A method for detecting microRNA-21 by photothermal analysis

Abstract

The application discloses a method for detecting microRNA-21 by photothermal analysis. The method mixes lock-step chain functionalized gold nanoparticles, substrate chain functionalized gold nanoparticles, microRNA-21, divalent aqueous manganese salt and water to generate HRP; H2S is generated by the reaction of thiosulfate and hydrogen peroxide catalyzed by HRP, and then the etching of Prussian blue by H2S affects the photothermal performance of Prussian blue, thereby constructing the linear relationship between the concentration of microRNA-21 and temperature, and realizing the signal detection of the target microRNA-21. The method ingeniously combines the PB with the 3DDNA Walker-based signal amplification, greatly improves the sensitivity, and realizes the low-concentration and high-selectivity detection of the target.

Description

TECHNICAL FIELD

[0001] The application relates to a MicroRNA-21 detection method, in particular to a photothermal analysis MicroRNA-21 detection method based on 3DDNAWalker and H2S etching Prussian blue, and belongs to the field of photothermal biological analysis. BACKGROUND

[0002] MicroRNA (miRNA) is a kind of non-coding single-stranded RNA molecule encoded by an endogenous gene, generally having a length of 18-25 nucleotides, which plays a crucial role in the biological processes of cell growth, proliferation, differentiation and apoptosis.

[0003] Traditional detection methods include electrochemistry, fluorescence, colorimetry, surface plasmon resonance (SPR) and electrochemiluminescence-based biological analysis methods, which exhibit satisfactory performance in the detection of miRNA, however, it is inevitable that most of these analysis techniques usually need to rely on expensive large instruments and complex sample preparation, which limits their more extensive application. Therefore, it is of important practical significance to develop a detection technology with simplicity, convenience, high sensitivity and selectivity.

[0004] In recent years, photothermal detection technology has attracted extensive attention due to its simple operation process and easy-to-read detection signal. Compared with other methods, photothermal detection method has obvious advantages such as simple operation, easy signal reading, etc. In the photothermal detection method, the temperature change sensing reading can be easily detected by a widely used thermometer, which relies on a photothermal probe under near-infrared (NIR) irradiation. Many photothermal agents and nanomaterials have been explored in photothermal detection, such as gold nanostructures, carbon-based materials and organic dye molecules. Among them, Prussian blue nanoparticles (PBNPs) as an ancient dye are a prototype of mixed valence transition metal hexacyanoferrite, which exhibits the advantages of low cost, convenient preparation and high molar extinction coefficient. PBNPs have attracted extensive attention in photothermal detection due to their good photothermal effect, although PBNPs have many advantages, however, the recent photothermal sensors based on PBNPs mainly rely on the in-situ generation of PBNPs or the combination of PBNPs with antigens and antibodies, therefore, there are inherent defects such as complex preparation, complicated surface process, high modification cost, etc. SUMMARY

[0005] The detection method of the MicroRNA-21 of the application is not for the purpose of diagnosing or treating diseases, but only for overcoming the defects of the existing detection technology and making improvements, and the purpose of the application is to provide a detection method of MicroRNA-21, which is based on the hybridization reaction of miRNA-21 and Locker, and ingeniously designs 3D DNAWalker signal amplification, hydrogen sulfide (H2S) generated by catalysis of horseradish peroxidase (HRP), and temperature change of etching reaction of PB by means of H2S, so as to realize high sensitivity and high selectivity detection of miRNA-21.

