miRNA precise addressing and ultrasensitive detection equipment and methods

Abstract

The present invention relates to a device and method for precise addressing and ultrasensitive detection of miRNAs. The method comprises: preparing an AFM probe, the AFM probe comprising an AFM tip and an AFM cantilever, the AFM tip being obtained by atomic layer deposition of a two-dimensional thin film on a diamond surface containing NV color centers; utilizing the AFM probe in combination with a magnetic field module and an optical system to locate and detect a target miRNA-coupled magnetic sphere, the surface of which is modified with a DNA probe and target miRNA with a modified substance. The AFM probe containing diamond NV color centers of the present invention utilizes a magnetic effect with the magnetic sphere to obtain an ODMR spectrum based on changes in magnetic field intensity, thereby achieving magnetic imaging. Furthermore, a fluorescence intensity variation spectrum is obtained by fluorescence resonance energy transfer and fluorescence enhancement between the diamond NV color center and the modified substance, thereby achieving precise addressing and ultrasensitive detection of miRNAs in vitro through the collaboration of the AFM and the optical system.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a device and method for precise addressing and ultra-sensitive detection of miRNA. Background Art

[0002] Laboratory methods for the labeling, diagnosis, and monitoring of characteristic biomolecules (markers) have gradually become a new trend in the early diagnosis of malignant tumors (cancer). Among them, the expression levels of miRNAs in serum, saliva, and urine have a significant correlation with the occurrence and progression of cancer. Traditional miRNA detection methods, such as Northern blot hybridization, are time-consuming and labor-intensive, with low sensitivity, poor specificity, large sample requirements, and poor reproducibility. Currently, early cancer diagnosis still urgently requires higher-level ultrasensitive measurement of low-abundance miRNAs and innovation in corresponding detection strategies.

[0003] Diamond NV color centers are the most common luminescent defects in diamond. They consist of a nitrogen atom replacing a carbon and a vacancy. The energy level structure of diamond NV color centers reveals spin-tripled states in both their ground and excited states. At room temperature, the zero phonon line of diamond NV color centers is located near 637 nm, with peak fluorescence at 700 nm and absorption peaks in the green band. Due to their high stability and low cytotoxicity, diamond NV color centers are suitable for biological cell imaging and intracellular electromagnetic field and temperature measurements.

[0004] In the existing technology, the patent with application number 202010224841.6 and titled "A scanning detection system based on diamond NV color center" improves the signal collection efficiency, but cannot achieve real-time and dynamic detection of in vitro miRNA information. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a miRNA precise addressing and ultra-sensitive detection device and method, the purpose of which is to solve the real-time and dynamic detection of miRNA information in vitro.

[0006] The technical solution adopted in the present invention is as follows:

[0007] In one aspect, the present invention provides a miRNA precise addressing and ultrasensitive detection device, comprising: an AFM system, a two-dimensional thin film generation system, an optical path system, a magnetic field module, and a microwave system;

[0008] The AFM probe of the AFM system includes an AFM tip and an AFM cantilever. The AFM tip is obtained by atomic layer deposition of a two-dimensional thin film on a diamond surface containing NV color centers. The AFM system is used to achieve addressing of the target miRNA-coupled magnetic sphere, i.e., the sample to be tested.

[0009] The two-dimensional thin film generation system is used to generate a two-dimensional thin film by atomic layer deposition on the surface of the diamond containing NV color centers;

[0010] The magnetic field module is used to apply a magnetic field to the sample detection area;

[0011] The optical system is integrated with the AFM system to emit excitation light and collect fluorescence signals of the diamond NV color center of the AFM tip scanning the sample to be tested, the dye molecules and the metal nanoparticles in the sample to be tested to achieve signal collection;

[0012] The microwave system is used to modulate the signal to improve detection accuracy;

[0013] Based on the principles of fluorescence resonance energy transfer (FRET) and metal enhancement of fluorescence (MEF), the various systems are used in conjunction with each other to simultaneously perform AFM detection during the magnetic imaging and fluorescence detection of the sample to be tested, thereby acquiring dynamic information of the sample to be tested, so as to achieve the purpose of utilizing the quantum properties of diamond NV color centers to regulate and realize the precise addressing and ultra-sensitive detection of one or more miRNAs in vitro.

