基于硼酸盐亲和辅助电化学调控ATRP的癌胚抗原超灵敏电化学适体传感研究.pdf

ARTICLEBoronate Affinity-assisted ElectrochemicallyControlled ATRP for Ultrasensitive ElectrochemicalAptasensing of Carcinoembryonic AntigenQiong Hu*,Shi-Qi Li,Yi-Yi Liang,Wen-Xing Feng,Yi-Lin Luo,Xiao-Jing Cao,Li Niu*Guangdong Engineering Technology Research Center for Sensing Materials and Devices,Guangzhou Key Laboratory of SensingMaterials and Devices,Center for Advanced Analytical Science,School of Chemistry and Chemical Engineering,GuangzhouUniversity,Guangzhou,510006,PR ChinaAbstractAs an acidic glycoprotein,carcinoembryonic antigen(CEA)is of great value as a broad-spectrum tumor marker inthe differential diagnosis and surveillance of malignant tumors.In this work,we report an electrochemical aptasensorfor the ultrasensitive and highly selective detection of CEA,taking advantage of the dual amplification by the boronateaffinity-assisted electrochemically controlled atom transfer radical polymerization(BA-eATRP).Specifically,the BA-eATRP-based electrochemical aptasensing of CEA involves the capture of target antigens by nucleic acid aptamers,the covalent crosslinking of ATRP initiators to CEA antigens via the selective interactions between the phenylboronicacid(PBA)group and the cis-diol group of the monosaccharide residues,and the collection of the ferrocene(Fc)re-porters via the eATRP of ferrocenylmethyl methacrylate(FcMMA).As CEA is decorated with hundreds of cis-diolgroups,the BA-based crosslinking can result in the labeling of each CEA with hundreds of ATRP initiators;furthermore,the eATRP of FcMMA results in the surface-initiated growth of long-chain ferrocenyl polymers,leadingto the tethering of each ATRP initiator-conjugated site with hundreds to thousands of Fc reporters.Such that,the BA-eATRP can result in the efficient labeling of each CEA with a plenty of Fc reporters.Under the optimized conditions,the BA-eATRP-based strategy enables the highly selective aptasensing of CEA at a concentration as low as0.34 pg$mL?1,with a linear range of 1.0?1000 pg$mL?1.Besides,this aptasensor has been successfully applied to thequantitative analysis of CEA in human serum.The BA-eATRP-based electrochemical aptasensor is cost-effective andsimple in operation,holding broad application prospect in the ultrasensitive and highly selective detection of CEA.Keywords:Boronate affinity;Atom transfer radical polymerization;Electrochemical aptasensor;Carcinoembryonicantigen;Tumor marker;Signal amplification1.IntroductionCarcinoembryonicantigen(CEA)isacelladhesion-related acidic glycoprotein 1.As aoncofetal antigen,CEA is normally secreted by thegastrointestinal tissue during fetal developmentand occurs at very low levels in the serum ofhealthy adults 1.However,the level of CEA isoftenelevatedintheserumofindividualssuffering from various neoplastic conditions(e.g.,colorectal cancer,gastric cancer,pancreatic can-cer,lung cancer,breast cancer,and medullarythyroidcancer)2andsomenon-neoplasticconditions(e.g.,cirrhosis,pancreatitis,ulcerativecolitis,hypothyroidism,and Crohns disease)3.Due to the poor sensitivity and specificity,CEA isunable to act as a tumor marker for the diagnosisand screening of a specific cancer.However,it isof great value as a broad-spectrum tumor markerin the differential diagnosis and surveillance ofmalignant tumors 1,4.As the cutoff value of CEAfor clinical application is 5 ng$mL?15,sensitiveand selective detection of CEA is highly desired.In recent decades,a diversity of methods havealready been established for CEA detection,suchas fluorescent 6,7,photoelectrochemical(PEC)Received 5 August 2022;Received in revised form 8 September 2022;Accepted 15 September 2022Available online 19 September 2022*Corresponding author,Qiong Hu,Tel:[phone],E-mail address:.