Micro-fluidic chip for detecting breast cancer tumor markers as well as preparation method and application of micro-fluidic chip

Abstract

The invention discloses a micro-fluidic chip for detecting breast cancer tumor markers and a preparation method and application thereof, and belongs to the technical field of microfluidics, the preparation method of the micro-fluidic chip comprises the following steps: preparing up-conversion nanoparticles with a core-shell structure through a solvothermal method and a homogeneous wrapping method; the preparation method comprises the following steps: carrying out surface ligand-free treatment on an up-conversion nanoparticle with a core-shell structure, and modifying the up-conversion nanoparticle with ssDNA complementary to a breast cancer tumor marker to obtain the UCNPs-ssDNA fluorescent nanoprobe; the preparation method comprises the following steps: spin-coating a ZnO inverse opal photonic crystal with a UCNPs-ssDNA fluorescent nanoprobe to obtain a photonic crystal / UCNP composite structure; and dispensing the MoS2 nanosheet solution on the composite structure of the photonic crystal / UCNP to obtain the micro-fluidic chip. Compared with the traditional detection means, the micro-fluidic chip has the advantages of low cost, real-time visual detection, high detection sensitivity and the like.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics technology, in particular to a microfluidics chip for detecting breast cancer tumor markers and a preparation method and application thereof. Background Art

[0002] Real-time visual detection of microRNA (miRNA) content in living cells is of great significance in clinical research and molecular biology. Traditional miRNA detection methods, such as Northern hybridization, microarray technology, and quantitative polymerase chain reaction, have serious drawbacks in terms of cost, detection complexity, sample preparation, and analysis time. The application of fluorescent nanotechnology in biosensor manufacturing has opened a new door for miRNA detection. However, fluorescent miRNA biosensors are difficult to be widely used in real-time, visual detection due to their high background signals, insufficient sensitivity, and durability.

[0003] miRNA plays an important role in gene expression regulation and is the transmitter of genetic information that connects cell genotype and phenotype. miRNA detection plays an important role in tumor cell analysis and cell heterogeneity research. Developing a fast, easy-to-use, real-time visual miRNA detection strategy has become an inevitable trend. There are many methods for detecting miRNA, including quantum dot-based fluorescence detection, sequencing, organic fluorescent dyes, quantitative polymerase chain reaction (q-PCR), etc., but these methods face some obstacles in practice. For example, organic fluorescent dyes have poor photostability, high noise, low fluorescence quantum efficiency of quantum dots, high background fluorescence, high cost of sequencing technology, etc., and miRNA is a small endogenous non-coding RNA (~19-25nt), which is incompatible with qPCR. In contrast, the fluorescence resonance energy transfer method has higher sensitivity, lower environmental requirements, can be detected at the single cell level, and can also be combined with various instruments and technologies. Generally, FRET-based miRNA detection usually includes fluorescence enhancement and fluorescence quenching, that is, signal generation or signal quenching after hybridization of target miRNA with its complementary DNA. Therefore, obtaining nanofluorescent probes with high fluorescence signal intensity, multicolor tunability, strong photostability, and no background light interference is the key to real-time visualized biosensor detection.

[0004] Rare earth ion doped upconversion nanoparticles (UCNPs) have significant advantages such as excellent photostability, low potential toxicity, no background light interference, and almost no damage to biological tissues. In addition, with the development of single nanoparticle upconversion spectroscopy and the advancement of single particle detection technology, people have conducted interesting research on light tracking, micro-photosensors and various biological applications. The significant photostability, multicolor sharp band emission, good biocompatibility and sufficient anti-Stokes emission efficiency of single rare earth doped upconversion nanoparticles (UCNPs) have been fully exploited, showing the broad prospects of single particle biosensors. UCNP-based biosensors have the ability to detect ultra-trace target molecules with high precision and high signal-to-noise ratio. Based on UCNP-microfluidic chips, not only can the detection sensitivity of miRNA be improved, but also the above problems can be overcome. However, the detection of tumor markers using such UCNP-microfluidic chips is still in its infancy, because it is still a daunting challenge to control the intensity and color change of upconversion emission in UCNPs with fixed composition to realize fluorescence change biosensors. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a microfluidic chip for detecting tumor markers of breast cancer, so as to solve the problems raised in the above background technology.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] A method for preparing a microfluidic chip for detecting breast cancer tumor markers comprises the following steps:

[0008] The core-shell structured upconversion nanoparticles are prepared by a solvothermal method and a homogeneous encapsulation method; the matrix of the core-shell structured upconversion nanoparticles is NaYF4 and is doped with Yb 3+ 、Tm 3+ and Ho 3+ ;

[0009] After the core-shell structured upconversion nanoparticles were treated with ligand-free surface, they were modified with ssDNA complementary to breast cancer tumor markers to obtain UCNPs-ssDNA fluorescent nanoprobes;

[0010] The UCNPs-ssDNA fluorescent nanoprobe was spin-coated on the ZnO inverse opal photonic crystal to obtain a photonic crystal / UCNP composite structure;

[0011] The MoS2 nanosheet solution was drop-coated on the composite structure of photonic crystal / UCNP to obtain a microfluidic chip.

[0012] Preferably, the steps of preparing the core-shell structured upconversion nanoparticles by a solvothermal method and a homogeneous encapsulation method specifically include:

[0013] A thulium source, a ytterbium source and a yttrium source are heated and mixed with oleic acid and octadecene solution to obtain a mixed solution A;

[0014] Sodium hydroxide and ammonium fluoride powders are dissolved in a methanol solution, and then slowly added into a mixed solution A, fully stirred, and heated to obtain a mixed solution B;

[0015] The mixed solution B is centrifuged to obtain upconversion nanoparticle precipitation;

[0016] Taking an ytterbium source, a yttrium source, a holmium source, and the upconversion nanoparticle precipitate, heating and mixing them with oleic acid and octadecene solution to obtain a mixed solution C;

[0017] Sodium hydroxide and ammonium fluoride powders are dissolved in a methanol solution, and then slowly added into the mixed solution C, fully stirred, and heated to obtain a mixed solution D;

[0018] The mixed solution D is centrifuged to obtain upconversion nanoparticles with a core-shell structure.

