IVD Technology Magazine
IVDT Article Index

Originally published July, 1997

Electrochemical biosensors for affinity assays

Part 1

Mark S. Vreeke

With the commercial success of blood glucose monitors, IVD firms are exploring ways to realize the potential of biosensor technologies.

Note: this is the first part of a two-part article. Part 2 is also available for on-line reading.

While most of the diagnostic market remains stagnant, the point-of-care testing market is booming, growing at an annual rate of 25%, from $600 million in 1994 to a projected $1 billion in 1997.¹ Biosensors promise low-cost, rapid, and simple-to-operate analytical tools. They therefore represent a broad area of emerging technology ideally suited for point-of-care analysis. The following article provides a summary of biosensors, electrochemical detection, and their combination to yield an electrochemical affinity assay.

Figure 1. Schematic of the generic biosensor strategy.

Biosensors are analytical tools combining a biochemical recognition component with a physical transducer (Figure 1).^2­5 The biological sensing element can be an enzyme, antibody, DNA sequence, or even microorganism. The biochemical component serves to selectively catalyze a reaction or facilitate a binding event. The selectivity of the biochemical recognition event allows for the operation of biosensors in a complex sample matrix, i.e., a body fluid. The transducer converts the biochemical event into a measurable signal, thus providing the means for detecting it. Measurable events range from spectral changes, which are due to production or consumption of an enzymatic reaction's product/substrate, to mass change upon biochemical complexation. Examples of biochemical recognition and transducer are provided in Table I.

Table I. Biosensor strategies. (FET = field-effect transistor; NADH = nicotinamide adenine dinucleotide.)

This article discusses the development of a specific type of biosensor, an amperometric enzyme electrode. With enzyme electrodes, the biochemical recognition element is provided by an enzyme that selectively catalyzes reaction of a substrate to produce an electroactive product. Amperometric detection transduces the flux of electroactive product to a measured current. A previously established relationship between transducer output and analyte concentration (calibration curve) allows for quantitation of the analyte.

Enzyme Function

Enzymes are nature's catalysts. Like all catalysts, they increase the rate at which a reaction reaches equilibrium by providing a low-activation energy reaction pathway. They usually operate in approximately neutral pH at mild temperatures, generate no by-products, and are highly selective. Enzyme-catalyzed reactions can be selective for one substrate or a group of substrates. They are also stereoselective and stereospecific. These characteristics have resulted in frequent use of enzymes in analytical applications.

Several thousand enzymes have been isolated, and several hundred are available commercially. They are classified by the reactions they catalyze. With amperometric enzyme electrodes, oxidoreductase enzymes are most frequently used. Oxidoreductases catalyze the oxidation (removal of electrons) or reduction (addition of electrons) of the enzyme substrate. Since oxidoreductases are most closely associated with electrochemical processes, their turnover is easiest to observe by electrochemical detection.

Enzymes, like all proteins, are made of amino acid chains folded into specific three-dimensional structures. They range in size from 10,000 to several million daltons. Besides amino acids, many enzymes also contain prosthetic groups--nicotinamide adenine dinucleotide (NADH), flavin (FAD), heme, Mg⁺², and Ca⁺²--that enhance enzyme activity. With oxidoreductases, the prosthetic groups serve as temporary traps of electrons or electron vacancies.

The prosthetic groups can be near the surface or deep within the enzyme's protein structure. In the latter case, the trapped charge is not easily transferred to an electrode. According to Marcus theory, which is used to explain electrochemical reaction rate, electron transfer decays exponentially with distance. Therefore, electron transfer from an active site near the center of a 100-kD protein to an electrode is highly improbable.

From a biological perspective, concealment of the active site is often necessary for selective targeting or redox reactions toward a specific synthetic or degradative route. For electrochemical biosensor applications, however, the difficulty of electron transfer into or out of the active site poses a real problem. Mediators capable of accessing the active site are frequently used to assist in the transduction of the enzyme activity into a measurable amperometric response.

Amperometric Detection⁶

High sensitivity, selectivity, and ability to operate in turbid solutions are advantages of electrochemical biosensors. Amperometric detection is based on measuring the oxidation or reduction of an electroactive compound at a working electrode (sensor). A potentiostat is used to apply a constant potential to the working electrode with respect to a second electrode (reference electrode). A potentiostat is a simple electronic circuit that can be constructed using a battery, two operational amplifiers, and several resistors. The applied potential is an electrochemical driving force that causes the oxidation or reduction reaction. The potential of the reference electrode is well defined through equilibrium, as in the following reaction:

Provided Cl^­ concentration is fixed, the subsequent reaction produces a stable potential.