[0006] In order to realize the above technical purpose, the application provides a photothermal analysis method for detecting MicroRNA-21, which comprises the following steps:

[0007] 1) mixing and reacting the Locker walking chain functionalized gold nanoparticles, the substrate chain functionalized gold nanoparticles, the MicroRNA-21, the divalent water-soluble manganese salt and water, and then magnetically separating to obtain a solution containing HRP;

[0008] 2) adding a thiosulfate solution and a hydrogen peroxide solution into the solution containing HRP to react, so as to obtain a solution containing hydrogen sulfide;

[0009] 3) reacting the solution containing hydrogen sulfide with Prussian blue, and then performing photothermal detection on the obtained reaction solution to obtain a temperature response value;

[0010] 4) performing photothermal detection on the standard MicroRNA-21 solution with different concentrations according to steps 1) to 3) to obtain a series of temperature response values, and constructing a standard curve of the concentration of the standard MicroRNA-21 solution and the temperature difference;

[0011] 5) performing photothermal detection on the MicroRNA-21 solution to be detected according to steps 1) to 3) to obtain a temperature response value, and calculating the concentration of the MicroRNA-21 solution to be detected according to the standard curve.

[0012] The principle of the technical scheme of the application for photothermal analysis detection of MicroRNA-21 is that when the target (MicroRNA-21) exists, the MicroRNA-21 hybridizes with the Locker on the Locker walking chain functionalized gold nanoparticles (WPs) to release the walking chain (Walker), and the separated Walker can combine with the substrate chain (Track) on the substrate chain functionalized gold nanoparticles (TPs). The Walker has a DNAzyme sequence capable of recognizing Mn 2+ , so that the Mn 2+As a cofactor, the Walker assists in cleaving the HRP-modified DNA fragment on the track, releasing the HRP. The hybridization reaction between the Walker and adjacent tracks causes the relative movement of WPs around TPs, resulting in the release of a large amount of HRP and thus achieving a large signal accumulation. After magnetic separation, a solution containing a large amount of HRP is obtained. This solution is reacted with thiosulfate solution and hydrogen peroxide solution, using HRP to catalyze the generation of H2S. PB itself has a photothermal effect and strong light absorption capacity, which can convert light into heat under near-infrared light irradiation. PB can be etched by hydrogen sulfide and lose its photothermal activity. When a solution containing H2S is added to a quantitative PB solution, PB and H2S undergo an etching reaction, reducing the PB content, decreasing the temperature change, and increasing the temperature difference. The temperature change value is linearly correlated with the concentration of miRNA-21 within a certain range, enabling signal detection of the target analyte, thus achieving the photothermal detection of miRNA-21.

[0013] As a preferred method, walker-chain functionalized gold nanoparticles, substrate chain functionalized gold nanoparticles, MicroRNA-21, divalent water-soluble manganese salt, and water are reacted at 30–40 °C for 1.0–3.0 h. Walker nanoparticles possess the ability to recognize Mn. 2+ The DNA zyme sequence, therefore, Mn 2+ As a cofactor, the Walker assists in cleaving the HRP-modified DNA fragment on the Track, releasing the HRP. The Walker then hybridizes with adjacent Tracks, causing relative movement of WPs around TPs, resulting in the release of a large amount of HRP. The divalent water-soluble manganese salt is preferably manganese dichloride.

[0014] As a preferred embodiment, the chain-locking functionalized gold nanoparticles are prepared by the following method: After incubating the walking chain with a locking chain, tris(2-chloroethyl) phosphate is added for incubation, followed by incubation with gold nanoparticles, thus obtaining chain-locking functionalized gold nanoparticles. First, the walking chain and locking chain are incubated to lock the walking chain; then, tris(2-chloroethyl) phosphate is added for incubation to reduce the formation of disulfide bonds in the DNA; finally, gold nanoparticles are added for further incubation, successfully modifying the surface of the gold nanoparticles to obtain chain-locking functionalized gold nanoparticles.

[0015] As a preferred method, the walking chain and the locking chain are incubated at 80-100℃ for 5-20 min, then tris(2-chloroethyl) phosphate is added and incubated at 30-40℃ for 1.0-2.0 h, and then gold nanoparticles are added and incubated at 30-40℃ for 10-15 h.

[0016] As a more preferred solution, the DNA sequence of the locking strand is: 5'-CCG CGG TCA ACA TCA GTC TGA TAA GCT A-3'.

