Lab-on-a-Chip
Dr. Thara Srinivasan
Lecture 20
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Picture credit: Anderson et al.
Lecture Outline
• Reading from reader
• Auroux, P.-A., Manz, A. et al. , “Micro Total Analysis
Systems,” (2002) pp. 2637-52.
• Krishnan, M., et al., “Microfabricated Reaction and
Separation Systems,” (2001) pp. 92-98.
• Quake, S., R, and A. Scherer, “From Micro- to
Nanofabrication Using Soft Materials,” (2001) pp. 1552-69.
• Today’s Lecture
• Lab-on-a-Chip Concept and Examples
• Application to Proteomics
• Lab-on-a-Chip Subunits
• Sample handling
• Reactors
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• Separation Methods
• Detection
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Lab-on-a-Chip
• Micro total analysis system (µ-TAS) • Saves reagents and
• Vision proposed by Manz, Widmer labor
and Harrison in early ’90’s
• Increases testing
• Perform sample addition, throughput
pretreatment and transport, chemical
reactions, separation, and detection • Creates portable
on a microscope slide or credit card systems
size chip
• Annual conference, MicroTAS, had
700 attendees in ‘02
• Applications
• Genomics and proteomics
• Environmental assays
• Medical diagnostics
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• Drug discovery
• Chemical production
• Cellular analysis
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Affymetrix
Lab-on-a-Chip
• Multiple operations
performed
• Cell lysis
• Sample
concentration
• Enzymatic
reactions such as
reverse
transcription, PCR,
DNAse digestion
and terminal
transferase
labeling
• Dilution,
hybridization, and
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washing
• Dye staining
U. Srinivasan © Anderson et al. 4
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U of M Lab-on-a-Chip
• Mastrangelo and Burns
groups’ integrated device
• Nanoliter liquid
injector
• Sample mixing
and positioning
system
• Temperature-
controlled PCR
reaction
chamber
• Electrophoretic
separation
• Fluorescent
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photodetector
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Microscope-on-a-Chip
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Proteomics
• A “proteome” is the set of proteins encoded by a gene
• Proteomics
• Identifying all the proteins made by a given cell, tissue or organism
• Determining how the proteins network among themselves
• Finding out precise 3D structures of the proteins
• Proteins more complex than genes
• DNA: 4 bases, proteins: 20 amino acids
• Even with a protein’s sequence, its function and networks still unknown
• 3D shape of folded protein difficult to predict
• All human cells have same genome, but differ in which genes are
active and which proteins are made
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• ~40,000 human genes, each gene can encode several proteins (typical
cell makes 100,000’s proteins)
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Scientific American April 2002
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Necessary Subunits for µ-TAS
• Sample handling
• Extraction
• Mixers
• Valves
• Pumps
• Reactors
• Separation
• Detection
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Sample Extraction
• Means for extracting samples from dilute
solutions required
• At macroscale, centrifugal force is used
• For microfluidics, sample extraction is interface to
macroscale
• Most of the power consumption is spent at this
step
• Methods include
• Filtration
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• Chromatography
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Extraction Using Filters
• Microfabricated filters
• Mechanically robust to withstand high pressure drops
for filtering µm-sized particles
• Very uniform pore sizes determined by
• Photolithography
• Sacrificial layer thickness
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C.-M. Ho group, UCLA Keller et al., UCB
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Solid-Phase Extraction
• As in chromatography,
• Desired components
bind reversibly to a
coated porous solid
and are later flushed
out by a change in
solvent
• Hydrophobic coatings
bind nonpolar
compounds in aqueous
flow
• Bead chambers
• Hydrophobic beads
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trapped in a flow
chamber
Harrison group, Stemme group,
U. Srinivasan © Univ. of Alberta Sweden 12
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Extraction Using Porous Polymers
• Porous polymers increase available surface
area for binding interactions
• Fill channels with polymerization mixture ~
monomers, initiator, and porogenic solvent
• Irradiate chip with UV light through
photomask
• Surface chemistry may be varied widely
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Fréchet group, UCB
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Extraction by Diffusion
• Mixing in low Re flows is nearly reversible
• Two flows that have been stirred together may be
“unstirred”—except for any mixing by diffusion—by
reversing the driving force
• Can we use irreversibility of diffusive mixing in
reversibly stirred flows to separate chemical
species based on size?
