Anal. Chem. 1994,66, 3139-3144
Glucose Biosensor Based on a Sol-Gel-Derived Platform Upvan Narang, Paras N. Prasad,' and Frank V. Bright' Department of Chemistry and Photonics Research Laboratory, Acheson Hall, State University of New York at Buffalo, Buffalo, New York 14214 Kumaran Ramanathan, N. Deepak Kumar, B. D. Malhotra,' M. N. Kamalasanan, and Subhas Chandra' National Physical Laboratory, Dr. K. S. Krishnan Marg, New Delhi 710012, India
Sol-gel-derived glasses have emerged as a new class of materials well suited for the immobilization of biomolecules. As a consequence, they are also finding new applicationsas platforms for chemical sensors. Room temperature (or lower) processing conditions, chemical inertness, negligible swelling effects, tunable porosity, and the high purity of sol-gel-derived glasses make them ideal for many types of sensor applications. We report here on the characterizationof tetraethyl orthosilicate(TEOS-) derived thin sol-gel films, doped with glucose oxidase (COX), as a sensing platform for a prototypical biosensor. COX was immobilized in/on a thin TEOS sol-gel film using physisorption, microencapsulation, and a new sol-gekC0x: sol-gel sandwich configuration. Amperometric and photometric detection modes are used to study the response profiles and in turn quantify glucose. The results clearly demonstrate that the sandwich configuration exhibits a fast response and high enzyme loading. This particular scheme is stable for at least 2 months under ambient storage conditions. Low temperature processing conditions have made it possible to routinely incorporate and maintain organic species in sol-gel-derived materials.14 To date, sol-gel processed materials have been used routinely for the development of ceramic thin films for conductive, optical, protective, mechanical, and electrooptic applications.l+ Encapsulation of a molecular recognition element within the porous sol-gel matrix together with the fact that little if any dopant leaches from matrix has lead to the investigation of sol-gels for chemical sensing s c h e m e ~ . ~ -More ' ~ recently, immobilization (1) Brinker, C. J.; Scherer, G. W. Sol-Gel Science; Academic Press: New York, 1989. (2) Chemical Processingof Advanced Materials; Hench, L. L., West J. K., Us.; Wiley: New York, 1992. (3) Lev, 0. Analusis 1992, 20, 543. (4) Klein, L. C. Annu. Reu. Mater. Sei. 1993, a,437. ( 5 ) Narang, U.; Jordan, J. D.; Bright, F. V.; Prasad, P. N. J. Phys. Chem., in press. (6) Narang, U.; Wan& R.; Bright, F. V.; Prasad, P. N. J . Phys. Chem. 1994,98, 17.
(7) Narang, U.; Bright, F. V.; Prasad, P. N. Appl. Spectrosc. 1993, 47, 229. (8) Kuselman, I.; Lev, 0. Talanta 1993, 40, 749. (9) Narang, U.; Dunbar, R. A,; Bright, F. V.; Prasad, P. N. Appl. Spectrosc. 1993, 5 5 ,
1700.
(10) Braun, S.; Rappoport, S.;Zusman, R.; Avnir, D.; Ottolenghi, M. Mater. Lett. 1990, 10, 2. ( I 1) Ellerby, L. M.; Nishida, C. R.; Nishida, F.; Yamanaka, S. A,; Dunn, B.; Valentine J. S.; Zink, J. I. Science 1992, 225, 1113. (12) Braun, S.; Shtelzer, S.; Rappoport, S.;Avnir, D.; Ottolenghi, M. J. NonCryst. Solids 1992, 1471148, 139. (13) Yamanaka, S. A.; Nishida, F.; Ellerby, L. M.; Nishida, C. R.; Dum, B.; Valentine J. S.; Zink, J. I. Chem. Mater. 1992, 4, 495. (14) Tatsu, Y.; Yamashita, K.; Yamaguchi, M.; Yamamura, S.;Yamamoto. H.; Yoshikawa, S. Chem. Letr. 1992, 1615. (15) Lev, 0.;Glezer V. J . Am. Chem. Soc. 1993, 115, 2533. (16) Audebert, P; Demaille, C.; Sanchez, C. Chem. Mater. 1993, 5 , 911.
