is described in application Ser. No.
similar methods can be used for determination of genetic
difference , assays for determining nucleic acid melting 08 / 977,528, filed Nov. 25, 1997 (now U . S . Pat. No. 5 , 965,
points, and the like . Additional details on these procedures 410 ). In brief , energy is provided to heat fluids , e. g .,
are found herein . 15 samples , analytes, buffers and reagents, in desired locations
PCR of the substrates in an efficient manner by application of
In addition to its applicability to sequencing. PCR is electric current to fluids in microchannels . Thus, the present
desirably practiced using the arrays of the invention. In invention optionally uses power sources that pass electrical
particular, PCR templates and /or reagents (e. g ., a thermo current through the fluid in a channel for heating purposes ,
stable polymerase ) can be fixed to particles, as described 20 aswell as for material transport . In exemplary embodiments,
herein and using techniques available in the art. Reagents the fluid passes through a channel of a desired cross -section
and / or templates can be passed over arrays, where PCR is ( e . g ., diameter ) to enhance thermal transfer of energy from
performed . This format is especially useful where several the current to the fluid . The channels can be formed on
PCR products are to be screened simultaneously (or sequen - almost any type of substrate material such as , for example ,
tially ). A variety of such PCR assays, e .g ., for diagnostic 25 amorphous materials ( e .g ., glass , plastic , silicon ), compos
applications ( e .g ., detection of viruses such as HIV , HBV, ites, multi-layered materials , combinations thereof, and the
HCV , etc ., detection of infectious organisms (bacteria , para - like . In general, electric current passing through the fluid in
sites, etc . ), detection of genetic abnormalities (genetic dis - a channel produces heat by dissipating energy through the
eases, cancer, etc .), as well as for research applications ( e . g ., electrical resistance of the fluid . Power dissipates as the
screening of drugs , drug targets , genes effected in vivo or in 30 current passes through the fluid and goes into the fluid as
vitro by drugs or potential drugs , results of forced evolution energy as a function of time to heat the fluid . The following
methods) as well as many others are well known in the mathematical expression generally describes a relationship
literature and adaptable to the present invention . between power, electrical current, and fluid resistance , i.e .,
Examples of techniques sufficient to direct persons of skill POWER = I2R where POWER = power dissipated in fluid ;
through in vitro amplification methods, including the poly - 35 l = electric current passing through fluid ; and R = electric
merase chain reaction (PCR ) the ligase chain reaction resistance of fluid .
(LCR ), Q -replicase amplification and other RNA poly - The above equation provides a relationship between
merase mediated techniques ( e. g., NASBA ) are found in power dissipated (“ POWER ) to current (“ I” ) and resistance
Berger, Sambrook , and Ausubel, as well as Mullis et al., (“ R ” ). In some of the embodiments, which are directed
( 1987 ) U .S . Pat. No . 4 ,683, 202 ; PCR Protocols A Guide to 40 toward moving fluid in channels , e . g ., to provide mixing,
Methods and Applications (Innis et al. eds) Academic Press electrophoretic separation , or the like , a portion of the power
Inc. San Diego , Calif. ( 1990 ) ( Innis ); Arnheim & Levinson goes into kinetic energy of moving the fluid through the
(Oct. 1 , 1990 ) C & EN 36 - 47 ; The Journal OfNIH Research channel. However, it is also possible to use a selected
( 1991) 3 , 81- 94 ; (Kwoh et al. ( 1989 ) Proc. Natl. Acad . Sci. portion of the power to controllably heat fluid in a channel
USA 86 , 1173 ; Guatelli et al. (1990 ) Proc . Natl. Acad . Sci. 45 or selected channel regions. A channel region suitable for
USA 87 , 1874 ; Lomeli et al . (1989 ) J. Clin . Chem 35 , 1826 ; heating is often narrower or smaller in cross-section than
Landegren et al., (1988 ) Science 241, 1077 - 1080 ; Van Brunt other channel regions in the channel structure , as a smaller
( 1990 ) Biotechnology 8 , 291- 294 ; Wu and Wallace , ( 1989 ) cross -section provides higher resistance in the fluid , which
Gene 4 , 560 ; Barringer et al. ( 1990 ) Gene 89, 117 , and increases the temperature of the fluid as electric current
Sooknanan and Malek ( 1995 ) Biotechnology 13: 563- 564 . 50 passes through . Alternatively, the electric current is
Improved methods of cloning in vitro amplified nucleic increased across the length of the channel by increased
acids are described in Wallace et al., U . S . Pat. No. 5 ,426 , voltage, which also increases the amount of power dissi
039. Improved methods of amplifying large nucleic acids by pated into the fluid to correspondingly increase fluid tem
PCR are summarized in Cheng et al. ( 1994 ) Nature 369: perature .
684-685 and the references therein , in which PCR amplicons 55 The introduction of electrical current into fluid causes
of up to 40 kb are generated . One of skill will appreciate that heat ( Joule heating ). In the examples of fluid movement
essentially any RNA can be converted into a double stranded herein where thermal effects are not desired, the heating
DNA suitable for restriction digestion , PCR expansion and effect is minimal because, at the small currents employed ,
sequencing using reverse transcriptase and a polymerase . heat is rapidly dissipated into the chip itself. By substantially
See, Ausubel, Sambrook and Berger, all supra. 60 increasing the current across the channel, rapid temperature
It will be appreciated that these benchtop uses for PCR are changes are induced that can be monitored by conductivity .
adaptable to microfluidic systems. Indeed , PCR amplifica - At the same time, the fluid can be kept static in the channel
tion is particularly well suited to use in the apparatus , by using alternating instead of direct current. Because nano
methods and systems of the invention . liter volumes of fluid have tiny thermal mass, transitions
Thermocycling amplification methods, including PCR 65 between temperatures can be extremely short. Oscillations
and LCR , are conveniently performed in microscale devices , between any two temperatures above 0° C . and below 100°
making iterative fluidic operations involving PCR well C . in 100 milliseconds have been performed . Additional
 US 10 , 138 ,517 B2
43 44
applications of joule heating to sequencing methodologies is The procedure is referred to as DNA sequencing using
set forth in " Closed Loop Biochemical Analyzers ” (WO differential extension with nucleotide subsets (DENS). See ,
98 /45481) . Raja et al. ( 1997 ) Nucleic Acids Research 25 ( 4 ):800 - 805.
Melting Point Analysis of Nucleic Acids These modular primer strategies, for sequencing or PCR , are
In an embodiment similar to sequencing by hybridization , 5 readily adapted to the present methods and arrays . For
the systems, devices arrays and methods of the present example , template or primer nucleic acids can be fixed ,
invention can be used to detect variations in nucleic acid directly or indirectly, to the array members of the present
sequences by determining the strength of the hybridization invention . The corresponding template or primer is flowed
between the targeted nucleic acid and probes that are puta into contact with the array member, along with any other
tive perfect complements to the target. By identifying the 10 components of the sequencing or PCR reaction (or other
difference in stability between the imperfect and perfect reaction , if appropriate ) and the reaction performed under
hybrids under conditions of increasing hydrogen bond appropriate conditions to the reaction . Products are detected
stress , one can identify those nucleic acids that contain a e . g ., by washing the products from the array and detecting
variation . the products at a downstream detector.
In practice , a microfluidic device is configured to accept 15 Liquid Crystal Assay Systems.
a sample containing an amplified nucleic acid or polynucle - In still another embodiment, binding of a protein to a
otide sequence of interest, convert it to single -stranded form , target component such as a nucleic acid can be detected by
facilitate hybridization with a nucleic acid probe, such as an the use of liquid crystals. Liquid crystals have been used , for
oligonucleotide , and then subject the hybridization mixture example , to amplify and transduce receptor -mediated bind
to a chemical or temperature gradient that distinguishes 20 ing of proteins at surfaces into optical outputs. Spontane
between perfectly matched targets and those that differ by at ously organized surfaces can be designed so that a protein ,
least one base pair (mismatch ). Either the probe or the upon binding to a nucleic acid hosted on the surface of an
template can be fixed to an array component. In some array member herein , triggers changes in the orientations of
embodiments, one or more loci or targeted areas of the 1 - to 20 -micrometer -thick films of supported liquid crystals ,
sample polynucleotide are first amplified by techniques such 25 thus corresponding to a reorientation of - 10 % to 100 meso
as PCR or sandwich hybridization . In other embodiments, gens per protein . Binding -induced changes in the intensity
unamplified polynucleotide is provided to the device and of light transmitted through the liquid crystal are easily seen
amplified therein . with the naked eye and can be further amplified by using
Hybridization of the probe results in a perfect hybrid with surfaces designed so that protein -nucleic acid binding causes
no mismatches when the sample polynucleotide contains the 30 twisted nematic liquid crystals to untwist (see, e. g., Gupta et
complementary sequence , i.e ., no variation , or in a hybrid al. ( 1998 ) Science, 279 : 2077 - 2080 ) . This approach to the
with mismatches if the sample polynucleotide differs from detection of protein /nucleic acid interactions does not
the probe, i.e ., contains a sequence variation . The stability of require labeling of the analyte , does not require the use of
the imperfect hybrid differs from the perfect hybrid under electroanalytical apparatus, provides a spatial resolution of
conditions of increasing hydrogen bond stress . A variety of 35 micrometers, and is sufficiently simple that it is useful in
methods are available for subjecting the hybrids to increas biochemical assays and imaging of spatially resolved chemi
ing hydrogen bond stress, sufficient to distinguish between cal libraries .
perfectly matched probe/target hybrids and imperfect Diagnostic /Screening Assays
matches . For example , the hybrids are optionally subjected In one aspect of the present invention , diagnostic assays
to a temperature gradient, or alternatively, can be subjected 40 are provided . As discussed supra, assays can take the form
to increasing concentrations of a chemical denaturant, e . g ., of nucleic acid detection or sequencing assays which screen
formamide , urea , and the like, or increasing pH . By moni- for the presence or absence or type of a nucleic acid . The
toring hybridization between one or more array component presence or type of a nucleic acid in a biological sample is
and one or more unknown nucleic acid , it is possible to an indicator for the presence of, e .g ., an infectious organism
determine percent sequence complementarity . 45 (e .g ., virus,bacteria , fungal cell or the like ) in the biological
The assay is optionally repeated several times, varying the sample . Thus , detection of a nucleic acid provides an indi
concentration of denaturant or temperature with each suc - cation that, e .g ., a patient is infected with such an infectious
cessive assay. By monitoring the level of hybridization , one organism .
can determine the concentration of denaturant at which the Similarly, the presence of certain mRNAs (e.g., mRNAs
probe -target hybrid is denatured . This level is then compared 50 from oncogene products ) and/or specific sequences in
to a standard curve , to determine whether one or more genomic DNA are correlated with a variety of disease states ,
variations are present in the nucleic acid . including, e . g ., cancer. Thus, any of the various assay
Other Sequencing Strategies formats described herein can be used for the detection of
Other sequencing methods which reduce the number of specific nucleic acids which correspond to particular disease
steps necessary for template preparation and primer selec - 55 states .Many such correlations are well established in the art.
