阅读说明：本技术 一种拉曼增强基底及其制备方法和检测miRNA的方法 (Raman enhanced substrate, preparation method thereof and method for detecting miRNA (micro ribonucleic acid) ) 是由 周宏� 丁可欣 刘静 刘树峰 于 2020-06-10 设计创作，主要内容包括：本发明提供了一种拉曼增强基底及其制备方法和检测miRNA的方法,涉及miRNA检测技术领域。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。(The invention provides a Raman enhanced substrate, a preparation method thereof and a method for detecting miRNA, and relates to the technical field of miRNA detection. According to the invention, a layer of 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 generate more active 'hot spots', and is a good Raman enhancement substrate. The Raman sensor is constructed on the basis of the Raman enhancement substrate and is used for detecting miRNA. The invention improves the detection specificity by modifying the capture probe with a special structure, and amplifies the signal by combining with the Toehold-mediated strand displacement amplification reaction, thereby realizing the high-sensitivity and specific Raman detection of the target miRNA.)

1. A preparation method of a Raman enhanced substrate is characterized by comprising the following steps: placing the cleaned and dried indium tin oxide glass chip in electrolyte, and acting for 50 periods by using cyclic voltammetry; the electrolyte comprises the following components in molar concentration: 0.03 to 0.1M phosphate solution, 0.03 to 0.1M KCl and 2.0 to 2.5mM HAuCl₄·4H₂O。

2. The method of claim 1, wherein the cleaning comprises: and (3) putting indium tin oxide glass into 2-propanol containing 2M KOH, boiling for 18-25 min, and then putting the indium tin oxide glass into an ultrasonic bath of ethanol water solution for ultrasonic cleaning for 3-5 min.

3. The method according to claim 1, wherein after the indium tin oxide glass chip is placed in the electrolyte, the electrolyte is filled with nitrogen gas and maintained at 60 ℃.

4. The method according to claim 3, wherein the electrolyte is used in an amount of 4cm²Indium tin oxide glass chips/5 mL of electrolyte.

5. The method of claim 1, wherein the cyclic voltammetry is a potential cycling between-0.8V and 0.3V at a rate of 0.05V/s.

6. The Raman-enhanced substrate prepared by the preparation method of any one of claims 1 to 5, wherein the Raman-enhanced substrate is a three-dimensional gold nano-film deposited on the surface of an indium tin oxide glass chip.

7. A method for constructing a Raman sensor based on a Raman-enhanced substrate prepared by the preparation method of any one of claims 1 to 5 or the Raman-enhanced substrate of claim 6, comprising the following steps: (1) reacting sulfhydryl-modified DNA1, auxiliary DNA2 and auxiliary DNA3 in a molar ratio of 3.0:3.2:3.2 in a Tris-HCl buffer solution for 10min, cooling to 18-25 ℃, and standing for not less than 60min to obtain a double helix capture probe DNA mixed solution; the nucleotide sequence of the sulfhydryl modified DNA1 is shown as SEQ ID NO.1, the nucleotide sequence of the auxiliary DNA2 is shown as SEQ ID NO.2, and the nucleotide sequence of the auxiliary DNA3 is shown as SEQ ID NO. 3; the temperature of the reaction is 85 ℃;

(2) mixing the double helix capture probe DNA mixed solution with TCEP and incubating for 1h to obtain an incubation solution; the molar ratio of the TCEP to the mixed amount of the sulfhydryl-modified DNA1, the auxiliary DNA2 and the auxiliary DNA3 is 1000: (9-10);

(3) and dropwise adding the incubation liquid on the surface of the Raman enhancement substrate, reacting for 6 hours at 0 ℃, washing for 3 times by using a phosphate buffer, drying by using purified nitrogen, then placing in mercaptohexanol for reacting for 2 hours, washing for 3 times by using the phosphate buffer, and drying by using the purified nitrogen to obtain the Raman sensor.

8. The method according to claim 7, wherein the phosphate buffer solution of step (3) has a molarity of 0.01M and a pH of 7.0.

