Nanodiamond array preparation and miRNA ultra-sensitive detection method

Abstract

The invention discloses a nano-diamond array preparation and miRNA ultra-sensitive detection method, and the nano-diamond array preparation comprises the following steps: self-assembling densely arranged single-layer polystyrene (PS) microsphere templates on a silicon wafer plated with a gold film, and thinning the particle size of PS microspheres by adopting a reactive ion etching process to obtain non-densely arranged PS microsphere templates; carrying out thermal evaporation on an aluminum film and ultrasonically removing the PS microspheres to obtain a micropore array; then modifying the surface of the nano-diamond containing the NV color center with a single-chain DNA probe, and self-assembling the diamond modified by the DNA probe to the nano-pore array through a gold-sulfur bond; the miRNA detection comprises the following steps: firstly, preparing a target miRNA coupled probe magnetic sphere, dropwise adding the target miRNA coupled probe magnetic sphere to the nano-diamond array, then scanning the target miRNA coupled probe magnetic sphere on the surface of the nano-diamond array through the scanning system, the microwave module and the optical path system, and collecting an optical detection magnetic resonance spectrum. The invention is expected to be applied to the field of early diagnosis of cancers.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a method for preparing a nano-diamond array and ultra-sensitive detection of miRNA. Background Art

[0002] Dynamic detection of biological molecules such as proteins and nucleic acids has gradually developed into a new trend for early diagnosis, monitoring and prognosis of malignant tumors (cancer). Among them, the expression of miRNA in human serum, saliva and other body fluids has a significant quantitative relationship in the occurrence and evolution of cancer. At present, miRNA detection based on fluorescence, electrochemistry and colorimetry has several shortcomings such as long detection cycle, low detection limit, poor specificity and harsh detection environment. Therefore, it is urgent to improve the existing early cancer diagnosis methods and explore the innovation of detection principles to achieve ultra-sensitive measurement of low-abundance miRNA. Nitrogen vacancy center (NV color center) is a luminescent defect in the atomic structure of diamond, which contains a nitrogen atom that replaces a carbon atom and a hole. The energy level structure of diamond NV color center shows that its ground state and excited state are both spin triplet. At room temperature, the zero phonon line of diamond NV color center is located near 637nm, the fluorescence is strongest at 680nm, and the absorption is strongest in the green light band. By applying a magnetic field and broadband microwaves to the NV center, the NV center spin state has a rapid response characteristic to the magnetic field and manifests itself as a change in fluorescence intensity. At the same time, diamond NV centers are suitable for electromagnetic field measurements of biomolecules due to their high stability and low cytotoxicity.

[0003] In the prior art, the Chinese invention patent "Application No.: 202010224841.6" discloses a scanning detection system based on diamond NV color centers, which improves the photon collection efficiency of NV color centers, but cannot achieve large-area and rapid sample scanning. Therefore, it has significant limitations in the high-throughput and rapid detection applications of in vitro miRNA. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for preparing a nanodiamond array and detecting ultra-sensitive miRNA, in which magnetic nano-labels are distributed directionally on the surface of nanodiamonds arranged regularly at a certain spacing to increase the scanning step distance of the magnetic nano-labels, thereby expanding the detection range and speed of miRNA samples.

[0005] Technical solution: To solve the above technical problems, the present invention provides a method for preparing a nanodiamond array and ultra-sensitive detection of miRNA, comprising:

[0006] Step 1: Prepare silicon wafers and perform ultrasonic cleaning with acetone, anhydrous ethanol and deionized water;

[0007] Step 2: thermally evaporating a gold film on the surface of the silicon wafer after ultrasonic cleaning;

[0008] Step 3: self-assembling a tightly packed single-layer PS microsphere array on the gold film on the surface of the silicon wafer; comprising:

[0009] (a) taking 300-500 μL of a water suspension of PS microspheres with a mass fraction of 5%-10% and a particle size of 0.5-3 μm and mixing it with an equal volume of anhydrous ethanol and dispersing it evenly by ultrasonication;

[0010] (b) ultrasonically cleaning the silicon wafer with acetone, anhydrous ethanol and deionized water, and then plasma treating the silicon wafer for 1 to 3 minutes after drying;

