Anal. Chem. 1999, 71, 1068-1076
Amperometric Determination of Total Cholesterol at Gold Electrodes Covalently Modified with Cholesterol Oxidase and Cholesterol Esterase with Use of Thionin as an Electron Mediator Takahiro Nakaminami, Shin-ichiro Ito, Susumu Kuwabata, and Hiroshi Yoneyama*
Department of Applied Chemistry, Faculty of Engineering, Osaka University, Yamada-oka 2-1, Suita, Osaka 565-0871, Japan
Immobilization of cholesterol oxidase (EC 1.1.3.6) (ChOx) on a gold electrode was attempted by cross-linking using glutaraldehyde between ChOx molecules and a selfassembled monolayer of 2-aminoethanethiolate. The resulting electrode (ChOx/Au) exhibits an amperometric response to free cholesterol in the presence of thionin as an electron mediator, and a steady-state response is obtained ∼60 s after injection of cholesterol into the electrolyte solution. Coimmobilization of cholesterol esterase (EC 3.1.1.13) (ChE) and ChOx (ChE/ChOx/Au) allows the amperometric determination of both esterified cholesterol and free cholesterol. Cyclic voltammetry of the ChE/ChOx/Au and the dependence of the amperometric response to cholesterol on the concentration of thionin suggest that thionin is encapsulated in the enzyme film on the electrode surface. Apparent Michaelis constants of the ChOx/Au and the ChE/ChOx/Au electrodes suggest that the amperometric response was controlled by penetration of the reaction substrate into the films of the enzyme(s). The concentration of total (free and esterified) cholesterol in human serum samples, determined by using the techniques developed in the present study, is in good agreement with that determined by the wellestablished technique using colorimetry. Cholesterol and its fatty acid ester are important compounds for human beings since they are components of nerve and brain cells and are precursors of other biological materials, such as bile acid and steroid hormones.1-3 However, accumulation of them in blood due to excessive ingestion results in fatal diseases, such as arteriosclerosis, cerebral thrombosis, and coronary diseases.4-7 It is desired to develop techniques which allow convenient and (1) Yeagle, P. L. Biology of Cholesterol; CRC Press: Boca Raton, FL, 1988. (2) Myant, N. B. The Biology of Cholesterol and Related Steroids; William Heinemann Medical Books: London, 1981. (3) Bittman, R. Cholesterol: Its Functions and Metabolism in Biology and Medicine; Plenum: New York, 1997. (4) Laboratory Standardization Panel of the National Cholesterol Education Program. Clin. Chem. 1988, 34, 193-201. (5) Sugano, M.; Beynen, A. C. Dietary Proteins, Cholesterol, Metabolism and Atherosclerosis; Karger: New York, 1990. (6) Grundy, S. M.; Goodman, D. S.; Rifkind, B. M.; Cleeman, J. I. Arch. Intern. Med. 1989, 149, 505-10. (7) Noble, D. Anal. Chem. 1993, 65, 1037A-41A.
