NL2028411A

NL2028411A - RAMAN-ENHANCED SUBSTRATE, FABRICATION METHOD THEREOF, AND METHOD FOR DETECTING miRNA

Info

Publication number: NL2028411A
Application number: NL2028411A
Authority: NL
Inventors: Zhou Hong; Ding Kexin; Liu Jing; Liu Shufeng
Original assignee: Qingdao Univ Of Science And Technology
Priority date: 2020-06-10
Filing date: 2021-06-08
Publication date: 2021-08-30
Also published as: NL2028411B1; WO2021248691A1; CN111518874A

Abstract

The present disclosure provides a Raman-enhanced substrate, a fabrication method thereof, and a method for detecting miRNA, and relates to the technical field of miRNA detection. According to the present disclosure, a layer of three-dimensional gold nanofiower film is produced on the surface of an indium tin oxide (ITO) glass chip through electroreduction method, the fabricated unique three-dimensional gold nanofiower film structure can generate more active 'hot spots' and is an excellent Ramanenhanced substrate. According to the present disclosure, a Raman sensor is constructed based on the Raman-enhanced substrate and is used to detect miRNA. The detection specificity is improved by modifying a capture probe with a special structure, and a Toehold-mediated strand displacement reaction is combined to amplify a signal, so that highly sensitive and specific Raman detection of the target miRNA can be realized.

Description

RAMAN-ENHANCED SUBSTRATE, FABRICATION METHOD THEREOF, AND METHOD FOR DETECTING miRNA

TECHNICAL FIELD

[01] The present disclosure belongs to the technical field of miRNA detection, and specifically relates to a Raman-enhanced substrate, a fabrication method thereof, and a method for detecting miRNA.

BACKGROUND ART

[02] MicroRNAs (miRNAs) are short non-coding small RNA molecules 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 a plurality of major diseases, especially cancers. Therefore, miRNA, as a typical tumor marker, is receiving more and more attention. The content of miRNA is often abnormal in patients with tumors. For example, the expression levels of miRNA-155 and miRNA-210 are significantly upregulated in the blood of patients with diffuse large B-cell lymphoma (DLBCL). The expression level of miRNA-21 is significantly upregulated in the sera of patients with ovarian cancer. In addition, the expression level of miRNA not only can detect the occurrence of tumors, but also can be used as an indicator for evaluating the prognosis of patients with tumors. In non-small cell lung cancer (NSCLC), patients with high expression of let-7 have significantly longer survival time than those with low expression thereof, while patients with low expression of miRNA-17a have significantly longer survival time than those with high expression thereof. Therefore, the investigation of miRNA molecular markers will be conducive to the exploration of the molecular mechanism of tumor formation and development in an all-round way, and provide guidance for the diagnosis and treatment of tumors.

[03] Ultra-sensitive miRNA detection is of great significance for the early diagnosis of cancers and the development of targeted anti-cancer drugs. However, due to the small size of miRNA fragments, low expression thereof in cells, and high sequence homology thereof, the ultra-sensitive miRNA detection faces a plurality of challenges. Conventional detection methods, such as microarray technology, quantitative fluorescent reverse transcription PCR and bioluminescence assay, are used for the quantitative detection of miRNA. However, these methods are expensive, time-

consuming, and complicated to operate, limiting the application of these technologies to some degree.

SUMMARY

[04] In view of this, an objective of the present disclosure is to provide a Raman- enhanced substrate, a fabrication method thereof, and a method for detecting miRNA, so as to realize highly sensitive and specific Raman detection of target miRNA.

[05] To achieve the above objective, the present disclosure provides the following technical solutions:

[06] The present disclosure provides a method for fabricating a Raman-enhanced substrate, including the following steps: placing a cleaned and dried indium tin oxide (ITO) glass chip in an electrolyte and reacting for 50 cycles via cyclic voltammetry; the electrolyte includes the following molar concentrations of components: 0.03-0.1 M phosphate solution, 0.03-0.1 M KCl, and 2.0-2.5 mM HAuCl4-4H:O.

[07] Preferably, the cleaning may include the steps of: placing ITO glass in 2- propanol containing 2 M KOH, boiling for 18-25 min, and ultrasonically cleaning in an ultrasonic bath of aqueous ethanol solution for 3-5 min.

