Fluorescent sensor for detecting miRNA based on hybridization chain reaction as well as preparation method and application of fluorescent sensor

Abstract

The invention discloses a fluorescence sensor for detecting miRNA based on hybridization chain reaction as well as a preparation method and application of the fluorescence sensor. The fluorescence sensor comprises a Com1 chain which can be specifically combined with a target miRNA and is completely complementarily paired with the target miRNA; a Com2 chain capable of complementarily pairing with the Com1 chain part and used as an initiator chain of a hybridization chain reaction; the hairpin DNA comprises an H1 chain and an H2 chain, and the H1 chain is modified with a fluorophore and a corresponding fluorescence quenching group. Different from a traditional hybridization chain reaction amplification structure, a pair of nucleotide complementary double strands is added to the fluorescence sensor to serve as an intermediate structure for triggering the hybridization chain reaction, so that the fluorescence sensor can be independently assembled into the sensor for use and can also be flexibly modified on a carrier such as a silicon nanowire through the intermediate structure; therefore, different detection application scenes can be adapted. The fluorescence sensor disclosed by the invention is good in biocompatibility, high in specificity and high in sensitivity, has a good and wide application prospect, and provides a brand new thought for detecting miRNA.

Description

Technical Field

[0001] The present invention relates to the technical field of biosensors, and more specifically to a fluorescent sensor for detecting miRNA based on hybridization chain reaction (HCR), and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a common, complex and insidious neurodegenerative disease, accounting for 50-70% of all dementia cases. Due to its high incidence, irreversibility and high disability rate in the elderly population, it has become a serious social and public health problem.

[0003] Currently, AD lacks specific and sensitive early diagnosis methods, and traditional biomarkers such as amyloid protein (Aβ) and microtubule-associated protein (tau) have certain limitations in diagnostic accuracy and disease staging. In addition, the pathological process of AD involves changes in multiple molecular and cellular mechanisms, so we urgently need to find new biomarkers to improve the diagnosis level, monitor disease progression and evaluate treatment effects.

[0004] MicroRNAs (miRNAs) are a class of non-coding RNAs with a length of approximately 19-23 nucleotides, which play an important role in epigenetic and post-transcriptional regulation of gene expression. More and more studies have shown that miRNAs are abnormally expressed in samples such as brain tissue, cerebrospinal fluid (CSF) and peripheral blood of AD patients, making them a potential research hotspot for AD biomarkers. As one of the miRNAs, miR-146a-5p has attracted researchers' attention for its potential in AD diagnosis due to its functions in inflammation regulation and immune response.

[0005] Therefore, how to reliably and accurately detect these trace miRNAs with biological significance in complex matrices is an urgent problem that needs to be solved. Summary of the invention

[0006] One object of the present invention is to provide a fluorescent sensor for detecting trace amounts of miRNA under mild enzyme-free conditions based on hybridization chain reaction (HCR) signal amplification.

[0007] The second object of the present invention is to provide a method for preparing the above-mentioned fluorescent sensor for detecting miRNA based on hybridization chain reaction.

[0008] The third object of the present invention is to provide a silicon nanowire-based fluorescent sensor for detecting miRNA, which utilizes an added pair of complementary double-stranded nucleotides as an intermediate to trigger the HCR reaction, so that the sensor can be flexibly modified on a silicon nanowire carrier, such as to adapt to the application scenario of miRNA detection in a single cell.

[0009] The fourth object of the present invention is to provide a method for preparing the above-mentioned silicon nanowire-based fluorescent sensor for detecting miRNA.

[0010] The fifth object of the present invention is to provide application of the above two fluorescent sensors in detecting miRNA.

[0011] The fluorescent sensor of the present invention has high sensitivity and specificity, and can detect low-abundance target miRNA. In addition, it also has high spatial resolution. For some tissue samples, such as brain tissue slices, it can locate and detect target miRNA at the cellular level or even at the subcellular level, which is helpful to study the distribution and expression of target miRNA in nerve cells, as well as their relationship with cell morphology and functional changes, and better understand the pathophysiological process of some diseases.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] In a first aspect, the present invention provides a fluorescent sensor for detecting miRNA based on hybridization chain reaction, the fluorescent sensor comprising:

[0014] The Com1 chain can specifically bind to the target miRNA and is completely complementary to the target miRNA;

[0015] The Com2 strand is capable of partially complementary pairing with the Com1 strand and is used as the initiator strand of the hybridization chain reaction;

[0016] Hairpin DNA H1 chain and H2 chain, wherein the H1 chain is modified with a fluorescent group and a corresponding fluorescent quenching group;

[0017] The Com1 chain and the Com2 chain hybridize to form a dsC1C2 double-stranded chain, and the target miRNA competitively binds to the Com2 chain, resulting in the release of the Com2 chain, thereby triggering a hybridization chain reaction between the hairpin DNA H1 chain and the H2 chain to generate a fluorescent signal.

[0018] Furthermore, the fluorescent group may be 6-FAM, Cy3 or ROX, and the corresponding fluorescent quenching group may be BHQ1 or BHQ2.