0003-2700/94/03663 139$04.50/0 0 1994 American Chemical Soclety
coupled with the retention of the affinity or activity of biomolecules within the matrix1&18 have made sol-gels a potential vehicle for the development of new chemical biosensors. There are several literature reports on the immobilization of enzymes, and glucose oxidase (GOx) in particular, within a sol-gel-derived matrix.12-16 Braun et al.I2 reported on the properties of a 8 X 2 mm disk of tetramethyl orthosilicate(TMOS-) derived xerogel doped with glucose oxidase, peroxidase, and a chromogenic dye for detection of glucose. This disk-based device did indeed respond to glucose, but information on enzyme activity, stability, detection limits, and response times were not presented. Y amanaka et al.13 demonstrated the feasibility of GOxdoped sol-gel processed materials for glucose sensing. In this work, the activity of the encapsulated enzyme (GOx) was investigated by use of a photometric detection scheme. Although activity information on the immobilized GOx was provided, these authors first fabricated GOx-doped sol-gel monoliths (10 X 5 x 2 mm) which were subsequently ground into a fine powder. In order to quantify GOx activity, the ground sol-gel material was incubated with a reagent solution and glucose added. The apparent turnover number (/cat = 250 f 80) was similar to the value for native GOx in solution (293).19720However, the recovered dissociation constant (Km 1 0.05 f 0.05 M) was 2-fold greater than native GOx in solution (0.026 M),19v20suggesting that glucose binding to the sol-gel-encapsulated GOx was weaker. The authors showed that the response was strongly dependent on the storage conditions and 5-10-fold slower compared to native GOx in solution. Information on detection limits and long-term stability were not reported. Tatsu et al.14 prepared TEOS-based sol-gel monoliths doped with COXand reported on their performance as glucosesensing elements. In a flow injection analysis scheme, the monolith-based sensor exhibited a peak response time on the order of 4 min. Saturation of the signal response was observed over 400 mg/dL, and the GOx activity was shown to vary over time, depending on the actual storage temperature. The activity of the entrapped GOx was found to be 20-fold greater when stored at -20 OC compared to room temperature. The (17) WU, S.; Ellerby, L. M.; Cohan, J. S.; Dunn, B.; El-Sayed, M. A.; Valentine, J. S.; Zink, J. I. Chem. Mater. 1993, 5, 115. (18) Wang, R.; Narang, U.; Bright, F. V.; Prasad, P. N. Anal. Chem. 1993, 65, 2671. (19) Bright, H.J.; Gibson, Q. H. J. Biol. Chem. 1967, 242, 994. (20) Rogers, M. J.; Brandt, K. G. Biochemistry 1971, 10, 4624.
AnalyticalChemistry, Vol. 66, No. 19, October 1, 1994 3139
sol-gel-encapsulated GOx was reported to remain active for at least 2 months when stored desiccated at 4 OC. Lev and GlezerI5prepared platinum electrodes coated with sol-gel-derived films of V2O5 doped with GOx. Cyclic voltammetry was used as the detection mode for glucose. Saturation of the signal was observed beyond 10 mM 8-Dglucose. Sanchez and co-workers16used cyclicvoltammetry to study the activity of GOx doped within a TMOS-derived sol-gel matrix. In this scheme the sol-gel was doped with GOx and a mediator, (hydroxymethy1)ferrocene. This mixture was then coated onto the distal end of a glassy carbon electrode. Detection limits, dynamic range, and response times were not reported. Enzyme activity was estimated to be 7 0 4 0 % in the fresh polymeric silica sol, but no information on the activity as the coating aged was provided. Although GOx apparently functions within a sol-gel matrix, the majority of the previous reports did not (1) use a consistent set of preparation protocols, (2) completely characterize the immobilized GOx, (3) discuss the issue of long-term stability and effects of storage conditions, (4) study the analytical response time, (5) present information on the glucose detection limits, ( 6 ) evaluate how the sol-gel composition affects the GOx activity and ultimate sensor response, or (7) determine how the immobilization protocol influences the system’s analytical figures of merit. That is, the previous reports did not fully quantify the analytical performance of the system. In this paper, we report on the characterization of thin sol-gel films derived from TEOS that are doped with GOx as a prototype for sol-gel-based biosensor development. The initial film fabrication/processing conditions were varied systematically in an effort to determine the optimum analytical performance of the GOx-doped materials. We also investigated three different schemes to “immobilize” the enzyme, GOx: (1) physisorption of GOx onto the precast sol-gel film; (2) microencapsulation within