tion have been developed . One proposed variation on Microfluidic devices are very efficient at capturing mol
sequencing technology involves the use ofmodular primers ecules in a fluid stream , e.g ., in functionalized channels , or
for use in PCR and DNA sequencing . For example , on particle sets within the channels . After capturing a
Ulanovsky and co -workers have described themechanism of particular molecule , e . g ., a sample that is present only in
the modular primer effect (Beskin et al. ( 1995 ) Nucleic 60 small concentrations, the devices are used to detect the
Acids Research 23 ( 15 ) : 2881- 2885 ) in which short primers molecule , e . g ., its presence or type . For example , a number
of 5 -6 nucleotides can specifically prime a template -depen - of nucleic acids or cells are optionally flowed through a
dent polymerase enzyme for template dependent nucleic microfluidic device for capture, e. g., by a set of particles.
acid synthesis . A modified version of the use of the modular Reagents that bind to a molecule of interest, e . g ., by hybrid
primer strategy , in which small nucleotide primers are 65 ization , are optionally flowed across the captured molecules
specifically elongated for use in PCR to amplify and and a molecule of interest is identified , e. g ., by its binding
sequence template nucleic acids has also been described . specificity . This is especially useful when samples of low
 US 10 ,138,517 B2
45 46
concentration are captured because the sample is optionally fication steps can be performed in the microfluidic system ,
positioned in a defined location for further analysis or or external to the microfluidic system .
detection , e .g ., by specific binding moieties . Probes can be fixed to array members to create a probe
In addition to screening for diseases, the arrays of the array, and expression products (or in vitro amplified nucleic
invention can also be used to select for the presence of 5 acids corresponding to expression products ) can be labeled
desirable traits. For example, in agriculture ,many correla and hybridized with the array . For convenience, it may be
helpful to use several arrays simultaneously (e .g., in the
tions between desirable traits and particular genomic or same or in separate microfluidic devices ), or to use arrays of
RNA sequences are well established . For example , crops large or small numbers of members, depending on the
such as corn , soybean , cotton , potatoes, tomatoes, wheat,
millet, and many others are routinely selected , in part, by 10 number of expression products to be detected .
It will be appreciated that probe design is influenced by
selecting for the presence or absence of nucleic acids which the intended application . For example , where several allele
are correlated with desirable traits such as yield , disease specific probe -target interactions are to be detected in a
resistance , herbicide resistance , drought tolerance and the single assay, e.g., on a single array, it can be desirable to
like . 15 have similar melting temperatures for all of the probes (or
Expression Profiling course this is not necessary , as joule or zone heating can be
One particularly useful aspect of array technology is the used to maintain different portions of an array at different
ability to profile expression of one or more expression temperatures). Accordingly, the length of the probes are
products for one or more biological sample . By profiling optionally adjusted so that the melting temperatures for all
expression of RNAs and /or proteins, it is possible to deter- 20 of the probes on the array are closely similar ( it will be
mine whether certain disease-related genes (e.g., oncogenes, appreciated that different lengths for different probes may be
infectious organisms, or the like ) are expressed . In addition , needed to achieve a particular Tm where different probes
it is possible to use expression profiles to determine com - have different GC contents ). Although melting temperature
binatorial genetic and polygenetic effects on expression and is a primary consideration in probe design , other factors are
to predict phenotypes resulting from polygenetic effects . For 25 also optionally used to further adjust probe construction ,
example , where expression of several genes causes a phe - such as elimination of self-complementarity in the probe
notype, it is possible to monitor expression of these genes on (which can inhibit hybridization of a target nucleotide ).
the arrays of the invention . For example , polygenic effects One way to compare expression products between two
often underlie disease or herbicide resistance in plants , cell populations is to identify mRNA species which are
pesticide resistance in insects , and disease states in animals 30 differentially expressed between the cell populations (i.e .,
( including humans). present at different abundances between the cell popula
Avariety of sources ofbiological material can be profiled , tions ). In addition to the techniques noted above, another
including animal sources (including human, vertebrate , preferred method is to use subtractive hybridization (Lee et
mammalian , insect, etc.) and plant sources (e. g ., wild or al. ( 1991 ) Proc . Natl. Acad . Sci. (U . S . A .) 88 : 2825) or
domesticated plants, such as crop plants ). The biological 35 differential display employing arbitrary primer polymerase
sources can be from tissues , cells , whole organisms, cell chain reaction (PCR ) (Liang and Pardee ( 1992 ) Science
cultures , tissue cultures , or the like . A variety of profiling 257 : 967 ). Each of these methods has been used by various
methods are adaptable to the present system , including investigators to identify differentially expressed mRNA spe
hybridization of expressed or amplified nucleic acids to a cies . See , Salesiotis et al. ( 1995 ) Cancer Lett. 91: 47; Jiang
nucleic acid array, hybridization of expressed polypeptides 40 et al. ( 1995 ) Oncogene 10 :1855 ; Blok et al. (1995 ) Prostate
to a protein array , hybridization of peptides or nucleic acids 26 : 213 ; Shinoura et al. ( 1995 ) Cancer Lett. 89: 215 ; Murphy
to an antibody array , subtractive hybridization , differential et al. ( 1993 ) Cell Growth Differ 4 :715 ; Austruy et al. ( 1993 )
display and others . Cancer Res. 53:2888; Zhang et al. ( 1993) Mol. Carcinog.
In one preferred embodiment, the expression products 8 : 123; and Liang et al. (1992 ) Cancer Res. 52 :6966 ). The
which are detected in the methods of the invention are 45 methods have also been used to identify mRNA species
RNAs, e . g ., mRNAs expressed from genes within a cell which are induced or repressed , e . g ., by drugs or certain
derived from the biological source to be profiled . A number nutrients ( Fisicaro et al. ( 1995 ) Mol. Immunol. 32 : 565 ;
of techniques are available for detecting RNAs, which can Chapman et al . (1995 ) Mol. Cell . Endocrinol. 108 : 108 ;
be utilized or adapted to the arrays of the invention . For Douglass et al. ( 1995 ) J. Neurosci. 15 : 2471; Aiello et al.
example , northern blot hybridization is widely used for 50 (1994 ) Proc . Natl. Acad . Sci. ( U . S . A .) 91:6231; Ace et al.
RNA detection , and is generally taught in a variety of ( 1994 ) Endocrinology 134 : 1305 .
standard texts on molecular biology , including: Berger and for the technique of differential display, Liang and Pardee
Kimmel, Sambrook , Ausubel (all supra ), etc . Furthermore , ( 1992 ), supra provide theoretical calculations for the selec
one of skill will appreciate that essentially any RNA can be tion of 5 ' and 3 ' arbitrary primers . Correlation of observed
converted into a double stranded DNA using a reverse 55 results to the theory is also provided . In practice, 5' primers
transcriptase enzyme and a polymerase . See, Ausubel, Sam - of less than about 9 nucleotides may not provide adequate
brook and Berger, id . Thus, detection of mRNAs can be specificity (slightly shorter primers of about 8 to 10 nucleo
performed by converting , e.g ., mRNAs into DNAs, which tides have been used in PCR methods for analysis of DNA
are subsequently detected in , e . g ., a “ Southern blot” format. polymorphisms. See also , Williams et al. ( 1991 ) Nucleic
Furthermore , DNAs can be amplified to aid in the detec - 60 Acids Research 18 , 6531). The primer ( s ) optionally com
tion of rare molecules by any of a number of well known prise 5 '-terminal sequences which serve to anchor other PCR
techniques, including: the polymerase chain reaction (PCR ), primers (distal primers ) and/ or which comprise a restriction
the ligase chain reaction (LCR ), Q -replicase amplification site or half- site or other ligatable end . Where a restriction
and other RNA polymerase mediated techniques ( e . g ., site or amplification template for a second primer is incor
NASBA ). Examples of these techniques are found in Berger, 65 porated , the primers are optionally longer than those
Sambrook , and Ausubel, id ., as well as in those references described above by the length of the restriction site , or
noted supra regarding in vitro amplification . These ampli - amplification template site. Standard restriction enzyme
 US 10 , 138,517 B2
47 48
sites include 4 base sites , 5 base sites , 6 base sites, 7 base be used to simultaneously and specifically detect large
sites, and 8 base sites . An amplification template site for a numbers of proteins . Either antibody or two dimensional gel
second primer can be of essentially any length , for example , electrophoresis can readily be adapted to microfluidic sys
the site can be about 15 - 25 nucleotides in length . Any of tems.
primers, templates, or other reactants (e . g ., enzymes ) can be 5 One typical technology for detecting specific proteins
fixed to array particle members . involves making antibodies to the proteins . By specifically
The amplified products are optionally labeled and are detecting binding of an antibody and a given protein , the
typically resolved e . g ., by electrophoresis on a polyacryl- presence of the protein can be detected . In addition to
amide gel or other sieving matrix in the microfluidic system ; available antibodies , one of skill can easily make antibodies
the location (s ) where label is present are recovered from the 10 using existing techniques, or modify those antibodies which
sieving matrix , typically by elution or electrokinetic meth - are commercially or publicly available . In addition to the art
ods. The resultant recovered product species can be sub - referenced above , general methods of producing polyclonal
cloned into a replicable vector with or without attachment of and monoclonal antibodies are known to those of skill in the
linkers , amplified further, and/ or detected , or even art. See , e .g ., Paul ( ed .) (1993 ) Fundamental Immunology ,
sequenced directly . As noted , direct sequencing of PCR 15 Third Edition Raven Press , Ltd ., New York Coligan ( 1991)
generated amplicons by selectively incorporating boronated Current Protocols in Immunology Wiley /Greene, N . Y .; Har
nuclease resistant nucleotides into the amplicons during low and Lane ( 1989 ) Antibodies: A Laboratory Manual Cold
PCR and digestion of the amplicons with a nuclease to Spring Harbor Press, N . Y .; Stites et al. ( eds.) Basic and
produce sized template fragments has been performed (Por - Clinical Immunology (4th ed .) Lange Medical Publications ,
ter et al. (1997 ) Nucleic Acids Research 25( 8 ): 1611 - 1617 ) 20 Los Altos , Calif., and references cited therein ; Goding
and is applicable to the present invention . ( 1986 ) Monoclonal Antibodies : Principles and Practice ( 2d
It is expected that one of skill can use , e. g ., differential ed .) Academic Press, New York , N . Y .; and Kohler and
display for expression profiling. In addition , companies such Milstein ( 1975 ) Nature 256 :495 -497 . Other suitable tech
as CuraGen Corp . (New Haven Conn .) provide robust niques for antibody preparation include selection of libraries
expression profiling based upon differential display tech - 25 of recombinant antibodies in phage or similar vectors . See ,
niques . See , e .g., WO 97 / 15690 by Rothenberg et al ., and Huse et al. (1989 ) Science 246 : 1275 - 1281 ; and Ward et al.
these methods are readily adapted to a microfluidic format. ( 1989 ) Nature 341:544 - 546 . Specific monoclonal and poly
Expression Profiling of Proteins clonal antibodies and antisera will usually bind with a KD of
In addition to nucleic acid formats, the arrays of the at least about 0 .1 uM , preferably at least about 0 .01 uMor
invention can easily be adapted to screening other biological 30 better , and most typically and preferably , 0 .001 uMor better.
