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 Journal of The Electrochemical Society, 2021 168 116504

Editors’ Choice—Review—From Polarography to

Electrochemical Biosensors: The 100-Year Quest for Selectivity
and Sensitivity
William R. Heineman,1,z Peter T. Kissinger,2 and Kenneth R. Wehmeyer1
1
Department of Chemistry, University of Cincinnati, Cincinnati, Ohio 45221-0172, United States of America
2
Department of Chemistry, Purdue University, West Lafayette, Indiana 47907-2084, United States of America

This is a story of the 100-year path for voltammetric electroanalytical chemistry from the serendipitous discovery of polarography
by Jaroslav Heyrovsky in 1922 to the miniature biosensors of today. In spite of issues with the dropping mercury electrode (DME),
polarography was dominant for almost 50 years due to the good quantitative results it produced. Then, significant developments led
to today’s methods with drastic improvements in detectable concentration and amount, selectivity, ease of use, and breadth of
application. Important steps forward include the shift from the DME to solid electrodes, the strategic modification of electrode
surfaces chemically and with membranes, electrochemistry in thin layers of solution with the associated decrease in sample amount
from milliliters to microliters and below, liquid chromatography with electrochemical detection, more powerful and smaller
instrumentation, microfabrication of electrodes, pulse techniques that improved concentration limits of detection by discriminating
against double layer charging, spectroelectrochemistry for enhanced selectivity by electrochemically changing a spectroscopic
signal, cyclic voltammetry for the general utility that makes it the work-horse of voltammetry, and biosensors that dramatically
expanded the applicability of voltammetry through the use of nature’s biological catalysts (enzymes) and capture agents
(antibodies, aptamers).
© 2021 The Author(s). Published on behalf of The Electrochemical Society by IOP Publishing Limited. This is an open access
article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives 4.0 License (CC BY-
NC-ND, http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reuse, distribution, and reproduction
in any medium, provided the original work is not changed in any way and is properly cited. For permission for commercial reuse,
please email: [email protected]. [DOI: 10.1149/1945-7111/ac33e3]

Manuscript submitted August 31, 2021; revised manuscript received October 13, 2021. Published November 11, 2021. This paper
is part of the JES Focus Issue on Modern Electroanalytical Research in the Society for Electroanalytical Chemistry (SEAC).

electrode consisting of dripping mercury entails, and the odd response

signal for an electrode with a continuously expanding surface area.
This is a story of the serendipitous discovery of polarography almost
Additionally, although mercury has an excellent negative potential
a century ago and the major impact across multiple research areas it is
range, it has a very limited positive range because of the ease of
having today. Since electrochemistry is a surface phenomenon, analytical
mercury oxidation. Consequently, early work was restricted primarily
measurements on ultra-small volumes are relatively easy to make with
to reductions, making polarography essentially a one-way street for
miniaturized devices. This is ideal for today’s development of sensors for
electrons to reduce species at the electrode surface. Never so true that
a wide variety of applications where small size and portability are
research is often determined by what can be done, not by what should
important: point of care, point of use, wearable, in vivo measurements
be done. It would almost seem at the time as if oxidation electro-
such as in the eye and brain, continuous in vivo monitoring of glucose
chemistry did not even exist. To add to the cumbersome nature of the
for diabetes control with data sent to the user’s smart phone, and who
technique, usually dissolved oxygen had to be removed by bubbling
knows what next. This inherent advantage provided a driving force for
the sample with nitrogen to eliminate interference from oxygen
the limitations encountered in the early days of voltammetry to be
reduction. Nonetheless, a significant—yet perhaps underappreciated
steadily overcome by a series of important advances (Fig. 1) that
at the time—feature of this electrode was the formation of a pristine
ultimately led to modern day sensors with significantly improved limit of
new electrode surface every few seconds in the form of a new drop.
detection and selectivity and broadly expanded applicability. In retro-
This minimized the Achilles heel of electrochemistry: electrode
spect, it is clear that electroanalytical chemistry was destined to play a
fouling caused by adsorbing species blocking electron transfer with
major role in the development of chemical sensors.
