Quantum precision detection method for low-abundance miRNA

Abstract

The invention discloses a quantum precision detection method for low-abundance miRNA (micro Ribonucleic Acid). According to the method, a miRNA sensing strategy is established through capture and enrichment of fluorescent nano-diamond by magnetic particles; the method comprises the following analysis steps: introducing an electric field to regulate and control the charge state population degree of a fluorescent nano-diamond NV color center, applying radio frequency microwaves to control the quantum spin state of the NV color center, and collecting and analyzing the fluorescence difference before and after electric field regulation and control and microwave control so as to accurately judge the intensity of a detection signal; according to the method, sensitive detection and accurate concentration quantification of low-abundance miRNA can be realized, and a detection means can be provided for finer division of tumor diseases in the early stage.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a quantum precision detection method for low-abundance miRNA. Background Art

[0002] As a typical tumor disease-related marker, the level of miRNA in human plasma or body fluids has a significant corresponding relationship with the development of the disease. For the diagnosis and treatment of tumor diseases that endanger human health, the earlier the implementation of medical treatment often means a relatively higher cure rate. However, in the early stage of the disease, the corresponding tumor marker miRNA usually exists in the human body at ultra-low concentrations, which brings considerable difficulties to the determination and implementation of medical treatment. Traditional fluorescence detection methods, such as the use of gold particles, dye molecules and quantum dots, have the disadvantages of weak signal, low stability and large fluctuations in low-abundance detection, while nanodiamonds with NV color centers can effectively optimize the current shortcomings of fluorescence detection by relying on their own high photon yield characteristics and controllable electron spin characteristics. By regulating the charge state and quantum state of the ensemble NV cluster, the concentration of low-abundance miRNA can be accurately quantified, which is expected to achieve a more detailed division of the early stages of tumor diseases.

[0003] Chinese patent "Application No.: CN201380064943.4" reports a nano-diamond particle and a method for manufacturing the same, as well as a fluorescent molecular probe and a method for structural analysis of proteins. By using nano-diamond particles containing NV color centers as fluorescent molecular probes, using the fluorescent molecular probes to label target proteins, and measuring ODMR spectra, a fine and real-time structural analysis of proteins in vivo can be achieved. Chinese patent "Application No.: 202010724310.3" reports a method and device for measuring the temperature of biological tissues or cells. Diamond particles of micro-nano size containing spin defects are introduced into biological tissues through biochemical methods, and microwave resonance is used to detect the energy level changes of the diamond defect spin ground state to achieve real-time dynamic temperature tracking detection of target biological tissues or cells at the micro-nano level. However, the actual operation of the above analysis method is complicated, and there are high requirements for the concentration of the marker, so its detection is still greatly limited. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a quantum precision detection method for low-abundance miRNA. This method utilizes the excellent fluorescence properties and controllable properties of NV color centers to reduce the interference caused by environmental factors during fluorescence detection and significantly improve the detection accuracy.

[0005] Technical solution: A quantum precision detection method for low-abundance miRNA of the present invention is achieved in the following manner: the detection method adopts a detection platform to perform precise quantitative detection of fluorescent nanodiamonds in a magnetic collector under the driving action of an external alternating electric field and an external radio frequency microwave source; the detection platform includes a silver substrate, a magnetic collector, electrodes and a microwave antenna.

[0006] The silver substrate is used to carry magnetic collection objects, and surface plasmon resonance and mirror reflection are used to enhance the fluorescence signal of the magnetic collection objects; the magnetic collection objects include a collection of a coupled structure of probed magnetic particles, miRNA and probed fluorescent nanodiamonds; the electrode is used to provide a variable electric field on the silver substrate to achieve a change in the charge state distribution degree of the fluorescent nanodiamond NV color center in the collection object; the microwave antenna is used to radiate radio frequency microwaves on the silver substrate to achieve a change in the electron spin state of the fluorescent nanodiamond NV color center in the collection object.

[0007] The silver substrate can be equivalently replaced by a gold substrate.

[0008] The method for preparing a collection containing a coupled structure of magnetic particles, miRNA and fluorescent nanodiamonds comprises the following steps:

[0009] S1. Preparation of probe-based magnetic particles;

[0010] S2. Preparation of probe fluorescent nanodiamonds;

[0011] S3. Take the probe-based magnetic particle solution and the probe-based fluorescent nanodiamond solution in deionized water, add the miRNA to be tested and shake and incubate on a shaker, use a magnet to adsorb and wash three times to obtain a collection containing the coupled structure of the probe-based magnetic particles, miRNA and probe-based fluorescent nanodiamond.

