基于SiC@Ag基底和银-生物素-链霉亲和素纳米聚集体双重SERS放大的miRNA-106a检测
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摘要
基于SiC@Ag基底与Ag纳米颗粒的表面增强拉曼散射效应,提出了利用银-生物素-链霉亲和素纳米聚集体二次表面增强拉曼散射放大的超灵敏miRNA-106a检测方案.首先,将地高辛修饰的捕获DNA与固定在SiC@Ag基底上的抗地高辛链接,制备SiC@Ag@anti-digoxin/digoxin-DNA基底;将4-巯基苯甲酸(4MBA)标记的银纳米颗粒与修饰有氨基和生物素的探针DNA链接,制备Ag@4MBA@DNA-biotin探针.然后将制备的基底、探针与待测miRNA-106a组成"三明治"结构,获得表面增强拉曼散射信号放大.最后,依次加入链霉亲和素和制备的探针,形成银-生物素-链霉亲和素纳米聚集体,实现检测信号的二次放大.实验结果表明,利用SiC@Ag基底和银-生物素-链霉亲和素纳米聚集体双重表面增强拉曼散射放大,可以实现miRNA-106a的超灵敏检测,检测极限达到0.579fmol/L,对于肿瘤的早期诊断具有应用潜力.
Based on the surface-enhanced Raman scattering enhancement effect of SiC@Ag substrate and Ag nanoparticles,an ultrasensitive miRNA-106 adetection protocol was proposed by using the second surface-enhanced Raman scattering amplification of the silver-biotin-streptavidin nanoaggregates.First,SiC@Ag@anti-digoxin/digoxin-DNA substrate was prepared by specifically binding of a capture DNA modified with a digoxigen group and an anti-digoxin immobilized on a SiC@Ag substrate.The Ag@4 MBA@DNA-biotin probe was prepared by linking the amino/biotin-modified probe DNA with the 4-mercaptobenzoic acid(4 MBA)immobilized silver nanoparticles.Then,the " sandwich" structure was constructed by Ag@4 MBA@DNA-biotin probe,target miRNA-106 aand SiC@ Ag@anti-digoxin/digoxin-DNA substrate to amplify the surface-enhanced Raman scattering signal.Finally,the streptavidinand the extra probes were alternately added into the "sandwich structure"to form the silver-biotinstreptavidin nanoaggregates and achieve secondary amplification of the detection signal.The experimental results show that the ultrasensitive detection of miRNA-106 ahas been implemented by using the dual surface-enhanced Raman scattering amplifications of SiC@ Ag substrate and silver-biotin-streptavidin nanoaggregates,arriving an ultralow detection limit of 0.579 fmol/L,which has a potential for early diagnosis of tumors.
Based on the surface-enhanced Raman scattering enhancement effect of SiC@Ag substrate and Ag nanoparticles,an ultrasensitive miRNA-106 adetection protocol was proposed by using the second surface-enhanced Raman scattering amplification of the silver-biotin-streptavidin nanoaggregates.First,SiC@Ag@anti-digoxin/digoxin-DNA substrate was prepared by specifically binding of a capture DNA modified with a digoxigen group and an anti-digoxin immobilized on a SiC@Ag substrate.The Ag@4 MBA@DNA-biotin probe was prepared by linking the amino/biotin-modified probe DNA with the 4-mercaptobenzoic acid(4 MBA)immobilized silver nanoparticles.Then,the " sandwich" structure was constructed by Ag@4 MBA@DNA-biotin probe,target miRNA-106 aand SiC@ Ag@anti-digoxin/digoxin-DNA substrate to amplify the surface-enhanced Raman scattering signal.Finally,the streptavidinand the extra probes were alternately added into the "sandwich structure"to form the silver-biotinstreptavidin nanoaggregates and achieve secondary amplification of the detection signal.The experimental results show that the ultrasensitive detection of miRNA-106 ahas been implemented by using the dual surface-enhanced Raman scattering amplifications of SiC@ Ag substrate and silver-biotin-streptavidin nanoaggregates,arriving an ultralow detection limit of 0.579 fmol/L,which has a potential for early diagnosis of tumors.
引文
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[2]SMITH K C.Aging,carcinogenesis,and radiation biology:the role of nucleic acid addition reactions[M].New York:Springer Science&Business Media,2013.
[3]ANKER P,MULCAHY H,CHEN Xu-qi,et al.Detection of circulating tumour DNA in the blood(plasma/serum)of cancer patients[J].Cancer and Metastasis Reviews,1999,18(1):65-73.
[4]AMERES S L,ZAMORE P D.Diversifying microRNA sequence and function[J].Nature Reviews Molecular Cell Biology,2013,14(8):475-488.
[5]SIRAVEGNA G,MARSONI S,SIENA S,et al.Integrating liquid biopsies into the management of cancer[J].Nature Reviews Clinical Oncology,2017,14(9):531-548.
[6]GASPARELLO J,ALLEGRETTI M,TREMANTE E,et al.Liquid biopsy in mice bearing colorectal carcinoma xenografts:gateways regulating the levels of circulating tumor DNA(ctDNA)and miRNA(ctmiRNA)[J].Journal of Experimental&Clinical Cancer Research,2018,37(1):124-134.
