Development Of Fluorescent Molecularly Imprinted Polymers-magnetic Covalent Organic Frameworks Composite Materials And Their Application In The Field Of Pharmaceutical Analysis

Objectives:（1）To develop the blue carbon dots（CDs）with the properties of simple preparation method and high sensitivity by using the flowers of Osmanthus fragrans（Thunb.）Lour.as precursors.The CDs were applied for the determination of Fe³⁺,ascorbic acid（AA）in water and plasma samples.Further,the CDs were applied for the cell imaging.（2）To develop novel composite materials based on CDs and magnetic molecularly imprinted polymers（MIPs）for the fluorometric separation and detection of2,4,6-trinitrophenol（TNP）.（3）To develop magnetic covalent organic frameworks（MCOFs）with the properties of good adsorption ability.The MCOFs were applied as sorbents for magnetic solid phase extraction（MSPE）of fluoroquinolones used in the the field of pharmaceutical analysis.（4）To develop composite materials with the properites of high selectivity,high adsorption ability and high sensitivity based on the MCOFs,MIIPs and CDs.The composite materials were further used for the enrichment and analysis of trace TNP in water samples.Methods:（1）In this work,dried flowers of Osmanthus fragrans Lour.were applied as green precursors to synthesize CDs by using a green hydrothermal method.The dosage of precursors,reaction temperature,reaction time et al were optimized to obtain CDs with high quantum yields（QYs）.The morphology and functional group analysis of CDs were characterized by using TEM,FT-IR,XRD,XPS and EDS.And the optical properties of CDs were characterized by using UV-vis and fluorescence spectrum.Further,CDs were applied for the sensitivity detection of Fe³⁺and the potential fluorescence quenching mechanism between CDs and Fe³⁺were applied by using UV-vis and fuorescence lifetime analysis.Moreover,as AA can partial recovery the quenching of Fe³⁺to CDs,a fluorescent“off-on”sensor for the detection of AA were prepared.Finally,in order to explore the cytotoxicity and biocompatibility of the CDs,A549 cells were applied as research object,CDs were applied for MTT analysis and cell imaging.（2）By using anhydrous citric acid as the carbon source,AEAPMS as surface modification agent,blue CDs were prepared.Further,CDs and Fe₃O₄ as nuclear,MIPs as a specific recognition sites,CDs-magnetic MIPs were prepared by reverse microemulsion method composites（CDs/Fe₃O₄@MIPs and CDs/Fe₃O₄@NIPs）.The CD/Fe₃O₄@MIPs were characterized by TEM,EDS,FT-IR,zeta potentiometric analysis,XRD and VSM.After that,the fluorescent detection conditions of TNP detection were optimized.And the selectivity and sensitivity of polymers were also explored.Fluorescent life test and fluorescence quenching rate constants at different temperatures were further applied to explore the quenching mechanism of polymers.Finally,the composite polymers were applied to the detection and analysis of TNP in real water samples.（3）A core-shell structured MCOFs was prepared by using the Fe₃O₄ nanoparticles as magnetic core,4,4”-diamino-p-terphenyl and1,3,5-tris（p-formylphenyl）benzene as two building blocks.The MCOFs were characterized by SEM、TEM、EDS、XRD、VSM、TGA and et al to ensure the successful preparation of MCOFs.Further,the MCOFs were used as the sorbents for MSPE,the effects of sorbent dosage,extraction time,pH value,ionic strength,desorption solvent and desorption time for MSPE were investigated.By combining MSPE with HPLC-DAD analysis,a rapid and sensitive method was developed for the determination of fluoroquinolones（FQs）.And the adsorption mechanism of MCOFs to FQs was also explored.Finally,MCOFs were applied to the separation and analysis of FQs including ciprofloxacin,enrofloxacin,lomefloxacin,gatifloxacin,levofloxacin and pefloxacin in human plasma,milk and pork samples.（4）Firstly,by using2,3,5,6-tetrafluoroterephthalaldehyde and 1,3,5-tris（4-aminophenyl）benzene as two building blocks,Fe₃O₄ nanoparticles as magnetic core,MCOFs with the properties of good adsorption ability were prepared.Combined with HPLC-DAD,the static adsorption ability of MCOFs were investigated.Further,by using MCOFs as supporting materials and sorbents,MIPs as selective sorbents（TNP as template）and CDs as fluorescent sensor element,MCOFs@MIPs@CDs composite materials were prepared.The MCOFs@MIPs@CDs composite materials were characterized by SEM、TEM、XRD、VSM and et al.Moreover,the fluorescence properties of the MCOFs@MIPs@CDs composite materials were analyzed by using fluorescence method.Finally,the composite materials were applied to the analysis and detection of TNP.Results:（1）CDs with a QYs of approximately 18.53%were successfully prepared by using 2 g of flowers of O.fragrans as raw materials,240°C as the reaction temperature and 5 h as the reaction time.The TEM image of the CDs revealed that the average diameter of the CDs was about 2.23 nm,and it can be clearly seen that the size distribution was relatively uniform and the size differentiation was