MiRNA quantum detection method based on plasmon enhancement

Abstract

The invention discloses a miRNA (micro Ribonucleic Acid) quantum detection method based on plasmon enhancement. The method comprises the following steps: modifying gold nanocubes on the surface of a fluorescent nano-diamond to generate the nano-diamond with a local surface plasmon resonance enhancement effect; the preparation method comprises the following steps: preparing a plasmon enhanced nano-diamond probe and a silicon dioxide microsphere probe, and hybridizing target miRNA to form a sandwich structure compound; injecting the compound into a micro-fluidic chip with a micro-column array structure, so as to entrap and enrich the compound with the sandwich structure, and filtering to remove the uncombined plasmon enhanced nano-diamond probe; the compound is subjected to fluorescence signal detection, and quantum signal detection is combined, so that qualitative or quantitative analysis of the target miRNA is realized. The background of the free probe is reduced through microfluidic filtration, the detection repeatability is improved, the nanodiamond reading signal intensity is improved by combining plasmon enhancement, and the method has the advantages of being high in sensitivity, high in specificity, convenient to operate, suitable for trace sample detection and the like.

Description

Technical Field

[0001] This invention belongs to the field of biological detection, specifically relating to a quantum detection method for miRNA based on plasmon enhancement. Background Technology

[0002] miRNAs, as endogenous small non-coding RNAs, are closely related to many major diseases, including cancer, cardiovascular disease, and immune disorders. However, the wide concentration range and complex matrix of miRNAs in clinical samples often lead to strong background interference, limited linear intervals, and insufficient reproducibility. Existing miRNA detection methods include electrophoresis, hybridization, PCR, and sequencing, but molecular amplification methods often rely on specialized equipment and complex procedures, which are not conducive to rapid and on-site detection. Conventional fluorescent dyes also suffer from problems such as photobleaching and signal attenuation due to repeated excitation, affecting the reliability of long-term and repeated measurements.

[0003] Fluorescent nanodiamond (NV) centers exhibit superior photostability, withstanding prolonged, high-power laser irradiation without photobleaching. As a luminescent defect structure, the fluorescence of an NV center can be modulated by microwaves, manifesting as changes in fluorescence intensity. This allows for the acquisition of optically detected magnetic resonance (ODMR) signals, making NV centers a high-performance quantum sensor capable of detecting physical quantities such as magnetic fields, electric fields, and temperature. However, the relatively limited emissivity of a single NV center restricts the detection limit based on fluorescence intensity readout.

[0004] A major challenge in high-sensitivity detection is how to effectively extract extremely low concentrations of specific signals from high background noise in complex biological sample environments. Existing methods typically rely on repeated washing or removal of unconnected nanodiamonds on solid substrates to achieve purification, but these methods may still introduce non-specific adsorption and residual background in complex systems or trace samples. Summary of the Invention

[0005] To address the aforementioned issues, this invention discloses a miRNA quantum detection method based on plasmon enhancement. This method reduces the background of free probes and improves detection repeatability through microfluidic filtration, and enhances the readout signal intensity of nanodiamonds by combining plasmon enhancement. It has advantages such as high sensitivity, strong specificity, convenient operation, and suitability for the detection of trace samples.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A plasmon-enhanced miRNA quantum detection method, comprising: Step 1: Modify the surface of fluorescent nanodiamond (FND) with gold nanocubes (AuNC) to obtain nanodiamonds (FND@AuNC) with localized surface plasmon resonance enhancement effect; specifically including: (1) Place a beaker containing 8 mL of distilled water in a magnetically stirred water bath at 25-32°C, add a magnetic stir bar, 364.44 mg of cetyltrimethylammonium bromide (CTAB), and 2 mL of 1.25 mM chloroauric acid aqueous solution in sequence. After stirring evenly, quickly add 600 µL of 10 mM cold sodium borohydride aqueous solution and stir for 3 min to obtain 2 nm gold nanoclusters. Then let it stand at 25-32°C for 3 h.

