A FSAP detection probe and its preparation method and application

Abstract

The present invention discloses a FSAP detection probe and a preparation method and application thereof, the method comprising: passing a protective gas in an octadecene solvent, then adding silver acetate and octanethiol and heating to 160-170°C to obtain a mixed solution; injecting an ODE-Se precursor and rapidly cooling to 110-130°C for reaction to obtain near-infrared hydrophobic Ag2Se quantum dots; coating and modifying the hydrophobic Ag2Se quantum dots with polyacrylic acid OPA grafted with octylamine in a chloroform solution to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface; and performing an amidation reaction with a PD-L1 aptamer with an amino group at one end to obtain FSAP. The FSAP detection probe can detect the level of PD-L1 in extracellular vesicles without the need to separate PD-L1-positive extracellular vesicles in advance, and avoids the interference of free PD-L1, thereby greatly improving the detection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of medical cross-technology related to fluorescence anisotropy detection technology and extracellular vesicle PD-L1 levels, and in particular to a FSAP detection probe and a preparation method and application thereof. Background Art

[0002] Malignant tumors have high mortality and recurrence rates and are still one of the major diseases that threaten human health and life. In recent years, tumor immunotherapy has become another method of tumor treatment after traditional surgery, radiotherapy, and chemotherapy, bringing hope to patients with advanced malignant tumors. American immunologist James Allison and Japanese biologist Tasuku Honjo won the 2018 Nobel Prize in Physiology or Medicine for "discovering a therapy for negative immune regulation to treat tumors." Among them, immunotherapy targeting programmed cell death 1ligand 1 (PD-L1) and its corresponding receptor PD-1 is a star molecule in the current field of immunotherapy. Immunotherapy "indirectly" acts on tumor cells by reactivating the patient's own anti-tumor immunity. Therefore, immunotherapy takes effect slowly and often requires several months of treatment before efficacy evaluation. This will undoubtedly cause a large number of patients who do not respond to immunotherapy to miss the opportunity to choose other treatment options. Therefore, predicting whether a patient responds to immunotherapy before the start of immunotherapy or in the early stages of treatment is a difficult problem that needs to be solved urgently in the current field of immunotherapy.

[0003] The expression of PD-L1 is believed to be useful for predicting the efficacy of immunotherapy. Currently, PD-L1 immunohistochemical staining is used clinically to judge tumor progression. However, due to the large spatial heterogeneity of tumor tissue, the immunohistochemical method based on tissue biopsy cannot accurately reflect the overall PD-L1 expression of the tumor. In our previous study, we found that tumor cells can secrete extracellular vesicles carrying PD-L1 into the patient's body fluids (such as peripheral blood) or tissues, and the PD-L1 level in the patient's peripheral blood extracellular vesicles is closely related to tumor progression, which can be used to predict the efficacy of immunotherapy. At present, the detection of PD-L1 levels on circulating extracellular vesicles mainly relies on traditional ELISA technology. The existence of PD-L1 in the blood includes the surface of tumor cell membranes, the surface of extracellular vesicles released by tumor cells, and the free form of PD-L1. However, only the PD-L1 on the surface of extracellular vesicles can play a role in disease diagnosis and efficacy prediction. ELISA testing requires ultracentrifugation of plasma samples to separate extracellular vesicles. However, ultracentrifugation separation not only requires expensive ultracentrifuges, but also has cumbersome and time-consuming steps. More importantly, the recovery rate of extracellular vesicles in this method is low (accounting for 5% of total extracellular vesicles), and free PD-L1 will also be mixed with it. Therefore, ultracentrifugation can lead to the loss of a large number of PD-L1-positive extracellular vesicles, which cannot be detected by the ELISA method.

