Enzymatic reaction modulation of G-quadruplex formation for the sensitive homogeneous fluorescence sensing of cholinesterase and organophosphate pesticides

In this work, we present the reported T-Hg2+-T hairpin structure and enzymatic reaction-modulated formation of the G-quadruplex, which is further employed for the fluorescence assays of butyrylcholinesterase (BChE) and organophosphate pesticides (OPs, parathion) in real samples. BChE can catalyze the reaction of acetylthiocholine (ATCh) to produce thiocholine (TCH), which has a thio-ligand to combine with mercury ions (Hg2+) in T-Hg2+-T. The released single-stranded DNA can associate with N-methyl mesoporphyrin IX (NMM) and K+ to form a G-quadruplex, producing a strong fluorescence signal. Therefore, the concentration of the released probe DNA was proportional to the amount of BChE, enabling the conversion of the target reaction events into a detectable signal from the G-quadruplex-NMM. Parathion, as an inhibitor of BChE activity, can be used to prevent the generation of thiocholine and therefore leads to less or no release of free probe DNA. As a result, most of the probe DNA maintains its T-Hg2+-T hairpin structure in the reaction solution, which leads to a significant fluorescence signal decrease. Therefore, by using the signal-off mode, the simple and homogeneous fluorescence detection of OPs is readily achieved. Under optimal conditions, the detection limits of BChE and OPs are 0.15 ng mL(-1) and 0.025 ng mL(-1) respectively. Besides, BChE in serum samples has been detected with satisfactory results. The proposed method retains several unique advantages, including being simple, cost-effective, highly sensitive and selective for label-free analysis.