- Email: [email protected]
Enhancing biosensor performance using multienzyme systems
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reviews
Enhancing biosensor performance using multienzyme systems Ulla Wollenberger, Florian Schubert, Dorothea Pfeiffer and Frieder W. Scheller Enhancing the performance of biosensors, in terms of increasing the range of
analytes that may be detected, and the sensitivity and specificity of the detection event, would improve the prospects for commercializing this technology. Coupling the catalytic activities of several enzymes is one approach being used to address these issues. Sequences of enzymes, where ligand binding triggers the activation of enzymes, or where biocatalytic pre-concentration of intermediates permits
augmentation of the signal, may be used. In addition, enzymatic recycling of the analyte can be used to increase the sensitivity by several orders of magnitude. The combination of the high specificity of enzymatic catalysis with the simplicity ofamperometric detectors is proving to be a successful route to creating low-cost sensors with applications in biotechnology, medicine and environmental monitoring. This approach, initiated by Clark and Lyons ~ is, at present, the bestestablished branch ofbiosensor research. Biosensors are devices that can detect or respond to environmental chemicals through specific interaction with a biological sensing component that is in close contact with a physical transducer. The biological component translates the specific molecular recognition of the analyte into a change in an easily measurable chemical or physical parameter. This change is sensed by the transducer which generates an electrical output signal (Fig. 1; tKef~ 2, 3). Analyte recognition is usually performed by immobilized biocomponents such as enzymes, cells, organelles, tissues, receptors, antibodies or antigens. The most frequently used transducers are electrochemical, optical and thermal detectors, but piezoelectric, and surface acoustic-wave methods may also be used 4. Currently, the most successful commercial developments are based o n electrochemical methods that utilize enzymes, and this review focuses on these amperometric enzyme electrodes. Amperometric enzyme electrodes are based on the oxidation or reduction ofredox species at the electrode surface, which is maintained at an appropriate electriU. kVollenberger is at the Fraunhofer Institute for Silicon Technology, Dillenburger Strasse 53, D-1000 Berlin 33, Germany. F. Schubert is at the Physikalisck-Tedmisdm Bundesanstalt, Abbestrasse 2-12, D-IO00 Berlirt 10, Germany. D. Pfe!ffer and F. W. Scheller are at the Max-DelbriMe Center _for Molecular Medicine, Robert-RJssleStrasse 10, O- 1115 Berlin, Germany.
© 1993,ElsevieSci r encePublishersLtd(UK)
Electronics
Biocomponent
Transducer Sample
l"
T leltric~al signal
Analyte ~ > Co- 0
substrates
b
~
i
-
Other ~
L
compou/~
Recognition
"-1// Transduction Reaction
Amplification and
data processing
Figure 1
Schematicillustrationof an enzymebiosensor.
cal potential. The function of the enzyme reaction that mediates the specific catalytic conversion of the analyte is to produce or consume a redox-active compound, and the current that is thus generated bears a stoichiometric relationship to the concentration of the analyte. Nature, however, provides only a few such enzymes that are able to generate or consume electrochemically active species, thus limiting the number ofanalytes that can be measured with mono-enzymatic electrodes. One way of overcoming this problem is to couple the
TIBTECHJUNE1993(VOL11)
256
reviews Electrode
b
( 1 ) - N A D H accumulation
(2) +NADH accumulation
01 4- S 2
S
01 + S
S2
4,
4,
.L
.L
L
Steady-state current
0
Peak current Time
Time
100-
-~
50-
i
NA
E n z y m e sequences with a c c u m u l a t i o n o f intermediates The sensitivity of electroanalytical measurements can be enhanced by accumulating the electrochemically active species at the electrode surface before 'stripping' for measurement. This principle can be adapted readily to enzyme membrane-electrodes based on sequentially acting enzymes. Such sensors combine biocatalytic pre-concentration of an intermediate with an enzymatic indicator system to measure the accumulated intermediate s . Both oxygen electrodes and chemically modified electrodes have been used as the basic sensor device, which is then adapted. The principle on which such measurement is based is shown in Fig. 2a. The addition of analyte (S) and an excess o f an appropriate substrate (C1) leads to the formation of the intermediate (I) through the action o f the 'generator' enzyme (El). The intermediate accumulates in the membrane owing to its low rate of diffusion. W h e n this reaction approaches equilibrium, substrate ($2) is injected to initiate the conversion [catalysed by the 'indicator' enzyme (E2) ] o f the accumulated intermediate into the electroactive product (P*). The determination o f glycerol, For example, involves coimmobilization o f glycerol dehydrogenase with the lactate dehydrogenase/lactate oxidase indicator sequence which, in the presence of oxygen, oxidizes the N A D H formed during the conversion of glycerol to dihydroxyacetone (Eqns 1-3).
glycerol dehydrogenase Glycerol + NAD + ~- dihydroxyacetone + NADH
accumulation
(1) - N A D H accumulation
I
I
F
10
50
100
lactate dehydrogenase Pyruvate + NA1)H ~ lactate + NAD +
(2)
I
200
lactate oxidase
Glycerol (~M 1-1) Figure 2 (a) Principle of an accumulation biosensor. (b) Current-time curves for a sensor operated without and with intermediate accumulation. The difference in the steady-state and peak currents represents the amplification of the sensor response. (c) Glycerol measurement without and with NADH accumulation on an enzyme electrode containing immobilized glycerol dehydrogenase, lactate dehydrogenase, and lactate monooxygenase. NADH is stripped by pyruvate addition after 6 min of accumulation.
catalytic activities of different enzymes either in sequence, in competing pathways, or in cycles. In this way, not only does a much wider range o f analyte species become accessible to measurement by the bioelectroanalytical approach but, in addition, the selectivity and sensitivity of the biosensor may be enhanced through appropriate choice of the coupling strategy. This article reviews the principles involved in coupling enzymatic reactions for the purpose of improving the analytical performance ofbiosensors. TIBTECHJUNE1993(VOL11)
Lactate + 02
~- pyruvate
+
H202
(3) The improved response that may be achieved by pre-concentration of the intermediate (in this case the co-factor N A D H ) is illustrated in Fig. 2b. The reaction between cofactor (NAD +) and analyte (glycerol) reaches equilibrium, and the addition of the initiator (pyruvate) results in a sudden increase in the current. This is due to the rapid diffusion ofpyruvate into the membrane and its reaction with the accumulated NADH. Although the same steady-state current as in the conventional measuring approach (Fig. 2b) is eventually reached when the accumulated supply of N A D H is exhausted and the current is limited by the N A D H production rate, evaluation o f the peak in the current-time curve (Fig. 2b) provides an enhanced signal for glycerol determination. Allowing N A D H to
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reviews Table 1. Enzyme sensors using the accumulation principle Accumulator (El)
Glucose
Glucose dehydrogenase NADH
Lactate dehydrogenase/ lactate monooxygenase
Pyruvate
40
Glucose 6phosphate
Glucose-6-phosphate dehydrogenase
NADH
Lactate dehydrogenase/ lactate monooxygenase
Pyruvate
17
NADH-PEG
Lactate dehydrogenase/ lactate monooxygenase
Pyruvate
1.7
Hypoxanthine Xanthine dehydrogenase
Intermediate Detector (E2)
Initiator
Amplification factor
Analyte
Formate
Formatedehydrogenase NADH
Lactate dehydrogenase/ lactate monooxygenase
Pyruvate
6
Glycerol
Glyceroldehydrogenase NADH
Lactate dehydrogenase/ lactate oxidase
Pyruvate
64
NADP+
Alkaline phosphatase
Lactate dehydrogenase
Lactate
NAD+
accumulate for six minutes increased the sensitivity of the measurement of glycerol by a factor o f 64 (Fig. 2c). This principle has also been applied to sensors used to determine glucose, glucose 6-phosphate, hypoxanthine, N A D P + and formate (Table 1). The extent to which an intermediate may be accumulated, however, is limited by the point o f equilibrium of the reaction. Differences that are observed in the amplification factors (Table 1) may be attributed to the equilibrium constants of the generator enzymes, and the diffusion behaviour of the intermediate and initiator substrates. The greatest enhancement due to accumulation is obtained when the intermediate is a large molecule (i.e. diffuses slowly), and the initiator is a small molecule. This principle of measurement, based on intermediate accumulation, is applicable to any analyte that may be converted in a sequential reaction, provided that one o f the enzymes (the terminal enzyme) is co-factor-dependent and can generate an electrochemical signal at the transducer. An important point in the practical application of this approach (as in any enzymatic analysis) is the need to eliminate any effects on the signal that are due to interfering molecular species. For this reason, the sample is allowed to equilibrate with the reaction medium to establish the 'background' current before initiating the reaction that measures the analyte. This eliminates any contribution from interfering species to the signal and improves the selectivity o f the analysis.
