A homogeneous entropy-driven amplification system combined with colorimetric silver nanoclusters for the detection of microRNA

Abstract

The application discloses a kind of enzyme-free, entropy-driven combination DNA-AgNCs detection microRNA-21 fluorescence ratio type biosensor.The analysis method is composed of two parts, the first part is the target cycle design without enzyme, L-DNA, S, Trigger chain is mixed and annealed to form stable three-strand DNA substrate in PB and CH3COONa hybridization buffer according to 1:1:1 molar ratio, when target exists, Trigger is released in the triggered entropy-driven cycle amplification system;The second part is the ratio fluorescence signal probe part, Y2-DNA or Y3-DNA is mixed in PB buffer according to DNA:AgNO3:NaBH4=1:6:6 molar ratio, obtain Y2-AgNCs and Y3-AgNCs.Y2-AgNCs, Y3-AgNCs and equal concentration Y1-DNA are mixed with equal volume, form Y type ratio fluorescence signal probe.When target microRNA-21 exists, trigger entropy-driven reaction to cause strand displacement and release Trigger chain, it is hybridized with Y1 chain in Y type structure, open Y type structure, Y2-AgNCs, Y3-AgNCs chain dimer separates, realize the controllable change of fluorescence emission from red fluorescence to yellow fluorescence, through the change of the ratio of two fluorescence intensities, realize the high sensitivity detection of target microRNA-21.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biochemical analysis method, and particularly relates to an analysis and detection method for super-sensitive detection of microRNA-21. BACKGROUND

[0002] MicroRNAs (miRNAs) are non-coding endogenous RNA molecules existing in eukaryotic cells, composed of 18-24 nucleotides, involved in the degradation or blocking of target mRNA at the post-transcriptional level, and play a very key role in regulating physiological processes such as development, cell proliferation, differentiation and death of human cells. In recent years, more and more studies have shown that miRNAs can be used as biomarkers for diagnosis of various cancers. In the detection method of miRNAs, nano-material assisted fluorescence method has attracted more and more attention due to its short detection time, high sensitivity and simple operation. DNA-AgNCs is a new type of fluorescent nanomaterial, which overcomes the problems of traditional organic fluorescent dyes such as easy photobleaching, poor solubility and poor stability, and gradually applies to the fields of biosensing and imaging with excellent light stability and biocompatibility, sub-nanometer size and fluorescence adjustable depending on the scaffold DNA sequence. The early fluorescence biosensor based on DNA-AgNCs is almost designed based on the fluorescence quenching (turn-off) or enhancement (turn-on) effect at a single specific wavelength, which will be disturbed by environmental factors or instruments, reducing the reliability of the measurement results. The ratio type AgNCs fluorescence biosensor simultaneously monitors the relative fluorescence changes of two completely separated wavelengths instead of monitoring the intensity changes of a single emission wavelength, which can greatly improve the detection sensitivity. As a kind of nucleic acid amplification strategy, entropy-driven signal amplification can be widely used in miRNA detection combined with fluorescence, colorimetric and other detection methods. Therefore, based on the effective signal amplification of entropy-driven reaction, combined with the ratio type AgNCs fluorescence signal method, a unique new idea is provided for the detection of miRNA. SUMMARY

[0003] Based on this idea, in order to improve the detection sensitivity, we combined the target-triggered enzyme-free entropy-driven signal amplification system with Y-type DNA conformation conversion and color-changing AgNCs based on DNA template to construct an enzyme-free and label-free ratio type fluorescence biosensor, and provided a high-sensitivity ratio detection analysis method for the target microRNA-21.

[0004] The application specifically provides the following technical solutions:

[0005] 1. A fluorescence ratio-type biosensor for detecting miRNA-21 without enzyme and entropy-driven binding DNA-AgNCs, which is composed of two parts. The first part is an enzyme-free target cycle design. L-DNA, S, and Trigger chain are mixed in a molar ratio of 1:1:1 in a hybridization buffer of PB and CH3COONa and annealed to form a stable triple-stranded DNA substrate. When the target is present, the Trigger released in the triggered entropy-driven amplification system; the second part is a ratio fluorescence signal probe part. Y2-DNA or Y3-DNA is mixed in a molar ratio of DNA: AgNO3: NaBH4 = 1:6:6 in a PB buffer. After shaking, Y2-AgNCs and Y3-AgNCs are obtained after overnight incubation. Y2-AgNCs, Y3-AgNCs, and Y1-DNA with the same concentration are mixed in equal volume to form a Y-type signal probe. When the target miRNA-21 is present, the triggered entropy-driven reaction causes strand displacement to release the Trigger chain, which hybridizes with the Y1 chain in the Y-type structure, opening the Y-type structure, and separating the Y2-AgNCs and Y3-AgNCs chain dimers, realizing the controllable transition of fluorescence emission from red fluorescence to yellow fluorescence. Through the change of the intensity ratio of red fluorescence to yellow fluorescence, high-sensitivity detection of the target miRNA-21 is realized.