*Corresponding author,Li Niu,Tel:[phone],E-mail address:.https:/doi.org/10.13208/j.electrochem.22180011006-3471/2023 Xiamen University and Chinese Chemical Society.This is an open access article under the CC BY-NC license(http:/creativecommons.org/licenses/by-nc/4.0/).8e10,surface-enhanced Raman scattering(SERS)11,12,colorimetric 13,14,electrochemilumine-scent15e18,andelectrochemicalbiosensors19e21,using such as antibodies 22,23,imprintedpolymers 24,25,and aptamers 26e28 as thecapture elements.Among them,electrochemicalaptasensing of CEA has attracted much attentionby virtue of the advantages of low detection cost,minimal matrix/background interference,simpleinstrument,and high sensitivity and selectivity29,30.Nevertheless,mostofthepreviouslydevelopedelectrochemicalCEAaptasensorsnecessitated the use of sophisticated enzymaticreactions(e.g.,DNAzymes)31,32ornano-composites(e.g.,nanoclusters)33,34 for signalamplification,which is not practical for clinicalapplications.By virtue of the simplicity,high efficiency,andcost-effectiveness,the grafting of polymer chainsthrough reversible deactivation radical polymeri-zation,such as atom transfer radical polymeriza-tion(ATRP)35,36andreversibleaddition-fragmentation chain-transfer(RAFT)polymeriza-tion 37,38,has attracted growing attention as anamplification strategy in the sensitive detection ofbiotargets 39,40.Among them,ATRP involves theuse of the alkyl halide initiators(R-X)and thelower oxidation state transition-metal activators(typically the Cu(I)complexes)41,42.Specifically,reduction of the initiators by the activators gener-ates the radical species,which then react withmonomers to form long-chain polymers 42.In theelectrochemically controlled ATRP(eATRP),theCu(I)activators are generated from the electro-chemical reduction of the Cu(II)deactivators 43,holding the merits of the improved control overpolymergrowth,betterbiocompatibility,andhigher O2tolerance 40,44.On the basis of the dual amplification by theboronateaffinity(BA)-assistedeATRP(BA-eATRP),we report herein a cost-effective and easy-to-operate electrochemical aptasensor for the ul-trasensitive and highly selective detection of CEA.As the capture elements,the DNA aptamers wereattached to a gold electrode via the gold-sulfurself-assembly chemistry.After the capture of CEAantigens,the oligosaccharide chains of CEA wereleveraged for the covalent crosslinking of ATRPinitiators via the selective interactions between thephenylboronic acid(PBA)group and the cis-diolgroup of the monosaccharide residues 45,46.Further,potentiostatic reduction of the Cu(II)de-activators resulted in the generation of the Cu(I)activators 40,44,triggering the eATRP of ferro-cenylmethyl methacrylate(FcMMA)and thus thesurface-initiated growth of long-chain ferrocenylpolymers.As each CEA is decorated with hun-dreds of cis-diol groups for the BA-based cross-linkingofATRPinitiatorswhileeachATRPinitiator-conjugatedsitecanbetetheredwithhundreds to thousands of Fc reporters,the BA-eATRP can result in the labeling of each CEA witha plenty of Fc reporters.As a result,the BA-eATRP-based electrochemical CEA aptasensor ishighly sensitive and selective.Moreover,it is cost-effective and east-to-operate,thus holding greatpromise in clinical applications.2.Experimental2.1.BA-eATRP-based electrochemical aptasensingof CEAPrior to use,gold electrode(Au)was cleaned asdescribed elsewhere 47,48.After incubating theAu with 10 mL of 0.5 mmol$L?1aptamer(Apt,10 mmol$L?1NaH2PO4/Na2HPO4(PBS,pH7.4)at37?C for 30 min,the Apt/Au was rinsed with ul-trapure H2O,incubated with 6-mercapto-1-hex-anol(MH,1.0 mmol$L?1in 60%ethanol)at 37?Cfor 15 min,and rinsed with ethanol and ultrapureH2O.After incubating the MH/Apt/Au with 10 mLof CEA(10 