[0007] (2) Place a beaker containing 2 mL of distilled water in a magnetically stirred water bath at 25-32℃, and add the magnetic stir bar, 128 mg of hexadecyltrimethylammonium chloride (CTAC), 2 mL of 0.5 mM chloroauric acid aqueous solution, and 1.5 mL of 0.1 mM ascorbic acid aqueous solution in sequence, and stir until homogeneous; take 5 µL of the 2 nm gold nanoclusters prepared in step (1) and add them to the beaker all at once in 2 mL of distilled water, stir for 15 min to obtain gold nanoseeds. Centrifuge twice, first disperse in 1 mL of distilled water, and then redisperse in 1 mL of 20 mM CTAC aqueous solution.

[0008] (3) Place a beaker containing 2 mL of distilled water in a magnetically stirred water bath at 25-32°C, and add magnetic particles, 64 mg CTAC, 10 µL of 20-120 mM sodium bromide aqueous solution, 130 µL of 10 mM ascorbic acid aqueous solution, and 9-300 µL of the 10 nm gold nanoseeds prepared in step (2) in sequence. Stir until homogeneous. Add 2 mL of 0.5 mM chloroauric acid aqueous solution and stir for 25 min to obtain gold nanocubes. After centrifugation and washing, disperse in 1 mL of distilled water.

[0009] (4) Take 150µL of 10mM mercaptoethylamine hydrochloride aqueous solution and add it to the gold nanocube aqueous solution. Mix and incubate for 4h. Centrifuge, wash and redisperse in 1mL aqueous solution to obtain aminated gold nanocubes. (5) 2 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDS), 2 mg of N-hydroxysuccinimide (NHS), and 100 µL of 1 mg / mL nanodiamond aqueous solution with a particle size of 100~200 nm were added sequentially to 1 mL of distilled water and incubated on a shaker for 4 h; after centrifugation and washing, the solution was redispersed in 1 mL of distilled water.

[0010] (6) Take 1 mL of aminated gold nanocube from (4) and 1 mL of surface carboxyl activated nanodiamond obtained in step (5), mix them, and incubate them on a shaker for 2 hours; after centrifugation and washing, redisperse them in 1 mL of distilled water to obtain plasmon-enhanced nanodiamond.

[0011] The fluorescent nanodiamonds have an average particle size of approximately 100-200 nm, and the gold nanocubes have a characteristic size of approximately 20-70 nm.

[0012] Step 2: Preparation of plasmon-enhanced nanodiamond probes 1 nmol of a nucleic acid probe with thiol-terminated ends was added to 1 mL of plasmon-enhanced nanodiamond solution and reacted for 4 h. After centrifugation and washing, the plasmon-enhanced nanodiamond probe was obtained.

[0013] Step 3: Preparation of silica microsphere probes 1 nmol of a biotin-terminated nucleic acid probe was added to 1 mL of a 30 mg / L solution of streptavidin-modified silica microspheres (2–50 µm) and reacted for 4 h. After centrifugation and washing, the silica microsphere probe was obtained.

[0014] Step 4: Fabrication of Micropillar Array Microfluidic Chips (1) Take an appropriate amount of SU-82075 photoresist and spin coat it onto a 4-inch polished silicon wafer. Set the spin coater parameters: low speed 9s 600r / s (speed), high speed 30s 4000r / s. After spin coating, remove the residual photoresist on the back of the polished silicon wafer and heat it at 95℃ for 10min. After the silicon wafer is exposed by a UV mask, place it on a 95℃ heating stage and heat it for 10min. After cooling, immerse it in the developer for 5min and finally rinse it with alcohol.

[0015] (2) Weigh 20g of PDMS adhesive and 2g of curing agent, mix and stir for 10min, pour the mixture into a petri dish containing a silicon wafer mold, and then put the petri dish into a vacuum box to evacuate until there are no bubbles in the mixture; heat at 65℃ for 2h to cure, then peel the PDMS layer off the silicon wafer mold to obtain the microfluidic chip layer.