[0006] The application method of the above-mentioned ratio sensor for detecting miRNA-21, comprising the following steps:

[0007] 1) Y2-DNA or Y3-DNA is mixed with AgNO3 in a PB buffer, and after vigorous shaking for 5s, it is placed in an ice water bath at 4℃ for 1h of dark incubation;

[0008] 2) NaBH4 solution is added to the solution after incubation in step 1), and after vigorous shaking for 20s, it is placed in an ice water bath at 4℃ for overnight incubation in the dark to prepare Y2-AgNCs / Y3-AgNCs monomers;

[0009] 3) Y2-AgNCs and Y3-AgNCs in step 2) are mixed with Y1-DNA with the same concentration in equal volume to form a Y-type signal probe;

[0010] 4) L-DNA, S, and Trigger chain are annealed to 25℃ in a hybridization buffer of PB and CH3COONa to form a triple-stranded DNA substrate after 2h of reaction;

[0011] 5) The entropy-driven amplification system obtained in step 4) and the Y-type signal probe obtained in step 3) are mixed in equal volume and equal concentration for fluorescence testing;

[0012] 6) Fuel-DNA is added to the triple-stranded DNA substrate obtained in step 4) and mixed thoroughly, and after dilution, it is divided into 10 PCR tubes.

[0013] 7) Add different concentrations of miRNA-21 to each PCR tube of step 6), and add buffer to the control group. React at 25℃ for 30 min.

[0014] 8) Add the Y-shaped structure signal probe obtained in step 3) to each PCR tube of step 7), mix well, and react at 25℃ for 2 h. Perform fluorescence test to obtain the specific detection result of miRNA-21.

[0015] Further, the molar ratio of DNA: AgNO3 in step 1) is kept at 1:6, the molar ratio of Y2-DNA (Y3-DNA), AgNO3 and NaBH4 in step 2) is always kept at 1:6:6, and the molar ratio of L-DNA, S, Trigger chain in step 4) is always kept at 1:1:1.

[0016] Further, the PB buffer in step 1) contains 20mM Na2HPO4 and NaH2PO4, pH = 7.4.

[0017] Further, the concentration of AgNO3 solution in step 1) is 1.8μM, and the concentration of NaBH4 solution is 1.8μM.

[0018] Further, the annealing process in step 4) is heated at 95℃ for 5 min, reduced to 65℃ at a rate of -5℃ / min, and then reduced to 25℃ at a rate of -1℃ / min.

[0019] Further, the range of miRNA-21 in step 7) is 0.5-100nM.

[0020] Further, the final three-stranded DNA substrate in step 8) is 300nM, the Fuel-DNA is 600nM, and the Y-shaped structure signal probe is 300nM.

[0021] Further, the fluorescence spectrum parameters in steps 5) and 8) are set to excitation slit and emission slit of 10nm, and scanning voltage of 700V.

[0022] The application has the beneficial effects that the fluorescent AgNCs monomers and the AgNCs dimers assembled based on the Y-type DNA structure realize the controllable switching between the yellow fluorescence of the monomers and the red fluorescence of the dimers through the conformational transformation of the Y-type structure. With the increase of the concentration of the target miRNA-21 to be detected, the red fluorescence of the dimers at 630 nm gradually weakens, and the yellow fluorescence of the monomers at 570 nm gradually strengthens, and the ratio of F630nm / F570nm is used to quantitatively detect miRNA-21. In addition, the detection performance of the sensor is successfully improved by introducing the entropy-driven target cycle signal amplification reaction. The results show that the ratio of the fluorescence intensity of the system F630nm / F570nm gradually decreases with the increase of the concentration of miRNA-21, the linear range is 0-100nM, the detection limit is 10.24pM, and the sensor has good specificity; in addition, the biosensor also shows good detection performance in the real sample 1% human serum. Since the entropy-driven amplification system and the Y-type structure signal probe system are independent of each other, the scheme has good universality and modularization characteristics, and only the sequence of the entropy-driven amplification system needs to be modified, and the high-sensitivity detection of another target can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to make the purpose, technical scheme and beneficial effects of the application more clear, the application provides the following drawings:

[0024] Figure 1 A schematic diagram of a homogeneous entropy-driven target cycle signal amplification system combined with a color-changing AgNCs to construct a ratio fluorescence biosensor platform;

[0025] Figure 2 UV-vis spectra of AgNCs monomers and dimers;

[0026] Figure 3 Fluorescence spectra of AgNCs monomers and dimers;

[0027] Figure 4 Gel electrophoresis diagram for verifying the formation and opening of Y structure;

[0028] Figure 5 Gel electrophoresis diagram for verifying the feasibility of the entropy-driven target cycle signal amplification reaction;

[0029] Figure 6 Gel electrophoresis diagram for the combination of the entropy-driven target cycle signal amplification reaction and the Y-type structure;

[0030] Figure 7 Gel electrophoresis diagram for the optimization of the substrate chain and the Fuel chain in the entropy-driven amplification reaction;

[0031] Figure 8Gel electrophoretogram for reaction time optimization in entropy-driven target cycle signal amplification reaction;

[0032] Figure 9 Analysis graph of different miRNA-21 concentrations and fluorescence signal intensity;

[0033] Figure 10 Selective analysis graph;

[0034] Figure 11 Analysis graph for target detection in 1% serum of real sample. DETAILED DESCRIPTION

[0035] The experimental principle is as shown in Figure 1 Without the target miRNA-21 to be detected, the triple-stranded DNA substrate does not release the Trigger strand, and since Y1, Y2 and Y3 form a Y-shaped structure, and Y2 and Y3 are very close to each other, the two monomers of AgNCs form a dimer to emit red fluorescence at 630 nm; when the target miRNA-21 exists, the entropy-driven reaction is triggered to release the Trigger strand, which hybridizes with the Y1 strand in the Y-shaped structure to open the Y-shaped structure, and the Y2 and Y3 strand dimer separates to emit yellow fluorescence at 570 nm, realizing the controllable conversion of fluorescence emission from red fluorescence to yellow fluorescence. Through the change of the ratio of red fluorescence intensity to yellow fluorescence intensity, high-sensitivity detection of the target miRNA-21 to be detected is realized.

[0036] The preferred embodiments of the present application will be described in detail below with reference to the accompanying drawings.

[0037] Example 1: Pretreatment of DNA

[0038] First, the powder-shaped primer DNA ordered from Shanghai Shengong was centrifuged at 4000 rpm / min for 1 min before use, and was dissolved in ultrapure water to obtain a 100 μM DNA solution according to the requirements. When used, PB buffer was used for preparation, and the buffer composition was 20 mM Na2HPO4, 20 mM NaH2PO4, and PH = 7.4.

[0039] The L-DNA sequence is 5'-ACCTAA CGT GTC TCT AGT GGG ATC AAC ATC AGT CTG ATA AGCTA-3';

[0040] The S sequence is 5'-TCAGAC TGATGT TGATCC C-3';

[0041] The Trigger sequence is 5'-GAT GAC TAG AGACAC GTT AGG TCG GCT GCG CT-3';

[0042] Fuel-DNA sequence is 5'-TCAGACTGATGTTGATCCCAC TAGAGCACGT T-3';

[0043] miRNA-21 sequence is 5'-UAGCUUAUCAGACUGAUGUUGA-3';

[0044] Y1 sequence is 5'-CAT TAG CGC AGC CGACCT AAC GTG TCT CTAGTC ATC-3';

[0045] Y2 sequence is 5'-GAG ACA CGT TAG GTC AGA CTA GAA AAC CCC CTA ATT CCC CC-3';

[0046] Y3 sequence is 5'-CCC CCT TAATCC CCC AAT TCT AGT CTG CGG CTG CGC TAA TG-3'.

