Cholesterol Oxidase nanoparticle preparation, characterization, and use in the improvement of a potentiometric cholesterol biosensor

The commercial cholesterol oxidase nanoparticles (NPs) aggregates were created by the desolvation, cross linking by Glutaraldehyde, and Cysteamine dihydrochloride functionalization. FESEM, U.V, and Fourier transform infrared spectroscopy were used to analyse these enzyme nanoparticles (ENPs). The Cholesterol Oxidase NPs' FESEM pictures revealed that they ranged in size from 9 to 100nm, with an average of 22 nm. In comparison to natural enzyme molecules, the Enzyme NPs were extra steady, and had a longer shelf existence. By using glutaraldehyde coupling, the Enzyme NPs were immobilized on chitosan (CHIT) activated cellulose acetate (CA) membrane with 28.56% starting action of free cholesterol oxidase nanoparticles and a conjugation yield of 1.52mg/cm2. To create a potentiometric cholesterol biosensor, an O-ring-mounted CA membrane was attached to the bottom end of a Fluoride ion -selective electrode (FLISE). Electrode was then coupled to a digital pH metre. At pH- 6.5 and 40°C, the biosensor responded at its best within 10 s. The biosensor was used to test the potentiometric determination of cholesterol in sera from people who seemed to be in good health and those who had cardiac issues. With a wide operating range of 0.003- 0.090 mM/Land a sensitivity of -58 mV/decade, biosensor had a LOD of 0.032µM/L. The added cholesterol in serum had a 106.33% analytical recovery. The current biosensor's coefficients of variations (CVs) were 1.92% for within batches and 2.32% for between batches. The serum cholesterol readings obtained using the reference technique enzymicmethod and the current biosensor had a strong correlation (r = 0.9999). L-alanine and pyruvate caused very little interference to the biosensor, although L-glutathione and glutamine did so somewhat. This interference was reduced by the use of a particular ion selective electrode. Cholesterol oxidase kept in 0.5M sodium phosphate buffer PBS at pH 7.0 at 4 °C, the Enzyme NPs-bound CA membrane was utilised up to 7-8 times daily for a total of 160 days.