Choline phosphoric acid modified quantum dot composite material as well as preparation method and application thereof

Abstract

The invention relates to the field of medical detection, in particular to a choline phosphoric acid modified quantum dot composite material and a preparation method and application thereof. The method comprises the following steps: (1) carrying out reaction on oil-soluble quantum dots and sulfydryl polyethylene glycol acrylamide to obtain surface double-bond modified quantum dots; and (2) in the presence of an initiator, carrying out polymerization reaction on the surface double bond modified quantum dots, a cross-linking agent and the choline phosphoric acid derivative as shown in the formula (1). According to the invention, oil-soluble quantum dots, sulfydryl polyethylene glycol acrylamide and choline phosphate derivatives are used to prepare the choline phosphate modified quantum dot composite material with the ability of specific recognition and combination of phosphatidylcholine and fluorescence color development, and the composite material can be used for marking and combination of colorectal cancer exosomes. Further, the kit can be used for detecting colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of medical testing, specifically to a choline phosphate modified quantum dot composite material, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is a malignant tumor that occurs in the colon or rectum of the lower digestive tract and is one of the leading causes of cancer death. Therefore, early diagnosis of colorectal cancer is expected to reduce the number of deaths from this disease. Currently, colonoscopy is the gold standard for colorectal cancer diagnosis, being the most effective and accurate method. However, due to its invasiveness and patient discomfort, it is not suitable for large-scale screening. In addition, there are fecal occult blood tests (FIT) and multi-target fecal FIT-DNA tests, which detect hemoglobin and DNA to determine colorectal cancer, but these methods suffer from low sensitivity, high false positive rates, and high cost. Therefore, there is an urgent need to develop a highly sensitive, simple, and convenient colorectal cancer screening method.

[0003] Developing colorectal cancer screening methods first requires identifying a highly accurate biomarker, typically a representative protein or nucleic acid. However, these substances are present at the site of colorectal cancer and are difficult to obtain through simple methods. Colorectal cancer exosomes carry proteins, lipids, nucleic acids, and other bioactive molecules derived from colorectal cancer cells. They are miniature representatives of colorectal cancer cells and are easily obtained by being excreted in feces and urine. Therefore, colorectal cancer can be diagnosed by detecting the proteins and nucleic acids on colorectal cancer exosomes.

[0004] The expression of exosome surface membrane proteins CD147 and A33 in colorectal cancer cells is significantly increased compared to exosomes secreted by normal cells, and can serve as biomarkers for the detection of colorectal cancer. However, how to label exosomes from colorectal cancer and detect the high expression of exosome membrane proteins CD147 and A33 remains a challenge. Summary of the Invention

[0005] The purpose of this invention is to overcome the problems of low sensitivity, high false positive rate and high cost in the existing technology for colorectal cancer detection, and to provide a choline phosphate modified quantum dot composite material, its preparation method and application.

[0006] To achieve the above objectives, the present invention provides a method for preparing choline phosphate-modified quantum dot composite materials, the method comprising the following steps:

[0007] (1) Reaction of oil-soluble quantum dots with mercapto-polyethylene glycol acrylamide yields quantum dots with surface double bond modification;

[0008] (2) In the presence of an initiator, surface double bond modified quantum dots, crosslinking agent and choline phosphate derivative shown in formula (1) are subjected to polymerization reaction;

[0009]

[0010] Preferably, the oil-soluble quantum dots are selected from oil-soluble CdSe / ZnS quantum dots and / or oil-soluble CdZn / SeS quantum dots;

[0011] Preferably, the hydrated particle size of the oil-soluble quantum dots is 20-30 nm.

[0012] Preferably, in step (1), the molecular weight of the mercaptopolyethylene glycol acrylamide is 1000D-3000D;

[0013] Preferably, the weight ratio of the oil-soluble quantum dots to the mercapto-polyethylene glycol acrylamide is 1:5-20.

[0014] Preferably, in step (1), the reaction conditions include: a temperature of 10-25°C and a time of 8-12 hours.

[0015] Preferably, in step (2), the weight ratio of the choline phosphate derivative represented by formula (1) to the oil-soluble quantum dots is 20-200:1; and / or

[0016] The weight ratio of the crosslinking agent to the choline phosphate derivative shown in formula (1) is 1:20-30; and / or;

[0017] The crosslinking agent is N,N-methylenebisacrylamide.

