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Electrochemical biosensors
Niina J. Ronkainen,*a H. Brian Halsallb and William R. Heinemanb
Received 3rd November 2008
First published as an Advance Article on the web 1st February 2010
DOI: 10.1039/b714449k
Electrochemical biosensors combine the sensitivity of electroanalytical methods with the inherent
bioselectivity of the biological component. The biological component in the sensor recognizes its
analyte resulting in a catalytic or binding event that ultimately produces an electrical signal
monitored by a transducer that is proportional to analyte concentration. Some of these sensor
devices have reached the commercial stage and are routinely used in clinical, environmental,
industrial, and agricultural applications. The two classes of electrochemical biosensors,
biocatalytic devices and affinity sensors, will be discussed in this critical review to provide an
accessible introduction to electrochemical biosensors for any scientist (110 references).
1. Introduction
1.1 Background
Sensors are devices that register a physical, chemical, or bio-
logical change and convert that into a measurable signal.1 The
sensor contains a recognition element that enables the selective
response to a particular analyte or a group of analytes, thus
Fig. 1 A schematic of a biosensor with electrochemical transducer.
minimizing interferences from other sample components
(Fig. 1). Another main component of a sensor is the transducer
or the detector device that produces a signal. A signal processor recognition element (enzymes, proteins, antibodies, nucleic
collects, amplifies, and displays the signal. acids, cells, tissues or receptors) that selectively reacts with
Electrochemical biosensors, a subclass of chemical sensors, the target analyte and produces an electrical signal that is
combine the sensitivity, as indicated by low detection limits, of related to the concentration of the analyte being studied.
electrochemical transducers with the high specificity of bio- Electrochemical biosensors can be divided into two main
logical recognition processes. These devices contain a biological categories based on the nature of the biological recognition
process i.e. biocatalytic devices and affinity sensors.2 Bio-
a
Department of Chemistry, Benedictine University, 5700 College Road, catalytic devices incorporate enzymes, whole cells or tissue
Lisle, IL 60532-0900, USA. E-mail: NRonkainen@ben.edu; slices that recognize the target analyte and produce electro-
Fax: +1 630 829 6547; Tel: +1 630 829 6549 active species. Special emphasis will be placed on enzyme
b
Department of Chemistry, University of Cincinnati, electrodes for the detection of glucose, lactose, and xanthine.
P.O. Box 210172, Cincinnati, OH 45221-0172, USA.
E-mail: brian.halsall@uc.edu, william.heineman@uc.edu; Affinity sensors rely on a selective binding interaction between
Fax: +1 513 556 9239; Tel: +1 513 556 9274, +1 513 556 9210 the analyte and a biological component such as an antibody,
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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1747–1763 | 1747
nucleic acid, or a receptor. Immunosensors and DNA hybridi- other detectable outcome.2 Enzymes, globular proteins com-
zation biosensors with electrochemical detection will be posed mainly of the 20 naturally occurring amino acids that
discussed as examples of affinity sensors. catalyze biochemical reactions, are the oldest and still most
Biosensors constitute an interdisciplinary field that is commonly used biorecognition element in biosensors.2,4
currently one of the most active areas of research in analytical Enzymes can increase the rate of a reaction significantly
chemistry. Using biosensors typically eliminates the need for relative to an uncatalyzed reaction. The enzyme–substrate
sample preparation. The biosensor’s performance is usually interactions can be characterized by kinetic studies. Para-
experimentally evaluated based on its sensitivity, limit of meters such as origin and availability of the biological com-
detection (LOD), linear and dynamic ranges, reproducibility ponent, its operational and storage stability as well as
or precision of the response, selectivity and its response to immobilization procedure should be considered when preparing
interferences.1 Other parameters that are often compared a biocatalytic sensor.5 Also, sensitivity of the biorecognition
include the sensor’s response time (i.e. the time after adding element to experimental conditions such as pH, temperature,
the analyte for the sensor response to reach 95% of its final and stirring should be minimal and variation between measure-
value), operational and storage stability, ease of use and ments should be as low as possible.3 Because of their complex
portability. Ideally, the sensing surface should be regenerable molecular structures, enzymes often have exquisite specificity
in order for several consecutive measurements to be made. For for their substrate molecule and can detect individual sub-
many clinical, food, environmental, and national defense stances in a complex mixture, such as urine or blood, very
applications, the sensor should be capable of continuously selectively. This removes the need for time-consuming, labor-
monitoring the analyte on-line. However, disposable, single-use intensive, and interference-prone sample pretreatment and
biosensors are satisfactory for some important applications separation steps used in composite methods. The arrangement
such as personal blood glucose monitoring by diabetics. of amino acids at the active site of the enzyme, often found at
the centroid of the protein, bind with the specific sub-
strate making the enzyme selective for one type of substrate
1.1.1 Biocatalytic sensors. Although many types of bio-
molecule.4 Many enzymes also incorporate small nonprotein
recognition elements have been used in biosensing devices,
chemical groups, such as cofactors or prosthetic groups, into
electrochemical biosensors primarily use enzymes due to their
the structures of their active site that help determine substrate
high biocatalytic activity and specificity.3 Biocatalytic sensors
specificity.4 The inherent selectivity of enzymes often circumvents
using enzymes as the recognition element often have relatively
the signals produced by interfering species that are sometimes
simple designs and do not require expensive instrumentation.
