Microfluidic chip for multi-channel detection of tumor markers as well as preparation method and application of microfluidic chip

Abstract

The invention discloses a microfluidic chip for multi-channel detection of tumor markers and a preparation method and application thereof, and belongs to the technical field of microfluidics, the preparation method of the microfluidic chip comprises the following steps: carrying out surface ligand-free treatment on upconversion nanoparticles of a core-shell structure, then wrapping with polydopamine, and carrying out ultrasonic treatment on the upconversion nanoparticles to obtain the microfluidic chip. Then dissolving the upconversion nanoparticles in ultrapure water to obtain a polydopamine coated upconversion nanoparticle solution; spin-coating the ZnO inverse opal photonic crystal with the up-conversion nanoparticle solution wrapped by the polydopamine to obtain a photonic crystal / single up-conversion nanoparticle composite structure; and dispensing at least two noble metal nanoparticle solutions modified by the ovarian cancer tumor marker aptamer on the photonic crystal / single up-conversion nanoparticle composite structure to obtain the micro-fluidic chip. Compared with the traditional detection means, the micro-fluidic chip has the advantages of low cost, visual real-time detection, reusability and high detection sensitivity.

Description

Technical Field

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

[0002] Rapid and accurate quantitative analysis of tumor markers is of great significance for early warning, screening, early diagnosis and prognosis analysis of tumors. Studies have found that the occurrence and development of many cancers are closely related to the proliferation of multiple tumor markers rather than the proliferation of a single tumor marker. The combined detection of multiple tumor markers plays an important role in the diagnosis of recurrence or metastasis in patients after tumor surgery. Therefore, in order to diagnose cancer more accurately and improve diagnostic reliability, it is necessary to quantitatively detect multiple tumor markers simultaneously. Currently, analytical techniques used for the quantification of multiple tumor markers include electrochemical immunoassay, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, etc. Although these methods have unique advantages, their preparation processes are complex, they require sophisticated experimental instruments and equipment, electrochemical signals interfere with each other, and the detection sensitivity is low. It has become an inevitable trend to develop a fast, easy-to-use, real-time, and reusable visualization strategy that can simultaneously detect multiple tumor markers. The fluorescence resonance energy transfer method has attracted much attention due to its high sensitivity and simplicity. It can be used for detection at the single cell level and can also be combined with a variety of instruments and techniques. These probes are usually excited by visible light photons, which brings serious interference to the spontaneous luminescence of biological samples.

[0003] Most of the existing detection technologies for ovarian cancer tumor markers are based on electrochemical immunoassay, enzyme-linked immunosorbent assay, quantitative polymerase chain reaction, etc. Although these methods have unique advantages, their preparation process is complex, and they have extremely high requirements for laboratory environment and equipment. Electrochemical signals interfere with each other, and the detection sensitivity is low. In addition, most of the above methods can only detect one tumor marker at a time. If you want to detect multiple indicators, you need to design different detection methods for different tumor markers, and joint testing cannot be achieved. This not only greatly reduces the detection efficiency, brings a poor medical experience to patients, but also causes a huge waste of resources. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing a microfluidic chip for multi-channel detection of tumor markers to solve the problems raised in the above background technology.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] A method for preparing a microfluidic chip for multi-channel detection of tumor markers comprises the following steps:

[0007] 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+ ;

[0008] After the core-shell structured upconversion nanoparticles are subjected to ligand-free surface treatment, they are coated with polydopamine and then dissolved in ultrapure water to obtain a polydopamine-coated upconversion nanoparticle solution;

[0009] The upconversion nanoparticle solution wrapped with polydopamine is spin-coated on the ZnO inverse opal photonic crystal to obtain a photonic crystal / single upconversion nanoparticle composite structure;

[0010] At least two noble metal nanoparticle solutions modified with ovarian cancer tumor marker aptamers are drop-coated on a photonic crystal / single upconversion nanoparticle composite structure to obtain a microfluidic chip; the ovarian cancer tumor markers include at least cancer antigen CA125 and human epididymis protein HE4.

[0011] Preferably, the steps of preparing the core-shell structured upconversion nanoparticles by a solvothermal method and a homogeneous encapsulation method specifically include:

[0012] 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;

[0013] 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;

[0014] The mixed solution B is centrifuged to obtain upconversion nanoparticle precipitation;

[0015] 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;

[0016] 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;

[0017] The mixed solution D is centrifuged to obtain upconversion nanoparticles with a core-shell structure.

[0018] Preferably, the step of subjecting the core-shell structured upconversion nanoparticles to surface ligand-free treatment specifically comprises:

[0019] adding the upconversion nanoparticles with a core-shell structure into cyclohexane to obtain a cyclohexane solution of the upconversion nanoparticles;

[0020] Hydrochloric acid is added to the cyclohexane solution of the upconversion nanoparticles for ultrasonic treatment, followed by centrifugation using ethanol, and the product after centrifugation is dissolved in deionized water to obtain an aqueous solution of the upconversion nanoparticles without ligands.

