Anal. Chem. 1990, 62, 2377-2380

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Oxygen Optrode for Use in a Fiber-optic Glucose Biosensor Maria C. Moreno-Bondi' and Otto S. Wolfbeis* Analytical Division, Institute of Organic Chemistry, Karl-Franzens University, 8010 Graz, Austria Marc J. P. Leiner and Bernhard P. H. Schaffar AVL-List GmbH, Biomedical Division, Kleist-Strasse 48, 8020 Graz, Austria

An optlcal flber oxygen sensor, based on the dynamic quenchlng of the luminescence of trls( 1,lO-phenanthrol1ne)ruthenium( I I ) catlon by molecular oxygen, Is presented. The complex Is adsorbed onto slllca gel, Incorporated In a slllcone matrix possessing a high oxygen permeability, and placed at the tlp of the optlcal fiber. Oxygen has been monltored continuously In the 0-750 Torr range, wlth the detectlon llmR belng as low as 0.7 Torr. The device has been applkd to the development of a fast respondlng and highly sensltlve flberoptlc glucose blosensor based on this highly sensltlve oxygen transducer. The sensor relates oxygen consumption (as a result of enzymatic oxldatlon) to glucose concentration. The enzyme Is Immobilized on the surface of the oxygen optrode; carbon black Is used as an optical Isolation In order to prevent ambient llght and sample fluorescence to interfere. Measurements have been performed In a flow-through cell In air-equlllbrated glucose standard solutions of pH 7.0. The ellects of enzyme lmmoblllzatlon procedures (including enzyme Immobilization on carbon black) as to response times (around 6 min), analytlcal ranges (0.06-1 mM glucose), reproduclblllty in sensor construction, and long-term stablllty have been studied as well.

INTRODUCTION Given the widespread demand for glucose sensing devices, the development of glucose biosensors displaying the required sensitivity, long-term stability, and fast response has been a main target in sensor research during the last few years. Glucose sensing devices can be divided into three main classes, namely the electrochemical, optical, and enthalpy sensors (1, 2). In most cases, the sensing scheme includes the use of glucose oxidase (GOx), an enzyme catalyzing the oxidation of glucose according to

+

-+ GOx

+

D-glucose O2 D-gluconolactone H,O, D-g~uconolactone HzO D-gluconic acid Therefore, by measuring the decrease in oxygen partial pressure (PO,) when glucose is oxidized by the enzyme gives an indirect indication of glucose concentration. In the amperometric enzyme electrode ( 3 ) ,an oxygen electrode is covered with a layer of immobilized GOx. Numerous modifications of this sensing scheme are known ( I ) . Uwira et al. (4) briefly report on the development of an optical glucose sensor. In this device, changes in PO, are measured via quenching of the fluorescence of an oxygen-sensitive dye. However, this system is not compatibile with conventional fiber-optic components. Other optical approaches have been made by Goldfinch and Lowe (5) who used bromocresol green immobilized on cellophane in order to measure the decrease in pH produced during the GOx-catalyzed oxidation of glucose by following the Present address: Departamento de Quimica Analitica, Facultad de Ciencias Quimicas, Universidad Complutense, 28040 Madrid, Spain.

