Quantum dot coded microsphere life design and regulation method based on phasor analysis method

Abstract

The invention provides a quantum dot coded microsphere life design and regulation method based on a phasor analysis method. The preparation method comprises the following steps: preparing quantum dot coded polymer microspheres with different service lives by virtue of an SPG membrane method; according to the method, dispersion phases corresponding to quantum dot coded polymer microspheres with different service lives are combined according to different proportions, a fluorescence lifetime imaging microscope and a phasor analysis method are combined, and combined coding of signals with different service lives is obtained through hierarchical regulation and control. According to the life combination design and regulation and control method of the fluorescent life coding microspheres, a phasor analysis method and a semi-compasses vector rule are used as guidance, and coding clusters with different lives are obtained in semi-compasses through hierarchical design and accurate regulation and control of fluorescent life signals. Good life decoding is realized for coding microspheres with different lives. Based on a strategy of a phasor diagram, rapid and accurate decoding of a multi-component life combination signal can be realized, the method is applied to multi-factor detection, and practical application of the life signal in optical multiplexing is promoted.

Description

Technical Field

[0001] The invention relates to the field of micro-nano material preparation, and in particular to a lifespan design and regulation method of quantum dot coding microspheres based on a phase analysis method. Background Art

[0002] Fluorescence lifetime is an inherent property of the time dimension. It has good test stability and strong resistance to background interference. It is almost unaffected by factors such as energy transfer and photobleaching. Therefore, it has become a new imaging mode in fields such as biological imaging and multiplexing. However, the test of fluorescence lifetime generally requires long-term collection and analysis of lifetime data. Currently common lifetime data analysis methods require multiple algorithm iterations and are also affected by other factors, such as instrument response function, fluorophore composition, and background noise. As a result, it is impossible to accurately design and control fluorescence lifetime signals, especially multi-exponential decay of multiple components. This hinders the practical application of fluorescence lifetime signals in biological analysis, especially in multiplexing.

[0003] The phasor analysis method is a method to simplify the lifetime information of the original fluorescence lifetime imaging diagram, which can convert the decay signal of each pixel in the image into a point on the phasor diagram. This conversion relationship is presented on the phasor diagram as a semicircular curve with a radius of 0.5 and a center of (G=0.5, S=0), which is called a semicircle. In the phasor semicircle, the pixels of the single exponential decay behavior are located on the semicircle, and the pixels of the multi-exponential decay behavior are located inside the semicircle. Multiple pixels with similar fluorescence decay characteristics are located in similar areas in the phasor diagram, forming a "phasor cluster", which is convenient for data visualization and cluster analysis. An important feature of the phasor diagram is that the phasor endpoints corresponding to the multi-exponential decay process are located in the set composed of the lifetime phasor endpoints corresponding to each single exponential decay component, and the distance from the endpoint is inversely proportional to the intensity of its contribution. These features play an important role in quantifying, visualizing and clustering fluorescence lifetime data.

[0004] At present, the methods of fluorescence lifetime signal processing based on phasor analysis mainly focus on the unmixing analysis of complex fluorescence lifetime signals in biological samples. However, the current problem is that there is a lack of a direct, accurate and visual lifetime processing method that can accurately design, predict and analyze known lifetime combination signals. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method for designing and regulating the life span of quantum dot-encoded microspheres based on phase analysis. Therefore, technicians in this field are committed to developing a method that can accurately design, predict and analyze complex lifespan combination signals, so as to obtain a fast, flexible, simple, accurate and intuitive lifespan decoding method, so as to build a fast and accurate liquid biochip detection platform and provide a powerful tool for disease diagnosis.

[0006] The technical solution provided by the present invention is as follows: <First aspect> A method for preparing quantum dot polymer microspheres comprises the following steps: S1, adding a zinc source and a manganese source in different proportions to a solvent to form a mixed solution, degassing, adding a selenium precursor solution after heating, and then adding a zinc precursor solution to obtain a Mn:ZnSe core solution with different manganese ion doping amounts; S2, degassing the Mn:ZnSe core solutions with different manganese ion doping amounts obtained in step S1, respectively adding zinc / sulfur precursors after heating, and keeping the temperature for a period of time to obtain core-shell quantum dot solutions with different lifespans; S3, dissolving core-shell quantum dot solutions and polymer solutions with different lifespans in an organic solvent to form a dispersed phase; dispersing a surfactant in water to form a continuous phase; passing through an SPG membrane to form an oil-in-water emulsion, and obtaining quantum dot-encoded polymer microspheres with different lifespans after solidification.

[0007] In S1, the ratios of zinc source to manganese source are 40-53:1-3, 10-15:1-3, 4-8:1-3, 1-5:1-3 and 1-3:1-3.

[0008] and / or, the zinc source comprises at least one of zinc oxide, zinc acetate, zinc stearate, zinc acetate and zinc sulfate; and / or, the manganese source comprises at least one of manganese oxide, manganese stearate, manganese acetate, and manganese dichloride; And / or, the concentration of the zinc source in the mixed solution formed by mixing the zinc source and the manganese source is 0.01-0.03 mol / L, and the concentration of the manganese source is 0.0005-0.001 mol / L.

[0009] Step S1 includes the following steps: S11, preparing a zinc precursor: mixing a zinc source, oleic acid and octadecene, stirring and heating to 120-130° C. under a protective atmosphere for vacuum treatment, then reintroducing the protective atmosphere and heating to 280-300° C. for 20-30 min, and then cooling to 250-260° C. to obtain a zinc precursor solution; Preparation of selenium precursor: mixing selenium powder, octadecene and oleylamine, and ultrasonically dispersing to obtain a selenium precursor solution; the concentration of the selenium precursor solution is 0.01-0.03 g / L; S12, preparing Mn: ZnSe core quantum dot stock solution: respectively mixing zinc source, octadecene and different amounts of manganese source, stirring under protective atmosphere and heating to 120-130°C for vacuum treatment, then reintroducing protective atmosphere and heating to 280-300°C, injecting selenium precursor, keeping the temperature at 300-320°C for 5-10 min, injecting zinc precursor solution, keeping the temperature at 300-320°C for 5-10 min, cooling to 75-85°C to obtain Mn: ZnSe core quantum dot stock solutions with different amounts of manganese ions doped; S13, purifying Mn:ZnSe core quantum dots: mixing the core quantum dot stock solution obtained in step S12 with n-hexane, adding acetone or anhydrous ethanol to precipitate the quantum dots, collecting the precipitate by centrifugation, and washing it several times with a mixture of n-hexane and acetone to obtain purified Mn:ZnSe core solutions with different manganese ion doping amounts.

[0010] In step S11, the zinc source includes zinc oxide.

[0011] In step S12, the zinc source S11 is zinc stearate; and the manganese source includes manganese stearate.

[0012] In S2, the Zn(OA)2-OT mixed precursor solution is prepared by the following method: adding a zinc source to a solvent for degassing, heating to 300-305 o C, keep warm for 10-30min, cool down to 130-160 o C and then add the sulfur source.

