An enzyme-free miRNA multi-target detection method based on PhC-coupled FRET

Abstract

The present invention relates to a method for enzyme-free miRNA multi-target detection based on PhC-coupled FRET, belonging to the technical fields of analytical chemistry and biomedicine. The present invention designs photonic crystals PhC with different reflected light colors as carriers for multi-target detection, combined with hairpin sequences that specifically recognize targets, and can synchronously optically detect multiple miRNAs, such as the detection of overexpressed miRNA-21, miRNA-155, and miRNA-10b in breast cancer. Specifically, by immobilizing the fluorescent donor hairpin chain on the PhC, when the target miRNA is specifically recognized, the miRNA triggers an enzyme-free amplification reaction, resulting in the formation of a double strand between the fluorescent donor hairpin and the fluorescent acceptor hairpin, thereby generating fluorescence resonance energy transfer FRET. By correlating the FRET ratio with the miRNA concentration, the detection of the miRNA concentration in the sample can be achieved, and the specific miRNA is determined by the reflected light colors of different PhCs. This method has high sensitivity, sequence specificity, and multi-target detection ability, and can be used for the determination of miRNA content in untreated serum.

Description

Technical Field

[0001] The present invention relates to a method for enzyme-free miRNA multi-target detection by PhC-coupled FRET, belonging to the technical fields of analytical chemistry and biomedicine. Background Art

[0002] Breast cancer is a breast pathological state characterized by high morphological and molecular heterogeneity. "Global Cancer Statistics 2020" pointed out that in 2020, breast cancer surpassed lung cancer for the first time to become the most diagnosed malignant tumor, and it is the main cause of disability and death in women. As a type of cancer with a significantly higher early survival rate than advanced patients, about 70%-80% of patients with early non-metastatic breast cancer can be cured. Making a diagnosis at an early stage and increasing the detection rate of breast cancer means that more breast cancer patients can be detected and treated early, thus reducing the impact of breast cancer on human health. Therefore, it is very urgent to develop a rapid, simple and highly accurate diagnostic method for breast cancer.

[0003] MicroRNA (miRNA) is a class of short-chain non-coding single-stranded RNAs, ranging in length from 19 to 25 nucleotides, and plays an important role in regulating gene expression, cell differentiation and cell proliferation. At the same time, miRNA is considered an ideal biomarker for early tumor diagnosis because it plays a crucial role in various physiological activities related to various cancers, such as breast cancer, lung cancer, gastric cancer, prostate cancer, colon cancer, brain cancer and esophageal cancer. In breast cancer patients, miRNA shows obvious overexpression. Specifically, miRNA-21, miRNA-155 and miRNA-10b have been confirmed to be highly overexpressed in breast cancer patients. However, there is no report on the combined use of these miRNAs for the diagnosis or detection of breast cancer. Simultaneous detection of multiple miRNAs is of great significance for understanding the functions of miRNAs and achieving reliable clinical diagnosis of breast cancer.

[0004] Most multi-target detections rely on molecular binding or recognition, and molecules need to be encoded to distinguish different processes. The most popular molecular encoding method is the planar array, where probe molecules are immobilized on a substrate with position coordinates as the encoding elements. However, this method is usually limited by the molecular binding kinetics on the substrate, resulting in an extended assay time. Recently, suspension arrays have emerged as a promising encoding method, which utilize elements such as fluorescent dyes, quantum dots, vibrational signals, discrete metal layers, photonic crystals, or radio frequencies. Compared with planar arrays, suspension arrays have the advantages of fast detection speed, high sensitivity, and good repeatability. In addition, suspension array detection requires a smaller sample volume and lower cost. Among various suspension arrays, spectral encoding suspension arrays have been widely used due to their simple encoding and detection mechanisms. Although fluorescent dyes and quantum dots are the main spectral encoding elements, fluorescent dyes are prone to quenching or fading, while quantum dots often exhibit biotoxicity. In addition, other commonly used encoding elements, such as electrochemical tags and Raman dyes, face limitations such as a limited number of distinguishable tags in signal response and potential spectral overlap between concurrent signals. Therefore, there is an urgent need for non-fluorescent encoding elements that combine single-signal response methods to achieve multi-target detection.