[08] Preferably, after the ITO glass chip is placed in the electrolyte, the electrolyte may be filled with nitrogen and held at 60°C.

[09] Preferably, the amount of the electrolyte may be 4 cm? ITO glass chip/5 mL electrolyte.

[10] Preferably, the cyclic voltammetry may be potential cycling between -0.8 V and

0.3 V atarate of 0.05 V/s.

[11] The present disclosure further provides a Raman-enhanced substrate fabricated by the foregoing fabrication method, where the Raman-enhanced substrate is a layer of three-dimensional gold nanoflower film deposited on the surface of an ITO glass chip.

[12] The present disclosure further provides a method for constructing a Raman sensor based on the Raman-enhanced substrate fabricated by the foregoing fabrication method or the Raman-enhanced substrate, including the following steps: step 1, allowing sulthydryl-modified DNAI, auxiliary DNA2 and auxiliary DNA3, in a molar ratio of

3.0:3.2:3.2, to react in Tris-HCI buffer for 10 min, cooling to 18-25°C, and letting a reaction mixture stand for no less than 60 min to obtain a double helix capture probe DNA mixture; where the sulfhydryl-modified DNAI has a nucleotide sequence as shown in SEQ ID NO. 1, the auxiliary DNA2 has a nucleotide sequence as shown in SEQ ID NO. 2, and the auxiliary DNA3 has a nucleotide sequence as shown in SEQ ID NO. 3; the reaction is conducted at 85°C;

[13] step 2, mixing the double helix capture probe DNA mixture with tris(2- carboxyethyl)phosphine (TCEP) and incubating a resulting mixture for 1 h to obtain an incubation buffer; where the TCEP and a mixture of the sulthydryl-modified DNA 1, the auxiliary DNA2 and the auxiliary DNA3 have a molar ratio of 1,000: (9-10); and

[14] step 3, dropping the incubation buffer onto the surface of the Raman-enhanced substrate, reacting at 0°C for 6 h, and rinsing the Raman-enhanced substrate with phosphate buffer solution (PBS) thrice; after drying with purified nitrogen, placing the Raman-enhanced substrate in mercaptohexanol (MCH) to react for 2 h, rinsing the Raman-enhanced substrate thrice with PBS, and drying with purified nitrogen to obtain the Raman sensor.

[15] Preferably, the PBS in step 3 may have a molar concentration of 0.01 M and a pH value of 7.0.

[16] The present disclosure further provides a method for detecting miRNA using the Raman sensor constructed by the foregoing construction method, including the following steps: separately dropping different concentrations of miRNA-21 and rhodamine 6G (R6G)-modified Raman probe DNA mixture on the Raman sensor, reacting for 85 min, rinsing the Raman sensor with PBS, placing the Raman sensor under a 633 nm laser to measure surface Raman intensity, plotting a standard curve as a function of the target compound concentration versus Raman intensity, and calculating a miRNA concentration according to the standard curve.

[17] Preferably, the standard curve may be I = 1770.18IgC + 27169.55, where Lis a Raman signal in the presence of a target compound, and C is a concentration of the target compound, with a linear correlation coefficient of R = 0.991.

[18] The present disclosure provides a method for fabricating a Raman-enhanced substrate. A layer of three-dimensional gold nanoflower film is produced on the surface of an ITO glass chip through electroreduction method; the fabricated unique three- dimensional gold nanoflower film structure can generate more active 'hot spots' and is an excellent Raman-enhanced substrate. According to the present disclosure, a Raman sensor 1s constructed based on the Raman-enhanced substrate and is used to detect miRNA. Taking miRNA-21 as an example, the present disclosure uses a three-

dimensional nanogold/ITO chip as a Raman-enhanced substrate, hybridizes sulfhydryl- modified DNA1 and two auxiliary DNAs (DNA2 and DNA3) to form a double helix DNA as a capture probe modifying on the surface of the Raman-enhanced substrate to form a Raman sensor. When target miIRNA-21 is added to the Raman sensor, the capture probe specifically binds to the target miRNA-21 and releases auxiliary DNA2; at this time, in the presence of substantial Raman probes (R6G-modified DNA) in the solution, the auxiliary DNA3 and target strand miRNA-21 are substituted by a Toehold-mediated strand displacement reaction, thereby releasing the target strand miRNA-21 (FIG. 2). The released target strand continues to open other capture probes on the surface of the substrate. Such circulation realizes the recycling of the target strand miRNA-21, and finally binds more Raman probes with R6G onto the surface of the substrate. Finally, the Raman signal of R6G is enhanced by the substrate to achieve highly sensitive and selective detection of the target miRNA-21.

