CN113648942A - Carboxylated quantum dot coding fluorescent microsphere and preparation method and application thereof - Google Patents

Abstract

The invention relates to a carboxylated quantum dot fluorescent coding microsphere, which is a carboxylated crosslinked polystyrene microsphere containing quantum dots, wherein the quantum dots are core-shell quantum dots ZnCdSe/ZnS, and the carboxylated quantum dot fluorescent coding microsphere has fluorescent response under the excitation wavelength of 300-600 nm, preferably 300-500 nm, and most preferably 400 nm. Also provides a preparation method and application thereof. The carboxylated quantum dot fluorescent coding microsphere has uniform and controllable particle size, and the quantum dot has strong fluorescent stability and is not easy to quench. By performing functional modification on the surface of the microsphere such as chemical molecules, antibodies, antigens, polypeptides, nucleic acids, aptamers and the like, the quantum dot encoding microsphere with different luminescent wavelengths can form multiple indexes, and can be used for simultaneous qualitative and quantitative analysis of multiple indexes such as diagnosis, prognosis, curative effect prediction, curative effect tracking and the like of diseases and the application of related in vitro diagnostic reagents, medical instruments, biochips and the like.

Description

Carboxylated quantum dot coding fluorescent microsphere and preparation method and application thereof

Technical Field

The invention belongs to the field of inorganic-polymer composite materials, and particularly relates to a fluorescent microsphere coded by carboxylated quantum dots, and a preparation method and application thereof.

Background

The liquid phase chip detection technology based on microsphere has the basic principle that microsphere is used as a reaction carrier, and the composite such as DNA-DNA, DNA-protein, protein-protein, polypeptide-polypeptide, polypeptide-protein and the like is formed on the surface of the microsphere according to the rule of interaction among various biomolecules, and then the composite on the surface of the microsphere is quantitatively analyzed by utilizing flow cytometry. The microsphere-based liquid phase chip technology is widely applied to the fields of gene detection, protein expression detection, disease diagnosis, prognosis and the like due to the faster kinetic binding rate, high flux and high flexibility.

In flow cytometry, different types of microspheres can be distinguished by collecting fluorescence information of the microspheres, and the fluorescence information can be accurately designed and detected, namely, the encoding and decoding of fluorescence can be realized. The quantum dots can generate strong fluorescence under the irradiation of exciting light due to the nanometer size effect. Compared with organic dye molecules, the quantum dot fluorescence emission spectrum has the characteristics of good symmetry, narrow half-peak width, difficult quenching of fluorescence, photobleaching resistance and the like, so that the quantum dot fluorescence emission spectrum becomes an ideal fluorescence coding material.

Extracellular vesicles are a class of nano-or micro-phospholipid bilayer vesicle structures that are released from cells to the outside of cells. The extracellular vesicles, because of their endosomal origin, carry a variety of proteins, lipids and nucleic acids from the cells of origin, are involved in the transfer of substances and communication of information between cells and are capable of reflecting to some extent the biochemical characteristics of the original cell or tissue. Compared with tissue aspiration biopsy widely used in clinic, the liquid biopsy based on blood has the advantages of low invasiveness, real-time dynamic monitoring, quick response and the like, and has wide application prospects in cancer diagnosis, prognosis and disease course monitoring.

In the aspect of extracellular vesicle detection, the liquid-phase chip detection method based on the fluorescence-encoded microspheres can well exert the advantages of the detection method, and by performing functional modification on the surfaces of the microspheres such as chemical molecules, antibodies, antigens, polypeptides, nucleic acids, aptamers and the like, the quantum dot-encoded microspheres with different light-emitting wavelengths can form multiple indexes, and can be used for simultaneous qualitative and quantitative analysis of multiple indexes such as diagnosis, prognosis, curative effect prediction, curative effect tracking and the like of diseases and the application of related in-vitro diagnosis reagents, medical instruments, biochips and the like.

Disclosure of Invention

Therefore, the present invention aims to overcome the defects in the prior art and provide a bio-functionalized quantum dot fluorescence-encoded microsphere, which embeds quantum dots of different types and quantities in the microsphere.

In order to achieve the above object, a first aspect of the present invention provides a carboxylated quantum dot fluorescent coding microsphere, which is a carboxylated crosslinked polystyrene microsphere containing quantum dots, wherein the quantum dots are core-shell quantum dots ZnCdSe/ZnS, and the carboxylated quantum dot fluorescent coding microsphere has a fluorescent response at an excitation wavelength of 300 to 600nm, preferably 300 to 500nm, and most preferably 400 nm; preferably, the particle size of the carboxylated quantum dot fluorescent coding microsphere is 1-20 μm; and/or

The emission wavelength of the quantum dots is 500 nm-750 nm.

