| Molecular imprinting technology is a technology that uses molecularly imprinted polymers(MIPs)to simulate the interaction between enzyme-substrate or antibody-antigen to specifically identify imprinted molecules(also called template molecules).Based on the advantages of simple preparation,good stability and tolerance to harsh environment,MIPs have been widely reported as a template molecular specific recognition element for the preparation of high-performance electrochemical sensors.In recent years,the main research direction in the field of molecular imprinting is to combine this technology with other materials or analytical methods to develop novel sensors with better sensitivity and selectivity.However,since the functional monomers in the MIPs are mainly the small molecule compounds,which have fewer sites to interact with the target molecules,due to various reasons,such as swelling deformation of recognition sites,the specificity of MIPs decreased,which greatly limited the application of molecular imprinting materials.Therefore,the development of functional monomers that can interact with template molecules at multiple sites can create conditions for the preparation of highly specific molecularly imprinted electrochemical sensors,Since the groups of recognition template molecules in natural antibodies are all on certain rigid peptide chains,if a rigid chain structure is introduced into the imprinted polymer and a certain amino acid structure is introduced into the chain structure,it may form an effect similar to antibody recognition of antigen.In this paper,chitosan oligosaccharide was used as a precursor,and a small rigid peptide chain structure(-CONHCH(R)-CONH-)was formed by introducing amino acid units into the amino group of chitosan oligosaccharide,so that the molecular chain of chitosan oligosaccharide with rigid structure was in the "T" shape cross in space.Thus,the polar groups,peptide-chain amide groups and flexible residues of amino acid side chains on chitooligosaccharide form a structural unit with multi-site action on a specific template molecule.Since functional monomers in MIPs need to be involved in polymerization,three different types of N-acylated chitooligosaccharides were designed according to the hydrophobicity of template molecules:for hydrophilic template molecules(e.g.,ribavirin),as long as they can construct MIPs from pyrrole,the corresponding design was N-(pyrrole-l-propionyl)-amino acyl-chitooligosaccharide;for weak hydrophilic template molecules(e.g.,chloramphenicol)and hydrophobic template molecules(e.g.,curcumin,bisphenol A),due to the need to initiate polymerization in solvent,the corresponding design was N-(4-pentenyl)-amino acyl-chitooligosaccharide;for template molecules with higher hydrophobicity(take Sudan I as an example),due to their low solubility in the polymerization solution,in order to increase the number of imprinted sites in the polymer,N-(4-pentenyl(lipoyl))-amino acyl-chitooligosaccharide,which can self-assemble on the surface of gold nanoparticles(AuNPs),and enrich the template molecules in the polymerization solution by its larger effect on template molecules,was designed.In order to verify the feasibility of this design,amino acids suitable for each template molecule were screened by quantitative calculation,and functional monomer oligomers(FMO)with strong molecular interaction with the template molecule were constructed.In the construction of molecularly imprinted electrochemical sensor,N-(pyrrole-1-propionyl)-amino acylchitooligosaccharide and pyrrole were used for electrochemical copolymerization for hydrophilic template molecules,and a novel bi-functional monomers molecularly imprinted membrane electrochemical sensor with improved sensitivity and specificity was obtained;for weak hydrophilic and hydrophobicity template molecules,the double polymerization process was designed in the sensor fabrication,that is,the bulk polymerization formed the primary imprinted film,following the electrochemical secondary polymerization,the advantages of bulk polymerization and electrochemical polymerization were combined to obtain the electrochemical sensor of composite molecular imprinted film with high sensitivity and specificity.The main contents and results are as follows:(1)For hydrophilic template molecules,pyrrole is often used as a functional monomer for direct electrochemical polymerization of molecularly imprinted sensors.However,because polypyrrole itself has a rigid chain structure and carries less polar groups,the position space for the template molecule in polypyrrole is not highly matched with the configuration of the template molecule itself during imprinting process,and a large thickness is required to achieve the imprinting effect,so the