基于三明治SERS结构和酶剪切技术的肿瘤标志物miRNA-21的高灵敏检测
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摘要
采用Langmiur-Bloggt膜技术和磁控溅射技术制备Ag覆盖聚苯乙烯小球六方密堆积阵列(Ag/PS HCA)基底,再将合成的海胆状Au纳米粒子与4-巯基苯甲酸(4-Mercaptobenzoic acid,4MBA)链接得到Au@4MBA探针,然后将单链寡核苷酸DNA21分别与基底和Au@4MBA探针链接,构建Au@4MBA-DNA21-Ag/PS HCA三明治结构,在DNA21与miRNA-21杂交后使用双链特异性剪切酶(DSN)剪切DNA磷酸二酯键,最后进行表面增强拉曼散射信号检测.实验结果表明,基于上述三明治表面增强拉曼散射结构和酶剪切技术进行肿瘤标志物miRNA-21的检测,在100pmol·L~(-1)到1fmol·L~(-1)的浓度范围内,检测极限达到0.853fmol·L~(-1),具有极高的灵敏度和优良的特异性.
An Ag-coated polystyrene pellet hexagonal close packed array(Ag/PS HCA)substrate was prepared by L-B film method and magnetron sputtering technique.And Au@4MBA probes were obtained by linking the synthesized urchin-like Au nanoparticles with 4-mercaptobenzoic acid(4MBA).Then thesingle-stranded oligonucleotide DNA21 was linked with the substrate and the Au@4MBA probes,respectively,to construct the Au@4MBA-DNA21-Ag/PS HCA sandwich structure.After DNA21 and miRNA-21 hybridization,the DNA phosphodiester bond is cleaved using a double-strand specific cleavage enzyme(DSN),and finally SERS signal detection was performed.The experimental results show that the detection scheme of the tumor marker miRNA-21 based on the above-mentioned sandwich SERS structure and enzymatic cleavage technology has extremely high sensitivity and excellent specificity,and the detection limit reaches 0.853 fmol· L~(-1).In addition,compared with real-time fluorescence quantitative polynucleotide chain reaction(RT-qPCR),our scheme not only has the same results also has higher sensitivity.
An Ag-coated polystyrene pellet hexagonal close packed array(Ag/PS HCA)substrate was prepared by L-B film method and magnetron sputtering technique.And Au@4MBA probes were obtained by linking the synthesized urchin-like Au nanoparticles with 4-mercaptobenzoic acid(4MBA).Then thesingle-stranded oligonucleotide DNA21 was linked with the substrate and the Au@4MBA probes,respectively,to construct the Au@4MBA-DNA21-Ag/PS HCA sandwich structure.After DNA21 and miRNA-21 hybridization,the DNA phosphodiester bond is cleaved using a double-strand specific cleavage enzyme(DSN),and finally SERS signal detection was performed.The experimental results show that the detection scheme of the tumor marker miRNA-21 based on the above-mentioned sandwich SERS structure and enzymatic cleavage technology has extremely high sensitivity and excellent specificity,and the detection limit reaches 0.853 fmol· L~(-1).In addition,compared with real-time fluorescence quantitative polynucleotide chain reaction(RT-qPCR),our scheme not only has the same results also has higher sensitivity.
引文
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[25]LWei-feng,GU Chen-jie,ZENG Shu-wen,et al.One-pot synthesis of multi-branch gold nanoparticles and investigation of their SERS performance[J].Biosensors,2018,8(4):113-123.
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[27]HE R X,LIANG R,PENG P,et al.Effect of the size of silver nanoparticles on SERS signal enhancement[J].Journal of Nanoparticle Research,2017,19(8):267-276.
[2]DUFFY M J,LAMERZ R,HAGLUND C,et al.Tumor markers in colorectal cancer,gastric cancer and gastrointestinal stromal cancers:European group on tumor markers 2014guidelines update[J].International Journal of Cancer,2014,134(11):2513-2522.
[3]CHEN Yan.The principal of the clinical application of tumor markers[J].Medical Latoratory Science and Clinices,2006,17(1):1-2+12.陈燕.肿瘤标志物临床应用原则[J].医学检验与临床,2006,17(1):1-2+12.
[4]FAN Zhen-fu,CHEN Zhi-zhou,XU Zhi-xiong,et al.Immunoradiometric and immunoenzymemetric assays for tumorassociated antigen CA19-9[J].Labeled Immunoassays and Clinical Medicine,1994,1(2):72-75.范振符,陈智周,许智雄,等.肿瘤相关抗原CA19-9免疫放射分析(IRMA)与免疫酶标分析(IEMA)[J].标记免疫分析与临床,1994,1(2):72-75.
[5]ENGVALL E,PERLMANN P.Enzyme-linked immunosorbent assay,ELISA:III.Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes[J].The Journal of Immunology,1972,109(1):129-135.
[6]ZHAO L,SUN L,CHU X.Chemiluminescence immunoassay[J].TrAC Trends in Analytical Chemistry,2009,28(4):404-415.
