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a convergence of fields
Cite this: Chem. Soc. Rev., 2015,
44, 5320
Darius G. Rackus,†ab Mohtashim H. Shamsi†ab and Aaron R. Wheeler*abc

Electrochemistry, biosensors and microfluidics are popular research topics that have attracted
widespread attention from chemists, biologists, physicists, and engineers. Here, we introduce the basic
concepts and recent histories of electrochemistry, biosensors, and microfluidics, and describe how they
Received 1st November 2014 are combining to form new application-areas, including so-called ‘‘point-of-care’’ systems in which
DOI: 10.1039/c4cs00369a measurements traditionally performed in a laboratory are moved into the field. We propose that this
review can serve both as a useful starting-point for researchers who are new to these topics, as well as
www.rsc.org/csr being a compendium of the current state-of-the art for experts in these evolving areas.

Introduction diagram in Fig. 1, these intersections can be sub-divided into

four application areas: (A) electrochemistry and microfluidics,
We live in an era in which the traditional disciplines of chemistry, (B) electrochemical biosensors, (C) microfluidic biosensors,
biology, physics, and engineering are intersecting and combining and (D) microfluidic electrochemical biosensors. Here, we
to form a dizzying array of new sub-fields and application-areas. focus on the rich interplay between electrochemistry, bio-
For example, electrochemistry, biosensors, and microfluidics sensors, and microfluidics, with an emphasis on how they are
have become increasingly linked together, making it difficult to combining to form new application-areas, including so-called
conceive of them as separate entities. As shown in the Venn ‘‘point-of-care’’ diagnostics, in which measurements tradition-
ally performed in a laboratory are moved into the field.
a
Department of Chemistry, University of Toronto, 80 St. George St., Toronto, The scientific literature is replete with in-depth discussions
ON M5S 3H6, Canada. E-mail: aaron.wheeler@utoronto.ca of electrochemistry,1,2 biosensors,3–5 and microfluidics.6–8 This
b
Donnelly Centre for Cellular and Biomolecular Research, University of Toronto,
review is unique in that we focus on how these sub-fields
160 College St., Toronto, ON M5S 3E1, Canada
c
Institute of Biomaterials & Biomedical Engineering, Rosebrugh Building,
overlap and work together, in two sections. First, we include
164 College St., Toronto, ON M5S 3G9, Canada three tutorials, defining the key terms and explaining the basic
† Equal contributors. principles for electrochemistry, biosensors, and microfluidics.

Darius Rackus obtained his MSc in Mohtashim Shamsi obtained

Chemistry and Biological Science his doctoral degree under the
in 2012 from the University of supervision of Dr Bernie Kraatz
Durham and is currently pur- and earned his PhD in the area of
suing a PhD in Analytical electrochemical DNA biosensors
Chemistry at the University of from the University of Toronto
Toronto under the supervision of in 2012. He joined the Wheeler
Professor Aaron Wheeler. Current group as a postdoctoral fellow in
research activities involve inte- 2012, where his research focus
grating novel electrochemical has been integration of electro-
sensors with digital microfluidics. chemical sensors in digital
Research interests include bio- microfluidic chips. In fall 2015,
Darius G. Rackus sensors, clinical chemistry, and Mohtashim H. Shamsi he will join the faculty in the
miniaturizing assays. Department of Chemistry and
Biochemistry at Southern Illinois University, Carbondale (SIUC).
His research focus will encompass the interfaces between
electrochemistry, biosensing and microfluidics.

5320 | Chem. Soc. Rev., 2015, 44, 5320--5340 This journal is © The Royal Society of Chemistry 2015
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Second, we review the current state-of-the-art in the overlapping rapidly; thus, great attention is paid to electrode size, geometry,
areas (A, B, C, D), as outlined in Fig. 1. We propose that the material, and surface structure. While electrodes used in electro-
fourth of these areas, microfluidic electrochemical biosensors chemical measurements have traditionally had dimensions on
(D), is a particularly attractive subject, with great promise for the order of millimetres, micrometre-scale ‘‘microelectrodes’’
point-of-care diagnostics and other advances that are shaping and ‘‘ultramicroelectrodes’’ have recently become popular in
the world that we live in. applications related to microfluidics and biosensing. Electro-
des with these dimensions offer advantages such as the ability
to measure small currents in the range of picoamperes to
Electrochemistry
nanoamperes (pA–nA), rapid response to changes in applied
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Electrochemistry1,2 is one of the oldest branches of chemistry, potential, low ohmic reduction in electric potential, efficient
and has historically been used for studying heterogeneous diffusional mass transport, and steady-state response at diffusion-
electron transfer kinetics11 (most commonly at a metal/solution controlled potential. These advantages allow the efficient electro-
interface), with applications in metallurgy,12 corrosion science,13 chemical study of organic systems, sensitive detection of ultralow
semiconductors,14 fuel cells,15 self-assembled coatings,16 and concentrations of analytes, and measurements of BmL sample
electrochemical sensors.7 The latter application, electrochemical volumes.
sensing, has attracted wide attention because of two important When an electrode with excess charge on its surface comes
advantages: inexpensive instrumentation and miniaturization. in contact with ions in solution, an electrical double-layer of
These advantages are typified by the use of systems relying on ions (B5–20 nm thick) is formed at the surface. The layer
inexpensive potentiostats (i.e., a control circuit used to apply closest to the electrode is called the inner layer, for which the
electric potentials and measure small currents) and electro- excess charge on the electrode surface is balanced by an equal
chemical cells formed by screen printing (i.e., a scalable manu- number of oppositely charged ions in solution. The second
facturing technique capable of forming electrodes at low cost). layer, known as the diffuse layer, is a group of oppositely
These advantages constitute the driving forces behind the develop- charged ions with concentration that decreases exponentially
ment of (now ubiquitous) point-of-care glucose monitors and as a function of distance from the inner layer. An electric
alcohol sensors.17 potential exists between the two layers, defined by the amount
We review here some of the fundamental terms and basic of charge and the distance between them. In an electrochemical
principles of electrochemistry. Electrochemical phenomena are process, the species under investigation moves from bulk solution
often measured using a cell comprising three electrodes: (1) a to the electrical double layer by one (or more) of three modes of
working electrode (WE) where the redox reactions of interest mass transfer: (1) diffusion, the movement under the influence
occur and are measured, (2) a counter electrode (CE) that is of a concentration gradient between the bulk solution and the
controlled by the potentiostat to set the WE potential and electrode surface region, (2) migration, the movement under the
balance current, and (3) a reference electrode (RE) that provides influence of the potential gradient between the electrode surface
feedback of the WE potential to the potentiostat. The WE and and the bulk solution, and (3) convection, the forced movement
CE are in direct contact with the solution being studied and the by means of mechanical force (e.g., stirring).
RE is often in indirect electrical contact by means of a con- For most electrochemical applications, the analyte partici-
ductive salt bridge. For analytical purposes, electrons should pates in a reduction–oxidation (or redox) reaction as conse-
transfer across the solution/solid interface smoothly and quence of an electric potential, E, that is measured between the
WE and RE. The electric potential and the concentrations of
species being oxidized or reduced (CO, CR) vary according to the
Aaron Wheeler earned his PhD in Nernst equation (eqn (1)):
Chemistry at Stanford University
RT CO
in 2003. After a postdoctoral E ¼ E0 þ ln (1)
nF CR
fellowship at UCLA, he joined
the faculty at the University of where E0 is the ‘‘standard’’ potential for the reaction, R is
Toronto in 2005, where he is the the universal gas constant, T is temperature, n is number of
Canada Research Chair in Bio- electron transfers involved in the reaction and F is Faraday’s
analytical Chemistry. Wheeler’s constant. In the most straightforward form of electrochemical
research group develops micro- sensing, potentiometry, the Nernst equation is used to discern
fluidic tools to solve problems in CO and/or CR in a passive system (i.e., one with no external
chemistry, biology, and medicine. potential applied). A powerful application of potentiometry is
Wheeler has been recognized with the use of an ion-selective electrode for highly selective measure-
Aaron R. Wheeler a number of honors including the ment of E for one species by means of a synthetic ion-specific
W.A.E. Mcbryde Medal from the coating. There are many other electrochemical techniques, but
Canadian Society for Chemistry, the Arthur F. Findeis Award from three of them, amperometry, voltammetry and electrochemical
the American Chemical Society, and the Joseph Black Award from impedance spectroscopy, are most commonly used with micro-
the Royal Society of Chemistry. fluidics and/or for biosensing applications.

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Fig. 1 Electrochemistry, biosensors, and microfluidics. (a) Electrochemistry – (i) representative amperometry response plot, (ii) representative cyclic
voltammogram, and (iii) representative electrochemical impedance spectroscopy Nyquist plot. (b) Biosensors – schematic of a representative biosensor,
coupling biomolecular recognition to signal transduction. (c) Microfluidics – (i) picture of a microchannel-based device reprinted from Balagaddé et al.9
[Science, 2005, 309, 137–140], reprinted with permission from AAAS, (ii) picture of a droplet microfluidic device reprinted with permission from Dolomite
Microfluidics, Charlestown, MA, USA, (iii) picture of a paper microfluidic device adapted from Martinez et al.,10 copyright (2008) National Academy
of Sciences, USA, (iv) frame from a video depicting actuation of coloured droplets on a DMF platform.

