Determination Of Butyrylcholinesterase,Organophosphorus Pesticides And Glutathione By Mn(Ⅱ)-based Electron Spin Resonance

Electron spin resonance（ESR）spectroscopy as a highly sensitive spectroscopic technique has been widely used in many fields such as biology,environmental science,chemistry,physics,medicine and food science.Compared with conventional detection methods,ESR has the advantages of high sensitivity,being capable of direct detecting metal ions and not being limited to the sample state,and can be used for both qualitative and quantitative analysis.In this dissertation,we synthesized well-dispersed and stable MnO₂ nanosheets for the measurement of butyrylcholinesterase,organophosphorus pesticide and biological small molecule GSH with high sensitivity.（1）Based on the ESR signal of Mn²⁺,we developed a new method for detecting butyrylcholinesterase（BCh E）and organophosphorus pesticides（OPs）.MnO₂nanosheets were synthesized with manganese chloride and hydrogen peroxide.With the help of catalysis of BCh E,S-butyrylthiocholine iodide（BTCh）was hydrolyzed into thiocholine which has a reducing–SH group.In the presence of the thiocholine,MnO₂nanosheets were decomposed and Mn⁴⁺in MnO₂ nanosheets was reduced into Mn²⁺.Mn²⁺is paramagnetic ion and gives a good ESR signal.In contrast,MnO₂ nanosheets have no ESR signal and need not be separated from Mn²⁺.Mn²⁺can be determined directly by ESR spectroscopy and no further sensing probe is needed,as makes the present method much simpler than those using extra probes.The ESR signal of Mn²⁺is proportional to the catalytic activity of BCh E.Organophosphorus pesticides which inhibit the activity of BCh E can also be detected by probing the ESR signal of Mn²⁺.Since there is no ESR signal of MnO₂ nanosheets,the background signal in the absence of BCh E was close to zero.The limit of detection（LOD）of BCh E was as low as 0.042U L^-1.The standard curve for determining organophosphorus pesticide paraoxon was established by measuring the inhibition of BCh E by paraoxon,and the LOD of paraoxon was 0.076 ng m L^-1.The spiked Chinese cabbage extract samples were analyzed and the experimental results indicated that the recoveries were from 96.5 to102.8%.In order to further prove the practicability of this method,the planted Chinese cabbage was sprayed with the paraoxon solution and the residue amount of paraoxon in the extract was determined.The result obtained by the present method was consistent with that obtained by high performance liquid chromatography（HPLC）.（2）We designed a ratiometric probe based on the intensity ratio of the ESR signal of ABTS free radical(ABTS^·+)to Mn²⁺and applied it to the detection of GSH.Nanozymes are widely used because of their easy storage,low cost and high stability,thus we synthesized MnO₂ nanosheets with oxidase-like properties by redox reaction.In our designed reaction system,MnO₂ nanosheets catalyze the generation of O₂^·-from dissolved oxygen and oxidize ABTS to ABTS^·+which has a stable ESR signal.Meanwhile,GSH also undergoes a redox reaction with MnO₂ nanosheets to yield Mn²⁺with stable ESR signal,as decreases the concentration of MnO₂,inhibits the production of O₂^·-and reduces the ABTS^·+generation.Therefore,the ESR signal ratio of ABTS^·+to Mn²⁺decreases with the incarese of the concentration of GSH.The experimental results indicated that the LOD for GSH was as low as 0.18μmol L^-1.The proposed ratiometric ESR probe could reduce the influence from the background signal and improve the accuracy and sensitivity of the detection.