Journal of Biotechnology, 15 (1990) 201-218

201

Elsevier BIOTEC 00445

Biosensors based on cell and tissue material G.A. R e c h n i t z 1 a n d M.Y. H o 2 l Department of Chemistry, University of Hawaii, 2545 The Mall, Honolulu, H I 96822, U.S.A. and 2 National Cancer Institute, Frederick, MD 21701, U.S.A.

(Received22 June 1989; accepted 14 August 1989)

Enzyme electrode; Amino acid; Biomonitoring; Bioanalysis

Introduction

Present trends in biotechnology suggest that monitoring fermentation processes and antibody production by means of biosensors may be of crucial importance in the future. Although many technical problems need yet to be overcome, biosensors offer a prospect of measuring biomolecules important in biotechnology with convenience and speed, as well as at low cost. The concept of biosensors can be traced back to the idea of using enzymes immobilized at the tip of an electrochemical sensor (Clark and Lyons, 1962), and the first enzyme electrode was described (Updike and Hicks, 1967) for the determination of glucose in biological samples. Since then, the field of biosensors has proliferated, recently resulting in a book devoted to the subject (Turner et al., 1987). Biosensors are devices which combine mediators, e.g. molecular recognition elements usually of biological origin, with some type of transducer or detector. Fig. 1 shows a typical configuration for a biosensor. The mediator is in intimate contact with the detector and serves to convert species of interest to products which can generate analytical signals registered by the detector, thus allowing the determination of substrates. Biosensors derive their specificity and sensitivity from nature. Ideally, biosensors can be used directly in sample matrices without pretreatment. Apparent advantages, from the synthesis of two disciplines include short response times, simplicity, specificity, portability, possible miniaturization and on-line monitoring. Thus, these devices have attracted (Biosensors, 1987a, b) considerable Correspondence to: G.A. Rechnitz, Department of Chemistry, University of Hawaii, 2545 The Mall, Honolulu, HI 96822, U.S.A.

0168-1656/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

202

DETECTOR

u

MEDIATORS

Fig. 1. Basic configuration of a biosensor. interest in the areas of: (a) h u m a n health care; (b) veterinary health care; (c) bioprocess control; (d) defense. The field of biosensor research is a very diverse one and often d e m a n d s knowledge from several disciplines as is reflected b y the possible c o m b i n a t i o n s of biological c o m p o n e n t s (e.g. enzymes, bacterial cells, a n i m a l / p l a n t tissue, organelles, organisms, receptors, antibodies, nucleic acid and organic molecules) and detectors (e.g. potentiometric, amperometric, conductimetric, optical, calorimetric, acoustic and mechanical) to appreciate the vastness of the subject. However, the following discussions will be limited to biosensors with electrochemical sensors as detectors. Emphasis will be placed on potentiometric electrodes, since they have been employed most extensively as detectors.

Potentiometric electrodes Potentiometric m e m b r a n e electrodes, such as pH, solid-state, p o l y m e r and gassensing electrodes, have been used as internal sensing elements for biosensors. A typical classification of these ion-selective electrodes is shown in Table 1. Gas-sensing electrodes are by far the most p o p u l a r choice due to the inherent selectivity of gas-permeable m e m b r a n e s (Arnold, 1983a). Special attention will be given here to the c a r b o n dioxide and a m m o n i a gas-sensing electrodes since they are i m p o r t a n t to the present state of the art.

TABLE 1 Classification of ion-selective electrodes based on the compositions of their sensor membranes (a) Glass electrodes, e.g. H ÷, Na ÷, monovalent cations. (b) Solid-state electrodes, e.g. F-, Ag+/S 2 , CI-, Br-, I-, CN-, SCN , Cd 2+, Cu 2+, pb2+. (c) Liquid ion-exchange membranes electrodes, Ca 2+, CI-, divalent cations, BF4 , N O 3 , CIO4-, K +. (d) Electrodes with coating over membranes of ion-selective electrodes, e.g. CO2, NH 3, H2S, NO2, SO2.