[0018] Preferably, in step (2), the initiator is ammonium persulfate and tetramethylethylenediamine;

[0019] Preferably, in step (2), the polymerization reaction takes 2-3 hours.

[0020] A second aspect of the present invention provides a choline phosphate-modified quantum dot composite material prepared according to the method described above.

[0021] A third aspect of this invention provides the application of the choline phosphate modified quantum dot composite material described above in the detection of exosomes in colorectal cancer.

[0022] A fourth aspect of the present invention provides a method for labeling colorectal cancer exosomes, the method comprising: mixing the choline phosphate modified quantum dot composite material described above with exosomes.

[0023] The fifth aspect of this invention provides a kit for detecting exosome marker membrane proteins in rectal cancer, the kit comprising a lateral chromatography test strip and the choline phosphate modified quantum dot composite material described above;

[0024] The lateral chromatography test strip includes: a base plate, a sample pad, a detection membrane, and an absorbent pad. The sample pad, the detection membrane, and the absorbent pad are sequentially adhered to the base plate and connected by overlapping. The detection membrane is provided with detection lines and control lines.

[0025] The detection line is coated with colorectal cancer marker antibodies, and the quality control line is coated with exosome marker antibodies.

[0026] Preferably, the colorectal cancer marker antibody is selected from anti-CD147 antibody and / or anti-A33 antibody;

[0027] Preferably, the exosome marker antibody is selected from at least one of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] 1. This invention uses quantum dots, mercapto-polyethylene glycol acrylamide, and choline phosphate derivatives to prepare a choline phosphate-modified quantum dot composite material that simultaneously possesses the ability to specifically recognize and bind phosphatidylcholine and exhibit fluorescence color development. This composite material can be used to label and bind colorectal cancer exosomes, and thus can be used in the detection of colorectal cancer.

[0030] 2. The kit for detecting exosome marker membrane proteins of rectal cancer described in this invention utilizes the labeling ability of choline phosphate modified quantum dot composite material on colorectal cancer exosomes, combined with lateral chromatography test strips and antibodies coated on the test strips, to rapidly and accurately detect colorectal cancer marker membrane proteins contained in exosomes.

[0031] 3. The kit described in this invention is a simple, rapid, and structurally simple tool with high sensitivity and specificity for colorectal cancer biomarkers, and can be used for the screening and detection of colorectal cancer. Attached Figure Description

[0032] Figure 1 This is a TEM comparison image of the choline phosphate modified quantum dot composite material prepared in Example 1 of Test Example 1 and the oil-soluble quantum dots used in Example 1;

[0033] Figure 2 The fluorescence intensity diagrams are of the choline phosphate modified quantum dot composite materials prepared in Example 1 at different concentrations in Test Example 1;

[0034] Figure 3This is a colocalization diagram of the choline phosphate modified quantum dot composite material and exosomes in Test Example 2;

[0035] Figure 4 This is a schematic diagram of the lateral chromatography test strip described in an embodiment of the present invention;

[0036] Figure 5 This is a schematic diagram illustrating the detection principle of a kit for detecting exosome marker membrane proteins in rectal cancer according to a specific embodiment of the present invention.

[0037] Explanation of reference numerals in the attached figures

[0038] 1. Base plate 2. Sample pad

[0039] 3. Detection membrane 4. Absorbent pad

[0040] 31 Testing Line 32 Quality Control Line

[0041] 100 Lateral Chromatography Test Strips Detailed Implementation

[0042] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0043] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0044] This invention provides a method for preparing choline phosphate modified quantum dot composites (CP-QDs), the method comprising the following steps:

[0045] (1) Reaction of oil-soluble quantum dots (QDs) with mercaptopolyethylene glycol acrylamide yields quantum dots with surface double bond modification;

[0046] (2) In the presence of an initiator, surface double bond modified quantum dots, crosslinking agent and choline phosphate derivative shown in formula (1) are subjected to polymerization reaction;

[0047]

[0048] In the method described in this invention, the thiol groups contained in mercapto-polyethylene glycol acrylamide can bind to oil-soluble quantum dots, and the polyethylene glycol chain segments help improve the hydrophilicity of the material. Based on this, by reacting oil-soluble quantum dots with mercapto-polyethylene glycol acrylamide in step (1), surface double-bond modified aqueous quantum dots can be obtained. In step (2), the amide groups, crosslinking agents, and choline phosphate derivatives contained in the surface double-bond modified aqueous quantum dots can undergo free radical polymerization to obtain a choline phosphate modified quantum dot composite material. This composite material has the ability to specifically bind phosphatidylcholine on the exosome membrane of colorectal cancer through covalent bonds and to exhibit fluorescence color development. It can be applied to the detection, labeling, and separation of colorectal cancer exosomes, and can further be used in the detection of colorectal cancer.

