Enhancement of the Stability of Wired Quinoprotein Glucose

Chapter 3

Enhancement of the Stability of Wired Quinoprotein Glucose Dehydrogenase Electrode 1

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Ling Ye , Ioanis Katakis , Wolfgang Schuhmann , Hanns-Ludwig Schmidt , Johannis A. Duine , and Adam Heller Downloaded by MICHIGAN STATE UNIV on November 27, 2013 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0556.ch003

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Department of Chemical Engineering, The University of Texas at Austin, Austin, TX 78712 Lehrstuhl für Allgemeine Chemie und Biochemie, Technische Universität München, Vöttingerstrasse 40, D—85350 FreisingWeihenstephan, Germany Department of Microbiology and Enzymology, Delft University of Technology, Delft, Netherlands 2

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Quinotrotein glucose dehydrogenase (EC1.1.99.17) was electrically wired to a glassy carbon electrode with an osmiun complex containing redox polymer. The current output of the electrode reached 1.8 mA cm at 70 m M glucose and it was insensitive to oxygen and nearly insensitive to pH change in 6.3 to 8.3 pH range. The half life of the electrode was about 8 hours. The main cause of the current decay was the leaching of the enzyme from the redox polymer/enzyme film. The stability of the electrode was improved by increasing the extent of the crosslinking of the film and/or overcoating the electrode. The overcoated electrodes had typical half life of 33 hours. -2

In our previous paper (7), we reported the high current density wired quinoprotein glucose dehydrogenase G D H electrode. In addtion to the extraordinarily high current density, the total insensitivity to oxygen and the wide pH operation range make the wired G D H electrode very attractive. However the reported half life time of the G D H electrode was only 8 hours. In the present paper, we report the results of the study for enhacing the operational stability of the wired G D H electrode. Materials and Methods Glucose oxidase (EC 1.1.3.4, from Aspergillus niger) and bovine serum albumin (BSA, fraction V) purchased from Sigma were used as received. 1 M glucose (from Sigma) stock solution was made with deionized water, allowed to mutarotate for more than 24 hours at room temperature and was stored at 4°C. Poly(ethylene glycol 400 diglycidyl ether) (PEGDE) was purchased from Polysciences and was used as received. The other chemicals were reagent or better grade. A pH 7.3 phosphate buffer solution (PBS), containing 0.15 M NaCl and 20 m M Na2HP0 , was used for all electrochemical measurements. Deionized water was used throughout the experiments. Potassium hexachloroosmate was purchased from Strem Chemicals and used as received. Polyallylamine (PAL) was purchased from Polysciences and used as received. 4

0097-6156/94/0556-0034$08.00/0 © 1994 American Chemical Society

In Diagnostic Biosensor Polymers; Usmani, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

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Wired Quinoprotein Glucose Dehydrogenase Electrode

The redox polymers, POs-EA, was polyvinylpyridine partially N-complexed with [osmium bis(bipyridine) c h l o r i d e ] ' 2 + and quaternized with bromoethylamine. The synthesis followed the published procedures (7). The structure and the composition of POs-EA is shown in Fig. 1. The elemental analysis and UV-VIS spectroscopy showed that the repeating unit of the POs-EA had 1.1 ethylamine pendant functions and 1.8 unsubstituted pyridine rings per osmium complexed pyridine. +

n = 1

Downloaded by MICHIGAN STATE UNIV on November 27, 2013 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0556.ch003

POs-EA

» m=1.8,

p=l.l

Fig. 1. Structure of the redox polymer POs-EA. G D H apo-enzyme and PQQ were obtained as described by Van der Meer(2 ). 500 μ\ of the apo-enzyme (lmg m l in a phosphate buffer, pH 7.0) was reconstituted by addtion of 10 μ\ of the PQQ (5 mg ml" in 10 mM HEPES with 3 m M CaCl2, pH 8.0), and the mixture was incubated at room temperature for 2 hours. The enzymatic activity of the reconstituted holo-enzyme was assayed spectrophotometrically by monitoring the decoloration of Wurster's Blue (3). The reconstituted G D H (in its reconstitution solution) can be stored at 4°C for more than two months without measurable loss of activity. The isoelectric focusing electrophoresis was done using the procedure of Katakis, Davidson and Heller (Katakis, I.; Davidson, L . ; Heller, A . in preparation for publication). Rotating disk electrodes were made by press fitting 3 mm diameter glassy carbon rods into Teflon housing. Stationary disk electrodes were prepared by sealing the glassy carbon rods into glass tubings with a fast curing epoxy. The surfaces of the electrodes were polished with rough, then increasingly fine sand paper, then with 5 and 1 μτη alumina to a mirror finish. The electrodes were sonicated for ca. 5 minutes and rinsed with deionized (DI) water after each polishing step. The enzyme electrodes were prepared by applying sequentially 1 μ\ of the polymer POs-EA (5 mg ml" in DI H 0 ) , 0.5 μ\ of G D H ( 1 mg m l in the reconstituted solution ) and 1 μ\ of freshly made solution of PEGDE 400 ( 2 mg m l in DI H2O) onto the electrode surface, unless otherwise indicated. The components were mixed, allowed to dry, then cured for 24 hours at room temperature. Before the electrochemical measurements, the electrodes were soaked in phosphate buffer solution (PBS) for 30 minutes and rinsed with DI water. - 1