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Scheme 1. Electrochemical Detection of Cholesterol and Cholesterol Ester
rapid determinations of cholesterol, and an amperometric cholesterol sensor whose detection principle is schematically shown in Scheme 1 may be one of promising candidates.8-14 In our previous paper,15 we reported that some redox-active phenazine and phenothiazine derivatives, such as thionin, work as good electron acceptors for cholesterol oxidase (ChOx) in place of O2, which is commonly used as a natural electron acceptor for ChOx.9-14 It was also shown that the use of those redox species as the electron mediators allows accurate electrochemical determination of cholesterol without any interference of several existing compounds dissolved in blood, such as ascorbate and urate,16-18 because the reduced electron mediators are oxidized at as low as 0 V vs SCE. However, the previous study was performed using ChOx-dissolved electrolyte solutions, and the time to obtain steady response currents was more than 10 min, which was found to be determined by the time required to attain an equilibrium in the (8) Kajiya, Y.; Tsuda, R.; Yoneyama, H. J. Electroanal. Chem. 1991, 301, 15564. (9) Motonaka, J.; Faulkner, L. R. Anal. Chem. 1995, 65, 3258-61. (10) Trettnak, W.; Lionti, I.; Mascini, M. Electroanalysis 1993, 5, 753-63. (11) Yon Hin, B. F. Y.; Lowe, C. R. Sens. Actuators 1992, B7, 339-42. (12) Masoom, M.; Townshend, A. Anal. Chim. Acta 1985, 174, 293-7. (13) Karube, I.; Hara, K.; Matsuoka, H.; Suzuki, S. Anal. Chim. Acta 1982, 139, 127-32. (14) Bertrand, C.; Coulet, P. R.; Gautheron, D. C. Anal. Lett. 1979, 12, 147788. (15) Nakaminami, T.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1997, 69, 236772. (16) Maidan, R.; Heller, A. Anal. Chem. 1992, 64, 2889-96. (17) Bindra, D. S.; Wilson, G. S. Anal. Chem. 1989, 61, 2566-70. (18) Ikeda, T.; Katasho, I.; Senda, M. Anal. Sci. 1985, 1, 455-7. 10.1021/ac9805450 CCC: $18.00
© 1999 American Chemical Society Published on Web 01/29/1999
enzymatic reaction in solution bulk. In the present study, attempts were made to immobilize ChOx and cholesterol esterase (ChE) onto a gold electrode substrate. Since accumulated immobilization of enzyme molecules restricts the detection reaction to the electrode surface, steady states of the reaction are thought to be obtained in a short time.19,20 Several approaches have been proposed for immobilizing enzyme molecules onto electrode substrates, involving physical entrapments of enzyme molecules within electropolymerized conductive polymers,21-25 electrostatic complexation between an enzyme and a polymer adhered on electrode substrates,26-28 construction of ordered multilayers of enzyme utilizing specific interaction of biotin/avidin or antigen/ antibody couples,29-32 covalent attachments of enzyme molecules to functionalized monolayers on electrodes,33-37 and cross-linking of an enzyme and a polymer backbone.16,38,39 In this paper, immobilization of enzyme(s) was performed by cross-linking between enzyme molecules with the use of a bifunctional agent, glutaraldehyde, because the procedure required for the immobilization is quite simple and a glucose oxidase-immobilized electrode prepared by using similar cross-linking methods showed a high activity for determination of glucose.20,40 It will be shown here that the electrode on which ChOx is immobilized exhibits the amperometric response to free cholesterol with use of thionin as an electron mediator, and that immobilization of ChE together with ChOx allows determinations of the total amount of esterified cholesterol and free cholesterol. The technique for individual determination of free cholesterol and esterified cholesterol using the prepared electrodes is described, and its validity will be demonstrated in measurements of real serum samples. EXPERIMENTAL SECTION Chemicals and Reagents. Cholesterol oxidase (EC 1.1.3.6) (ChOx) and cholesterol esterase (EC 3.1.1.13) (ChE) from Pseudomonas sp. were commercially available from Wako Pure Chemicals and used without further purification. Human serum (19) Battaglini, F.; Calvo, E. J. J. Chem. Soc., Faraday Trans. 