Technical Notes Anal. Chem. 1995,67,770-774
Controlled Deposition of Glucose Oxidase on Platinum Electrode Based on an Avidin/Biotin System for the Regulation of Output Current of Glucose Sensors Tomonori Hoshi, Jun-ichi Anmi, and Tetsuo Osa* Pharmaceutical Institute, Tohoku University, Aobayama, Sendai 980-77,Japan
A facile method for the regulation of enzyme loading on an electrode surface has been studied using avidin and biotinylated glucose oxidase (GOx). It was demonstrated that an alternate and repeated deposition of avidin and biotinylated GOx gives a protein thin film probably composed of avidin monolayers and biotinylated GOx monolayers which are connectedwith each other through strong affinity between avidin and biotin moieties of the enzyme (binding constant, 1OI5 M-I). Amperometric response of the glucose sensors constructed by this method was controlled stepwise and rather precisely by regulating the number of GOx layers deposited (or the loading of GOx). For example, the output current of the sensors to 1 mM glucose was enhanced to ca. 1300 and 2800 nA after deposition of 10 and 20 layers of GOx, respectively, as compared with 1 10 nA for the monolayer GOx sensor. The enhanced response contributed to the extension of the dynamic range of the sensors, especially at a lower glucose concentration. The response time of the sensors was satisfactorily fast (ca. 20 s), irrespective of the number of GOx layers. Much attention has been devoted to the molecular-level modification of electrode surface with enzymes for the develop ment of biosensors.1-3 We4and other groups5-' have reported a novel technique to immobilize enzymes on electrode surfaces using an avidin/biotin system, in which biotinylated enzymes are anchored to the avidin-modified electrodes through a strong affinity between avidin and biotin (binding constant, 1 x 1015M-l, Figure 1).8-11 In this procedure, enzymes are deposited on the electrodes as a monolayer film, resulting in rapid response (1) Bourdillon, C.; Bourgeois, J. P.; Thomas, D.]. Am. Chem. Soc. 1980,202, 4231-4235. (2) Johnson. K. W. Sets. Actuators B 1991.5.85-90. (3) Bianco, P.: Haladjian. J.; Bourdillon, C. ]. Electyoanal. Chem. 1990, 293, 151-163. (4) Lee. S.; Anla,J.: Osa.T.Sets. Actuators B 1993, 12, 153-158. (5) Pantano, P.; Morton, T. H.; Kuhr. W. G. ]. Am. Chem. Soc. 1991, 223, 1832-1833. (6) Pantano. P.; Kuhr, W. G. Anal. Chem. 1993, 65,623-630. (7) Achtnich. U. R; Tiefenauer. L X.;Andres. R Y. Biosens. Bioelectron. 1992, 7.279-290. (8) Green, N. M. Biochem.]. 1966, 201. 774-780.
770 Analytical Chemistry, Vol. 67,No. 4, February 15, 1995
Avidin
Biotin
0
Figure 1. Complex formation between avidin and biotin.
biosensors. However, such monolayer-modified biosensors often suffer from a low response problem arising from insufficient enzyme activity (or lack of enzyme load), because output currents of enzyme sensors depend basically on the total activity of the enzyme immobilized on the electrode. This problem is particularly serious for miniaturized biosensors because the available surface area on which enzymes can be immobilized is limited strictly. In fact, it has been reported that the output current of miniature enzyme sensors prepared by the avidin/biotin system is a few nanoamperes or lower. In this context, we have reported recently that a thick membrane of avidin can be prepared on the Pt electrode surface by an electrodeposition technique and that much biotinylated GOx is immobilized to the avidin-modified electrode. 12.13 Glucose sensors thus prepared showed a high and rapid response to glucose. However, it is somewhat difficult to control the sue of output current of the sensors precisely or to control the enzyme load, because the electrodeposited avidin membranes do not have any ordered structure or morphology but rather exhibit a shaggy surface. The present paper reports a facile method for the regulation of the sue of the output current of glucose sensors based on a (9) Wilchek, M.; Bayer, E. A, Eds. Methods in Enzymology; Academic Press: San Diego. CA. 1990. Vol. 184. (10) LAO, S.;Walt, D. R Anal. Chem. 1989, 62. 1069-1072. (11) Gunaratna. P. C.; Wilson. G. S. Anal. Chem. 1990.62.402-407. (12) Anzai. J.; Hoshi. T.; Osa.T. Chem. Lett. 1993, 1231-1234. (13) Hoshi. T.; Anzai. J.; Osa, T. Anal. Chim. Acta 1994,289,321-327.