changes in light absorption of a pH-sensitive dye. Trettnak et al. (6) describe a fiber-optic fluorosensor for glucose that uses a p H sensor as the transducer. However, the response of biosensors based on pH transducers strongly depends on the buffer capacity of the medium. In the recent approach of Weigel et al. (7),a GOx film was spread onto a germanium crystal. Fourier transform infrared attenuated total reflection (FTIR-ATR) spectroscopy was applied to measure the 1153-cm-' absorption band of gluconic acid, the intensity of which can be related to glucose concentration. Schultz et al. (8)have described an immunoaffinity glucose sensor based on the reversible competitive binding of glucose and fluorescein-labeled dextran for the sugar binding sites of concanavalin-A. This device is very slow in response. The intrinsic fluorescence of GOx is another parameter that has been utilized in a glucose sensing device described by Trettnak et al. (9),following the observation that the intrinsic fluorescence of the enzyme slightly changes upon interaction with glucose. While glucose biosensors based on PO, and, in particular, hydrogen peroxide transduction ( 1 0 , l l )are to be preferred over those based on pH transduction for a number of reasons, the current sensitivity limits of oxygen sensors (in terms of signal resolution) determine the glucose detection limits. Existing optical glucose biosensors, for instance, cannot be applied for determination of "normal" glucose levels in urine simply because the existing oxygen optrodes are not sensitive enough. More recently, the new indicator tris(2,2'-bipyridine)ruthenium(II) dichloride, [Ru(bpy),,+], has been applied to monitor oxygen via a fiber-optic device (12-14). Its quenching constant still is too poor for use in biosensors for urine glucose levels. We report here the development of a new PO, sensor based on the quenching of the luminescence of a closely related metal complex, tris(1,lO-phenanthro1ine)ruthenium(I1) [Ru(phen),,+] immobilized in a silicone matrix. The use of this sensing material substantially improves the characteristics of the optical sensors described to date. The material also has been applied to highly sensitive glucose determination by coupling it to the above GOx reaction.

EXPERIMENTAL SECTION Instrumentation. Optical measurements were performed with two different arrangements. System I consists of an Oriel 3090 fiber-optic photometer (Chelsea Instruments, London) equipped with a 150-W xenon lamp pulsed at 9 Hz. After passing a 450-nm interference filter (band-pass 10 nm), light was guided through the input bundle of a bifurcated optical fiber to a flow-through cell and luminescence was collected and guided back to the photometer through the output bundle. A 570-nm cut-off filter (Schott, Mainz, FRG) was used to remove the scattered light. The 1.5-m light guide is made of poly(methy1methacrylate) (PMMA) (Faseroptik Henning, Allenburg, FRG), with an internal diameter of 3.5 mm per single bundle (30 fibers per bundle) and 4.5 mm at the common end. The home-made flow-throughcell was machined from stainless steel and possesses a volume of ca. 20 pL. The biosensor forms one wall of the flow-throughcell and is separated from the optical fiber end by a 4 mm PMMA spacer (for a more detailed description of the cell see Trettnak et al. (IO)). The buffer and