[0019] The dsC1C2 double strand formed by hybridization of the Com1 strand and the Com2 strand described in the present invention is the main part of the fluorescent sensor, while the hairpin DNA H1 strand and H2 strand are the HCR signal amplification parts, thereby achieving quantitative detection of the target miRNA.

[0020] Exemplarily, taking the target miRNA as miR-146a-5p (shown in SEQ ID No. 1) as an example, the fluorescent sensor of the present invention comprises:

[0021] Sensor body: According to the sequence structure of the target miR-146a-5p, a Com1 chain that is fully complementary to it is designed, and then a Com2 chain that is partially complementary to Com1 is designed, and the Com2 chain is used as the initiator chain of the hybridization chain reaction. The Com1 chain and the Com2 chain hybridize to form a dsC1C2 double-stranded structure.

[0022] HCR signal amplification part: According to the sequence structure of Com2 chain, two pairs of hairpin DNA H1 chain and H2 chain are designed.

[0023] It should be noted that in the present invention, the design principle of "partial complementary pairing" means that the number of complementary base pairs between the Com1 chain and the target miR-146a-5p is greater than the number of complementary base pairs between the Com1 chain and the Com2 chain, so that the target miR-146a-5p can competitively bind to the Com2 chain, resulting in the unwinding of the Com1 chain and the Com2 chain, releasing the Com2 chain, thereby triggering the HCR reaction.

[0024] Preferably, the sequence of the Com1 strand is shown in SEQ ID No. 2; the sequence of the Com2 strand is shown in SEQ ID No. 3; the sequence of the hairpin DNA H1 strand is shown in SEQ ID No. 4, and the sequence of the hairpin DNA H2 strand is shown in SEQ ID No. 5. The structures of the hairpin DNA H1 strand and H2 strand are the same, and when forming a hairpin structure, the number of complementary base pairs is 18 pairs, the number of stem-loop base pairs is 6 pairs, the number of exposed base pairs at the hairpin tail is 6 pairs, and the total length is 48 base pairs.

[0025] Preferably, from the 5'-3' direction, the 15th base of the H1 chain is modified with a 6-FAM fluorescent group, and the 41st base is modified with a BHQ1 quenching group. Since the hairpin structures of the H1 chain and the H2 chain in the present invention are asymmetric, optimizing the distance between the fluorescent group and the corresponding fluorescent quenching group can ensure that they can interact when forming the hairpin structure, and can also ensure that after forming the HCR hybridization long chain, the fluorescent group (such as FAM) and the corresponding fluorescent quenching group (such as BHQ1) are alternately distributed, thereby maximizing the fluorescence intensity and being less affected by the fluorescent quenching group.

[0026] In a second aspect, the present invention provides a method for preparing the above-mentioned fluorescent sensor for detecting miRNA based on hybridization chain reaction, the preparation method comprising:

[0027] 1) Com1 chain and Com2 chain are dissolved in a buffer solution to prepare a mother solution, and an equal amount of the mother solution is annealed at 95°C to form a dsC1C2 double chain, and then naturally cooled to room temperature to obtain a dsC1C2 double chain solution;

[0028] 2) The H1 chain and the H2 chain are dissolved in a buffer solution to prepare a mother solution, which is annealed at 95° C. to form a hairpin structure, and then naturally cooled to room temperature to obtain a hairpin DNA H1 chain solution and a hairpin DNA H2 chain solution, respectively.

[0029] In a third aspect, the present invention provides a silicon nanowire-based fluorescent sensor for detecting miRNA, the fluorescent sensor comprising:

[0030] The Com1 chain can specifically bind to the target miRNA and is completely complementary to the target miRNA;

[0031] A Com2 chain capable of partially complementing and pairing with the Com1 chain and used as an initiator chain for hybridization chain reaction, wherein the 3' end of the Com2 chain is modified with a functional group;

[0032] Hairpin DNA H1 chain and H2 chain, wherein the H1 chain is modified with a fluorescent group and a corresponding fluorescent quenching group;

[0033] A single silicon nanowire substrate;

[0034] Among them, the Com1 chain hybridizes with the Com2 chain to form a dsC1C2 double chain, and is covalently bound to the surface of a single silicon nanowire substrate through the functional group modified at the 3' end of the Com2 chain; the target miRNA competitively binds to the Com1 chain with the Com2 chain, resulting in the exposure of the Com2 chain base on the single silicon nanowire substrate, thereby triggering a hybridization chain reaction between the hairpin DNA H1 chain and the H2 chain to generate a fluorescent signal.

[0035] Furthermore, the functional groups include but are not limited to amino, carboxyl or aldehyde groups.

[0036] Furthermore, the length of the single silicon nanowire substrate is 50-70 μm.

[0037] Furthermore, the fluorescent group is 6-FAM, Cy3 or ROX, and the corresponding fluorescence quenching group is BHQ1 or BHQ2.