switches, pathways may also be regulated by the availability o f intermediates that have been generated by continuous cycling reactions. Exploiting the principle of enzyme cascades (i.e. the sequential activation of enzymes triggered, for example, by a ligand binding to a receptor) is illustrated in Fig 3a. The binding of the activator (initiator) transforms the inactive enzyme Eli to its active form E h which, in turn, activates a second inactive enzyme, and so on. At the end o f the cascade, an enzyme is activated which produces the electroactive species P*. In glycogenolysis, which occurs in the liver and is a natural example of an enzyme cascade, binding of epinephrine activates adenylate cyclase as the first enzyme in a series ofphosphorylation reactions. The cascade o f these reactions results in amplification o f the signal by five orders of magnitude 7. The sensing principle, based on enzyme activation, may be demonstrated by considering the determination o f glycogen phosphorylase B, and its allosteric effector, AMP (tKe£ 8). Owing to their role in catalysing the sequential phosphorolytic removal of terminal glycosyl residues from the non-reducing end of glycogen, glycogen phosphorylases are key enzymes in the regulation of glycogenolysis. The products o f the reaction are or-glucose-l-phosphate and a truncated glycogen molecule (Eqn 4).
Sequential activation of enzymes Nature provides a variety o f ways of regulating metabolic pathways very specifically. The phenomena ofallosteric control and covalent modification o f proteins play key roles in this regulation 6. Exploiting the principles of metabolically controlled 'switches' and enzyme cascades, whereby repression or activation of enzymes can occur when a threshold value of a particular metabolite is reached, is of'paramount interest in designing analytical procedures. In addition to
Glucose n + Pi
phosphorylase active glucos%_l + c~-glucosel-phosphate
(4)
Glycogen phosphorylase B is active only in the presence o f AMP, which acts as an allosteric activator. The proportion of the enzyme that is active can be determined by the rate of phosphorylation o f glycogen, by quantifying the rate of glucose l-phosphate formation in the presence or absence of AMP. This TIBTECH JUNE 1993 (VOl- 1 t)
258
rg12igW5 a
Initiator reaction can be monitored using an electrode that measures the formation o f hydrogen peroxide and which has the enzymes o f the following sequence (Eqns 5-7) immobilized in the electrode membrane:
1 Eli
Ela
alkaline phosphatase
E2i
(x-glucose l-phosphate
E2a
~- 0~-glucose + Pi
(5) mutarotase
E3i
E3a
o~-glucose ~
~]
[~
Electrode
0
I
I
I
50
100
150
I
200
AMP (pM 1-1) Figure 3 (a) Schematic illustrationof sequentialactivation of enzymes in biosensors.A and C representco-reactants.(b) AMP-concentrationdependenceobtainedwith a glycogen phosphorylaseB/alkalinephosphatase/mutarotase/glucoseoxidaseelectrode in presenceof glycogen, oxygen, and phosphate.
[-~
5] Figure 4 Substrate-recyclingbiosensor. TIBTECHJUNE1993(VOL11)
~ D Electrode
(6)
glucose oxidase [3-glucose
O.
~- [3-glucose
+ 0 2
~-
gluconolactone -6 H202
(7)
Glucose 1-phosphate is dephosphorylated by alkaline phosphatase to yield ~x-glucose (Eqn 5). The oxidation of [3-glucose (Eqn 7) after mutarotation (Eqn 6) results in an increase in the anodic current at the sensor probe due to H202 oxidation. This threeenzyme sensor can be used to measure glycogen phosphorylases A and B at concentrations of 0.005-0.200 U m 1-I. The fact that AMP is required for glycogen phosphorylase B activity can be used to measure the concentration of AMP. For this purpose, glycogen phosphorylase B was entrapped with its substrate, glycogen, in a separate compartment in front o f the three-enzyme layer. The sensor was able to respond to micromolar concentrations of AMP, and the calibration plot o f current versus AMP concentration was linear within the range 5-75 I*M 1-1 (Fig. 3b). Concentrations above 150 t~M pl gave no increase in response; either all the phosphorylase molecules were activated (i.e. a kinetically controlled electrode), or the phosphorylase reaction had become limited by the rate at which glycogen can diffuse within the membrane. The observed increase in the apparent K Mvalue, from 2 ~M 1-I in solution, to 50 ~M ~i in the membrane, strongly supports the latter assumption of diffusion control of the reaction. Since the effect of AMP is to activate the enzyme, the A M P response functions to amplify probe semitivity to a considerably greater extent than that which can be achieved with glucose l-phosphate. The sensor arrangement outlined above could be used repeatedly for ~ 20 sequential A M P measurements (response time 20s, measuring cycle three minutes) without the need to add additional reagents; thereafter, sensitivity declined owing to exhaustion of the substrate, glycogen. Exploiting the allosteric activation of enzymes in sensor membranes opens up new possibilities in sensing. N o t only can it be used to increase sensitivity, but activation by covalent modification of physiologically important proteins also makes it possible to measure the concentration of the activators o f allosteric
259
reviews enzymes, as well as the concentration of substrates, inhibitors and enzyme prosthetic groups, and enzyme activity. In addition to the activation o f allosteric enzymes in an enzyme cascade and the accumulation of intermediates, biosensor sensitivity can also be enhanced by analyte recycling.
Amplification by analyte recycling With bare electrodes, the lower detection limit of amperometric electrodes is - lOOnM1 <. The introduction o f a layer incorporating the enzyme over the electrode surface decreases the sensitivity o f the sensor by one to two orders of magnitude, owing to the additional resistance to diffusion of the analyte. Thus, for measurement o f analyte concentrations in the nanomolar range, a way o f increasing the sensitivity of the enzyme electrode is required. Analyte recycling is the best studied method which uses enzymatic systems to ampli6f the response to very low analyte concentrations 9. The enhancement in semitivity is provided by shuttling the analyte between enzymes acting in a reaction cycle with consumption ofco-substrates and accumulation o f by-products. In Fig. 4, S~ and S2 can be substrates or co-substrates of the enzymes E 1 or E2, respectively. Assuming that the activity o f the enzyme E~ is sufficiently high in the presence o f its co-substrate C 1 and the analyte S (S1 or $2), (the concentration o f which is far below its Michaelis-Menten constant), amplification is achieved through switching on E e by addition of its co-substrate C 2. The analyte S is then shuttled between the two enzymes and, as a consequence, the conversion o f c o substrates into products is amplified, relative to the concentration o f analyte present in the membrane, beyond the normal stoichiometry o f the single reactions. The overall reaction o f both reactants is thus:
enzyme electrodes has been achieved for ~ 15 pairs of coupled enzymes (Table 2; 1Kef~ 3, 11). Using this approach, the limit o f detection was reduced to nanomolar concentrations for some substrates, such as lactate, pyruvate and hydroquinone 13. In addition to analyte recycling electrodes, there have also been reports on the use of the enzyme cycles coupled to fibre-optic probes 27,28, and in a column combined with a thermistor 29,3°, and with a flowinjection system with amperometric detection 31 33.