[0012] Specifically, they are:

[0013] S1. Prepare probe-based magnetic particles: dilute 50-100 μL of streptavidin-coated magnetic beads in deionized water, add a DNA probe modified with biotin at the 3' end and incubate for 4-6 hours, centrifuge and wash, then resuspend in 100-400 μL of deionized water to obtain a probe-based magnetic particle solution;

[0014] S2. Preparation of probe-based fluorescent nanodiamonds: Take 50-100 μL of 100 nm surface carboxylated fluorescent nanodiamonds, add 1-3 mg 1-ethyl-(3-dimethylaminopropyl) carbodiimide and 1-3 mg N-hydroxysuccinimide, mix and let stand for 1-3 hours, add a DNA probe modified with an amino group at the 3' end, incubate at room temperature for 4-6 hours, wash three times by centrifugation, and resuspend in 200-500 μL deionized water to obtain a probe-based fluorescent nanodiamond solution;

[0015] S3. Take 2-15 μL of probe-type magnetic particle solution and 1-3 μL of probe-type fluorescent nanodiamond solution in 1-2 mL of deionized water, add the miRNA to be tested and incubate on a shaker for 3-5 hours, and wash three times using a magnet to obtain a collection containing a coupled structure of probe-type magnetic particles, miRNA and probe-type fluorescent nanodiamond.

[0016] The present invention also discloses a method for accurately and quantitatively detecting fluorescent nanodiamonds in the magnetic collection object using the electrode, comprising:

[0017] The electrode changes the electric field state of the silver substrate surface under the driving action of an external alternating electric field, thereby changing the proportion of the negative charge state and the zero charge state of the nitrogen vacancy color center of the fluorescent nanodiamond in the magnetic collector, and collecting the change of the fluorescence intensity in the magnetic collector under the action of the electric field.

[0018] The present invention also discloses using the microwave antenna to accurately and quantitatively detect the fluorescent nanodiamonds in the magnetic collection object, including:

[0019] The antenna radiates 2-3 GHz (preferably 2.87 GHz) pulsed microwaves under the action of an external radio frequency microwave source to change the quantum spin state population of the fluorescent nanodiamond nitrogen vacancy color center in the magnetic collector, and collects the changes in fluorescence intensity in the magnetic collector under the action of microwaves.

[0020] Furthermore, the electrodes and microwave antennas are combined to achieve fluorescence regulation of the magnetic collection material, and the data is analyzed to quantitatively detect the miRNA concentration.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) In the process of detecting miRNA, the present invention uses the surface plasmon resonance (SPR) effect of the metal substrate to improve the luminescence efficiency of fluorescent nanodiamonds; at the same time, the mirror reflection effect of the metal substrate can enhance the excitation and emission of fluorescent nanodiamonds. Under the premise that miRNA is not amplified, the concentration of miRNA is detected based on the fluorescence intensity in the collection, and the detection limit can reach the fM level; (2) The present invention uses electrodes to control the proportion of negative and zero states of the NV color center charge in the fluorescent nanodiamond, and uses microwave radio frequency to control the distribution of the quantum spin state of the NV color center in the fluorescent nanodiamond. By collecting and analyzing the degree of fluorescence change of the collection before and after the above two control methods, linear detection of low-abundance miRNA in a wide concentration range can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is an experimental principle diagram of the method for sensitive detection of low-abundance miRNA and accurate concentration quantification of the present invention; wherein: 1 is a silver substrate, 2 is a magnetic collector, 3 is an electrode, and 4 is a microwave antenna. DETAILED DESCRIPTION

[0023] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0024] Example 1

[0025] 1. Preparation of probe magnetic particles, the specific steps are:

[0026] Take 50 μL of streptavidin-coated magnetic beads and dilute them in deionized water, add the DNA probe modified with biotin at the 3' end and incubate for 4 hours, wash by centrifugation and resuspend in 200 μL of deionized water to obtain a probe-coated magnetic particle solution.

[0027] 2. Preparation of probe fluorescent nanodiamonds, the specific steps are:

[0028] Take 50 μL of 100 nm surface carboxylated fluorescent nanodiamond, add 2 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg N-hydroxysuccinimide, mix well and let stand for 1 hour, then add the DNA probe modified with amino group at the 3' end, incubate at room temperature for 4 hours, centrifuge and wash three times, and resuspend in 400 μL deionized water to obtain the probe-modified fluorescent nanodiamond solution.