[0038] For example, when the target miRNA is miR-146a-5p (shown in SEQ ID No.1), the sequence of the Com1 chain is shown in SEQ ID No.2, the sequence of the Com2 chain is shown in SEQ ID No.3, and the 3' end of the Com2 chain is modified with -(CH2) n-NH2 to obtain a Com2 chain with an amino group modified at the 3' end, wherein n is an integer from 6 to 8, and the specific structure is: CTGAATTCCATGGGTTACTAGTCG-(CH2) n -NH2; when modifying the amino group, add a carbon chain of a certain length "-(CH2) n -" can ensure that the properties of the DNA itself are not affected after the DNA chain is covalently modified to the material surface through the functional group; the sequence of the H1 chain is shown in SEQ ID No.4, and the sequence of the H2 chain is shown in SEQ ID No.5; the Com1 chain and the Com2 chain modified with an amino group at the 3' end are hybridized to form a dsC1C2-NH2 double-stranded structure.

[0039] Preferably, from the 5'-3' direction, the 15th base of the H1 chain is modified with a 6-FAM fluorescent group, and the 41st base is modified with a BHQ1 fluorescent quenching group.

[0040] In a fourth aspect, the present invention provides a method for preparing the above silicon nanowire-based fluorescent sensor for detecting miRNA, the preparation method comprising:

[0041] thermally oxidizing the silicon nanowire array to obtain an oxidized silicon nanowire array;

[0042] The surface of the oxidized silicon nanowire array is subjected to hydroxylation and amination treatment in sequence, and then the silicon nanowires are peeled off to obtain a single amination silicon nanowire;

[0043] Performing aldehyde treatment on the aminated single silicon nanowire to obtain aldehyde-treated single silicon nanowire;

[0044] The 3' end of the Com2 chain is modified with an amino group, hybridized with the Com1 chain to form a dsC1C2-NH2 double chain, and then covalently bound to the surface of the aldehyde-modified single silicon nanowire substrate, dispersed in a buffer solution, and a single silicon nanowire solution loaded with a dsC1C2 double chain is obtained;

[0045] The H1 chain and the H2 chain were dissolved in a buffer solution to prepare a mother solution, annealed at 95° C. to form a hairpin structure, and naturally cooled to room temperature to obtain a hairpin DNA H1 chain solution and a hairpin DNA H2 chain solution, respectively.

[0046] According to a specific implementation method of the present invention, the method for preparing the silicon nanowire-based fluorescent sensor for detecting miRNA includes:

[0047] 1) Preparing silicon nanowire arrays by silver ion assisted chemical etching;

[0048] 2) performing thermal oxidation treatment on the silicon nanowire array obtained in step 1), wherein the thermal oxidation treatment process is calcining at 800-900° C. for 180-240 min in an oxygen atmosphere;

[0049] 3) performing a hydroxylation treatment on the silicon nanowire array obtained in step 2), wherein the hydroxylation treatment comprises soaking the silicon nanowire array in a mixed solution of concentrated sulfuric acid and hydrogen peroxide, heating and refluxing, washing it, soaking it in a mixed solution of water, ammonia water and 30% hydrogen peroxide, washing it again, and drying it to obtain a hydroxylated silicon nanowire array;

[0050] Preferably, the heating reflux time is 1-2h.

[0051] Preferably, the soaking time is 2-10 hours.

[0052] Preferably, the volume ratio of the concentrated sulfuric acid to the hydrogen peroxide is 3-5:1, and the concentration of the concentrated sulfuric acid is 18.4 mol / L.

[0053] Preferably, the volume ratio of H2O, NH3·H2O and 30% H2O2 is 5:1:1.

[0054] 4) performing an amination treatment on the hydroxylated silicon nanowire array obtained in step 3), wherein the amination treatment is to place the hydroxylated silicon nanowire array in anhydrous toluene, add 3-aminopropyltrimethoxysilane, react at a constant temperature, take it out, clean it, peel off the silicon nanowires, and dissolve it in anhydrous ethanol to obtain an amination single silicon nanowire;

[0055] Preferably, the volume ratio of anhydrous toluene to (3-methylaminopropyl)trimethoxysilane is 250:1; and the isothermal reaction is carried out at 80-100° C. for 24-36 hours.

[0056] 5) performing a aldehyde treatment on the aminated single silicon nanowire obtained in step 4), wherein the aldehyde treatment comprises mixing the aminated single silicon nanowire with glutaraldehyde, reacting with shaking at room temperature overnight in an ethanol system, and after the reaction is completed, repeatedly washing the silicon nanowire by suction filtration, and finally dispersing it in a buffer solution to obtain the aldehyde-aminated single silicon nanowire;

[0057] Preferably, the mass concentration of the glutaraldehyde is 50%, and the volume ratio of glutaraldehyde to (3-methylaminopropyl)trimethoxysilane is 2.5:1.

[0058] 6) loading the dsC1C2 double chain onto the aldehyde-modified single silicon nanowire obtained in step 5), wherein the loading is performed by mixing the aldehyde-modified silicon nanowire and the dsC1C2-NH2 double chain solution with a PBS solution of sodium cyanoborohydride, and reacting at room temperature overnight by shaking; after the reaction is completed, centrifuging and discarding the supernatant, repeatedly washing, and finally dispersing in a buffer solution to obtain a single silicon nanowire solution loaded with the dsC1C2 double chain;

[0059] The dsC1C2-NH2 double-stranded solution is prepared by the following method: firstly, the Com1 chain and the Com2 chain with the 3'-terminal amino group modified are respectively dissolved in a buffer solution to prepare a mother solution, an equimolar amount of the mother solution is annealed at 95° C. to form a dsC1C2-NH2 double-stranded chain, and naturally cooled to room temperature to obtain a dsC1C2-NH2 double-stranded solution;

[0060] Preferably, the concentration of the aldehyde-modified silicon nanowires is 0.2-0.4 mg / ml, the concentration of the dsC1C2-NH2 double chain is 2-3 μM, and the molar concentration ratio of sodium cyanoborohydride to dsC1C2-NH2 double chain is 1:20.

