WO2021248691A1 - 一种拉曼增强基底及其制备方法和检测miRNA的方法 - Google Patents

一种拉曼增强基底及其制备方法和检测miRNA的方法 Download PDF

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WO2021248691A1
WO2021248691A1 PCT/CN2020/110189 CN2020110189W WO2021248691A1 WO 2021248691 A1 WO2021248691 A1 WO 2021248691A1 CN 2020110189 W CN2020110189 W CN 2020110189W WO 2021248691 A1 WO2021248691 A1 WO 2021248691A1
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raman
mirna
auxiliary
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tin oxide
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周宏�
丁可欣
刘静
刘树峰
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青岛科技大学
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    • C12Q1/6813—Hybridisation assays
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    • C12Q1/6825—Nucleic acid detection involving sensors
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    • C25D3/00—Electroplating: Baths therefor
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C25D5/00—Electroplating characterised by the process; Pretreatment or after-treatment of workpieces
    • C25D5/18—Electroplating using modulated, pulsed or reversing current
    • C—CHEMISTRY; METALLURGY
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    • C25D5/00—Electroplating characterised by the process; Pretreatment or after-treatment of workpieces
    • C25D5/60—Electroplating characterised by the structure or texture of the layers
    • C25D5/625—Discontinuous layers, e.g. microcracked layers
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    • C25D7/00—Electroplating characterised by the article coated
    • C25D7/12—Semiconductors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons

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  • the invention belongs to the technical field of miRNA detection, and specifically relates to a Raman enhanced substrate, a preparation method thereof, and a method for detecting miRNA.
  • miRNAs are short non-coding small RNAs that are widely present in eukaryotic cells and can regulate the expression of target gene mRNA.
  • the abnormal expression of miRNA is closely related to the occurrence of many major diseases, especially cancer. Therefore, miRNA, as a typical tumor marker, is receiving more and more attention.
  • the content of miRNA is often abnormal in tumor patients.
  • the expression levels of miRNA-155 and miRNA-210 are significantly increased in the blood of patients with diffuse large B-cell lymphoma (DLBCL).
  • the expression level of miRNA-21 is significantly increased in the serum of patients with ovarian cancer.
  • the expression level of miRNA can not only detect the occurrence of tumors, but also can be used as an indicator for evaluating the prognosis of tumor patients.
  • NSCLC non-small cell lung cancer
  • the survival time of patients with high expression of let-7 is significantly longer than that of patients with low expression
  • the survival time of patients with low expression of miRNA-17a is significantly longer than that of patients with high expression. Therefore, the study of miRNA molecular markers will help to explore the molecular mechanism of tumor formation and development in an all-round way, and provide guidance for tumor diagnosis and treatment.
  • Ultra-sensitive miRNA detection is of great significance for the early diagnosis of cancer and the development of targeted anti-cancer drugs.
  • the ultra-sensitive detection of miRNA faces many challenges. challenge.
  • Traditional detection methods such as microarray technology, quantitative fluorescent reverse transcription PCR, and biofluorescence assay are used for the quantitative detection of miRNA, but these methods are expensive, time-consuming, and complicated to operate, which limits these technologies to a certain extent.
  • the purpose of the present invention is a Raman-enhanced substrate, a preparation method thereof, and a method for detecting miRNA, so as to achieve highly sensitive and specific Raman detection of target miRNA.
  • the present invention provides a method for preparing a Raman-enhanced substrate, which includes the following steps: placing a cleaned and dried indium tin oxide glass chip in an electrolyte and using cyclic voltammetry for 50 cycles; the electrolyte includes the following Molar components: 0.03-0.1M phosphate solution, 0.03-0.1M KCl and 2.0-2.5mM HAuCl 4 ⁇ 4H 2 O.
  • the cleaning method includes: putting indium tin oxide glass in 2-propanol containing 2M KOH, boiling for 18-25 minutes, and then ultrasonically cleaning it in an ultrasonic bath of ethanol aqueous solution for 3-5 minutes.
  • the electrolyte is filled with nitrogen and maintained at a 60°C environment.
  • the amount of the electrolyte is 4 cm 2 of indium tin oxide glass chip/5 mL of electrolyte.
  • the cyclic voltammetry is to circulate the potential between -0.8V and 0.3V at a rate of 0.05V/s.
  • the present invention also provides a Raman-enhanced substrate prepared by the above-mentioned preparation method.
  • the Raman-enhanced substrate is a three-dimensional gold nano-film deposited on the surface of an indium tin oxide glass chip.
  • the present invention also provides a Raman-enhanced substrate prepared based on the above-mentioned preparation method or a method for constructing a Raman sensor of the Raman-enhanced substrate, which includes the following steps: (1) The molar ratio is 3.0:3.2:3.2.
  • Sulfhydryl-modified DNA1, auxiliary DNA2, and auxiliary DNA3 were reacted in Tris-HCl buffer for 10 minutes, cooled to 18-25°C, and allowed to stand for no less than 60 minutes to obtain a double helix capture probe DNA mixture; the sulfhydryl-modified DNA
  • the nucleotide sequence of DNA1 is shown in SEQ ID NO.1
  • the nucleotide sequence of auxiliary DNA2 is shown in SEQ ID NO.2
  • the nucleotide sequence of auxiliary DNA3 is shown in SEQ ID NO.3
  • the temperature of the reaction is 85°C;
  • the molar concentration of the phosphate buffer in step (3) is 0.01M, and the pH value is 7.0.