[0014] Further technical solutions are:

[0015] Each system structure includes:

[0016] The optical system includes: a laser, an avalanche photodiode, a photon counter, an Olympus inverted microscope, a spectrometer, a pinhole and a dichroic mirror;

[0017] The objective lens of the Olympus inverted microscope is inverted, with a transparent sample stage above it. The excitation light emitted by the laser is emitted to the surface of the sample to be tested through the objective lens, and the optical path system collects the emission spectrum, fluorescence lifetime and magnetic resonance parameters of the sample to be tested through the objective lens;

[0018] The AFM system includes an atomic force microscope, which is a cantilever type;

[0019] The integration of the optical system and the AFM system involves placing a transparent sample stage in the atomic force microscope chamber. The optical system emits a laser below the sample and collects signals, while the AFM tip simultaneously scans the sample above it. The signals collected by the AFM and optical systems enable dynamic, real-time acquisition of the fluorescence and magnetic resonance characteristics of diamond NV centers.

[0020] The microwave system includes a microwave source, a microwave switch, a high-power amplifier, and a circulator. After connection and debugging, the microwave system generates a microwave field from the microwave source, which is capacitively coupled to the Ω-type resonator to provide a uniform peak field measurement area. The quantum properties of the diamond NV color center are modulated at a fixed frequency to achieve NV color center qubit initialization and specific detection and analysis.

[0021] The magnetic field module is a permanent magnet or an electromagnet, which can influence the self-selection characteristics of the NV color center by adjusting the direction and magnitude of the magnetic field and the distance between the magnetic field module and the NV color center to achieve magnetic imaging function;

[0022] The two-dimensional thin film generation system is an atomic layer deposition platform.

[0023] In another aspect, the present invention provides a method for precise addressing and ultrasensitive detection of miRNA, comprising:

[0024] Prepare an AFM probe, wherein the AFM probe includes an AFM tip and an AFM cantilever, wherein the AFM tip is obtained by atomic layer deposition of a two-dimensional thin film on a diamond surface containing NV color centers;

[0025] The AFM probe is combined with a magnetic field module and an optical system to locate and detect the target miRNA-coupled magnetic ball, the surface of which is modified with a DNA probe with a modified substance and the target miRNA.

[0026] Further technical solutions are:

[0027] The method for preparing the AFM probe comprises:

[0028] a) Using reactive ion etching (RIE) to etch bulk diamond containing NV color centers, a diamond with a conical tip is obtained;

[0029] b) Placing a diamond with a conical tip in an atomic layer deposition reaction chamber to modify a two-dimensional thin film on the surface;

[0030] c) processing the main portion of the rear end of the conical tip by a diamond etching process to obtain a complete diamond AFM tip;

[0031] d) Assembling the diamond AFM tip and the AFM cantilever into a complete probe.

[0032] The thickness of the two-dimensional film is 0.5-100 nm.

[0033] The AFM probe is combined with a magnetic field module and an optical system to locate and detect the target miRNA coupled magnetic sphere, including:

[0034] Spin-coating the target miRNA-coupled magnetic spheres onto the sample area to be tested on a quartz glass slide;

[0035] The magnetic field module applies a magnetic field to the sample area to be tested, and the direction of the magnetic field is parallel to the NV axis;

[0036] The laser of the optical system generates excitation light that acts on the NV color center of the AFM probe. The AFM probe is used to scan the sample to be tested to obtain the magnetic field strength at different positions. The magnetic field strength changes are used to perform magnetic imaging on the magnetic spheres modified with miRNA to achieve the positioning of the miRNA-coupled magnetic spheres. At the same time, the optical system collects fluorescence signals within the motion range of the AFM probe, and uses the spectral scanning imaging function to reflect the fluorescence changes within the motion range of the AFM probe in real time, thereby achieving ultra-sensitive detection of miRNA.

[0037] The method utilizes the AFM probe in combination with the magnetic field module and the optical system to locate and detect the target miRNA coupled magnetic sphere, further comprising:

[0038] The signal is modulated and amplified using a microwave system.

[0039] Preparation of target miRNA-coupled magnetic beads, including:

[0040] preparing a magnetic ball solution with surface modified with streptavidin and a probe DNA solution;

[0041] 5-10 μl of the probe DNA solution is added to 100-150 μl of the magnetic ball solution and incubated at room temperature for 12-15 hours; the incubated solution is centrifuged and washed with a buffer solution to remove loosely connected probe DNA to obtain a treated magnetic ball solution;

[0042] Take 10-20 μl of the target miRNA solution and mix it with 100-150 μl of the treated magnetic ball solution, incubate it at room temperature, and wash it with a centrifugal buffer to remove excess miRNA to obtain the target miRNA-coupled magnetic balls;

[0043] Both ends of the probe DNA are modified with biotin and a modifier respectively, and the modifier includes a dye molecule and a metal nanoparticle.

