Diamond NV color center-based miRNA multiple detection method

Abstract

The invention discloses a miRNA multiple detection method based on a diamond NV color center, which comprises the following steps: firstly, preparing a plasma nano-diamond: modifying amino on the surface of the nano-diamond containing the NV color center, then coupling silver nano-seeds through an electrostatic adsorption effect, and growing silver nano-particles with different particle sizes in situ; plasma nano-diamonds with different ultraviolet absorption peaks are obtained, and then the surfaces of the nano-diamonds and the plasma nano-diamonds are modified with single-chain DNA probes. The multiple detection of miRNA comprises the following steps: firstly, carrying out amination modification on the surface of a quartz substrate and modifying different DNA capture probes, then dropwise adding target miRNA, a nano-diamond DNA probe and a plasma nano-diamond DNA probe, and finally collecting a fluorescence spectrum and an optical detection magnetic resonance spectrum of nanoparticles on the surface of a sample through a microwave module, an optical path system and an infrared radiation system. According to the fluorescence spectrum shape and the optical detection magnetic resonance spectrum resonance frequency, various miRNAs are identified, and simultaneous and in-situ detection of various cancer markers can be realized.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a method for multiplex detection of miRNA based on diamond NV centers. Background Technology

[0002] Simultaneous quantitative detection of multiple miRNA biomarkers in trace samples is of great significance for efficient early diagnosis, monitoring, and prognosis of cancer. Currently, multiplex biomarker methods based on fluorescence, surface-enhanced Raman scattering, and electrochemiluminescence principles suffer from several shortcomings, including low detection limits, spectral overlap, and poor reproducibility. Therefore, there is an urgent need to explore innovative detection mechanisms to achieve multiplex detection of low-abundance biomarkers. Nitrogen-vacancy centers (NV centers) are luminescent defects in the atomic structure of nanodiamonds, containing a nitrogen atom that has substituted a carbon atom and a hole. Both the ground and excited states in their energy level structure are spin-triple states. At room temperature, nanodiamond NV centers exhibit the strongest absorption in the green light band, with a fluorescence spectrum ranging from 575 to 800 nm. The fluorescence spectrum can be dynamically controlled by designing plasmonic nanostructures. The spin state of the NV center exhibits rapid response characteristics to broadband microwave, magnetic field, and temperature fields, manifesting as changes in fluorescence intensity. Due to the high optical stability of diamond NV centers, they are suitable for multimodal measurement of biomolecules.

[0003] In the prior art, Chinese invention patent CN202411378220.8 discloses a Salmonella fluctuation immunoassay method based on nanodiamond NV centers, which improves the fluorescence intensity of NV centers, but cannot achieve simultaneous quantitative detection of multiple biomolecules, thus having significant limitations in high-throughput and rapid in vitro miRNA detection applications. In the prior art, Chinese invention patent CN202311305432.9 discloses a multi-virus joint detection method and system based on diamond NV centers, which enriches different analytes into different regions by setting two T lines, but it cannot achieve in-situ detection. Furthermore, the test strip preparation material (nitrocellulose) has a high fluorescence background and introduces non-specific adsorption problems, reducing sensitivity. Summary of the Invention

[0004] To address the aforementioned problems, this invention discloses a method for multiplex detection of miRNAs based on diamond NV centers. By dynamically controlling the fluorescence spectral shape of the nitrogen-vacancy centers and their photothermal effect on infrared light by altering the diameter and shape of gold nanoparticles on the surface of the diamond nanoparticles, the method achieves different photodetector magnetic resonance frequencies for the NV centers. Furthermore, by modifying different molecular probes onto the surfaces of different plasma-modified diamond nanoparticles, the simultaneous detection of multiple biomolecules can be realized. To achieve the above objectives, the technical solution of the present invention is as follows: A method for multiplex detection of miRNAs based on diamond NV centers includes: Step 1: Synthesize plasma nanodiamonds; Step 2: Preparation of nanodiamond DNA probes; Step 3: Preparation of plasma nanodiamond DNA probes; Step 4: Prepare a quartz substrate for coupling the target miRNA and DNA probe; Step 5: Using a microwave system, an optical system, and an infrared light source, the target miRNA-coupled nanodiamonds and plasma nanodiamonds on the cover slide surface are scanned and located, and fluorescence spectra and photodetector magnetic resonance spectra are acquired.

