Studies On Molecular Imprinting Technology For The Analysis Of New Psychoactive Substance Cathinones

Background:Nowadays,illicit drug abuse is still a serious worldwide problem.With the development of society and science and technology,the world drug problem is also undergoing a series of significant problems:the non-medical use of synthetic opioids has aggravated the public health crisis,the increasing use of stimulants,the increased risk of injecting drug users due to COVID-19,the signs of continued expansion of the amphetamine type stimulant market,and the continuous change of routes and production processes by producers/traffickers and repeated prohibitions.The rapid change of substance abuse and the changing trend of drug abuse undoubtedly pose a difficult and multifaceted challenge to the supervision of substance abuse.At present,the method used to detect trace target in matrix is expensive and time-consuming,which greatly limits its application and popularization.In addition,rapid,accurate and low-cost identification of trace substances of abuse is also a hot and difficult point in forensic research,which is particularly urgent and important for monitoring and evaluating the drug situation in China.Fluorescent biosensors based on nanomaterials are characterized by rapidity,sensitivity,strong anti-interference ability and visualization,and have unique advantages for quantitative detection and functional analysis of complex systems.In recent years,molecular imprinted polymers（MIPs）technology as a new probe construction technology has made new progress;It has been successfully applied in many analytical fields due to its advantages such as predetermined structure and activity,strong specificity recognition,wide applicability,simple preparation process and high stability.The combination of fluorescent nanomaterials and molecular imprinting technology not only gives full play to their advantages,but also opens up new ideas for their application in forensic toxicology analysis and detection.Objective:Develop magnetic fluorescence probe based on Fe₃O₄and carbon quantum dots,as well as the combination technology of molecular imprinting magnetic solid phase microextraction materials and LC-MS/MS,to verify its analytical and detection performance for trace methcathinone in sewage;Based on Cd Te and MgF₂quantum dots,a ratio fluorescence probe was synthesized by molecular imprinting technology for the analysis and detection of N-Ethylpentylone in forensic medicine.Methods:（1）the high-quality magnetic Fe₃O₄NPs employed to as the carrier of the fluorescent signal molecule and magnetic collecting functional body were synthesized by high temperature coprecipitation approach followed by the addition of TEOS to isolate Fe₃O₄NPs.Subsequently,the methcathinone（template molecule）and Si-CDs（fluorescent signal molecule）were uniformly imprinted on the surface of Fe₃O₄NPs via sol-gel polymerization in the presence of APTES,TEOS and NH₃·H₂O which was used as functional monomer,crosslinker,catalyst respectively.After the templates were eluted,the cavities with high specificity and affinity to methcathinone were exposed and the construction of the final MFMIPs probe was completed.Among which,the Fe₃O₄NPs can make the probe realize recycling and reuse under the help of external magnetic field.The experimental conditions were optimized to achieve the ideal detection effect,and methcathinone was used in the detection and analysis of real sewage samples to verify its effect.（2）Nano-sized magnetic Fe₃O₄particles were synthesized by high temperature coprecipitation method.With the assistance of functional monomers,cross-linkers and catalysts,methcathinone template molecules were imprinted on the surface of diamagnetic particles.After the imprinting,the template molecules were eluted to prepare magnetic molecularly imprinted polymer materials（MMIPs）for methcathinone extraction in sewage.Due to the strong magnetic properties of Fe₃O₄particles,the target was enriched and eluted rapidly by magnetic solid-phase microextraction of MMIPs.The concentrated pretreated samples were combined with LC-MS/MS method for trace detection of methcathinone in sewage.Meanwhile,non-imprinted polymers（MNIPs）were prepared for comparative experiment.（3）1-[3,4-（methylenedioxy）phenyl]-2-ethylamino-1-pentanone（N-Ethylpentylone,NEP）was used as a template molecule for the synthesis of Cd Te coated with MIPs（Cd Te@MIPs）by sol-gel method.Due to the electron transfer between the NEP molecule and Cd Te@MIPs,the Cd Te@MIPs covered by the specific recognition cavity can bind to the template molecule NEP and lead to fluorescence burst.Meanwhile,the bare MgF₂was used as the internal standard reference signal to improve the stability of the method;the fluorescence signal remained stable due to the lack of specific recognition cavities on the surface of MgF₂,making it almost unresponsive to NEP.Therefore,by integrating the combination of Cd Te@MIPs and NEP,a ratiometric probe with low cost,high sensitivity and high precision with excellent performance was developed for the analytical study of NEP detection in wastewater.Results:（1）A multifunctional magnetic fluorescence MFMIPs probe was developed for quantifying methcathinone by anchored fluorescent MIPs-CDs on the surface of Fe₃O₄NPs.The proposed probe exploits the inherent high specific recognition of MIPs combining with the magnetism of Fe₃O₄NPs and fluorescent signal of CDs.Under optimal condition,the developed magnetic MFMIPs probe could be applied for the direct detection of methcathinone,showing a good linearity in the range of 0.5 to100 n M with detection limit at 0.2 n M compared to interferents.Especially,the proposed probe would be recycled under external magnetic field and reused at least five times.（2）The synthesized magnetic Fe₃O₄molecular imprinted nanomaterials MMIPs have abundant imprinted recognition sites and can bind methcathinone well.The prepared nanomaterials have short adsorption equilibrium time（t=20 min）,high adsorption capacity（P=3.12 mg/g）,good selectivity,and can achieve rapid magnetic separation from sewage samples（about 60 seconds）.The developed new psychoactive substance（methcaoxone）of MMIPs showed significant advantages such as shorter operation time（40 min from sample processing to result）,lower organic solvent consumption,and more environmentally friendly when compared to the enrichment and elution of conventional SPE materials.In addition,the established M-SPE method based on MMIPs has been successfully applied to the analysis of methcazenone in spiked wastewater samples,with good linearity,high recovery and precision,low detection limit and limit of quantification.MMIPs combined with LC-MS/MS was used for the specific separation and detection of methcathidone in Xiangjiang River water,daily tap water and sewage collected from Yuebeiyuan Sewage treatment pump station in Yuelu District.The results show that the measured results of the M-SPE/LC-MS/MS method are consistent with those of the traditional methods.（3）In the developed dual-emission probe,Cd Te@MIPs were used as the receptor and response unit for NEP,and MgF₂was used as the reference signal to improve the stability of the method.Using the ratiometric sensing technique,a good linear response to zebularine was achieved in the range of 0-200 n M with an LOD of0.6 n M;Cd Te@MIPs/MgF₂showed significant selectivity for NEP compared with other analogues or interferents,including river methamphetamine,methcathinone,ketamine,cathinone,morphine,cocaine,etc.In addition,the method was detected by spiked analysis of wastewater samples,and the recoveries of NEP were96.12%-105.05%with RSDs of 3.59%-4.33%.Conclusion:In this study,three different detection methods were developed based on molecular imprinting technology:Fe₃O₄and carbon quantum dots magnetic fluorescence probe,magnetic molecular imprinting solid-phase microextraction-liquid chromatography/tandem mass spectrometry and Cd Te@MIPs/MgF₂ratio fluorescence probe,and were used for the detection and analysis of methcathinone and cantharidin,respectively.Compared with traditional methods,the three developed methods not only improve the sensitivity of detection,but also greatly reduce the detection cost（including equipment firmware,labor costs,etc.）,which can meet the daily identification needs of forensic toxicological analysis.In addition,this research combines theory with practice,seeking ideas from the interdisciplinary integration;The combination of magnetic nanomaterials and molecular imprinting technology in the application of toxicological analysis and detection not only has great potential and good application prospects,but also opens up the idea of new technologies and materials in forensic research.