the sol-gel film matrix; (3) a sol-ge1:GOx:sol-gel sandwich configuration. Sensor performance was compared for each immobilization scheme. Thin films were prepared by casting directly onto glass plates and a simple photometric detection scheme was used. Also, indium tin oxide (ITO) coated glass plates were fabricated, using the sandwich architecture, and used for amperometric detection. Glucose oxidase was chosen as our model enzyme for several reasons. First, there are several reports that it remains active within a sol-gel-glass matrix.12-16 Second, GOx is available commercially and is inexpensive. Third, GOx is an extremely stable enzyme and can survive wide excursions of pH, ionic strength, etc. Fourth, there is a substantial literature on glucose sensors based on GOx using a plethora of immobilization scheme^.^'-^^ Finally, the characteristics of GOx in bulk solution are well documented.19~20~26~28 (21) Taylor, R. F. Protein Immobilizatior FundamentalsandApplicarions;Marcel Dekker, Inc.: New York, 1991; Chapter 8, p 263. (22) Chemical Sensors and Microinstrumentation;Murray, R. W., Dessey,R. E., Heineman, W. R., Janata, J., Seitz, W. R., Eds.;ACS Symposium Series 403; American Chemical Society: Washington, DC,1989; Chapters 5-7, and 16. (23) Ito, K.;Shoichiro, I.; Kaori, A.; Naruse, H.; Ohkura, K.; Ichihashi, H.; Kamei, H.; Kondo, T. In Fundamentals and Applications of Chemical Sensors; Schuetzle, D., Hammerle, R., Eds.; ACS Symposium Series 309; American Chemical Society: Washington, DC, 1986; Chapter 23. (24) Janata, J. Principles of Chemical Sensors; Plenum Press: New York, 1989. (25) Ramanathan, K.; Annapoori, S.; Malhotra, B. D. Sens. Actuators B, in press. (26) Quentin, H.; Bennett, E. P.; Swoboda, 2.;Massey, V. J . Biol. Chem. 1964, 239, 3927. (27) Reach, G.; Wilson, G. S. Anal. Chem. 1992, 64, 381A.
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Table 1. Effects of Sol-QekWater Ratlo on Flnal Fllm Thlcknese*b
vol of water (mL) 0.0
0.5 1.o 1.5 2.0 >2.0
thickness (rm) 1.oo 0.70 0.35 0.20 0.10 poor film quality
a A 0.5-mL aliquot of stock sol-gel solution mixed with x mL of water. Precision in the film thickness is *lo0 A.
EXPERIMENTAL SECTION Materials. The following chemicals were used: tetraethyl orthosilicate (TEOS), 0-D-glucose, glucose oxidase (GOx) type X-S (EC 1.1.3.4 from Aspergillus Niger; Sigma), horse radish peroxidase (HRP) (Sisco Research Laboratories); HCl, K2HP04, KHzPOc2H20, methanol (Fisher Scientific Co.), glass slides (Bluestar, Polar Ind. Co.), and ITO-coated glass slides (Balzers). All reagents were used as received without further purification, and aqueous solutions were prepared in doubly distilled-deionized water (Millipore R O lOTS water purification system). Equipment. pH values were determined with a glass electrode and a pH meter (Control Dynamics). UV-visible absorbance data were acquired using a Shimadzu Model 160A spectrophotometer. Cyclic voltammetry was carried out using an Electrochemical Interface Model EC 8617, and the output was recorded directly on a Hewlett-Packard Model H P 7440A ColorPro plotter. Amperometric responses were recorded using a Keithley electrometer (Model EC 61 7), and the plots were taken on an Omnigraphic recorder series 2000 (Digital Electronics). Film thicknesses were determined using a surfometer (Model SF 200, Planner Industrial). Sol-Gel Film Preparation. Sensor performance was studied using three different thin film preparation protocols. A stock sol-gel solution was prepared by mixing 4.5 mL of TEOS, 1.4 mL of H20, and 100 p L of 0.1 M HCl in a glass vial. After 3 h of mixing, a clear sol-gel solution resulted. The pH of the stock sol-gel solution was 5.0. This solution was used throughout the experiments and was diluted as required (vide infra). In order to prepare an actual thin film, we first rinsed all glass slides (plain glass or ITO-coated glass) with 1-propanol and then pipeted 200 pL of an appropriate sol-gel solution (e.g., diluted) onto the glass slides. The glass slides were then spun at 3000 rpm for 30 s. All films were then stored and dried under ambient conditions. The final film thickness depends on the viscosity of the sol-gel solution from which it is spin cast. In the current work, we used water as the diluent. A specific casting solution was prepared by mixing 0.5 mL of the stock sol-gel solution with between 0.0 and 2.0 mL of water. Nonuniform films resulted if more than 2.0 mL of water was added. Using this scheme, we were able to reproducibly (f100 A) prepare spincast films with thicknesses ranging fromO.lOto 1.00pm (Table 1). All films wereoptically transparent with novisiblesurface cracks. (28) Bergmeyer, Y. Methods of Enzymatic Analysis, 2nd ed.; Allied Press: New York, 1974.