[0005] Furthermore, the synthesis of plasma nanodiamonds described in step one is as follows: (1) Add 200~400 µL of 0.5 mg / mL silver nitrate aqueous solution and 1.4~4.2 mg sodium citrate to 10 mL of distilled water, add 120 µL of 0.5 mg / mL sodium borohydride aqueous solution under ice-water bath conditions, and stir vigorously for 5 minutes to obtain silver nanoseeds; (2) Mix 100~300 µL of 1 mg / mL nanodiamond aqueous suspension with a particle size of 100 nm, 100~300 µL of 20 mg / mL EDC, 100~300 µL of 20 mg / mL NHS aqueous solution and 100~300 µL of 10 mg / mL polyethyleneimine aqueous solution and incubate for 4 hours; after centrifugation and washing, redisperse in 500~1500 µL of distilled water to obtain surface-aminated nanodiamonds; (3) Take 50~150 µL of surface-aminated nanodiamonds and 100~300 µL of silver nanoseeds obtained in step (1), mix them and sonicate for 1 hour, then stir and incubate for 3 hours; after centrifugation and washing, redisperse them in 500~1500 µL of distilled water to obtain nanodiamonds with silver nanoseeds attached to the surface. (4) Add 500~1500 µL of nanodiamonds with silver nanoseeds attached to the surface, 0.3~0.9 mg of sodium citrate, and 20~100 µL of 1% tetrachloroauric acid aqueous solution to 9.5 mL of distilled water. Under vigorous stirring, add 40~360 µL of 0.5 mg / mL silver nitrate solution and 100~300 µL of 17.64 mg / mL ascorbic acid solution in sequence. After reacting for 2 minutes, centrifuge and redisperse in 1 mL of distilled water to obtain plasma nanodiamonds of different particle sizes.

[0006] Furthermore, step two involves the preparation of nanodiamond DNA probes; specifically as follows: Mix 100–300 µL of 1 mg / mL aqueous suspension of 100 nm nanodiamond nanoparticles, 100–300 µL of 20 mg / mL EDC, 100–300 µL of 20 mg / mL NHS aqueous solution, and 1–3 nmol of DNA probe and incubate for 8 hours; after centrifugation and washing, redisperse in 100–300 µL of distilled water to obtain the nanodiamond probe; Furthermore, step three involves the preparation of the plasma nanodiamond DNA probe; specifically as follows: 0.2–0.6 nmol of SH-PEG-NHS and 0.2–0.6 nmol of streptavidin (SA) were added to 500–1500 µL of deionized water and incubated at room temperature for 2 h. The mixture was then washed three times with deionized water by centrifugation. Subsequently, plasmonic nanodiamonds were mixed with SH-PEG-SA and reacted with shaking for 4 h. Then, 50–150 µL of 1% BSA solution was added and incubated for 2 h. The resulting mixture was washed three times by centrifugation and redispersed in 1–3 mL of deionized water to obtain streptavidin-coupled plasmonic nanodiamonds. Finally, two DNA probes with molar amounts of 1–3 nmol were added to two centrifuge tubes containing 1 mL of plasmonic nanodiamond solutions with different UV absorption peaks and streptavidin coupling, and reacted for 4 h. After centrifugation, two different plasmonic nanodiamond DNA probes were obtained; the DNA probes were modified with biotin at their ends.

[0007] Furthermore, step four involves preparing a silver nanoisland array that couples the target miRNA and DNA probe; specifically as follows: (a) Immerse the quartz substrate in a mixed solution of concentrated sulfuric acid and hydrogen peroxide (volume ratio 3:1), let it stand for 30 minutes, rinse with deionized water and immerse it in a 10% APTES ethanol solution, and react for 8 hours in the dark; remove the quartz substrate and rinse with deionized water to obtain an aminated quartz substrate. (b) The surface-aminated quartz substrate was immersed in 1 mg / mL NHS-PEG-biotin solution and incubated for 4 hours; after rinsing with deionized water, it was immersed in 0.1 mg / mL streptavidin solution and reacted for 4 hours; after rinsing with deionized water again, streptavidin-modified quartz substrate was obtained. (c) 20-60 µL of a 1 µM DNA probe solution was dropped onto the surface of a streptavidin-modified quartz substrate, incubated for 2 hours, and then washed with distilled water; the DNA probe was modified with biotin at its end. (d) 20-60 µL of nanodiamond DNA probe and plasma nanodiamond probe with a total concentration of 1 µM were added to the sample surface obtained in step (c) and incubated at room temperature for 1 hour. The sample was then washed with PBS buffer and dried to obtain a quartz substrate coupled with the target miRNA and DNA probe.