mmol$L?1PBS,pH7.4)at 37?C for45 min,the CEA/MH/Apt/Au was rinsed with ul-trapure H2O,incubated with(4-(2-bromo-2-phe-nylacetylamino)phenyl)boronicacid(PBABr,0.5 mmol$L?1in 10 mmol$L?1PBS containing 10%DMF(pH9.5)at 37?C for 15 min,and rinsed withethanol and ultrapure H2O.The PBABr/CEA/MH/Apt/Au was soaked into the polymerization solu-tion(obtained by adding 0.1 mL of FcMMA(10 mmol$L?1in DMF),0.1 mL of CuIIBr/Me6TREN(10mmol$L?1inH2O),7.5mLofKPF6(0.1 mol$L?1),and 1.0 mL of KBr(1.0 mol$L?1)into1.3 mL of DMF),followed by the eATRP of FcMMAat room temperature(ca.25?C)for 40 min using i-tcurve.After removal of the possible Cu(0)residuesvia the anodic oxidation at 0.19 V(vs.saturatedcalomel electrode(SCE)for 30 s,the Fc/PBABr/CEA/MH/Apt/Au was tested by square wave vol-tammetry(SWV;potential increase,4.0 mV;po-tential amplitude,25 mV;frequency,15 Hz)in0.5 mol$L?1LiClO447,48.3.Results and discussion3.1.PrincipleoftheBA-eATRP-basedelectrochemicalCEA aptasensorThe principle of the electrochemical CEA apta-sensor is shown in Scheme 1.The aptamer wasused as the recognition element and it was tetheredvia the Au-S chemistry.As CEA is a glycoproteinJournal of Electrochemistry,2023,29(6),2218001(2 of 9)49,the ATRP initiators for the later polymerizationwerecovalentlycrosslinkedtotheaptamer-capturedantigensviathefacile,selectivein-teractionsbetweenthecis-diolgroupsofthemonosaccharideresiduesandthePBAgroup50e52.Afterward,the eATRP of FcMMA resultedin the surface-initiated growth of the Fc-conjugatedpolymer chains and consequently the collection ofFc reporters 48.As each CEA is decorated withhundreds of cis-diol groups for the BA-basedcrosslinking of ATRP initiators while each long-chain polymer can be composed of a plenty of Fcreporters,the BA-eATRP enabled the dual-ampli-ficationdetection.Thus,theBA-eATRP-basedelectrochemical CEA aptasensor is ultrasensitiveand highly selective.3.2.Effect of eATRP potentialThe surface-initiated growth of the Fc-conju-gated polymer chains via the eATRP of FcMMAwas initiated and sustained through the electro-chemical reduction of the Cu(II)deactivators.Withthis in mind,the redox properties of the Cu(II)deactivator were investigated.As displayed inFig.1A,the cyclic voltammetric(CV)curve of theCu(II)deactivator at the PBABr/CEA/MH/Apt/Aupresented a reduction peak at?0.58 V,corre-sponding to the reduction of the Cu(II)deactivatorsinto the Cu(I)activators 43,44.For the CuIIBr/L/CuI/L redox couple(L Me6TREN),the Nernstequation can be expressed as Eapp E(RT/nF)*lnCuIIBr/L/CuI/L*Br?.It is obvious that theconcentration of the Cu(I)activator is highlydependent on the eATRP potential(Eapp)43,44.Specifically,a higher Eappwould result in a lowerconcentration of the Cu(I)activator,while a morenegative Eappwould result in the generation ofmore Cu(I)activators 43.As the initiating radicals(R?)were generated from the reduction of theATRP initiators by the Cu(I)activators 43,the rateof the surface-initiated growth of polymer chainswas dependent on the Eapp43,44.With this inmind,the effect of eATRP potential on the detec-tion signal was investigated.The peak currentcorresponding to the anodic oxidation of the Fcreporters,as displayed in Fig.1B,exhibited thehighest value at the eATRP potential of?0.54 V,Fig.1.(A)CV curve of the PBABr/CEA/MH/Apt/Au in the FcMMA-free polymerization solution(the dashed vertical line indicates the reductionpeak).