[0016] (3) The cleaned microfluidic chip layer and glass slide were placed in an oxygen plasmon refining machine and treated with 80W power for 6 minutes to activate the surface; after removal, the microfluidic chip and glass slide were quickly bonded together and dried at 120℃ for 4 hours to obtain a micropillar array microfluidic chip. The microfluidic chip includes an inlet / outlet channel, a filtration enrichment region, and a detection region. The filtration enrichment region is equipped with a micropillar array filtration structure for enriching the microsphere carrier and filtering out unbound plasmon-enhanced nanodiamond probes. The detection region is used to detect fluorescence and quantum signals of the microsphere carrier.

[0017] The micropillar array filter structure of the microfluidic chip is a circumferentially distributed micropillar structure with an adjacent micropillar spacing of approximately 1~30 µm. The micropillar spacing is set to be smaller than the size of the microsphere carrier and much larger than the size of the plasmon-enhanced nanodiamond, so as to achieve the enrichment and filtration removal of unbound plasmon-enhanced nanodiamond probes by the microsphere carrier.

[0018] The flow test was performed by injecting dye solution to verify the sealing, waterproofing and structural integrity of the microchannels. All channels were rinsed sequentially with anhydrous ethanol and phosphate buffered saline (PBS). Finally, 1% bovine serum albumin (BSA) was introduced for 30 min to block the flow, followed by rinsing with PBS. The microchannels were then ready for use.

[0019] Step 5: Detection process of miRNA on the chip Using a syringe pump, continuously inject 1X phosphate buffer into the chip prepared in step four at a constant flow rate of 0.1 mL / min, and rinse for 10 minutes to thoroughly remove impurities from the chip.

[0020] Plasmon-enhanced nanodiamond probes, silica microsphere probes, and miRNA were thoroughly mixed to form a sandwich-structured complex of “SiO2-probe2-miRNA-probe1-FND@AuNC” with silica microspheres as the carrier. The complex was injected into the microfluidic chip at a constant flow rate of 1µL / min using a syringe pump. The sample supply was stopped when the detection area was observed to be filled with silica microspheres under an optical microscope.

[0021] Using a syringe pump, 1X phosphate buffer was continuously injected into the chip at a constant flow rate of 1µL / min, followed by rinsing for 10 minutes to further remove unbound target miRNA and free plasma nanodiamonds from the detection area.

[0022] Step 6: Using quantum detection equipment, combined with quantum signal detection, the detection area in the microcolumn array microfluidic is scanned and located, fluorescence signal is detected, and fluorescence spectrum and photodetector magnetic resonance spectrum are collected to achieve qualitative or quantitative detection of target miRNA.

[0023] The fluorescence signal is the photoluminescence intensity of the nanodiamond NV color center under 532nm laser excitation; the quantum signal detection is a photodetector magnetic resonance signal detection based on the NV color center, and includes applying microwave excitation and a 532nm laser and collecting fluorescence changes under microwave modulation to extract the quantum readout signal.

[0024] The beneficial effects of this invention are as follows: (1) Local surface plasmon resonance effect is introduced by modifying the surface of fluorescent nanodiamonds with gold nanocubes. When the size, morphology and incident light frequency of the gold nanocubes are matched, the free electrons on their surface will oscillate collectively, resulting in strong absorption and scattering of light, and generating a significant local electromagnetic field enhancement around the particles, which can significantly enhance the contrast of fluorescence and quantum signals of the nearby nanodiamonds.

[0025] (2) The microfluidic chip with micropillar array structure designed in this invention can precisely control the fluid in the micrometer-scale flow channel, and can highly integrate multiple steps such as sample reaction, filtration and enrichment into a small chip, which has the advantages of low reagent consumption, low background noise and high degree of automation. Attached Figure Description

[0026] Figure 1 This is a flowchart of a plasmon-enhanced miRNA quantum detection method according to the present invention.

[0027] List of identifiers in attached diagrams: 1. Aminated gold nanocubes, 2. Plasmon-enhanced nanodiamonds, 3. Plasmon-enhanced nanodiamond probes, 4. miRNA, 5. Silica microspheres, 6. Silica microsphere probes, 7. Sandwich-structured complex with silica microspheres as carriers, 8. Microfluidic chip, 9. On-chip detection area, 10. On-chip micropillar filtration enrichment area. Detailed Implementation

[0028] 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.