[0013] In S2, the Zn(OA)2-OT mixed precursor solution is prepared by the following method: adding a zinc source to a solvent for degassing, heating to 300-305 o C, keep warm for 10-30min, cool down to 130-160 o C followed by the addition of a sulfur source; Wherein, the sulfur source is at least one of n-octyl mercaptan, thioacetamide and sulfur powder; and / or, the zinc source comprises at least one of zinc acetate, zinc stearate, zinc acetate and zinc sulfate; and / or, the concentration of the zinc source is 0.01-0.03 mol / L, and the concentration of the manganese source is 0.0005-0.001 mol / L; And / or, the solvent is a mixed solution of n-octadecene and oleic acid, and the volume ratio of n-octadecene to oleic acid is 0.5-1.5:0.5-1.5.

[0014] As an embodiment of the present invention, step S2 includes the following steps: S21, preparing a Zn(OA)2-OT mixed precursor solution: mixing zinc oxide, oleic acid and octadecene, stirring under a protective atmosphere and heating to 100-120°C for vacuum treatment, then introducing a protective atmosphere and heating to 300-320°C and keeping the temperature for 10-20 min, cooling to 120-150°C, injecting n-octyl mercaptan and keeping the temperature at 120-150°C to obtain a Zn(OA)2-OT precursor solution; S22. Preparation of Mn:ZnSe / ZnS core-shell structure quantum dots: The Mn:ZnSe core solution with different manganese ion doping amounts obtained in step S1 is dissolved in octadecene, and the temperature is raised to 100-120°C under a protective atmosphere for vacuum treatment. Subsequently, a protective atmosphere is introduced, and the temperature is raised to 300-320°C for growth of a ZnS shell layer. The Zn(OA)2-OT precursor solution of step S21 is injected into the core. After keeping the temperature at 300-320°C for 30-60 min, Mn:ZnSe / ZnS core-shell structure quantum dot solutions with different manganese ion doping amounts are obtained.

[0015] The protective atmosphere mentioned above includes inert gases such as nitrogen and argon.

[0016] In step S3, the polymer includes at least one of polystyrene-maleic anhydride copolymer, polystyrene, polylactic acid, styrene-acrylic acid copolymer, polyvinyl chloride, polymethyl methacrylate, and polyethyl acetate.

[0017] In S3, the concentration of the polymer in the dispersed phase is 0.5-2 g / mL.

[0018] In S3, the concentration of the Mn:ZnSe / ZnS core-shell structure quantum dots in the dispersed phase is 0-50 mg / mL.

[0019] In S3, the organic solvent includes at least one of toluene, ethylbenzene, chloroform, dichloromethane, acetone, acetonitrile, and ether.

[0020] In S3, the surfactant includes one of polyvinyl alcohol, sodium dodecylbenzene sulfonate, fatty alcohol polyoxyethylene ether sulfate, and sodium dodecyl sulfate.

[0021] In S3, the concentration of the surfactant in the continuous phase is 0.001-0.01 g / mL.

[0022] <Second Aspect> A method for controlling different lifetime combination signals of quantum dot coded microspheres, the method comprising the following steps: The dispersed phases corresponding to the quantum dot-encoded polymer microspheres with different lifetimes described above are combined in different proportions, combined with fluorescence lifetime imaging microscopy and phase analysis, and a method for encoding different lifetime signal combinations is obtained through hierarchical regulation.

[0023] The photon number of the dispersed phase of the mixed quantum dot solution with different lifetimes and the polymer under the same excitation intensity is consistent. The mixing ratio of the quantum dot solution with different lifetimes in the dispersed phase is adjusted, and the position of the quantum dot microspheres with different lifetimes in the semicompass is designed, predicted and analyzed according to the vector addition principle in the semicompass.

[0024] The hierarchical control method comprises the following steps: Step 1, core-shell quantum dot solutions with different lifespans are mixed with polymer solutions respectively, and the concentration is adjusted using a diluent under the same excitation light so that the number of photons emitted is consistent; Step 2: Prepare a microsphere suspension with the same photon number and different lifespans by SPG membrane emulsification method under the action of continuous phase from a dispersed phase solution with the same photon number and different lifespans; place the microsphere suspension with the same photon number and different lifespans under a lifetime imaging microscope for detection, collect the lifetime signals and convert them into clusters on the phase semicircle, which are defined as quantum first-order lifetime quantum dot coded microspheres; Step 3, the dispersed phases corresponding to the two different primary lifetime quantum dot coded microspheres are mixed in different volume ratios to form a dispersed phase solution, and a secondary lifetime coded microsphere suspension is prepared under the action of a continuous phase by an SPG membrane emulsification method; the secondary lifetime coded microsphere suspension is placed under a lifetime imaging microscope for detection, and the lifetime signal is collected and converted into clusters on a phase semicircle, each cluster representing different secondary lifetime information; Step 4: The dispersed phase corresponding to the primary lifetime quantum dot coded microspheres and the dispersed phase corresponding to the secondary lifetime coded microspheres are mixed in different volume ratios to form a dispersed phase solution, and a tertiary lifetime coded microsphere suspension is prepared under the action of a continuous phase by an SPG membrane emulsification method; the tertiary lifetime coded microsphere suspension is placed under a lifetime imaging microscope for detection, and the lifetime signal is collected and converted into clusters on a phase semicircle, each cluster representing different tertiary lifetime information; Step 5, selecting at least two dispersed phases corresponding to coded microspheres of different levels and mixing them in different volume ratios to form a dispersed phase solution, and preparing a multi-level lifetime coded microsphere suspension under the action of a continuous phase by an SPG membrane emulsification method; placing the three-level lifetime coded microsphere suspension under a lifetime imaging microscope for detection, collecting lifetime signals and converting them into clusters on a phase semicircle, each cluster representing different multi-level lifetime information; Step 6: Analyze and decode the lifetime signal in biological detection through the positions of each lifetime cluster in the phasor diagram.

[0025] <Third Aspect> The application of different lifetime combination signal control methods of quantum dot-encoded microspheres as described above in multi-factor detection.

[0026] <Fourth Aspect> Application of quantum dot-encoded microspheres with different lifetimes in tumor marker detection.

[0027] <Fifth Aspect> A method for detecting tumor markers for non-disease diagnosis purposes, comprising the following steps: Step 1, incubate quantum dot-encoded microspheres with different lifespans and capture proteins corresponding to different tumor markers at room temperature, and couple the capture proteins on the surface of the microspheres (one protein is coupled to each microsphere); Step 2, adding the microspheres coupled with the capture protein into the reaction container, adding different concentrations of antigen standard solution for incubation, then adding biotinylated secondary antibody solution for secondary incubation, and washing to remove unbound components; Step 3: Based on step 2, streptavidin-phycoerythrin (SAPE) is added for incubation, and the mixture is resuspended in washing buffer; Step 4, adding the microsphere suspension mixture obtained in step 3 dropwise onto a carrier having a micropore unit array, so that the microspheres are embedded in the micropores, and detecting by confocal microscopy; Step 5: Under specific excitation light, the fluorescence intensity of different microspheres is detected, and the fluorescence information is converted into lifetime information by phase analysis to achieve decoding of the antigen coupled to the microspheres; Step 6: gating different lifetime clusters in the phasor diagram and assigning different pseudo-colors to generate phasor imaging diagrams for qualitative judgment of different tumor markers; Step 7: Under another excitation light, the fluorescence intensity of phycoerythrin coupled to the marker is detected, and quantitative analysis is performed based on the fluorescence intensity of phycoerythrin to achieve quantitative detection of different markers.

[0028] In step 1, quantum dot-encoded microspheres with different lifetimes have distinguishable fluorescence lifetime characteristics.