[0029] 3. Preparation of the coupling structure of magnetic particles, miRNA and fluorescent nanodiamonds. The specific steps are as follows:

[0030] Take 10 μL of probe-type magnetic particle solution and 2 μL of probe-type fluorescent nanodiamond solution in 1 mL of deionized water, add the miRNA to be tested and incubate on a shaker for 4 hours, wash three times using a magnet, and resuspend in 100 μL of deionized water to obtain a collection solution containing a coupling structure of probe-type magnetic particles, miRNA and probe-type fluorescent nanodiamond.

[0031] 4. Fluorescence detection of MiRNA, the specific steps are as follows:

[0032] Take 20 μL of the magnetic collection solution and drop it on a silver substrate and dry it at room temperature. Then place it under the objective lens of a fluorescence spectrometer with a confocal system, use 532 nm excitation light, and collect fluorescence signals near 680 nm to achieve sensitive detection of miRNA.

[0033] 5. Electric field regulation of nanodiamond fluorescence in magnetic collection to quantify low-abundance miRNA concentration. The specific steps are as follows:

[0034] The electrodes are connected to an electrochemical workstation to provide an alternating electric field, which changes the electric field state on the silver substrate surface and thus changes the proportion of the negative charge state and the zero charge state of the nitrogen vacancy color center of the fluorescent nanodiamond in the magnetic collection. The fluorescence spectrometer is used to record the change in fluorescence intensity in the magnetic collection under the change of the electric field, and the data is analyzed to quantify the concentration of low-abundance miRNA.

[0035] Example 2

[0036] 1. Preparation of probe magnetic particles, the specific steps are:

[0037] 100 μL of streptavidin-coated magnetic beads were diluted in deionized water, and a DNA probe modified with biotin at the 3' end was added and incubated for 6 hours. After centrifugation and washing, the beads were resuspended in 200 μL of deionized water to obtain a probe-coated magnetic particle solution.

[0038] 2. Preparation of probe fluorescent nanodiamonds, the specific steps are:

[0039] Take 100 μL of 100 nm surface carboxylated fluorescent nanodiamond, add 2 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 mg N-hydroxysuccinimide, mix well and let stand for 1 hour, then add the DNA probe modified with amino group at the 3' end, incubate at room temperature for 6 hours, centrifuge and wash three times, and resuspend in 400 μL deionized water to obtain the probe-modified fluorescent nanodiamond solution.

[0040] 3. Preparation of the coupling structure of magnetic particles, miRNA and fluorescent nanodiamonds. The specific steps are as follows:

[0041] Take 10 μL of probe-type magnetic particle solution and 2 μL of probe-type fluorescent nanodiamond solution in 1 mL of deionized water, add the miRNA to be tested and incubate on a shaker for 4 hours, wash three times using a magnet, and resuspend in 100 μL of deionized water to obtain a collection solution containing a coupling structure of probe-type magnetic particles, miRNA and probe-type fluorescent nanodiamond.

[0042] 4. Fluorescence detection of MiRNA, the specific steps are as follows:

[0043] Take 20 μL of the magnetic collection solution and drop it on a gold substrate and dry it at room temperature. Then place it under the objective lens of a fluorescence spectrometer with a confocal system, use 532 nm excitation light, and collect fluorescence signals near 680 nm to achieve sensitive detection of miRNA.

[0044] 5. Microwave-controlled nanodiamond fluorescence in magnetic collection to quantify low-abundance miRNA concentrations. The specific steps are as follows:

[0045] The antenna radiates 2.87 GHz pulsed microwaves under the action of an external radio frequency microwave source to change the quantum spin state population of the fluorescent nanodiamond nitrogen vacancy color center in the magnetic collection, and collects the change in fluorescence intensity in the magnetic collection under the action of microwaves. The change in fluorescence intensity in the magnetic collection under the change of electric field is recorded by a fluorescence spectrometer, and the data is analyzed to quantitatively detect the concentration of low-abundance miRNA.

[0046] Combined with Examples 1 and 2, it can be obtained that the use of gold and silver materials on the substrate can achieve the same effect. Therefore, the low-abundance miRNA sensitive detection method based on quantum precision measurement of the present invention, by introducing an electric field to regulate the charge state distribution of the fluorescent nanodiamond NV color center and applying radio frequency microwaves to manipulate the quantum spin state of the NV color center, collects and analyzes the fluorescence difference before and after electric field regulation and microwave manipulation to achieve accurate judgment of the detection signal intensity, and realizes the capture and enrichment of fluorescent nanodiamonds by magnetic particles to establish the sensing of miRNA; this method can realize sensitive detection and precise quantification of low-abundance miRNA, and can provide detection means for more detailed division of the early stages of tumor diseases, and has great application prospects in the field of early tumor detection.