203

--reference

element

outer b o d y - -

sensing

J

element solution

Fig. 2. Configuration of a gas-sensing electrode.

Like other gas-sensing membrane electrodes for ammonia, hydrogen sulfide, sulfur dioxide and nitrogen dioxide, the carbon dioxide electrode consists of a hydrophobic gas-permeable membrane and a p H electrode separated by a thin layer of internal electrolyte (Orion, a). Its basic configuration is shown in Fig. 2. The differences between the several gas-sensing electrodes are mainly apparent with respect to their internal tilting solutions and their operating p H ranges. The key principles of such gas-sensing membrane electrodes can be exemplified by those of the carbon dioxide electrode. Typically, the gas-sensing electrode and an external reference electrode are immersed in a sample solution. Dissolved carbon dioxide in the sample solution diffuses across the gas-permeable membrane into the internal electrolyte layer until the partial pressures of carbon dioxide on both sides of the hydrophobic membrane are equal. The carbon dioxide that diffuses across the membrane affects the internal filling solution according to: CO 2 + H 2 0 ~ H + + HCO 3 .

(1)

The internal p H electrode thus follows the p H changes in the internal electrolyte layer. Hence the carbon dioxide concentration in the sample solution can be quantitated by the Nernst equation, which is: E = E 0 + s log[CO2],

(2)

where E, measured potential; E 0, reference potential; s, constant; [CO2], concentration of carbon dioxide. The gas concentrations are related to their partial pressures through Henry's law. Such gas-sensing electrodes are quite selective since their membranes are permeable only to gases. However, for the case of the dioxide electrode, it has been found that volatility of the interferents affects the transient electrode characteristics (Kobos et al., 1982). While at equilibrium, the extent of interferences can be quantitated by the acidity of the interfering compounds (Lopez, 1984; Morf et al.,

204

1985). For the ammonia electrode, volatile amines (Fraticelli and Meyerhoff, 1981) and amine basicity (Lopez and Rechnitz, 1982) determine the extent of interference. Sample solutions have to be totally aqueous since the hydrophobicity of these gas-permeable membranes would be compromised by organic solvents and lead to membrane failure. For organic samples, an air-gap electrode can be a useful alternative to time consuming pretreatment steps (Ruzicka and Hassan, 1974). The high selectivity of gas-sensing electrodes has accounted for the voluminous literature on gas-sensing type biosensors (Arnold and Solsky, 1986; Arnold and Meyerhoff, 1984; Arnold, 1986). Response times can be defined as the time required to approach the steady-state potential within I mV (Guilbault et al., 1976). The response times of gas probes vary from less than half a minute to several minutes depending on the concentration of the dissolved gas and to a lesser extent, the stir-rate, p H and temperature. Response times are also dependent on the thickness and composition of the electrolyte layer, as well as the type and thickness of membrane material employed. Their recovery times can be quite long, but Arnold (1983) has shown that, for an ammonia gas sensor, a teflon membrane with pore size of 0.02 /zm is a good choice. Keeley and Walters (1983) further improved recovery times by conditioning the electrodes in buffers of nonphysiological pH, but some time is then needed to recondition the electrodes to sample conditions. The usefulness of gas-sensing electrodes is often hampered by p H limitations imposed by samples or by the biocatalysts used in the construction of biosensors. Compromises often have to be made in order to satisfy both the p H requirements of the electrodes and that of the biological components with the latter usually having stringent pH limitations due to their biological nature. The pH requirements of the gas-sensing electrodes themselves arise from the fact that the electrodes are responsive only to the gaseous form of the measured substrate. For carbon dioxide, low p H values favor carbon dioxide in the carbon dioxide/bicarbonate equilibrium (Orion, b) and high p H values favor the ammonia form in the a m m o n i a / a m m o n i u m equilibrium (Orion, c). The osmolarities of sample solutions and the internal electrolyte solutions have to be similar (Arnold and Rechnitz, 1984). If they are very different, water vapor will, like the gaseous substrate, diffuse through the membrane until water vapor pressures are equal on both sides of the membrane. This diffusion affects the stability of the baseline potential. If the internal electrolyte solution contains a relatively insoluble salt such as ammonium picrate, the water vapor diffusion will result in either concentration or dilution of the internal filling solution and thus affect the ammonium concentration which will in turn cause the baseline potential to drift. High sample osmolarity has also been shown to increase the electrode response times with aging of the electrode. This behavior is thought to be caused by the precipitation of an ammonium salt which is deposited as a solid layer on the membrane. The use of the more soluble ammonium chloride rather than ammonium picrate (which is present in the commercially available internal filling solutions) circumvents this problem (Arnold and Reehnitz, 1984). For the carbon dioxide electrode, the internal electrolyte solution contains the very soluble sodium bi-