[0044] The magnetic ball solution is a solution containing magnetic balls of one diameter or multiple diameters;

[0045] For a solution containing magnetic spheres of various diameters, the probe DNA solution is a solution containing various DNAs, and the target miRNA-coupled magnetic spheres are a coupled magnetic sphere solution containing various target miRNAs. Various DNAs are respectively mixed with magnetic spheres of various diameters and are respectively paired with various target miRNAs.

[0046] The beneficial effects of the present invention are as follows:

[0047] The present invention simultaneously implements AFM detection during the fluorescence detection process of the sample, which can realize the dynamic information acquisition of the sample. The target miRNA is coupled to the DNA probe and the target miRNA modified with the modified substance on the surface of the magnetic sphere. The AFM probe of the diamond NV color center obtains an ODMR spectrum according to the change of the magnetic field intensity through the magnetic effect between the magnetic sphere and the diamond NV color center, realizing magnetic imaging, and obtaining the fluorescence resonance energy transfer and fluorescence enhancement between the diamond NV color center and the modified substance to obtain a fluorescence intensity change spectrum, thereby realizing ultra-sensitive detection and addressing of miRNA in vitro through the cooperation of AFM and optical path system. The present invention optimizes and controls the quantum properties of the diamond NV color center AFM needle tip through atomic layer deposition of thin films, thereby improving the sensitivity of signal detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic structural diagram of the detection device of Example 1 of the present invention.

[0049] In the figure: 1. Microwave source; 2. Microwave switch; 3. High-power amplifier; 4. Circulator; 5. Ω-type resonator; 6. Conical tip; 7. Main body; 8. Atomic force microscope; 9. Transparent sample stage; 10. Olympus inverted microscope; 11. Laser; 12. Dichroic mirror; 13. Pinhole; 14. Spectrometer; 15. Avalanche photodiode; 16. Photon counter; 17. Magnetic field module; 18. Sample to be tested. DETAILED DESCRIPTION

[0050] The specific embodiments of the present invention are described below with reference to the accompanying drawings.

[0051] The present application provides a miRNA precise addressing and ultrasensitive detection device, comprising: an AFM system, a two-dimensional thin film generation system, an optical path system, a magnetic field module, and a microwave system;

[0052] The AFM probe of the AFM system includes an AFM tip and an AFM cantilever, wherein the AFM tip is obtained by atomic layer deposition of a two-dimensional thin film on a diamond surface containing NV color centers;

[0053] The two-dimensional thin film generation system is used to generate a two-dimensional thin film by atomic layer deposition on the surface of the diamond containing NV color centers;

[0054] The magnetic field module is used to apply a magnetic field to the sample detection area;

[0055] The optical system is used to generate excitation light to act on the NV color center of the AFM tip of the scanning sample, and collect signals to obtain an optical detection magnetic resonance spectrum;

[0056] The microwave system is used to modulate and amplify the signal.

[0057] The following specific example 1 further illustrates the technical solution of the miRNA precise addressing and ultra-sensitive detection device of the present application.

[0058] Example 1

[0059] like Figure 1 As shown, the detection equipment includes: AFM system, optical system, microwave system, magnetic field module 17, and two-dimensional thin film generation system. The integration and use of these systems enables dynamic real-time signal acquisition of samples.

[0060] The AFM system includes an atomic force microscope 8, which is a cantilever beam type;

[0061] The optical system includes: a laser 11, an avalanche photodiode 15, a photon counter 16, an Olympus inverted microscope 10, a spectrometer 14, a pinhole 13, a dichroic mirror 12, etc.; the components of the optical system are connected and assembled in sequence, wherein the objective lens of the Olympus inverted microscope 10 is inverted, with a transparent sample stage 9 above it. The excitation light emitted by the laser 11 is emitted to the sample surface through the objective lens, and the optical system collects the emission spectrum, fluorescence lifetime and magnetic resonance parameters of the sample through the objective lens;

[0062] The microwave system consists of a microwave source 1, a microwave switch 2, a high-power amplifier 3, and a circulator 4. After the microwave system is connected and debugged in sequence, the microwave source 1 generates a microwave field, which is capacitively coupled to the Ω-type resonator 5, providing a uniform peak field measurement area. The fluorescence of the diamond NV color center is modulated at a fixed frequency to achieve NV color center qubit initialization and specific detection and analysis.