found in complex samples. However, enzyme activity is often
Such sensors are typically easy to use, compact, and inexpensive
further modulated by other components such as activators and
devices. Different detection configurations can be used such as
inhibitors.4 Researchers also had to find ways to manage the
stationary sample solution vs. flow conditions or bulk sample
enzyme adsorption that could lead to electrode fouling as well
solution vs. a microdrop detected using a microelectrode.
as denaturation and loss of enzyme’s catalytic activity on the
Biocatalytic sensors can also be easily adapted to automatic
electrode surface.5 Biocatalytic biosensors will be described in
clinical lab and/or industrial analysis. Personal blood glucose
more detail in Section 2.
monitoring devices are the most successful commercial applica-
Many biochemical analytes of interest are not amenable to
tion of biocatalytic sensors.
detection by enzyme electrodes due to the lack of sufficiently
Biocatalytic sensors incorporate biological components
selective enzymes being available for the analyte or the analyte
such as enzymes, whole cells or tissue slices that recognize
not being commonly found in living systems.1,5 That is when
the target analyte and produce electroactive species or some
affinity biosensors are considered as an alternative method.
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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1747–1763 | 1749
industrial processes more easily than steady-state batch
systems, since the flow conditions allow the solution to be
changed more easily in multistep assay procedures, and are
ideal for on-line monitoring.
Electrochemical sensors are part of an electrochemical cell
that consists of either three electrodes or two electrodes.
A typical three electrode electrochemical cell consists of a
working (or indicator) electrode of a chemically stable solid,
conductive material, such as platinum, gold, or carbon
(e.g. graphite); a reference electrode, usually consisting of
silver metal coated with a layer of silver chloride (Ag/AgCl);
and a platinum wire auxiliary electrode. The reference
electrode is usually further removed from the site of the redox Fig. 2 Diagram of a screen printed electrode (SPE). Ref., reference
reaction in order to maintain a known and stable reference electrode; Aux., auxiliary electrode; and Work., working electrode.
potential.3 One advantage of this system is that the charge
from electrolysis passes through the auxiliary electrode instead Interdigitated array (IDA) electrodes are good amperometric
of the reference electrode, which protects the reference electrochemical transducers in biosensors (Fig. 3). IDAs are
electrode from changing its half-cell potential. A two electrode made of two pairs of working electrodes consisting of parallel
system has only the working and reference electrodes. If the strips of metal fingers that are interdigitated and separated by
current density is low enough (omA cm 2) then the reference insulating material.6,28 One electrode array serves as an anode
electrode can carry the charge with no adverse effect.5 Both for oxidation and the other as a cathode for reduction as shown
three electrode systems and two electrode systems are used for in Fig. 3 for one anode finger and the adjacent cathode fingers.
sensors. However, two electrodes are generally preferred for The main advantage of using an IDA is the redox cycling of the
disposable sensors because long-term stability of the reference electroactive enzyme product or mediator that occurs when
is not needed and the cost is lower. different potentials are applied to the two electrodes causing
These electrodes can be easily miniaturized, so dimensions oxidation–reduction cycling when the electrode reaction is
on the order of micrometres are common, while nanometre reversible. The redox cycling provides lower limits of detection
sizes have been demonstrated.18–20 Nanowires, nanoparticles, because the current due to oxidation of each redox active
and carbon nanotubes are now being incorporated into bio- molecule contributes multiple times to the detection current.6,28
sensors. Shrinking electrode dimensions may lead to higher As a result, the signal-to-noise ratio is improved significantly
sensitivity.3 Very small sample volumes (on the order of and a lower detection limit is obtained. Signal enhancement
microlitres and less) are required to detect with such small increases as the spacing and width of the metal fingers decrease
electrodes due to their small surface areas, and this is a signifi- because the diffusion distances for the redox species are shorter.