  • the present invention also provides a method for detecting miRNA using the Raman sensor constructed by the above construction method, which comprises the following steps: respectively dripping miRNA-21 and rhodamine 6G (R6G) modification at different concentrations on the Raman sensor Raman probe DNA mixture, after the reaction for 85 minutes, rinse with phosphate buffer, place it under a 633nm laser to measure the surface Raman spectrum intensity, and draw a standard curve based on the relationship between the target concentration and the Raman spectrum intensity. The standard curve is used to calculate the concentration of miRNA.
  • R6G rhodamine 6G
  • the present invention provides a method for preparing a Raman-enhanced substrate.
  • a three-dimensional gold nano-film is generated on the surface of an indium tin oxide (ITO) glass chip by an electro-reduction method, and the prepared unique three-dimensional gold nano-structure can produce more Active "hot spots" are a good Raman enhancement base.
  • the present invention constructs a Raman sensor based on the Raman enhanced substrate, and is used for detecting miRNA.
  • the present invention takes miRNA-21 as an example, uses a three-dimensional gold nanometer/ITO chip as a Raman enhancement substrate, and hybridizes sulfhydryl-modified DNA1 and two auxiliary DNAs (DNA2 and DNA3) to form a double helix DNA as a capture probe.
  • Raman enhances the substrate surface to form a Raman sensor.
  • the capture probe specifically binds to the target miRNA-21 and releases auxiliary DNA2; at this time, a large amount of Raman signal probes in the solution
  • a Toehold-mediated strand displacement reaction replaces the auxiliary DNA3 and the target strand miRNA-21, thereby releasing the target strand miRNA-21 ( Figure 2).
  • the released target strand continues to open other capture probes on the surface of the substrate.
  • Such a cyclic effect realizes the recycling of the target strand miRNA-21, and finally binds more Raman signal probes with rhodamine 6G on the surface of the substrate.
  • the Raman signal of Rhodamine 6G is enhanced by the substrate to achieve highly sensitive and selective detection of target miRNA-21.
  • Figure 1 is a scanning electron microscope image of constructing three-dimensional gold nanometers on the surface of an indium tin oxide (ITO) chip;
  • Figure 2 is a schematic diagram of miRNA Raman detection based on surface-enhanced Raman signal substrate and Toehold-mediated strand displacement amplification reaction;
  • Figure 3 shows the Raman response curve and the working curve, where A is the Raman response curve and B is the working curve;
  • Figure 4 is a selective experiment diagram.
  • the present invention provides a method for preparing a Raman-enhanced substrate, which includes the following steps: placing a cleaned and dried indium tin oxide glass chip in an electrolyte and using cyclic voltammetry for 50 cycles; the electrolyte includes the following Molar components: 0.03-0.1M phosphate solution, 0.03-0.1M KCl and 2.0-2.5mM HAuCl 4 ⁇ 4H 2 O.
  • the indium tin oxide glass chip is placed before the electrolyte, and the indium tin oxide glass chip is first cleaned and dried.
  • the cleaning preferably includes: putting the indium tin oxide glass into 2-propane containing 2M KOH
  • the alcohol is boiled for 18-25 minutes, and then placed in an ultrasonic bath of ethanol aqueous solution for ultrasonic cleaning for 3 to 5 minutes.
  • the boiling time of the present invention is preferably 20 min.
  • the volume percentage of ethanol in the ethanol aqueous solution of the present invention is preferably 75%.
  • the ultrasonic frequency of the ultrasonic cleaning of the present invention is preferably 100KHZ, and the time of ultrasonic cleaning is preferably 5min.
  • the cleaned indium tin oxide glass is dried, and the drying is preferably drying in an environment of 60°C.
  • the indium tin oxide glass is preferably cut into small pieces of 1 cm ⁇ 4 cm.
  • the electrolyte of the present invention preferably includes the following components in molar concentrations: 0.05M phosphate solution, 0.05M KCl, and 2.4mM HAuCl 4 ⁇ 4H 2 O.
  • the cyclic voltammetry of the present invention preferably circulates the potential between -0.8V and 0.3V at a rate of 0.05V/s.
  • the dosage of the electrolyte of the present invention is preferably 4 cm 2 of indium tin oxide glass chip/5 mL of electrolyte.
  • the color of the ITO electrode changed from colorless to yellow, indicating that the dimensional gold nanometer was successfully deposited on the electrode.
  • the present invention generates a layer of three-dimensional gold nano-film on the surface of indium tin oxide (ITO) glass chip by electro-reduction method, and the prepared unique three-dimensional gold nano-structure can generate more active "hot spots", which is a good stretch Man strengthens the base.
  • the present invention also provides a Raman-enhanced substrate prepared by the above-mentioned preparation method.
  • the Raman-enhanced substrate is a three-dimensional gold nano-film deposited on the surface of an indium tin oxide glass chip.
  • the present invention also provides a Raman-enhanced substrate prepared based on the above-mentioned preparation method or a method for constructing a Raman sensor of the Raman-enhanced substrate, which includes the following steps: (1) The molar ratio is 3.0:3.2:3.2.
