Fusion of binary split allosteric aptasensor for the ultra-sensitive and super-rapid detection of malachite green

The complicated labeling procedure and high cost of split aptasensors have hitherto limited their application in the detection of hazardous substances. Herein we report the first examples of label-free aptasensors based on the fusion of a binary split G-quadruplex and malachite green (MG) aptamer, transducing recognition events into fluorescent signals through the allosteric regulation of the aptamer to achieve selective and sensitive detection. Specifically, RNA MGA was successfully converted into DNA MGA with comparable affinity and improved stability, thereby overcoming the limitations of poor stability and high expense. We subsequently split the DNA MGA and attached them to a G-rich DNA sequence at the 5' and 3' ends, to construct the binary split allosteric aptasensor. The performance of the binary split aptasensor for MG detection was significantly improved based on proposed allosteric regulation strategy, and the reconfiguration capability of the aptamers upon binding with targets was proven, providing the binary split aptasensor with superior sensitivity and selectivity. This sensing method has a wide dynamic detection range of 5 nmol·L^-1 to 500 μmol·L^-1, with a low limit of detection (LOD) of 4.17 nmol·L^-1, and achieves the ultra-sensitive and super-rapid detection of MG. This newly proposed aptasensor is equipped with the advantages of being label-free, time saving and economical. More importantly, this successful construction of a fused aptasensor expands the principles of split aptasensor design and provides a universal platform for the detection of environmental contaminants.