[0011] (c) applying 3-10 μL of a dispersion of PPS microspheres / ethanol on the surface of a plasma-treated silicon wafer, and spin coating the surface at 500-1000 rpm on a coating machine for 10-30 seconds; after drying, the surface is slowly immersed in an aqueous solution containing 10%-30% ethanol, and a monolayer of a PPS microsphere film is suspended on the surface of the liquid;

[0012] (d) A silicon wafer coated with a gold film is immersed in the solution, and the PS microsphere film is slowly withdrawn from the liquid after being close to it, and the PS microspheres on the liquid surface are transferred to the silicon wafer.

[0013] Step 4: Etch the densely arranged PS microsphere array by reactive ion etching process, wherein the oxygen flow rate is 10-50 sccm, the RF power is 10-50 mW, the vacuum degree is 0.1-0.5 mTorr, and the etching time is 5-30 minutes.

[0014] Step 5: thermally evaporate a 20-100 nm aluminum film on the surface of the sample prepared in step 4; then place the sample in anhydrous ethanol and perform ultrasonic treatment to remove the PS microspheres to obtain a micropore array with exposed gold film;

[0015] Step 6: using concentrated sulfuric acid and nitric acid to pickle the surface of nanodiamonds with an average particle size of 40 to 750 nm and containing NV color centers;

[0016] Step 7. Modify the surface of the acid-washed nanodiamond with DNA probes: take 100-500 μL of the acid-washed fluorescent nanodiamond into a centrifuge tube, then add 10-50 μL of EDC aqueous solution and 10-50 μL of NHS aqueous solution; shake well, add 1-5 nmol of DNA probes modified with amino and thiol groups at both ends, and incubate at room temperature overnight.

[0017] Step 8: Add the surface modified probe nanodiamonds to the surface of the microwell array, incubate at room temperature, immerse in 3% Tween-20 solution for 10 to 30 minutes to remove the nanodiamonds not connected to the gold film, and dry to obtain a nanodiamond array with an average particle size of 40 to 750 nm.

[0018] The present invention also provides a nanodiamond array miRNA ultra-sensitive detection method, comprising the following steps:

[0019] 1-5 nmol of probe DNA with terminal amino modification, 2-10 mg of EDC, and 2-10 mg of NHS were added to 100-500 μL of a magnetic ball solution with a concentration of 1 nmol / mL and containing carboxyl groups on the surface, and incubated overnight at room temperature; the incubated solution was centrifuged and washed with a buffer to remove the probe DNA that was not firmly connected, and a DNA probe magnetic ball solution was obtained; 10 μL of the target miRNA solution was mixed with 100-500 μL of the DNA probe magnetic ball solution and incubated, and the unreacted miRNA was centrifuged and washed with a buffer to obtain a target miRNA-coupled magnetic ball. The target miRNA-coupled probe magnetic ball was dropped onto the surface of the nanodiamond array for incubation, and then the target miRNA-coupled magnetic ball on the surface of the nanodiamond array was scanned and positioned using a scanning system, a microwave module, and an optical path system, and a light detection magnetic resonance spectrum was collected.

[0020] The microwave system includes a microwave generator and a microwave antenna, and the optical path system includes a laser, a single photon counter, an Olympus inverted microscope and a dichroic mirror. The scanning system includes a piezoelectric scanning stage.

[0021] like Figure 2 As shown, the magnetic ball coupled with the target miRNA is connected to the nanodiamond array through base complementary pairing; the laser emits 532nm laser, which is transmitted to the surface of the nanodiamond array through the dichroic mirror and the optical path system of the Olympus microscope. At the same time, the microwave source transmits 2.7-2.95GHz microwaves to the microwave antenna to regulate the energy level of the nitrogen vacancy color center inside the nanodiamond. The nitrogen vacancy color center produces fluorescence under the excitation of the 532nm laser, and when the external magnetic field exists, the fluorescence at the frequency of 2.87GHz decays. The photon signal is transmitted to the single photon counting module by the optical path system of the Olympus inverted microscope and the dichroic mirror to obtain a light detection magnetic resonance spectrum at a coordinate position. The light detection magnetic resonance spectrum at different positions of the sample can be obtained by scanning with a piezoelectric displacement stage. According to the 2.87GHz fluorescence change in the light detection magnetic resonance spectrum of each coordinate point, the position of the miRNA coupled magnetic ball can be determined, and ultra-sensitive detection of miRNA can be achieved.

