Ultrasensitive multiplexed immunoassay of autophagic biomarkers based on Au/rGO and Au nanocages amplifying electrochemcial signal.

A novel sandwich-assay electrochemical immunosensor for simultaneous determination of autophagic biomarkers was introduced for the first time, the gold-reduced grapheme oxide nanocomposite (Au/r-GO) set as a good conductive platform with super high specific area, and provided more binding sites for the both antibodies of Beclin-1 and LC3B-II. While Au nanocages (AuNCs) served as good conductive platform to encapsulate a large amount of redox probe and secondary antibodies for signal amplification, due to the abundant reactive oxygen functional groups on its surface. Through differential pulse voltammetry (DPV) measurements, two separate signals can be detected directly in a single run, which represent the existence of Belin-1 and LC3B-II. Under optimized conditions, the electrochemical immunosensor exhibited good sensitivity and selectivity for the simultaneous determination of Beclin-1 and LC3B-II with linear ranges of 0.1-100 ng/mL. The detection limit for Beclin-1 and LC3B-II is 0.02 and 0.03 ng/mL respectively. This method was also applied for the analysis of Beclin-1 and LC3B-II levels in experimental cellular protein lysates, and the results were in good agreement with those of enzyme linked immunosorbent assay. This approach gives a promising simple, sensitive and quantitative strategy for the detection of autophagy.