[0005] A photonic crystal (PhC) is a periodic dielectric structure with the property of a photonic band gap (PBG). The PBG refers to the phenomenon that wavelengths within a specific frequency range cannot propagate through the periodic structure. Colloidal PhC is a substance with a three-dimensional ordered structure formed by dispersed micron or submicron inorganic or organic particles. These three-dimensional ordered structures enable Bragg diffraction of visible light (400 - 700 nm), showing different colors. The encoding ability of colloidal PhC stems from the periodicity of its structure, making it resistant to fading, bleaching, quenching, and chemical instability. These properties improve the reliability of colloidal PhC encoding. Detecting biological targets using fluorescence resonance energy transfer ( FRET) has several advantages, including cost-effectiveness, simple procedures, good stability, high detection sensitivity, and compatibility with various nanomaterial platforms. Efficient FRET depends on precisely controlling the distance between the donor and acceptor fluorescent dyes because their absorption and emission spectra must overlap during the energy transfer process. Based on the advantages described above, if the two techniques can be combined for miRNA detection, it may provide a new way for efficient disease detection. Of course, there are currently some reports on combining PhC with FRET for miRNA detection, but they are all enzyme-containing systems, and most enzymes are used in signal amplification strategies, which may face problems of poor stability and repeatability, resulting in inaccurate detection results. Summary of the Invention

[0006] To solve the above technical problems, based on the fluorescence resonance energy transfer (FRET) system coupled with the photon crystal labeling strategy, the present invention establishes a multi-target detection method. The FRET ratio is highly sensitive to minute changes in the separation between the donor and acceptor. Meanwhile, combined with the enzyme-free target amplification strategy, this technology is designed to be more stable, simple, and rapid in detection, with better detection performance. Combined with different PhC designs, it can be used for the multi-target detection of miRNAs, and serum detection verifies that this method has high sensitivity, high specificity, and accuracy.

[0007] The first object of the present invention is to provide an application of an miRNA detection system in the preparation of diagnostic products, and the application includes the following steps:

[0008] S1. Prepare the donor probe and the acceptor probe into hairpin structures respectively;

[0009] S2. Fix the donor probe on the photon crystal, incubate it with the acceptor probe and the miRNA target with a known concentration, and perform enzyme-free amplification;

[0010] S3. After the amplification is completed, perform fluorescence resonance energy transfer detection to establish a relationship curve between the fluorescence intensity ratio and the content of the miRNA target;

[0011] S4. Repeat steps S1 to S3 for the test sample, calculate the content of the miRNA target through the fluorescence intensity ratio of the test sample, and obtain the diagnostic result based on the content of the miRNA target;

[0012] The miRNA target includes one or more miRNAs, the donor probe includes one or more first probes, the acceptor probe includes one or more second probes, and different first probes and second probes are designed according to different miRNA targets respectively; the photon crystal is one or more, and the colors of the reflected light of multiple photon crystals are different;

[0013] The donor probe is fixed on the photon crystal. When detecting multiple miRNA targets, different first probes are respectively fixed on the photon crystals with different colors of reflected light;

[0014] The first probe is provided with a linking sequence, a first sequence, a second sequence, a third sequence, a fourth sequence and a linking sequence. The second probe is provided with a sequence A, a sequence B and a sequence C, and: the second sequence is complementary to the sequence C, the third sequence is complementary to the sequence B, the miRNA target is complementary to the third sequence and the fourth sequence, the length of the first sequence is 10 - 14 bases, the length of the sequence A is 9 - 13 bases, and the binding free energy (ΔG) of the second probe (sequence B + sequence C) and the first probe (second sequence + third sequence) is lower than the binding free energy of the miRNA target and the first probe (third sequence + fourth sequence);

[0015] One end of the first probe near the first sequence is modified with a donor fluorescent molecule, and the other end is connected to a photonic crystal. One end of the second probe near the sequence C is modified with an acceptor fluorescent molecule.

[0016] Further, the linking sequence is 1 - 5 arbitrary bases. Preferably, it is 1 - 5 identical bases, such as 1A - 5A, 1T - 5T, 1G - 5G, 1C - 5C.

[0017] Further, the miRNA target includes one or more of miRNA - 21, miRNA - 155, miRNA - 10b;

[0018] When the miRNA target includes miRNA - 21, the first sequence shown in SEQ ID NO.1 is included in the donor probe, and / or the second sequence shown in SEQ ID NO.4 is included in the acceptor probe,

[0019] When the miRNA target includes miRNA - 155, the first sequence shown in SEQ ID NO.2 is included in the donor probe, and / or the second sequence shown in SEQ ID NO.5 is included in the acceptor probe,

[0020] When the miRNA target includes miRNA - 10b, the first sequence shown in SEQ ID NO.3 is included in the donor probe, and / or the second sequence shown in SEQ ID NO.6 is included in the acceptor probe.