[0008] Furthermore, step five is detailed below: 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 infrared light source is an 808nm laser. The laser emits 532nm laser light, which is transmitted to the surface of the nanodiamond through the optical path system of the dichroic mirror and the Olympus inverted microscope. Simultaneously, the microwave source transmits 2.82~2.92 GHz microwaves to the microwave antenna to modulate the energy levels of the nitrogen-vacancy color centers inside the nanodiamond and plasma nanodiamond. The nitrogen-vacancy color centers produce fluorescence with a spectral range of 575~800nm ​​under the excitation of the 532nm laser. Because silver nanoparticles have a fluorescence enhancement effect on NV color centers in a specific frequency band, the fluorescence spectral shapes of nanodiamond and plasma nanodiamond are different. Furthermore, because plasma nanodiamonds with different ultraviolet absorption peaks have different absorption intensities to the 808nm infrared light source, their surface temperatures differ, resulting in different resonance frequencies in the photodetector magnetic resonance spectrum. 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 the photodetector magnetic resonance spectrum at a coordinate position. The photodetector magnetic resonance spectrum at different positions of the sample can be obtained by scanning with a piezoelectric displacement stage.

[0009] The beneficial effects of this invention are as follows: (1) The plasma nanodiamond of the present invention has a dynamically regulated surface plasmon resonance effect, which can realize the fluorescence enhancement and spectral shape regulation of the nitrogen vacancy color center of nanodiamond; (2) The plasma nanodiamond of the present invention can adjust the infrared light absorption capacity according to the diameter and shape of the gold particles, thereby realizing the dynamic control of the resonance frequency of the internal nitrogen vacancy color center photodetector magnetic resonance spectrum. (3) Compared with fluorescent or surface-enhanced Raman scattering signal tags, the present invention can accurately identify the background fluorescence generated by biological sample contaminants, thereby improving detection accuracy.

[0010] (4) This invention does not rely on special surface metal structure design and is also applicable to non-rigid and non-standard planar biosensing platforms such as paper-based microfluidics, microneedles, and hydrogels. Attached Figure Description

[0011] Figure 1 This is a flowchart of a miRNA multiplex detection method based on diamond NV centers as described in this invention.

[0012] List of identifiers in attached diagrams: 1. Nanodiamond, 2. Silver nanoparticles, 3. Plasma nanodiamond, 4. Nanodiamond probe, 5. Spherical plasma nanodiamond probe, 6. Star-shaped plasma nanodiamond probe, 7. Surface-aminated quartz sheet, 8. NHS-PEG-Biotin modified quartz sheet, 9. Streptavidin modified quartz sheet, 10. DNA probe modified quartz sheet, 11. Quartz sheet coupled with target miRNA, 12. Quartz sheet coupled with nanodiamond probe and plasma nanodiamond probe, 13. Microwave source, 14. Microwave antenna, 15. 532 nm laser, 16. Dichroic mirror, 17. Single photon counting module, 18. Olympus inverted microscope, 19. 808 nm laser. Detailed Implementation

[0013] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0014] Implementation Case 1: A miRNA multiplex detection method based on diamond NV centers ( Figure 1 This method can detect two miRNAs by fluorescence spectral shape, and includes the following steps: Step 1: Synthesis of plasma nanodiamonds; including: (1) Add 200 µL of 0.5 mg / mL silver nitrate aqueous solution and 2.8 mg sodium citrate to 10 mL of distilled water, add 120 µL of 0.5 mg / mL sodium borohydride aqueous solution under ice-water bath conditions, and stir vigorously for 5 minutes to obtain silver nanoseeds; (2) Mix 100 µL of 1 mg / mL nanodiamond aqueous suspension with a particle size of 100 nm, 100 µL of 20 mg / mL LEDC, 100 µL of 20 mg / mL NHS aqueous solution and 100 µL of 10 mg / mL polyethyleneimine aqueous solution and incubate for 4 hours; after centrifugation and washing, redisperse in 500 µL of distilled water to obtain surface-aminated nanodiamonds; (3) Take 50 µL of surface-aminated nanodiamonds and 100 µL of silver nanoseeds obtained in step (1) and mix them together and sonicate for 1 hour, then stir and incubate for 3 hours; after centrifugation and washing, redisperse in 500 µL of distilled water to obtain nanodiamonds with silver nanoseeds attached to the surface. (4) Add 500 µL of nanodiamonds with silver nanoseeds on the surface and 0.3 mg of sodium citrate to three glass bottles containing 9.5 mL of distilled water. Then add 40 µL, 80 µL and 120 µL of silver nitrate solution with a mass fraction of 0.5 mg / mL to the three glass bottles respectively. Add 300 µL of ascorbic acid solution with a mass fraction of 17.64 mg / mL under vigorous stirring. After reacting for 2 minutes, centrifuge and redisperse in 1 mL of distilled water to obtain plasma nanodiamonds with ultraviolet absorption peaks at ~410 nm, ~480 nm and ~560 nm.

