RESUMO
A new label-free method was developed for SERS detection of human apolipoprotein A4. Rolling circle amplification (RCA) was used, which could induce the production of AuNPs (poly adenine and adsorption gold nanoparticles). When there were two DNA labeled antibodies and target protein, MB1 (molecular beacon 1) was unfolded and the substrate was modified in the homogeneous solution, and the proximate complex was formed. The unfolded molecular beacon worked as a primer in the hybridization with the RCA template to start RCA, which could produce many long sequences of DNA containing amounts of adenines. The AuNPs were bound with the long-repeated adenine in the RCA product, causing accumulation of AuNPs on the surface of the electrode. It was indicated that the spectral characteristics of adenine at 736 cm-1 strongly dominated the SERS spectrum of DNA. Adenine worked as an internal marker for detecting human apolipoprotein A4 by using label-free SERS method. When the conditions were optimal, the detection of human apolipoprotein A4 was carried out from 10 pg mL-1 to 1000 ng mL-1, and the detection limit was low (4.1 pg mL-1). Meanwhile, the specificity was also excellent because the antibody could specifically bind with the corresponding antigen. In addition, since adenine was dominant in SERS spectra and the affinity between AuNPs and poly adenine was high, the detection procedure could be performed without any sophisticated modification. This method might provide a promising strategy for diagnosis in clinical practice.