0003-2700/90/0362-2377$02.50/0 0 1990 American Chemical Society

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glucose solutions are pumped through the cell a t a typical total flow rate of 1.0 mL min-'. An autosampler (Ventilomat R08, Fa. Ziegler, Graz, Austria) was utilized to calibrate the glucose sensor using standard solutions. System I1 was used for calibration of the oxygen sensor performances, in order to compare the latter results with those obtained with the Oriel device. This home-made system has been described in some detail (13).Basically, it consists of a 6-W, 4.6-V tungsten halogen lamp focused onto a 460-nm interference filter and launched into a bifurcated fiber-optic waveguide bundle (Volpi AG, CH-8952 Schlieren, Switzerland), the common end of which Figure 1. Cross-section through the sensing layer of the fiber-optic (10 mm 0.d.) is thermostated and contains the sensing layer. glucose sensor consisting of a polyester film as a support (P), a silicone Luminescence and scattered light return through the second half matrix with suspended dyed kieselgel particles (R), a carbon black of the fiber bundle, pass a 550-nm cut-off filter, and are detected optical isolation (C), and a GOx/glutardialdehyde gel (E). with a photodiode (BPW 21, Siemens, D-8000Munich, FRG). The collected data are then transferred to, and stored in, an IBM-XT 10 , personal computer, which also governs the automated measurement procedure including gas supply and sample flow. Chemicals. Oxygen, air, and nitrogen of +99.99% purity where obtained from cylinders. Oxygen and nitrogen are mixed by using a RMD 280 mass flow controller (Air Liquide, Vienna) to yield mixtures of defined oxygen percentage. The gas mixtures are bubbled through an aqueous buffered solution before entering the flow cell for measurements with moist gases. The precision of the mixing device is specified to be within fl% of the actual value. All experiments were performed at barometric pressure of about 736 Torr and room temperature of 25 f 2 "C. Tris(1,lO-phenanthroline)ruthenium(II)dichloride was purchased from Aldrich (Steinheim, FRG). Glucose oxidase (EC 1.1.3.4,Type VII-S, from Aspergillus niger),with a specificactivity of 200 units mg-' of solid was obtained from Sigma (Munich, 1 0 4 0 12 16 20 24 FRG). Glucose oxidase (from Penicillium amagasakiense) with 0 2 [%I a specific activity of 320 units mg-' of solid was from Nagase Biochemicals (Tokyo, Japan). Glutaraldehyde (a 25% solution Figure 2. Effect of oxygen on the relative luminescence intensity of in water) from Sigma has been used to cross-link the GOx. Imthe oxygen sensor (+), and corresponding Stern-Volmer plot ( O ) , munodyne immunoaffinity membranes, made of Nylon 6-6 having corrected for false light according to eq 1, at a total gas pressure of a porosity of 200 nm, an approximate thickness of 150 pm, and 736 Torr. chemically activated carboxyl groups on the surface, were obtained from Pall Co. (Glen Glove, NY). Stokes' shift (fluorescence A, = 603 nm, corrected), and a Glucose solutions have been prepared by diluting a 20 mM stock sufficiently long excited-state lifetime (0.6 KS in oxygen-free in 0.1 M phosphate buffer of pH 7.0 (when affinity membranes aqueous solution) at room temperature (15, 16). However, were used) or 0.02 M phosphate buffer of pH 7.0 containing 0.9% it has an emission quantum yield of 0.042 only (15). Similarly, (w/v) NaCl and 0.1% sodium azide (referred to as phosphate the [R~(diphenyl-l,lO-phenanthroline)~~+] complex was found buffered saline, PBS) when combined PO,-glucose measurements (16) to exhibit poor luminescence and moderate quenchability were performed. All other chemicals were of analytical reagent as well. While this was found t o be of sufficient sensitivity grade. for determination of oxygen over the usual range encountered Sensing Membranes. Oxygen sensors have been prepared according to a published procedure (14) but using R ~ ( p h e n ) ~ ~ + in blood gas analysis, its quenching constant is too small to detect traces of glucose when used in a urine glucose biosensor as the oxygen-sensitive dye. Glucose oxidase was immobilized by two different methods. In the first one, a 2-cm disk of the with a n oxygen transducer. immunoaffinity membrane was immersed into a solution of 2 mg We therefore looked for a dye with improved Stern-Volmer of the enzyme in 1 mL of pH 7 phosphate buffer for 24 h. The quenching constants and found ruthenium tris(1,lOmembrane was then washed with phosphate buffer and stored phenanthroline) dichloride [ R ~ ( p h e n ) , ~ +t o] have improved in the refrigerator at 4 "C in the same buffer containing sodium indicator properties particularly when adsorbed on a hydroazide as a bactericide. The membrane was placed on top of the philic support. T h e excitation and emission wavelengths of oxygen sensor membrane and kept in position with an O-ring. this complex in water are 447 n m (e 18100 M-l cm-l) and 604 The second procedure is a modification of the one described n m (corrected), respectively, and the luminescence quantum by Schaffar et al. (11). In this case, the oxygen sensing layer was yield has been determined to be 0.058 (17). The luminescence covered with a thin layer of carbon black (ca. 1pm) before curing lifetime in oxygen-free aqueous solutions is 1.0 KS at room and allowed to polymerize for 1week at room temperature. The resulting black oxygen optrodes were washed with 70% ethanol temperature. Therefore, the indicator is subject to more as a desinfectant. Squares (2 X 5 cm) of these foils were first efficient quenching than the bipyridine complex. It has been soaked with PBS and then treated with a mixture of 6 mg of the immobilized in a silicone matrix as described in the expericorresponding enzyme in 240 fiL of PBS and 30 pL of a 2.5% mental part to give a sensor material useful for oxygen sensing. glutardialdehyde solution. The solution is allowed to evaporate Analytical Features of the O2Sensor. Silicone is conwhile the cross-linking reaction occurs on the carbon black surface sidered t o be a n ideal material for the preparation of most at room temperature. When the enzyme film is completely dry, oxygen sensors. It can be easily spread in thin layers and has 2-cm disks were punched out and stored in the refrigerator a t 4 the best permeability for gases when compared to other "C in PBS, with 0.1% sodium azide being added when not in use. polymers (18, 19). Figure 2 is a plot of the relative signal Figure 1 shows a cross section of this type of glucose sensing membrane. change of the Ru(phen)32+-basedoxygen sensor as a function of the oxygen concentration, together with a Stern-Volmer RESULTS A N D D I S C U S S I O N type representation. This curve has been obtained by using Choice of the Indicator. The [ R u ( b ~ y ) ~complex ~+] eq 1, which takes into account the contributions of the sodisplays a fairly strong absorption in the visible region of the spectrum (A,, 452 nm, t 13600 M-' cm-', in water), a large

