Anal. Chem. 1909, 65, 865-668
Design of a Glucose Minisensor Based on Streptavidin-Glucose Oxidase Complex Coupling with Self-Assembled Biotinylated Phospholipid Membrane on Solid Support Maja bejdarkov8,’ Marian Rehik, and Matthias Otto Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, 900 28 Ivanka pri Dunaji, Slovakia
A dmpk and fast procedure to prepare a glucose-senrltlve mlnklectrode k presented. I t k based on a blotln-modified phoophollpld Mlayer to whlch a streptavldin+lumse oxldase complex kcoupkd. Thearray k basedon the ekcbochemlcal detection of enzymatkalty generated hydrogen peroxlde at the potential +670 mV. I n the case of an alr-saturated buffering rolutlon the response to glucose was measured up to 50 mmd-L-l, wlth a Ilnear portlon up to 7 mmol-L-l. The Influence of oxygon tendon, pH, and temperature as well as POrdMy IntWorIngsubstanceswas Investigated. The prospect usage for the measuremen! of blood and urine was tested.
INTRODUCTION The characteristics and performances of enzyme-based electrochemical sensors for the measurement of glucose have been extensively studied and reviewed.’-7 Considerable effort has been devoted to develop various techniques for immobilizing enzymes to suitable supporta.*~9A new approach in the development of the biosensors is a self-assembled phospholipid bilayer membrane on solid support, fnst reported by Tien and Salamon.10 A phospholipid bilayer matrix is a natural environment for various enzymes, receptors, and carriers, most of them being highly specific for a given compound. The coating of the platinum electrode with an amorphous lipid layer made of a lipid mixture (asolectin) was (1) Mascini, M.; Selleri,S.;Riviello, M. J. Pharm. Biomed. Anal. 1989, 7,1507-1512. (2) Palleschi, G.; Faridnia, M. H.; Lubrano, G. J.; Guilbault, G . G. Appl. Biochem. Biotechnol. 1991,31,21-35. (3) Bartlett, P. N.; Tebbutt, P.; Tyrrell, C. H. Anal. Chem. 1992, 64, 138-142. (4) Amine,A.; Kaufmann, J.-M.;Patriarche,G. J. A n d L e t t . 1991,24, 1293-1315. (5) Stoecker, P. W.; Yacynych, A. M. Sel. Electrode Rev. 1990, 12, 137-160. (6) Janata, J. Anal. Chem. 1990,62,33R43R. (7) Wang, J. Electroanalysis 1991, 3, 255-259. (8) Barker,S.A. InBiosensol-Fundamenta1andApplications;Turner, A. P. F., Karube, I., Wilson, G. S., Eds.; Oxford University Press: New York, 1987; pp 85-97. (9) Wileon,G. S.;Th&onot,D. R. InBiosensors: APractical Approach; Case, A. E. G., Ed.; Oxford University Press: New York, 1990; pp 1-16. (10) Tien, H. T.; Salamon, 2.Bioelectrochem. Bioenerg. 1989,22,211218. 0003-2700/93/0385-0665$04.00/0
deacribed by Amine.” The final product of the oxidases is also hydrogen peroxide, which can be electrochemically detected. Recently, we deacribed a hydrogen peroxideloxygen biosensor based on a self-assembled phospholipid bilayer on the solid support.12 Incorporation of hydrophilic enzymes-glucose oxidase in our case-into a hydrophobic matrix represents a difficult task. Our approach is based on the exploitation of chemical interactions between biotin and streptavidin. The interaction of biotin to streptavidin protein is extremely strong (K,= 1 X 1015 M-1) which is similar in ita stability to a covalent bond.13-15 In this paper we demonstrate the feasibility of such a sensor constructed by the formation of self-assembled lipid bilayers with biotin-modified phospholipid and subsequent coupling using streptavidin-modified glucose oxidase. Due to both the high specificity and affinity of the biotinstreptavidin interaction, this system can be represented as a unique universal approach for the making of different kinds of biosensors. In this paper it is shown that this glucose biosensor has an excellent stability with a small background current and good reproducibility with fast response time.
