A nanodiamond array preparation and miRNA ultrasensitive detection method

Abstract

The present invention discloses a method for preparing a nanodiamond array and ultrasensitive detection of miRNA. The nanodiamond array preparation comprises: self-assembling a densely packed monolayer polystyrene (PS) microsphere template on a gold-coated silicon wafer, then thinning the PS microspheres using a reactive ion etching process to obtain a non-densely packed PS microsphere template, then thermally evaporating an aluminum film and ultrasonically removing the PS microspheres to obtain a micropore array; then modifying the surface of the nanodiamond containing NV color centers with a single-stranded DNA probe, and the DNA probe-modified diamond self-assembles into the nanopore array through gold-sulfur bonds. MiRNA detection comprises: first preparing a probe magnetic sphere coupled to the target miRNA and dropping it onto the nanodiamond array, then scanning the target miRNA-coupled magnetic sphere on the surface of the nanodiamond array using a scanning system, a microwave module, and an optical path system, and collecting a light detection magnetic resonance spectrum. The present invention is expected to be applied to the field of early cancer diagnosis.

Description

Technical Field

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

[0002] Dynamic detection of biomolecules such as proteins and nucleic acids has gradually developed into a new trend in the early diagnosis, monitoring, and prognosis of malignant tumors (cancer). Among them, the expression of miRNAs in human body fluids such as serum and saliva has a significant quantitative relationship with the development and progression of cancer. Currently, miRNA detection based on fluorescence, electrochemistry, and colorimetry suffers from several shortcomings, including long detection cycles, low detection limits, poor specificity, and harsh detection environments. Therefore, there is an urgent need to improve existing early cancer diagnosis methods and explore innovative detection principles to achieve ultrasensitive measurement of low-abundance miRNAs. A nitrogen-vacancy center (NV) is a luminescent defect in the atomic structure of diamond. It consists of a nitrogen atom that replaces a carbon atom and a vacancy. The energy level structure of a diamond NV center shows that both its ground and excited states are spin-tripled. At room temperature, the zero phonon line of a diamond NV center is located near 637 nm, with the strongest fluorescence at 680 nm and the strongest absorption in the green band. By applying a magnetic field and broadband microwaves to the NV center, the NV center's spin state rapidly responds to the magnetic field, manifesting as changes in fluorescence intensity. Diamond NV centers are also suitable for electromagnetic field measurements of biomolecules due to their high stability and low cytotoxicity.

[0003] In the existing technology, 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 nanotags are distributed directionally on the surface of nanodiamonds arranged at a certain interval to increase the scanning step distance of the magnetic nanotags, 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 ultrasensitive detection of miRNA, comprising:

[0006] Step 1: Prepare the silicon wafer and ultrasonically clean it 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 densely packed monolayer PS microsphere array on the gold film on the silicon wafer surface; comprising:

[0009] (a) 300-500 μL of a 5%-10% mass fraction of PS microspheres with a particle size of 0.5-3 μm was mixed with an equal volume of anhydrous ethanol and ultrasonically dispersed uniformly;

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

[0011] (c) Applying 3-10 μL of a polystyrene microsphere / ethanol dispersion to a plasma-treated silicon wafer surface is spin-coated at 500-1000 rpm for 10-30 seconds on a spin coater. After drying, the wafer is slowly immersed in a 10%-30% ethanol aqueous solution, resulting in a monolayer polystyrene microsphere film suspended on the surface of the liquid.

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

[0013] Step 4: Etch the densely packed PS microsphere array by reactive ion etching process, wherein the oxygen flow rate is 10-50 sccm, the radio frequency 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, thereby obtaining a micropore array with the gold film exposed;

[0015] Step 6: using concentrated sulfuric acid and nitric acid to pickle the surface of the nanodiamond 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 a DNA probe: Place 100-500 μL of the acid-washed fluorescent nanodiamond in a centrifuge tube, then add 10-50 μL of an EDC aqueous solution and 10-50 μL of an NHS aqueous solution; shake well, then add 1-5 nmol of a DNA probe modified with amino groups and thiol groups at both ends, and incubate overnight at room temperature.

