Multi-DNAzymes-functionalized gold nanoparticles for ultrasensitive chemiluminescence detection of thrombin on microchip

Highlights: • Ultrasensitive chemiluminesence assay realized by dual signal amplification strategy. • Multichannel microchip increased reaction efficiency and reduced detection time. • Thrombin in serum could be sensitively, selectively and rapidly determined. - Abstract: Herein, a chemiluminescence assay with dual signal amplification has been developed based on multi-DNAzymes-functionalized gold nanoparticles (AuNPs) using in situ rolling circle amplification (RCA) for ultrasensitive detection of thrombin on microchip. In this assay, AuNPs was functionalized by aptamer and multi-RCA primer for amplification, and thrombin was sandwiched between the aptamer modified on the microchannel and the aptamer linked AuNP. The further amplification was realized by in situ RCA to expand specific oligonucleotides chains on the AuNPs and produce particular multi-DNAzymes. Enhanced chemiluminescence signal was achieved by the catalytic effect of DNAzymes in the luminol-H₂O₂ system. The sensitivity of detection was greatly improved by the dual amplification of multi-RCA primer modified AuNPs, and RCA. The whole strategy was applied for ultrasensitive and specific detection of thrombin. The chemiluminesce assay of thrombin performed a good linear range of 1–25 pM and the limit of detection was as low as 0.55 pM. The successful determination of thrombin in real human serum sample indicated a great potential in clinical study.