cant advantage when the sample sizes are limited.21,22 Further- Typical signal enhancements provided by the IDA are about
more, electrochemical detectors and their required control 3–10 and can be up to 1000 depending on the dimensions of
instrumentation can be easily miniaturized at a relatively low the IDA.28 IDA electrodes have been used as detectors in
cost by micromachining, making possible the manufacture of electrochemical immunoassays.29 An IDA with dimensions on
field-portable instruments for biosensing. Since the limiting the nanoscale was used for immunoassay detection of a virus.30
current in voltammetry is temperature-dependent, the detec-
tion cell should be maintained at a constant temperature for
running calibrants and samples in order to obtain accurate
and precise results.23
Screen-printed electrodes (SPEs), patterned minielectrode
systems with working, reference and auxiliary electrodes, have
gained popularity in electrochemical biosensors due to their
low cost and ease and speed of mass production using thick
film technology.6 An SPE for detecting oxygen is shown in
Fig. 2. SPEs can also be miniaturized easily making them an
attractive transducer choice for microfluidic systems and
portable meters. The patterned working electrode is typically
made of conductive carbon ink that results in a rough surface
that makes difficult the exact determination of electrode
area.24 Gold coated and gold-based SPE sensors have been
used in stripping voltammetry to determine trace levels of lead,
copper, cadmium, and mercury in water samples.25 Nafion Fig. 3 Cycling of a redox active species at the interdigitated array
coated SPE biosensors with immobilized butyrylcholinesterase electrode (IDA). Alkaline phosphatase (ALP) hydrolyzes o-phosphate
have also been developed to detect low levels of pesticides.26 from a p-aminophenyl phosphate under alkaline conditions. R is the
Disposable SPEs have also been used in immunochemical reduced p-aminophenol (PAP). O is the oxidized p-quinone imine
sensors and to measure blood glucose.27 (PQI).
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Field effect transistors have been adapted to chemical amplification in a biosensor.5 The shelf life and stability of
sensors (ChemFETs) by incorporation into an electrochemical an enzyme generally determine the lifespan of the biosensor.
cell.37,38 They can also be made into biosensors by coating the The use of enzyme electrodes as biosensors will continue to
sensing surface with a biological agent such as described above increase because they are simple and inexpensive to construct,
for penicillin.39 The light addressable potentiometric sensor they provide rapid analysis, they easily regenerate, and they
(LAPS) determines the surface potential optically by means of are reusable.2,5 However, the number of available enzyme-
the photovoltaic effect.40 The LAPS can also be used as a based biosensors is still smaller than the number of potential
biosensor by adding a biological element to its surface, such as analytes. Another disadvantage of enzyme electrodes is that
an oligonucleotide.41 the enzyme layer in the biosensor has to be replaced periodi-
cally since it gradually loses activity. Also, clever electro-
1.2.5 Miniaturized electrochemical transducers. Miniaturi- chemical detection strategies or membranes are sometimes
zation is a growing trend in analytical chemistry. In order to required to prevent interference from other redox active
design and manufacture small biosensors, the transducer or species at certain detection potentials.
the electrode needs to be small and portable. The manufacturing Development of biocatalytic sensors for medical appli-
capabilities for depositing microelectrodes on surfaces are cations, primarily blood glucose monitoring starting in the
good and microelectrodes can easily be deposited on a micro- late 1960s, was the main driving force for this research area.5
fluidic chip or other solid surface using vapor deposition.6 Enzyme-based biosensors can be historically divided into three
Usually the electrode is part of a bigger device such as a generations. First-generation biosensors were oxygen-based
handheld meter or a microfluidic system. whereas second-generation are mediator-based. Third-generation
Microelectrodes are defined as electrodes with a diameter in biosensors are so-called directly coupled enzyme electrodes.