I
1.5 I
t I
Sol-Gel
W
u
C
I
I
Sol-Gel
:1.0 L ul 0
n
c
a -0
- 0.5 .0
Figure 1. Simplified schematic of the sol-gel:GOx:sol-gel sandwich film architecture.
z 0
300
Physisorption. Thin sol-gel-derived films were prepared as described above and then immersed in a GOx solution (20 mg/mL, prepared in phosphate buffer, pH 6.0). After 24 h these films were removed from the GOx solution and washed three to four times with 100 mL of buffer. This protocol removed the majority of the loosely physisorbed GOx. Microencapsulation. To 0.5 mL of the stock sol-gel solution was added 0.5 mL of GOx (20 mg/mL, prepared in phosphate buffer, pH 6.0). The pH of the sol-gel solution after the addition of GOx solution was 6.0. Films of varying thicknesses (vide supra), doped with GOx, were cast using the water-sol-gel dilution scheme discussed above. Sandwich Configuration. To 0.5 mL of the stock solution was added 1 mL of methanol. A 0.30-pm-thick neat sol-gel film was then spin cast onto the appropriate substrate. After drying under ambient conditions for 6 h, the coated substrate was cured at 200 OC for 10 min, forming a dry, thin sol-gel film. After removal from thecuring oven, the coated substrates were allowed to equilibrate in air at room temperature for 30 min. A 0.5-mL aliquot of GOxsolution (20 mg/mL, prepared in phosphate buffer, pH 6.0) was coated onto the surface of the cast sol-gel film and allowed to air dry in a covered Petri dish. After 24 h a second sol-gel layer (no methanol) was cast on top of the sol-ge1:GOx surface, resulting in a solge1:GOx:sol-gel sandwich (Figure 1). We found that the lower sol-gel layer was necessary in order to yield a uniform coating of GOx and form a uniform sol-gel top layer. We speculate that this arises because of the high O H content of the sol-gel lower film,14 which leads to increased adsorption of GOx to the lower film. Because the diffusion of the analyte must proceed through the top sol-gel layer into the GOx, the thickness of the upper sol-gel-derived film becomes extremely important. We used the water dilution scheme (vide supra) to adjust the thickness of the upper sol-gel-derived thin film. Determination of Glucose Oxidase Activity. The activity of the immobilized GOx was determined using the o-dianisidine colorimetric assay:28
P-D-glucose + H,O
-
+ 0,
H202+ DH,
gluconic acid + H202
2H20
+D
where the upper and lower reactions are catalyzed by GOx and HRP, respectively, DH2 is the reduced dye, and D is the oxidized dye. The change in absorbance of the oxidized dye at 460 nm was used as a measure of the enzymatic activity. The assay was performed in a 3-mL quartz cuvette containing
4 00
500
600
Wavelength (nm)--
Figure 2. Normalized UV-visible absorbance spectra of the color developed on exposure of a sol-gei:GOx:soi-gel sandwich film to the dye (cMianisidine), HRP, and 10 % @+glucose reaction mixture. Results for the sol-ge1:GOx:sol-gel sandwich configuration (- -) and native GOx in solution (-1.