[0019] Preferably, the step of subjecting the core-shell structured upconversion nanoparticles to surface ligand-free treatment specifically comprises:

[0020] adding the upconversion nanoparticles with a core-shell structure into cyclohexane to obtain a cyclohexane solution of the upconversion nanoparticles;

[0021] Hydrochloric acid was added to the cyclohexane solution of the upconversion nanoparticles for ultrasonic treatment, followed by centrifugation using ethanol, and the product after centrifugation was dissolved in deionized water to obtain a ligand-free UCNPs solution.

[0022] Preferably, the step of modifying with ssDNA complementary to a breast cancer tumor marker to obtain a UCNPs-ssDNA fluorescent nanoprobe specifically comprises:

[0023] The ligand-free UCNPs solution and ssDNA are mixed and stirred at a molar ratio of 1:(10-50), and then washed, sonicated, and centrifuged to obtain a UCNPs-ssDNA fluorescent nanoprobe.

[0024] Preferably, the breast cancer tumor marker is miRNA-155; the nucleotide sequence of the ssDNA complementary to the breast cancer tumor marker is shown in the sequence listing SEQ ID NO.1.

[0025] Preferably, the method for preparing the ZnO inverse opal photonic crystal comprises the following steps:

[0026] A glass sheet is vertically inserted into an aqueous solution containing polymethyl methacrylate (PMMA) latex spheres, and PMMA microspheres are self-assembled using a vertical deposition self-assembly process, and then dried; during the drying process, driven by the surface tension of the liquid, the PMMA microspheres slowly self-assemble into a highly ordered colloidal array on the glass substrate, and then sintered to obtain a PMMA opal template;

[0027] dissolving zinc nitrate and citric acid in a solvent to form a precursor solution;

[0028] The precursor solution is infiltrated into the voids of the PMMA opal template by capillary force and dried to obtain a ZnO inverse opal photonic crystal.

[0029] Preferably, the pore size of the ZnO inverse opal photonic crystal is 200-800 nm.

[0030] Preferably, the concentration of the MoS2 nanosheet solution is 0.09-900 μg / mL.

[0031] Another object of the embodiment of the present invention is to provide a microfluidic chip manufactured by the above-mentioned manufacturing method.

[0032] Another object of an embodiment of the present invention is to provide a use of the above-mentioned microfluidic chip in preparing a kit for detecting breast cancer tumor markers.

[0033] The preparation method of the microfluidic chip provided by the present invention can enhance the fluorescence intensity of a single upconversion nanoparticle by using an inverse opal structure, thereby improving the detection sensitivity of the microfluidic chip. The microfluidic chip prepared by the present invention can perform visual detection of breast cancer tumor markers, thereby improving the accuracy of cancer detection; and the microfluidic chip can be connected with complementary ssDNA of different tumor markers, and the fluorescence resonance energy transfer effect can be used to detect other tumor markers except breast cancer, and has extremely strong universality. Compared with traditional detection methods, the microfluidic chip prepared by the present invention has the advantages of low cost, real-time visual detection, and high detection sensitivity; at the same time, the upconversion nanomaterial prepared by the present invention has low material toxicity, no background fluorescence, and weak self-absorption ability, and is an excellent material for fluorescence recovery type microfluidic chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A schematic diagram of a process for preparing a microfluidic chip for detecting tumor markers of breast cancer provided by an embodiment of the present invention;

[0035] Figure 2 TEM image of the ZnO inverse opal photonic crystal prepared in the embodiment of the present invention;

[0036] Figure 3 TEM images and absorption spectra of UCNPs-ssDNA prepared in the embodiment of the present invention; in the figure, a is a TEM image of UCNPs-ssDNA prepared in the embodiment of the present invention; b is an absorption spectra of UCNPs-ssDNA prepared in the embodiment of the present invention and UCNPs without ssDNA modification;

[0037] Figure 4 SEM top view of the composite structure of photonic crystal / UCNP prepared in an embodiment of the present invention;

[0038] Figure 5 This is a fluorescence recovery relationship diagram of the microfluidic chip prepared in an embodiment of the present invention under different concentrations of miRNA-155. DETAILED DESCRIPTION

[0039] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0040] The embodiment of the present invention proposes a simple strategy to achieve upconversion emission color switching in UCNP using local field modulation of the photon cavity, and based on a fluorescence resonance energy transfer effect, realizes real-time and visual detection of breast cancer tumor markers. The UCNP-microfluidic chip combines ssDNA-modified UCNP and inverse opal photonic crystal structure, and has excellent breast cancer marker detection capabilities, with a detection limit of up to 1pM. Combined with an advanced single particle imaging system, the fluorescence image of the UCNP can be obtained, the changes in the fluorescence intensity of the nanoparticles can be observed in real time, the changes in the concentration of miRNAs can be analyzed, and visual detection can be achieved.

[0041] Specifically, Figure 1 As shown, in one embodiment of the present invention, a method for preparing a microfluidic chip for detecting breast cancer tumor markers is provided, which comprises the following steps:

[0042] S1. Prepare core-shell structured upconversion nanoparticles (UCNPs) by solvothermal method and homogeneous encapsulation method; the matrix of the core-shell structured upconversion nanoparticles is NaYF4 and doped with Yb 3+ 、Tm 3+ and Ho 3+ ;

[0043] S2, after the surface of the core-shell structured upconversion nanoparticles is treated without ligand, the ssDNA complementary to the breast cancer tumor marker is used for modification to obtain 0.001-100 mM UCNPs-ssDNA fluorescent nanoprobe;

[0044] S3, spin coating the UCNPs-ssDNA fluorescent nanoprobe on the ZnO inverse opal photonic crystal to obtain a photonic crystal / UCNP composite structure;

[0045] S4. Drop-coat the MoS2 nanosheet solution on the photonic crystal / UCNP composite structure to obtain a microfluidic chip.