Linear recycling with oxidase and dehydrogenase When oxidases are coupled with their respective dehydrogenase electrode, the reaction system includes species that are readily detectable electrochemically; the change in co-reactant concentration can thus be measured directly at the electrode onto which the recycling enzyme pair is immobilized. In most cases, consumption o f the co-reactant has been followed (Table 2). The system incorporating the enzyme couple lactate dehydrogenase/lactate oxidase (Eqns 9, 10) has been studied in detail 14. This consists of a gelatinentrapped enzyme couple and an oxygen electrode as transducer. In the presence of N A D H , the pyruvate formed in the lactate oxidase reaction is converted by the dehydrogenase back to lactate, which is then rc~ oxidized by lactate oxidase. lactate oxidase Lactate + 0 2
~ pyruvate + H 2 0 2
(9)
lactate dehydrogenase Pyruvate + N A D H
lactate + NAD +
(8)
(10)
By measuring the change in concentration of one of the co-reactants or products (for example, P*), either directly, or by an additional analytical reaction step, the recycling system may be used to amplify the response of the system to measure either analyte S1 or S2. In the recycling mode, it is not possible to distinguish between the analytes. When one molecule of product is fbrmed per substrate molecule reacting, the concentration o f the co-products (Pl, P2) increases, and the concentration o f the co-substrates (Cl, C2) decreases linearly with time, while the concentration of the cycling substrates (S~ and $2) remains constant. The number of cycles for which the substrate is turned over in a given time is a function of the substrate concentration. This recycling system, based on the concept of a linear enzymatic amplification of the signal, has been put to practical use by coupling dehydrogenases with oxidases or transaminases, or by coupling the catalytic activities of several kinases. The early applications of the principles of analyte recycling in enzyme electrodes were aimed at cofactor regeneration 1°. Enzymatic analyte recycling in
Oxygen consumption in the enzyme membrane is thus enhanced, yielding an increase in the sensitivity to lactate by a factor o f up to 4100. Under steady-state conditions, the ratio of the sensitivities in the linear measuring range of amplified and unamplified systems (termed the amplification factor, G) is:
C1 + C2
~ P~ + P2
L 2 klk 2 G= 2D (k 1 + k2)
(11)
where L is the membrane thickness; kt, the first-order rate constant; and D, the diffusion coefficient34. When enzymes of very high activities are incorporated into an enzyme layer with a high, characteristic diffhsion time (L2/D), the amplification possible is very large. Experiments investigating the influence o f enzyme loading (i.e. the amount of the enzyme activity in the membrane) on the amplification o f the lactate signal showed, in agreement with the theory, that a 4100fold amplification was feasible using a gelatin matrix with a characteristic diffusion time o f ~ 90s. With this TIBTECHJUNE 1993 (VOL 1i)
260
reviews Table 2. Substrate recycling in biosensors Enzyme couple
Glucose
Glucose oxidase/glucose dehydrogenase Oxygenelectrode
10
12
Lactate
Lactate oxidase/lactate dehydrogenase
Oxygen electrode
48 000 4100 250
13 14 15
Lactate/ pyruvate
Cytochrome b2/ lactate dehydrogenase
Platinum electrode
10
12
NADH/NAD+
Peroxidase/glucose dehydrogenase
Oxygen electrode
60
12
Glutamate
Glutamate dehydrogenase/ alanine aminotransferase Glutamate oxidase/ glutamate dehydrogenase Glutamate oxidase/ alanine aminotransferase
Modified carbon electrode Oxygen electrode
15
16
20
17
Hydrogen peroxide electrode
500
18
Hexokinase/pyruvate kinase
Oxygen electrode with lactate dehydrogenase Lactate monooxygenase modified carbon electrode with glucose-6-phosphate dehydrogenase
220
19
1200a
20
800
21
ADP/ATP
Transducer
Amplification factor
Analyte
ADP
Myokinase/pyruvate kinase
Oxygen electrode with pyruvate oxidase
Ethanol
Alcohol oxidase/alcohol dehydrogenase
Oxygen electrode
Benzoquinone/ Cytochrome b2/laccase hydroquinone
Ref.
22
Oxygen electrode
500
23
Malate/ oxalacetate
Malate dehydrogenase/lactate monooxygenase
Oxygen electrode
3
24
L-Leucine
Leucine dehydrogenase/aminoacid oxidase
Oxygen electrode
40
25
Phosphate
Nucleoside phosphorylase/ alkaline phosphatase
Oxygen electrode with xanthine oxidase
20
26
atn combinationwithintermediateaccumulation.
amplification, lactate concentrations as low as 1 nM 1-1 could be determined with reasonable precision. The response to lactate could be increased still further, up to 48000-fold, when the immobilization was performed using polyurethane, which permits higher enzyme loading 13. However, the reproducibility and stability of this large amplification is poor, owing to the number o f enzymes used. In general, the specificity of recycling sensors is poorer compared with m o n o - e n z y m e sensors. TIBTECH JUNE 1993 (VOL 11)
Linear recycling with kinases Recycling systems need not be limited to reactions in which electroactive compounds are produced. In such cases, the recycling enzyme pair may be c o m bined with an indicator enzyme (or sequence of enzymes) which transforms one of the cycle constituents(usually a product) into a measurable species. Kinase reactions, owing to their, in general, favourable equilibrium constants are well suited to such systems. However, none of the cycle constituents for kinase-
261
reviews mediated reactions are directly measurable at a favourable potential, and the products o f the cycle have therefore been coupled with o x y g e n - c o n s u m i n g enzyme reactions for their determination. T o enable sensitive measurement o f A D P and ATP, a biosensor was constructed by combining an oxygen electrode and a gelatin layer in which were immobilized h e x o kinase and pyruvate kinase (shuttling A D P / A T P b e t w e e n each other), and a lactate dehydrogenase and lactate m o n o o x y g e n a s e sequence (which was also used for intermediate-accumulation experiments) to transform the e n o r m o u s a m o u n t o f p y r u v a t e formed in the recycling reaction into measurable oxygen consumption 19. W i t h this recycling system, a 220-fold amplification was possible (Table 2). This system has also been used with the lactate dehydrogenase/lactate m o n o o x y g e n a s e 'detector' enzymes substituted by pyruvate oxidase or glucose-6-phosphate dehydrogenase 20.
Exponential recycling with kinases In contrast to linear amplification recycling systems, an exponential recycling system is characterized by the use o f an e n z y m e that forms m o r e than one molecule o f product per molecule ofsubstrate (i.e. the total amount o f intermediates and by-products increases exponentially with time). Theoretical considerations showed that the concentration o f any o f the cycling intermediates, or by-products, at any given time, is a linear function o f the initial substrate concentration 35. An example illustrating this principle is the A D P / A T P cycling that occurs with the m y o k i n a s e / p y r u v a t e kinase/pyruvate oxidase-modified oxygen sensor 21. In one cycle, myokinase forms two molecules o f A D P per molecule A T P , which is derived from the phosphorylation o f A D P by pyruvate kinase (Eqns 12, 13): pyruvate ADP+ kinase phosphoenolpyruvate ~- A T P + pyruvate 02)
myokinase ATP + AMP
~
2 ADP
(13)
T h e amounts o f A T P and ADP, which are initially present at very low concentrations, increase e x p o n e n tially with cycling time, provided that A M P and phosphoenolpyruvate concentrations are sufficiently high, so as not to limit the reaction. T h e e n o r m o u s a m o u n t o f pyruvate f o r m e d in the cycle is reflected by the oxygen c o n s u m p t i o n in the immobilized pyruvate oxidase layer. W i t h an optimized sensor configuration, an increase in the sensitivity o f detection o f A D P by a factor o f 800 was obtained.
