聚多巴胺固定的聚%282-甲基-2-噁唑啉%29_聚%284-乙烯吡啶%29混合刷对胃蛋白酶的可控吸附.pdf

CHINESE JOURNAL OF CHEMICAL PHYSICSVOLUME 36,NUMBER 3JUNE 27,2023ARTICLEPolydopamine Anchored Poly(2-methyl-2-oxazoline)/Poly(4-vinyl pyridine)Mixed Brushes with Switchable Properties for Pepsin AdsorptionKang He,Fei Hu,Chaoshi Chen,Muhammad Atif,Yanmei WangCAS Key Laboratory of Soft Matter Chemistry,Department of Polymer Science and Engineering,University of Science and Technology of China,Hefei 230026,China.(Dated:Received on June 21,2021;Accepted on September 27,2021)Mixed polymer brushescoating based on poly(2-methyl-2-oxazoline)/poly(4-vinyl pyridine)(PMOXA/P4VP)waspreparedbysimultaneously graft-ing amine-terminatedPMOXA and thiol-terminated P4VP onto poly(dopamine)(PDA)-modified substrates in this work.Thecoatings were characterized by X-ray photoelectron spectroscopy,ellipsometry,zeta poten-tial measurements,and the static water contact angle tests.The results indicated that it isfeasible to control the components of the coating by adjusting the feed ratio of PMOXA toP4VP.Moreover,the zeta potential and the water contact angle of mixed brushes modifiedsurfaces could be tuned by changing the environmental pH value and surface compositions.Finally,fluorescein isothiocyanate-labelled pepsin assay and surface plasmon resonance wereperformed to investigate the responsive adsorption/desorption of pepsin by PMOXA/P4VPmixed brushes.The results showed that by adjusting the fraction of PMOXA or P4VP,thePMOXA/P4VP mixed brushes coated surfaces could adsorb a high amount of pepsin atpH=3,and achieve a desorption efficiency of over 92%at pH=7.Key words:Binary mixed brush,Zeta potential,Controlling protein adsorption,Pepsin,Surface plasmon resonanceI.INTRODUCTIONControlling protein adsorption/desorption is a press-ing issue in many biotechnology fields,such as biosen-sors,medical device coatings,and drug delivery,etc.15.Biomaterial surfaces modified with stimuli-responsive polymer brushes that show the ability to con-trol protein adsorption and desorption are thus highlydesired 6,7.However,the protein-interface interac-tions are quite complex and diverse,involving van derAuthor to whom correspondence should be addressed.E-mail:Waals forces,electrostatic interactions,hydrophobic in-teractions,and conformational entropy 812.There-fore,it is difficult for surfaces modified with stimuli-responsive polymer brushes alone to realize the con-trolled or switchable protein adsorption 1316.In order to realize the controlled protein adsorption,mixed polymer brushes composed of polymer with anti-fouling property and stimulus-response polyelectrolytehave been proposed.Bratek-Skicki has fabricatedthe mixed poly(ethylene oxide)(PEO)and poly(2-(dimethylamine)ethylmethacrylate)(PDMAEMA)brushes 17,in which PEO plays an anti-fouling part,the PDMAEMA as the stimulus-response polyelec-trolyte could be responsive to pH and ionic strengthDOI:10.1063/1674-0068/cjcp2106103339c 2023 Chinese Physical Society340Chin.J.Chem.Phys.,Vol.36,No.3Kang He et al.change.It was demonstrated that human serumalbumin(HSA)and human fibrinogen adsorbed onmixed PEO/PDMAEMA brushes could be completelydesorbed when changing pH and ionic strength,whileon PDMAEMA homobrushes,only 61.0%of adsorbedHSA and 23.0%of adsorbed human fibrinogen couldbe released.Dupont-Gillain et al.have investigatedthe adsorption/desorption capability of HSA on mixedbrushes of PEO/poly(acrylic acid)(PAA)inducedby pH and ionic strength 18.It is observed thatthe PEO/PAA mixed brushes are able to achieve adesorption percentage of 86%after protein adsorption,displaying a great improvement of desorption per-centage compared with PAA homobrushes(desorptionpercentage is only 30%).It can be concluded that theintroduction of