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Electrochemical Biosensors
Electrochemical Biosensors
Electrochemical Biosensors
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
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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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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
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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.
such as a rabbit or a mouse with high specificity and affinity.4 detecting electrochemical immunoassays have been constructed.7,8
Such polyclonal Abs are heterogeneous with respect to their In homogeneous immunoassays, which have no separation
binding domain, and may be refined by a selection process to step to isolate the antibody–antigen complex from the unbound
yield monoclonal Abs—MAbs—all of whose members of a assay constituents, electrochemical detection is not affected by
particular MAb clone are identical. Abs and MAbs can be sample components such as chromophores, fluorophores, and
developed for a wide range of substances. Theoretically, if an particles that often interfere with spectrophotometric detec-
Ab can be raised against a particular analyte, an immuno- tion. Therefore electrochemical measurements can be made on
sensor could be developed to detect for that substance. Immuno- colored or turbid samples such as whole blood, without
sensors are well known among analytical methods for their interference from fat globules, red blood cells, hemoglobin,
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1.2.2 Impedance. Electrochemical impedance spectroscopy participating in the biologically mediated redox reaction must
(EIS), described by Lorenz and Schulze in 1975,31 measures the be easily accessible to the analyte solution and in close
resistive and capacitive properties of materials upon perturba- proximity to the electrode surface. As discussed before, redox
tion of a system by a small amplitude sinusoidal ac excitation mediators have been used to help overcome the accessibility
signal typically of 2–10 mV.5,32 The frequency is varied over a and proximity limitations but cause the detection to be limited
wide range to obtain the impedance spectrum. The in-phase and by the mediator’s mass transfer rate. Furthermore, some
out-of-phase current responses are then determined to obtain additional redox active species such as urate and ascorbate
the resistive and capacitive components of impedance, respec- that are often present in the sample matrix can contribute to
tively. Impedance methods are powerful because they are the amperometric signal if the detection potential is not care-
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capable of sampling electron transfer at high frequency and fully chosen. Being directly able to impedimetrically monitor
mass transfer at low frequency. Impedimetric detection is the Ab–Ag binding helps by-pass the aforementioned limita-
primarily used for affinity biosensors.27 It can be used to tions. EIS is also insensitive to most environmental distur-
monitor immunological binding events such as antibody bances. However, biosensors using impedance detection
(Ab)–antigen (Ag) binding on an electrode surface, for example, have to be carefully designed to minimize nonspecific binding
where the small changes in impedance are proportional to the of the analyte. Nonspecific binding in affinity sensors will be
concentration of the measured species, the Ag. discussed further in Section 3.1.7. Using nanomaterials
The surface of the electrode can be modified by a highly such as gold nanoparticles and carbon nanotubes in electro-
specific biological recognition element. In one approach the chemical impedance sensors is advantageous due to the increased
recognition elements are incorporated in a conductive polymer electrode surface area, improved electrical conductivity of
film formed on the surface of a working electrode by electro- the sensing interface, chemical accessibility to the analyte, and
chemical deposition (Fig. 4). During the detection step, a electrocatalysis.32 Recent applications of impedance spectro-
known voltage is applied to the electrode and the resulting scopy in affinity sensors will be described in Section 3.1.8.
current is measured. The electron transfer resistance at the
interface between the electrode and the solution changes
1.2.3 Conductometry. Conductometric detection monitors
slightly when analyte binds. Directly monitoring the formation
changes in the electrical conductivity of the sample solution, or
of an antibody–antigen conjugated layer provides a label-
a medium such as nanowires, as the composition of the
free detection system with many potential advantages such
solution/medium changes in the course of the chemical reac-
as higher signal-to-noise ratio, ease of detection, lower assay
tion. Conductometric biosensors often include enzymes whose
cost, faster assays and shorter detector response times. How-
charged products result in ionic strength changes, and thus
ever, regenerating the sensing surface for a subsequent measure-
increased conductivity. Conductometry has been used as the
ment in an impedance biosensor is typically very time-consuming
detection mode in biosensors for environmental monitoring
and not reproducible.27 This continues to be the biggest
and clinical analysis. A conductometric tyrosinase biosensor
limitation of immunosensors involving Ab–Ag complexes with
was developed to measure ppb amounts of pollutants such as
high affinity constants. The regeneration conditions can also
diuron, and atrazine and its metabolites.33 Conductometric
damage and release the immunoreagent bound to the surface
immunosensors have also been developed to detect foodborne
of the transducer.27
pathogens such as enterohemorrhagic Escherichia coli O157:H7
Electrochemical biosensors using impedance spectroscopy
and Salmonella spp., which are of concern to biosecurity.34
to detect analytes have recently gained popularity among
The sensitive, low volume biosensor consists of an immuno-
the biosensor community.3 EIS has some advantages over
sensor that is based on an electrochemical sandwich immuno-
the widely used amperometric detection. The active site
assay, and a reader device for measuring the signal.34 Drug
detection of methamphetamine in human urine has also been
done using conductometry.35
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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-
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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.