The second aspect of the present invention provides a preparation method of the carboxylated quantum dot fluorescent coding microsphere described in the first aspect, and the preparation method may include the following steps:

(1) preparing polystyrene seed microspheres by a dispersion polymerization method;

(2) preparing carboxylated crosslinked polystyrene microspheres by a seed swelling polymerization method;

(3) and preparing the quantum dot fluorescent coding microspheres.

The production method according to the second aspect of the present invention, wherein the step (1) comprises: dissolving a styrene monomer in a mixed solution of ethanol and 2-methoxyethanol, reacting in the presence of nitrogen by using polyvinylpyrrolidone as a dispersant and azodiisobutyronitrile as an initiator, and centrifuging, washing and drying after the reaction is finished to obtain the polystyrene seed microspheres.

The preparation method according to the second aspect of the present invention, wherein the reaction temperature is 60 to 90 ℃, preferably 70 to 90 ℃, and most preferably 70 ℃; and/or

The reaction time is 10 to 48 hours, preferably 10 to 24 hours, and more preferably 10 to 15 hours. The production method according to the second aspect of the present invention, wherein the step (2) includes: dispersing the polystyrene seed microspheres prepared in the step (1) into a sodium dodecyl sulfate aqueous solution, adding a good solvent of polystyrene for swelling, adding the sodium dodecyl sulfate aqueous solution, a polystyrene monomer, olefine acid, a crosslinking agent ethylene glycol dimethacrylate and an initiator of ethylene glycol peroxide for continuing swelling, finally adding a dispersant of polyvinylpyrrolidone for reacting under the protection of nitrogen, and after the reaction is finished, centrifugally washing and drying; preferably, the good solvent of the polystyrene is selected from one or more of the following: chloroform, cyclohexane, toluene; and/or

The olefinic acid is selected from one or more of the following: methacrylic acid, crotonic acid and hexenoic acid.

The production method according to the second aspect of the invention, wherein the step (3) includes: dispersing the microspheres obtained in the step (2) in a swelling agent for swelling, then adding a quantum dot solution into a microsphere dispersion solution, performing ultrasonic dispersion, and washing to obtain the quantum dot fluorescence encoding microspheres;

preferably, the swelling agent is a mixed solvent of chloroform and n-butanol;

more preferably, the volume fraction of chloroform in the mixed solvent is 1-10%.

The third aspect of the invention provides a biochip, which comprises the carboxylated quantum dot fluorescence-encoded microspheres of the first aspect or the carboxylated quantum dot fluorescence-encoded microspheres prepared by the preparation method of the second aspect;

preferably, the biochip is a suspension phase chip.

The fourth aspect of the present invention provides a method for preparing the biochip according to the third aspect, the method comprising the steps of:

(1) preparing carboxylated quantum dot fluorescence-encoded microspheres according to the preparation method of any one of claims 2 to 6;

(2) coupling the carboxylated quantum dot fluorescent coding microspheres and an antibody through amidation reaction to obtain the quantum dot fluorescent coding microsphere array functionally modified by the antibody.

In a fifth aspect, the present invention provides a method for multi-target assay of extracellular vesicle surface protein, the method comprising performing the multi-target assay of extracellular vesicle surface protein using the biochip of the third aspect by flow cytometry.

The sixth aspect of the present invention provides an application of the carboxylated quantum dot fluorescence-encoded microsphere of the first aspect, the carboxylated quantum dot fluorescence-encoded microsphere prepared by the preparation method of the second aspect, or the biochip of the third aspect in the preparation of medical products for diagnosis, prognosis, efficacy prediction and/or efficacy tracking of diseases;

preferably, the medical product is selected from one or more of the following: in vitro diagnostic reagents and medical instruments.

The fluorescent microsphere prepared by the invention has uniform particle size, carboxyl modification on the surface, better swelling resistance and good fluorescence stability.

Further, the quantum dots are oil-soluble core-shell quantum dots ZnCdSe/ZnS.

Further, the emission wavelength of the quantum dots is between 500nm and 750 nm.

Further, the bio-functionalized quantum dot fluorescent coding microsphere can be prepared by the following method:

1) the polystyrene seed microsphere is prepared by a dispersion polymerization method. Dissolving styrene monomer in mixed solution of ethanol and 2-methoxy ethanol, taking polyvinylpyrrolidone (PVP) as a dispersing agent and Azobisisobutyronitrile (AIBN) as an initiator, and reacting in the presence of nitrogen. After the reaction is finished, centrifuging, washing and drying.

2) Preparing the carboxylated crosslinked polystyrene microspheres by a seed swelling polymerization method. Dispersing the polystyrene seed microspheres obtained in the step 1 into a Sodium Dodecyl Sulfate (SDS) aqueous solution, adding a good solvent of polystyrene for swelling, adding the Sodium Dodecyl Sulfate (SDS) aqueous solution, a polystyrene monomer, olefine acid, a cross-linking agent Ethylene Glycol Dimethacrylate (EGDMA) and an initiator Benzoyl Peroxide (BPO) into the solution for continuing swelling, and finally adding a dispersant polyvinylpyrrolidone (PVP) for reaction under the protection of nitrogen. After the reaction is finished, the reaction product is centrifugally washed and dried.