the micrometre scale, and are made as disks or cylinders from Electrodes coated with glucose oxidase (GOx) have been
carbon fibers or metal microwires.18,19 The first measurements widely used in detection of glucose since the pioneering work
using microelectrodes to measure the concentration of oxygen of Clark and Lyons in the 1950s and 1960s (Fig. 5).50 These
in biological tissues were made in early 1940s,42 and they have amperometric sensors became known as the first-generation
since been used to measure electroactive species in critical biosensors or Clark oxygen electrodes and were soon imple-
places such as inside a mammalian brain.18 Measurements mented by Updike and Hicks, who constructed the first func-
with voltammetric microelectrodes have been made even inside tional biocatalytic sensor for glucose.51 In the first-generation
a very small, live biological cell.43 This is because the important biosensors, an oxidase enzyme is immobilized behind a semi-
reactions occur at the microelectrode surface instead of bulk permeable membrane at the surface of a Pt electrode.
solution, and the very small sensing surface area of a micro- GOx is a readily available, inexpensive, and stable enzyme
electrode can be easily inserted into very small drops or spaces from Aspergillis niger that is among the most important
without causing much disturbance or damage. Carbon fiber enzymes in biosensor applications and industrial processes.
microelectrodes have been used to detect 190 zmol of catechol- GOx is highly specific for b-D-glucose, which can be detected
amine release from a single, stimulated rat nerve cell,44 to via the following reactions.2,5,52
directly monitor catecholamines released from adrenal cells in
culture,45 and to measure the release of serotonin from neuronal
b-D-Glucose + GOx–FAD - GOx–FADH2
vesicles achieving a 4.8 zmol detection limit.46 Microelectrodes
+ d-D-gluconolactone (1)
have also been used as detectors in microvolume electro-
chemical immunoassays.22 The nanoamp to picoamp currents
GOx–FADH2 + O2 - GOx–FAD + H2O2 (2)
generated at microelectrodes are so small that they are virtually
nondestructive,18 and amplification of the small currents produced
H2O2 - 2e + O2 + 2H+ (3)
is typically required in order to observe the signals.6
2. Biocatalytic sensors
2.1 Introduction to enzyme-based electrodes
Enzyme electrodes are electrochemical probes with a thin
layer of immobilized enzyme on the surface of the working
electrode.47,48 The enzyme is the most critical component of
the enzyme electrode since it provides the selectivity for the
sensor and catalyzes the formation of the electroactive product
for detection.49 The electroactive product can be monitored
directly using amperometry, in which the produced current is
measured in response to an applied, constant voltage. Alter-
natively, the disappearance of the redox active reactant in an
enzyme-catalyzed reaction can be monitored by the electrode.
The activity of the immobilized enzyme depends on solution
parameters and electrode design. The rapid enzymatic Fig. 5 Oxygen-dependent first-generation biosensor with ampero-
catalysis can also sometimes provide significant signal metric detection.
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the transducer.2 Vmax is the maximal velocity of a reaction that Therefore the lifetime of a sensor prepared using adsorption is
occurs at high substrate concentrations when the enzymes are rather limited. However, adsorption is very easy because it does
saturated. By having an immobilized enzyme with a high Vmax not require any reagents or clean-up and is less disruptive to the
the electrochemical transducer responding to a reaction catalyzed enzymes.1 The formation of intermolecular interactions with
by the enzyme has a broader range where the signal is propor- the surface may compete with similar interactions stabilizing the
tional to substrate concentration for reliable quantitation of the enzyme, and is often a prelude to denaturation. This is probably
analyte. Km, the Michaelis constant, is the substrate concen- why adsorption usually works best in the short term, because
tration at which the reaction velocity is half-maximal. Enzymes the protein deformation increases with time. Adsorption is
with low Km reach maximal catalytic efficiency at low substrate often sufficient for short-term studies. The stability of immobi-
concentrations. The immediate environment around the immobi- lized enzymes with respect to time, temperature, and pH is
lized enzyme can be carefully designed to enhance the enzyme typically greater making enzyme electrodes preferable to soluble
activity and the overall biosensor performance. enzyme assays.5,52 Covering the immobilized enzyme layer with
The easiest approach is to physically entrap a solution of a membrane or a polymer coating also helps to minimize
the enzyme between preformed membranes on the electrode interferences by physically blocking some interfering species
surface.2 The inner membrane protects the electrode surface from approaching the electrode surface.52
from interfering substances and electrode fouling due to