[0063] Among them, the magnetic field module 17 is a permanent magnet or an electromagnet, which affects the self-selection characteristics of the NV color center by adjusting the direction and size of the magnetic field, the distance between the module and the NV color center, etc., so as to realize the magnetic imaging function.

[0064] Among them, the two-dimensional thin film generation system is an atomic layer deposition platform;

[0065] Among them, the use process of the integration of the optical path system and the AFM system is as follows: the sample stage 9 is placed in the chamber of the atomic force microscope 8, and a sample area to be tested is provided on the sample stage 9. The sample to be tested 18 is coated in the sample area to be tested, and the optical path system emits laser below the sample, and the AFM tip scans it above the sample. Based on the principles of fluorescence resonance energy transfer (FRET) and metal enhancement of fluorescence (MEF), the optical path system collects signals, and the microwave system amplifies and modulates the signals, so that the fluorescence and magnetic resonance characteristic information of the diamond NV color center are dynamically and real-time collected through the linkage of the AFM system, the optical path system and other systems.

[0066] The AFM system is used to address the miRNA-coupled magnetic sphere, i.e., the sample to be tested 18; the optical system emits excitation light and collects fluorescence signals of the diamond NV center of the AFM tip, dye molecules, and metal nanoparticles in the sample to achieve signal acquisition; the microwave system realizes signal modulation; based on the principles of fluorescence resonance energy transfer (FRET) and metal enhancement of fluorescence (MEF), the various systems are linked to each other to simultaneously realize AFM detection during the magnetic imaging and fluorescence detection of the sample, thereby realizing dynamic information acquisition of the sample, so as to achieve the purpose of precise addressing and ultra-sensitive detection of one or more miRNAs in vitro by regulating the quantum properties of the diamond NV center.

[0067] The present application also provides a method for precise addressing and ultrasensitive detection of miRNA, comprising:

[0068] preparing an AFM probe, wherein the AFM probe comprises an AFM tip and an AFM cantilever, wherein the AFM tip is obtained by atomic layer deposition of a two-dimensional thin film on a diamond surface containing NV color centers;

[0069] The AFM probe is combined with a magnetic field module and an optical system to locate and detect the target miRNA-coupled magnetic ball, the surface of which is modified with a DNA probe with a modified substance and the target miRNA.

[0070] Wherein, the preparation of the AFM probe comprises:

[0071] a) Using reactive ion etching (RIE) to etch bulk diamond containing NV color centers, a diamond with a conical tip is obtained;

[0072] b) Placing a diamond with a conical tip in an atomic layer deposition reaction chamber to modify a two-dimensional thin film on the surface;

[0073] c) processing the main portion of the rear end of the conical tip by a diamond etching process to obtain a complete diamond AFM tip;

[0074] d) Assembling the diamond AFM tip and the AFM cantilever into a complete probe.

[0075] Wherein, the thickness of the two-dimensional film is 0.5-100 nm.

[0076] This approach further modifies the quantum properties of NV centers by modifying the diamond tip with a two-dimensional thin film. By adjusting the thickness of the two-dimensional thin film, the fluorescence signal of the diamond NV center can be adjusted in three ways: NV center depth, energy transfer, and protective layer thickness. This improves the transverse and longitudinal relaxation times of the NV center, the fluorescence intensity and lifetime, and its isolation from the external environment, ultimately improving the quantum coherence time and fluorescence detection efficiency of the diamond NV center.

[0077] The AFM probe is combined with a magnetic field module and an optical system to locate and detect the target miRNA coupled magnetic sphere, including:

[0078] Spin-coating the target miRNA-coupled magnetic spheres onto the sample area to be tested on a quartz glass slide;

[0079] The magnetic field module applies a magnetic field to the sample area to be measured;

[0080] The laser of the optical system generates excitation light that acts on the NV color center of the AFM probe. The AFM probe is used to scan the sample to be tested to obtain the magnetic field strength at different positions. The magnetic sphere modified with miRNA is magnetically imaged through the change in magnetic field strength to achieve the positioning of the miRNA-coupled magnetic sphere. At the same time, due to the FRET and MEF between the modifier in the sample to be tested and the NV color center, the fluorescence properties excited by the NV color center will change. The optical system collects the fluorescence signal within the movement range of the AFM probe, and uses the spectral scanning imaging function to reflect the fluorescence changes within the movement range of the AFM probe in real time, thereby achieving ultra-sensitive detection of miRNA.