3) Preparing quantum dot fluorescent coding microspheres: and (3) dispersing the microspheres obtained in the step (2) in a chloroform-n-butanol mixed solvent for swelling. And then adding the quantum dot solution into the microsphere dispersion liquid, and performing ultrasonic dispersion. And finally, cleaning the product once by using normal hexane, cleaning for 3 times by using absolute ethyl alcohol, and cleaning for 3 times by using ultrapure water to obtain the final product.

4) Extraction, characterization and quantitative detection of protein expression of extracellular vesicles: 1) the cell culture supernatant was collected and then filtered through a 0.2 μm filter, and the filtered liquid was centrifuged at 120000g to 200000g for 2 hours by ultracentrifugation. The supernatant is discarded and centrifuged again at 120000-200000 g for 2 hours. The resulting pellet was centrifuged and resuspended in 100 to 500. mu.L for storage. The protein of the extracted extracellular vesicles was quantified by BCA protein quantification. The specific steps and the dosage are determined according to the specific kit. Dropping the extracted extracellular vesicles on a copper mesh special for TEM, standing for 1min, sucking off liquid, washing with ultrapure water, adding 1% phosphotungstic acid or uranyl acetate, standing for 30s, sucking away, and drying. And characterizing by a biological transmission electron microscope. And (3) taking a certain amount of extracellular vesicle suspension, diluting the extracellular vesicle suspension to 1mL by using PBS, and representing the size by using a dynamic light scattering technology, or analyzing the size and the exosome concentration by using a nanoparticle tracking analysis technology. And (3) quantitatively detecting the extracellular vesicular protein marker by using flow cytometry. According to the protein quantitative result, 1-20 mu g of protein extracellular vesicles and antibody functionalized quantum dot fluorescent coding microspheres are mixed and incubated overnight. Blocking nonspecific binding sites on the microspheres with 2% -10% BSA solution. Then, a mixed solution of fluorescently-labeled CD63, CD9 and CD81 antibodies is added to stain the extracellular vesicles bound on the microspheres. And finally, detecting by using a flow cytometer with a corresponding fluorescence channel and comparing with a blank control result to determine the expression amount.

The particle size of the carboxyl functionalized polystyrene microsphere prepared by the invention is 1-5 microns. By controlling the concentration of the styrene monomer, the concentration of the initiator, the concentration of the dispersing agent and the stirring speed, the carboxylated crosslinked polystyrene microspheres with uniform particle size can be prepared. The concentration and the type of the quantum dot solution are changed, so that fluorescent microspheres with different fluorescence information can be obtained, and further, the fluorescent microspheres can be distinguished in flow cytometry.

The invention further provides a preparation method of the carboxylated polystyrene microspheres, wherein the mass fraction of the styrene monomer in the system is controlled to be 10-35%. According to the mass percentage, the polyvinylpyrrolidone is controlled to be 1-10% of the styrene monomer, and the azodiisobutyronitrile is controlled to be 0.05-0.2% of the styrene monomer. The benzoyl peroxide is controlled to be 1-5% of the styrene monomer, and the ethylene glycol dimethacrylate is controlled to be 10-20% of the styrene monomer.

The method firstly prepares the carboxylated polystyrene microspheres by a seed swelling method to obtain the carboxylated polystyrene microspheres with uniform particle size, and then prepares the quantum dot doped fluorescent coding microspheres by the swelling method on the basis. The method separates the three processes of nucleation, growth and quantum dot modification of the microspheres, and can better control the particle size of the microspheres. More importantly, the two processes of microsphere synthesis and quantum dot swelling and doping are separated, so that the fluorescent encoding of the polystyrene microspheres can be simply and easily realized (as in example 3), and the same batch of samples have the same particle size, and each fluorescent encoding microsphere does not need to be synthesized separately. Compared with the prior art, pore-foaming agents such as toluene and the like are not used in the synthetic method of the carboxylated polystyrene microspheres, which has important influence on the microsphere appearance and the subsequent quantum dot coding method, and the synthetic polystyrene microspheres retain complete surface appearance.

When the quantum dot fluorescent coding microsphere is prepared, a good solvent of polystyrene is used for fully swelling the polystyrene microsphere to form a nano-scale gap, then the oil-soluble quantum dot with the oleic acid ligand can be loaded on the surface of the microsphere through coordination, and finally the microsphere is shrunk in the poor solvent to fix the quantum dot.

