High Current Density “Wired” Quinoprotein Glucose Dehydrogenase Electrode Ling Ye,’J Martin Hiimmerle$ Arjen J. J. Olsthoorn,s Wolfgang Schuhmannj Hans-Ludwig Schmidt$ Johannis A. Duine,g and Adam Hellert Department of Chemical Engineering, University of Texas a t Austin, Austin, Texas 78712, Lehrstuhl fiir Allgemeine Chemie und Biochemie, Technische Universitht Miinchen, 0-8050 Freising- Weihemtephan, Germany, and Department of Microbiology and Enzymology, Delft University of Technology, 2628 BC Delft, The Netherlands
in contrast to other PQQ-containing enzymes where dialysis Glucose eloctrockr were propared by “wlrlng” qulnoprotdn against EDTA-containingbuffers easily removesthern.l2The glucose ckhydrogenaw,GDH (EC 1.1.99.17) to glassy carbon enzyme has a very high catalytic activity: approximately 8 with an omlwn complex contalnlng redox-conductlng epoxy of glucose is oxidized per minute per milligram of protein notwork. ~ c u r n n t d r n d t y a t 7 0 m M g ~ ~ a k mmol n with Wurster’s blue’ and approximately 3 with 2,tl-dichloreached 1.8 mA an-? when 15 pg cm-?of the enzyme having rophenol indophenol.6 Taking the latter value, this is up to an acthdty of 250 units mg-l was applkd to the electrode. 20 times the activity of pure glucose oxidase. In contrast, the Under the same condltlonr, eMrockr made with glucose rates obtained with the presumed natural electron acceptor oxMaw (QOX) of rlmHar actlvlty (250 u n L mg-I) had a of this GDH,13 cytochrome b ~ 2are , rather low. maxbnum current donslty of 0.66 mA an-?. The maxlmum Redox centers of flavoprotein enzymes such as glucose cwrentd.ndty wasreachedwW8% GDH Inthe redoxpolymer oxidase (GOX) have been electrically connected to glaeey fllm. The currenl denrlty was ahnod flat through the 8.3-8.8 carbon, graphite, and gold electrodes through a 3-dimensional pH range and was not altered when the solution was elthw redox conducting network, to which the enzyme is covalently aerated or argon purged. I t docrossed at 25 O C to hatf its bound. One of these networks is made of a water-soluble lnltlal value In 8 h. osmium complex containing redox polymer POs-EA14J5
INTRODUCTION Many quinoproteins (enzymes containing a quinone cofactor) have been isolated and characterized in recent years.’ These enzymes have either pyrroloquinolinequinone (PQQ), topaquinone (TPQ), or tryptophanyltryptophanquinone (“Q) as cofactor.2 Glucose dehydrogenase belongs to the group of PQQ-containing quinoproteins? T w o quite different types of quinoproteinglucose dehydrogenase exist, one soluble and the other membrane-bound.4 The soluble type has so far only been detected in Acinetobacter calcoaceticw strains, while the membrane-bound one is widely distributed among Gram-negative bacteria, including A. calcoaceticus.5 The soluble type enzyme, used in the here describedinvestigations has been characterized by several research groups6-8 and it is referred to here as GDH (EC 1.1.99.17). Its gene has been cloned0 and an efficient expression of the apo-enzyme was obtained in a recombinant Escherichia coli strain.10 Reconstitution of apo- to holo-enzyme requiresthe presence of PQQ3 and Ca2+.11 The latter two are firmly bound to the protein, of Texas a t Austin. Universitat Munchen. 8 Delft University of Technology. (1)Duine, J. A,; Frank, J.; Jongejan, J. A. Adu. Enzymol. 1987, 59,
(Figure 1)that binds the protein moiety of enzymes, such as GOX, forming a redox polymer/enzyme complex.l6 Ethylamine functions of POs-EA and lysylamines of GOX are cross-linked with a water-soluble diepoxide, poly(ethy1ene glycol diglycidyl ether) (PEGDE). The cross-linking, i.e. epoxy-curing reaction, can be carried out on the electrode surface itself. The cured film, though insoluble in water, is highly hydrophilic and is permeable to water-soluble ions and molecules. Because the enzyme in the film is electrically connected to the electrode and because the film is permeable to glucose and gluconate, a catalytic glucose electrooxidation current density of 0.8mA cm-2 is obtained in 3-mm-diameter electrodes at a 1:l polymer to enzyme (weight/weight)ratio16 and 2 mA cm-2 in 7-pm-diameter microelectrodes.17 POsEA-based epoxies also connect redox centers of other flavoprotein oxidases to electrodes.18 Amperometric GDH sensors that are 0 2 insensitive have been rep0rted.19,~oThese sensorsemployed diffusional redox shuttles, such as the 1,l’-dimethylferriciniumcation, with which ca. 60 PA cm-2 current density was reached. Here we report that 02-insensitiveglucose electrodes reaching a 1.8 mA cm-2 current density can be made with GDH connected through the PEGDE cross-linked redox epoxy POs-EA.