[0029] Quantum dot coded microspheres with different lifetimes include quantum dot coded microspheres with a primary lifetime, quantum dot coded microspheres with a secondary lifetime, quantum dot coded microspheres with a tertiary lifetime, or quantum dot coded microspheres with multiple lifetimes.

[0030] The preparation method of primary lifetime information quantum dot encoding microspheres comprises the following steps: The above-mentioned Mn:ZnSe / ZnS core-shell quantum dot solutions with different lifetimes are mixed with the above-mentioned polymer solution respectively, and the concentration is adjusted by using a diluent under the same excitation light to make the number of photons emitted consistent; the dispersed phase solutions with consistent photon numbers and different lifetimes are prepared by the SPG membrane emulsification method under the action of the continuous phase to obtain microsphere suspensions with consistent photon numbers but different lifetimes; the microsphere suspensions with consistent photon numbers but different lifetimes are placed under a lifetime imaging microscope for detection, and the lifetime signals are collected and converted into clusters on the phase semi-compass, which are defined as quantum first-order lifetime quantum dot encoded microspheres.

[0031] The preparation method of the secondary lifetime quantum dot coded microspheres is as follows: two dispersed phases corresponding to the two different primary lifetime quantum dot coded microspheres are mixed in different volume ratios to form a dispersed phase solution, and a secondary lifetime coded microsphere suspension is prepared under the action of a continuous phase by an SPG membrane emulsification method. The secondary lifetime coded microsphere suspension is placed under a lifetime imaging microscope for detection, and the lifetime signal is collected and converted into clusters on a phase semicircle, each cluster representing different secondary lifetime information.

[0032] The preparation method of the three-level lifetime quantum dot coded microspheres is as follows: the dispersed phase corresponding to the first-level lifetime quantum dot coded microspheres and the dispersed phase corresponding to the second-level lifetime coded microspheres are mixed in different volume ratios to form a dispersed phase solution, and the three-level lifetime coded microsphere suspension is prepared under the action of the continuous phase by the SPG membrane emulsification method. The three-level lifetime coded microsphere suspension is placed under a lifetime imaging microscope for detection, and the lifetime signal is collected and converted into clusters on the phase semicircle, and each cluster represents different three-level lifetime information.

[0033] The preparation method of multi-level lifetime quantum dot coded microspheres is as follows: at least two dispersed phases corresponding to coded microspheres of different levels are selected and mixed in different volume ratios to form a dispersed phase solution, and a multi-level lifetime coded microsphere suspension is prepared under the action of a continuous phase by an SPG membrane emulsification method. The three-level lifetime coded microsphere suspension is placed under a lifetime imaging microscope for detection, and the lifetime signal is collected and converted into clusters on a phase semicircle, each cluster representing different multi-level lifetime information.

[0034] In step 4, the central surface of the micropore unit array has a 20x20 micropore unit, each micropore unit consists of 20 micropores, the micropore spacing is 100 mm, each micropore has a diameter of 9 mm and a depth of 3 mm. The confocal microscope detection uses a 20× objective lens and a 1000 Hz laser to ensure high-precision detection of microspheres.

[0035] In step 5, the phasor analysis method includes converting the fluorescence imaging data into lifetime clusters in the phasor diagram and decoding them by the cluster positions.

[0036] As an embodiment of the present invention, the tumor markers include four different tumor markers for breast cancer (CEA, CA125, CA153 and CA199).

[0037] <Sixth Aspect> A detection system for implementing the above-mentioned detection method comprises: quantum dot-encoded polymer microspheres with different lifespans, capture proteins, antigen standard solution, biotinylated secondary antibody solution, streptavidin-phycoerythrin (SAPE), washing buffer, microwell unit array, confocal microscope, and data processing and analysis software.

[0038] The phasor analysis method can directly convert the lifetime information of each pixel point in the fluorescence intensity imaging diagram into a point in the phasor diagram.

[0039] The phasor diagram is a semicircle with G as the horizontal axis, ranging from 0-1, and S as the vertical axis, ranging from 0-0.5.

[0040] Each point in the semicircle represents a corresponding life information, and the life shows a decreasing trend from left to right, wherein the (0,0) coordinate represents an infinite life, and the (0,1) coordinate represents an infinitely small life.

[0041] The life information on the semicircular compass follows the vector addition law In a geometric sense, the phase endpoints corresponding to the two life mixed materials should be located on the connecting line of the life phase endpoints corresponding to their respective life materials, and the distance from the two end points is determined by the proportion of the two components.

[0042] Through fluorescence lifetime imaging microscopy, the microspheres in the microwells can be imaged, and the lifetime information can then be directly converted into cluster information in the phasor diagram to achieve decoding. At the same time, the fluorescence intensity of the detection molecules on the biomarkers can be imaged to achieve quantitative detection of the biomarkers.

[0043] Another aspect of the present invention provides a method for designing, predicting and regulating a quantum dot-encoded microsphere lifetime combination signal based on a phasor method. The quantum dot lifetime-encoded microspheres include polymers and quantum dot fluorescent materials with different lifetimes. The particle size of the fluorescent-encoded microspheres is 1 μm-20 μm. Through hierarchical coding, a total of six levels of lifetime regulation coding strategies can be obtained.

[0044] According to another aspect of the present invention, a fluorescence lifetime analysis method based on the combination of fluorescence lifetime imaging microscopy and phase analysis as described above is also provided, and the method can be used to accurately design, predict and analyze the combined lifetime signals of different lifetime coded microspheres.

[0045] The preparation process of this lifespan regulation method is simple and fast, and it solves the current problems of designing, controlling and analyzing the often complex lifespan signals. Using this method to perform rapid and high-throughput multivariate quantitative detection of proteins, nucleic acids, etc. can greatly improve the speed and intuitiveness of detection, and has very important significance and broad application prospects in liquid biochip detection technology.

[0046] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention uses different methods to prepare different types of polymer microspheres, and prepares microspheres encoded with quantum dots of different lifetimes by changing the lifetime of the quantum dots in the microspheres.

[0047] 2. The present invention utilizes fluorescence lifetime imaging microscopy and phasor analysis technology, combined with the vector addition law in the semi-compass, to mix quantum dot solutions of different lifetimes in proportion, and through hierarchical regulation, realizes the design, prediction and analysis of lifetime combination signals.

[0048] 3. This method is a non-fitting and visual fluorescence lifetime analysis method, which is much faster than the traditional fitting method. It can also present pixels with similar decay characteristics in the form of "phasor clusters", has powerful data visualization functions, and is also convenient for clustering analysis of data, providing a useful reference for research in related fields.

