An amperometric cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase

Cholesterol determination is important for the diagnosis and management of hypercholesterolemia, associated with pathological conditions like atherosclerosis, cardiovascular diseases, coronary artery diseases and transient ischaemic heart attacks1. Among the various methods available for determination of cholesterol level in serum such as chemical/colorimetric method, spectrophotometery, thin layer chromatography, gas-liquid chromatography, high-performance liquid chromatography, fluorimetric method, polarographic method and gas chromatography/mass spectrometry (GC/MS), biosensing methods employing immobilized enzymes are the most simple, rapid, specific and sensitive methods. In these biosensors, cholesterol esterase, cholesterol oxidase and peroxidase have been immobilized onto octyl-agarose gel2, poly (2-hydroxyethyl methacrylate) (p(HEMA))/ polypyrrole) membrane3, prussian-blue (PB)4, multiwalled carbon nanotubes (MWCNTs)5, porous silicon6, electrochemically prepared polyaniline film7, amino-undecanethione self assembled monolayer8, cellulose acetate (CA) membrane9, polyvinyl chloride (PVC) reaction cell10, a screen printed electrodes based on carbon nanotubes and cytochrome P₄₅₀11, dithiobisuccinimidyl propionate self assembled monolayer12, Pt incorporated fullerenes like ZnO nanosphere13, poly (pyrrole) vs poly (3,4-ethylenedioxythiophene) film14, polyaniline/multiwalled carbon nanotubes15 and pencil graphite16. All these supports (organic and inorganic) had one or more disadvantages such as low stability to microbial attack, high cost of the support material or the immobilization procedure, leaching of immobilized enzymes, reduced substrate + enzyme complex formation due to steric hindrance, requirement of high working potential, long response time, metabolite interference and low storage stability10. Epoxy resin membrane, a highly cross-linked network, a long-lived, water permeable membrane has high affinity for enzyme, low cost, easy synthesis, high temperature stability, chemical resistance and porous in nature and thus an ideal material for constructing an enzyme electrode. The application of epoxy resin membrane as a support for immobilization of enzyme has resulted into improved sensitivity and stability of biosensors for uric acid17, ascorbic acid18 and polyphenols19 in our laboratory. It is expected that the use of this membrane would lead to the improved sensitivity and stability of the cholesterol biosensor also. Hence, the present study was aimed to construct an amperometric cholesterol biosensor employing epoxy resin membrane bound cholesterol oxidase.

Material & Methods

The work was carried out in Biochemistry Research Laboratory at M.D. University, Rohtak (Haryana), India.

Sources of chemicals: 4- Aminophenazone from Sigma, USA, cholesterol oxidase from Streptomyces sp., cholesterol, alumina oxide (60-325MESH BSS) and triton X- 100 from SRL, Mumbai, were used. Epoxy resin “Araldite” was purchased from the local market. The kit of enzymic colorimetric method for cholesterol determination was from Erba Transasia, Daman, India. All other chemicals were of analytical grade.

Preparation of enzyme solution: Commercial choles-terol oxidase from Streptomyces sp. (2 mg, containing 26 U) was dissolved in 1.7 ml 0.02 M sodium phosphate buffer, pH 7.0, and stored at 4°C until use. Cholesterol (50 mg) was dissolved in 1.0 ml of triton X 100 by slowly heating and stirring until solution was clear. Sodium phosphate buffer (0.05 M, pH- 7.0) was added to it to get final concentration of 500 mg/dl. Solutions of different concentrations ranging from 50 to 500 mg/dl were prepared and stored at 4°C.

Assay of free cholesterol oxidase20: Reaction mixture consisting of 1.8 ml sodium phosphate buffer (0.05 M, pH 7.0) and 0.1 ml cholesterol oxidase was pre-incubated at 37°C for 2 min. The reaction was started by adding 0.1 ml cholesterol solution (500 mg/dl). After incubating at 37°C for 10 min, 1.0 ml colour reagent was added and kept at room temperature for 15 min to develop the colour and absorbance was read at 520 nm (A₅₂₀) against control in Spectronic-20D (Thermo, USA) and content of H₂O₂ generated in reaction was calculated from standard curve between H ₂O ₂concentration and A₅₂₀. The soluble protein in dissolved enzyme was determined by Lowry method21.