Amperometry is an electrochemical analysis mode in which Voltammetry is the most extensively used technique in
current is measured while a constant external electric poten- electrochemistry, partly because it can probe the reversibility
tial is applied between the WE and CE. The current I is of the system under study. Like amperometry, in voltammetry,
recorded as a function of time t, as shown (for a system with an electric potential E is applied between the WE and CE and
no convection or migration) in Fig. 1(a)i. Because electron the resulting current I is measured. Unlike amperometry, in
transfer can only occur in very close proximity to the electrode, voltammetry, E is varied as a function of time. For example, in
the current at the WE is proportional to the flux of analyte to the (most common) format of cyclic voltammetry, E is swept
the electrode surface which depends linearly upon the concen- in a linear cycle at scan rate v. As shown in Fig. 1(a)ii, as
tration gradient of the analyte between the surface and the E becomes more positive, the analyte becomes oxidized, and as
bulk solution. Initially, only the analyte near the double-layer E becomes more negative, the analyte becomes reduced, with
is depleted (i.e., oxidized or reduced), resulting in high current. each step (oxidation and reduction) associated with a peak
As the current continues to flow, the region of reduced analyte current, ip. The relationship between ip and v is given by the
concentration extends further into the solution; thus, the Randles–Sevcik equation (eqn (2)):
concentration gradient declines with time, which causes the
ip = (2.69  105) ACD1/2n3/2v1/2 (2)
current to decline. Since the progression of the concentration
gradient depends on the concentration of analyte in the bulk where A is the electrode area, C is the concentration of analyte
solution, the time dependence of the magnitude of the current in bulk solution, D is the diffusion coefficient of the analyte, and n
that is measured can be related to the concentration of analyte is the number of electrons involved in the reaction. The magnitude
in solution. of ip can be used to determine analyte concentration, and the

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potentials at which the analyte is oxidized/reduced can be used for (a) to (b), regardless of the physical location of the individual
qualitative identification. Peak currents can be enhanced by means components.
of redox cycling whereby the species of interest that is being An ideal biosensor is selective, rapid, reusable (or reversible),
oxidized or reduced at the electrode surface is regenerated (and portable, and requires minimal sample processing prior to
thus measured repeatedly) either chemically, enzymatically, or analysis. There are few biosensors that achieve this in practice
electrochemically. – trade-offs are almost always required. Fig. 1(b) depicts a generic
Voltammetric methods also include linear sweep voltam- biosensor scheme. As shown, the interaction between analyte
metry, differential pulse voltammetry, square-wave voltammetry, molecules and the biorecognition element (in this example, a
AC voltammetry, and stripping voltammetry. Stripping voltam- layer of bioreceptor molecules) causes transduction of a measur-
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metry, in particular, is used widely in sensing applications for able physicochemical change such as current flow, heat transfer,
trace detection. Analytes are electrochemically immobilized or change in mass or refractive index.
(deposited or adsorbed) on the electrode surface by reduction, Biorecognition elements in biosensing can be broadly
oxidation, or other adsorption processes, allowing for accu- classified in terms of the nature of their interaction with
mulation of low-concentration species as a function of time. analyte molecules. An affinity biosensor operates as a function
Subsequent sweeping of the potential then strips off the analyte, of permanent or semi-permanent binding between the bio-
resulting in a peak current that correlates with the original recognition element and the analyte. This class of biosensors
analyte concentration. includes immunosensors (antibody–antigen binding), nucleic
Electrochemical impedance spectroscopy (EIS) is a powerful acid biosensors (probe and complementary nucleic acid target
electrochemical method that has recently become popular in binding), and aptamer biosensors (ligand and synthetic oligo-
biosensing because of its capability to detect binding events on nucleotide receptor binding). In contrast, in catalytic biosensors,
a transducer surface. In EIS, a DC potential EDC and a small the interaction between the analyte and the biorecognition
sinusoidal AC perturbation (EAC, B5–10 mV amplitude) are element is impermanent, and involves a chemical reaction
applied between the WE and the RE. The magnitude and the that forms an easily detected product. This class of biosensors
phase angle f of the resulting current I are recorded as a includes enzymatic biosensors, cell-based biosensors, and bio-
function of the AC frequency. The current magnitude can be sensors relying on catalytically active polynucleotides (DNAzymes).20
converted into impedance Z using Ohm’s law (Z = V/I), and Catalytic systems are particularly useful for trace analysis because of
impedance is expressed as a complex number Z = Zre + jZim the inherent amplification; i.e., the presence of a single analyte
where real impedance Zre has f = 0 (i.e., is independent of molecule can result in a large number of products to be detected.
frequency) and imaginary impedance Zim has f a 0 (i.e., is Signal transduction techniques in biosensing can be broadly
dependent on frequency). The data generated by EIS is often classified in terms of whether the process requires labels, or
presented as a Nyquist plot of Zim relative to Zre at different whether the process is label-free. Biosensors that require labels
frequencies. As shown in Fig. 1(a)iii, at very high frequencies are designed to transduce the analytical signal from a desig-
(left side of the plot) and very low frequencies (right side of the nated reporter molecule (not the analyte molecule itself). The
plot) there is no contribution from Zim; at the low-frequency label-format allows for flexible implementation of biosensing
limit, Zre = Rct, the charge-transfer resistance. Rct represents the in a wide range of detection schemes, but a trade-off is the
diffusion-controlled limit for the redox reaction rate at the time, cost, and additional steps associated with incorporating
electrode surface (in biosensing, this property can be related to reporter molecules into the process. In label-free biosensors,
analyte concentration), and the maximum of the ‘‘semicircular’’ the signal is transduced directly from the presence of the
response at moderate frequencies is related to the capacitance of analyte molecule itself. Some electroanalysis techniques (described
the double-layer. EIS is thus often used in characterizing the above) as well as surface plasmon resonance and mass-sensitive
properties and behaviour of modified electrode surfaces and to techniques (described below) fall into this category. Label-free
extract the capacitive and resistive components of such surfaces biosensors require very specific formats, but are advantageous in
using equivalent circuit models.18 that reporter molecules are not required.
Biosensors (particularly when viewed in context of the
definition used here) are remarkably diverse, comprising a wide
Biosensors range of combinations of biorecognition and transduction. Here
The concept of a ‘‘biosensor’’ has been defined in many we review common examples of each of these fundamental
different ways in the literature.3–5 Nearly all parties can agree aspects of biosensing.
that the definition should include (a) a biomolecular recogni- Biorecognition. Enzyme-based biosensors are catalytic sensors
tion element to confer selectivity, and (b) a signal transduction in which the bioreceptors comprise enzyme molecules in solution
element to enable quantitative or semi-quantitative analysis. or tethered to a surface. Enzyme-based biosensors are typically
There are some who also insist that the biorecognition element implemented in direct or indirect format. In the direct format,
should be positioned physically adjacent to the transducer.19 the analyte promotes the activity of an enzyme (either acting as a
We appreciate the reasons for this definition and agree that it is co-factor for the enzyme or in concert with an affinity binding
useful in some circumstances, but here we employ a broad event to localize the enzyme near the analyte), which catalyzes the
definition of biosensor that incorporates any method coupling formation of a measurable product (i.e., analyte concentration is