1994, 90, 987-95. (20) Kuwabata, S.; Okamoto, T.; Kajiya, Y.; Yoneyama, H. Anal. Chem. 1995, 67, 1684-90. (21) Kajiya, Y.; Sugai, H.; Iwakura, C.; Yoneyama, H. Anal. Chem. 1991, 63, 49-54. (22) Kajiya, Y.; Matsumoto, H.; Yoneyama, H. J. Electroanal. Chem. 1991, 319, 185-94. (23) Kuwabata, S.; Martin, C. R. Anal. Chem. 1994, 66, 2757-62. (24) Bartlett, P. N.; Whitaker, R. G. J. Electroanal. Chem. 1987, 224, 37-48. (25) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1988, 60, 2473-8. (26) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1989, 111, 2357-8. (27) Heller, A. J. Phys. Chem. 1992, 96, 3579-87. (28) Mizutani, F.; Sato, Y.; Yabuki, S.; Hirata, Y. Chem. Lett. 1996, 251-2. (29) Hoshi, T.; Takeshita, H.; Anzai, J.; Osa, T. Anal. Sci. 1995, 11, 311-2. (30) Hoshi, T.; Anzai, J.; Osa, T. Anal. Chem. 1995, 67, 770-4. (31) Bourdillon, C.; Demaille, C.; Mouiroux, J.; Saveant, J.-M. J. Am. Chem. Soc. 1994, 116, 10328-36. (32) Bourdillon, C.; Demaille, C.; Mouiroux, J.; Saveant, J.-M. Acc. Chem. Res. 1996, 29, 529-36. (33) Lion-Dagan, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 1994, 116, 7913-4. (34) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114, 10965-6. (35) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Adv. Mater. 1993, 5, 912-5. (36) Willner, I.; Katz, E.; Willner, B. Electroanalysis 1997, 9, 965-77. (37) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992, 64, 625-30. (38) Gregg, B.; Heller, A. J. Phys. Chem. 1991, 95, 5976-80. (39) Calvo, E. J.; Etchenique, R.; Danilowicz, C.; Diaz, L. Anal. Chem. 1996, 68, 4186-93. (40) Kajiya, Y.; Okamoto, T.; Yoneyama, H. Chem. Lett. 1993, 2107-10.
Scheme 2. Immobilization of ChOx and ChE on an Au Electrode Substrate
was purchased from Sigma. Warning: Human serum is considered to be potentially biohazardous and should be handled with safety precautions! All other chemicals used were of analytical grade and obtained from Wako Pure Chemicals except for thionin, i.e., 3,7diaminophenothiazinium (Aldrich), cystamine (Aldrich), cholesterol linoleate (Tokyo Chemical Industry), and N-(9-acridinyl)maleimide (Dojindo Laboratories). Water used for preparation of all aqueous solutions was purified by double distillation of deionized water. Electrode Substrate. An Au plate was used as an electrode substrate for immobilization of the enzymes. Prior to immobilization, the Au plate (radius 0.44 cm) was polished successively with alumina slurries of 1.0 and 0.3 µm, subjected to ultrasonication in deionized water for 30 min, soaked overnight in a piranha solution, which is a mixture of 30% hydrogen peroxide aqueous solution and 97% sulfuric acid (25/75, v/v),41 and then mounted in a Teflon electrode holder, which restricted to the exposed area of 0.27 cm2. Note: Piranha solution is a strong oxidant and must be used with extreme caution! Vapor-deposited Au on glass plates of 17.5 cm2 were also used as the Au substrates in the experiments for determination of the amount of immobilized ChOx or ChE on electrodes and for observation of cross sections of the prepared enzyme films with a scanning electron microscope (Hitachi, S-800). Immobilization Procedure. Immobilization of ChOx onto the Au electrode substrate was accomplished using the procedures shown in Scheme 2. The Au electrode was immersed in dimethyl sulfoxide containing 1 mmol dm-3 cystamine for 8 h to deposit a self-assembled monolayer of 2-aminoethanethiolate, and 30 mm3 (111 mm3/1 cm2 of electrode area) of 20 mmol dm-3 phosphate buffer (pH 7.0) containing 2.5 µmol dm-3 ChOx and 5 wt % (∼0.4 mol dm-3) glutaraldehyde were cast onto the resulting electrode surface. The electrode was then kept in a glass vessel filled with dry N2 at room temperature (20 ( 2 °C) for 4 h except where noted to allow cross-linking between amino residues (lysine or arginine) of the protein shell of ChOx and the amino group of (41) Hayes, W. A.; Shannon, C. Langmuir 1996, 12, 3688-94.