[0021] Preferably, the step of encapsulating with polydopamine and then dissolving in ultrapure water to obtain a polydopamine-encapsulated upconversion nanoparticle solution specifically comprises:

[0022] Adding surfactant CO-520 to an aqueous solution of upconversion nanoparticles without ligands, followed by adding ammonium hydroxide solution, and performing stirring and ultrasonic treatment; then, injecting dopamine hydrochloride aqueous solution at a certain speed and stirring, then adding excess ethanol to precipitate nanoparticles, and washing with ethanol and ultrapure water to obtain polydopamine-coated upconversion nanoparticles;

[0023] The polydopamine-wrapped upconversion nanoparticles are redispersed in ultrapure water to obtain a polydopamine-wrapped upconversion nanoparticle solution.

[0024] Preferably, the method for preparing the ZnO inverse opal photonic crystal comprises the following steps:

[0025] 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;

[0026] dissolving zinc nitrate and citric acid in a solvent to form a precursor solution;

[0027] 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.

[0028] Preferably, the pore size of the ZnO inverse opal photonic crystal is 200-800 nm.

[0029] Preferably, the method for preparing the noble metal nanoparticle solution modified with the ovarian cancer tumor marker aptamer comprises the following steps:

[0030] The disulfide bonds of the ovarian cancer tumor marker aptamer were cleaved using a thiol reducing agent;

[0031] The noble metal nanoparticles are added to the ovarian cancer tumor marker aptamer after cleavage, and then a NaCl solution is added, stirred, and then centrifuged. The precipitate is resuspended in a buffer solution to obtain a noble metal nanoparticle solution modified with the ovarian cancer tumor marker aptamer.

[0032] Preferably, the noble metal nanoparticles are Au nanoparticles.

[0033] Another object of an embodiment of the present invention is to provide a microfluidic chip manufactured by the above-mentioned manufacturing method.

[0034] 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 simultaneously detecting multiple ovarian cancer tumor markers.

[0035] The preparation method of the microfluidic chip for multi-channel detection of tumor markers provided by the present invention can realize the visual joint detection of multiple ovarian cancer tumor markers based on a fluorescence resonance energy transfer method by designing a photonic crystal / single upconversion nanoparticle composite structure. Compared with traditional detection methods, the microfluidic chip of the present invention has the advantages of low cost, visual real-time detection, reusability, and high detection sensitivity. At the same time, the material of the upconversion nanoparticles prepared by the present invention has low 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

[0036] Figure 1 A schematic diagram of a process for preparing a microfluidic chip for multi-channel tumor marker detection provided by an embodiment of the present invention;

[0037] Figure 2 TEM image of the ZnO inverse opal photonic crystal prepared in the embodiment of the present invention;

[0038] Figure 3 TEM image of upconversion nanoparticles wrapped with polydopamine prepared in an embodiment of the present invention;

[0039] Figure 4 TEM images of precious metal nanoparticles modified with ovarian cancer tumor marker aptamers prepared in the embodiments of the present invention; in the figure, a is a TEM image of Au nanoparticles modified with aptamer CA125; b is a TEM image of Au nanoparticles modified with aptamer HE4;

[0040] Figure 5 This is a SEM top view of a photonic crystal / single upconversion nanoparticle composite structure prepared in an embodiment of the present invention;

[0041] Figure 6 This is the up-conversion emission spectrum of the microfluidic chip prepared in the embodiment of the present invention in the presence of different concentrations of CA124 and HE4 antigens. DETAILED DESCRIPTION

[0042] 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.

[0043] Lanthanide-doped upconversion nanoparticles are widely used in the biomedical field due to their unique photon upconversion properties, photostability, no background light interference, and almost no damage to biological tissues. Their upconversion luminescence can be adjusted by changing the lanthanide dopant, and then designed into an ideal luminescent nanoprobe. Through the local field modulation of the photonic crystal structure, the electric field intensity around the rare earth-doped upconversion nanoparticles can be changed, and the multi-color emission of the nanoparticles can be achieved for the simultaneous quantitative detection of multiple tumor markers.

[0044] In particular, the microfluidic chip for multi-channel detection of tumor markers based on single particles can not only eliminate the statistical average effect of fluorescent probes based on integrated nanoparticles, but also realize the multiple recycling of a single microfluidic chip because the single particle can be firmly embedded in the inverse opal photonic crystal, greatly reducing the detection cost. The dual-channel microfluidic chip for ovarian cancer tumor markers based on a single rare earth upconversion nanoparticle can greatly improve the detection sensitivity of tumor markers and realize real-time dynamic visualization detection.