relatively small.The XRD pattern of the CDs indicated that the CDs contained graphite-like structures.FT-IR,XPS and XRD were applied to explore the functional groups on the surface of CDs and the corresponding element analysis,and the results showed that the structure of CDs contained C=C,NH₂,and-COOH groups,etc.The fluorescence performance analysis results showed that under the excitation of 340 nm,CDs had the highest fluorescence emission at the wavelength of 420 nm.Furthermore,the CDs were applied for the sensitive detection of Fe³⁺under optimal detection conditions.The linear response of Fe³⁺ranged from 10 nM to 50μM with a limit of detection as low as 5 nM.Furthermore,the CDs were used as a fluorescent sensor for the detection of Fe³⁺in water samples（including tap water,tuojiang river water and minjiang river water）and human serum,and the recovery range was 93.76-113.80%.In addition,the potential fluorescence quenching mechanism between CDs and Fe³⁺was identified as the inner filter effect by using UV-vis and fuorescence lifetime analysis.Moreover,the CD-Fe³⁺system can be used as a fluorescent“off-on”sensor for the detection of AA with a limit of detection of 5μM.What is more,the results of MTT experiments and cell imaging showed that CDs had almost no toxicity on A549 cells and CDs can go through cytomembrane to cytoplasm,resulting that cells emitted blue light.Hence,CDs can be used for cell imaging.Howover,the blue fluorescence of cells became very weak after incubation with Fe³⁺for 30 min.The fluorescence was partially recovered after treatment with AA for 30 min.These results demonstrated that the CDs can be used as a fluorescent on–off sensor for the qualitative analysis of Fe³⁺,and the CD-Fe³⁺system can be applied as a fluorescent off–on sensor for the qualitative analysis of AA in biological systems.（2）CDs/Fe₃O₄@MIPs composite materials were successfully prepared.The results of TEM demonstrated that the composite materials had a core-shell structure with average diameter of 200 nm.EDS results further confirmed the existence of Fe₃O₄ and CDs in the structure of composite materials,demonstrating the successfully preparation of composite materials.The results of FT-IR spectra of Fe₃O₄,CDs/Fe₃O₄@NIPs and CDs/Fe₃O₄@MIPs（after the removal of templates）demonstrated that molecularly imprinted silica is successfully grafted onto the surface of Fe₃O₄ and CDs.In addition,FT-IR spectra of CDs/Fe₃O₄@MIPs（after removal of the template）were similar to that of CDs/Fe₃O₄@NIPs,indicating that the polymerization has been successfully synthesized and the templates had been almost completely removed.The results of Zeta potential analysis,XRD analysis and VSM analysis further indicated that Fe₃O₄ and CDs existed in the polymers,further indicating the successful preparation of composite materials.Moreover,it can be easily obtained from the results of fluorescence analysis that the maximum excitation wavelength was 370 nm while the corresponding maximum emission wavelength was about 470 nm.And it had good fluorescence stability.In order to find out the best detection condition for TNP,the following parameters were optimized:（a）solvent for CDs’dispersion,（b）pH value,（c）ionic strength and（d）concentrations of CDs/Fe₃O₄@MIPs.And the best experimental conditions were found as follows:（a）solvent for CDs’dispersion is water;（b）best pH value:as different pH values have little effect on the detection results,the CDs/Fe₃O₄@MIPs can be applied to detect TNP in ultrapure water;（c）optimal ionic strength:No ionic was added.（d）optimal concentration of CDs/Fe₃O₄@MIPs is 10μg·mL^-1.Under the optimal detection conditions,the CDs/Fe₃O₄@MIPs had a good line relationship which is obtained in the concentration range of 1 nM-100μM.The LOD is found to be 0.5 nM.The imprinted factor（IF）of CDs/Fe₃O₄@MIPs and CDs/Fe₃O₄@NIPs was calculated as 3.6.The above results demonstrated that combined with molecular imprinting technique,the sensitivity and selectivity of TNP detection can be significantly improved.The results of quenching mechanism showed that TNP quenching of composite materials belonged to the static quenching.The results of selectivity experiments indicated that the quenching effect of TNP was much more pronounced than those of its analogues and metal ions,demonstrating that the composite materials had good selectivity for TNP.Finally,the CDs/Fe₃O₄@MIPs were applied to the detection of TNP in three kinds of real water samples,and the relative recoveries of TNP ranged from 89.4%to108.5%.The results indicated that the composite materials can be effectively used for the detection of TNP in real samples.