-
0.1 M phosphate buffer (pH 6) at 30 OC using 10% p-Dglucose as the enzyme substrate. The 0-D-glucose solution was allowed to mutarotate for 24 h at 30 "C before its use for the GOx activity assay. Prior to monitoring the absorbance, the contents of the reaction mixture were mixed thoroughly for uniform color distribution. Identical concentrations of H R P and dye were used whenever comparative studies were done. Voltammetric Response of Films. A three-electrode cell was used, and an ITO-coated glass plate served as the working electrode. Only the sol-ge1:GOx:sol-gel sandwich scheme was used in this portion of the work. Platinum foil was used as the counter electrode and Ag/AgCl as the reference electrode. Potential scans were carried out by ramping the potential from -0.5 to +0.7, V vs Ag/AgCl at a scan rate of 50 mV s-l. Phosphate buffer (0.1 M, pH 6) was used as the supporting electrolyte. Similar scans were performed in the presence of 10 mM @-D-glucose. However, prior to any potential sweep, the sol-gel electrodes were allowed to equilibrate with the electrolyte for approximately 60 min. Between the potential sweeps, the electrode was equilibrated for 2 min. Amperometric Response of Films. The amperometric response of the GOx immobilized thin films was carried out using a three-electrode cell configuration similar to the one used for the cyclic voltammetry experiments. Again, the solge1:GOx:sol-gel sandwich film configuration was used exclusively and the working electrode was polarized at +0.7 V with respect to Ag/AgCl. The amperometric current was monitored for 100 s after the injection of glucose into the cell. Prior to initiating the current vs time run, the sample was stirred for 2 s. Between measurements, the current was allowed to decay to the background value.
RESULTS AND DISCUSSION Optical Characterization of the Films. General Features. After the preparation of the sol-gel-derived thin films, we first tested each immobilization scheme for their enzymatic activity using the dye oxidation method.28 Figure 2 shows typical normalized absorbance spectra that result when the o-dianisidine and H R P reagent solution2* and 0-D-glucose Analytical Chemistty, Vol. 66, No. 19, October 1, 1994
3141
are exposed to a sol-ge1:GOx:sol-gel sandwich film (---)and native GOx (-). The recovered spectral contours of the oxidized dye are very similar and suggest that there is no difference in the behavior when the dye is oxidized by the GOx immobilized in the sol-ge1:GOx:sol-gel sandwich or by native GOx in buffer. Similar behavior was seen when we investigated physisorbed and microencapsulated GOx films (data not shown). These results suggest that thesol-gel matrix does not affect the ability of the immobilized GOx to effect the o-dianisidine oxidation. Similar results have also been observed by Yamanaka et a1.I3in the GOx immobilized within sol-gel monoliths. The advantage of the above methods of enzyme immobilization is the ease in performing the activity assay and negligible leaching of GOx in the encapsulated and sandwich configuration. The GOx-immobilized sol-gel films could be directly placed in the reaction mixture and the change in absorbance monitored as a function of time used to estimate the enzymatic activity. As mentioned in the Experimental Section, films of varying thickness were prepared by diluting the stock sol-gel solution with water prior to spin casting of the sol-gel films. Only water was used for dilution to ensure maximum retention of the GOx activity. Other solvents such as methanol and ethanol have been found to denature enzymes, resulting in decreased enzymatic activity.29 Physisorbed GOx Films. The response of the physisorbed GOx-based sol-gel thin films was recorded by immersing each film in a solution containing the dye, HRP, and 10% &Dglucose. The final absorbance measured for each film sample was within f O . O O 1 A . This result suggests strongly that the physisorbed GOx is not affected significantly by physisorption to the sol-gel-derived film surface. We also noted that the actual film thickness did not affect the physisorbed GOx performance. However, theenzymatic activity associated with physisorbed GOx sol-gel films ceased after 2 days. Analysis of the rinse solutions showed the presence of GOx and thus indicates that there is leaching of the GOx from the sol-gel film surface. This scheme was not investigated further due to poor stability. Encapsulated GOx Films. The response of GOx encapsulated within a sol-gel-derived thin film was monitored by immersing a film in a solution containing dye, HRP, and 10% P-D-glucose and following the change in solution absorbance (0-dianisidine oxidation) with time. Figure 3 shows that there is a significant change in absorbance with time; however, a stable response is not achieved for at least 2 h. Similar results were achieved for all films studied, and the response profiles were essentially independent of film thickness. Interestingly, a stable response for the same system using native GOx is achieved in about 30 s . ~ O The microencapsulation scheme was not evaluated further because the long response time was deemed unacceptable. Sol-Ge1:GOx:Sol-Gel Sandwich Films. Figure 4 presents typical response profiles for the sol-ge1:GOx:sol-gel sandwich films that have been aged under ambient conditions for 1 week ( 0 )and 2 months (v). These particular sandwich films were prepared by first casting a 0.30-pm “bottom” film, coating (29) Shiono, S.;Hanazato, Y.; Nakako, M.; Maeda, M. GBFMonogr. Ser. 1987, IO, 291. (30) McCann, J. World Biotech. Rep. 1987, 1 , 41.