the analyte. Resistance to surface fouling, excellent reproducibility of
the dropping action and the associated stirring of the adjacent solution
Early Discovery
providing high-precision measurements, excellent negative potential
Jaroslav Heyrovsky is credited with discovering modern day range, and ability to rapidly analyze a multicomponent sample at low
voltammetric electrochemistry with his invention of polarography in concentrations (for the time) enabled polarography’s half-century
19221 for which he received the Nobel Prize in 1959.2 In polaro- domination of voltammetric electroanalytical chemistry. In addition to
graphy, the potential applied to a dropping mercury electrode (DME) its development as one of the few techniques for instrumental analysis
is scanned and the resulting current displayed as a polarogram (current at the time, polarography was used extensively to study mechanisms
vs potential plot—see Fig. 1). Qualitative identification of an analyte of electrode reactions and adsorption on mercury of an increasingly
is possible from the half-wave potential on the rising part and long list of organic, inorganic and biological compounds.3
quantitation is achieved from the limiting current after the current Many a chemist encountered the “polarography experiment” as
plateaus.3 Polarography was the first instrumental technique capable students in the traditional instrumental analysis course and marveled
of simultaneous multianalyte analysis with a good Limit of Detection at its excellent performance while attempting to avoid the disaster of
(LOD) for the time—high micromolar. It revolutionized the determi- a major mercury spill. The authors were all one of those students—
nation of metal ions such as Pb2+, Cd2+, Zn2+, Cu2+ and was soon amazed at the results and relieved that we were not the ones to spill
applied to electroactive organic compounds. As such, polarography 5–10 lbs. of mercury on the floor—a joy to clean up!
became widely used in spite of significant disadvantages. The sensing
(working) electrode was the quirky DME, which today seems almost The Age of Electroanalytical Chemistry
ludicrous given mercury’s toxicity, the relatively high maintenance an
The 60 s through the 80 s could well be viewed as a golden age in
electroanalytical chemistry. Multiple significant advances in meth-
z
E-mail: [email protected] odology left the traditional polarography experiment in the dust and
 Journal of The Electrochemical Society, 2021 168 116504

Figure 1. The development of electroanalytical chemistry in the past 100 years since the invention of polarography.

paved the road that led to where the field is now. These advances led exchanged between the electrode and the electroactive species in
to dramatic improvements in LOD (both concentration and amount), solution, it can be influenced by the “condition” of the electrode
selectivity, applicability, versatility and user friendliness. The high- surface. One advantage of the DME is a new, pristine surface is
lights of these advances (as viewed by the authors!) follow and are formed every few seconds, minimizing the deleterious effects of
illustrated with an approximate timeline in Fig. 1. extraneous material from the sample adsorbing on the electrode
surface and interfering with the desired electron transfer process. In
Solid electrodes.—Early electrochemical research using solid elec- other words, extraneous material on the electrode surface was at the
trodes, such as the noble metals Pt and Au, to extend the potential into time viewed primarily as a problem to be avoided. However,
the positive range for oxidations was often met with the frustration of intentional “modification,” if done correctly, could expand the
irreproducible results caused by several issues. These included electrode capability of electroanalytical chemistry applications. Royce
fouling from adsorption on the electrode, the sensitivity of electron Murray was one of the first to demonstrate this with the DME by
transfer and electrode mechanisms to the nature of the surface such as adsorbing an optically active film on the electrode surface to
the effect of oxide formation on electrodes in the positive potential selectively change the electrochemistry of two enantiomers.13 The
