Immunoassay Based On Super-Resolution Multicolor Fluorescence Co-Localization

Immunoassay has high sensitivity and strong specificity,which is an important analytical method in modern life science and has broad application prospects in biological detection.However,non-specific adsorption in immunoassays is always inevitable.Although various methods have been developed to reduce non-specific adsorption,false positive events have always been one of the important factors affecting the reliability of immunoassay results.Super-resolution technology breaks through the diffraction limit of traditional fluorescence microscopy,allowing people to observe finer biological structures.Single-molecule localization microscopy(SMLM)is one of these technologies,which brings new ideas and technical support for reducing false positive events in immunoassays.Herein,at first,a new kind of immunoassay based on super-resolution multicolor fluorescence co-localization has been proposed.According to this technique,Alexa Fluor 488 modified goat anti-rabbit IgG(A488@Goat anti Rabbit IgG)and Alexa Fluor 647 modified goat anti-rabbit IgG(A647@Goat anti Rabbit IgG)formed a sandwich composite structure in the presence of rabbit IgG.Then,SMLM was used to detect the co-localization coefficients of A488 and A647 fluorescent sites to achieve quantitative analysis of the antigens.The detection limit of this technology can be as low as 0.1 pg / m L.More importantly,this method can reduce the interference caused by false positive events to a certain extent.However,due to the inconsistency of the number of fluorescent probes in different view fields during SMLM imaging,the fluorescent probes in different regions have different capture efficiencies for the antigens,which may easily cause analysis errors.Therefore,in order to solve this problem,we further proposed a super-resolution multicolor fluorescence co-localization immunoassay based on ordered gold nanoarrays.The ordered gold nanoarrays are used as the substrates in immune reactions,which effectively reduces the analysis errors mentioned above.In this technique,aptamers are used instead of antibodies.We successfully achieved the determination of false positive binding sites and the detection of tumor cell exosomes.The detection limit of this technique for exosomes can be as low as 10 particles/μL.This new type of immunoassay has the advantages of high sensitivity,good specificity and strong universality,and holds a good application prospect in the detection of disease markers.