[0061] 7) The H1 chain and the H2 chain are dissolved in a buffer solution to prepare a mother solution, which is annealed at 95° C. to form a hairpin structure, and then naturally cooled to room temperature to obtain a hairpin DNA H1 chain solution and a hairpin DNA H2 chain solution, respectively.

[0062] In a fifth aspect, the present invention provides the use of the above-mentioned fluorescent sensor for detecting miRNA based on hybridization chain reaction or the fluorescent sensor for detecting miRNA based on silicon nanowires in detecting miRNA.

[0063] Furthermore, the application of silicon nanowire-based fluorescent sensors for detecting miRNA in detecting miRNA in single cells is also protected.

[0064] The fluorescent sensor of the present invention detects the content of target miRNA, especially miR-146a-5p, by imaging with a confocal fluorescence microscope and exciting with a laser having a wavelength of 488 nm, through changes in fluorescence intensity.

[0065] The beneficial effects of the present invention are as follows:

[0066] (1) High selectivity: Through specially designed hairpin structures and triggering segments, it can specifically recognize the target miRNA and reduce the interference of nonspecific signals.

[0067] (2) High sensitivity: HCR is an efficient molecular amplification tool that can achieve in situ amplification reactions, thereby improving the sensitivity of detection and detecting trace amounts of miRNA.

[0068] (3) It does not require the assistance and participation of enzymes, which greatly reduces the experimental cost, simplifies the experimental operation, and has good reaction reproducibility.

[0069] (4) It is an isothermal amplification technology, which simplifies the instrument requirements and only requires simple temperature control equipment to complete the reaction, making it easy to operate.

[0070] (5) The present invention can not only specifically detect trace substances through the HCR signal amplification method, but also flexibly modify the sensor onto the silicon nanowire carrier by designing the nucleotide double chain, which can be applied to the detection of miRNA in single cells. Therefore, the present invention provides a new concept for the construction of fluorescent sensors for detecting trace nucleic acid-related small molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0072] Figure 1 The structure and working principle diagram of the fluorescent sensor for detecting miR-146a-5p based on HCR are shown;

[0073] Figure 2 The normalized linear fitting results of the fluorescent sensor for detecting miR-146a-5p based on HCR for the response test of 0-4000pM miR-146a-5p are shown;

[0074] Figure 3 The results of the selective testing of the fluorescent sensor for detecting miR-146a-5p based on HCR for common ions and negative control RNA in blood are shown;

[0075] Figure 4 A scanning electron microscope image (top view) of a silicon nanowire array is shown;

[0076] Figure 5 A scanning electron microscope image (side view) of a silicon nanowire array is shown;

[0077] Figure 6 Confocal images showing the SiNWs-based miR-146a-5p HCR fluorescence sensor after adding different concentrations of miR-146a-5p;

[0078] Figure 7 The normalized linear fitting results of the SiNWs-based miR-146a-5p HCR fluorescence sensor response test to different concentrations of miR-146a-5p are shown. DETAILED DESCRIPTION

[0079] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0080] The present invention is a fluorescent sensor for detecting target miRNA based on HCR reaction, which uses the RNA chain segment Com1 of a specific sequence as the recognition group of the target miRNA, and the Com2 chain as a partial complementary chain segment of the Com1 chain and an HCR initiator chain segment. Figure 1 As shown, after the Com1 chain and the Com2 chain hybridize into a double-stranded structure, when the target miRNA exists, since the Com1 chain and the target miRNA have more complementary base pairs and the binding is more stable, the Com1 chain and the Com2 chain unwind and bind to the target miRNA. The released Com2 chain acts as an initiator of the hybridization chain reaction, inducing the hairpin DNA H1 chain and the H2 chain to open the hairpin structure to form a HCR hybrid long chain. The fluorescence of the fluorescent group on the H1 chain that was originally quenched by the fluorescent quenching group is restored and used as the sensor detection signal. Finally, the concentration of the target to be measured is confirmed by comparing with the standard curve of the target miRNA.

[0081] The following describes it in detail with reference to specific embodiments.

[0082] Example 1 A fluorescent sensor for detecting miR-146a-5p based on HCR

[0083] 1. Preparation of a fluorescent sensor for detecting miR-146a-5p based on HCR, comprising the following steps:

[0084] 1) Synthesize RNA and DNA sequences, each sequence is shown in Table 1 below:

[0085] Table 1 All DNA and RNA segment sequences of the present invention

[0086]

[0087] Note: In the sequence table, "t" represents uracil (U) in RNA sequence and thymine (T) in DNA sequence.