region, and difficulties in reproducibly preparing and cleaning elec- advent of solid electrodes greatly expanded the possibilities for
trodes. Nonetheless, solid electrodes such at Pt, Au and carbons such as controlling the electrode process by opening the large arsenal of
glassy carbon began to be used to extend voltammetry into the positive chemistries for the covalent attachment of a variety of materials to
potential range to access materials that oxidized. Ralph Adams’ book surface functional groups on an electrode. An explosive growth
Electrochemistry at Solid Electrodes, published in 1969,4 made a major followed where electrochemists could now alter electrode properties
impact on the field. Solid electrodes could be miniaturized5 which by molecularly designing surfaces with all sorts of materials such as
paved the way for novel applications such as voltammetry in brain polymers, microporous solids, and biological compounds.14
tissue.6–8 Such “ultra-micro” electrodes enabled the development of Practical outcomes were better selectivity by preventing electron
scanning electrochemical microscopy for obtaining high resolution transfer with interferences and improved LOD by preconcentrating
electrochemical images of a surface to probe its topography and surface analytes at the electrode surface. An early example is an electrode
reactivity.9 Dissolved oxygen was not a problem in the positive chemically modified with a film of negatively charged Nafion
potential range, which eliminated the cumbersome deoxygenation polymer for in vivo detection of positively charged neurotransmitters
process for those studying oxidation. Very importantly, the at times in the presence of an excess concentration of negatively charged
troublesome oxygen reduction wave was turned into a huge success ascorbate in the brain.8,15 Here the interfering ascorbate is kept from
when Leland Clark invented the Clark Oxygen Electrode for the rapid the electrode surface by electrostatic repulsion whereas the analyte
and easy determination of oxygen.10 This simple device revolutionized neurotransmitter is preconcentrated at the surface by electrostatic
the measurement of oxygen—one of most important analytes, which at attraction into the polymer. Chemical attachment of biological
the time was very difficult to determine. The Clark oxygen electrode molecules such as enzymes, antibodies and aptamers to the electrode
had a major impact on the medical community for monitoring dissolved surface has played an enormous role in the development of
oxygen in blood, which was critical in developing the now common- electrochemical biosensors (vide infra). Membranes were also
place heart-lung machine.11,12 shown to improve selectivity. An important example is the pre-
At this point it became clear that solid electrodes opened up viously mentioned Clark oxygen electrode where an oxygen perme-
exciting new vistas for research and many of us put our DMEs on the able membrane over a Pt electrode made oxygen easy to detect in
shelf and never looked back. This shift in thinking paved the way for blood by excluding proteins that would ordinarily interfere by
what might be considered the most important practical application of adsorbing on the surface and blocking electron transfer.10,12
voltammetry—the glucose biosensor.
Thin layer electrochemistry.—Occasionally a particularly im-
Chemically/membrane modified electrodes.—Since electro- portant discovery is made by accident, and such was the case for thin
chemistry is a heterogeneous process in which electrons are layer electrochemistry. Fundamental studies of adsorption of
 Journal of The Electrochemical Society, 2021 168 116504

molecules on electrode surfaces was a hot topic in the 1960’s that wavelength. Hydrodynamic electrochemistry likewise enabled
Fred Anson was interested in. One procedure for verifying adsorp- methods at a fixed potential with very low volume flow cells
tion of an electroactive molecule on a solid electrode, such as (volumes in the microliter range). The electrode interfacial property
platinum, was to cause adsorption by soaking the electrode in a of being a “physical capacitor” (C) suggested great virtue in not
solution of the molecule of interest, remove the electrode, rinse it scanning the electrode-solution interfacial potential (V or E) at all.