[0004] There is an urgent need to develop a kit that is not affected by free PD-L1 and does not require centrifugation to detect the level of plasma extracellular vesicle PD-L1. Summary of the invention

[0005] The purpose of the present invention is to provide a FSAP detection probe and its preparation method and application. After the FSAP detection probe is mixed and incubated with a sample for half an hour, a fluorescence instrument cuvette is added to measure the change value of fluorescence anisotropy before and after the binding, so as to detect the level of PD-L1 in extracellular vesicles. This method does not require the separation of PD-L1 positive extracellular vesicles in advance, and avoids the interference of free PD-L1, which greatly improves the detection efficiency.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a method for preparing a FSAP detection probe is provided, the method comprising:

[0008] A protective gas is passed through an octadecene solvent, and then silver acetate and octyl mercaptan are added and heated to 160-170° C. to obtain a mixed solution; an ODE-Se precursor is injected into the mixed solution and the temperature is rapidly reduced to 110-130° C. to react, thereby obtaining near-infrared hydrophobic Ag2Se quantum dots;

[0009] The hydrophobic Ag2Se quantum dots are coated and modified with polyacrylic acid OPA grafted with octylamine in a chloroform solution, and then the excess OPA is removed by centrifugation to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface;

[0010] The Ag2Se-OPA quantum dots are subjected to an amidation reaction with a PD-L1 aptamer having an amino group at one end to obtain a FSAP detection probe.

[0011] Furthermore, the preparation method of the ODE-Se precursor comprises: dissolving selenium powder in octadecene solvent under a protective gas atmosphere, heating to 200-220° C. until a transparent solution is obtained, and then maintaining the temperature for a period of time to obtain the ODE-Se precursor.

[0012] Furthermore, the molar ratio of the silver acetate, the ODE-Se precursor and the octanethiol is 1:1:(18-22).

[0013] Furthermore, the hydrophobic Ag2Se quantum dots are coated and modified with polyacrylic acid OPA grafted with octylamine in a chloroform solution, and then the excess OPA is removed by centrifugation to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface, comprising:

[0014] Obtaining octylamine-grafted polyacrylic acid OPA;

[0015] Dissolving the hydrophobic Ag2Se quantum dots and the OPA in chloroform respectively to obtain a quantum dot chloroform solution and an OPA chloroform solution;

[0016] The OPA chloroform solution is added dropwise to the quantum dot chloroform solution to mix well, and then the chloroform is evaporated to dryness and dissolved in phosphate buffer;

[0017] The excess OPA is removed by ultra-speed density gradient centrifugation to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface.

[0018] Furthermore, the mass ratio of the octylamine-grafted polyacrylic acid OPA to the hydrophobic Ag2Se quantum dots is (2-3):1.

[0019] Furthermore, in the quantum dot chloroform solution, the mass volume ratio of the hydrophobic Ag2Se quantum dots to chloroform is (8-12) mg / ml; in the OPA chloroform solution, the mass volume ratio of the OPA to chloroform is (8-12) mg / ml.

[0020] Further, the Ag2Se-OPA quantum dots are subjected to an amidation reaction with a PD-L1 aptamer having an amino group at one end to obtain a FSAP detection probe, comprising:

[0021] Ag2Se-OPA quantum dots were dispersed in PBS buffer, EDC was first added to activate the surface carboxyl groups, and then a PD-L1 aptamer with an amino group at one end was added for amidation reaction. After washing, the FSAP detection probe was obtained.

[0022] Furthermore, the conditions of the amidation reaction are: 22-28° C., 140-160 rpm, and reaction time of 3-5 hours.

[0023] In the second aspect of the present invention, a FSAP detection probe obtained by the method described above is provided.

[0024] In the third aspect of the present invention, a detection kit is provided, wherein the detection kit comprises the FSAP detection probe.

[0025] In the third aspect of the present invention, provided is the use of the FSAP detection probe or the detection kit in the preparation of a PD-L1 positive extracellular vesicle detection kit.