[0049] In a preferred embodiment, in step (1), the oil-soluble quantum dots are selected from oil-soluble CdSe / ZnS quantum dots and / or oil-soluble CdZn / SeS quantum dots. The oil-soluble CdSe / ZnS quantum dots comprise CdSe / ZnS quantum dots and a hydrophobic ligand coated on the surface of the CdSe / ZnS quantum dots. The hydrophobic ligand can be oleic acid. The CdSe / ZnS quantum dots have a core-shell structure, with a CdSe core and a ZnS shell. The maximum emission wavelength of the oil-soluble CdSe / ZnS quantum dots is 525±5 nm.

[0050] More preferably, the hydrated particle size of the oil-soluble quantum dots is 20-30 nm.

[0051] In a preferred embodiment, in step (1), the molecular weight of the mercaptopolyethylene glycol acrylamide is 1000D-3000D.

[0052] According to some preferred embodiments of the present invention, the specific process of step (1) includes: mixing oil-soluble quantum dots with an organic solvent, adding mercapto-polyethylene glycol acrylamide to react, then adding ethanol and n-hexane for centrifugation, and then resuspending the precipitate in water. The organic solvent may be chloroform.

[0053] In a preferred embodiment, the reaction conditions in step (1) include a temperature of 10-25°C and a time of 8-12 hours.

[0054] According to some preferred embodiments of the present invention, the specific process of step (2) includes: in the presence of a protective gas and an initiator, a crosslinking agent and a choline phosphate derivative of formula (1) are added dropwise to the material obtained in step (1) to carry out a polymerization reaction, and then dialysis purification is performed.

[0055] In a preferred embodiment, in step (2), the weight ratio of the choline phosphate derivative represented by formula (1) to the oil-soluble quantum dots is 20-200:1, more preferably 50-100:1; specifically, it can be 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0056] In a preferred embodiment, the crosslinking agent may be N,N-methylenebisacrylamide. The weight ratio of the crosslinking agent to the choline phosphate derivative shown in formula (1) is 1:20-30; specifically, it may be 1:20, 1:22, 1:25, 1:28 or 1:30.

[0057] In this invention, the protective gas can be an inert gas commonly used in the art, such as argon.

[0058] In the method described in this invention, there are no special requirements for the selection of the initiator in step (2). It can be an initiator commonly used in the art for amide polymerization reactions, such as ammonium persulfate and tetramethylethylenediamine. There are no special requirements for the amount of the initiator; it can be added according to the conventional feeding amount in the art.

[0059] In a preferred embodiment, in step (2), the polymerization reaction takes 2-3 hours and the polymerization reaction temperature is 20-30°C.

[0060] In a preferred embodiment, the dialysis purification time in step (2) is 4-12 hours.

[0061] A second aspect of the present invention provides a choline phosphate-modified quantum dot composite material prepared according to the method described above.

[0062] A third aspect of this invention provides the application of the choline phosphate modified quantum dot composite material described above in the detection of exosomes in colorectal cancer.

[0063] The choline phosphate-modified quantum dot composite material described in this invention not only specifically recognizes colorectal cancer exosomes but also exhibits excellent fluorescence colorimetric ability. Based on this, the choline phosphate-modified quantum dot composite material can specifically bind to the abundant phosphatidylcholine on the exosome membrane surface through covalent interactions. This specific approach allows the exosomes to be linked to the quantum dots, thereby labeling the exosome membrane surface with fluorescently colored quantum dots, thus enabling the detection and analysis of colorectal cancer exosomes.

[0064] A fourth aspect of the present invention provides a method for labeling colorectal cancer exosomes, the method comprising: mixing the choline phosphate modified quantum dot composite material described above with exosomes.