205

carbonate salt. However, the ionic strength of the buffer system used has to match that of the internal filling solutions in order to minimize the osmolarity pressure gradient across the membrane. Ion-selective electrodes can tolerate a certain degree of turbidity in samples.

Biocatalytic membrane electrodes

Enzyme electrodes Enzymes are very efficient catalysts and their inherent selectivity makes them very attractive as part of biosensors for use in samples with complex matrices (Mascini and Guilbault, 1986). Enzymes are protein molecules with catalytic activities controlled by pH, ionic strength, temperature and the presence of co-factors. Enzyme stability is usually the deciding factor in determining the lifetimes of enzyme based biosensors. Whenever possible, commercial sources of enzymes are used since enzyme purification is tedious and technically challenging (Muller et al., 1978). Nevertheless, many novel enzyme electrodes have been developed (Table 2) (Guilbault and Shu, 1972; Jensen and Rechnitz, 1979; Deng and Enke, 1980; Arnold and Rechnitz, 1980; Nikolelis et al., 1981; Kovach and Meyerhoff, 1982; Pau and Rechnitz, 1984; Fonong and Rechnitz, 1984), It can be seen that lifetimes vary from hours to weeks depending on the particular enzyme employed.

TABLE 2 Some examples of enzyme electrodes Substrate

Biocatalyst

Internal sensing element

Adenosine

NH 3

9d

NH 3

10 d

Glutamine

adenosine deaminase L-alanine dehydrogenase glutaminase

NH 3

1d

Guanine

guanase

NH 3

42 d

Glueonate

gluconate kinase/ 6-phospho-Dgluconate dehydrogenase histidine decarboxylase salicylate dehydroxylase L-tyrosine decarboxylase

NH 3

4d

CO 2

30 d

CO2

12 d

COz

10 h

L-Alanine

Histidine Salicylate Tyrosine

Lifetimes

References

Deng and Enke (1980) Pan and Rechnitz (1984) Arnold and Rechnitz (1980) Nikolelis et al. (1981) Jensen and Rechnitz (1979)

Kovach and Meyerhoff (1982) Fonong and Rechnitz (1984) Guilbault and Shu (1972)