[0045] Specifically, Figure 1 As shown, in one embodiment of the present invention, a method for preparing a microfluidic chip for multi-channel detection of tumor markers is provided, which comprises the following steps:

[0046] S1. Prepare upconversion nanoparticles with core-shell structure that can generate 550nm and 650nm light by 980nm light excitation through solvothermal method and homogeneous encapsulation method; the matrix of the upconversion nanoparticles with core-shell structure is NaYF4, and it is doped with Yb 3+ 、Tm 3+ and Ho 3+ ;

[0047] S2, after surface-free ligand treatment of the core-shell structured upconversion nanoparticles, polydopamine is used to encapsulate the nanoparticles, and then the nanoparticles are dissolved in ultrapure water to obtain a polydopamine-encapsulated upconversion nanoparticle solution with a molar concentration of 0.005-5 mol / L;

[0048] S3, spin coating the upconversion nanoparticle solution wrapped with polydopamine on the ZnO inverse opal photonic crystal to obtain a photonic crystal / single upconversion nanoparticle composite structure;

[0049] S4. Drop-coat at least two noble metal nanoparticle solutions modified with ovarian cancer tumor marker aptamers on a photonic crystal / single upconversion nanoparticle composite structure to obtain a microfluidic chip as a single-particle multi-channel upconversion fluorescent probe for simultaneous detection of multiple ovarian cancer tumor markers; ovarian cancer tumor markers include at least cancer antigen CA125 and human epididymal protein HE4.

[0050] 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:

[0051] 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;

[0052] 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;

[0053] S13, centrifuging the mixed solution B to obtain upconversion nanoparticle precipitation;

[0054] 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;

[0055] 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;

[0056] S16, centrifuging the mixed solution D to obtain upconversion nanoparticles with a core-shell structure.

[0057] In a preferred embodiment of the present invention, the step of subjecting the core-shell structured upconversion nanoparticles to surface ligand-free treatment specifically comprises:

[0058] S21, adding the upconversion nanoparticles with a core-shell structure into cyclohexane to obtain a cyclohexane solution of the upconversion nanoparticles;

[0059] 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 an aqueous solution of ligand-free upconversion nanoparticles.

[0060] In a preferred embodiment of the present invention, the steps of encapsulating with polydopamine and then dissolving in ultrapure water to obtain a polydopamine-encapsulated upconversion nanoparticle solution specifically include:

[0061] S23. Add surfactant CO-520 to the aqueous solution of ligand-free upconversion nanoparticles, and then add ammonium hydroxide solution, and stir and ultrasonicate; then, inject dopamine hydrochloride aqueous solution at a certain speed and stir, then add excess ethanol to precipitate the nanoparticles, and wash with ethanol and ultrapure water to obtain polydopamine-coated upconversion nanoparticles; wherein the surfactant CO-520 can specifically be commercially available Igepal CO-520.

[0062] S24. Re-dispersing the polydopamine-wrapped upconversion nanoparticles in ultrapure water to obtain a polydopamine-wrapped upconversion nanoparticle solution.

[0063] In a preferred embodiment of the present invention, the method for preparing the ZnO inverse opal photonic crystal comprises the following steps:

[0064] 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;

[0065] S32, dissolving zinc nitrate and citric acid in a solvent to form a precursor solution;

[0066] 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.

[0067] In a preferred embodiment of the present invention, the pore size of the ZnO inverse opal photonic crystal is 200-800 nm.

[0068] In a preferred embodiment of the present invention, the method for preparing a noble metal nanoparticle solution modified with an ovarian cancer tumor marker aptamer comprises the following steps:

[0069] S41, cleaving the disulfide bond of the ovarian cancer tumor marker aptamer using a thiol reducing agent (TCEP);

[0070] S42. Add precious metal nanoparticles to the cleaved ovarian cancer tumor marker aptamer, then add NaCl solution, stir, and then centrifuge. Take the precipitate and resuspend it in 0.01-10mL Tris-HCl buffer to obtain a precious metal nanoparticle solution modified with the ovarian cancer tumor marker aptamer.

[0071] In a preferred embodiment of the present invention, the noble metal nanoparticles are Au nanoparticles.