[0049] 4. This method can realize fast and intuitive decoding of fluorescence lifetime signals in multivariate analysis. The rapid analysis of multi-component lifetime signals through the phasor diagram strategy will promote the practical application of lifetime signals in multivariate detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings: Figure 1 TEM images of six quantum dots with different lifetimes obtained in Example 1 of the present invention; Figure 2 The SEM images of polymer microspheres encoded with quantum dots of different lifetimes and the lifetime decay curves of microspheres with different lifetimes of Example 2; wherein a is the SEM image of the quantum dot-encoded microspheres; b is the lifetime decay curves of microspheres with different lifetimes; Figure 3 is a phasor cluster diagram of six primary lifetime quantum dot-encoded microspheres with different lifetimes obtained in Example 2 of the present invention; Figure 4 It is a phasor cluster diagram of the quantum dot coded microspheres obtained in Example 3 of the present invention from the secondary lifetime coding to the sixth lifetime coding; a is the secondary coding, b is the third level coding, c is the fourth level coding, d is the fifth level coding, and e and f are both the sixth level coding; Figure 5 The results of detecting four breast cancer markers CEA, CA125, CA153 and CA199 by using quantum dot encoded microspheres with different lifetimes obtained in Example 4 of the present invention in combination with fluorescence lifetime microscopy and phase analysis; a is a schematic diagram of the detection process; b is a fluorescence imaging diagram of four different lifetime encoded microspheres; c is a result diagram of phase analysis of Figure b; Figure d is a phase pseudo-color diagram obtained by assigning different pseudo-colors to the four clusters in Figure c, which corresponds one-to-one to the position of the microspheres in Figure a, but the lifetimes are distinguishable; Figure e is a fluorescence imaging diagram of the detection protein coupled to SAPE on all the microspheres in Figure b. DETAILED DESCRIPTION

[0051] The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0052] In the following embodiments and comparative examples: polymer A polymer is a long chain of molecules, or macromolecules, built by linking repeating chemical units. Each molecule of a polymer can be made up of hundreds, thousands, or even millions of repeating units.

[0053] The polymers mainly include trioxymethylene, silicone oligomers, siloxane oligomers, polyethylene wax oligomers, polyethylene, polypropylene, polyacrylamide, polyvinyl chloride, polystyrene, resin, polyester, rubber, styrene-maleic anhydride copolymer, styrene-acrylic acid copolymer, polyacrylic acid, polymethyl methacrylate, polyethyl acetate, polystyrene methacrylic acid copolymer, maleic anhydride-1-octadecene copolymer, polydimethylsiloxane, polyethoxylated trimethylolpropane triacrylate, polyhydroxyethyl methacrylate, polyvinyl chloride ... Glycol diacrylate, polyethylene glycol, poly (N-isopropyl acrylamide), protein, starch, polylactic acid, cellulose, polyethylene [—CH2—CH2—]n, nylon 6 [—NH(CH2)5CO—]n, phenolic resin, urea-formaldehyde resin, vulcanized rubber, phenolic resin, amine-formaldehyde resin, epoxy resin, polyester resin, chemical fiber, polyamide, polyether, vinyl polymer, cellulose acetate, acrylate, polycarbonate, polyetheretherketone, polysulfone, polyphenylene sulfide, terephthalate, fluororubber, nitrile rubber, etc.

[0054] In some embodiments, the polymer forming the dispersed phase of the present invention can be polystyrene-maleic anhydride copolymer, polystyrene, polylactic acid, styrene-acrylic acid copolymer, polyvinyl chloride, polymethyl methacrylate, polyethyl acetate, and the like.

[0055] Organic solvents Organic solvents are a large class of organic compounds that are widely used in life and production. They have a small molecular weight and are found in coatings, adhesives, paints and detergents. Commonly used organic solvents include styrene, perchloroethylene, trichloroethylene, ethylene glycol ether and triethanolamine. Organic solvents can dissolve some organic compounds that are insoluble in water (such as grease, wax, resin, rubber, dye, etc.). They are characterized by being liquid at room temperature and pressure and having high volatility. During the dissolution process, the properties of the solute and the solvent do not change. Solvents cannot react chemically with solutes and are inert.

[0056] Inert organic solvents mainly include benzene, toluene, xylene, o-xylene, m-xylene, p-xylene, hexamethylbenzene, ethylbenzene, n-propylbenzene, isopropylbenzene, biphenyl, diphenylmethane, triphenylmethane, styrene, phenylacetylene, naphthalene, tetralin, anthracene, phenanthrene, pyrene, chloroform, N-methylpyrrolidone, diphenyl ether, acetone, methyl butyl ketone, methyl isobutyl ketone, cyclohexanone, isophorone, diacetone alcohol, silicone, tetraethylene glycol dimethyl Ether, ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, nitrobenzene, carbon tetrachloride, ethylene dichloride, chlorobenzene, dichlorobenzene, dichloromethane, chloropicrin, methyl bromide, dichloropropane, dibromoethylene, chloroform, dimethyl sulfoxide, acetone, acetonitrile, dimethylformamide, carbon tetrachloride, cyclohexane, cyclohexanone, toluene cyclohexanone, pyridine, phenol, carbon tetrachloride, isooctane, ethyl ether, pentane, hexane, propylene oxide, etc.

[0057] In some embodiments, the organic solvent used to form a dispersed phase between the polymer and the organic solvent in the present invention is one of toluene, ethylbenzene, chloroform, dichloromethane, acetone, acetonitrile, and diethyl ether.

[0058] Dispersed Phase The present invention uses polymer and quantum dots as solutes as dispersed phases, and an inert organic solvent as solvent, thereby configuring to form a dispersed phase. The polymer here can be any polymer, and the polymer and quantum dots together form the solute in the dispersed phase. The so-called inert organic solvent is a solvent that can neither give nor accept protons.

[0059] quantum dots Quantum dots are semiconductor nanostructures that bind excitons in three spatial directions. Sometimes called "artificial atoms", "superlattices", "superatoms" or "quantum dot atoms", they are a new concept proposed in the 1990s. Quantum dots are semiconductor nanocrystals with a radius smaller than or close to the Bohr exciton radius. Due to their unique fluorescent nano effect, their excitation spectrum is wide and continuously distributed, and their emission spectrum is narrow and symmetrical. The emission wavelength can be regulated by changing the particle size and composition. They have strong fluorescence intensity, slow bleaching rate and high sensitivity. They are widely used in photocatalysis, photosensors, luminescent materials, fluorescent probe labels and other fields. In particular, quantum dots composed of group IIB and group VIA elements (such as CdSe) have important value in biomedical fluorescent probe markers and sensors due to their excellent fluorescence emission properties in the visible and near-infrared spectral regions. Quantum dots have separate quantized energy spectra. The corresponding wave function is located in the quantum dot in space, but extends to several lattice periods. A quantum dot has a small number (1-100) of integer electrons, holes or electron-hole pairs, that is, the charge it carries is an integer multiple of the elementary charge. Quantum dots mainly include CdSe / ZnS, CdSe / CdS, CdTeSe / ZnS, CdTe / CdSe / ZnS, CdTe, Ag2S, Ag2Se, InAs, InP, HgTe, PbS, PbSe, CuInS2, CuInSe2, CdSeT e. CdHgTe, InP / ZnS, InAs / CdSe, CdTe / CdS, CdTe / ZnS, CdTe / CdSe, ZnTe / CdTe, ZnTe / CdSe, ZnTe / CdS, CdSeTe / CdS, Cu:CdS / ZnS, Mn: ZnSe / ZnS, CuInS2 / ZnS, CuInSe2 / ZnS, CsPbCl3, CsPbBr3, CsPbI3, CsPb(Cl / Br)3, CsPb(Br / I)3, CH3NH3PbI3, CH3NH3PbBr3, CdSe, CdS, ZnSe, ZnTe, ZnO, HgSe, HgS, CaAs, InCaAs, CdSe / ZnSe, CdS / ZnS, CdS / Ag2S, CdS / Cd(OH)2, CdSe / ZnS e. CdS / HgS, CdS / HgS / CdS, ZnS / CdS, ZnS / CdS / ZnS, ZnS / HgS / ZnS / CdS, CdSe / CuSe, C dSeTe / CdS / ZnS, CdSe / CdS / ZnS, CdS:Mn, ZnS:Mn, CdS:Cu, ZnS:Cu, CdS:Tb, ZnS:Tb, etc.

