Biosensors: Potentiometric and Amperometric Analytical, biological, and clinical chemists have for decades concerned themselves with the development of instruments and techniques capable of determining the identity and concentration of chemical substances that affect living things. Recently this endeavor has focused on biosensors, small portable or disposable sensing devices that can be used to detect biologically important substances in vivo or in vitro. Current research efforts on biosensors are proceeding on many different types of sensor principles. In this arti-
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cle, progress on potentiometric and amperometric designs is discussed. In the Oct. 1 issue of A N A L Y T I C A L C H E M -
ISTRY, coverage of the field of biosensors continues with information on optical and piezoelectric designs. An emphasis in these articles is on the prospects for real-world applications and commercialization. Electrochemical sensors are of two types: potentiometric and amperometric. In potentiometric sensors, such as pH, Na + , and K + electrodes, the zerocurrent potential (relative to a reference) developed at a selective membrane or electrode surface in contact with a sample solution is related to analyte concentration. With amperometric sensors, the electrode potential is maintained at a constant level sufficient for oxidation or reduction of the species of interest (or a substance electrochemically coupled to it); the current that flows is then proportional to analyte concentration. Both types are being investigated for biosensing applications. Potentiometric electrodes The approach to the design of potentiometric electrodes t h a t has been adopted by Garry Rechnitz of the University of Delaware involves the design of biosensors that incorporate chemoreceptor structures (such as intact nerve cells), cellular materials from plant and animal sources, and immunoagents. One such receptor is de-
rived from blue crab antennules, which, in the living crustacean, enable the animal to follow food concentration gradients in murky waters. Rechnitz has demonstrated the detection of amino acids and nucleotides with electrodes on which blue crab sensor elements have been immobilized. Electrodes based on specialized plant structures include a "bananatrode," formed by the incorporation of banana slices onto an electrode surface. Because bananas contain an enzyme that metabolizes dopamine to melanin with the consumption of oxygen, Rechnitz has been able to apply the bananatrode to the detection of dopamine. In addition, he recently announced the development of a biosensor for antigen monitoring using immobilized monoclonal antibodies. Although real-world applications of sensors derived from plant and animal sources have been hampered by fragility, reproducibility, and stability problems, commercialization may be on the horizon, with development currently proceeding on fish freshness and milk bacteria sensors based on related principles. Rechnitz's message is, "If you're going to make a new biosensor, look at nature first to see if you can imitate it." Solvent-polymeric membrane electrodes are already commercially available and are routinely used for the selective detection of several ions (such as K+, Na + , Ca 2+ , NH 4 +, H+, and C0 3 2 ~) in complex biological matrices (1). Solvent-polymeric refers to a semirigid but liquid-state plasticized polyvinyl chloride (PVC) matrix that provides mechanical strength and also permits diffusion of species to binding sites in the membrane, such as neutral carriers or ion exchangers. Charge separation, and hence a change in potential, is generated across the interface when these binding sites selectively extract charged species into the membrane phase, leaving co-ions behind in the sample phase. Sensor selectivity is dictated by the specificity of these ionbinding or partitioning reactions. One of the best known examples of such an electrode is the K + electrode based on valinomycin, an antibiotic molecule with an electron-rich pocket into which
K + ions are selectively extracted. Recent research efforts have concentrated on expanding the range of species that can be determined with solvent-polymeric membrane electrodes to blood gases and additional anions. According to Mark Meyerhoff of the University of Michigan, "We've focused on making very simple, inexpensive, catheter-size electrodes for blood gases because the current electrodes are very expensive and fragile, with glass electrodes inside. And although considerable work has been done on the development of new carriers for cations, anion sensing has been a real
A severe difficulty in the widespread application of electrochemical techniques based on faradaic reactions is the problem of electrode poisoning . . . %% void for this type of electrode. There are anion sensors based on solid-state electrodes, but these don't work very well for biological samples." Research by Meyerhoff on the use of metalloporphyrin molecules as anion carriers or exchangers in PVC membranes has shown that potentiometric anion selectivity can be altered by changing the metal center or by steric control over which anions can be accommodated as axial ligands (perpendicular to the plane of the metal) within the porphyrin structure. Abnormal pressures of blood gases can be indicative of respiratory or metabolic disorders, necessitating frequent blood gas analysis for many surgical and intensive care patients. This type of test is normally run on discrete blood samples using commercially available blood gas analyzers. However, in a new generation of potentiometric gas sensors developed in Meyerhoff s laboratory for blood gases such as CO2, neu-