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proportional to signal). In the indirect format, the analyte inhibits has a one base-pair mismatch with the probe. This level of
the activity of the enzyme, resulting in reduced rates of formation selectivity is required to identify single nucleotide polymorphisms
of a measurable product21 (i.e., analyte concentration is inversely (SNPs); there is great interest in using SNP detection to identify
proportional to signal). patients with genetic diseases.
Immunosensors are affinity-based biosensors that rely on Aptamer-based biosensors feature an alternative form of
the binding of an antibody to its specific antigen. Immuno- affinity biorecognition relying on synthetic oligonucleotide
sensors are implemented in a variety of schemes, including (single-stranded DNA or RNA molecules) probes; in contrast
(a) direct format, featuring binding of an unlabeled antigen to conventional nucleic acid sensors (which bind only their
to an unlabeled antibody (requiring label-free transduction), complements), aptamers can be designed to bind any type of
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(b) competitive format, featuring competition for binding of an target. Aptamers are prepared by a combinatorial approach
unlabeled (target) antigen and a labeled (exogenous) antigen to called systematic evolution of ligands by exponential enrichment
an antibody, (c) ‘‘sandwich’’ format featuring an antigen with (SELEX).23 SELEX is an iterative process in which (1) a pool of
two epitopes (i.e., antibody-recognition sites) that binds to an oligonucleotides with varying sequences is generated, and (2) the
immobilized primary antibody and also to a labeled- or enzyme- population that binds best to a given target is selected and
modified secondary antibody (when the secondary antibody isolated. The best binding sequence(s) then serve(s) as the basis
is enzyme-modified, the technique is known as an ‘‘enzyme- to generate new sequences, and steps (1)–(2) are iterated for
linked immunosorbent assay,’’ ELISA – this is likely the most several rounds to generate a product with high specific binding
common immunosensor format), and (d) inhibition format to the desired target. Aptamers modified with electroactive
featuring competition between an analyte and a primary anti- indicators, fluorescent tags, nanoparticles and enzymes have
body for binding to a labeled (or enzyme-modified) secondary been used for amplified detection24,25 of a wide range of targets
antibody. Immunosensors are likely the most common form including amino acids, antibiotics, co-factors, drugs, metal ions,
of biosensors, primarily a function of the flexibility of the nucleic acids, and organic dyes.
biorecognition; antibodies can be raised to selectively bind Transduction. Electrochemical transducers used in biosensing
proteins (including enzymes and other antibodies), small exploit the redox activity of a solute in solution – either the analyte
molecules (including hormones, toxins, environmental con- itself, an electroactive label attached to the analyte, or a catalyti-
taminants), cells (including surface-markers on pathogenic cally generated electroactive reporter. The electrons generated
bacteria), and many other classes of antigen. in the redox process are detected as current, which is related
Nucleic acid-based biosensors are affinity sensors that exploit to the number of redox species involved in the process. In some
the sequence-specific Watson–Crick base pairing between nucleic instances, an electron transfer mediator is used to shuttle
acids and their complements. The most common form of nucleic electrons from the electroactive species to the electrode surface
acid sensors are formed from a single-stranded DNA (ss-DNA) (e.g., from the redox centre of an enzyme to the electrode).
probe that is immobilized onto the surface of a transducer. Electrochemical measurement systems, which are ubiquitous
Upon recognition of its complementary ss-DNA or RNA analyte in modern society in the form of portable glucose monitors,
(or target) by hybridization, transduction is facilitated by optical, represent the most common form of transduction used in
electrochemical, or mass-sensitive techniques. A well-known biosensing. As described in the preceding section, the most
example of a (highly multiplexed) nucleic acid-based biosensor common electrochemical biosensors transduce signals by
is the DNA microarray, which enables semi-quantitative analysis means of amperometry, voltammetry, or EIS. Amperometric
of gene expression for thousands of sequences in one shot.22 and voltammetric sensors are often used in catalytic mode; for
There are a number of variations on the simple DNA-probe–DNA- example, in amperometric glucose sensors, the WE is coated
target theme. One variation uses peptide nucleic acid (PNA) with a layer of glucose oxidase. When glucose in a blood sample
probes, in which the negatively charged sugar-phosphate back- encounters the enzyme, hydrogen peroxide is formed, which is
bone of DNA is replaced by a neutral pseudopeptide chain. then oxidized at the WE to generate a current that is propor-
PNA probes have higher binding affinities (relative to their tional to the amount of glucose in blood. EIS, on the other
analogous ss-DNA probes) for ss-DNA targets, and the reduced hand, is often used for affinity biosensing, in which biorecep-
charges on these probes confer advantages for some forms tors such as antibodies or nucleic acids are attached to the WE
of electroanalysis. Another variation is the sandwich assay, in surface. In this scheme, the charge transfer resistance experi-
which an immobilized probe binds a region of an analyte, and a enced by an electroactive reporter as it diffuses through the
second, labeled probe binds a different region of the analyte. film of bioreceptors is a measure of the amount of bound
A third variation known as a ‘‘molecular beacon’’ features analyte and the charge on the surface.26 A disadvantage of EIS
probe-sequences that self-bind to form stem-and-loop or hair- is its high sensitivity towards nonspecific adsorption.
pin structures. Complementary targets compete for binding Optical techniques represent another very common form of
with such structures (requiring the probe to undergo a change signal transduction used in biosensing.20 While many analytes
in conformation) which can enable very sensitive detection of are optically active, optical transduction in biosensing often
small numbers of targets. The most useful nucleic acid bio- requires a label – one that binds to the analyte or one that is
sensors allow for differentiation between the binding of a target generated catalytically. The simplest optical sensing technique
that is perfectly complementary to the probe and a target that is absorbance (also known as colourimetry when used with

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visible wavelengths), in which the relative intensity of light wave (SAW) sensors and quartz crystal microbalance (QCM)
before and after passing through a sample correlates with the sensors. In the former (SAW), an AC excitation signal is applied
concentration of absorbing species (also known as chromo- to the surface of a piezoelectric substrate to generate a SAW with
phores) in the sample. In the related (and more sensitive) characteristic velocity v. After propagating across a sensing area,
technique of fluorescence (or fluorimetry), the light that is the properties of the wave are interrogated and compared to the
detected is emitted from fluorescent species (also known as excitation signal. The velocity of the wave depends on the density
fluorophores) after the initial absorption (or excitation). The of molecules on the surface of the substrate; thus, the technique
wavelengths of light that are absorbed and emitted match can be used to probe the mass of analyte molecules bound to
differences between electronic energy levels of the chromo- bioreceptors on the surface.31 QCM is similarly mass-sensitive,
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phores and fluorophores. In some cases, a fluorophore may evaluating the shift in resonant frequency Df for a standing wave
be ‘‘quenched’’ by the presence of other species that allow the applied through the bulk of a piezoelectric substrate, which
excited fluorophores to relax non-radiatively. Some biosensors (like SAW) depends on the mass of analyte molecules bound to
are designed to take advantage of this effect, such that when the surface.32 The resonant frequency is inversely proportional to
analytes bind, a fluorophore and quencher become more or less the crystal thickness and the addition of mass on the surface can
associated (which changes the amount of emitted light). be treated as an extension of the crystal thickness. A third mass
A related effect known as Förster resonance energy transfer sensitive transducer is the microcantilever, which derives its
(FRET), involves two fluorophores, a ‘‘donor’’ and an ‘‘acceptor.’’ origins from atomic force microscopy. Binding of the analyte to
The relative intensities of emitted light generated from the two a biological recognition layer on the microcantilever induces
fluorophores varies as a function of the distance between them, surface stress resulting in nanomechanical motion. This motion
making FRET a useful tool for probing recognition events that can be monitored optically or by a piezoresistive readout system.33
result in a change in probe conformation (as in molecular A limitation of mass-sensitive detectors is an inherent sensitivity
beacons, described above). A disadvantage of absorbance and to non-specific adsorption onto the surface.
fluorescence transduction is the requirement of an external light
source; an alternative known as chemiluminescence, often used Microfluidics
in catalytic biosensors to generate chemiluminescent reporter Microfluidics is a technology that facilitates the manipulation
molecules, does not require an external light source. All optical of small volumes of fluids in the range of mL–aL (106 to
analysis techniques require an optical transducer such as a 1018 liters).6–8,34 Microfluidics is most often implemented in
photodiode or photomultiplier tube (PMT), and they often planar substrates bearing enclosed channels with lengths,
require lenses, reflectors, and other optical components. widths, and depths on the B10 mm, B100 mm, and B10 mm
Surface plasmon resonance (SPR) is a transducer that is scales, respectively. The technology was popularized in the early
often used in label-free affinity biosensing. In SPR, light from 1990s35,36 for applications related to chemical separations, but
an external source is reflected off of a metal film, generating an in the intervening years it has been applied to an incredible
evanescent wave that penetrates a short distance into the film. array of applications, ranging from genomics37 and synthesis38,39
When the energy of the light matches that of the surface to music40 and mazes.41,42 A particularly attractive vision for the
plasmons (i.e., coherent oscillations of electrons at the metal- microfluidics community has been the development of integrated
external medium interface), the energy from the incident light is ‘‘lab on a chip’’ systems that reproduce laboratory-scale processes
transferred into the surface plasmons.27 This effect is typically with reduced cost, less time, and with substantially smaller
evaluated by monitoring the wavelength or angle at which the footprints than their conventional counterparts.43 A highly
energy is transferred, observed as a ‘‘dip’’ in the intensity of the regarded journal with the same name was founded in 2000,
reflected beam. The surface plasmon resonance energy depends and now publishes 4600 papers in 24 issues per year.
on the refractive index of the medium adjacent to the metal film; The micrometer dimensions that are common in microfluidics
thus, if the surface is modified with receptor molecules and the result in fluidic phenomena that exhibit increased importance
receptors bind analyte molecules, the shift in resonance angle or of viscosity, surface tension, and diffusion when compared to
wavelength provides a real-time signal that corresponds with conventional systems. These properties are often represented in
analyte concentration.28 Nanostructuring the thin metal surface terms of dimensionless parameters, including Reynold’s number
improves signal by exciting the various modes of SPR and long- (Re, eqn (3)), Capillary number (Ca, eqn (4)), and Péclet number
range surface plasmons.29 Surfaces can be imaged by SPR using (Pe, eqn (5))
a technique known as SPR imaging (SPRi). SPRi is performed
by holding both the wavelength and the angle constant and rlv
Re ¼ (3)
measuring the reflectance across a sample surface.30 m
Mass-sensitive detectors represent a final class of transducers
that are commonly used in biosensing. The most common forms mv
Ca ¼ (4)
of these sensors rely on piezoelectric substrates – that is, materials g
(including some ceramics and quartz crystals) that can convert
electrical into mechanical energy and vice versa. The two most vl
Pe ¼ (5)
common mass-sensitive analysis techniques are surface acoustic D