EXPERIMENTAL SECTION Materials. Crude ox brain extract was prepared according to Folch et a l . 1 6 and stored under nitrogen at -20 O C . D-BiotinN-hydroxysuccinimideester andstreptavidin were obtained from Boehringer Mannheim, FRG. Glucose oxidase (EC 1.1.3.4.,240 units/mg, solid) from Aspergillus niger, glutaraldehyde (50 % solution in water),tris(hydroxymethyl)aminomethane,and urea were obtained from Serva Heildelberg, FRG. D-Galactose was obtained from Merck Darmstadt, FRG. D-Glucose, potassium chloride, and all other chemicals were of an analytical reagent grade (Lachema Brno, CSFR). Double-distilled water was used in the preparation of all solutions. In all experimenta stainless steel wire (Osteofix Chomutov, CSFR) of 0.3-mm diameter was used. (11) Amine, A.; Kaufmann, J.-M.; Patriarche, G. J.; Guilbault, G . G. Anal. Lett. 1989,22,2403-2411. (12) Otto, M.; Snejdhkovh, M.; R e h a , M. Anal. Lett. 1 9 9 2 , s . 653662. (13) Vaknin, D.;Als-Nielsen, J.;Piepenstock, M.;Lbsche,M. Biophys. J. 1991,60, 1545-1552. (14) Bayer, E. A.; Wilchek, M. Method Biochem. Anal. 1980,26,1-45. (15) Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1-32. (16) Folch, J.: Lees, M.: SloaneStanlev, G. H. J.Biol. Chem. 1957.226, 497-505. 0 1493 American Chemical Society
666
ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993
Buffering solutions usually consisted of 0.1 M Tris-HClmixed with 0.1 M KC1, 1:l v/v, pH 7. During each measurement the buffering system was stirred. Minielectrode Fabrication. A. Coating of the Stainless Steel Wire. The electrochemicalpolymerization of the stainless steel wire with allylamine, 2-allylphenol,and 2-butoxyethanol at a constant potential of 4 V for 1h was carried out according to Potje-Kamloth et al." The quality of electropolymerizationwas checked regularly by means of a highly sensitive ohmmeter (BM 595, Tesla Brno, CSFR); the resistivity was about lo7 Q. The thickness of the poly(oxypheny1ene) film covering the stainless steel wire was about 5 pm. B. Sensor Preparation. The technique for the forming of self-organid bilayer lipid membranes on solid substratesis based on the interactions between a nascent metallic surface and amphipatic lipid molecules. Biotinylated phospholipid (BPL) was prepared from crude brain extract and synthetic D-biotinN-hydroxysuccinimide ester according to Rivnay et al.l8 Streptavidin conjugation with glucose oxidase (GOX)via glutaraldehyde was prepared following a modification of the procedure for avidinantibody conjugates.18 Reaction products were checked using TLC and electrophoresis in the case of BPLl9and streptavidinmodified GOX,2O respectively. The membrane was self-assembled on a freshly cut coated wire using 2 % biotinylated phospholipid in n-decane (w/v).The electrical measurement of a current at +670 mV in the 0.1 M Tris-KC1 buffering system indicates the formation of a biotinylated phospholipid membrane. As soon as the value of the current was stable (1X 10-9 A), the covered wire was immersed into 2.7 pmo1.L-l water solution of Streptavidin-GOX (S-GOX) conjugate. During this step the link between S-GOX and BPL A. is formed, the current decreased on A second possibility for the preparation of the membrane was to use the BPL and S-GOX (4:1, v/v) complex after ita mixing. Two layers, organic and water, were formed. The coated wire was cut in upper hydrophobicmilieu. We also prepared ultrathin modified phospholipid bilayer on a solid support using this method. No differencesbetween these two methods of membrane preparation were observed. Electrochemical Apparatus. The direct current was measured using the picoamperemeter NC 0102 (MikrotechnaPraha, CSFR) in conjunction with the polarographic analyser PA 3 (Laboratorni pristroje Praha, CSFR). In all experiments the potentialwas set at +670 mV, and measurements were performed in aFaradaycage. A two-electrodesystem was used, the saturated calomel electrode being the reference electrode.