[0021] Further, regarding the modification of the fluorescent molecule, preferably:

[0022] When the first sequence is as shown in SEQ ID NO.1, the donor fluorescent molecule is modified at the 3' end, and / or when the second sequence is as shown in SEQ ID NO.4, the acceptor fluorescent molecule is modified at the 5' end;

[0023] When the first sequence is as shown in SEQ ID NO.2, the donor fluorescent molecule is modified at the 5'-end, and / or when the second sequence is as shown in SEQ ID NO.5, the acceptor fluorescent molecule is modified at the 3'-end;

[0024] When the first sequence is as shown in SEQ ID NO.3, the donor fluorescent molecule is modified at the 3'-end, and / or when the second sequence is as shown in SEQ ID NO.6, the acceptor fluorescent molecule is modified at the 5'-end.

[0025] Further, the diagnostic product is a breast cancer diagnostic product. Specifically, the result of the test sample is compared with the result of the healthy sample. If the miRNA content (or FRET ratio) of the test sample is higher than that of the healthy sample, it indicates that the subject may be a breast cancer patient or a high-risk population.

[0026] Further, the selection of the donor-acceptor fluorescent molecule pair includes but is not limited to FAM-ROX, CFP (cyan fluorescent protein)-YFP (yellow fluorescent protein), BFP (blue fluorescent protein)-GFP, BFP-YFP, Cy3-Cy5, FITC (fluorescein isothiocyanate)-Rhodamine, etc.

[0027] Further, the incubation is carried out at 20-30°C.

[0028] Further, the concentration of the miRNA is 1 pM - 10000 pM. In the present invention, when measuring the miRNA content of untreated human serum, the minimum sample volume only needs 1 μL, and miRNA-21, miRNA-155, and miRNA-10b in different serum samples can be qualitatively and quantitatively analyzed.

[0029] Further, the photonic crystal reflects light of different colors by means of adjusting the photonic band gap, nano-scale, etc.

[0030] The second object of the present invention is to provide a miRNA detection probe composition, which includes a donor probe and an acceptor probe in the miRNA detection probe composition, and the donor probe and the acceptor probe are hairpin structures;

[0031] The donor probe includes one or more first probes, the acceptor probe includes one or more second probes, and different first probes and second probes are designed according to different miRNAs;

[0032] The first probe is provided with a linker sequence, a first sequence, a second sequence, a third sequence, a fourth sequence, and a linker sequence. The second probe is provided with a sequence A, a sequence B, and a sequence C, and: the second sequence is complementary to the sequence C, the third sequence is complementary to the sequence B, the miRNA is complementary to the third sequence and the fourth sequence, the length of the first sequence is 10 - 14 bases, the length of the sequence A is 9 - 13 bases, and the binding free energy (ΔG) of the second probe (sequence B + sequence C) to the first probe (second sequence + third sequence) is lower than the binding free energy of the miRNA to the first probe (third sequence + fourth sequence);

[0033] One end of the first probe near the first sequence is modified with a donor fluorophore, and one end of the second probe near the sequence C is modified with an acceptor fluorophore.

[0034] Furthermore, the miRNA target includes one or more of miRNA-21, miRNA-155, and miRNA-10b;

[0035] When the miRNA target includes miRNA-21, the first sequence shown in SEQ ID NO.1 is included in the donor probe, and / or the second sequence shown in SEQ ID NO.4 is included in the acceptor probe,

[0036] When the miRNA target includes miRNA-155, the first sequence shown in SEQ ID NO.2 is included in the donor probe, and / or the second sequence shown in SEQ ID NO.5 is included in the acceptor probe,

[0037] When the miRNA target includes miRNA-10b, the first sequence shown in SEQ ID NO.3 is included in the donor probe, and / or the second sequence shown in SEQ ID NO.6 is included in the acceptor probe.