Mono-enzymatic substrate recycling Recently, a novel amplification scheme has been developed, based on the fact that N A D H recycling is performed by only one enzyme 36. In the presence o f
N A D +, lactate dehydrogenase catalyses the oxidation ofglyoxylate to oxalate, with concomitant formation o f N A D H . In the presence o f pyruvate, glyoxylate, and a limiting a m o u n t ofpyridine nucleotide, the cofactor is cyclically regenerated. With such a system, a 170-fold increase in lactate formation enabled as little as 50nM 1-1 to be detected for N A D H . This kind of m o n o - e n z y m a t i c cycle might facilitate the design of novel, simple enzymatic amplification schemes. Alternatively, it may be possible to enhance this amplification by a second recycling e n z y m e pair in a double amplification system, thus yielding a still further i m p r o v e m e n t in the sensitivity o f the sensor. Conclusions
Sequentially coupled enzyme reactions are widely used in analysers based on the use o f soluble reagents. Signal amplification by a cyclic e n z y m e couple is already exploited in a commercial e n z y m e - i m m u n o assay system (Novoclone). In contrast, a m o n g biosensors that are based on immobilized biomolecules, m o n o - e n z y m e electrodes dominate the practical applications, and only a few sequentially coupled enzyme electrodes have been commercialized. T h e difference in reaction optima is a serious p r o b l e m in combining several immobilized enzymes within one c o m p a r t ment, as for example, in the three-enzyme systems for creatinine analysis. Further progress, both for soluble and immobilized coupled enzymes, is expected to result from the creation o f chimeric enzyme molecules, either by gene fusion or by 'site to site' linkage o f the interacting e n z y m e s using post-translational, chemical modification m e t h o d s . In this way, the b i o c h e m i c a l signal m a y be effectively 'channelled', w h i c h m a y lead to high sensitivity and specificity o f b i o s e n s o r s . References
1 Clark, L. C. and Lyons, C. (1962) Ann. NYAcad. Sci. 102, 29-45 2 Turner, A. P. F., Karube, 1. and Wilson, G. S. (eds) (1987) Biosenso~ - Fundamentals and Applications, Oxford UniversityPress 3 Scheller,F. and Schubert, F. (1989). Biosensoren, Akademie Verlag 4 Hall, E. A. H. (1990) Biosensors, Open UniversityPress 5 Schubert, F., Lutter,J. and Scheller, F. (1991) Anal. Chim. Acta 243, 17-21 6 Koshland,D. E.,Jr (1987) Trends Biochem. Sci. "[2,225 229 7 Stryer,L. (1988) Biochemistry, Freeman Co. 8 Wollenberger,U. and Scbeller,F. Biosensors and Bioelectronics (in press) 9 Lowry, O. H. and Passonneau,J.V. (1972) A Flexible System of Enzymatic Anal}sis, Academic Press 10 Davies, P. and Mosbach, K. (1974) Biochim. Biophys. Acta 370, 329-338 11 Schubert, F., Wollenberger, U., Pfeiffer,13. and Scheller, F. (1991) in Advances in Biosensors Vol. 1 (Turner, A. P. F., ed.), pp. 77 105, JA! Press 12 Schubert, F., Kirstein,D., Schr6der, K. L. and Scheller, F. W. (1985) Anal. Chim. Acta 169, 391-396 13 Scheller, F. W., Schubert, F., Pfeiffer, D., Wollenberger, U., Renneberg, R., Hintsche, R. and Ktihn, M. (1992) GBFMonouaphs 17, 3-10 14 Wollenberger,U., Schubert, F., Scheller, F. W., Danielsson, B. and Mosbach, K. (1987) Stud. Biophys. 119, 167-170 15 Mizutani, F, Yamanaka,T., Tanabe, Y. and Tsuda, K. (1985) Anal. Chim. Acta 177, 153-166 TIBTECH JUNE 1993 (VOL 11)
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reviews 16 Schubert, F., Kirstein, D., Schellcr, F., Appelqvist, P,., Gorton, L. and Johansson, G. (1986)Anal. Lett. 19, 1273-1288 17 Wollenberger, U., Scheller, F., Pavlova, M., Mtiller, H. G., Risinger, L. and Gorton, L. (1989) GBF Monographs 13, 33~6 18 Yao, T., Yamamoto, H. and Wasa, T. (1989) in Proc. ISE Meeting, Kyoto, pp. 1014~1015, Kodanasha 19 Wollenberger, U., Schubert, F., Scheller, F. W., Danielsson, B. and Mosbach, K. (1987) Anal. Lett. 20, 657 668 20 Yang, X., PlCeiffer,D., Johansson, G. and Scheller, F. W. (1991) Electroanalysis 3, 659 663 21 Scheller, F., Wollenberger, U., Schubert, F., Pfeiffer, D., Makower, A. and O'Neil, C. in Proceedings N A T O advanced workshop on use of immobilized biological components for detection, medical, food and environmental analysis, Brixen, Italy (in press) 22 Hopkins, T. P,.. (1985) Int. Biotechnol. Lab. 3, 20-25 23 Scheller, F., Wollenberger, U., Schubert, F., Pfeiffbr, D. and Bogdanovskaya, V. A. (1987) GBF Monographs 10, 39-49 24 Scheller, F., P, enneberg, R. and Schubert, F. (1988) Methods Enzytool. 137, 29-43 25 Scheller, F., Pfeiffer, D., Hintsche, R., Dransfeled, 1., Wollenberger,
U. and Schubert, F. (1990) Ann. NTAcad. &i. 613, 68-78 26 Wollenberger, U., Schubert, F. and Scheller, F. (1992) Senso~:~Actuat. B7, 412-415 27 Schubert, F. (1993) Sensors Actuat. Bli, 531-535 28 Wang, A.J. and Arnold, M. A. (1992) Anal. Chem. 64, 1051-1055 29 Scheller, F. W., Siegbahn, N., Danielsson, B. and Mosbach, K. (1985) Anal. Chem. 57, 1740-1743 30 Kirstein, D., Danielsson, B., Scheller, F. W. and Mosbach, K. (1989) Biosensors 4, 231-239 31 Asouzu, M. U., Nonidez, W. K. and Ho, M. H. (1990) Anal. Chem. 62,708~12 32 Hansen, E. H., Arndal, A. and Norgaard, L. (1990) Anal. Lett, 23, 225-240 33 Yang, X., Pfeiffer, D.,Johansson, G. and Scheller, F. W. (1991) Anal. Lett. 24 (1991) 1401 1417 34 Kulys,J. J., Sorochinskii, V. V. and Vidzivnaite, P,.. A. (1986) Biosensots 2, 135 146 35 Kopp, L. E. and Miech, P,.. P. (1972)J. Biol. Chem. 247, 3558-3563 36 Schubert, F., Scheller, F. and Krasteva, N. (1990) Electroanalysis 2, 347-351
TIBTECH Editorial Policy Trends in Biotechnology is a news, reviews and commentary journal designed to keep its international readershi p up-to-date with the current developments in biotechnology. TIBTECH occupies a niche between primary research journals and conventional review journals - it is not a vehicle for the publication of original research data or methods. There is a strong emphasis on an integrated approach to communicating significant new advances in biotechnology, and discussing their commercial potential. This necessitates combining essential background with state-of-the-art information to make the topics addressed accessible and valuable to newcomers and experts in the field alike. All articles are subject to peer-review and are prepared to strict standards to ensure clarity, scientific accuracy and readability. Commissioning does not guarantee publication. Reviews: Balanced and concise presentations of specific areas of research. The scope of such reviews is more limited than those of conventional review journals, but provides sufficient information to place the latest developments in a particular field in perspective. Focus: Short minireview-style articles, which focus on a detailed examination of a narrow topic, thus providing a qu ck publication response to recognition of significant new directions of research. Forum: A platform for debate and analytical discussion of newly reported advances -,onferences. Includes subsections: Biotopics: A column providing a lighter, more personal treatment of topics ranging from senous science and novel techniques, to finance and ethical questions.