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Figure 3. Calibration graph (obtained for sensors based on the use of the immunoaffinity membrane: (0)GOx with a specific activity of 320 units mg-’; (0)GOx of 200 units mg-’.

called false light (10). This term (If)includes all components responsible for a constant background of light not associated with quenchable indicator fluorescence and which cannot be removed by electronic or manual subtraction. As expected, the K, shown by the Ru(phen)32+complex is larger by a fador of 2.2 than the values described (13) for the same type of sensor using the Ru(bpy)32+. Data reproducibility is mostly limited by the instrument performance. This is mainly due to the difficulty of getting a calibration point for a perfectly oxygen-free sample. The standard deviation for the determination of 7.3% oxygen in nitrogen is &0.7% and &0.5% for 21.9% oxygen at barometric pressure. The detection limit as defined by the analyte concentration giving a fluorescence of 3 times the standard deviation of the background is calculated to be 0.7 Torr when dry gases are used. The response time for a complete signal change to occur is about 40 s. Preparation of the Glucose Sensor. In order to make the oxygen sensor a glucose biosensor, it has to be covered with a membrane containing immobilized GOx. The oxygen sensor then acts as a transducer for the rate at which oxygen is consumed during enzymatic oxidation. Hence, the response is the result of a dynamic balance in diffusion of glucose and oxygen into the sensor, and consumption of oxygen in the reaction, resulting in a steady-state decreased oxygen level and, consequently, an increase in fluorescence. Two different immobilization procedures have been studied with respect to their effect on the sensor performance. When immunoaffiiity membranes were used to immobilize GOx, the response time turned out to become very long. This may be attributed to the characteristics of the sensing layer, formed by a nylon membrane with the immobilized GOx deposited onto the oxygen sensor described before. This configuration is the limiting factor for the development of sensors prepared in this fashion because the glucose and oxygen transport across the membranes is very slow due to the thickness of the layers (particularly the 150-pm nylon membrane), yielding a 32-min response time in the most unfavorable case. Figure 3 shows a calibration curve of two glucose sensors prepared from two different glucose oxidases. Not surprisingly, the enzyme with higher activity gives a more pronounced signal change, but a t the same time the saturation point of the sensor is reached earlier because oxygen is consumed in the membrane at a faster rate during the enzymatic reaction or the enzyme is saturated earlier. The analytical range for the 320 units mg-l enzyme is from 0.1 to 1.0 mM glucose. The standard deviation for 10 replicate measurements was f3.5%. T h e useful range is between pH 5.5 and 7.5. However, it should be pointed out that if the membranes are treated with pH 9.0 phosphate buffer, its activity decreases dramatically and irreversibly. The analytical range for the 200 units mg-l GOx lies between 0.3 and 5 mM glucose and the standard deviation for the measurements with this membrane was calculated to be &4.8%.