specificity and sensitivity of this kind of sensor is generally low.In order to improve the recognition specificity of polypyrrole to template molecules,N(pyrrole-1-propionyl)-aminoacyl-chitooligosaccharide was designed as the functional monomer.Ribavirin(RBV)was selected as the template molecule,and then the functional monomer N-(pyrrole-l-propionyl)-alaninoyl-chitooligosaccharide(PPACO),which has strong molecular interaction with RBV,was screened by quantitative calculation.The strong interaction between functional monomer and RBV was confirmed by UV scanning.Using RBV as template molecule,pyrrole and PPACO as biffunctional monomer,the molecular imprinted membrane(MIM)of RBV was obtained by direct electrochemical polymerization on GCE.The modified electrode is capable to adsorb RBV specifically after eluting the template molecules in the imprinted membrane.The performance of the sensor was measured by differential pulse voltammetry(DPV).The sensor has high sensitivity and selectivity to RBV under optimized conditions.The results show that the detection range of the sensor for RBV is 0.5-5.0 ?M with a detection limit of 50 nM(S/N=3).The sensor has good reproducibility and stability.In addition,the sensor has been successfully used to monitor the RBV content in RBV granules with a satisfactory recovery(96.5%-99.2%)with a relative standard deviation(RSD)of less than 3.5%,which has the potential of practical application.(2)For weak hydrophilic template molecules,although the method mentioned in(1)can also be used to prepare sensors,due to their greater solubility in organic solvents,bulk polymerization in solvents was considered to prepare molecularly imprinted films.Chloramphenicol(CAP,XlogP=1.1)was selected as the target molecule and N-(4-pentenyl)amino acyl-chitooligosaccharide was used as the functional monomer oligomer.N-(4pentenyl)-isoleucyl-chitooligosaccharide(PICO)with strong molecular interaction with CAP was screened by quantitative calculation.Besides,this strong interaction between functional monomer and CAP was confirmed by UV scanning.Firstly,the CAP primary MIM with good permeability was prepared by a bulk polymerization on a GCE surface using CAP,PICO and ethylene glycol dimethacrylate(EGDMA)as the template molecule,functional monomer oligomer and crosslinking agent,respectively.Then,the electrode modified with primary MIM was scanned by cyclic voltammetry in the pyrrole solution saturated by chloramphenicol,and the polypyrrole imprinted film was formed in the gap between the primary imprinted polymer.The MIM(PPr)/GCE sensor is capable of absorbing CAP specifically after eluting the template molecules from the composite imprinted membrane.The performance of the sensor was measured by DPV in order to optimize the preparation parameters of the membrane.The results showed that the detection range of CAP was 0.04-4.0 ?M and the detection limit was 15.0 nM(S/N=3).The sensor has a good reproducibility and stability to retain more than 90%value of the fresh performance after storage at room temperature for 10 days.The recovery rate of actual samples detected by this sensor is satisfactory(94.0%-103.4%),and the RSD is less than 5%.(3)For hydrophobicity template molecules,molecularly imprinted electrochemical sensors can be prepared by using the method mentioned in(2)in principle.However,for some template molecules that are easy to be oxidized under positive voltage conditions,the secondary polymerization needs to be initiated under negative voltage conditions.Curcumin(CUR,XlogP=3.1)was selected as the target molecule and N-(4-pentenyl)-amino acylchitooligosaccharide was used as the functional monomer oligomer.N-(4-pentenyl)-alanylchitooligosaccharide(PACO)with strong molecular interaction with CUR was screened by quantitative calculation.The primary CUR molecular-imprinting membrane was prepared by a bulk polymerization on a GCE surface using CUR,PACO and EGDMA as the template molecule,functional monomer oligomer and crosslinking agent,respectively.And then through electrochemical polymerization in methylene bisacrylamide(MBA)solution(the polymer was abbreviated as PM)containing ammonium persulfate(APS),the composite imprinted film modified electrode was obtained.After removing the template molecules in the composite molecular imprinting film,the MIM(PM)/GCE sensor was gained.The performance of the sensor was measured by DPV.Under the optimized conditions,the sensor has high sensitivity and selectivity to CUR,and the detection range of CUR was 0.01-2.0 ?M with a detection limit of 5.0 nM(S/N=3).This sensor showed excellent reproducibility and stability,and more than 90%of its original response was retained after storage in a sealed capsule at room temperature for 30 days.The sensor was successfully applied to monitor the content of CUR in turmeric extract.The recoveries were 94.0%-106.5%,and the RSD was less than 3.5%.