In eqn (1) the prosthetic group of the enzyme, FAD, is reduced biosensor could be measured in a potential range where other
and glucose is oxidized to d-D-gluconolactone. Molecular possible sample components such as ascorbate, urate, and
oxygen acts as the oxidizing agent to produce hydrogen paracetamol are not oxidized or reduced thereby minimizing
peroxide (eqn (2)). During the oxidation of H2O2 at a working interferences.5 Incorporating redox mediators also allowed
electrode two electrons are transferred directly to the electrode other oxidoreductase enzymes such as peroxidases and dehydro-
(eqn (3)), resulting in the current response of the enzyme genases to be used as the biorecognition element in the sensor
electrode. These first-generation sensors required the ample thereby expanding the list of possible target analytes.
and constant presence of ambient oxygen as a co-substrate for Third-generation biosensors have the biorecognition com-
the enzyme to function optimally. However, oxygen is not very ponent coupled with the electrode by co-immobilizing the
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soluble in aqueous solutions and can therefore limit the enzyme and the mediator at an electrode surface. This can
currents produced in the presence of the analyte. be achieved by direct electrical contact between the enzyme
Direct redox reactions between enzymes and electrodes are and the electrode, immobilizing the enzyme and mediator in a
very rare because most proteins tend to denature at the conducting polymer, or ‘wiring’ the enzyme to the electrode by
electrode surface and many direct electron transfer reactions immobilizing it in a redox polymer (Fig. 6) as first described by
are slow and irreversible.1 However, a limited number of Heller et al.56,57 The co-immobilization prevents the mediators
enzymes such as horseradish peroxidase have proven capable from diffusing out of the biosensor film. The co-immobilized
of direct electron transfer between the enzyme active site’s mediators, or the flexible surrounding redox polymer, help to
prosthetic group and the electrode.53 The active site of an transport electrons between the enzyme’s active site and the
enzyme that allows the selective targeting of an analyte is working electrode surface in an array of rapid electron relays
usually buried within the enzyme’s tertiary protein structure, and hence generate high current densities.2 The enzymes
near the centroid of the protein.4 Therefore, the electrons immobilized in flexible redox polymers that are covalently
produced in the enzyme-catalyzed reaction cannot always be attached to the electrode have been called ‘wired enzymes’.
easily and rapidly transferred to the electrode surface thereby The 3rd-generation sensors are ideal for repeated measure-
limiting the electrical communication between the enzyme and ments since neither mediator nor enzyme need to be added.
the transducer. The widely accepted Marcus theory of electron This self-contained nature also lowers the cost per measure-
transfer states that electron transfer decays exponentially with ment and opens up possibilities for continuously monitoring
distance.54,55 Therefore enzymes often require some assistance the analytes.
with electron transfer to the transducer surface.
Artificial redox mediators are small, soluble molecules 2.2 Preparing enzyme electrodes
capable of undergoing rapid and reversible redox reactions,
which shuttle electrons between the redox center at the active 2.2.1 Methods for immobilizing enzymes to electrode surfaces.
site of the enzyme and the electrode surface. Mediators have Enzyme electrodes have been studied extensively and various
replaced O2 molecules as the electron shuttle (eqn (4)) in physical and chemical schemes have been used to immobilize
glucose sensors. Mediators are re-oxidized at relatively low enzymes on the electrochemical transducer. The objective is to
potentials and generate a current when they come in contact have an intimate contact between the enzyme and the trans-
with the working electrode (eqn (5)). ducer’s sensing surface without blocking the active site of the
enzyme or drastically altering the enzyme geometry.2 Immobili-
GOx–FADH2 + 2MediatorOx - GOx–FAD zation methods are considered successful if the biosensors pre-
pared are stable, reusable, and maintain the selectivity of the
+ 2MediatorRed + 2H+ (4)
enzyme. Although immobilization may alter the conformation of
2MediatorRed - 2MediatorOx + 2e (5) the enzyme, thereby reducing its activity, many methods have
been successful. Some immobilization methods even improve
Mediators should ideally be nontoxic, independent of the enzyme stability by minimizing enzyme unfolding. The enzyme
pH, stable in both the oxidized and reduced forms, and should have high Vmax and low Km values when immobilized on
unreactive with oxygen.1 Although many organic compounds
are capable of acting as enzyme mediators, organometallic
redox compounds are the most common.1,2 Examples of
previously used mediators include quinones, organic conducting
salts, dyes, ruthenium complexes, ferrocene, and ferricyanide
derivatives. Mediated enzyme electrodes had a much better
sensor performance than the first-generation biosensors
mainly due to eliminating the O2 dependence and being able
to control the concentration of the oxidizing agent in the
biosensor.1 Hand selecting the oxidizing agent for the sensor
also allowed more suitable oxidation potentials to be used for
the amperometric sensors. These mediated enzyme electrodes
were named second-generation biosensors.