  • Sulfhydryl-modified DNA1, auxiliary DNA2, and auxiliary DNA3 were reacted in Tris-HCl buffer for 10 minutes, cooled to 18-25°C, and allowed to stand for no less than 60 minutes to obtain a double helix capture probe DNA mixture; the sulfhydryl-modified DNA
  • the nucleotide sequence of DNA1 is shown in SEQ ID NO.1
  • the nucleotide sequence of auxiliary DNA2 is shown in SEQ ID NO.2
  • the nucleotide sequence of auxiliary DNA3 is shown in SEQ ID NO.3
  • the temperature of the reaction is 85°C;
  • sulfhydryl modified DNA1, auxiliary DNA2 and auxiliary DNA3 with a molar ratio of 3.0:3.2:3.2 are reacted in Tris-HCl buffer at 85°C for 10 minutes, and then cooled to 18-25°C, and then allowed to stand for no less than 60 minutes.
  • the double helix capture probe DNA mixture is obtained; the nucleotide sequence of the sulfhydryl modified DNA1 is shown in SEQ ID NO. 1, the nucleotide sequence of the auxiliary DNA 2 is shown in SEQ ID NO. 2, and the auxiliary DNA 3
  • the nucleotide sequence of is shown in SEQ ID NO.3; the temperature of the reaction is 85°C.
  • the Tris-HCl buffer solution of the present invention preferably further includes 0.1M NaCl, and the pH value is 7.4.
  • three DNA strands can hybridize with each other to form a double helix capture probe DNA during the static state.
  • miRNA-21 is used as an example to construct a Raman enhanced substrate and a Raman sensor, wherein the nucleotide sequence of sulfhydryl modified DNA1 is SEQ ID NO. 1: 5'-(SH)-TTTTTTGAA ATG GTGGAAAGGTAGGGTCAACATCAGTCTGATAAGCTA-3 ';
  • auxiliary DNA2 The nucleotide sequence of auxiliary DNA2 is SEQ ID NO. 2: 5'-TCAGACTGATGTTGACCCTATATCCATAAATT-3';
  • auxiliary DNA3 The nucleotide sequence of auxiliary DNA3 is SEQ ID NO. 3: 5'-CCTTTCCACCATTTC-3.
  • the present invention mixes the double helix capture probe DNA mixture with TCEP and incubates for 1 hour to obtain an incubation solution; the TCEP is mixed with sulfhydryl modified DNA1, auxiliary DNA2, and auxiliary DNA3
  • the molar ratio of the amount is 1000: (9-10).
  • the incubation temperature of the present invention is preferably 25°C.
  • the addition of the TCEP of the present invention can break the disulfide bond on the sulfhydryl DNA1.
  • the present invention adds the incubation solution dropwise to the surface of the Raman-enhanced substrate. After reacting for 6 hours in an environment of 0°C, it is washed with phosphate buffer solution 3 times, dried with purified nitrogen, and then placed The reaction was carried out in mercaptohexanol (MCH) for 2 hours, washed with phosphate buffer solution 3 times, and dried with purified nitrogen to obtain the Raman sensor.
  • the 0°C environment in the present invention is preferably an ice bath environment.
  • the molar concentration of the phosphate buffer of the present invention is preferably 0.01M, and the pH value is 7.0.
  • the MCH of the present invention is preferably a freshly prepared solution of 1 mM.
  • the addition of the MCH of the present invention can make the above-mentioned double helix capture probe DNA stand upright on the surface of the Raman enhanced substrate in an orderly manner, and can also seal the surface of the Raman substrate Other active sites to prevent non-specific adsorption of other biomolecules.
  • the present invention also provides a method for detecting miRNA using the Raman sensor constructed by the above construction method, which comprises the following steps: respectively dripping miRNA-21 and rhodamine 6G (R6G) modification at different concentrations on the Raman sensor Raman probe DNA mixture, after the reaction for 85 minutes, rinse with phosphate buffer, place it under a 633nm laser to measure the surface Raman spectrum intensity, and draw a standard curve based on the relationship between the target concentration and the Raman spectrum intensity. The standard curve is used to calculate the concentration of miRNA.
  • the nucleotide sequence of the Rhodamine 6G (R6G) modified Raman probe DNA is preferably as shown in SEQ ID NO. 4: 5'-TCA GAC TGA TGTTGACCC TAC CTT TCC ACC ATTTC-(R6G)-3'.
  • the present invention When detecting miRNA, the present invention first draws a standard curve. Taking the detection of miRNA-21 in the embodiment as an example, it specifically includes: taking 10 ⁇ L of a set of concentration gradient target miRNA-21 and Rhodamine 6G modified Raman probe DNA (1.0 ⁇ M) the mixed solution was added dropwise to the Raman sensor and reacted for 85 minutes at room temperature, and then washed twice with 10mM, pH7.4 phosphate buffer solution; finally, under the 633nm laser, the Raman spectrometer was used to measure the sensor’s Surface-enhanced Raman spectrum intensity.
  • Kit instruments laser confocal Raman analyzer (RamLab-010, Renishaw, UK), electrochemical workstation (CHI660B, Shanghai Chenhua Instrument Co., Ltd.), ultrapure water machine (Sybergy UV, Merck Millipore) .
