CN104614359A - 一种基于新型纳米拉曼球的microRNA的检测方法 - Google Patents
一种基于新型纳米拉曼球的microRNA的检测方法 Download PDFInfo
- Publication number
- CN104614359A CN104614359A CN201410773198.7A CN201410773198A CN104614359A CN 104614359 A CN104614359 A CN 104614359A CN 201410773198 A CN201410773198 A CN 201410773198A CN 104614359 A CN104614359 A CN 104614359A
- Authority
- CN
- China
- Prior art keywords
- raman
- spheres
- mirna
- nano
- hybrid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001069 Raman spectroscopy Methods 0.000 title claims abstract description 71
- 238000001514 detection method Methods 0.000 title claims abstract description 20
- 239000002679 microRNA Substances 0.000 title claims abstract description 19
- 108700011259 MicroRNAs Proteins 0.000 title description 12
- 239000000523 sample Substances 0.000 claims abstract description 16
- 230000035945 sensitivity Effects 0.000 claims abstract description 10
- 239000011324 bead Substances 0.000 claims abstract description 7
- 238000005096 rolling process Methods 0.000 claims abstract description 5
- 108020004414 DNA Proteins 0.000 claims abstract description 4
- 102100033072 DNA replication ATP-dependent helicase DNA2 Human genes 0.000 claims abstract description 3
- 101000927313 Homo sapiens DNA replication ATP-dependent helicase DNA2 Proteins 0.000 claims abstract description 3
- 108091070501 miRNA Proteins 0.000 claims abstract 7
- 102000053602 DNA Human genes 0.000 claims abstract 2
- 206010028980 Neoplasm Diseases 0.000 claims description 19
- 108020004707 nucleic acids Proteins 0.000 claims description 11
- 102000039446 nucleic acids Human genes 0.000 claims description 11
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 230000003321 amplification Effects 0.000 claims description 6
- 238000005516 engineering process Methods 0.000 claims description 6
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 6
- 239000002077 nanosphere Substances 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 4
- 229910021645 metal ion Inorganic materials 0.000 claims description 2
- 230000005684 electric field Effects 0.000 claims 1
- 238000009396 hybridization Methods 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 4
- 230000004048 modification Effects 0.000 abstract description 2
- 238000012986 modification Methods 0.000 abstract description 2
- 210000004881 tumor cell Anatomy 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract 1
- 230000010076 replication Effects 0.000 abstract 1
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical class [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 description 2
- 238000000975 co-precipitation Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- 230000009946 DNA mutation Effects 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000001973 epigenetic effect Effects 0.000 description 1