Because the quantum dots have strong fluorescence, the quantum dot fluorescence encoding has high sensitivity to the quantum dot doping amount. The method provided by the invention overcomes the problems of uneven quantum dot loading capacity, easy leakage and difficulty in accurate and quantitative doping of the porous polystyrene microspheres due to the difference between the number of pores and the pore diameter of the porous polystyrene microspheres, and establishes a fluorescence encoding technology by using fluorescence emission wavelength and fluorescence intensity parameters to realize accurate fluorescence encoding of the polystyrene microspheres.

In conclusion, the quantum dot fluorescent coding microsphere synthesized by the method has the advantages of good monodispersity, smooth surface, accurate and controllable fluorescence intensity, large coding quantity and the like.

The obtained carboxylated quantum dot fluorescent coding microspheres can be used for surface capture antibody functional modification and can be used for multi-target detection of extracellular vesicles. Can be used for judging whether breast cancer is benign or malignant.

The carboxylated quantum dot fluorescent coding microspheres obtained by the invention can be used for surface functional modification of chemical molecules, antibodies, antigens, polypeptides, nucleic acids, aptamers and the like, and quantum dot coding microspheres with different light-emitting wavelengths can form multiple indexes, and can be used for simultaneous qualitative and quantitative analysis of multiple indexes of diagnosis, prognosis, curative effect prediction, curative effect tracking and the like of diseases, and application of related in-vitro diagnostic reagents, medical instruments, biochips and the like.

The carboxyl functionalized polystyrene microsphere of the present invention may have the following beneficial effects, but is not limited to:

the particle size of the carboxyl functionalized polystyrene microsphere prepared by the invention is 1-20 microns. The fluorescence of the quantum dot fluorescent microsphere is not easy to quench and photobleach. The microspheres have good swelling resistance. By controlling the concentration of the styrene monomer, the concentration of the initiator, the concentration of the dispersing agent and the stirring speed, the carboxylated crosslinked polystyrene microspheres with uniform particle size can be prepared. The concentration and the type of the quantum dot solution are changed, so that fluorescent microspheres with different fluorescence information can be obtained, and further, the fluorescent microspheres can be distinguished in flow cytometry.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows the design concept and preparation method of polystyrene microspheres in examples 1 to 3.

FIG. 2 shows a scanning electron micrograph of the polystyrene seed microspheres of example 1.

FIG. 3 shows a scanning electron micrograph of carboxylated polystyrene microspheres from example 2.

Fig. 4 shows a scanning electron micrograph of the quantum dot modified polystyrene microsphere of example 2.

FIG. 5 shows a Fourier transform infrared spectrum characterization of carboxylated polystyrene microspheres from example 2.

FIG. 6 shows the change of fluorescence intensity of supernatant after the quantum dot fluorescence-encoded microspheres in example 3 are stored in PBS solution for 36 days.

Fig. 7 shows a scatter plot of fluorescence intensity distribution of FSC, SSC, QD525, QD605 channels in flow cytometry for quantum dot fluorescence encoded microspheres in example 3.

FIG. 8 shows the fluorescence spectrum of a quantum dot fluorescence-encoded microsphere prepared in example 3 under excitation of 400 nm.

FIG. 9 shows a TEM image of extracellular vesicles extracted from the cell culture solution in example 4.

Fig. 10 shows the particle size distribution of extracellular vesicles extracted from the cell culture fluid measured by the nanoparticle particle-size-tracking instrument in example 4.

Fig. 11 shows a scatter plot of fluorescence intensity distribution of FSC, SSC, QD525, QD605 channels in flow cytometry for the nine quantum dot fluorescence encoded microspheres of example 5.

FIG. 12 shows the expression of 9 extracellular vesicular protein markers in five cell lines as measured by flow cytometry using the 9 quantum dot fluorescence-encoded microspheres of example 7.

FIG. 13 shows the expression of 9 extracellular vesicular protein markers in 32 breast cancer patients and 6 control groups, as measured by flow cytometry using the 9 quantum dot fluorescence-encoded microspheres of example 6.

Fig. 14 shows the results of ROC analysis of CD47, CD133, and EGFR in serum-derived extracellular vesicles from clinical samples, with AUC calculations plotted in the figure.

Detailed Description

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.

Unless otherwise indicated, the human breast cancer cell lines used in the examples below were purchased from the cell center of the institute of basic medicine, academy of medical sciences, china.

All reagents used in the following examples were analytical reagents unless otherwise indicated.

