A universal electrochemical sensing system for small biomolecules using target-mediated sticky ends-based ligation-rolling circle amplification

A novel versatile electrochemical platform for ultrasensitive detection of small biomolecules was developed using ligation-rolling circle amplification (L-RCA) with analyte-mediated sticky ends. In order to achieve DNA cyclization, we designed two ss-DNA probes: the leftpart probe could form a “hairpin” structure by denaturing; the rightpart probe could also form a “hairpin” structure based on analyte-activated conformation change. Then the two probes with the same sticky ends (G-AATTC) could be ligated in the presence of Escherichia coli DNA ligase, forming a circular template for rolling circle amplification (RCA), which could be triggered by adding the primer probe and Phi29 DNA polymerase. Electrochemical impedance spectroscopy (EIS) was employed as the detection method. Overall, the proposed L-RCA-based sensing system not only exhibits excellent analytical characteristics with a detection limit of 320 pM and a linear range of 5 orders of magnitude (1 nM-10 μM), but also provides a universal design idea of L-RCA, which broadens the use of DNA amplification method and holds great promise in ultrasensitive bioassay in the future.