  • HAuCl 4 ⁇ 4H 2 O chloroauric acid
  • TCEP tris (2-carboxyethyl) phosphine
  • MCH mercaptohexanol
  • ITO glass chip Cut a piece of ITO glass into small pieces (1cm ⁇ 4cm), put it in 2-propanol containing 2M KOH, boil it for 20 minutes, clean it thoroughly, and then clean it with ethanol and water in an ultrasonic bath and dry it at 60°C.
  • the nucleotide sequence of sulfhydryl modified DNA1 is SEQ ID NO. 1: 5'-(SH)-TTTTTTGAAATGGTGGAAAGGTAGGGTCAACATCAGTCTGATAAGCTA-3';
  • auxiliary DNA2 The nucleotide sequence of auxiliary DNA2 is SEQ ID NO. 2: 5'-TCAGACTGATGTTGACCCTATATCCATAAATT-3';
  • auxiliary DNA3 The nucleotide sequence of auxiliary DNA3 is SEQ ID NO. 3: 5'-CCT TTC CAC CAT TTC-3.
  • Rhodamine 6G (R6G) modified Raman probe DNA nucleotide sequence is SEQ ID NO. 4: 5'-TCA GAC TGA TGTTGACCC TAC CTT TCC ACC ATTTC-(R6G)-3'.
  • the sensor has good selectivity for the Raman detection method of miRNA-21; wherein the target miRNA-21 nucleotide sequence is SEQ ID NO. 5: 5'-UAG CUU AUC AGA CUG AUG UUG A-3';
  • nucleotide sequence of single-base mismatch miRNA-21 is SEQ ID NO.6: 5'-UAG CUU AUC AGA CCG AUG UUGA-3';
  • the nucleotide sequence of multi-base mismatch miRNA-21 is SEQ ID NO. 7: 5'-UAG CUA AUC AGA CCG AUG UAG A-3'.
  • This patent prepares a surface-enhanced Raman substrate, improves the specificity of detection by modifying a capture probe with a special structure, and combines the Toehold-mediated strand displacement amplification reaction to amplify the signal, which can achieve high-sensitivity and specificity of the target miRNA. Man detection.

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Abstract

本发明提供一种拉曼增强基底及其制备方法，所述拉曼增强基底为在氧化铟锡玻璃芯片表面沉积一层三维金纳米化薄膜。

Description

一种拉曼增强基底及其制备方法和检测miRNA的方法
本申请要求于2020年06月10日提交中国专利局、申请号为202010523507.0、发明名称为“一种拉曼增强基底及其制备方法和检测miRNA的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于miRNA检测技术领域，具体涉及一种拉曼增强基底及其制备方法和检测miRNA的方法。
背景技术
MicroRNAs(miRNAs)是一种短片段非编码的小分子RNA，广泛存在于真核细胞中，可以调控靶基因mRNA的表达。miRNA的异常表达与许多重大疾病特别是癌症的发生密切相关。因此，miRNA作为一种典型的肿瘤标志物，正受到越来越多的关注。miRNA的含量在肿瘤患者体内往往呈现异常，比如miRNA-155和miRNA-210的表达水平在弥漫大B细胞淋巴瘤(DLBCL)患者的血液中显著增高。miRNA-21的表达水平在卵巢癌患者的血清中显著增高。此外，miRNA的表达水平不仅能检测肿瘤的发生，还可以作为肿瘤患者预后疗效评估的指标。在非小细胞肺癌(NSCLC)中，高表达let-7的患者生存时间明显长于较低表达的患者，而低表达miRNA-17a的患者的生存时间明显长于高表达的患者。因此，研究miRNA分子标志物有助于全方位的探讨肿瘤形成和发展的分子机理，并为肿瘤的诊断和治疗提供指导。