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DIAGNOSTIC BIOSENSOR POLYMERS

The electrochemical measurements were carried out in a conventional 3electrode electrochemical cell. A Model 400 bipotentiostat from E G & G Princeton Applied Research was used for the amperometric measurements, and a K I P P & Z O N E N BD101 strip chart recorder was used to record the data. The rotator used was from Pine Instruments. A l l steady-state currents were measured at 0.4 V vs SCE. Results and Discussion Figure 2 shows the current dependence of a G D H electrode on glucose concentration. The steady state current at saturating glucose concentration reached 1.8 mA cm" , three times the current density for the similar glucose oxidase electrode. To the best of our knowledge, this was the highest current density ever


2

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glucose concentration, mM Fig. 2 Current dependence of the G D H electrode on glucose concentration. G D H loading=10% by weight; PEG concentration=12% by weight; E=0.4 V vs SCE; Rotation rate=1000 R P M ; Argon atmosphere. reported for mediated glucose electrodes. This was not very surprising if we notice that the natural electron acceptor for G D H is chytochrom bs62, a rather big molecule, while that for G O X is oxygen. From the x-ray crystal structure of G O X (4), we know that the F A D redox centers of G O X are buried about 15 Â deep in side the insulating glycoprotein shell. Since oxygen is such a small molecule, it can diffuse easily into the active site of G O X and take electrons from FADH2 efficiently. But for the redox polymer used, a strong complex formation with GOX is required in order to shorten the distance between the osmium centers and FADH2 for the electron exchange to occur (Katakis, I.; Davidson, L . ; Heller, A . in preparation for publication). However, in the case of G D H , since its natural electron acceptor, chytochrome 0502, is a large molecule, the PQQ redox center of G D H is most likely loacated close to the surface of the enzyme molecule, i.e., the distance for the electron transfer between the osmium centers of the redox polymer and PQQH2 is short. If the Marcus theory holds in this case, i.e., the electron transfer rate decreases exponentially with increasing distance, the electron transfer from PQQH2 is faster than from FADH2 to the osmium center of the redox polymer. This faster electron transfer is translated to the higher current acheived


3. YE ET AL.


with wired G D H electrode. This agrees with the reported results for apparent bimolecular electron transfer rate constant from the reduced active centers to free diffusing mediators: this rate constant of electron transfer from reduced G D H to ferrocene monocarboxylic acid, 96.0xl0 L m o l s , is about 50 times that from reduced GOX, 2.01x10 L m o l s" (5). In fact, direct electron transfer from PQQ enzymes to the surface of a solid electrode has been reported (6). We also observed some direct electron transfer from G D H to high surface area graphite electrodes. The sensitivity of the G D H electrode was 120 μΑ c n r m M " at 6 m M glucose concentration. The apparent K m of the electrode obtained from the Eadie-Hofstee plot is 7 mM. In addition to the extraodinarily high current density, another advantage of G D H electrode over G O X electrode is that G D H electrode is totally oxigen insensitive, as reported in our preveous paper (7). However the half life time of the G D H electrode was only 8 hours (dotted line in Fig. 4). We performed some preliminary studies to elucidate the mechanism responsible for the fast decay of the current. Figure 3 shows the results of isoelectric focusing (IEF) electrophoresis. The electric field is indicated by the positive and negative signs. Samples were premixed and deposited directly on the gel. 10 μ% of each compound was used for 5

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Fig. 3 Isoelectic focusing electrophoresis. Samples deposited on lane 1 to 7: 1, G O X ; 2, B S A ; 3, G D H ; 4, G O X and G D H ; 5, G O X , G D H and POsE A © ; 6, BSA and G D H ; 7, BSA, G D H and POs-EA.



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each lane. From lane 1 to 7, the samples deposited on the gel are l : G O X ; 2: B S A ; 3: G D H ; 4: G O X and G D H ; 5: GOX, G D H and POs-EA(I); 6: BSA and G D H ; 7: B S A , G D H and POs-EA. As expected, the negative charged G O X and B S A migrated toward the anode (the positive pole), and the positive charged G D H and POs-EA migrated toward the cathode (the negative pole). When G O X and G D H are deposited together (lane 4), they migrated to the opposite direction and formed clear bands seperately, indicating that there was no detectable complex formation under the experimental conditions. However, when GOX, G D H and POs-EA were deposited together (lane 5), the G O X band disappeared, indicating that G O X and POs-EA formed a strong complex which was not separable by the electrophoresis. The clear band of G D H at the top of lane 5 indicates that G D H did not form a detecable complex with either G O X or POs-EA. The same results were obtained with BSA (lane 6 and 7), i.e., BSA formed complex with POs-EA but G D H did not complex with either. As we see from Figure 3, G D H has an isoelectric point of 9.6. Because it is highly positively charged and all of the amino groups are protonated at neutrual pH, it can not react with the diepoxide which was used as the cross linker to form the redox polymer/enzyme hydrogel. It also does not complex with the redox polymer, as shown by the IEF results in Figure 3. Essentially, for a G D H electrode made with POs-EA and the diepoxide cross linker, G D H was simply entrapped in the three dimensional network of the cross linked POs-EA. If this entrapment is not sufficient, as it is the case for a losely cross linked, highly porous hydrogel, G D H is very likely to leach out from the redox polymer/enzyme hydrogel, especially when there is a repulsion force between the redox polymer and G D H since both are highly positively charged at pH 7.3. If our speculation is correct, we should be able to stabilize the current output of the G D H electrode by preventing the leaching of GDH. Figure 4 shows the time dependence of the currents of different G D H electrodes. The dotted line was the decay curve of normal G D H electrode made 100