[0015] Step 2: Preparation of nanodiamond DNA probes; including: 100 µL of 1 mg / mL aqueous suspension of 40 nm nanodiamond nanoparticles, 100 µL of 20 mg / mL EDC, 100 µL of 20 mg / mL NHS aqueous solution, and 1 nmol of DNA probe (5'-NH2-CCCCCTAGACACCGTGTACCCCTATCAC-3') were mixed and incubated for 8 hours; after centrifugation and washing, the mixture was redispersed in 100 µL of distilled water to obtain the nanodiamond DNA probe. Step 3: Preparation of plasma-enhanced nanodiamond DNA probes; including: 0.2 nmol of SH-PEG-NHS and 0.2 nmol of streptavidin (SA) were added to a centrifuge tube containing 1000 µL of deionized water and incubated at room temperature for 2 h. The solution was then washed three times with deionized water to obtain the SH-PEG-SA solution. Subsequently, the plasmonic nanodiamonds with a UV absorption peak of 480 nm prepared in step one were mixed with the SH-PEG-SA and reacted with shaking for 4 h. Then, 100 µL of 1% BSA solution was added and incubated for 2 h. The resulting mixture was washed three times with centrifugation and redispersed in 2 mL of deionized water to obtain streptavidin-conjugated plasmonic nanodiamonds. Finally, 1 nmol of a DNA probe (5'-biotin-CCCCCTAGACACCGTGTTCAACATCAGT-3') was added to a centrifuge tube containing 1 mL of the streptavidin-conjugated plasmonic nanodiamond solution and reacted for 4 h. After centrifugation, the plasmonic nanodiamond DNA probe was obtained. The DNA probe was modified with biotin at its end.

[0016] Step 4: Prepare a quartz substrate for coupling the target miRNA and DNA probe; including: (a) Immerse the quartz substrate in a mixed solution of concentrated sulfuric acid and hydrogen peroxide (volume ratio 3:1), let it stand for 30 minutes, rinse with deionized water and immerse it in a 10% APTES ethanol solution, and react for 8 hours in the dark; remove the quartz substrate and rinse with deionized water to obtain an aminated quartz substrate. (b) The surface-aminated quartz substrate was immersed in 1 mg / mL NHS-PEG-biotin solution and incubated for 4 hours; after rinsing with deionized water, it was immersed in 0.1 mg / mL streptavidin solution and reacted for 4 hours; after rinsing with deionized water again, streptavidin-modified quartz substrate was obtained. (c) 20 µL of 1 µM DNA probe solution (5'-CTGATAAGCTACCCCC-SH-3', 5'-GATTAGCATTAACCCCC-SH-3', and 5'-GCTAGTGCCAAACCCCC-SH-3') was added dropwise to the surface of a streptavidin-modified quartz substrate, incubated for 2 hours, and then washed with distilled water; the DNA probes were modified with biotin at their ends. (d) 20 µL of nanodiamond DNA probe and plasma nanodiamond probe with a total concentration of 1 µM and 20 µL of miRNA (miRNA-21, miRNA-155 and miRNA-96 are all 1 fM) were added to the sample surface obtained in step (c) and incubated at room temperature for 1 hour. The sample was then washed with PBS buffer and dried to obtain a quartz substrate coupled with the target miRNA and DNA probe.

[0017] Step 5: Using a microwave system, an optical system, and an infrared light source, the target miRNA-coupled nanodiamonds and plasma nanodiamonds on the cover slide surface are scanned and located, and fluorescence spectra and photodetector magnetic resonance spectra are acquired.

[0018] The microwave system includes a microwave generator 13 and a microwave antenna 14, and the optical path system includes a laser 15, a single-photon counter 17, an Olympus inverted microscope 18, and a dichroic mirror 16. The infrared light source is an 808nm laser. Figure 1As shown, a 532nm laser emits light, which is transmitted to the surface of the nanodiamond through the optical path system of the dichroic mirror 16 and the Olympus inverted microscope 18. Simultaneously, a microwave source 13 transmits 2.82~2.92 GHz microwaves to a microwave antenna to modulate the energy levels of the NV color centers inside the nanodiamond 1 and the plasma nanodiamond 3. The NV color centers produce fluorescence with a spectral range of 575~800nm ​​under 532nm laser excitation. Due to the fluorescence enhancement effect of silver nanoparticles on the NV color centers in a specific frequency band, the fluorescence spectral shapes of the nanodiamond and the plasma nanodiamond are different. Furthermore, because the plasma nanodiamond 3 with different ultraviolet absorption peaks has different absorption intensities to the 808nm infrared light source, there are differences in surface temperature, resulting in different resonance frequencies in the photodetector magnetic resonance spectrum. The photon signal is transmitted by the Olympus inverted microscope optical path system and the dichroic mirror to the single-photon counting module to obtain the photodetector magnetic resonance spectrum at one coordinate position. The photodetector magnetic resonance spectrum at different positions of the sample can be obtained by scanning with a piezoelectric displacement stage.