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

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

[0019] 1–5 nmol of amino-terminally modified probe DNA, 2–10 mg of EDC, and 2–10 mg of NHS were added to 100–500 μL of a 1 nmol / mL solution of magnetic spheres containing surface carboxyl groups and incubated overnight at room temperature. The incubated solution was centrifuged and washed with a buffer solution to remove loosely attached probe DNA, yielding a DNA probe magnetic sphere solution. 10 μL of the target miRNA solution was mixed with 100–500 μL of the DNA probe magnetic sphere solution and incubated. The mixture was then centrifuged and washed with a buffer solution to remove unreacted miRNA, yielding target miRNA-coupled magnetic spheres. The target miRNA-coupled magnetic spheres were then added dropwise to the surface of a nanodiamond array and incubated. A scanning system, microwave module, and optical path system were then used to scan and locate the target miRNA-coupled magnetic spheres on the nanodiamond array surface and acquire optical detection magnetic resonance spectra.

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

[0021] like Figure 2 As shown, a magnetic sphere coupled to a target miRNA is connected to a nanodiamond array through base complementary pairing. A laser emits 532nm laser light, which is transmitted to the surface of the nanodiamond array via a dichroic mirror and the optical system of an Olympus microscope. Simultaneously, a microwave source transmits 2.7-2.95GHz microwaves to a microwave antenna to modulate the energy levels of nitrogen vacancy color centers within the nanodiamonds. The nitrogen vacancy color centers produce fluorescence under 532nm laser excitation, and in the presence of an external magnetic field, the fluorescence at a frequency of 2.87GHz decays. The photon signal is transmitted by the optical system of the Olympus inverted microscope and the dichroic mirror to a single-photon counting module, which generates a photodetection magnetic resonance spectrum at a coordinate position. The photodetection magnetic resonance spectrum at different locations of the sample can be obtained by scanning with a piezoelectric translation stage. The position of the miRNA-coupled magnetic sphere can be determined based on the 2.87GHz fluorescence changes in the photodetection magnetic resonance spectrum at each coordinate point, enabling ultrasensitive detection of miRNA.

[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 to 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, which can effectively isolate the background fluorescence of the substrate material compared to directly collecting the fluorescence signal, thereby improving the signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart of a method for preparing a nanodiamond array and ultrasensitive miRNA detection 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 principle of ultra-sensitive detection of miRNA using the nanodiamond array of the present invention, wherein 8 is a carboxyl magnetic sphere, 9 is a DNA probe, 10 is the target miRNA, 11 is the miRNA-coupled nanodiamond array, 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 ), including the following steps:

[0027] Step 1: Prepare the silicon wafer and ultrasonically clean it 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 silicon wafer surface. This includes:

[0030] (a) 500 μL of a 10% by mass fraction (mass fraction) aqueous suspension of PS microspheres (3 μm in size) 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 the wafer for 3 minutes after drying;

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

[0033] (d) A silicon wafer coated with a gold film is immersed in a solution containing a suspended polystyrene microsphere film. 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 densely packed 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, thereby obtaining a micropore array with the gold film exposed;

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

[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 into 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 for 30 minutes, then add 1 nmol of a DNA probe modified with an amino group and a thiol group at both ends (5'-SH-CTGATAAGCTACCCCC-NH2-3') and incubate at room temperature overnight.

[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, and then immerse in 3% Tween-20 solution for 10 minutes 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 target miRNA-coupled probe magnetic balls, including: taking 1 nmol of terminally amino-modified probe DNA (5'-NH2-CCCCCTAGACACCGTGTTCAACATCAGT-3', Shanghai Sangon Biotechnology Co., Ltd.), 2 mg of EDC, and 2 mg of NHS, adding them to 100 μL of a 1 nmol / mL magnetic ball solution (HY-K0225, MCE) with a surface carboxyl group, and incubating overnight at room temperature; using a buffer solution to centrifuge and wash the incubated solution to remove the probe DNA that is not firmly connected to obtain a DNA probe magnetic ball solution; taking 10 μL of a 1 nmol / L target miRNA (5'-UAGCUUAUCAGACUG AUGUUGA-3') solution and mixing it with 100 μL of the DNA probe magnetic ball solution, incubating at 37°C, and centrifuging and washing with a buffer solution to remove unreacted miRNA to obtain target miRNA-coupled magnetic balls.