The current response can be defined mathematically using Faraday's law:

where the current in amperes (I) represents the electrochemical oxidation or reduction rate of the analyte at the working electrode, da/dt is the oxidation or reduction rate in mols^­1, F is Faraday's constant, and n is the number of electrons transferred. The reaction rate depends on both the rate of electron transfer at the electrode surface and analyte mass transport.

With most redox reactions, the rate of electron transfer can be accelerated by increasing the potential at which the electrode is poised. As the potential is increased, the reaction reaches the point where the rate is limited by the mass transport of reactant to the electrode. When the reaction at the electrode surface is sufficiently fast, the concentration of analyte at the electrode is zero, and a maximum overall rate of reaction is reached. This overall rate is limited by the rate of mass transfer given by the following equation:

where dC/dX is the flux of C (electroactive species) to the electrode surface, A is the electrode area, and D is the diffusion coefficient. The rate of mass transport to the electrode surface depends on the bulk concentration of analyte, the electrode shape and area, and diffusion and convection conditions.

Use of amperometric enzyme electrodes is complicated by the enzymatic generation of the electroactive component from the analyte (enzyme substrate). The concentration of the electroactive component at the surface of the electrode is affected by diffusion of the product through the enzyme layer, the activity of the enzyme, and diffusion of analyte.

History of Amperometric Biosensors

The history of amperometric biosensors is linked with that of blood glucose monitoring, which has a world market in excess of $2 billion. Blood glucose is the most common analyte measured after electrolytes and blood gases. The largest growth in blood glucose measurement has been with the home monitoring devices.

Most published work on amperometric enzyme biosensors has targeted blood glucose monitoring. Amperometric biosensors have been divided into three generations. The first-generation biosensors were proposed by Clark and Lyons and implemented by Updike and Hicks, who coined the term enzyme electrode.^7,8 Typically, an oxidase enzyme, i.e., glucose oxidase (GOX), is immobilized behind a dialysis membrane at the surface of a platinum electrode. The enzyme's function is to selectively oxidize analyte by the reduction of O_² to H_²O_²; i.e., GOX selectively catalyzes the following two reactions:

GOX-FAD and GOX-FADH^₂ represent the oxidized and reduced states of the glucose oxidase enzyme's flavin active site.

The consumption of O^₂, or, as first described by Guilbault and Lubrano, the formation of H^₂O^₂, is subsequently measured at a platinum electrode. In the 1970s, Yellow Springs Instruments (Yellow Springs, OH) was the first company to successfully market an amperometric biosensor, a benchtop glucose analyzer (see Table II). Today most commercial benchtop amperometric biosensors rely on reactions catalyzed by oxidase enzyme and subsequent detection of H^₂O^₂ on platinum electrodes. A universal problem with this sensor arrangement is the loss in selectivity between the biorecognition event and the amperometric H^₂O^₂ detection. The highly oxidizing potential (700 mV versus Ag/AgCl) necessary for H^₂O^₂ oxidation results in substantial interference from the oxidation of other compounds in complex matrices.

Table II. Enzyme electrode devices.

Second-generation biosensors use an artificial electron mediator, which replaces O^₂ as the electron shuttle. Ferrocene, quinones, quinoidlike dyes, organic conducting salts, and viologens have been used as mediators. Most oxidase enzymes are not selective with respect to oxidizing agent, allowing substitution of a variety of artificial oxidizing agents, as in the following reaction:

Eliminating the O^₂ dependence of the first-generation method facilitated control of the enzymatic reaction and sensor performance. Specifically, the selection of mediators with appropriate redox potentials allows poising of the working electrode in a potential range where other components in the sample matrix are not oxidized or reduced.

Low O^₂ solubility in aqueous solutions and the difficulty associated with controlling the O^₂ partial pressure were disadvantages of biosensors based on the O^₂/H^₂O^₂ reaction. When a highly soluble artificial mediator is used, the enzyme turnover rate is not limited by the cosubstrate (O^₂) concentration. Use of mediators other than O^₂ allows exploitation of other oxidoreductase enzymes, including peroxidases and dehydrogenases. Unlike oxidases, these enzymes cannot use O^₂ as an electron-accepting cosubstrate.

Second-generation biosensors have been commercialized, mostly in single-use testing format. MediSense (Waltham, MA) was the first company to launch a second-generation product. Again the application was blood glucose monitoring, but this device was for home use. The mediation was provided by a ferrocene species. In March 1996, Abbott Laboratories (Abbott Park, IL) acquired MediSense for $876 million. Other second-generation amperometric biosensors have subsequently come onto the market.

Third-generation sensors are marked by the progression from use of a freely diffusing mediator (O^₂ or artificial) to a system where enzyme and mediator are coimmobilized at an electrode surface, making the biorecognition component an integral part of the electrode transducer. Coimmobilization of enzyme and mediator can be accomplished by redox mediator labeling of the enzyme followed by enzyme immobilization, enzyme immobilization in a redox polymer, or enzyme and mediator immobilization in a conducting polymer. There are even reported cases of direct electrical contact of enzyme to electrode. Whether this is direct electrical connection or mediation by surface functionalities is a matter of debate.