adsorption. The outer membrane also provides some selecti- 2.2.2 Optimizing enzyme electrodes. Although many enzyme
vity based on the pore size or chemical nature of the polymer, electrodes have been fabricated and some sensors have reached
stabilizes the sensor response by moderating the substrate the commercial stage, some factors that prevent their wider
diffusion to the enzyme layer, and provides a biocompatible adaptation and successful routine use still remain. Research
outer surface for the sensor.5 In physical immobilization continues in trying to overcome the dependence of enzyme
methods the native composition of the enzyme is preserved activity on the solution conditions such as temperature, pH,
since the methods do not involve the formation of covalent ionic strength, and buffer composition.60 Ideally the solution
bonds.34 Chemical methods involve the formation of covalent conditions should remain constant between samples and during
bonds between the functional groups of the enzyme and the the measurements. The enzyme electrode should also have a
electrode material.5 Common enzyme immobilization methods wide linear range. Enzymes become saturated with their sub-
include enzyme entrapment against the electrode using a strate at high concentrations due to their active sites becoming
preformed membrane; encapsulation; inclusion in a gel or the limiting reagent, thus causing the signal response to no
electropolymerized film; incorporation in a carbon paste; longer be proportional to the analyte concentration. The
and using biospecific interactions such as biotin–avidin binding, amount of enzyme incorporated into the sensor can however
adsorption, cross-linking, and covalent attachment (Fig. 7).58,59 be adjusted based on the expected sensor application. The
Covalent bonding provides the most stable immobilization of catalytic biosensor should also be biocompatible since blood
proteins followed by cross-linking and encapsulation.1 Covalent and other biological fluids are the most common sample
bonding to the transducer links functional groups on the matrices for enzyme electrodes. Many blood components foul
enzyme such as NH2, COOH, OH, and SH that are not the electrode in a matter of minutes unless special precautions
necessary for the catalytic activity of the enzyme. The coupling are taken in designing the sensor’s outermost surface properties
reactions need to be done under mild conditions (low ionic and permeability to prevent the adsorption of sample com-
strengths, low temperatures, and near physiological pHs) and ponents on the electrode surface.60 Product design requirements
often in the presence of the enzyme–substrate in order to protect also include optimization of sample introduction, sample size,
the catalytic activity of the enzyme.1 Adsorption is the least the sensor’s reproducibility, selectivity, sensitivity, stability, cost,
stable of the common immobilization methods.1 The forces and ease of use.5 The storage stability of enzymes immobilized
linking the biorecognition element to the transducer in adsorp- on electrode surface varies from hours to months depending on
tion are primarily very weak van der Waals forces with occa- the sensor preparation and design, and the storage environment.
sional hydrogen bonds that are not very stable or permanent.1
2.3 Examples of biocatalytic sensors
2.3.1 Glucose sensors. Enzyme electrodes are produced
commercially and are routinely used in biomedical appli-
cations such as glucose testing in clinical laboratories and
personal monitoring by diabetic patients.2,5,48 Low cost blood
glucose home monitoring kits consisting of handheld battery
operated meters and disposable glucose test strips based on
glucose oxidase (GOx) or glucose dehydrogenase enzyme
electrodes are sold off the shelf worldwide. Biosensors for this
application must be easy to use, reliable, and inexpensive.1 In a
typical sensor, a single drop of blood is placed on a disposable
PVC sample strip on which the dry reagents have been
deposited using a method similar to ink-jet printing techno-
Fig. 7 Common methods of immobilizing enzymes onto an electrode logy. The test strip also contains two electrodes, one holding
surface. the enzyme and a mediator for the amperometric detection
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Live microorganisms have also been coupled with electro- easy to use once fully developed and optimized, and the
chemical transducers (i.e. electrodes) to monitor biotechno- reductions in chemicals used, waste disposal, expensive instru-
logical processes such as brewing, food manufacturing, ments and maintenance also help lower the overall cost per
waste-water treatment, energy production, and pharma- analysis. The four key factors involved in the design of a
ceutical synthesis.1,2,74 Bacteria are often immobilized on sensitive immunoassay are its format, the type of label, the
transducers by microencapsulation where an inert membrane method of detection, and being able to minimize nonspecific
is used to trap the microbe on the electrode surface.1 Changes binding (NSB).27 These factors will be discussed further in
in the respiration activity of the microorganism, induced by Sections 3.1.4–3.1.7.