[0038] Furthermore, regarding the modification of the fluorophores, preferably:

[0039] When the first sequence is as shown in SEQ ID NO.1, the donor fluorophore is modified at the 3' end, and / or when the second sequence is as shown in SEQ ID NO.4, the acceptor fluorophore is modified at the 5' end;

[0040] When the first sequence is as shown in SEQ ID NO.2, the donor fluorophore is modified at the 5' end, and / or when the second sequence is as shown in SEQ ID NO.5, the acceptor fluorophore is modified at the 3' end;

[0041] When the first sequence is as shown in SEQ ID NO.3, the donor fluorophore is modified at the 3'-end, and / or when the second sequence is as shown in SEQ ID NO.6, the acceptor fluorophore is modified at the 5'-end.

[0042] The third object of the present invention is to provide a miRNA detection system, which comprises a miRNA detection probe composition and a photonic crystal:

[0043] The donor probe is immobilized on the photonic crystal, and is immobilized at one end of the first probe away from the donor fluorophore; the photonic crystal is one or more, and the colors of the light reflected by the multiple photonic crystals are different.

[0044] The fourth object of the present invention is to provide the use of the miRNA detection probe composition or the miRNA detection system in the preparation of miRNA detection products.

[0045] The fifth object of the present invention is to provide a diagnostic product, which contains the miRNA detection probe composition or the miRNA detection system.

[0046] Furthermore, according to the types of miRNAs detected, the diagnostic products include, but are not limited to, diagnostic products for cancers (including, but not limited to, breast cancer, cervical cancer, prostate cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, etc.), cardiovascular diseases, neurodegenerative diseases, viral infections, muscle diseases, liver diseases, autoimmune diseases, etc.

[0047] By means of the above solutions, the present invention has at least the following advantages:

[0048] The present invention designs a miRNA (multi-target) detection method by coupling a fluorescence resonance energy transfer system with a photonic crystal labeling strategy, which can be used for the highly sensitive and highly specific detection of at least miRNA-21, miRNA-155 and miRNA-10b. By preparing different PhCs for multi-target qualitative analysis, the donor probes modified on the surfaces of different PhCs specifically recognize the target miRNAs, and the miRNA opens the hairpin structure of the donor probe, thereby forming a partial double-stranded complementary structure between the donor probe and the miRNA, and at the same time exposing the foothold of the donor probe. The acceptor probe can bind to the foothold and form a double strand with the donor probe, so that the miRNA is competitively displaced and the signal amplification process is carried out. This cyclic process is repeated in the cyclic miRNA recognition system and the enzyme-free amplification reaction, realizing efficient signal amplification. The constructed enzyme-free assisted target amplification system does not require the addition of auxiliary enzymes and fuel chains during the reaction process, and can spontaneously complete the signal release and signal amplification processes after recognizing the target, greatly simplifying the detection process, reducing the detection cost and improving the detection sensitivity.

[0049] The above description is only an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and be able to implement it according to the content of the specification, the following describes the preferred embodiments of the present invention in conjunction with detailed drawings as follows. Description of the Drawings

[0050] Figure 1 It is a schematic diagram of a method for detecting multiple target miRNAs by coupling a photonic crystal based on a fluorescence resonance energy transfer system.

[0051] Figure 2 It is the detection standard curve of miRNA-21.

[0052] Figure 3 It is the detection standard curve of miRNA-155.

[0053] Figure 4 It is the detection standard curve of miRNA-10b.

[0054] Figure 5 It is the application result of the multi-target detection method in actual serum samples.

[0055] Figure 6 It is the comparison of the detection effects of different probes. Detailed Embodiment

[0056] The present invention will be further described below in conjunction with the drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.

[0057] The solutions and principles involved in the present invention are as follows:

[0058] The present invention provides a method for detecting multiple miRNA targets in various cancers including breast cancer by coupling a fluorescence resonance energy transfer system with a photonic crystal labeling strategy: First, SiO2 with different diameters was used as the aqueous phase, and dimethyl silicone oil with a viscosity of 10 cs was used as the oil phase to synthesize PhCs with different reflected lights in a droplet microfluidic system. The surface of the PhC was modified with NH2 using an ethanol solution containing a silane coupling agent ((3-Aminopropyl)triethoxysilane, APTE). After washing with PBS buffer, the amino-modified PhC was added to the PBS buffer containing glutaraldehyde to form an aldehyde-amine condensation reaction between the amino group and the aldehyde group on the PhC surface, thereby modifying the aldehyde group on the PhC surface. The donor hairpin sequence was added to the aldehyde group-modified PhC, and there was a strong FAM fluorescence signal on the PhC surface. The donor sequence was connected to the PhC surface through an amino-aldehyde bond, and the donor sequence was capable of specifically recognizing miRNA and being opened by miRNA. NaBH4 was used to block the excess sites on the PhC surface after immobilizing the probe to avoid non-specific adsorption of DNA. In the presence of miRNA, the specifically recognized donor sequence could be opened and expose the toehold, and the receptor sequence could bind to the toehold of the donor sequence and compete with the target miRNA, thereby realizing the target enzyme-free amplification process and forming a donor-receptor double strand for FRET. At this time, under the excitation of the fluorescence donor excitation wavelength, the fluorescence intensity of the fluorescence receptor increased, and the fluorescence intensity of the fluorescence donor decreased. Finally, a linear relationship between the FRET ratio and the miRNA concentration was established, and the miRNA content in the sample was calculated using this standard curve.