[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. The method comprises the following steps:

[0042] Step 1: Prepare the silicon wafer and ultrasonically clean it 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 monolayer PS microsphere array on the gold film on the silicon wafer surface. This includes:

[0045] (a) 300 μL of a 5% (mass fraction) aqueous suspension of PS microspheres with a particle size of 0.5 μm 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 the PS microsphere / ethanol dispersion was applied to the plasma-treated silicon wafer surface and spin-coated at 1000 rpm for 30 seconds on a spin coater. After drying, the wafer was slowly immersed in a 30% ethanol aqueous solution, resulting in a monolayer of PS microsphere film suspended on the surface of the liquid.

[0048] (d) A silicon wafer coated with a gold film is immersed in a solution containing a suspended polystyrene microsphere film. 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 densely packed 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, thereby obtaining a micropore array with the gold film exposed;

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

[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 for 30 minutes, then add 5 nmol of a DNA probe modified with amino and thiol groups at both ends (5'-SH-CTGATAAGCTACCCCC-NH2-3') and incubate at room temperature overnight.

[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, and then immerse in 3% Tween-20 solution for 30 minutes to remove the nanodiamonds not connected to the gold film. After drying, a nanodiamond array with an average particle size of 40 nm is obtained.

[0054] Step 9. Prepare target miRNA-coupled probe magnetic balls, including: taking 5 nmol of terminally amino-modified probe DNA (5'-NH2-CCCCCTAGACACCGTGTTCAACATCAGT-3', Shanghai Sangon Biotechnology Co., Ltd.), 10 mg of EDC, and 10 mg of NHS, adding them to 500 μL of a 1 nmol / mL solution of magnetic balls (HY-K0225, MCE) with surface carboxyl groups, and incubating them overnight at room temperature; using a buffer solution to centrifuge and wash the incubated solution to remove the probe DNA that is not firmly connected to obtain a DNA probe magnetic ball solution; taking 10 μL of a 1 pmol / L target miRNA (5'-UAGCUUAUCAGAC UGAUGUUGA-3') solution and mixing it with 500 μL of the DNA probe magnetic ball solution, incubating them at 37°C, and centrifuging and washing with a buffer solution to remove unreacted miRNA to obtain target miRNA-coupled magnetic balls.

[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 ultrasonically clean it 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 silicon wafer surface. This includes:

[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 the PS microsphere / ethanol dispersion was applied to the plasma-treated silicon wafer surface and spin-coated at 700 rpm for 20 seconds on a spin coater. After drying, the wafer was slowly immersed in a 20% ethanol aqueous solution, resulting in a monolayer of PS microspheres suspended on the surface of the liquid.

[0063] (d) A silicon wafer coated with a gold film is immersed in a solution containing a suspended PS microsphere film. 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 densely packed PS microsphere array by reactive ion etching to form a non-densely 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, thereby obtaining a micropore array with the gold film exposed;

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

[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 into 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 for 30 minutes, then add 3 nmol of a DNA probe modified with an amino group and a sulfhydryl group at both ends (5'-SH-CTGATAAGCTACCCCC-NH2-3') and incubate at room temperature overnight.

[0068] Step 8: Add the surface-modified DNA probe nanodiamonds to the surface of the microwell array prepared in step 5, incubate at room temperature, and then immerse in 3% Tween-20 solution for 20 minutes 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 target miRNA-coupled probe magnetic balls, including: taking 3 nmol of terminally amino-modified probe DNA (5'-NH2-CCCCCTAGACACCGTGTTCAACATCAGT-3', Shanghai Sangon Biotechnology Co., Ltd.), 6 mg of EDC, and 6 mg of NHS, adding them to 300 μL of a 1 nmol / mL magnetic ball solution (HY-K0225, MCE) with a surface carboxyl group, and incubating overnight at room temperature; using a buffer solution to centrifuge and wash the incubated solution to remove the probe DNA that is not firmly connected to obtain a DNA probe magnetic ball solution; taking 10 μL of a target miRNA (5'-UAGCUUAUCAGACUGA UGUUGA-3') solution with a concentration of 1 fmol / L and mixing it with 300 μL of the DNA probe magnetic ball solution, incubating at 37°C, and centrifuging and washing with a buffer solution to remove unreacted miRNA to obtain target miRNA-coupled magnetic balls.

