Design And Application Of Molecular Imprinting Based Ratiometric Fluorescence Sensing System For Proteins Analysis

Molecular imprinting technique has been widely used for the detection of ions,small molecules and macromolecular proteins due to its predetermined structure-activity and selectivity to target substances in the detection process.Among the methods for synthesizing imprinted polymers,the sol-gel process and the surface imprinting technique are diffusely used for their remarkable advantages.The abrasion resistance and physical rigidity of porous materials synthesized by sol-gel process are high.In addition,the preparation method of the sol-gel process is simple and can be carried out even at low temperature,which greatly reduces the influence of temperature on the activity of proteins and is beneficial to the imprinting of proteins.Using the surface imprinting technique to synthesize imprinted polymers can reduce the migration resistance and increase the binding force of template molecules.This is beneficial to the elution of template molecules,and effectively avoids the problem that template molecules are not easily eluted and leaked due to the deep entrapment.As the recognition site is at or near the surface of the imprinted polymer,the detection efficiency to targets isgreatly improved.Fluorescence sensing technique is widely applied to various fields due to its sensitive response and fast analysis speed to the target substance.The advantages of the ratiometric fluorescence sensing system are particularly outstanding.Compared with the single fluorescence emission peak system,the ratiometric fluorescence sensing system can effectively reduce the interference of external factors,which could improve the accuracy of test results by introducing reference substances as a control.In this thesis the ovalbumin,bovine hemoglobin and Cu(?)ions were detected by ratiometric fluorescence sensing system.(1)Preparation of ovalbumin molecularly imprinted polymer post-modified by fluorescein isothiocyanate and detection of ovalbumin.The work was carried out in two steps.First,the ovalbumin(OVA)molecularly imprinted polymer was synthesized by sol-gel process,and then fluorescein isothiocyanate(FITC)was post-modified on the surface of the polymer.The post-modification method,performed in mild reaction conditions,is beneficial to protect the activity of OVA.Meanwhile this method enhances the sensitivity of FITC to OVA.Finally,ratiometric fluorescence sensing system was constructed by adding carbon quantum dots as reference substance in an appropriate proportion.OVA has hydrophobic regions,which can change the environment around FITC and change its fluorescence intensity.The imprinted polymer has specific recognition sites of OVA.The imprinting factor(IF)was 2.5.After adding of ovalbumin to the fluorescence sensing system,the fluorescence intensity of MIP@FITC at 520 nm was enhanced,while the fluorescence intensity of carbon dots at 454 nm was almost unchanged.The fluorescence intensity ratio of two peaks showed good linear relationship with the concentration of ovalbumin in the range of 0.05~2 ?mol/L.The detection limit was 15 nmol/L and the linear correlation coefficient was 0.9972.Under ultraviolet light,the color of the test strip exhibited a distinct change from blue to green with increasing concentration of OVA.In the experiment of the actual samples of human urine,the recovery rates were between 92~104%,and the relative standard deviations were in the range of 3.3~3.9 %.The proposed method had good selectivity and practical application prospects for ovalbumin detection.(2)Preparation of bovine hemoglobin imprinted polymer with double fluorescence emission peaks and detection of bovine hemoglobin.An imprinted polymer system with composite fluorescent materials as the ratiometric fluorescent signal was constructed.Firstly cadmium telluride quantum dots(CdTe QDs)were embedded in silica and modified by 3-aminopropyltriethoxysilane(APTES),which changed them from negative charge to positive charge.Then it was grafted onto the surface of negatively charged graphene(GQDs)by electrostatic interaction to synthesize a composite fluorescent material with dual fluorescence emission peaks.The fluorescences of CdTe QDs and GQDs respectively are red and blue.Bovine hemoglobin molecularly imprinted polymers(BHb-MIPs)were prepared by sol-gel method with composite fluorescent nanomaterials as the substrate.The imprinting layer was located on the surface of BHb-MIPs,contained a large number of specific recognition sites consistent with bovine hemoglobin(BHb)in size,shape and functional groups.Therefore,the BHb-MIPs could recognize BHb rapidly and efficiently.When different concentrations of BHb solutions were added,the fluorescence intensity at 660 nm was regularly quenched,while the fluorescence intensity at 460 nm fluctuated little.The fluorescence intensity ratios of two peaks showed good linear relationship with the concentrations of BHb in the range of 0.05~4 ?mol/L,with the detection limit of 25 nmol/L.Under UV lamp,the color of the solution exhibited a significant change from red to blue.The system had good selectivity for BHb.showing a good application prospect in the test of human urine samples.The relative standard deviations were in the range of 3.1~4.2 %.The recoveries were between 95~101 %.(3)Functional ZnS:Mn(?)quantum dot modified with L-cysteine and 6-mercaptonicotinic acid as a fluorometric probe for copper(?)Manganese-doped ZnS quantum dots(ZnS:Mn(?)QDs)were synthesized and modified with L-Cys and MNA.This prevents the aggregation of the QDs and makes them available for the interaction with Cu(?)ions via Cu(?)-S interaction.As a result,the fluorescence of the QDs is quenched by Cu(?)due to an electron transfer mechanism.The QDs display two emission peaks under 325 nm excitation,one(being red)peaking at 593 nm,the other(blue)at 412 nm.The red fluorescence is strongly quenched,while the blue fluorescence is not affected.An easily distinguishable color change from orange red to purple can be observed in fluorescence as the concentration of Cu(?)is increased.The probe was selective over commonly encountered other ions.The ratio of fluorescence intensities at 593 and 412 nm increased linearly in the 5~500 nmol/L Cu(?)concentration range,and the detection limit was 1.2 nmol/L.