超灵敏miRNA检测对于癌症的早期诊断和靶向抗癌药物的开发具有重要意义，然而，由于miRNA片段体积小、在细胞中表达低、序列同源性高，因此对miRNA的超敏检测面临诸多挑战。传统的检测方法如微阵列法技术、定量荧光逆转录PCR和生物荧光测定法等被用于miRNA的定量检测，但是，这些方法成本高、耗时长且操作复杂，在一定程度上限制了这些技术的应用。
发明内容
有鉴于此，本发明的目的在于一种拉曼增强基底及其制备方法和检测miRNA的方法，实现目标miRNA的高灵敏、特异性拉曼检测。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种拉曼增强基底的制备方法，包括以下步骤：将清洗、干燥的氧化铟锡玻璃芯片置于电解液中，利用循环伏安法作用50个周期；所述电解液包括以下摩尔浓度的成分：0.03～0.1M磷酸盐溶液、0.03～0.1M KCl和2.0～2.5mM HAuCl 4·4H 2O。
优选的，所述清洗的方法，包括：将氧化铟锡玻璃，放入含有2M KOH的2-丙醇煮沸18～25min，然后置于乙醇水溶液的超声波浴中超声清洗3～5min。
优选的，将所述氧化铟锡玻璃芯片置于电解液中后，向所述电解液充氮气和保持60℃环境。
优选的，所述电解液的用量为4cm 2氧化铟锡玻璃芯片/5mL电解液。
优选的，所述循环伏安法为电位在-0.8V到0.3V之间以0.05V/s的速度循环。
本发明还提供了利用上述制备方法制备得到的拉曼增强基底，所述拉曼增强基底为在氧化铟锡玻璃芯片表面沉积一层三维金纳米化薄膜。
本发明还提供了一种基于上述制备方法制备得到的拉曼增强基底或所述拉曼增强基底的拉曼传感器的构建方法，包括以下步骤：(1)将摩尔比为3.0:3.2:3.2的巯基修饰的DNA1、辅助DNA2和辅助DNA3在Tris-HCl缓冲液中反应10min，冷却至18～25℃后，静置不少于60min，得双螺旋捕获探针DNA混合液；所述巯基修饰的DNA1的核苷酸序列如SEQ ID NO.1所示，所述辅助DNA2的核苷酸序列如SEQ ID NO.2所示，辅助DNA3的核苷酸序列如SEQ ID NO.3所示；所述反应的温度为85℃；
(2)将所述双螺旋捕获探针DNA混合液与TCEP混合后孵育1h，得孵育液；所述TCEP与巯基修饰的DNA1、辅助DNA2和辅助DNA3混合量的摩尔比为1000：(9～10)；
(3)将所述孵育液滴加于所述拉曼增强基底的表面，在0℃环境中反应6h后，用磷酸缓冲液冲洗3次，纯化氮烘干后，再置于巯基己醇中反应2h，磷酸缓冲液冲洗3次，纯化氮烘干，得拉曼传感器。
优选的，步骤(3)所述磷酸缓冲液的摩尔浓度为0.01M，pH值为7.0。
本发明还提供了一种利用上述构建方法构建得到的拉曼传感器检测miRNA的方法，包括以下步骤：分别向所述拉曼传感器上滴加不同浓度的miRNA-21和罗丹明6G(R6G)修饰的拉曼探针DNA混合液，反应85min后，用磷酸缓冲液冲洗，置于633nm激光下测量表面拉曼光谱强度，根据目标物浓度与拉曼光谱强度的关系作图得标准曲线，根据所述标准曲线计算miRNA的浓度。
优选的，所述标准曲线为I＝1770.18lg C+27169.55，其中I为有目标物时的拉曼信号，C为目标物的浓度，线性相关系数R＝0.991。
本发明提供了一种拉曼增强基底的制备方法，在氧化铟锡(ITO)玻璃芯片表面通过电还原方法生成一层三维金纳米化薄膜，制备的该独特的三维金纳米化结构可以产生更活跃的“热点”，是一种良好的拉曼增强基底。本发明基于所述拉曼增强基底构建拉曼传感器，并用于检测miRNA。本发明以miRNA-21为例，利用三维金纳米化/ITO芯片作为拉曼增强基底，将巯基修饰的DNA1和两条辅助DNA(DNA2和DNA3)杂交反应形成双螺旋DNA作为捕获探针修饰在拉曼增强基底表面形成拉曼传感器，在拉曼传感器上加入目标miRNA-21时，捕获探针以特异性结合目标miRNA-21并释放出辅助DNA2；此时，在溶液中大量拉曼信号探针(罗丹明6G修饰的DNA)存在下，通过Toehold介导的链置换反应取代辅助DNA3和目标链miRNA-21，从而释放目标链miRNA-21(图2)。释放的目标链则继续打开基底表面的其他捕获探针，如此的循环作用实现了目标链miRNA-21的循环使用，并且最终在基底表面结合更多的带有罗丹明6G的拉曼信号探针，最后通过基底增强罗丹明6G的拉曼信号来实现对目标miRNA-21的高灵敏高选择性检测。
附图说明
图1为在氧化铟锡(ITO)芯片表面构建三维金纳米化的扫描电镜图；
图2为基于表面增强拉曼信号基底和Toehold介导的链置换放大反应的miRNA拉曼检测原理图；