[0046] In a preferred embodiment of the present invention, the steps of preparing core-shell structured upconversion nanoparticles by a solvothermal method and a homogeneous encapsulation method specifically include:

[0047] S11, taking a thulium source, a ytterbium source, and a yttrium source, and heating and mixing them with oleic acid and octadecene solution to obtain a mixed solution A;

[0048] S12, dissolving sodium hydroxide and ammonium fluoride powder in a methanol solution, then slowly adding the mixture to the mixed solution A, stirring thoroughly, and heating to obtain a mixed solution B;

[0049] S13, centrifuging the mixed solution B to obtain upconversion nanoparticle precipitation;

[0050] S14, taking an ytterbium source, a yttrium source, a holmium source, and the upconversion nanoparticle precipitate, and heating and mixing them with oleic acid and octadecene solution to obtain a mixed solution C;

[0051] S15, dissolving sodium hydroxide and ammonium fluoride powder in a methanol solution, then slowly adding the mixture to the mixed solution C, stirring thoroughly, and heating to obtain a mixed solution D;

[0052] S16, centrifuging the mixed solution D to obtain upconversion nanoparticles with a core-shell structure.

[0053] In a preferred embodiment of the present invention, the step of performing ligand-free surface treatment on the core-shell structured upconversion nanoparticles specifically comprises:

[0054] S21, adding the upconversion nanoparticles with a core-shell structure into cyclohexane to obtain a cyclohexane solution of the upconversion nanoparticles;

[0055] S22. Add hydrochloric acid to the cyclohexane solution of the upconversion nanoparticles for ultrasonic treatment, then use ethanol for centrifugation, and dissolve the product after centrifugation in deionized water to obtain a ligand-free UCNPs solution.

[0056] In a preferred embodiment of the present invention, the step of using ssDNA complementary to a breast cancer tumor marker for modification to obtain a UCNPs-ssDNA fluorescent nanoprobe specifically includes:

[0057] The ligand-free UCNPs solution and ssDNA are mixed and stirred at a molar ratio of 1:(10-50), and then washed, sonicated, and centrifuged to obtain a UCNPs-ssDNA fluorescent nanoprobe.

[0058] In a preferred embodiment of the present invention, the breast cancer tumor marker is miRNA-155; the nucleotide sequence of the ssDNA complementary to the breast cancer tumor marker is shown in the sequence table SEQ ID NO.1, specifically as follows: 5'-AAAAAAAAAACCCCTATCACGATTAGCATTAA-SH-3'.

[0059] In a preferred embodiment of the present invention, the method for preparing the ZnO inverse opal photonic crystal comprises the following steps:

[0060] S31, vertically inserting a glass sheet into an aqueous solution containing polymethyl methacrylate (PMMA) latex balls, self-assembling PMMA microspheres by a vertical deposition self-assembly process, and drying; during the drying process, driven by the surface tension of the liquid, the PMMA microspheres slowly self-assemble into a highly ordered colloidal array on the glass substrate, and then sintering is performed to obtain a PMMA opal template;

[0061] S32, dissolving zinc nitrate and citric acid in a solvent to form a precursor solution;

[0062] S33, infiltrating the precursor solution into the voids of the PMMA opal template by capillary force, and performing drying treatment to obtain a ZnO inverse opal photonic crystal.

[0063] In a preferred embodiment of the present invention, the pore size of the ZnO inverse opal photonic crystal is 200-800 nm.

[0064] In a preferred embodiment of the present invention, the concentration of the MoS2 nanosheet solution is 0.09-900 μg / mL.

[0065] In another embodiment of the present invention, a microfluidic chip prepared by the above preparation method is provided, which is a structural probe based on inverse opal photonic crystal / single upconversion nanoparticle, wherein the photonic crystal structure is a ZnO inverse opal photonic crystal with a microsphere size of 200-800nm; the upconversion nanoparticle is NaYF 4 :10%-30%Yb 3+ ,0.05%-30%Tm3+ @NaYF4:10%-30%Yb 3+ ,0.1%-30%Ho 3+ (abbreviated as Tm@Ho), ssDNA complementary to the breast cancer tumor marker miRNA-155 was used to modify the ligand-free Tm@Ho, and the obtained UCNPs-ssDNA was used as a nanofluorescent probe. MoS2 nanosheets with a concentration of 0.09-900 μg / mL were dripped into the microfluidic chip. In the absence of targeted miRNA-155, MoS2 nanosheets as energy collectors can bind to ssDNA through van der Waals forces, thereby triggering fluorescence resonance energy transfer between UCNPs-ssDNA and MoS2 nanosheets, causing the upconversion emission of UCNPs to be quenched; however, in the presence of targeted miRNA-155, miRNA-155 can hybridize with ssDNA on UCNPs to inhibit the close contact between UCNPs-ssDNA and MoS2 nanosheets, blocking the fluorescence resonance energy transfer between them, and restoring the upconversion fluorescence of UCNPs. At the same time, with the increase of the concentration of targeted miRNA-155, the single-particle sensor has a good linear relationship in the range of 1.0 pM-0.6 nM, and gradually tends to saturation with the increase of concentration.