(4)For the hydrophobicity template molecules that need to be polymerized under negative voltage for secondary polymerization,in order to further improve the sensitivity of the sensor,carbon nanotubes that can improve the conductivity of the electrochemical sensor were to be introduced during fabrication,and imprinted polymers with the same template were used as secondary polymers.N-(4-pentenyl)-amino acyl-chitooligosaccharide was selected as functional monomer oligomer,and bisphenol A(BPA,XlogP=3.2)with similar hydrophobic parameters to CUR was selected as the target molecule.PICO with strong molecular interaction with BPA was screened as functional monomer oligomer through quantitative calculation.The interaction between the functional monomer and BPA was confirmed by UV scanning.The primary molecularly imprinted membrane with high permeability was in situ formed on multiwalled carbon nanotubes(MWCNTs)modified GCE surface by using BPA,PICO and EGDMA as the template,functional monomer oligomer and crosslinking agent,while the secondary MIM was subsequently prepared by electrochemical polymerization in the presence of BPA,acrylamide and MBA as template,monomer and crosslinking agent,respectively.After BPA in the MIMs was eluted,a composite molecularly imprinted electrochemical sensor(MIM(MIPs)/MWCNTs/GCE)was constructed for highly sensitive monitoring of BPA by means of MWCNTs-enhanced sensitivity and MIMs-mediated selectivity.DPV was used to measure the performance of the constructed sensor.Under the optimal conditions,the detection range of BPA by the sensor is.0.04-8.0 ?M,as well as a detection limit of 8.0 nM(S/N=3).Moreover,in the range of 0.5-2.0 ?M,the current response of the sensor could add up to 12%by the addition of secondary imprinting polymerization.The developed sensor showed good reproducibility and stability,and was applied to detect BPA in water samples with desirable recoveries ranging from 92.0%-107.0%,and the RSD was less than 5%.(5)For template molecules with higher hydrophobicity,the number of imprinted sites in the polymer cannot be increased due to their low solubility in the bulk polymerization solution.In order to improve the concentration of imprinted sites,the functional monomer oligomer layer was self-assembled on the surface of gold nanoparticles(AuNPs),and the template molecules in the polymerization solution were enriched by taking advantage of the strong molecular interaction between functional monomer oligomers and template molecules.After the self-assembly layer was fixed by bulk polymerization,the non-imprinted region of the selfassembly layer was sealed by electrochemical polymerization,thus forming a highly sensitive molecularly imprinted film.In view of this,N-(4-pentenyl(lipoyl))-amino acylchitooligosaccharide was designed to not only self-assemble on the surface of AuNPs but also participate in bulk polymerization.In order to improve the hydrophilicity of the primary membrane,polar amino acids were selected to construct functional monomer oligomers.Using N-(4-pentenyl(lipoyl))-amino acyl-chitooligosaccharide and N-(4-pentenyl)-amino acylchitooligosaccharide as functional monomer oligomers,Sudan I(SDI,XlogP=4.1)was selected as the target molecule,N-(4-pentenyl(lipoyl))-isoleucyl-chitooligosaccharide(P(L)ICO)and N-(4-pentenyl)-asparagyl-chitooligosaccharide(PASCO)with strong molecular interaction with SDI were screened by quantitative calculation.The strong interaction between the two functional monomers and SDI was confirmed by UV scanning.After being deposited by AuNPs,a P(L)ICO layer was self-assembled on GCE surface.Subsequently,the MIM of SDI was first polymerized on GCE surface by using SDI,PASCO and EGDMA as the template,functional monomer oligomer and crosslinking agent,respectively.Then the primary molecularly imprinted film was electrolytically polymerized in MBA solution containing APS as initiator to form composite imprinted film.After removing the template molecule form composite MIMs,the modified electrode(MIM(PM)/Fn-AuNPs/GCE)could specifically adsorb SDI.DPV was used to measure the performance of the constructed sensor,and the experimental parameters affecting the performance of the sensor were optimized.Under the optimal conditions,the detection range of SDI was 0.02-3.5 ?M with a detection limit of 4.0 nM(S/N=3).The sensor has good reproducibility and stability.More than 90%value of the fresh performance can be retained after 10 days in sealed capsules at room temperature.The results of the sensor were satisfactory(97.0%-106.6%),and the RSD was less than 5%. |
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