BRIEF DESCRIPTION OF THE DRAWINGS

[19] FIG. 1 is a scanning electron micrograph of three-dimensional gold nanoflowers constructed on the surface of an ITO chip;

[20] FIG. 2 illustrates the principle of Raman detection of miRNA based on a surface- enhanced Raman substrate and Toehold-mediated strand displacement reaction; [ZI] FIG. 3 shows Raman response curves and a working curve, where A shows the Raman response curves, and B shows the working curve;

[22] FIG. 4 illustrates a selectivity test.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[23] The present disclosure provides a method for fabricating a Raman-enhanced substrate, including the following steps: placing a cleaned and dried ITO glass chip in an electrolyte and reacting for 50 cycles via cyclic voltammetry; the electrolyte includes the following molar concentrations of components: 0.03-0.1 M phosphate solution, 0.03-

0.1 M KCl, and 2.0-2.5 mM HAuCl4 4H:0.

[24] In the present disclosure, before the ITO glass chip is placed in the electrolyte, the ITO glass chip is cleaned and dried. The cleaning may preferably include the steps of: placing ITO glass in 2-propanol containing 2 M KOH, boiling for 18-25 min, and ultrasonically cleaning in an ultrasonic bath of aqueous ethanol solution for 3-5 min. In the present disclosure, the boiling may preferably be conducted for 20 min. In the present disclosure, the volume percent fraction of ethanol in the aqueous ethanol solution may preferably be 75%. In the present disclosure, the sonication may preferably be conducted at 100 KHz for 5 min. In the present disclosure, the cleaned ITO glass is dried, and the 5 drying may preferably be conducted at 60°C. In the present disclosure, before the cleaning and drying, the ITO glass may preferably be cut into 1 x 4 cm small pieces.

[25] Inthe present disclosure, after the ITO glass chip is placed in the electrolyte, the electrolyte may preferably be filled with nitrogen and held at 60°C. In the present disclosure, the electrolyte may preferably include the following molar concentrations of components: 0.05 M phosphate solution, 0.05 M KCl, and 2.4 mM HAuCl4-4H;0. In the present disclosure, the cyclic voltammetry may preferably be potential cycling between -0.8 V and 0.3 V at a rate of 0.05 V/s. In the present disclosure, the amount of the electrolyte may preferably be 4 cm? ITO glass chip/5 mL electrolyte. In the present disclosure, after 50 cycles of cyclic voltammetry, the color of the ITO electrode may turn from colorless to yellow, indicating that three-dimensional gold nanoparticles are successfully deposited on an electrode. According to the present disclosure, a layer of three-dimensional gold nanoflower film may be produced on the surface of an ITO glass chip through electroreduction method; the fabricated unique three-dimensional gold nanoflower film structure may generate more active 'hot spots’ and be an excellent Raman-enhanced substrate.

[26] The present disclosure further provides a Raman-enhanced substrate fabricated by the foregoing fabrication method, where the Raman-enhanced substrate is a layer of three-dimensional gold nanoflower film deposited on the surface of an ITO glass chip.

[27] The present disclosure further provides a method for constructing a Raman sensor based on the Raman-enhanced substrate fabricated by the foregoing fabrication method or the Raman-enhanced substrate, including the following steps: step 1, allowing sulfhydryl-modified DNA1, auxiliary DNA2 and auxiliary DNA3, in a molar ratio of

3.0:3.2:3.2, to react in Tris-HCI buffer for 10 min, cooling to 18-25°C, and letting a reaction mixture stand for no less than 60 min to obtain a double helix capture probe DNA mixture; where the sulthydryl-modified DNA1 has a nucleotide sequence as shown in SEQ ID NO. 1, the auxiliary DNA2 has a nucleotide sequence as shown in SEQ ID NO. 2, and the auxiliary DNA3 has a nucleotide sequence as shown in SEQ ID NO. 3; the reaction is conducted at 85°C;

