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CN-121992076-A - MiRNA quantum detection method based on plasmon enhancement

CN121992076ACN 121992076 ACN121992076 ACN 121992076ACN-121992076-A

Abstract

The invention discloses a miRNA quantum detection method based on plasmon enhancement. The method comprises the steps of modifying gold nanocubes on the surfaces of fluorescent nanodiamonds to generate a local surface plasmon resonance enhancement effect, preparing plasmon enhancement nanodiamond probes and silicon dioxide microsphere probes to enable target miRNAs to hybridize to form a sandwich structure compound, injecting the compound into a micro-fluidic chip with a micro-column array structure to achieve interception and enrichment of the sandwich structure compound and filtering to remove unbound plasmon enhancement nanodiamond probes, and detecting fluorescent signals of the compound and combining quantum signal detection to achieve qualitative or quantitative analysis of the target miRNAs. The invention reduces the background of the free probe and improves the detection repeatability through microfluidic filtration, combines plasmon enhancement to improve the read-out signal intensity of the nano diamond, and has the advantages of high sensitivity, strong specificity, convenient operation, suitability for detecting micro samples and the like.

Inventors

  • LIU LEI
  • YU JIA
  • CHEN YUNFEI

Assignees

  • 东南大学

Dates

Publication Date
20260508
Application Date
20260203

Claims (10)