[0066] The microfluidic chip obtained by the above preparation method is used to detect the breast cancer tumor marker miRNA-155 in vitro, and uses an atomic force microscope and an in-situ fluorescence lifetime imaging system to track the position of a single particle and record its fluorescence spectrum and lifetime information. In the presence of targeted miRNA-155, miRNA-155 can hybridize with ssDNA on UCNPs to inhibit the close contact between UCNPs-ssDNA and MoS2 nanosheets, block the fluorescence resonance energy transfer between them, and restore the upconversion fluorescence of UCNPs. At the same time, as the concentration of targeted miRNA-155 increases, within the range of 1.0pM-0.6nM, the single particle sensor has a good linear relationship and gradually tends to saturation with the increase of concentration. The inverse opal photonic crystal structure can regulate the upconversion fluorescence intensity of a single nanoparticle through a local light field, thereby improving the detection sensitivity of the microfluidic chip, and the change of miRNA-155 concentration can be visualized in real time through a time-resolution imaging device.

[0067] In summary, the microfluidic chip based on photonic crystal / single upconversion nanoparticles designed in the embodiment of the present invention can realize real-time visual quantitative detection of breast cancer tumor marker miRNA-155, and has the advantages of low cost, good stability, real-time observation and high detection sensitivity.

[0068] The following embodiments are some specific implementation cases of the present invention in practical applications, but are not limited thereto.

[0069] Embodiment 1: This embodiment provides a method for preparing a microfluidic chip, which comprises the following steps:

[0070] Step 1: Synthesis of upconversion nanoparticles NaYF4:10% Yb by a classical solvothermal method 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho 3+ . Take 0.0015g of thulium chloride hexahydrate powder, 0.388g of ytterbium chloride hexahydrate powder, and 2.620g of yttrium chloride hexahydrate powder in a three-necked bottle, add 60mL of oleic acid and 150mL of octadecene solution, fix it in an oil bath, under nitrogen protection at 160℃, stir at 700rpm until the drugs in the three-necked bottle are completely dissolved, and then cool to 30℃.

[0071] Step 2: Take 1.0g of sodium hydroxide and 1.48g of ammonium fluoride powder and dissolve them in 60mL of methanol solution. Slowly add the methanol mixed solution into the mixed solution obtained in step 1, stir thoroughly for 1h, and then raise the temperature from room temperature to 125°C. During the heating process, bubbles will continue to emerge from the three-necked flask. After there is no foam in the three-necked flask, connect the condenser, slowly raise the temperature of the solution in the three-necked flask to 300°C, stir thoroughly for 2h, stop heating and lower the temperature to 30°C.

[0072] Step 3: The mixed solution obtained in step 2 was divided into centrifuge tubes, 150 mL of ethanol solution was added to the centrifuge tubes, and centrifuged at 10000 rpm for 20 min. The supernatant was poured out, 160 mL of cyclohexane solution was added, and the precipitate was dissolved by ultrasonication. Then 200 mL of ethanol solution was added and centrifuged again. This step was repeated twice to obtain NaYF4:10%Yb 3+ ,0.05%Tm 3+ precipitation.

[0073] Step 4: Prepare core-shell upconversion nanoparticles by homogeneous encapsulation. Take 0.388g of ytterbium chloride hexahydrate powder, 2.590g of yttrium chloride hexahydrate powder, and 0.004g of holmium chloride hexahydrate powder in a three-necked bottle, add the upconversion nanoparticle precipitate obtained in step 3, and then add 60mL of oleic acid and 150mL of octadecene solution. Fix the oil bath pot, under nitrogen protection at 160℃, stir at 700rpm until the drugs in the three-necked bottle are completely dissolved, and then cool to 30℃. After repeating steps 2-3, NaYF4:10%Yb 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho3+ The prepared precipitates of upconversion nanoparticles were dissolved in cyclohexane to obtain a cyclohexane solution of upconversion nanoparticles with a molar concentration of 0.005 mol / L.

[0074] Step 5: Washing NaYF4:10% Yb with hydrochloric acid solution 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho 3+ Surface ligands of nanoparticles. Take 10 mL of NaYF4:10% Yb 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho 3+ 1.6 mol hydrochloric acid was added to the cyclohexane solution and ultrasonic treatment was performed for 5 min. Then, ethanol was centrifuged twice. After the first centrifugation, 10 mL of NaYF4:10% Yb 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho 3+ 30 mL of ethanol was added to the mixed solution of cyclohexane and hydrochloric acid, and the mixture was centrifuged at 9000 rpm for 30 min. The obtained product was centrifuged again under the same conditions, and the product after centrifugation was dissolved in deionized water to obtain a ligand-free UCNPs solution with a molar concentration of 0.005 mol / L.

[0075] Step 6: Mix the ligand-free UCNPs solution with ssDNA (nucleotide sequence as shown in SEQ ID NO.1 in the sequence table) at a molar ratio of 1:10 and stir at room temperature for 2.5 hours. Then, wash the above product with dd-Water at 4°C, centrifuge at 6500rpm for 15 minutes each time, for a total of 3 times. After that, ultrasonicate in an ice water bath for 30 minutes and centrifuge at 300rpm for 5 minutes to remove the precipitate. Finally, the UCNPs-ssDNA fluorescent nanoprobe was synthesized and stored at 4°C.

[0076] Step 7: Prepare ZnO inverse opal photonic crystals with pore sizes of 200-800nm ​​by using the sol-gel method combined with the PMMA microsphere vertical self-assembly template method. First, monodisperse PMMA latex spheres with a particle size of 200nm were synthesized by emulsifier-free emulsion polymerization technology. The cleaned glass sheet was vertically inserted into an aqueous solution containing 5% PMMA latex spheres. The 200nm PMMA microspheres were self-assembled in an oven at 35°C using a vertical deposition self-assembly process, and the drying lasted for 2 days. During the evaporation process, driven by the surface tension of the liquid, the PMMA colloidal spheres slowly self-assembled into a highly ordered colloidal array on the glass substrate. After deposition, they were sintered at 120°C for 40 minutes to obtain a PMMA opal template to improve its physical strength.