components as well , including cells , antibodies, antibody As used herein , an “ antibody” refers to a protein consisting
ligands and the like. The presence or absence of such cells , of one or more polypeptide substantially or partially
antibodies and antibody ligands are also known to correlate encoded by immunoglobulin genes or fragments of immu
with desirable or undesirable features. For example, one noglobulin genes. The recognized immunoglobulin genes
common assay for the detection of infectious organisms 35 include the kappa, lambda, alpha, gamma, delta , epsilon and
involves an ELISA assay or western blot to detect the mu constant region genes, as well as myriad immunoglobu
presence of antibodies in a patient to a particular infectious lin variable region genes . Light chains are classified as either
agent ( e . g ., an HIV virus) . Such immunological assays are kappa or lambda . Heavy chains are classified as gamma, mu,
also adaptable to the arrays of the present invention , e .g ., by alpha, delta , or epsilon , which in turn define the immuno
fixing an antibody or antibody target to an array member and 40 globulin classes, IgG , IgM , IgA , IgD and IgE , respectively .
exposing the array member to the corresponding antibody or A typical immunoglobulin (antibody) structural unit is
antibody target. Thus, immunological reagents (i.e ., those known to comprise a tetramer. Each tetramer is composed of
used in an assay in which an antibody is a target or reagent) two identical pairs of polypeptide chains, each pair having
can be flowed across the arrays of the invention . one “ light” (about 25 KD ) and one “ heavy” chain (about
In addition to profiling RNAs ( or corresponding cDNAs) 45 50 -70 kD ). The N -terminus of each chain defines a variable
as described above, it is also possible to profile proteins. In region of about 100 to 110 or more amino acids primarily
particular, various strategies are available for detecting responsible for antigen recognition . The terms variable light
many proteins simultaneously . As applied to the present chain (VL ) and variable heavy chain ( VH ) refer to these
invention , detected proteins, corresponding to expression light and heavy chains respectively . Antibodies exist as
products , can be derived from one of at least two sources . 50 intact immunoglobulins or as a number of well characterized
First, the proteins which are detected can be either directly fragments produced by digestion with various peptidases .
isolated from a cell or tissue to be profiled , providing direct Thus, for example , pepsin digests an antibody below the
detection ( and , optionally , quantification ) of proteins present disulfide linkages in the hinge region to produce F ( ab ) '2 , a
in a cell. Second , mRNAs can be translated into cDNA dimer of Fab which itself is a light chain joined to VH -CH1
sequences, cloned and expressed . This increases the ability 55 by a disulfide bond . The F (ab )'2 may be reduced under mild
to detect rare RNAs, and makes it possible to immediately conditions to break the disulfide linkage in the hinge region
associate a detected protein with its coding sequence . For thereby converting the (Fab ')2 dimer into an Fab 'monomer.
purposes of the present invention , even an out of frame The Fab ' monomer is essentially an Fab with part of the
peptide is an indicator for the presence of a corresponding hinge region (see , Fundamental Immunology, W . E . Paul,
RNA . 60 ed ., Raven Press , N . Y . ( 1993 ), for a more detailed descrip
A variety of hybridization techniques, including western tion of other antibody fragments ). While various antibody
blotting, ELISA assays, and the like are available for detec - fragments are defined in terms of the digestion of an intact
tion of specific proteins. See , Ausubel, Sambrook and antibody, one of skill will appreciate that such Fab ' frag
Berger, supra . See also , Antibodies : A Laboratory Manual, ments may be synthesized de novo either chemically or by
( 1988) E . Harlow and D . Lane, Cold Spring Harbor Labo - 65 utilizing recombinant DNA methodology . Thus , the term
ratory , Cold Spring Harbor, N . Y . Non -hybridization based antibody , as used herein also includes antibody fragments
techniques such as two- dimensional electrophoresis can also either produced by the modification of whole antibodies or
 US 10 ,138 ,517 B2
49 50
synthesized de novo using recombinant DNA methodolo - samples, and is suitable for identification of many proteins
gies. Antibodies include single chain antibodies, including in a sequential or parallel fashion . For example, Hutchens et
single chain Fv (sFv) antibodies in which a variable heavy al. U .S . Pat . No . 5,719,060 , describe methods and apparatus
and a variable light chain are joined together (directly or for desorption and ionization of analytes for subsequent
through a peptide linker ) to form a continuous polypeptide . 5 analysis by mass spectroscopy and /or biosensors . In the
For purposes of the present invention , antibodies or present invention , components can be released from array
antibody fragments can be arrayed , e. g ., by coupling to an members in a sequential fashion and prepared for mass
amine moiety fixed to a solid phase particle array member, spectrometry .
in a manner similar to that described above for construction Two and three dimensional gel based approaches can also
of nucleic acid arrays. As above for nucleic acid probes, the 10 be used for the specific and simultaneous identification and
antibodies can be labeled , or proteins corresponding to quantification of large numbers of proteins from biological
expression products can be labeled . In this manner, it is samples . Multi-dimensional gel technology is well-known
possible to couple hundreds, or even thousands , of different and described e. g., in Ausubel, supra , Volume 2 , Chapter 10 .
antibodies to members of an array. In one embodiment, a As applied to microfluidic systems, intersecting channels
bacteriophage antibody display library is screened with a 15 can comprise different separation media . After flowing com
polypeptide encoded by a cell , or obtained by expression of ponents through a first media , components can be flowed
mRNAs, differential display, subtractive hybridization or the through a second media in a second channel. The compo
like. Combinatorial libraries of antibodies have been gen - nents can be labeled , e. g., by flowing staining reagents into
erated in bacteriophage lambda expression systems which contact with the components . The labeled components are
are screened as bacteriophage plaques or as colonies of 20 then flowed past a suitable detector (or the entire microflu
lysogens (Huse et al. ( 1989) Science 246 : 1275 ; Caton and idic system can be imaged simultaneously , e .g ., using a CCD
Koprowski ( 1990 ) Proc . Natl . Acad . Sci. (U .S . A .)87:6450 ; array ). Image analysis of multi-dimensional protein separa
Mullinax et al (1990 ) Proc . Natl. Acad . Sci. (U . S . A .)87: tion channels provides an indication of the proteins that are
8095 ; Persson et al. ( 1991) Proc . Natl . Acad . Sci. (U . S . A . ) expressed e. g ., in a cell or tissue type .
88 :2432 ). Various embodiments of bacteriophage antibody 25 In addition to identifying expression products , such as
display libraries and lambda phage expression libraries have proteins or RNA , it is also possible to screen for large
been described (Kang et al. ( 1991 ) Proc. Natl. Acad . Sci. numbers of metabolites in cell or tissue samples . The
(U .S . A .)88: 4363; Clackson et al. ( 1991) Nature 352 :624 ; presence , absence or level of a metabolite can be treated as
McCafferty et al. (1990 ) Nature 348 :552; Burton et al. a character for comparison purposes in the same way that
( 1991) Proc . Natl. Acad . Sci. (U . S . A .) 88 : 10134 ; Hoogen - 30 nucleic acids or proteins are discussed herein . Metabolites
boom et al. ( 1991) Nucleic Acids Res . 19 :4133; Chang et al. can be monitored by any of currently available microfluidic
( 1991 ) J. Immunol. 147 : 3610 ; Breitling et al. ( 1991 ) Gene method , including chromatography, uni or multi dimen
104 :147 ; Marks et al . (1991 ) J. Mol. Biol. 222:581; Barbas sional gel separations, hybridization to complementary mol
et al. ( 1992 ) Proc. Natl. Acad . Sci. (U . S . A .) 89: 4457 ; ecules, or the like .
Hawkins and Winter ( 1992 ) J. Immunol. 22 : 867 ; Marks et 35 Immunoassays
al. ( 1992) Biotechnology 10 :779 ;Marks et al . (1992 ) J. Biol. A particular protein or other biological component can be
Chem . 267: 16007; Lowman et al (1991) Biochemistry quantified by a variety of immunoassay methods which can
30 :10832 ; Lerner et al. (1992 ) Science 258 :1313 . be practiced using the arrays of the invention . For a review
The patterns of hybridization which are detected on the of immunological and immunoassay procedures in general,
array provide an indication of the presence or absence of 40 see Stites and Terr ( eds.) 1991 Basic and Clinical Immu
expressed protein sequences . As long as the library or array n ology ( 7th ed .); Maggio ( ed.) ( 1980 ) Enzyme Immunoassay
against which a population of proteins are to be screened can CRC Press , Boca Raton , Fla.; Tijan (1985) “ Practice and
be correlated from one experiment to the next (e .g ., by Theory of Enzyme Immunoassays," Laboratory Techniques
noting the x -y coordinates of the library or array member, or in Biochemistry and Molecular Biology , Elsevier Science
by noting the position of markers within the arrays (e .g., 45 Publishers B . V ., Amsterdam ; Harlow and Lane, supra ; Chan
where the arrays comprise mobile members )), no sequence ( ed .) ( 1987 ) Immunoassay : A Practical Guide Academic
information is required to compare expression profiles from Press , Orlando , Fla.; Price and Newman (eds.) ( 1991) Prin
one representative sample to another. In particular, the mere ciples and Practice of Immunoassays Stockton Press , N . Y .;
presence or absence (or degree ) of label provides the ability Ngo ( ed . ) ( 1988 ) Non isotopic Immunoassays Plenum Press ,
to determine differences . One advantage of using arrayed 50 N . Y . and Fundamental Immunology , Third Edition , W . E .
libraries of antibodies for protein detection is that the Paul, ed ., Raven Press , N . Y . ( 1993 ).
individual library members can be uncharacterized . Immunoassays often utilize a labeling agent to specifi
More generally, peptide and nucleic acid hybridization to cally bind to and label the binding complex formed by the
arrays or libraries (or even simple two dimensional gels ) can capture agent and the analyte . The labeling agentmay itself
be treated in a manner analogous to a bar code label. Any 55 be one of the moieties comprising the antibody /analyte
diverse library or array can be used to screen for the complex . Thus, the labeling agent may be a labeled analyte
presence or absence of complementary molecules, whether or a labeled anti- analyte antibody. Alternatively , the labeling
RNA , DNA , protein , or a combination thereof. By measur agentmay be a third moiety , such as another antibody, that
ing corresponding signal information between different specifically binds to the antibody /analyte complex, or to a
sources of test material ( e . g ., different hybrid or inbred 60 modified capture group ( e . g ., biotin ) which is covalently
plants, or different tissues, or the like), it is possible to linked to the analyte or anti - analyte antibody . Any of these
determine differences in expression products for the differ - components can be e . g ., fixed to array members or can be
ent source materials . As set forth below , this process is present in reagent trains which are flowed across array
facilitated by various high throughput integrated systems set m embers .
forthbelow . 65 In one embodiment, the labeling agent is an antibody that
In addition to array based approaches ,mass spectrometry specifically binds to a capture agent ( e .g ., an antibody which
is in use for identification of large sets of proteins in binds either the analyte, an analyte -antibody complex or an
 US 10 ,138 ,517 B2
51
antibody which binds the analyte ). Such labeling agents are In one embodiment, the capture agent is immobilized on
well known to those of skill in the art, and most typically a solid substrate . The amount of e . g ., polypeptide bound to
comprise labeled antibodies that specifically bind antibodies the capture agent is determined either by measuring the
of the particular animal species from which the capture amount of analyte present in an analyte - antibody complex ,
agent is derived ( e . g ., an anti- idiotypic antibody ). Thus, for 5 or alternatively by measuring the amount of remaining
example , where the capture agent is a mouse derived anti- uncomplexed analyte or antibody. The amount ofmaterial in
marker gene antibody , the label agent may be a goat anti- a sample to be assayed can also be detected by providing
mouse IgG , i.e ., an antibody specific to the constant region exogenous labeled marker gene to the assay .