  1. 1. The miRNA quantum detection method based on plasmon enhancement is characterized by comprising the following steps of: (1) Modifying a gold nanocube on the surface of the fluorescent nano diamond to obtain the enhanced nano diamond with localized surface plasmons; (2) Preparing a plasmon enhanced nano-diamond probe and a silicon dioxide microsphere probe, wherein the plasmon enhanced nano-diamond probe and the silicon dioxide microsphere probe are respectively paired with different complementary sections of a target miRNA; (3) Preparing a micro-column array micro-fluidic chip; (4) Mixing target miRNA with the plasmon enhanced nano diamond probe and the silicon dioxide microsphere probe to form a sandwich structure compound taking the silicon dioxide microsphere as a carrier; (5) Injecting the compound into a micro-fluidic chip with a micro-column array filtering structure, enriching the silicon dioxide microsphere carrier, and filtering to remove unbound plasmon enhanced nano-diamond probes; (6) And (3) carrying out fluorescence signal detection on the enriched silica microsphere carrier, and combining quantum signal detection to realize qualitative or quantitative detection on the target miRNA.
  2. 2. The method of claim 1, wherein the fluorescent nanodiamond comprises NV color-centered light-emitting defect structures.
  3. 3. The method of claim 1, wherein the preparation of the plasmonic-enhanced nanodiamond comprises the steps of: (1) Placing a beaker with 8mL of distilled water in a magnetic stirring water bath kettle at 28 ℃, sequentially adding magnetons and 364.44mg CTAB,2mL 1.25mM chloroauric acid water solution, stirring uniformly, rapidly adding 600 mu L of 10mM cold sodium borohydride water solution, stirring for 3min to obtain 2nm gold nanoclusters, and then standing for 3h at 28 ℃; (2) Placing a beaker with 2mL of distilled water in a magnetic stirring water bath kettle at 28 ℃, sequentially adding magnetons, 128mg CTAC,2mL 0.5mM mL of 0.1mM ascorbic acid aqueous solution, uniformly stirring, taking 5 mu L of the 2nm gold nanoclusters prepared in the step (1) into 2mL of distilled water, adding the 2nm gold nanoclusters into the beaker at one time, stirring for 15min to obtain 10nm gold nano seeds, centrifuging 20600g of the gold nano seeds twice for 30min, dispersing the gold nano seeds in 1mL of distilled water firstly, and then redispersing the gold nano clusters in 1mL of 20mM CTAC aqueous solution; (3) Placing a beaker with 2mL of distilled water in a magnetic stirring water bath kettle at a temperature of 28 ℃, sequentially adding magneton, 64mg of CTAC,10 mu L of 120mM sodium bromide aqueous solution, 130 mu L of 10mM ascorbic acid aqueous solution and 300 mu L of the 10nm gold nano seeds prepared in the step (2), uniformly stirring, adding 2mL of 0.5mM chloroauric acid aqueous solution, stirring for 25min to obtain gold nano cubes, centrifuging and washing, and dispersing in 1mL of distilled water; (4) Adding 150 mu L of 10mM cysteamine hydrochloride aqueous solution into the gold nanocubes aqueous solution, mixing and incubating for 4 hours, and performing centrifugal washing to redisperse in 1mL of aqueous solution to obtain aminated gold nanocubes; (5) 2mg EDS,2mgNHS,100 mu L of 1mg/mL nano-diamond aqueous solution with the particle size of 100nm is sequentially added into 1mL of water, incubated for 4 hours on a shaking table, and then is redispersed in 1mL of distilled water after centrifugal washing; (6) Mixing 1mL (4) of nano diamond with activated surface carboxyl and 1mL of gold nano cube obtained in the step (5), incubating for 2 hours on a shaking table, and obtaining the plasmon enhanced nano diamond by re-dispersing in 1mL of distilled water after centrifugal washing.
  4. 4. The method according to claim 1, wherein the preparation of the plasmon-enhanced nanodiamond probe comprises the steps of adding 1nmol of the nucleic acid probe with the terminal modified with the sulfhydryl group into 1ml of 1mM plasmon-enhanced nanodiamond solution for reaction for 4 hours, and obtaining the plasmon-enhanced nanodiamond probe after centrifugal washing and dispersion.
  5. 5. The method of claim 1, wherein the preparation of the silica microsphere probe comprises the steps of adding 1nmol of the nucleic acid probe with biotin modified at the tail end into 1ml of 10-100 mg/L of the aqueous solution of the silica microsphere with streptavidin modified at the surface for reaction for 4 hours, and centrifugally washing and dispersing to obtain the silica microsphere probe, wherein the average particle size of the silica microsphere is 2-50 μm.
  6. 6. The method of claim 1, wherein the preparation of the micro-pillar array microfluidic chip comprises: (1) Spin-coating a proper amount of SU-82075 photoresist on a 4-inch polished silicon wafer, setting parameters of a photoresist homogenizing machine, namely 9s 600r/s at low speed and 4000r/s at high speed, removing residual photoresist on the back of the polished silicon wafer after photoresist homogenizing is finished, heating and drying for 10min at 95 ℃, setting exposure of 150-200 mJ/cm < 2 >, exposing the silicon wafer through an ultraviolet mask for 2 min, placing the silicon wafer on a 95 ℃ heating platform for 10min, immersing the silicon wafer in a developing solution for 5min after cooling, and finally washing the silicon wafer cleanly with alcohol; (2) Weighing 20g of PDMS glue and 2g of curing agent, mixing and stirring for 10min, pouring the mixed solution into a culture dish provided with a silicon wafer die, placing the culture dish into a vacuum box for vacuumizing until no bubble exists in the mixed solution, heating and curing for 2h at 65 ℃, and stripping the PDMS layer from the silicon wafer die to obtain a microfluidic chip layer; (3) Placing the cleaned microfluidic chip layer and the glass slide in an oxygen plasmon cleaning machine, and treating for 6min at 80W power to activate the surface; after the micro-fluidic chip is taken out, the micro-fluidic chip is quickly bonded with a glass slide, and the micro-fluidic chip is heated and dried for 4 hours at 120 ℃ to obtain a micro-column array micro-fluidic chip; (4) And (3) performing flow test by injecting dye solution, verifying the tightness, waterproofness and structural integrity of the micro-channel, sequentially flushing all channels with absolute ethyl alcohol and PBS, finally introducing 1% BSA to seal 30min, and flushing with PBS for later use.
  7. 7. The method of claim 1, wherein the microfluidic chip comprises a sample inlet and outlet channel, a filtration and enrichment region, and a detection region, wherein the filtration and enrichment region is provided with a micro-column array filtration structure for enriching microsphere carriers and filtering to remove unbound plasmon enhanced nanodiamond probes, and the detection region is used for detecting fluorescent signals and quantum signals of the microsphere carriers.
  8. 8. The method of claim 7, wherein the micro-column array filter structure of the micro-fluidic chip is a micro-column structure distributed in a circumferential array, the adjacent micro-columns of the circumferential array micro-column structure are spaced by 1-30 μm, and the micro-column gaps are smaller than the size of the microsphere carrier and far larger than the size of the plasmon enhanced nanodiamond, so as to enrich the microsphere carrier and filter and remove unbound plasmon enhanced nanodiamond probes.
  9. 9. The method according to claim 1, wherein the average particle size of the fluorescent nanodiamond is 100-200 nm, and the characteristic size of the gold nanocubes is 20-70 nm.
  10. 10. The method of claim 1, wherein the fluorescence signal is a photoluminescence intensity of the nanodiamond NV color center under 532nm laser excitation, wherein the quantum signal detection is a photo-detection magnetic resonance signal detection based on the NV color center, and wherein the method comprises applying microwave excitation and 532nm laser and collecting fluorescence changes under microwave modulation to extract a quantum readout signal.

