Aptamer and rolling circle amplification-involved sandwich assay for platelet-derived growth factor-BB with absorbance analysis

Abstract

This paper described an aptamer-based absorbance assay combining rolling circle amplification (RCA) for platelet-derived growth factor-BB (PDGF-BB). PDGF-BB was specifically captured by an antibody on a microplate, and then bound with an anti-PDGF-BB aptamer connected with a primer sequence, forming a sandwich complex. The primer sequence was extended by RCA reaction to generate a long singlestranded DNA with repeated copies. The amplified DNA product was hybridized with many biotinylated short DNA probes, and then labeled with streptavidin-horseradish peroxidase conjugate (SA-HRP). The HRP catalyzed the conversion of a substrate to a colored product, which was measured by simple absorbance analysis to achieve the detection of PDGF-BB. By involving RCA and HRP catalysis amplification, sensitive detection of PDGF-BB with a good selectivity was obtained; the detection limit reached 3.1 pM.

Functional nucleic acid biosensors utilizing rolling circle amplification

“…Alternatively, enzymes such as horseradish peroxidase (HRP) can be used to avoid the issues with monitoring of pH changes noted above. HRP can reduce chromogenic substrates such as 3,3 0 ,5,5 0 -tetramethylbenzidine (TMB) [266][267][268][269] in the presence of peroxide to generate colorimetric or electrochemical outputs for RCA-based assays. Importantly, the RCA reaction must reach completion before detection can occur as the peroxide can denature the polymerase or other enzymes (e.g., nicking enzymes) used in the RCA reaction.…”

“…As is the case for optical sandwich assays, electrochemical sandwich assays typically require protein targets, as it is necessary to have at least two binding epitopes available to form the sandwich structure. Given that most proteins have only a single aptamer, which binds to a specific epitope, the majority of sandwich-based FNA biosensors that utilize RCA employ dual antibody/aptamer interactions to detect their target, 197,217,235,261,266,276,334,[343][344][345]390,391 including electrochemical sensors. 108,294,299,301,318,319,331,377,387,392 We note that most of these examples utilize the detection aptamer as a ligation template to initiate PLP-based RCA or as a primer for RCA, and all current electrochemical sandwich assay examples utilize linear RCA with turn-on assay outputs (see Table 6).…”

A label-free electrochemical aptasensor based on leaf-like vanadium disulfide-Au nanoparticles for the sensitive and selective detection of platelet-derived growth factor BB

“…The development of an accurate, highly selective and sensitive assay for the detection of cancer-related proteins has received fervent attention due to the ever-increasing demands of proteomic strategies for clinical diagnosis and therapeutic analysis. 1,2 Platelet derived growth factor-BB (PDGF-BB) is a cancerrelated protein, and it has been reported to be involved in many cell transformation processes and in tumor growth. 3,4 The detection of PDGF-BB is therefore of particular signicance in biomedical elds.…”

DNA aptamer-based rolling circle amplification product as a novel immunological adjuvant

“…To achieve this, the reverse complimentary sequences of the eight candidate aptamer sequences were used to design 4 templates for RCA procedure with each RCA template consisting of reverse complementary sequences of two distinct aptamers (Supplementary Table S1 ). RCA consists of long tandemly repeated (several hundreds) single-stranded DNA sequence, which is reverse complementary of the original circular single-stranded DNA template 28 , 29 . Over the run of the RCA procedure, the reaction produced the RCA products of high molecular weight (Supplementary Figure S3 ).…”

“…In this study, for the first part of the study, we developed DNA aptamers by systemic evolution of ligands by exponential enrichment (SELEX) procedure 20 , 21 . Then, pairs of the aptamer candidates were used produce rolling circle amplification (RCA) products 27 – 29 . These RCA products, termed aptamer-based RCA (aptamer RCA), consist of thousands of tandemly repeated sequences of the selected aptamers to mimic the naturally occurring receptor-ligand binding more closely.…”