The present invention relates to nanobiocomposite bienzymatic amperometric cholesterol biosensor coupled with cholesterol oxidase (ChOx) and horseradish peroxidase (HRP) immobilised on nanocomposite film deposited on ITO, electrode for estimation of free cholesterol with higher accuracy in blood serum samples. The bienzyme based nano composite bioelectrodes (ChOx-HRP/NSPANI-AuNP-GR/lTO) offer better performance in terms of detection limit, sensitivity, and response time than single enzyme system. This is attributed to the presence of HRP along with ChOx to enhance the overall biochemical reaction. The ChOx-HRP/NSPANI-AuNP-GR/ITO nanocomposite bioelectrode exhibits minimum interference, very low Km value, low response time and excellent reusability. The large specific surface area, excellent conductivity, stable and reliable redox properties of NSPANI-AuNP-GR nano composite film allow the rapid transmit of electron and enhance current response for the immobilized enzymes.
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