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where r is fluid density, l is a characteristic length in the system, technique is in throughput rather than individual droplet control.
v is mean fluid velocity, m is dynamic fluid viscosity, g is surface The application for droplet microfluidics that has likely attracted
tension, and D is coefficient of diffusion. In general terms, Re, Ca, the most attention is ‘‘digital PCR,’’ in which a sample is dispensed
and Pe are low for microfluidic systems, meaning that viscous into millions of droplets, allowing massively parallel amplification
forces dominate inertial forces (resulting in laminar flow), inter- and measurement, which affords orders of magnitude greater
facial forces dominate viscous forces, and diffusion dominates precision and sensitivity relative to conventional PCR.62
convection. These phenomena are important to consider when Paper-based microfluidics. Paper-based microfluidics is an
designing microfluidic systems for biosensors and electro- alternative scheme for miniaturized fluid handling in which
chemistry. For more, there are a number of reviews44,45 and liquid samples are passively wicked (or ‘‘pumped’’) by lateral
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textbooks46,47 that describe fluid phenomena at micron length flow through paper substrates. The Whitesides group popular-
dimensions in great detail. ized this phenomenon as being a member of the ‘‘micro-
Microfluidic platforms can be classified into a number of fluidics’’ family in 2007,63 but similar ideas have been used
different categories, including (1) channel-based microfluidics, for many decades64 and indeed, products relying on lateral flow
(2) paper-based microfluidics and (3) digital microfluidics. are widely available to consumers in the form of pregnancy
There are many alternative classifications (e.g., one might tests. Paper microfluidic devices (in their modern format) are
include a SlipChip48 category, or split a ‘‘droplet microfluidics’’ implemented by forming hydrophobic/hydrophilic patterns to
category out from ‘‘channel-based microfluidics’’), but these guide fluid movement through paper (Fig. 1(c)iii). A variety of
categories suffice for the purposes of this review. We describe creative techniques have been developed to form such patterns
each of them below. including wax printing,65 inkjet printing,66 photolithography,67
Channel-based microfluidics. In its original conception, flexographic printing,68 and many others;69 paper can also be
the technology of ‘‘microfluidics’’ was coincident with ‘‘micro- cut to a specific geometry to guide fluid movement.70 Paper
channels,’’ and channel-based microfluidics continues to microfluidics has become popular because of the very low cost,
represent (by far) the most widely practiced category of micro- ease of fabrication, flexibility, disposability, and the conveni-
fluidics. Initial work with microchannels focused on the trans- ence of liquid transport without applying an external driving
lation of the electrokinetic flow techniques (e.g., electroosmosis force. For these reasons, there is great enthusiasm for using
and electrophoresis) used in capillary electrophoresis to networks paper microfluidics for point-of-care diagnostic assays,71 with
of microchannels to effect chemical separations.49 Electrokinetic particular interest in their use as a low-cost platform for
flow is particularly advantageous because there is very little delivering medical diagnostics in resource-limited settings.72
external equipment required (i.e., only a high-voltage power The paper microfluidic concept has been implemented in
supply), but the range of reagents and solvents that can be used formats ranging from simple dipstick assays in which a single
is limited. Other forms of fluid manipulation soon followed, reagent adsorbed on a paper substrate changes colour after
including various types of pressure-driven flow controlled by contacting an analyte (e.g., pH strips), to sophisticated micro-
external pumps, centrifugal forces,50 or on-chip peristaltic fluidic paper-based analytical devices (mPADs) relying on multi-
pumps.51 These techniques are amenable for working with a layer substrates including some or all of (a) a membrane
wide range of reagents, but they each (to varying degrees) require modified with biorecognition agents, (b) a pad designed to
ancillary equipment to operate. Microchannel device materials absorb sample, (c) conjugate pads which are preloaded with
are an important concern – for example, polymer materials that conjugated particles – e.g., gold nanoparticles, (AuNPs), etc.,
can be molded such as polydimethyl(siloxane) (PDMS) are (d) a wicking or an absorbent pad to provide capillary driving
straightforward to fabricate, but have limited chemical compati- forces, and (e) a backing that provides mechanical stability.73,74
bility, while hard materials like glass and silicon have greater Detection is typically implemented by electrochemistry or
chemical compatibility but typically require access to specialized colourimetry (consistent with low-tech, portable applications),
instrumentation and are more time-consuming to fabricate.52 but paper microfluidic systems have also been reported that
Microchannel-devices are operated in either continuous mode use chemiluminescence75 and electrogenerated chemilumines-
(often with integrated valves and pumps as in Fig. 1(c)i, or droplet- cence.76 Despite these advances, paper-based microfluidics lags
mode, as in Fig. 1(c)ii). The laminar flow-characteristics of the the other categories of microfluidics in quantitative perfor-
former (with its inherently low Re) has been exploited for a wide mance; there is room for improvement in selectivity, specificity,
range of applications involving intricate chemical gradients.53,54 sensitivity, and linear dynamic range.73,77
The latter, which is implemented by combining immiscible Digital microfluidics. Digital microfluidics (DMF) is a third
solvents in microchannels to form emulsions (often aqueous category of microfluidics, in which samples are manipulated
droplets in an oil carrier-phase), has recently exploded in popu- as discrete droplets on a flat surface.7,78–80 The most common
larity, with seemingly endless examples of applications.55–57 implementation of digital microfluidics relies on electrostatic
Microchannel droplet systems can be operated at extremely high- forces generated on arrays of electrodes coated with a hydrophobic
throughput (generating more than 103 droplets per second58), and insulating layer. In this format, droplets can be made to move,
are particularly well-suited for sorting applications.59 There has merge, mix, split, and be dispensed from reservoirs. These opera-
been some progress developing methods that allow for individual tions and others can be called iteratively (as in computer program-
droplet addressing and manipulation,60,61 but the strength of the ming) to execute sophisticated, multi-step assays. While the other

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forms of microfluidics are compatible with similar operations, for separations were used for such applications,102,103 which was
DMF is unique in enabling them on devices with a generic followed by the incorporation of voltammetric methods to detect a
format (Fig. 1(c)iv) that can be used and reused for very variety of organic and inorganic analytes.104
different applications. Moreover, the capability to address each Typically, microfluidic systems incorporating electro-
individual droplet allows for complete control over reagent/ chemical detection are implemented by patterning electrodes
sample state, position, and activity. on a flat glass or silicon substrate, and then a polymer-based
The most common digital microfluidic systems rely on electro- substrate bearing microchannels is adhered to this substrate.105
static forces, which are often described in terms of ‘‘electrowetting,’’ Glass and silicon are common substrates for electrode fabrication,
in which Laplace pressures are applied as consequence of but inexpensive alternatives such as compact discs can also be
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asymmetric changes in droplet shape.81 The electrowetting used.106 In some cases, discrete REs and CEs are inserted into
analogy is useful for modeling conductive liquids with high channel inlets. REs can also be incorporated within channels by
surface tensions, but droplet actuation can be described more means of a salt bridge,107,108 as depicted in Fig. 2(a). The per-
generally using the Maxwell Stress Tensor82 or electromecha- formance of electrodes within a microchannel depends on a variety
nical lumped-sum models.83 Historically, DMF devices were of parameters including electrode position and flow rate.109
rigid, formed from hard materials such as glass and silicon, but Examples of separations coupled with electrochemical detec-
a recent trend is the formation of paper-based devices using tion include the isolation of hydrolysis products,110 the monitoring
inkjet printing.84,85 One barrier to entry for users adopting DMF of biomolecule release from individual cells,111 and the quantita-
is the need for a custom, highly parallelized control system tion of bioactive molecules in serum.112 The main challenge in
capable of handling high voltages. The recent development of integrating electrochemical detection with electrophoretic separa-
an ‘‘open-source’’ control system86 mitigates this to some extent. tions has been isolating the separation and detection modules.
As an alternative to electrostatic control, alternative DMF systems Several strategies have been employed to overcome this problem,
can be realized using magnetic,87 optical,88 acoustic,89 or thermo- including the placement of the WE within the channel,113 the use
capillary90 forces. of a decoupler that shunts electrophoretic current away from
Digital microfluidics is complementary to the other forms of the electrochemical cell,114 floating the potentiostat ground,115
microfluidics. For example, DMF is advantageous in that there wirelessly isolating the potentiostat,116 or using an in-channel
are no pumps, valves, interconnects, or fittings; on the other salt-bridge.108
hand, the throughput of DMF is much lower than that of Microchannel devices have also been found useful for cell
droplets-in-channels (note that DMF throughput may improve culture affording reduced volumes and precise control over the
significantly with the development of devices formed from extracellular environment. This makes them ideal for studying
arrays of thin film transistors91). DMF is particularly well suited cell-to-cell signaling including neurotransmission. For example,
for applications involving solids (e.g., tissue,92 dried blood,93 microfluidics and electrochemistry have become popular in
hydrogels,94 monoliths95), as there are no microchannels that neuroscience because of the electroactive nature of (some)
might become clogged. Indeed, methods that use magnetic forces neurotransmitter analytes.117,118
to control large (solid) boluses of magnetic particles combined Reusable electrodes can be incorporated with microfluidic
with electrostatic droplet control over droplet position is emerging systems using a modular approach. Using 3D printing, Erkal
as a powerful method for implementing immunoassays.96–100 et al.119 fabricated microchannel devices with threaded receiving
ports where electrodes could be integrated. This allows the user
to remove and polish the electrodes in between experiments
A convergence of fields when electrode surface-fouling is observed. This modular
approach to integration was coupled with flow injection analysis
Here we review a selection of representative applications in of dopamine and monitoring ATP concentrations in cell culture
each of the overlapping areas defined in the Venn diagram in studies.
Fig. 1: (A) electrochemistry and microfluidics, (B) electrochemical Paper microfluidics. Coupling of electrochemistry with paper
biosensors, (C) microfluidic biosensors, and (D) microfluidic microfluidics is attractive as it combines two low-cost techno-
electrochemical biosensors. logies for the prospect of inexpensive diagnostics and point-of-
care testing. Two strategies for integrating electrochemical
A. Electrochemistry & microfluidics detection with paper microfluidics are (1) printing electrodes
Among all the analytical techniques coupled with microfluidics, from conductive inks using screen printing (Fig. 2(b))120,123 or
electrochemical detection is the simplest to integrate, which inkjet printing,124 and (2) coupling external electrodes to the paper
makes it ideal for point-of-care applications (as reviewed by Sassa microfluidic devices. The simplest approach for the latter is to affix
et al.101). In this section we discuss the incorporation of electro- a three-electrode assembly directly to a paper device.125–127 Alter-
chemical detection with the various modalities of microfluidics. natively, microwire electrodes can be incorporated in both conven-
Microchannels. The integration of electrochemical detection tional microchannels and paper/lateral flow devices, which can be
with microchannels dates back to the early days of micro- affixed using adhesive tape (Fig. 2(c)).121
fluidics when microchannels were primarily used for electro- Paper microfluidic devices integrated with electrochemical
phoretic separations. Originally, amperometric detection methods detection have been used for separations,127,128 and quantitation

