Preparation Of Magnetic Molecularly Imprinted Polymers And Gold Nanoclusters And The Application In Sensing Aristolochic Acid Ⅰ

Objectives:1.Aristolochic acids I（AAI）,a kind of nitrophenanthrene carboxylic acid,which is found primarily in Aristolochiaceae plant including Aristolochia and Asarum species.AAI is a toxic compound that can cause a variety of diseases,such as kidney failure and cancers.Although the current detection strategy for monitoring AAI has been reported,the application of these methods is limited.The existing technical methods focus on the concentration of AAI in herbal remedies and botanical products,only limited methods have been reported for its direct detection in biological samples.As a result,further exploration to optimize the detection is urgent required.In this paper,molecularly imprinted polymers were prepared by using molecularly imprinted technology for rapid enrichment of AAI,which simplified the steps of pre-treatment and reduced the time cost of determining AAI by HPLC detection.2.Protein was introduced as templating agent to the synthesis of gold nanoclusters,and a novel detection strategy was constructed based on the linear relationship between the concentration of AAI and the fluorescence intensity of gold nanoclusters for the rapid,easy and effective sensing of AAI.Methods:1.Molecularly imprinted polymers for AAI were successfully prepared by surface molecular imprinting method using functionalized Fe₃O₄ nanoparticles as the magnetic materials.In this work,some essential factors such as functional monomer,desorption solvents and the molar ratios of template-functional monomer-crosslinker were investigated to achieve high binding properties with efficient recognition.Scanning electron microscopy and fourier transform infrared spectroscopy were applied to characterize the magnetic molecularly imprinted polymers.The adsorption capacity of the molecularly imprinted polymers for AAI were confirmed by static adsorption experiments and dynamic adsorption experiments.2.Gold nanoclusters were synthesized by utilizing bovine serum albumin as reducing agent and stabilizing agent with a one-pot procedure.The optical properties and morphology of the gold nanoclusters were characterized by fluorescence spectrophotometer,ultraviolet spectrophotometer,fourier transform infrared spectrometer and transmission electron microscope.To obtain the sensitive response for AAI,the parameters affecting the fluorescence performance were optimized including types of solvent,p H value,detection temperature and detection time.As the fluorescence intensity at the maximum emission wavelength of gold nanoclusters can be quenched by AAI,the calibration curve was conducted based on the concentration of AAI and the quenching degree of gold nanoclusters.The present approach was successfully applied in sensing AAI in the rat urine samples.Results:1.An imprinted layer was successfully formed on the surface of Fe₃O₄ after modification,and the obtained molecularly imprinted polymer maintain the inherent properties of the magnetic element.The experimental research showed that the optimal preparation condition was that AAI as the template,methacrylic acid（MAA）as the functional monomer,2,2-azobisisobutyronitrile（AIBN）as the initiator,ethylene glycol dimethyl acrylate（EGDMA）as the crosslinking agent and acetonitrile-methanol（3:1,v/v）as the porogen.In addition,the molar ratio of template,functional monomer and crosslinking agent was 1:4:20.The imprinting factor of the prepared magnetic molecularly imprinted polymer is 3.11.2.The prepared gold nanoclusters show deep brown in daylight and exhibit red fluorescence under UV light（365nm）.In this paper,the synthesized gold nanoclusters with an average diameter of 7 nm,and exhibited a strong fluorescence emission at around 660 nm（with an excitation wavelength of 328nm）.The fluorescence of the system can be quenched after the combination of AAI and gold nanoclusters in phosphate buffer solution with p H of 7.4.The standard curves of gold nanoclusters towards AAI was defined as follows:F₀-F/F₀=0.02194C_AAI+0.02848（correlation coefficient was 0.9901）,and the detection limit for AAI was 0.08μg/m L at a signal-to-noise ratio of 3.This method is suitable for the detection of AAI in rat urine samples,showing recoveries ranging from 96.1%to 120.9%and the detection can be completed quickly within10 s.The result of fluorescent probe detection was in consistent with that from the HPLC method（recoveries ranging from 104.9%to 130.2%）.Conclusions:1.In present study,the prepared novel magnetic molecularly imprinted polymers contain more specific recognition sites,thus,the polymers were able to perform high affinity and selectivity for AAI.These results demonstrated that the prepared magnetic molecularly imprinted polymers were proven to be an effective material for the enrichment of AAI in real samples.2.A quantitative detection method for AAI was developed based on the obtained gold nanoclusters and the development of effective,easy-to-operate and without any derivatization detection methods for AAI showed satisfactory results.3.In this work,the detection strategies for AAI were explored based on the optimization of pre-treatment steps and establishment of a new method,which provided a new sight for the detection of AAI in other materials and had important significance for realizing the modernization of Chinese medicine detection.