Multiplexed electrochemical immunoassay for two immunoglobulin proteins based on Cd and Cu nanocrystals

Abstract

Herein, a simple and feasible electrochemical immunosensing method for simultaneous voltammetric detection of two immunoglobulin proteins, human IgG (HIgG) and rabbit IgG (RIgG), was developed using two distinguishable signal-generation tags on the same electrode. The immunosensor was prepared by immobilizing two Fab antibody fragments on a gold electrode. After this, Cu and Cd nanocrystals, as nanotags, were synthesized and functionalized with identical detection antibodies. Transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR) were employed to characterize the Cu and Cd nanocrystals. The covalently modified electrode with the Fab antibody fragments through the Au-thiolate bond (to dispel the non-specific adsorption) was investigated via scanning electron microscopy (SEM). After the sandwiched immunoreaction, the antibody-modified nanocrystals were captured on the immunosensor, which could be interrogated in pH 3.5 HCl using square-wave anodic stripping voltammetry. Experimental results also indicated that the multiplexed immunoassay enabled the simultaneous detection of HIgG and RIgG in a single run with the similar linear range from 0.01 to 10 ng mL⁻¹, and the limits of detection (LODs) towards two analytes could be as low as 3.4 pg mL⁻¹ (at 3σ). Acceptable assay results on precision, reproducibility, specificity, and method accuracy were also acquired. Importantly, the newly designed strategy avoided cross-talk and enzymatic introduction as compared to conventional electrochemical immunoassays, thus exhibiting a promising potential in clinical applications.