By carefully selecting a mediator and a suitable redox Fig. 6 Third-generation catalytic biosensor containing enzymes
potential, the transduction event at the second-generation wired to the electrode through a conducting redox polymer.
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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-
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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
of glucose, and the other serving as a reference electrode. Senslab (Germany) and Arkray (Japan).70 These sensors
The current produced when a potential is applied gives a read- require only 0.5 mL and 5 mL blood samples, respectively.
out to a liquid crystal display on the glucose meter. The The concentration of lactate in blood is also a sensitive
commercially sold glucose test strips are second- or third- measure of oxygen deprivation from ischemia, trauma, and
generation biosensors and no longer rely on oxygen as the hemorrhage, which can lead to life-threatening shock, and its
oxidizing agent. Ferricyanide is a commonly used mediator for measurement has therefore become a vital component in
the second-generation sensors. Eqn (1), (4), and (5) describe medical monitoring.70 Blood lactate levels are used as indi-
the sequence of reactions for such a sensor when GOx is used cators of conditions such as acidosis or bacterial meningitis.71
as the enzyme. The commercially sold blood glucose meters Conventional photometric assays for lactate are slow and not
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typically have a range of 1.1–33.3 mM glucose with a precision suited for continuous lactate monitoring systems that are
of 3–8% and test time of about 30 seconds or less.1 being developed for medical applications. Bench top lactate
Some invasive and minimally invasive implantable glucose biosensors are also routinely used to measure lactic acid in
sensors that have an intimate contact between the biological milk and other foods.
fluids or tissues and the biocatalytic sensor have been developed.5,61 Four different enzymes have been used as the biorecognition
The minimally invasive blood glucose sensors are inserted component in lactate biosensors: lactate dehydrogenase, lactate
subcutaneously into the arm or belly of a patient. More oxidase, lactate monooxidase, and cytochrome b2.1 Some of the
invasive, intravascular sensors that measure glucose levels in electrochemical lactate sensors include mediators such as
hospitalized diabetes patients are also being developed. Some NAD+/NADH and ferricyanide.1 All of these enzymes help
problems such as pain, skin irritation, limited lifetime of the ultimately to produce a current at the working electrode that is
sensor, and accuracy of the data continue to slow down their measured amperometrically.
wider use.62
2.4 Interference-based enzyme electrodes
2.3.2 Xanthine sensors. Xanthine oxidase (XO) catalyzes the
Interference-based enzyme electrodes are probes used for quanti-
oxidation of xanthine to uric acid (eqn (6)). Amperometric
tative analysis based on the changes in the rate of catalytic
biosensors using immobilized XO are highly specific for xanthine,
reactions when enzyme effectors bind, such as inhibitors or
which can be measured by the following redox reaction:
activators.72 Usually, the binding of an inhibitor to the enzyme’s
Xanthine + O2 + XO - uric acid + H2O2 + XO (6) active site or another site that causes the conformation of the
enzyme to be altered, results in a decreased electrochemical
Xanthine is an intermediate of purine metabolism and is
response because substrate cannot freely access the catalytic site.
produced after adenosine triphosphate (ATP) decomposition.