[0072] In another embodiment of the present invention, a microfluidic chip prepared by the above-mentioned preparation method is provided, which is a fluorescent nanoprobe based on an inverse opal photonic crystal / single upconversion nanoparticle structure, wherein the photonic crystal structure is a ZnO inverse opal photonic crystal with a microsphere size of 200-800nm; the upconversion nanoparticle is NaYF4: 10%-30% Yb 3+ ,0.05%-30%Tm 3+ @NaYF4:10%-30%Yb 3+ ,0.1%-30%Ho 3+ (abbreviated as Tm@Ho), can convert the excitation light into visible light through the anti-Stokes process under 980nm laser irradiation. After the local light field regulation of the inverse opal photonic crystal structure, the upconversion emission of a single upconversion nanoparticle at 550nm and 650nm is significantly enhanced. 125 ) and human epididymis protein HE4 (abbreviated as Apt4) aptamers were used to label precious metal Au nanoparticles with absorption peaks at 550nm and 650nm, respectively. 550 -Apt 125 and Au 650 -Apt4 as energy receptor, NaYF4 coated with polydopamine PDA: 10%-30% Yb 3+ ,0.05%-30%Tm 3+ @NaYF4:10%-30%Yb 3+ ,0.1%-30%Ho 3+ Upconversion nanoparticles (abbreviated as Tm@Ho@PDA) are used as energy donors. 550 -Apt 125 and Au 650 After Apt4 is dropped into the composite structure of ZnO inverse opal photonic crystal / Tm@Ho@PDA, Au 550 -Apt 125 and Au 650-Apt4 is simultaneously adsorbed on the Tm@Ho@PDA surface through π-π stacking and hydrogen bonding interactions, and fluorescence resonance energy transfer occurs at this time, and the upconversion emission of a single rare earth upconversion nanoparticle at 550nm and 650nm is significantly suppressed. Since the aptamers of CA125 and HE4 can split the connection between Au nanospheres-aptamer-upconversion nanoparticles and are easier to conjugate with CA125 and HE4 antigens, when the microfluidic chip is used to detect CA125 and HE4 antigens, as the antigen concentration increases, the fluorescence resonance energy transfer effect is gradually weakened, and the upconversion emission of a single nanoparticle at 550nm and 650nm gradually recovers. The recovery process is a linear relationship, so the antigen concentration of CA125 and HE4 can be accurately obtained by the size of the recovered fluorescence intensity, realizing the quantitative detection of dual-channel ovarian cancer tumor markers.

[0073] The microfluidic chip prepared by the above preparation method is used to detect ovarian cancer tumor markers CA125 and HE4 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. The upconversion nanoparticles and the noble metal Au nanospheres are energy donors and acceptors, respectively. When the Tm@Ho@PDA upconversion nanoparticles are respectively bonded to the Au modified with the aptamer, 550 -Apt 125 and Au 650 When the noble metal nanoparticles of -Apt4 are connected together, fluorescence resonance energy transfer occurs, resulting in quenching of the upconversion fluorescence of the nanoparticles. As the concentrations of CA125 and HE4 antigens increase, the antigens split the structure between the upconversion nanoparticles-aptamers-Au nanospheres, resulting in the recovery of the upconversion fluorescence of the nanoparticles. Within a certain range, the recovery of the fluorescence intensity is linearly related to the concentrations of CA125 and HE4 antigens, achieving dual-channel real-time detection of ovarian cancer tumor markers CA125 and HE4. The anti-opal photonic crystal structure can not only regulate the upconversion fluorescence intensity of a single nanoparticle through a localized light field, but also provide a stable environment for a single particle. After the sample is continuously flushed with a buffer, the microfluidic chip can be reused repeatedly.

[0074] In summary, the dual-channel ovarian cancer detection 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 ovarian cancer tumor markers CA125 and HE4, and can be used multiple times, with low cost, high stability and detection sensitivity, and the advantages of large-scale production and preparation. More importantly, the microfluidic chip can be connected with aptamers of different tumor markers to realize dual-channel detection of tumor markers other than ovarian cancer, which has extremely strong universality.

[0075] The following embodiments are some specific implementation cases of the present invention in practical applications, but are not limited thereto.

[0076] Embodiment 1: This embodiment provides a method for preparing a microfluidic chip, which comprises the following steps:

[0077] 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+ , can be excited by 980nm light to produce visible light emission at 550nm and 650nm, providing two obvious emission peaks for dual-channel detection. Specifically, 0.0015g of thulium chloride hexahydrate powder, 0.388g of ytterbium chloride hexahydrate powder, and 2.620g of yttrium chloride hexahydrate powder were taken in a three-necked bottle, 60mL of oleic acid and 150mL of octadecene solution were added, and the mixture was fixed in an oil bath pot, and stirred at 700rpm under nitrogen protection at 160℃ until the drugs in the three-necked bottle were completely dissolved, and then cooled to 30℃.

[0078] 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.

[0079] 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.

[0080] Step 4: Prepare core-shell upconversion nanoparticles by homogeneous encapsulation. Specifically, 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 it in an oil bath, under nitrogen protection at 160°C, stir at 700rpm until the medicine in the three-necked bottle is completely dissolved, and then cool to 30°C. After repeating steps 2-3, NaYF4:10%Yb 3+,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%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 0.005 mol / L.

[0081] 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 a 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 an aqueous solution of ligand-free upconversion nanoparticles with a molar concentration of 0.005 mol / L.

