Boronate Affinity-Assisted Electrochemically Controlled ATRP for Ultrasensitive Electrochemical Aptasensing of Carcinoembryonic Antigen

Abstract

As an acidic glycoprotein, carcinoembryonic antigen (CEA) is of great value as a broad-spectrum tumor marker in the differential diagnosis and surveillance of malignant tumors. In this work, we report an electrochemical aptasensor for the ultrasensitive and highly selective detection of CEA, taking advantage of the dual amplification by the boronate affinity-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 phenylboronic acid (PBA) group and the cis-diol group of the monosaccharide residues, and the collection of the ferrocene (Fc) reporters via the eATRP of ferrocenylmethyl methacrylate (FcMMA). As CEA is decorated with hundreds of cis-diol groups, 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, leading to 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 as 0.34 pg·mL⁻¹, with a linear range of 1.0−1,000 pg·mL⁻¹. Besides, this aptasensor has been successfully applied to the quantitative analysis of CEA in human serum. The BA-eATRP-based electrochemical aptasensor is cost-effective and simple in operation, holding broad application prospect in the ultrasensitive and highly selective detection of CEA.

Keywords

boronate affinity / atom transfer radical polymerization / electrochemical aptasensor / carcinoembryonic antigen / tumor marker / signal amplification

References

[1] Fletcher R H. Carcinoembryonic antigen[J]. Ann. Intern. Med., 1986, 104(1): 66-73.

[2] Hall C, Clarke L, Pal A, Buchwald P, Eglinton T, Wakeman C, Frizelle F. A review of the role of carcinoembryonic antigen in clinical practice[J]. Ann. Coloproctol., 2019, 35(6): 294-305.

[3] Benchimol S, Fuks A, Jothy S, Beauchemin N, Shirota K, Stanners C P. Carcinoembryonic antigen, a human tumor marker, functions as an intercellular adhesion molecule[J]. Cell, 1989, 57(2): 327-334.

[4] Huang L T, Zeng Y Y, Liu X L, Tang D P. Pressure-based immunoassays with versatile electronic sensors for carcinoembryonic antigen detection[J]. ACS Appl. Mater. Interfaces, 2021, 13(39): 46440-46450.

[5] Qiu Z L, Shu J, Tang D P. Bioresponsive release system for visual fluorescence detection of carcinoembryonic antigen from mesoporous silica nanocontainers mediated optical color on quantum dot-enzyme-impregnated paper[J]. Anal. Chem., 2017, 89(9): 5152-5160.

[6] Yu Q L, Wang X, Duan Y X. Capillary-based three-dimensional immunosensor assembly for high-performance detection of carcinoembryonic antigen using laser-induced fluorescence spectrometry[J]. Anal. Chem., 2014, 86(3): 1518-1524.

[7] Xing T Y, Zhao J, Weng G J, Zhu J, Li J J, Zhao J W. Specific detection of carcinoembryonic antigen based on fluorescence quenching of hollow porous gold nanoshells with roughened surface[J]. ACS Appl. Mater. Interfaces, 2017, 9(42): 36632-36641.

[8] Fan G C, Zhu H, Du D, Zhang J R, Zhu J J, Lin Y. Enhanced photoelectrochemical immunosensing platform based on CdSeTe@CdS:Mn core-shell quantum dots-sensitized TiO₂ amplified by CuS nanocrystals conjugated signal antibodies[J]. Anal. Chem., 2016, 88(6): 3392-3399.

[9] Guan X X, Deng X X, Song J, Wang X Y, Wu S. Polydopamine with tailorable photoelectrochemical activities for the highly sensitive immunoassay of tumor markers[J]. Anal. Chem., 2021, 93(17): 6763-6769.

[10] Li J J, Zhang Y, Kuang X, Wang Z L, Wei Q. A network signal amplification strategy of ultrasensitive photoelectrochemical immunosensing carcinoembryonic antigen based on CdSe/melamine network as label[J]. Biosens. Bioelectron., 2016, 85: 764-770.

[11] Carneiro M C, Sousa-Castillo A, Correa-Duarte M A, Sales M G F. Dual biorecognition by combining molecularly-imprinted polymer and antibody in SERS detection. Application to carcinoembryonic antigen[J]. Biosens. Bioelectron., 2019, 146: 111761.