[0077] Step 8: Dissolve Zn(NO3)2·6H2O and citric acid in water and alcohol solution to form a 0.5mM homogeneous precursor solution, with citric acid as a chelating agent in the mixed solution. Then, the prepared precursor solution is infiltrated into the voids of the PMMA opal template by capillary force. The resulting product is dried in air at room temperature for 24h, heated to 500℃ at a heating rate of 1℃ / min, then kept in air at 120℃ for 3h, and then dried at room temperature for 3h to obtain a ZnO inverse opal photonic crystal with a pore size of 200nm.

[0078] Step 9: Take 10 μL of the UCNPs-ssDNA fluorescent nanoprobe prepared in step 6 and drop it on the 200 nm ZnO inverse opal photonic crystal prepared in step 8 to prepare a photonic crystal / UCNP composite structure. Use Tris-HCl buffer (pH = 8.0) to dissolve the MoS2 nanosheets to obtain a 0.09 μg / mL MoS2 nanosheet solution, which is then drop-coated on the photonic crystal / UCNP composite structure. In the absence of targeted miRNA-155, the MoS2 nanosheets can be used as energy collectors to bind to ssDNA through van der Waals forces, thereby triggering fluorescence resonance energy transfer between UCNPs-ssDNA and MoS2 nanosheets, causing the upconversion emission of UCNP to be quenched, and obtaining a microfluidic chip.

[0079] Step 10: Add the targeted detection miRNA-155 to the microfluidic chip obtained in step 9, and record the upconversion fluorescence recovery of UCNP after reacting at room temperature for 1 hour. The microfluidic chip obtained by the above preparation method is used to detect the breast cancer tumor marker miRNA-155 in vitro, and an atomic force microscope and an in-situ fluorescence lifetime imaging system are used to track the position of the single particle and record its fluorescence spectrum and lifetime information. In the presence of targeted miRNA-155, miRNA-155 can hybridize with the ssDNA on the UCNP to inhibit the close contact between UCNPs-ssDNA and MoS2 nanosheets, block the fluorescence resonance energy transfer between them, and restore the upconversion fluorescence of UCNP. At the same time, as the concentration of targeted miRNA-155 increases, within the range of 1.0pM-0.6nM, the single particle sensor has a good linear relationship and gradually tends to saturation with the increase of concentration. The inverse opal photonic crystal structure can regulate the upconversion fluorescence intensity of a single nanoparticle through a local light field, thereby improving the detection sensitivity of the microfluidic chip. The changes in the concentration of miRNA-155 can be visualized in real time through a time-resolution imaging device.

[0080] Embodiment 2: This embodiment provides a method for preparing a microfluidic chip, which comprises the following steps:

[0081] Step 1: Synthesis of upconversion nanoparticles NaYF4:20% Yb by a classical solvothermal method 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ . Take 0.003g of thulium chloride hexahydrate powder, 0.775g of ytterbium chloride hexahydrate powder, and 2.30g of yttrium chloride hexahydrate powder in a three-necked bottle, add 60mL of oleic acid and 150mL of octadecene solution, fix it in an oil bath, under nitrogen protection at 160℃, stir at 700rpm until the drugs in the three-necked bottle are completely dissolved, and then cool to 30℃.

[0082] Step 2: Take 1.0g of sodium hydroxide and 1.48g of ammonium fluoride powder and dissolve them in 60mL of methanol solution. Slowly add the methanol mixed solution into the mixed solution obtained in step 1, stir thoroughly for 1h, and then raise the temperature from room temperature to 125°C. During the heating process, bubbles will continue to emerge from the three-necked flask. After there is no foam in the three-necked flask, connect the condenser, slowly raise the temperature of the solution in the three-necked flask to 300°C, stir thoroughly for 2h, stop heating and lower the temperature to 30°C.

[0083] Step 3: The mixed solution obtained in step 2 was divided into centrifuge tubes, 150 mL of ethanol solution was added to the centrifuge tubes, and centrifuged at 10000 rpm for 20 min. The supernatant was poured out, 160 mL of cyclohexane solution was added, and the precipitate was dissolved by ultrasonication. Then 200 mL of ethanol solution was added and centrifuged again. This step was repeated twice to obtain NaYF4:20%Yb 3+ ,0.1%Tm 3+ precipitation.

[0084] Step 4: Prepare core-shell upconversion nanoparticles by homogeneous encapsulation. Take 0.775g of ytterbium chloride hexahydrate powder, 2.327g of yttrium chloride hexahydrate powder, and 0.04g of holmium chloride hexahydrate powder in a three-necked bottle, add the upconversion nanoparticle precipitate obtained in step 3, and then add 60mL of oleic acid and 150mL of octadecene solution. Place in a fixed oil bath, under nitrogen protection at 160°C, stir at 700rpm until the drugs in the three-necked bottle are completely dissolved, and then cool to 30°C. After repeating steps 2-3, NaYF4:20%Yb 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ The prepared precipitates of upconversion nanoparticles were dissolved in cyclohexane to obtain a cyclohexane solution of upconversion nanoparticles with a molar concentration of 0.05 mol / L.

[0085] Step 5: Washing NaYF4:20% Yb with hydrochloric acid solution 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ Surface ligands of nanoparticles. Take 10 mL of NaYF4:20% Yb 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ 1.6 mol hydrochloric acid was added to the cyclohexane solution and ultrasonic treatment was performed for 5 min. Then, ethanol was centrifuged twice. After the first centrifugation, 10 mL of NaYF4:20% Yb 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ 30 mL of ethanol was added to the mixed solution of cyclohexane and hydrochloric acid, and the mixture was centrifuged at 9000 rpm for 30 min. The obtained product was centrifuged again under the same conditions, and the product after centrifugation was dissolved in deionized water to obtain a ligand-free UCNPs solution with a molar concentration of 0.05 mol / L.