[0088] 2) Preparation of Com1 chain, Com2 chain, H1 chain, and H2 chain master liquor: Centrifuge the centrifuge tubes containing each nucleic acid chain segment obtained in step 1) at 5000 rpm for 2 minutes. Then gently open the lid and add an appropriate amount of TE buffer (pH=7.4, 0.01M) or ultrapure water to obtain a 100μM master liquor. Cover and shake for ten minutes to mix evenly. After centrifugation, dilute the tubes and store them in a 4℃ or -20℃ refrigerator for use.

[0089] 3) Annealing to prepare dsC1C2 double-strand solution, hairpin DNA H1 strand solution and H2 strand solution: Place a beaker containing aqueous solution on a heating table, heat to 95°C, and place the centrifuge tube containing the above DNA / RNA solution (V Com1 :V Com2 =1:1). After being kept at constant temperature for 5 min, the mixture was slowly cooled to room temperature and then stored in a refrigerator at 4°C for later use.

[0090] 2. Detection of response to miR-146a-5p, including the following steps:

[0091] A buffer solution system (PH=7.2-7.4) of 10 mM Tris, 150 mM NaCl, and 10 mM MgCl2 was accurately prepared and used as the detection solution system of the fluorescence sensor.

[0092] FL-4600 fluorescence spectrometer (Hitachi) was selected as the detection instrument, 485 nm was selected as the excitation wavelength, and the fluorescence intensity at the maximum emission peak was collected. The maximum emission peak fluorescence intensity of the detection solution system without miR-146a-5p was F0, and the maximum emission peak fluorescence intensity of the detection solution system containing different concentrations of miR-146a-5p was F, and the F / F0 value was used as the detection signal. Specifically:

[0093] 1) Add 1 mL of Tris buffer solution containing 3.5 nM dsC1C2 double strand, 10 nM hairpin DNA H1 strand and 10 nM hairpin DNA H2 strand into a 1 cm × 1 cm cuvette, keep the temperature at 25°C for 1 hour, and measure the maximum emission peak fluorescence intensity of the solution system as F0.

[0094] 2) Accurately add different volumes of miR-146a-5p standard solution to make the final concentrations of 200pM, 400pM, 600pM, 800pM, 1000pM, 1500pM, 2000pM, 2500pM, 3000pM, 3500pM, and 4000pM, respectively, and react at 25°C for 2h. The maximum emission peak fluorescence intensity of the solution system was measured to be F; each concentration was repeated three times.

[0095] 3) After sorting the data, the linear fitting results are as follows Figure 2 As shown, we can get:

[0096] Relative fluorescence intensity F / F0 = 0.0045 × C miR-146a-5p (pM) + 1.22, the calculated detection limit LOD = 3.98pM, R 2 =0.999. This indicates that the sensor has good performance.

[0097] Example 2 Anti-interference test of HCR fluorescence sensor of miR-146a-5p

[0098] Accurately prepare 1.5M Na + , 500mM K + 、100mMMg 2+ , 300 mM Ca 2+ Standard solution: accurately prepare 100 μM miR-146a-5p and NC-RNA master solution, wherein NC-RNA is a negative control nucleic acid chain for the miR-146a-5p chain segment, and its sequence is shown in SEQ ID No.6.

[0099] 1 mL of Tris buffer solution containing 3.5 nM dsC1C2 chain, 10 nM hairpin DNA H1 chain and 10 nM hairpin DNA H2 nucleic acid chain was added to a 1 cm × 1 cm cuvette and kept at 25 ° C for 1 h. The maximum emission peak fluorescence intensity of the solution system was measured to be F0. 100 μM miR-146a-5p and 1.5 M Na + , 500mM K + 、100mMMg 2+ , 300 mM Ca 2+ , NC-RNA standard solution, so that the final concentrations were 2000pM, 150mM, 5mM, 1mM, 3mM, 2000pM, respectively, and the reaction was carried out at a constant temperature of 25°C for 2h. The maximum emission peak fluorescence intensity of the solution system was measured to be F; each concentration was repeated three times.

[0100] After arranging the data, the test results are as follows Figure 3 As shown by Figure 3 It can be seen that there is no obvious response to the above-mentioned various ions commonly found in blood and random miRNA negative control nucleic acid chains, and the detection specificity of the sensor is good.

[0101] Example 3 Recovery test of miR-146a-5p HCR fluorescence sensor in human plasma

[0102] Accurately prepare 100 μM miR-146a-5p stock solution, add 1 mL of 3.5 nM dsC1C2, 10 nM hairpin DNA H1 chain and 10 nM hairpin DNA H2 nucleic acid chain to the dilute solution of blocked normal human serum in a 1 cm × 1 cm cuvette, keep constant temperature at 25 ° C for 1 hour, and measure the maximum emission peak fluorescence intensity of the solution system as F0;

[0103] Since the detection range of the drawn working curve is 0-4000pM, three points, namely the low point, the mid point and the high point, were selected for recovery rate test. The low point selected three times of the minimum concentration (100pM) detectable by the working curve: 300pM, the mid point selected the midpoint of the detection range of the working curve: 2000pM, and the high point selected 75% of the maximum concentration (4000pM) detectable by the working curve: 3000pM.