In the embodiment of the invention, the quantum dots are purchased from Wuhan Jia source quantum dot technology development limited company. The apparatus used for the experiment: nanoparticle tracers (NTA, NanoSight Technology, Malvern, UK), Scanning electron microscopes (Scanning electron microscope, Hitachi S4800, Japan), transmission electron microscopes (transmission electron microscope, HT7700, Hitachi High-Tech, Japan), flow cytometers (FCM, Invitrogen Attune NxT, US)

EXAMPLE 1 Synthesis of polystyrene seed microspheres

The reaction was carried out in a 100mL three-necked flask equipped with a condensing reflux apparatus, with nitrogen as the shielding gas, magnetic stirring at 100rpm, AIBN as the initiator, and a mixed solution of ethanol and 2-methoxyethanol as the reaction medium. Taking 40g of mixed solution of ethanol and 2-methoxyethanol, adding 0.4g of polyvinylpyrrolidone (PVP), and fully stirring for dissolving. Then, 8g of the purified styrene monomer, 0.08g of Azobisisobutyronitrile (AIBN) was added and mixed well. Introducing nitrogen into the reaction system for 15min to remove oxygen in the system. Heating in oil bath, and heating to 70 ℃ for reaction for 12 h. After the polymerization reaction, the mixture was slowly cooled to room temperature. The product was centrifuged at 5000rpm for 5 min. The supernatant was discarded and washed three times with ethanol and purified water. The resulting product was dried in a vacuum oven at 60 ℃ for 24 h.

The particle size was 1 μm as statistically measured by a scanning electron microscope, and the scanning electron micrograph thereof is shown in FIG. 2.

Example 2 Synthesis of carboxylated crosslinked polystyrene Quantum dot fluorescent microspheres

The reaction was carried out in a 250mL three-necked flask equipped with a condensed reflux vessel, under nitrogen, with magnetic stirring at 200 rpm. 0.1g of cyclohexane is added into 10g of SDS solution (wt0.25 percent), and emulsified for standby after 30min of ultrasonic treatment. Weighing 1g of polystyrene seed microspheres with the particle size of 1 micron, adding 30g of SDS solution into the polystyrene seed microspheres, performing ultrasonic treatment for 10min to fully disperse the polystyrene seed microspheres, and adding the polystyrene seed microspheres into a three-necked bottle; finally, the two are mixed and swelled at 30 ℃ for 6 h. 80g of SDS solution was added to the reaction system, and 8.5g of styrene, 1g of Ethylene Glycol Dimethacrylate (EGDMA), 0.5g of methacrylic acid (MAA) and 0.1g of Benzoyl Peroxide (BPO) were further added to swell at 30 ℃ for 10 hours. 2g of PVP and 100 mu L of methylene blue aqueous solution with the concentration of 5mg/mL are added into the reaction system, 50mL of water is added, and the polymerization reaction is carried out for 12 hours by heating the reaction system to 80 ℃ in an oil bath. After the reaction was completed, it was slowly cooled to room temperature. Repeatedly centrifuging and washing the product in a centrifuge at 3000rpm for many times to remove microspheres subjected to secondary nucleation in the system; and finally, drying the microspheres in a vacuum oven at 60 ℃ for 24 hours to obtain the carboxylated crosslinked polystyrene microspheres, wherein a scanning electron microscope photo of the crosslinked polystyrene microspheres is shown in figure 3, and a Fourier infrared spectrum characterization result is shown in figure 5. The carboxylated polystyrene microspheres obtained from 0.1g of the carboxylated polystyrene microspheres are re-dispersed in 1mL of deionized water, washed three times in a centrifuge at 3000rpm for 5min, and the supernatant is removed. Then, the microspheres are dispersed in 4mL of mixed solution of chloroform and n-butanol (volume ratio is 5:95), and ultrasonic dispersion is carried out for 30 min. And (3) taking 500 mu L of oil-soluble shell-core ZnCdSe/ZnS quantum dot solution with the emission wavelength of 595nm, adding the mixed solution of the chloroform and the n-butanol in which the microspheres are dispersed, and performing ultrasonic dispersion for 30 min. And repeatedly washing and centrifuging for 5 times by using absolute ethyl alcohol, washing and centrifuging for 3 times by using deionized water, and re-suspending the precipitate in ultrapure water to obtain the carboxylated quantum dot fluorescence crosslinked polystyrene microsphere. The scanning electron micrograph is shown in FIG. 4. Because the cross-linking agent EGDMA is added during the synthesis of the carboxylated polystyrene microspheres, the prepared carboxylated quantum dot fluorescent cross-linked polystyrene microspheres have good swelling resistance, and scanning electron microscope photos show that the quantum dot polystyrene microspheres prepared by the method keep good spherical morphology after swelling.

Example 3 preparation of Quantum dot fluorescent encoding microspheres by adjusting the addition amount of Quantum dots

Carboxylated crosslinked polystyrene microspheres were obtained in the same manner as in example 2. Centrifuging 1000L of microsphere dispersion in a centrifuge at 3000rpm for 5 min; the precipitate was redispersed in 5mL of a 10:90 volume ratio mixture of chloroform and n-butanol and dispersed by sonication for 30 min. The microsphere mixture was then divided into 5 equal portions, to which 0. mu.L, 10. mu.L, 20. mu.L, 30. mu.L, 40. mu.L of a quantum dot (QD605) solution emitting at 605nm was added, respectively. Thereafter, each of the mixed solutions was divided into five equal parts, to which 0. mu.L, 20. mu.L, 30. mu.L, 40. mu.L, and 50. mu.L of a quantum dot (QD525) solution having an emission wavelength of 525nm was added, respectively. And ultrasonically dispersing the mixed solution for 30 min. And finally, repeatedly washing and centrifuging the product for 5 times by using absolute ethyl alcohol, washing and centrifuging the product for 3 times by using deionized water, and resuspending the precipitate in ultrapure water. The 25 kinds of prepared fluorescent coding microspheres are respectively marked as 1-25 according to the doping concentration of the quantum dots from low to high. In the flow cytometer, the corresponding fluorescence channel is selected, and the distribution of different fluorescence-encoded microspheres is determined according to the fluorescence intensity of the two channels, with the result shown in fig. 7.