[0022] Beneficial effects: (1) The nanodiamond array of the present invention adopts a self-assembly process, which has the advantages of simple process and no need for precision photolithography equipment, compared with the use of semiconductor micro-nano processing technology to directly etch a columnar array structure on the surface of a block diamond; (2) The nanodiamond array has a larger spacing, which is conducive to increasing the step scanning distance of the piezoelectric displacement stage, thereby improving the detection speed of biological samples; (3) The nanodiamond array miRNA detection method of the present invention collects the fluorescence change rate caused by the magnetic label in the biological sample. Compared with directly collecting the fluorescence signal, it can effectively isolate the background fluorescence of the substrate material, thereby improving the signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The flowchart of the method for preparing a nanodiamond array and ultrasensitive detection of miRNA according to the present invention, wherein: 1 is a nanodiamond containing NV color centers, 2 is a carboxylated nanodiamond, 3 is a DNA probe nanodiamond, 4 is a PS microsphere, 5 is a silicon wafer, 6 is a gold film, and 7 is an aluminum film;

[0024] Figure 2 This is a schematic diagram of the ultra-sensitive detection principle of miRNA using the nanodiamond array of the present invention, wherein 8 is a carboxyl magnetic sphere, 9 is a DNA probe, 10 is a target miRNA, 11 is a nanodiamond array coupled to miRNA, 12 is a piezoelectric displacement stage, 13 is a microwave antenna, 14 is a microwave source, 15 is a laser, 16 is a dichroic mirror, 17 is a single photon counting module, and 18 is an Olympus inverted microscope. DETAILED DESCRIPTION

[0025] The specific embodiments of the present invention are described in detail below.

[0026] Example 1: Preparation of a nanodiamond array and ultrasensitive miRNA detection method ( Figure 1 ), comprising the following steps:

[0027] Step 1, prepare the silicon wafer and perform ultrasonic cleaning with acetone, anhydrous ethanol and deionized water respectively;

[0028] Step 2: thermally evaporating a gold film on the surface of the silicon wafer after ultrasonic cleaning;

[0029] Step 3: Self-assemble a tightly packed single-layer polystyrene microsphere (PS microsphere) array on the gold film on the surface of the silicon wafer. Including:

[0030] (a) 500 μL of a 10% mass fraction, 3 μm particle size PS microsphere aqueous suspension was mixed with an equal volume of 98% anhydrous ethanol and ultrasonically dispersed to obtain a PS microsphere / ethanol dispersion;

[0031] (b) ultrasonically cleaning the silicon wafer in step 2 with acetone, anhydrous ethanol and deionized water respectively, and then plasma treating for 3 minutes after drying;

[0032] (c) 3 μL of PS microsphere / ethanol dispersion was applied to the surface of the plasma-treated silicon wafer and spin-coated at 500 rpm for 10 seconds on a spin coater; after drying, the film was slowly immersed in an aqueous solution containing 10% ethanol, and a single layer of polystyrene microsphere film was suspended on the surface of the liquid;

[0033] (d) A silicon wafer coated with a gold film is immersed in a solution in which a polystyrene microsphere film is suspended. The PS microsphere film is slowly withdrawn from the liquid after approaching it, and the PS microspheres on the liquid surface are transferred to the silicon wafer.