gently, immerse it in a solution of supporting electrolyte, and then We know from basic physics that the current passing a capacitor
run a voltammogram to detect the layer of adsorbed molecules. In depends on the variation of the voltage across it (i = C dV/dt). To a
one such experiment, a signal was obtained and initially attributed to first degree, this parasitic current is zero at a fixed electrode
adsorption.16 But subsequent experiments proved this was actually potential. Operating this way enabled concentration detection limits
due to a small amount of the initial solution that had seeped into a for redox active analytes far below anything achievable with optical
tiny gap between the solid electrode and the glass capillary in which detectors of the day. The analytes were brought to the electrode
it was sealed where it was not rinsed off. Interestingly, the shape of hydrodynamically and the background currents were low and steady-
the voltammogram for an adsorbed molecule (no diffusion to the state. Over time, applications were extended to environmental and
electrode) is very similar to that of a molecule dissolved in a thin food assays and to reducible as well as oxidizable analytes. In some
layer of solution adjacent to the electrode (restricted diffusion to the cases, the chromatography column could be left out with a scheme
electrode). Anson reported this result17 and then added thin layer labeled flow injection analysis (FIA). The flow approach enabled the
electrochemistry to his repertoire of research projects. Others took use of multiple electrodes in series and parallel19
notice including Charles Reilley who initiated an ambitious research A feature of flow methods is the ease of automation to a level
program in this area that was on-going when two of the authors unknown in conventional electrochemical cells. Automated pumps,
(WRH and PTK) were PhD students there. This influenced the samplers and data processing enabled LCEC to use tools originally
inventions of thin layer spectroelectrochemistry by Murray, developed for UV absorption detectors. Liquid chromatography with
Heineman and O’Dom and LCEC by Kissinger and Adams (vide mass spectrometry detection (LCMS) was still two decades in the
infra). Of special importance, thin layers provided the way to do future. Meanwhile, LCEC revolutionized neurotransmitter measure-
electrochemistry on very small, but with well-defined amounts of ments in many laboratories and those data were used to develop best
sample, which has proven important in the development of the selling drugs for depression, schizophrenia and neuropathic pain.
personal glucose sensor, the largest market in electroanalytical While still in use, many applications of LCEC were repurposed with
chemistry. These events are presented in more detail below. LCMS/MS.
Just as cyclic voltammetry and pulse techniques allow for study
Liquid chromatography with electrochemical detection (LCEC).— of the product(s) of a redox reaction on the fly, hydrodynamic
One process question about electrochemistry relates to the electrochemistry does the same. Products can be studied at steady-
immersing of the sensor electrode in a sample vs bringing the state downstream from where they are created. This idea was first
sample (or selected analyte components) to the sensor. Nearly all made practical with spinning disk electrodes with an outlying ring to
analytical chemistry methods have similar alternatives. In the capture products moved radially. Later, flow cells in various
broadest conception, we use the term “sample preparation” which configurations and numbers of electrodes (2 or more of the same
may entail isolation of specific analyte(s), preconcentration, or different materials) have been used. For mass spectrometry fans,
elimination of interferences, a change in pH, and derivatization this EC/EC approach is analogous to the more widely applicable
to enhance selectivity and/or response. Terms of that art often are MS/MS. In both cases, a product is made and then explored on the
described by extraction, as in liquid-liquid extraction or liquid- fly with the same instrument. This is useful to understand reaction
solid extraction. mechanics but also to improve analytical selectivity in complex
In the early 70 s, one of us (PTK) was assigned (by the samples.
aforementioned Prof. Adams) to consider the alternative of mon-
itoring oxidizable neurotransmitters in the brain by sensing directly Instrumentation.—Developments in electronics and instrumen-
in vivo or by first isolating those substances from the tissue.18 The tation played a key role in improvements in electrochemistry
issue remains the same in 2022 as it was 50 years ago in 1972. First, methodology. Our generation was fortunate to see electronics
let’s relieve one concern by noting there are no pain sensors in brain transition from vacuum tubes to integrated circuits and on to digital
tissue. For in vivo sensing we have advantages of temporal and from analog. We and our students took full advantage. Milliamps
spatial resolution while requiring adequate chemical selectivity. became microamps and microamps became nanoamps and pi-
For in vitro sensing we lose much of the temporal and spatial coamps. Minutes became seconds and milliseconds on down.
resolution while gaining selectivity and reduced concentration limits Given that electrochemistry interfaces directly with electronics,
of quantitation. As described above, electrodes can be modified with no intermediate magnets or optics, it was relatively easy to
chemically or physically (membranes, size) to enhance performance. take technologies designed for military and industrial applications
The ultimate in vitro sample preparation technique that evolved and put them to work for electrochemical measurements. Along
rapidly through the decade of the 1970s was mechanized liquid the way, digital progressed from mainframes, to minicomputers
chromatography (LC) which today is as common as a pH meter was (1960s), to personal computers (1970s), to tablets and to smart
then. In the reversed-phase variant, the compatibility with electro- phones (post 2000).