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Fig. 2 Electrochemical detection in microfluidics. (a) Cartoon of a microfluidic channel with a polyelectrolytic gel salt bridge isolating the RE from the
main channel. The Au WE is patterned onto the channel substrate. Wires in a 1 M KNO3 solution are separated from the microchannel by a salt bridge.
Reprinted with permission from Kang et al.,108 copyright 2012 American Chemical Society. (b) Photograph of screen printed electrodes on a paper
microfluidic device. The microfluidic device divides the sample into three aliquots which are then analyzed for glucose, lactate, and uric acid. Reprinted
with permission from Dungchai et al.,120 copyright 2009 American Chemical Society. (c) Cartoon of a folded paper microfluidic device with hollow
channels. Electrodes are incorporated by taping microwires to the device. Ag adhesive and Cu tape are used for electrical contacts. Reprinted with
permission from Fosdick et al.,121 copyright 2014 American Chemical Society. (d) Photograph and schematic of electrodes incorporated into the bottom
plate of a DMF device. Reactions and sample preparation take place in a general purpose area on the DMF device. A three-electrode electrochemical cell
is patterned and insulating coatings are removed. Adapted with permission from Dryden et al.,122 copyright 2013 American Chemical Society.

of metals74,125,129 and bioactive molecules such as glucose, lactate, of droplet operations compatible with one-plate DMF devices.
cholesterol, and uric acid.120,130 In one unique example, Renault These challenges can be overcome by using a two-plate DMF
et al.131 used screen printed electrodes formed in a multilayer format where detection electrodes are either incorporated into
paper device bearing microchannels. This device was shown to be the top or bottom plate of a DMF device.
versatile for a variety of voltammetric and amperometric analyses. Recently, two examples of integrating electrochemical ana-
Pressure driven flow, not typically used in paper microfluidics, was lysis electrodes in the bottom plate of a two-plate DMF device
exploited to couple convection with electrochemical detection. were reported. Dryden et al.122 integrated a three-electrode
Digital microfluidics. In DMF, electrochemical detection can system within a gold layer used to form DMF actuation electro-
be implemented using external electrodes or by patterning the des, as shown in Fig. 2(d). The photoresist SU-8 was used as the
electrodes into the device, itself. The first report of electro- dielectric insulator on the bottom plate so that apertures could
chemistry coupled with DMF was a one-plate device used to be opened over the electrochemical electrodes for sensing. The
detect the product of Greico’s reaction in an ionic liquid droplet RE was electroplated with silver to provide a stable reference
microreactor.132 The device used two suspended gold wires as a potential. This device was used for the quantification of acet-
ground electrode and the WE, respectively. However, this and aminophen by linear sweep voltammetry. In an alternative
other one-plate DMF/electrochemistry systems133,134 suffer approach, Yu et al.135 used reactive ion etching (RIE) to expose
from the challenges of manual positioning of the electro- sensing electrodes on the bottom plate of a DMF device. The
chemical electrodes, liquid evaporation and the limited range electrochemical cell comprised Au interdigitated WE and CE

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and a small rectangular Au RE, all embedded within a driving For example, Patolsky et al.144 developed an amperometric
electrode. glucose sensor that featured glucose oxidase (GOx) tethered
A second approach to integrating electrochemical detection to a Au electrode surface via single-walled carbon nanotube
in a two-plate DMF device is to pattern sensing electrodes on (SWCNT) mediators. The SWCNTs were terminally functiona-
the top plate, which contains a thin conductive layer (often lized with flavin adenine dinucleotide (FAD) which was attached
transparent ITO). This circumvents the need to pattern the to apo-glucose oxidase, as illustrated in Fig. 3(a). Zayats et al.145
dielectric insulator (a challenge for using electrodes embedded developed an analogous voltammetric glucose biosensor system
in the bottom plate, as above) and only the hydrophobic layer using AuNP linker/mediators, which were covalently modified
requires patterning. Shamsi et al.97 used patterned ITO for the with glucose dehydrogenase (GDH) that was reconstituted
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sensing electrodes. Most of the top plate was covered with a around a pyrroloquinoline quinone (PQQ). Oxidation of glucose
hydrophobic Teflon-AF layer, except the sensing electrodes and electron transfer from the PQQ redox centre to the electrode
which were exposed through a lift-off process. The exposed surface was mediated by the AuNP.
ITO was electroplated with Au to serve as the WE and Ag as a Affinity electrochemical biosensors. The most common
pseudo-RE. Alternatively, Yu et al.136 used Au electrodes for biorecognition elements used in affinity electrochemical bio-
grounding and sensing on the top plate of a DMF device. In this sensors are antibodies and nucleic acids that bind with high
configuration, the ground electrode was patterned as a thin specificity to their antigens and complementary probes, respec-
trace to maintain visibility. This format restricts droplet move- tively. A third (emerging) class of electrochemical biosensors
ment to patterns that match the ground electrode. use biomimetic probes such as aptamers to detect a diverse
range of analytes.
B. Electrochemical biosensing Electrochemical immunosensors & immunoassays. An enor-
Electrochemistry has been linked to biosensing since the con- mous amount of literature has been produced in the area
cept of the latter was first proposed. In fact, the first biosensor, of electrochemical immunosensors. One should be aware of
described by Clark and Lyons137 in 1962, was an electrochemical potentiometric,146 conductometric,147 and impedance148 immuno-
oxygen sensor, with selectivity for glucose conferred by a layer of sensors; we limit our discussion here to the most common format:
glucose oxidase. Electrochemical biosensors can be categorized voltammetric sandwich-type immunoassays.
either by the type of electrochemical technique or on the basis As described in the preceding sections, in sandwich immuno-
of the biorecognition element (i.e., catalytic or affinity). There assays, an immobilized primary antibody binds an analyte, which
are good reviews138,139 covering the principles, architecture, is then bound again by a secondary antibody. When imple-
and applications of electrochemical biosensors. Here, we pre- mented with electrochemical detection, the secondary antibody
sent a selection of some of our favourites that exemplify the is typically covalently linked to an enzyme that produces or
most common strategies and techniques used in electrochemical consumes a redox active species to be detected at the electrode
biosensing. surface. While traditional electrode materials (e.g. glassy carbon,
Catalytic electrochemical biosensors. Catalytic electro- Au, etc.) are common, alternative materials have recently become
chemical biosensors function on the basis of an interaction popular. For example, Pampalakis and Kelley149 reported an
between a catalyst (often an enzyme) and a target analyte, which electrochemical immunosensor in which the primary antibody
results in a reaction that consumes or produces an electroactive for prostate specific antigen (PSA) was immobilized on electrodes
species to be detected at the electrode through change in formed from Au nanowires. Alkaline phosphatase (ALP)
current140 or potential.138 It is important that the reaction covalently attached to a secondary antibody was used to hydro-
occur in close proximity to the electrode surface, as distance lyze 2-phospho-L-ascorbic acid to ascorbic acid, which in turn
can cause signal attenuation.141 A variety of methods have been reduced Ag+ ions to Ag metal to be detected by stripping
used to immobilize enzyme molecules on electrode surfaces, voltammetry. Paper substrates with screen printed electrodes
including physical adsorption, covalent attachment, and encap- have also been reported for electrochemical ELISAs developed
sulation in sol–gel or redox-active polymer layers.142,143 for low-cost applications.150
Transduction of electrons from soluble redox-active sub- A significant advantage of electrochemical immunoassays
strates to the electrode can be aided (a) by directly tethering implemented in sandwich format is amplification, which
the enzyme’s redox core to the electrode surface, or (b) by means enables quantification of trace analytes. While all ELISAs
of electron transfer mediators. The latter strategy is more flexible benefit from inherent enzymatic amplification (i.e., one analyte
and is becoming increasingly popular; common mediators molecule can cause the turnover of many enzyme substrate
include ferrocene (and derivatives), ferricyanide, methylene blue molecules), electrochemical based ELISAs allow for additional
(MB), benzoquinone, and N-methyl phenazine.138 The phenom- amplification through redox cycling of the electroactive enzyme
enon of diffusion of mediators away from the electrode surface product. Redox cycling can be done chemically with reductants
(which reduces the magnitude of signal that can be measured) like NADH132 or hydrazine,133 enzymatically (using a second
has led researchers to investigate methods for anchoring both enzyme, different from the secondary-antibody-conjugate151,152),
the mediator and enzyme to the electrode. This has been made or electrochemically134,135 using interdigitated electrode arrays
possible by nanomaterial linkers that can also transport (IDA). Signal enhancement by as much as 50-fold from redox
electrons, such as carbon nanotubes and Au nanoparticles. cycling has been reported.153