（3）Various characterization indicated that the MCOFs were successfully prepared by using Fe₃O₄nanoparticles as magnetic core,4,4”-diamino-p-terphenyl and1,3,5-tris（p-formylphenyl）benzene as two building blocks.The results of morphology analysis showed that the MCOFs had relatively homogeneous spherical structure.The results of BET analysis demonstrated that MCOFs had high specific surface area(123.51 m²?g^-1)and uniform pore size distribution（the average particle size was 3.12 nm）.The results of VSM and stability experiment showed that MCOFs had good magnetic(36.8 emu?g^-1),good thermal stability and chemical stability.Further,static adsorption experiment had been applie to explore the adsorption model of the MCOFs.The results showed that MCOFs had good adsorption ability for the selected FQs(The saturation adsorption capacities for levofloxacin,pefloxacin,ciprofloxacin,lomefloxacin,enrofloxacin and gatifloxacin were calculated to be 41.8,58.5,27.5,48.9,48.3,69.9 mg·g^-1,respectively).In addition,it can be found tha the adsorption behavior of MCOFs was accorded with the Freundlich adsorption model through the analysis of the results of static adsorption experiment.Dynamic adsorption experiment showed that the MCOFs had faster dynamic adsorption performance.Further,MCOFs were used as sorbents in MSPE for the enrichment of FQs.The optimal conditions of MSPE were as followed:（a）the amount of MCOFs was 14 mg;（b）the extraction time:60 min;（c）pH of sample solution:pH=6.0;（d）desorption solvent:methanol（with 1%NH₃·H₂O）;（f）the contents of NH₃·H₂O in desorption solvent:1%,（e）desorption time:20min and（f）desorption times:3 times.Finally,MSPE was used in the analysis of FQs in blood,milk,pork samples.It can be found that this material showed a high recoveries（78.65-103.52%）,the repeatability and stability were good.The above results showed that the MCOFs can be selectively used in a variety of objects in the enrichment of FQs,and they had good application prospect.（4）By using 2,3,5,6-tetrafluoroterephthalaldehyde and 1,3,5-tris（4-aminophenyl）benzene as two building blocks,Fe₃O₄ nanoparticles as magnetic core,MCOFs were successfully prepared.Further,by using MCOFs as supporting materials and sorbents,MIPs as selective sorbents（TNP as template）and CDs as fluorescent sensor element,MCOFs@MIPs@CDs composite materials were successfully prepared.Various kinds of characterization showed that the MCOFs and MCOFs@MIPs@CDs composite materials were successful preparation.Static adsorption experiments showed that the MCOFs had good adsorption ability to TNP(166.85 mg?g^-1).Dynamic adsorption experiments showed that the saturated adsorption time of about 30 min.MCOFs@MIPs@CDs composite materials had good fluorescence performance and stability.Under the optimal detection conditions,MCOFs@MIPs@CDs composite materials showed good sensitivity to TNP,the LOD was 100 pM and the IF was8.4.Moreover,compared with MCOFs,MCOFs@MIPs@CDs had shorter detection time（1 min）.Selective experimental results further proved that the MCOFs@MIPs@CDs had high selectivity for TNP.By using fluorescence lifetime analysis,UV-vis spectra and fluorescence life quenching rate constant at different temperatures,it can be found that TNP can quench the composite materials through static quenching mechanism.Finally,MCOFs@MIPs@CDs composite materials can be quickly,accurately detected and monitored TNP in actual water samples（recoveries was 88.73%-103.43%）.Conclusions:（1）By using dried flowers of Osmanthus fragrans Lour.as precursors,CDs with blue fluorescence can be preapred by a green hydrothermal method.The CDs can be used as fluorescent sensors for the direct detection of Fe³⁺and for the indirect detection of AA effectively.Moreover,the CDs had low toxicity,good biocompatibility and had great application prospect in the field of biological imaging and even in disease diagnosis.The successful preparation and application of CDs laid a good foundation in the field of pharmaceutical analysis by using CDs.（2）By using CDs,Fe₃O₄ as co-nuclear,MIPs as specific recognition sites,new fluorescent magnetic composite materials（CDs/Fe₃O₄@MIPs）with spherical and uniform core-shell structure were prepared with good magnetic,fluorescent properties and selectivity.The preparation of CDs/Fe₃O₄@MIPs were successfully combined CDs with MIPs and the composite materials can be used in the field of pharmaceutical analysis to the high sensitivity and selective detection of TNP.（3）MCOFs with high surface area and good adsorption capacity were successfully applied as the sorbents for the MSPE of FQs.MSPE which based on MCOFs combining with HPLC-DAD had great application potential to the enrichment and detection of trace drug quickly and accurately.The preparation of MCOFs with good adsorption capacity laid the foundation for the further preparation of fluorescent-MCOFs composite materials.（4）The preparation of composite materials which based on the fluorescent CDs,MIPs and MCOFs,highly improved the selectivity and sensitivity of the TNP detection.The detection time had been shortened.The preparation of the composite materials and the related research results provided reference for the detection of trace concentrations of drugs in drug analysis,and conducived to the further development of the fluorescence analysis in the field of pharmaceutical analysis.