[0065] In a preferred embodiment, the method for labeling colorectal cancer exosomes includes the following steps: mixing a colorectal cancer exosome sample with an aqueous solution of the choline phosphate-modified quantum dot composite material, and then incubating at 10-25°C for 1-2 hours. The incubation can be carried out on a shaker at a speed of 200-400 rpm.

[0066] More preferably, the mass ratio of the colorectal cancer exosome sample to the choline phosphate modified quantum dot composite material is 5-10:1, and the colorectal cancer exosome sample is measured by total protein content.

[0067] In this invention, there are no special requirements for the method of obtaining the colorectal cancer exosome samples; it is a conventional method in the field.

[0068] The fifth aspect of this invention provides a kit for detecting exosome marker membrane proteins in rectal cancer, the kit comprising a lateral chromatography test strip and the choline phosphate modified quantum dot composite material described above;

[0069] Among them, in conjunction with reference Figure 4 The lateral chromatography test strip 100 includes: a base plate 1, a sample pad 2, a detection membrane 3, and an absorbent pad 4. The sample pad 2, the detection membrane 3, and the absorbent pad 4 are sequentially adhered to the base plate 1 and connected by overlapping. The detection membrane 3 is provided with a detection line 31 and a quality control line 32.

[0070] The detection line 31 is coated with colorectal cancer marker antibodies, and the control line 32 is coated with exosome marker antibodies.

[0071] Preferably, the colorectal cancer marker antibody is selected from anti-CD147 antibody and / or anti-A33 antibody.

[0072] Preferably, the exosome marker antibody is selected from at least one of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody.

[0073] In this invention, the base plate 1 can be a low fluorescence background base plate, the absorbent pad and the sample pad can be of the types commonly used in the art, and the detection membrane 3 can be selected from nitrocellulose membranes.

[0074] In a preferred embodiment, the detection membrane 3 is provided with a detection line 31 near the sample pad 2 and a quality control line 32 near the absorbent pad 4.

[0075] In this invention, in conjunction with reference to Figure 5The main process for detecting exosomes using the kit for detecting exosome marker membrane proteins in rectal cancer is as follows: the choline phosphate modified quantum dot composite material, exosome sample and chromatography buffer are mixed evenly to obtain a test solution. The test solution is dropped onto sample pad 2. Based on capillary action, the solution will sequentially pass through the detection membrane (detection line 31 first, then control line 32) and absorbent pad 4. After 10-20 minutes, the regions of detection line 31 and control line 32 are observed. The test kit can be observed using ultraviolet light, with the naked eye, or with a fluorescence imaging instrument (such as a dry immunofluorescence analyzer or a gel imaging system). The detection principle of the kit is as follows: the choline phosphate modified quantum dot composite material can label exosomes; the colorectal cancer marker antibody attached to the test line 31 can recognize and bind to the colorectal cancer marker in the exosome through antigen-antibody interaction; the exosome marker antibody on the control line 32 can recognize and bind to the exosome marker. If the exosome sample contains colorectal cancer markers, the binding of the antibody to the antigen will capture the colorectal cancer marker on the test line 31. The test result is determined by the color development or luminescence of the test line 31 and the control line 32. If both the test line 31 and the control line 32 show color, the result is positive, indicating that the exosome sample contains a colorectal cancer marker corresponding to the antibody attached to the test line 31. If only the control line 32 shows color, the result is negative. If the control line 32 does not show color, the result is invalid regardless of whether the test line 31 shows color.

[0076] In this invention, the chromatography buffer can be any chromatography buffer conventionally used in the art. For example, it can be a mixture of PBS buffer and BSA.

[0077] The present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited thereto.

[0078] The oil-soluble CdSe / ZnS quantum dots used in the following examples were purchased from Wuhan Jiayuan Quantum Dot Technology Development Co., Ltd., and the product name is oil-soluble quantum dot Q1525.