[0081] The optical path system collects sample signals to obtain optical detection magnetic resonance spectra to show the additional Zeeman splitting caused by the local magnetic field of the magnetic nanoparticles.

[0082] Also includes:

[0083] The signal is modulated and amplified using a microwave system.

[0084] This approach achieves precise addressing and ultrasensitive detection of miRNA in vitro through the synergistic combination of magnetic imaging and fluorescence signal detection. Simultaneous AFM detection of the sample during fluorescence detection enables dynamic information acquisition.

[0085] The preparation of target miRNA-coupled magnetic spheres includes:

[0086] preparing a magnetic ball solution with surface modified with streptavidin and a probe DNA solution;

[0087] 5-10 μl of the probe DNA solution is added to 100-150 μl of the magnetic ball solution and incubated at room temperature for 12-15 hours; the incubated solution is centrifuged and washed with a buffer solution to remove loosely connected probe DNA to obtain a treated magnetic ball solution;

[0088] Take 10-20 μl of the target miRNA solution and mix it with 100-150 μl of the treated magnetic ball solution, incubate it at room temperature, and wash it with a centrifugal buffer to remove excess miRNA to obtain the target miRNA-coupled magnetic balls;

[0089] Both ends of the probe DNA are modified with biotin and a modifier respectively, and the modifier includes a dye molecule and a metal nanoparticle.

[0090] Wherein, the magnetic ball solution is a solution containing magnetic balls of one diameter or multiple diameters;

[0091] For a solution containing magnetic spheres of various diameters, the probe DNA solution is a solution containing various DNAs, and the target miRNA-coupled magnetic spheres are a coupled magnetic sphere solution containing various target miRNAs. Various DNAs are respectively mixed with magnetic spheres of various diameters and are respectively paired with various target miRNAs.

[0092] The target miRNA-coupled magnetic spheres prepared by the above scheme have one or more miRNAs, thereby realizing the simultaneous identification and detection of multiple miRNAs.

[0093] The following specific examples 2 and 3 further illustrate the technical solutions of the miRNA precise addressing and ultrasensitive detection method of the present application.

[0094] Example 2

[0095] 1. AFM probe preparation, the specific steps are:

[0096] 1) Using a reactive ion etching process, etching a block of diamond containing NV color centers to obtain a conical tip 6, specifically comprising:

[0097] A SiNx layer is deposited on a diamond substrate by plasma-enhanced chemical vapor deposition (PECVD); a negative electron resist (HSQ) layer is then spin-coated on the SiNx, which is used for electron beam exposure; patterning is performed using an electron beam lithography (EBL) system, and the HSQ is then dissolved away in 25% tetramethylammonium hydroxide (TMAH); the SiNx layer is etched using a reactive ion etching (RIE) system to transfer the pattern; the corresponding nanodiamond structure is obtained by inductively coupled plasma (ICP) etching, and finally, HF wet etching is used to remove the HSQ or SiNx residue;

[0098] 2) placing a block of diamond with a conical tip 6 in an atomic layer deposition reaction chamber and modifying a two-dimensional thin film on its surface;

[0099] 3) A complete diamond AFM tip (including the tapered structure 6 and the main body 7 extending from the rear end of the tapered structure 6) is obtained by a diamond etching process;

[0100] 4) The two-dimensional thin film modified diamond NV color center tip and the cantilever of the AFM probe are assembled into a complete probe through the AFM probe assembly process.

[0101] After the assembled AFM probe is installed, the AFM system is switched to tapping mode for standby use.