chemistry was nearly perfect. There was excellent hydrodynamic The commercialization of modular solid-state operational ampli-
control. Mobile phases compatible with organic electrochemical fiers enabled electrochemists to easily assemble circuits for new
measurements were a very good fit. Electrochemical sensors were techniques such as cyclic voltammetry from individual electronic
made better by the separations that “cleaned up” samples. Thin-layer components. Electrochemists of our generation knew some electro-
electrochemistry, heavily developed in the 1960s (a topic for the nics, reconfigured operational amplifier circuits to switch from one
PhD theses of two of the authors—WRH and PTK), became technique to another, obtained our voltammograms on x-y recorders
electrochemical (specifically amperometric) detectors when operated and measured current peak heights with a ruler. This all changed
hydrodynamically as flow cells. This thin layer electrode application when the first electroanalytical instrument integrated with a com-
was not contemplated at all through the 1960s and neither was its puter to provide an array of techniques was developed by Larry
wide application as home glucose sensors now available in every Faulkner’s group.20 This so-called cybernetic potentiostat provided
pharmacy. 16 electrochemistry techniques plus automatic measurement and
Polarography (then “voltammetry”) provided the virtue of an compensation of resistance. With only 36 K ROM for Control
electrochemical analog with spectroscopy (response v. energy). Software and 22 K RAM for Data collection, it completely set a
Colorimetric and fluorometric methods often use detection at a fixed new direction for instrumentation. After discussion among Larry
 Journal of The Electrochemical Society, 2021 168 116504

Faulkner, Peter Kissinger, and William Heineman at a breakfast square wave voltammetry because of its relatively fast scan rate. In
associated with the Pittsburgh Conference in 1983, Bioanalytical general, the LOD can be improved by 10–1000x by using a pulse
Systems (BAS) embarked on a path to commercialize the first fully technique for scanning the potential compared to a linear potential
integrated computer controlled electroanalytical workstation, the scan. Combining pulse techniques that had low concentration LODs
BAS100. As Faulkner puts it—“In my view, an important part of with the very small volume thin layer cell enabled the amount of
this was giving users simplified access to practically the whole array analyte needed for detection to be significantly reduced as demon-
of techniques in use at the time, thereby encouraging investigators to strated for metal ions.34
use the best techniques for their purposes, not just those (usually one
or two) that they had.”21 Spectroelectrochemistry.—Ted Kuwana’s 1964 report on an
In summary, the electronic instrumentation enabled measure- optically transparent electrode (OTE) at which electrochemistry
ments a millionfold faster at a millionfold lower concentration with and absorption spectroscopy were performed simultaneously stimu-
electrodes a millionfold smaller than anything Galvani, Volta, Davy, lated interest in what was termed spectroelectrochemistry.35 The
or Faraday could possibly have tried in their day. It was a fortunate OTE consisted of a thin film of conductive tin oxide coated on glass
gift to our generation, gratefully received. through which visible light was shined to record spectral data of the
solution soluble products formed during electrode reactions. The
Miniaturization and microfabrication.—Miniaturization of the technique was first explored for fundamental studies of mechanisms
working electrode into the low micrometer dimensional domain of electrode reactions where rate constants of homogenous chemical
(sometimes termed ultramicroelectrode) using materials such as an reactions coupled to the heterogeneous electrode reaction could be
8 μm carbon fiber changed the shape of cyclic voltammograms, measured.36 Digital simulation of concentration-distance profiles
enabled measurements to be made on unusually high resistive in the diffusion layer developed by Stephen Feldberg provided