+ University
1 Technische
169-212.
(2) Duine, J. A. Eur. J.Biochem. 1991,200, 271-284. (3) Duine, J. A.; Frank, J.; Van Zeeland, J. K. FEBS Lett. 1979,108,
443-446. Ameyama, M. FEBS (4) Mataushita, K.; Shinagawa, E.; Adachi, 0.; Microbiol. Lett. 1988,55, 55-59. (5) Duine, J. A. Energy generation and the glucose dehydrogenase
pathway in Acinetobecter. In The Biology of Acinetobacter; Tower, K. J., Ed.; Plenum Press: New York, 1991; pp 295-312. (6) Hauge, J. J. Biol. Chem. 1964,239, 3630-3639. (7) Dokter, P.; Frank, J.; Duine, J. A. Biochem. J.1986,239,163-167. (8)Geiger, 0.; Goerisch, H. Biochemistry 1986,25, 6043-6048. (9) Cleton-Jansen, A. M.; Groen, N.; Vink, K., Van de Putte, P. Mol. Gen. Genet. 1989,217,430-434. (10) Van der Meer, R. A.; Groen, B. W.; Van Kleef, M. A. G.;Frank, J.; Jongejan, J. A.; Duine, J. A. Methods Enzymol. 1990, 188, 260-283. (11) Geiger, 0.; Goerisch, H. Biochem. J. 1989,261, 415-421. 0003-2700/93/0385-0238$04.00/0
MATERIALS AND METHODS Glucose oxidase (EC1.1.3.4, from Aspergillus niger) purchased from Boehringer and gluconic acid purchased from Sigma were (12) Duine, J. A.; Frank, J.; Jongejan, J. A. Anal. Biochem. 1983,133, 239-245. (13) Dokter, P.; Van Wielink, J. E.; Van Kleef, M. A. G.;Duine, J. A. Biochem. J . 1988,254, 131-138. (14) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991,95,5970-5975. (15) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991,95, 5976-6980. (16) Heller, A. J. Phys. Chem. 1992,96, 3579-3587. (17) Pishko, M. V.; Michael, A. C.; Heller, A. Anal. Chem. 1991, 63, 2268. (18) Katakis, I.; Heller, A. Anal. Chem. 1992, 64, 1008-1013. (19) D’Costa, E. J.; Higgins, 1. J.; Turner, A. P. F. Eiosensors 1986,2, 71-87.
(20) Yokoyama, K.; Sode, K.;Tamiya,E.; Karube, I.Anal. Chin. Acta 1989,218, 137-142.