The prepared quantum dot coded fluorescent microspheres have the fluorescence stability. Storing 23 different quantum dot fluorescence encoding microspheres in PBS buffer solution, and checking fluorescence intensity in supernatant of the stored solution after storing for a period of time to indicate desorption and leakage of quantum dots doped in the microspheres. Fig. 6 shows the fluorescence intensity results of the supernatant on day 36 of storage, where the control group was blank PBS buffer. The result shows that the fluorescence intensity of the supernatant of other samples has no obvious change except that the fluorescence intensity of the supernatant of a small amount of samples is increased, and the supernatant of other samples shows better long-term stability.

FIG. 8 shows the fluorescence spectrum of a quantum dot fluorescence-encoded microsphere prepared in example 3 under excitation of 400 nm. Fig. 8 shows that the ratio of the doping level of QD525 to the doping level of QD605 is 1: 1 of the quantum dot fluorescent coding microspheres.

As shown in FIG. 8, the prepared carboxylated quantum dot encoded fluorescent microspheres have strong fluorescence at the wavelength positions of 525nm and 605nm under the excitation wavelength of 405 nm.

Example 4 extraction of extracellular vesicles from cell culture supernatant

Collecting cell culture supernatants of the breast cancer cell lines MCF 7, SKBR3, MDA-MB-468, MDA-MB-231 and normal breast epithelial MCF-10A, filtering the cell culture supernatant with a 0.2 μm filter membrane, centrifuging with an ultracentrifuge after filtering, and centrifuging at 110000g for 2 h. The supernatant was discarded, the PBS resuspended and then centrifuged again at 110000g for 2h, and the resulting pellet resuspended in 100. mu.L PBS. And dripping 10 mu L of the obtained sample on a 400-mesh ultrathin carbon film supporting copper net, washing with water twice, carrying out negative staining by 1% uranyl acetate, and shooting under the accelerating voltage of 80kV of a transmission electron microscope, wherein the result shows that the extracellular vesicles are successfully extracted. The topography is shown in fig. 9. The size distribution and concentration of extracellular vesicles obtained by diluting 50. mu.L with 1000. mu.L PBS and detecting with a nanoparticle tracer are shown in FIG. 10.

Example 5 preparation of Quantum dot fluorescent-encoded microsphere arrays

Coupling the carboxylated quantum dot fluorescent coding microspheres and the antibody through amidation reaction to obtain the antibody functionalized modified quantum dot fluorescent coding microsphere array.

A total of nine quantum dot fluorescence encoded microspheres coupled to CD24, CD44, CD47, CD133, CXCR4, HER2, EGFR, EpCAM, and N-Cadherin, respectively:

respectively suck 5X 10³-1×10⁶Preferably 5 × 10⁴And (3) putting the stock solution of the quantum dot fluorescent coding microspheres into a 1.5mL centrifuge tube, removing the stock solution of the microspheres by using a magnetic sorting frame, and washing the microspheres for three times by using MES buffer solution. And activating the carboxyl on the surface of the microsphere by using an EDC/NHS carboxyl activation method. And (5) carrying out shaking table incubation and activation at room temperature for 15 min. The supernatant was discarded, washed three times with PBS buffer solution, and excess EDC and NHS were removed. And adding the antibodies with saturation concentration into the corresponding microspheres respectively, wherein the dosage of the antibodies is 1-20 mu g, preferably 2-10 mu g, and the optimal dosage of each antibody is different. Wherein the saturation concentration of the CD44 antibody is 2.5. mu.g/100. mu.L, the saturation concentration of the CD24 antibody is 2.5. mu.g/100. mu.L, the saturation concentration of the CD133 antibody is 5. mu.g/100. mu.L, the concentration of the CD47 antibody is 2.5. mu.g/100. mu.L, the saturation concentration of the CXCR4 antibody is 5. mu.g/100. mu.L, the saturation concentration of the EpCAM antibody is 2.5. mu.g/100. mu.L, the concentration of the HER2 antibody is 5. mu.g/100. mu.L, the concentration of the EGFR antibody is 5. mu.g/100. mu.L and the concentration of the N-Cadherin antibody is 2.5. mu.g/100. mu.L. Vortex well and incubate at 37 ℃ for 2h with a shaker, then wash 3 times with PBS buffer to remove unbound antibody. Then adding 200 μ L2% -10% BSA solution into the microspheres, and incubating in a shaker at 37 ℃ to 1 ℃Blocking for 3h, preferably for 1h with 5% BSA. And discarding the supernatant, washing the supernatant for 2 times by using a PBS (phosphate buffer solution), and then suspending the supernatant into the PBS to obtain the quantum dot fluorescent coding microsphere immune array modified by the specific capture antibody.