[0026] In the application, the specific operations are as follows:

[0027] The FSAP detection probe was placed in a fluorescence instrument cuvette to detect its fluorescence anisotropy value. The FSAP detection probe was then incubated with PD-L1-positive extracellular vesicles of known concentration and placed in a fluorescence instrument cuvette to measure its fluorescence anisotropy value again. The difference between the fluorescence anisotropy values ​​obtained before and after was calculated to construct a linear relationship between the change in fluorescence anisotropy value and the concentration level of PD-L1 in extracellular vesicles.

[0028] The FSAP detection probe is placed in a fluorescence instrument cuvette, and its fluorescence anisotropy value is detected by using a fluorescence instrument. The FSAP detection probe is then incubated with the PD-L1 positive extracellular vesicles to be tested and the fluorescence anisotropy value is measured again. After calculating the difference before and after, the concentration of the positive extracellular vesicle PD-L1 is calculated according to the linear relationship.

[0029] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0030] 1. The present invention provides a FSAP detection probe and its preparation method and application. The detection probe designed by the present invention does not need to be pre-separated and processed for the extracellular vesicle sample to be tested when used. The current extracellular vesicle PD-L1 quantitative method is the ELISA method, which requires the extracellular vesicles in the blood to be separated by ultracentrifugation before detection. This method not only takes a lot of time, but also causes serious loss of extracellular vesicles during the separation and purification process, which has a great impact on the detection of extracellular vesicle PD-L1 levels.

[0031] 2. For patients in the early stages of tumors, since the tumor tissue is small in the early stage, the abundance of PD-L1 in extracellular vesicles is low, and the low-sensitivity ELISA method is difficult to detect. Using FSAP probes for separation-free detection of plasma samples can detect more PD-L1-positive extracellular vesicles.

[0032] 3. The detection kit designed by the present invention is simple and quick. The FSAP fluorescence anisotropy detection probe is simple to prepare, easy to use, and highly sensitive. It can quickly detect the concentration level of extracellular vesicle PD-L1 in conjunction with a fluorescence instrument cuvette, and the detection process only takes 30 minutes.

[0033] 4. The FSAP fluorescence anisotropy detection probe in the detection kit designed by the present invention utilizes the characteristics of near-infrared second-region quantum dots, which are strong in resistance to matrix spontaneous fluorescence interference and resistance to light bleaching. It is suitable for complex matrix detection, has good photostability, and can maintain its detection ability for a long period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 Schematic diagram of using Ag2Se quantum dots to construct FSAP fluorescence anisotropy detection probe in the detection kit.

[0036] Figure 2 : The fluorescence anisotropy values ​​of the samples after FSAP, FSAP incubated with free PD-L1, FSAP incubated with extracellular vesicle PD-L1, and FSAP co-incubated with free PD-L1 and extracellular vesicle PD-L1 were measured. (Appendix Figure 2 ,FP: free form PD-L1,sEV: extracellular vesicle PD-L1)

[0037] Figure 3 : (A) The FSAP detection probe was placed for 21 days, and the fluorescence intensity was monitored, showing that it had good fluorescence stability; (B) The ability of the FSAP detection probe to detect PD-L1-positive extracellular vesicles within 21 days was monitored, and it was found that the detection ability of the probe remained good.

[0038] Figure 4:(A) Specificity of FSAP detection probe. The fluorescence anisotropy value of the detection probe constructed by random sequence aptamers on the left was almost unchanged after incubation with PD-L1-positive extracellular vesicles. After incubation with PD-L1-positive extracellular vesicles on the right, the fluorescence anisotropy value increased significantly compared with before; (B) (a) TEM image of FSAP detection probe binding to PD-L1-positive extracellular vesicles; (b) TEM image of PD-L1-positive extracellular vesicles after incubation with random sequence probes;

[0039] Figure 5 :(A) The fluorescence emission spectra of FSAP detection probe in PBS buffer and artificial plasma simulated fluid (SBF) were stable, and the position of the fluorescence emission peak did not change; (B) FSAP detection probe quantitatively detected PD-L1-positive extracellular vesicles in PBS and SBF environments, respectively, and still had good detection ability in the more complex matrix SBF, with a detection limit of up to 375 particals / μL.