[12] Chon H, Lee S, Son S W, Oh C H, Choo J. Highly sensitive immunoassay of lung cancer marker carcinoembryonic antigen using surface-enhanced Raman scattering of hollow gold nanospheres[J]. Anal. Chem., 2009, 81(8): 3029-3034.

[13] Wang J, Cao Y, Xu Y Y, Li G X. Colorimetric multiplexed immunoassay for sequential detection of tumor markers[J]. Biosens. Bioelectron., 2009, 25(2): 532-536.

[14] Zhao L J, Wang J, Su D D, Zhang Y Y, Lu H Y, Yan X, Bai J, Gao Y, Lu G Y. The DNA controllable peroxidase mimetic activity of MoS₂ nanosheets for constructing a robust colorimetric biosensor[J]. Nanoscale, 2020, 12(37): 19420-19428.

[15] Zhou Y, Chen S H, Luo X L, Chai Y Q, Yuan R. Ternary electrochemiluminescence nanostructure of Au nanoclusters as a highly efficient signal label for ultrasensitive detection of cancer biomarkers[J]. Anal. Chem., 2018, 90(16): 10024-10030.

[16] Wang N N, Feng Y Q, Wang Y W, Ju H X, Yan F. Electrochemiluminescent imaging for multi-immunoassay sensitized by dual DNA amplification of polymer dot signal[J]. Anal. Chem., 2018, 90(12): 7708-7714.

[17] Yang L, Zhu W, Ren X, Khan M S, Zhang Y, Du B, Wei Q. Macroporous graphene capped Fe₃O₄ for amplified electrochemiluminescence immunosensing of carcinoembryonic antigen detection based on CeO₂@TiO₂[J]. Biosens. Bioelectron., 2017, 91: 842-848.

[18] Wu M S, Shi H W, He L J, Xu J J, Chen H Y. Microchip device with 64-site electrode array for multiplexed immunoassay of cell surface antigens based on electrochemiluminescence resonance energy transfer[J]. Anal. Chem., 2012, 84(9): 4207-4213.

[19] Gu X, She Z, Ma T, Tian S, Kraatz H B. Electrochemical detection of carcinoembryonic antigen[J]. Biosens. Bioelectron., 2018, 102: 610-616.

[20] Ji Y L, Guo J X, Ye B X, Peng G H, Zhang C, Zou L N. An ultrasensitive carcinoembryonic antigen electrochemical aptasensor based on 3D DNA nanoprobe and Exo III[J]. Biosens. Bioelectron., 2022, 196: 113741.

[21] Liang H H, Luo Y, Li Y Y, Song Y H, Wang L. An immunosensor using electroactive COF as signal probe for electrochemical detection of carcinoembryonic antigen[J]. Anal. Chem., 2022, 94(13): 5352-5358.

[22] Hu Q, Ma K F, Mei Y Q, He M H, Kong J M, Zhang X J. Metal-to-ligand charge-transfer: Applications to visual detection of β-galactosidase activity and sandwich immunoassay[J]. Talanta, 2017, 167: 253-259.

[23] Wang D F, Li Y Y, Lin Z Y, Qiu B, Guo L H. Surface-enhanced electrochemiluminescence of Ru@SiO₂ for ultrasensitive detection of carcinoembryonic antigen[J]. Anal. Chem., 2015, 87(12): 5966-5972.

[24] Qi J, Li B W, Zhou N, Wang X Y, Deng D M, Luo L Q, Chen L X. The strategy of antibody-free biomarker analysis by in-situ synthesized molecularly imprinted polymers on movable valve paper-based device[J]. Biosens. Bioelectron., 2019, 142: 111533.

[25] Liu Z, Lei S, Zou L N, Li G P, Xu L L, Ye B X. A label-free and double recognition-amplification novel strategy for sensitive and accurate carcinoembryonic antigen assay[J]. Biosens. Bioelectron., 2019, 131: 113-118.

[26] Li J, Xu L Q, Shen Y J, Guo L, Yin H, Fang X H, Yang Z J, Xu Q, Li H B. Superparamagnetic nanostructures for split-type and competitive-mode photoelectrochemical aptasensing[J]. Anal. Chem., 2020, 92(12): 8607-8613.

