An ACRCA-microchip integrated with immune-reaction and RCA for trace norovirus antigen detection

Protein detection plays a crucial role in early disease diagnosis and therapeutic efficacy assessment. However, it has consistently faced the challenge of single-molecule level sensitivity, lacking signal amplification capability like nucleic acid. Herein, we establish an extremely strong antibody-coupled rolling circle amplification (ACRCA) strategy based on electrochemical microchip platform. This approach achieves specific antigen-antibody detection signals on the single-molecule level by exponentially amplified RCA in a one-tube reaction, which are then quantitatively read by a sensitive electrochemical microchip. To validate the performance, we applied this technique for super high-sensitive detection of foodborne norovirus (NoV) antigen in a one-tube assay. Surprisingly, we discovered that the linear detection range was from 10 fg/mL to 100 pg/mL, and the detection limit was 10 fg/mL (0.169 fM). Furthermore, the NoV can be obviously distinguished from other foodborne viruses (HAV, HEV and RV). The ACRCA-microchip or upgraded version (for high-throughput detection) has great potential for disease diagnostics, antibody screening and organoid research.