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Extraction by Diffusion
• As two parallel
laminar flows
contact, diffusion
extracts certain
components
• Components with
higher diffusivity
extracted
• Micronics H-filter
pull elements out
of sample into
diluent
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Necessary Subunits for µ-TAS
• Sample handling
• Preparation
• Mixers
• Pumps
• Valves
• Reactors
• Separation
• Detection
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Mixing
• Mixing of particles, cells and molecules often
determines the system efficiency
• PCR, DNA hybridization, cell lyses…
• Diffusion, the mechanism of mixing at the microscale, still
requires relatively long times for thorough mixing.
• How to assist mixing?
• Repeated lamination of
flows increases contact
area and decreases
diffusion length
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U. Srinivasan © C.-M. Ho Group, UCLA 17
• Chaotic flows
can be very
efficient mixers
• Changing
surface
topography of
microchannel
floor induces
chaotic flows
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Stroock et al.,
Whitesides Group, Harvard 18
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Necessary Subunits for µ-TAS
• Sample handling
• Preparation
• Mixers
• Pumps
• Valves
• Reactors
• Separation
• Detection
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Pumping Mechanisms
• Pressure gradients
• Electrokinetic forces
• Surface tension forces
• Electrowetting
• Thermocapillary
• Surface acoustic waves
• Magnetohydrodynamic
• Dielectrophoresis
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U. Srinivasan ©
C. M.Ho 20
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Centrifugal Forces
• Gyros, Sweden
• When CD spins,
centrifugal force causes
liquids on their surface to
move outwards.
• The force can drive
liquids through
microchannels…
• …even breaking through
hydrophobic barriers in
the channels, releasing
different chemicals
selectively
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Electrowetting
• Electrical potential can control surface tension on a
dielectric solid surface
• Asymmetric contact angles generate internal pressure imbalance,
leading to movement
• Fluidic operations can be done on discrete droplets
• Low voltages: 25 V DC for v = 30 mm/s; 100V AC for v = 200 mm/s
εε 0V 2
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cosθ (V ) = cosθ 0 +
CJ Kim group, UCLA 2γ LV t
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Thermocapillary
Pumping
• Thermocapillary effect
• Local heating reduces surface tension, pulling liquid
towards cooler surface
• Surface temperature manipulated by embedded heaters
• Results
• v = 600 µm/s for liquid PDMS
+ Low operating voltage (2-3 V)
+ Works with polar and non-polar
liquids
• Thermocapillary mixer ~1000×
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faster than diffusion
Troian group,
U. Srinivasan © Princeton U. 23
Thermocapillary
Mixer
• ~1000× faster than diffusion
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Troian group,
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Surface Acoustic Waves
• More on ultrasonic fluidic devices
at http://www-
bsac.eecs.berkeley.edu/fluidics/
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U. Srinivasan © White group, BSAC Sandia Labs 25
Necessary Subunits for µ-TAS
• Sample handling
• Preparation
• Mixers
• Pumps
• Valves
• Reactors
• Separation
• Detection
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Elastomer Valves
• A good valve needs flexibility and a valve seat that closes
completely
• Microfabricated poly-Si valves: microactuator forces limited, so stiffness limits
minimum size
• For elastomers, Young’s Modulus can be tuned over 2 orders of magnitude…
• PDMS valves and pumps
made by replica molding
• Crossed channel layout;
channels 100 µm wide, 10 µm
high
• When P is applied to upper
channel, membrane deflects,
closing lower channel
• Response time 1 ms, applied P
= 100kPa
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• Dead volume is zero for on-off
valve
Unger et al.,
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Valves and Pumping
• Peristaltic pumping with
elastomer valves
• 3 valves on a single channel (closing
pattern: 101, 100, 110, 010, 011, 001)
• 2.35 nL/s at 75 Hz, 1 mN force
• Avoids drawbacks of EO pumping
• Dependence on medium
• Electrolytic bubble formation
• Difficulty setting voltages when many
junctions present
• Flow stops and gas vents
• Hydrophobic patches
• Hydrophobic membrane vents
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• Thermally-generated bubbles
Unger et al., Quake group,
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U. Srinivasan © Caltech
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Necessary Subunits for µ-TAS
• Sample handling
• Preparation
• Mixers
• Pumps
• Valves
• Reactors
• Separation
• Detection
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Jensen group, MIT
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Immunoassay Reactor
• Immunoassays
• Important analytical method for
clinical diagnostics,
environmental analyses, and
biochemical studies.