[0017] As a more preferred solution, the DNA sequence of the walking strand is: 5'-SH-TTT TTT TTT TTT TTT TTT TTT TTT TTT TCT GAT GTT GAC CGC GGC CAG GCT AGC TAC AAC GAC CTG GAC GA-3'.

[0018] As a preferred solution, the substrate strand functionalized gold nanoparticles are prepared by the following method: after the substrate strand is incubated with tris(2-chloroethyl) phosphate, gold nanoparticles are added for incubation and SA-HRP enzyme is added for incubation, and the substrate strand functionalized gold nanoparticles are obtained.

[0019] As a more preferred solution, the substrate strand is incubated with tris(2-chloroethyl) phosphate at 30-40℃ for 1.0-2.0h, then gold nanoparticles are added for incubation at 30-40℃ for 10-15h, and then SA-HRP enzyme is added for incubation at 30-40℃ for 10-40min; the sequence of the substrate strand is: 5'-SH-TTT TTT TTT TTT TTT CGT CCA GG-rA-rU-GGC CGC GG-biotin-3'.

[0020] As a more preferred solution, the thiosulfate solution and the hydrogen peroxide solution are added dropwise in the HRP-containing solution and reacted at 30-40℃ for 10-40min. The concentration of the thiosulfate solution is 5.0-6.0nM. The concentration of the hydrogen peroxide solution is 2.0-5.0nM, and both are added to the HRP-containing solution according to the same volume ratio. The thiosulfate is preferably sodium thiosulfate. The most preferred method for generating H2S is: 20μL of a 5.6nM concentration of Na2S2O3 solution and 20μL of a 3.0mM concentration of H2O2 solution are added dropwise into the HRP-containing solution and mixed for incubation for 30min, and H2S is generated by the catalytic action of HRP.

[0021] As a more preferred solution, the Prussian blue is cubic Prussian blue. Cubic Prussian blue has high reactivity.

[0022] As a more preferred solution, the hydrogen sulfide-containing solution is reacted with Prussian blue at 30-40℃ for 0.5-1.5h.

[0023] The cubic Prussian blue of the application is prepared by the following method: first, prepare an HCl solution, then dissolve PVP and K4[Fe(CN)6]·3H2O into the HCl solution, ultrasonic treatment, make it fully dissolved, obtain a clear solution under magnetic stirring, then, transfer the mixture to a Teflon-lined autoclave, after cooling, continuously wash the product with distilled water and dihydrogen ethanol, finally, place the product in a vacuum drying crucible.

[0024] In the photothermal detection process of the application, the laser wavelength is 808nm, the power density is 2.63W·cm -2 , and the irradiation time is 2min.

[0025] The Prussian blue (PB) of the application is a cubic structure with smooth surface and has strong light absorption capacity, which can convert light into heat under near-infrared light irradiation.

[0026] The synthesis method of the gold nanoparticles of the application is as follows: 0.6ml of 0.035M sodium citrate and 20ml of water are added to a round-bottom flask, then the above solution is quickly added to 0.2ml of 0.025M HAuCl4 solution boiling, keep boiling and continuous stirring until the color of the solution changes from yellow to purple, and then cool the solution to room temperature.

[0027] The synthesis method of the WPs of the application is as follows: 50mL of 2mM Walker and 50mL of 2mM Locker are combined in 90℃ Tris-HCl buffer (pH=8.0) for 10min, then cooled to room temperature to obtain the combination of the two, then 10mL of TCEP (10mM) is added at 37℃ and incubated for 90min, then 200μL of AuNPs (pH9.0) dispersion prepared by the above method is added at 37℃ and incubated for 12h, 10μL of 0.1%Tween 20 solution is added to the above mixture and incubated for another 30min. Finally, 90mL of 2M NaCl solution is added to the mixture in six times at an interval of 30min, and reacted at 37℃ for 24h, after centrifugation, the obtained WPs are re-dispersed in 50μL of deionized water.