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2-aminoethanethiolate. When ChE was immobilized together with ChOx, 1.7 µmol dm-3 ChE was added to the above-mentioned cast solution. The prepared electrodes were thoroughly rinsed with phosphate buffer (pH 7.0) to remove weakly adsorbed enzyme and unreacted glutaraldehyde molecules. In this paper, the electrode modified with ChOx alone and that with both ChE and ChOx will be denoted as ChOx/Au and ChE/ChOx/Au, respectively. Estimation of the Amount of Immobilized Enzyme. The amount of ChOx immobilized on an Au electrode was determined by measuring the fluorescence intensity emitted from flavin adenine dinucleotide (FAD) contained in ChOx as a prosthetic group after extraction of FAD from the ChOx. For this purpose, the ChOx/Au and the ChE/ChOx/Au were soaked in 8 mol dm-3 urea aqueous solution under agitation at 70 °C for 2 days in the dark to release FAD from ChOx into the solution.20,42,43 Though this procedure is usually used for extracting FAD from dissolved ChOx, it must be applicable to the enzyme immobilized on the electrode, since it was found that the extraction efficiency was the same between the dissolved ChOx and the ChOx cross-linked in a solution, as determined by the fluorescence intensity measurements described below. The resulting solution was microfiltered using a membrane filter (Millipore, JGWP 090) having a pore diameter of 0.2 µm in order to remove insoluble denaturated proteins which were contained in the solution. The filtrate was analyzed using a high-performance liquid chromatograph system which consisted of a fluorescence detector (Jasco, FP 920), a pump (Jasco, PU 980), a column (Tosoh, TSK-GEL ODS-80TM), and a digital recorder (Shimadzu, C-R3A). The eluent used was a mixed solvent of acetonitrile and water (10/90, v/v) containing 0.1 vol % H3PO4, and its flow rate was chosen to be 0.5 cm3 min-1.44 The fluorescence intensity at 524 nm was measured with excitation at 365 nm. As a reference, 8 mol dm-3 urea solution in which commercially available FAD was dissolved was used. The amount of ChE immobilized on the ChE/ChOx/Au electrode was determined by using fluorophore-labeled ChE. Before immobilization, ChE was covalently labeled by fluorogenic reagent N-(9-acridinyl)maleimide, which possesses a specific property for binding to thiol groups.45,46 For this purpose, 20 mmol dm-3 phosphate buffer (pH 7.0) containing 50 µmol dm-3 N-(9acridinyl)maleimide and 3 µmol dm-3 ChE was agitated overnight and then gel-filtered with use of Sephadex G-25 (Pharmacia) to remove unreacted N-(9-acridinyl)maleimide. The emission intensity of the prepared solution at 430 nm obtained with excitation at 360 nm showed that 9.14 molecules of N-(9-acridinyl)maleimide were bound to one molecule of ChE, suggesting that ChE possessed approximately nine reactive cysteine residues per molecule. Using the obtained N-(9-acridinyl)maleimide-labeled ChE solution, ChE/ChOx/Au was prepared. To determine the amount of immobilized ChE, the prepared electrode was immersed in an aqueous solution containing 0.1 mol dm-3 HCl and 8 mol dm-3 urea at 50 °C overnight, resulting in cleavage of (42) Kamei, T.; Takiguchi, Y.; Suzuki, H.; Matsuzaki, M.; Nakamura, S. Chem. Pharm. Bull. 1978, 26, 2799-804. (43) Uwajima, T.; Yagi, H.; Terada, O. Agric. Biol. Chem. 1974, 38, 1149-56. (44) General Catalog for Liquid Chromatography; GL Sciences: Tokyo, 1995. (45) Takahashi, H.; Nara, Y.; Meguro, H.; Tuzimura, K. Agric. Biol. Chem. 1979, 43, 1439-45. (46) Meguro, H.; Takahashi, C.; Matsui, S.; Ohrui, H. Anal. Lett. 1983, 16, 162532.
