Sensors and Actuators B, II (1993) 531-535 531

A fiber-optic enzyme sensor for the determination of adenosine diphosphate using internal analyte recycling*

Florian Schubert Physikolisch-Technische Bundesatwtalt, Abbestraw 2-12, D-loo0 Berlin 10 (Germany)

Abstract

An optical multienzyme biosensor for the sensitive determination of adenosine diphosphate (ADP) is presented. Hexokinase, pyruvate kinase, and glucose-6-phosphate dehydrogenase are coimmobiliied in a polyurethane mem- brane and tixed to a commercial fiber-optic probe. ADP is continuously phosphorylated/dephosphorylated in a cycle catalyzed by the kinases. The glucose-o-phosphate formed in the hexokinase reaction is oxidized by the dehydroge- nase, leading to the formation of NADH, the fluorescence of which is detected by the optical transducer and used as the measuring signal. Comparison of the responses of the sensor to adenosme triphosphate (ATP) and ADP shows an ampliication factor of 90 for ADP measurement. The detection limit is thus lowered to 0.1 pM, the useful measuring range extending up to 20 pM. The response of the sensor to other compounds involved in the reaction system has also been investigated.

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In the last few years, the coupling of biochemical recognition elements with optical transducers in optical biosensors has attracted increased interest (see [l-3] for review). Among them, fiber-optic sensors (optrodes) offer distinct advantages, such as minute size, no need for a reference, no electrical contacts, spatial flexibility, and remote analysis. Particularly, many attempts have been made to combine biocatalytic reactions with opti- cal-fiber probes capable of detecting oxygen, pH, intrin- sic enzyme fluorescence, or the fluorescence of reduced pyridine nucleotide coenzymes. Making use of fluores- cence of NAD(P)H at around 460 mn when excited in the upper ultraviolet region, the latter approach has been employed in the development of optical sensors incorporating the appropriate dehydrogenases for the determination of lactate and pyruvate [4,5], ethanol [6], and glucose [7J

There are, however, hardly any reports on a fiber-op- tic biosensor using more than one enzyme, although the advantages of adopting coupled enzyme reactions for numerous purposes in biosensors have been well docu- mented (e.g., [8,9]). With regard to the restricted sensi- tivity of fiber-optic enzyme probes, the method of internal analyte recycling for the enhancement of the sensitivity of biosensors [lo] appears to be of particular interest here. It is based on two coimmobilized en- zymes, one of which catalyzes the conversion of the

*Dedicated to Professor Frieder W. Scheller on the occasion of his 50th birthday.

0925-4OQ5/93/$6.00

analyte to a product and the other the conversion of that product back to the analyte. One of the coreac- tants is detected directly or via additional enzyme reac- tions. The recycling results in the formation (or consumption) of significantly more coreactant than the amount of analyte present in the immobilized enzyme layer. This principle may lead to sensitivities in the nanomolar range. It has been successfully implemented in a number of enzyme electrodes, e.g., for glucose, lactate, ethanol, glutamate, and NADH (see [9] for review). Other measuring systems based on internal analyte recycling have been assembled using appropri- ate enzyme reactors combined with electrochemical [ll, 121, thermometric [ 13,141, or chemihtminescence [15] detection. It appears that up to now no optical biosensor has been studied in conjunction with this powerful method for sensitivity enhancement. The present paper reports a fiber-optic biosensor using a recycling system for purine nucleotides, specifically adenosine diphosphate/adenosine triphosphate (ADP/ ATP), consisting of the immobilized enzyme pair hex- okinase and pyruvate kinase. During the cyclic conversion of the analyte:

ADP + phosphoenolpyruvate + ATP + pymvate ATP + glucose + ADP + glucosed-phosphate

glucose-6-phosphate is formed, which is oxidized in the subsequent reaction catalyzed by glucose-6-phosphate dehydrogenase:

glucose-6-phosphate + NAD + + 6-phosphogluconate + NADH + H+

@ 1993 - Else& Sequoia. All rights reserved

532

The NADH formed is detected fluorometrically by the fiber-optic probe. The kinase system, which is well suited for such recycling because of its favorable equi- libria [ 161, has been previously used for ADP measure- ment, but with entirely dierent signal-generation schemes. In these studies, factors of signal amplification have been observed which amount to 30 in an enzyme thermistor [ 141, 1200 in an enzyme reactor with electro- chemical detection and a controlled-stop recycling regime [ 121, and 220 in a highly complex four-enzyme electrode [ 171. The aim of the present study is to demonstrate the feasibility of the system in an optical biosensor and assess the optimum conditions for operation of the sensor.

