Rolling Circle Amplification Immunoassay Combined with Gold Nanoparticle Aggregates for Colorimetric Detection of Protein

A highly sensitive and novel colorimetric rolling circle amplification (RCA) immunoassay for detecting C-reactive protein (CRP) has been developed. In the assay, a CRP capture antibody was immobilized on magnetic beads and a CRP detection antibody was conjugated with single-stranded DNA (ssDNA) using N-[epsilon-maleimidocaproyloxy] sulfosuccinimide ester. Along with the addition of CRP, a sandwich structure was formed. Subsequently, the ssDNA was used as a primer to initiate the RCA reaction in the presence of the circular template, phi29 DNA polymerase and deoxynucleotide triphosphates. The RCA product obtained by magnetic separation, and long tandem repeated sequences mediated the aggregation of gold nanoparticles (AuNPs), which could be observed by the naked eye or quantified using absorption spectra with a detection limit of 30 fg mL(-1) and a linear response range from 10 ng mL(-1) to 1 pg mL(-1). This assay offers the advantages of isothermal conditions, low cost and label-free quantification that could be hopeful for ultrasensitive and robust visual protein detection.