- 238000012921 fluorescence analysis Methods 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 230000009711 regulatory function Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明制备了一种新型的具有强拉曼信号的纳米杂化拉曼球，以该拉曼球为主体，设计了一种拉曼探针，该探针实现了对miRNA的高灵敏度、高选择性的检测。在拉曼球上修饰生物条码DNA1和DNA2。利用磁珠上修饰的捕获DNA捕获检测对象miRNA，miRNA与捕获链DNA3杂交，然后DNA3发生滚环复制得到一条长的DNA链，该长DNA链能分段与多条DNA2特异性杂交，亦即捕获多个纳米杂化拉曼球，产生超强拉曼信号。本发明通过成功制备纳米杂化拉曼球和巧妙的探针设计实现了高灵敏的miRNA拉曼分析，该探针可准确识别肿瘤细胞和正常细胞，稳定性和可信性高，在临床应用方面具有良好的前景。
Description
技术领域
本发明涉及一种基于金属-有机物杂化纳米拉曼球的拉曼探针制备及其对核酸类肿瘤标志物（如microRNA-21）的高灵敏度检测，主要内容为制备新型纳米拉曼球，并以此为核心设计制备新型拉曼探针，并结合表面增强拉曼光谱技术（SERS）对核酸类肿瘤标志物进行高灵敏度检测，并能清楚的判断肿瘤细胞和正常细胞。
背景技术
核酸类肿瘤标志物在肿瘤的预警、诊断和治疗中起着至关重要的作用，其多样化的特点为研发高灵敏度的检测方法提供了便利。核酸类肿瘤标志物包括：RNA 标志物、肿瘤相关病毒 DNA、DNA 突变及DNA 甲基化。其中microRNA (miRNA) 是一类长度约为20-24个核苷酸的内生小RNA，对生命体有多种重要的调节作用。研究显示，miRNA参与细胞的增殖、凋亡和分化，表观遗传学沉默、转录因子失调等会导致miRNA 的表达下降，进而导致肿瘤的发生，例如弥漫性大 B 细胞淋巴瘤病人血清 中miRNA-21 水平明显高于正常人，血清 miRNA-92 的水平也被用作结直肠癌的肿瘤标志物。因此，miRNA是一种很有发展前景的核酸类肿瘤标志物，对肿瘤的早期诊断和治疗有重要的意义。
针对核酸类肿瘤标志物的检测方法经历了数十年的发展，现已开发出多种检测技术，如酶联免疫吸附测定（ELISA）、放射免疫法、荧光法、比色法和电化学法等。每种方法在灵敏度、选择性、稳定性、检测时间、成本、操作性等方面都各有其优缺点。然而，癌症早期患者血清中肿瘤标志物浓度仅为10-12-10-16M，目前的临床免疫测定法能测定的最低浓度仅为pM，无法满足现代医学早期诊断的要求。目前科研工作者重点研发了基于荧光、电化学和表面等离子共振的生物传感器，已经取得了较大的进展。
表面增强拉曼散射（SERS）近年来在生物和化学传感方面的应用较为活跃。相对荧光分析、电化学分析和酶联免疫分析，SERS具有以下优势：有效避免光致褪色、背景低、有效增强分子的拉曼强度，灵敏度高。因此SERS有望实现对核酸肿瘤标志物的高灵敏度高选择性的测定。
为了简化核酸肿瘤标志物的检测步骤提高其检测灵敏度和选择性，该专利制备表面修饰银纳米粒子的金属-有机物杂化纳米拉曼球，结合生物条码技术和滚环放大技术制备基于该拉曼球的纳米探针。只有在核酸肿瘤标志物（以miRNA-21为例）存在时，磁珠才能捕获具有强拉曼信号的纳米杂化拉曼球，才能检测到拉曼信号，以此实现对核酸肿瘤标志物的高灵敏度高选择性的检测。
发明内容
本发明的目的是：以miRNA-21为模型，制备一种能快速、高灵敏度、高选择性检测miRNA的拉曼探针，为实现肿瘤的早期预警和诊断提高依据。
本发明提出的可对miRNA-21进行拉曼检测的纳米探针制作路线图如图2所示。以纳米杂化拉曼球为主题，结合生物条码和滚环放大技术，制得该拉曼探针。
本发明通过以下技术方案来实现：
(1)具有强拉曼活性的纳米杂化拉曼球由三部分组成：由拉曼活性分子和Cu2+发生沉淀共价结合生成纳米球，对纳米球和银纳米粒子（AgNPs）表面进行表面功能化修饰，使上述两者通过氢键结合，得到AgNPs修饰的纳米杂化拉曼球，在拉曼球表面做生物条码；
(2)以磁珠（MB）为载体，将待测miRNA负载在磁珠上，磁珠上的miRNA与捕获链杂化，捕获链发生滚环放大后将捕获若干个生物条码修饰的纳米杂化拉曼球，实现信号的放大，将最终样品滴在金片上即可测得相应拉曼信号，如图2所示。
本发明的主要创新性和优越性为：
本发明基于拉曼活性分子和金属离子的共沉淀作用制备了纳米球，在该纳米球上修饰了AgNPs，制备了AgNPs修饰的拉曼球，并以此为主体制备了拉曼探针，实现了对miRNA-21的高灵敏拉曼检测。与已有的检测方法相比，本发明具备以下优点：
(1) 本发明采用共沉淀法制备了具有拉曼活性的新型纳米杂化拉曼球；
(2) 本发明设计的拉曼球表面通过氢键修饰有大量的AgNPs，能非常可观的增强拉曼球的拉曼信号；
(3) 本发明利用条码技术和滚环放大技术使一条miRNA链能链接一条超长捕获链，从而能捕获若干个拉曼球，进一步放大信号；
(4) 本发明所述的拉曼探针制备和操作方法简便快捷，灵敏度高，检测限达10-16，能达到肿瘤预警和早期诊断的要求。
附图说明
图1 为拉曼球的TEM图。
图2为基于金属-有机物纳米杂化拉曼球的拉曼探针的制备过程示意图。
图3 为正常细胞和Hela细胞中miRNA-21拉曼检测的结果示意图。
实施例
以下为实施本发明的具体示例，其作用在于进一步阐明本发明的内容，使阅读者更容易理解，但不构成对本发明要求的保护范围的限定或限制。
实施例一
结合图2，合成具有强拉曼信号的纳米杂化拉曼球。在40-60μl (0.04M)拉曼活性物质的DMSO溶液中，逐滴加入1mL二次水，室温搅拌过夜，然后快速加入20-30μl 0.15M的Cu(OAc)2，95℃反应1h。离心去除上清，分散于1mL PBS （pH7.4）中，得到纳米杂化拉曼球。将DNA1和DNA2以90:1的比例，37℃与纳米杂化拉曼球反应4h，离心去除上清，重新分散于1mL PBS （pH7.4）中，得到生物条码修饰的纳米杂化拉曼球，4℃保存。
实施例二
结合图2，利用拉曼探针进行细胞内miRNA的拉曼检测。以HeLa细胞和正常细胞为基本模型(0.5mL，1×106mL-1)，超声破碎，然后与生物条码修饰的纳米杂化拉曼球、DNA3修饰的磁珠、T4连接酶、DNA聚合酶混合，调节pH为7.4，温育2h。然后将样品滴在金片上，进行拉曼检测。比较两者信号，可明显看出HeLa细胞中miRNA的水平明显高于正常细胞的水平。
专利中所用DNA序列
Claims (4)
1.一种具有强拉曼信号的纳米杂化拉曼球，其技术核心是能产生拉曼信号的有机分子与金属离子发生共价沉淀形成纳米球，在其表面修饰AgNPs，利用表面等离子体的共振放大实现巨大的电场增强效应，制备强拉曼信号的纳米杂化拉曼球。
2.一种可对miRNA进行高灵敏度拉曼检测的拉曼探针，其特征为以AgNPs修饰的拉曼球为信号核心，待测miRNA负载在磁珠上并与捕获链杂化，捕获链发生滚环放大后通过与修饰在拉曼球上的DNA2杂化捕获若干个拉曼球，实现拉曼信号的二次放大。
3.根据权利要求1和2所述的拉曼探针，其优势在于纳米杂化拉曼球本身的拉曼信号由于表面AgNPs的作用就很强；其高特异性在于待测miRNA能特异性与捕获链杂化，从而能将多个拉曼球聚集到一条DNA链上，实现信号的进一步富集。