[0029] Example 1: A quantum detection method for miRNA based on plasmon enhancement ( Figure 1 ), including the following steps: Step 1: Preparation of plasmonic-enhanced nanodiamonds, including: (1) Place a beaker containing 8 mL of distilled water in a magnetically stirred water bath at 28 °C, add a magnetic stir bar, 364.44 mg CTAB, and 2 mL of 1.25 mM chloroauric acid aqueous solution in sequence, stir well, and then quickly add 600 µL of 10 mM cold sodium borohydride aqueous solution. Stir for 3 min to obtain 2 nm gold nanoclusters. Then let stand at 28 °C for 3 h.

[0030] (2) Place a beaker containing 2 mL of distilled water in a magnetically stirred water bath at 28 °C, and add the magnetic stir bar, 128 mg CTAC, 2 mL of 0.5 mM chloroauric acid aqueous solution, and 1.5 mL of 0.1 mM ascorbic acid aqueous solution in sequence, and stir evenly; take 5 µL of the 2 nm gold nanoclusters prepared in step (1) into 2 mL of distilled water, add it to the beaker all at once, stir for 15 min, and obtain 10 nm gold nanoseeds. Centrifuge twice at 20600 g (centrifugal force) for 30 min, first disperse in 1 mL of distilled water, and then redisperse in 1 mL of 20 mM CTAC aqueous solution.

[0031] (3) Place a beaker containing 2 mL of distilled water in a magnetically stirred water bath at 28 °C, and add magnetic particles, 64 mg CTAC, 10 µL of 120 mM sodium bromide aqueous solution, 130 µL of 10 mM ascorbic acid aqueous solution, and 300 µL of the 10 nm gold nanoseeds prepared in step (2) in sequence, and stir evenly; add 2 mL of 0.5 mM chloroauric acid aqueous solution, stir for 25 min to obtain gold nanocubes; after centrifugation and washing, disperse in 1 mL of distilled water.

[0032] (4) Take 150µL of 10mM cysteine ​​hydrochloride aqueous solution and add it to the gold nanocube aqueous solution. Mix and incubate for 4h. Centrifuge, wash and redisperse in 1mL aqueous solution to obtain aminated gold nanocubes. (5) 2 mg EDS, 2 mg NHS, and 100 µL of 1 mg / mL nanodiamond aqueous solution with a particle size of 100 nm were added sequentially to 1 mL of water and incubated on a shaker for 4 h. After centrifugation and washing, the solution was redispersed in 1 mL of distilled water.

[0033] (6) Take 1 mL of aminated gold nanocube from step (4) and 1 mL of surface carboxyl-activated nanodiamond obtained in step (5), mix them, and incubate them on a shaker for 2 hours; after centrifugation and washing, redisperse them in 1 mL of distilled water to obtain plasmon-enhanced nanodiamond. Step 2: Preparation of plasmon-enhanced nanodiamond probes 1 nmol of a probe (5'-SH C6-CCCCCTAGACACCGTGTTCAACATCAGT-3') with a thiol-terminated end was added to 1 mL of plasmon-enhanced nanodiamond solution and reacted for 4 h. After centrifugation and washing, the plasmon-enhanced nanodiamond probe was obtained.

[0034] Step 3: Preparation of silica microsphere probes 1 nmol of a biotin-terminated probe (5'-CTGATAAGCTACCCCC-Biotin-3') was added to 1 mL of a 30 mg / L solution of 20 µm silica microspheres modified with streptavidin and reacted for 4 h. After centrifugation and washing, the silica microsphere probe was obtained.

[0035] Step 4: Fabrication of Micropillar Array Microfluidic Chips (1) Spin coat an appropriate amount of SU-8 2075 photoresist onto a 4-inch polished silicon wafer. Set the spin coater parameters as follows: low speed 9s 600r / s, high speed 30s 4000r / s. After spin coating, remove any residual photoresist from the back of the polished silicon wafer and heat it at 95℃ for 10min. Set the exposure dose to 150–200 mJ / cm². After exposing the silicon wafer to a UV mask for 2min, place it on a 95℃ heating platform for 10min. After cooling, immerse it in the developer for 5min and finally rinse it clean with alcohol.