[0079] Example 1

[0080] Preparation of choline phosphate modified quantum dot composite material S1:

[0081] (1) Oil-soluble CdSe / ZnS quantum dots (TEM image as shown) Figure 1 The hydrated CdSe / ZnS quantum dots (with a hydrated particle size of 20-30 nm) were dissolved in chloroform, and mercaptopolyethylene glycol acrylamide (molecular weight of 2000 D) was added. The mixture was ultrasonically mixed for 5 min and then reacted with shaking at room temperature (25 °C) for 10 h. Then anhydrous ethanol and n-hexane were added, the precipitate was centrifuged, and the precipitate was resuspended in ultrapure water to obtain aqueous quantum dots with surface double bonds modified. The weight ratio of oil-soluble CdSe / ZnS quantum dots to mercaptopolyethylene glycol acrylamide was 1:10.

[0082] (2) The surface double bond modified aqueous quantum dots obtained in step (1) were mixed with water. Then, under argon protection, the choline phosphate derivative and crosslinking agent (N,N-methylenebisacrylamide) shown in formula (1) were added dropwise as initiators. The mixture was allowed to react fully at 25°C for 3 hours, and then purified by dialyzing for 8 hours to obtain the choline phosphate modified quantum dot composite material S1 (TEM image shown). Figure 1 ); wherein, the weight ratio of the choline phosphate derivative shown in formula (1) to the oil-soluble CdSe / ZnS quantum dots is 70:1, and the weight ratio of the crosslinking agent to the choline phosphate derivative shown in formula (1) is 1:25.

[0083] Example 2

[0084] Preparation of choline phosphate modified quantum dot composite material S2:

[0085] (1) Dissolve oil-soluble CdSe / ZnS quantum dots in chloroform, add mercaptopolyethylene glycol acrylamide (molecular weight 2000D), sonicate for 5 min, and shake at room temperature (25℃) for 10 h; then add anhydrous ethanol and n-hexane, centrifuge to precipitate, and then add ultrapure water to resuspend the precipitate to obtain surface double bond modified aqueous quantum dots; wherein, the weight ratio of oil-soluble CdSe / ZnS quantum dots to mercaptopolyethylene glycol acrylamide is 1:20;

[0086] (2) The surface double bond modified aqueous quantum dots obtained in step (1) are mixed with water, and then under argon protection, the choline phosphate derivative and crosslinking agent (N,N-methylenebisacrylamide) shown in formula (1) are added dropwise as initiators. The reaction is carried out at 25°C for 3 hours, and then purified by dialyzing for 8 hours to obtain the choline phosphate modified quantum dot composite material S3; wherein, the weight ratio of the choline phosphate derivative shown in formula (1) to the oil-soluble CdSe / ZnS quantum dots is 100:1, and the weight ratio of the crosslinking agent to the choline phosphate derivative shown in formula (1) is 1:20.

[0087] Example 3

[0088] Preparation of choline phosphate modified quantum dot composite material S3:

[0089] (1) Dissolve oil-soluble CdSe / ZnS quantum dots in chloroform, add mercaptopolyethylene glycol acrylamide (molecular weight 2000D), sonicate for 5 min, and shake at room temperature (25℃) for 10 h; then add anhydrous ethanol and n-hexane, centrifuge to precipitate, and then add ultrapure water to resuspend the precipitate to obtain surface double bond modified aqueous quantum dots; wherein, the weight ratio of oil-soluble CdSe / ZnS quantum dots to mercaptopolyethylene glycol acrylamide is 1:5;

[0090] (2) The surface double bond modified aqueous quantum dots obtained in step (1) are mixed with water, and then under argon protection, the choline phosphate derivative and crosslinking agent (N,N-methylenebisacrylamide) shown in formula (1) are added dropwise as initiators. The reaction is carried out at 25°C for 2 hours, and then purified by dialyzing for 8 hours to obtain the choline phosphate modified quantum dot composite material S3; wherein, the weight ratio of the choline phosphate derivative shown in formula (1) to the oil-soluble CdSe / ZnS quantum dots is 50:1, and the weight ratio of the crosslinking agent to the choline phosphate derivative shown in formula (1) is 1:30.