[7]PERTSCHUK L P,TOBIN E H,BRIGATI D J,et al.Immunofluorescent detection of estrogen receptors in breast cancer.Comparison with dextran-coated charcoal and sucrose gradient assays[J].Cancer,1978,41(3):907-911.
[8]DIAMANDIS E P.Immunoassays with time-resolved fluorescence spectroscopy:principles and applications[J].Clinical Biochemistry,1988,21(2):139-150.
[9]KUBISTA M,ANDRADE J M,BENGTSSON M,et al.The real-time polymerase chain reaction[J].Molecular Aspects of Medicine,2006,27(2-3):95-125.
[10]PAULETTI G,GODOLPHIN W,PRESS M F,et al.Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization[J].Oncogene,1996,13(1):63-72.
[11]GASSER R B,HU M,CHILTON N B,et al.Single-strand conformation polymorphism(SSCP)for the analysis of genetic variation[J].Nature Protocols,2006,1(6):3121-3128.
[12]BROWN T.Southern blotting[J].Current Protocols in Molecular Biology,1993,21(1):2-9.
[13]BANTZ K C,MEYER A F,WITTENBERG N J,et al.Recent progress in SERS biosensing[J].Physical Chemistry Chemical Physics,2011,13(24):11551-11567.
[14]GAO Yong-feng,FENG Yuan-hui,ZHOU Lu,et al.Ultrasensitive SERS-based immunoassay of tumor marker in serum using Au-Ag alloy nanoparticles and Ag/AgBr hybrid nanostructure[J].Nano,2018,13(1):1850001-1850012.
[15]WANG Zhe,YANG Hong-qin,WANG Miao-hao,et al.SERS-based multiplex immunoassay of tumor markers using double SiO2@Ag immune probes and gold-film hemisphere array immune substrate[J].Colloids and Surfaces A:Physicochemical and Engineering Aspects,2018,546:48-58.
[16]LIANG Zhao-heng,ZHOU Jun,PETTI L,et al.SERS-based cascade amplification bioassay protocol of miRNA-21by using sandwich structure with biotin-streptavidin system[J].Analyst,2019,144(5):1741-1750.
[17]ZHANG Hao,FU Chao-peng,YU Yi,et al.Magnetic-based SERS approach for highly sensitive and reproducible detection of cancer-related serum microRNAs[J].Analytical Methods,2018,10:624-633.
[18]CHENG L,SHARPLES R A,SCICLUNA B J,et al.Exosomes provide a protective and enriched source of miRNA for biomarker profiling compared to intracellular and cell-free blood[J].Journal of Extracell Vesicles,2014,3(1):17-27.
[19]WANG Zhao-xia,BIAN Hai-bo,WANG Ji-rong,et al.Prognostic significance of serum miRNA-21expression in human non-small cell lung cancer[J].Journal of Surgical Oncology,2011,104(7):847-851.
[20]LIU Lin,GAO Yan-ping,LIU Hui-ping,et al.An ultrasensitive electrochemical miRNAs sensor based on miRNAsinitiated cleavage of DNA by duplex-specific nuclease and signal amplification of enzyme plus redox cycling reaction[J].Sensors and Actuators B:Chemical,2015,208:137-142.
[21]WENG Ke-gui,WANG Ying,JIANG Yong.Effect of microRNA-21on the proliferation and invasion of non-small cell lung cancer through targeting Wnt/β-catenin pathway[J].Chinese Journal of Histochemistry and Cytochemistry,2018,27(6):514-520.翁克贵,王颖,蒋勇.miRNA-21靶向调控Wnt/β-catenin对非小细胞肺癌细胞增殖与侵袭的影响[J].中国组织化学与细胞化学杂志,2018,27(6):514-520.
[22]LIN Hao,CHEUNG H Y,XIU Fei,et al.Developing controllable anisotropic wet etching to achieve silicon nanorods,nanopencils and nanocones for efficient photon trapping[J].Journal of Materials Chemistry A,2013,1(34):9942-9946.
[23]KELLY P J,ARNELL R D.Magnetron sputtering:a review of recent developments and applications[J].Vacuum,2000,56(3):159-172.
[24]XIAO Xing-ning,ZHU Long-jiao,LI Xiang-yang,et al.Research progress on duplex-specific nuclease mediated biosensors[J].Biotechnology Bulletin,2018,34(9):45-53.肖星凝,朱龙佼,李相阳,等.关于双链特异性核酸酶介导的生物传感器研究进展[J].生物技术通报,2018,34(9):45-53.
[25]LWei-feng,GU Chen-jie,ZENG Shu-wen,et al.One-pot synthesis of multi-branch gold nanoparticles and investigation of their SERS performance[J].Biosensors,2018,8(4):113-123.
[26]SAVEROT S,GENG X,LENG W,et al.Facile,tunable,and SERS-enhanced HEPES gold nanostars[J].RSCAdvances,2016,6(35):29669-29673.
[27]HE R X,LIANG R,PENG P,et al.Effect of the size of silver nanoparticles on SERS signal enhancement[J].Journal of Nanoparticle Research,2017,19(8):267-276.