[0059] Example 1 Construction of the detection system

[0060] The sequences involved in this example are shown in the following table.

[0061]

[0062] The specific steps are as follows:

[0063] (1) Preparation of PhC

[0064] In a microfluidic device connected by a "T" - shaped tube, 10 cs dimethyl silicone oil was used to shear different SiO2 aqueous phases into droplets. Among them, the flow rate of 10 cs dimethyl silicone oil was set at 6 mL / h, and the flow rate of SiO2 aqueous solution was 1 mL / h. Then, these droplets were collected in a container filled with 500 cs highly viscous dimethyl silicone oil. After overnight evaporation at 80 °C and treatment with n - hexane to wash away the residual silicone oil, calcination was carried out at 550 °C for 4 h to obtain PhC. Among them, the diameters of SiO2 were 212.9 nm, 240.8 nm, and 255.7 nm respectively. In this invention, the color of the emitted light was changed by varying the nanoscale, and the reflected light colors presented by different PhCs were blue, green, and red respectively.

[0065] (2) Immobilization of donor probes

[0066] After PhC was soaked overnight in piranha solution (30% hydrogen peroxide and 70% sulfuric acid, v / v), it was rinsed with water and then dried with a nitrogen stream. PhC was treated with a 5% ethanol solution of APTE for 4 h to introduce NH2 groups on the PhC surface. Then, aldehyde group modification was carried out on the PhC surface. The amino - functionalized PhC was dispersed in PBS buffer containing 2.5% glutaraldehyde and reacted at 37 °C for 4 h, and then washed with PBS buffer. After the donor probe was denatured at 95 °C for 5 min, it was gradually annealed to room temperature to form a hairpin structure. PhC and the donor probe were reacted in PBS buffer at 4 °C for 12 h, and then the remaining aldehyde groups on the PhC surface were blocked with a 20 mM glycine (Glycine, Gly) blocker at 37 °C for 2 h. The PhC modified with the probe was stored at 4 °C for later use. Among them, the fluorescence donor sequence HP for detecting miRNA - 21 1-1 immobilized on blue PhC, and the fluorescence acceptor sequence is HP 1-2 , the fluorescence donor sequence HP for detecting miRNA - 155 2-1 immobilized on green PhC, and the fluorescence acceptor sequence is HP 2-2 , the fluorescence donor sequence HP for detecting miRNA - 10b 3-1 immobilized on red PhC, and the fluorescence acceptor sequence is HP 3-2 .

[0067] (3) Determination of multiple miRNAs

[0068] The donor probe-modified PhC was added to PBS buffer. Subsequently, the receptor probe was heated at 95 °C for 5 min for denaturation, and then slowly annealed to room temperature to form a hairpin structure, and then introduced into the reaction system. 1 μL of miRNA samples with different concentrations was added and incubated at room temperature. After incubation, it was washed with PBS buffer and the fluorescence intensity was measured. The fluorescence values were statistically analyzed through the color camera channel and the corresponding fluorescence values, and the relationship between the FRET ratio (F2 / F1) and miRNA concentration was calculated. Among them, the donor probes for detecting miRNA-21, miRNA-155, and miRNA-10b are HP 1-1 , HP 2-1 , and HP 3-1 , and the receptor probes are HP 1-2 , HP 2-2 , and HP 3-2 . When measuring the fluorescence intensity, two channels were set: for channel 1 (F1), the excitation wavelength was 495 nm and the emission wavelength was 515 - 545 nm; for channel 2 (F2), the excitation wavelength was 495 nm and the emission wavelength was 595 - 625 nm.