[0091] Example 4

[0092] Preparation of choline phosphate modified quantum dot composite material S4:

[0093] (1) Dissolve oil-soluble CdSe / ZnS quantum dots in chloroform, add mercaptopolyethylene glycol acrylamide (molecular weight 2000D), sonicate for 5 min, and shake at room temperature (25℃) for 12 h; then add anhydrous ethanol and n-hexane, centrifuge to precipitate, and then add ultrapure water to resuspend the precipitate to obtain surface double bond modified aqueous quantum dots; wherein, the weight ratio of oil-soluble CdSe / ZnS quantum dots to mercaptopolyethylene glycol acrylamide is 1:15;

[0094] (2) The surface double bond modified aqueous quantum dots obtained in step (1) are mixed with water. Then, under argon protection, the choline phosphate derivative and crosslinking agent (N,N-methylenebisacrylamide) shown in formula (1) are added dropwise as initiators. The reaction is carried out at 25°C for 3 hours and then purified by dialyzing for 10 hours to obtain the choline phosphate modified quantum dot composite material S4. The weight ratio of the choline phosphate derivative shown in formula (1) to the oil-soluble CdSe / ZnS quantum dots is 80:1, and the weight ratio of the crosslinking agent to the choline phosphate derivative shown in formula (1) is 1:25.

[0095] Example 5

[0096] Preparation of choline phosphate modified quantum dot composite material S5:

[0097] (1) Dissolve oil-soluble CdSe / ZnS quantum dots in chloroform, add mercaptopolyethylene glycol acrylamide (molecular weight 3000D), sonicate for 5 min, and shake at room temperature (25℃) for 10 h; then add anhydrous ethanol and n-hexane, centrifuge to precipitate, and then add ultrapure water to resuspend the precipitate to obtain surface double bond modified aqueous quantum dots; wherein, the weight ratio of oil-soluble CdSe / ZnS quantum dots to mercaptopolyethylene glycol acrylamide is 1:20;

[0098] (2) The surface double bond modified aqueous quantum dots obtained in step (1) are mixed with water. Then, under argon protection, the choline phosphate derivative and crosslinking agent (N,N-methylenebisacrylamide) shown in formula (1) are added dropwise as initiators. The reaction is carried out at 25°C for 3 hours and then purified by dialyzing for 8 hours to obtain the choline phosphate modified quantum dot composite material S5. The weight ratio of the choline phosphate derivative shown in formula (1) to the oil-soluble CdSe / ZnS quantum dots is 60:1, and the weight ratio of the crosslinking agent to the choline phosphate derivative shown in formula (1) is 1:30.

[0099] Test Example 1

[0100] The fluorescence intensity of the choline phosphate-modified quantum dot composite material prepared in Example 1 at different concentrations was detected, and the results are as follows: Figure 2 As shown. By measuring the ultraviolet absorption spectrum, the extinction coefficient of the oil-phase CdSe / ZnS quantum dots at 400 nm is known, thus determining the molar concentration of the quantum dots.

[0101] from Figure 2 It can be seen that the choline phosphate modified quantum dot composite material of the present invention has fluorescence color development ability.

[0102] Test Example 2

[0103] Choline phosphate-modified quantum dot composite material for labeling exosomes.

[0104] Colorectal cancer exosome samples were obtained as follows: hct116 cells were cultured, serum-free cell supernatant was collected, centrifuged at 10,000g for 30 min, the supernatant was retained and filtered at 0.22 μm; then ultracentrifuged at 110,000g for 1 h to obtain a precipitate; the precipitate was resuspended in PBS and washed once at the same speed (i.e., 110,000g) and time (i.e., 1 h), and the resulting precipitate was resuspended in a small amount of PBS to obtain the final exosome suspension, which was stored at -80℃ for subsequent experiments.

[0105] Add 1 μL of APC anti-mouse CD63 fluorescent antibody (labeled exosomes) to 50 μL of exosome sample, incubate with shaking at 4 °C for 20 min, purify by ultrafiltration, then add 2.5 μL of choline phosphate modified quantum dot composite material (CP-QDs) solution prepared in Example 1, incubate at 20 °C for 2 h, place in a confocal dish and take a fluorescence image using a TIRF microscope.

[0106] Figure 3 The results showed that the green fluorescence of CP-QDs was almost identical to the red fluorescence of labeled exosomes, indicating that the choline phosphate modified quantum dot composite material of the present invention can label colorectal cancer exosomes.

[0107] Application Example 1

[0108] A kit for detecting exosome marker membrane proteins in rectal cancer, the kit comprising a lateral chromatography test strip and a choline phosphate modified quantum dot composite material S1 prepared in Example 1;

[0109] The lateral chromatography test strip 100 includes: a base plate 1, a sample pad 2, a detection membrane 3 (nitrocellulose membrane) and an absorbent pad 4. The sample pad 2, the detection membrane 3 and the absorbent pad 4 are sequentially adhered to the base plate 1 and connected by overlapping. The detection membrane 3 is provided with a detection line 31 near the sample pad 2 and a quality control line 32 near the absorbent pad 4.