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Fig. 3 Electrochemical biosensors. (a) Cartoon sequence (left-to-right) depicting the generation of a catalytic voltammetric biosensor. A layer of
SWCNTs is attached to the surface of an electrode, where it is subsequently modified with an enzyme co-factor (FAD), which associates with an enzyme
(apo-GOx), which converts glucose to gluconic acid. Each SWCNT acts as an electron mediator, permitting efficient transfer of electrons to the
electrode. Reprinted with permission from Patolsky et al.,144 copyright 2004 with permission from Wiley-VCH Verlag GmbH & Co. KGaA. (b) Cartoon
depicting a competitive voltammetric immunoassay, in which an electroactive reporter (Fe2+) is blocked from interacting with a WE by the formation of
an immunosandwich complex. Additionally, the small dimensions of the nanochannels prevent cells (blue and red) from interfering with the electrode
surface. The subset of cartoons, (A–C, right), depicts the growth of the immunosandwich complexes and demonstrates how they physically impede Fe2+
transfer to the electrode surface. Reprinted with permission from De La Escosura-Muñiz and Merkoçi,155 copyright 2011 John Wiley and Sons. (c) Cartoon
of a sandwich-type voltammetric biosensor for nucleic acid detection. The capture probe is attached to the electrode surface via an alkane linker. Upon
binding of target to the immobilized capture probe, a second signaling probe labeled with ferrocene binds to the target. Molecular wires within the SAM
transduce the electrons to the electrode surface. Reprinted from Umek et al.,158 copyright 2001 with permission from Elsevier. (d) Cartoon of a ‘‘signal-
off’’ E-DNA voltammetric sensor. Binding of complementary DNA (cDNA) to the probe prohibits electron transfer from the ferrocene (Fc) label. Adapted
with permission from Fan et al.,159 copyright 2003 National Academy of Sciences, U.S.A.

Immobilizing the recognition layer on the transducer detection. A similar strategy using electrochemical impedance
surface is a widely used strategy in electrochemical biosensing. spectroscopy through porous channels has been reported for
However, immobilization blocks the electrode surface thus the detection of dengue virus particles.157
limiting the active surface area for an efficient electron transfer. Electrochemical nucleic acid biosensors. The electro-
To overcome this phenomenon, Bhimji et al.154 immobilized chemical detection of DNA was pioneered by Paleček,160 who
the recognition element adjacent to the sensing electrode studied the redox properties of DNA at a mercury electrode.
instead of on the surface of the electrode – HIV-1/2 antibodies Since those initial studies, a variety of label-free and labeled
were immobilized on a planar region near a nanostructured sensors for nucleic acids have been developed, typically relying
microelectrode (NME). Likewise, De La Escosura-Muñiz and on a complementary probe immobilized on an electrode
Merkoçi155,156 reported an immunosensor system with the surface.
capture antibody located in nanochannels above the electrode One popular sensing technique for nucleic acid biosensors
surface as illustrated in Fig. 3(b). Blockage of the nanopores by is EIS. These biosensors typically employ a solution-based redox
the immuno-sandwich prevents the redox reporter from acces- probe such as [Fe(CN)6]3/4 or [Ru(NH3)6]3+/2+ that diffuses
sing the electrode surface, resulting in an ‘‘indirect’’ mode of through a layer of biorecognition element–analyte complexes to

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reach the an electrode surface to carry out the charge transfer interacts with the electrode surface resulting in a decrease in
process. The charge-transfer resistance, Rct, has been widely voltammetric response. As target concentration increases, the
used to discern the signal across the range of frequencies current decreases; this is considered a ‘‘signal-off’’ type sensor.
before and after biorecognition. EIS has been successfully used In contrast, ‘‘signal-on’’ sensors based on a displacement
for DNA hybridization detection with various target lengths161 strategy have also been developed, where binding of the target
and single nucleotide polymorphisms.162–164 For example, at the distal region of the probe strand releases the redox
Cheng et al.165 formed a biorecognition layer composed of reporter which can then be oxidized or reduced voltammetri-
DNA probes on AuNPs on an ITO electrode. Dual detection cally at the electrode surface.176 In these sensors, femtomolar
with EIS and localized SPR was possible with this electrode detection of the target oligonucleotide is achieved by using a
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substrate. hybridized probe.

Electroactive intercalators, groove binders, and electrostatic Aptamer based electrochemical biosensors. As described in
reporters have been used in conjunction with voltammetry for the preceding sections, aptamers are synthetic DNA oligomers
nucleic acid biosensors.166–168 By associating with a double with a high binding affinity for a specific, non-nucleic acid
helix, these reporters become preconcentrated at the electrode analyte. Binding typically causes a change in aptamer confor-
surface and can be detected by voltammetry. Intercalator mation which can be detected by variety of approaches, including
surface densities at the electrode surface have been reported ‘‘signal-on’’177 and ‘‘signal-off’’178 formats. Recently, Kelley and
on the order of 50 pmol cm2 which limits the absolute coworkers179 developed a so-called ‘‘universal’’ strategy relying on
electrochemical signal that can be detected. Signals can be displacement of a semi-complementary neutralizer strand, which
improved by the addition of a redox cycling species, such as neutralizes a negatively charged aptamer probe. Binding of the
potassium ferricyanide, which is negatively charged and does target molecule (protein, DNA, drug etc.) displaces the loosely
not associate with DNA.166 The reporter is electrochemically bound neutralizer resulting in an increase in negative charge at
reduced and then oxidized by reducing the ferricyanide anion. the electrode surface, which concentrates the Ru(NH3)63+ at the
A large body of work using Ru(NH3)63+ as the reporter and electrode surface that enhances the electrocatalytic cycle resulting
Fe(CN)63 as the redox cycler has been described by Kelley and in amplified voltammetric signal.
co-workers.169–174 For example, using this electrocatalytic redox
cycling strategy in conjunction with NMEs and PNA probes, C. Microfluidic biosensing
Lam et al.175 were able to identify bacteria from unpurified There is great enthusiasm for the coupling of biosensing and
lysate in less than 30 min. The use of PNA probes reduces the microfluidics, benefitting from the high selectivity of bio-
background signal and increases the sensitivity of detection as sensors and the small sample volumes, multiplexing, fast
the Ru(NH3)63+ does not localize to the electrode surface in the turnaround times, and automation offered by microfluidics.
presence of PNA alone. Here we describe representative examples of microfluidic bio-
Another strategy is the sandwich assay, which forms the sensors implemented in microchannels, paper, and DMF
basis of a large selection of electrochemical nucleic acid bio- formats (excluding electrochemical based biosensors, which
sensors. After the target binds an immobilized ‘‘capture’’ probe are discussed in Section D) for a variety of analytes.
that is shorter in length than the target, a second electroactively Biosensing in microchannels. There is a large body of
labeled ss-DNA ‘‘signaling’’ probe that is complementary to literature describing the use of microchannels for biosensing
another region of the target binds, thus enabling electro- applications, often in formats that allow for multiplexed analysis.
chemical detection. For example, Umek et al.158 described a For example, Heo and Crooks180 reported a microchannel-based
sandwich DNA sensor that relies on a self-assembled mono- system for parallel detection of glucose and galactose. Hydrogels
layer (SAM) consisting of a mixture of DNA–alkanethiol capture with entrapped enzymes for a catalytic fluorescent assay were
probe, molecular wires, and a polyethylene glycol (PEG) termi- positioned in a microchannel array. Fluorescence detection
nated alkanethiol spacer/insulator (Fig. 3(c)). The PEG-terminated mediated by horse-radish peroxidase (HRP) and Amplex Red
alkanethiol prevents non-specific adsorption and insulates the gave a limit of detection of 0.8 mM for glucose – well below the
electrode surface, minimizing background signal. Upon binding 4.2–6.4 mM range of normal blood glucose.181 Another example
of the target analyte to the immobilized probe, a second probe was reported by Mitsakakis et al.,182 who developed a SAW-based
labeled with ferrocene moieties binds, resulting in an oxidation sensor that was addressed in four different regions by a network
signal from the molecular wires in the SAM. of microfluidic channels (Fig. 4(a)). The channels were used to
Another popular strategy for electrochemical DNA sensing, functionalize the sensor surface with capture antibodies for
developed by Plaxco and co-workers,159 is based on conforma- four cardiac biomarkers. To enable multianalyte detection on a
tion change in the probe after hybridization with the target single sensor, the sample was sequentially added to each
sequence. Sensors using this strategy, which is similar to FRET- channel. The detection limit was 1 nM and the total assay time
based molecular beacons, are referred to as E-DNA sensors. In was less than 30 min. For comparison, clinical assays for the
the absence of the target, a ferrocene label is kept in proximity cardiac biomarker C-reactive protein have a detection limit of
to the electrode surface. Hybridization between the oligo- approximately 0.8–2 nM.183
nucleotide target and probe opens up the hairpin structure While SAW-based biosensors, requiring only one plane of elec-
and decreases the frequency with which the ferrocene label trodes, are fairly straightforward to integrate with microchannels,