Sensors using this operating principle have been developed for
The physiological conversion of xanthine by xanthine oxidase
pesticides such as organophosphate and carbamate, respiratory
is of increasing medical interest.63 Moreover, xanthine sensors
poisons such as cyanide and azide, as well as toxic heavy metals.2
are frequently used in food industries to determine the freshness
Enzymes that have been used in these enzyme electrodes include
of fish. The need for maintaining an acceptable quality of
tyrosinase, horseradish peroxidase, and acetylcholinesterase. Due
fish sold to consumers requires rapid and reliable analytical
to the operating mechanism and the uses these sensors have, they
methods that detect the products formed in their degradation
have also been called enzyme inhibition biosensors or toxin
processes. After the death of a fish, nucleotides such as ATP
biosensors.2,72
are most affected by degradation and give rise to the formation
of inosine, which is transferred to hypoxanthine by action of
2.5 Biosensors based on tissue and bacteria
the enzyme nucleoside phosphorylase.64 Hypoxanthine causes
a bitter taste in the degrading meat.65 XO catalyzed oxidation Some biocatalytic sensors incorporate cellular materials such
of hypoxanthine to xanthine and conversion of xanthine to as plant tissues as the recognition component.2 These bio-
uric acid occurs in two steps.64 The quantitation of xanthine or catalytic electrodes function in a manner similar to that for
hypoxanthine can therefore be used to determine the freshness conventional enzyme electrodes (i.e., enzymes present in the
of fish.64,65 Other existing methods for detecting xanthine or tissue or cell produce or consume electrochemically detectable
hypoxanthine such as anion-exchange chromatography, thin species). Whole cells and tissue slices are sometimes a better
layer chromatography, precipitation and capillary electro- source of enzymatic activity compared to isolated enzymes as
phoresis are complicated and very time-consuming. Therefore some enzymes are expensive or not commercially available in
biocatalytic sensors with amperometric detection continue to the pure state.2 Also, many isolated enzymes have limited
be developed to monitor the freshness of fish meat.66–68 stability and lifetime compared to enzymes in their native
environment. However, the sensor response may be slower for
2.3.3 Lactate sensors. Lactate, an ester of lactic acid, is a these sensors because there is more tissue material for the
product of fermentation and is produced during cellular substrate to diffuse through.1
respiration as glucose is broken down. Its concentration in Bananatrode, a banana tissue containing electrode, was one
blood rises from the normal value of 0.9 mM to about 12 mM of the early uses of tissue in a biosensor.63 The banana tissue,
due to strenuous exercise such as running, which results in which is rich with polyphenol oxidase (PPO), can be mixed in a
anaerobic metabolism.69 Small handheld electrochemical carbon paste matrix to yield a fast responding and sensitive
lactate meters for use in sports medicine capable of inter- dopamine sensor. The amperometric probe has high biocatalytic
mittent ‘‘spot’’ lactate monitoring are being manufactured by activity, good time stability, and favorable selectivity.73
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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
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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
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the principles described above have been commercialized.92 measurements down to 3000 copies of target DNA or
Single cartridges for cardiac markers creatine kinase MB zmols.103 Nanoparticle labels such as colloidal gold have also
(CK-MB), cardiac troponin I (cTnI) and B-type natriuretic been used to quantitate binding.2 Label-free electrochemical
peptide (BNP) use alkaline phosphatase as the enzyme measurement of hybridization induced changes in capacitance
label. or conductivity at the transducer surface have been used.5 The
Affinity biosensors using impedance spectroscopy with gold nucleotide base guanine can be oxidized at the electrode and
(Au) nanoparticles as the solid support for the biorecognition the signal amplified by a redox mediator such as Ru(bpy)32+.
element have been developed for the IgE antibody to a protein Like other biological macromolecules with complex
allergen from dust mites,93,94 human immunoglobulin (hIgG),95 structures, the experimental conditions, such as tempera-
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and carcinoembryonic antigen (CEA),96 a glycoprotein that is ture, ionic strength, and time allowed for hybridization, have
produced only during fetal development. Au nanoparticles of to be controlled in order to achieve high selectivity and
several different sizes are now commercially available and their sensitivity.
use in biosensors has become very popular.32 These Au
nanoparticles are also biocompatible. Biomolecules immobi- 3.2.3 Aptamer production. Single-stranded, 15–40 bases
lized on Au nanoparticles are usually stable and able to retain long DNA or RNA oligonucleotide sequences that are used
their biological activity. Au nanoparticles are typically used to as the biorecognition component called aptamers in biosensors
form a single layer or a three-dimensional network on a are rapidly screened in the SELEX (systematic evolution of
conductive electrode surface or are incorporated into a ceramic ligands by exponential enrichment) process for their ability to
sol–gel or polymer film.32 selectively bind low molecular weight organic, inorganic or
Impedance sensors using carbon nanotubes (CNTs) as the protein targets.104,105 In solution, the synthetic nucleotide
sensor interface on which the capture Ab is immobilized have chains form intramolecular interactions that fold the aptamer
also been reported.97–99 CNTs contain allotropes of carbon molecules into a complex three-dimensional shape. The unique
arranged in sheets that have been rolled up into highly shape of the aptamer allows it to bind tightly and selectively
conductive, hollow tubes of various nanometre dimensions. with its target molecule. Aptamers can either bind to small
CNTs have been incorporated in the sensing layer of sections of macromolecules, such as proteins, or they can
impedance biosensors due to their exceptionally high con- engulf a small molecular target.