[0028] The preparation method of the TPs of the application is as follows: 10μL of TCEP (10mM) is added to 50mL of Track (5mM) and reacted at 37℃ for 1.5h, 100μL of AuNPs (pH9.0) prepared by the above method is added at 37℃ and incubated for 12h, 10μL of 0.1%Tween 20 is added to the above mixture and incubated for another 30min, then 5mL of SA-HRP is added and reacted at 37℃ for half an hour, centrifuged to obtain TPs, which are dispersed in 50μL of deionized water.

[0029] The specific preparation method of the cubic Prussian blue of the application is as follows: first, 10 mL of 0.1M HCl solution is configured, then 300 mg of PVP and 22 mg of K4[Fe(CN)6]·3H2O are accurately weighed and dissolved into the HCl, ultrasonic treatment is performed to make it fully dissolved, a clear solution is obtained under magnetic stirring for 30 min, next, the mixture is transferred to a Teflon-lined autoclave and heated at 80℃ for 24 hours, after cooling to room temperature, the product is washed with distilled water and ethanol three times respectively, finally, the product is placed in a vacuum dried crucible and driven at 60℃ for 12 hours.

[0030] The application provides a photothermal analysis method for detecting MicroRNA-21, comprising the following steps:

[0031] (1) HRP generation process based on 3D DNA Walker: 10 μL of WP dispersion, 10 μL of TPs dispersion, 10 μL of miRNA-21, 10 μL of MnCl2 solution (2mM) and 10 μL of deionized water are mixed, and reaction is carried out at 37℃ for 2.5h, when miRNA-21 exists, it hybridizes with Locker on WPs to release Walker, then, the separated Walker can combine with Track on TPs, the Walker with DNAzyme sequence can recognize Mn 2+ , and therefore Mn 2+ As a cofactor, the Walker assists in cleaving the HRP-modified DNA fragment on the Track to release HRP, the Walker can hybridize with the adjacent Track to cause the relative movement of WPs around TPs, resulting in the release of a large amount of HRP and the realization of large signal accumulation.

[0032] (2) H2S generation process: the product solution obtained by DNA Walker reaction is subjected to magnetic separation to obtain a solution containing a large amount of HRP, then 20 μL of Na2S2O3 (5.6nM) solution and 20 μL of H2O2 (3.0mM) solution are added dropwise into the supernatant and mixed for incubation for 30 min, the HRP in the supernatant plays a catalytic role to generate H2S.

[0033] (3) miRNA-21 photothermal detection: 20 μL of H2S solution with different concentrations obtained is mixed with 70 μL of water and 10 μL of PBNPs at 37℃ for 30 min, and then transferred into a 0.2 mL centrifuge tube. Next, the centrifuge tube is irradiated with a laser with a power of 2.63Wcm -2 , and a wavelength of 808nm for 2 min. The temperature difference before and after irradiation is recorded, and a standard curve can be drawn according to the logarithm of the concentration of the standard sample; the above detection is carried out by replacing the miRNA-21 standard solution with the test solution, and the concentration result is obtained through the standard curve.

[0034] Compared with the prior art, the technical scheme of the present application has the beneficial technical effects:

[0035] 1) The present application improves the detection sensitivity by a biomolecule-based signal amplification strategy. The three-dimensional DNA Walker signal amplification reaction is triggered by the target. The hybridization of the target and Locker on the WP leads to the release of Walker. Then, the separated Walker can bind to Track on the TP, and the DNAzyme sequence on the Walker can recognize Mn 2+ Therefore, Mn 2 + As a cofactor, the Walker helps to cleave the HRP-modified DNA fragment on the Track, releasing HRP. The Walker can hybridize with the adjacent Track, causing the relative movement of the WPs around the TPs, resulting in the release of a large amount of HRP, thereby achieving large signal accumulation and achieving the purpose of signal amplification.