of the mouse antibody. Other proteins capable of specifically A hapten inhibition assay is another preferred competitive
binding immunoglobulin constant regions, such as strepto - 10 assay. In this assay, a known analyte is fixed on an array
coccal protein A or protein G are also used as the labeling member . A known amount of antibody is added to the
agent. These proteins are normal constituents of the cell sample, and the sample is then contacted with the fixed
walls of streptococcal bacteria . They exhibit a strong non analyte . In this case , the amount of antibody bound to the
immunogenic reactivity with immunoglobulin constant fixed analyte is proportional to the amount of analyte in the
regions from a variety of species. See , generally Kronval, et 15 sample. Again the amount of immobilized antibody is
al., ( 1973 ) J. Immunol., 111 : 1401- 1406 , and Akerstrom , et detected by quantitating either the immobilized fraction of
al., ( 1985) J. Immunol., 135 :2589 -2542 . antibody or the fraction of the antibody that remains in
Throughout the assays , incubation and/ or washing steps solution . Detection may be direct where the antibody is
may be required after each combination of reagents . Incu - labeled , or indirect where a labeled moiety is subsequently
bation steps can vary from about 0 .5 seconds to several 20 added which specifically binds to the antibody as described
hours. However, the incubation time will depend upon the above .
assay format, analyte, volume of solution , concentrations, Many other immunoassay formats are known and can be
and the like . One advantage of the present invention is that practiced by fixing one or more component of the assay to
incubation times can ordinarily be short, because , in micro - an array member in a microfluidic array of the invention ,
fluidic systems, small fluid volumes are ordinarily used . 25 e .g ., using the coupling techniques described supra .
Usually, the assays are carried out at ambient temperature , Downstream Separations
although they can be conducted over a range of tempera For all of the sequencing and PCR methods noted above,
tures, such as 5 C to 45 C . as well as for many other methods noted herein , products
(i) Non Competitive Assay Formats can be electrophoresed , e .g ., following release from an array ,
Immunoassays for detecting an analyte can be, e. g ., 30 e . g ., to facilitate separation and detection of the products .
competitive or noncompetitive . Noncompetitive immunoas - microfluidic systems which combine fluid handling and
says are assays in which the amount of captured analyte is electrophoresis are described , e.g., in U .S . Ser. No . 09 /093 ,
directly measured . In a “ sandwich ” assay, for example, the 832 “ MICROFLUDIIC MATRIX LOCALIZATION APPA
capture agent (e .g ., an antibody ) is bound directly to an array RATUS AND METHODS” Burd Mehta and Kopf-Sill filed
member where it is fixed or immobilized . These immobi- 35 Jun . 8 , 1998 (now U .S . Pat . No. 6 ,306 ,590 ). In brief, this
lized antibodies then capture analytes present in a test application , which describes , e. g ., anaphasic and especially
sample . The analytes which are immobilized are optionally multiphasic microfluidic systems, a channel comprising a
bound by a labeling agent, such as a second antibody bearing liquid phase intersects a channel comprising a sieving
a label. Alternatively, the second marker gene antibody may matrix . Applied to the present invention, products are
lack a label , but it may, in turn , be bound by a labeled third 40 optionally washed from arrays , where they are flowed into
antibody specific to antibodies of the species from which the contact with a sieving matrix . The product components
second antibody is derived . In another format, a sandwich typically “ stack ” at the fluid -sieving matrix interface , and
assay is unnecessary because either the antibody or analyte are then electrophoresed through the sieving matrix . The
are labeled , with the complementary component typically products can then be detected during or after electrophoresis,
being fixed to an array member. 45 e . g ., by placing a detection element within or proximal to the
Sandwich assays for an analyte are optionally con - sieving matrix . The products can also be purified in the
structed . As described above , the antibody or antibody sieving matrix and electrokinetically (or by pressure mecha
ligand bound to an array member specifically binds to the nisms) moved into contact with subsequent reactants or
corresponding element present in a sample . A labeled anti- additional array members for further processing or for use as
body then binds to analyte -antibody complexes . Free labeled 50 reactants in subsequent reactions ( e. g ., as templates in
antibody is washed away, e . g ., by electrophoresis , electroos - sequencing reactions, as targets for amplification , as probes
mosis , electrokinesis or pressure based fluid movement and to detect amplified products or other targets, etc . ).
the remaining bound labeled complex is detected (e . g ., using In uniphasic separatory systems, array components are
a gamma detector where the label is radioactive, or an optionally dispersed within a sieving matrix . Reactions such
optical arrangement where the label is fluorescent or lumi- 55 as sequencing , PCR , LCR , or the like can be conducted on
nescent). or proximal to the array members , with products being
( ii ) Competitive Assay Formats released into the sieving matrix for separation .
In competitive assays, the amount of analyte present in the In addition to the multiphasic and uniphasic microfluidic
sample is measured indirectly by measuring the amount of systems of the ' 832 application , a variety of microfluidic
an added ( exogenous ) analyte displaced ( or competed away ) 60 electrophoretic applications are described , e . g ., in “ Closed
from a capture agent by the analyte present in the sample . In Loop Biochemical Analyzers ” (WO 98 /45481), as well as
one competitive assay, a known amount of analyte is added other references available in the art .
to the sample and the sample is contacted with a capture Type Switchable Arrays
agent, in this case an antibody that specifically binds the In one aspect, the arrays are particle type switchable . In
analyte . The amount of analyte bound to the antibody is 65 this embodiment, arrays are modified by flowing reagents
inversely proportional to the concentration of analyte pres across all , or a portion of the particle sets of the arrays. The
ent in the sample . reagents chemically interact with ( e.g ., covalently modify,
 US 10 ,138 ,517 B2
53 54
hybridize to , or the like ) particle sets of the array, thereby changes in pH , light levels , or the like . These reagents can
altering one ormore sets of the array . Thus, particle sets can be maintained in an inactive state and activated when they
be fixed in place (or flowable ) and switchable from one type are brought into contact with an array member. Similarly,
of particle set to another. For example , in one aspect, the array members can be maintained in an inactive state and
ordered array of a plurality of sets of particles is produced 5 activated by exposure to a temperature change , a change in
by flowing a substantially homogeneous or heterogeneous pH , light, or the like.
set of particles into a particle modification region and Similarly , reagents can be trapped or “ caged” by being
flowing a plurality of particle modification reagents across complexed to a particle and released or “ uncaged ” from the
the substantially homogeneous or heterogeneous set of par - particle by exposure to an activating reagent or reaction
ticles. The reagents react with the substantially homoge - 10 condition . Using these approaches , reagents can be delivered
neous or heterogeneous set of particles to create a plurality in trapped or inactive packets to a reaction site where they
of sets of different particles. For example , the homogeneous are released and / or activated .
or heterogeneous sets of particles optionally include a plu - For example , a sequencing reagent such as a nucleic acid
rality of particles which have one or more molecular tags template can be fixed to a particle , e . g ., by synthesizing or
( e . g ., streptavidin , avidin , biotin , an antibody, an antibody 15 fixing a single - stranded oligonucleotide on the surface of the
ligand , a nucleic acid , a nucleic acid binding molecule , etc . ). particle and hybridizing a complimentary nucleic acid to the
The plurality of particle modification reagents can include fixed single stranded nucleic acid . The complimentary
one or more anti -tag ligand. The plurality of particle modi nucleic acid can be released by exposure to , e.g., heat , or
fication reagents can be flowed sequentially across the exposure to a base ( e. g ., dilute NaOH ), or exposure to a
substantially homogeneous set of particles, thereby binding 20 denaturant (e .g ., guanidine HCL ). For example , the particle
the anti -tag ligand to the tag and producing sets of different is optionally flowed to a point where a sequencing reaction
particles , each set having a different bound particle modifi- is to be performed , and the complimentary strand then
cation reagent such as a nucleic acid . Tags and tag ligands released by heat or exposure to base or a denaturant.
can be attached to particles or particle modification reagents Alternatively , a sequencing reaction , e . g ., by PCR exonu
directly or through a linker, through covalent or non - cova - 25 clease digestion , is performed on one or more DNA template
lent interactions. attached to a particle, which particle is fixed in the channel,
The ability to switch the type of a particle member of an e. g ., by a particle retention element, by another set of
array provides an elegant method for making arrays within particles, or the like. The DNA is then optionally released
microfluidic systems. In particular, arrays are made in situ after sequencing for separation or further manipulation .
by exposure to reagents , avoiding the necessity ofmoving 30 Similarly , proteins can be associated ( e . g ., by bonding to
and tracking different particle sets to different array loca - fixed ligands or antibodies ) and released (e . g ., by exposure
tions . to heat or a base or a denaturant) from the particle . Other
Moreover, particle type switchability provides for the molecules such as polymers or large organic molecules can
creation of microfluidic logic circuits. In particular, the similarly be caged and released from particle members.
presence or absence of a signal from an array location is 35 Similarly, reagents such as enzymes ( e .g ., thermostable
equivalent to a bit of information in a typical computer enzymes ), sequencing reagents , or other molecules can be
system and it is possible to reprogram the array simply by held in an inactive form (e. g ., due to heat or presence of
flowing appropriate reagents to appropriate array positions. blocking groups on the molecules , e . g ., those set forth in
It should be appreciated that, in at least one sense, these Greene , et al. ( 1991 ) Protective Groups In Organic Chem
switchable arrays are superior to existing silicon -based 40 istry, 2nd Ed., John Wiley & Sons, New York , N . Y .) and
computer design . In particular, rather than being limited to flowed into contact with array members. The reagents or
a simple digital " on /off " binary programming language , it is other molecules can then be activated , e . g ., by exposure to
possible to obtain analogue information from the arrays . light, changes in temperature , exposure to an acid , base , or
This is because degrees of signal intensity from array a denaturant, or the like . Thus , in one embodiment, reagents
locations can be discerned . Thus, a very rich non -binary 45 are held in a reagent train in an inactive state and are
programming language can be used in programming and activated only upon exposure to an appropriate activation
interpreting switchable microfluidic arrays . Of course, swit- agent (heat, light, base , acid , denaturant, electric or magnetic
chable arrays can incorporate both ordinary binary silicon field , etc .).
based switches, as well as microfluidic switches, providing Modulation of Hydrodynamic Resistance with Particle Sets ;
for design heuristics that incorporate both binary and non - 50 Iterative Fluid Manipulations
binary programming. The hydrodynamic resistance in a channel can be
In addition to the creation of logic circuits and array increased by packing the channel with particles. In addition ,
construction , type -switchable arrays can be used as chemical electroosmotic flow can be altered significantly by packing
synthesis and purification machines. In particular, array the capillary with particles having different surface charges
members can have chemical components synthesized in a 55 and thus different zeta potential. In one aspect of the present
solid phase fashion on the array members, and subsequently invention, particle manipulation in channels can be used to
used to purify complementary molecules in a manner similar dynamically control the resistance to pressure or electroki
to affinity chromatography. Alternatively, following synthe- netic flow to enhance the flexibility ofmicrofluidic opera
sis , chemical moieties can be cleaved from the array for tions .