0003-2700/95/0367-0770$9.00/0 0 1995 American Chemical Society
-
1) avidin
-
1) avidin 2) B-GOx
2) B-GOx
E
:Avidin,
1) avidin
2) B-GOx
: B-GOx
Figure 2. Stepwise deposition of avidin and biotinylated GOx (B-GOx) on a Pt electrode surface.
stepwise and repeated deposition of avidin monolayer and biotinylated GOx monolayer on the electrode surface. An alternating and repeated deposition of avidin and biotinylated GOx may give a multilayer structure as shown schematically in Figure 2, because avidin contains four biotin-binding sites per molecular, and GOx used are tagged with ca. 5.1 biotin residues. One can expect that an amperometric response of such sensor will depend stepwise and precisely on the number of GOx monolayers deposited. EXPERIMENTAL SECTION
Reagents. Anhydrous dextrose (glucose) was obtained from Wako (Osaka, Japan). Glucose standard solutions were prepared by dissolving glucose in distilled water. GOx (EC 1.1.3.4) from AspergiZZus niger and biotinylated GOx (5.1 biotin residues per GOx molecule) were obtained from Sigma (St. Louis, MO). Avidin was obtained from Calzyme Laboratories (San Luis Obispo, CA). Fluorescein3 isothiocyanate (FITC)-conjugated avidin (5.5 fluorescein residues per avidin molecule) was purchased from Molecular Probes (Eugene, OR). Dulbecco's phosphate buffered saline (PBS) was used to prepare enzyme and avidin solutions. Dichlorodimethylsilane was obtained from Tokyo Kasei Vokyo, Japan). All other reagents were of the highest grade available and were used without further purification. The PBS was prepared by dissolving 8.0 g of NaCl, 0.2 g of KCl, 0.2 g of KH2P04, and 1.15 g of Na2HP04 in 1000 mL of distilled water. The pH of this buffer is ca. 7.4 without further adjustment. Apparatus. A potentiostat equipped with a function generator (Nikko Keisoku NPGF-250W Atsugi, Japan) was used to supply a fixed potential to the electrode. The working electrode used was a Teflon-supported platinum disk with a 3.0 mm diameter. Ag/AgCl electrode with a liquid junction of 3.3 M KCl saturated with AgCl was used as a reference electrode, and the auxiliary electrode was a platinum wire. The electrode materials, Ag and Pt (>9!3.99%), were purchased from Tanaka Vokyo, Japan). The surface of the Pt electrode was polished thoroughly with alumina powder and sonicated in distilled water and PBS successively before use. Absorption spectra were measured using a JASCO Ubest-30 spectrophotometer. Fabrication of Glucose Sensors. The Pt electrode was immersed in an avidin solution (10 mg/mL) for 30 min at room temperature (to deposit avidin on the electrode surface by simple adsorption) and subsequently in PBS for 12 h (to wash out any weakly adsorbed avidin). It has been reported that avidin is adsorbed to Au and Ag surfaces through a hydrophobic or ligating interaction to form a monolayer (metal-sulfur bonding is not
involved in the binding of avidin).14 Therefore, it is reasonable to assume that an avidin monolayer can be formed on the Pt electrode surface. The avidin-modified electrode was immersed in a biotinylated GOx solution (50 pg/mL) for 30 min to immobilize the GOx on the avidin-modified electrode through avididbiotin complexation. In order to deposit the second layer of avidin and GOx, the GOx-modified electrode was immersed in the avidin solution for 30 min, washed in PBS for 10 min, and then immersed in the biotinylated GOx solution for 30 min. The same procedure was repeated 20 times to further deposit enzyme layers. The procedure for constructing the enzyme layer is shown schematically in Figure 2. Determination of Glucose Concentration. The electrochemical response of the GOx-modified electrode was measured with a conventional threeelectrode system at 0.6 V. The GOx catalyzes the oxidation reaction of glucose to produce H202, and the H202 can be oxidized at the Pt surface at this potential. glucose
cox + 0, d-gluconolactone + H,O, H,O, - 0, + 2H+ + 2e-