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peptide bonds in proteins and dissolution of the enzyme film. After the solution pH was adjusted to ∼7 by adding NaOH aqueous solution, fluorescence intensities were measured using a spectrophotofluorometer (Hitachi, F3010). Molecular weights of ChOx and ChE used in this study were estimated both by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (PerSeptive Biosystems, Voyager) using sinapinic acid as a matrix compound47 and by sodium dodecyl sulfate polyacrylamide gel electrophoresis.48,49 Measurements of Amperometric Response and Cyclic Voltammetry. Amperometric responses of the ChOx/Au and the ChE/ChOx/Au to cholesterol and cholesterol linoleate were measured by polarizing the electrodes at 0 V vs SCE in 20 mmol dm-3 potassium phosphate buffer (pH 7.0) containing 1 mmol dm-3 thionin and 30 vol % 2-propanol. As shown in our previous paper, 2-propanol worked well as a solubilizer for free cholesterol without causing much damage to ChOx.15 In this study, 2-propanol was found to work as a solubilizer also for esterified cholesterol. Electrochemical measurements were carried out using a potentiostat (Bioanalytical Systems, BS-1), a one-compartment electrochemical cell equipped with a platinum foil of 2 cm2 as a counter electrode, and a saturated calomel electrode (SCE) as a reference electrode. The cell was placed in an incubator controlled at 30 °C during the course of measurements. The electrolyte solution was deoxygenated by bubbling N2 for 20 min prior to the measurements. When constant background currents were obtained, an aliquot of 0.1 mol dm-3 cholesterol or 50 mmol dm-3 cholesterol linoleate dissolved in 2-propanol was added to the electrolyte solution so as to give desired concentrations, followed by agitating for 5 s. The time course of the oxidation currents in quiescent solution was then monitored on an electric polyrecorder (Toa Denpa, EPR-151A). Steady currents obtained are termed here as the current response (ir) and the time required to get the steady currents as the response time (tr). Cyclic voltammetry of the ChE/ChOx/Au was performed using an electrochemical analyzer (Bioanalytical Systems, BAS-100B/ W) connected to a personal computer (Gateway 2000, 4DX-33), or using conventional combinations of a potentiogalvanostat (Nikko Keisoku, NPGS-2501), a function generator (Nikko Keisoku, HB-104), and an X-Y recorder (Graphtech, WX-1000). When measurements of the rotating disk electrodes were carried out, a rotating disk electrode system (Bioanalytical Systems, RDE-1) was used. Amperometric Determination of Total Cholesterol in Human Serum. The concentration of cholesterol and esterified cholesterol in a real human serum sample was determined using the following procedures. A phosphate buffer solution (pH 7.0) of 6.0 cm3 containing thionin and 2-propanol was mixed with a serum of 3.0 cm3 so as to give 1 mmol dm-3 thionin and 30 vol % 2-propanol. Two kinds of working electrodes, i.e., ChOx/Au and ChE/ChOx/Au, were placed above the prepared solution and the solution was gently bubbled with dry N2 gas for a minimum of 20 min. The ChOx/Au was then first immersed in the solution, followed by polarizing at 0 V vs SCE, until steady oxidation currents were obtained. The current response to free form of (47) Patterson, S. D.; Katta, V. Anal. Chem. 1994, 66, 3727-32. (48) Shapiro, A. L.; Vinuela, E.; Maizel, J. V., Jr. Biochem. Biophys. Res. Commun. 1967, 28, 815-20. (49) Weber, K.; Osborn, M. J. Biol. Chem. 1969, 244, 4406-12.
cholesterol (ifChOx) was obtained by subtracting the background currents obtained without serum (