[28] step 2, mixing the double helix capture probe DNA mixture with TCEP and incubating a resulting mixture for 1 h to obtain an incubation buffer; where the TCEP and a mixture of the sulfhydryl-modified DNA 1, the auxiliary DNA2 and the auxiliary DNA3 have a molar ratio of 1,000: (9-10); and

[29] step 3, dropping the incubation buffer onto the surface of the Raman-enhanced substrate, reacting at 0°C for 6 h, and rinsing the Raman-enhanced substrate with PBS thrice; after drying with purified nitrogen, placing the Raman-enhanced substrate in MCH to react for 2 h, rinsing the Raman-enhanced substrate thrice with PBS, and drying with purified nitrogen to obtain the Raman sensor.

[30] In the present disclosure, sulthydryl-modified DNAI, auxiliary DNA2 and auxiliary DNA3, in a molar ratio of 3.0:3.2:3.2, are allowed to react in Tris-HCI buffer at 85°C for 10 min, cooled to 18-25°C, and let stand for no less than 60 min to obtain a double helix capture probe DNA mixture; where the sulfhydryl-modified DNA1 has a nucleotide sequence as shown in SEQ ID NO. 1, the auxiliary DNA2 has a nucleotide sequence as shown in SEQ ID NO. 2, and the auxiliary DNA3 has a nucleotide sequence as shown in SEQ ID NO. 3; the reaction is conducted at 85°C. In the present disclosure, the Tris-HCI buffer may further preferably include 0.1 M NaCl and may be at pH 7.4. In the present disclosure, the standing may enable mutual hybridization of the three DNA strands to form a double helix capture probe DNA. In the examples of the present disclosure, miIRNA-21 is taken as an example to construct the Raman-enhanced substrate and the Raman sensor, where the sulfhydryl-modified DNA 1 has a nucleotide sequence as shown in SEQ ID NO. 1. 5-(SH)-TTTTTTGAAATG GTGGAAAGGTAGGGTCAACATCAGTCTGATAAGCTA-3';

[31] the auxiliary DNA2 has a nucleotide sequence as shown in SEQ ID NO. 2: 5'- TCAGACTGATGTTGACCCTATATCCATAAATT-3';

[32] the auxiliary DNAS3 has a nucleotide sequence as shown in SEQ ID NO. 3: 5'- CCTTTCCACCATTTC-3'.

[33] After the double helix capture probe DNA mixture is obtained, in the present disclosure, the double helix capture probe DNA mixture is mixed with TCEP and incubated for 1 h to obtain an incubation buffer, the TCEP and a mixture of the sulfhydryl-modified DNA 1, the auxiliary DNA2 and the auxiliary DNA3 have a molar ratio of 1,000: (9-10). In the present disclosure, the incubation may preferably be conducted at 25°C. The addition of the TCEP in the present disclosure may break disulfide bonds on the sulthydryl DNA 1.

[34] After the incubation buffer is obtained, in the present disclosure, the incubation buffer is dropped onto the surface of the Raman-enhanced substrate, reacted at 0°C for 6 h, and rinsed with PBS thrice; after drying with purified nitrogen, the Raman-enhanced substrate is placed in MCH to react for 2 h, rinsed thrice with PBS, and dried with purified nitrogen to obtain the Raman sensor. The 0°C environment in the present disclosure may preferably be an ice bath environment. The PBS of the present disclosure may preferably have a molar concentration of 0.01 M and a pH value of 7.0. The MCH of the present disclosure may preferably be a freshly prepared 1 mM solution. The addition of the MCH of the present disclosure may not only make the foregoing double helix capture probe DNA stand upright on the surface of the Raman-enhanced substrate in an orderly manner, and but also block other active sites on the surface of the Raman substrate to prevent non-specific adsorption of other biological molecules.

[35] The present disclosure further provides a method for detecting miRNA using the Raman sensor constructed by the foregoing construction method, including the following steps: separately dropping different concentrations of miRNA-21 and R6G- modified Raman probe DNA mixture on the Raman sensor, reacting for 85 min, rinsing the Raman sensor with PBS, placing the Raman sensor under a 633 nm laser to measure surface Raman intensity, plotting a standard curve as a function of the target compound concentration versus Raman intensity, and calculating a miRNA concentration according to the standard curve. Herein, the R6G-modified Raman probe DNA preferably has a nucleotide sequence as shown in SEQ ID NO. 4: 5'-TCA GAC TGA TGTTGACCC TAC CTT TCC ACCATTTC-(R6G)-3'.