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Figure 4. Calibration graph (expressed as the % increase in fluorescence over the value of air-saturated buffer) for the glucose biosensor with an oxygen sensor as a transducer and GOx immobilized directly on carbon black which also acts as an optical isolation to prevent ambient light and sample fluorescence to interfere. Standard deviations are given for 12 replicate measurements.

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When compared with sensors of this type known in the literature (10, 201, the use of Ru(phen)z+ as the oxygensensitive indicator obviously leads to a tremendous enhancement in relative signal change even a t low glucose concentrations. Nevertheless, since long response times turned out to be a limitation of this sensor, we decided to apply a different immobilization procedure to produce thinner sensor layers and achieve faster oxygen diffusion. Both enzymes were cross-linked with glutardialdehyde onto the oxygen-sensitive layer covered with carbon black, as described in the Experimental Section. The biosensor layer made from Penicillium amagasakiense yielded an active membrane, while the other one showed no sensitivity to glucose changes, pointing to the possibility that the active centers of the enzyme were somehow affected during the immobilization procedure. The calibration curve of this new sensor type which incorporates the oxygen-sensitive complex and the enzyme in the same membrane is depicted in Figure 4. The maximum signal change measured with this device is a 2.2-fold increase of the optrode luminescence intensity in air-equilibrated buffer upon exposure to 2 mM (or more) glucose solutions. In terms of both relative signal change and detection limits, this is a 50% increase over the values obtained with optical biosensors of this kind so far (11). The signal-to-noise ratio is 2W1, and the response times are about 2 min for a 90% signal change (tw) and around 6 min for tlW. The time required to reach the base line again is approximately 7 min. The dynamic range of the sensor is 0.06-1.0 mM glucose, similar to that obtained with the sensor using the immunoaffinity membrane. However, the former provides response times substantially shorter and the signal change is larger as can be observed from Figures 3 and 4. The sensor performance is insensitive to flow rates between 0.9 and 1.7 mL min-’. Figure 5 shows the response of the sensor to 1 mM glucose solutions of different pH. The maximum signal is obtained in the pH 7.C-7.5 range, which is in agreement with previous

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Table I. Effect of Sensor Lifetime to the Response of the

Glucose Biosensor toward 1.0 and 0.8 mM Glucose Solutions increase in fluorescence intensitv,” arbitrarv units time, weeks

1 mM glucose

0.8 mM glucose

1 2 4

203 (*9) 194 (k7) 187 (f3)

110 (*6) 111 (f4) 85 (*0.7)

Standard deviations of five measurements given in parentheses.

findings on immobilized enzyme (21) whose optimum activity is a t pH 5.6 in free form. No signal changes were observed when 100 mM saccharose, fructose, lactose, or galactose were tested. The reproducibility in the fabrication of the biosensor is demonstrated by the calibration curves for two different membranes in Figure 6. It is evident that both sensors exhibit a very similar behavior. However, it is essential to precisely follow the standardized procedure for enzyme gel preparation in order to obtain reproducible results. When stored at room temperature in PBS containing sodium azide, the sensors lost 16-18% of their activity within 4 weeks when used daily. They were found still to be operative 12 months after preparation and storage a 4 “C. The activity of the enzyme does not decrease in a constant rate; in fact, during the period monitored only a slight decrease of the sensor response was observed as appears from the data in Table I. During this time the biosensor was flashed at 9 Hz for ca. 150 h but no photobleaching was observed.

CONCLUSIONS The advantages displayed by this biosensor over other optical biosensors described in the literature include (a) an excitation and emission wavelength within the visible range, (b) a large Stokes’ shift which facilitates the separation of

fluorescence from straylight, and (c) an improved quenching efficiency. The dynamic range of this biosensor makes it adequate for glucose determinations in urine, where normal glucose levels are at around 0.8 mM (22). Any increase in this value is indicative of a pathological situation and appears to be detectable with this biosensor. Experiments are underway to couple this optical fiber system to flow injection analysis of urine glucose.