solutions by virtue of the very small current reducing iR drop working curves that allowed rate constants to be determined
issues, and made electrodes less intrusive for in vivo studies.22–24 quantitatively.37,38 Mediators were shown to effectively couple
The development of microfabrication techniques that drove the redox active proteins to the electrode and that opened the door to
miniaturization of electronic devices in the 1960 s also provided new spectroelectrochemical studies of biologically relevant materials
strategies for the design of electrodes and electrochemical cells.25 with their redox centers surrounded by protein such as cytochrome
Instead of having free-standing electrodes immersed in a solution of c and cytochrome c oxidase.36 Heineman was influenced by the thin
analyte and supporting electrolyte, electrochemical cells in which all layer electrochemistry occurring in the Reilley lab (vide supra) when
three electrodes were screen printed as strips of conducting metal his advisor Royce Murray suggested that he and postdoc George
such as platinum on a solid substrate opened the door to mass O’Dom turn a piece of gold micromesh into an OTE.39 The result
production of miniaturized cells.26 These cells could be transformed was the optically transparent thin layer electrode (OTTLE), which
into sensors for specific analytes by coating with appropriate became widely used for studying redox active inorganic, organic,
materials. For example, a thin film of an ionically conductive and biological (often using mediators to facilitate electron transfer)
polymer replaced the usual solution with supporting electrolyte to compounds by providing spectra of different oxidation states,
form a sensor for oxygen detection in air.27 Coating with glucose reduction potential (E°′) and electron stoichiometry (n), all in one
oxidase and a mediator to enhance electron transfer with the working relatively easy experiment.40 Since then, spectroelectrochemistry has
electrode made a glucose sensor, which ultimately led to a revolution expanded to include a variety of spectroscopic techniques such as
in glucose self-monitoring, vide infra. Arrays of interdigitated UV absorption, infrared, fluorescence and electrogenerated chemi-
electrodes could be formed with dual working electrodes set at luminescence (ECL), Raman scattering, EXAFS; OTEs made of a
different potentials to enable redox cycling—a more efficient use of range of materials such as vapor-deposited films of gold and
the electroactive species that improves LOD.28 Arrays of sensors for platinum and carbon nanotubes; and more conventional, nontran-
multiple analytes could now be fabricated.29 A potentiostat circuit sparent electrodes using reflectance and parallel beam optical
can even be part of a biosensor chip with data transferred to a cell techniques.
phone.30 The success of electrochemical devices relying on techni- The excellent LOD and selectivity of electrochemiluminescence
ques for miniaturizing electronic circuits caused some electrical (ECL) immunoassay, pioneered by Bard and Whitesides,41 propelled
engineers to expand their research into the development of electro- it to a widely used analytical technique. In ECL typically a tris-
chemical sensors and stimulated collaborations between chemists ruthenium(bipyridine) complex (Ru(byp)32+) is oxidized at the
and engineers. electrode to generate a product that undergoes a high-energy electron
transfer reaction with an amine compound resulting in emission of a
Pulse techniques.—An important consideration in achieving the luminescent photon from the excited state of the ruthenium complex.
lowest possible LOD is minimizing the nemesis of voltammetric The result is an exceptionally good LOD (due in part to the absence
techniques—charging current. As described above, in order to move of scattered light from an excitation beam such as in conventional
the potential of the working electrode, non-faradaic charging current fluorescence) and selectivity, especially when used in an affinity
must be injected, much like changing the voltage of a capacitor. The assay format with highly selective binding agents such as antibodies.