[36] When miRNA is detected by the present disclosure, a standard curve is first plotted, taking the detection of miRNA-21 in the example as an example, specifically including steps of: taking 10 uL of a set of concentration gradients of target miRNA-21 and R6G-modified Raman probe DNA (1.0 uM) mixture, respectively adding dropwise to the Raman sensor and reacting at room temperature for 85 min, and rinsing the Raman sensor twice with 10 mM pH 7.4 PBS; finally, under the 633 nm laser, measuring a surface-enhanced Raman intensity of the sensor on a Raman spectrometer, plotting a standard curve as a function of the target compound concentration versus the Raman intensity, and calculating the linear regression equation as I = 1770.18IgC + 27169.55 according to the standard curve, where I is a Raman signal in the presence of a target compound, and C is a concentration of the target compound, with a linear correlation coefficient of R = 0.991.

[37] The Raman-enhanced substrate, the fabrication method and use thereof provided by the present disclosure will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present disclosure.

[38] Kit and instrument include: Laser Microscopic Confocal Raman Spectrometer (RamLab-010, Renishaw, UK), Electrochemical Workstation (CHI660B, Shanghai Chenhua Instrument Co., Ltd.), and Water Purification System (Sybergy UV, Merck Millipore).

[39] HAuCl4 4H:0 (chloroauric acid), tris(2-carboxyethyl)phosphine (TCEP) and mercaptohexanol (MCH) are purchased from Sigma-Aldrich; DNAs and RNAs are synthesized by Takara Biotechnology (Dalian) Co., Ltd. (where DNA1 is sulfhydryl- modified).

[40] Example 1

[41] A piece of ITO glass was cut into a small piece (1 * 4 cm), put in 2-propanol containing 2 M KOH for boiling for 20 min, cleaned thoroughly, cleaned with ethanol and water in an ultrasonic bath, and dried at 60°C. The cleaned ITO glass chips were immersed in 5.0 mL of electrolyte containing 0.05 M phosphate solution (pH = 2), 0.05 M KCI and 2.4 mM HAuCly 4H:0; the electrolyte was filled with nitrogen and held at 60°C, and cyclic voltammetry (potential between -0.8 V and 0.3 V at a rate of 0.05 V/s) was used to act for 50 cycles. Under this condition, the color of the ITO electrode turned from colorless to yellow, indicating the successful deposition of three-dimensional gold nanoparticles on the electrode (FIG. 1). Finally, these ITO chips were used as Raman- enhanced substrates for microwashing and stored at 4°C for subsequent experiments.

[42] Example 2

[43] 20 mL of Tris-HCI buffer (0.1 M NaCl, pH 7.4) containing 3.0 uM sulthydryl- modified DNA 1, 3.2 uM auxiliary DNA2 and 3.2 uM auxiliary DNA3 was heated at 85°C for 10 min, and cooled to room temperature for at least 60 min to enable mutual hybridization of the three DNA strands to form a double helix capture probe DNA. 1.0 mM TCEP was added to the above probe mixture, incubated at 25°C for 1 h to break disulfide bonds on the sulfhydryl-modified DNA1; finally, 10 uL of capture probe DNA solution was dropped on the surface of the well-prepared Raman substrate chips in an ice bath to react for 6 h, and the chips were washed with 0.01 M pH 7.0 PBS thrice; after drying with purified nitrogen, the chips were immersed into 1 mM fresh MCH to react for 2 h, washed with 0.01 M pH 7.0 PBS thrice, and dried with nitrogen to obtain a Raman sensor for subsequent experiments.

[44] Herein, the sulthydryl-modified DNA 1 has a nucleotide sequence as shown in SEQ ID NO. 1: 5'-(SH)- TTTTTTGAAATGGTGGAAAGGTAGGGTCAACATCAGTCTGATAAGCTA-3'

[45] the auxiliary DNA2 has a nucleotide sequence as shown in SEQ ID NO. 2: 5'- TCAGACTGATGTTGACCCTATATCCATAAATT-3";

[46] the auxiliary DNA3 has a nucleotide sequence as shown in SEQ ID NO. 3: 5'- CCT TTC CAC CAT TTC-3".