9. A method for detecting miRNA by using a Raman sensor constructed by the construction method of claim 7 or 8, which is characterized by comprising the following steps: respectively dripping mixed solution of miRNA-21 and rhodamine 6G modified Raman probe DNA with different concentrations on the Raman sensor, reacting for 85min, washing with phosphate buffer, measuring the surface Raman spectrum intensity under 633nm laser, drawing a standard curve according to the relation between the target concentration and the Raman spectrum intensity, and calculating the miRNA concentration according to the standard curve.

10. The method of claim 9, wherein the standard curve is I-1770.18 lg C +27169.55, where I is the raman signal in the presence of the target, C is the concentration of the target, and the linear correlation coefficient R is 0.991.

Technical Field

The invention belongs to the technical field of miRNA detection, and particularly relates to a Raman enhanced substrate, a preparation method thereof and a method for detecting miRNA.

Background

MicroRNAs (miRNAs) are short-fragment non-coding small-molecule RNAs widely existing in eukaryotic cells and can regulate and control the expression of target gene mRNA. Abnormal expression of mirnas is closely associated with the development of many significant diseases, particularly cancer. Therefore, miRNA is receiving increasing attention as a typical tumor marker. The content of miRNA is often abnormal in tumor patients, for example, the expression level of miRNA-155 and miRNA-210 is obviously increased in the blood of Diffuse Large B Cell Lymphoma (DLBCL) patients. The expression level of miRNA-21 is obviously increased in the serum of ovarian cancer patients. In addition, the expression level of miRNA can not only detect the occurrence of tumor, but also be used as the index of the prognosis curative effect evaluation of tumor patients. In non-small cell lung cancer (NSCLC), patients with high expression of let-7 survived significantly longer than those with lower expression, while patients with low expression of miRNA-17a survived significantly longer than those with high expression. Therefore, the research on the miRNA molecular marker is helpful for comprehensively discussing the molecular mechanism of tumor formation and development and provides guidance for the diagnosis and treatment of tumors.

Ultrasensitive miRNA detection is of great significance for early diagnosis of cancer and development of targeted anti-cancer drugs, however, due to the small size of miRNA fragments, low expression in cells and high sequence homology, hypersensitive detection of miRNA is faced with many challenges. Traditional detection methods such as microarray technology, quantitative reverse transcription PCR (fluorescent quantitative polymerase chain reaction), biological fluorimetry and the like are used for quantitative detection of miRNA, but the methods are high in cost, long in time consumption and complex in operation, and the application of the technologies is limited to a certain extent.

Disclosure of Invention

In view of the above, the invention aims to provide a raman-enhanced substrate, a preparation method thereof and a method for detecting miRNA, so as to realize high-sensitivity and specific raman detection of target miRNA.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a preparation method of a Raman enhanced substrate, which comprises the following steps: placing the cleaned and dried indium tin oxide glass chip in electrolyte, and acting for 50 periods by using cyclic voltammetry; the electrolyte comprises the following components in molar concentration: 0.03 to 0.1M phosphate solution, 0.03 to 0.1M KCl and 2.0 to 2.5mM HAuCl₄·4H₂O。

Preferably, the method of cleaning comprises: and (3) putting indium tin oxide glass into 2-propanol containing 2M KOH, boiling for 18-25 min, and then putting the indium tin oxide glass into an ultrasonic bath of ethanol water solution for ultrasonic cleaning for 3-5 min.

Preferably, after the indium tin oxide glass chip is placed in the electrolyte, nitrogen is filled into the electrolyte and the environment at 60 ℃ is maintained.

Preferably, the dosage of the electrolyte is 4cm²Indium tin oxide glass chips/5 mL of electrolyte.

Preferably, the cyclic voltammetry is a method in which the potential is cycled between-0.8V and 0.3V at a rate of 0.05V/s.

The invention also provides the Raman enhancement substrate prepared by the preparation method, wherein the Raman enhancement substrate is formed by depositing a layer of three-dimensional gold nano film on the surface of an indium tin oxide glass chip.