[0060] In some embodiments, the fluorescent coding material that forms a dispersed phase with the polymer of the present invention can be quantum dots (CdSe / ZnS, CdSe / CdS, CdTeSe / ZnS, CdTe / CdSe / ZnS, CdTe, Ag2S, Ag2Se, InAs, InP, HgTe, PbS, PbSe, CuInS2, CuInSe2, CdSeTe, CdHgTe, InP / ZnS, InAs / CdSe, CdTe / CdS, CdTe / ZnS, CdTe / CdSe, ZnTe / CdTe, ZnTe / CdSe, ZnTe / CdS, CdSeTe / CdS, Cu:CdS / ZnS, Mn: ZnSe / ZnS, CuInS2 / ZnS, CuInSe2 / ZnS, CsPbCl3, CsPbBr3, CsPbI3, CsPb(Cl / Br)3, CsPb(Br / I)3, CH3NH3PbI3, CH3NH3PbBr3), etc.

[0061] Detection Detection means to examine or test a substance or material for the presence or absence of a substance or material, such as, but not limited to, a chemical substance, an organic compound, an inorganic compound, a metabolite, a drug or a drug metabolite, an organic tissue or a metabolite of an organic tissue, a nucleic acid, a protein, or a polymer. In addition, detection means to test the amount or presence of a substance or material. Furthermore, assay also means immunoassay, chemical assay, enzyme assay, nucleic acid assay, etc.

[0062] Analyte The quantum dot-encoded microspheres with different life spans prepared by the present invention can be used to detect proteins or nucleic acids. The purpose of these tests is to detect whether the liquid sample contains protein or nucleic acid-type analytes. For example, the analyte can be a protein or nucleic acid test. For nucleic acids, nucleic acids of viruses, bacteria or fungi can be detected, such as AIDS nucleic acid detection, hepatitis B virus nucleic acid detection, hepatitis C virus nucleic acid detection, influenza A virus universal nucleic acid detection, influenza A H1N1 (2009) nucleic acid detection, Middle East Respiratory Syndrome (MERS) coronavirus nucleic acid detection, adenovirus nucleic acid detection, Legionella pneumophila (LP) nucleic acid detection, respiratory syncytial virus detection, Chlamydia pneumoniae (CP) nucleic acid detection, dengue virus universal nucleic acid detection, fever with thrombocytopenia syndrome bunyavirus nucleic acid detection, influenza B virus nucleic acid detection, new coronavirus 2019-nCov nucleic acid detection, Mycobacterium tuberculosis (TB) nucleic acid detection, Mycoplasma pneumoniae (MP) nucleic acid detection, human papillomavirus nucleic acid detection, Chlamydia trachomatis nucleic acid detection, etc. Of course, nucleic acids related to tumors can also be detected.

[0063] Protein testing is mainly used for tumor screening, primary hepatocellular carcinoma, gonadal embryonal carcinoma (testicular cancer, ovarian cancer, malignant teratoma, etc.), viral hepatitis, cirrhosis, pancreatic cancer, gastric cancer, adenocarcinoma, myeloma, liver function, hepatolenticular degeneration, cardiovascular disease, lipid level, ovarian serous cystadenocarcinoma, mucinous cystadenocarcinoma, prostate cancer, medullary thyroid carcinoma, small cell lung cancer, liver fibrosis, cirrhosis, heart disease, gastric ulcer, congenital afibrinogenemia, congenital fibrinogen abnormality or insufficiency, sudden placental abruption and massive bleeding, pheochromocytoma, medullary thyroid carcinoma, etc.

[0064] The substances analyzed during protein detection can also include carcinoembryonic antigen CEA, alpha-fetoprotein (AEP), carbohydrate antigen 125 (CA125), prostate-specific antigen (PSA), calcitonin (CT), chorionic gonadotropin (HCG), albumin, alpha globulin, beta globulin, gamma globulin, immunoglobulin, placental globulin, serum protein, ceruloplasmin, C-reactive protein, total protein, hemoglobin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, direct and indirect bilirubin, urine bilirubin, urobilinogen, blood bile acid, glutamyl transpeptidase, alkaline phosphatase, hyaluronic acid, laminin, collagen, pepsin, nephropathy, hemochromatosis, ferritin, calprotectin, troponin, anti-thyroglobulin antibody, fibrinogen, chromogranin, etc.

[0065] Example 1 Preparation of core-shell quantum dot solutions doped with transition metal elements of different lifetimes The quantum dots prepared in this embodiment are Mn:ZnSe / ZnS, and the specific preparation steps are as follows: (1) 406.9 mg zinc oxide, 7.05 mL oleic acid and 12.95 mL octadecene were placed in a three-necked glass flask, heated to 120 °C under nitrogen protection and magnetic stirring, and evacuated for 1 h. After nitrogen was introduced again, the temperature was raised to 280 °C, kept at this temperature for 20 min, and then lowered to 250 °C to obtain a zinc oleate precursor.

[0066] (2) Take 156 mg of selenium powder, 5.6 mL of octadecene and 2.4 mL of oleylamine in a centrifuge tube and mix them by ultrasonication for 5 min to obtain a selenium precursor.

[0067] (3) Take 243 mg of zinc stearate, 20 mL of octadecene, and (5 mg, 10 mg, 20 mg, 40 mg, 80 mg, 160 mg) of manganese stearate respectively and place them in a three-necked glass flask. Heat to 120 °C under nitrogen protection and magnetic stirring, and evacuate for 1 h. Pass nitrogen and heat to 300 °C, and quickly inject selenium precursor. Keep at 300 °C for 5 min, inject 1.6 mL of zinc oleate precursor solution dropwise, cool to 200 °C, inject 1.6 mL of zinc oleate precursor dropwise, heat to 260 °C and keep for 10 min, inject 1.6 mL of zinc oleate precursor dropwise again, inject 1.6 mL of zinc oleate precursor for the third time after 10 min, keep for 5 min, and then cool to 75-85 °C to obtain Mn:ZnSe core quantum dot stock solution with different manganese ion doping amounts.

[0068] (4) Add 25 mL of n-hexane to the Mn:ZnSe core quantum dot stock solutions with different manganese ion doping amounts in step (3), mix well, add a large amount of acetone to precipitate the quantum dots in the stock solution, and centrifuge at 10,000 rpm for 5 min. Then, wash three times with a mixture of n-hexane and acetone in a volume ratio of 2:7, at a centrifugal speed of 10,000 rpm and a centrifugal time of 5 min / time. Collect the precipitate, disperse the purified Mn:ZnSe core quantum dots with different manganese ion doping amounts in toluene, and store at 4 °C for later use.

[0069] (5) Take 781 mg of zinc oxide, 9 mL of oleic acid and 22.5 mL of octadecene, place them in a three-necked glass flask, heat them to 100 °C under nitrogen protection and magnetic stirring, start vacuuming and continue to heat them to 120 °C, introduce nitrogen after vacuuming for 30 min, heat them to 315 °C and keep them warm for 15 min. Finally, cool the reaction solution to 150 °C and inject 3.2 mL of n-octyl mercaptan (OT) to obtain a mixed precursor solution of zinc oleate and OT (Zn(OA)2-OT precursor solution).