206 An early potentiometric enzyme electrode was described by Guilbault and Montalvo (1969) for the determination of urea. Sometimes, more than one enzyme can be used in conjunction with a sensor (Renneberg et al., 1986) and such multi-enzyme sensors broaden the range of substrates that enzyme-electrodes can measure while retaining high sensitivity and selectivity. The sensitivity of these sensors can also be enhanced by recycling of the co-immobilized co-factor (Pau and Rechnitz, 1984). Bacterial electrodes Owing to the high cost and limited lifetime of enzyme electrodes, efforts have been made to search for altemative biocatalysts to be used in the construction of biosensors. Bacterial cells contain enzymes that can catalyze the conversion of substrates into products detectable by electrochemical sensors. Since the enzymes are already contained in optimal environments provided by nature, the use of bacteria and other microorganisms may help in extending the lifetime of biosensors (Rechnitz et al., 1978). Other benefits associated with the use of bacterial cells as biocatalysts include reduced cost, regeneration of activity (Kobos and Rechnitz, 1977), and the ability to effect complex reactions, which simplifies the analytical systems (Kobos and Pyon, 1981) and provides for induction of enzyme activity (Dipaolantonio and Rechnitz, 1982). The major drawback of such biocatalytic membrane electrodes stems from the fact that bacterial cells contain a variety of enzymes, which may compromise the selectivities of these biosensors. Selectivities can, in certain cases, be improved with the use of enzyme and transport inhibitors (Dipaolantonio, 1983). Bacterial electrodes sometimes have low sensitivities and long response times due to insufficient biocatalytic activity and the low slopes of bacterial electrodes can often be attributed to insufficient co-factor levels accessible to the enzyme (Ho and Rechnitz, 1985). Bacterial contamination can also introduce selectivity problems with electrode aging, but this can be eliminated with the use of appropriate inhibitors (Corcoran and Kobos, 1983). Table 3 shows some examples of contemporary potentiometric bacterial electrodes (Rechnitz et al., 1977; Rechnitz et al., 1978; Kobos and Rechnitz, 1977; Jensen and Rechnitz, 1978; Riechel and Rechnitz, 1978; Kobos et al., 1979; Hikuma et al., 1980; Waiters et al., 1980; Grobler and Rechnitz, 1980; Kobos and Pyon, 1981; Dipaolantonio et al., 1981; Dipaolantonio and Rechnitz, 1982; Dipaolantonio and Rechnitz, 1983; Kawashima et al., 1984; Kobos, 1986). Plant/animal tissue electrodes Plant (Kuriyama and Rechnitz, 1981) and animal tissue (Rechnitz et al., 1979) electrodes were developed as a result of the successful use of bacteria as the biocatalyst in biosensors (Tables 4 and 5). Like the bacterial electrodes, they offer low cost, prolonged lifetime, simple construction, reduced co-factor requirements and high catalytic activity (Arnold and Rechnitz, 1981b; Sidwell and Rechnitz, 1986). Indeed, a comparison of enzyme, mitochondrial, bacterial and tissue electrodes for glutamine determination has shown that tissue electrode can be superior

207 TABLE 3 Some examples of bacterial electrodes Substrate

Biocatalyst

Internal sensing element

L-Arginine

Streptococcus f aecium

NH 3

20 d

L-Aspartate

bacterium cadaveris

NH 3

10 d

L-Cysteine

H2S

6d

CO2

21 d

Glutamine

Proteus morganii Escherichia coli Sarcina flava

NH 3

14 d

L-Histidine

Pseudornonas

NH 3

21 d

NH 3

> 7d

NH 3

14 d

NH 3

30 d

CO2

14 d

NH 3

3d

L-Glutamate

Lifetimes

sp. NAD + Nitrate Nitrilo-triacetic acid Pyruvate Serine Sulphate

Escherichia coli / N A D a s e Azotobacter vinelandii Pseudomonas

sp. Streptococcus faecium Clostridium acidiurici Desulfovibrio desulfuricans

Sulphide

10 d

Sugars

bacteria from dental plaque

CO2

3d

Tyrosine

Aeromonas phenologenes Pichia membranae faciens

NH 3

8d

CO2

50 d

Uric acid

References

Rechnitz et al. (1977) Kobos and Rechnitz (1977) Jensen and Rechnitz (1978) Hikuma et al. (1980) Rechnitz et al. (1978) Waiters et al. (1980) Riechel and Rechnitz (1978) Kobos et al. (1979) Kobos and Pyon (1981) Dipaolantonio and Rechnitz (1983) Dipaolantonio et al. (1981) Kobos (1986) Grobler and Rechnitz (1980) Dipaolantonio and Rechnitz (1982) Kawashima et al. (1984)