The invention also provides a Raman enhancement substrate prepared based on the preparation method or a construction method of the Raman sensor of the Raman enhancement substrate, which comprises the following steps: (1) reacting sulfhydryl-modified DNA1, auxiliary DNA2 and auxiliary DNA3 in a molar ratio of 3.0:3.2:3.2 in a Tris-HCl buffer solution for 10min, cooling to 18-25 ℃, and standing for not less than 60min to obtain a double helix capture probe DNA mixed solution; the nucleotide sequence of the sulfhydryl modified DNA1 is shown as SEQ ID NO.1, the nucleotide sequence of the auxiliary DNA2 is shown as SEQ ID NO.2, and the nucleotide sequence of the auxiliary DNA3 is shown as SEQ ID NO. 3; the temperature of the reaction is 85 ℃;

(2) mixing the double helix capture probe DNA mixed solution with TCEP and incubating for 1h to obtain an incubation solution; the molar ratio of the TCEP to the mixed amount of the sulfhydryl-modified DNA1, the auxiliary DNA2 and the auxiliary DNA3 is 1000: (9-10);

(3) and dropwise adding the incubation liquid on the surface of the Raman enhancement substrate, reacting for 6 hours at 0 ℃, washing for 3 times by using a phosphate buffer, drying by using purified nitrogen, then placing in mercaptohexanol for reacting for 2 hours, washing for 3 times by using the phosphate buffer, and drying by using the purified nitrogen to obtain the Raman sensor.

Preferably, the phosphate buffer solution in step (3) has a molarity of 0.01M and a pH of 7.0.

The invention also provides a method for detecting miRNA by using the Raman sensor constructed by the construction method, which comprises the following steps: respectively dripping mixed solution of miRNA-21 and rhodamine 6G (R6G) modified Raman probe DNA with different concentrations on the Raman sensor, reacting for 85min, washing with phosphate buffer, measuring surface Raman spectrum intensity under 633nm laser, drawing a standard curve according to the relation between the concentration of the target and the Raman spectrum intensity, and calculating the concentration of miRNA according to the standard curve.

Preferably, the standard curve is 1770.18lg C +27169.55, wherein I is the raman signal in the presence of the target, C is the concentration of the target, and the linear correlation coefficient R is 0.991.

The invention provides a preparation method of a Raman enhancement substrate, wherein a layer of 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 generate more active 'hot spots', and is a good Raman enhancement substrate. The Raman sensor is constructed on the basis of the Raman enhancement substrate and is used for detecting miRNA. Taking miRNA-21 as an example, a three-dimensional gold nanocrystallization/ITO chip is used as a Raman enhancement substrate, sulfhydryl modified DNA1 and two auxiliary DNAs (DNA2 and DNA3) are subjected to hybridization reaction to form double-helix DNA which is used as a capture probe to be modified on the surface of the Raman enhancement substrate to form a Raman sensor, and when target miRNA-21 is added to the Raman sensor, the capture probe is specifically combined with the target miRNA-21 and releases the auxiliary DNA 2; at this time, the helper DNA3 and the target strand miRNA-21 were displaced by a Toehold-mediated strand displacement reaction in the presence of a large amount of raman signaling probe (rhodamine 6G-modified DNA) in the solution, thereby releasing the target strand miRNA-21 (fig. 2). The released target chain continues to open other capture probes on the surface of the substrate, the cyclic use of the target chain miRNA-21 is realized through the cyclic action, more Raman signal probes with rhodamine 6G are finally combined on the surface of the substrate, and finally the high-sensitivity and high-selectivity detection of the target miRNA-21 is realized through the enhancement of the Raman signal of the rhodamine 6G by the substrate.

Drawings

FIG. 1 is a scanning electron microscope image of three-dimensional gold nanocrystallization constructed on the surface of an Indium Tin Oxide (ITO) chip;

FIG. 2 is a diagram of miRNA Raman detection based on a surface-enhanced Raman signal substrate and a Toehold-mediated strand displacement amplification reaction;

FIG. 3 is a Raman response curve and a working curve, wherein A is the Raman response curve and B is the working curve;

FIG. 4 is a graph of a selectivity experiment.