[0034] Step 4: Etch the closely arranged PS microsphere array by reactive ion etching process; wherein the oxygen flow rate is 20 sccm, the RF power is 50 mW, the vacuum degree is 0.3 mTorr, and the etching time is 30 minutes;

[0035] Step 5: thermally evaporate a 100 nm aluminum film on the surface of the sample prepared in step 4; then place the sample in anhydrous ethanol and perform ultrasonic treatment to remove the PS microspheres to obtain a micropore array with exposed gold film;

[0036] Step 6: Use concentrated sulfuric acid and nitric acid to pickle the surface of nanodiamond (MDNV1μmHi10mg, Adamas Nanotechnologies) with an average particle size of 750nm and containing NV color centers;

[0037] Step 7: Modify the surface of the acid-washed nanodiamond with a DNA probe: Take 100 μL of the acid-washed fluorescent nanodiamond solution with a concentration of 1 mg / mL in a centrifuge tube, then add 10 μL of a 100 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) aqueous solution and 10 μL of a 100 mg / mL N-hydroxysuccinimide (NHS) aqueous solution; shake well for 30 minutes and add 1 nmol of a DNA probe (5'-SH-CTGATAAGCTACCCCC-NH 2 -3') and incubated overnight at room temperature.

[0038] Step 8: Add the surface-modified DNA probe nanodiamonds to the surface of the microwell array prepared in step 5, incubate at room temperature, immerse in 3% Tween-20 solution for 10 minutes, and wash to remove the nanodiamonds not connected to the gold film. After drying, a nanodiamond array with an average particle size of 750 nm is obtained.

[0039] Step 9: Prepare the target miRNA-coupled probe magnetic ball, including: taking 1 nmol of the probe DNA with terminal amino modification (5'-NH 2 -CCCCCTAGACACCGTGTTCAACATCAGT-3', Shanghai Shenggong Biotechnology Co., Ltd.), 2mg EDC, and 2mgNHS were added to 100μL of a 1nmol / mL magnetic ball solution (HY-K0225, MCE) with a surface carboxyl group, and incubated overnight at room temperature; the incubated solution was centrifuged and washed with a buffer solution to remove the probe DNA that was not firmly connected to obtain a DNA probe magnetic ball solution; 10μL of a 1nmol / L target miRNA (5'-UAGCUUAUCAGACUG AUGUUGA-3') solution was mixed with 100μL of the DNA probe magnetic ball solution and incubated at 37°C, and the unreacted miRNA was removed by centrifugation and washed with a buffer solution to obtain the target miRNA-coupled magnetic ball.

[0040] Step 10: Add the target miRNA-coupled probe magnetic balls onto the surface of the nanodiamond array for incubation, and then use the scanning system, microwave module and optical path system to scan the target miRNA-coupled magnetic balls on the surface of the nanodiamond array, and finally obtain the optical detection magnetic resonance spectrum of the sample with a target miRNA concentration of 1 nmol / L.

[0041] Example 2: A method for preparing a nanodiamond array and detecting ultrasensitive miRNA, comprising the following steps:

[0042] Step 1: Prepare silicon wafers and perform ultrasonic cleaning with acetone, anhydrous ethanol and deionized water;

[0043] Step 2: thermally evaporating a gold film on the surface of the silicon wafer after ultrasonic cleaning;

[0044] Step 3: Self-assemble a tightly packed single-layer PS microsphere array on the gold film on the surface of the silicon wafer. Including:

[0045] (a) 300 μL of a 5% mass fraction, 0.5 μm PS microsphere aqueous suspension was mixed with an equal volume of 98% anhydrous ethanol and ultrasonically dispersed to obtain a PS microsphere / ethanol dispersion;

[0046] (b) The silicon wafer was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water respectively, and then plasma treated for 1 minute after drying;

[0047] (c) 10 μL of PS microsphere / ethanol dispersion was applied to the surface of the plasma-treated silicon wafer and spin-coated at 1000 rpm for 30 seconds on a spin coater; after drying, the film was slowly immersed in an aqueous solution containing 30% ethanol, and a monolayer of PS microsphere film was suspended on the surface of the liquid;

[0048] (d) A silicon wafer coated with a gold film is immersed in a solution in which a polystyrene microsphere film is suspended. The PS microsphere film is slowly withdrawn from the liquid after approaching it, and the PS microspheres on the liquid surface are transferred to the silicon wafer.