subsequent use in other microfluidic assays ( or even for 60 In particular, microbeads or other particles are transported
purification and use outside of the microfluidic system ). This into microchannels by any of the flow methods described
ability to act as a biochemical reactor is a preferred aspect herein . The particles are captured (by a physical barrier,
of the present invention . electric field , magnetic field , porous matrix , sintered glass
Reagent Caging and Triggering frit, a fixed set of particles , etc ., as also described supra ). By
The present invention provides for reagent triggering 65 localizing particles to a selected location , it is possible to
upon contact with an array member. For example , certain alter the local zeta potential, surface charge , etc . This , in turn
reagents are activated by heat or cold , or are activated by alters flow characteristics in the region , providing for altera
 US 10 ,138 ,517 B2
55 56
tions in microfluidic operations , e .g ., changes in the rate , or Because microfluidic manipulations are performed in a
even direction , of fluid flow . Thus, in addition to interacting partially or fully sealed environment, contamination and
with components of various reactions, particle sets can also fluidic evaporation in the systems are minimized .
be used to modulate fluid flow in a selected region . In the methods, a first reagent from the plurality of
Similarly , a powerful application of this dynamic micro - 5 sources of reagent or mixtures of reagents is selected . A first
fluidic control is to create a “ smart ” microfluidic system . sample material and the first reagent or mixture of reagents
Applications which use particles in assays are described is introduced into the first reaction channel, whereupon the
herein . The combination of flow modulation by microbead first sample material and the first reagent or mixture of
localization and e . g ., chemical assays on the beads provides reagents react. Typically , one of the first sample material or
for a great deal of flexibility in designing and controlling 10 first reagent or reagent mixture are bound to a microparticle
assays . In addition , with appropriate software feedback in an array. This reaction can take a variety ofdifferent forms
control, subsequent assay steps are selectable based upon the depending on the nature of the reagents . For example , where
results of initial assays . This ability to reconfigure flow
conditions and assay components by manipulating particles the reagents bind to one another, such as where the reagents
in response to assay results is a very potent new way of 15 are an antibody or cell receptor and a ligand , or an amino
performing iterative reactions . acid and a binding ligand , the reaction results in a bound
For example , the integrated assay and flow control fea - component such as a bound ligand ( e .g ., bound to an array
tures provide very high throughput methods of assessing member). Where the reagents are sequencing reagents, a
biochemical components and performing biochemical primer extension product results from the reaction . Where
manipulations. A wide variety of reagents and products are 20 the reagents include enzymes and enzyme substrates, a
suitably assessed , including libraries of chemical or biologi- modified form of the substrate typically results. Where two
cal compounds or components, nucleic acid templates , PCR reacting chemical reagents are mixed , a third product chemi
reaction products , and the like. In the integrated systems it cal typically results.
is possible to use the results of a first reaction or set of In the methods , a reaction product of the first sample
reactions to select appropriate flow conditions, reagents, 25 material and the first reagent or mixture of reagents is
reactants, products , or the like, for additional analysis . For analyzed in the context of an array. This analysis can take
example , the results of a first sequencing reaction can be any of a variety of forms, depending on the application . For
used to select primers , templates or the like for additional example , where the product is a primer extension product,
sequencing, or to select related families of compounds for the analysis can take the form of separating reactants by size,
screening in high - throughput assay methods. These primers 30 or
or by location on the array and detecting the sized reactants
or templates ( e .g ., as components of an array ) are then and translating the resulting information to give the
accessed by the system and the process continued .
In one aspect, the invention provides integrated methods sequence of a template nucleic acid . Similarly , because
of analyzing and manipulating sample materials for fluidic microscale fluidic devices of the invention are optionally
analysis on arrays. In the methods, an integrated microflu - 35 suitable for heating and cooling a reaction , a PCR reaction
idic system including a microfluidic device comprising an utilizing PCR reagents ( thermostable polymerase , nucleo
array is provided . The device has at least a first reaction tides, templates , primers , buffers and the like ) can be per
channel and at least a first reagent introduction channel, formed and the PCR reagents detected . Where the reaction
typically etched , machined , printed , or otherwise manufac results in the formation of a new product, such as an
tured in or on a substrate . Optionally, the device can have a 40 enzyme-substrate product, a chemical species, or an immu
second reaction channel and/ or reagent introduction chan - nological component such as a bound ligand , the product is
nel, a third reaction channel and /or reagent introduction typically detected by any of a variety of detection tech
channel or the like, up to and including hundreds or even niques , including fluorescence , autoradiography , micros
thousands of reaction and /or reagent introduction channels . copy, spectroscopy, or the like .
The reaction channel and reagent introduction channels are 45 Based upon the reaction product, a second reagent or
in fluid communication , i.e ., fluid can flow between the mixture of reagents is selected and a second sample material
channels under selected conditions. The device has a mate - is assessed , optionally following manipulation of particle
rial transport system for controllably transporting a material sets to modify flow conditions. For example , where the
through and among the reagent introduction channel and product is a DNA sequence , a sequencing primer and/ or
reaction channel and for positioning array components . For 50 template for extension of available sequence information is
example , the material transport system can include electro - selected . Where the product is a new product such as those
kinetic, electroosmotic, electrophoretic or other fluid above , an appropriate second component such as an enzyme,
manipulation aspects (micro -pumps and microvalves, fluid ligand , antibody, receptor molecule , chemical, or the like, is
switches, fluid gates , etc .) which permit controlled move selected to further test the binding or reactive characteristics
ment and mixing of fluids and movement of array members. 55 of an analyzed material. The second reagent or mixture of
The device also has a fluidic interface in fluid communica - reagents is introduced into the first reaction channel, or
tion with the reagent introduction channel. Such fluidic optionally into a second (or third or fourth . . . or nth )
interfaces optionally include capillaries, channels, pins, reaction channel in the microfluidic device . The second
pipettors , electropipettors, or the like , for moving fluids, and sample material and the second reagent or mixture of
optionally further include microscopic , spectroscopic , fluid 60 reagents react, forming a new product, which is analyzed as
separatory or other aspects . The fluidic interface samples a above . The results of the analysis can serve as the basis for
plurality of reagents or mixtures of reagents from a plurality the selection and analysis of additional reactants and addi
of sources of reagents or mixtures of reagents and introduces tional flow conditions for similar subsequent analysis . The
the reagents or mixtures of reagents into the reagent intro - second sample material, reagents, or mixtures of reagents
duction channel. Essentially any number of reagents or 65 can comprise the same or different materials. For example ,
reagent mixtures and or array members can be introduced by a single type of DNA template is optionally sequenced in
the fluidic interface, depending on the desired application . several serial reactions. Alternatively, completing a first
 US 10 , 138 ,517 B2
57 58
sequencing reaction , as outlined above, serves as the basis 6 -mer primers can be made to hybridize specifically to a
for selecting additional templates (e .g ., overlapping clones, target, e .g ., where the primers are modular and hybridize to
PCR amplicons, or the like ). a single region of a nucleic acid . All possible 5 or 6 mers can
Accordingly , in a preferred aspect, the invention provides be synthesized for selection in the methods herein , or any
methods of sequencing a nucleic acid by an iterative process 5 subset of 5 or 6 mers can also be selected . In some
on an array . For example , in one typical method , the embodiments, the primers are transferred to themicrofluidic
biochemical components of a sequencing reaction (e . g ., a apparatus , e . g ., by a capillary , an electropipettor, or using
target nucleic acid , a first and optionally, second sequencing sipping technology, from a microtiter plate or from and array
primer, a polymerase (optionally including thermostable of oligos. The primers are used to hybridize to bound targets
polymerases for use in PCR ), dNTPs , and ddNTPs ) are 10 fixed to array members (see also , above, under discussions
mixed in a microfluidic device in contact with one or moreof modular primer strategies ). In other embodiments , the
array member under conditions permitting target dependent
polymerization of the dNTPs. Polymerization products are primers are located on a region of a microfluidic device, chip
optionally separated in the microfluidic device to provide a or other substrate .
sequence of the target nucleic acid , or as in the case of 15 In another, similar aspect, the devices, systems arrays and
pyrophosphate methods described above , can be read methods of the invention are useful in performing fluidic
directly from the array , depending on the position of com - operations that require a large number of successive fluid
ponents in the array ( similarly, sequencing by hybridization manipulations , 1.e ., in performing a number of preparative
methods do not require separation of products, with results and analytical reactions or operations on a given sample . By
being determined by position on an array ). Typically, 20 " successive fluid manipulations ” is generally meant a fluidic
sequencing information acquired by this method is used to operation that involves the successive treatment of a given
select additional sequencing primers and /or templates or fluid sample volume, i.e ., combination / reaction with reac
probes, and the process is reiterated , optionally following tants, incubation , purification /separation , analysis of prod
movement of array components to modulate flow condi- ucts , and the like. Where successive fluid manipulations are
tions . 25 performed at the bench scale , e . g ., the performance of
In one integrated sequencing system , methods of sequenc- numerous, different manipulations on a particular sample
ing a target nucleic acid are provided in which an integrated such as combination with reagents, incubation , separation
microfluidic system comprising a microfluidic device hav - and detection , such manipulations can also become cumber
ing an array is utilized in the sequencing method . The some as the number of steps increases, as with each step , the
integrated microfluidic device has at least a first sequencing 30 possibility of introducing an error into the operation or
reaction channel and at least a first sequencing reagent experiment increases. This complexity, and the consequent
introduction channel, the sequencing reaction channel and increased possibility of errors increases substantially as the
sequencing reagent introduction channel being in fluid com - number of samples to be passed through the operation
munication and at least one of the channels comprising one increases. Thus, the devices or systems of the present
or more array component. The integrated microfluidic sys - 35 invention are also particularly useful in performing fluidic
tem also has a material transport system for controllably operations which require successive fluid manipulations of a
transporting sequencing reagents through the sequencing given sample or fluid of interest, e . g ., more than 2 steps or
reagent introduction channel and sequencing reaction chan - different manipulations, typically greater than 5 steps or
nel and a fluidic interface in fluid communication with the different manipulations, preferably greater than 10 steps or
sequencing reagent introduction channel for sampling a 40 different fluid manipulations. The systems are also useful
plurality of sequencing reagents , ormixtures of sequencing and readily capable of performing fluidic operations that
reagents , from a plurality of sources of sequencing reagents include greater than 20, 50 , 100, 1000 steps or different fluid
or mixtures of sequencing reagents and introducing the manipulations on a given fluid volume.
sequencing reagents or mixtures of sequencing reagents into In a related , but alternate aspect, the devices , arrays ,
the sequence reagent introduction channel. For example , the 45 systems and methods of the invention are useful in perform
system set forth in U . S . Pat. No. 5 ,779 , 868 can be used . As ing fluidic operations that require a large number of parallel
discussed above, the interface optionally includes capillar - fluid manipulations, i.e., to screen biological samples , screen
ies , pins, pipettors and the like . In the method , a first test compounds for drug discovery , e . g ., as set forth in
sequencing primer sequence complementary to a first sub - WO / 98 / 00705 and WO 98 /00231 and incorporated herein by
sequence of a first target nucleic acid sequence is introduced 50 reference . To carry out these operations , a substrate will
into the sequence reagent introduction channel. The first typically employ parallel channels and /or channel networks ,
primer is hybridized to the first subsequence and the first interconnected by one or more common channels , with at
primer is extended with a polymerase enzyme along the least one particle array dispersed within the device . Fluids
length of the target nucleic acid sequence to form a first required for the subject reaction , e .g ., samples or reagents,
extension product that is complementary to the first subse - 55 are directed along one or more of the common channels , and
quence and a second subsequence of the target nucleic acid . are delivered to each of the parallel channels.