(B)Effect of eATRP potential on the detection signal.CEA,1.0 ngmL?1.Scheme 1.Principle of the BA-eATRP-based electrochemical CEA aptasensor.Journal of Electrochemistry,2023,29(6),2218001(3 of 9)which was hence adopted in the subsequentexperiments.3.3.Characterizations of the electrochemical CEAaptasensorThe feasibility of the BA-eATRP-based electro-chemical CEA aptasensor was investigated basedon the SWV curves derived from various elec-trodes.As seen in Fig.2A,the perfectly modifiedelectrode(i.e.,the Fc/PBABr/CEA/MH/Apt/Au)can be featured by a high oxidation peak at ca.0.29 V,while no oxidation peak could be observedforthePBABr/CEA/MH/Apt/Au(i.e.,withouteATRP).Thisobservationvalidatedthattheoxidation current observed at ca.0.29 V wasderived from the electrochemical oxidation of theFc reporters.Likewise,almost no oxidation peakcould be observed for the electrodes modified inthe absences of aptamers,CEA antigens,or thePBABr agents.The high reliability of the detectionsignal validated the feasibility of the BA-eATRP-based electrochemical CEA aptasensor.CV of theFc/PBABr/CEA/MH/Apt/Au was further appliedto investigate the redox behavior of the Fc re-porters(Fig.2B).As the Fc reporters were surface-tethered,the peak currents showed well-definedlinear correlation with the scan rate(Figure S1),confirming the surface-confined charge-transferprocess 47,48.Fig.3 clearly shows that there is ahigh density of polymer chains on the Fc/PBABr/CEA/MH/Apt/Au.Apart from the P elementsobserved for the DNA aptamer in the energydispersive X-ray spectrum(EDS)mappingasshown in Fig.4A,the EDS mappings of the B(Fig.4B)and Br elements(Fig.4C)proved thepresence of PBABr,while that of the Fe element(Fig.4D)unequivocally demonstrated the presenceof the Fc reporters.The preparation of the Fc/PBABr/CEA/MH/Apt/Au was monitored by electrochemical impedancespectroscopy(EIS,Fig.5A)and CV(Fig.5B).Forthe Au,the charge-transfer resistance(Rct)was thesmallest(Fig.5A,a),while the redox currents werethe highest(Fig.5B,a).Owing to the steric hin-drance of the aptamer layer,and the electrostaticrepulsion between the phosphate groups and theFe(CN)63e/4e44,the modification of aptamers(i.e.,the Apt/Au)resulted in the increase of the Rct(Fig.5A and b)and the decrease of the redoxcurrents(Fig.5B,b).As the blocking of the residualsites would cause an increase in the steric hin-drance 43,47,the MH/Apt/Au showed a higherRct(Fig.5A,c)and the lower redox currents(Fig.5Band c)relative to the Apt/Au.As displayed,thecapture of CEA antigens by aptamers(i.e.,theCEA/MH/Apt/Au)resulted in a slightly increasedRct(Fig.5A,d)and slightly decreased redox cur-rents(Fig.5B,d)45,46.As the affinity crosslinkingbetween the cis-diol groups and the PBA groupsresulted in the formation of the negatively-chargedphenylborates 53,54,the PBABr/CEA/MH/Apt/Au featured a significantly increased Rct(Fig.5A,e)andsignificantlydecreasedredoxcurrents(Fig.5B,e)relative to the CEA/MH/Apt/Au 45,46.AsthedenselypackedFcreporterscouldFig.2.(A)SWV curves of various electrodes.(B)CV curves of the Fc/PBABr/CEA/MH/Apt/Au at various scan rates.CEA,1.0 ngmL?1.Fig.3.SEMimageoftheFc/PBABr/CEA/MH/Apt/Au.CEA,1.0 ngmL?1.Journal of Electrochemistry,2023,29(6),2218001(4 of 9)significantly facilitate the charge transfer betweenthe electrode and the Fe(CN)63e/4e38,44,47,theFc/PBABr/CEA/MH/Apt/Aushowedasignifi-cantly decreased Rct(Fig.5A,f)and the significantlyincreasedredoxcurrents(Fig.5B,f)whencompared with the PBABr/CEA/MH/Apt/Au.Asthe increase of the Rctwas accompanied by thedecrease of the redox currents,while the decreaseof the Rctwas accompanied by the increase of theredox currents 48,the EIS results agreed wellwith the CV results 45,46,which validated thesuccessful preparation of the Fc/PBABr/CEA/MH/Apt/Au.3.4.Analytical performance of the electrochemicalCEA aptasensorUnder the optimal conditions,the analyticalperformanceoftheBA-eATRP-basedelectro-chemical CEA aptasensor was investigated.FromFig.6,it can be seen that the peak current increasedwith the increase of CEA concentration,indicatingFig.4.EDS mappings of(A)P,(B)B,(C)Br,and(D)Fe e