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FOCUS tral carrier-based membranes are used as inner transducers in conventional gas-sensing arrangements. An example of this type of design is a gas-sensing catheter suitable for continuous in vivo monitoring (2). A polymeric membrane-based flowthrough sensor for creatinine in blood that uses an immobilized enzyme to convert creatinine to ammonia is presently undergoing commercialization. The sensor is based on a dual-electrode sensor for CO2 in blood that operates in a differential measurement mode. The CO2 sensor will be incorporated into an instrument system that is used to monitor patients in open-heart surgery. "The most successful electrodes for in situ biological measurements have been the solvent-polymeric membrane types," says Meyerhoff. "The question is how to extend the menu to measure additional analytes using these membranes." Another potentiometric electrode concept attracting interest for biosensing applications is the chemically selective field effect transistor (chemFET), an F E T device that has been modified to respond to the presence of ions or other species through charge separation at an interface. A chemFET is an insulated gate F E T in which the metal gate contact has been replaced by a chemically modified layer capable of extracting or donating charge with respect to an aqueous sample solution. The resulting charge separation generates a potential that modulates the FET's source-to-drain current, and it is this modulation that serves as an indicator of analyte concentration. Types of chemFETs include the following: ion-selective FETs, which respond to ions in solution; enzyme FETs, in which immobilized enzymes are used to measure enzyme substrates or species that are coupled to enzymatic reactions; immunochemical FETs, which generate charge separation via antibodyantigen interactions; and suspended gate FETs, the operation of which is based on changes in work function and dipole orientation that result from the interaction of the sensing element with various gases. For example, palladium, which dissolves hydrogen, is used in the sensing element of a suspended gate F E T for the detection of hydrogen gas. A device related to the suspended gate F E T is a recently developed ammonia-sensitive metal oxide semiconductor (MOS) that uses a catalytic metal such as iridium as part of the sensing element (4). Whereas chemFET responses are monitored by measuring voltage-induced changes in the conductivity of the channel region of
the FET, the ammonia-sensitive MOS device is based on the measurement of capacitance changes in this region. The Ir-MOS sensor, which operates either in the gas phase or, with the addition of a gas-permeable membrane, in aqueous solution, has already proven useful for the determination of ammonia in various biological and nonbiological solutions, such as whole blood, serum, rainwater, and river water.
When working with cells that have micrometer dimensions, you don't want to destroy everything you're trying to make measurements on. % % A major advantage of the chemFET is its small size, which creates opportunities for multiplexing (combining different ion-selective gates on the same chip) and for incorporation into miniaturized analytical systems involving microliter solution volumes (e.g., flowinjection analysis on a microscale). Another advantage is a potential for low production costs using microlithographic techniques. Before this cost advantage can be realized, however, a major problem with encapsulation must be solved. The encapsulation problem involves a need to expose the chemically selective layer to a sample solution while simultaneously preventing the electrical transducer from shorting out from exposure to moisture. "People claim to have solved the encapsulation problem," comments one observer, "but then you ask about manufacturing feasibility. You start out with a chemFET that costs less than a dollar, but after encapsulation the cost becomes $50 to 100. Conventional pH electrodes can be purchased at comparable cost, so what has been gained? What we need to find is a more manufacturable way to encapsulate and still keep the cost down." Amperometric electrodes Chemically modified electrodes have stimulated considerable interest over the last few years as researchers have attempted to exert more direct control over the character of the electrode surface. "Designing molecular films on electrodes gives you the flexibility to make a reaction more sensitive or selective, based upon the rational application of chemistry," explains