TIBTECH JUNE 1993 (VOL 11)
reviews
Enhancing biosensor performance using multienzyme systems Ulla Wollenberger, Florian Schubert, Dorothea Pfeiffer and Frieder W. Scheller Enhancing the performance of biosensors, in terms of increasing the range of
analytes that may be detected, and the sensitivity and specificity of the detection event, would improve the prospects for commercializing this technology. Coupling the catalytic activities of several enzymes is one approach being used to address these issues. Sequences of enzymes, where ligand binding triggers the activation of enzymes, or where biocatalytic pre-concentration of intermediates permits
augmentation of the signal, may be used. In addition, enzymatic recycling of the analyte can be used to increase the sensitivity by several orders of magnitude. The combination of the high specificity of enzymatic catalysis with the simplicity ofamperometric detectors is proving to be a successful route to creating low-cost sensors with applications in biotechnology, medicine and environmental monitoring. This approach, initiated by Clark and Lyons ~ is, at present, the bestestablished branch ofbiosensor research. Biosensors are devices that can detect or respond to environmental chemicals through specific interaction with a biological sensing component that is in close contact with a physical transducer. The biological component translates the specific molecular recognition of the analyte into a change in an easily measurable chemical or physical parameter. This change is sensed by the transducer which generates an electrical output signal (Fig. 1; tKef~ 2, 3). Analyte recognition is usually performed by immobilized biocomponents such as enzymes, cells, organelles, tissues, receptors, antibodies or antigens. The most frequently used transducers are electrochemical, optical and thermal detectors, but piezoelectric, and surface acoustic-wave methods may also be used 4. Currently, the most successful commercial developments are based o n electrochemical methods that utilize enzymes, and this review focuses on these amperometric enzyme electrodes. Amperometric enzyme electrodes are based on the oxidation or reduction ofredox species at the electrode surface, which is maintained at an appropriate electriU. kVollenberger is at the Fraunhofer Institute for Silicon Technology, Dillenburger Strasse 53, D-1000 Berlin 33, Germany. F. Schubert is at the Physikalisck-Tedmisdm Bundesanstalt, Abbestrasse 2-12, D-IO00 Berlirt 10, Germany. D. Pfe!ffer and F. W. Scheller are at the Max-DelbriMe Center _for Molecular Medicine, Robert-RJssleStrasse 10, O- 1115 Berlin, Germany.
© 1993,ElsevieSci r encePublishersLtd(UK)
Electronics
Biocomponent
Transducer Sample
l"
T leltric~al signal
Analyte ~ > Co- 0
substrates
b
~
i
-
Other ~
L
compou/~
Recognition
"-1// Transduction Reaction
Amplification and
data processing
Figure 1
Schematicillustrationof an enzymebiosensor.
cal potential. The function of the enzyme reaction that mediates the specific catalytic conversion of the analyte is to produce or consume a redox-active compound, and the current that is thus generated bears a stoichiometric relationship to the concentration of the analyte. Nature, however, provides only a few such enzymes that are able to generate or consume electrochemically active species, thus limiting the number ofanalytes that can be measured with mono-enzymatic electrodes. One way of overcoming this problem is to couple the
TIBTECHJUNE1993(VOL11)
256
reviews Electrode
b
( 1 ) - N A D H accumulation
(2) +NADH accumulation
01 4- S 2
S
01 + S
S2
4,
4,
.L
.L
L
Steady-state current
0
Peak current Time
Time
100-
-~
50-
i
NA
E n z y m e sequences with a c c u m u l a t i o n o f intermediates The sensitivity of electroanalytical measurements can be enhanced by accumulating the electrochemically active species at the electrode surface before 'stripping' for measurement. This principle can be adapted readily to enzyme membrane-electrodes based on sequentially acting enzymes. Such sensors combine biocatalytic pre-concentration of an intermediate with an enzymatic indicator system to measure the accumulated intermediate s . Both oxygen electrodes and chemically modified electrodes have been used as the basic sensor device, which is then adapted. The principle on which such measurement is based is shown in Fig. 2a. The addition of analyte (S) and an excess o f an appropriate substrate (C1) leads to the formation of the intermediate (I) through the action o f the 'generator' enzyme (El). The intermediate accumulates in the membrane owing to its low rate of diffusion. W h e n this reaction approaches equilibrium, substrate ($2) is injected to initiate the conversion [catalysed by the 'indicator' enzyme (E2) ] o f the accumulated intermediate into the electroactive product (P*). The determination o f glycerol, For example, involves coimmobilization o f glycerol dehydrogenase with the lactate dehydrogenase/lactate oxidase indicator sequence which, in the presence of oxygen, oxidizes the N A D H formed during the conversion of glycerol to dihydroxyacetone (Eqns 1-3).
glycerol dehydrogenase Glycerol + NAD + ~- dihydroxyacetone + NADH
accumulation
(1) - N A D H accumulation
I
I
F
10
50
100
lactate dehydrogenase Pyruvate + NA1)H ~ lactate + NAD +
(2)
I
200
lactate oxidase
Glycerol (~M 1-1) Figure 2 (a) Principle of an accumulation biosensor. (b) Current-time curves for a sensor operated without and with intermediate accumulation. The difference in the steady-state and peak currents represents the amplification of the sensor response. (c) Glycerol measurement without and with NADH accumulation on an enzyme electrode containing immobilized glycerol dehydrogenase, lactate dehydrogenase, and lactate monooxygenase. NADH is stripped by pyruvate addition after 6 min of accumulation.
catalytic activities of different enzymes either in sequence, in competing pathways, or in cycles. In this way, not only does a much wider range o f analyte species become accessible to measurement by the bioelectroanalytical approach but, in addition, the selectivity and sensitivity of the biosensor may be enhanced through appropriate choice of the coupling strategy. This article reviews the principles involved in coupling enzymatic reactions for the purpose of improving the analytical performance ofbiosensors. TIBTECHJUNE1993(VOL11)
Lactate + 02
~- pyruvate
+
H202
(3) The improved response that may be achieved by pre-concentration of the intermediate (in this case the co-factor N A D H ) is illustrated in Fig. 2b. The reaction between cofactor (NAD +) and analyte (glycerol) reaches equilibrium, and the addition of the initiator (pyruvate) results in a sudden increase in the current. This is due to the rapid diffusion ofpyruvate into the membrane and its reaction with the accumulated NADH. Although the same steady-state current as in the conventional measuring approach (Fig. 2b) is eventually reached when the accumulated supply of N A D H is exhausted and the current is limited by the N A D H production rate, evaluation o f the peak in the current-time curve (Fig. 2b) provides an enhanced signal for glycerol determination. Allowing N A D H to
257
reviews Table 1. Enzyme sensors using the accumulation principle Accumulator (El)
Glucose
Glucose dehydrogenase NADH
Lactate dehydrogenase/ lactate monooxygenase
Pyruvate
40
Glucose 6phosphate
Glucose-6-phosphate dehydrogenase
NADH
Lactate dehydrogenase/ lactate monooxygenase
Pyruvate
17
NADH-PEG
Lactate dehydrogenase/ lactate monooxygenase
Pyruvate
1.7
Hypoxanthine Xanthine dehydrogenase
Intermediate Detector (E2)
Initiator
Amplification factor
Analyte
Formate
Formatedehydrogenase NADH
Lactate dehydrogenase/ lactate monooxygenase
Pyruvate
6
Glycerol
Glyceroldehydrogenase NADH
Lactate dehydrogenase/ lactate oxidase
Pyruvate
64
NADP+
Alkaline phosphatase
Lactate dehydrogenase
Lactate
NAD+
accumulate for six minutes increased the sensitivity of the measurement of glycerol by a factor o f 64 (Fig. 2c). This principle has also been applied to sensors used to determine glucose, glucose 6-phosphate, hypoxanthine, N A D P + and formate (Table 1). The extent to which an intermediate may be accumulated, however, is limited by the point o f equilibrium of the reaction. Differences that are observed in the amplification factors (Table 1) may be attributed to the equilibrium constants of the generator enzymes, and the diffusion behaviour of the intermediate and initiator substrates. The greatest enhancement due to accumulation is obtained when the intermediate is a large molecule (i.e. diffuses slowly), and the initiator is a small molecule. This principle of measurement, based on intermediate accumulation, is applicable to any analyte that may be converted in a sequential reaction, provided that one o f the enzymes (the terminal enzyme) is co-factor-dependent and can generate an electrochemical signal at the transducer. An important point in the practical application of this approach (as in any enzymatic analysis) is the need to eliminate any effects on the signal that are due to interfering molecular species. For this reason, the sample is allowed to equilibrate with the reaction medium to establish the 'background' current before initiating the reaction that measures the analyte. This eliminates any contribution from interfering species to the signal and improves the selectivity o f the analysis.