Experimental

Reagents Glucosed-phosphate dehydrogenase (EC 1.1.1.49,

from Leuconostoc mesenteroides 155 U/mg using NAD+), hexokinase (EC 2.7.1.1, from baker’s yeast, 65 U/mg), and pyruvate kinase (EC 2.7.1.40, from rab- bit muscle, 500 Ujmg) were purchased as lyophilized powders from Sigma, Saint Louis, MO. The sodium salts of nicotinamide adenine dinucleotides, reduced and oxi- dized (NADH and NAD+), were obtained from AWD Dresden, Germany. ADP, ATP, and phosphoenolpyru- vate were obtained from Reanal Budapest, Hungary. Polyurethane and poly( phenyl) polyisocyanate cross- linker (Systanat MR) were supplied by Synthesewerk Schwarzheide, Germany. All other reagents were of analytical-reagent grade and were used without further purification.

Apparatus and procebres As the basis of the sensor device a commercially

available fluorescence probe, the Fluorosensor@ from Ingold, Steinbach, Germany [5, 181, was used. Its func- tion is based on the fact that NADH fluoresces at 460 nm when excited at around 360 nm, whereas its oxidized ,form, NAD+, does not. In the Fluorosensor light from a low-pressure mercury lamp is passed through interference filters to produce a light beam at 360 mn. This light is guided through the probe tip into the surrounding medium by a quartz fiber bundle. The fluorescent light is collected at the probe tip and guided by another fiber bundle and through appropriate filters to a photomultiplier tube, which transfers the signal to a processing unit. The sensor is capable of measuring NADH concentrations up to a least 400 FM. It was connected to a y-t recorder.

Enzyme membranes were prepared by entrapment of the biocatalysts in a polyurethane network as described previously [ 191. In a narrow test tube 147 U of glucose-6- phosphate dehydrogenase (G6P-DH), 85 U of hexoki-

nase, and 125 U of pyruvate kinase were thoroughly mixed with a spatula and suspended in 50 ul of polyurethane solution (Syspur, 0.5% in acetone) in an ultrasonic bath. Aliquots of 10 ul were then transferred to a dialysis membrane (Nephrophan, CK Bitterfeld, Germany, thickness 20 pm) and allowed to dry for 30min at room temperature. Five microliters of 0.5% (v/v) polyisocyanate crosslinker (Systanat MR) in ace- tone was dropped on each of the dried layers and, after drying for 5 min, the formed membranes were covered with another dialysis membrane. The membranes were stored at 4 “C until taken for use.

The enzyme sensor was assembled by fixing a sand- wich membrane to the tip of the Fluorosensor by means of a rubber O-ring. The measuring cell was made of black plastic material (Delrin) to avoid the effects of ambient light. It was screwed to the sensor body in such a way that the sensing area was tightly fixed and exposed to the inner cell volume in a horizontal position. The cell was thermostatted at 25 “C and equipped with a mag- netic stirring bar to ensure perfect mixing of the solution. A hollow needle was glued vertically into the cell body at the bottom level to permit removal of the solution by means of a suction pump. The cell was furthermore covered with a rubber lid through which a central hole of 2 mm diameter had been drilled for injection of buffer and sample solutions.

The buffer solution for the sensor was composed of 0.05 M Tris-HCl, 10 mM MgC&, and 10 mM KC1 at pH 7.5 [ 161. As background solution, 2 ml of buffer and the required cofactors were pipetted into the cell, and when the fluorescence output had reached a constant level, the sample (usually 20 pl) was injected with a microliter syringe. The increase of the fluorescence intensity was re- corded until at least 90% of the stationary level was attain- ed. Then the measuring cell was emptied, washed twice with buffer, and filled with 2 ml buffer for the next assay.

