Development of a biosensor platform based on ITO sheets modified with 3-glycidoxypropyltrimethoxysilane for early detection of TRAP1

Abstract

The aim of this research was to design an electrochemical immunosensor for determination of tumour necrosis factor receptor-associated protein-1(TRAP1) antigen, a heat shock protein linked to tumour necrosis factor. The indium-tin oxide covered polyethylene terephthalate (ITO-PET) electrode surface was cleaned and was prepared for the introduction of hydroxyl groups on its surface by using NH4 OH/H2 O2 /H2 O. As a silanization agent for covalent attachment of anti-TRAP1 on the surface of the ITO working electrode, 3-glycidoxypropyltrimethoxysilane (3-GOPS) was used. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to characterize the immobilization steps. A variety of parameters, 3-GOPS and anti-TRAP1 concentrations, and anti-TRAP1 and TRAP1 incubation durations were optimized. After determining the optimum conditions, characterization studies such as repeatability, reproducibility, regeneration, square wave voltammetry, and single frequency impedance were performed. The electrochemical immunosensor has presented an extremely wide determination range for TRAP1 from 0.1 pg/mL to 100 pg/mL.

Keywords: 3-GOPS; ITO-PET; TRAP1; biosensor; immunosensor.

Figure 1

(A) Equivalent circuit model used for electrochemical impedance extraction. (B) Illustration of the immobilization steps for the TRAP1 immunosensor on the ITO-PET surface.

Figure 2

Immobilization steps of the biosensor [(A) Electrochemical impedance spectra obtained for the fabrication steps. (B) Cyclic voltammograms obtained for the fabrication steps.]

Figure 3

Optimization steps for the biosensor [(A) The effect of the 3-GOPS concentration on the biosensor performance. (B) The effect of anti-TRAP1 concentration on the biosensor performance. (C) The effect of anti-TRAP1 incubation duration on the biosensor. (D) The effect of TRAP1 incubation duration on the biosensor.]

Figure 4

Characterization experiments of the biosensor [(A) EIS experiments for standard calibration plot of TRAP1. (B) CV experiments for standard calibration plot of TRAP1. (C) Linear calibration graph for TRAP1 obtained by EIS. (D) Linear calibration graph for TRAP1 obtained by SWV.]

Figure 5

(A) Reproducibility of the biosensor. (B) Storage stability of the biosensor. (C) Regeneration experiment results for the biosensor.

Figure 6

Single frequency impedance experiment for TRAP1 biosensor.