0 1993 Amerlcan Chernlcal Society
ANALYTICAL CHEMISTRY, VOL. 65, NO. 3, FEBRUARY 1, ~OS-EA
n=l, m=1.8, p i l . 1
lQB3 298
1
2000
1
1500
1000
\
/CH2
500
NH2 HCI
Flgure 1. Structure of the osmlum complex containlng redox polymer POs-EA.
used as received. Glucose (1M) (from Merck, Darmstadt, FRG) stock solution was made with deionized water, allowed to mutarotate for more than 24 h at room temperature and was stored at 4 'C. Poly(ethy1ene 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 mM NazHPOd, was used for all electrochemical measurements, unless otherwise indicated. When used in the flow system, the carrier buffer was partially degassed by reducing the pressure aboveit with a water aspirator for ca. 20 min. Deionized water, filtered with a hollow fiber microfiltration capsule (0.2 pm, MICROGON, Laguna Hills, CA, was used throughout the experiments. Potassium hexachloroosmatewas purchased from Strem Chemicals and used as received. The redox polymer was poly(viny1pyridine)partially N-complexed with [osmium bis(bipyridine) chloride]+/2+and quaternized with bromoethylamine (POs-EA,Figure 1). The synthesis procedure was similar to that described by Gregg and Heller.I4 c i s - b i s ( 2 , 2 ' - b i p y r i d ~ ~ ~ ~ ~ c ~ o r o(0.6 o sg,m1.05 i u mmol) and poly(4-vinylpyridine)(0.33g, 3.15 mequiv of monomerunits) were heated under nitrogen to reflux in 18 mL of ethylene glycol for 3 h. The intermediate polymer was collected by dripping the reaction mixture into rapidly stirred ethyl acetate (400mL). The sticky polymer was redissolved in a minimum amount of methanol. The intermediate polymer was precipitated from the methanol solution by adding the meth'anol solution dropwise to 800 mL of rapidly stirred ether and then filtered and dried. The intermediate polymer (0.3 g), bromide (2.418 g, 7.5 mmol) were dissolved in 15 mL of ethylene glycol/DMF (1:l)and heated for 24 h at 100 OC under nitrogen with stirring. After being precipitated from acetone, the crude polymer was dissolvedwith a small amount of water and passed through a Sephadex column (G-50,Pharmacia) with 0.2 M NaCl as the eluant. The collected solution was stirred with 12 g of Bio-Red AGl-X4 ion-exchange resin (CI form) for 24 h, filtered, and desalted by ultrafiltration. The final polymer product was obtained by evaporating the water in a vacuum oven at 80 "C. The elemental analysis and UV-vis spectroscopy showed that the repeating unit of the polymer had 1.1ethylamine pendant functions and 1.8unsubstituted pyridine rings per osmium complexed pyridine. GDH apo-enzyme and PQQ were obtained as described by Van der Meer.Io Wurster's blue was synthesized according to the procedure of Michaelis and Grannick.21The apo-enzyme (2 mg mL-') was dissolved in 10 mM HEPES buffer, pH 8.0. PQQ (6.6 mg mL-') was dissolved in 10 mM HEPES (pH 8.0) with 3 mM CaClZ. A 10-pLaliquot of the PQQ solution was then added to 100 MLof the enzyme solution and mixed thoroughly. The mixture was incubated at room temperature for 15min; then the enzymatic activity of the reconstituted holo-enzymewas assayed spectrophotometricallyby monitoringthe decoloration of Wurster's blue? The measured activity was 250 units mg-l. The PQQGDH solution was stored at 4 "C. The reconstituted GDH can be stored at 4 OC for more than 2 months without measurable loss of activity. Rotating disk electrodes were made by press fitting 3-mmdiameter glassy carbon rods into Teflon housing. Stationary ~~
0 0
20
40
60
80
glucose concentration, mM Flgwe 2. Comparisonof the current output and the sensitivity for GQH and QOX electrodes: (open square) GDH electrode; (solid diamonds) GOXelectrode. Condltions: 0.4 V vs SCE; 1000 rpm; air atmosphere.