图3为拉曼响应曲线和工作曲线，其中A为拉曼响应曲线，B为工作曲线；
图4为选择性实验图。
具体实施方式
下面结合说明书附图和实施例对本发明进一步说明。
本发明提供了一种拉曼增强基底的制备方法，包括以下步骤：将清洗、干燥的氧化铟锡玻璃芯片置于电解液中，利用循环伏安法作用50个周期；所述电解液包括以下摩尔浓度的成分：0.03～0.1M磷酸盐溶液、0.03～0.1M KCl和2.0～2.5mM HAuCl 4·4H 2O。
本发明将所述氧化铟锡玻璃芯片置于电解液前，首先对所述氧化铟锡玻璃芯片进行清洗和干燥，所述清洗优选包括：将氧化铟锡玻璃放入含有2M KOH的2-丙醇煮沸18～25min，然后置于乙醇水溶液的超声波浴中超声清洗3～5min。本发明所述煮沸的时间优选为20min。本发明所述乙醇水溶液中乙醇的体积百分含量优选为75％。本发明所述超声清洗的超声频率优选为100KHZ，超声清洗的时间优选为5min。本发明将清洗后的氧化铟锡玻璃进行干燥，所述干燥优选为在60℃环境中烘干。本发明在进行所述清洗和干燥前，优选将氧化铟锡玻璃裁剪为1cm×4cm的小块。
本发明将所述氧化铟锡玻璃芯片置于电解液中后，优选向所述电解液充氮气和保持60℃环境。本发明所述电解液优选包括以下摩尔浓度的成分：0.05M磷酸盐溶液、0.05M KCl和2.4mM HAuCl 4·4H 2O。本发明所述循环伏安法优选为电位在-0.8V到0.3V之间以0.05V/s的速度循环。本发明所述电解液的用量优选为4cm 2氧化铟锡玻璃芯片/5mL电解液。在本发明中，经过循环伏安法作用50个周期后，ITO电极颜色由无色变为黄色，说明维金纳米在电极上沉积成功。本发明在氧化铟锡(ITO)玻璃芯片表面通过电还原方法生成一层三维金纳米化薄膜，制备的该独特的三 维金纳米化结构可以产生更活跃的“热点”，是一种良好的拉曼增强基底。
本发明还提供了利用上述制备方法制备得到的拉曼增强基底，所述拉曼增强基底为在氧化铟锡玻璃芯片表面沉积一层三维金纳米化薄膜。
本发明还提供了一种基于上述制备方法制备得到的拉曼增强基底或所述拉曼增强基底的拉曼传感器的构建方法，包括以下步骤：(1)将摩尔比为3.0:3.2:3.2的巯基修饰的DNA1、辅助DNA2和辅助DNA3在Tris-HCl缓冲液中反应10min，冷却至18～25℃后，静置不少于60min，得双螺旋捕获探针DNA混合液；所述巯基修饰的DNA1的核苷酸序列如SEQ ID NO.1所示，所述辅助DNA2的核苷酸序列如SEQ ID NO.2所示，辅助DNA3的核苷酸序列如SEQ ID NO.3所示；所述反应的温度为85℃；
(2)将所述双螺旋捕获探针DNA混合液与TCEP混合后孵育1h，得孵育液；所述TCEP与巯基修饰的DNA1、辅助DNA2和辅助DNA3混合量的摩尔比为1000：(9～10)；
(3)将所述孵育液滴加于所述拉曼增强基底的表面，在0℃环境中反应6h后，用磷酸缓冲液冲洗3次，纯化氮烘干后，再置于MCH中反应2h，磷酸缓冲液冲洗3次，纯化氮烘干，得拉曼传感器。
本发明将摩尔比为3.0:3.2:3.2的巯基修饰的DNA1、辅助DNA2和辅助DNA3在Tris-HCl缓冲液中85℃反应10min，然后冷却至18～25℃后，静置不少于60min，得双螺旋捕获探针DNA混合液；所述巯基修饰的DNA1的核苷酸序列如SEQ ID NO.1所示，所述辅助DNA2的核苷酸序列如SEQ ID NO.2所示，辅助DNA3的核苷酸序列如SEQ ID NO.3所示；所述反应的温度为85℃。本发明所述Tris-HCl缓冲液中优选还包括0.1M NaCl，pH值为7.4。本发明在所述静置时，可使三条DNA链相互杂交形成双螺旋捕获探针DNA。本发明实施例中以miRNA-21为例构建拉曼增强基底和拉曼传感器，其中巯基修饰的DNA1的核苷酸序列为SEQ ID NO.1：5'-(SH)-TTTTTTGAA ATG GTGGAAAGGTAGGGTCAACATCAGTCTGATAAGCTA-3'；
辅助DNA2的核苷酸序列为SEQ ID NO.2： 5'-TCAGACTGATGTTGACCCTATATCCATAAATT-3'；
辅助DNA3的核苷酸序列为SEQ ID NO.3：5'-CCTTTCCACCATTTC-3。
得双螺旋捕获探针DNA混合液后，本发明将所述双螺旋捕获探针DNA混合液与TCEP混合后孵育1h，得孵育液；所述TCEP与巯基修饰的DNA1、辅助DNA2和辅助DNA3混合量的摩尔比为1000：(9～10)。本发明所述孵育的温度优选为25℃。本发明所述TCEP的加入可使巯基DNA1上的二硫键断裂。
得孵育液后，本发明将所述孵育液滴加于所述拉曼增强基底的表面，在0℃环境中反应6h后，用磷酸缓冲液冲洗3次，纯化氮烘干后，再置于巯基己醇(MCH)中反应2h，磷酸缓冲液冲洗3次，纯化氮烘干，得拉曼传感器。本发明所述0℃环境优选为冰浴环境。本发明所述磷酸缓冲液的摩尔浓度优选为0.01M，pH值为7.0。本发明所述MCH优选为1mM的新鲜配制溶液，本发明所述MCH的加入可使上述的双螺旋捕获探针DNA在拉曼增强基底表面有序的直立起来，并且还可以封闭拉曼基底表面的其它活性位点，防止其它生物分子发生非特异性的吸附。
