Cholesterol and its fatty acid esters 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. [P. L. Yeagle, Biology of Cholesterol (CRC Press): Its function and metabolism in biology and medicine: (Plenum: New York, 1972)]. Cholesterol determination in blood is clinically important for the diagnosis of heart diseases since accumulation of cholesterol and its fatty acid esters in blood due to excessive ingestion can be fatal (D. Noble Anal. Chem., 1993, vol 65, pp1037A–41A). The normal range of blood serum values extends from 3 to 6 mM for total cholesterol while in the hyperlipidamic condition the level can increase to 10 mM.
It is therefore desired to develop techniques that allow convenient and rapid determination of cholesterol. Various methods have been employed in the art for stabilization and immobilization of enzymes within carbon paste or covalently linking it to the surface of glassy carbon electrode or immobilizing it within a polymer film for the preparation of enzyme electrode.
Methods of immobilization of biorecognition elements have been reported for use in chemical sensing [R. F. Taylor, Protein Immobilizing Fundamentals and Applications: Marcel Dicker, New York (1975) Chapter 8, 263–303 and H. H. Weetall, Immobilized Enzyme; Antigen, Antibodies and Peptides Preparation and Characterization; Marcel Dicker, New York (1975) Chapter 6, 263–303]. The methods reported in literature can generally be classified into one of the following categories (1) physiorption (2) covalent attachment or (3) entrapment, among which physisorption is the simplest immobilization approach.
Several disadvantages arise with these methods of immobilization such as problems associated with the large size of the biorecognition elements (e.g. proteins and enzymes). Physisorption produces a range of biorecognition element orientations and apparent biding affinities. Besides physisorption generally leads to a population of biorecognizing elements that is completely unresponsive to target analyte. The immobilized species is completely unresponsive to target analyte. The immobilized species will often leach/desorb from sensing interface because there are no covalent bonds. Covalent schemes generally lead to more stable and uniform (interim of biorecognition orientation) interface and enzyme leaching is minimized. Unfortunately covalent attachment involves one or more chemical transformation and tends to be time consuming and expensive.
Electrochemically polymerised conducting polymers have also received considerable attention over the last two decades. The remarkable switching capacity of these materials between the conducting oxidised (doped) and he insulating reduced (undoped0 state is the basis of many applications. For example, polyconjugated conducting polymers have been proposed for biosensing applications because of advantageous characteristics such as direct and easy deposition on the sensor electrode by electrochemical oxidation of monomer, control of thickness by deposition of charge and redox conductivity and polyelectrolyte characteristics of the polymer useful for sensor applications.
Polypyrrole is commonly used for these applications since it meets with the above stated requirements. Polypyrrole has several advantageous properties such as easy water solubility, easily oxidisable, low cost of monomer, and the high chemical stability of the polymer [Diaz et al, J. Electroanal Chem 1981, 129, 115].
Yabuki et al [J. Chem. Soc. Commun, 1989, 41, 945] report an electrochemically synthesised polypyrrole membrane with the electropolymerised polypyrrole working as an efficient molecular interface for electron transfer. The result polypyrrole (PPY) polymer was clarified with a flavin adenine dinucleotide (FAD) entrapped PPY membrane with a smooth and reversible oxidation and reduction of FAD, the prosthetic group of glucose oxidase. Yabuki et al [J. Electroanal Chem, 1990, 277, 179] also report the use of nicotinamide adenine dinucleotide in a mediator-entrapped polymer membrane electrode.
It is known in the art that the conductivity of polypyrrole deposited, for example on textile, from an aqueous solution of polypyrrole undergoing oxidative polymerisation depends of the dopant anion such as anthraquinone-2-sulfonic acid which gives the least surface resistance [Theibelmont et al, Polym Degrad Stab 1994, 43, 293; Kuhn et al, Synth Metab, 1993, 55, 3707; Gregory et al, Synth Metab, 1989, 28, 823]. The redox species such as phenazene, thionine and ferricyanide allow accurate electrochemical determination of cholesterol without any interference of several dissolved compounds in blood such as ascorbic acid or uric acids.
It is therefore highly desirable to develop biosensors that allow conventional and rapid determination of cholesterol.