[0104] The above-prepared 100 μM miR-146a-5p standard solution was accurately added to make the final concentrations 300 pM, 2000 pM, and 3000 pM, respectively. The reaction was carried out at a constant temperature of 25°C for 2 h, and the maximum emission peak fluorescence intensity of the solution system was measured to be F; each concentration was repeated three times.

[0105] After arranging the data, the test results are shown in Table 2. From the results in Table 2, it can be seen that in the dilute solution of blocked normal human serum, the recovery rate of the detected object miR-146a-5p is good.

[0106] Table 2 Recovery test results in blocked normal human serum

[0107]

[0108] Example 4 Preparation and testing of silicon nanowire (SiNWs)-based fluorescence sensor for detecting miR-146a-5p

[0109] 1. Preparation of SiNWs-based fluorescent sensor for detecting miR-146a-5p

[0110] 1) Preparation method of silicon nanowire array: The silicon nanowire array is prepared by a metal silver-assisted chemical solution etching method. Specifically, an N-type P-doped silicon wafer is selected. <100> , use a diamond pen to cut into 2cm×1cm long silicon wafers, wash with sufficient acetone, ethanol and ultrapure water for 10 minutes in turn; immerse the cleaned silicon wafer in a mixed solution of 5mM AgNO3 and 4.8MHF to deposit silver ions, take out the silicon wafer after 8 minutes, wash it with deionized water, put it into a mixed solution of 0.2M H2O2 and 4.6M HF, and keep it in a constant temperature water bath at 50℃ for 60 minutes. After etching, wash it repeatedly with deionized water and soak it in aqua regia overnight. Finally, take out the silicon wafer, wash it, and dry it to obtain a silicon nanowire array. Figure 4 and Figure 5 .

[0111] 2) Thermal oxidation of silicon nanowire arrays:

[0112] The silicon nanowire array in step 1) is placed flatly in a porcelain boat with its front side facing upwards, and the porcelain boat is placed in a tube furnace, heated to 900° C. in an oxygen atmosphere and kept at a constant temperature for 200 min, and then cooled naturally to room temperature.

[0113] 3) Hydroxylation of silicon nanowire arrays:

[0114] Add 90mL of concentrated sulfuric acid and 30mL of 30% H2O2 to a 250mL round-bottom flask, place the silicon nanowire array in step 2) into the above mixed solution, and heat at 90°C for 1.5h. Take out the silicon nanowire array, wash it repeatedly with deionized water for several times, then put the silicon nanowire array face up into a mixed solution containing 20mL of deionized water, 4mL of ammonia water and 4mL of 30% H2O2, soak for 2h, and then rinse it repeatedly with deionized water for several times until the washing solution is neutral, to obtain a hydroxylated silicon nanowire (SiNWs-OH) array, and put it in a vacuum drying oven to dry for later use.

[0115] 4) Surface amination of silicon nanowires:

[0116] Add 50 mL of redistilled anhydrous toluene to the hydroxylated silicon nanowire array in step 3), dehydrate the toluene solution again under a nitrogen atmosphere, then add 200 μL of 3-aminopropyltrimethoxysilane (APTES) and heat at 90°C for 24 hours. After the reaction is completed, take out the silicon nanowire array, wash it repeatedly with anhydrous toluene and anhydrous ethanol, carefully scrape off the silicon wires on the silicon nanowire array with a scalpel, and disperse and store them in anhydrous ethanol solution to obtain an amino silicon nanowire array (SiNWs-NH2).

[0117] 5) Surface aldehyde modification of silicon nanowires:

[0118] Take 200 μL (1 mg / ml) of the amino silicon nanowires in step 4) and dilute it to 0.2 mg / ml, add 30 μL of 50% wt glutaraldehyde (GA), and shake at room temperature for reaction overnight.

[0119] After the reaction was completed, the silicon nanowires were repeatedly washed three times by suction filtration, and finally dispersed in a 0.2 mg / ml PBS solution to obtain aldehyde-modified silicon nanowires (SiNWs-CHO).

[0120] 6) Silicon nanowire surface modified with dsC1C2 double chain:

[0121] 110 μL 0.2 mg / ml SiNWs-CHO PBS solution, 10 μM dsC1C2-NH2 double-strand solution and 100 μL 0.3 M NaBH3CN PBS solution were shaken and reacted at room temperature overnight. The dsC1C2-NH2 double-strand solution was prepared by the following method: first, the Com1 chain and the Com2 chain with amino group modified at the 3' end (the 3' end of the Com2 chain was modified with -(CH2)7-NH2, and its structure was CTGAATTCCATGGGTTACTAGTCG-(CH2)7-NH2) were dissolved in PBS buffer solution to prepare a mother solution, and an equimolar amount of the mother solution was annealed at 95°C to form a dsC1C2-NH2 double chain, and naturally cooled to room temperature to obtain a dsC1C2-NH2 double-strand solution;

[0122] After the reaction was completed, the mixture was centrifuged at 13,000 rpm for 10 min, the supernatant was discarded, and the mixture was carefully washed three times and finally dispersed in 1000 μL PBS solution to obtain a single silicon nanowire (SiNWs-dsC1C2) solution loaded with a dsC1C2 double chain.