magnitude of the charging current ultimately limits the faradaic This format also greatly expands the applicability because of the
current signal of interest that can be measured accurately. One availability of antibodies for a wide range of important analytes.42
strategy is to simply operate at a constant potential as described Roche Diagnostics Elecsys and Meso Scale Discovery have both
above in the LCEC section. Another strategy is to pulse the potential successfully commercialized ECL immunoassay for single and
and allow the charging current associated with the pulse to decay multianalyte analysis for a wide variety of biomolecules.43
before sampling the current. This strategy takes advantage of the More recently, the concept of using spectroelectrochemistry to
more rapid decay of the nonfaradaic capacitance current than the improve selectivity of absorption and fluorescence spectroscopy by
faradaic current which decays at t−1/2. Thus, to obtain a voltammo- electrochemically reducing/oxidizing the target analyte to change its
gram the voltage scan is not just simply a linear ramp, but a series of spectral signal has been demonstrated.44,45 The change in absor-
pulses or pulses and ramp combined with current sampled at precise bance or fluorescence is used for quantification and selectively
points and plotted versus potential to provide the voltammogram.31 distinguishes the target analyte from potential interferences with
Robert and Janet Osteryoung were major proponents of such spectra that overlap but do not change. This technique takes
techniques including pulse voltammetry, differential pulse voltam- advantage of the ease in distinguishing a changing signal from a
metry, and square wave voltammetry.32,33 Although originally constant signal. LOD can be improved by coating the OTE with a
developed to be used in polarography with the DME, today pulse chemically selective film that preconcentrates the analyte at the
techniques are commonly used with solid electrodes, especially electrode surface to enhance the spectral change such as in
 Journal of The Electrochemical Society, 2021 168 116504

attenuated total reflection spectroscopy.46 Detection of ferrocyanide biological material—glucose oxidase—as the key feature. Also, it
in a very complex sample—stored nuclear waste—demonstrated the did not detect the analyte glucose by its direct electrode redox
extraordinary selectivity that can be achieved for an analyte with reaction as did most electroanalytical methods at that time, but rather
appropriate spectral and electrochemical properties and the rugged- was based on electrolysis of a reactant or product of the enzyme
ness of the materials.47 Raman scattering spectroelectrochemistry is catalyzed reaction. Selectivity was based on the ability of glucose
also proving very useful for analysis and can provide even more oxidase to distinguish glucose from other possible substrates in the
selectivity than absorption and fluorescence.48 sample and a membrane positioned in front of the electrode to
prevent the electrolysis of other electroactive components in the
Cyclic voltammetry.—Perhaps the most generally useful tech- sample at the detection potential.56–58 In this “first generation”
nique developed in the 60s is cyclic voltammetry, CV, which is biosensor for detecting glucose in blood the membrane prevented the
widely used to obtain general electrochemical properties of a oxidation of blood components uric acid, ascorbic acid, and lactic
material.49 CV has seen less use for quantitative analysis because acid and, later, acetaminophen when Tylenol was introduced.
the charging current problem referred to earlier restricts the LOD to Yellow Springs Instruments commercialized a glucose analyzer
levels much higher than achievable by other techniques discussed based on this sensor for medical applications.
here. However, it has been very successfully applied by Wightman The glucose sensor has come a long way since its invention.59–65
and others to the study of neurochemical dynamics in the form of Significant advances in the development of second generation
fast scan cyclic voltammetry (FSCV) at microelectrodes.50–52 FSCV sensors included screen printed disposable test strips for self-testing
takes advantage of signal averaging of many scans and background and replacing oxygen with mediators such as ferricene and ferri-
subtraction of the charging current to improve the LOD. Potential cyanide to eliminate oxygen concentration dependence and improve
waveforms can be adjusted to give better resolution of peaks for selectivity.66–68 Heller introduced the innovative concept of “wiring”
codetection of two analytes such as guanosine and adenosine in glucose directly to the electrode and also promoted the thin-layer
real-time.53 concept that enables measurements to be made reproducibly on a
very small sample volume.69–71 Third generation sensors use direct
Stripping voltammetry.—Stripping voltammetry achieves a very electron transfer between the electrode and glucose oxidase to
low LOD by preconcentrating the analyte at the electrode before achieve faster response and better sensitivity. This is accomplished
performing the potential scan for analysis.54 It is most widely known with engineered enzymes or fixing the enzyme to a porous polymer
for the detection of metal ions such as Pb2+. Here the Pb2+ is first electrode.64 The small volume advantage of electrochemical detec-
deposited at the electrode by its reduction to lead metal Pb at a tion enabled micro fabricated glucose sensors to displace the
constant potential while the sample solution is stirred to enhance spectroscopic based sensors for glucose self-testing for diabetes.