[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 densely packed monolayer polystyrene microsphere arrays on a gold film on a silicon wafer surface; (3) Etching the densely packed PS microsphere array; (4) A 20-100 nm aluminum film was thermally evaporated on the surface of the PS microsphere array prepared in step (3), and the array was placed in anhydrous ethanol and ultrasonically treated to remove the PS microspheres, thereby obtaining a microporous array with exposed gold film; (5) Modifying the pretreated nanodiamond surface with a DNA probe; adding the DNA probe dropwise to the surface of the microporous array described in step (4); incubating the nanodiamond surface at room temperature; immersing the nanodiamond surface in a surfactant solution for cleaning; and drying the nanodiamond array; The nano-diamond contains nitrogen vacancy color centers and has an average particle size of 40 nm to 750 nm. The pretreatment includes: pickling the nano-diamond with concentrated sulfuric acid and nitric acid.

2. The method for preparing a nanodiamond array according to claim 1, wherein: The method for preparing the self-assembled and tightly packed single-layer polystyrene microsphere array in step (2) comprises the following steps: (a) Mix 300–500 μL of a 5%–10% mass fraction of PS microspheres in water with a particle size of 0.5–3 μm with an equal volume of anhydrous ethanol and disperse them evenly by ultrasonication. (b) The silicon wafer was ultrasonically cleaned with acetone, anhydrous ethanol, and deionized water, dried, and then plasma treated for 1–3 minutes to obtain a PS microsphere / ethanol dispersion. (c) 3–10 µL of the PS microsphere / ethanol dispersion was applied to the plasma-treated silicon wafer surface and spin-coated at 500–1000 rpm for 10–30 seconds. After drying, the wafer was slowly immersed in an aqueous solution containing 10%–30% ethanol, resulting in a monolayer of PS microsphere film suspended on the surface of the liquid. (d) A silicon wafer coated with a gold film is immersed in the solution of step (c). The PS microsphere film is brought close to the wafer and then slowly withdrawn from the liquid. The PS microspheres on the liquid surface are transferred to the rigid silicon substrate.

3. The method for preparing a nanodiamond array according to claim 1, wherein: The preparation method of the PS microsphere array in step (3) includes 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 10~50 sccm, the radio frequency power is 10~50 mW, the vacuum degree is 0.1~0.5mTorr, and the etching time is 5~30 minutes.

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

5. The method for preparing a nanodiamond array according to claim 1, wherein: The cleaning treatment in step eight includes: immersing in a 3% Tween-20 solution for 10 to 30 minutes.

6. A method for ultrasensitive detection of miRNA using a nanodiamond array prepared by the method according to any one of claims 1 to 5, characterized in that: The following steps are involved: Prepare target miRNA-coupled probe magnetic balls and drop them onto the surface of the nanodiamond array for incubation. Then use a scanning system, microwave module and optical system to scan and locate the target miRNA-coupled magnetic balls on the surface of the nanodiamond array and detect signals.

7. The nanodiamond array miRNA ultrasensitive detection method according to claim 6, characterized in that: The microwave module 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.

8. The nanodiamond array miRNA ultrasensitive detection method according to claim 6, wherein Preparation of target miRNA-coupled probe magnetic spheres; comprising the following steps: adding 1-5 nmol of probe DNA solution, 2-10 mg of EDC, and 2-10 mg of NHS to 100-500 μL of a 1 nmol / mL magnetic sphere solution containing surface carboxyl groups, and incubating overnight at room temperature; centrifugally washing the incubated solution with a buffer solution to remove probe DNA that is not firmly connected, thereby obtaining a DNA probe magnetic sphere solution; mixing 10-50 μL of the target miRNA solution with 100-500 μL of the DNA probe magnetic sphere solution, and then incubating the mixture; centrifuging and washing the mixture with a buffer solution to remove unreacted miRNA, thereby obtaining target miRNA-coupled magnetic spheres; wherein the probe DNA is terminally modified with an amino group.

Images