switches, pathways may also be regulated by the availability o f intermediates that have been generated by continuous cycling reactions. Exploiting the principle of enzyme cascades (i.e. the sequential activation of enzymes triggered, for example, by a ligand binding to a receptor) is illustrated in Fig 3a. The binding of the activator (initiator) transforms the inactive enzyme Eli to its active form E h which, in turn, activates a second inactive enzyme, and so on. At the end o f the cascade, an enzyme is activated which produces the electroactive species P*. In glycogenolysis, which occurs in the liver and is a natural example of an enzyme cascade, binding of epinephrine activates adenylate cyclase as the first enzyme in a series ofphosphorylation reactions. The cascade o f these reactions results in amplification o f the signal by five orders of magnitude 7. The sensing principle, based on enzyme activation, may be demonstrated by considering the determination o f glycogen phosphorylase B, and its allosteric effector, AMP (tKe£ 8). Owing to their role in catalysing the sequential phosphorolytic removal of terminal glycosyl residues from the non-reducing end of glycogen, glycogen phosphorylases are key enzymes in the regulation of glycogenolysis. The products o f the reaction are or-glucose-l-phosphate and a truncated glycogen molecule (Eqn 4).
Sequential activation of enzymes Nature provides a variety o f ways of regulating metabolic pathways very specifically. The phenomena ofallosteric control and covalent modification o f proteins play key roles in this regulation 6. Exploiting the principles of metabolically controlled 'switches' and enzyme cascades, whereby repression or activation of enzymes can occur when a threshold value of a particular metabolite is reached, is of'paramount interest in designing analytical procedures. In addition to
Glucose n + Pi
phosphorylase active glucos%_l + c~-glucosel-phosphate
(4)
Glycogen phosphorylase B is active only in the presence o f AMP, which acts as an allosteric activator. The proportion of the enzyme that is active can be determined by the rate of phosphorylation o f glycogen, by quantifying the rate of glucose l-phosphate formation in the presence or absence of AMP. This TIBTECH JUNE 1993 (VOl- 1 t)
258
rg12igW5 a
Initiator reaction can be monitored using an electrode that measures the formation o f hydrogen peroxide and which has the enzymes o f the following sequence (Eqns 5-7) immobilized in the electrode membrane:
1 Eli
Ela
alkaline phosphatase
E2i
(x-glucose l-phosphate
E2a
~- 0~-glucose + Pi
(5) mutarotase
E3i
E3a
o~-glucose ~
~]
[~
Electrode
0
I
I
I
50
100
150
I
200
AMP (pM 1-1) Figure 3 (a) Schematic illustrationof sequentialactivation of enzymes in biosensors.A and C representco-reactants.(b) AMP-concentrationdependenceobtainedwith a glycogen phosphorylaseB/alkalinephosphatase/mutarotase/glucoseoxidaseelectrode in presenceof glycogen, oxygen, and phosphate.
[-~
5] Figure 4 Substrate-recyclingbiosensor. TIBTECHJUNE1993(VOL11)
~ D Electrode
(6)
glucose oxidase [3-glucose
O.
~- [3-glucose
+ 0 2
~-
gluconolactone -6 H202
(7)
Glucose 1-phosphate is dephosphorylated by alkaline phosphatase to yield ~x-glucose (Eqn 5). The oxidation of [3-glucose (Eqn 7) after mutarotation (Eqn 6) results in an increase in the anodic current at the sensor probe due to H202 oxidation. This threeenzyme sensor can be used to measure glycogen phosphorylases A and B at concentrations of 0.005-0.200 U m 1-I. The fact that AMP is required for glycogen phosphorylase B activity can be used to measure the concentration of AMP. For this purpose, glycogen phosphorylase B was entrapped with its substrate, glycogen, in a separate compartment in front o f the three-enzyme layer. The sensor was able to respond to micromolar concentrations of AMP, and the calibration plot o f current versus AMP concentration was linear within the range 5-75 I*M 1-1 (Fig. 3b). Concentrations above 150 t~M pl gave no increase in response; either all the phosphorylase molecules were activated (i.e. a kinetically controlled electrode), or the phosphorylase reaction had become limited by the rate at which glycogen can diffuse within the membrane. The observed increase in the apparent K Mvalue, from 2 ~M 1-I in solution, to 50 ~M ~i in the membrane, strongly supports the latter assumption of diffusion control of the reaction. Since the effect of AMP is to activate the enzyme, the A M P response functions to amplify probe semitivity to a considerably greater extent than that which can be achieved with glucose l-phosphate. The sensor arrangement outlined above could be used repeatedly for ~ 20 sequential A M P measurements (response time 20s, measuring cycle three minutes) without the need to add additional reagents; thereafter, sensitivity declined owing to exhaustion of the substrate, glycogen. Exploiting the allosteric activation of enzymes in sensor membranes opens up new possibilities in sensing. N o t only can it be used to increase sensitivity, but activation by covalent modification of physiologically important proteins also makes it possible to measure the concentration of the activators o f allosteric
259
reviews enzymes, as well as the concentration of substrates, inhibitors and enzyme prosthetic groups, and enzyme activity. In addition to the activation o f allosteric enzymes in an enzyme cascade and the accumulation of intermediates, biosensor sensitivity can also be enhanced by analyte recycling.
Amplification by analyte recycling With bare electrodes, the lower detection limit of amperometric electrodes is - lOOnM1 <. The introduction o f a layer incorporating the enzyme over the electrode surface decreases the sensitivity o f the sensor by one to two orders of magnitude, owing to the additional resistance to diffusion of the analyte. Thus, for measurement o f analyte concentrations in the nanomolar range, a way o f increasing the sensitivity of the enzyme electrode is required. Analyte recycling is the best studied method which uses enzymatic systems to ampli6f the response to very low analyte concentrations 9. The enhancement in semitivity is provided by shuttling the analyte between enzymes acting in a reaction cycle with consumption ofco-substrates and accumulation o f by-products. In Fig. 4, S~ and S2 can be substrates or co-substrates of the enzymes E 1 or E2, respectively. Assuming that the activity o f the enzyme E~ is sufficiently high in the presence o f its co-substrate C 1 and the analyte S (S1 or $2), (the concentration o f which is far below its Michaelis-Menten constant), amplification is achieved through switching on E e by addition of its co-substrate C 2. The analyte S is then shuttled between the two enzymes and, as a consequence, the conversion o f c o substrates into products is amplified, relative to the concentration o f analyte present in the membrane, beyond the normal stoichiometry o f the single reactions. The overall reaction o f both reactants is thus:
enzyme electrodes has been achieved for ~ 15 pairs of coupled enzymes (Table 2; 1Kef~ 3, 11). Using this approach, the limit o f detection was reduced to nanomolar concentrations for some substrates, such as lactate, pyruvate and hydroquinone 13. In addition to analyte recycling electrodes, there have also been reports on the use of the enzyme cycles coupled to fibre-optic probes 27,28, and in a column combined with a thermistor 29,3°, and with a flowinjection system with amperometric detection 31 33.