4.根据权利要求1、2和3，该拉曼探针能高特异性、高灵敏度、高生物相容性的识别检测实际样品中的核酸肿瘤标志物，如miRNA-21。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410773198.7A CN104614359A (zh) | 2014-12-16 | 2014-12-16 | 一种基于新型纳米拉曼球的microRNA的检测方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410773198.7A CN104614359A (zh) | 2014-12-16 | 2014-12-16 | 一种基于新型纳米拉曼球的microRNA的检测方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104614359A true CN104614359A (zh) | 2015-05-13 |
Family
ID=53148904
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410773198.7A Pending CN104614359A (zh) | 2014-12-16 | 2014-12-16 | 一种基于新型纳米拉曼球的microRNA的检测方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104614359A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021248691A1 (zh) * | 2020-06-10 | 2021-12-16 | 青岛科技大学 | 一种拉曼增强基底及其制备方法和检测miRNA的方法 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206357A (zh) * | 2011-03-28 | 2011-10-05 | 复旦大学 | 一种sers标签微球及其制备方法 |
| CN102323412A (zh) * | 2011-08-09 | 2012-01-18 | 中国科学院合肥物质科学研究院 | 一种拉曼编码微球的用途及利用拉曼编码微球检测肿瘤标志物的方法 |
| CN102423670A (zh) * | 2011-08-09 | 2012-04-25 | 中国科学院合肥物质科学研究院 | 一种拉曼编码微球及其制备方法 |
| CN103127890A (zh) * | 2013-03-07 | 2013-06-05 | 复旦大学 | 一种拉曼增强活性微球及其制备方法和应用 |
-
2014
- 2014-12-16 CN CN201410773198.7A patent/CN104614359A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206357A (zh) * | 2011-03-28 | 2011-10-05 | 复旦大学 | 一种sers标签微球及其制备方法 |
| CN102323412A (zh) * | 2011-08-09 | 2012-01-18 | 中国科学院合肥物质科学研究院 | 一种拉曼编码微球的用途及利用拉曼编码微球检测肿瘤标志物的方法 |
| CN102423670A (zh) * | 2011-08-09 | 2012-04-25 | 中国科学院合肥物质科学研究院 | 一种拉曼编码微球及其制备方法 |
| CN103127890A (zh) * | 2013-03-07 | 2013-06-05 | 复旦大学 | 一种拉曼增强活性微球及其制备方法和应用 |
Non-Patent Citations (5)
| Title |
|---|
| SUJUAN YE ET AL.: "Enzyme-based signal amplification of surface-enhanced Raman scattering in cancer-biomarker detection", 《TRENDS IN ANALYTICAL CHEMISTRY》 * |
| SUJUAN YE ET AL.: "Surface-enhanced Raman scattering assay combined with autonomous DNA machine for detection of specific DNA and cancer cells", 《CHEMICAL COMMUNICATION》 * |
| YING LI ET AL.: "Proximity-dependent isothermal cycle amplification for small-molecular detection based on surface enhanced Raman scattering", 《BIONSENSORS AND BIOELECTRONICS》 * |
| 何鹏: "基于酶循环放大技术的生物传感器分析研究及其在肿瘤标志物检测中的应用", 《万方学文论文》 * |
| 叶素娟: "基于DNA循环放大技术的表面增强拉曼散射传感分析方法研究及在肿瘤标志物检测中的应用", 《万方学位论文》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021248691A1 (zh) * | 2020-06-10 | 2021-12-16 | 青岛科技大学 | 一种拉曼增强基底及其制备方法和检测miRNA的方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jin et al. | ExoAPP: exosome-oriented, aptamer nanoprobe-enabled surface proteins profiling and detection | |
| Xiong et al. | Recent progress in detection and profiling of cancer cell‐derived exosomes | |
| Li et al. | Emerging nanotechnologies for liquid biopsy: The detection of circulating tumor cells and extracellular vesicles | |
| Xia et al. | A visible and colorimetric aptasensor based on DNA-capped single-walled carbon nanotubes for detection of exosomes | |