• Antigens and antibodies are
fixed onto a solid support
• ELISA = Enzyme-Linked
ImmunoSorbent Assay
• Point of care testing using
microfluidics
• Enhanced reaction efficiency
• Simplified procedures
• Reduced assay time
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• Lower sample & energy
consumption
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Sato et al., University of Tokyo 30
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Clinical Diagnosis On-Chip
• Diagnosis of colon cancer by
detection of human carcino-
embryonic antigen (CEA) in serum
on-chip
• Polystyrene beads coated with
antibody in microchannel, antigen-
antibody complex detected optically
• Liquid handling significantly
simplified
• Assay time reduced to ~1% (45 h to
35 min)
• Compared to conventional ELISA,
detection limit dozens of times lower
• High throughput analysis using
branching channels for simultaneous
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analysis
U. Srinivasan © Sato et al., University of Tokyo 31
Necessary Subunits for µ-TAS
• Sample handling
• Preparation
• Mixers
• Pumps
• Valves
• Reactors
• Separation
• Detection
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Separation by Electrophoresis
• Current standard method for protein sizing
• Sodium Dodecyl Sulfate-PolyAcrylamide Gel
Electrophoresis (SDS-PAGE)
• SDS denatures proteins and gives them charge;
PAGE separates by size
• Protein electrophoresis on chip
• Steps: sample loading (protein + SDS), dye labeling
(staining), separation, SDS dilution and destaining,
and detection
• Staining and SDS dilution steps occur in 100’s ms,
104 × faster than macroscale
• Sequential analysis of 11 samples, sizing accuracy
>5%, sensitivity 30 nM
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Video clip at
http://www.chem.agilent.com/scripts/generic.asp?lPage=1566&indcol=N&prodcol=Y
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Separation by Isoelectric Focusing
• Isoelectric focusing (IEF) is electrophoresis in a pH
gradient (cathode at higher pH)
• A protein’s isoelectric point (pI) is the pH at which it has neutral
charge
• Charged species stop moving when EP pushes them to their pI
• Linear pH gradient built up using ampholytes
• IEF concentrates and separates
lower pH, (+)
Higher pH, (-)
Dilute acid
Dilute base
• Issues
+ IEF downscales well since
resolution is independent of
channel length, in contrast to CE
• EP focusing effect counteracted dpH dµ
∆pI min = 3 D
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by diffusion, yielding Gaussian L V
band distribution dx dpH
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IEF On-Chip
• Advantages
• Sample mobilization unnecessary
• No injection plug so separation does not depend on initial
sample shape
• Short channel length gives rapid analysis and…
• Full field detection by imaging with inexpensive CCD
• Challenge
• High field with shorter separation length leads to increased
Joule heating
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Separation by
Entropic Traps
• Channels with nanoscale constrictions
• Require long DNA to repeatedly change
conformation, costing entropic free
energy
• Longer DNA has higher mobility
• Separation
• No sieving medium needed
• 5-kbp sample at 80 V/cm in 30 min
• Longer channels for better separations;
resolution not as good as CE
• Sample concentration
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• At low E, DNA is trapped into band
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Craighead group, Cornell 36
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Separation by Diffusion
• Using 2-D “obstacle course” and electric
field in –y direction
• Asymmetric obstacles rectify Brownian
motion (diffusion) of molecules
• Faster-diffusing species move more in
+x direction
• Results
• Obstacles: 1.5×6 µm² at 45° angle
• No sieving medium; low E (1.4 V/cm);
may be applied to DNA, proteins, cells,
etc.