Detailed Description

The invention provides a preparation method of a Raman enhanced substrate, which comprises the following steps: placing the cleaned and dried indium tin oxide glass chip in electrolyte, and acting for 50 periods by using cyclic voltammetry; the electrolyte comprises the following components in molar concentration: 0.03 to 0.1M phosphate solution, 0.03 to 0.1M KCl and 2.0 to 2.5mM HAuCl₄·4H₂O。

In the invention, before the indium tin oxide glass chip is placed in the electrolyte, the indium tin oxide glass chip is firstly cleaned and dried, and the cleaning preferably comprises the following steps: and (3) putting the indium tin oxide glass into 2-propanol containing 2M KOH, boiling for 18-25 min, and then putting the indium tin oxide glass into an ultrasonic bath of ethanol water solution for ultrasonic cleaning for 3-5 min. The boiling time of the invention is preferably 20 min. The volume percentage content of ethanol in the ethanol aqueous solution is preferably 75%. The ultrasonic frequency of the ultrasonic cleaning is preferably 100KHZ, and the ultrasonic cleaning time is preferably 5 min. The cleaned indium tin oxide glass is dried, and the drying is preferably carried out in an environment of 60 ℃. According to the invention, the indium tin oxide glass is preferably cut into small pieces of 1cm by 4cm before the washing and drying.

After the indium tin oxide glass chip is placed in the electrolyte, the electrolyte is preferably filled with nitrogen and kept at 60 ℃. The electrolyte of the invention preferably comprises the following components in molar concentration: 0.05M phosphate solution, 0.05MKCl and 2.4mM HAuCl₄·4H₂And O. The cyclic voltammetry of the present invention is preferably a potential cycling between-0.8V to 0.3V at a rate of 0.05V/s. The dosage of the electrolyte of the invention is preferably 4cm²Indium tin oxide glass chips/5 mL of electrolyte. In the invention, after 50 cycles of cyclic voltammetry,the color of the ITO electrode is changed from colorless to yellow, which indicates that the Virginine nano-scale is successfully deposited on the electrode. According to the invention, a layer of 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 generate more active 'hot spots', and is a good Raman enhancement substrate.

The invention also provides the Raman enhancement substrate prepared by the preparation method, wherein the Raman enhancement substrate is formed by depositing a layer of three-dimensional gold nano film on the surface of an indium tin oxide glass chip.

The invention also provides a Raman enhancement substrate prepared based on the preparation method or a construction method of the Raman sensor of the Raman enhancement substrate, which comprises the following steps: (1) reacting sulfhydryl-modified DNA1, auxiliary DNA2 and auxiliary DNA3 in a molar ratio of 3.0:3.2:3.2 in a Tris-HCl buffer solution for 10min, cooling to 18-25 ℃, and standing for not less than 60min to obtain a double helix capture probe DNA mixed solution; the nucleotide sequence of the sulfhydryl modified DNA1 is shown as SEQ ID NO.1, the nucleotide sequence of the auxiliary DNA2 is shown as SEQ ID NO.2, and the nucleotide sequence of the auxiliary DNA3 is shown as SEQ ID NO. 3; the temperature of the reaction is 85 ℃;

(2) mixing the double helix capture probe DNA mixed solution with TCEP and incubating for 1h to obtain an incubation solution; the molar ratio of the TCEP to the mixed amount of the sulfhydryl-modified DNA1, the auxiliary DNA2 and the auxiliary DNA3 is 1000: (9-10);

(3) and dropwise adding the incubation liquid on the surface of the Raman enhancement substrate, reacting for 6h at 0 ℃, washing for 3 times by using a phosphate buffer, drying by using purified nitrogen, then placing in MCH for reacting for 2h, washing for 3 times by using the phosphate buffer, and drying by using the purified nitrogen to obtain the Raman sensor.

The method comprises the steps of reacting sulfydryl modified DNA1, auxiliary DNA2 and auxiliary DNA3 in a Tris-HCl buffer solution at 85 ℃ for 10min, cooling to 18-25 ℃, standing for not less than 60min, and obtaining a double helix capture probe DNA mixed solution; the nucleotide sequence of the sulfhydryl modified DNA1 is shown as SEQ ID NO.1, the nucleotide sequence of the auxiliary DNA2 is shown as SEQ ID NO.2, and the nucleotide sequence of the auxiliary DNA3 is shown as SEQ ID NO. 3; the temperature of the reaction was 85 ℃. The Tris-HCl buffer solution preferably further comprises 0.1M NaCl, and the pH value is 7.4. In the standing process, three DNA strands can be hybridized with each other to form the double helix capture probe DNA. In the embodiment of the invention, miRNA-21 is taken 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) -TTTTTTGAAATG GTGGAAAGGTAGGGTCAACATCAGTCTGATAAGCTA-3';

the nucleotide sequence of the auxiliary DNA2 is SEQ ID NO. 2: 5'-TCAGACTGATGTTGACCCTATATCCATAAATT-3', respectively;

the nucleotide sequence of the auxiliary DNA3 is SEQ ID NO. 3: 5' -CCTTTCCACCATTTC-3.