[0036] (2) Weigh 20g of PDMS adhesive and 2g of curing agent, mix and stir for 10min, pour the mixture into a petri dish containing a silicon wafer mold, and then place the petri dish into a vacuum chamber to evacuate until there are no air bubbles in the mixture. After curing at 65℃ for 2h, peel the PDMS layer off the silicon wafer mold to obtain the microfluidic chip layer.

[0037] (3) The cleaned microfluidic chip layer and glass slide were placed in an oxygen plasmon cleaner and treated with 80W power for 6 minutes to activate the surface. After removal, the microfluidic chip and glass slide were quickly bonded and dried at 120℃ for 4 hours to obtain the micropillar array microfluidic chip.

[0038] (4) The flow test was performed by injecting dye solution to verify the sealing, waterproofing and structural integrity of the microchannel. All channels were rinsed with anhydrous ethanol and PBS in sequence. Finally, 1% BSA was introduced to seal for 30 min, and then rinsed with PBS for later use.

[0039] Step 5: Detection process of miRNA-21 on the chip (1) Using a syringe pump, continuously inject 1X phosphate buffer into the chip prepared in step four at a constant flow rate of 0.1 mL / min and rinse for 10 minutes to thoroughly remove impurities from the chip.

[0040] (2) Thoroughly mix the plasmon-enhanced nanodiamond probe, silica microsphere probe and miRNA. Use a syringe pump to inject the mixture into the chip at a constant flow rate of 1µL / min. Stop the sample supply when the detection area is observed to be filled with silica microspheres under an optical microscope.

[0041] (3) Using a syringe pump, continuously inject 1X phosphate buffer into the chip at a constant flow rate of 1µL / min, rinse for 10 minutes to further remove unbound target miRNA and free plasma-enhanced nanodiamonds in the detection area.

[0042] Step 6: Using the microwave module and optical path system, scan and locate the detection area in the microcolumn array microfluidic and acquire fluorescence spectrum and photodetector magnetic resonance spectrum. Example 2: A quantum detection method for miRNA based on plasmon enhancement ( Figure 1 ), including the following steps: Step 1: Preparation of plasmonic-enhanced nanodiamonds, including: (1) Place a beaker containing 8 mL of distilled water in a magnetically stirred water bath at 28 °C, add a magnetic stir bar, 364.44 mg CTAB, and 2 mL of 1.25 mM chloroauric acid aqueous solution in sequence, stir well, and then quickly add 600 µL of 10 mM cold sodium borohydride aqueous solution. Stir for 3 min to obtain 2 nm gold nanoclusters. Then let stand at 28 °C for 3 h.

[0043] (2) Place a beaker containing 2 mL of distilled water in a magnetically stirred water bath at 28 °C, and add the magnetic stir bar, 128 mg CTAC, 2 mL of 0.5 mM chloroauric acid aqueous solution, and 1.5 mL of 0.1 mM ascorbic acid aqueous solution in sequence, and stir until homogeneous; take 5 µL of the 2 nm gold nanoclusters prepared in step (1) into 2 mL of distilled water, add it to the beaker all at once, and stir for 15 min to obtain 10 nm gold nanoseeds. Centrifuge twice at 20600 g for 30 min, first disperse in 1 mL of distilled water, and then redisperse in 1 mL of 20 mM CTAC aqueous solution.

[0044] (3) Place a beaker containing 2 mL of distilled water in a magnetically stirred water bath at 28 °C, and add magnetic particles, 64 mg CTAC, 10 µL of 20 mM sodium bromide aqueous solution, 130 µL of 10 mM ascorbic acid aqueous solution, and 9 µL of the 10 nm gold nanoseeds prepared in step (2) in sequence, and stir evenly; add 2 mL of 0.5 mM chloroauric acid aqueous solution, stir for 25 min, and obtain 42 nm gold nanocubes; after centrifugation and washing, disperse in 1 mL of distilled water.