[0036] 2) The present application utilizes the good photothermal effect of Prussian blue (PBNPs), which can convert light into heat under near-infrared light irradiation. By utilizing the temperature change of the etching reaction between PBNPs and H2S, the detection of the target is achieved, thereby constructing a sensor-free photothermal detection method based on H2S etching PBNPs.

[0037] 3) The present application ingeniously links the concentration of the target and the change of temperature by generating H2S from the mixed solution of Na2S2O3 and H2O2 catalyzed by HRP, successfully constructing a detection method for MicroRNA-21 based on 3D DNA Walker and H2S etching PB photothermal analysis.

[0038] 4) The signal amplification detection method constructed by the present application for miRNA-21 detection has a good linear relationship between the temperature difference and the logarithm of the target concentration in the concentration range of 0.2 nM-200 pM, with the advantages of wide linear range and low detection limit. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Figure 1 is a schematic diagram of the principle of a photothermal analysis based on Prussian blue (PBNPs) combined with a 3D DNA Walker signal amplification strategy for a MicroRNA-21 detection method.

[0040] Figure 2 (A) and (B) are scanning electron microscope and transmission electron microscope images of smooth cubic PBNPs, respectively. Figure 2(C) and (D) are scanning electron microscope images and transmission electron microscope images of PBNPs after reaction with H2S, respectively.

[0041] Figure 3 For the standard sample test results of Example 1, A is the concentration of the detection target of Example 1 and the corresponding thermal image; B is the logarithmic linear graph of the temperature difference and the concentration of the detection target of Example 1, C is the selectivity test results of Example 1, and D is the stability test results of Example 1. DETAILED DESCRIPTION

[0042] The technical solutions of the present application will be further described in detail below through specific implementation examples, but the protection scope of the claims of the present application cannot be limited thereby.

[0043] Optimization of experimental conditions:

[0044] Taking miRNA with a concentration of 2nM as an example and adopting the control variable method in the experimental exploration, the optimal conditions of the reaction time of DNA walker, the concentration of manganese dichloride, and the reaction time of hydrogen sulfide and Prussian blue are discussed. In the experiment, it can be seen that when the reaction time of DNA walker is 2.5h, the temperature change begins to stabilize, in order to save the reaction time, it can be concluded that the optimal reaction time of DNA walker is 2.5h. Mn 2+ As a cofactor, it affects the catalytic activity of DNAzyme. When the concentration of manganese dichloride is in the range of 0.2nM-2.0nM, the temperature difference increases the most when the concentration of manganese dichloride solution is 1.0nM, so the optimal concentration of manganese dichloride solution is 1.0nM. The change of temperature is through the photothermal effect of Prussian blue, so the reaction time of hydrogen sulfide and Prussian blue is discussed. When the reaction time is 1.0h, the temperature difference increases most obviously, so the optimal reaction time is 1.0h. The following example conditions are all under the optimal conditions.

[0045] Example 1

[0046] (1) Add 0.6ml of 0.035M sodium citrate and 20ml of water to a round-bottom flask. Then quickly add the above solution to 0.2ml of 0.025M HAuCl4 solution boiling, keep boiling and constant stirring until the color of the solution changes from yellow to purple, which indicates the formation of AuNPs. Finally, cool the solution to room temperature and store at 4℃ in the dark before use.

