Computer-Aided Design Of Molecularly Imprinted Polymers Of Clenbuterol And Its Metabolites

Since the beginning of this century,there have been several food safety incidents caused by the large-scale illegal addition of“clenbuterol”,which has caused widespread concern in the society.“Clenbuterol”is usually referred to as Clenbuterol(CLB)in China.In view of the current lack of CLB rapid detection methods,this study attempts to develop molecularly imprinted sensors for rapid detection of CLB and its metabolites,providing new ideas and technical accumulation for the rapid detection of CLB.By studying the metabolic pathway of clenbuterol in mice and existing results of the laboratory,in addition to the CLB,phenylephrine(PE),4-Aminohippuric acid(AHA),4-Hydroxymandelic acid(HMA)were taken as dummy template molecule for CLB,3,5-Dichloro-4-Amino Mandelic acid and 3,5-Dichloro-4-Aminohippuric acid,respectively.Computer-aided design of molecular imprinting systems were performed using the quantum chemistry software Gaussian.Based on the DFT density functional theory,the imprinting systems using CLB,PE,HMA and AHA as template molecules were simulated at B3LYP/6-31+G(d,p)level.Steady-state of template-monomer complexes were constructed based on the MEP electrostatic potential map and the NPA charge distribution.The results showed that the best functional monomer was MAA,and the optimum template/monomer imprinting ratio was 1:3,1:3,1:5,1:5,and the crosslinker was selected as EDGMA.The aprotic solvent showed lower solvation energy under implicit solvent model and thus,was more suitable for the designed molecular imprinting systems.Further AIM analysis of the wave function information of the simulated molecularly imprinted complexes illustrated the chemical nature of the imprinting process and facilitated a better understanding of the imprinting process.The selectivity simulations of designed molecular imprinting systems theoretically verified the specificity of each molecularly imprinted cavities.MIPs with CLB,HMA and AHA as template molecules were synthesized on the surface of QCM gold electrode by surface imprinting method.The surface morphology of the synthesized MIP film was observed by atomic force microscopy,which confirmed the successful polymerization of the MIP film on the surface of the gold electrode and the roughness was measured to be about 53 nm.The film thickness was measured by ellipsometry,and the measured data was 31 nm.The quality factor Q of the MIP-QCM sensors were measured by an impedance analyzer.The results showed that the Q factor of the QCM sensors used in this experiment could reach more than 140,000,meeting the requirements of subsequent testing.After successfully preparing the sensors,the performance of a single sensor of CLB-MIP-QCM,HMA-MIP-QCM and AHA-MIP-QCM was tested.The outcome showed that the signal response value of the sensor at10 min had a good linear relationship with the negative logarithm(-lg C)of the standard sample solution concentration prepared by its corresponding template molecules.The R² values of the fitted linear equations were respectively 0.9935,0.9918,and 0.9927.The repeat performance of MIP-QCM was calibrated after 20 consecutive tests.It was found that the number of extreme reuses was up to 17 times.Three sensors were built into an array to test the mixed standard solution,when the collected array signals were processed by multiple linear regression,the signal data showed serious collinearity problems.Although the problem of collinearity had been proven to be overcome via the usage of ridge regression method,which is a biased estimation method by abandoning partial information,partial least squares regression(PLS)method was tried to process the signal data.The calibration curve was established by PLS method integrating principal component analysis and correlation analysis,which realized accurate quantification under standard laboratory conditions.The calibration model was y=-13.658+0.91x₁-0.37x₂-0.74x₃,R²=0.999.