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0.09 I
t
0
-
80
40
Time (min.)
120
16
Flguro 3. Change in absorbance at 460 nm vs time profile for GOx encapsulated within a 0.30-pm-thick TEOSderived sol-gel film on exposure to the dye (edianisidine), HRP, and 10 % j3+giucose reaction mixture. 0.80
o g c d for one week
v v
a 0
-/ 0
I
4
o g e d for t w o months
I
I
8
42
I
I
I
20
16
Time (mid-
Figure 4. Change in absorbance at 460 nm vs time profile for W x Immobilizedwithin a 0.30/0.10 TEOSderlved sol-gel sandwich film on exposure to the dye (edlanisidine), HRP, and 10 % @glucose reaction mixture.
on GOx, and then casting a 0.10-pm “top” film. (Throughout the remainder of this paper we abbreviate this as a 0.30/0.10 sandwich film.) The actual response was measured using the protocol outlined for the encapsulated GOx (vide supra). These results show that a stable response is achieved in 4 min for a I-week-old film. The response time increases to 12 min after the film has agedfor 2 months. Weattribute theobserved increase in response time to the decrease in the pore structure of the sol-gel matrix upon aging and drying,* causing a subsequent increase in the diffusion time of analyte and products within the evolving sol-gel network. In an effort to determine the effect of film thickness on the response characteristics, we held the bottom film thickness constant at 0.30 pm and varied the thickness of the top film while holding the enzyme loading level constant. Maximum absorbance was observed for the film where the top layer was 0.10 pm thick (cast from 0.5 mL of stock solution 2 mL
+
of H2O). Response times became longer if the upper films were made more thick (e.g., 15 min at 0.20 pm), and no measurable response was seen for a 1.00-pm-thick top film even after 3 h. Prior to moving onto the electrochemical experiments, we first investigated the optical response characteristics of the 0.30/0.10 sandwich film to varying concentrations of p-Dglucose. The analytical response (AA) vs glucose concentration (G, mM) is given by AA = 8.790 (f0.003) X 10-4 G 5.570 (f0.003) X (r2= 0.9992). The linear range (5-35 mM) encompasses the physiological glucose concentration in the blood of diabetics.3’ This result suggests that this scheme may be used for future glucose biosensor development. The absorbance values for p-D-glucose concentration beyond 35 mM could not be accurately determined because a fraction of the colored dye partitioned into the sol-gel matrix. This partitioning process becomes pronounced at higher concentrations of 8-D-glucose (135 mM), introducing significant errors in the absorbance measurements. Based on the above results, all further investigations focused on using the sandwich configuration scheme. Stability of Sol-Ge1:GOx:Sol-Gel Films. After obtaining good enzyme loadings with the sandwich scheme, we studied the long-term stability of these films for chemical sensing platforms. Toward this end, we investigated a series of identical 0.30/0.10 sandwich films that were stored under dry ambient conditions up to 2 months. In these experiments, we incubated the films in a solution containing the dye, HRP, and 10% 0-D-glucose and recorded the maximum absorbance after 30 min. We observed that the sol-ge1:GOx:sol-gel sandwich platform functions well and there is only a 5% decrease in response under room-temperature storage conditions. These results suggest that GOx within the sol-gel: G0x:sol-gel architecture is moderately stable. Activity of GOx within the Sol-Ge1:GOx:Sol-Gel Films. The activity of GOx within a 0.30/0.10 sandwich film (Figure 4) is 0.600 f 0.005 and 0.200 f 0.005 IU mg-l for l-weekand 2-month-old preparations, respectively. As a benchmark, the activity of GOx encapsulated in TEOS-derived sol-gel monolith aged under ambient conditions was only 0.1 IU mg-’.l4 Our