Proximity Ligation Assay with Three-Way Junction-Induced Rolling Circle Amplification for Ultrasensitive Electronic Monitoring of Concanavalin A

A new signal amplification strategy based on target-induced proximity ligation assay accompanying three-way junction-based rolling chain amplification was designed for ultrasensitive detection of concanavalin A (Con A) by coupling with a sequential injection mode. To construct such a proximity ligation assay system, two types of magnetic sensing probes including glucosamine/DNA1-conjugated magnetic bead (GA-MB-DNA1) and glucosamine/DNA2-labeled magnetic bead (GA-MB-DNA2) were first synthesized and prepared through a typical carbodiimide coupling. In the presence of target Con A, GA-MB-DNA1 and GA-MB-DNA2 were ligated together based on the interaction between Con A and the conjugated glucosamine on the MB, thereby resulting in the formation of a three-way DNA junction because of partial base pairing on the DNA1/DNA2. With the aid of ligase and polymerase, the formed three-way DNA junction could be used as the primer to produce numerous repeated oligonucleotide sequences through rolling circle amplification (RCA) reaction. The formed long oligonucleotide strand could cause the intercalation of numerous positively charged methylene blue molecules with a negatively charged DNA backbone. During the electrochemical measurement, each of the intercalated indicators could produce an electrochemical signal within the applied potentials, resulting in the amplification of detectable electronic signal. By monitoring the change in the signal, we could indirectly determine the concentration of target Con A in the sample. Under the optimal conditions, the developed sensing platform exhibited high sensitivity for detection of Con A with a wide dynamic range of 1.96 pM to 98 nM and a low limit of detection (LOD) of 1.5 pM at the 3sB level. Intra-assay and interassay coefficients of variation were less than 8.9% and 9.7%, respectively. In addition, the methodology was validated by assaying Con A spiked samples including newborn cattle serum and peanuts, and the recovery in all cases was 88.8–134.7%.