A ratiometric fluorescent aptamer assay based on RCA amplification mult-G-quadruplex

Ratiometric assay with dual signals can effectively avoid false positives of single signals. Therefore, a dual-signal assay was designed to detect Aflatoxin B1 (AFB1) using the affinity of the aptamers to the target as well as the binding force between the DNA strands. The first fluorescent signal was obtained by binding AFB1 to aptamer to shed the complementary sequence with the FAM (6-carboxyfluorescein). The G-quadruplex is a common DNA conformation capable of emitting light with special fluorescent dyes. At room temperature, the G-quadruplex is opened by the shed complementary strand to form double-stranded DNA, disrupting its original structure. The NMM (N-methylmesoporphyrin IX) dye is unable to embed the G-quadruplex and emit light. Taking advantage of the DNA conformational transition, NMM dye was introduced as the second fluorescence signal. Based on the above principle, a dual fluorescence signal ratiometric assay was constructed for the detection of AFB1 by the ratio of FAM and NMM fluorescence intensity. This study provides a prospective strategy for developing a dual-signal construct ratio assay.