[0086] Step 6: Mix the ligand-free UCNPs solution with ssDNA (nucleotide sequence as shown in the sequence table SEQ ID NO.1) at a molar ratio of 1:20 and stir at room temperature for 2.5 hours. Then, wash the above product with dd-Water at 4°C, centrifuge at 6500rpm for 15 minutes each time, for a total of 3 times. After that, ultrasonicate in an ice water bath for 30 minutes and centrifuge at 300rpm for 5 minutes to remove the precipitate. Finally, the UCNPs-ssDNA fluorescent nanoprobe was synthesized and stored at 4°C. The TEM image of the UCNPs-ssDNA fluorescent nanoprobe is shown below. Figure 3 As shown in a; the absorption spectra of Tm@Ho UCNPs without ssDNA modification and the UCNPs-ssDNA fluorescent nanoprobe prepared as shown Figure 3 As shown in b, the UCNPs-ssDNA fluorescent nanoprobe prepared above has an obvious characteristic absorption peak of ssDNA at 260nm.

[0087] Step 7: Prepare ZnO inverse opal photonic crystals with pore sizes of 200-800nm ​​by using the sol-gel method combined with the PMMA microsphere vertical self-assembly template method. First, monodisperse PMMA latex spheres with a particle size of 600nm were synthesized by emulsifier-free emulsion polymerization technology. The cleaned glass sheet was vertically inserted into an aqueous solution containing 5% PMMA latex spheres. The 600nm PMMA microspheres were self-assembled in an oven at 35°C using a vertical deposition self-assembly process, and the drying lasted for 2 days. During the evaporation process, driven by the surface tension of the liquid, the PMMA colloidal spheres slowly self-assembled into a highly ordered colloidal array on the glass substrate. After deposition, they were sintered at 120°C for 40 minutes to obtain a PMMA opal template to improve its physical strength.

[0088] Step 8: Dissolve Zn(NO3)2·6H2O and citric acid in water and alcohol solution to form a 1mM homogeneous precursor solution, with citric acid as a chelating agent in the mixed solution. Then, the prepared precursor solution is infiltrated into the voids of the PMMA opal template by capillary force. The resulting product is dried in air at room temperature for 24 hours, heated to 500°C at a heating rate of 1°C / min, then kept in air at 120°C for 3 hours, and then dried at room temperature for 3 hours to obtain a ZnO inverse opal photonic crystal with a pore size of 600nm, and its TEM image is shown in the figure below. Figure 2 shown.

[0089] Step 9: Take 10 μL of the UCNPs-ssDNA fluorescent nanoprobe prepared in step 6 and drop it on the 200 nm ZnO inverse opal photonic crystal prepared in step 8 to prepare a photonic crystal / UCNP composite structure, the SEM top view of which is shown in FIG. Figure 4As shown. MoS2 nanosheets were dissolved in Tris-HCl buffer (pH = 8.0) to obtain a 50 μg / mL MoS2 nanosheet solution, which was then drop-coated on the photonic crystal / UCNP composite structure. In the absence of targeted miRNA-155, MoS2 nanosheets as energy collectors can bind to ssDNA through van der Waals forces, thereby triggering fluorescence resonance energy transfer between UCNPs-ssDNA and MoS2 nanosheets, causing the upconversion emission of UCNP to be quenched, and a microfluidic chip was obtained.

[0090] Step 10: Add the target detection miRNA-155 to the microfluidic chip obtained in step 9, react at room temperature for 2 hours, and record the upconversion fluorescence recovery of UCNP. The results are as follows: Figure 5 As shown; Figure 5 The figure reflects the fluorescence recovery of the microfluidic chip prepared in the embodiment of the present invention under the condition of different concentrations of miRNA-155, where F0 represents the initial quenched upconversion fluorescence, and F represents the intensity after fluorescence recovery; it can be seen from the figure that when the concentration of miRNA-155 is 5pM-1.5nM, the fluorescence recovery degree of the microfluidic chip prepared in the embodiment of the present invention shows a good linear relationship, and as the concentration of miRNA-155 gradually increases, the fluorescence recovery gradually tends to saturation. The microfluidic chip obtained by the above preparation method is used to detect the breast cancer tumor marker miRNA-155 in vitro, and an atomic force microscope and an in-situ fluorescence lifetime imaging system are used to track the position of a single particle and record its fluorescence spectrum and lifetime information. In the presence of targeted miRNA-155, miRNA-155 can hybridize with ssDNA on UCNP to inhibit the close contact between UCNPs-ssDNA and MoS2 nanosheets, blocking the fluorescence resonance energy transfer between them, while restoring the upconversion fluorescence of UCNP. At the same time, as the concentration of targeted miRNA-155 increases, within the range of 1.0pM-0.6nM, the single particle sensor has a good linear relationship and gradually tends to saturation with the increase of concentration. The inverse opal photonic crystal structure can regulate the upconversion fluorescence intensity of a single nanoparticle through a localized light field, thereby improving the detection sensitivity of the microfluidic chip, and the change of miRNA-155 concentration can be visualized in real time through a time-resolution imaging device.

[0091] Embodiment 3: This embodiment provides a method for preparing a microfluidic chip, which comprises the following steps:

[0092] Step 1: Synthesis of upconversion nanoparticles NaYF4:30% Yb by a classical solvothermal method 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho3+ . Take 0.88g of thulium chloride hexahydrate powder, 1.163g of ytterbium chloride hexahydrate powder, and 1.165g of yttrium chloride hexahydrate powder in a three-necked bottle, add 60mL of oleic acid and 150mL of octadecene solution, fix it in an oil bath, under nitrogen protection at 160℃, stir at 700rpm until the drugs in the three-necked bottle are completely dissolved, and then cool to 30℃.