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Royce Murray of the University of North Carolina. The concepts that are being investigated in this area fall into five major categories (Reference 5): • Electrocatalysis involves electron transfer mediation by an immobilized catalyst or molecular film between the electrode and the substrate or analyte solution, resulting in faster electrode reactions at less extreme applied potentials. • Preconcentration involves the use of a molecular film that exhibits a significant partition effect for a substrate, thus presenting the electrode surface with a higher concentration of that substrate. • Membrane barriers are extremely thin, pinhole-free coatings that exhibit unfavorable partition toward undersired solution constituents, such as substances that act as interferences. • Electroreleasing involves the expulsion of species or reagents from an electrode film by electrode-driven changes in film oxidation state, the purpose being to release controlled microdosages of these substances into the solution phase. • Microstructures are miniaturized electrodes with polymeric molecular films. This includes the concept of using microlithography to miniaturize electrodes and polymer films, as in Jiri Janata's work on chemFETs. According to Murray, "We're learning the chemistry and microfabrication technology to make chemical sensors of unique sizes and physical designs. This may be important for making sensors that are small, simple, and disposable, including in vivo sensors." One of the most promising aspects of this research effort involves the use of microstructuring or miniaturization to design entirely solid-state electrochemical cells that require no liquids to function. "You can add the sample as a liquid and let it be absorbed by the polymer film, which is a semirigid ionically conducting phase, or you can add the sample as a gas," says Murray. "This type of electrode, which could be manufactured by lithographic techniques, would be simple enough to be used by consumers and is potentially disposable. A severe difficulty in the widespread application of electrochemical techniques based on faradaic reactions is the problem of electrode poisoning; keeping electrodes clean takes a lot of skill and fussing about. Never having to use an electrode more than once totally short-cuts that problem. That's an element of where we're going with the solid state, and that's one thing that makes it important." Mark Wightman of Indiana University has also been designing ampero-
FOCUS metric microelectrodes (6), in this case for the determination of neuro transmitter substances in mammalian brain tissue. According to Wightman, several requirements must be met in developing a successful sensor for the in situ determination of these com pounds. "When working with cells that have micrometer dimensions, you don't want to destroy everything you're try ing to make measurements on," says Wightman. Therefore, the electrode must be sufficiently small to prevent damage to the cells surrounding the measurement location. To address this problem, Wightman's group has fabri cated electrodes from single-carbon fi bers with a radius of only 5 Χ 10 - 4 cm. For neurotransmitter determina tions, the analyst must be concerned with the temporal response of the mea surement system. "We're not only in terested in measuring basal levels of these substances," says Wightman. "We want to see how concentrations change in response to different stimu li." To make it possible for neurosensors to track relatively fast-changing chemical events in the brain, experi ments may be performed in a fast-scan mode (300 V/s).
In addition, the voltammetric probe must exhibit chemical selectivity. Wightman has addressed this problem through the coating of ionomeric films onto electrode surfaces. For example, a cation exchange membrane barrier can be used to exclude anions such as ascorbate from the electrode surface. Ascorbate, which is present at relatively high concentration in brain tissue, is an in terfèrent in the electrochemical detection of neurotransmitters. According to Wightman, stability and sensitivity problems with neurochemical microelectrodes still need to be overcome. "Ideally you'd like these in vivo sensors to be stable for a number of years," he says. "I don't think anybody has been able to do that yet. That's one of the big challenges for the future. In addition, we will need to have electrodes with sensitivity below 100 nM to see the true resting state of neurotransmitters in extracellular brain fluid. We haven't achieved that yet, but our sensitivity is getting better and we are approaching this goal." Although enzyme electrodes have not yet attained widespread commercialization, they have attracted a tremendous amount of research interest. The concept is to immobilize an en-
zyme atop a conventional ion-selective electrode. The electrode is then used to measure a substrate or product of the enzyme, either potentiometrically or amperometrically (7). A classic example is the glucose electrode using immobilized glucose oxidase. The enzyme electrode concept recently has been further extended by the immobilization of antibodies or antigens to form immunochemical sensors. The electrochemical enzyme immunoassay (8) is based on the labeling of antibody or antigen species with an enzyme that catalyzes the production of an electroactive product. There are two basic types of immunoassay: heterogeneous, in which antibodybound antigen must be separated from free antigen at some point in the procedure, and homogeneous, in which no separation step is required. Because the homogeneous assays lack a separation step, they tend to be simpler and faster than heterogeneous assays but they also suffer from poorer detection limits and a higher susceptibility to interferences. "In terms of detection limits," explains William R. Heineman of the University of Cincinnati, "the best we've done—and although this is not