[0019] Since nanodiamonds and plasmonic nanodiamonds with ultraviolet absorption peaks at ~480 nm have similar photothermal effects under infrared lasers, the simultaneous detection of two miRNAs can only be achieved through fluorescence spectral shapes.

[0020] Implementation Case 2: A miRNA multiplex detection method based on diamond NV centers ( Figure 1 This method can detect two miRNAs using any fluorescence-ODMR mode, and includes the following steps: Step 1: Synthesis of plasma nanodiamonds; including: (1) Add 400 µL of 0.5 mg / mL silver nitrate aqueous solution and 2.8 mg sodium citrate to 10 mL of distilled water, add 120 µL of 0.5 mg / mL sodium borohydride aqueous solution under ice-water bath conditions, and stir vigorously for 5 minutes to obtain silver nanoseeds; (2) Mix 200 µL of 1 mg / mL nanodiamond aqueous suspension with a particle size of 100 nm, 200 µL of 20 mg / mL LEDC, 200 µL of 20 mg / mL NHS aqueous solution and 200 µL of 10 mg / mL polyethyleneimine aqueous solution and incubate for 4 hours; after centrifugation and washing, redisperse in 1000 µL of distilled water to obtain surface-aminated nanodiamonds; (3) Take 100 µL of surface-aminated nanodiamonds and 200 µL of silver nanoseeds obtained in step (1), mix them and sonicate for 1 hour, then stir and incubate for 3 hours; after centrifugation and washing, redisperse in 1000 µL of distilled water to obtain nanodiamonds with silver nanoseeds attached to the surface. (4) Add 1000 µL of nanodiamonds with silver nanoseeds on the surface and 0.3 mg of sodium citrate to three glass bottles containing 9.5 mL of distilled water. Then add 80 µL, 160 µL and 240 µL of silver nitrate solution with a mass fraction of 0.5 mg / mL to the three glass bottles respectively. Add 300 µL of ascorbic acid solution with a mass fraction of 17.64 mg / mL under vigorous stirring. After reacting for 2 minutes, centrifuge and redisperse in 1 mL of distilled water to obtain plasma nanodiamonds with ultraviolet absorption peaks at ~410 nm, ~480 nm and ~560 nm.

[0021] Step 2: Preparation of nanodiamond DNA probes; including: 200 µL of 1 mg / mL aqueous suspension of 100 nm nanodiamond nanoparticles, 200 µL of 20 mg / mL EDC, 200 µL of 20 mg / mL NHS aqueous solution, and 2 nmol of DNA probe (5'-NH2-CCCCCTAGACACCGTGTACCCCTATCAC-3') were mixed and incubated for 8 hours; after centrifugation and washing, the mixture was redispersed in 200 µL of distilled water to obtain the nanodiamond DNA probe. Step 3: Preparation of plasma-enhanced nanodiamond DNA probes; including: 0.3 nmol of SH-PEG-NHS and 0.3 nmol of streptavidin (SA) were added to a centrifuge tube containing 1000 µL of deionized water and incubated at room temperature for 2 h. The solution was then washed three times with deionized water to obtain the SH-PEG-SA solution. Subsequently, the plasmonic nanodiamonds with a UV absorption peak of ~560 nm prepared in step one were mixed with the SH-PEG-SA and reacted with shaking for 4 h. Then, 100 µL of 1% BSA solution was added and incubated for 2 h. The resulting mixture was washed three times with centrifugation and redispersed in 2 mL of deionized water to obtain streptavidin-conjugated plasmonic nanodiamonds. Finally, 3 nmol of a DNA probe (5'-biotin-CCCCCTAGACACCGTGTTCAACATCAGT-3') was added to 1 mL of the streptavidin-conjugated plasmonic nanodiamond solution and reacted for 4 h. After centrifugation, the plasmonic nanodiamond DNA probe was obtained. The DNA probe was modified with biotin at its end.

[0022] Step 4: Prepare a quartz substrate for coupling the target miRNA and DNA probe; including: (a) Immerse the quartz substrate in a mixed solution of concentrated sulfuric acid and hydrogen peroxide (volume ratio 3:1), let it stand for 30 minutes, rinse with deionized water and immerse it in a 10% APTES ethanol solution, and react for 8 hours in the dark; remove the quartz substrate and rinse with deionized water to obtain an aminated quartz substrate. (b) The surface-aminated quartz substrate was immersed in 1 mg / mL NHS-PEG-biotin solution and incubated for 4 hours; after rinsing with deionized water, it was immersed in 0.1 mg / mL streptavidin solution and reacted for 4 hours; after rinsing with deionized water again, streptavidin-modified quartz substrate was obtained. (c) 40 µL of 1 µM DNA probe solution was added to streptavidin-modified quartz substrate, incubated for 2 hours, washed with distilled water, and 20 µL of 1% bovine serum albumin was added to block the surface of silver nanoparticles.