[0082] Step 6: Add 0.65 mL of Igepal CO-520 to 10 mL of 0.005 mol / L ligand-free upconversion nanoparticles. Then add 75 L of 28 wt% ammonium hydroxide to the above mixture, stir gently for 20 min, ultrasonicate for 15 min, and stir for 30 min. After that, inject 50 L of 25 wt% dopamine hydrochloride aqueous solution at a rate of 3 L / min and stir for 1 h. Finally, add excess ethanol to the above mixture to precipitate the nanoparticles, and wash them three times with ethanol and ultrapure water. The obtained PDA-coated upconversion nanoparticles were redispersed in ultrapure water to obtain 0.005 mol / L PDA@NaYF4:10%Yb 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho 3+ The aqueous solution is kept as a standby.

[0083] 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, the PMMA opal template was obtained and sintered at 120°C for 40min to improve its physical strength.

[0084] 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.

[0085] Step 9: Using TCEP and NaCl as crosslinking agents, the amino-modified CA125 aptamer and HE4 aptamer were crosslinked with Au spheres (abbreviated as Au) with absorption peaks at 550nm and 650nm, respectively. 550 and Au 650 ) for covalent coupling. First, 5 nmol of TCEP was used to cleave the disulfide bonds of 1 nmol of CA125 aptamer and HE4 aptamer, and incubated at room temperature for 1 h. 550 and 0.3 mL of Au 650 The solution was added to the freshly cleaved CA125 aptamer and HE4 aptamer, respectively, and stirred at room temperature for 16 hours. Then, 10L of 2M NaCl solution was added to the two mixtures at a rate of 5 min / time until the final NaCl concentration reached 60mM. After continuous stirring for 24 hours, the products were collected by centrifugation and washed three times with ultrapure water to obtain Au nanoparticles modified with CA125 aptamer and HE4 aptamer, respectively. 550 -Apt 125 and Au 650 -Apt4. Finally, Au 550 -Apt 125 and Au 650-Apt4 were resuspended in 0.01 mL Tris-HCl (10 mM, 50 mM NaCl, pH = 7.4) buffer for later use. It is worth noting that the above experiments were all carried out in the dark.

[0086] Step 10: 0.005 mol / L PDA@NaYF4:10% Yb obtained in step 6 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho 3+ The aqueous solution was spin-coated (the spin coater was set at a low speed of 200 rpm for 2 s and a high speed of 2000 rpm for 40 s) on the ZnO inverse opal photonic crystal with a pore size of 200 nm obtained in step 8 to prepare a photonic crystal / single upconversion nanoparticle composite structure, and 0.01 mL of the aptamer-modified Au obtained in step 9 was 550 -Apt 125 and Au 650 -Apt4 noble metal nanoparticles were drop-coated on the photonic crystal / single upconversion nanoparticle composite structure as a single-particle dual-channel upconversion fluorescent probe for simultaneous detection of ovarian cancer tumor markers CA125 and HE4. At this time, the upconversion nanoparticles and the noble metal Au nanospheres are energy donors and acceptors, respectively. When PDA@NaYF4:10% Yb 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho 3+ When the upconversion nanoparticles are connected with the aptamer-modified Au550-Apt125 and Au650-Apt4 noble metal nanoparticles, fluorescence resonance energy transfer occurs, resulting in PDA@NaYF4:10%Yb 3+ ,0.05%Tm 3+ @NaYF4:10%Yb 3+ ,0.1%Ho 3+ The upconversion fluorescence of the nanoparticles is quenched.

[0087] Step 11: The microfluidic chip obtained by the above preparation method can be used for in vitro real-time detection of two ovarian cancer tumor markers CA125 and HE4. As the concentration of CA125 and HE4 antigens increases, the antigens split the structure between the upconversion nanoparticles-aptamer-Au nanospheres, resulting in the recovery of the upconversion fluorescence of the nanoparticles. Within a certain range, the recovery of the fluorescence intensity is linearly related to the concentration of CA125 and HE4 antigens, realizing dual-channel real-time detection of ovarian cancer tumor markers CA125 and HE4. The inverse opal photonic crystal structure can not only regulate the upconversion fluorescence intensity of a single nanoparticle through a localized light field, which helps to improve the detection sensitivity of the microfluidic chip, but also provide a stable environment for a single particle. After the sample is continuously flushed with a buffer solution, the microfluidic chip can be repeatedly used.

[0088] Embodiment 2: This embodiment provides a method for preparing a microfluidic chip, which comprises the following steps:

[0089] 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+ , can be excited by 980nm light to produce visible light emission at 550nm and 650nm, providing two obvious emission peaks for dual-channel detection. Specifically, 0.003g of thulium chloride hexahydrate powder, 0.775g of ytterbium chloride hexahydrate powder, and 2.30g of yttrium chloride hexahydrate powder were taken in a three-necked bottle, 60mL of oleic acid and 150mL of octadecene solution were added, and the mixture was fixed in an oil bath pot, and stirred at 700rpm under nitrogen protection at 160℃ until the drugs in the three-necked bottle were completely dissolved, and then cooled to 30℃.