Linear recycling with oxidase and dehydrogenase When oxidases are coupled with their respective dehydrogenase electrode, the reaction system includes species that are readily detectable electrochemically; the change in co-reactant concentration can thus be measured directly at the electrode onto which the recycling enzyme pair is immobilized. In most cases, consumption o f the co-reactant has been followed (Table 2). The system incorporating the enzyme couple lactate dehydrogenase/lactate oxidase (Eqns 9, 10) has been studied in detail 14. This consists of a gelatinentrapped enzyme couple and an oxygen electrode as transducer. In the presence of N A D H , the pyruvate formed in the lactate oxidase reaction is converted by the dehydrogenase back to lactate, which is then rc~ oxidized by lactate oxidase. lactate oxidase Lactate + 0 2
~ pyruvate + H 2 0 2
(9)
lactate dehydrogenase Pyruvate + N A D H
lactate + NAD +
(8)
(10)
By measuring the change in concentration of one of the co-reactants or products (for example, P*), either directly, or by an additional analytical reaction step, the recycling system may be used to amplify the response of the system to measure either analyte S1 or S2. In the recycling mode, it is not possible to distinguish between the analytes. When one molecule of product is fbrmed per substrate molecule reacting, the concentration o f the co-products (Pl, P2) increases, and the concentration o f the co-substrates (Cl, C2) decreases linearly with time, while the concentration of the cycling substrates (S~ and $2) remains constant. The number of cycles for which the substrate is turned over in a given time is a function of the substrate concentration. This recycling system, based on the concept of a linear enzymatic amplification of the signal, has been put to practical use by coupling dehydrogenases with oxidases or transaminases, or by coupling the catalytic activities of several kinases. The early applications of the principles of analyte recycling in enzyme electrodes were aimed at cofactor regeneration 1°. Enzymatic analyte recycling in
Oxygen consumption in the enzyme membrane is thus enhanced, yielding an increase in the sensitivity to lactate by a factor o f up to 4100. Under steady-state conditions, the ratio of the sensitivities in the linear measuring range of amplified and unamplified systems (termed the amplification factor, G) is:
C1 + C2
~ P~ + P2
L 2 klk 2 G= 2D (k 1 + k2)
(11)
where L is the membrane thickness; kt, the first-order rate constant; and D, the diffusion coefficient34. When enzymes of very high activities are incorporated into an enzyme layer with a high, characteristic diffhsion time (L2/D), the amplification possible is very large. Experiments investigating the influence o f enzyme loading (i.e. the amount of the enzyme activity in the membrane) on the amplification o f the lactate signal showed, in agreement with the theory, that a 4100fold amplification was feasible using a gelatin matrix with a characteristic diffusion time o f ~ 90s. With this TIBTECHJUNE 1993 (VOL 1i)
260
reviews Table 2. Substrate recycling in biosensors Enzyme couple
Glucose
Glucose oxidase/glucose dehydrogenase Oxygenelectrode
10
12
Lactate
Lactate oxidase/lactate dehydrogenase
Oxygen electrode
48 000 4100 250
13 14 15
Lactate/ pyruvate
Cytochrome b2/ lactate dehydrogenase
Platinum electrode
10
12
NADH/NAD+
Peroxidase/glucose dehydrogenase
Oxygen electrode
60
12
Glutamate
Glutamate dehydrogenase/ alanine aminotransferase Glutamate oxidase/ glutamate dehydrogenase Glutamate oxidase/ alanine aminotransferase
Modified carbon electrode Oxygen electrode
15
16
20
17
Hydrogen peroxide electrode
500
18
Hexokinase/pyruvate kinase
Oxygen electrode with lactate dehydrogenase Lactate monooxygenase modified carbon electrode with glucose-6-phosphate dehydrogenase
220
19
1200a
20
800
21
ADP/ATP
Transducer
Amplification factor
Analyte
ADP
Myokinase/pyruvate kinase
Oxygen electrode with pyruvate oxidase
Ethanol
Alcohol oxidase/alcohol dehydrogenase
Oxygen electrode
Benzoquinone/ Cytochrome b2/laccase hydroquinone
Ref.
22
Oxygen electrode
500
23
Malate/ oxalacetate
Malate dehydrogenase/lactate monooxygenase
Oxygen electrode
3
24
L-Leucine
Leucine dehydrogenase/aminoacid oxidase
Oxygen electrode
40
25
Phosphate
Nucleoside phosphorylase/ alkaline phosphatase
Oxygen electrode with xanthine oxidase
20
26
atn combinationwithintermediateaccumulation.
amplification, lactate concentrations as low as 1 nM 1-1 could be determined with reasonable precision. The response to lactate could be increased still further, up to 48000-fold, when the immobilization was performed using polyurethane, which permits higher enzyme loading 13. However, the reproducibility and stability of this large amplification is poor, owing to the number o f enzymes used. In general, the specificity of recycling sensors is poorer compared with m o n o - e n z y m e sensors. TIBTECH JUNE 1993 (VOL 11)
Linear recycling with kinases Recycling systems need not be limited to reactions in which electroactive compounds are produced. In such cases, the recycling enzyme pair may be c o m bined with an indicator enzyme (or sequence of enzymes) which transforms one of the cycle constituents(usually a product) into a measurable species. Kinase reactions, owing to their, in general, favourable equilibrium constants are well suited to such systems. However, none of the cycle constituents for kinase-
261
reviews mediated reactions are directly measurable at a favourable potential, and the products o f the cycle have therefore been coupled with o x y g e n - c o n s u m i n g enzyme reactions for their determination. T o enable sensitive measurement o f A D P and ATP, a biosensor was constructed by combining an oxygen electrode and a gelatin layer in which were immobilized h e x o kinase and pyruvate kinase (shuttling A D P / A T P b e t w e e n each other), and a lactate dehydrogenase and lactate m o n o o x y g e n a s e sequence (which was also used for intermediate-accumulation experiments) to transform the e n o r m o u s a m o u n t o f p y r u v a t e formed in the recycling reaction into measurable oxygen consumption 19. W i t h this recycling system, a 220-fold amplification was possible (Table 2). This system has also been used with the lactate dehydrogenase/lactate m o n o o x y g e n a s e 'detector' enzymes substituted by pyruvate oxidase or glucose-6-phosphate dehydrogenase 20.
Exponential recycling with kinases In contrast to linear amplification recycling systems, an exponential recycling system is characterized by the use o f an e n z y m e that forms m o r e than one molecule o f product per molecule ofsubstrate (i.e. the total amount o f intermediates and by-products increases exponentially with time). Theoretical considerations showed that the concentration o f any o f the cycling intermediates, or by-products, at any given time, is a linear function o f the initial substrate concentration 35. An example illustrating this principle is the A D P / A T P cycling that occurs with the m y o k i n a s e / p y r u v a t e kinase/pyruvate oxidase-modified oxygen sensor 21. In one cycle, myokinase forms two molecules o f A D P per molecule A T P , which is derived from the phosphorylation o f A D P by pyruvate kinase (Eqns 12, 13): pyruvate ADP+ kinase phosphoenolpyruvate ~- A T P + pyruvate 02)
myokinase ATP + AMP
~
2 ADP
(13)
T h e amounts o f A T P and ADP, which are initially present at very low concentrations, increase e x p o n e n tially with cycling time, provided that A M P and phosphoenolpyruvate concentrations are sufficiently high, so as not to limit the reaction. T h e e n o r m o u s a m o u n t o f pyruvate f o r m e d in the cycle is reflected by the oxygen c o n s u m p t i o n in the immobilized pyruvate oxidase layer. W i t h an optimized sensor configuration, an increase in the sensitivity o f detection o f A D P by a factor o f 800 was obtained.