Fig. 4 Comparison of the operational stability of G D H electrodes at 21.3°C. E=0.4 V vs SCE; Rotation rate=1000 R P M ; Argon atmosphere.



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only with POs-EA, G D H and the diepoxide as a eross linker. The dashed line is the decay curve of a G D H electrode with BSA in the film. When 40% by weight BSA was added to the film, the half life of the electrode was prolonged from 8 hours to 26 hours. But the current of the electrode was decreased by about 58%. The possible reasons for the stabilization of the G D H electrode by coimmobilized BSA are the following: First, since BSA is negatively charged at pH 7.3, it may serve as a shield in the film to decrease the repulsion between G D H and the positively charged redox polymer. Secondly, we have seen from the IEF results that BSA formed a complex with POs-EA. This might result in a less porous or more crosslinked network so that the leaching of G D H is decreased. Finally, the increase in the stability by coimmobilization of BSA could be due to the decrease in current density since the decay of the current was associated with the electrochemical turnover of the sensor (7). The solid line in Figure 4 is the decay curve for a G D H electrode which was overcoated by polyallylamine (PAL) as described else where (Katakis, I.; Ye, L . ; Duine, J.; Schmidt, H.-L.; Heller, A. in preparation for publication). The stability was futher improved to a half life time of 33 hours because that the top layer of crosslinked P A L prevented the leaching of the enzyme. It is worthwhile to point out that the P A L overcoating increased the half life time of the G D H electrode by a factor of 4 at the expense of only 22% decrease in current output. This suggests that the main cause for the fast decay of the uncoated electrode was the leaching of the enzyme. The results with different G D H electrodes are summarized in Table I. Here we can see that overcoating the G D H electrode with P A L did not only enhance the operational stability of the electrode, but also increased the apparent K m of the electrode to 14 mM from 7 mM of the corresponding uncoated electrode, resulting in a wider dynamic measurement range. Table I.

Properties for G D H Electrodes Initial current at 100 mM glucose μΑ cm"

Electrode composition (by weight)

Apparent Km mM

80% POs-EA 8% G D H 12% PEGDE

7±1

1800±200

8±1

2 ±0.2

413 ± 5 0

^ „ 26 ± 2

14 ± 2

1400 ± 1 0 0

„ 33 ± 2

40% POs-EA 40% BSA 4% G D H 16% PEGDE 80% POs-EA 8% G D H 12% PEGDE over coated with P A L

2

Half life time hours

0

Λ

0


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Conclusion The high current density achieved with wired quinoprotein glucose dehydrogenase electrode was due to the fast electron transfer between the osmium centers of the redox polymer and the reduced redox center of GDH, PQQH2 which is most likely located close to the surface of the enzyme. The main cause of the fast decay of the G D H electrode is the leaching of GDH. The leaching can be prevented by incorporating a negatively charged protein in the redox polymer/enzyme hydrogel, or overcoating the electrode. Overcoating the G D H electrode with polyallylamine not only increased the operational half life time of the GDH electrode from 8 to 33 hours, but also increased the apparent K m of the G D H electrode from 7 to 14 m M without significant loss of the current. Aknowledgment The work at the University of Texas at Austin was supported by the Office of Naval Research, the Welch Foundation and the National Science Foundation. Literature Cited (1) Ye, L.; Hämmerle, M.; Olsthoorn, A. J. J.; Schuhmann, W.; Schmidt, H.-L.; Duine, J. Α.; Heller, A. J. Anal. Chem. 1993, 65(3), 238-241. (2) Van der Meer, R.A.; Groen, B.W.; Van Kleef, M.A.G.; Frank, J.; Jongejan, J.A. and Duine, J.A. Meth. Enzymol. 1990, 188, 260-283. (3) Dokter, P.; Frank, J. and Duine, J.A. Biochem. J. 1986, 239, 163-167. (4) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153-172. (5) Davis, G. Biosensors 1985, 1, 161-178. (6)

Ikeda, T.; Matsushita, F.; Senda, M. Biosens. Bioelectr. 1991, 6, 299-304.

RECEIVED

February 7,

1994


Enhancement of the Stability of Wired Quinoprotein Glucose

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