[0123] 7) The H1 chain and the H2 chain were dissolved in PBS buffer solution to prepare mother solutions, annealed at 95° C. to form a hairpin structure, and naturally cooled to room temperature to obtain a hairpin DNA H1 chain solution and a hairpin DNA H2 chain solution, respectively.

[0124] 2. Response of SiNWs-based miR-146a-5p HCR fluorescence sensor to different concentrations of miR-146a-5p

[0125] The prepared SiNWs-dsC1C2 solution was imaged by confocal fluorescence microscopy.

[0126] Equimolar amounts of PBS buffer solution of hairpin DNA H1 chain and H2 chain were added, incubated at a constant temperature of 25°C for 1 hour, and the fluorescence intensity of the silicon nanowires was measured as F0 using a laser with a wavelength of 488nm; then a certain volume of miR-146a-5p standard solution was accurately added to make the final concentrations of 5nM, 15nM, 25nM, and 50nM, respectively, and the reaction was carried out at a constant temperature of 25°C for 1 hour, and the fluorescence intensity of the silicon nanowires was measured as F; each concentration group was repeated three times.

[0127] Test results such as Figure 6 , Figure 7 As shown:

[0128] Relative fluorescence intensity F / F0=0.0261×C 146a-5p (nM)+1.35

[0129] The calculated detection limit LOD = 0.69 nM R 2=0.999

[0130] This indicates that the SiNWs-based fluorescent sensor for detecting miR-146a-5p has good performance and can detect different concentrations of miR-146a-5p.

[0131] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A fluorescent sensor for detecting miRNA based on hybridization chain reaction, characterized in that: The fluorescence sensor includes: The Com1 chain can specifically bind to the target miRNA and is completely complementary to the target miRNA; The Com2 strand is capable of partially complementary pairing with the Com1 strand and is used as the initiator strand of the hybridization chain reaction; Hairpin DNA H1 chain and H2 chain, wherein the H1 chain is modified with a fluorescent group and a corresponding fluorescent quenching group; The Com1 chain and the Com2 chain hybridize to form a dsC1C2 double-stranded chain, and the target miRNA competitively binds to the Com2 chain, resulting in the release of the Com2 chain, thereby triggering a hybridization chain reaction between the hairpin DNA H1 chain and the H2 chain to generate a fluorescent signal.

2. The fluorescence sensor according to claim 1, characterized in that The fluorescent group is 6-FAM, Cy3 or ROX, and the corresponding fluorescence quenching group is BHQ1 or BHQ2; Preferably, the target miRNA is miR-146a-5p, and its sequence is shown in SEQ ID No.1; the sequence of the Com1 chain is shown in SEQ ID No.2; the sequence of the Com2 chain is shown in SEQ ID No.3; the sequence of the H1 chain is shown in SEQ ID No.4, and the sequence of the H2 chain is shown in SEQ ID No.5; Preferably, from the 5'-3' direction, the 15th base of the H1 chain is modified with a 6-FAM fluorescent group, and the 41st base is modified with a BHQ1 fluorescent quenching group.

3. The method for preparing a fluorescent sensor for detecting miRNA based on hybridization chain reaction according to claim 1 or 2, characterized in that: The preparation method comprises: 1) Com1 chain and Com2 chain are dissolved in buffer solution to prepare mother solution, and equimolar amount of mother solution is annealed at 95°C to form dsC1C2 double chain, and then cooled to room temperature naturally to obtain dsC1C2 double chain solution; 2) The H1 chain and the H2 chain are dissolved in a buffer solution to prepare a mother solution, which is annealed at 95° C. to form a hairpin structure, and then naturally cooled to room temperature to obtain a hairpin DNA H1 chain solution and a hairpin DNA H2 chain solution, respectively.

4. A silicon nanowire-based fluorescent sensor for detecting miRNA, characterized in that: The fluorescence sensor includes: The Com1 chain can specifically bind to the target miRNA and is completely complementary to the target miRNA; A Com2 chain capable of partially complementing and pairing with the Com1 chain and used as an initiator chain for hybridization chain reaction, wherein the 3' end of the Com2 chain is modified with a functional group; Hairpin DNA H1 chain and H2 chain, wherein the H1 chain is modified with a fluorescent group and a corresponding fluorescent quenching group; A single silicon nanowire substrate; Among them, the Com1 chain hybridizes with the Com2 chain to form a dsC1C2 double chain, and is covalently bound to the surface of a single silicon nanowire substrate through the functional group modified at the 3' end of the Com2 chain; the target miRNA competitively binds to the Com1 chain with the Com2 chain, resulting in the exposure of the Com2 chain base on the single silicon nanowire substrate, thereby triggering a hybridization chain reaction between the hairpin DNA H1 chain and the H2 chain to generate a fluorescent signal.

5. The fluorescence sensor according to claim 4, characterized in that: The functional group is an amino group, a carboxyl group or an aldehyde group; Preferably, the length of the single silicon nanowire substrate is 10-200 μm.