[0102] 2. Prepare the sample to be tested and miRNA-155 coupled magnetic beads. The specific steps are as follows:

[0103] 1) Prepare a magnetic sphere (200 nm in diameter) modified with streptavidin on its surface and a linear DNA probe modified with biotin and gold nanoparticles at both ends;

[0104] 2) Add 5 μl of probe DNA solution to 100 μl of magnetic ball solution and incubate at room temperature for 12 h;

[0105] 3) Wash three times with TTL buffer (100 mM Tris, 1 M LiCl, 0.1% Tween-20, pH 8.0) at 4°C by centrifugation to remove loosely attached probe DNA;

[0106] 4) 10 μl of the miRNA-155 solution to be tested was mixed with 100 μl of the magnetic sphere solution and incubated at room temperature for 2 h. The mixture was then centrifuged and washed three times with 0.01 M PBS (pH 7.4) buffer at 4°C to remove excess miRNA-155, thereby obtaining miRNA-155-coupled magnetic spheres.

[0107] 3. Sample testing, the specific steps are as follows:

[0108] 1) 5 μl of miRNA-155-coupled magnetic sphere solution was added dropwise to a quartz glass slide and spin-coated at 600 rpm for 5 min.

[0109] 2) Dry the slides on a 30°C heating plate for 10 minutes;

[0110] 3) Place the slide on the stage, first adjust the objective lens to about 1 mm from the sample, and focus the optical system;

[0111] 4) Adjust the AFM tip to the appropriate position;

[0112] 5) Adjust the magnetic field module parameters to ensure that the magnetic field direction is parallel to the NV axis and the magnetic field strength is 55G;

[0113] 6) Turn on the laser of the optical system, select a 510nm picosecond laser, and start AFM scanning at the same time;

[0114] 7) As the AFM tip scans the sample, the optical system collects the fluorescence signals of the corresponding diamond NV color centers, dye molecules, and metal nanoparticles to achieve signal acquisition, and records key information such as the sample's magnetic resonance parameters, fluorescence intensity, and fluorescence lifetime, thereby achieving precise addressing and ultrasensitive detection of miRNA-155.

[0115] Specifically, the AFM tapping mode is used to scan the sample area to obtain the magnetic field strength at different positions, and the magnetic spheres modified with miRNA are magnetically imaged through the changes in magnetic field strength, thereby achieving the positioning of the miRNA-coupled magnetic spheres.

[0116] Specifically, FRET and MEF between the modifiers and NV centers in the sample will cause changes in the fluorescence properties of the NV centers. The fluorescence intensity of the AFM tip will change due to the influence of the modifiers at different positions. The optical path system uses the spectral scanning imaging function to reflect the fluorescence changes within the movement range of the AFM tip in real time, thereby achieving precise positioning and ultra-sensitive detection of miRNA.

[0117] Example 3

[0118] 1. AFM probe preparation is the same as in Example 2.

[0119] 2. Prepare the samples to be tested, miRNA-155 and miRNA-182 coupled to magnetic beads. The specific steps are as follows:

[0120] 1) Prepare magnetic spheres (200 nm and 300 nm in diameter) modified with streptavidin on their surfaces and linear, hairpin-shaped probe DNA modified with biotin and fluorescent dye molecules at both ends;

[0121] The linear DNA corresponds to base complementary pairing with miRNA-155, and the dye molecule modified at one end of the linear DNA is Cy5;

[0122] The hairpin DNA achieves base complementary pairing with miRNA-182, and the dye molecule modified at one end of the hairpin DNA is Cy3;

[0123] 2) Take 5 μl of probe DNA solution and add it to 100 μl of magnetic ball solution, and incubate at room temperature for 12 hours;

[0124] 3) Wash three times with TTL buffer (100 mM Tris, 1 M LiCl, 0.1% Tween-20, pH 8.0) at 4°C by centrifugation to remove loosely attached probe DNA;

[0125] 4) Take 10 μl of the miRNA-155 and miRNA-182 solutions to be tested and mix them with 100 μl of the corresponding magnetic sphere solution, incubate at room temperature for 2 h, and wash three times with 0.01 M PBS (pH 7.4) buffer at 4°C to remove excess miRNA-155 and miRNA-182 to obtain miRNA-155 and miRNA-182-coupled magnetic spheres;

[0126] 5) Take 20 μl of miRNA-155 and miRNA-182 coupled magnetic sphere solutions respectively, mix them to obtain miRNA-155 and miRNA-182 coupled magnetic sphere solutions for later use.

[0127] 3. Sample testing, the specific steps are as follows:

[0128] 1) 5 μl of miRNA-155 and miRNA-182 coupled magnetic sphere solution was added dropwise onto a quartz glass slide and spin-coated at 600 rpm for 5 min.