in terms of shelf life ( A r n o l d a n d Rechnitz, 1982a) a n d electrode lifetime ( A r n o l d a n d Rechnitz, 1980). Recently, the use of flower petals ( U c h i y a m a a n d Rechnitz, 1987a, b), leaves (Smit a n d Rechnitz, 1984; Smit, 1986) a n d fruit (Sidwell a n d Rechnitz, 1985) i n the c o n s t r u c t i o n of p l a n t tissue electrodes has also b e e n reported. Since flowers, leaves a n d fruits are structures related to p l a n t growth, r e p r o d u c t i o n a n d n u t r i e n t storage, they have high c o n c e n t r a t i o n s of biocatalytic activity. T h e m a g n o l i a petal electrode offers a n a d d i t i o n a l u n i q u e feature i n that n o s u p p o r t m e m b r a n e is needed, resulting i n a significant decrease i n response times ( U c h i y a m a a n d Rechnitz, 1987a). T h e selectivity of tissue-based sensors is sometimes poor c o m p a r e d to that of e n z y m e electrodes due to the presence of m u l t i p l e enzymes i n the biocatalyst.

208 TABLE 4 Some examples of plant tissue electrodes Substrate

Biocatalyst

Internal sensing element

Lifetimes

Ascorbate

cucumber

02

Cysteine

cucumber leaf

NH 3

28 d

Dopamine

banana pulp

02

10 d

Glutamate

yellow squash

CO 2

Phosphate/ fluoride Pyruvate

potato t u b e r / glucose oxidase corn kernel

02 CO 2

7d

Urea

jack bean meal

NH 3

94 d

L-Glutamine/ L-Asparagine Tyrosine

magnolia flower

NH 3

14 d

sugar beet

02

5-6 d

7d 28 d

8d

References

Vincke et al. (1985) Srnit and Rechnitz (1984) Sidwell and Rechnitz (1985) Kuriyama and Rechnitz (1981) Schubert et al. (1984) Kuriyama et al. (1983) Arnold and Glazier (1984) Uchiyama and Rechnitz (1987a) Schubert et al. (1983)

However, such multienzyme systems can work to advantage; by merely changing the external experimental conditions, different substrates can be determined with the same biocatalytic material (Ma and Rechnitz, 1985). The appropriate use of enzyme inhibitors, activators (Arnold and Rechnitz, 1981a) and stabilizing agents (Arnold, 1982) can also be used to enhance the selectivity and lifetimes of tissue-based biosensors. Hybrid sensors have also been developed. Kubo and Karube (1986) used an amperometric 02 electrode with the immobilized enzyme creatinine deiminase plus

TABLE 5 Some examples of animal tissue electrodes Substrate

Biocatalyst

Internal sensing element

Lifetimes

References

Adenosine-5 'monophosphate Guanine

rabbit muscle

NH 3

28 d

rabbit liver

NH 3

14 d

Glucosamine-6phosphate Glutamine

porcine kidney

NH 3

21 d

porcine kidney

NH 3

30 d

Arnold and Rechnitz (1981b) Arnold and Rechnitz (1982a) Ma and Rechnitz (1985) Rechnitz et al. (1979)

209 nitrifying bacteria for the determination of creatinine. Similarly, a potato tuber and glucose-6-phosphate hydrolase hybrid sensor was used to determine phosphate and fluoride (Schubert et al., 1984). Tables 4 and 5 show some examples of recently reported plant and animal tissue electrodes (Rechnitz et al., 1979; Kuriyama and Rechnitz, 1981; Arnold and Rechnitz, 1981, 1982; Kuriyama et al., 1983; Smit and Rechnitz, 1984; Schubert et al., 1983, 1984; Arnold and Glazier, 1984; Sidwell and Rechnitz, 1985; Ma and Rechnitz, 1985; Vincke et al., 1985; Smit, 1986; Uchiyama and Rechnitz, 1987). Immunochemical sensors