Third-generation biosensors offer all the benefits of second-generation sensors, and some new ones as well. The latter arise from the self-contained nature of the sensor. Since neither mediator nor enzyme must be added, this design facilitates repeated measurements. Sensor use for multiple analyses minimizes cost pressures on sensor design. It also follows that such a sensor could allow for continuous analyte monitoring. TheraSense, Inc. (Alameda, CA), is researching continuous blood glucose monitoring using wired enzyme technology.

Figure 2. Mediated electron transfer. Enzyme-immobilized mediators ( ) allow electron transport between an enzyme active site and an electrode surface by shortening the electron tunneling steps. FADH^₂=flavin adenine dinucleotide.M

Figure 3. Structure of enzyme "wiring" polymers. Variation of X, Y, and Z allow the properties of the polymer to be customized.

Degani and Heller coined the term wired enzyme to describe enzymes with covalently attached redox mediators (Figure 2). The enzyme was in effect wired by the mediator to an electrode. The wired enzymes were able to transfer redox equivalents from the enzyme's active site through the mediator to an electrode.¹⁰ The wired-enzyme principle resulted in subsequent development of enzyme-immobilizing redox polymers (Figure 3).^11,12

Figure 4. The redox cycles occurring at a three-dimensional redox epoxy-wired enzyme electrode. The wired enzyme is a flavin (FAD) containing oxidase enzyme.

These polymers effectively transfer electrons from glucose-reduced GOX flavin sites to polymer-bound redox centers. A series of chain redox reactions within and between polymers transfer the equivalents to an electrode surface (Figure 4). The redox enzyme and wire are immobilized by cross-linking to form three-dimensional redox epoxy hydrogels. A large fraction of enzymes bound in the three-dimensional redox epoxy gel are wired to the electrode. These wires provided a general approach to third-generation biosensors, sensitive not only to glucose but also to sarcosine, L-lactate, D-amino acids, L-glycerophosphate, cellobiose, and choline.

References

1. "Diagnosis: Growth Accompanied by Anxiety," Med Ad News, 14(3):1, 1995.

2. Ho MYK, and Rechnitz GA, "An Introduction to Biosensors," in Immunochemical Assays and Biosensor Technology for the 1990s, Nakamura RM, Kasahara Y, and Rechnitz GA (eds), Washington, DC, American Society for Microbiology, pp 275­290, 1992.

3. Rechnits GA, "Biosensors," Chem Eng News, 5:24­36, 1988.

4. Scheller FW, Wollenberger U, Pfeiffer D, et al., "Overview of Biosensor Technology," in Advances in Molecular and Cell Biology," Bittar E, Danielson B, and Bülow L (eds), Biochemical Technology Series, vol 15B, Greenwich, CT, JAI Press, pp 353­363, 1996.

5. Hall EAH, "Biosensors in Context," in Biosensors, Hall EAH (ed), Buckingham, Open University Press, pp 6­7, 1990.

6. Bard AJ, and Faulkner LR, Electrochemical Methods, New York, John Wiley & Sons, 1980.

7. Clark LC, and Lyons C, "Electrode Systems for Continuous Monitoring in Cardiovascular Surgery," Ann NY Acad Sci, 102:29, 1962.

8. Updike SJ, and Hicks GP, "The Enzyme Electrode," Nature, 214:986, 1967.

9. Guilbault GG, and Lubrano GJ, "An Enzyme Electrode for the Amperometric Determination of Glucose," Anal Chim Acta, 64:439­455, 1972.

10. Heller A, "Electrical Connection of Enzyme Redox Centers to Electrodes," J Phys Chem, 96:3579­3587, 1988.

11. Pishko MV, Katakis I, Lindquist S-E, et al., "Electrical Communication between Graphite Electrodes and Glucose Oxidase/Redox Polymer Complexes," Molec Cryst Liq Cryst, 190:221, 1990.

12. Gregg BA, and Heller A, "Redox Polymer Films Containing Enzymes,
1: A Redox-Conducting Epoxy Cement: Synthesis, Characterization, and Electrocatalytic Oxidation of Hydroquinone," J Phys Chem, 95:5970­5975, 1991.

Mark S. Vreeke, PhD, is a product development scientist at TheraSense, Inc. (Alameda, CA). This work was completed at the Department of Chemical Engineering and Materials Science and Engineering Center of the University of Texas at Austin. Support was provided by an H. H. Dow Memorial Award, a Welch Fellowship, NSF, NIH, and the Department of Defense.

Continue to part 2 of this article.

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