[47] Example 3

[48] 10 pL of a set of concentration gradients of target miRNA-21 and R6G-modified Raman probe DNA (1.0 uM) mixture were added dropwise to the Raman sensor to react at room temperature for 85 min, and rinsed twice with 10 mM pH 7.4 PBS; finally, under the 633 nm laser, a surface-enhanced Raman intensity of the sensor was measured on a Raman spectrometer, a standard curve was plotted as a function of the target compound concentration versus the Raman intensity (FIG. 3), and the linear regression equation was calculated as I = 1770.18IgC + 27169.55 according to the standard curve (where 1 is a Raman signal in the presence of a target compound, and C is a concentration of the target compound), with a linear correlation coefficient of R = 0.991. Herein, the R6G- modified Raman probe DNA has a nucleotide sequence as shown in SEQ ID NO. 4: 5'- TCA GAC TGA TGTTGACCC TAC CTT TCC ACC ATTTC-(R6G)-3'.

[49] Example 4

[50] (1) Selectivity test: The selectivity of the sensor was verified by using target miRNA-21, single-base mismatch miRNA-21, multiple-base mismatch miRNA-21, and a blank group. As shown in FIG. 4, when there are excessive single-base mismatch miRNA-21 (5.0 nM) and multiple-base mismatch miRNA-21 (5.0 pM), there is a tiny change in Raman signal compared with the blank test (the target miRNA-21 is absent in the blank test). However, compared with the single-base mismatch miRNA (5.0 nM), the Raman intensity is significantly enhanced when a small amount of target miRNA-21 appears. These comparisons clearly indicate that due to the high sequence dependence of the Toehold-mediated strand displacement reaction, the sensor has excellent selectivity for the Raman detection method of miRNA-21; herein, the target miRNA-21 has a nucleotide sequence as shown in SEQ ID NO. 5: 5'-UAG CUU AUC AGA CUG AUG UUG A-3';

[51] the single-base mismatch miRNA-21 has a nucleotide sequence as shown in SEQID NO. 6: 5'-UAG CUU AUC AGA CCG AUG UUG A-3

[52] the multiple-base mismatch miRNA-21 has a nucleotide sequence as shown in SEQ ID NO. 7: 5'-UAG CUA AUC AGA CCG AUG UAG A-3".

[53] (2) Sample measurement: A sample containing the target miRNA-21 was mixed with R6G-modified Raman probe DNA (1.0 uM) and added to the Raman sensor. After reacting at room temperature for 85 min, the Raman sensor was rinsed with 10 mM pH

7.4 PBS twice; finally, under the 633 nm laser, a surface-enhanced Raman intensity of the sensor was measured on a Raman spectrometer; according to the Raman intensity (I = 2176) and using the linear regression equation (I = 1770.18lgC + 27169.55), the content of the target miRNA-21 in the sample was calculated to be 7.6 fM.

[54] The patent fabricates a surface-enhanced Raman substrate. The detection specificity is improved by modifying a capture probe with a special structure, and a Toehold-mediated strand displacement reaction is combined to amplify a signal, so that highly sensitive and specific Raman detection of the target miRNA can be realized.

[55] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, and such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Sequence Listing <110> Qingdao University of Science and Technology <120> RAMAN-ENHANCED SUBSTRATE, FABRICATION METHOD THEREOF, AND METHOD FOR DETECTING miRNA <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 48 <212> DNA <213> Artifical sequence <400> 1 ttttttgaaa tggtggaaag gtagggtcaa catcagtctg ataagcta 48 <210> 2 <211> 32 <212> DNA <213> Artifical sequence <400> 2 tcagactgat gttgacccta tatccataaa tt 32 <210> 3 <211> 15 <212> DNA <213> Artifical sequence file:///LNV INTERN/Home/S/Sahadat A/SEQLTXT txt[30-6-2021 15:11:00]

<400> 3 cctttccacc atttc 15 <210> 4 <211> 35 <212> DNA <213> Artifical sequence <400> 4 tcagactgat gttgacccta cctttccacc atttc 35 <210> 5 <211> 22 <212> RNA <213> Artifical sequence <400> 5 uagcuuauca gacugauguu ga 22 <210> 6 <211> 22 <212> RNA <213> Artifical sequence <400> 6 uagcuuauca gaccgauguu ga 22 <210> 7 <211> 22 file:///LNV INTERN/Home/S/Sahadat A/SEQLTXT txt[30-6-2021 15:11:00]

