10.

Bioelectrochembtry

and Bioenergetics,

28 (1992) 105-115

A section of .I Electroanal. Chem., and constituting Vol. 343 (1992) Elsevier Sequoia S.A., Lausanne

JEC BB 01506

An electrochemical

bienzyme membrane
l

sensor

for free cholesterol

Ana Maria C.F. Oliveira Brett

Departamento de Quimica, Universidade

l Maria Helena Gil and Ana Paula Piedade *,

de Coimbra, 3049 Coimbra Codex (Portugal)

(Received 29 November 1991)

Abstract
An electrochemical bienzyme membrane sensor for free cholesterol was developed. The membrane polyQetrafluoroethylene)_g.co-acrylic acid (Teflon-g.co-AA), contains catalase and cholesterol oxidase immobilized covalently and this bienzyme sequence transforms cholesterol and produces oxygen which is detected at a standard oxygen electrode. Preparation of graft membranes of Teflon was achieved using gamma irradiation at 400 Gy h- for different intervals of time. The single membrane containing immobilized catalase and cholesterol oxidase was characterized and the activity and stability of the enzymes studied. This amperometric biosensor for the determination of free cholesterol showed good reproducibility, a linear response for a range of cholesterol concentrations from 90 pM to 0.55 mM and a reasonable lifetime of 1 month.

INTRODUCTION

Cholesterol as a structural component of biological membranes is essential for life. When the metabolism of cholesterol is disturbed it accumulates as the cholesteryl ester in the artery walls and so the role of cholesterol in health is associated with the incidence of heart diseases. Cholesterol deposits lead to a disease, atherosclerosis, which is now an epidemic in western society owing to genetic and dietary factors, and social factors such as smoking. The determination of cholesterol has been the subject of many extensive studies. Many methods have, therefore, been developed. The most commonly used methods in routine clinical analysis are the Lieberman-Burchard, ferric perchlo-

l Dedicated to Professor Giulio Milazzo, a great person, scientist and wonderful friend, on the occasion of his 80th birthday. l * To whom correspondence should be addressed.

0302-4598/92/$05.00

0 1992 - Elsevier Sequoia S.A. All rights reserved

106

MEMBRANE

cNsterol
/catalasQ \

+ 0,

cholestc2rol

oxiciase

4% cho&tcnon

Fig. 1. Schematic

representation

of the sequence of the enzyme reactions of cholesterol oxidase and

catalase. acetate-H,SO, and enzymic methods [l-3]. Human serum contains a mixture of free and esterified cholesterol, both of which are measured to determine the total cholesterol level. The present tendency is to use the direct enzymic method for routine clinical cholesterol testing, employing cholesterol esterase and cholesterol oxidase. Owing to the high cost of enzymes, methods of enzyme immobilization have been developed so that less enzyme is required and enzymes can be reused. Electrochemical biosensors which employ immobilized enzymes and combine electrode and enzyme specificity [4] can play an important part in quantifying biological species. In this work we developed an electrochemical bienzyme membrane sensor for free cholesterol. A Clark-type oxygen electrode was used as transducer. The reactions of two enzymes, cholesterol oxidase and catalase, both immobilized directly onto the Teflon membrane after being grafted with poly(acrylic acid), were employed. The first enzyme oxidizes cholesterol and produces hydrogen peroxide, and the second oxidizes peroxide to oxygen (Fig. 1). The characteristics of the membrane-enzymes system were studied, i.e. the kinetic parameters, pH, activity yield and stability.
EXPERIMENTAL DETAILS

rate-ethyl

Chemicals Catalase (from bovine liver), cholesterol oxidase (CO) (from streptomyces species), cholesterol and l-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methop-toluenosulphonate (CMC) were obtained from Sigma Chemical Company (Poole, UK). Acrylic acid from Merck (Schuchardt, Germany), was used without purifica-