[0093] Step 2: Take 1.0g of sodium hydroxide and 1.48g of ammonium fluoride powder and dissolve them in 60mL of methanol solution. Slowly add the methanol mixed solution into the mixed solution obtained in step 1, stir thoroughly for 1h, and then raise the temperature from room temperature to 125°C. During the heating process, bubbles will continue to emerge from the three-necked flask. After there is no foam in the three-necked flask, connect the condenser, slowly raise the temperature of the solution in the three-necked flask to 300°C, stir thoroughly for 2h, stop heating and lower the temperature to 30°C.

[0094] Step 3: The mixed solution obtained in step 2 was divided into centrifuge tubes, 150 mL of ethanol solution was added to the centrifuge tubes, and centrifuged at 10000 rpm for 20 min. The supernatant was poured out, 160 mL of cyclohexane solution was added, and the precipitate was dissolved by ultrasonication. Then 200 mL of ethanol solution was added and centrifuged again. This step was repeated twice to obtain NaYF4:30% Yb 3+ ,30%Tm 3+ precipitation.

[0095] Step 4: Prepare core-shell upconversion nanoparticles by homogeneous encapsulation. Take 1.163g of ytterbium chloride hexahydrate powder, 1.165g of yttrium chloride hexahydrate powder, and 1.138g of holmium chloride hexahydrate powder in a three-necked bottle, add the upconversion nanoparticle precipitate obtained in step 3, and then add 60mL of oleic acid and 150mL of octadecene solution. Fix the oil bath pot, under nitrogen protection at 160℃, stir at 700rpm until the drugs in the three-necked bottle are completely dissolved, and then cool to 30℃. After repeating steps 2-3, NaYF4:30%Yb 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+ The prepared precipitates of upconversion nanoparticles were dissolved in cyclohexane to obtain a cyclohexane solution of upconversion nanoparticles with a molar concentration of 5 mol / L.

[0096] Step 5: Cleaning NaYF4:30% Yb using hydrochloric acid solution 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+ Nanoparticle surface ligands. Take 10 mL of NaYF4:30% Yb3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+ 1.6 mol hydrochloric acid was added to the cyclohexane solution and ultrasonic treatment was performed for 5 min. Then, ethanol was centrifuged twice. After the first centrifugation, 10 mL of NaYF4:30% Yb 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+ 30 mL of ethanol was added to the mixed solution of cyclohexane and hydrochloric acid, and the mixture was centrifuged at 9000 rpm for 30 min. The obtained product was centrifuged again under the same conditions, and the product after centrifugation was dissolved in deionized water to obtain a ligand-free UCNPs solution with a molar concentration of 5 mol / L.

[0097] Step 6: Mix the ligand-free UCNPs solution with ssDNA (nucleotide sequence as shown in SEQ ID NO.1 in the sequence table) at a molar ratio of 1:50 and stir at room temperature for 2.5 hours. Then, wash the above product with dd-Water at 4°C, centrifuge at 6500rpm each time, wash for 15 minutes, for a total of 3 times. After that, ultrasonicate in an ice water bath for 30 minutes, centrifuge at 300rpm for 5 minutes, and remove the precipitate. Finally, the UCNPs-ssDNA fluorescent nanoprobe was synthesized and stored at 4°C.

[0098] Step 7: Prepare ZnO inverse opal photonic crystals with pore sizes of 200-800nm ​​by using the sol-gel method combined with the PMMA microsphere vertical self-assembly template method. First, monodisperse PMMA latex spheres with a particle size of 800nm ​​were synthesized by emulsifier-free emulsion polymerization technology. The cleaned glass sheet was vertically inserted into an aqueous solution containing 5% PMMA latex spheres. The 800nm ​​PMMA microspheres were self-assembled in an oven at 35°C using a vertical deposition self-assembly process, and the drying lasted for 2 days. During the evaporation process, driven by the surface tension of the liquid, the PMMA colloidal spheres slowly self-assembled into a highly ordered colloidal array on the glass substrate. After deposition, they were sintered at 120°C for 40 minutes to obtain a PMMA opal template to improve its physical strength.

[0099] Step 8: Dissolve Zn(NO3)2·6H2O and citric acid in water and alcohol solution to form a 2mM homogeneous precursor solution, with citric acid as a chelating agent in the mixed solution. Then, the prepared precursor solution is infiltrated into the voids of the PMMA opal template by capillary force. The resulting product is dried in air at room temperature for 24h, heated to 500℃ at a heating rate of 1℃ / min, then kept in air at 120℃ for 3h, and then dried at room temperature for 3h to obtain a ZnO inverse opal photonic crystal with a pore size of 800nm.

[0100] Step 9: Take 10 μL of the UCNPs-ssDNA fluorescent nanoprobe prepared in step 6 and drop it on the 200 nm ZnO inverse opal photonic crystal prepared in step 8 to prepare a photonic crystal / UCNP composite structure. Use Tris-HCl buffer (pH = 8.0) to dissolve the MoS2 nanosheets to obtain a 900 μg / mL MoS2 nanosheet solution, which is then drop-coated on the photonic crystal / UCNP composite structure. In the absence of targeted miRNA-155, the MoS2 nanosheets can be used as energy collectors to bind to ssDNA through van der Waals forces, thereby triggering fluorescence resonance energy transfer between UCNPs-ssDNA and MoS2 nanosheets, causing the upconversion emission of UCNP to be quenched, and obtaining a microfluidic chip.