Example 6 quantitative determination of extracellular vesicle membrane proteins Using carboxylated polystyrene fluorescent-encoded microspheres

The carboxylated quantum dot fluorescent coding microsphere can be coupled with various molecules by utilizing carboxyl functional groups of which the surfaces are beneficial to modification. In the embodiment, the antibody is coupled to the surface of the microsphere, so that the microsphere can be used for specifically capturing extracellular vesicles, and multi-target detection is performed on each protein of the extracellular vesicles in a flow cytometer.

1) Saturation curve determination of capture antibodies coupled to quantum dot encoded microspheres:

according to the recommendation, 1-20. mu.g antibody corresponds to 1X 10⁶Starting the microsphere, the procedure of 4) was followed, and 5X 10 microspheres were added⁴Adding the fluorescent coding microspheres into antibodies with different working concentrations, and incubating for 2h in a shaking table at 37 ℃, wherein each antibody corresponds to one fluorescent coding microsphere. The supernatant was discarded, PBS buffer was added to wash away unbound antibody, and 2% -10% BSA was added to block excess binding sites to avoid non-specific adsorption. After washing 3 times with PBS buffer, the cells were incubated with a secondary antibody diluted to the working concentration with a fluorescent label for 40min at room temperature on a shaker. The supernatant was discarded and unbound fluorescent secondary antibody was washed off with PBS. The microsphere-antibody complexes were resuspended in PBS buffer and assayed by flow.

2) Determination of saturation curves of extracellular vesicles coupled to quantum dot fluorescence-encoded microspheres functionalized with capture antibodies:

according to the calculation of the vesicle protein concentration, 2.5X 10⁴The fluorescent microspheres coupled with different capture antibodies were incubated overnight with extracellular vesicles of different total protein concentrations in a shaker at 37 ℃. The dosage of the extracellular vesicles derived from the cell line culture supernatant is 1-50 μ g, preferably 25 μ g. Washing with PBS buffer for 3 times to remove unbound extracellular vesicles, and adding 100 μ L of fluorescently labeled CD9/CD63/CD81 mixed antibody solution diluted to working concentrationThe solution was incubated at 37 ℃ for 40min with shaking. The supernatant was discarded, washed three times with PBS, and resuspended in 400. mu.L of PBS buffer for determination of flow-positive binding rate.

Fig. 11 shows a scatter plot of fluorescence intensity distribution of FSC, SSC, QD525, QD605 channels in flow cytometry for the nine quantum dot fluorescence encoded microspheres of example 5. FIG. 11 shows that the fluorescently encoded microspheres used in example 5 can be accurately distinguished in flow cytometry based on their fluorescent encoding.

Example 7 targeting of extracellular vesicle membrane proteins derived from tumor serum using carboxylated polyphenylene Quantum dot-encoded microspheres Quantity detection

The antibody functionalized quantum dot fluorescent coding microspheres prepared in example 5 are used for measuring the breast cancer cell line and the extracellular vesicles derived from the culture supernatant of normal mammary epithelial cells and the serum of a breast cancer patient, and the corresponding results are shown in fig. 12 and 13.

FIG. 12 shows the expression of 9 extracellular vesicular protein markers in five cell lines as measured by flow cytometry using the 9 quantum dot fluorescence-encoded microspheres of example 6. FIG. 13 shows the expression of 9 extracellular vesicular protein markers in 32 breast cancer patients and 6 control groups, as measured by flow cytometry using the 9 quantum dot fluorescence-encoded microspheres of example 6. The quantum dot fluorescence coding microsphere determination can find that the expression quantity of protein markers related to dryness and proliferation in extracellular vesicles derived from normal epithelial cells is low in the verification of breast cancer cell lines and normal epithelial cells of the breast, and the expression quantity of the protein markers related to dryness and proliferation on the breast cancer cell lines such as MDA-MB-231 and MBA-MB-468 is high. For breast cancer patients and control group serum source extracellular vesicles assay known: the expression of the serum-derived extracellular vesicle 9 proteins of the breast patients is different from that of the control group, wherein CD47, CD133 and EGFR can be used for judging the breast cancer malignancy and malignancy.

