Reusable and mediator-free cholesterol biosensor based on cholesterol oxidase immobilized onto TGA-SAM modified smart bio-chips.

Mohammed M Rahman,Vince Grolmusz

doi:10.1371/journal.pone.0100327

Abstract

A reusable and mediator-free cholesterol biosensor based on cholesterol oxidase (ChOx) was fabricated based on self-assembled monolayer (SAM) of thioglycolic acid (TGA) (covalent enzyme immobilization by dropping method) using bio-chips. Cholesterol was detected with modified bio-chip (Gold/Thioglycolic-acid/Cholesterol-oxidase i.e., Au/TGA/ChOx) by reliable cyclic voltammetric (CV) technique at room conditions. The Au/TGA/ChOx modified bio-chip sensor demonstrates good linearity (1.0 nM to 1.0 mM; R = 0.9935), low-detection limit (∼0.42 nM, SNR∼3), and higher sensitivity (∼74.3 µAµM−1cm−2), lowest-small sample volume (50.0 μL), good stability, and reproducibility. To the best of our knowledge, this is the first statement with a very high sensitivity, low-detection limit, and low-sample volumes are required for cholesterol biosensor using Au/TGA/ChOx-chips assembly. The result of this facile approach was investigated for the biomedical applications for real samples at room conditions with significant assembly (Au/TGA/ChOx) towards the development of selected cholesterol biosensors, which can offer analytical access to a large group of enzymes for wide range of biomedical applications in health-care fields.

Highlights

Development of cholesterol biosensors in therapeutic diagnostics has gained much attention in health care and biomedical fields
It is used for covalent bond formation to immobilize the cholesterol oxidase (ChOx) enzyme on the thioglycolic acid (TGA)-selfassembled monolayer (SAM) via peptide conjugation in presence of activating agent (EDC)
ChOx enzyme is immobilized onto TGA-SAM electrode by amide-bond formation between the terminal-unbound carboxylic acids (-COOH) group of TGA SAM and the amine groups (-NH2) of ChOx enzymes

Summary

Introduction

Development of cholesterol biosensors in therapeutic diagnostics has gained much attention in health care and biomedical fields. The term ‘‘immobilized ChOx enzymes’’ refers to ‘‘ChOx enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities, and which can be used repeatedly and continuously.’’ As a consequence of ChOx enzyme immobilization, some properties of the enzyme molecule, such as its catalytic activity, stability, become altered with respect to those of its soluble counterpart [9,10,11] This modification of the properties may be caused either by changes in the intrinsic activity of the immobilized enzyme or by the fact that the interaction between the immobilized selective enzyme and the substrate takes place in a microenvironment that is different from the bulk solution. The concept of stabilization has been an important driving force for immobilizing ChOx enzymes [17,18,19]

Methods

Results

Conclusion