Another approach to biosensor development involves the use of immuno-reagents. An antigen can be covalently bonded to an ionophore and the resulting conjugate then incorporated in a polyvinyl chloride (PVC) polymer matrix membrane (Solsky and Rechnitz, 1979). Antibody sensing polymer membrane electrodes based on this concept for the determination of digoxin (Keating and Rechnitz, 1984), prostaglandins (Connell et al., 1983) and cortisol (Keating and Rechnitz, 1983) have been reported. When the antigens are themselves ionophoric, they can be incorporated directly into the PVC membrane without any conjugation procedure (Bush and Rechnitz, 1987a), as was demonstrated by the construction of a sensor for the antiarrhythmic drug, quinidine (Bush and Rechnitz, 1986), where quinidine has a proton carrier property. Recently, a fully reversible biosensor for antigen monitoring was constructed for the model hapten 2,4-dinitrophenol (Bush and Rechnitz, 1987b). It has an operating lifetime of 17 d and may perhaps be extended to other small haptens. It is thought that the potential changes observed with these antibody membrane electrodes are due to the modulation of the ionophoric properties upon selective binding of the antibody to the antigen immobilized in the membrane. Crawley and Rechnitz (1985) gave a detailed study on the response characteristics of such ionophore modulation immunoassays. Aizawa et al. (1979) used a different approach to construct an immunosensor. Here, the antibody is immobilized onto a membrane and placed next to an 02 probe, and the extent of immuno-reaction is determined enzymatically. The specificities of immunosensors are determined in part by the affinity constants of the antigen-antibody interaction. A compromise is thus needed in the design of reversible immunosensors. While a large affinity constant will give rise to high specificity, too high an affinity constant will result in an irreversible immunosensor unless protein denaturing conditions are used. Furthermore, non-specific adsorption has to be minimized. Eddowes (1987) derived a theoretical consideration for the binding of antigens to immobilized antibody layers and found that these immunosensors are useful for the determination of analytes in the micromolar to nanomolar region because of the fundamental limitation of these devices. Chemoreceptor based electrode

Belli and Rechnitz (1986) recently developed a new type of biosensor which is based on the chemoreceptors of blue crabs. Response times of this sensor are

210 extremely short. Thus, this receptor based electrode represents a new branch of biosensors to be explored in the future.

Response characteristics of biocatalytic membrane electrodes Steady state Concentration of analytes to be determined can be related to the measured signals at steady state, where steady state is a condition at which the rates of diffusion of the electro-active products to and from the electrode surface are equal (Kobos, 1980). Numerous parameters affect the steady-state behavior (Carr and Bowers, 1980; Arnold and Rechnitz, 1982a), including: (a) diffusion of substrates to the electrode surface; (b) substrate diffusion within the biocatalytic layer; (c) conversion of substrates to products; and (d) various product transport diffusion processes from the electrode surface to the bulk solution. Excellent mathematical descriptions and treatments of the steady-state behavior were provided by Carr and Bowers (1980). In practice, the bulk solution is stirred to ensure that mass transport processes are not rate limiting. At steady state, the electrode potential is constant and the substrate concentrations are related to the steady-state potentials in a logarithmic manner. The slopes of the calibration curves of these biocatalytic membrane electrodes sometimes fall below the theoretical Nernstian values because of insufficient biocatalytic activity. Response times Response times are usually of the order of several minutes, perhaps longer for bacterial electrodes, and are dependent on the type of electrode used, biocatalyst concentration, thickness of the biocatalytic layer, pH, membrane permeability, temperature and stir-rate (Kobos, 1980). Hameka and Rechnitz (1983) gave a detailed theoretical discussion on the mechanism of biosensors with an emphasis on the time-dependent approach to steady state and found that the biocatalytic layer thickness and the effective diffusion constants of the substrate solution are the most important factors in determining response times. The latter parameter is related to stir-rate. The overall time-determining factor in the use of these gas probe-based biocatalytic membrane electrodes is usually the long time needed to return to baseline potential between sample measurements. Guilbault and co-workers (1985) show, however, that the recovery time of a urea probe can be improved by changing the gas-probe design. The barrier effect of the immobilized enzyme layer can also affect dynamic electrode response (Bradley and Rechnitz, 1984; Bradley, 1986). Contrary to the popular assumption that these biosensors are non-consumptive devices, it has been found that under certain experimental conditions, the bulk substrate concentration does change. The substrate consumption is, however, not affected by the stir-rate or temperature (Arnold and Rechnitz, 1982b).