107

tion. All other chemicals were of analytical grade and were obtained commercially. Poly(tetrafluoroethylene) (Teflon) membranes were supplied by Metrohm, EA 1092/2. Apparatus and procedures Copolymer preparation Portions of Teflon (0.02 g) were immersed in 30 cm3 acrylic acid solution (10% in water) and irradiated at 400 Gy h- for different periods of time, at room temperature, in the presence of air. The homopolymer was removed by Soxhlet extraction with water to constant mass. The graft copolymers were dried in vacua at 313 K to constant weight. The percentage of graft add-on is denoted as percentage grafting = w, - II&, w, xl00 I(C)

where W, is the final of mass of dried, extracted copolymer and Wiccj is the corrected initial mass of Teflon. This correction allows for mass changes noted in the accompanying blank experiments. Assessment of the aqueous solutions: sorption capacity Copolymer samples (0.02 g) were immersed in distilled water or in 0.05 M phosphate buffer pH 7.0 (10 cm3) at 277 K and 293 K for 24 h. Excess water was removed with filter paper. The resultant moistened membrane was, in each instance, weighed every minute for 10 min. The initial sorption capacity was obtained graphically after extrapolation to zero time. Each sample was dried to constant mass under reduced pressure at 373 K. The percentage of solution uptake is given by percentage sorption =
wi(w) wf(c)

x100

wrCc, where Wicw, is the initial wet mass at zero time and WKs is the final dry mass corrected to allow for changes in blanks. Determination of contact angles Contact angles were determined with a Contact-Meter (Livereel, Consett, UK). A drop of water or of 0.05 M phosphate buffer pH 7.0 was put on the surface of the membrane and the contact angle was determined at 293 K. Immobilization of cholesterol oxidase and catalase In order to couple the enzymes to the -COOH groups of the grafted poly(acrylic acid), CMC was used as activating agent 151. In one type of experiment, the membrane (20 mg> was treated in 0.05 M phosphate buffer pH 7.0 (10 cm3), containing 5.0 mg catalase and 5.0 mg CMC, for 18 h at 273 K. Then the system containing the immobilized catalase was treated with 10 cm3 of 0.05 M phosphate

108

buffer pH 7.0 containing 5.0 mg cholesterol oxidase and 5.0 mg CMC for 18 h at 273 K. The activity of both enzymes was determined separately. In a second type of experiment, both enzymes were simultaneously coupled. In this case the membrane (20 mg) was immersed in 10 cm3 of a 0.05 M phosphate buffer pH 7.0 containing 5.0 mg catalase, 5.0 mg cholesterol oxidase and 10.0 mg CMC for 18 h at 273 K. Determination of the cholesterol oxidase and catalase activities The catalase activity was determined following the method of Beers and Sizer using hydrogen peroxide as the substrate [6]. The cholesterol oxidase activity was determined using cholesterol as substrate and by the following procedure: to 2.9 cm3 of 0.05 M phosphate buffer pH 7.0 was added 0.05 cm3 of Triton X-100 solution (2.9% in water), 0.05 cm3 of cholesterol solution (6 mM in Lso-propanol) and 0.01 cm3 of cholesterol oxidase solution; the absorbance of the mixture was followed at 240 nm against a blank. All measurements were done on a Jasco 7800 spectrophotometer. Electrochemical measurements For the electrochemical measurements the Teflon membrane containing immobilized catalase and cholesterol oxidase was placed over the gold cathode and silver auxiliary-reference electrode assembly of a standard Clark-type oxygen electrode (Metrohm EA-541) in the usual way and connected to a Metrohm E627 0, meter. The electrode was then introduced in 25 cm3 of the buffer solution containing Triton X-100 (with concentrations of O.l%-1.5% <v/v)) to which was added aliquots of 6.0 mM cholesterol in tie-propanol.
RESULTS AND DISCUSSION

Support characterization Copolymers with different percentages of grafting were used to immobilize the enzymes. The percentage of grafting was determined by weight and the presence of acrylic acid was confirmed by IR spectroscopy, as previously [7]. The membranes used had 22% and 35% of grafting respectively. In Table 1 the results obtained for the aqueous solution sorption and contact angle for Teflon and Teflon-g.co-AA 22% are shown. We observe that the presence of -COOH groups increases the sorption of water and of buffer and also the affinity of the aqueous solutions for the polymeric support; consequently the enzyme coupling yield will be better. Immobilization of the enzymes Catalase was covalently immobilized on the Teflon-g.co-AA 22%, followed by the fixation of cholesterol oxidase. In this experiment a system with 480 U g-