protein repellent PEO could effectivelycompensatefortheimperfectproteindesorptionefficiency of the environment-responsive polymer.However,oxidative degradation and enzymatic cleav-age of PEO could result in the decrement of stability forPEO coating after its long-term use 19,20.Recently,poly(2-methyl-2-oxazoline)(PMOXA)has drawn moreattention for its similar protein resistant ability to PEO,while it might be less prone to degradation than PEO2125.Pan et al.have prepared PMOXA/PAAmixed brushes through polydopamine(PDA)anchorfor bovine serum albumin(BSA)and lysozyme ad-sorption/desorption 26,27.It is demonstrated thatthe desorption percentage of protein could be improvedhighly from 50.5%for PAA homobrushes alone to 87.0%for PMOXA/PAA mixed brushes.Besides PAA,poly(4-vinyl pyridine)(P4VP),a kindof weakly basic polyelectrolyte,has been extensivelyinvestigated for its pH-dependent protonation of itspyridine ring along the chain and the consequent pH-dependent swelling-deswelling behavior 2830.WhenpH value of environment is lower than the isoelectricpoint of P4VP(pI4.8)31,the electrostatic repulsionbetween positively charged repeating pyridine groupsproduces a certain degree of swelling of the brush;whenpH value is higher than pI value of P4VP,polymerchains collapse due to the neutralization of pyridinegroups,as well as the hydrophobic interaction betweenvinyl groups existing in polymer chains 32,33.On the basis of the swelling-deswelling behavior ofP4VP upon pH change and the stable anti-fouling prop-erty of PMOXA,PDA anchored PMOXA/P4VP bi-nary mixed brushes with the property of controlledprotein adsorption were designed in this work to in-vestigate the controlled or switchable protein adsorp-tion.Usually,the pepsin with extremely low isoelectricpoint(pI2.2)is a reliable indicator for the diagnosis oflaryngopharyngeal reflux disease 34,therefore in thiswork pepsin was chosen as a model protein to studythe interaction between surfaces and proteins.Firstly,PMOXA/P4VP binary mixed brushes were preparedby simultaneously grafting amine-terminated PMOXA(PMOXA-NH2)and thiol-terminated P4VP(P4VP-SH)with designed chain length to PDA modified sil-icon/glass/gold substrates.The coating composition,surface hydrophilicity,thickness,and surface chargewere measured by X-ray photoelectron spectroscopy(XPS),static water contact angle(WCA),variable an-gle scanning ellipsometry(VASE),and zeta potential,respectively.Furthermore,the behavior of pepsin ad-sorption/desorption on the coatings surfaces was eval-uated by fluorescence microscopy and surface plasmonresonance(SPR).II.EXPERIMENTSA.Materials2-Methyl-2-oxazoline(MOXA,99%,Sigma-AldrichChemical Company,USA)and 4-vinyl pyridine(4VP,95%,Tixiai,Shanghai,China)were dried over calciumhydride overnight and distilled under atmospheric andreduced pressure conditions,respectively.Dopaminehydrochloride,potassium phthalimide,methyl triflu-oromethanesulfonate(MeOTf,98%),potassium chlo-ride(KCl,99.98%),and pepsin(pI2.2)from porcinegastric mucosa were purchased from Sigma-Aldrichandusedasreceived.2,2-Azobisisobutyronitrile(AIBN,Tianjin Guangfu Fine Chemical ResearchInstitute,Tianjin,China)was recrystallized fromethanol.Tris(hydroxyl methyl)-aminomethane(Tris),hydrazine hydrate,ethanolamine,ethanol,hydrochlo-ricacid(HCl),chloroform(CHCl3),acetonitrile,and isopropyl alcohol were obtained from SinopharmChemical Reagents(Shanghai,China).ACN andIPA were dried over calcium hydride for 6 h anddistilled before use.S-1-dodecyl-S-(,-dimethyl-acetic acid)trithiocarbonate(DDMAT)and fluoresceinisothiocyanate-labeled pepsin(FITC-pepsin)were pre-pared according to previous literatures 35,36.Thegold SPR sensors were received by coating glass slideswith an adhesion promoting chromium