[0129] 2) Dry the slides on a 30°C heating plate for 10 minutes;

[0130] 3) Place the slide on the stage, first adjust the objective lens to about 1 mm from the sample, and focus the optical system;

[0131] 4) Adjust the AFM tip to the appropriate position;

[0132] 5) Adjust the magnetic field module parameters to ensure that the magnetic field direction is parallel to the NV axis and the magnetic field strength is 55G;

[0133] 6) Turn on the laser, select a 510nm picosecond laser, and start AFM scanning at the same time;

[0134] 7) During AFM tip scanning, the optical system collects the fluorescence signals of the corresponding diamond NV color centers, dye molecules, and metal nanoparticles to realize signal acquisition, and records key information such as the sample's magnetic resonance parameters, fluorescence intensity, and fluorescence lifetime. Magnetic spheres of different particle sizes, DNA of different shapes, and different dye molecules will result in different spectral information. By analyzing this spectral information, precise addressing and ultrasensitive detection of miRNA-155 and miRNA-182 can be achieved simultaneously.

[0135] This application simultaneously implements AFM detection during sample fluorescence detection, enabling dynamic information acquisition of the sample. The surface of a magnetic sphere is modified with modified DNA probes and miRNA. The AFM diamond NV color center probe generates an ODMR spectrum based on changes in magnetic field intensity through the magnetic effect between the probe and the sphere, achieving magnetic imaging. The diamond NV color center generates a fluorescence intensity variation spectrum through fluorescence resonance energy transfer and fluorescence enhancement with the modified substance. The synergistic effect of the AFM and optical system enables ultrasensitive detection and addressing of miRNA in vitro.

[0136] Those skilled in the art will understand that the foregoing descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will be able to modify the technical solutions described in the foregoing embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A miRNA precise addressing and ultrasensitive detection device, characterized in that: include: AFM system, two-dimensional thin film generation system, optical path system, magnetic field module (17) and microwave system; The AFM probe of the AFM system includes an AFM tip and an AFM cantilever. The AFM tip is obtained by atomic layer deposition of a two-dimensional thin film on a diamond surface containing NV color centers. The AFM system is used to achieve addressing of the target miRNA-coupled magnetic sphere, i.e., the sample to be tested. The two-dimensional thin film generation system is used to generate a two-dimensional thin film by atomic layer deposition on the surface of the diamond containing NV color centers; The magnetic field module is used to apply a magnetic field to the sample detection area; The optical system is integrated with the AFM system to emit excitation light and collect fluorescence signals of the diamond NV color center of the AFM tip scanning the sample to be tested, the dye molecules and the metal nanoparticles in the sample to be tested to achieve signal collection; The microwave system is used to modulate and amplify the signal; The various systems are used in conjunction with each other to simultaneously perform AFM detection during the magnetic imaging and fluorescence detection of the sample to be tested, thereby acquiring dynamic information of the sample to be tested, so as to achieve the purpose of precise addressing and ultra-sensitive detection of one or more miRNAs in vitro by regulating the quantum properties of diamond NV color centers.

2. The miRNA precise addressing and ultrasensitive detection device according to claim 1, characterized in that: The structure of each system is: The optical system includes: a laser (11), an avalanche photodiode (15), a photon counter (16), an Olympus inverted microscope (10), a spectrometer (14), a pinhole (13) and a dichroic mirror (12); The objective lens of the Olympus inverted microscope (10) is inverted, with a transparent sample stage (9) above it. The excitation light emitted by the laser (11) is emitted to the surface of the sample to be tested through the objective lens, and the optical path system collects the emission spectrum, fluorescence lifetime and magnetic resonance parameters of the sample to be tested through the objective lens; The AFM system comprises an atomic force microscope (8), wherein the atomic force microscope (8) is a cantilever beam type; The process of integrating the optical system with the AFM system is as follows: a transparent sample stage (9) is placed in the chamber of the atomic force microscope (8); when the optical system emits laser light below the sample to be measured and collects signals, the AFM tip simultaneously scans the sample to be measured above the sample, and the signals collected by the AFM system and the optical system are used to realize dynamic and real-time collection of the fluorescence and magnetic resonance characteristic information of the diamond NV color center; The microwave system comprises a microwave source (1), a microwave switch (2), a high power amplifier (3) and a circulator (4); after the microwave system is connected and debugged, the microwave source (1) generates a microwave field, which is capacitively coupled to the Ω-type resonator (5), providing a uniform peak field measurement area, and modulating the quantum properties of the diamond NV color center through a fixed frequency to achieve NV color center quantum bit initialization and specific detection and analysis; The magnetic field module (17) is a permanent magnet or an electromagnet, and the self-selection characteristics of the NV color center are affected by adjusting the direction and magnitude of the magnetic field and the distance between the magnetic field module (17) and the NV color center, so as to realize the magnetic imaging function; The two-dimensional thin film generation system is an atomic layer deposition platform.