[0049] Step 4: Etch the closely arranged PS microsphere array by reactive ion etching process; wherein the oxygen flow rate is 10 sccm, the RF power is 10 mW, the vacuum degree is 0.1 mTorr, and the etching time is 15 minutes;

[0050] Step 5: thermally evaporate a 20 nm aluminum film on the surface of the sample prepared in step 4; then place the sample in anhydrous ethanol and perform ultrasonic treatment to remove the PS microspheres to obtain a micropore array with exposed gold film;

[0051] Step 6: Use concentrated sulfuric acid and nitric acid to pickle the surface of nanodiamond (MDNV40nmHi10mL, Adamas Nanotechnologies) with an average particle size of 40nm and containing NV color centers;

[0052] Step 7: Modify the surface of the acid-washed nanodiamond with a DNA probe: Take 500 μL of the acid-washed fluorescent nanodiamond solution with a concentration of 1 mg / mL into a centrifuge tube, then add 50 μL of a 100 mg / mL EDC aqueous solution and 50 μL of a 100 mg / mL NHS aqueous solution; shake well for 30 minutes and add 5 nmol of a DNA probe with amino and thiol groups modified at both ends (5'-SH-CTGATAAGCTACCCCC-NH 2 -3') and incubated overnight at room temperature.

[0053] Step 8: Add the surface-modified DNA probe nanodiamonds to the surface of the microwell array prepared in step 5, incubate at room temperature, immerse in 3% Tween-20 solution for 30 minutes to remove the nanodiamonds not connected to the gold film, and dry to obtain a nanodiamond array with an average particle size of 40 nm.

[0054] Step 9: Prepare the target miRNA-coupled probe magnetic ball, including: taking 5 nmol of the probe DNA with terminal amino modification (5'-NH 2-CCCCCTAGACACCGTGTTCAACATCAGT-3', Shanghai Shenggong Biotechnology Co., Ltd.), 10 mg EDC, and 10 mg NHS were added to 500 μL of a 1 nmol / mL magnetic sphere (HY-K0225, MCE) solution containing carboxyl groups on the surface, and incubated overnight at room temperature; the incubated solution was centrifuged and washed with a buffer solution to remove the probe DNA that was not firmly connected to obtain a DNA probe magnetic sphere solution; 10 μL of a 1 pmol / L target miRNA (5'-UAGCUUAUCAGAC UGAUGUUGA-3') solution was mixed with 500 μL of the DNA probe magnetic sphere solution and incubated at 37°C, and the unreacted miRNA was removed by centrifugation and washed with a buffer solution to obtain the target miRNA-coupled magnetic sphere.

[0055] Step 10: Add the target miRNA-coupled probe magnetic balls onto the surface of the nanodiamond array for incubation, and then use the scanning system, microwave module and optical path system to scan the target miRNA-coupled magnetic balls on the surface of the nanodiamond array, and finally obtain the optical detection magnetic resonance spectrum of the sample with a target miRNA concentration of 1 pmol / L.

[0056] Example 3: A method for preparing a nanodiamond array and detecting ultrasensitive miRNA, comprising the following steps:

[0057] Step 1, prepare the silicon wafer and perform ultrasonic cleaning with acetone, anhydrous ethanol and deionized water respectively;

[0058] Step 2: thermally evaporating a gold film on the surface of the silicon wafer after ultrasonic cleaning;

[0059] Step 3: Self-assemble a tightly packed single-layer polystyrene microsphere (PS microsphere) array on the gold film on the surface of the silicon wafer. Including:

[0060] (a) 400 μL of a 7.5% mass fraction, 1 μm particle size PS microsphere aqueous suspension was mixed with an equal volume of 98% anhydrous ethanol and ultrasonically dispersed to obtain a PS microsphere / ethanol dispersion;

[0061] (b) The silicon wafer was ultrasonically cleaned with acetone, anhydrous ethanol and deionized water respectively, and then plasma treated for 2 minutes after drying;

[0062] (c) 5 μL of PS microsphere / ethanol dispersion was applied to the surface of the plasma-treated silicon wafer and spin-coated at 700 rpm for 20 seconds on a spin coater; after drying, the film was slowly immersed in an aqueous solution containing 20% ​​ethanol, and a single layer of PS microsphere film was suspended on the surface of the liquid;

[0063] (d) A silicon wafer coated with a gold film is immersed in a solution in which a PS microsphere film is suspended. The PS microsphere film is slowly withdrawn from the liquid after approaching it, and the PS microspheres on the liquid surface are transferred to the silicon wafer.