[0047] Example 2 Preparation and characterization of DNA-AgNCs

[0048] A certain volume of Y2-DNA or Y3-DNA with a certain concentration was mixed with 500 μΜ of AgN03 solution in 20 mM PB buffer (pH = 7.4) at a molar ratio of DNA: AgN03 = 1:6, then shaken vigorously for 5 s, placed in an ice water bath at 4°C for 1 h of incubation in the dark. After 1 h, 500 μΜ of freshly prepared NaBH4 solution with a molar ratio of 1:1 to AgN03 was added, shaken vigorously for 20 s, incubated overnight in the dark in an ice water bath at 4°C, and then subjected to fluorescence testing. The molar ratio of Y2-DNA (Y3-DNA), AgN03 and NaBH4 was always kept at 1:6:6. Y2-AgNCs, Y3-AgNCs were mixed with Y1-DNA at the same concentration and volume in 20 mM PB + 20 mM CH3COONa buffer to form Y-type signal probes. Hybridization was characterized by gel electrophoresis, which was performed at 100 V for about 60 minutes, and then the gel was stained with Gelred.

[0049] To verify the successful preparation of fluorescent AgNCs, we used ultraviolet-visible spectrophotometer (UV-vis) and fluorescence spectrum for characterization. By analyzing the UV-visible spectrum of the synthesized AgNCs monomers and dimers, as shown in Fig. 2a, the characteristic absorption peaks of AgNCs monomers and dimers were observed at 320 nm and 340 nm, respectively. The characteristic absorption peak of Y2-AgNCs and Y3-AgNCs was observed at 340 nm, which was consistent with the characteristic absorption peak of the dimers. The characteristic absorption peak of Y-type signal probe was observed at 320 nm, which was consistent with the characteristic absorption peak of the monomers. The results showed that the Y-type signal probe was successfully prepared. Figure 2As shown, both of them have strong absorption peaks at 260 nm, and the absorption intensity of the dimer is significantly higher than that of the monomer, which is mainly due to the interaction of the DNA template. In addition, the dimer has a characteristic absorption peak at 410 nm, which is significantly different from the monomer. Under ultraviolet lamp irradiation, the monomer is yellow fluorescent and the dimer is red fluorescent. By measuring the excitation wavelength of AgNCs monomer and dimer respectively, it is found that the monomer gets an emission wavelength of 570 nm at the best excitation wavelength of 500 nm, and the dimer gets an emission wavelength of 630 nm at the best excitation wavelength of 565 nm. Figure 3 The fluorescence spectra of the monomer and the dimer at the excitation wavelengths of 500 nm and 565 nm respectively. The emission wavelength is consistent with the corresponding wavelength of the observed fluorescence color in the above figure. The experimental results confirm the successful preparation of AgNCs.

[0050] Example 3: Polyacrylamide gel electrophoresis

[0051] Verify the formation of Y-type DNA structure and opening after adding trigger chain. Prepare 10% polyacrylamide gel electrophoresis, run the gel at 100V voltage, then image it on the gel imager. As shown in Figure 4 Band 1-3 is three single-stranded DNA constituting the Y-type structure; band 4 is double-stranded DNA hybridized by Y1 and Y2-AgNCs; band 5 is double-stranded DNA hybridized by Y1 and Y3-AgNCs; in addition, the requirement of this experiment is that Y2-AgNCs and Y3-AgNCs do not hybridize in the absence of Y1, and this process is also verified in band 6, that is, Y2-AgNCs and Y3-AgNCs alone do not hybridize; band 7 is the Y-type structure formed; band 8 is the addition of Trigger based on 7, which also indicates that the Y-type structure is opened.

[0052] Verify the feasibility of entropy-driven target cycle signal amplification reaction. As shown in Figure 5 Band 1-4 is L chain, Fuel chain, Trigger chain, and S chain in turn; band 5 is the three-stranded DNA substrate of L+Trigger+S; band 6 is (L+Trigger+S)+miRNA-21, it can be seen that strand displacement occurs, and the addition of miRNA-21 displaces the S chain; band 7 is L+Fuel; band 8 is the control group of (L+Trigger+S)+Fuel; band 9 is the experimental group of (L+Trigger+S)+miRNA-21+Fuel.