ACKNOWLEDGMENT The authors gratefully acknowledge the help of W. Trettnak in the preparation of the immobilized membranes and Dr. G. Orellana for suggesting the use of Ru(phen)?+ as an oxygen indicator and reading the manuscript.

LITERATURE CITED (1) Turner, A. P. F.; Karube, I.; Wilson G. S.Biosensors; Oxford University Press: Oxford, 1987. (2) Mlecular Luminescence Spectroscoy: Methods and Applications; Schulman, S. G., Ed.; Wiley: New York, 1988; Vol 2, Chapter 3. (3) Clark, L. G.; Lyons, C. Ann. N . Y . Acad. Sci. 1962, 102, 29. (4) Uwira, N.: Opitz, N.; Lubbers, D. W. A&. Exp. Med. Bioi. 1984, 169, 915. ( 5 ) Goldfinch, M. J.; Lowe, C. R. Anal. Biochem. 1984, 138, 430. (6) Trettnak, W.; Leiner, M. J. P.; Wolfbeis, 0.S. Blosensors 1969, 5 , 245. (7) Weigel, Ch.; Keliner, R. R o c . SHE-Inr. Soc. Opt. fng. 1989. 1745, 134. (8) Schultz, J. S.;Mansouri, S.;Goldstein, L. J. Diabetes Care 1982, 5 , 245. (9) Trettnak, W.; Wolfbeis, 0.S. Anal. Chim. Acta 1989, 227, 195. (10) Trettnak, W.; Leiner, M. J. P.; Wolfbeis, 0. S. Analyst 1988, 113, 1519. (11) Schaffar, B. P. H.; Wolfbeis, 0.S.Blosensors Bioelectronics 1990, 5 , 137. (12) Wolfbeis, 0.S.;Weis, L. J.; Leiner, M. J. P.; Ziegler, W. E. Anal. Chem. 1988, 6 0 , 2028. (13) Wolfbeis, 0.S.;Leiner, M. J. P.; Posch, H. E. Mikrochim. Acta 1986. I I I , 359. (14) Lippitsch, M. E.; Pusterhofer,J.; Leiner, M. J. P.; Wolfbeis, 0. S.Anal. Chim. Acta 1988, 205, 1. (15) Juris, A.; Balzani, V.; Barigeletti, F.; Campagna, S.;Belser, P.; von Zelewski, A. Coord. Chem. Rev. 1988, 8 4 , 85. (16) Hoffman, M. 2.; Bolletta, F.: Mcggi, L.; Hug, G. L. J . Phys. Chem. Ref. Data 1989, 18, 219. (17) Bacon, J. R.; Demas, J. N. Anal. Chem. 1987, 59, 2780. (18) Cox, M. E.; Dunn, 8.J . folym. Sci. 1988, 24, 821 and 2395. (19) Bandrup, J.; Immergut, E. H. The Polymer Handbook; Wiiey: New York, 1975; p 111-232 ff. (20) Dremel, B. A. A.; Schaffar, B. P. H.; Schmid, R. D. Anal. Chim. Acta 1989, 225, 293. (2 1) Hartmeier, W. Immobilsierfe Biokatalysatoren; Springer Verlag: Berlin, 1986. (22) Peter, G. Lehrbuch der Kiinschen Chemie, edition medizin; Verlag Chemie: Weinheim-Deerfield Beach-Basel, 1982; p 172f.

RECEIVED for review May 15, 1990. Accepted July 16, 1990. M. C. Moreno-Bondi thanks the Community Council of Madrid for a grant.

Oxygen optrode for use in a fiber-optic glucose biosensor.

An optical fiber oxygen sensor, based on the dynamic quenching of the luminescence of tris(1,10-phenanthroline)-ruthenium(II) cation by molecular oxyg...
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