[0045] (4) Take 150µL of 10mM cysteine ​​hydrochloride aqueous solution and add it to the gold nanocube aqueous solution. Mix and incubate for 4h. Centrifuge, wash and redisperse in 1mL aqueous solution to obtain aminated gold nanocubes. (5) Add 2 mg EDS, 2 mg NHS, and 100 µL of 1 mg / mL nanodiamond aqueous solution with a particle size of 200 nm to 1 mL of water in sequence, and incubate on a shaker for 4 h; after centrifugation and washing, redisperse in 1 mL of distilled water.

[0046] (6) Take 1 mL of aminated gold nanocube from step (4) and 1 mL of surface carboxyl-activated nanodiamond obtained in step (5), mix them, and incubate them on a shaker for 2 hours; after centrifugation and washing, redisperse them in 1 mL of distilled water to obtain plasmon-enhanced nanodiamond. Step 2: Preparation of plasmon-enhanced nanodiamond probes 1 nmol of a probe (5'-SH C6-CCCCCTAGACACCGTGTAGCAAAAATGT-3') with a thiol-terminated end was added to 1 mL of plasmon-enhanced nanodiamond solution and reacted for 4 h. After centrifugation and washing, the plasmon-enhanced nanodiamond probe was obtained.

[0047] Step 3: Preparation of silica microsphere probes 1 nmol of a biotin-terminated probe (5'-GCTAGTGCCAAACCCCC-Biotin-3') was added to 1 mL of a 30 mg / L solution of 20 µm silica microspheres modified with streptavidin and reacted for 4 h. After centrifugation and washing, the silica microsphere probe was obtained.

[0048] Step 4: Fabrication of Micropillar Array Microfluidic Chips (1) Spin coat an appropriate amount of SU-82075 photoresist onto a 4-inch polished silicon wafer. Set the spin coater parameters as follows: low speed 9s 600r / s, high speed 30s 4000r / s. After spin coating, remove any residual photoresist from the back of the polished silicon wafer and heat it at 95℃ for 10min. Set the exposure dose to 150–200 mJ / cm². After exposing the silicon wafer to a UV mask for 2min, place it on a 95℃ heating platform for 10min. After cooling, immerse it in the developer for 5min and finally rinse it clean with alcohol.

[0049] (2) Weigh 20g of PDMS adhesive and 2g of curing agent, mix and stir for 10min, pour the mixture into a petri dish containing a silicon wafer template, and then place the petri dish into a vacuum chamber to evacuate until there are no air bubbles in the mixture. After curing at 65℃ for 2h, peel the PDMS layer off the silicon wafer template to obtain the microfluidic chip layer.

[0050] (3) The cleaned microfluidic chip layer and glass slide were placed in an oxygen plasmon cleaner and treated with 80W power for 6 minutes to activate the surface. After removal, the microfluidic chip and glass slide were quickly bonded and dried at 120℃ for 4 hours to obtain the micropillar array microfluidic chip.

[0051] (4) The flow test was performed by injecting dye solution to verify the sealing, waterproofing and structural integrity of the microchannel. All channels were rinsed with anhydrous ethanol and PBS in sequence. Finally, 1% BSA was introduced to seal for 30 min, and then rinsed with PBS for later use.

[0052] Step 5: Detection process of miRNA-96 on the chip (1) Using a syringe pump, continuously inject 1X phosphate buffer into the chip prepared in step four at a constant flow rate of 0.1 mL / min and rinse for 10 minutes to thoroughly remove impurities from the chip.

[0053] (2) Thoroughly mix the plasmon-enhanced nanodiamond probe, silica microsphere probe and miRNA. Use a syringe pump to inject the mixture into the chip at a constant flow rate of 1µL / min. Stop the sample supply when the detection area is observed to be filled with silica microspheres under an optical microscope.

[0054] (3) Using a syringe pump, continuously inject 1X phosphate buffer into the chip at a constant flow rate of 1µL / min, rinse for 10 minutes to further remove unbound target miRNA and free plasma-enhanced nanodiamonds in the detection area.

