Ultrasensitive Electrochemical Immunosensor Based On Rolling Circle Amplification

Objective：In this work, we tried to combine the specificity of electrochemistryimmunoassay with the signal amplification function of the RCA technique tofabricate an electrochemical immunosensor.Methods：Chitosan–TiC nanocomposite suspension was prepared. Theelectrochemistry property of the Chitosan–TiC nanocomposite andAuNPs/Chitosan–TiC nanocomposite modified electrodes were also studied.The factors influenced the property of the immunosensor was optimized.A chitosan–titanium carbide (TiC) nanocomposition flm was frstelectrodeposited onto a tin-doped indium oxide (ITO) electrode at a constantpotential. Gold (Au) nanoparticles were then electrodeposited on the surfaceof the chitosan–TiC flm by cyclic voltammetry (CV). The captureantibody(anti-HE4) was adsorbed onto the Au and TiC nanoparticles. After aspecifc sandwich immunoreaction among the capture antibody, HE4, andbiotinylated secondary antibody, biotinylated primer DNA was immobilized on the secondary antibody by biotin–streptavidin system. Appropriateamounts of circular template DNA and biotinylated primer DNA were usedfor rolling circle amplifcation (RCA) under optimal conditions. The RCAproducts provided a large number of sites to link DNA detection probes.Doxorubicin hydrochloride intercalated the CG–GC steps between the RCAproducts and the DNA detection probes, which was monitored bydifferential pulse voltammetry (DPV) based on the current signal ofdoxorubicin hydrochloride. Stepwise changes in the microscopic features ofthe surfaces and electrochemical properties upon the formation of each layerwere confrmed by scanning electron microscopy (SEM), atomic forcemicroscopy (AFM), and electrochemical impedance spectroscopy (EIS).Results：With the above-mentioned amplifcation factors, the current respondedto HE4linearly in the concentration range of3~300pM under optimaldetection conditions, with a detection limit of0.06pM.Conclusion：The method shows a broad dynamic range and low detection limit. Thegreatly enhanced sensitivity relies on a series of signal amplificationtechniques: nanobiotechnology, RCA technique, and biotin-streptavidinsystem. This system was successfully employed for the detection of HE4with good accuracy and renewable ability. Meanwhile, this method is provento be a suitable method for the detection of HE4.