the target analyte, results in a lower surface concentration of
electroactive metabolites (e.g., oxygen), which can be detected 3.1.1 Biorecognition and immunochemical reactions. IgG
by the electrochemical transducer.2,74 Some microorganisms antibodies (Ab), large Y-shaped glycoproteins of MW E
also produce electroactive metabolites that can be monitored 150 kDa, are produced by a host in response to the presence
directly.1 Using microbes in biosensors gained popularity of a foreign molecule called antigen (Ag).5 Antigens are any-
because they are typically cheaper to obtain than isolated thing that the body recognizes as foreign such as chemical
enzymes, are less sensitive to inhibition by other sample com- compounds, proteins, and particulate matter (dust, pollen,
ponents, and are more tolerant of slight temperature and pH etc.).75 Abs are produced by specialized B lymphocyte cells
variations than enzymes.1,5 Some of their disadvantages include of the immune system and can usually be found in blood
longer recovery times after exposure to the analyte of interest, serum, tissue fluids, and membranes of vertebrates.75 Antigens
longer response times, hysteresis effects, and possible loss in commonly have relatively high molecular weights, are recog-
selectivity due to containing many types of enzymes.1,5,74 nized as nonself or foreign by the immune system and have a
certain level of chemical complexity.75 For example, synthetic
homopolymers composed of a single sugar or amino acid tend
3. Affinity biosensors to lack immunogenicity regardless of their large size due to a
lack of structural complexity.75 The production of Abs against
3.1 Immunoassays and immunosensors
low molecular weight analytes (MW o 1000 g mol 1) called
Immunoassays gained popularity for biomedical applications haptens is more challenging and often requires coupling the
in the 1970s because of the impressively low detection limits hapten to a carrier protein with a spacer molecule before an
and high selectivities for analyzing complex samples that could immune response can be provoked in the host animal.75,76
be achieved with relatively simple procedures and instru- IgGs have four polypeptide chains (two identical heavy
mentation. The availability of highly selective antibodies for chains with MW of 50 000 or higher and two identical and
an increasingly wide variety of important analytes was also an smaller light chains with MW of about 25 000) that are held
important factor in the growth of the method over the together by disulfide bonds and noncovalent interactions such
following decades. The development of more sensitive labels as hydrogen bonds as seen in Fig. 8.75 Each chain has several
and detection devices also improved the sensitivity of the different domains. Each Ab molecule has two identical binding
assays even further. Once immunoassays became more com- sites and is therefore called bivalent. The highly selective
mon, the development of more convenient immunosensors antigen-binding site is formed at the tips of each of the Y
that are easier and faster to use gained momentum. arms where a heavy-chain variable domain (VH) and a light-
Most applications of immunoassays (IA) and immuno- chain variable domain (VL) come close together.75 These
sensors with electrochemical detection were initially developed complementarity-determining regions (CDRs) are the domains
at research laboratories due to the level of expertise required, that differ most in their sequence and structure between
time, and the high initial cost of developing and optimizing a different antibodies. Parts of VL and VH contribute to the
new immunoassay.27 However, the cost of immunological finger like loops that interact with the antigens.75 The non-
reagents continues to decrease with recent developments in covalent interaction between the Ab and Ag is highly specific,
molecular biology techniques. Many of the early radioimmuno- which makes antibodies an excellent biorecognition element
assays were developed for biomedical applications such as for affinity biosensors. However, unlike in enzyme–substrate
detecting hormones and disease related proteins, but appli- interactions, Ab–Ag binding does not lead to an irreversible
cations in environmental, agricultural, processed food and chemical alteration in either the Ab or the Ag.75 The non-
beverage areas, and to detect harmful chemical and biological covalent interactions that are cumulative and form the basis
agents in national defense, have become more common.6,27 for the binding interaction include hydrogen bonding, ionic
The advantages of IAs such as exceptionally high specificity bonds, hydrophobic interactions, and van der Waals forces. A
of Ab for Ag, small sample volumes, low detection limits, little very close fit resulting from a high degree of complementarity
or no sample preparation, reduced use of chemicals, little between the Ab and the Ag is required for the noncovalent
waste, and ease of automation, far outweigh their limitations, interactions to form since they operate over very short dis-
thus making the IAs an attractive alternative to the more tances. Sometimes the exceptional selectivity can be a dis-