本发明还提供了一种利用上述构建方法构建得到的拉曼传感器检测miRNA的方法，包括以下步骤：分别向所述拉曼传感器上滴加不同浓度的miRNA-21和罗丹明6G(R6G)修饰的拉曼探针DNA混合液，反应85min后，用磷酸缓冲液冲洗，置于633nm激光下测量表面拉曼光谱强度，根据目标物浓度与拉曼光谱强度的关系作图得标准曲线，根据所述标准曲线计算miRNA的浓度。其中，罗丹明6G(R6G)修饰的拉曼探针DNA的核苷酸序列优选如SEQ ID NO.4所示：5'-TCA GAC TGA TGTTGACCC TAC CTT TCC ACC ATTTC-(R6G)-3’。
本发明在检测miRNA时，首先绘制标准曲线，以实施例中检测miRNA-21为例，具体包括：取10μL一组浓度梯度的目标miRNA-21和罗丹明6G修饰的拉曼探针DNA(1.0μM)混合液，分别滴加到拉曼传感器上室温下反应85分钟后，然后用10mM，pH7.4的磷酸缓冲溶液冲洗 两次；最后，在633nm激光下，用拉曼光谱仪测量了传感器的表面增强拉曼光谱强度，根据目标物浓度与拉曼光谱强度的关系作图得标准曲线，并根据标准曲线计算线性回归方程为I＝1770.18lg C+27169.55，其中I为有目标物时的拉曼信号，C为目标物的浓度，线性相关系数R＝0.991。
下面结合实施例对本发明提供的拉曼增强基底及其制备方法和应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
试剂盒仪器：激光共焦拉曼分析仪(RamLab-010，英国雷尼绍公司)、电化学工作站(CHI660B，上海辰华仪器有限公司)、超纯水机(Sybergy UV，默克密理博)。
HAuCl 4·4H 2O(氯金酸)、TCEP(三(2-羧乙基)膦)和MCH(巯基己醇)购于Sigma-Aldrich公司，DNA和RNA由大连宝生物合成(其中DNA1为巯基修饰)。
实施例1
将一块ITO玻璃切成小块(1cm×4cm)，放入含有2M KOH的2-丙醇煮沸20min，彻底清洗干净，然后用乙醇和水在超声波浴中清洗后60℃烘干。将清洗后的ITO玻璃芯片浸入5.0mL含有0.05M磷酸盐溶液(pH＝2)、0.05M KCl和2.4mM HAuCl 4·4H 2O的电解液中，在电解液充氮气和60℃的条件下，采用循环伏安法(电位在-0.8V到0.3V之间以0.05V/s的速度)作用50个周期。在此条件下，ITO电极颜色由无色变为黄色，说明维金纳米在电极上沉积成功(图1)。最后，将这些ITO芯片作为拉曼增强基底进行微洗并在4℃条件下保存，以备后续实验。
实施例2
20mL含有3.0μM巯基修饰的DNA1、3.2μM辅助DNA2和3.2μM辅助DNA3的Tris-HCl缓冲液(0.1M NaCl，pH值7.4)在85℃下加热10分钟，然后冷却至室温至少60分钟使三条DNA链相互杂交形成双螺旋捕获探针DNA。在上述探针混合物中加入1.0mM TCEP，25℃孵育1h，使巯基DNA1上的二硫键断裂，最后取10μL捕获探针DNA溶液滴加到制备好的拉曼基底芯片表面于冰浴反6小时后用0.01M，pH7.0磷酸缓冲溶液洗涤芯片3次，然后用纯化氮烘干后将芯片沉入1mM新鲜的MCH 中反应2小时，然后用0.01M，pH7.0磷酸缓冲溶液洗涤芯片3次，再用氮气烘干后制得拉曼传感器以备后续实验。
其中，巯基修饰的DNA1的核苷酸序列为SEQ ID NO.1：5'-(SH)-TTTTTTGAAATGGTGGAAAGGTAGGGTCAACATCAGTCTGATAAGCTA-3'；
辅助DNA2的核苷酸序列为SEQ ID NO.2：5'-TCAGACTGATGTTGACCCTATATCCATAAATT-3'；
辅助DNA3的核苷酸序列为SEQ ID NO.3：5'-CCT TTC CAC CAT TTC-3。
实施例3
取10μL一组浓度梯度的目标miRNA-21和罗丹明6G(R6G)修饰的拉曼探针DNA(1.0μM)混合液，分别滴加到拉曼传感器上室温下反应85分钟后，然后用10mM，pH7.4的磷酸缓冲溶液冲洗两次；最后，在633nm激光下，用拉曼光谱仪测量了传感器的表面增强拉曼光谱强度，根据目标物浓度与拉曼光谱强度的关系作图得标准曲线(图3)，并根据标准曲线计算线性回归方程为I＝1770.18lg C+27169.55(I为有目标物时的拉曼信号，C为目标物的浓度)，线性相关系数R＝0.991。其中，罗丹明6G(R6G)修饰的拉曼探针DNA核苷酸序列为SEQ ID NO.4：5'-TCA GAC TGA TGTTGACCC TAC CTT TCC ACC ATTTC-(R6G)-3’。
实施例4