[0023] (d) 40 µL of nanodiamond DNA probe and plasma nanodiamond probe (5'-CTGATAAGCTACCCCC-SH-3', 5'-GATTAGCATTAACCCCC-SH-3') and 20 µL of miRNA (miRNA-21 and miRNA-155 both at 0.1 pM) were added to the sample surface obtained in step (c) and incubated at room temperature for 1 hour. The sample was then washed with PBS buffer and dried to obtain a quartz substrate conjugated with the target miRNA and DNA probe.

[0024] Step 5: Using a microwave system, an optical system, and an infrared light source, the target miRNA on the coverslip surface is scanned and located using nanodiamond-coupled nanodiamond probes and plasma nanodiamond probes, and fluorescence and photodetector magnetic resonance spectra are acquired. The nanodiamond probes and the plasma nanodiamond probes with UV absorption peaks at ~560 nm can be distinguished based on their fluorescence spectral shapes. Furthermore, the plasma nanodiamond probes exhibit a higher photothermal effect under infrared laser light, generating a smaller ODMR resonance frequency than the nanodiamond probes. Therefore, they can also be combined with ODMR spectroscopy to achieve dual-mode detection of two miRNAs.

[0025] Implementation Case 3: A miRNA multiplex detection method based on diamond NV centers ( Figure 1 This method, combined with fluorescence and ODMR, can simultaneously detect three miRNAs. The steps include: Step 1: Synthesis of plasma nanodiamonds; including: (1) Add 400 µL of 0.5 mg / mL silver nitrate aqueous solution and 2.8 mg sodium citrate to 10 mL of distilled water, add 120 µL of 0.5 mg / mL sodium borohydride aqueous solution under ice-water bath conditions, and stir vigorously for 5 minutes to obtain silver nanoseeds; (2) Mix 300 µL of 1 mg / mL nanodiamond aqueous suspension with a particle size of 100 nm, 300 µL of 20 mg / mL LEDC, 300 µL of 20 mg / mL NHS aqueous solution and 300 µL of 10 mg / mL polyethyleneimine aqueous solution and incubate for 4 hours; after centrifugation and washing, redisperse in 1500 µL of distilled water to obtain surface-aminated nanodiamonds; (3) Take 150 µL of surface-aminated nanodiamonds and 300 µL of silver nanoseeds obtained in step (1), mix them and sonicate for 1 hour, then stir and incubate for 3 hours; after centrifugation and washing, redisperse in 1500 µL of distilled water to obtain nanodiamonds with silver nanoseeds attached to the surface. (4) Add 1500 µL of nanodiamonds with silver nanoseeds on the surface and 0.3 mg of sodium citrate to three glass bottles containing 9.5 mL of distilled water. Then add 120 µL, 240 µL and 360 µL of silver nitrate solution with a mass fraction of 0.5 mg / mL to the three glass bottles respectively. Add 300 µL of ascorbic acid solution with a mass fraction of 17.64 mg / mL under vigorous stirring. After reacting for 2 minutes, centrifuge and redisperse in 1 mL of distilled water to obtain plasma nanodiamonds with ultraviolet absorption peaks at ~410 nm, ~480 nm and ~560 nm.

[0026] Step 2: Preparation of nanodiamond DNA probes; including: 300 µL of 1 mg / mL aqueous suspension of 100 nm nanodiamond nanoparticles, 300 µL of 20 mg / mL EDC, 300 µL of 20 mg / mL NHS aqueous solution, and 3 nmol of DNA probe (5'-NH2-CCCCCTAGACACCGTGTACCCCTATCAC-3') were mixed and incubated for 8 hours; after centrifugation and washing, the mixture was redispersed in 300 µL of distilled water to obtain the nanodiamond DNA probe. Step 3: Preparation of plasma-enhanced nanodiamond DNA probes; including: 0.6 nmol of SH-PEG-NHS and 0.6 nmol of streptavidin (SA) were added to two centrifuge tubes containing 1000 µL of deionized water and incubated at room temperature for 2 h. The solutions were then washed three times with deionized water to obtain the SH-PEG-SA solution. Subsequently, plasmonic nanodiamonds with UV absorption peaks at ~410 nm and ~560 nm were mixed with SH-PEG-SA, respectively, and the mixture was shaken for 4 h. Then, 150 µL of 1% BSA solution was added, and the mixture was incubated for 2 h. The resulting mixed solutions were washed three times by centrifugation and redispersed in 3 mL of deionized water to obtain plasmonic nanodiamonds with two streptavidin-coupled formulations. Finally, 3 nmol DNA probes (5'-biotin-CCCCCTAGACACCGTGTTCAACATCAGT-3' and 5'-biotin-CCCCCTAGACACCGTGTAGCAAAAATGT-3') were added to two centrifuge tubes containing 1 mL of streptavidin-conjugated plasmonic nanodiamond solution, respectively, and reacted for 4 h. After centrifugation, two types of plasmonic nanodiamond DNA probes were obtained. The DNA probes were modified with biotin at their ends.