Description

MiRNA quantum detection method based on plasmon enhancement Technical Field The invention belongs to the field of biological detection, and particularly relates to a miRNA quantum detection method based on plasmon enhancement. Background MiRNA is used as endogenous small molecule non-coding RNA and is closely related to various major diseases such as tumor, cardiovascular diseases, immune disorder and the like. However, the clinical samples have large miRNA concentration span and complex matrix, which often results in strong background interference, limited linear interval and insufficient repeatability. The existing miRNA detection means comprise electrophoresis, hybridization, PCR, sequencing and the like, but the molecular amplification method usually depends on professional equipment and complex flow, which is unfavorable for rapid and on-site detection, and the conventional fluorescent dye also has the problems of signal attenuation and the like caused by photobleaching and repeated excitation, thereby influencing the reliability of long-term and repeated measurement. The fluorescent nano diamond NV color center has better light stability, and can bear long-time and high-power laser irradiation without photo bleaching. The NV color center is a luminescence defect structure, fluorescence of which can be modulated by microwaves and shows a change in fluorescence intensity, so that the NV color center can be used for acquiring a photodetection magnetic resonance (ODMR) signal, and the NV color center is a quantum sensor with excellent performance and can be used for detecting physical quantities such as a magnetic field, an electric field, temperature and the like. However, the fluorescence photon emission rate of a single NV color center is relatively limited, which limits the detection limit based on fluorescence intensity readout to some extent. A challenge in high sensitivity detection is how to efficiently extract very low concentrations of specific signals from high background noise in the context of complex biological samples. The existing scheme generally relies on repeated cleaning or removal of unconnected nano-diamond on a solid substrate to achieve purification, but non-specific adsorption and residual background may still be brought in complex systems or micro-samples. Disclosure of Invention In order to solve the problems, the invention discloses a miRNA quantum detection method based on plasmon enhancement, which reduces the background of a free probe and improves the detection repeatability through microfluidic filtration, combines plasmon enhancement to improve the read-out signal strength of nano-diamond, and has the advantages of high sensitivity, strong specificity, convenience in operation, suitability for detecting micro-samples and the like. In order to achieve the above purpose, the technical scheme of the invention is as follows: a plasmon enhancement-based miRNA quantum detection method, comprising: Step one, a gold nanocube (AuNC) is modified on the surface of a Fluorescent Nanodiamond (FND) to obtain the nanodiamond (FND@AuNC) with a localized surface plasmon resonance enhancement effect, which specifically comprises the following steps: (1) Placing a beaker with 8mL of distilled water in a magnetic stirring water bath at 25-32 ℃, sequentially adding magnetons, 364.44mg of cetyltrimethylammonium bromide (CTAB), 2mL of 1.25mM chloroauric acid aqueous solution, stirring uniformly, rapidly adding 600 mu L of 10mM cold sodium borohydride aqueous solution, stirring for 3min to obtain 2nm gold nanoclusters, and standing for 3h at 25-32 ℃. (2) Placing a beaker with 2mL of distilled water in a magnetic stirring water bath at 25-32 ℃, sequentially adding magneton, 128mg of cetyltrimethylammonium chloride (CTAC), 2mL of 0.5mM aqueous chloroauric acid solution and 1.5mL of 0.1mM aqueous ascorbic acid solution, uniformly stirring, taking 5 mu L of the 2nm gold nanoclusters prepared in the step (1) in 2mL of distilled water, adding the 2nm gold nanoclusters into the beaker at one time, and stirring for 15min to obtain gold nano seeds. Centrifugation was performed twice, first in 1mL distilled water, and then in 1mL 20mM CTAC aqueous solution. (3) Placing a beaker with 2mL of distilled water in a magnetic stirring water bath kettle with the temperature of 25-32 ℃, sequentially adding magnetons, 64mg of CTAC,10 mu L of 20-120 mM sodium bromide aqueous solution, 130 mu L of 10mM ascorbic acid aqueous solution and 9-300 mu L of the 10nm gold nano seeds prepared in the step (2), uniformly stirring, adding 2mL of 0.5mM chloroauric acid aqueous solution, stirring for 25min to obtain gold nano cubes, centrifuging and washing, and dispersing in 1mL of distilled water. (4) Adding 150 mu L of 10mM mercaptoethylamine hydrochloride water solution into the gold nanocubes water solution, mixing and incubating for 4 hours, and performing centrifugal washing and redispersion in 1mL of water solution to obta