Series of glucose standard solutions were injected into 10 mL of the measuring buffer solution (0.1 M phosphate, pH 6.8) with stirring at 400 rpm. All measurements were carried out at 20 "C. Deposition of FITC-Avidin and Biotinylated GOx on a Quartz Plate. A quartz slide (5 cm x 1cm x 0.1 cm) was first chemically modified to make the surface hydrophobic. For this purpose, the slide was treated with a mixture of sulfuric acid and chromic acid for more than 24 h, washed with distilled water, and dried under reduced pressure. The quartz slide was modified with dichlorodimethylsilane (5% solution in toluene) for 24 h at room temperatureI5 and washed with toluene, acetone, and distilled water. This silylated quartz slide was immersed in an avidin solution of 10 mg/mL for 30 min. After being rinsed with PBS for 12 h, the quartz slide was immersed in a biotinylated GOx solution of 50 pg/mL for 30 min, rinsed with PBS, and immersed in a FITGavidin solution of 1mg/mL for 30 min. This process provides both sides of the quartz slide with an avidin/ biotinylated GOx/FITC-avidin layer. The deposition of biotinylated GOx and FITC-avidin was repeated 20 times, and the absorption spectra were recorded after every two depositions. (14) Ebersole. R C.; Miller, J. A; M o m , J. R; Ward, M. D.J.Am. Chem. Soc. 1990, 112,3239-3241. (15) Wolfbeis. 0. S.; Schaffar, B. P. H.And. Chim. Acta 1987, 198, 1-12.
Analytical Chemistry, Vol. 67, No. 4, February 15, 1995
771
' 3000
0'05
2
O O O
2500
0 0
\
0
5
10
15
0
z
5
2000
0
1500
d
500
0 0
0
20
Figure 3. Successive deposition of biotinylated GOx and FITCavidin on a quartz slide, monitored by absorbance at 495 nm.
RESULTS AND DISCUSSION Multilayer Formation. Prior to the construction of glucose sensors, we immobilized FITC-avidin and biotinylated GOx alternately on a quartz slide to ascertain the formation of a multilayer structure of the proteins. An increase in absorbance at 495 nm, originating from the FITC moiety of FITC-avidin, was monitored after every two depositions of FITC-avidin and biotinylated GOx layers on both sides of the quartz slide (Figure 3). The absorbance increased in proportion to the number of layers deposited (deposition number), suggesting the formation of a multilayer structure on the quartz slide. We observed concomitantly a slight increase in absorption around 380 nm originating from a cofactor flavin adenine dinucleotide (FAD)in biotinylated GOx, though the absorbance was too small to be plotted, supporting the formation of alternating multilayers of FITC-avidin and biotinylated GOx. Using a molar extinction coefficient of 176 000 M-I cm-I for FITC-avidin at 495 nm and assuming that the thickness of each layer is 4.0 nm, the density of FITC-avidin in each layer was found to be ca. 3.86 x 10l2mol/cm2 (or occupied area, ca. 25.9 nm2per FITC-avidin molecule). This result indicates that FITC-avidin is immobilized in each layer as a roughly monomolecular layer considering the molecular dimensions of avidin (6.0 nm x 5.5 nm x 4.0 nm) .I6 Although further study is needed to define the well-organized multilayer structure of avidin and enzyme, it is concluded at least that the proteins are accumulated on the Pt electrode surface in proportion to the deposition number. Glucose Sensors. Avidin and biotinylated GOx were deposited stepwise up to 20 layers on a Pt electrode, and the effect of the deposition number on the amperometric response of the glucose sensor was studied. Figure 4 plots the output current (AZ) of the glucose sensor to 1 mM glucose as a function of the deposition number. It is clear that the response current depends on the number of GOx layers deposited. We carried out the same experiment several times independently,and the Nvalues tended to show some scatter after the 15th deposition. On the contrary, when native GOx bearing no biotin residue was used in place of biotinylated GOx, N was negligibly small and did not increase, even after several treatments with avidin and GOx. This shows clearly that the repeated deposition of avidin and biotinylated GOx relies on the specific binding between avidin and biotin. Thus, it is safe to say that nonspecific binding of proteins does not play a role in the repeated deposition of avidin and biotinylated GOx. (16) Green, N. M.; Joynson,M. A Biochem. J. 1970,118, 71-72.