[0040] Figure 6 :(A) FSAP probe is applied to clinical plasma sample detection; (B) FSAP can successfully distinguish plasma samples from healthy volunteers and OSCCP patients. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0042] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0043] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods. Steps S1, S2, S3, etc. of the present invention do not represent a strict order relationship, and the order can be adjusted appropriately as needed.

[0044] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0045] According to a typical embodiment of the present invention, a method for preparing a FSAP detection probe is provided, the method comprising:

[0046] Step S1, passing protective gas into octadecene solvent, then adding silver acetate and octyl mercaptan and heating to 160-170° C. to obtain a mixed solution; injecting ODE-Se precursor into the mixed solution and rapidly cooling to 110-130° C. to react, to obtain near-infrared hydrophobic Ag2Se quantum dots;

[0047] In the step S1,

[0048] The preparation method of the ODE-Se precursor comprises: dissolving selenium powder in octadecene solvent under a protective gas atmosphere, heating to 200-220° C. until a transparent solution is obtained, and then maintaining the temperature for a period of time to obtain the ODE-Se precursor.

[0049] The molar ratio of the silver acetate, the ODE-Se precursor and the octanethiol is 1:1:(18-22). This molar ratio range is conducive to the preparation of quantum dots with high crystallinity and complete reaction.

[0050] Step S2, coating and modifying the hydrophobic Ag2Se quantum dots with polyacrylic acid OPA grafted with octylamine in a chloroform solution, and then removing the excess OPA by centrifugation to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface;

[0051] The step S2 specifically includes:

[0052] S201, obtaining octylamine-grafted polyacrylic acid OPA;

[0053] S202, dissolving the hydrophobic Ag2Se quantum dots and the OPA in chloroform respectively to obtain a quantum dot chloroform solution and an OPA chloroform solution;

[0054] S203, adding the OPA chloroform solution dropwise to the quantum dot chloroform solution and mixing, then evaporating the chloroform and dissolving it in phosphate buffer;

[0055] S204, removing the excess OPA through ultra-speed density gradient centrifugation to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface.

[0056] The mass ratio of the octylamine-grafted polyacrylic acid OPA to the hydrophobic Ag2Se quantum dots is (2-3): 1. This mass ratio range is conducive to ensuring that all quantum dots are converted into hydrophilic quantum dots. If the mass ratio is too small, some oil-soluble quantum dots may not be successfully converted into water-soluble. If it is too large, too much OPA may be introduced, resulting in uneven particle size.

[0057] In the quantum dot chloroform solution, the mass volume ratio of hydrophobic Ag2Se quantum dots to chloroform is (8-12) mg / ml; that is, 8-12 mg of hydrophobic Ag2Se quantum dots are dissolved in 1 ml of chloroform;

[0058] In the OPA chloroform solution, the mass volume ratio of OPA to chloroform is (8-12) mg / ml, that is, 8-12 mg OPA is dissolved in 1 ml chloroform solution.

[0059] The chloroform used in the present invention is purchased pure chloroform.

[0060] Step S3, subjecting the Ag2Se-OPA quantum dots to an amidation reaction with a PD-L1 aptamer having an amino group at one end to obtain a FSAP detection probe.

[0061] The step S3 comprises:

[0062] Ag2Se-OPA quantum dots were dispersed in PBS buffer, EDC was first added to activate the surface carboxyl groups, and then a PD-L1 aptamer with an amino group at one end was added for amidation reaction. After washing, the FSAP detection probe was obtained.

[0063] The conditions of the amidation reaction are: 22-28° C., 140-160 rpm, and reaction time of 3-5 hours.

[0064] According to another typical embodiment of the present invention, a FSAP detection probe obtained by the method described is provided.

[0065] According to another typical embodiment of the present invention, provided is the use of the FSAP detection probe in the preparation of a PD-L1 positive extracellular vesicle detection kit.