[0090] 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.

[0091] 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%Tm3+ precipitation.

[0092] Step 4: Prepare core-shell upconversion nanoparticles by homogeneous encapsulation. Specifically, 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, fix it in an oil bath, under nitrogen protection at 160°C, stir at 700rpm until the medicine in the three-necked bottle is 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.

[0093] Step 5: Washing NaYF4:20% Yb with hydrochloric acid solution 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ Nanoparticle surface ligands. 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 a 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 an aqueous solution of ligand-free upconversion nanoparticles with a molar concentration of 0.05 mol / L.

[0094] Step 6: Add 0.65 mL of Igepal CO-520 to 10 mL of 0.05 mol / L ligand-free upconversion nanoparticles. Then add 75 L of 28 wt% ammonium hydroxide to the above mixture, gently stir for 20 min, ultrasonicate for 15 min, and stir for 30 min. After that, inject 50 L of 25 wt% dopamine hydrochloride aqueous solution at a rate of 3 L / min and stir for 1 h. Finally, add excess ethanol to the above mixture to precipitate the nanoparticles, and wash them three times with ethanol and ultrapure water. The obtained PDA-coated upconversion nanoparticles were redispersed in ultrapure water to obtain 0.05 mol / L PDA@NaYF4:20%Yb 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ The aqueous solution was used as a standby, PDA@NaYF4:20% Yb 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3 + ,1%Ho 3+ TEM images of upconversion nanoparticles are shown in Figure 2. Figure 3 shown.

[0095] 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, the PMMA opal template was obtained and sintered at 120°C for 40 minutes to improve its physical strength.

[0096] 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.

[0097] Step 9: Using TCEP and NaCl as crosslinking agents, the amino-modified CA125 aptamer and HE4 aptamer were crosslinked with Au spheres (abbreviated as Au) with absorption peaks at 550nm and 650nm, respectively. 550 and Au 650 ) for covalent coupling. First, 5 nmol of TCEP was used to cleave the disulfide bonds of 1 nmol of CA125 aptamer and HE4 aptamer, and incubated at room temperature for 1 h. 550 and 3.3 mL of Au 650 The solution was added to the freshly cleaved CA125 aptamer and HE4 aptamer, respectively, and stirred at room temperature for 16 hours. Then, 10L of 2M NaCl solution was added to the two mixtures at a rate of 5 min / time until the final NaCl concentration reached 60mM. After continuous stirring for 24 hours, the products were collected by centrifugation and washed three times with ultrapure water to obtain Au nanoparticles modified with CA125 aptamer and HE4 aptamer, respectively. 550 -Apt 125 and Au 650 -Apt4, where Au 550 -Apt 125 The TEM image is as follows Figure 4 As shown in a, Au 650 -TEM image of Apt4 Figure 4 Finally, Au 550 -Apt 125 and Au 650 -Apt4 were resuspended in 0.1 mL Tris-HCl (10 mM, 50 mM NaCl, pH=7.4) buffer for later use. It is worth noting that the above experiments were all carried out in the dark.

[0098] Step 10: 0.05 mol / L PDA@NaYF4:20% Yb obtained in step 6 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ The aqueous solution was spin-coated (the spin coater was set to a low speed of 200 rpm for 2 s and a high speed of 2000 rpm for 40 s) on the ZnO inverse opal photonic crystal with a pore size of 600 nm obtained in step 8 to prepare a photonic crystal / single upconversion nanoparticle composite structure, the SEM top view of which is shown in FIG. Figure 5 As shown; 0.1 mL of the aptamer-modified Au obtained in step 9 550 -Apt 125 and Au 650-Apt4 noble metal nanoparticles were drop-coated on the photonic crystal / single upconversion nanoparticle composite structure as a single-particle dual-channel upconversion fluorescent probe for simultaneous detection of ovarian cancer tumor markers CA125 and HE4. At this time, the upconversion nanoparticles and the noble metal Au nanospheres are energy donors and acceptors, respectively. When PDA@NaYF4:20%Yb 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ When the upconversion nanoparticles are connected with the aptamer-modified Au550-Apt125 and Au650-Apt4 noble metal nanoparticles, fluorescence resonance energy transfer occurs, resulting in PDA@NaYF4:20%Yb 3+ ,0.1%Tm 3+ @NaYF4:20%Yb 3+ ,1%Ho 3+ The upconversion fluorescence of the nanoparticles is quenched.