[27] Zeng X X, Ma S S, Bao J C, Tu W W, Dai Z H. Using graphene-based plasmonic nanocomposites to quench energy from quantum dots for signal-on photoelectrochemical aptasensing[J]. Anal. Chem., 2013, 85(24): 11720-11724.

[28] Zhang Y H, Li M J, Wang H J, Yuan R, Wei S P. Supersensitive photoelectrochemical aptasensor based on Br,N-codoped TiO₂ sensitized by quantum dots[J]. Anal. Chem., 2019, 91(16): 10864-10869.

[29] Ma C, Liu H Y, Zhang L N, Li H, Yan M, Song X R, Yu J H. Multiplexed aptasensor for simultaneous detection of carcinoembryonic antigen and mucin-1 based on metal ion electrochemical labels and Ru(NH₃)₆³⁺ electronic wires[J]. Biosens. Bioelectron., 2018, 99: 8-13.

[30] Yang H Q, Xu Y, Hou Q Q, Xu Q Z, Ding C F. Magnetic antifouling material based ratiometric electrochemical biosensor for the accurate detection of CEA in clinical serum[J]. Biosens. Bioelectron., 2022, 208: 114216.

[31] Zhai X J, Wang Q L, Cui H F, Song X, Lv Q Y, Guo Y. A DNAzyme-catalyzed label-free aptasensor based on multifunctional dendrimer-like DNA assembly for sensitive detection of carcinoembryonic antigen[J]. Biosens. Bioelectron., 2021, 194: 113618.

[32] Wang Q L, Cui H F, Song X, Fan S F, Chen L L, Li M M, Li Z Y. A label-free and lectin-based sandwich aptasensor for detection of carcinoembryonic antigen[J]. Sens. Actuators, B, 2018, 260: 48-54.

[33] Paniagua G, Villalonga A, Eguílaz M, Vegas B, Parrado C, Rivas G, Díez P, Villalonga R. Amperometric aptasensor for carcinoembryonic antigen based on the use of bifunctionalized Janus nanoparticles as biorecognition-signaling element[J]. Anal. Chim. Acta, 2019, 1061: 84-91.

[34] Guo C P, Su F F, Song Y P, Hu B, Wang M H, He L H, Peng D L, Zhang Z H. Aptamer-templated silver nanoclusters embedded in zirconium metal-organic framework for bifunctional electrochemical and SPR aptasensors toward carcinoembryonic antigen[J]. ACS Appl. Mater. Interfaces, 2017, 9(47): 41188-41199.

[35] Wu Y F, Liu S Q, He L. Electrochemical biosensing using amplification-by-polymerization[J]. Anal. Chem., 2009, 81(16): 7015-7021.

[36] Yuan L, Wei W, Liu S Q. Label-free electrochemical immunosensors based on surface-initiated atom radical polymerization[J]. Biosens. Bioelectron., 2012, 38(1): 79-85.

[37] He P, Zheng W, Tucker E Z, Gorman C B, He L. Reversible addition-fragmentation chain transfer polymerization in DNA biosensing[J]. Anal. Chem., 2008, 80(10): 3633-3639.

[38] Hu Q, Han D X, Gan S Y, Bao Y, Niu, L. Surface-initiated-reversible-addition-fragmentation-chain-transfer polymerization for electrochemical DNA biosensing[J]. Anal. Chem., 2018, 90(20): 12207-12213.

[39] Wu Y F, Wei W, Liu S Q. Target-triggered polymerization for biosensing[J]. Acc. Chem. Res., 2012, 45(9): 1441-1450.

[40] Hu Q, Gan S Y, Bao Y, Zhang Y W, Han D X, Niu L. Controlled/“living” radical polymerization-based signal amplification strategies for biosensing[J]. J. Mater. Chem. B, 2020, 8(16): 3327-3340.

[41] Wang J S, Matyjaszewski K. Controlled/“living” radical polymerization. Atom transfer radical polymerization in the presence of transition-metal complexes[J]. J. Am. Chem. Soc., 1995, 117(20): 5614-5615.

[42] Magenau A J, Strandwitz N C, Gennaro A, Matyjaszewski K. Electrochemically mediated atom transfer radical polymerization[J]. Science, 2011, 332(6025): 81-84.