In the present report the values of the measuring signal are given as relative fluorescence in volts, accord- ing to the Fluorosensor output.

Results and discussion

The principle of the sensor is shown in Fig. 1. It relies on three enzyme reactions combined with the fluores- cence indication of NADH by the fiber-optic trans- ducer. In the presence of phosphoenolpyruvate and glucose, the final analyte, ADP, is shuttled between pyruvate kinase and hexokinase, the latter reaction leading to the formation of glucosed-phosphate. The oxidation of glucosed-phosphate by NAD+ is cata- lyzed by coimmobilized G6P-DH. The radiation ema- nating from the optical-fiber bundle causes the formed NADH to fluoresce and the resulting emission is de- tected as the response of the probe. The measurement

533

AOP

PEP

NAD’

Fig. 1. Schematic representation of the fiber-optic biosensor for ADP and the biocatalytic reactions taking place in the enzyme membrane: HK, hexokinase; PK, pyruvate kinase; GBP-DH, glu- cose-6-phosphate dehydrogenase; PEP, phosphoenolpyruvate; ADP, adenosine diphosphate; ATP, adenosine triphosphate.

L

0.2 0.L 0.6 0.8 1.0 1.2 glucose-6-phosphote,mM

Fig. 2. Calibration graph of the fiber-optic sensor for glucose-& phosphate. The concentration of NAD+ was 1 mM.

of ADP thus requires excess amounts of phos- phoenolpyruvate, glucose, and NAD+.

In principle the present sensor is also capable of measuring glucose&-phosphate and ATP. In order to test the functioning of the immobilized G6P-DH, which acts as the ‘terminal’ enzyme of the biochemical reac- tion system, the response of the sensor to glucose-d phosphate was first studied (Fig. 2). In the presence of 1 mM NAD+ the increase of the fluorescence intensity depends linearly on the concentration of this substrate up to 0.5 mM in the measuring cell. The sensitivity to glucose&phosphate increases with increasing concen- tration of the nicotinamide cofactor to attain a plateau at 0.5 mM (Fig. 3). The response time for these mea- surements was in the order of 12 min. The next step was to demonstrate the activity of the immobilized kinases towards ATP and ADP. Figure 4 shows a typical recorder trace for the determination of ATP with subse- quent injection of phosphoenolpyruvate to achieve the recycling of this analyte. With NAD+ and glucose present in the solution, addition of 50 PM ATP resulted in an increase in the sensor signal of 0.17 V. When the signal-time curve had reached a steady state, phospho- enolpyruvate (PEP) was added and the sensor re- sponded with a steep signal increase, indicating the recycling by pyruvate kinase of the ADP formed in the reaction catalyzed by hexokinase. The sensor signal levelled off after about 20min. In the experiment

? -X-X-X--X

c

- 1.5 NAD*,mM

Fig. 3. Dependence of the response of the fiber-optic sensor to glucose-6-phosphate (final concentration 0.25 mM) on the concen- tration of NAD+.

50pM PEP ATP

Fig. 4. Response curve of the sensor to successive injections of ATP and phosphoenolpyruvate (PEP).

-I

0.5 1 1.5 2

NAD+, mM Fig. 5. Effect of NAD+ concentration on the sensor response to ATP (final concentration 0.25mM) without amplification ( x ) and ADP (0, final concentration 5 pM; I mM phosphoenolpyru- vate). NAD+ and glucose concentrations were I mM and 5 mM, respectively.

shown, the signal for ATP was thus increased by a factor of 15. Because both NAD+ and glucose are necessary for the reactions involved in the (unam- plified) response to ATP, the effect of their concentra- tions on the signal has been established. In the presence of 1 mM nicotinamide cofactor, the signal for 250 FM ATP increased with glucose concentration up to 0.4 mM. No further change was observed up to 5 mM. The dependence on the concentration of NAD+ is shown in Fig. 5. A cofactor concentration of 1- 1.5 mM appears to be sufficient for optimum response.

534

0.2 0.4 0.6 0.6 1 12 phosphoenolpyruvate, mM

Fig. 6. Effect of phosphoenolpyruvate concentration on the sen- sor response to ADP (final concentration 1OpM). NAD+ and glucose concentrations were 1 mM and 5 mM, respectively.