[0101] Step 10: Add the targeted detection miRNA-155 to the microfluidic chip obtained in step 9, and record the upconversion fluorescence recovery of UCNP after reacting at room temperature for 3 hours. The microfluidic chip obtained by the above preparation method is used to detect the breast cancer tumor marker miRNA-155 in vitro, and an atomic force microscope and an in-situ fluorescence lifetime imaging system are used to track the position of the single particle and record its fluorescence spectrum and lifetime information. In the presence of targeted miRNA-155, miRNA-155 can hybridize with the ssDNA on the UCNP to inhibit the close contact between UCNPs-ssDNA and MoS2 nanosheets, block the fluorescence resonance energy transfer between them, and restore the upconversion fluorescence of UCNP. At the same time, as the concentration of targeted miRNA-155 increases, within the range of 1.0pM-0.6nM, the single particle sensor has a good linear relationship and gradually tends to saturation with the increase of concentration. The inverse opal photonic crystal structure can regulate the upconversion fluorescence intensity of a single nanoparticle through a local light field, thereby improving the detection sensitivity of the microfluidic chip. The changes in the concentration of miRNA-155 can be visualized in real time through a time-resolution imaging device.

[0102] Based on the above-mentioned ideal embodiments of the present invention, the relevant staff can make various changes and modifications without departing from the technical concept of the present invention through the above-mentioned description. The technical scope of the present invention is not limited to the contents of the specification.

Claims

1. A method for preparing a microfluidic chip for detecting breast cancer tumor markers, characterized in that: The following steps are involved: The core-shell structured upconversion nanoparticles are prepared by a solvothermal method and a homogeneous encapsulation method; the matrix of the core-shell structured upconversion nanoparticles is NaYF4 and is doped with Yb 3+ 、Tm 3+ and Ho 3+ ; After the core-shell structured upconversion nanoparticles were treated with ligand-free surface, they were modified with ssDNA complementary to breast cancer tumor markers to obtain UCNPs-ssDNA fluorescent nanoprobes; The UCNPs-ssDNA fluorescent nanoprobe was spin-coated on the ZnO inverse opal photonic crystal to obtain a photonic crystal / UCNP composite structure; The MoS2 nanosheet solution was drop-coated on the composite structure of photonic crystal / UCNP to obtain a microfluidic chip.

2. The method for preparing a microfluidic chip for detecting breast cancer tumor markers according to claim 1, characterized in that: The steps of preparing core-shell structured upconversion nanoparticles by solvothermal method and homogeneous encapsulation method specifically include: A thulium source, a ytterbium source and a yttrium source are heated and mixed with oleic acid and octadecene solution to obtain a mixed solution A; Sodium hydroxide and ammonium fluoride powders are dissolved in a methanol solution, and then slowly added into a mixed solution A, fully stirred, and heated to obtain a mixed solution B; The mixed solution B is centrifuged to obtain upconversion nanoparticle precipitation; Taking an ytterbium source, a yttrium source, a holmium source, and the upconversion nanoparticle precipitate, heating and mixing them with oleic acid and octadecene solution to obtain a mixed solution C; Sodium hydroxide and ammonium fluoride powders are dissolved in a methanol solution, and then slowly added into the mixed solution C, fully stirred, and heated to obtain a mixed solution D; The mixed solution D is centrifuged to obtain upconversion nanoparticles with a core-shell structure.

3. The method for preparing a microfluidic chip for detecting breast cancer tumor markers according to claim 1, characterized in that: The step of performing ligand-free treatment on the surface of the core-shell structured upconversion nanoparticles specifically comprises: adding the upconversion nanoparticles with a core-shell structure into cyclohexane to obtain a cyclohexane solution of the upconversion nanoparticles; Hydrochloric acid was added to the cyclohexane solution of the upconversion nanoparticles for ultrasonic treatment, followed by centrifugation using ethanol, and the product after centrifugation was dissolved in deionized water to obtain a ligand-free UCNPs solution.

4. The method for preparing a microfluidic chip for detecting breast cancer tumor markers according to claim 3, characterized in that: The steps of modifying ssDNA complementary to a breast cancer tumor marker to obtain a UCNPs-ssDNA fluorescent nanoprobe specifically include: The ligand-free UCNPs solution and ssDNA are mixed and stirred at a molar ratio of 1:(10-50), and then washed, sonicated, and centrifuged to obtain a UCNPs-ssDNA fluorescent nanoprobe.

5. The method for preparing a microfluidic chip for detecting breast cancer tumor markers according to claim 1 or 4, characterized in that: The breast cancer tumor marker is miRNA-155; the nucleotide sequence of the ssDNA complementary to the breast cancer tumor marker is shown in the sequence table SEQ ID NO.

1.

6. The method for preparing a microfluidic chip for detecting breast cancer tumor markers according to claim 1, characterized in that: The preparation method of the ZnO inverse opal photonic crystal comprises the following steps: A glass sheet is vertically inserted into an aqueous solution containing PMMA latex balls, and PMMA microspheres are self-assembled using a vertical deposition self-assembly process, and then dried; during the drying process, driven by the surface tension of the liquid, the PMMA microspheres slowly self-assemble into a highly ordered colloidal array on the glass substrate, and then sintered to obtain a PMMA opal template; dissolving zinc nitrate and citric acid in a solvent to form a precursor solution; The precursor solution is infiltrated into the voids of the PMMA opal template by capillary force and dried to obtain a ZnO inverse opal photonic crystal.

7. The method for preparing a microfluidic chip for detecting breast cancer tumor markers according to claim 6, characterized in that: The pore size of the ZnO inverse opal photonic crystal is 200-800nm.

8. The method for preparing a microfluidic chip for detecting breast cancer tumor markers according to claim 1, characterized in that: The concentration of the MoS2 nanosheet solution is 0.09-900 μg / mL.

9. A microfluidic chip made by the preparation method according to any one of claims 1 to 8.

10. Use of the microfluidic chip according to claim 9 in preparing a kit for detecting tumor markers of breast cancer.

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