Electrochemical sandwich immunoassay for human epididymis-specific protein 4 using a screen-printed electrode modified with graphene sheets and gold nanoparticles, and applying a modular magnetic detector device produced by 3D laser sintering

Abstract

The authors describe an electrochemical sandwich immunoassay for the human epididymis-specific protein 4 (HE4). A commercially available electromagnetic detector device and a screen-printed electrode (SPE) modified with graphene sheets and gold nanoparticles were used to fabricate the detector. First, a nanocomposite suspension consisting of graphene sheets and gold nanoparticles was deposited onto the SPE. This results in an enlarged electrochemically active area and improves electron transfer. Next, biotinylated monoclonal antibody against HE4 (anti-HE4) was bound to streptavidin-modified magnetic beads via biotin-avidin binding. Nonspecific binding sites were blocked with bovine serum albumin. Stepwise changes in the microscopic features of the surfaces and electrochemical properties upon the formation of each layer were studied by scanning electron microscopy, transmission electron microscopy and cyclic voltammetry. After magnetic separation and washing, the biotinylated anti-HE4 beads were deposited on the SPE, which then was inserted into the electromagnetic modular detector. Following the immunoreaction with HE4, a sandwich-type immunoassay was performed on the SPE using horseradish peroxidase (HRP)-labeled HE4 as a tracer label. Electrochemical detection was carried out after addition of H2O2 as a substrate for HRP. Under optimal conditions, differential pulse voltammetry can detect HE4 in the 0 to 400 pM concentration range with a detection limit of 0.5 pM (at an S/N ratio of 3). The system was applied to the determination of HE4 with good accuracy and selectivity.