(1)选择性实验：使用目标miRNA-21、单碱基错配miRNA-21、多碱基错配miRNA-21以及空白组来验证该传感器的选择性。如图4所示，当存在过量的单碱基错配miRNA-21(5.0nM)和多碱基错配miRNA-21(5.0nM)时，与空白试验相比，拉曼信号的变化非常微小(空白试验中不存在目标miRNA-21)。然而，即使与单碱基错配miRNA(5.0nM)相比，当出现少量的目标miRNA-21时，拉曼强度也显著增强。这些对比清楚地表明，由于Toehold链置换反应的高度序列依赖性，使得该传感器对于miRNA-21的拉曼检测方法具有良好的选择性；其中，目标miRNA-21核苷酸序列为SEQ ID NO.5：5'-UAG CUU AUC AGA CUG AUG UUG A-3'；
单碱基错配miRNA-21核苷酸序列为SEQ ID NO.6：5'-UAG CUU AUC AGA CCG AUG UUGA-3'；
多碱基错配miRNA-21核苷酸序列为SEQ ID NO.7：5'-UAG CUA AUC AGA CCG AUG UAG A-3'。
(2)样品测定：将含目标miRNA-21的样品与罗丹明6G修饰的拉曼探针DNA(1.0μM)混合后加到拉曼传感器上，室温下反应85分钟后，然后用10mM，pH7.4的磷酸缓冲溶液冲洗两次；最后，在633nm激光下，用拉曼光谱仪测量了传感器的表面增强拉曼光谱强度，根据拉曼强度(I＝2176)，再利用线性回归方程(I＝1770.18lg C+27169.55)计算样品中目标miRNA-21含量为7.6fM。
本专利制备表面增强拉曼基底，通过修饰特殊结构的捕获探针来提高检测的特异性，并且结合Toehold介导的链置换放大反应对信号进行放大，可实现目标miRNA的高灵敏、特异性拉曼检测。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (11)

  1. 一种拉曼增强基底的制备方法，其特征在于，包括以下步骤：将清洗、干燥的氧化铟锡玻璃芯片置于电解液中，利用循环伏安法作用50个周期；所述电解液包括以下摩尔浓度的成分：0.03～0.1M磷酸盐溶液、0.03～0.1M M KCl和2.0～2.5mM HAuCl 4·4H 2O。
  2. 根据权利要求1所述制备方法，其特征在于，所述清洗的方法，包括：将氧化铟锡玻璃，放入含有2M KOH的2-丙醇煮沸18～25min，然后置于乙醇水溶液的超声波浴中超声清洗3～5min。
  3. 根据权利要求2所述制备方法，其特征在于，在进行所述清洗和干燥前，将氧化铟锡玻璃芯片裁剪为1cm×4cm的小块。
  4. 根据权利要求1所述制备方法，其特征在于，将所述氧化铟锡玻璃芯片置于电解液中后，向所述电解液充氮气和保持60℃环境。
  5. 根据权利要求4所述制备方法，其特征在于，所述电解液的用量为4cm 2氧化铟锡玻璃芯片/5mL电解液。
  6. 根据权利要求1所述制备方法，其特征在于，所述循环伏安法为电位在-0.8V到0.3V之间以0.05V/s的速度循环。
  7. 利用权利要求1～6任一项所述制备方法制备得到的拉曼增强基底，其特征在于，所述拉曼增强基底为在氧化铟锡玻璃芯片表面沉积一层三维金纳米化薄膜。
  8. 一种基于利用权利要求1～7任一项所述制备方法制备得到的拉曼增强基底或权利要求7所述拉曼增强基底的拉曼传感器的构建方法，其特征在于，包括以下步骤：(1)将摩尔比为3.0:3.2:3.2的巯基修饰的DNA1、辅助DNA2和辅助DNA3在Tris-HCl缓冲液中反应10min，冷却至18～25℃后，静置不少于60min，得双螺旋捕获探针DNA混合液；所述巯基修饰的DNA1的核苷酸序列如SEQ ID NO.1所示，所述辅助DNA2的核苷酸序列如SEQ ID NO.2所示，辅助DNA3的核苷酸序列如SEQ ID NO.3所示；所述反应的温度为85℃；
    (2)将所述双螺旋捕获探针DNA混合液与TCEP混合后孵育1h，得孵育液；所述TCEP与巯基修饰的DNA1、辅助DNA2和辅助DNA3混合量的摩尔比为1000：(9～10)；
    (3)将所述孵育液滴加于所述拉曼增强基底的表面，在0℃环境中反应6h后，用磷酸缓冲液冲洗3次，纯化氮烘干后，再置于巯基己醇中反应2h，磷酸缓冲液冲洗3次，纯化氮烘干，得拉曼传感器。
  9. 根据权利要求8所述构建方法，其特征在于，步骤(3)所述磷酸缓冲液的摩尔浓度为0.01M，pH值为7.0。
  10. 一种利用权利要求8或9所述构建方法构建得到的拉曼传感器检测miRNA的方法，其特征在于，包括以下步骤：分别向所述拉曼传感器上滴加不同浓度的miRNA-21和罗丹明6G修饰的拉曼探针DNA混合液，反应85min后，用磷酸缓冲液冲洗，置于633nm激光下测量表面拉曼光谱强度，根据目标物浓度与拉曼光谱强度的关系作图得标准曲线，根据所述标准曲线计算miRNA的浓度。
  11. 根据权利要求10所述方法，其特征在于，所述标准曲线为I＝1770.18lg C+27169.55，其中I为有目标物时的拉曼信号，C为目标物的浓度，线性相关系数R＝0.991。
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