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, sandpaper,thenwith5-,l-,and0.3-pmaluminatoarnirrorfii. The electrodes were sonicated for ca. 5 min and rinsed with deionized (DI)water after each polishing step. The enzyme electrodes were prepared by applying sequentially 1p L of the polymer PVPOs-EA (10 mg mL-' in DI HzO), 0.5 pL of GDH (2 mg mL-' in the reconstituted solution) or GOX (2 mg mL-* in 10 mM HEPES, pH 8),and 0.5 pL of PEGDE 400 (3 mg mL-' in DI HzO) to the electrodesurface. The componentswere mixed, allowed to dry, and then cured for more than 24 h at room temperature. Before the electrochemical measurements, the electrodes were soaked in phosphate buffer solution (PBS) for 30 min and rinsed with DI water. The electrochemical measurements were carried out either in a conventional electrochemicalcell or in a flow injection analysis system.22 A Model 400 bipotentiostat from EG&G Princeton AppliedResearch was used for the amperometric measurements, and a KLPP & ZONEN BD41 strip chart recorder was used to record the data. The rotator used was a Model ASR 2 analytical rotator from Pine Instruments. All steady-state currents were measured at 0.4 V vs SCE.
RESULTS AND DISCUSSION Current Response. The variation of the current density with glucose concentration of POs-EA/PEGDE-wired GDH and GOX electrodes is seen in Figure 2. When the electroactive film contains 8% of the enzyme, the current density of the GDH electrode substantially exceeds that of the GOX electrode at +12 mM glucose and levels off at 1.8 mA cm-2 a t near 70 mM glucose. This current density is about 3 times that reached with GOX. The sensitivity of the wired GDH electrode at 5 mM glucose is 165 pA cm-2 M-l. The higher current density of the GDH electrode, relative to that of the GOX electrode, derives from the faster rate of electron transfer from the PQQHz centers than from FADHz centers to the osmium complex in the redox polymer/enzyme network. The rate constant for the electron transfer to diffusional shuttles from reduced GDH is also much faster than from reduced GOX.19,23The rate constant for the electron transfer from reduced GDH to ferrocenemonocarboxylic acid is 96.0 X l@ L mol-' s-', while it is only 2.01 x lo5L mol-' a-1 from reduced GOX.23 That the 3-dimensional redox network is effective in collecting from GDH and transferring them to the surface of the electrode is seen from the magnitude of the increase in current density relative to that with electron shuttling with 1,l'-dimethylferrocene. For the latter, D'Costa, Higgins,
~
(21)Michaelis, L.; Grannick, S. J . Am. Chem. SOC.1943, 65, 17471755.
100
(22) Schuhmann, W. Sens. Actuators B 1991,4,41-49. (23) Davis, G. Biosensors 1986,1,161-178.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 3,FEBRUARY 1, 1993
OI
1
6
a
7
I
1s
9
Is+O
8ol] l
-
40
Is
IS+lOP
15
lp= 1 mM gluconate Figure5. Responseof GDH electrodes to pulses of 1mM glucose wlth different concentrations of gluconic acid added, measured In a flow injection system.
I
0.10 I
-
60
ls+lp
Is
concentration IS= 1 mM glucose
PH Figure 3. Effect of pH on the current output of the GDH and GOX electrodes. Solution of 0.15 M NaCI, 20 mM Na2P0,, and 1 mM glucose was titrated to the desired pH wlth HCI.
loo{ - =
Ip
'
0.06-
C
I-
€
5
i
I
z
006-
20j I-
C
0
I 2 Oo4I 002 0 1 2 3 4 5 6 Glucose concentration, mM
Figure 4. Percentage of the current retained when the atmosphere is changed from argon to oxygen: (solid square) GDH electrode; (open square) GOX electrode.