[0110] The detection line 31 is coated with a colorectal cancer marker antibody (anti-CD147 antibody), and the control line 32 is coated with exosome marker antibodies (anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody).

[0111] Application Example 2

[0112] A kit for detecting exosome marker membrane proteins in rectal cancer, the kit comprising a lateral chromatography test strip and a choline phosphate modified quantum dot composite material S1 prepared in Example 1;

[0113] The lateral chromatography test strip 100 includes: a base plate 1, a sample pad 2, a detection membrane 3 (nitrocellulose membrane) and an absorbent pad 4. The sample pad 2, the detection membrane 3 and the absorbent pad 4 are sequentially adhered to the base plate 1 and connected by overlapping. The detection membrane 3 is provided with a detection line 31 near the sample pad 2 and a quality control line 32 near the absorbent pad 4.

[0114] The detection line 31 is coated with colorectal cancer marker antibodies (anti-A33 antibody), and the control line 32 is coated with exosome marker antibodies (anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody).

[0115] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing choline phosphate modified quantum dot composite materials, characterized in that, The method includes the following steps: (1) Reaction of oil-soluble quantum dots with mercapto-polyethylene glycol acrylamide yields quantum dots with surface double bond modification; (2) In the presence of an initiator, surface double bond modified quantum dots, crosslinking agent and choline phosphate derivative shown in formula (1) are subjected to polymerization reaction; 2. The method according to claim 1, characterized in that, The oil-soluble quantum dots are selected from oil-soluble CdSe / ZnS quantum dots and / or oil-soluble CdZn / SeS quantum dots; Preferably, the hydrated particle size of the oil-soluble quantum dots is 20-30 nm.

3. The method according to claim 1 or 2, characterized in that, In step (1), the molecular weight of the mercaptopolyethylene glycol acrylamide is 1000D-3000D; Preferably, the weight ratio of the oil-soluble quantum dots to the mercapto-polyethylene glycol acrylamide is 1:5-20.

4. The method according to claim 1, characterized in that, In step (1), the reaction conditions include a temperature of 10-25°C and a time of 8-12 hours.

5. The method according to any one of claims 1-4, characterized in that, In step (2), the weight ratio of the choline phosphate derivative represented by formula (1) to the oil-soluble quantum dots is 20-200:1; and / or The weight ratio of the crosslinking agent to the choline phosphate derivative shown in formula (1) is 1:20-30; and / or; The crosslinking agent is N,N-methylenebisacrylamide.

6. The method according to claim 1 or 5, characterized in that, In step (2), the initiator is ammonium persulfate and tetramethylethylenediamine; Preferably, in step (2), the polymerization reaction takes 2-3 hours.

7. The choline phosphate modified quantum dot composite material prepared by the method according to any one of claims 1-6.

8. The application of the choline phosphate modified quantum dot composite material according to claim 7 in the detection of exosomes in colorectal cancer.

9. A method for labeling exosomes from colorectal cancer, characterized in that, The method includes mixing the choline phosphate modified quantum dot composite material of claim 7 with exosomes.

10. A kit for detecting exosome marker membrane proteins in rectal cancer, characterized in that, The kit includes a lateral chromatography test strip and the choline phosphate modified quantum dot composite material as described in claim 7; The lateral chromatography test strip (100) includes: a base plate (1), a sample pad (2), a detection membrane (3) and an absorbent pad (4). The sample pad (2), the detection membrane (3) and the absorbent pad (4) are sequentially adhered to the base plate (1) and connected by overlapping. The detection membrane (3) is provided with a detection line (31) and a quality control line (32). The detection line (31) is coated with colorectal cancer marker antibodies, and the control line (32) is coated with exosome marker antibodies; Preferably, the colorectal cancer marker antibody is selected from anti-CD147 antibody and / or anti-A33 antibody; Preferably, the exosome marker antibody is selected from at least one of anti-CD63 antibody, anti-CD9 antibody, and anti-CD81 antibody.

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