After a double helix capture probe DNA mixed solution is obtained, the double helix capture probe DNA mixed solution and TCEP are mixed and incubated for 1h to obtain an incubation solution; the molar ratio of the TCEP to the mixed amount of the sulfhydryl-modified DNA1, the auxiliary DNA2 and the auxiliary DNA3 is 1000: (9-10). The temperature of the incubation according to the invention is preferably 25 ℃. The addition of TCEP described in the present invention can cleave the disulfide bond on the sulfhydryl DNA 1.

After obtaining the incubation liquid, the method comprises the steps of dropping the incubation liquid on the surface of the Raman enhancement substrate, reacting for 6 hours at 0 ℃, washing for 3 times by using a phosphate buffer, drying purified nitrogen, then placing in Mercaptohexanol (MCH) for reacting for 2 hours, washing for 3 times by using the phosphate buffer, and drying the purified nitrogen to obtain the Raman sensor. The environment at 0 ℃ is preferably an ice bath environment. The phosphate buffer solution of the present invention preferably has a molarity of 0.01M and a pH of 7.0. The MCH is preferably a fresh preparation solution with the concentration of 1mM, and the addition of the MCH can enable the double-helix capture probe DNA to stand up on the surface of the Raman enhancement substrate orderly, and can also block other active sites on the surface of the Raman substrate to prevent other biomolecules from being adsorbed non-specifically.

The invention also provides a method for detecting miRNA by using the Raman sensor constructed by the construction method, which comprises the following steps: respectively dripping mixed solution of miRNA-21 and rhodamine 6G (R6G) modified Raman probe DNA with different concentrations on the Raman sensor, reacting for 85min, washing with phosphate buffer, measuring surface Raman spectrum intensity under 633nm laser, drawing a standard curve according to the relation between the concentration of the target and the Raman spectrum intensity, and calculating the concentration of miRNA according to the standard curve. The nucleotide sequence of the Raman probe DNA modified by rhodamine 6G (R6G) is preferably shown as SEQ ID NO. 4: 5'-TCA GAC TGA TGTTGACCC TAC CTT TCC ACCATTTC- (R6G) -3'.

When detecting miRNA, the invention firstly draws a standard curve, taking the detection of miRNA-21 in the embodiment as an example, and specifically comprises the following steps: respectively dripping 10 mu L of a group of target miRNA-21 and rhodamine 6G modified Raman probe DNA (1.0 mu M) mixed solution with concentration gradient on a Raman sensor for reaction for 85 minutes at room temperature, and then washing twice by using 10mM phosphate buffer solution with pH 7.4; finally, under laser of 633nm, the surface enhanced raman spectrum intensity of the sensor is measured by a raman spectrometer, a standard curve is drawn according to the relation between the concentration of the target object and the raman spectrum intensity, and a linear regression equation is calculated according to the standard curve, wherein I is 1770.18lg C +27169.55, I is a raman signal when the target object exists, C is the concentration of the target object, and a linear correlation coefficient R is 0.991.

The raman-enhanced substrate provided by the present invention, the preparation method and the application thereof are described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Kit instrument: laser confocal raman analyzer (RamLab-010, renisha, uk), electrochemical workstation (CHI660B, shanghai chenhua instruments ltd), ultra pure water machine (Sybergy UV, merck michigan).

HAuCl₄·4H₂O (chloroauric acid), TCEP (tris (2-carboxyethyl) phosphine), and MCH (mercaptohexanol) were purchased from Sigma-Aldrich, and DNA and RNA were biosynthesized from Dalian (where DNA1 was thiol-modified).