[0047] (2) 50 mL of 2 mM Walker and 50 mL of 2 mM Locker were combined in 90 °C Tris-HCl buffer (pH = 8.0) for 10 min, then cooled to room temperature to obtain the combination of both. Before functionalization, 10 mL of TCEP (10 mM) was added at 37 °C and incubated for 90 min to reduce the formation of disulfide bonds. Next, 200 μL of AuNPs (pH 9.0) dispersion prepared in step (1) was added at 37 °C for 12 h, and 10 μL of 0.1% Tween 20 solution was added to the above mixture for another 30 min. Finally, 90 mL of 2 M NaCl solution was added to the mixture in six equal portions at an interval of 30 min, and reacted at 37 °C for 24 h. After centrifugation, the resulting WPs were re-dispersed in 50 μL of deionized water; 10 μL of TCEP (10 mM) was added to 50 mL of Track (5 mM) and reacted at 37 °C for 1.5 h to reduce the formation of disulfide bonds in DNA. Next, 100 μL of AuNPs (pH 9.0) dispersion prepared in step (1) was added at 37 °C for 12 h, and 10 μL of 0.1% Tween 20 was added to the above mixture for another 30 min. Finally, 5 mL of SA-HRP was added and reacted at 37 °C for half an hour to obtain TPs. Similarly, it was dispersed in 50 μL of deionized water, and the resulting WPs and TPs were stored at 4 °C in the dark before use.

[0048] (3) 300 mg of PVP and 22 mg of K4[Fe(CN)6]·3H2O were added to 10 ml of HCl (0.1 M) solution, and then a clear solution was obtained by ultrasonic treatment under magnetic stirring for 30 minutes. Next, the mixture was transferred to a Teflon-lined autoclave and heated at 80 °C for 24 hours. After cooling to room temperature, the product was washed with distilled water and dihydroethanol three times, respectively. Finally, the product was placed in a vacuum-dried crucible and driven at 60 °C for 12 hours.

[0049] (4) 10 μL of microRNA with different concentrations were mixed with 10 μL of WPs and TPs prepared in step (2), 10 μL of MnCl2 and 10 μL of deionized water, and reacted at 37°C for 2.5 h. A solution of Na2S2O3 (20 μL, 5.6 nM) and a solution of H2O2 (20 μL, 3 mM) were added to the supernatant obtained after centrifugation, and reacted for half an hour to obtain H2S with different concentrations. After 20 μL of H2S solution with different concentrations, 70 μL of water and 10 μL of 50 mg / L PBNPs were mixed at 37°C for half an hour, they were transferred to a 0.2 mL centrifuge tube. The centrifuge tube was irradiated with a laser with a wavelength of 808 nm for 2 min for photothermal detection, and the temperature difference before and after irradiation was recorded by an infrared temperature imager. Due to the signal amplification effect of DNA Walker, a large amount of HRP was generated, which played a catalytic role to generate a large amount of H2S, and the etching reaction of H2S with PBNPs occurred, the temperature difference was significantly reduced, and the temperature difference had a certain relationship with the concentration of miRNA-21, so as to realize the sensitive detection of miRNA-21; the above detection was carried out by using the test solution instead of the miRNA-21 standard solution, and the concentration result was obtained by the standard curve.

[0050] The DNA sequences used in Example 1 were purchased from Shenguo Bioengineering (Shanghai) Co., Ltd., and are as follows:

[0051]

[0052] Figure 1 The principle and process schematic diagram of the photothermal analysis MicroRNA-21 detection method based on 3D DNA Walker and H2S etching Prussian blue (PBNPs) involved in the present application.

[0053] Figure 2 The scanning electron microscope and transmission electron microscope images of PBNPs and PBNPs etched by H2S. The scanning electron microscope shows that PBNPs have a smooth surface and a cubic structure with an average diameter of 160 nm. After the etching reaction of PBNPs with H2S, the smooth surface disappears, which proves that the etching reaction with H2S is successful.

[0054] Figure 3 In Table A, the temperature difference shows a trend of increasing with the increase of the concentration of miRNA-21. For example, Figure 3 Table B shows that in the concentration range of 0.2 nM to 200 pM of miRNA-21, the temperature difference has a good linear relationship with the logarithm of the concentration of the target. In addition, in order to prove the practical application of the present application in life, the selectivity and stability of the method for miRNA-21 were also investigated. The experimental results show that Figure 3The detection method has good selectivity for miRNA-21. In addition, the relative standard deviations (RSD) of five detection results are all less than 3%, and the detection method has good stability Figure 3 The detection method has good selectivity for miRNA-21. In addition, the relative standard deviations (RSD) of five detection results are all less than 3%, and the detection method has good stability

[0055] The above only describes the best embodiments of the present application, and any equivalent changes and modifications made within the scope of the present application should be included in the scope of the present application.