Again , any of these components are optionally coupled to As used herein , " parallel fluid manipulations” means the
one or more array member. The sequence of the first substantially concurrent movement and /or direction , incu
extension product is determined and, based upon the bation /reaction , separation or detection of discrete fluid
sequence of the first extension product, a second primer 60 volumes to a plurality of parallel channels and /or channel
sequence complementary to a second subsequence of the networks, or chambers of a microfluidic device , i.e., greater
target nucleic acid sequence is selected , hybridized and than about 10 distinct parallel channels or chambers, typi
extended as above . cally greater than 20 distinct channels or chambers, prefer
In the sequence methods herein , it is sometimes advan - ably greater than about 50 distinct channels or chambers,
tageous to select sequencing primers from a large set of 65 and often greater than about 100 distinct channels or cham
sequencing primers , rather than synthesizing primers to bers. As used herein , the term “ parallel” refers to the ability
match a particular target nucleic acid . For example , 5 or to concomitantly or substantially concurrently process two
 US 10 ,138 ,517 B2
59 60
or more separate fluid volumes, and does not necessarily particle is a particle that, if the assay or other use for the
denote a specific channel or chamber structure or layout. array member is working properly will not result in a
Ultra high - throughput analysis systems are provided , for significant accumulation , release or display of a detectable
example for performing nucleic acids-based diagnostic and signal. A " sample ” particle comprises an assay or reaction
sequencing applications, e . g ., in a reference laboratory set- 5 element of interest. A " test" particle is a particle that
ting . The system typically has several components: a speci comprises an unknown element to be tested , or which
men and reagents handling system ; an " operating system ” interacts with such an element in an assay or reaction ,
for processing integrated microchip experimentation steps; depending on the specified context of the invention . A
application - specific analysis devices ; a signal detection sys- 6" tracking” particle is a particle which displays a detectable
tem , and multiple software components that allow the user 10 signal, or which displays a known absence of signal within
to interact with the system , and run processing steps, inter- a labeled array, and which is used to track the position of
pret data , and report results . array elements.
Integrated Systems for Assay Normalization It will be appreciated that many useful particle types will
One similar application of the integrated systems and meet more than one of the above criteria . For example a
arrays of the invention is the titration of assay components 15 negative control particle can also be a blank , a tracking
into the dynamic range of an assay . For example , an assay element, or the like . Furthermore , the particles can be placed
can first be performed where one or more components of the in any selected relative positional conformation . For
assay are not within the range necessary for adequate example , blank particles can be interspersed with different
performance of the assay , e. g ., if the assay is performed sample particles to maintain separation and prevent con
using a concentration which is too high or too low for some 20 tamination between the different sample sets . Calibration
components , the assay may not provide quantitative results . particles can be interspersed with sample particles in any
This need to titrate assay components into the dynamic manner to provide signals for calibration of assays , reac
range of an assay typically occurs where one or more tions, etc . Many such variations will be apparent upon
component of the assay is present at an unknown activity or review .
concentration . Ordinarily , the assay must be run at several 25 The ability to titrate and optimize assays is useful for
concentrations of components , i. e., the assay is run a first diagnostic assays, for determining concentrations or activi
time, components are diluted , the assay is run a second time, ties of selected components in a system (proteins, enzymes ,
etc . until the assay can be performed within the dynamic nucleic acids, small molecules, etc .). Furthermore, the pres
range of the assay. It will be appreciated that this iterative ent integrated systems provide for rational selection of assay
approach can involve several unknown concentrations 30 conditions as data is acquired . For example , in one embodi
simultaneously, requiring considerable trial and error. ment, a diagnostic assay needs to be performed using several
In the integrated array systems of the invention , an assay components which are present at initially unknown concen
can be performed at as many concentrations of components trations or activities . A first series of concentration or
as necessary to titrate the assay components into the activity assays is performed on the array to determine the
dynamic range of the assay, with the results of each assay 35 activity or concentration of particular components , e . g .,
being used to optimize additional assay points . Similarly, enzyme, protein , inhibitor, co -factor , nucleic acid , or the
titration curves, which are often the result of multiple assay like . After these assays are performed and the concentrations
runs with different component concentrations are deter - or activities of some or all of the components for the
mined by performing repeated assays with different concen diagnostic assay are determined , the integrated system
trations of components . Different concentrations of assay 40 selects appropriate amounts of the assay components, per
components in separate assays can be monitored serially or forms any necessary dilutions, combines the assay compo
in parallel . In brief , one simply runs the assay at one or more nents and performs the diagnostic assay. Similarly , further
array location , detecting the results . If assays are simulta - data points can be collected by adjusting the concentrations
neously (or even separately ) run at additional array locations or amounts of diagnostic assay components and re - running
with known components , it is possible to use these known 45 the assay. All of the fluid manipulations can be performed
components as normalization elements for the array and the rapidly and the integrated system is able to assess and
assay . Thus , arrays can include positive or negative control compile the results of individual data points or individual
elements ( reagents , templates, etc . ), calibration components assays to select which additional assays need to be per
( e. g ., an array can include a labeled array member and an formed for assay verification .
unlabeled array member for calibration purposes , or even 50 In its most basic form , assay optimization involves the
include members comprising gradations of label intensity ). identification of factors affecting a reaction result, followed
For example, in one aspect, the invention comprises any by the systematic variation of each of these variables until
of "blank ," " dummy," " calibration ," " control," " positive optimal reaction conditions are identified . This is generally
control,” “ negative control," " sample," " test" or " tracking” termed an “ OFAT” method for “ one factor at a time.” Thus ,
particles , e . g ., interspersed with each other or with other 55 assuming a simple two reagent reaction , one would first
elements of an array . Blank particles are particles which do identify the factors affecting the outcome, e . g., concentration
not comprise a label . Dummy particles are either blank , or ofreagent A , concentration of reagent B and , e.g., tempera
comprise a known signal component. Calibration particles ture. One would then run the assay where one factor was
comprise a selected quantity of labeled component, or of a varied while the others remained constant. For example, one
component to be labeled . For example , calibration particles 60 would perform the same reaction at numerous different
(or any of the other particles herein ) can comprise one or concentrations of reagent A , while maintaining the concen
more quantum dot (Warren and Nie ( 1998 ) Science, 281: tration of reagent B and the temperature. Next, reagent B
2016 - 2018 ). Control particles comprise one ormore selected would be varied while reagent A and temperature remained
known element. Positive control particles comprise a known constant, and finally , the temperature would be varied .
component which , if the assay or other use for the array 65 Even in this simplest form , the number and complexity of
member is working properly , will result in accumulation or necessary reactions is apparent. When one considers that
display of a detectable signal. Conversely , a negative control most reactions will have far more than three variables, and
 US 10 ,138 ,517 B2
62
that these variable will not be independent of each other, the residues , or the like ). Thousands of compounds are easily
possibility of manually performing these assays, or even screened using this method , in a short period of time ( e . g .,
performing them in currently available automated formats less than an hour ).
becomes a daunting prospect. For example , while robotic An advantage of the integrated nature of the present
systems using microwell plates can perform large numbers 5 system is that it provides for rational selection of structurally
or functionally homologous compounds or components as
ofmanipulations to optimize assay parameters , such systems the
are very expensive. Further, as these systems are typically foundassay progresses . For example , where one compound is
to have binding activity in an array based assay , the
limited to the bench scale volumes described above , they
require large volumes of reagents and large amounts of 10 selection
formed
of a second compound to be tested can be per
based upon structural similarity to the first active
space in which to operate . compound . Similarly , where a compound is shown to have
In contrast, the devices , systems and methods of the
present invention permit the optimization of large numbers activity in a cell ( e .g ., up - regulation of a gene of interest ) a
second compound affecting the same cellular pathway ( e . g.,
of different assays , by providing an extremely low volume, calcium or inositol phosphate second messenger systems,
automatable and sealed format in which such optimization 15 etc .) can be selected from the group of available compounds
can occur rapidly and automaticallyich. For
such example
optimization
, the for testing . In this way, it is possible to focus screening
devices can run a first fluidic operation by combining a assays from purely random at the outset to increasingly
preselected volume of a first reactant with a preselected focused on likely candidate compounds as the assays prog
volume of a second reactant, at a desired or preselected ress .
temperature for a desired or preselected amount of time. The 20 Integrated Microfluidic Array Systems
device then repeats the assay , but varies at least one of the Device Integration
volume of the first or second reactants , the temperature , or Although the devices and systems specifically illustrated
the amount of time allowed for the reaction . This is repeated herein are generally described in terms of the performance
until a desired number of varied reactions are performed , of a few or one particular operation, it will be readily
i.e., generating sufficient data to permit an estimation of 25 appreciated from this disclosure that the flexibility of these
optimal assay conditions which will produce an optimal systems permits easy integration of additional operations
result of the reaction within a desired range of statistical into these devices. For example , the devices and systems
significance. 11 optimal assay conditions , include those con described will optionally include structures , reagents and
systems for performing virtually any number of operations
ditions that are required to achieve the desired result ofn theof 30
reaction . Such desired results can include maximization of 30both upstream and downstream from the operations specifi
reaction yields, but also includes assay conditions which are cally described herein . Such upstream operations include
optimized for sensitivity to one variable , e.g., inhibitor sample handling and preparation operations, e.g ., cell sepa
ration , extraction , purification , amplification , cellular acti
concentration, and the like. vation , labeling reactions, dilution , aliquoting , and the like .