conventional quantitative analytical methods like chromato- advantage when an Ab is selective only for one isomer of the
graphy and mass spectrometry.6 Many IA formats also allow Ag when a sensor should ideally measure the total amount of
the simultaneous analysis of multiple samples, which improves the Ag type.1
efficiency and makes the assays relatively fast and cost effec- The unique antibody-binding region of the CDR is also
tive. The immunoassays and affinity biosensors are relatively called the paratope, and recognizes and binds with high
3.1.2 Antibody production. The production of the anti- 3.1.5 Formats for enzyme immunoassays. Enzyme immuno-
bodies (Abs) against a specific antigen (Ag) can be fairly assays (EIAs) were first introduced by Engvall, Perlmann,
difficult and time-consuming.77 A small host animal such as Van Weemen, and Schuurs in 1971 as an alternative to
a mouse, a rabbit, or a chicken is injected with small sub-lethal radioimmunoassays.27 The previously used radioactive label
doses of Ag to challenge their humoral immune system to indicating that an Ab–Ag complex had formed was replaced
produce the specific Abs against the foreign invader. Some- by a safer, selective and less expensive enzyme label at the cost
times larger mammals such as goats are preferred as the host of less sensitivity and more complexity.27 In EIAs the activity
because the amount of blood serum that can be collected is of the enzyme label in generating electroactive product is
greater. Mice are usually used in the initial stages of mono- measured. Enzymes are also highly selective for their given
clonal Ab (MAb) production. substrate, and can provide a large signal amplification due to a
MAbs are produced by a single Ab-producing cultured cell high turnover rate, which yields low limits of detection. How-
line (containing clones of a single parent cell) in a bioreactor ever, as discussed in Section 1.1.1 the activity of the enzyme
and are identical in the primary structure.75 MAbs can also be labels can be affected by reaction conditions that have to be
produced in microbial systems and transgenic mice.78,79 These controlled during the detection step. Like radioimmunoassays,
homogeneous Abs that are known for their high specificity enzyme immunoassays can be time-consuming due to including
and affinity are used as the primary or capture Ab in most multiple incubation and washing steps. Many variations of
research, diagnostic, and sensing applications. MAbs have an immunoassays have been developed that allow sensitive quanti-
inherent specificity toward a single epitope that allows fine tation of either Ag or Ab. The two main immunoassay (IA)
detection and quantitation of small differences in Ag. Poly- formats are homogeneous and heterogeneous.27 Homogeneous
clonal Abs are a heterogeneous mixture of immunoglobulin assays, which do not contain separation steps, are faster and
molecules secreted against a specific antigen, each recognizing easier, but have poorer limits of detection. Homogeneous assays
a different epitope.75 They have varying affinities for the Ag are also more susceptible to interferences by other species in the
and are often used as the secondary Ab in immunoassays. sample than IAs with other formats.27 Heterogeneous assays
The small size of mice prevents their use for sufficient include a physical separation step to isolate the antibody–
quantities of polyclonal, serum antibodies.77 Animals usually antigen complex from the unbound constituents followed by a
used for polyclonal Ab production include chickens, goats, wash step to remove any unbound materials. The separation
rats, guinea pigs, hamsters, sheep, camels, llamas, and horses. step in a heterogeneous assay makes the procedure longer, but
Rabbit is by far the most commonly used laboratory animal results in significantly better limits of detection.
for Ab production. The soluble antibodies produced by the Homogeneous and heterogeneous EIAs can be done either
host in these immune system challenges are then recovered competitively or noncompetitively.27 Competitive immunoassays,
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also known as limited reagent assays, are often used when the
antigen is small and has only one epitope.75 In a competitive
assay a limited amount of Ab is used, which is insufficient to
bind with all the Ag molecules in the sample. A fixed, known
amount of labeled Ag is mixed with the unknown sample
and allowed to incubate. Unlabeled Ag and the labeled Ag
compete for binding to the limited number of capture Ab sites.
Rinses are required to separate the unbound Ag from the
bound prior to the detection step. A decrease in signal
response indicates the presence of the Ag in the sample
being analyzed. The ratio of limited Ab reagent to the added
labeled Ag must remain constant between samples to obtain
Fig. 9 Sandwich enzyme immunoassay steps. Ab, antibody; Ag,
quantitative results. antigen; Ab*, enzyme-labeled secondary antibody; S, substrate; P,
Noncompetitive assays are also called excess reagent assays product; and shaded oval, nonspecific binding blocker.