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integration of other mass sensitive transducers such as QCM For nucleic acid detection, multiple microchannels are
(which typically require electrodes sandwiching the resonator useful for spatial multiplexing and also for eliminating cross-
crystal) in channels – pose a challenge. In one proof-of-concept contamination between sensors or sensor regions. However, it
example, Kato et al.184 integrated a high-frequency QCM with a is also possible to multiplex within a single microchannel; for
silicon microchannel for the detection of human IgG. Encapsu- example, Peng et al.185 developed a sensitive nucleic acid
lating a quartz resonator within a microchannel reduced its microfluidic biosensor on the basis of fluorescence quenching
fragility; metal electrodes were not needed, reducing the thick- by Ag nanoparticles. DNA probes with terminal fluorescent
ness of the crystal thus increasing the resonant frequency of the labels were covalently attached to an Ag nanoparticle modified
system. The authors demonstrated the detection of 6 mg mL1 of glass surface. A microarray printer was used to spot DNA probes
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human IgG in pure water using this sensor system. onto the surface, which was then enclosed by annealing within

Fig. 4 Microfluidic biosensors. (a) Illustration of a SAW biosensor with parallel microfluidic channels. Electrical contacts are available for signal input and
output. The single sensor is capable of measuring four different samples. Reprinted from Mitsakakis and Gizeli,182 copyright 2011 with permission from
Elsevier. (b) Cartoon of a microfluidic device (top left) and mechanism of LFDA (top right and below). The probe-micro RNA-biotinylated DNA sandwich
starts the formation of the streptavidin–biotin dendrimer complex. Laminar flow enables the continual addition of fluorescent streptavidin (green) and
biotinylated anti-streptavidin antibodies (red). Reprinted from Arata et al.187 under Creative Commons license attribution. (c) Photographs of a paper
microfluidic device used for the determination of glucose and protein. Hydrophobic patterning directs the flow of liquids by capillary action. The devices
have two regions, one for a glucose assay and another for a protein assay. Reprinted with permission from Martinez et al.,63 copyright 2007 Wiley-VCH
Verlag GmbH & Co. KGaA. (d) Video stills of magnetic bead separations on DMF. Engaging a permanent magnet allows the supernatant to be separated
from the magnetic particles. Parallel processing of multiple samples can be implemented on large arrays of DMF electrodes. Adapted with permission
from Choi et al.,99 copyright 2013 American Chemical Society.

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a microchannel. The flow in the channel was useful for allowing were demonstrated, and a chemiluminescent signal was detected
fast response times – without flow, incubation overnight is from the catalyzed activity of alkaline phosphatase conjugated
necessary for low concentration detection but with flow, detec- reporter antibodies. IgE immunoassays using fluorescently
tion times were decreased to as little as six minutes. With this labeled IgE aptamers have also been performed on magnetic
microfluidic biosensor, reagent consumption is significantly nanoparticles.190 Ng et al.96 presented the first DMF particle-
reduced, requiring sample volumes of only 4 mL. Another based immunoassay without the need for an oil carrier fluid. The
example of multiplexing by microfluidics for nucleic acid same group also incorporated motorized magnets99 into a
detection was reported by Lechuga et al.186 In this work, shoebox-sized instrument, and recently demonstrated its efficacy
20 micromechanical cantilever sensors were integrated with for screening a panel of patient samples to diagnose rubella
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microchannels to enable straightforward sample delivery. The immunity.100 Fig. 4(d) demonstrates how this instrument can be
authors also reported that individual sensors can be addressed used to separate multiple aliquots of magnetic particles from
by using individual channels. supernatant.
Beyond multiplexing and handling small sample volumes, The DMF format can also be used to enhance nucleic acid
physical properties inherent to microfluidics can be used to biosensor performance. Malic et al.191 coupled an SPRi based
enhance the capabilities of biosensors. For example, laminar nucleic acid sensor with DMF. In this report, DMF offered two
flow in low-Re conditions can be used to enhance signal ampli- advantages: (1) the ability to address independent spots with
fication for the detection of micro RNA.187 Laminar flow- droplets, making hybridization detection flexible and recon-
assisted dendritic amplification (LFDA) is a technique whereby figurable, and (2) the use of the DMF architecture to apply
two amplification reagents are simultaneously supplied and electric fields to enhance probe deposition. The authors
confined to a detection zone by means of laminar flow, as reported that a negative DC voltage led to a build-up of positive
depicted in Fig. 4(b). The amplification reagents are fluores- charge on the substrate, which in turn, increased the probe
cently labeled streptavidin and biotinylated anti-streptavidin density of negatively charged DNA.
which bind the surface-captured micro RNA target. Their con-
tinual addition propagates a dendritic structure resulting D. Microfluidic electrochemical biosensing
in lower detection limits, which are crucial as micro RNA Microfluidic electrochemical biosensing is a new application
concentrations in tissues are on the order of 1–10 pM.188 area that is emerging from the convergence of the three sub-fields
Paper-based biosensing. Paper-based biosensors are a straight- of electrochemistry, biosensing, and microfluidics. We propose
forward route to selective, low-cost diagnostics. For example, that microfluidic electrochemical biosensors may be particularly
Martinez et al.63 presented a qualitative colourimetric assay for fertile ground for the development of the next generation of
glucose and protein. The device (Fig. 4(c)) split the sample into portable analysis systems, whether they are applied to disease
two regions for parallel testing and colourimetric results could be diagnostics, to cell culture and analysis, or to many other
compared to artificial standards. More complicated assays such applications. Examples listed here are categorized in terms of
as ELISA are a challenge to implement with paper because of the microfluidic format, with a final section describing commer-
need for multiple steps (i.e. sample delivery, washing steps, and cially available systems (of various formats).
antibody delivery). For ideal end-user operation, a single step is Microchannel-based electrochemical biosensing. The micro-
preferable – sample introduction. One such single-step paper- channel format confers numerous analytical advantages (and
based immunoassay was developed by Apilux et al.189 To over- also some disadvantages) for electrochemical biosensing. As an
come the challenge of timing reagent delivery, channels of example of the former, the confinement of electroactive species
varying lengths were patterned on nitrocellulose paper using near the detector in a small volume in a microchannel allows
an inkjet printer and dipropylene glycol methyl ether acetate significant improvements in sensitivity, allowing for ampero-
‘‘ink.’’ This allowed reagents to be delivered to test sites at the metric detection of DNA in an enzyme-linked hybridization
appropriate times and only required a sample introduction assay at the 100 pM level.192 As an example of the latter,
step. Quantitative results for the detection of human chorionic Lamberti et al.193 studied the effect of flow on indirect catalytic
gonadotropin (hCG) were determined by photographing the electrochemical biosensors using a simple GOx based system.
paper immunosensor and analyzing the intensities of various Oxidation of glucose by immobilized GOx in a microchannel
colour channels. The paper immunosensor had a limit of yielded gluconic acid and H2O2, which was detected electro-
detection as low as 0.81 ng mL1, which is 2–12 times lower chemically. Results of this study showed that flow-rate is a
than the detectable levels of hCG determined by commercial critical parameter for biosensor performance and must be
pregnancy strips. optimized to prevent the washing away of electron transfer
DMF-based biosensing. For complex bioassays, DMF is a mediators and electroactive products. For the most part, however,
useful microfluidic format because of its configurability and the microchannel format has become increasingly popular for
flexible control. Heterogeneous immunoassays making use of electrochemical biosensing because of numerous structural
magnetic particles are a popular choice for conducting immuno- advantages, including improvements in throughput, integration,
assays on DMF. Early reports of DMF-based immunoassays used portability, and analysis time.
a permanent magnet in a fixed region and aqueous droplets Microfluidic electrochemical biosensors are particularly useful
surrounded by silicone oil.98 ELISAs for interlukin-6 and insulin for improving throughput, which is important for applications in