[0070] (6) The toluene solutions of Mn:ZnSe core quantum dots with different manganese ion doping amounts in (4) were centrifuged at low speed, and the supernatant was dissolved in 10 mL of octadecene. The temperature was raised to 100°C under nitrogen protection and magnetic stirring, and the solution was evacuated for 30 min. Nitrogen was introduced and the temperature was raised to 310°C to grow the ZnS shell layer. 9 mL of the Zn(OA)2-OT precursor solution in step (5) was injected with a syringe pump at an injection rate of 6 mL / h, and the solution was kept at 310°C for 30 min to obtain Mn:ZnSe / ZnS core-shell structure quantum dots with different manganese ion doping amounts. When the temperature was lowered to 80°C, the solution was centrifuged and washed with n-hexane and acetone (the volume ratio of the two was 2:7). The centrifugal washing method was the same as step (4). Finally, the purified quantum dots were dissolved in toluene and stored at 4°C. The TEM image is shown as follows. Figure 1 shown.

[0071] (7) By Figure 1 It can be found that by changing the doping amount of manganese stearate, six quantum dot solutions with different lifetime decay curves can be obtained, named QDt 11 -QD 16 .

[0072] Example 2 (1) Take six different lifetime-coded quantum dot solutions (QDt 11 -QD 16 ) 0.05 mg and 0.125 g of styrene-maleic anhydride copolymer (PSMA) were dissolved in 2 mL of toluene to form different dispersed phases; (2) Dissolve 0.25 g of sodium dodecyl sulfate (SDS) in 200 mL of water to form a continuous phase; (3) An SPG membrane with a specification of Φ10xL20mm (effective length 10mm) and a membrane pore size of 5μm was used. An SPG membrane emulsification device (purchased from Beijing Jiasheng Xingye Technology Co., Ltd., imported from Japan, model: MN-20) was used, in which the continuous phase body was 200ml and the volume of the dispersed phase was 2ml. Under a nitrogen pressure of 3KPa, the dispersed phase was squeezed through the SPG membrane. Under the shear force of the continuous phase, uniform and stable water-in-oil droplets were formed on the surface of the SPG membrane, and six microsphere suspensions with different lifetime codes were obtained.

[0073] (4) Each microsphere suspension was then centrifuged at 6500 rpm, washed five times with deionized water, and freeze-dried to obtain 5 μm polymer microspheres encoded with quantum dots of different lifetimes. The SEM images are shown in Figure 2. Figure 2 As shown; (a) SEM image of quantum dot encoded microspheres and (b) lifetime decay curves of microspheres with different lifetimes).

[0074] (5) By Figure 2From the lifetime decay curves, it can be found that six quantum dot-encoded polymer microspheres with different lifetime decay curves were successfully obtained.

[0075] Example 3: First-level life coding (1) The quantum dot solutions (QDt 11 -QD 16 ) and PSMA to prepare a dispersed phase (0.1 mM QD / g PSMA), which was diluted with toluene and excited with 405 nm excitation light to make the number of photons emitted uniform; (2) Six dispersed phase solutions with the same photon number but different lifespans are emulsified by the SPG membrane method. Under the shear force of the continuous phase, uniform and stable water-in-oil droplets are formed on the surface of the SPG membrane, thereby obtaining a suspension of microspheres with the same photon number but different lifespans. (3) The suspension of microspheres with the same photon number and different lifetimes obtained in step (2) was centrifuged at 6500 rpm, washed five times with deionized water, and freeze-dried (vacuum degree 8 Pa, temperature -70 o C) obtaining polymer microspheres encoded with different quantum dots; (4) Take 10 μL of microsphere suspension (redissolve the freeze-dried powder from step (3) in PBST solvent to a concentration of 1 mg / mL), drop it on a clean coverslip, and observe it under a lifetime imaging microscope. The objective lens is a 60x water lens, the excitation light is 405 nm, the excitation frequency is 1000 Hz, the detector is an APD detector, and the fluorescence channel is 615 / 40 nm.

[0076] Microspheres with regular morphology and high luminescence intensity were selected for fluorescence imaging, and the lifetime signals were collected using the FastFLIM FLIMbox of ISS. After imaging, the fluorescence imaging images were directly converted into six clusters on the phasor semicircle by SimFCS-64 software, which were defined as the primary lifetime encoding of quantum dot-encoded microspheres and named t B11 ,t B12 ,t B13 ,t B14 ,t B15 and t B16 ,like Figure 3 shown.

[0077] Example 4 Multi-level life coding The secondary life code is the first code B11 and t B16 The corresponding dispersed phases were mixed in volume ratios of 5:1, 4:2, 3:3, 2:4 and 1:5 to form a variety of dispersed phases; the secondary lifetime coded microspheres were obtained by SPG membrane emulsification method, such as Figure 4In a, the secondary lifetime encoding cluster is named t B21 -t B25 ; will t B13 and t B14 The corresponding dispersed phases are mixed in a volume ratio of 1:1 to form a mixed dispersed phase, and the secondary lifetime coded microspheres are obtained by SPG membrane emulsification method, such as Figure 4 In a, the secondary lifetime encoding cluster is named t B26 ; will t B12 and t B13 The corresponding dispersed phases were mixed in a volume ratio of 1:1 to form a mixed dispersed phase. The secondary lifetime coded microspheres were obtained by SPG membrane emulsification method, and their clusters were named t B27 .

[0078] Table 1

[0079] The fluorescence imaging images were directly converted into clusters on the phasor semicircle through SimFCS-64 software, representing different lifetime information.

[0080] (2) Level 3 life coding The first level code is t B13 and secondary code t B21 The corresponding dispersed phases were mixed in a volume ratio of 1:1 to form a mixed dispersed phase, and three-level lifetime coded microspheres were obtained by SPG membrane emulsification method, such as Figure 4 In b, the lifetime encoding cluster is named t B31 ; The first level code is t B14 and secondary code t B25 The corresponding dispersed phases were mixed in a volume ratio of 1:1 to form a mixed dispersed phase, and three-level lifetime coded microspheres were obtained by SPG membrane emulsification method, such as Figure 4 In b, the lifetime encoding cluster is named t B32 ; The fluorescence imaging images were directly converted into clusters on the phasor semicircle through SimFCS-64 software, representing different lifetime information.

[0081] (3) Four-level life coding The secondary code is t B26 and t B23 The corresponding dispersed phases were mixed in a volume ratio of 2:1 to form a mixed dispersed phase, and four-level lifetime coded microspheres were obtained by SPG membrane emulsification method, such as Figure 4 In c, the lifetime encoding cluster is named t B41 ; The fluorescence imaging images were directly converted into clusters on the phasor semicircle through SimFCS-64 software, representing different lifetime information.

[0082] (4) Five-level life coding The secondary code is t B26 and three-level coding t B31 The corresponding dispersed phases were mixed in a volume ratio of 1:1 to form a mixed dispersed phase, and five-level lifetime coded microspheres were obtained by SPG membrane emulsification method, such as Figure 4 In d, the five-level lifetime encoding cluster is named t B51 The fluorescence imaging images were directly converted into clusters on the phasor semicircle by SimFCS-64 software, representing different lifetime information.