[0069] (4) Plotting the miRNA concentration standard curve

[0070] According to the relationship between the fluorescence signal and miRNA-21 concentration, the corresponding linear relationship curve was plotted. As Figure 2 shown, the fluorescence intensity increased with the increase of miRNA-21 concentration, and its linear regression equation was y = 0.0201*log x + 0.338, R 2 was 0.992, where y represents the FRET ratio and x represents the miRNA-21 concentration (pM), and the detection limit of this method was 6.36 fM.

[0071] According to the relationship between the fluorescence signal and miRNA-155 concentration, the corresponding linear relationship curve was plotted. As Figure 3 shown, the fluorescence intensity increased with the increase of miRNA-155 concentration, and its linear regression equation was y = 0.0172*1og x + 0.359, R 2 was 0.992, where y represents the FRET ratio and x represents the miRNA-155 concentration (pM), and the detection limit of this method was 8.52 fM.

[0072] According to the relationship between the fluorescence signal and miRNA-10b concentration, the corresponding linear relationship curve was plotted. As Figure 4 shown, the fluorescence intensity increased with the increase of miRNA-10b concentration, and its linear regression equation was y = 0.0333*log x + 0.369, R 2It is 0.998, where y represents the FRET ratio and x represents the miRNA-10b concentration (pM). The detection limit of this method is 6.06 fM.

[0073] Example 2 Multi-target Detection Application

[0074] The following shows the application of the detection system of the present invention in the detection of actual serum samples. The sample is the serum of breast cancer patients without treatment.

[0075] Three PhCs with different characteristic reflection peaks, namely 456.73 nm (blue), 530.07 nm (green), and 632.79 nm (red), are used to quantitatively analyze these three miRNAs simultaneously ( Figure 5 ). The donor probes HP 1-1 , HP 2-1 , and HP 3-2 are used to modify the blue PhC, green PhC, and red PhC respectively to specifically recognize three different targets. Subsequently, the acceptor probes HP 1-2 , HP 2-2 , and HP 3-2 are introduced and co-incubated under the same conditions. By specifically binding the target to the corresponding probe and through enzyme-free amplification, it is observed that the fluorescence intensity of channel 1 on the PhC connected to the matching miRNA target decreases, and the fluorescence intensity of channel 2 increases. At the same time, the FRET ratio (F2 / F1) value increases, and the results are as Figure 5 shown.

[0076] These results indicate that the combination of miRNA-triggered enzyme-free amplification and PhC technology has high specificity, sensitivity, and anti-interference ability, and is expected to be used for the clinical detection of multiple miRNAs. This strategy has the potential to become a new platform for analyzing multiple biomarkers and promoting disease diagnosis.

[0077] Example 3

[0078] The sequence information involved in this example is as follows:

[0079]

[0080] The target detected in this example is miRNA-155. Two groups of experiments are set up. Group 1: Donor hairpin HP 2-1 and acceptor hairpin HP 2-2 . Group 2: Donor hairpin HP 2-1-2 and acceptor hairpin HP 2-2-2 . The detection process is the same as that in the above example.

[0081] The results are as Figure 6As shown, the FRET ratio of Group 1 is significantly higher than that of Group 2, indicating that the modification of donor molecules and acceptor molecules has a certain impact on the detection effect.

[0082] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of the present invention.