mass transport to the surface. Then the stirring is stopped and the The volume of blood required was steadily reduced from a drop
potential is scanned to oxidize the Pb back to Pb2+ where the peak (25 μL) to submicroliter volumes by taking advantage of the thin-
current is proportional to the original concentration of Pb2+ in the layer concept described earlier. The “holy grail” of continuous
sample. Stripping voltammetry was first demonstrated using a glucose monitoring with an in vivo biosensor was effectively
mercury drop or a mercury film electrode where the Pb dissolved reached in 2018 with the FDA news release: ‘FDA approves first
in the liquid mercury. Solid electrodes such as carbon can be used. continuous glucose monitoring system with a fully implantable
Using pulse techniques described above for the stripping step glucose sensor and compatible mobile app for adults with
improves the LOD. Stripping voltammetry is commonly used as a diabetes’.72 Now such sensor systems where results are sent from
rapid test for lead poisoning by detecting the level in blood. Here the the sensor to a cell phone for easy monitoring by the user are
blood sample is first mixed with a reagent to free lead bound to commercially available. This billion-dollar market is perhaps the
protein. single largest use of electrochemical sensors in the world.
Development of the glucose sensor is an especially interesting
Biosensors.—Nature has evolved a myriad of selective biochem- example of how various strategies for achieving selectivity were
ical interactions upon which life is based. The introduction of explored and how sensitivity in terms of the amount of required
biological components into electrochemistry was a giant step glucose was steadily improved to benefit the patient by minimizing
forward for expanding the range of accessible analytes, as well as the volume of blood required.
improving selectivity and LOD. Coincident and also important was The large number of oxidase enzymes enabled this concept to be
the concept of indirect analysis where the target analyte itself is not expanded for the detection of numerous other substrates.73 For
electrolyzed by the electrochemical technique, but rather another example, a biosensor based on lactate oxidase is used for determina-
compound whose concentration is in some way proportional to the tion of lactate in blood, which is useful for establishing intensive
analyte. This meant the analyte itself did not need to be electro- training regimens such as in swimming, marathon running and horse
active, which removed one of the major hurdles to the broader racing
application of electroanalysis.
Electrochemical affinity assays/biosensors.—The concept of
Enzyme biosensors.—Leland Clark is referred to as the father of combining selective “capture” of an analyte by a biological binding
biosensors—sensors that involve a biological material in the agent with electrochemical detection was another major step
detection step—for his invention with Lyons of the glucose forward. The idea stems from radioimmunoassay (RIA), which
biosensor.11,55 They showed that glucose can be determined using was invented and developed into a powerful technique by Solomon
its reaction with oxygen as catalyzed by the enzyme glucose Berson and Rosalyn Yalow, with Yalow receiving the 1977 Nobel
oxidase (G.O.) Prize. RIA combined the excellent selectivity of antibody binding,
which is used by the body’s immune system for defense against
G.O.
glucose + O 2 + 2H + → gluconate + H2 O 2 “non-self,” with the very sensitive detection of a radioisotope. RIA
was recognized as a powerful analytical technique and rapidly
by electrochemically monitoring either the reduction of oxygen or became widespread. However, after an extremely successful run,
the oxidation of hydrogen peroxide. The decrease in the oxygen problems with the safety and disposal issues of radioisotopes
reduction current is inversely proportional to the concentration of eventually stimulated the development of other labels such as
glucose whereas the increase in the hydrogen peroxide oxidation enzymes, radicals, chromophores, and electroactive species. The
current shows direct proportionality to glucose. The sensor was first electrochemical studies of the interaction between an antibody
novel for two reasons. It consisted of an electrode coated with a and its target compound were reported in the 1950’s.74 About 35
 Journal of The Electrochemical Society, 2021 168 116504

years later, the first immunoassays and immunosensors with it. Later Clark realized that this reaction could be the basis for a
electrochemical detection were reported independently by several glucose sensor. And so it goes in science!
groups.42,75–81 Most assays and sensors involve electrochemical
detection of the analyte with an electroactive label or an enzyme ORCID
label that catalyzes production of an electroactive compound
William R. Heineman https://orcid.org/0000-0003-2428-5445
involved with the assay. Other capture-based sensors using DNA
fragments, or aptamers were introduced later. The success of these References
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