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FOCUS necessarily representative, it does show what can be achieved—is the determi nation of about 10~18 mol of immunoglobulin-G with a heterogeneous sand wich immunoassay on 20-μΙι samples. We're at the limit set by the formation constant between the antibody and antigen. Therefore, it is difficult to go down to lower concentrations, but we can go to smaller samples. In some in stances, this could be advantageous. If we lower the volume to the nL range using microelectrodes, I can envision getting down to 10 _19 -10~ 20 mol of ma terial." One of the restrictions of electro chemistry over the years was that a ma terial had to be electroactive. If it wasn't, you had to make it electroactive in one way or another. An advantage of the electrochemical immunoassay is that the analyte doesn't have to be elec troactive as long as the analyte or an antibody to the analyte can be labeled with an enzyme. In biological applications, electrode fouling from matrix constituents tends to be a troublesome problem. In het erogeneous electrochemical immuno assays, the biological matrix is washed away in the separation step and fouling is not a problem. However, the fouling
Almost every major instrument, pharmaceutical, and biotech firm has a project or two going on biosensors, although they may not want you to know it. %% problem has proved to be more of a challenge in the case of homogeneous assays. One recent approach involves the use of gamma radiation to form size-exclusion polymer networks on electrode surfaces (9) to protect them from fouling by blood plasma proteins. George Wilson of the University of Kansas (formerly of the University of Arizona) has demonstrated an electro chemical immunoassay system amena ble to total automation (10), in which antibodies and antigens are attached covalently to a chromatographic sup port material to form a reproducible immunosorbent of high biological ac
tivity and stability. The immunosor bent is packed into a flow-through microreactor in a flow-injection analysis system with fluorometric or thin-layer amperometric detection. Using this system, Wilson has achieved subpicomole detection limits with precisions of 2-3% for determinations of immunoglobulin-G in serum for both competi tive and sandwich assays. The immu nosorbent reactor is stable for up to 500 assays. The biological activity of covalently immobilized antibodies is strongly af fected by their orientation on surfaces. Through control of this orientation pa rameter with new coupling chemistries, Wilson has been able to achieve immo bilized antibody activities of better than 70%, a considerable improvement over the 1-10% activities that are com monly experienced in this line of re search. A new "uphill transport membrane sensor" recently introduced by Yoshio Umezawa of Hokkaido University (11) uses the biological principle of active transport to enhance the sensitivity and selectivity of the electrochemical response. In biological systems, the transport of metabolites or nutrients against their concentration gradients
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FOCUS (i.e., toward areas of higher concentration) is accomplished by active transport across cell membranes. Umezawa has been able to mimic this uphill transport phenomenon by incorporating lipophilic liquid membranes into electrodes. The uphill transport of ions or molecules through these artificial membranes is driven by an external source of energy, such as complexation, pH gradients, concentration gradients of other ions, redox energy, or UV-visible radiation. This process selectively boosts the virtual concentration of specific analytes by uphill transport against their concentration gradients across the liquid membrane into an inner electrode filling solution, the volume of which is purposely made very small. Biosensors: Research curiosity or commercial reality? As research proceeds on amperometric and potentiometric biosensors, commercial products are beginning to emerge. For example, a new glucose sensor for home use by diabetics based on a collaborative effort between H. Allen 0 . Hill's group at the University of Oxford and a group at the Cranfield Institute of Technology is expected to be on the market by the end of the year. The sensor, which is based on a technique originally published in ANALYTICAL CHEMISTRY (22), is a ballpoint pen-shaped device (13) with solid-state preprogrammed electronics and a liquid crystal display readout on the side. After insertion of a disposable, singleuse electrode into the lower part of the sensor, a drop of blood is placed on the free end of the electrode, which is coated with glucose oxidase. Glucose oxidase then catalyzes the oxidation of glucose in the sample to gluconic acid, with mediation of electron transfer to the underlying electrode by a ferrocene derivative. The glucose meter will be marketed in the U.S. by Baxter Travenol and manufactured in Abingdon, England, by Genetics International. Another commercial development of note is the recent introduction of solid-