Claims

1. A quantum precision detection method for low-abundance miRNA, characterized in that: The detection method adopts a detection platform to perform precise quantitative detection of fluorescent nanodiamonds in a magnetic collection object under the driving action of an external alternating electric field and an external radio frequency microwave source; the detection platform comprises a silver substrate (1), a magnetic collection object (2), an electrode (3) and a microwave antenna (4); The silver substrate (1) is used to carry the magnetic collection object, and the fluorescent signal of the magnetic collection object is enhanced by using surface plasmon resonance and mirror reflection; The magnetic collection (2) comprises a collection formed by connecting the probe-based magnetic particles, miRNA and the probe-based fluorescent nanodiamonds; The electrode (3) is used to provide a variable electric field on the silver substrate to achieve a change in the charge state distribution degree of the fluorescent nanodiamond NV color center in the collection; The microwave antenna (4) is used to radiate radio frequency microwaves on the silver substrate to achieve regulation of the electron spin state of the fluorescent nanodiamond NV color center in the collection.

2. The method for quantum precision detection of low-abundance miRNA according to claim 1, characterized in that: The silver substrate (1) is equivalently replaced by a gold substrate.

3. The method for quantum precision detection of low-abundance miRNA according to claim 1, characterized in that: The preparation of a collection containing a coupled structure of magnetic particles, miRNA and fluorescent nanodiamonds comprises the following steps: S1. Preparation of probe-based magnetic particles; S2. Preparation of probe fluorescent nanodiamonds; S3. Take the probe-based magnetic particle solution and the probe-based fluorescent nanodiamond solution in deionized water, add the miRNA to be tested and shake and incubate on a shaker, use a magnet to adsorb and wash three times to obtain a collection containing the coupled structure of the probe-based magnetic particles, miRNA and probe-based fluorescent nanodiamond.

4. The method for quantum precision detection of low-abundance miRNA according to claim 3, characterized in that: S1 is specifically to prepare probe-type magnetic particles: 50-100 μL of streptavidin-coated magnetic beads are diluted in deionized water, a DNA probe modified with biotin at the 3' end is added and incubated for 4-6 hours, and the mixture is centrifuged and washed and then resuspended in 100-400 μL of deionized water to obtain a probe-type magnetic particle solution.

5. The method for quantum precision detection of low-abundance miRNA according to claim 3, characterized in that: S2 is specifically, preparing probe-type fluorescent nanodiamonds: taking 50-100 μL of 100 nm surface carboxylated fluorescent nanodiamonds, adding 1-3 mg 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 1-3 mg N-hydroxysuccinimide, mixing and standing for 1-3 hours, adding a DNA probe modified with an amino group at the 3' end, incubating at room temperature for 4-6 hours, centrifuging and washing three times, and resuspending in 200-500 μL deionized water to obtain a probe-type fluorescent nanodiamond solution.

6. The method for quantum precision detection of low-abundance miRNA according to claim 3, characterized in that: S3 specifically comprises taking 2-15 μL of probe-based magnetic particle solution and 1-3 μL of probe-based fluorescent nanodiamond solution into 1-2 mL of deionized water, adding the miRNA to be tested and incubating on a shaker for 3-5 hours, and washing three times by magnet adsorption to obtain a collection containing a coupled structure of probe-based magnetic particles, miRNA and probe-based fluorescent nanodiamonds.

7. The method for quantum precision detection of low-abundance miRNA according to claim 1, characterized in that: The electrode is used to perform precise quantitative detection of the fluorescent nanodiamonds in the magnetic collection under the driving action of an external alternating electric field, including: the electrode changes the electric field state of the silver substrate surface, thereby changing the proportion of the negative charge state and the zero charge state of the nitrogen vacancy color center of the fluorescent nanodiamond in the magnetic collection, and collecting the change of the fluorescence intensity in the magnetic collection under the action of the electric field.

8. The method for quantum precision detection of low-abundance miRNA according to claim 1, characterized in that: The microwave antenna is used to perform precise quantitative detection of the fluorescent nanodiamonds in the magnetic collection under the action of an external radio frequency microwave source, including: the antenna radiates 2-3GHz pulse microwaves to change the quantum spin state population of the nitrogen vacancy color center of the fluorescent nanodiamond in the magnetic collection, and collects the changes in fluorescence in the magnetic collection under the action of microwaves.

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