An aptamer-based chemiluminescence method for ultrasensitive detection of platelet-derived growth factor by cascade amplification combining rolling circle amplification with hydroxylamine-enlarged gold nanoparticles

摘要

An ultrasensitive and selective chemiluminescence (CL) assay system for the detection of platelet-derived growth factor (PDGF)-BB was developed by taking advantage of the powerful signal amplification capability of rolling circle amplification (RCA) and a hydroxylamine-amplified gold nanoparticle (Au NP)-based CL technique. Rabbit anti-human PDGF-BB polyclonal antibody was covalently coupled on the surface of a 96-well plate that offers the reactive N-oxysuccinimide (NOS) group. In the presence of PDGF-BB, the aptamer-primer oligonucleotides were bound to the surface-captured antigen, resulting in an analyte-specific single-stranded DNA that could serve as the prime for the RCA reaction. The captured aptamer-primer was extended through the RCA reaction forming a single-stranded tandem repeated biotinylated copy of the circular template. After binding of the RCA product with streptavidin-gold (SA-Au NPs), the assembled Au NPs were enlarged by a NH2OH-HAuCl4 redox reaction allowing gold metal to be catalytically deposited onto the surface of the Au NPs. After an oxidative gold metal dissolution, a huge number of Au3+ ions were released from the enlarged Au NPs and were determined by a simple and sensitive luminol CL reaction. With two successive amplification steps, the proposed CL assay system exhibits not only high sensitivity with the detection limit of PDGF-BB as low as 0.06 pM (corresponding to 3 amol in 50 mu L of sample solution), but also high specificity by taking advantage of using aptamers as recognition elements. In addition, PDGF-BB has been determined in diluted serum indicating the applicability of this assay.