[0027] Step 4: Prepare a quartz substrate for coupling the target miRNA and DNA probe; including: (a) Immerse the quartz substrate in a mixed solution of concentrated sulfuric acid and hydrogen peroxide (volume ratio 3:1), let it stand for 30 minutes, rinse with deionized water and immerse it in a 10% APTES ethanol solution, and react for 8 hours in the dark; remove the quartz substrate and rinse with deionized water to obtain an aminated quartz substrate. (b) The surface-aminated quartz substrate was immersed in 1 mg / mL NHS-PEG-biotin solution and incubated for 4 hours; after rinsing with deionized water, it was immersed in 0.1 mg / mL streptavidin solution and reacted for 4 hours; after rinsing with deionized water again, streptavidin-modified quartz substrate was obtained. (c) 60 µL of 1 µM DNA probe solution (5'-CTGATAAGCTACCCCC-SH-3', 5'-GATTAGCATTAACCCCC-SH-3' and 5'-GCTAGTGCCAAACCCCC-SH-3') was added to the silver nano island array. After incubation for 2 hours, the array was washed with distilled water and 30 µL of 1% bovine serum albumin was added to block the surface of the silver nanoparticles.

[0028] (d) 60 µL of nanodiamond DNA probe and plasma nanodiamond probe with a total concentration of 1 µM and 20 µL of miRNA (miRNA-21, miRNA-155 and miRNA-96 are all 0.01 nmol / L) were added to the sample surface obtained in step (c) and incubated at room temperature for 1 hour. The sample was then washed with PBS buffer and dried to obtain a quartz substrate coupled with the target miRNA and DNA probe.

[0029] Step 5: Using a microwave system, optical system, and infrared light source, the target miRNA coupled with nanodiamonds and plasma nanodiamonds on the surface of the quartz substrate is scanned and located, and fluorescence and photodetector magnetic resonance spectra are acquired. The nanodiamond probes and the two types of plasma nanodiamond probes can be distinguished based on the shape of their fluorescence spectra. Furthermore, the two types of plasma nanodiamonds with different ultraviolet absorption peaks exhibit different photothermal effects under infrared laser light, producing different ODMR resonance frequencies. This allows for the simultaneous detection of three miRNAs.

[0030] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. A method for multiplex detection of miRNAs based on diamond NV centers, characterized in that: include: Step 1: Synthesize plasma nanodiamonds; Step 2: Prepare nanodiamond DNA probes; Step 3: Preparation of plasma nanodiamond DNA probes; Step 4: Prepare a quartz substrate for coupling the target miRNA and DNA probe; Step 5: Use a microwave system, an optical system, and an infrared light source to scan and locate the target miRNA-coupled nanodiamonds and plasma nanodiamonds on the surface of the quartz substrate, and collect fluorescence spectra and photodetector magnetic resonance spectra.

2. The miRNA multiplex detection method based on diamond NV centers according to claim 1, characterized in that: The synthesis of plasma nanodiamonds described in step one is as follows: (1) Add 200~400µL of 0.5mg / mL silver nitrate aqueous solution and 1.4~4.2 mg sodium citrate to 10 mL of distilled water, add 120 µL of 0.5 mg / mL sodium borohydride aqueous solution under ice-water bath conditions, and stir vigorously for 5 minutes to obtain silver nanoseeds; (2) Mix 100~300 µL of 1 mg / mL nanodiamond aqueous suspension with a particle size of 100 nm, 100~300 µL of 20 mg / mL LEDC, 100~300 µL of 20 mg / mL NHS aqueous solution and 100~300 µL of 10 mg / mL polyethyleneimine aqueous solution and incubate for 4 hours; after centrifugation and washing, redisperse in 500~1500 µL of distilled water to obtain surface-aminated nanodiamonds; (3) Take 50~150 µL of surface-aminated nanodiamond and 100~300 µL of silver nanoseeds obtained in step (1), mix them and sonicate for 1 hour, then stir and incubate for 3 hours; After centrifugation and washing, the nanodiamonds with silver nanoseeds attached to their surface were redispersed in 500-1500 µL of distilled water. (4) Add 500~1500 µL of nanodiamonds with silver nanoseeds attached to the surface, 0.3~0.9 mg of sodium citrate, and 20~100 µL of silver nitrate aqueous solution with a concentration of 0.5 mg / mL to 9.5 mL of distilled water. Add 100~300 µL of ascorbic acid solution with a mass fraction of 17.64 mg / mL under vigorous stirring. After reacting for 2 minutes, centrifuge and redisperse in 1 mL of distilled water to obtain plasma nanodiamonds with ultraviolet absorption peaks at 410~560 nm.