results clearly demonstrate that our TEOSderived sandwich scheme yields a significantly higher activity of GOx compared to GOx-encapsulated TEOS sol-gel monoliths. The apparent dissociation constant (K’m) for the GOx-glucose reaction in our sandwich films was found to be 0.015 f 0.005 M, which can be compared to 0.05 f 0.05 M reported by Yamanaka et al.13 in TMOS-derived sol-gel monoliths. These results indicate a stronger affinity of GOx to /3-D-glucose in our TEOS-derived sol-gel sandwich films compared to a TMOS-derived sol-gel monolith. ElectrochemicalCharacterizationof Sol-Cel:GoX:Sol-Cel Films. Redox Properties of theSol-Ge1:GOx:Sol-Gel Films. In order to understand the redox characteristics of GOx immobilized in the 0.30/0.10 sandwich films,32we performed
+
(31) Scheller, F.; Schubert, F.; Pfeiffer, D.; Wollenberger, U.;Renneberg, R.; Hintsche, R.; Kuhn, M. In Biosensors: Fundamentals, Technologies and Applications; Scheller. F., Schmid, R. D., a s . ; VCH, Weinheim, Germany 1992; Vol. 17, Chapters 1-3. (32) A limited number of ITO-coated glass slides restricted our studies somewhat
to only one thickness of the bottom film. One would predict that even better response times would be realized with more thin-bottom films.
(A)
I
A
a
7
Y
H
5 4
3
I
2 I
h
Q:
i
Y
c(
-1
0
t4
E/V Vs Ag/AgCi Figure 5. Cyclic voltammograms for a 0.30/0.10 TEOSderhred soigel sandwlch film: (A) blank solution (1) and in the presence of 10 mM @glucose (20 min); (B) blank solution (1) and in 10 mM &D-glucose after (2) 5, (3) 10, (4) 15, and (5) 20 min.
experiments using cyclic voltammetry. Figure 5A presents the cyclic voltammogram of a GOx sol-gel sandwich film recorded in phosphate buffer in a three-electrode setup. At a scan rate of 50 mV s-l there is a finite amount of charge flowing between the GOx sol-gel electrode and the platinum auxiliary electrode. Although no characteristic peaks were observed in the potential window between -0.5 and +0.8 V in the anodic sweep, there was a finite increment in current at potentials between +0.3 and +0.7 V in the presenceof 10 mM P-D-glucose. This rise in the anodic current is attributed to the direct oxidation of H202 on the surface of the underlying ITO-coated glass electrode. The cathodic sweep showed welldefined peaks at about +0.2 V with respect to a Ag/AgCl reference electrode, but one also observes significant shifts in the peak position as a function of time upon repeated scans at 50 mV s-l (Figure 5B). These results are suggestive of changes in the electrode processes occurring on the surface of the GOx sol-gel films. This issue is currently unresolved, and further investigations are in progress to address this question. Amperometric Response of the Sol-Ge1:GOx:Sol-Gel Films. In order to use the GOx sandwich platform for chemical sensing, we used an amperometric scheme based on the detection of hydrogen peroxide at the I T 0 electrode (2.5 X 2.5 cm). The setup for amperometric response of the GOx sandwich films is described in the Experimental Section. Analytical Chemistty, Vol. 66,No. 19, October 1, 1994
3143
4 50
Table 2. Stabllty Comparkn of Soffiel:GOx:8d-oel Sandwlch Film Architecture with Prevlous Literature Reports.
f
(A)
e
scheme/material
d
covalent attachment poly(vinylbutyral)b cellulose acetate' nylonc carbon surfaceC poly(viny1 chloride)c entrapment ferrocene-modified pyrrole polymeld polyaniline' polypyrroleC acrylamide gelC this worke
C
b
a
I
0
25
50
75
400
Time ( S e d 1
150 t
H
0
40
20
30
40
[Glucose] (mM)Figure 6. Amperometrlc response of a 0.30/0.10TEOSderived solgel sandwich film to @+-glucose: (A) current vs tlme profiles at a potential of +0.7 V with respectto Ag/AgCI referenceafter the addition of (a) 5, (b) 10, (c) 15, (d) 20, (e) 25, and (f) 30 mM @+-glucoseto the reaction cell; (B)amperometric responsevs@+glucoseworking curve.