RESULTS AND DISCUSSION Recently12 we have demonstrated the effectiveness of a new approach toward electrode surface modification involving the use of self-assembled lipid bilayers for a hydrogen peroxide/oxygen biosensor. In this paper we explore the possibility of the biosensor fabrication based on the selfassembled bilayer formed from BPL and its subsequent reaction with S-GOX. The formation of a phospholipid bilayer was tested by measuring of the value for current in buffering solution. Unmodified coated fresh cut wire (without membrane) had a high background (10-6A) while the wire covered with BPL bilayer rather low (le9 A). The complete biosensor (with S-GOX)shown the lowest current A). The effect of anodic current as a function of the glucose concentration is presented in Figure 1. A range up to 50 mmo1.L-1 glucose was investigated. A linear response was observed up to 7 mmo1.L-l. The detection limit was 0.1 (17) Potje-Kamloth,K.;Janata, J.;Jasowicz, M.Ber.Bunsenges.Phys. Chem. 1989,93, 1480-1485. (18) Rivnay, B.; Bayer, E. A.; Wilchek, M. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press: New York, 1987; Vol. 149, pp 121-123. (19) Bayer, E.A.; Rivnay, B.; Skuletsky, E. Biochim. Biophys. Acta 1979,550,464-473. (20) Laernmli, U. K. Nature 1970,227, 680-685.
>
350
1
250
50
'
0
I
I
I
I
I
3
6
9
12
15 -1
CO,"cose
[mmol.l
1
Flgurr 1. Calibration curve of glucose electrode in 0.1 M Tris/HCI-KCi buffer, pH 7 (bar Indicates SD, n = 10).
U
a
u
500 450 400
1c
350 300 2 50
200 150 100
50
Q
I
I
I
I
I
I
0
10
20
30
40
50
60
70
c [rnmol.l-']
Flguro2. Effect of varlous pressures of oxygen in the bufferlng system as a function of a glucose concentratkm [(O)bubblng wlth nltrogsn, (0)alr normal conditions, (V)bubbling with oxygen] (bar indicates SD, n = 10).
mmol-L-1of glucose under air saturation conditions. Response time TWS, was about 45 a. The crucial point in the construction of a glucose oxidase biosensor is that the Michael-Menten constant is oxygendependent. We therefore measured the response of the glucose concentration as a function of different partial pressures of oxygen in the buffer system. It is clear from Figure 2 that the oxygen-enrichedmedium strongly influenced the calibration curve with a linear response up to 15mmo1.L-1 of glucose. Accordingly, measurements taken after bubbling nitrogen through the solution showed a diminished response. These changes are most probably connected with an insufficient oxygen content in the solution. Another explanation for the behavior of the system might be due to an increase in the concentration of gluconic acid close to the membrane21 and the subsequent inhibition of the enzyme. In Figure 3it is shownthat the pH optimum for immobilized glucose oxidase on our biosensor is around pH 7. Values (21) Fortier, G.;Bbliveau, R.; Leblond, E.; Bblanger, D. Anal. Lett. 1990,23, 1607-1619.
667
ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993 150
-3
Y
-
I
d
a
v
125
200 180
M
I
160
a 100
140 75
120 100
50
80 25 60
0 6
5
0
8
7
10
20
-1
PH
250
a a
Y
50
40
cg,ucosc [mmol.l
Flgurr 9. Effect of the pH on the glucose probe (final glucose concentration was 3.1 mmol.L-l).
-
30
1
Flgurr 5. Response to the external addition of the glucose into the diluted human blood (bar Indicates SD, n = 4).
h
I
I
50
20
I
I
I
I
25
30
35
40
Temperature
u
450 ~.
0
I
I
I
I
5
10
15
20
25 -1
[OC]
cg,ucoss[mmol.l
1
Flgurr 4. Effect of the temperature on the various glucose concentratbns [(0)10 mmoi*L-I, (0)25 mmol.L-', (V)50 mmol.L-', bar indlcates SD, n =4).
