Regenerable Molecularly Imprinted Polymers Based On Reversible Chemical Bonds

Part 1 Recycling and regeneration of molecularly imprinted nanoparticles using reversible boronate ester bondsObjectives: The formation and breakage of reversible boronate ester bonds were controlled by pH.By adjusting the levels of cis-diol and pH,we could control the formation of reversible boronate ester bonds formed between cis-diol and boronic acid and boroxine bonds formed between boronic acid-functionalized nanoparticles themselves to adjust the aggregation and sedimentation of boronic acid-modified nanoparticles.A viable approach to recover and regenerate functional nanoparticles without involving heavy-duty instrument was established.Methods: Boronic acids modified inorganic silica nanoparticles(Si@BA)and organic molecularly imprinted polymer(MIP@BA)nanoparticles were prepared using different synthetic methods.Then scanning electron microscopy(SEM),Fourier transform infrared spectroscopy(FT-IR),and dynamic light scattering(DLS)were applied to evaluate physicochemical characteristics of these two boronic acid-coated functional nanoparticles.Alizarin red s(ARS),a cis-diol containing fluorogenic dye,was used as a general fluorescent reporter to detect the existence of boronic acid on the surfaces of these two nanoparticles.The pH depending binding characters of MIP@BA were studied.These two types of nanoparticles represent hard and soft materials decorated with different levels of boronic acid on their surfaces.Polyvinyl alcohol(PVA),a synthetic polymer with repeating pendant cis-diol groups,was used as a particle aggregation reagent to realize controlled nanoparticle separation.Impacts of pH,the molecular weight and concentration of PVA were investigated.The reversibility of the boronate bond was also verified.Finally,through the optimized pH,molecular weight and concentration of PVA,the MIP@BA were recovered and regenerated.The specific adsorption function to propranolol of regenerated MIP nanoparticles was tested.Results: SEM images showed the diameter of Si@BA and MIP@BA were 297.8nm and170 nm,respectively.FT-IR and fluorescence emission spectrum results showed that two kinds of boronic acid-functionalized nanoparticles were successfully synthesized.For nanoparticles aggregation and sedimentation after crosslinking with PVA,we could conclude that the optimal pH for the boronic acid-mediated particle sedimentation was pH 9.These two boronate nanoparticles had different optimal molecular weight and concentration of PVA,the optimal condition to precipitate the Si@BA nanoparticles was determined to be 0.005 mg/m L PVA with a molecular weight of 9000-10000.For the MIP nanoparticles,the optimal concentration and molecular weight of PVA was found to be 0.5 μg/m L and MW = 146000-186000,respectively.With adding fructose in formed boronate ester bonds solution,we could observe the previously precipitated particles disassemble and turn into a stable particle suspension,which verified the reversibility of boronate ester bonds.Using the optimal separation condition and adjusting pH to 4,the bound templates were removed from MIP and MIP nanoparticles still kept sedimentation.By removing the supernatant,MIP nanoparticles were recovered and regenerated.After recycling and regenerating for 5 times,the functional MIP nanoparticles still showed good specific affinity to templates.Conclusions: In this work,a new approach to facilitate separation and recycling of functional nanoparticles based on pH-controlled reversible covalent bonds between boronic acid modified nanoparticles and flexible PVA polymer chains was developed.Using pH-induced formation of boronate ester versus boroxine bonds,both hard inorganic nanoparticles and soft MIP particles can be easily recovered through simple particle sedimentation without involving specialized heavy instrument.The new method can be useful when nanoparticle separation and recovery needs to be achieved in certain underdeveloped or remote areas or to handle a large sample volume.Part 2: Preparation and characterization of fluorescent molecularly imprinted polymers(f MIPs)based on reversible