<212> RNA

<213> Artifical sequence

<400> 7 uagcuaauca gaccgaugua ga 22 file:///LNV INTERN/Home/S/Sahadat A/SEQLTXT txt[30-6-2021 15:11:00]

Claims

“11 - Conclusions

A method of manufacturing a reinforced raman substrate, comprising the steps of: placing a cleaned and dried glass indium tin oxide (“indium tin oxide”, ITO) chip in an electrolyte and reacting for 50 cycles via cyclic voltammetry ; wherein the electrolyte comprises the following molar concentrations of components: 0.03 - 0.1 M phosphate solution, 0.03 - 0.1 M KCl and 2.0 - 2.5 mM HAuCl:4 4H:0.

The manufacturing method according to claim 1, wherein the cleaning comprises the steps of: placing ITO glass in 2-propanol containing 2 M KOH, boiling for 18-25 min and ultrasonic cleaning in an ultrasonic bath of aqueous ethanol solution for 3 — 5 min.

The manufacturing method according to claim 1, wherein after the glass ITO chip is placed in the electrolyte, the electrolyte is filled with nitrogen and maintained at 60°C.

The manufacturing method according to claim 3, wherein the amount of the electrolyte is 4 cm ? glass ITO chip/5 mL of electrolyte.

The manufacturing method of claim 1, wherein the cyclic voltammetry potentially cycles between -0.8 V and 0.3 V at a rate of 0.05 V/s.

Reinforced raman substrate manufactured by the manufacturing method according to any one of claims 1 to 5, wherein the reinforced raman substrate is a layer of three-dimensional gold nanoflower foil deposited on the surface of an ITO glass chip.

A method of constructing a raman sensor based on the reinforced raman substrate manufactured by the manufacturing method according to any one of claims 1 to 5 or the reinforced raman substrate according to claim 6, which

The following steps include: step 1, allowing sulfhydryl-modified DNAL1, additional DNA2 and additional DNA3, in a molar ratio of 3.0:3.2:3.2, to react in Tris-HCl buffer for 10 min, cooling to 18-25°C and standing a reaction mixture for not less than 60 min to obtain a probe double helix capture DNA mixture; wherein the sulfhydryl-modified DNAI has a nucleotide sequence as shown in SEQ ID NO.1, wherein the additional DNA2 has a nucleotide sequence as shown in SEQ ID NO. 2, and wherein the additional DNA3 has a nucleotide sequence as shown in SEQ ID NO. 3; wherein the reaction is carried out at 85°C; step 2, mixing the probe double helix capture DNA mixture with tris(2-carboxyethyl)phosphine (TCEP) and incubating a resulting mixture for 1 hour to obtain an incubation buffer; wherein the TCEP and a mixture of the sulfhydryl-modified DNA L1, the supplemental DNA2, and the supplemental DNA3 has a molar ratio of 1,000 : (9-10); and step 3, dropping the incubation buffer onto the surface of the enhanced raman substrate reacting at 0°C for 6 hours, and rinsing the enhanced raman substrate three times with phosphate buffer solution (PBS); after drying with purified nitrogen, placing the enhanced raman substrate in mercaptohexanol (MCH) to react for 2 h, rinsing the enhanced raman substrate three times with PBS, and drying with purified nitrogen to obtain the raman sensor.

The construction method of claim 7, wherein the PBS in step 3 has a molar concentration of 0.01 M and a pH of 7.0.

A method for detecting miRNA using the raman sensor constructed by the construction method according to claim 7 or 8, comprising the steps of: dropping different concentrations of miRNA-21 and rhodamine 6G-modifiedraman DNA separately mixture of a probe on the raman sensor, reacting for 85 min, rinsing the raman sensor with PBS, placing the raman sensor under a 633 nm laser to measure surface raman intensity, plotting a standard curve as a function of target compound concentration versus Raman intensity, and calculating a

-13-miRNA concentration according to the standard curve.

The method of claim 9, wherein the standard curve is 1 = 1770.181 gC + 27169.55, where I is a Raman signal in the presence of a target compound, and C is a concentration of the target compound, with a linear correlation coefficient of R = 0.99].