Drug Screening Assays 35
Similarly , downstream operations may include similar
In addition to sequencing , the integrated microfluidic operations, including , e .g., separation of sample compo
systems and arrays of the invention are broadly useful in a nents, labeling of components, assays and detection opera
variety of screening assays where the results ofmixing one tions, electrokinetic or pressure -based injection of compo
or more components are to be determined , and particularly, nents into contact with particle sets , or materials released
where the results determined are used to select additional 40 from particle sets , or the like .
reagents to be screened . Assay and detection operations include, without limita
As described more fully below , the integrated microflu - tion , probe interrogation assays, e.g., nucleic acid hybrid
idic system of the invention can include a very wide variety ization assays utilizing individual probes , free or tethered
of storage elements for storing reagents to be assessed within the channels or chambers of the device and/or probe
These include well plates, matrices, membranes and the like . 45 arrays having large numbers of different, discretely posi
The reagents are stored in liquids (e . g ., in a well on a tioned probes, receptor/ligand assays , immunoassays , and
microtiter plate ), or in lyophilized form ( e . g., dried on a the like. Any of these elements can be fixed to array
membrane ), and can be transported to an array component of members , or fixed , e . g ., to channel walls , or the like .
the microfluidic device using conventional robotics , or using Loading of Array Components and Reagents
an electropipettor as described below . 50 Array members and reagents can be loaded into micro
Because of the breadth of the available sample storage fluidic structures , e . g ., by placing the reagent or array
formats for use with the present invention , virtually any set member in a well fluidly coupled to a microfluidic channel
of reagents can be sampled and assayed in an integrated network . The reagent or array member is then flowed
system of the present invention . For example, enzymes and through the microchannel network as described supra , e. g.,
substrates , receptors and ligands, antibodies and ligands , 55 by pressure ( positive or negative ) or by electrokinesis , or by
proteins and inhibitors, cells and growth factors or inhibi- moving a magnetic field relative to the array member i. e.,
tors , viruses and virus binding components ( antibodies, where the array member is magnetic ).
proteins, chemicals, etc .) immunochemicals and immuno - Alternatively, array or particle members can be stored
globulins, nucleic acids and nucleic acid binding chemicals, external to the microfluidic system in a system of wells ,
proteins, or the like, reactant chemicals ( acids, bases, 60 plates, or even as dried components stored on a surface .
organic molecules, hydrocarbons, silicates, etc .) can all be Thus, the integrated systems of the invention optionally
assayed using the integrated systems of the invention . For include such external storage elements . In one aspect, the
example , where a molecule which binds a protein is desired , present invention includes a microwell plate ( e. g., a 96 , 384
potential binding moieties ( chemicals , peptides , nucleic or more well plate ) having array members stored within
acids, lipids, etc.) are sequentially mixed with the protein in 65 wells of the plate .
a reaction channel, and binding is measured (e .g .,by change To introduce array members or reagents into the micro
in electrophoretic mobility, quenching of fluorescent protein fluidic system , either pressure -based , electrokinetic or cen
 US 10 , 138 ,517 B2
63 64
trifugal approaches can be used . For example , electropi- trating subject materials and buffer fluids in a complex
pettors (which can include one or multiple " sipper " microfluidic system . Current control is also preferred for
channels ) can be used to access wells , plates or to re -hydrate moderating temperature effects ( e . g ., joule heating ) within
soluble or suspendable dried components from dry storage the channels .
media . A variety of access systems for coupling reagent 5 The present invention also provides novel methods of
storage and microfluidic systems are described in Knapp et controlling fluid flow , particle flow , or the like in microflu
al. “ Closed Loop Biochemical Analyzers ” WO 98 /45481. As idic channels. By flowing reagents, samples, and /or particle
applied to the present invention , these coupling devices and arrays through a microfluidic network using a system of split
strategies are also used for flowing particle sets into micro - wells , contamination by components ofprevious reactions is
fluidic systems. 10 avoided . In an assay involving many components, the com
Instrumentation ponents typically vary in charge . Therefore when transport
In the present invention , the materials in the arrays are ing materials using electrokinetic techniques, components
optionally monitored and/ or detected so that velocity can bemay move in different directions depending on their charge ,
determined . From velocity measurements , decisions are then especially in buffers in which the electroosmotic mobility is
made regarding subsequent fluidic operations. 15 low . Therefore , a sample introduction step may not properly
The systems described herein generally includemicroflu - introduce all components to the reaction area and a washing
idic devices, as described above , in conjunction with addi step may not clear away all reaction components to the waste
tional instrumentation for controlling fluid transport, flow reservoir( s ) before the next reaction or phase . Therefore ,
rate and direction within the devices, detection instrumen - some components may carry over from a first reaction to a
tation for detecting or sensing results of the operations 20 second reaction in a sequential system , e. g ., a high through
performed by the system , processors, e.g ., computers , for put system .
instructing the controlling instrumentation in accordance For example, in a pyrosequencing reaction in which the
with preprogrammed instructions, receiving data from the DNA template is immobilized on beads, all sequencing
detection instrumentation , and for analyzing, storing and reagents are optionally present in a reaction mixture along
interpreting the data , and providing the data and interpreta - 25 with reagents used to transform any generated pyrophos
tions in a readily accessible reporting format. phate into chemiluminescence . The DNA sequencing
Controllers reagents , e . g ., dNTPs , and the pyrophosphate are negatively
A variety of controlling instrumentation is optionally charged , but some enzymes can be positively charged . The
utilized in conjunction with the microfluidic devices present system clears away both positively and negatively
described above, for controlling the transport and direction 30 charged components before the next base extension reaction
of fluids and / or materials within the devices of the present begins , thus avoiding contamination between samples .
invention , e.g ., by pressure -based or electrokinetic control. To provide more complete washing of oppositely charged
For example , in many cases , fluid transport and direction components , e . g ., from a microfluidic system , a pair of wells
are controlled in whole or in part, using pressure based flow is provided on each side of a reaction region . Reactions of
systems that incorporate external or internal pressure 35 interest, e .g ., pyrosequencing , occur, e . g ., in a network of
sources to drive fluid flow . Internal sources include micro - microscale channels or capillaries, in the reaction area . One
fabricated pumps, e. g., diaphragm pumps, thermal pumps, well of each pair is used as a waste well and the other is used
lamb wave pumps and the like that have been described in as a reagent source or reservoir, e .g ., for reagents of a certain
the art. See, e . g ., U . S . Pat. Nos. 5 ,271,724 , 5 , 277 ,556 , and charge . For example , see FIG . 18 , Panel A . Well 1810
5 ,375 ,979 and Published PCT Application Nos. WO 40 comprises a source ofpositive and neutral reagents and well
94 /05414 and WO 97/02357. In such systems, fluid direction 1830 comprises a source for negative reagents (or those
is often accomplished through the incorporation of micro - reagents whose total electrokinetic mobility is negative).
fabricated valves , which restrict fluid flow in a controllable Wells 1820 and 1840 comprise waste wells. Reagents are
manner. See, e .g ., U .S . Pat. No . 5, 171, 132. optionally loaded into the microfluidic system , e.g., reaction
As noted above , the systems described herein can also 45 area 1850 , by applying a positive voltage to well 1810
utilize electrokinetic material direction and transport sys - relative to well 1830 . Substantially zero current is applied at
tems. As such , the controller systems for use in conjunction wells 1820 and 1840. All reagents are loaded simultaneously
with the microfluidic devices typically include an electrical using this method . Positive and neutral reagents flow from
power supply and circuitry for concurrently delivering well 1810 to the reaction area 1850 and negative reagents
appropriate voltages to a plurality of electrodes that are 50 flow from well 1830 to reaction area 1850. Typically , the
placed in electrical contact with the fluids contained within reagents are loaded quickly to avoid generation of reaction
the microfluidic devices . Examples of particularly preferred products during loading. Alternatively, the positive and/ or
electrical controllers include those described in , e . g ., pub - neutral reagents are loaded separately from the negative
lished PCT application WO 98 /00707 and in U .S . Pat. No. reagents . Positive and neutral reagents only are loaded by
5 ,800 ,690 . In brief , the controller uses electric current 55 applying a positive voltage atwell 1810 relative to well 1840
control in the microfluidic system . The electrical current and substantially zero current at wells 1830 and 1820 . The
flow at a given electrode is directly related to the ionic flow negative reagents alone are loaded by applying a positive
along the channel( s ) connecting the reservoir in which the voltage at well 1820 relative to well 1830 and substantially
electrode is placed . This is in contrast to the requirement of zero current at wells 1840 and 1810 . Reagents are removed
determining voltages at various nodes along the channel in 60 from reaction area 1850 in a similar manner. A positive
a voltage control system . Thus the voltages at the electrodes voltage is applied to well 1820 relative to well 1840 and
of the microfluidic system are set responsive to the electric substantially zero voltage at wells 1810 and 1830 . This step
currents flowing through the various electrodes of the sys - does not use either of the reagent wells , 1810 and 1830 ,
tem . This current control is less susceptible to dimensional thereby preventing contamination as reagents are removed
variations in the process of creating the microfluidic system 65 or rinsed from the device .
in the device itself . Current control permits far easier opera Alternative channel configurations are provided in FIG .
tions for pumping, valving , dispensing , mixing and concen 18 , panels B and C . Reaction areas 1850b and 18500
 US 10 ,138 ,517 B2
65 66
illustrate possible channel configurations . Other configura In the microfluidic systems described herein , a variety of
tions as described herein are also optionally used . Wells detection methods and systems are employed , depending
18106 and 1810c are optionally used for positive and/or upon the specific operation that is being performed by the
neutral reagents and wells 1830b and 1830c optionally system . A microfluidic system can also employ multiple
comprise negative reagents . Introduction of reagents option - 5 different detection systems for monitoring the output of the
ally comprises applying a positive voltage at well 1810b or system . Detection systems of the present invention are used
1810c relative to 1830b or 1830C . Alternatively, a positive to detect and monitor the materials in the detection window .
voltage is applied at well 1810b or 1810c relative to one or Once detected , the flow rate and velocity of particles in the
more of wells 1860b , 1860c , 1820b , 1820c, 1840b , and channels is optionally measured and controlled as described
1840c. Negative reagents are optionally introduced by 10 above.
applying a positive voltage at any of wells 1860b , 1860c , Examples of detection systems include optical sensors ,
1820b, 1820c, 1840b , and 1840c relative to well 1830b or temperature sensors, pressure sensors , pH sensors, conduc
1830c. Removal of reagents is achieved , e . g ., by applying a tivity sensors , and the like. Each of these types of sensors is
positive voltage at 1860b or 1860c relative to 1840b or readily incorporated into the microfluidic systems described
1840c . Other possible methods of flowing reagents through 15 herein . In these systems, such detectors are placed either
the devices of FIG . 18 will be readily apparent upon further within or adjacent to the microfluidic device or one or more
review . channels , chambers or conduits of the device , such that the
Typically , the controller systems are appropriately con - detector is within sensory communication with the device ,
figured to receive a microfluidic device as described herein . channel, or chamber. The phrase " within sensory commu
In particular, the controller and / or detector ( as described in 20 nication " of a particular region or element, as used herein ,
greater detail, below ), includes a stage upon which the generally refers to the placement of the detector in a position
device of the invention is mounted to facilitate appropriate such that the detector is capable of detecting the property of
interfacing between the controller and /or detector and the the microfluidic device , a portion of the microfluidic device ,
device . Typically, the stage includes an appropriate mount- or the contents of a portion of the microfluidic device , for
ing/alignment structural element, such as a nesting well, 25 which that detector was intended . For example , a pH sensor
alignment pins and /or holes , asymmetric edge structures (to placed in sensory communication with a microscale channel
facilitate proper device alignment), and the like. Many such is capable of determining the pH of a fluid disposed in that
configurations are described in the references cited herein . channel. Similarly , a temperature sensor placed in sensory
The controlling instrumentation discussed above is also communication with the body of a microfluidic device is
used to provide for electrokinetic injection or withdrawal of 30 capable of determining the temperature of the device itself.