and are better suited for large analyte molecules with several
epitopes.75 The Ag sample is incubated with an excess of Ab 3.1.7 Nonspecific binding. Nonspecific binding (NSB)
reagent. All the Ag molecules form a complex with antibodies, involves the adsorption of conjugated enzyme or other labels
but not all of the Ab-binding sites are occupied. To detect the used for immunoassay to materials other than the analyte.27
amount of Ag attached to an Ab, a labeled secondary Ab This phenomenon, which increases the background signal, is
is added which binds to another, available epitope on the the major determinant of the detection limit of the IA and
bound Ag. This leads to the formation of a sandwich complex therefore including procedures that minimize NSB in immuno-
(Ab:Ag:Ab*). Unbound excess reagent is washed away after assays is critical. NSB can be reduced with blockers such as a
each incubation step. The electrochemical signal produced nonionic surfactant, Tween 20, protein blockers such as
during the detection step is directly proportional to the bovine serum albumin (BSA), polyethylene glycol,83 gelatin,84
amount of Ag in the unknown sample. casein,85 and proprietary blended commercial products. Self-
Sandwich IAs are often referred to as enzyme-linked immuno- assembling monolayers of oligo(ethylene glycol)86–88 and
sorbent assays (ELISA) because the antibody or the antigen is dextran layers89 have also been used successfully to prevent
immobilized on a solid surface such as a bead, membrane, a NSB on affinity biosensor surfaces. These NSB blocker re-
polystyrene well, or an electrode surface. Fig. 9 shows the main agents are commercially available and widely used in affinity
steps in a sandwich enzyme immunoassay. Having the immuno- biosensors.
reactants of the ELISA immobilized makes it easy to separate With plastic surfaces, such as polystyrene used to make
bound from unbound material during the assay washing steps.27 beads and microtiter wells, hydrophobic interactions usually
dominate the adsorption process.8 The adsorption is entropi-
cally driven and can usually be minimized by physically coating
3.1.6 Enzyme labels and substrates. The enzyme label the exposed areas of the reaction vessel by surface treatments
chosen for the IA with electrochemical detection should have such as a mixture of bovine serum albumin and a detergent
a high catalytic activity for the corresponding substrate and be such as Tween 20.90 Sulfonate ion-pairing reagents have been
fairly stable in the sample matrix. It should also be readily found to reduce NSB on positively charged surfaces.8 Deter-
available in a purified and soluble form at a reasonable cost. gents and proteins can be added to the buffer to block NSB
The enzyme label should contain surface functional groups with bead-based immunoassays.14,15 A 13-fold reduction
that can be used to form conjugates with other molecules as in detection limit has been seen in blocked electrochemical
needed without impairing its catalytic activity or compromising immunoassays compared to the unblocked assays.90 Contact
the biorecognition events. The redox active product that is between NSB blocking agents and the electrode transducer
formed by the enzyme catalysis should have a low redox should be avoided because the blockers may adsorb on the
potential to minimize interference from other components in electrode surface, fouling it.27
the sample, while the substrate should be electroinactive at the
measuring potential to keep the background signal low.27 It is 3.1.8. Applications of immunoassays. Immunoassays and
usually not necessary to remove oxygen from the sample if the enzyme sensors have been incorporated into portable instru-
observed reaction is an oxidation occurring between +200 ments capable of quickly measuring multiple analytes. A good
and +900 mV. The lower end of the range is more desirable example is the i-STATt, which is able to make measurements
because the more positive values may result in electrolysis of on small volumes (17–95 mL) of whole blood.91 The i-STATt
the solvent. Several enzymes satisfy the above requirements analyzer is based on single-use disposable cartridges con-
and are used in electrochemical IAs and immunosensors. The taining a microfabricated biosensor array. The system auto-
most commonly used enzyme labels are alkaline phosphatase matically calibrates the sensors and analyzes the sample.
(ALP), b-galactosidase (b-Gal), horseradish peroxidase Ion-selective electrodes are used to determine Na+, K+,
(HRP), and glucose oxidase (GOx).1,2,27 GOx has a lower Cl , Ca2+, pH and pCO2. Amperometric enzyme biosensors
activity than the other enzyme labels and is typically used in are used to determine glucose, lactate and creatinine using the
amperometric immunoassays where the product is detected principles described above. Recently, cartridges capable of
directly. sandwich immunoassay with electrochemical detection using
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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1747–1763 | 1759
Fig. 10 Commercially available electrochemical DNA sensor (eSensors) by Osmetech: (a) detection principle, (b) assay genotyping principle, (c)
disposable biosensor printed circuit. (Published with permission of copyright holder, Clinical Micro Sensors, Inc. dba Osmetech Molecular
Diagnostics.)
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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1747–1763 | 1761
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