109 TABLE 1 Aqueous solution sorption and contact angle for Teflon and Teflon-g.co-AA water and 0.05 M phosphate buffer pH 7.0 Polymer Solution Sorption (%) 277 K Teflon Teflon-g.co-AA 22% Water Phosphate buffer Water Phosphate buffer 0.6 0.3 24.0 38.0 293 K 1.0 0.8 32.0 39.0 22% in the presence of Contact angle (deg.) 87 86 70 51

copolymer of catalase and 300 U g- copolymer of cholesterol oxidase was obtained. These values were determined using chemical methods. The catalase activity did not decrease after coupling of the cholesterol oxidase, as indicated in Table 2. In a second experiment, both enzymes were simultaneously coupled to the membranes of Teflon-g.co-AA 22% and 35% of grafting. We found that better results for the enzyme coupling yield were obtained when Teflon-g.co-AA 35% was used, but the enzyme activity yield and the total activity of the system membrane-enzymes was higher for the immobilization on Teflon-g.co-AA 22%. So, all further experiments for system characterization were carried out with the enzymes immobilized on the membrane with 22% of grafting. A system with a 360 U gg membrane of cholesterol oxidase activity and 450 U g- membrane of catalase activity was obtained (6% and 0.2% of yield of activity respectively).
Kinetic parameters

The kinetic parameters Kh and Vmaxwere determined for both enzymes in the free form and in the membrane-enzymes system by chemical methods. The results indicated in Table 3 show that Kd values of the enzymes, after being immobilized, increase. This suggests that the affinity of the enzymes for the substrates decreases. However, these results are in agreement with values described in the literature [8] and are promising for the application of this system to free cholesterol analysis.

TABLE 2 Immobilization of catalase followed by immobilization of cholesterol oxidase (CO) on Teflon-g.co-AA 22% in 0.05 M phosphate buffer 7.0, using CMC as activating agent at 277 K for 18 h Enzyme Catalase Cholesterol oxidase Before CO coupling After CO coupling ug- 480 480 300 Activity yield (%) 0.2 0.2 5.0

110 TABLE 3 Chemical determination of the kinetic parameters of the enzymes (catalase and cholesterol oxidase) in free and immobilized form Enzyme G!I _ (mM) Free Immobilized Free Immobilized 0.024 0.091 23.0 46.9 V max U (mgE)- 66.7

ug- 400 1250

Cholesterol oxidase Catalase

1250

Optimization of the assay conditions for free cholesterol determination

In order to optimize the assay conditions, the influence of pH, buffer identity, Triton X-100 concentrations and rate of stirring on the electrode response were analysed. Figure 2 shows that there is no optimum pH for the system in the pH range 5-9, in contrast to the enzymes in the free form where each enzyme has a optimum pH of 7.0. Figure 2 also shows that better results are obtained when phosphate buffer is used, similarly to the enzymes in solution. Triton X-100 surfactant was added to increase the cholesterol solubility. The results in Fig. 3 show that the best response was obtained for Triton X-100 concentrations lower than 0.5% (v/v) of detergent. A rate of stirring of 250 rotation min- gave a better biosensor response (Fig. 4).

,v

o-o-o-o_
A

Cl

PH

Fig. 2. Influence of the pH value and buffer identity on the biosensor response: o 0.05 M phosphate buffer, 0 0.05 M acetate buffer, A 0.05 M citrate buffer, day 1. Stirring rate 250 rot. min-, [Triton X-100] = 0.1% (v/v), [cholesterol] = 0.325 mM.

111
A

[O&ng.drFi3 _

5-

0.06

O.l2

I ale

[Triton X100] I %v.v-

Fig. 3. Influence of the Triton X-100 concentration on the biosensor response in 0.05 M phosphate buffer pH 7.0, day 1. Stirring rate 250 rot. min-, [cholesterol] = 0.325 mM.

Electrochemical

determination of free cholesterol

Cholesterol can be determined at an oxygen electrode by the amount of oxygen released when the Teflon membrane containing catalase and cholesterol oxidase is placed over the electrode, in a free cholesterol solution: in the membrane

CH3
CH, + 0,

Cholesterol oxidase

HO Cholesterol

/CH3

ow

-a3 + I-w,

Cholestenone

112

5-

I 1000

.I

vd.rotatiin/rpm

Fig. 4. Influence of the rate of stirring on the biosensor response, in 0.05 M phosphate buffer pH 7.0, day 1. [Triton X-1001 = 0.1% (v/v), [cholesterol] = 0.325 mM.