[0053] Verify the combination of entropy-driven target cycle signal amplification reaction and Y-type structure. After determining the ratio of three-stranded DNA substrate and Fuel chain in entropy driving as 1:2, we combine the entropy-driven target cycle signal amplification system with the Y-type structure signal probe. As shown inFigure 6 As shown in the figure, strip 1 is Y structure; strip 2 is a control group of (L+Trigger+S)+Fuel; strip 3 is an experimental group of (L+Trigger+S)+miRNA-21+Fuel; strip 4 is a control group combined with 1:1 Y structure on the basis of strip 2; and strip 5 is an experimental group combined with 1:1 Y structure on the basis of strip 3. The gray value of strip 4 and strip 5 is compared, and compared with the control group in strip 4, the experimental group in strip 5 is mostly released because of the three-strand DNA substrate L+Trigger+S, so that the Y structure is opened more, the strip yield of L+Fuel, Y1+Trigger, Y2-AgNCs+Y3-AgNCs is more than that of the product in the control group, and the target miRNA-21 strip released in circulation is also clear, thus proving the feasibility of the combination of entropy-driven amplification reaction and Y structure. The result of polyacrylamide gel electrophoresis verifies the feasibility of the scheme.

[0054] Optimization of experimental conditions in example 4

[0055] The present application optimizes the ratio of three-strand DNA substrate to Fuel chain. The ratio is set as (L+Trigger+S):Fuel=1:1, 1:1.5 and 1:2 respectively. As shown in Figure 7 A, strips 1, 3 and 5 are control groups without target, only L, Trigger, S and Fuel chains exist, and strips 2, 4 and 6 are experimental groups under three ratios, respectively, in which the target chain is added to the three-strand DNA substrate at a ratio of 1:1. Combined with Figure 7 B, the gray value of L+Fuel product in the lane is compared, and it is found that when (L+Trigger+S):Fuel=1:2, the yield of L+Fuel in the control group is the least, the yield of L+Fuel in the experimental group is the most, and the difference between the experimental group and the control group is the largest, so the best reaction ratio is (L+Trigger+S):Fuel=1:2, which can release the Trigger chain under the same conditions and improve the reaction efficiency.

[0056] The present application optimizes the reaction time in the entropy-driven target cycle signal amplification reaction, and the control groups and experimental groups with reaction times of 30 min, 60 min, 120 min and 180 min are analyzed by running gel. As shown in Figure 8 A, strips 1, 3, 5 and 7 are control groups with reaction times of 30 min, 60 min, 120 min and 180 min, respectively, and strips 2, 4, 6 and 8 are experimental groups under each reaction time. Combined with Figure 8 B, the gray value of L+Fuel product in the lane is compared, and it is found that the best reaction time of the system entropy-driven is 30 min.

[0057] Sensor analysis of miRNA

[0058] The detection process of the whole miRNA-21 includes two parts, first complete the entropy-driven amplification reaction and then combine with Y-shaped structure signal probe. First, add different concentrations of miRNA-21 (final concentration of 0, 0.5, 1, 2, 3, 5, 10, 30, 50, 100 nM) and molar ratio of 1:2 Fuel-DNA to the pre-formed three-stranded DNA substrate, and react at 25℃ for 30 min. After 30 min, mix with the same volume and concentration of pre-formed Y-shaped structure signal probe uniformly, and react at 25℃ for another 2 h. The performance of the sensor for detecting miRNA-21 is explored by detecting the change of fluorescence intensity in the whole system.

[0059] As shown in Figure 9 A, with the increase of the concentration of miRNA-21, the red fluorescence intensity of AgNCs dimer at 630 nm gradually weakens, and the yellow fluorescence of AgNCs monomer at 570 nm gradually enhances. As shown in Figure 9 B, the ratio of F630nm / F570nm decreases with the increase of the concentration of miRNA-21. When the concentration of miRNA-21 is low, the concentration of miRNA-21 C miRNA-21 / nM is taken as the abscissa, and the ratio of two fluorescence intensities F630nm / F570nm is taken as the ordinate, as shown in the insert of Figure 9 B, within the range of 0-3 nM, F630nm / F570nm is linearly related to C miRNA-21 / nM, and the lowest detection limit calculated is 10.24 pM.

[0060] Specificity analysis

[0061] In order to evaluate the specificity of the invention for detecting miRNA-21, control experiments were performed by measuring the fluorescence signals of miRNA-21 and other sequences (miRNA-141, Let-7a, MDR1, random sequence) with the same concentration. The results are shown in Figure 10 Compared with the fluorescence signal response of miRNA-21, when the concentration of other molecules is measured, the observed fluorescence signal is roughly the same as that of the blank control without target, which indicates that the method has good selectivity.