ductivity and increased active surface area. CNT towers have The selection process for aptamers has been around since
been used in impedance detection of mouse IgG and prostate 1990.104,105 An aptamer for a desired target molecule is chosen
cancer cells.98,99 from a large pool of random DNA and RNA sequences
generated using automated oligonucleotide synthesis methods
3.2 DNA hybridization biosensors by successive cycles of binding to the immobilized target
molecule, followed by removing unbound material, and repli-
3.2.1 Background. Nucleic acid layers can also be used as cating the bound nucleic acid strands for another round of
the biorecognition element coupled with electrochemical trans- SELEX using the polymerase chain reaction (PCR). Chosen
ducers in affinity biosensors. Electrochemical DNA hybridi- aptamers after several cycles of SELEX can also be chemically
zation biosensors are useful in the diagnosis of genetic or modified to increase their stability and affinity for a target
infectious diseases, in environmental monitoring, to detect molecule. Once the sequence of nucleic acids in an aptamer for
microorganism contaminants in food and beverages, and for a specific target is known, the aptamer can be synthesized in
national defense applications, among others.5 large quantities. Like other biological molecules, aptamers
are sensitive to their environment and have to be protected
3.2.2 Detection mechanism. Complementary DNA base- from high temperatures and DNAase enzymes. A variety of
pairing is the basis for the biorecognition process in hybridi- strategies for developing aptamer-based electrochemical bio-
zation biosensors. Short, 20–40 basepair single-stranded DNA sensors are possible.106
segments with the ability to selectively bind with target analyte
are immobilized on the electrode surface.5 The DNA frag- 3.2.4 Applications of DNA sensors. Osmetech has commer-
s
ments have to be immobilized in a way that retains their cialized an electrochemical sensor (eSensor ) based on the
stability, reactivity, accessibility to target analyte and optimal selective reaction between a DNA capture probe immobilized
orientation.5 Sensor surface coverage by DNA probes is also on the electrode surface and target DNA in the sample.107,108
important in minimizing nonspecific binding.5,100 An electrical The biosensor uses a sandwich type assay as shown in
signal is produced when target DNA binds to the comple- Fig. 10A. Self-assembled monolayer (SAM) technology is used
mentary sequence of the capture or probe DNA in a process to create the chemical layer attached to the gold electrode. The
called hybridization. An electrochemical signal can result from monolayers are mixed SAMs, each comprised of a sequence-
an electroactive indicator that binds preferentially to the DNA specific capture probe (or probes) and an insulator com-
duplexes instead of single-stranded DNA probes such as ponent. The DNA capture probe is immobilized on the gold
ferrocenyl naphthalene diimide (FND).100 Electrochemical using an alkane thiol linker that projects it beyond a layer of
measurement of a catalytic product from a captured enzyme shorter alkane thiols. The shorter layer covers the surface
label such as horseradish peroxidase or alkaline phosphatase between the DNA capture probes and thereby minimizes
can also be used as a measure of hybridization.101,102 The interference from redox active materials in the sample and
enzymatic amplification of the binding event allows nonspecific adsorption, by blocking their access. Exposing the
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c The Royal Society of Chemistry 2010 Chem. Soc. Rev., 2010, 39, 1747–1763 | 1759
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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.)
electrode to the sample results in hybridization between the environmental, industrial, pharmaceutical, defense, and
capture probe and the complementary strand of the target security applications due to their superior sensitivity and
DNA. The capture probe is designed to be shorter than the selectivity. Although many electrochemical sensors are still
complementary target strand, leaving a segment on the target in the development and testing phases, some have reached
DNA where a signal probe containing an electroactive label the consumer market as handheld devices, portable units used
can bind. The label, ferrocene, is detected by measuring the for field measurements or are routinely used in a laboratory
peak current for its oxidation by a positive potential scan in ac setting. Recent developments in nanotechnology and mate-
voltammetry. The layer of alkane thiols is sufficiently thin as to rial science as well as being able to custom engineer the
not interfere with the electrochemistry. The current is propor- biorecognition component will further push the develop-
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tional to the target DNA concentration in the sample. As ment of useful and reliable biosensor devices. The sometimes
shown in Fig. 10B, genotyping can be done using different limited shelf life and stability of the biorecognition component
ferrocene labels with distinguishable electrochemical poten- as well as nonspecific binding continue to be the biggest
tials for each label. The biochip consists of a microarray of limitations of biosensors. However, many strategies have
72 working electrodes, a Ag/AgCl reference electrode and two helped with overcoming or minimizing these problems.
auxiliary electrodes (Fig. 10C). Each working electrode of the
array can be interrogated independently which allows multiple
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