at the electrode 0,+4H++4e2H,O

and there is a net oxygen consumption in the first enzyme reaction which is twice that produced by peroxide oxidation. This suggests that the oxygen electrode measures oxygen produced in the oxidation of peroxide by catalase and that the oxygen reacting with cholesterol comes from the sample solution. Such an explana-

Time lmin
Fig. 5. Biosensor response time for different concentrations of cholesterol in 0.05 M phosphate buffer pH 7.0, day 1: 0, 0.185 mM, o 0.275 mM, A 0.325 mM, A 0.55 mM. Stirring rate 250 rot. min-, [T&on X-100] = 0.1% (v/v).

113

[q/mg.drii T

o@
10

20 Number of saqies

Fig. 6. Reproducibility test for the biosensor, day 1. [cholesterol] = 0.325 mM, other conditions as in Fig. 5.

tion is reasonable since there is no cholesterol in the electrolyte solution enclosed between the gold electrode and the membrane. Using the optimized assay conditions, the free cholesterol solution was analysed electrochemically in 0.05 M phosphate buffer at pH 7.0. In Fig. 5 we see that a steady-state value was obtained after 2 min at 293 K The typical data of Fig. 6 show that the reproducibility and precision of the biosensor can be considered

[cholesterol]lmM
Fig. 7. Biosensor response to cholesterol on
l

day 1,

day 15, A day 35, conditions as in Fig. 5.

114

8-

0.2

014

06

0.8 peroxidel/mM
l

[hydrogen

Fig. 8. Biosensor response to hydrogen peroxide on

day 1, 0 day 15, A day 35, conditions as in Fig. 5.

good. Statistical analysis of the data leads to a mean value of 3.54 mg dm3 of 0, with a relative standard deviation of 3%. The average value falls exactly on the calibration curve of Fig. 7. Calibration curves for cholesterol and hydrogen peroxide are shown in Figs. 7 and 8 respectively. They indicate that a linear response is obtained for a range of concentrations of cholesterol from 90 p.M to 0.55 mM and the detection limit is 50 ~.LM. The results obtained also show that the enzymes are still active after 35 days of storage at 293 K in distilled water.

CONCLUSIONS

We can summarize this study as follows. (1) The introduction of carboxylic acid groups onto the Teflon provides a good environment for immobilization of the enzymes. (2) The use of a single membrane, with immobilized catalase and cholesterol oxidase placed over the standard oxygen electrode, enables quick determinations of hydrogen peroxide and free cholesterol with already existent commercial portable equipment. (3) The covalent immobilization of catalase and cholesterol oxidase on the Teflon membrane showed a lifetime of 1 month, the membrane being left on the electrode at room temperature in distilled water. (4) The cholesterol sensor will measure free cholesterol in the linear range from 90 IJ.M to 0.55 mM.

115

(5) The response of the sensor is reproducible sample solution every 3 min.
ACKNOWLEDGMENTS

and allows analysis of about one

We acknowledge the grant given by JNICT, Junta National de Investiga@o Cientifica e Tecnolbgica, to A.P. Piedade. We also thank C. Cavaco and M.E. Andrade from LNETI, Portugal, for preparation of the Teflon-g.co-acrylic acid membranes.
REFERENCES 1 2 3 4 5 6 7 8 H.M. Flegg, Ann. Clin. B&hem., 10 (1973) 79. W. Richmond, Clin. Chem., 19 (1973) 130. P.N. Tarbutton and CR. Gunter, Clin. Chem., 28 (1974) 724. F.W. Scheller, in A. Turner, I. Karube and G. Wilson (Eds.1, Biosensors, Oxford University Press, Oxford, 1987, Chapter 18. C.G. Beddows, M.H. Gil and J.T. Guthrie, J. Appl. Polym. Sci., 35 (1988) 135. R.F. Beers and I.W. Sizer, J. Biol. Chem., 195 (19521 133. M. Alves da Silva, M.H. Gil, A.P. Piedade, J.S. Redinha, A.M. Oliveira Brett and J.M. Caridade Costa, J. Polym. Sci. A: Polym. Chem., 29 (1991) 269. U. Wollenberger, M. Kiihn, F. Scheller, V. Deppurmeyer and M. JHnchen, Bioelectrochem. Bioenerg., 11 (1983) 307.