772 Analytical Chemistry, Vol. 67,No. 4, February 15, 1995
5
0
The number of deposition
15
10
The number of deposition Figure 4. Effect of successive deposition of biotinylated GOx layers on the output current of glucose sensor. The average values of AI for two sensors were plotted. Glucose concentration, 1 mM.
I
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P
1041 A
\
A A
A
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c
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Q v)
d
A
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'
n
o
n
o
o
o
0
n 0
'
o
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0 0
-log(Glucose / M) Figure 5. Calibration curves for the glucose sensors bearing 20 layers (A),10 layers (O), and a monolayer (0)of biotinylated GOx membrane.
It should be noted that the AI value is highly enhanced by the repeated deposition of enzyme. For example, the AI values were ca. 1300 and 2800 nA for glucose sensors moditied with 10 and 20 layers of GOx, respectively, as compared with 110 nA for the monolayer-modfied sensor. The slope of the plot (i.e., A(AI) per deposition) in Figure 4 seems to change slightly after the seventh deposition, the reason of which is not obvious at present stage. A plausible explanation for this is that the structure of the enzyme layer gradually deviates from the idealized growing structure shown in Figure 2 after the seventh deposition. One of the problems in the present procedure is that each deposition is somewhat time-consuming. Optimizing of the deposition conditions is still needed. In any case, the size of the output current of the sensor can be controlled arbitrarilyby changing the number of GOx layers deposited. It is also interesting to reveal the effect of the enzyme deposition on the calibration graph and response time of the glucose sensor. Figure 5 illustrates calibration graphs of glucose sensors loaded with a monolayer, 10 layers and 20 layers of biotinylated GOx. The monolayer-modified sensor gave a useful calibration to glucose concentration ranging from 3 x to 1 x M, below which the output current was too small (less than 1 nA) for practical use. On the other hand, the detection limit was extended to 1 x M or lower by the deposition of
I
a
I
\
\ I
0
Time Flgure 6. Typical response curves of glucose sensors with a monolayer (a), 10 layers (b), and 20 layers (c) of biotinylated GOx to 1 mM glucose.