Claims

1. A method for detecting MicroRNA-21 by photothermal analysis, characterized in that: Includes the following steps: 1) Locked walking chain functionalized gold nanoparticles, substrate chain functionalized gold nanoparticles, MicroRNA-21, divalent water-soluble manganese salt and water were mixed and reacted at 30~40°C for 1.0~3.0 h, and then separated by magnetic separation to obtain an HRP-containing solution. 2) Add thiosulfate solution and hydrogen peroxide solution to HRP solution and react at 30~40°C for 10~40 min to obtain hydrogen sulfide solution; 3) After reacting a solution containing hydrogen sulfide with Prussian blue, the resulting reaction solution was subjected to photothermal detection to obtain the temperature response value; 4) Perform photothermal detection on standard MicroRNA-21 solutions of different concentrations according to steps 1) to 3) to obtain a series of temperature response values, and construct a standard curve between the concentration of standard MicroRNA-21 solution and the temperature difference; the Prussian blue is cubic Prussian blue; 5) Perform photothermal detection on the MicroRNA-21 solution to be tested according to steps 1) to 3), obtain the temperature response value, and calculate the concentration of the MicroRNA-21 solution to be tested according to the standard curve.

2. The method for photothermal analysis and detection of MicroRNA-21 according to claim 1, characterized in that: The locked walking chain functionalized gold nanoparticles were prepared by the following method: after incubating the walking chain with the locked chain, tris(2-chloroethyl) phosphate was added for incubation and then gold nanoparticles were added for incubation in sequence, thus obtaining the locked walking chain functionalized gold nanoparticles.

3. The method for photothermal analysis and detection of MicroRNA-21 according to claim 2, characterized in that: The walking chain and locking chain were incubated at 80-100°C for 5-20 min, then tris(2-chloroethyl) phosphate was added and incubated at 30-40°C for 1.0-2.0 h, and then gold nanoparticles were added and incubated at 30-40°C for 10-15 h.

4. The method for photothermal analysis and detection of MicroRNA-21 according to claim 3, characterized in that: The DNA sequence of the locked strand is: 5'-CCG CGG TCA ACA TCA GTC TGA TAA GCT A-3'; The DNA sequence of the walking chain is: 5'-SH-TTT TTT TTT TTT TTT TTT TTT TTT TTT TCT GATGTT GAC CGC GGC CAG GCT AGC TAC AAC GAC CTG GAC GA-3'.

5. The method for photothermal analysis and detection of MicroRNA-21 according to claim 1, characterized in that: The substrate chain-functionalized gold nanoparticles were prepared by the following method: the substrate chain was incubated with tris(2-chloroethyl) phosphate, followed by incubation with gold nanoparticles and then with SA-HRP enzyme, to obtain the substrate chain-functionalized gold nanoparticles.

6. The method for photothermal analysis and detection of MicroRNA-21 according to claim 5, characterized in that: The substrate chain was incubated with tris(2-chloroethyl) phosphate at 30-40°C for 1.0-2.0 h, followed by the addition of gold nanoparticles and incubation at 30-40°C for 10-15 h, and then the addition of SA-HRP enzyme and incubation at 30-40°C for 10-40 min. The sequence of the substrate chain is: 5'-SH-TTT TTT TTTTTT TTT CGT CCA GG-rA-rU-GGC CGC GG-biotin-3'.

7. The method for photothermal analysis and detection of MicroRNA-21 according to claim 1, characterized in that: A solution containing hydrogen sulfide is reacted with Prussian blue at 30-40°C for 0.5-1.5 hours.

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