Mono-enzymatic substrate recycling Recently, a novel amplification scheme has been developed, based on the fact that N A D H recycling is performed by only one enzyme 36. In the presence o f
N A D +, lactate dehydrogenase catalyses the oxidation ofglyoxylate to oxalate, with concomitant formation o f N A D H . In the presence o f pyruvate, glyoxylate, and a limiting a m o u n t ofpyridine nucleotide, the cofactor is cyclically regenerated. With such a system, a 170-fold increase in lactate formation enabled as little as 50nM 1-1 to be detected for N A D H . This kind of m o n o - e n z y m a t i c cycle might facilitate the design of novel, simple enzymatic amplification schemes. Alternatively, it may be possible to enhance this amplification by a second recycling e n z y m e pair in a double amplification system, thus yielding a still further i m p r o v e m e n t in the sensitivity o f the sensor. Conclusions
Sequentially coupled enzyme reactions are widely used in analysers based on the use o f soluble reagents. Signal amplification by a cyclic e n z y m e couple is already exploited in a commercial e n z y m e - i m m u n o assay system (Novoclone). In contrast, a m o n g biosensors that are based on immobilized biomolecules, m o n o - e n z y m e electrodes dominate the practical applications, and only a few sequentially coupled enzyme electrodes have been commercialized. T h e difference in reaction optima is a serious p r o b l e m in combining several immobilized enzymes within one c o m p a r t ment, as for example, in the three-enzyme systems for creatinine analysis. Further progress, both for soluble and immobilized coupled enzymes, is expected to result from the creation o f chimeric enzyme molecules, either by gene fusion or by 'site to site' linkage o f the interacting e n z y m e s using post-translational, chemical modification m e t h o d s . In this way, the b i o c h e m i c a l signal m a y be effectively 'channelled', w h i c h m a y lead to high sensitivity and specificity o f b i o s e n s o r s . References
1 Clark, L. C. and Lyons, C. (1962) Ann. NYAcad. Sci. 102, 29-45 2 Turner, A. P. F., Karube, 1. and Wilson, G. S. (eds) (1987) Biosenso~ - Fundamentals and Applications, Oxford UniversityPress 3 Scheller,F. and Schubert, F. (1989). Biosensoren, Akademie Verlag 4 Hall, E. A. H. (1990) Biosensors, Open UniversityPress 5 Schubert, F., Lutter,J. and Scheller, F. (1991) Anal. Chim. Acta 243, 17-21 6 Koshland,D. E.,Jr (1987) Trends Biochem. Sci. "[2,225 229 7 Stryer,L. (1988) Biochemistry, Freeman Co. 8 Wollenberger,U. and Scbeller,F. Biosensors and Bioelectronics (in press) 9 Lowry, O. H. and Passonneau,J.V. (1972) A Flexible System of Enzymatic Anal}sis, Academic Press 10 Davies, P. and Mosbach, K. (1974) Biochim. Biophys. Acta 370, 329-338 11 Schubert, F., Wollenberger, U., Pfeiffer,13. and Scheller, F. (1991) in Advances in Biosensors Vol. 1 (Turner, A. P. F., ed.), pp. 77 105, JA! Press 12 Schubert, F., Kirstein,D., Schr6der, K. L. and Scheller, F. W. (1985) Anal. Chim. Acta 169, 391-396 13 Scheller, F. W., Schubert, F., Pfeiffer, D., Wollenberger, U., Renneberg, R., Hintsche, R. and Ktihn, M. (1992) GBFMonouaphs 17, 3-10 14 Wollenberger,U., Schubert, F., Scheller, F. W., Danielsson, B. and Mosbach, K. (1987) Stud. Biophys. 119, 167-170 15 Mizutani, F, Yamanaka,T., Tanabe, Y. and Tsuda, K. (1985) Anal. Chim. Acta 177, 153-166 TIBTECH JUNE 1993 (VOL 11)
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reviews 16 Schubert, F., Kirstein, D., Schellcr, F., Appelqvist, P,., Gorton, L. and Johansson, G. (1986)Anal. Lett. 19, 1273-1288 17 Wollenberger, U., Scheller, F., Pavlova, M., Mtiller, H. G., Risinger, L. and Gorton, L. (1989) GBF Monographs 13, 33~6 18 Yao, T., Yamamoto, H. and Wasa, T. (1989) in Proc. ISE Meeting, Kyoto, pp. 1014~1015, Kodanasha 19 Wollenberger, U., Schubert, F., Scheller, F. W., Danielsson, B. and Mosbach, K. (1987) Anal. Lett. 20, 657 668 20 Yang, X., PlCeiffer,D., Johansson, G. and Scheller, F. W. (1991) Electroanalysis 3, 659 663 21 Scheller, F., Wollenberger, U., Schubert, F., Pfeiffer, D., Makower, A. and O'Neil, C. in Proceedings N A T O advanced workshop on use of immobilized biological components for detection, medical, food and environmental analysis, Brixen, Italy (in press) 22 Hopkins, T. P,.. (1985) Int. Biotechnol. Lab. 3, 20-25 23 Scheller, F., Wollenberger, U., Schubert, F., Pfeiffbr, D. and Bogdanovskaya, V. A. (1987) GBF Monographs 10, 39-49 24 Scheller, F., P, enneberg, R. and Schubert, F. (1988) Methods Enzytool. 137, 29-43 25 Scheller, F., Pfeiffer, D., Hintsche, R., Dransfeled, 1., Wollenberger,
U. and Schubert, F. (1990) Ann. NTAcad. &i. 613, 68-78 26 Wollenberger, U., Schubert, F. and Scheller, F. (1992) Senso~:~Actuat. B7, 412-415 27 Schubert, F. (1993) Sensors Actuat. Bli, 531-535 28 Wang, A.J. and Arnold, M. A. (1992) Anal. Chem. 64, 1051-1055 29 Scheller, F. W., Siegbahn, N., Danielsson, B. and Mosbach, K. (1985) Anal. Chem. 57, 1740-1743 30 Kirstein, D., Danielsson, B., Scheller, F. W. and Mosbach, K. (1989) Biosensors 4, 231-239 31 Asouzu, M. U., Nonidez, W. K. and Ho, M. H. (1990) Anal. Chem. 62,708~12 32 Hansen, E. H., Arndal, A. and Norgaard, L. (1990) Anal. Lett, 23, 225-240 33 Yang, X., Pfeiffer, D.,Johansson, G. and Scheller, F. W. (1991) Anal. Lett. 24 (1991) 1401 1417 34 Kulys,J. J., Sorochinskii, V. V. and Vidzivnaite, P,.. A. (1986) Biosensots 2, 135 146 35 Kopp, L. E. and Miech, P,.. P. (1972)J. Biol. Chem. 247, 3558-3563 36 Schubert, F., Scheller, F. and Krasteva, N. (1990) Electroanalysis 2, 347-351
TIBTECH Editorial Policy Trends in Biotechnology is a news, reviews and commentary journal designed to keep its international readershi p up-to-date with the current developments in biotechnology. TIBTECH occupies a niche between primary research journals and conventional review journals - it is not a vehicle for the publication of original research data or methods. There is a strong emphasis on an integrated approach to communicating significant new advances in biotechnology, and discussing their commercial potential. This necessitates combining essential background with state-of-the-art information to make the topics addressed accessible and valuable to newcomers and experts in the field alike. All articles are subject to peer-review and are prepared to strict standards to ensure clarity, scientific accuracy and readability. Commissioning does not guarantee publication. Reviews: Balanced and concise presentations of specific areas of research. The scope of such reviews is more limited than those of conventional review journals, but provides sufficient information to place the latest developments in a particular field in perspective. Focus: Short minireview-style articles, which focus on a detailed examination of a narrow topic, thus providing a qu ck publication response to recognition of significant new directions of research. Forum: A platform for debate and analytical discussion of newly reported advances -,onferences. Includes subsections: Biotopics: A column providing a lighter, more personal treatment of topics ranging from senous science and novel techniques, to finance and ethical questions.
TIBTECH JUNE 1993 (VOL 11)