[0055] Step 6: Using the microwave module and optical path system, scan and locate the detection area in the microcolumn array microfluidic and acquire fluorescence spectrum and photodetector magnetic resonance spectrum.

[0056] This invention employs a combination of plasmon enhancement and microfluidic technology to fabricate plasmon-enhanced nanodiamonds and a microfluidic chip with a micropillar array structure. Detection was performed based on the fluorescence and quantum signals of the nanodiamonds, resulting in high signal contrast, low background interference, convenient operation, and suitability for the analysis of trace samples.

[0057] 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 quantum detection of miRNA based on plasmon enhancement, characterized in that, Includes the following steps: (1) Modify gold nanocubes on the surface of fluorescent nanodiamonds to obtain nanodiamonds with localized surface plasmon enhancement; (2) Prepare plasmon-enhanced nanodiamond probes and silica microsphere probes, wherein the plasmon-enhanced nanodiamond probes and silica microsphere probes are respectively paired with different complementary segments of the target miRNA; (3) Fabrication of micropillar array microfluidic chips; (4) The target miRNA is mixed with the plasmon-enhanced nanodiamond probe and silica microsphere probe to form a sandwich structure complex with silica microspheres as the carrier. (5) The composite is injected into a microfluidic chip with a micropillar array filter structure to enrich the silica microsphere carrier and filter out unbound plasmon-enhanced nanodiamond probes. (6) Fluorescence signal detection is performed on the enriched silica microsphere carrier, and combined with quantum signal detection, to achieve qualitative or quantitative detection of target miRNA.

2. The method according to claim 1, characterized in that, The fluorescent nanodiamond contains NV color center luminescent defect structures.

3. The method according to claim 1, characterized in that, The preparation of the plasmon-enhanced nanodiamond includes the following steps: (1) Place a beaker containing 8 mL of distilled water in a magnetically stirred water bath at 28 °C, add a magnetic stir bar, 364.44 mg CTAB, and 2 mL of 1.25 mM chloroauric acid aqueous solution in sequence, stir evenly, and then quickly add 600 µL of 10 mM cold sodium borohydride aqueous solution. Stir for 3 min to obtain 2 nm gold nanoclusters; then let stand at 28 °C for 3 h. (2) Place a beaker containing 2 mL of distilled water in a magnetically stirred water bath at 28 °C, and add magnetic stir bar, 128 mg CTAC, 2 mL of 0.5 mM chloroauric acid aqueous solution, and 1.5 mL of 0.1 mM ascorbic acid aqueous solution in sequence, and stir evenly; take 5 µL of the 2 nm gold nanoclusters prepared in step (1) into 2 mL of distilled water, add it to the beaker all at once, stir for 15 min to obtain 10 nm gold nanoseeds; centrifuge twice at 20600 g for 30 min, first disperse in 1 mL of distilled water, and then redisperse in 1 mL of 20 mM CTAC aqueous solution; (3) Place a beaker containing 2 mL of distilled water in a magnetically stirred water bath at 28 °C, and add magnetic particles, 64 mg CTAC, 10 µL of 120 mM sodium bromide aqueous solution, 130 µL of 10 mM ascorbic acid aqueous solution, and 300 µL of the 10 nm gold nanoseeds prepared in step (2) in sequence, and stir evenly; add 2 mL of 0.5 mM chloroauric acid aqueous solution, stir for 25 min to obtain gold nanocubes; after centrifugation and washing, disperse in 1 mL of distilled water; (4) Take 150µL of 10mM cysteine ​​hydrochloride aqueous solution and add it to the gold nanocube aqueous solution. Mix and incubate for 4h. Centrifuge, wash and redisperse in 1mL aqueous solution to obtain aminated gold nanocubes. (5) 2 mg EDS, 2 mg NHS, and 100 µL of 1 mg / mL nanodiamond aqueous solution with a particle size of 100 nm were added sequentially to 1 mL of water and incubated on a shaker for 4 h; after centrifugation and washing, the solution was redispersed in 1 mL of distilled water. (6) Take 1 mL of the surface carboxyl-activated nanodiamond from (4) and 1 mL of the gold nanocube obtained in step (5), mix them, and incubate them on a shaker for 2 hours; after centrifugation and washing, redisperse them in 1 mL of distilled water to obtain plasmon-enhanced nanodiamond.