3. The method for multiplex detection of miRNA based on diamond NV centers according to claim 1, characterized in that: Step two describes the preparation of nanodiamond DNA probes; the details are as follows: Mix 100–300 µL of 1 mg / mL aqueous suspension of 100 nm nanodiamond nanoparticles, 100–300 µL of 20 mg / mL LEDC, 100–300 µL of 20 mg / mL NHS aqueous solution, and 1–3 nmol of DNA probe and incubate for 8 hours; after centrifugation and washing, redisperse in 100–300 µL of distilled water to obtain the nanodiamond probe.

4. The method for multiplex detection of miRNA based on diamond NV centers according to claim 1, characterized in that: Step three describes the preparation of the plasma nanodiamond DNA probe; the details are as follows: 0.2–0.6 nmol of SH-PEG-NHS and 0.2–0.6 nmol of streptavidin SA were added to 500–1500 µL of deionized water and incubated at room temperature for 2 h. The mixture was then washed three times by centrifugation with deionized water. Subsequently, plasmonic nanodiamonds were mixed with SH-PEG-SA and reacted with shaking for 4 h. Then, 50–150 µL of 1% BSA solution was added and incubated for 2 h. The resulting mixed solution was washed three times by centrifugation and redispersed in 1–3 mL of deionized water to obtain plasmonic nanodiamonds coupled with streptavidin. Finally, two DNA probes with a molar amount of 1–3 nmol were added to two centrifuge tubes containing 1 mL of plasmonic nanodiamond solutions with different UV absorption peaks and coupled with streptavidin and reacted for 4 h. After centrifugation, two different plasmonic nanodiamond DNA probes were obtained. The DNA probes were modified with biotin at their ends.

5. The method for multiplex detection of miRNA based on diamond NV centers according to claim 1, characterized in that: Step four describes the preparation of the quartz substrate for coupling the target miRNA and DNA probe; the details are as follows: (a) Immerse the quartz substrate in a mixed solution of concentrated sulfuric acid and hydrogen peroxide, let it stand for 30 minutes, rinse it with deionized water, and then immerse it in a 10% APTES ethanol solution. React for 8 hours in the dark. Remove the quartz substrate and rinse it with deionized water to obtain an aminated quartz substrate. (b) The surface-aminated quartz substrate was immersed in 1 mg / mL NHS-PEG-biotin solution and incubated for 4 hours; after rinsing with deionized water, it was immersed in 0.1 mg / mL streptavidin solution and reacted for 4 hours; after rinsing with deionized water again, streptavidin-modified quartz substrate was obtained. (c) 20-60 µL of a 1 µM DNA probe solution was dropped onto the surface of a streptavidin-modified quartz substrate, incubated for 2 hours, and then washed with distilled water; the DNA probe was modified with biotin at its end. (d) 20-60 µL of nanodiamond DNA probe and plasma nanodiamond probe with a total concentration of 1 µM were added to the sample surface obtained in step (c) and incubated at room temperature for 1 hour. The sample was then washed with PBS buffer and dried to obtain a quartz substrate coupled with the target miRNA and DNA probe.

6. The miRNA multiplex detection method based on diamond NV centers according to claim 1, characterized in that: Step five is as follows: 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 infrared light source is an 808nm laser, which emits 532nm laser light. This light is transmitted to the surface of the nanodiamond array via the optical path system of the dichroic mirror and the Olympus inverted microscope. Simultaneously, the microwave source transmits light at a wavelength of 2.82~2.92nm. GHz microwaves are transmitted to a microwave antenna to modulate the energy levels of NV centers of nanodiamonds and plasma nanodiamonds. The NV centers produce fluorescence in the spectral range of 575~800nm ​​under 532nm laser excitation. Due to the plasma resonance effect of silver nanoparticles and their inherent luminescence properties near 580nm, the spectral shapes of plasma nanodiamonds and nanodiamonds at 580nm are different. Furthermore, due to the different absorption intensities of plasma nanodiamonds with different ultraviolet absorption peaks to the 808nm infrared light source, their surface temperatures differ, resulting in different resonance frequencies in the photodetector magnetic resonance spectrum. The photon signal is transmitted to the single-photon counting module by the Olympus inverted microscope optical path system and dichroic mirror to obtain the photodetector magnetic resonance spectrum at a coordinate position. The photodetector magnetic resonance spectrum at different positions of the sample is obtained by scanning with a piezoelectric displacement stage.

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