10-and 20-layer enzyme membranes, resulting in a wider dynamic range of the sensors. It is often the case that response time of enzyme sensors depends significantly on the thickness of the immobilized enzyme layer. Therefore, we checked the response time of the sensors carefully and found that it remained virtually unchanged, independent of the number of GOx layers. Figure 6 shows amperometric response of the sensors modified with monolayer and 10and 20-layer enzyme membranes to 1mM glucose. These sensors responded rapidly to glucose and reached a steady-state current in ca. 20 s or faster. These results suggest that the enzyme layers are so thin that they do not influence significantly the mass transfer of analyte and reaction products of the enzymatic reaction. In this situation, the response time of the sensors would be determined by the rate of enzymatic reaction. In order to characterize the response of the GOx-modified sensors, the Eadie-Hofstee form of the Michaelis-Menten equation (1)l7J8was employed to obtain the apparent Michaelis constants and the maximum current,
I = I,,
- K,"PP
(I,Q
(1)
where I is the steady-state current, I,, is the maximum current under stationary substrate conditions,KmaPP denotes the apparent Michaelis constant, and Cis the concentrationof glucose. Figure 7 shows the Eadie-Hofstee plots for the glucose sensors modified with monolayer and 10-and 20-layer GOx membranes. The I,, and KmaPPvalues were obtained from the extrapolation of linear portions of the plots and their slopes Vable 1). I,, values of 2.2, 18, and 26 p A were obtained for monolayer and 10-and 20layer enzyme sensors, respectively. This is reasonable because I- should reflect total enzyme activity of electrode. On the other hand, Kmapp values ranged from 6.2 to 18 mM. The I,, and KmaPP values were almost independent of the stirring rate of the sample solution. The observed values are consistent with literature results for glucose sensors. For example, Castner and W~ngardl~reported KmaPpvalues of 6, 14,and 36 mM for glucose sensors prepared by using albumin-, allylamine-, and silanemodified electrodes, respectively. In our case, the slight dependence of Kmapp values on the number of GOx layers may be (17) Scott, D. L.; Bowden, E. F. Anal. Chem. 1994, 66, 1217-1223. (18) Kaku, T.;Karan, H.I.; Okamoto, Y.Anal. Chem. 1994, 66, 1231-1235. (19) Castner, J. F.; Wingard, L. B., Jr. Biochemisty 1984, 23, 2203-2210.
1
I
I
2
I
I
1
4
3
(Current / Glucose concentration) / A
mM-l
Figure 7. Electrochemical Eadie-Hofstee plot of 20 layers (A), 10 layers (U), and a monolayer (0) of GOx sensors. The plots were based on the steady-state current of the sensors to 3-1 00 mM glucose under the same conditions as in Figure 5. Table 1. Enzyme-Substrate Kinetic Parameters Estimated from Eadle-Hofstee Plot'
no. of GOx layers 1 10 20
I,,/pA c m 2
KmaW/mM
2.1 18 26
18 12
6.2
Each value was determined using three electrodes.
originating from a slight difference in diffusional resistance of the GOx/avidin layer of the sensors, in view of the fact that the values derived from the Eadie-Hofstee plot do not characterize the enzyme itself but the whole enzyme sensor. The concentration of 02,HzOz, and even glucose inside GOx/avidin layers is probably different from that in the bulk solution and dependent on the thickness of the GOx/avidin layers. The long-term stability of the 20-layer-modified sensor was tested for more than 2 months. After the sensor was rinsed in the working buffer thoroughly, the AZvalue to 1mM glucose was recorded once a day, and the sensor was stored in the buffer at 4 "C when not in use. The AZvalue remained almost unchanged for the first 10 days and then decreased gradually. About 50% response of the original AZ value was maintained after 2 months. CONCLUSIONS The present study shows that enzyme layers can be constructed on an electrode surface using avidin and biotinylated GOx, simply by immersing the electrode in the avidin and biotinylated GOx solution, alternately. The enzyme loading or the number of GOx layers can be controlled arbitrarily by regulating the deposition number, resulting in stepwise and precise control of the size of the output current of the glucose sensors thus prepared. This would be especially useful for the construction of sensitive microsensors. It should be emphasized that not only does the present technique provide enzyme layers of controlled activity, but also, the layered structure of enzyme molecules can be arranged to some extent. It may also be possible to prepare bienzyme layers composed of monolayers of GOx and another biotinylated enzyme, by which further improvements of the sensor performance, such as elimination of interference by ascorbic acid and uric acid and extention Analytical Chemistry, Vol. 67, No. 4, February 15, 1995
773
of dynamic range, seem feasible. Studies along this line are now in progress. ACKNOWLEDGMENT
The present work was supported in part by Grant-in-Aid No. 05235102 from the Ministry of Education, Science and Culture of Japan.
774 Analytical Chemistry, Vol. 67,No. 4, February 15, 1995
Received for review August 11, 1994. Accepted November
23, 1994.@ AC940805N
Abstract published in Advance ACS Abstracts, January 1, 1995.