| Kim et al. | Three-dimensional hierarchical plasmonic nano-architecture based label-free surface-enhanced Raman spectroscopy detection of urinary exosomal miRNA for clinical diagnosis of prostate cancer | |
| Wang et al. | Advanced on-site and in vitro signal amplification biosensors for biomolecule analysis | |
| Zhang et al. | Application of electrochemical biosensors in tumor cell detection | |
| Zhu et al. | Colorimetric detection of immunomagnetically captured rare number CTCs using mDNA-wrapped single-walled carbon nanotubes | |
| Choi et al. | Nanomaterial-based fluorescence resonance energy transfer (FRET) and metal-enhanced fluorescence (MEF) to detect nucleic acid in cancer diagnosis | |
| Chen et al. | CRISPR/Cas12a-based electrochemical biosensor for highly sensitive detection of cTnI | |
| Cao et al. | Nonenzymatic chemiluminescence detection of circulating tumor cells in blood based on Au@ luminol nanoparticles, hybridization chain reaction and magnetic isolation | |
| CN104597240A (zh) | 石墨烯/类过氧化物酶双信号放大检测白血病的生物传感方法 | |
| Bhardwaj et al. | Recent advancement in the detection of potential cancer biomarkers using the nanomaterial integrated electrochemical sensing technique: a detailed review | |
| Wu et al. | Self-templated formation of aptamer-functionalized copper oxide nanorods with intrinsic peroxidase catalytic activity for protein and tumor cell detection | |
| Ding et al. | Magnetic-assisted self-assembled aptamer/protein hybrid probes for efficient capture and rapid detection of cancer cells in whole blood | |
| CN104089936B (zh) | 基于生物传感器对荧光标记的mcf肿瘤标志物检测的方法 | |
| Liao et al. | Nurturing the marriages of urinary liquid biopsies and nano‐diagnostics for precision urinalysis of prostate cancer | |
| Huang et al. | The application of nucleic acid probe–based fluorescent sensing and imaging in cancer diagnosis and therapy | |
| CN105274195B (zh) | 一种检测癌症标志物微小核糖核酸的试剂盒 | |
| Li et al. | An excellent colorimetric aptasensor integrating multifunctional SNAs and TdT-induced dual signal amplification for rapid sensitive detection of exosomes | |
| Wu et al. | Biomolecules-mediated electrochemical signals of Cu2+: Y-DNA nanomachines enable homogeneous rapid one-step assay of lung cancer circulating tumor cells | |
| Shu et al. | A novel chemiluminescence biosensor based on dual aptamers bound nanoparticles with multi-site signal amplification for sensitive detection of carcinoembryonic antigen | |
| Niazi et al. | A universal ratiometric method for Micro-RNA detection based on the ratio of electrochemical/electrochemiluminescence signal, and toehold-mediated strand displacement amplification | |
| Li et al. | An interference-free and label-free sandwich-type magnetic silicon microsphere-rGO-based probe for fluorescence detection of microRNA | |
| Gan et al. | Biobarcode and sequence-responsive DNA nanocapsules for label-free and sensitive electrochemical detection of circulating tumor cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150513 |