To further obtain information on relevant tumor markers, we analyzed the 9 protein markers separately, and we determined the sensitivity, specificity and accuracy of each marker by Receiver Operating Characteristics (ROC) analysis, as shown in fig. 14, where CD133, CD47 and EGFR were found to have good diagnostic value. In our experiment, the specificity of CD133 is 84.38%, the sensitivity is 90.63% [ 95% confidence interval (81.48% -100%), the specificity of CD47 is 80.73%, the sensitivity is 68.75% (66.73% -94.73%), the specificity of EGFR is 83.33%%, and the sensitivity is 90.63% (71.14% -99.69%). CD47, CD133 and EGFR in the extracellular vesicle of the serum of the breast cancer patient can be used as high-sensitivity and specificity biomarkers for judging the benign and malignant breast cancer, and the combination of the three can further improve the sensitivity and specificity of detection.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims (10)

1. The carboxylated quantum dot fluorescent coding microsphere is characterized by being a carboxylated crosslinked polystyrene microsphere containing quantum dots, the quantum dots are core-shell quantum dots ZnCdSe/ZnS, and the carboxylated quantum dot fluorescent coding microsphere has fluorescent response under the excitation wavelength of 300-600 nm, preferably 300-500 nm, and most preferably 400 nm;

preferably, the particle size of the carboxylated quantum dot fluorescent coding microsphere is 1-20 μm; and/or

The emission wavelength of the quantum dots is 500 nm-750 nm.

2. The method for preparing the carboxylated quantum dot fluorescent coding microsphere according to claim 1, wherein the method comprises the following steps:

(1) preparing polystyrene seed microspheres by a dispersion polymerization method;

(2) preparing carboxylated crosslinked polystyrene microspheres by a seed swelling polymerization method;

(3) and preparing the quantum dot fluorescent coding microspheres.

3. The method of claim 2, wherein the step (1) comprises: dissolving a styrene monomer in a mixed solution of ethanol and 2-methoxyethanol, reacting in the presence of nitrogen by using polyvinylpyrrolidone as a dispersant and azodiisobutyronitrile as an initiator, and centrifuging, washing and drying after the reaction is finished to obtain the polystyrene seed microspheres.

4. The method of claim 3, wherein the reaction temperature is 60 to 90 ℃, preferably 70 to 90 ℃, and most preferably 70 ℃; and/or

The reaction time is 10 to 48 hours, preferably 10 to 24 hours, and more preferably 10 to 15 hours.

5. The production method according to any one of claims 2 to 4, wherein the step (2) includes: dispersing the polystyrene seed microspheres prepared in the step (1) into a sodium dodecyl sulfate aqueous solution, adding a good solvent of polystyrene for swelling, adding the sodium dodecyl sulfate aqueous solution, a polystyrene monomer, olefine acid, a crosslinking agent ethylene glycol dimethacrylate and an initiator of ethylene glycol peroxide for continuing swelling, finally adding a dispersant of polyvinylpyrrolidone for reacting under the protection of nitrogen, and after the reaction is finished, centrifugally washing and drying;

preferably, the good solvent of the polystyrene is selected from one or more of the following: chloroform, cyclohexane, toluene; and/or

The olefinic acid is selected from one or more of the following: methacrylic acid, crotonic acid and hexenoic acid.

6. The production method according to any one of claims 2 to 5, wherein the step (3) includes: dispersing the microspheres obtained in the step (2) in a swelling agent for swelling, then adding a quantum dot solution into a microsphere dispersion solution, performing ultrasonic dispersion, and washing to obtain the quantum dot fluorescence encoding microspheres;

preferably, the swelling agent is a mixed solvent of chloroform and n-butanol;

more preferably, the volume fraction of chloroform in the mixed solvent is 1-10%.

7. A biochip comprising the carboxylated quantum dot fluorescence-encoded microsphere described in claim 1 or the carboxylated quantum dot fluorescence-encoded microsphere prepared by the preparation method described in any one of claims 2 to 6;

preferably, the biochip is a suspension phase chip.

8. The method for preparing a biochip according to claim 7, wherein the method comprises the steps of:

(1) preparing carboxylated quantum dot fluorescence-encoded microspheres according to the preparation method of any one of claims 2 to 6;

(2) coupling the carboxylated quantum dot fluorescent coding microspheres and the antibody through amidation reaction to obtain the quantum dot fluorescent coding microsphere array functionally modified by the antibody.

9. A method for multi-target assay of extracellular vesicle surface protein, comprising performing a flow cytometry on a multi-target assay of extracellular vesicle surface protein using the biochip of claim 7.

10. Use of the carboxylated quantum dot fluorescence-encoded microspheres according to claim 1, the carboxylated quantum dot fluorescence-encoded microspheres prepared by the preparation method according to any one of claims 2 to 6, or the biochip according to claim 7 for the preparation of a medical product for the diagnosis, prognosis, prediction of therapeutic effect and/or tracking of therapeutic effect of a disease;

preferably, the medical product is selected from one or more of the following: in vitro diagnostic reagents and medical instruments.

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