211 Immobilization

The appropriate method of union between the electrochemical sensors and biological components can often minimize the drawbacks of biocatalytic membrane electrodes arising from biocatalyst instability (Arnold and Rechnitz, 1982b) and interferences (Mascini and Guilbault, 1986). Furthermore, costs can be reduced due to reusability of immobilized enzymes on support materials and increases in efficiency of substrate transfer for multi-enzyme sensors (Kricka and Thorpe, 1986). Another advantage of immobilized enzymes is that they do not stimulate antibody production which is an important consideration in the design of biosensors for in-vivo monitoring (Klein and Langer, 1986). There are numerous methods for enzyme immobilization, basically falling into two main categories: (a) physical methods, e.g., adsorption, entrapment, encapsulation; (b) chemical methods, e.g., covalent linkage. The most common method of immobilization for other biocatalysts is physical entrapment between barriers of membranes or polymeric matrices (Tor and Freeman, 1986). Scott (1987) offered an excellent review on immobilized cells. Another type of immobilization technique, suggested by Krull and Thompson (1985), is the use of lipid bilayer membranes. Limitations of physical methods focus primarily on the exclusion of highly charged substrates and the potentiating effect of product inhibition. Biocatalyst leaching can also be a problem in such methods of immobilization. Chemical immobilization of enzymes may create microenvironments that can: (a) either attract or repel substrates, products or inhibitors by introducing a charge or hydrophobic group onto the support. This can lead to stabilization or de-stabilization of enzymes. (b) change the pH optima of enzymes by introducing positively or negatively charged groups into the support matrix. Thus immobilization can affect storage, thermal and operational stability of the biocatalysts. Ulbrich et al. (1986) tried to explain the thermal inactivation of immobilized enzymes with a mathematical treatment of biphasic inactivation kinetics. Various supports have been shown to successfully anchor enzymes by reaction between amino groups and carboxyl groups. Nylon is mechanically strong and biodegradable. However, native nylon of high molecular weight has to be pretreated to generate reactive groups (Mascini et al., 1983). The hydrophilic environment of collagen membranes minimizes enzyme denaturation and increases the contact of substrates with the insolubilized enzymes (Coulet et al., 1974), thus prolonging the lifetime of the probe. Furthermore, the reactive conditions required for conjugation are so mild that enzyme activities are not lost during the process. Immobilization of enzymes on cellulose hollow fibre has been reported but cellulose membranes are very prone to bacterial attack.

Aminopyrine Aniline Aromatic amines Acetylcholine Choline Phosphatidylcholine Creatine Guanidine Guanosine Penicillin Spermine Creatinine Uric acid Urea Xanthine Hypoxanthine

D-Alanine L-Arginine L-Asparagine L-Aspartic acid L-Cysteine L-Glutarnine L-Glutamic acid Glutathione L-Histidine L- and DLeucine L-Lysine L- and DMethionine N-Acetylmethionine L- and DPhenylalanine Sarcosine Serine L-Tyrosine L-Tryptophan D-Valine

* Koryta (1986)

Amines, amides heterocycles

Amino acids Amygdahne Galactose Glucose Glucose-6phosphate Lactose Maltose Sucrose Starch

Carbohydrates Acetic acid Formic acid Gluconic acid Isocitric acid L-Ascorbic acid Lactic acid Malic acid Oxalic acid Pyruvic acid Succinic acid Nitrilo-triacetic acid

Carboxylic acid

Substances determined with biocatalytic membrane electrodes *

TABLE 6

NO

SO 2

NH 3 H2 CH 4

Gases AMP ATP NAD(P)H H202

Cofactors Fluoride Nitrite Nitrate Phosphate Sulphate Sulphite Hg 2+ Zn 2+

Inorganic ions Antibiotics Assimilable carbohydrates Assimilable substances Biological oxygen demand Freshness of meat Mutagens Vitamins