[0099] Step 11: The microfluidic chip prepared by the above method can be used for in vitro real-time detection of two ovarian cancer tumor markers CA125 and HE4; wherein the up-conversion emission spectra of the microfluidic chip in the presence of different concentrations of CA124 and HE4 antigens are as follows: Figure 6 As shown. As the concentration of CA125 and HE4 antigens increases, the antigens split the structure between the upconversion nanoparticles-aptamer-Au nanospheres, resulting in the recovery of the upconversion fluorescence of the nanoparticles. Within a certain range, the recovery of the fluorescence intensity is linearly related to the concentration of CA125 and HE4 antigens, realizing the dual-channel real-time detection of ovarian cancer tumor markers CA125 and HE4. The inverse opal photonic crystal structure can not only regulate the upconversion fluorescence intensity of a single nanoparticle through a local light field, which helps to improve the detection sensitivity of the microfluidic chip, but also provide a stable environment for a single particle. After the sample is continuously flushed with a buffer, the microfluidic chip can be reused repeatedly.

[0100] Embodiment 3: This embodiment provides a method for preparing a microfluidic chip, which comprises the following steps:

[0101] Step 1: Synthesis of upconversion nanoparticles NaYF4:30% Yb by a classical solvothermal method 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+, can be excited by 980nm light to produce visible light emission at 550nm and 650nm, providing two obvious emission peaks for dual-channel detection. Specifically, 0.88g of thulium chloride hexahydrate powder, 1.163g of ytterbium chloride hexahydrate powder, and 1.165g of yttrium chloride hexahydrate powder were taken in a three-necked bottle, 60mL of oleic acid and 150mL of octadecene solution were added, and the mixture was fixed in an oil bath pot, and stirred at 700rpm under nitrogen protection at 160℃ until the drugs in the three-necked bottle were completely dissolved, and then cooled to 30℃.

[0102] 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.

[0103] 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.

[0104] Step 4: Prepare core-shell upconversion nanoparticles by homogeneous encapsulation. Specifically, 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 it in an 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, NaYF4:30%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.

[0105] Step 5: Washing NaYF4:30% Yb with hydrochloric acid solution 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho3+ Nanoparticle surface ligands. Take 10 mL of NaYF4:30% Yb 3+ ,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 a 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 an aqueous solution of ligand-free upconversion nanoparticles with a molar concentration of 5 mol / L.

[0106] Step 6: Add 0.65 mL of Igepal CO-520 to 10 mL of 5 mol / L ligand-free upconversion nanoparticles. Then add 75 L of 28 wt% ammonium hydroxide to the above mixture, gently stir for 20 min, ultrasonicate for 15 min, and stir for 30 min. After that, inject 50 L of 25 wt% dopamine hydrochloride aqueous solution at a rate of 3 L / min and stir for 1 h. Finally, add excess ethanol to the above mixture to precipitate the nanoparticles, and wash them three times with ethanol and ultrapure water. The obtained PDA-coated upconversion nanoparticles were redispersed in ultrapure water to obtain 5 mol / L PDA@NaYF4:30%Yb 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+ The aqueous solution is kept as a standby.

[0107] 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, the PMMA opal template was obtained and sintered at 120°C for 40 minutes to improve its physical strength.

[0108] 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.

[0109] Step 9: Using TCEP and NaCl as crosslinking agents, the amino-modified CA125 aptamer and HE4 aptamer were crosslinked with Au spheres (abbreviated as Au) with absorption peaks at 550nm and 650nm, respectively. 550 and Au 650 ) for covalent coupling. First, 5 nmol of TCEP was used to cleave the disulfide bonds of 1 nmol of CA125 aptamer and HE4 aptamer, and incubated at room temperature for 1 h. 550 and 8.3 mL of Au 650 The solution was added to the freshly cleaved CA125 aptamer and HE4 aptamer, respectively, and stirred at room temperature for 16 hours. Then, 10L of 2M NaCl solution was added to the two mixtures at a rate of 5 min / time until the final NaCl concentration reached 60mM. After continuous stirring for 24 hours, the products were collected by centrifugation and washed three times with ultrapure water to obtain Au nanoparticles modified with CA125 aptamer and HE4 aptamer, respectively. 550 -Apt 125 and Au 650 -Apt4. Finally, Au 550 -Apt 125 and Au 650 -Apt4 were resuspended in 10 mL Tris-HCl (10 mM, 50 mM NaCl, pH = 7.4) buffer for later use. It is worth noting that the above experiments were all carried out in the dark.