[0083] (5) For level 6 life coding The secondary code is t B22 , t B24 and t B26 The corresponding dispersed phases were mixed in a volume ratio of 1:1:1 to form a mixed dispersed phase, and six-level lifetime coded microspheres were obtained by SPG membrane emulsification method, such as Figure 4 As shown in f, the six-level lifetime encoding cluster is named t B61 The three-level coding B31 and t B32 The corresponding dispersed phases were mixed at a volume ratio of 2:1 and 1:2 to prepare different mixed dispersed phases, and six-level lifetime coded microspheres were obtained by SPG membrane emulsification method, such as Figure 4 As shown in Figure e, the two six-level lifetime coding clusters are named t B62 and t B63 .

[0084] The fluorescence imaging images were directly converted into clusters on the phasor semicircle by SimFCS-64 software, representing different lifetime information. The regulation results of hierarchical lifetime clusters are shown in Figure 4 shown.

[0085] Application test case Application of quantum dot-encoded microspheres with different lifetimes in the detection of four tumor markers in breast cancer based on phase analysis and fluorescence lifetime imaging microscopy.

[0086] The specific steps are: (1) The four different lifetime coded microspheres (t B13 , t B14 , t B15 and t B16 ) The capture proteins corresponding to different tumor markers CEA, CA125, CA153 and CA199 were incubated at room temperature for 4 h, and four different capture proteins were coupled to the surface of the microspheres;

[0087] (2) Add the four capture protein-coupled microspheres in step (1) to a 96-well plate, and add 100 μL of antigen standard solution of different concentrations (CEA: 0.01-1000 nm / mL; CA125: 0.1-1000 U / mL; CA153: 0.0001-10 U / mL; CA199: 0.5-1000 U / mL) to each well. Then incubate at room temperature for 60 minutes, wash away the unbound antigen, add 100 μL of biotinylated secondary antibody IgG solution (provided by Shanghai Wanzijian Biotechnology Co., Ltd.), and incubate at room temperature for a further 60 minutes.

[0088] (3) Wash off the excess secondary antibody, add 100 μL of streptavidin-phycoerythrin (SAPE), and incubate at room temperature for 10 minutes. Finally, resuspend the mixture in 100 μL of washing buffer (PBS with 0.05% Tween-20).

[0089] (4) A microwell unit array with a center surface of 20x20 was prepared by chemical etching. Each microwell unit consisted of 20 microwells. The microwell spacing was 100 mm, each microwell had a diameter of 9 mm and a depth of 3 mm. The suspension mixture of four microspheres with different lifespans in step (3) was dripped onto the microwell unit array. The 5 mm microspheres were embedded in the microwells. All microspheres were detected by confocal microscopy with a 20× objective lens and a 1000 Hz laser.

[0090] (5) Under the excitation light of 405 nm and the detection channel of 615 / 40 nm, the fluorescence intensity of the four lifetime-coded microspheres was detected to obtain the fluorescence intensity diagram of the red light channel (FL-Red).

[0091] (6) Direct phasor analysis of fluorescence imaging is performed to directly convert fluorescence information into lifetime information, and four different lifetime clusters are obtained. The positions of the lifetime clusters in the phasor diagram are decoded (Cursor), and four antigens coupled to different lifetime-encoded microspheres (CEA, CA125, CA153, and CA199) can be decoded. (7) By using circles of different colors to "gate" the four different clusters in the phasor diagram and assigning four different pseudo-colors, reverse annotation can be achieved to obtain a phasor image. The four lifespan microspheres in the image appear in four different colors, corresponding to the four "gates", which enables qualitative judgment of the four markers (CEA, CA125, CA153 and CA199).

[0092] (8) Then, under the excitation of 532 nm excitation light, the fluorescence intensity (PE intensity) of phycoerythrin (PE) coupled to the four different markers was collected in the 575 / 40 nm channel and quantitatively analyzed to achieve quantitative detection of the four markers.

[0093] Figure 5 The results of detecting four breast cancer markers CEA, CA125, CA153 and CA199 by using quantum dot encoded microspheres with different lifetimes obtained in Example 4 of the present invention in combination with fluorescence lifetime microscopy and phase analysis; a is a schematic diagram of the detection process; b is a fluorescence imaging diagram of four different lifetime encoded microspheres; c is a result diagram of phase analysis of Figure b; Figure d is a phase pseudo-color diagram obtained by assigning different pseudo-colors to the four clusters in Figure c, which corresponds one-to-one to the position of the microspheres in Figure a, but the lifetimes are distinguishable; Figure e is a fluorescence imaging diagram of the detection protein coupled to SAPE on all the microspheres in Figure b.

[0094] The present invention provides a lifetime design and regulation method for quantum dot coded microspheres based on phasor analysis; the present invention prepares quantum dot coded polymer microspheres with different lifetimes by SPG film method; the dispersed phases corresponding to quantum dot coded polymer microspheres with different lifetimes are combined in different proportions, combined with fluorescence lifetime imaging microscopy and phasor analysis, and hierarchical regulation is used to obtain a method for combining and encoding different lifetime signals. The lifetime combination design and regulation method of fluorescence lifetime coded microspheres of the present invention uses phasor analysis and semi-compass vector law as guidance, and obtains coded clusters with different lifetimes in the semi-compass through hierarchical design and precise regulation of fluorescence lifetime signals. Good lifetime decoding is achieved for coded microspheres with different lifetimes. The strategy based on phasor diagram can realize fast and accurate decoding of multi-component lifetime combination signals, and is applied to multi-factor detection, which promotes the practical application of lifetime signals in optical multiplexing.

[0095] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing quantum dot polymer microspheres, characterized in that: The steps include: S1, adding a zinc source and a manganese source in different proportions to a solvent to form a mixed solution, degassing, adding a selenium precursor solution after heating, and then adding a zinc precursor solution to obtain a Mn:ZnSe core solution with different manganese ion doping amounts; S2, degassing the Mn:ZnSe core solutions with different manganese ion doping amounts obtained in step S1, respectively, heating them, respectively mixing the Zn(OA)2-OT precursor solutions, and keeping them warm for a period of time to obtain Mn:ZnSe / ZnS core-shell structure quantum dot solutions with different manganese ion doping amounts; that is, Mn:ZnSe / ZnS core-shell quantum dot solutions with different lifespans; S3. Dissolve the Mn:ZnSe / ZnS core-shell quantum dot solutions with different lifespans and the polymer solution in an organic solvent to form a dispersed phase; disperse the surfactant in water to form a continuous phase; pass through an SPG membrane to form an oil-in-water emulsion, and after curing, obtain quantum dot-encoded polymer microspheres with different lifespans.