state multispecies microsensors by Integrated Ionics, Inc. (Dayton, N.J.). The sensors consist of arrays of miniaturized chemically sensitive membranes that are incorporated into integrated circuit chips containing operational amplifiers and signal-processing electronics. Applications foreseen by the company include clinical analysis and on-line patient monitoring. Despite commercial developments such as these, and despite strong interest in biosensors by academic researchers, industrial people, and potential users, doubts have been expressed in some quarters about near-term prospects for the widespread appearance of commercial devices. As one academician says, "I am cautious in talking about commercial sensors, in that there are a tremendous number of problems in a practical sense that one has to solve to take an analytical device that has sophistication from the research laboratory to a marketable item." The negative opinions of others on biosensors frequently are expressed even more bluntly. An example is the following quote from a scientist whose company, a major U.S. corporation, is evaluating the feasibility of commercial biosensors: "Our perception is that, when you compare the R&D cost for development of a biosensor to the market for that biosensor, you find that there will not be a significant market. The estimated sales value for a biosensor is in the $10-100 million range. For that size market, the R&D cost is probably going to be prohibitive." (The scientist requested anonymity because the interview was not approved by his company, although he believes that his views reflect corporate thinking on the matter.) "People have been working on the drug delivery-glucose loop for 20 years," he continues. "The problems are astronomical, and no one's solved those problems yet. I have no argument with people continuing to work on that kind of thing, but most of the problems that those people face are compounded when you look at analytes at a lower
40th Summer Symposium on Analytical Chemistry This two-part series on biosensors was inspired by the 40th annual Summer Symposium on Analytical Chemistry, "Biosensors." The symposium, which is sponsored by the ACS Division of Analytical Chemistry and ANALYTICAL CHEMISTRY, was held June 28-July 1 at Indiana University. The attendance of nearly 300 individuals was especially high for a summer symposium, reflecting intense interest in biosensors among people in academia, government, and industry. The general chairman of the symposium was Gary Hieftje of Indiana University; the program chairman was William Heineman of the University of Cincinnati.
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concentration than glucose. For example, when you start talking about a biosensor for nitroglycerin that would go inside a person's body, you have biocompatibility problems, you have sensitivity problems, and you have selectivity problems, and most of what we've seen in the literature is what we call hype. People have been hyping this up for a long time. We think that you are not going to make a biosensor before you make a sensor for some other purpose, such as process control·— something that will go in a plant where, if it fails, it may cost a lot of money, but it won't kill anybody." This perspective is countered by an academic researcher who also requested anonymity: "I think academicians would agree that there is hype, that some sensors are being oversold too early, and that there are problems in adapting it to the end-user. I wouldn't quarrel with that. But I think it's an overstatement to say that biosensors aren't any good, that you shouldn't even bother, that there's no market, etc. "I get a little annoyed with these industrial people," he continued, "because on the one hand they say that biosensors aren't worth the trouble, but at the same time they come snooping around in your lab trying to get the latest advantage for their company. You can't have it both ways; it's just not honest. The actual fact is that almost every major instrument, pharmaceutical, and biotech firm has a project or two going on biosensors, although they may not want you to know it. To say there is no interest in biosensors is absolutely wrong. Never have we worked on anything where we've seen so much interest." Stu Borman References (1) Czaban, J. D. Anal. Chem. 1985, 57, 345-56 A. (2) Opdycke, W. N.; Meyerhoff, M. E. Anal. Chem. 1986,58,950-56. (3) Cassidy, J.; Pons, S.; Janata, J. Anal. Chem. 1986,58,1757-61. (4) Winquist, F.; Lundstrom, I.; Danielsson, B. Anal. Chem. 1986,58,145-48. (5) Murray, R. W.; Ewing, A. G.; Durst, R. A. Anal. Chem. 1987,59,379-90 A. (6) Wightman, R. M. Anal. Chem. 1981, 53,1125-34 A. (7) Frew, J. E.; Hill, H. O. Anal. Chem. 1987,59,933-44 A. (8) Heineman, W. R.; Halsall, H. B. Anal. Chem. 1985,57,1321-31 A. (9) De Castro, E. S. et al. Anal. Chem. 1987,59,134-39. (10) De Alwis, W. U.; Wilson, G. S. Anal. Chem. 1985, 57, 2754-56. (An additional paper on this topic has been accepted for publication in an upcoming issue of ANALYTICAL CHEMISTRY.) (11) Uto, M. et al. Anal. Chem. 1986, 58, 1798-1803. (12) Cass, A.E.G. et al. Anal. Chem. 1984, 56,667-71. (13) Matthews, D. R. Lancet, 1987, April 4,778-79.