[7]CUI L,ZHANG X,YE G,et al.Gastric juice MicroRNAs as potential biomarkers for the screening of gastric cancer[J].Cancer,2013,119(9):1618-1626.
[8]IVKOVIC T C,ARALICA G,CACEV T,et al.miR-106aoverexpression and pRB downregulation in sporadic colorectal cancer[J].Experimental and Molecular Pathology,2013,94(1):148-154.
[9]LI P,XU Q,ZHANG D,et al.Upregulated miR-106aplays an oncogenic role in pancreatic cancer[J].FEBS Letters,2014,588(5):705-712.
[10]MOLTZAHN F,OLSHEN A B,BAEHNER L,et al.Microfluidic-based multiplex qRT-PCR identifies diagnostic and prognostic microRNA signatures in the sera of prostate cancer patients[J].Tumor and Stem Cell Biology,2011,71(2):550-560.
[11]ZHOU Yun-lei,ZHANG Zhao-yan,XU Zhen-ning,et al.MicroRNA-21detection based on molecular switching by amperometry[J].New Journal of Chemistry,2012,36(10):1985-1991.
[12]WIKTOR A E,VAN DYKE D L,STUPCA P J.et al.Preclinical validation of fluorescence in situ hybridization assays for clinical practice[J].Genetics in Medicine,2006,8(1):16-23.
[13]ZHANG Ye,ZHAO Shao-jie,ZHENG Jin-kai,et al.Surface-enhanced Raman spectroscopy(SERS)combined techniques for high-performance detection and characterization[J].Trac Trends in Analytical Chemistry,2017,90(1):1-13.
[14]KNEIPP J,KNEIPP H,KNEIPP K.SERS-a single-molecule and nanoscale tool for bioanalytics[J].Chemical Society Reviews,2008,37(5):1052-1060.
[15]YUAN Yu-feng,PANWAR N,YAP S H K,et al.A high-sensitivity fiber grating pressure sensor and its pressure sensing characteristics[J].Coordination Chemistry Reviews,2017,337(1):1-33.
[16]ZONG Shen-fei,WANG Le,CHEN Chen,et al.Facile detection of tumor-derived exosomes using magnetic nanobeads and SERS nanoprobes[J].Analytical Methods,2016,8(25):5001-5008.
[17]ZHANG Chen-meng,GAO Yu-kun,YANG Nan,et al.Direct determination of the tumor marker AFP via silver nanoparticle enhanced SERS and AFP-modified gold nanoparticles as capturing substrate[J].Microchimica Acta,2018,185(2):90-95.
[18]GE Ming,CUI Yan,GU Ren-ao.Progress in labeled immunoassay based on SERS[J].Spectroscopy and Spectral Analysis,2008,28(1):110-116.葛明,崔颜,顾仁敖.SERS标记免疫检测研究进展[J].光谱学与光谱分析,2008,28(1):110-116.
[19]SUN Yu-die,LI Tao.Composition-tunable hollow Au/Ag SERS nanoprobes coupled with target-catalyzed hairpin assembly for triple-amplification detection of miRNA[J].Analytical Chemistry,2018,90(19):11614-11621.
[20]LEE P C,MEISEL D.Adsorption and surface-enhanced Raman of dyes on silver and gold sols[J].The Journal of Physical Chemistry,1982,86(17):3391-3395.
[21]ZHANG Ran,XU Bin-bin,LIU Xue-qing,et al.Highly efficient SERS test strips[J].Chemical Communications,2012,48(47):5913-5915.
[22]FAN Mei-kun,ANDRADEC G F S,BROLO A G.A review on the fabrication of substrates for surface enhanced Raman spectroscopy and their applications in analytical chemistry[J].Analytica Chimica Acta,2011,693(1-2):7-25.
[23]ZHOU Lu,ZHOU Jun,FENG Zhao,et al.Immunoassay for tumor markers in human serum based on Si nanoparticles and SiC@Ag SERS-active substrate[J].Analyst,2016,141(8):2534-2541.
[24]SIVASHANMUGAN K,LIAO J D,LIU B H,et al.Focused-ion-beam-fabricated Au nanorods coupled with Ag nanoparticles used as surface-enhanced Raman scattering-active substrate for analyzing trace melamine constituents in solution[J].Analytica Chimica Acta,2013,800(1):56-64.
[25]WANG Ai-jun,LV Jing-jing,ZHOU Dan-ling,et al.Facile synthesis of ultrathin worm-like Au nanowires for highly active SERS substrates[J].New Journal of Chemistry,2014,38(8):3395-3400.
[26]LVAREZ-PUEBLA R A.Effects of the excitation wavelength on the SERS spectrum[J].Journal of Physical Chemistry Letters,2012,3(7):857-866.
[27]SHRIVASTAVA A,GUPTA V B.Methods for the determination of limit of detection and limit of quantitation of the analytical methods[J].Chronicles of Young Scientists,2011,2(1):21-25.
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