• v = 1-15 µm/s, for a 10 cm sieve
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• Bandwidth = 200 µm for 15 kbp DNA
(RG = 0.31 µm)
Chou, Austin groups, Princeton,
U. Srinivasan © Craighead group, Cornell 37
Necessary Subunits for µ-TAS
• Sample handling
• Preparation
• Mixers
• Pumps
• Valves
• Reactors
• Separation
• Detection
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Detection: Chemiluminescence
• Chemiluminescence (CL) or electrochemiluminescence
(ECL)
• Ru(bpy)3+2 oxidized chemically or electrochemically to Ru(bpy)3+3
which…
• Reacts with amines, amino acids, glucose, PCR products, etc…
• …and emits light at 620 nm
• Advantages
• Laser not required
• Instruments much simpler than for LIF
• Low to zero background signal; sensitivity high
• Scaling benefits ~ microphotodetector for on-chip detection
• Challenges
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• Need for robust and/or universal probes
• Isolation of ECL electrodes from CE high voltage
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Electrochemical Detection
• Electrochemical detection (EC)
• Control potential of working electrode and monitor current as samples
pass by
• Applied potential is driving force for electrochemical reactions of
sample analytes, current reflects concentration of compounds
• Benefits and challenges
• On-chip detection; truly portable
• Chemistries need to be developed
• Rossier et al. integrated screen-printed carbon ink electrodes into
plastic microchannels and demonstrated detection limit of ~1 fmol
for ferrocenecarboxylic acid (2001). (EPL, Lausanne)
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Mass Spectrometry
• Mass spectrometry (MS) measures
mass-to-charge ratios (m/z) of species
fragments
• Electrospray ionization spectrometry
(ESI) is recent, powerful technique
• Dilute solution of analyte (10-4-10-5 M) is
sprayed from capillary tip at high potential
(3-4 kV)
• Liquid forms Taylor cone, fine jet of tiny
charged droplets which blow apart due to
charge repulsion
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• “Nanospray” uses smaller glass
Objective
capillaries for lower flows (20-50 nL/min)
New
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Proteomics-on-a-Chip
• Integrated chromatography + CE + ESI
• Photolithography and wet-etching of Corning 0211 glass
• Nanospray emitter placed into a flat-bottomed hole drilled into the exit of
separation channel
• Bead channel for sample
concentration
• 800 µm wide, 150 µm deep, 22
mm long, etched into the cover
plate (2.4 µL volume)
• Filled with bead suspension
slurry
• Low flow resistance of bead
channel allows sample loading
without perturbing CE channel.
• Results
• Flowrate ~ 2 µL/min
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• Throughput ~ 5 min/sample
• Sensitivity ~ 25 fmol (5 nM)
Harrison Group, U. of Alberta
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ESI On-Chip – 2
• Fabrication
• Polymer chip embossed from silicon
master
• Electrospray tip is flat parylene C
triangle (5 µm thick) sandwiched
between channel chip and sealing
cover
• Tip is wet by analyte, helping to
form and fix position of Taylor cone
• Results
• Low dead volume connection
• Stable ion current 30-40 nA
measured using 2-2.8 kV potentials
• Analyte liquid is completely confined
on triangular tip
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• Cone volume estimated as 0.06 nL
Craighead group,
Cornell U 43
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More Topics
• Cell culturing
• Cell handling
• Dielectrophoresis
• Optical tweezers
• Protein crystallization
• Interfacing between micro-macroworlds
• Materials and surfaces
• Microfluidic/nanofluidic components, modeling
• Applications
• Many more…
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