Flgwr 6. Response to the external addition of the glucose into the diluted human urine (bar indicates SD, n = 10).
lower than pH 6 and higher than pH 8 are less suitable for the measurement of glucose. The soluble glucose oxidase is known to have ita pH optimum about pH 5.5;22however, the pH range for immobilized GOX is somewhere between 5.8 and 8.0.23 The shift to neutral pH is probably due to a modification of glucose oxidase with streptavidin via glutaraldehyde. Nonmodified phospholipid membrane is more stable between pH 7 and 8.12 Therefore we measured the net response of modified membrane; the response is a function of both the enzyme activity and the membrane stability. The difference in the apparent pH of a bulk solution and pH near the enzyme-modified membrane can involve this shift, too. We measured the influenceof temperature on the response of biosensor between 24 and 37 "C (Figure 4). The values of the current were not significantly different at the level of p < 0.01. The stability of the biosensor was tested by measuring glucose for 2 weeks, using the same electrode with the same
membrane. The biosensors were stored under different conditions, i.e. in double distilled water, in physiological solution, or in the 0.1 M Tris-KC1 buffer, pH 7. The least and the most suitable solutions were distilled water and physiological solution, respectively. In general, the slow decrease of biosensor activity due to enzyme inactivation waa observed. The enzyme activity displaced 70% of the original activity after 2 weeks. The modified electrode may be used 1h following ita fabrication after it has been equilibrated in the buffering medium. One of the biggest problems facing a glucose seneor based on hydrogen peroxide detection is the elimination of interferences resulting from the presence of electrochemically active physiologicalcompounds. Unfortunately,the detection of hydrogen peroxide requires a rather high applied potential of +670 mV. At this potential several compounds might possibly interfere. The ideal hydrogen peroxide sensor would ignore everything except hydrogen peroxide. We tested the response of several potential interfering substances up to 10 mmo1.L-1. Of the group of tested substances including D-frUCtoSe, D-gdaCtoSe, D-lactose, saccharose,uric acid, urea,
_____
~~
(22) Bright, H. J.; Apple, M. J. B i d . Chem. 1969, 244, 3625-3632. (23) Guilbault, G. G.; Lubrano,G. J. A w l . Chim. Acta 1973,64,439446.
668
ANALYTICAL CHEMISTRY, VOL. 65, NO. 6, MARCH 15, 1993
L-glutathione, L-cysteine, cholesterol, and ascorbic acid, only the latter was found to interfere. Traces of ammonium ions increased the biosensor background. One way to minimize the influence of ascorbic acid is to increase the content of negatively charged phospholipids in the membrane, thereby electrically repulsing the ascorbicacid which bears a negative charge at physiological pH. We tested the usefulness of the biosensor in biological samples, i.e. diluted whole blood and urine. We performed some pilot experiments,one of which was to test the biosensor in human whole blood (diluted 10times with Tris-KC1buffer, pH 7). The results are shown in Figure 5. In our case, the background current of diluted blood was about 60 PA. We increased the level of glucose by external addition in the range up to 50 mmo1.L-l with a linear response up to 7 mmo1.L-'. Probably due to presence of glucose, lipases and ascorbic acid the background current dramatically increased in undiluted whole blood. In human urine, diluted 1:3 with 0.1 M Tris (Figure 6), the curve shifted due to presence of different metabolites, mainly ammonium ions. Standard deviations are therefore too high. The stability of the biosensor in diluted blood, plasma, and urine was minimally 8 h.
CONCLUSION The construction of a biosensor based on biotinylated phospholipid and streptavidin-modified glucose oxidase conjugate is highly advantageous with regard to its easy, cheap, and quick construction, its minisize, selectivity, short response time, and reproducibility, as well as its stability in both diluted blood and urine. The biotinylated phospholipid/streptavidinmodified GOX approach described here should be easily applicable to a large variety of other enzymes, carriers, receptors, etc.
ACKNOWLEDGMENT We thank Sylvia RajEgniovB. and Jbn Jasenovec for their laboratory and technical assistance. This work was supported by a grant from the Slovak Academy of Sciences (GA-SAV 17/91).
RECEIVED for review July 1, 1992. Accepted December 1, 1992.