[0110] Step 10: 5 mol / L PDA@NaYF4:30% Yb obtained in step 6 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+The aqueous solution was spin-coated (the spin coater was set at a low speed of 200 rpm for 2 s and a high speed of 2000 rpm for 40 s) on the ZnO inverse opal photonic crystal with a pore size of 800 nm obtained in step 8 to prepare a photonic crystal / single upconversion nanoparticle composite structure. 10 mL of the aptamer-modified Au obtained in step 9 was 550 -Apt 125 and Au 650 -Apt4 noble metal nanoparticles were drop-coated on the photonic crystal / single upconversion nanoparticle composite structure as a single-particle dual-channel upconversion fluorescent probe for simultaneous detection of ovarian cancer tumor markers CA125 and HE4. At this time, the upconversion nanoparticles and the noble metal Au nanospheres are energy donors and acceptors, respectively. When PDA@NaYF4:30% Yb 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+ Upconversion nanoparticles were respectively combined with aptamer-modified Au 550 -Apt 125 and Au 650 When the noble metal nanoparticles of -Apt4 are connected together, fluorescence resonance energy transfer occurs, resulting in PDA@NaYF4:30% Yb 3+ ,30%Tm 3+ @NaYF4:30%Yb 3+ ,30%Ho 3+ The upconversion fluorescence of the nanoparticles is quenched.

[0111] Step 11: The microfluidic chip obtained by the above preparation method can be used for in vitro real-time detection of two ovarian cancer tumor markers CA125 and HE4. As the concentration of CA125 and HE4 antigens increases, the antigens split the structure between the upconversion nanoparticles-aptamer-Au nanospheres, resulting in the recovery of the upconversion fluorescence of the nanoparticles. Within a certain range, the recovery of the fluorescence intensity is linearly related to the concentration of CA125 and HE4 antigens, realizing dual-channel real-time detection of ovarian cancer tumor markers CA125 and HE4. The inverse opal photonic crystal structure can not only regulate the upconversion fluorescence intensity of a single nanoparticle through a localized light field, which helps to improve the detection sensitivity of the microfluidic chip, but also provide a stable environment for a single particle. After the sample is continuously flushed with a buffer solution, the microfluidic chip can be repeatedly used.

[0112] 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 multi-channel detection of 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 are subjected to ligand-free surface treatment, they are coated with polydopamine and then dissolved in ultrapure water to obtain a polydopamine-coated upconversion nanoparticle solution; The upconversion nanoparticle solution wrapped with polydopamine is spin-coated on the ZnO inverse opal photonic crystal to obtain a photonic crystal / single upconversion nanoparticle composite structure; At least two noble metal nanoparticle solutions modified with ovarian cancer tumor marker aptamers are drop-coated on a photonic crystal / single upconversion nanoparticle composite structure to obtain a microfluidic chip; the ovarian cancer tumor markers include at least cancer antigen CA125 and human epididymis protein HE4.

2. The method for preparing a microfluidic chip for multi-channel detection of 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 multi-channel detection of 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 is added to the cyclohexane solution of the upconversion nanoparticles for ultrasonic treatment, followed by centrifugation using ethanol, and the product after centrifugation is dissolved in deionized water to obtain an aqueous solution of the upconversion nanoparticles without ligands.

4. The method for preparing a microfluidic chip for multi-channel detection of tumor markers according to claim 3, characterized in that: The steps of encapsulating with polydopamine and then dissolving in ultrapure water to obtain a polydopamine-encapsulated upconversion nanoparticle solution specifically include: Adding surfactant CO-520 to an aqueous solution of upconversion nanoparticles without ligands, followed by adding ammonium hydroxide solution, and performing stirring and ultrasonic treatment; then, injecting dopamine hydrochloride aqueous solution at a certain speed and stirring, then adding excess ethanol to precipitate nanoparticles, and washing with ethanol and ultrapure water to obtain polydopamine-coated upconversion nanoparticles; The polydopamine-wrapped upconversion nanoparticles are redispersed in ultrapure water to obtain a polydopamine-wrapped upconversion nanoparticle solution.

5. The method for preparing a microfluidic chip for multi-channel detection of 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.

6. The method for preparing a microfluidic chip for multi-channel detection of tumor markers according to claim 5, characterized in that: The pore size of the ZnO inverse opal photonic crystal is 200-800nm.

7. The method for preparing a microfluidic chip for multi-channel detection of tumor markers according to claim 1, characterized in that: The method for preparing a noble metal nanoparticle solution modified with an ovarian cancer tumor marker aptamer comprises the following steps: The disulfide bonds of the ovarian cancer tumor marker aptamer were cleaved using a thiol reducing agent; The noble metal nanoparticles are added to the ovarian cancer tumor marker aptamer after cleavage, and then a NaCl solution is added, stirred, and then centrifuged. The precipitate is resuspended in a buffer solution to obtain a noble metal nanoparticle solution modified with the ovarian cancer tumor marker aptamer.

8. The method for preparing a microfluidic chip for multi-channel detection of tumor markers according to claim 1 or 7, characterized in that: The noble metal nanoparticles are Au nanoparticles.

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 simultaneously detecting multiple ovarian cancer tumor markers.

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