and Turner,lg using a porous graphite electrode, observed 54 pA cm-2 at 4 mM glucose and reached a limiting current density near 5.3 mM glucose. In comparison, the wired GDH electrode has a current density of 724 p A cm-2a t 4 mM glucose and a limiting current density of 1.817 pA cm-2 near 70 mM glucose. It is thus evident that the redox network effectively connects not only FADHz/FAD ~ e n t e r s l ~but - ' ~also the PQQ centers to the electrodes. The enhanced limiting current density impliesthat in the PEGDE cross-linkedPOs-EAwired GOX electrodes the current density is not limited by electron transfer through the redox polymer but by the electron transfer from the enzyme redox centers to the polymer. This transfer is faster for PQQ-GDH than for FAD-GOX. pH Dependence. The pH dependence of the current densities for the GDH and the GOX electrodes is shown in Figure 3. The optimal activity of free GDH is at pH 9 when Wurster's blue is used as the electron acceptor? However, when the electron acceptor is oxidized phenazine methosulfate, the pH optimum is 7.24 Thus the current of the GDH electrode changes relatively little from pH 6.3 to 8.8. Absence of Current Suppression by Oxygen. The current of the GDH electrode is not suppressed by dissolved 02 (Figure 4). Comparison of stationary GDH and GOX electrodesby testing in the same solution, with a bipotentiostat holding their potentials at 0.4 V vs SCE and a mechanical stirrer operating at 500 rpm to facilitate even mass transfer to both electrodes, showed that only the current of the GOX (24) Olsthom, A. J. J.; Duine, J. A. Unpublished results.
I
0
2
6
4
8
10
Time,days Flgure 6. Stablllty of reconstttutedGDH Insolutionat roomtemperature. The activity of the enzyme was assayed spectroscopically.
0 0
10
20
30
40
time, hours Figure 7. Comparisonof the stablllty of the GDHelectrodes. Continuous operation was in a 10 mM glucose solution in a conventional electrochemlcal cell (open squares) and In a flow injection analyzer where 15 pL of 10 mM glucose pulses was injected once per hour (solid diamonds). Electrodes were at 0.4 V vs SCE.
electrode decreased as the atmosphere was changed from argon to oxygen. Product Inhibition. Product inhibition of the electrode was studied in the flow injection analysis system by periodically injecting samples of 1mM glucose to which increasing
ANALYTICAL CHEMISTRY, VOL. 65, NO. 3, FEBRUARY 1, 1993
concentrations of gluconate were added. The current of the GDH electrode is not affected by gluconate (Figure 5). Stability. The half-life of the dissolved enzyme in 10mM HEPES buffer (pH 7.3) at room temperature, determined by periodic spectrophotometric assay of ita activity (Figure 61, is 5 days. However, the decay of the current of the GDH electrodes was faster when tested continuously in 10 mM glucose at 25 "C,pH 7.3 (Figure 7, opensquares). Thecurrent dropped to its initial value in about 8 h. The operational stability of the "wired" GDH electrode is glucose concentration dependent, the output declining more rapidly at higher glucose concentrations. Figure 7 shows the rates of decline for GDH electrodes in a conventional electrochemicalcell where the glucose concentration was held at 10 mM and in a flow injection analyzer where a computercontrolled injection system injected fixed volumes of 10 mM glucose once every 60 min. Under the conditions of the experiment, it took less than 3 min for the sample to pass through the cell; i.e., the electrode was in 10 mM glucose for
241
only 3 min each hour. The rate of current loss is thus related to the amount of glucose electrooxidized (Figure 7). We conclude that PQQ-glucose dehydrogenase is effedively electrically wired through an osmium complex based redox polymer to glassy carbon. The extraordinarily high limiting current density, 1.8 mA cm-2, suggests fast electron transfer from PQQ to the osmium complex centers of the polymer.
ACKNOWLEDGMENT The work at the University of Texas was supported by the Office of Naval Research, the Welch Foundation, and the National Science Foundation. The work at the Technische Universitiit Miinchen was supported by the Bundesministerium fiir Forschung und Technologie (BMFT), Projekttriger BEO. RECEIVED for review June 25, 1992. Accepted October 29, 1992.