3. A method for precise addressing and ultrasensitive detection of miRNA, characterized in that: include: Prepare an AFM probe, wherein the AFM probe includes an AFM tip and an AFM cantilever, wherein the AFM tip is obtained by atomic layer deposition of a two-dimensional thin film on a diamond surface containing NV color centers; The AFM probe is combined with a magnetic field module and an optical system to locate and detect the target miRNA-coupled magnetic sphere, wherein the surface of the target miRNA-coupled magnetic sphere is modified with a modified DNA probe and a target miRNA; The AFM probe is combined with a magnetic field module and an optical system to locate and detect the target miRNA coupled magnetic sphere, including: Spin-coating the target miRNA-coupled magnetic spheres onto the sample area to be tested on a quartz glass slide; The magnetic field module applies a magnetic field to the sample area to be tested, and the direction of the magnetic field is parallel to the NV axis; The laser of the optical system generates excitation light that acts on the NV color center of the AFM probe. The AFM probe is used to scan the sample to be tested to obtain the magnetic field strength at different positions. The magnetic field strength changes are used to perform magnetic imaging on the magnetic spheres modified with miRNA to achieve the positioning of the miRNA-coupled magnetic spheres. At the same time, the optical system collects fluorescence signals within the motion range of the AFM probe, and uses the spectral scanning imaging function to reflect the fluorescence changes within the motion range of the AFM probe in real time, thereby achieving ultra-sensitive detection of miRNA.

4. The miRNA precise addressing and ultrasensitive detection method according to claim 3, characterized in that: The method for preparing the AFM probe comprises: a) Using reactive ion etching (RIE) to etch bulk diamond containing NV color centers, a diamond with a conical tip is obtained; b) Placing a diamond with a conical tip in an atomic layer deposition reaction chamber to modify a two-dimensional thin film on the surface; c) processing the main portion of the rear end of the conical tip by a diamond etching process to obtain a complete diamond AFM tip; d) The diamond AFM tip is assembled with the AFM cantilever to form a complete probe.

5. The miRNA precise addressing and ultrasensitive detection method according to claim 3, characterized in that: The thickness of the two-dimensional film is 0.5-100 nm.

6. The miRNA precise addressing and ultrasensitive detection method according to claim 3, characterized in that: The method utilizes the AFM probe in combination with the magnetic field module and the optical system to locate and detect the target miRNA coupled magnetic sphere, further comprising: The signal is modulated and amplified using a microwave system.

7. The miRNA precise addressing and ultrasensitive detection method according to claim 3, characterized in that: Preparation of target miRNA-coupled magnetic beads, including: preparing a magnetic ball solution with surface modified with streptavidin and a probe DNA solution; 5-10 μl of the probe DNA solution was added to 100-150 μl of the magnetic ball solution and incubated at room temperature for 12-15 h; the incubated solution was centrifuged and washed with a buffer solution to remove loosely connected probe DNA to obtain a treated magnetic ball solution; Take 10-20 μl of the target miRNA solution and mix it with 100-150 μl of the treated magnetic ball solution, incubate it at room temperature, and wash it with a centrifugation buffer to remove excess miRNA to obtain the target miRNA-coupled magnetic balls; Both ends of the probe DNA are modified with biotin and a modifier respectively, and the modifier includes a dye molecule and a metal nanoparticle.

8. The miRNA precise addressing and ultrasensitive detection method according to claim 7, characterized in that: The magnetic ball solution is a solution containing magnetic balls of one diameter or multiple diameters; For a solution containing magnetic spheres of various diameters, the probe DNA solution is a solution containing various DNAs, and the target miRNA-coupled magnetic spheres are a coupled magnetic sphere solution containing various target miRNAs. Various DNAs are respectively mixed with magnetic spheres of various diameters and are respectively paired with various target miRNAs.

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