Results & Discussion

Immobilization of cholesterol oxidase on epoxy resin membrane: Cholesterol oxidase from Streptomyces sp. was immobilized onto epoxy resin membrane with 88.46 per cent retention of initial activity of free enzyme and a conjugation yield of 0.575 mg/cm². The -OH groups of epoxy (based on dihydroxydiphenylpropane and epichlorohydrin) containing polymers react with bifunctional polyamine and -NH₂ groups of enzyme to form a -C-N- linked enzyme-epoxy amine resin composites. Earlier cholesterol oxidase has been immobilized onto various membranes such as collagen membrane23, nylon membrane24 and cellulose acetate membrane925 for cholesterol biosensor development.

Scanning electron microscopy (SEM): The SEM of epoxy resin membrane without bound enzyme had a uniform polymeric layer, while membrane with bound enzyme had many globular structures (Fig. 1). These observations confirmed immobilization of enzyme.

Close

Fig. 1

Scanning electron micrograph (SEM) of cholesterol oxidase bound epoxy resin membrane without enzyme (a) with immobilized cholesterol oxidase (b).

Export to PPT

FT-IR spectra of epoxy resin: The IR spectra of epoxy resin revealed the presence of characteristic absorption bands for Ar-C=C-H stretching and bending -CH₂ and -CH₃ asymmetrical and symmetrical, -C-Ar-O-C stretching, and epoxy CH₂-(O- CH-) ring stretching vibration. The presence of epoxy groups in IR spectra was proved from the presence of strong bands at frequencies of 3,056 cm^-1 (νC-H epoxy) and 915 cm^-1 (γC-O epoxy). The 1, 4-substitution of aromatic ring was seen at 830 cm^-1 for epoxy resin. The crosslinking of compound was confirmed by the identification of characteristic absorption peaks. The IR spectrum (Fig. 2) displays a strong broad band in the 3,600-3,200 cm^-1 region assigned to O-H stretching vibrations. The appearance of the band at 1,638 cm^-1 indicates the formation of OH groups. A strong bands at 1,605, 1,580, 1,510, 1,455 cm^-1 are assigned for Ar-C=C-H stretching vibrations. The two bands at 729 and 693 cm^-1 may be attributed to out of plan bending of aromatic rings. The disappearance of the bands at 3,056 and 915 cm^-1 indicates the opening of epoxy rings. The appearance of the band at 1,109 cm^-1 is characteristic for C-N stretching vibrations. The absence of the absorption of epoxy ring and presence of OH group and C-N group confirms the conversion of epoxy group into the corresponding polymer, as well as crosslinking process.

Close

Fig. 2

The partial FT-IR spectra of the crosslinked epoxy resin.

Export to PPT

Optimization of biosensor: The sensor showed optimum response within 25 sec at pH 7.0 and 45°C (Fig. 3). At pH 7.0 the epoxy resin membrane bound ChOx must be fully ionized and able to interact with cholesterol. There was a linear relationship between electrode response (current in μA) and cholesterol concentration upto a final concentration of 8.0 mM after which it was constant (Fig. 4). LB plot gave an apparent K_m of 5.0 mM and I_max of 9.09 μA (Fig. 4: inset). A comparison of kinetic and analytic property of the present biosensor with those of earlier amperometric cholesterol biosensors is summarized in Table I.

Close

Fig. 3

Effect of pH and incubation temperature on response of cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase.

Export to PPT

Close

Fig. 4

Effect of cholesterol concentration on cholesterol biosensor response based on epoxy resin membrane bound cholesterol oxidase (inset): Lineweaver-Burk plot of 1/I vs. 1/ cholesterol of epoxy resin membrane cholesterol oxidase.

Export to PPT

Table I A comparison of various amperometric cholesterol biosensors

Determination of serum free cholesterol by biosensor: An amperometric method for analysis of serum cholesterol was developed employing present biosensor. The following parameters were studied in order to evaluate the method:

Linearity: There was a linear relationship between current (μA) and cholesterol concentration ranging from 1 to 8 mM in the reaction mixture, which is comparable to earlier amperometric biosensors, using cholesterol oxidase immobilized onto octyl agarose gel (1-25.8 mM)2, co-immobilized with peroxidase on polyaniline film (1-12.9 mM)3, pencil graphite rod (1.29 to 10.3 mM)16 but higher than those immobilized on carbon paste electrode modified with hydroxymethyl ferrocene and hydrogen peroxide (0.15 mM)6, electropolymerized pyrrole (8 μM)26.