[0062] Real sample analysis

[0063] Add 50 nM of miRNA-21 to 1% human serum sample, and from Figure 11As can be seen, the analysis results in serum samples are substantially consistent with the analysis results in 20 mM PB + 20 mM CH3COONa buffer, indicating that it has potential for practical application.

[0064] Finally, it should be pointed out that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various modifications can be made in form and details without departing from the scope defined by the claims of the present application.

Claims

1. A ratio sensing method for detecting miRNA using a homogeneous entropy-driven amplification system combined with color-changing silver nanoclusters, characterized in that, The steps are as follows: 1) Mix Y2-DNA or Y3-DNA with AgNO3 using PB buffer, shake vigorously for 5 seconds, and then incubate in a 4°C ice-water bath in the dark for 1 hour. 2) Add NaBH4 solution to the solution after incubation in step 1), shake vigorously for 20 seconds, and then incubate overnight in an ice-water bath at 4°C in the dark to prepare Y2-AgNCs / Y3-AgNCs monomers. 3) Mix Y2-AgNCs and Y3-AgNCs from step 2) with an equal volume of Y1-DNA to form a Y-type signal probe; 4) Anneal the L-DNA, S, and Trigger strands to 25°C in PB and CH3COONa buffer and react for 2 hours to form a triple-stranded hybrid DNA substrate; 5) Mix the triple-stranded hybrid DNA substrate obtained in step 4) and the Y-type signal probe obtained in step 3) in equal volumes and concentrations, and perform fluorescence testing; 6) Take another triple-stranded hybridization DNA substrate obtained in step 4), add Fuel-DNA, mix thoroughly, dilute and divide into 10 PCR tubes; 7) Add different concentrations of miRNA-21 to each PCR tube in step 6), and add buffer to the control group. React at 25 °C for 30 min. 8) Add the Y-shaped signal probe obtained in step 3) to each PCR tube in step 7), mix thoroughly, react at 25°C for 2 h, and perform fluorescence detection to obtain the specific detection results of miRNA-21, where the L-DNA sequence is 5'-ACCTAA CGT GTC TCT AGT GGG ATC AAC ATC AGT CTG ATA AGC TA-3'; The S sequence is 5'-TCA GAC TGA TGT TGA TCC C-3'; The trigger sequence is 5'-GAT GAC TAG AGA CAC GTT AGG TCG GCT GCG CT-3'; The Fuel-DNA sequence is 5'-TCA GAC TGA TGT TGA TCC CAC TAG AGA CAC GTT-3'; The miRNA-21 sequence is 5'-UAG CUU AUC AGA CUG AUG UUG A-3'; The Y1 sequence is 5'-CAT TAG CGC AGC CGA CCT AAC GTG TCT CTA GTC ATC-3'; The Y2 sequence is 5'-GAG ACA CGT TAG GTC AGA CTA GAA AAC CCC CTA ATT CCC CC-3'; The Y3 sequence is 5'-CCC CCT TAA TCC CCC AAT TCT AGT CTG CGG CTG CGC TAA TG-3'.

2. The method according to claim 1, characterized in that, In step 1), the molar ratio of DNA to AgNO3 is maintained at 1:

6. In step 2), the molar ratio of Y2-DNA or Y3-DNA to AgNO3 and NaBH4 is maintained at 1:6:

6. In step 4), the molar ratio of L-DNA, S, and Trigger chains is maintained at 1:1:

1.

3. The method according to claim 1, characterized in that, Step 1) The PB buffer solution contains 20 mM Na2HPO4 and NaH2PO4, pH=7.

4.

4. The method according to claim 1, characterized in that, The concentration of the AgNO3 solution in step 1) is 1.8 µM.

5. The method according to claim 1, characterized in that, The concentration of the NaBH4 solution in step 2) is 1.8 µM.

6. The method according to claim 1, characterized in that, The annealing process described in step 4) involves heating at 95 °C for 5 min, cooling to 65 °C at a rate of -5 °C / min, and then cooling to 25 °C at a rate of -1 °C / min.

7. The method according to claim 1, characterized in that, The miRNA-21 range mentioned in step 7) is 0.5-100 nM.

8. The method according to claim 1, characterized in that, Steps 5) and 8) set the fluorescence spectral parameters to 10 nm for the excitation slit and 10 nm for the emission slit, and 700 V for the scanning voltage.

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