Development of electrochemical microbiosensor for detection of biomarkers

Abstract

This research is divided into two parts, the first part is the development of the electrochemical sensor for the determination of cholesterol using microelectrodes. Microelectrodes were fabricated from a 7 µm diameter carbon fiber (CF) and a 25 µm diameter platinum wire (Pt). Gold nanoparticles (AuNPs) were electrochemically deposited on the microelectrode surface in HAuCl₄ solution using one step by cyclic voltammetry. The deposited AuNPs on the microelectrode surfaces were characterized by scanning electron microscopy and cyclic voltammetry. For the microsensor, an AuNPs/CF microelectrode was used to detect cholesterol in the phosphate buffer solution containing cholesterol oxidase. Chronoamperometric measurements of cholesterol showed the linear response in the range of 50 to 500 µM with the detection limit of 10 µM. For the microbiosensor, cholesterol oxidase was covalently immobilized on the AuNPs/Pt microelectrode surface using 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC)/N-hydroxysulfosuccinimide (NHS) as a cross linker. Under the optimal conditions, chronoamperometric current was linearly increased with the increase of the logarithm for cholesterol concentrations in the range from 0.001 to 7 mM. The response time of the microsensor and microbiosensor is 10 seconds. In addition, the microbiosensor was successfully applied to a real sample of bovine serum. In the second part, CdSe/ZnS quantum dot (QD) based electrochemical immunoassay was used to detect the phosphorylated bovine serum albumin (BSA-OP) as a protein biomarker. The QDs were used as labels for amplifying electrochemical signals and were conjugated with a secondary anti-phosphoserine antibody in a sandwich immunoassay. In this assay, the BSA-OP was added to the primary BSA antibody coated polystyrene microwell, and then the QD labeled anti-phosphoserine antibody was added for completing immunorecognition. Finally, the bound QD was detected by electrochemical stripping analysis. The voltammetric response was linear over the range of 0.5 to 500 ng mL⁻¹ of BSA-OP, with a detection limit of 0.5 ng mL⁻¹.