Claims

1. Use of a miRNA detection system in the preparation of a miRNA detection product, characterized in that, The miRNA detection system includes a donor probe, a receptor probe, and a photonic crystal. The application includes the following steps: S1. Prepare the donor probe and the receptor probe into hairpin structures respectively; S2. Fix the donor probe on the photonic crystal, incubate it with the receptor probe and a miRNA target with a known concentration for enzyme-free amplification; S3. After the amplification is completed, perform fluorescence resonance energy transfer detection to establish a relationship curve between the fluorescence intensity ratio and the content of the miRNA target; S4. Repeat steps S1 to S3 for the test sample, calculate the content of the miRNA target through the fluorescence intensity ratio of the test sample, and obtain a diagnosis result based on the content of the miRNA target; The miRNA target includes one or more miRNAs. The donor probe includes one or more first probes, and the receptor probe includes one or more second probes. Different first probes and second probes are designed according to different miRNA targets respectively; The photonic crystal is one or more, and the colors of the reflected light of multiple photonic crystals are different; The donor probe is fixed on the photonic crystal. When detecting multiple miRNA targets, different first probes are respectively fixed on the photonic crystals with different colors of reflected light; The first probe is provided with a linking sequence, a first sequence, a second sequence, a third sequence, a fourth sequence, and a linking sequence. The second probe is provided with a sequence A, a sequence B, and a sequence C, and: the second sequence is complementary to sequence C, the third sequence is complementary to sequence B, the miRNA target is complementary to the third sequence and the fourth sequence, the length of the first sequence is 10-14 bases, the length of sequence A is 9-13 bases, and the binding free energy of the second probe to the first probe is lower than the binding free energy of the miRNA target to the first probe; One end of the first probe is modified with a donor fluorescent molecule, and the other end is connected to the photonic crystal. The second probe is modified with a receptor fluorescent molecule; The miRNA target includes one or more of miRNA-21, miRNA-155, and miRNA-10b; When the miRNA target is miRNA-21, the donor probe sequence is the first probe shown in SEQ ID NO.1, and the receptor probe sequence is the second probe shown in SEQ ID NO.4, When the miRNA target is miRNA-155, the donor probe sequence is the first probe shown in SEQ ID NO.2, and the receptor probe sequence is the second probe shown in SEQ ID NO.5, When the miRNA target is miRNA-10b, the donor probe includes the first probe shown in SEQ ID NO.3, and the receptor probe sequence is the second probe shown in SEQ ID NO.

6.

2. The application according to claim 1, wherein When the first probe is as shown in SEQ ID NO.1, the donor fluorescent molecule is modified at the 3' end, the second probe is as shown in SEQ ID NO.4, and the receptor fluorescent molecule is modified at the 5' end; When the first probe is as shown in SEQ ID NO.2, the donor fluorophore is modified at the 5'-end, the second probe is as shown in SEQ ID NO.5, and the acceptor fluorophore is modified at the 3'-end; When the first probe is as shown in SEQ ID NO.3, the donor fluorophore is modified at the 3'-end, the second probe is as shown in SEQ ID NO.6, and the acceptor fluorophore is modified at the 5'-end.

3. The application according to claim 1, wherein The selection of donor-acceptor fluorophore pairs includes FAM-ROX, CFP-YFP, BFP-GFP, BFP-YFP, Cy3-Cy5, FITC-Rhodamine.

4. A miRNA detection probe composition, characterized in that, The miRNA detection probe composition includes a donor probe and an acceptor probe, and the donor probe and the acceptor probe are in a hairpin structure; The donor probe includes one or more first probes, the acceptor probe includes one or more second probes, and different first probes and second probes are designed according to different miRNAs; The first probe is provided with a linking sequence, a first sequence, a second sequence, a third sequence, a fourth sequence and a linking sequence, and the second probe is provided with a sequence A, a sequence B and a sequence C, and: the second sequence is complementary to sequence C, the third sequence is complementary to sequence B, the miRNA is complementary to the third sequence and the fourth sequence, the length of the first sequence is 10-14 bases, the length of sequence A is 9-13 bases, and the binding free energy of the second probe to the first probe is lower than the binding free energy of the miRNA to the first probe; The first probe is modified with a donor fluorophore, and the second probe is modified with an acceptor fluorophore; The miRNA targets include one or more of miRNA-21, miRNA-155, miRNA-10b; When the miRNA target is miRNA-21, the donor probe sequence is the first probe shown in SEQ ID NO.1, and the acceptor probe sequence is the second probe shown in SEQ ID NO.4, When the miRNA target is miRNA-155, the donor probe sequence is the first probe shown in SEQ ID NO.2, and the acceptor probe sequence is the second probe shown in SEQ ID NO.5, When the miRNA target is miRNA-10b, the donor probe sequence is the first probe shown in SEQ ID NO.3, and the acceptor probe sequence is the second probe shown in SEQ ID NO.

6.

5. A miRNA detection system, characterized in that, The miRNA detection system contains the miRNA detection probe composition according to claim 4 and a photonic crystal; the donor probe is fixed on the photonic crystal, and the photonic crystal is one or more, and the colors of the light reflected by the multiple photonic crystals are different.

6. Use of the miRNA detection probe composition according to claim 4 or the miRNA detection system according to claim 5 in the preparation of miRNA detection products.

7. A diagnostic product for breast cancer, characterized in that, The diagnostic product contains the miRNA detection probe composition according to claim 4 or the miRNA detection system according to claim 5.

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