2. The preparation method according to claim 1, characterized in that: Step S1 includes the following steps: S11, preparing a zinc precursor: mixing a zinc source, oleic acid and octadecene, stirring and heating to 120-130° C. under a protective atmosphere for vacuum treatment, then reintroducing the protective atmosphere and heating to 280-300° C. for 20-30 min, and then reducing the temperature to 250-260° C. to obtain a zinc precursor; Preparing a selenium precursor: mixing selenium powder, octadecene and oleylamine, and subjecting the mixture to ultrasonic treatment to obtain a selenium precursor; S12, preparing Mn: ZnSe core quantum dot stock solution: zinc stearate, octadecene and different amounts of manganese stearate are mixed respectively, stirred under a protective atmosphere and heated to 120-130°C for vacuum treatment, then the protective atmosphere is reintroduced and the temperature is raised to 280-300°C, a selenium precursor is injected, and the temperature is kept at 300-320°C for 5-10 min, and then a zinc precursor solution is injected, and the temperature is kept at 300-320°C for 5-10 min, and the temperature is cooled to 75-85°C to obtain Mn: ZnSe core quantum dot stock solutions with different manganese ion doping amounts; S13, purifying Mn:ZnSe core quantum dots: mixing the core quantum dot stock solution obtained in step S12 with n-hexane, adding acetone or anhydrous ethanol to precipitate the quantum dots, collecting the precipitate by centrifugation, and washing it several times with a mixture of n-hexane and acetone to obtain purified Mn:ZnSe core solutions with different manganese ion doping amounts.

3. The preparation method according to claim 1, characterized in that: Step S2 includes the following steps: S21, preparing a Zn(OA)2-OT mixed precursor solution: mixing zinc oxide, oleic acid and octadecene, stirring under a protective atmosphere and heating to 100-120°C for vacuum treatment, then introducing a protective atmosphere and heating to 300-320°C and keeping the temperature for 10-20 min, cooling to 120-150°C, injecting n-octyl mercaptan and keeping the temperature at 120-150°C to obtain a Zn(OA)2-OT precursor solution; S22. Preparation of Mn:ZnSe / ZnS core-shell structure quantum dots: The Mn:ZnSe core solution with different manganese ion doping amounts obtained in step S1 is dissolved in octadecene, and the temperature is raised to 100-120°C under a protective atmosphere for vacuum treatment. Subsequently, a protective atmosphere is introduced, and the temperature is raised to 300-320°C for growth of the ZnS shell layer. The Zn(OA)2-OT precursor solution of step S21 is injected into the core. After keeping the temperature at 300-320°C for 30-60 min, Mn:ZnSe / ZnS core-shell structure quantum dot solutions with different manganese ion doping amounts are obtained.

4. The preparation method according to claim 1, characterized in that: The polymer includes at least one of polystyrene-maleic anhydride copolymer, polystyrene, polylactic acid, styrene-acrylic acid copolymer, polyvinyl chloride, polymethyl methacrylate, and polyethyl acetate.

5. A method for controlling signals of different lifetime combinations of quantum dot-encoded microspheres, characterized in that: The control method comprises the following steps: The dispersed phases corresponding to the quantum dot-encoded polymer microspheres with different lifetimes as described in claim 1 are combined in different proportions, combined with fluorescence lifetime imaging microscopy and phase analysis, and a method for encoding different lifetime signal combinations is obtained through hierarchical regulation.

6. The method for controlling signals of different lifetime combinations of quantum dot coded microspheres according to claim 5, characterized in that: The hierarchical control method comprises the following steps: A. A core-shell quantum dot solution with different lifetimes and a polymer solution are mixed to obtain a mixed dispersed phase. The concentration is adjusted using a diluent under the same excitation light so that the number of photons emitted by the mixed dispersed phase is consistent; B. Dispersed phase solutions with the same photon number but different lifespans are prepared by SPG membrane emulsification method under the action of continuous phase to obtain microsphere suspensions with the same photon number but different lifespans; A suspension of microspheres with the same photon number but different lifetimes is placed under a lifetime imaging microscope for detection, and the lifetime signals are collected and converted into clusters on the phase semicircle, which are defined as quantum first-order lifetime coded microspheres. C. The dispersed phases corresponding to two different primary lifetime coded microspheres are mixed in different volume ratios to form a dispersed phase solution, and a secondary lifetime coded microsphere suspension is prepared under the action of a continuous phase by an SPG membrane emulsification method; The secondary lifetime-encoded microsphere suspension is placed under a lifetime imaging microscope for detection, and the lifetime signals are collected and converted into clusters on a phase semicircle, each cluster representing different lifetime information; D. The dispersed phase corresponding to the primary lifetime coded microspheres and the dispersed phase corresponding to the secondary lifetime coded microspheres are mixed in different volume ratios to form a dispersed phase solution, and a tertiary lifetime coded microsphere suspension is prepared under the action of a continuous phase by an SPG membrane emulsification method; the tertiary lifetime coded microsphere suspension is placed under a lifetime imaging microscope for detection, and the lifetime signals are collected and converted into clusters on a phase semicircle, each cluster representing different lifetime information; E. Select at least two dispersed phases corresponding to coded microspheres of different levels and mix them in different volume ratios to form a dispersed phase solution. Prepare a multi-level lifetime coded microsphere suspension under the action of a continuous phase by SPG membrane emulsification method; place the three-level lifetime coded microsphere suspension under a lifetime imaging microscope for detection, collect lifetime signals and convert them into clusters on a phase semicircle, each cluster representing different lifetime information; F. Analyze and decode the lifetime signal in biological detection through the position of each lifetime cluster in the phasor diagram.

7. The control method of different lifetime combination signals according to claim 6, characterized in that: Step A includes the following steps: A1. Adding a zinc source and a manganese source in different proportions to a solvent to form a mixed solution, degassing, adding a selenium precursor after heating, and then adding a zinc precursor to obtain a Mn:ZnSe core solution with different manganese ion doping amounts; A2. Degas the Mn:ZnSe core solutions with different manganese ion doping amounts obtained in step A1, respectively, add zinc / sulfur precursors after heating, and keep warm for a period of time to obtain Mn:ZnSe / ZnS core-shell structure quantum dot solutions with different manganese ion doping amounts, that is, core-shell quantum dot solutions with different lifespans.

8. Use of the method for preparing quantum dot polymer microspheres according to any one of claims 1 to 3 in the detection of tumor markers.

9. A method for detecting tumor markers for non-disease diagnosis purposes, comprising the following steps: Step 1, incubating the quantum dot-encoded polymer microspheres of different lifespans prepared in claim 1 with capture proteins corresponding to different tumor markers at room temperature, respectively, to couple the capture proteins on the surface of the microspheres; Step 2, adding the microspheres coupled with the capture protein into the reaction container, adding different concentrations of antigen standard solution for incubation, then adding biotinylated secondary antibody solution for secondary incubation, and washing to remove unbound components; Step 3: Based on step 2, streptavidin-phycoerythrin is added for incubation, and the mixture is resuspended in washing buffer; Step 4, adding the microsphere suspension mixture obtained in step 3 dropwise onto a carrier having a micropore unit array, so that the microspheres are embedded in the micropores, and detecting by confocal microscopy; Step 5: Under specific excitation light, the fluorescence intensity of different microspheres is detected, and the fluorescence information is converted into lifetime information by phase analysis to achieve decoding of the antigen coupled to the microspheres; Step 6: gating different lifetime clusters in the phasor diagram and assigning different pseudo-colors to generate phasor imaging diagrams for qualitative judgment of different tumor markers; Step 7: Under another excitation light, the fluorescence intensity of phycoerythrin coupled to the marker is detected, and quantitative analysis is performed based on the fluorescence intensity of phycoerythrin to achieve quantitative detection of different markers.

10. The detection method according to claim 9, characterized in that: In step 5, the excitation light wavelength is 405 nm; And / or, in step 7, the light emission wavelength is 532nm.

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