material downstream of the region of interest to control an Particularly preferred detection systems include optical
upstream flow rate . The same instrumentation and tech - detection systems for detecting an optical property of a
niques described above are also utilized to inject a fluid into material within the channels and /or chambers of the micro
a downstream port to function as a flow control element. fluidic devices that are incorporated into the microfluidic
Detector 35 systemsdescribed herein . Such optical detection systems are
The devices herein optionally include signal detectors , typically placed adjacent to a microscale channel of a
e.g ., which detect fluorescence , phosphorescence, radioac - microfluidic device, and are in sensory communication with
tivity , pH , charge, absorbance , luminescence , temperature , the channel via an optical detection window that is disposed
magnetism or the like. The detectors optionally monitor a across the channel or chamber of the device . Optical detec
plurality of signals from the plurality of particle sets, either 40 tion systems include systems that are capable of measuring
simultaneously or sequentially . For example , the detector the light emitted from material within the channel, the
can monitor a plurality of optical signals which correspond transmissivity or absorbance of the material, as well as the
in position to sets of particles within the array . Example materials spectral characteristics . In preferred aspects, the
detectors include of photo multiplier tubes, a CCD array, a detector measures an amount of light emitted from the
scanning detector or galvo - scann or the like . Particles from 45 material, such as a fluorescent or chemiluminescent mate
the array which emit a detectable signal can be flowed past r ial . As such , the detection system will typically include
the detector, or, alternatively, the detector can move relative collection optics for gathering a light based signal transmit
to the array to determine particle position (or, preferably , the ted through the detection window , and transmitting that
detector can simultaneously monitor a number of spatial signal to an appropriate light detector. Microscope objec
positions corresponding to array members , e. g ., as in a CCD 50 tives of varying power, field diameter, and focal length are
array ). The detector can include or be operably linked to a readily utilized as at least a portion of this optical train . The
computer, e. g., which has software for converting detector light detectors are optionally photodiodes , avalanche pho
signal information into nucleic acid sequence information , todiodes , photomultiplier tubes , diode arrays, or in some
converting detector signal information into reaction kinetic cases, imaging systems, such as charged coupled devices
information , converting signal information into antibody 55 (CCDs ) and the like . In preferred aspects, photodiodes are
binding data , converting signal information into cell recep - utilized , at least in part , as the light detectors. The detection
tor binding data converting signal information into hybrid system is typically coupled to a computer (described in
ization data , or the like. greater detail below ) , via an analog to digital or digital to
Signals from arrays are optionally calibrated , e.g ., by analog converter, for transmitting detected light data to the
calibrating the microfluidic system by monitoring a signal 60 computer for analysis , storage and data manipulation .
from a known member of the array , e. g ., a calibration or In the case of fluorescent materials, the detector will
marker particle , or from a known particle set external to the typically include a light source which produces light at an
array . Similarly the relative positions of particle sets and appropriate wavelength for activating the fluorescent mate
signals from the array is monitored , e . g ., by determining the rial, as well as optics for directing the light source through
position of one ormoremembers of the array by monitoring 65 the detection window to the material contained in the
a signal from a known member of the array, thereby deter - channel or chamber. The light source can be any number of
mining the position of the known member of the array. light sources that provides an appropriate wavelength ,
 US 10 , 138 ,517 B2
67 68
including lasers, laser diodes and LEDs. Other light sources reagents are provided in a stabilized form , so as to prevent
required for other detection systems. For example, broad degradation or other loss during prolonged storage , e. g.,
band light sources are typically used in light scattering from leakage . A number of stabilizing processes are widely
transmissivity detection schemes, and the like . Typically , used for reagents that are to be stored , such as the inclusion
light selection parameters are well known to those of skill in 5 of chemical stabilizers (i.e ., enzymatic inhibitors, micro
the art. cides/bacteriostats, anticoagulants ), the physical stabiliza
The detector can exist as a separate unit, but is preferably tion of the material, e.g ., through immobilization on a solid
integrated with the controller system , into a single instru support, entrapment in a matrix (i.e., a gel), lyophilization,
ment. Integration of these functions into a single unit facili or the like.
tates connection of these instruments with the computer 10 The discussion above is generally applicable to the
(described below ), by permitting the use of few or a single aspects and embodiments of the invention described below .
communication port( s ) for transmitting information between
the controller , the detector and the computer. Moreover, modifications can be made to the method and
Computer apparatus described herein without departing from the spirit
As noted above , either or both of the controller system 15 and scope of the invention as claimed , and the invention can
and /or the detection system are coupled to an appropriately be put to a number of different uses including the following:
programmed processor or computer which functions to The use of a microfluidic system containing at least a first
instruct the operation of these instruments in accordance substrate and having a first channel and a second channel
with preprogrammed or user input instructions , receive data intersecting the first channel, at least one of the channels
and information from these instruments , and interpret , 20 having at least one cross- sectional dimension in a range
manipulate and report this information to the user. As such , from 0 . 1 to 500 um , in order to test the effect of each of a
the computer is typically appropriately coupled to one or plurality of test compounds on a biochemical system , the
both of these instruments ( e. g ., including an analog to digital system including an array .
or digital to analog converter as needed ). The use of a microfluidic system as described herein ,
The computer typically includes appropriate software for 25 wherein a biochemical system flows through one of said
receiving user instructions , either in the form of user input channels substantially continuously , providing for, e. g .,
into a set parameter fields, e .g ., in a GUI, or in the form of sequential testing of a plurality of test compounds.
preprogrammed instructions, e. g ., preprogrammed for a The use of an array in a microfluidic device as described
variety of different specific operations . The software then herein to modulate reactions within microchannels or micro
converts these instructions to appropriate language for 30 chambers
instructing the operation of the fluid direction and transport The use of electrokinetic injection in a microfluidic device
controller to carry out the desired operation . The computer as described herein to modulate or achieve flow in the
then receives the data from the one or more sensors /detec - channels.
tors included within the system , and interprets the data , The use of a combination of wicks, electrokinetic injec
either provides it in a user understood format, or uses that 35 tion and pressure based flow elements in a microfluidic
data to initiate further controller instructions, in accordance device as described herein to modulate or achieve flow of
with the programming , e . g ., such as in monitoring and materials to arrays , or array members to materials, e . g ., in
control of flow rates, temperatures, applied voltages , and the the channels of the device .
like . The use of split waste wells or split reagent wells to load
In the present invention , the computer typically includes 40 or unload reagents from a microfluidic device as described
software for the monitoring of materials in the channels , so herein .
that flow rate and velocity may be determined . Additionally An assay utilizing a use of any one of the microfluidic
the software is optionally used to control electrokinetic systems or substrates described herein .
injection or withdrawal of material. The electrokinetic or Microfluidic devices and bioassays which can be adapted
withdrawal is used to modulate the flow rate as described 45 to the present invention by the addition of arrays include
above. various PCT applications and issued U . S . Patents , such as,
Kits U . S . Pat. No . 5 ,699, 157 ( J. Wallace Paree) issued Dec . 16 ,
Generally, the microfluidic devices described herein are 1997 , U .S . Pat. No . 5 ,779 ,868 (J. Wallace Paree et al.) issued
packaged to includemany if not all of the necessary reagents Jul. 14 , 1998, U . S . Pat. No. 5, 800 ,690 (Calvin Y . H . Chow
for performing the device 's preferred function . For example , 50 et al.) issued Aug . 1, 1998, and U .S . Pat. No. 5 ,842,787
the kits can include any of microfluidic devices comprising ( Anne R .Kopf -Sill et al.) issued Dec . 1 , 1998 ; and published
arrays , particle array members, reagents ( e . g ., sequencing or PCT applications, such as, WO 98 /00231, WO 98 /00705 ,
PCR reagents ), sample materials , control materials , or the WO 98 /00707 , WO 98 / 02728 , WO 98 /05424, WO
like . Such kits also typically include appropriate instructions 98 /22811 , WO 98 /45481 ,WO 98 /45929,WO 98 /46438 , and
for using the devices and reagents, and in cases where 55 WO 98 /49548 , which are all incorporated herein by refer
reagents are not predisposed in the devices themselves, with ence.
appropriate instructions for introducing the reagents into the While the foregoing invention has been described in some
channels and / or chambers of the device . In this latter case , detail for purposes of clarity and understanding , it will be
these kits optionally include special ancillary devices for clear to one skilled in the art from a reading of this disclosure
introducing materials into the microfluidic systems, e . g ., 60 that various changes in form and detail can be made without
appropriately configured syringes /pumps, or the like ( of departing from the true scope of the invention . For example ,
course , in one preferred embodiment, the device itself all the techniques and apparatus described above may be
comprises a pipettor element, such as an electropipettor for used in various combinations . All publications and patent
introducing material into channels and chambers within the documents cited in this application are incorporated by
device ). In the former case , such kits typically include a 65 reference in their entirety for all purposes to the same extent
microfluidic device with necessary reagents predisposed in as if each individual publication or patent document were so
the channels / chambers of the device. Generally , such individually denoted .
 US 10, 138,517 B2
69 70
What is claimed is : 7 . The method of claim 1, wherein the first particle set
1. A method of modifying flow in a microchannel , the comprises particles of a dimension such that particles of the
method comprising: second particle set cannot pass therebetween .
flowing a first particle set into the microchannel; 8 . The method of claim 1 , wherein at least one of the first
performing a first assay in the microchannel; 5 particle set and the second particle set comprise one of
moving the first particle set within the microchannel and magnetic particles and affinity particles .
flowing a second particle set stacked against the first 9 . The method of claim 1 , wherein at least one of the first
one
particle set into the microchannel, thereby altering the ofparpolymer beads, silica beads , silicon beads, clay beads
hydrodynamic resistance in the microchannel; and, 10 ceramic beads, glass beads,magnetic beads, metallic beads,,
performing a second assay in the microchannel. inorganic compound beads, organic compound beads, mag
2 . The method of claim 1 , the method further comprising netic particles and affinity particles.
altering the surface charge on the first particle set, thereby 10. The method of claim 1 , wherein at least one of the first
modifying the zeta potential of the first particle set. particle set and the second particle set comprise at least one
3 . The method of claim 1 , the method further comprising
altering the surface charge on the first particle set, thereby 15 material
ecules
comprising a linking chemistry for linking mol
thereto .
modifying the zeta potential of the first particle set, wherein 11 . A method of performing assay in a microchannel, the
the zeta potential is altered in response to the results of the method comprising:
first assay or the second assay in the microchannel. stacking a first particle set against a second particle set;
4 . The method of claim 1 , the method further comprising
flowing the first particle set into the microchannel;
altering the surface charge on the first particle set, thereby - performing
modifying the zeta potential of the first particle set, wherein a first assay in the microchannel;
the zeta potential is altered in response to the results of the moving the first particle set within the microchannel and
first assay or the second assay in the microchannel, wherein flowing the second particle set into the microchannel,
the results of the first assay or the second assay are detected thereby altering the hydrodynamic resistance in the
using a system comprising a microprocessor and the hydro - 25 microchannel; and,
dynamic resistance or zeta potential are altered in response performing a second assay in the microchannel.
to a signal from the system . 12 . The method of claim 11 , further comprising control
5 . The method of claim 1 , wherein either the first assay or ling13the moving according to results of the first assay .
. The method of claim 11 , wherein steps of the second
the second assay comprises contacting a reagent with a† 30 assay
member of the first particle set or the second particle set » 14 . are selected based on the results of the first assay.
The method of claim 11 , further comprising control
respectively, or with a member of a third particle set .
6 . The method of claim 1 , wherein the first particle set acts ling the moving according to software feedback control .
as a matrix to prevent passage of the second particle set .