Phosphate buffer (0.1 M, pH 6.0) was used as the electrolyte. 0.30/0.10 sandwich films were used e x c l ~ s i v e l y ,and ~ ~ the response to fi-D-glucosewas monitored with time (Figure 6A). Prior to the actual response measurements, the contents of the cell were mildly stirred for 2 s and the temperature was maintained at 30 OC. A maximum current of 130 pA was obtained with 30 mM 0-D-glucose and the change was insignificant above this concentration. The response time was less than 30 s and reproducible to within 5% (n = 5 ) over the desired P-D-glucose concentration range (5-30 mM). Figure 6B shows a typical amperometric calibration curve using the 0.30/0.10 sandwich film. The system did not show any perceptible noise due the presence of the insulating sol-gel layers, suggesting a facile electrode reaction in the GOximmobilized sol-gel film. In this configuration, the detection limits for @-D-glucosewas 0.2 mM.
stability 3 weeks 3 months 6 months 3 months 2 weeks 2 days 20 days 20 days 0.5-1 month >2 months
The "stability" is determined different ways by different authors. Our estimates are most conservative (Le., a decrease in response of more than 10%). Reference 33. Reference 31. Reference 34. A 0.30/ 0.10 sol-ge1:GOx:sol-gel configuration.
Immobilization of GOx was carried out in three ways, namely, physisorption, microencapsulation, and a novel sandwich configuration. Due to high loading of the enzyme and a relatively fast response time, we conclude that the sandwich configuration shows the most promise for future biosensor development. Photometric and amperometricdetection modes were used to study the GOx and quantify fi-D-glucose. The new sol-ge1:GOx:sol-gel sandwich preparations were between 2- and 6-fold more active than previous sol-ge1:GOx preparations, and the stability of the glucose-GOx complex was at least 3-fold greater than other sol-gel entrapment schemes. Response times with the sol-ge1:GOx:sol-gel sandwich film were as short as 30 s, and detection limits were on the order of 0.2 mM. Working curves were linear from 5 to 35 mM, and precision was on the order of 5% RSD. The sandwich architecture was stable for at least 2 months, which compares favorably with previous GOx immobilization schemes (Table 2) * These results indicate the viability of sol-gel sandwich architecture for the development of various biosensing schemes.
ACKNOWLEDGMENT This work was supported in part by the National Science Foundation (Grant CHE-9300694). The work at the Photonics Research Laboratory was supported in part by the National Science Foundation (Grant DMR-9213907) and by the Office of Innovative Science and Technology of BMDO and the Air Force Office of Scientific Research, Directorate of Chemistry and Materials Science, through Contract F49620-90-C-0053. We also thank Dr. E. S. R. Gopal, Director, National Physical Laboratory, New Delhi, India, for his keen interest and encouragement during this work. The work at the National Physical Laboratory was supported by the Department of Science and Technology, India, under CONCLUSIONS their sponsored project (F121/TSD/DST/89). K.R. and We report on a series of new PrototYPical ~ ~ ~ S - d e r i v e d N.D.K. thank the Council of Scientific and Industrial sol-gel thin films for immobilization of biomolecules. In this Research, India, for financial assistance. particular work glucose oxidase was used as the model enzyme. (33) Gotoh, M.; Tamiya, E.; Seki,A,; Shimizu, I.; Karube, I. Anal. Leu. 1989,22, 309. (34) Foulds, N. C.; Lowe, C . R. Anal. Chem. 1988,60, 2473.
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Analytical Chemistry, Vol. 66, No. 19, October 1, 1994
for review April
71
lgg4.
Accepted June 6* lgg4."
Abstract published in Advance ACS Abstracts, July 15, 1994.