layer(thicknessDOI:10.1063/1674-0068/cjcp2106103c 2023 Chinese Physical SocietyChin.J.Chem.Phys.,Vol.36,No.3Poly(2-methyl-2-oxazoline)/Poly(4-vinyl pyridine)Mixed Brushes3415 nm)and a surface plasmon active gold(Au)layer(45nm).Silicon(111)wafers with natural oxidized layerwere derived from Zhejiang Crystal Photoelectric Tech-nology Company(Taizhou,China).Glass slides werepurchased from Shanghai Jinglun Industry Glass Com-pany(Shanghai,China).B.Synthesis of the polymersThe typical synthesis process of PMOXA and P4VPis shown in FIG.S1 in Supplementary materials(SM).PMOXA was synthesized by using cationic ring-openingpolymerization(CROP)of MOXA with MeOTf as aninitiator(the molar ratio of monomer to initiator was25:1),then end-capped with potassium phthalimide24.The successful preparation of PMOXA-NH2witha degree of polymerization of 28 was confirmed by1HNMR spectroscopy(FIG.S2 in SM).P4VP was synthesized by using reversible addition-fragmentation chain transfer(RAFT)polymerizationwith AIBN as initiator and DDMAT as a chain trans-fer agent(the molar ratio of monomer to chain trans-fer agent was 100:1)37.The successful preparationof P4VP-SH with a degree of polymerization of 95was confirmed by1H NMR spectroscopy(FIG.S3 inSM).The calculated theoretical contour chain lengthsof the PMOXA-NH2and P4VP-SH were 10.62 nm and23.90 nm,respectively(Table S1 in SM).C.Preparation of polymer brushesIn order to remove any organic contamination on rawsubstrates,silicon/glass wafers were cleaned by sonica-tion in ethanol,acetone and deionized water for 15 minsuccessively,then immersed in freshly prepared piranhasolution(7:3,V/V mixture of H2SO4(95%98%)andH2O2(30%),50C)for 4 h.The gold SPR sensorswere cleaned by freshly prepared piranha solution for1 h at 70C.And then,all substrates were rinsed withan excess of deionized water and dried under nitrogenatmosphere.Polymer brushes were prepared using the grafting-to method in this work.Firstly,the cleaned sub-strates were immersed in the freshly prepared Tris-HCl solution(pH=8.5)of dopamine hydrochloride(2 mg/mL)with continuous vibration at room tem-perature,in contact with atmospheric oxygen.After-wards,a 10 mg/mL solution of polymers containingPMOXA and P4VP was prepared by dissolving bothpolymers with the solution containing 30%Tris-HCl(pH=8.5)buffer and 70%ethanol.The mass ratios ofPMOXA/P4VP used in this work were 100/0(PMOXAhomobrushes),80/20,50/50,20/80,and 0/100(P4VPhomobrushes),respectively.Substrates modified byPDA were soaked in polymer solutions mentioned aboveat 50C for 24 h,then the samples were rinsed exten-sively with deionized water and dried by a stream ofnitrogen.Corresponding prepared surfaces were namedas PMOXA,PMOXA80/P4VP20,PMOXA50/P4VP50,PMOXA20/P4VP80,and P4VP,respectively.D.Characterization1.Nuclear magnetic resonance(1H NMR)The1H NMR measurements of polymers were per-formed by using an AVANCE 300 spectrometer(Brukerbiospin,Switzerland)(300 MHz)at room temperature.2.XPSThe chemical composition of the polymer modifiedsilicon substrates was investigated by a VG ESCALABMK II X-ray Photoelectron Spectrometer(XPS,VGScientific Instruments,East Grinstead,England)withan Al K X-ray source(energy of 1486.6 eV)at roomtemperature.The take-offangles of the photoelectronswere set to 90and the spot size was kept at 500 m.3.VASEThe dry thicknesses of brushes coating on the sili-con wafer were measured using a variable angle spec-troscopic ellipsometer(M-2000,Woollam Co.,Inc.,Lin-coln,NE)at room temperature.The ellipsometric datawere acquired in the spectral range of 3701000 nmat two different angles of incidence(65and 75forsilicon wafer).The analysis software,CompleteEASE4.81,was used to analyze all data.To fit the ellip-sometric data,the optical constants(refractive