[0064] Step 4: etching the closely packed PS microsphere array by reactive ion etching to form a non-close packed PS microsphere array; wherein the oxygen flow rate is 50 sccm, the RF power is 25 mW, the vacuum degree is 0.5 mTorr, and the etching time is 5 minutes;

[0065] Step 5: thermally evaporate a 50 nm aluminum film on the surface of the sample prepared in step 4; then place the sample in anhydrous ethanol and perform ultrasonic treatment to remove the PS microspheres to obtain a micropore array with exposed gold film;

[0066] Step 6: Use concentrated sulfuric acid and nitric acid to pickle the surface of nanodiamond (MDNV100nmHi10mL, Adamas Nanotechnologies) with an average particle size of 100nm and containing NV color centers;

[0067] Step 7: Modify the surface of the acid-washed nanodiamond with a DNA probe: Take 300 μL of the acid-washed fluorescent nanodiamond solution with a concentration of 1 mg / mL in a centrifuge tube, then add 30 μL of a 100 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) aqueous solution and 30 μL of a 100 mg / mL N-hydroxysuccinimide (NHS) aqueous solution; shake well for 30 minutes and add 3 nmol of a DNA probe (5'-SH-CTGATAAGCTACCCCC-NH 2 -3') and incubated overnight at room temperature.

[0068] Step 8: Add the surface-modified DNA probe nanodiamonds to the surface of the microporous array prepared in step 5, incubate at room temperature, immerse in 3% Tween-20 solution for 20 minutes, and wash to remove the nanodiamonds not connected to the gold film. After drying, a nanodiamond array with an average particle size of 100 nm is obtained.

[0069] Step 9: Prepare the target miRNA-coupled probe magnetic ball, including: taking 3 nmol of the probe DNA with terminal amino modification (5'-NH 2-CCCCCTAGACACCGTGTTCAACATCAGT-3', Shanghai Shenggong Biotechnology Co., Ltd.), 6mg EDC, and 6mgNHS were added to 300μL of a 1nmol / mL magnetic ball solution (HY-K0225, MCE) with a surface carboxyl group, and incubated overnight at room temperature; the incubated solution was centrifuged and washed with a buffer solution to remove the probe DNA that was not firmly connected to obtain a DNA probe magnetic ball solution; 10μL of a 1fmol / L target miRNA (5'-UAGCUUAUCAGACUGA UGUUGA-3') solution was mixed with 300μL of the DNA probe magnetic ball solution and incubated at 37°C, and the unreacted miRNA was removed by centrifugation and washed with a buffer solution to obtain the target miRNA-coupled magnetic ball.

[0070] Step 10: Add the target miRNA-coupled probe magnetic balls onto the surface of the nanodiamond array for incubation, and then use the scanning system, microwave module and optical path system to scan the target miRNA-coupled magnetic balls on the surface of the nanodiamond array, and finally obtain the optical detection magnetic resonance spectrum of the sample with a target miRNA concentration of 1 fmol / L.

Claims

1. A method for preparing a nanodiamond array, characterized in that The steps include: (1) thermally evaporating a gold film on the surface of the silicon wafer after ultrasonic cleaning; (2) Self-assembled tightly packed monolayer polystyrene microsphere arrays on a gold film on the surface of a silicon wafer; (3) Etching the densely packed PS microsphere array; (4) thermally evaporating a 20-100 nm aluminum film on the surface of the PS microsphere array prepared in step (3), placing it in anhydrous ethanol and performing ultrasonic treatment to remove the PS microspheres, thereby obtaining a microporous array with exposed gold film; (5) modifying the pretreated nanodiamond surface with DNA probes; dropping the probes onto the surface of the microporous array in step (4); immersing the probes in a surfactant solution for cleaning after incubation; and drying the nanodiamond array.