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which multiple samples must be analyzed quickly. For example, device architecture was primitive, incorporating only one or a
Wisitsoraat et al.194 and Ruecha et al.195 reported the develop- few microchannels. These designs can complicate the loading
ment of microfluidic chips bearing amperometric biosensors for of multiple reagents through a single input valve; to overcome
cholesterol relying on cholesterol oxidase (ChOx), both with this challenge, Kellner et al.202 reported a device in which
throughput of 60 samples per h. In the former report, ChOx multiple reagents were controlled using a syringe pump and a
was immobilized on a CNT WE within a microfluidic flow 9-port valve. The device, pictured in Fig. 5(b), contains all the
injection system. In the latter report, the ChOx element was necessary reagents on-chip for performing an amperometric
decoupled from the sensing electrode and introduced in the ELISA for either CEA or cancer antigen 15-3.
running buffer of a capillary electrophoresis setup. The reaction There are many other reports of microchannel-based electro-
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between cholesterol and ChOx produces H2O2, which was chemical biosensing. Space precludes listing them all; we
detected by a Au WE. include a few interesting (and unique) examples here. Berdat
Microfluidic electrochemical biosensors are also particularly et al.203 reported the detection of 1 nM DNA using an IDA in a
useful for integration of cell culture with analysis. For example, microfluidic channel. DNA probes were immobilized between
voltammetric and amperometric biosensors for H2O2 generated the interdigitated electrodes and hybridization with comple-
by cellular activities using HRP have been incorporated into mentary targets results in an increase in conductance, which
microfluidic systems.196 Yan et al.197 and Matharu et al.198 used could be calculated from the charge-transfer resistance as
HRP trapped in polyethylene glycol (PEG) hydrogels positioned determined by EIS measurements. In this report, the measure-
over Au electrodes. Both reports used the same device archi- ments were taken in de-ionized H2O and no labels or reporters
tecture, which is illustrated in Fig. 5(a). Yan et al.197 detected were necessary. For robust environmental sensing, Lin et al.204
H2O2 release from activated macrophages cultured in the implemented label-free detection of an arbitrary DNA oligo-
microfluidic chip while Matharu et al.198 probed the effects of nucleotide using an organic electrochemical transistor in a
ethanol and antioxidants by studying reactive oxide species flexible microfluidic device, pictured in Fig. 5(c). Hybridization
produced in hepatocytes cultured in the device. of target DNA was enhanced by pulsing a small positive
Microchannels are also useful for making electrochemical potential at the sensing electrodes which extends the detection
biosensing assays portable. Itoh et al.199 presented a droplet- limit to 10 pM. Swensen et al.205 reported an aptamer based
based microfluidic system for the rapid determination of fish system for the real-time detection of cocaine within a micro-
freshness, monitored as a function of adenosine triphosphate fluidic channel. Sensor surfaces can be regenerated by flowing
(ATP) concentration. ATP can be detected electrochemically by a blank sample205 or exposure to urea.206 The ability to measure
means of the enzymes glycerol kinase (GK) and glycerol-3- analyte concentrations in real-time is particularly useful for cell
phosphate oxidase (G3PO) with glycerol. The phosphorylation culture studies. For example, numerous methods have been
of glycerol to glycerol-3-phosphate by GK requires a stoichio- developed to detect cell secretion of signaling molecules,
metric amount of ATP. Oxidation of glycerol-3-phosphate by including tumor necrosis factor-a,206,207 interferon-g,206 and
G3PO produces H2O2, which can be quantified electrochemically. transforming growth factor-b.208 Kim et al.209 incorporated
The authors demonstrated a good correlation between their electrodes with a centrifugal microfluidic disc for the ampero-
microfluidic results and those generated by conventional HPLC. metric detection of C-reactive protein by a sandwich immuno-
Implementation in microchannels makes electrochemical assay on polystyrene beads. Coupling with the disc-format
biosensing fast. For example, Messina et al.200 developed an showed a 5-fold in improvement in the detection limit over a
immunosensor system in which primary antibodies for similar stationary assay.
interleukin-6 (IL-6) were immobilized on packed glass beads Paper-based electrochemical biosensing. The (modern renais-
contained within a central PMMA microchannel. Working, sance of the) ‘‘paper microfluidic’’ format is still quite new, and
reference, and CEs were positioned downstream of the packed there are only a few examples of integrated paper-based electro-
beads in secondary cross channels. The authors reported that chemical biosensors. Despite this, we propose that this is a
for sample sizes of 100 mL, flow rates above 6 mL min1 resulted particularly promising strategy for portable analysis, given the
in a decrease in amperometric signal. The total assay time was combination of miniaturized detection with straightforward
approximately 25 min. In another example, a microfluidic incorporation of dried reagents. For example, Zhao et al.210
immunoassay for cholera toxin subunit B was developed in developed a paper-based microfluidic cartridge coupled to a
which antibodies were conjugated to liposomes containing low-cost potentiostat for the determination of glucose, lactate,
ferri/ferrocyanide.201 Lysis of the liposomes released ferri/ and uric acid. All reagents including K3Fe(CN)6 and oxidase
ferrocyanide which could be detected downstream by ampero- enzymes were stored in dry form within the paper device. As
metry. Microfluidic channels were used to deliver the sample shown in Fig. 5(d), the device consists of a sample inlet and
and the lysis detergent and paramagnetic particles were used as screen printed electrodes over the test zone, which was directly
a support for the capture antibodies. The turnaround time for integrated with a potentiostat via metal clamps and Ag connec-
the assay was 1 h, significantly faster relative to comparable tions. Multiplexing circuitry was added to the USB controlled
techniques. potentiostat to enable detection at all eight test spots. This
In some of the early reports describing the integration of low-cost device was shown to have detection limits for all three
microfluidics with electrochemical biosensors, the microfluidic analytes within the appropriate clinically relevant ranges and to

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Fig. 5 Microfluidic electrochemical biosensors. (a) Schematic of a microfluidic device for real-time electrochemical monitoring of cells grown in a PEG
hydrogel matrix. Reprinted with permission from Yan et al.,197 copyright 2011 AIP Publishing. (b) Photograph of a computer controlled channel-based system
with reagent storage (1–5), on-chip valves (7 & 8) and electrochemical sensing area (9). Adapted with permission from Kellner et al.,202 copyright 2011 Wiley-
VCH Verlag GmbH & Co. KGaA. (c) Photograph of a flexible microfluidic channel-based device for nucleic acid detection. Reprinted with permission from Lin
et al.,204 copyright 2011 Wiley-VCH Verlag GmbH & Co. KGaA. (d) Photograph of a paper-fluidic device incorporated onto a low-cost potentiostat. The test
strips wick the sample into a detection zone with a three electrode setup. Adapted from Zhao et al.210 under Creative Commons license attribution.
(e) Cartoon of an electrochemical immunoassay implemented in a DMF device. The immune-sandwich is prepared on a magnetic bead and HRP converts the
substrate TMB to TMB+. Droplet movement separates the enzyme product from the magnetic particles and delivers the product to electrodes on the top
plate for detection. Adapted from the methods described by Shamsi et al.97 (f) Photograph of the Abbott Point of Care i-STATs, the leading commercial
microfluidic electrochemical-detection based point of care system. The handheld analyzer is compatible with a variety of microfluidic cartridges (inset).
Reprinted with permission from Abbott Laboratories, Abbott Park, IL. (g) Photograph of the Nano  mix eLab, a cartridge based hand-held microfluidic
electrochemical biosensor currently available for research purposes. Reprinted with permission from Nano  mix. Emeryville, CA.

outperform comparable commercial biosensors. Specifically, DMF-based electrochemical biosensing. DMF is also rela-
the reported limit of detection for glucose was 0.35 mM, tively new, and there are few examples of the application of this
compared to 0.83 mM for commercial glucose meters. technology to electrochemical biosensing. Despite this, we propose

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that DMF is particularly well-suited for coupling complex sample biosensors for nucleic acids, proteins, and antibodies, there
handling regimens with electrochemical biosensors. For example, seems to be an opportunity for new methods capable of quantify-
Shamsi et al.97 reported an amperometric ELISA using para- ing small molecules (suggesting a role for aptamer-based sensors).
magnetic particles and electrodes incorporated with a DMF Along with these opportunities, microfluidic electrochemical bio-
chip (Fig. 5(e)). The electrode layout of the DMF device made it sensors face competition as other techniques decrease in cost and
possible to implement an eight-step protocol including sample size. For example, bench-top and personal mass spectrometers are
and reagent loading steps, multiple supernatant/particle separa- available – eliminating the need for biorecognition elements in
tion steps, and multiple particle-washing steps. Thyroid stimulating order to achieve highly specific detection. Yet, in the face of this
hormone (TSH) was detected by means of a sandwich assay with competition, advancements in microfluidic electrochemical bio-
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a secondary antibody conjugated to HRP. Turnover of 3,30 ,5,50 - sensors may lead to supplementary and even complementary
tetramethylbenzidine (TMB) to TMB+ by HRP was detected by technologies. We propose, based on the trajectory of advances to
amperometry at an Au plated ITO electrode. date, that the overlapping areas of microfluidics, electrochemistry,
Commercial microfluidic electrochemical biosensing. The and biosensors will continue to evolve in unpredictable directions.
microfluidic electrochemical biosensor is an idea whose time It will be exciting to see what new ideas emerge from this
has come, and numerous versions of such devices have been development – we predict that the outcome will be interesting
commercialized for portable, point-of-care analysis. The largest and potentially important for the world that we live in.
market of such devices is likely the personal blood glucose
meter, with an estimated worldwide value near $10 billion.211
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