[43] Hu Q, Wang Q W, Sun G Z, Kong J M, Zhang X J. Electrochemically mediated surface-initiated de novo growth of polymers for amplified electrochemical detection of DNA[J]. Anal. Chem., 2017, 89(17): 9253-9259.

[44] Hu Q, Gan S Y, Bao Y, Zhang Y W, Han D X, Niu L. Electrochemically controlled ATRP for cleavage-based electrochemical detection of the prostate-specific antigen at femtomolar level concentrations[J]. Anal. Chem., 2020, 92(24): 15982-15988.

[45] Hu Q, Hu S H, Li S Q, Liu S J, Liang Y Y, Cao X J, Luo Y L, Xu W J, Wang H C, Wan J W, Feng W X, Niu L. Boronate affinity-based electrochemical aptasensor for point-of-care glycoprotein detection[J]. Anal. Chem., 2022, 94(28), 10206-10212.

[46] Hu Q, Wan J W, Wang H C, Cao X J, Li S Q, Liang Y Y, Luo Y L, Wang W, Niu L. Boronate-affinity cross-linking-based ratiometric electrochemical detection of glycoconjugates[J]. Anal. Chem., 2022, 94(26), 9481-9486.

[47] Hu Q, Su L F, Chen Z H, Huang Y Y, Qin D D, Niu L. Coenzyme-mediated electro-RAFT polymerization for amplified electrochemical interrogation of trypsin activity[J]. Anal. Chem., 2021, 93(27): 9602-9608.

[48] Hu Q, Su L F, Luo Y L, Cao X J, Hu S H, Li S Q, Liang Y Y, Liu S J, Xu W J, Qin D D, Niu L. Biologically mediated RAFT polymerization for electrochemical sensing of kinase activity[J]. Anal. Chem., 2022, 94(16): 6200-6205.

[49] Fu Z F, Liu H, Ju H X. Flow-through multianalyte chemiluminescent immunosensing system with designed substrate zone-resolved technique for sequential detection of tumor markers[J]. Anal. Chem., 2006, 78(19): 6999-7005.

[50] Zhou L L, Wang Y J, Xing R R, Chen J, Liu J, Li W, Liu Z. Orthogonal dual molecularly imprinted polymer-based plasmonic immunosandwich assay: A double characteristic recognition strategy for specific detection of glycoproteins[J]. Biosens. Bioelectron., 2019, 145: 111729.

[51] Wu X, Li Z, Chen X X, Fossey J S, James T D, Jiang Y B. Selective sensing of saccharides using simple boronic acids and their aggregates[J]. Chem. Soc. Rev., 2013, 42(20): 8032-8048.

[52] Zhang W, Liu W, Li P, Xiao H B, Wang H, Tang B. A fluorescence nanosensor for glycoproteins with activity based on the molecularly imprinted spatial structure of the target and boronate affinity[J]. Angew. Chem. Int. Ed., 2014, 53(46): 12489-12493.

[53] Li D J, Chen Y, Liu Z. Boronate affinity materials for separation and molecular recognition: Structure, properties and applications[J]. Chem. Soc. Rev., 2015, 44(22): 8097-8123.

[54] Ye J, Chen Y, Liu Z. A boronate affinity sandwich assay: An appealing alternative to immunoassays for the determination of glycoproteins[J]. Angew. Chem. Int. Ed., 2014, 53(39): 10386-10389.

[55] Wu L L, Wang Y D, Xu X, Liu Y L, Lin B Q, Zhang M X, Zhang J L, Wan S, Yang C Y, Tan W H. Aptamer-based detection of circulating targets for precision medicine[J]. Chem. Rev., 2021, 121(19): 12035-12105.

[56] Tan W, Donovan M J, Jiang J. Aptamers from cell-based selection for bioanalytical applications[J]. Chem. Rev., 2013, 113(4): 2842-2862.

[57] Zhu Z, Song Y, Li C, Zou Y, Zhu L, An Y, Yang C J. Monoclonal surface display SELEX for simple, rapid, efficient, and cost-effective aptamer enrichment and identification[J]. Anal. Chem., 2014, 86(12): 5881-5888.