Mild reduction-promoted sandwich aptasensing for simple and versatile detection of protein biomarkers

Abstract Sandwich format has drawn considerable attention in aptasensing in view of simple design, convenient and stable application, and easy coupling with signal amplification strategies; however, developing sandwich aptasensing methods is still problematic, because it is a rather challenging and troublesome task to find or screen suitable aptamer pairs. Herein, we made use of mild reduction to propose a novel sandwich electrochemical aptasensing method for protein biomarker detection that avoided the need of aptamer pairs. In our design, target protein was captured by surface-immobilized aptamer and exposed active thiol groups after mild reduction. Then, maleimide-functionalized Probe DNA was linked to protein through Michael addition between maleimide and active thiol groups, thus forming a sandwich structure that could be used for signal output. Moreover, considering that the sequence of Probe DNA was freely designed, DNA-based amplification strategies could be easily incorporated in the method so as to further improve the detection sensitivity. Hybridization chain reaction was utilized here as an example, which could accumulate signaling molecules and finally arouse amplified electrochemical responses. Results demonstrated improved sensitivity and specificity of our method even in a complex serum samples by using α-fetoprotein as a model, and also revealed the versatility of our method by using both α-fetoprotein and Golgi protein 73 as examples. Our method might pave a feasible way for constructing sandwich aptasensing methods, which would provide a new insight into high-throughput determination of disease-related protein biomarkers in the future.