Target-triggered parallel rolling circle amplification circuits for chemiluminescent imaging assay of proteins

This work designed a strategy of target-triggered parallel rolling circle amplification circuits (PRCAC) for chemiluminescent (CL) imaging assay of proteins. The PRCAC was performed in a homogeneous system containing a pair of affinity probes, a predesigned Block/initial-Primer (Block/iPrimer) hybrid and a tailored circular-DNA/cyclic-Primer hybrid (cDNA/cPrimer). In the presence of target protein, the iPrimer was firstly released through target-initiated proximity binding-induced strand displacement to recognize cDNA/cPrimer, which led to an iPrimer-activated rolling circle amplification (iRCA) and the release of cPrimer to recognize cDNA/cPrimer for triggering self-feeding multiple cPrimer-activated RCAs. The PRCAC could generate numerous G-quadruplex units for forming DNAzyme, which catalyzed the oxidation of luminol by H2O2 to produce strong CL signal, and thus led to an amplified CL protein imaging method. This method could detect both antigen and antibody through changing the specific affinity probes. Using NT-proBNP and anti-PCSK9 antibody as analytes, this method exhibited the linear ranges of 0.01-10 pg mL-1 and 0.1-1000 pg mL-1, respectively. The low limits of detection, excellent specificity, simple operation, relatively short analytical time and acceptable reliability demonstrated the applicability of the PRCAC in biomarker analysis.