We describe a sensitive aptamer-based sandwich assay for protein detection on microplate by using rolling circle amplification (RCA) coupled with thrombin catalysis. This assay takes advantage of RCA generating long DNA oligonucleotides with repeat thrombin-binding aptamer sequence, specific aptamer affinity binding to achieve multiple thrombin labeling, and enzyme activity of thrombin for signal generation. Protein target is specifically captured by antibody-coated microplate. Then, an oligonucleotide containing an aptamer for protein and a primer sequence is added to form a typical sandwich structure. Following a template encoded with complementary sequence of aptamer for thrombin, RCA reaction extends the primer sequence into a long oligonucleotide. Many thrombin molecules bind with the RCA product. Thrombin catalyzes the conversion of its chromogenic or fluorogenic peptide substrates into detectable products for final quantification of protein targets. We applied this strategy to the detection of a model protein target, platelet-derived growth factor-BB (PDGF-BB). Due to double signal amplifications from RCA and thrombin catalysis, this assay enabled the detection of PDGF-BB as low as 3.1 pM when a fluorogenic peptide substrate was used. This assay provides a new way for signal generation in RCA-involved assay through direct thrombin labeling, circumventing time-consuming preparation of enzyme-conjugate and affinity probes. This method has promise for a variety of analytical applications.
Keywords: Aptamer; Multiple enzyme labeling; Protein detection; Rolling circle amplification; Signal amplification; Thrombin.