Complex variables

Acetaldehyde Bifirubin Catechol Cholesterol Cholesterol ester Ethanol Glycerol Glycerol esters Methanol Phenol

Alcohols, phenols

213

Applications The ability of electrochemical sensors to provide real-time information and their relatively simple design have resulted in m a n y bioanalytical applications. Table 6 lists some of the analytes that have been determined with biocatalytic m e m b r a n e electrodes (Koryta, 1986). However, biosensors are still in their infancy with respect to commercialization (Rechnitz, 1987), with a few exceptions, such as the glucose sensors by Yellow Spring Instruments. The problems associated with the commercialization of this technology are enormous, and include instability, non-specificity (Lowe, 1984), and reproducibility in the manufacturing of the sensors (Russell and Rawson, 1986). Nevertheless, there are numerous reports and reviews (Arnold and Meyerhoff, 1984; Karube and Suzuki, 1984; Arnold and Solsky, 1986; Arnold, 1986) on the applications of biosensors. The scope of application of enzyme electrodes can even be extended from substrate determinations (Rahni et al., 1986) to that of inhibitors (Schubert et al., 1984) and coenzyme concentrations (Riechel and Rechnitz, 1978). The use of ion-selective electrodes in clinical chemistry has already been quite successful in blood gas analysis (Scott et al., 1986), electrolyte determinations (Tarcali et al., 1985; Khalil et al., 1986) and pharmaceutical analysis (Cosofret and Buck, 1984). Ciba Corning has recently developed an instrument (model 278) and claimed to be capable of monitoring pH, carbon dioxide, oxygen, sodium, potassium and chloride in clinical samples. Simpson and Kobos (1982, 1983) reported on the use of carbon dioxide sensors to determine antibiotics based upon their inhibition of carbon dioxide production from suspended bacterial cells. One potential application of leaf biosensors is the determination of herbicides based on the detoxification of herbicides by deamination (Sidwell and Rechnitz, 1986; Sidwell, 1986). In a non-clinical setting, such as the analysis of ions in food (Florence, 1986), in theoretical studies (Doe et al., 1983), and in environmental samples (Hara and Okazaki, 1984), ion-selective electrodes continue to be a valuable tool.

References Aizawa, M., Morioka, A., Suzuki, S. and Nagamura, Y. (1979) Enzyme immunosensor III-amperometric determination of human chorionic gonadotropin by membrane-bound antibody. Anal. Biochem. 94, 22-28. Arnold, M.A. (1982) Tissue-Based Biocatalytic Membrane Electrodes, University of Delaware Thesis. Arnold, M.A. (1983a) An introduction to biocatalytic membrane electrodes Am. Lab. 15, 34-40. Arnold, M.A. (1983b) Improved dynamic response of potentiometric ammonia sensors using pure teflon membranes. Anal. Chim. Acta 154, 33-39. Arnold, M.A. (1986) Potentiometric sensors using whole tissue sections, lon-sel. Electrode Key. 8, 85-113. Arnold, M.A. and Glazier, S.A. (1984) Jack bean meal as biocatalyst for urea biosensors. Biotechnol. Lett. 6, 313-318. Arnold, M.A. and Meyerhoff, M.E. (1984) Ion-selectiveelectrodes. Anal. Chem. 56, 20R-48R. Arnold, M.A. and Rechnitz, G.A. (1980) Comparison of bacterial, mitochondrial, tissue, and enzyme biocatalysts for glutamine selectivemembrane electrodes. Anal. Chem. 52, 1170-1174.

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Biosensors based on cell and tissue material.

Journal of Biotechnology, 15 (1990) 201-218 201 Elsevier BIOTEC 00445 Biosensors based on cell and tissue material G.A. R e c h n i t z 1 a n d M.Y...
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