4. The method according to claim 1, characterized in that, The preparation of the plasmon-enhanced nanodiamond probe includes the following steps: 1 nmol of nucleic acid probe with thiol-terminal modification is added to 1 mL of 1 mM plasmon-enhanced nanodiamond solution and reacted for 4 h. After centrifugation, washing and dispersion, the plasmon-enhanced nanodiamond probe is obtained.

5. The method according to claim 1, characterized in that, The preparation of the silica microsphere probe includes the following steps: 1 nmol of a nucleic acid probe with biotin-modified ends is added to 1 mL of an aqueous solution of silica microspheres with 10~100 mg / L surface-modified streptavidin and reacted for 4 h. After centrifugation, washing and dispersion, the silica microsphere probe is obtained. The average particle size of the silica microspheres is 2~50 µm.

6. The method according to claim 1, characterized in that, The fabrication of the micropillar array microfluidic chip includes: (1) Take an appropriate amount of SU-82075 photoresist and spin coat it onto a 4-inch polished silicon wafer. Set the spin coater parameters: low speed 9s 600r / s, high speed 30s 4000r / s. After spin coating, remove the residual photoresist on the back of the polished silicon wafer and heat it at 95℃ for 10min. Set the exposure amount to 150–200 mJ / cm². After exposing the silicon wafer to the UV mask for 2min, place it on a 95℃ heating platform for 10min. After cooling, immerse it in the developer for 5min and finally rinse it with alcohol. (2) Weigh 20g of PDMS adhesive and 2g of curing agent, mix and stir for 10min, pour the mixture into a petri dish containing a silicon wafer mold, and then put the petri dish into a vacuum box to evacuate until there are no bubbles in the mixture; heat and cure at 65℃ for 2h, then peel the PDMS layer off the silicon wafer mold to obtain the microfluidic chip layer; (3) The cleaned microfluidic chip layer and glass slide were placed in an oxygen plasmon cleaning machine and treated with 80W power for 6 minutes to activate the surface; after taking it out, the microfluidic chip and glass slide were quickly bonded and dried at 120℃ for 4 hours to obtain the micropillar array microfluidic chip. (4) The flow test was performed by injecting dye solution to verify the sealing, waterproofing and structural integrity of the microchannel. All channels were rinsed with anhydrous ethanol and PBS in sequence. Finally, 1% BSA was introduced to seal for 30 min, and then rinsed with PBS for later use.

7. The method according to claim 1, characterized in that, The microfluidic chip includes an inlet / outlet channel, a filtration enrichment region, and a detection region. The filtration enrichment region is equipped with a micropillar array filtration structure for enriching the microsphere carrier and filtering out unbound plasmon-enhanced nanodiamond probes. The detection region is used to detect fluorescence and quantum signals of the microsphere carrier.

8. The method according to claim 7, characterized in that, The micropillar array filter structure of the microfluidic chip is a circumferentially distributed micropillar structure. The spacing between adjacent micropillars in the circumferential array micropillar structure is 1~30 µm, and the micropillar gap is set to be smaller than the size of the microsphere carrier and much larger than the size of the plasmon-enhanced nanodiamond, so as to achieve the enrichment and filtration removal of unbound plasmon-enhanced nanodiamond probes by the microsphere carrier.

9. The method according to claim 1, characterized in that, The fluorescent nanodiamonds have an average particle size of 100-200 nm, and the gold nanocubes have a characteristic size of 20-70 nm.

10. The method according to claim 1, characterized in that, The fluorescence signal is the photoluminescence intensity of the nanodiamond NV color center under 532nm laser excitation; the quantum signal detection is a photodetector magnetic resonance signal detection based on the NV color center, and includes applying microwave excitation and a 532nm laser and collecting fluorescence changes under microwave modulation to extract the quantum readout signal.

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