index,extinction coefficient)of Si(n=3.865,k=0.020)andSiO2(n=1.465,k=0)were used to determine the SiO2layer thickness of the freshly cleaned silicon surfaces.Each polymer layer was represented as a slab of uniformthickness having sharp interfaces and optical propertiesdescribed by a Cauchy model.The average thicknessvalue was obtained by measuring one sample in at leastthree different positions,and the data were reported asmeansstandard deviation(SD).DOI:10.1063/1674-0068/cjcp2106103c 2023 Chinese Physical Society342Chin.J.Chem.Phys.,Vol.36,No.3Kang He et al.4.Zeta potentialThe surface zeta potential of the samples was in-vestigated by SurPASS electrokinetic analyzer(AntonPaar GmbH,Graz,Austria).The pH of the KCl(0.001 mol/L)solution was adjusted from 7 to 3 by theaddition of hydrochloric acid(0.05 mol/L).The averagesurface zeta potential value was recorded by measuringone sample in at least three times,and the data werereported as meansSD.5.WCAThe surface hydrophilicity of the coating surfaces wasmeasured by Optical Contact Angle&Interface Ten-sion Meter SL200KS(SOLON TECH,Shanghai,China,manufactured under authorized from USA KINO In-dustry Co.Ltd.).Before measurement,the sampleswere immersed into aqueous solutions of different pHfor 2 h(aqueous solutions with pH=29 were preparedby adding 1 mol/L hydrochloric acid or 1 mol/L sodiumhydroxide solution into deionized water under the anal-ysis of a pH meter).The average WCA value was ob-tained by measuring one sample in at least three differ-ent positions,and the data were reported as meansSD.Aqueous solutions with pH=29 were used in the ex-periments.6.Fluorescence microscopyTo evaluate the protein adsorption/desorption prop-erty of the coatings,FITC-pepsin was chosen as themodel protein.Bare and modified glass wafers were in-cubated in the FITC-pepsin solution(1.0 mg/mL,hy-drochloric acid solution of pH=3)for 3 h in dark,andthen the samples were washed with the aqueous solu-tion(pH=3)three times to remove the weakly boundproteins.Subsequently,the samples were immersed indeionized water(pH=7)for another 2 h,then rinsedwith the deionized water extensively and dried by astream of nitrogen.Lastly,the glass wafers were imagedunder an optical microscope,Olympus BX81(Olympus,Japan)equipped with a halogen lamp,filter U-MNG2(exit=470490 nm,emit510 nm)and camera typeDP72.Image J software was used to evaluate the fluo-rescence intensity from three different positions and themean values were calculated,with the data expressed asmeansSD.7.SPRBiacore T200 Instrument(Ge Healthcare Bio-ScienceAB,Uppsala,Sweden)was used to analyze protein ad-sorption/desorption quantitatively on the surfaces atroom temperature.Firstly,hydrochloric acid solutionof pH=3 was flowed through the sensor surface for 600 sto obtain the baseline signal,and then the protein so-lution(pepsin of 0.1 mg/mL,pH=3)was injected for900 s for the protein adsorption.After rinsing by pH=3aqueous solution for 600 s,the deionized water(pH=7)was passed onto the sensor surface for 240 s,and endedwith 600 s injection of pH=3 aqueous solution.Theflow rate was maintained at 20 L/min throughout theprocedure.The response signal from the SPR measure-ment was recorded in response units(RU).Generally,10 response unit equals 1.0 ng/cm2of protein adsorbedon the sensor surface.III.RESULTS AND DISCUSSIONA.Characterization of polymer-modified silicon wafersIn this work,PMOXA/P4VP binary mixed brusheswere prepared by“grafting to”method through theMichael addition reaction between the amine end groupof PMOXA-NH2or thiol end group of P4VP-SH andquinone existing in PDA.After 24 h of reaction,the drythickness of PMOXA homobrushes and P4VP homo-brushes determined by VASE was 3.2 nm and 3.4 nm,respectively.The results indicated that