Detection limit: The detection limit of the present amperometric biosensor was 0.1 mM with a sensitivity of 0.63 μA/mM, which is lower than biosensors using cholesterol oxidase co-immobilized with peroxidase on polyaniline (0.64 mM)7, cellulose acetate membrane, (0.32 mM)9, PVC reaction cell (0.11 mM)10 and higher than pencil graphite rod (0.09 mM)16, silica gel bound enzyme (0.003 mM)27 and chitosan-modified glassy carbon electrode (9.5×10^-3 mM)28.

Analytical recovery: In order to check the accuracy of the methods, the analytical recovery of added cholesterol in six serum samples of apparently healthy persons was determined. The mean analytical recovery of added cholesterol (2.84 mM and 4.13 mM) in serum was 91.4±2.8 and 92.3±3.1 per cent (n = 6), which is higher than pencil graphite rod (85 and 90%)16 but comparable to electrochemical method employing alkyl amine glass bound enzyme (95-102%) recovery for the added cholesterol at 192 mg/dl and 145 mg/dl29, amperometric method using co-immobilized enzyme using silica gel bound enzyme (95-101% recovery)30 and cellulose acetate membrane (98.86±0.78 & 97.79±0.73%)13 and chitosan-modified glassy carbon electrode (95-101%)28 (Fig. 5).

Close

Fig. 5

Effect of storage at 4°C on activity of cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase.

Export to PPT

Precision: To check the reproducibility and reliability of the procedure, the cholesterol content of the sample in one run (within batch) and after storage at -20°C for one week (between batch) were determined. The results showed that the cholesterol value of these determinations agreed with each other and within batch and between batch coefficients of variation (CV) were <2.0 per cent (1.59%) and <2.01 per cent (1.9%) for biosensor. These values are quite close to those reported for other methods such as electrochemical method employing pencil graphite rod (1.59 and 4.15%)16, cellulose acetate membrane (<3 and <5%)9, alkyl amine glass bound enzyme (1.6% for intra-batch and 3.2% for inter-batch)29, amperometric method using silica gel bound enzyme (<1.5% for all samples)30 and chitosan-modified glassy carbon electrode (4.0%)28. Low coefficient of variation indicates that the method is accurate, reproducible and reliable.

Accuracy: In order to know the accuracy of the present method, the values of cholesterol in 10 apparently healthy persons were determined by commercial enzymic colorimetric method (x) and compared with those obtained by present methods (y). The serum cholesterol values obtained by these two methods matched with each other and showed a good correlation (r = 0.991) with the following regression equation: y₂= (0.9909x-7.5776). The values obtained by both the methods were also in agreement with the earlier study with pencil graphite rod (r=0.99)16, cellulose acetate membrane (0.990)9 and chitosan-modified glassy carbon electrode (r=0.99)28.

Effect of serum metabolites: To study interference by metabolites, lactate (0.5 mM), glucose (5 mM), uric acid (0.2 mM), ascorbic acid (0.05 mM), acetone (0.17 mM) and bilirubin (5.1 to 17.0 mM) were added to the reaction mixture at their physiological concentrations before addition of cholesterol. Only uric acid and ascorbic acid showed slight increase (12 and 8%) in biosensor response (Table II), while the remaining had no effect. These observations were similar to earlier reports for biosensor having cholesterol oxidase co-immobilized with peroxidase on polyaniline film7. Earlier some interference by uric acid, glucose and lactate for polypyrrole bound enzyme3 and uric acid and ascorbic acid for cellulose acetate membrane bound enzyme has been reported9.

Table II Effect of various serum substances and metal salts on response of cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase

Effect of metal ions: The effect of some metal salts such as KCl, MgCl₂, NaCl, CaSO₄, and CuSO₄, each at a final concentration of 1.0 mM was tested on the response of biosensor. All metals, sodium and potassium ions had practically no effect. These observations were similar to enzyme immobilized on polyaniline film7 and biosensor consisting of co-immobilized enzyme onto cellulose acetate membrane9.

Storage stability and reusability: The enzyme electrode lost 50 per cent of its initial activity after its regular use for 150 times over a period of 6 months, when stored in 0.02 M sodium phosphate buffer, pH 7.0 at 4°C.

In conclusion, a method is described for construction of an amperometric biosensor for estimation of free cholesterol using epoxy resin membrane bound cholesterol oxidase. The biosensor worked efficiently within 25 sec at +0.5V and pH 7.0 and exhibited linearity with cholesterol concentration in the range 1-8 mM and showed temperature tolerance up to 45°C. The sensitivity of biosensor was 0.63 μA/mM with the detection limit of 0.1 mM. The half life of the biosensor was 6 months, when stored in reaction buffer at 4°C. The biosensor had reasonable sensitivity and stability.