2. The method for preparing a nano-diamond array according to claim 1, characterized in that: The method for preparing the self-assembled and tightly arranged single-layer polystyrene microsphere array in step (2) comprises the following steps: (a) taking 300-500 μL of a water suspension of PS microspheres with a mass fraction of 5%-10% and a particle size of 0.5-3 μm and mixing it with an equal volume of anhydrous ethanol and dispersing it evenly by ultrasonication; (b) ultrasonically cleaning the silicon wafer with acetone, anhydrous ethanol and deionized water, and then plasma treating the silicon wafer for 1 to 3 minutes after drying to obtain a PS microsphere / ethanol dispersion; (c) applying 3 to 10 μL of PS microsphere / ethanol dispersion on the surface of the plasma-treated silicon wafer, and spin coating it on a coating machine at a speed of 500 to 1000 rpm for 10 to 30 seconds; after drying, slowly immersing it in an aqueous solution containing 10% to 30% ethanol, and a monolayer of PS microsphere film is suspended on the surface of the liquid; (d) The silicon wafer coated with a gold film is immersed in the solution of step (c), and the PS microsphere film is slowly withdrawn from the liquid after being close to the PS microsphere film, and the PS microspheres on the liquid surface are transferred to the rigid silicon substrate.

3. The method for preparing a nano-diamond array according to claim 1, characterized in that: The preparation method of the PS microsphere array in step (3) comprises the following steps: reducing the particle size of the single-layer PS microspheres on the surface of the silicon wafer by a reactive ion etching system, wherein the oxygen flow rate is 10 to 50 sccm, the RF power is 10 to 50 mW, the vacuum degree is 0.1 to 0.5 mTorr, and the etching time is 5 to 30 minutes.

4. The method for preparing a nano-diamond array according to claim 1, characterized in that: The nano-diamond in step (5) contains nitrogen vacancy color centers and has an average particle size of 40nm to 750nm. The pretreatment includes: pickling the nano-diamond with concentrated sulfuric acid and nitric acid.

5. The method for preparing a nano-diamond array according to claim 1, characterized in that: In step (5), the surface modification of nanodiamond with DNA probe comprises the following steps: taking 100-500 μL of acid-washed nanodiamond, adding 10-50 μL of EDC solution and NHS solution respectively, shaking well and adding 1-5 nmol of DNA probe, wherein the 5' end of the DNA probe is modified with amino group and the 3' end is modified with thiol group.

6. The method for preparing a nano-diamond array according to claim 1, characterized in that: The cleaning treatment in step eight includes: immersing in a 3% Tween-20 solution for 10 to 30 minutes.

7. A nanodiamond array miRNA ultrasensitive detection method, characterized in that: The following steps are involved: The target miRNA-coupled probe magnetic balls are prepared and dropped onto the surface of the nanodiamond array for incubation. Then, a scanning system, a microwave module and an optical path system are used to scan and locate the target miRNA-coupled magnetic balls on the surface of the nanodiamond array and detect signals.

8. The nanodiamond array miRNA ultrasensitive detection method according to claim 7, characterized in that: The microwave system includes a microwave generator and a microwave antenna; the optical path system includes: a laser, a single photon counter, an Olympus inverted microscope and a dichroic mirror; the scanning system includes: a piezoelectric scanning stage; and the detected signal is a light detection magnetic resonance spectrum.

9. The nanodiamond array miRNA ultrasensitive detection method according to claim 7, characterized in that: The method comprises the following steps: adding 1-5 nmol of a probe DNA solution, 2-10 mg of EDC and 2-10 mg of NHS to 100-500 μL of a magnetic ball solution with a concentration of 1 nmol / mL and containing carboxyl groups on the surface, and incubating the solution overnight at room temperature; washing the incubated solution by centrifugation with a buffer to remove probe DNA that is not firmly connected, and obtaining a DNA probe magnetic ball solution; mixing 10-50 μL of a target miRNA solution with 100-500 μL of a DNA probe magnetic ball solution, and incubating the mixture; washing the mixture by centrifugation with a buffer to remove unreacted miRNA, and obtaining a target miRNA-coupled magnetic ball; the probe DNA terminal is modified with an amino group.

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