Colorimetric aptasensor for the detection of mercury based on signal intensification by rolling circle amplification

Techniques that are sensitive to detect mercury ion (Hg²⁺) are very important, due to its serious threat to public health and food security. In this work, a colorimetric aptasensor was fabricated for the detection of Hg²⁺ based on rolling circle amplification (RCA). The aptamer was immobilized onto the microplate and hybridized with its complementary strand (cDNA₁) which linked with a primer for triggering the RCA reaction of circular template. The successfully RCA process led to the formation of long ssDNA chains on the microplate, which created many hybridized DNA fragments for bio-cDNA₂. The tagged amount of horseradish peroxidase (HRP) was enhanced through the avidin/biotin binding between avi-HRP and bio-cDNA₂. In the addition of TMB-H₂O₂, HRP was catalyzed and generated an optical signal. However, in the presence of target, Hg²⁺ specifically and preferentially bound with aptamer and formed a strong and stable T-Hg²⁺-T complex, which led to the release of cDNA₁ and HRP cluster. Consequently, the optical signal decreased. Our results showed that the limit of detection (LOD) of this system was 1.6 nM with excellent specificity, and that the detection signals were enhanced by up to 18 times under RCA conditions when compared with detections without RCA. This method has been successfully used to detect Hg²⁺ in water samples with a recovery of 98%-105.74%.