The observed recycling effect was utilized for the sensitive determination of ADP. To warrant a large excess of glucose for that part of the recycling system catalyzed by hexokinase, a glucose concentration of 5 n&I was provided. With additional 1 mM NAD + and 1 mM phosphoenolpyruvate in the solution, aliquots of ADP solutions were added. After a reproducible lag phase of 1 - 1.5 min, the fluorescence intensity increased to approach a new steady state after 20-30 min, with the shorter response times at higher analyte concentra- tions. NAD+ intluences the magnitude of the probe response to ADP in a ,manner somewhat different for that to ATP (Fig. 5): at concentrations above 1 mM the signal decreases. Since no inhibiting effect of the nicoti- namide cofactor on the pyruvate kinase reaction is known, the reason for this behavior remains to be elucidated. The variation of the sensor response to 10 uM ADP with the concentration of phospho- enolpyruvate is depilcted in Fig. 6. No change in the fluorescence intensity was observed without the cosub- strate; with increasing phosphoenolpyruvate concentra- tion the signal increased, which again indicates the function of the three-enzyme system. Saturation was attained at 0.8mM. Routinely, 1 mM phospho- enolpyruvate was used for further analyses.

Figure 7 shows the calibration graphs obtained for the measurement of ADP and the unamplified measure- ment of ATP (i.e., in the absence of phosphoenolpyru- ate), both obtained under the established optimum conditions. The response to ATP is linear up to 0.6 mM with a lower limit of detection of about 10 uM. In contrast, for ADP the linear portion of the graph extends only up to 5 uM but has a slope about 90 times as large as that for ATP. This amplification permits ADP concentrations as low as 0.1 uM to be deter- mined. The amplification factor decreases with increas- ing concentration, the curve levelling off at 40 uM. The region up to 20 uM might be regarded as the useful range. Saturation is reached at the same signal magni-

- ADP. pM > 10 20 30 40 50

II

-- ATP, mM

Fig. 7. Calibration graphs for ADP (with phosphoenolpyruvate) and ATP (without phosphoenolpyruvate) using the fiber-optic sensor.

tude for both analytes, giving evidence for diffusion control of the sensor [ 171. After two days of operation the sensitivity of the enzyme probe for ADP sharply dropped, whereas it remained fairly constant for five and 12 days in the determination of ATP and glucose- B-phosphate, respectively. Hence it follows that the activity of the immobilized pyruvate kinase enzyme determines the overall stability of the sensor. A more stable pyruvate kinase immobilizate might be ob- tained by using another matrix for entrapment, such as gelatin [20].

A serious problem regarding the practicability of the sensor is its long response time, particularly with the ADP determination. With all concentrations tested, however, 90% of the fluorescence intensity change was completed after 12min. In order to shorten the measuring time a non-steady-state regime can there- fore be employed by taking the relative fluorescence after that time period as the measuring signal. Using this approach, the relative standard deviation for 10 successive assays of 2.5 uM ADP was determined to be 5.9%.

The present sensor demonstrates, to our knowledge for the first time, the applicability of the internal ana- lyte-recycling concept for signal amplification in a true optical biosensor. The amplification factor of 90 lies between those obtained in other ADP/ATP recycling systems [ 12, 14,171, where respective detection limits of 1 nM, 2 PM, and 0.25 pM have been realized. Stud- ies to overcome the shortcomings mentioned above, i.e., to increase the sensor stability by varying the immobilization method and to shorten the response time substantially by changing the sensor geometry, are under way.

The author wishes to thank Thomas Scheper for providing the Ingold Fluorosensor@ and for his con- tinued interest and support.

References

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Florian Schubert received the Ph.D. and Doctor of Sciences degrees in biotechnology from the Academy of Sciences of the former GDR in 1983 and 1990, respec- tively. He is currently at the Department of Medical Physics of Physikalisch-Techniscche Bundesanstalt. Be- fore this he was with the Central Institute of Molecular Biology in Berlin-Buch. He is coauthor of Biosemors (Elsevier, 1992). His research interests are electrochemi- cal and optical multienzyme sensors and their applica- tion in clinical chemistry.