6. The fluorescence sensor according to claim 4, characterized in that: The fluorescent group is 6-FAM, Cy3 or ROX, and the corresponding fluorescence quenching group is BHQ1 or BHQ2; Preferably, the target miRNA is miR-146a-5p, whose sequence is shown in SEQ ID No.1; the sequence of the Com1 chain is shown in SEQ ID No.2; the sequence of the Com2 chain is shown in SEQ ID No.3; the 3' end of the Com2 chain is modified with -(CH2) n -NH2 to obtain a Com2 chain with an amino group modified at the 3' end, wherein n is an integer of 6-8; the sequence of the H1 chain is shown in SEQ ID No.4, and the sequence of the H2 chain is shown in SEQ ID No.5; Preferably, from the 5'-3' direction, the 15th base of the H1 chain is modified with a 6-FAM fluorescent group, and the 41st base is modified with a BHQ1 fluorescent quenching group.

7. The method for preparing the silicon nanowire-based fluorescent sensor for detecting miRNA according to any one of claims 4 to 6, characterized in that: The preparation method comprises: thermally oxidizing the silicon nanowire array to obtain an oxidized silicon nanowire array; The surface of the oxidized silicon nanowire array is subjected to hydroxylation and amination treatment in sequence, and then the silicon nanowires are peeled off to obtain a single amination silicon nanowire; Performing aldehyde treatment on the aminated single silicon nanowire to obtain aldehyde-treated single silicon nanowire; The 3' end of the Com2 chain is modified with an amino group, hybridized with the Com1 chain to form a dsC1C2-NH2 double chain, and then covalently bound to the surface of the aldehyde-modified single silicon nanowire substrate, dispersed in a buffer solution, and a single silicon nanowire solution loaded with a dsC1C2 double chain is obtained; The H1 chain and the H2 chain were dissolved in a buffer solution to prepare a mother solution, annealed at 95° C. to form a hairpin structure, and naturally cooled to room temperature to obtain a hairpin DNA H1 chain solution and a hairpin DNA H2 chain solution, respectively.

8. The preparation method according to claim 7, characterized in that: Specifically include: 1) Preparing silicon nanowire arrays by silver ion assisted chemical etching; 2) performing thermal oxidation treatment on the silicon nanowire array obtained in step 1), wherein the thermal oxidation treatment process is calcining at 800-900° C. for 180-240 min in an oxygen atmosphere; 3) performing a hydroxylation treatment on the silicon nanowire array obtained in step 2), wherein the hydroxylation treatment comprises soaking the silicon nanowire array in a mixed solution of concentrated sulfuric acid and hydrogen peroxide, heating and refluxing, washing it, soaking it in a mixed solution of water, ammonia water and 30% hydrogen peroxide, washing it again, and drying it to obtain a hydroxylated silicon nanowire array; 4) performing an amination treatment on the hydroxylated silicon nanowire array obtained in step 3), wherein the amination treatment is to place the hydroxylated silicon nanowire array in anhydrous toluene, add 3-aminopropyltrimethoxysilane, react at a constant temperature, take it out, clean it, peel off the silicon nanowires, and dissolve it in anhydrous ethanol to obtain an amination single silicon nanowire; 5) performing a aldehyde treatment on the aminated single silicon nanowire obtained in step 4), wherein the aldehyde treatment comprises mixing the aminated single silicon nanowire with glutaraldehyde, reacting with shaking at room temperature overnight in an ethanol system, and after the reaction is completed, repeatedly washing the silicon nanowire by suction filtration, and finally dispersing it in a buffer solution to obtain the aldehyde-aminated single silicon nanowire; 6) loading the dsC1C2 double chain onto the aldehyde-modified single silicon nanowire obtained in step 5), wherein the loading is performed by mixing the aldehyde-modified silicon nanowire and the dsC1C2-NH2 double chain solution with a PBS solution of sodium cyanoborohydride, and reacting at room temperature overnight by shaking; after the reaction is completed, centrifuging and discarding the supernatant, repeatedly washing, and finally dispersing in a buffer solution to obtain a single silicon nanowire solution loaded with the dsC1C2 double chain; The dsC1C2-NH2 double-stranded solution is prepared by the following method: firstly, the Com1 chain and the Com2 chain with the 3'-terminal amino group modified are respectively dissolved in a buffer solution to prepare a mother solution, an equimolar amount of the mother solution is annealed at 95° C. to form a dsC1C2-NH2 double-stranded chain, and naturally cooled to room temperature to obtain a dsC1C2-NH2 double-stranded solution; 7) The H1 chain and the H2 chain are dissolved in a buffer solution to prepare a mother solution, which is annealed at 95° C. to form a hairpin structure, and then naturally cooled to room temperature to obtain a hairpin DNA H1 chain solution and a hairpin DNA H2 chain solution, respectively.

9. Use of the hybridization chain reaction-based fluorescent sensor for detecting miRNA according to any one of claims 1 to 2 or the silicon nanowire-based fluorescent sensor for detecting miRNA according to any one of claims 4 to 6 in detecting miRNA.

10. Use of the silicon nanowire-based fluorescent sensor for detecting miRNA according to any one of claims 4 to 6 in detecting miRNA in single cells.

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