chemical bondsObjectives: Two propranolol-f MIPs were prepared using reversible chemical bonds and irreversible chemical bonds,respectively.Binding characters and fluorescence properties of these two f MIPs were studied.Methods: First,propranolol-imprinted polymer was synthesized by reversible addition-fragmentation chain transfer(RAFT)polymerization using meth acrylic acid(MAA)as the functional monomer.Then dithioester end groups of cumyl dithiobenzoate(CDB)on the surface of the polymer were converted into thiol groups.After that,two types of f MIP were prepared using different methods.To prepare f MIP based on reversible chemical bonds(MIP-DDA),boronic acid was connected to thiol groups by reversible disulfide bonds.Then fluorescence molecule dansyl dopamine(DDA)was modified on the boronic acid-modified MIP by reversible boronate ester bonds.For the f MIP based on irreversible chemical bonds(MIP-NPM),MIP modified with thiol groups was connected with fluorescence molecules N-(1-pyrenyl)maleimide(NPM)by thiol-ene click reaction.The morphology,adsorption properties and fluorescence properties of two f MIPs were evaluated.Results: SEM showed that two f MIPs were uniform,particles with diameter about2.9 μm.The excitation and emission wavelength of MIP-DDA and MIP-NPM were Ex=340 nm,Em=530 nm,and Ex= 334 nm,Em= 375 nm respectively.Both MIP-DDA and MIP-NPM had higher binding capacity to propranolol than the corresponding MIP-NPM.The impact factor(IF)of MIP-DDA and MIP-NPM were 2.15 and 1.99 respectively.Binding kinetics showed that MIP-DDA required about 90 min to reach equilibrium in ethanol-phosphate buffer,while MIP-NPM needed only 5 min to reach equilibrium in acetonitrile-phosphate buffer.Conclusions: Two kinds of f MIP microspheres containing reversible covalent bonds or irreversible covalent bond were successfully prepared by linking fluorescent molecules to MIP prepared by RAFT method.These two f MIPs both had regular morphology and could bond target template selectively.Part 3: Dynamic FRET based on regenerable fluorescence molecularly imprinted polymers with reversible chemical bondsObjectives: To evaluate the capability of prepared MIP-DDA and MIP-NPM containing reversible or irreversible covalent bonds to form fluorescence resonance energy transfer(FRET)with fluorescent template.The impacts of breaking reversible chemical bonds on FRET characters and regeneration of MIP were also studied.Methods: The fluorescence template propranolol,fluorescent and non-fluorescent analogs of propranolol were added to MIP-DDA and MIP-NPM respectively.Time-dependent and dose–dependent fluorescence response were recorded to evaluate the possibility of f MIPs to form MIP-FRET with propranolol,selectivity of MIP-FRET was also studied.When the reversible chemical bonds of MIP-DDA were broken,FRET characters and the regeneration of MIP was investigated.Results: Both MIP-DDA and MIP-NPM could form MIP-FRET by bonding fluorescence template propranolol.Two MIP-FRET had time-dependent FRET response and dose-responsive FRET signals.With adding the analogs,MIP-FRET showed good selectivity.In MIP-DDA,fluorescent molecules DDA bond MIP with reversible disulfide bonds and boronate ester bonds When disulfide bonds were reduced to thiol groups by dithiothreitol(DTT),or boronate ester bonds were bond by competitor fructose,FRET intensity decreased as the fluorescent molecules DDA was removed from when the reversible chemical bonds were broken.Adding DDT not only decreased MIP-FRET of MIP-DDA but also regenerate MIP.The regenerated MIP could bond fluorescent molecule NPM with irreversible chemical bonds.The achieved MIP-NPM could form MIP-FRET when binding propranolol.Conclusions: MIP-FRET was successfully established using two f MIPs containing reversible chemical bonds and irreversible chemical bonds.By breaking the reversible chemical bonds on MIP-DDA,the intensity of MIP-FRET decreased and MIP regenerated due to the lost of fluorescent molecule bond with reversible disulfide bonds and boronate ester bonds.The regenerated MIP could react with NPM to prepared MIP-NPM,which could form MIP-FRET when bindng propranolol.