[0066] The detection probe designed by the present invention does not need to be pre-separated and processed for the extracellular vesicle sample to be tested when used. The current extracellular vesicle PD-L1 quantitative method is the ELISA method, which requires the extracellular vesicles in the blood to be separated by ultracentrifugation before detection. This method not only takes a lot of time, but also causes serious loss of extracellular vesicles during the separation and purification process, which has a great impact on the detection of extracellular vesicle PD-L1 levels.

[0067] Fluorescence anisotropy analysis is suitable for homogeneous system detection, does not require sample washing and other operations, and has a fast detection speed. Since the prepared near-infrared second-zone quantum dot probe has a small volume and mass, and the PD-L1-positive extracellular vesicles have a larger volume and mass than it, the change in the fluorescence anisotropy value before and after the probe binds to the PD-L1-positive extracellular vesicle can be used to achieve quantitative detection of the extracellular vesicle PD-L1, and since the mass volume of the free form of PD-L1 in the sample is extremely small, it just cleverly avoids the impact on the detection of extracellular vesicle PD-L1. The technology for preparing the probe is simple and convenient, and the detection process is fast. It only needs to incubate the detection probe with the sample for half an hour for detection. Compared with the traditional clinical ELISA method for quantifying PD-L1-positive extracellular vesicles, this method greatly shortens the sample pretreatment time, avoids the large loss of PD-L1-positive extracellular vesicles during the separation process, and is free from the interference of free form PD-L1, which provides a reference for the early diagnosis of malignant tumors and the prediction of disease progression, and has great application potential.

[0068] The following will describe in detail a FSAP detection probe of the present application and its preparation method and application in combination with embodiments, comparative examples and experimental data.

[0069] Example 1: FSAP detection probe and its preparation method and application

[0070] 1. FSAP detection probe and preparation method thereof

[0071] (1) A protective gas is passed through an octadecene solvent, and then 0.2 mmol of silver acetate and 700 μL of octanethiol are added and heated to 165° C. to obtain a mixed solution; 0.2 mmol of ODE-Se precursor is injected into the mixed solution and the temperature is rapidly lowered to 120° C. to react, thereby obtaining near-infrared hydrophobic Ag2Se quantum dots;

[0072] (2) 20 mg of the hydrophobic Ag2Se quantum dots were coated and modified with 50 mg of octylamine-grafted polyacrylic acid OPA in a chloroform solution, and then the excess OPA was removed by centrifugation, and the Ag2Se-OPA quantum dots with a concentration of 0.4 μM and hydrophilic surface and carboxyl groups were obtained by dispersion in PBS;

[0073] (3) 500 μL of the Ag2Se-OPA quantum dots and 50 μL (100 μM) of a PD-L1 aptamer having an amino group at one end were subjected to an amidation reaction at 25° C. to obtain a FSAP detection probe.

[0074] 2. Rapid and highly sensitive detection of plasma extracellular vesicle PD-L1

[0075] The isolated PD-L1 positive extracellular vesicles were placed in artificial plasma and incubated with the above-prepared FSAP detection probe for detection. According to the properties of the synthesized near-infrared second-zone Ag2Se quantum dots, the excitation wavelength of the fluorescence instrument was set to 450nm and the emission wavelength was set to 1020nm. The horizontal and vertical polarizers and horizontal and vertical analyzers of the instrument were selected to construct the relationship between the change value of fluorescence anisotropy and the positive extracellular vesicles of known concentration of PD-L1 (see Appendix). Figure 4 B), the linear relationship is Y = 0.03912X-0.09435, R 2 =0.994.

[0076] The unknown concentration of the sample was detected with the FSAP probe, and the concentration of extracellular vesicle PD-L1 was calculated to be 2.02×10 4 particles / μL.

[0077] Example 2: FSAP detection probe and its preparation method and application

[0078] 1. FSAP detection probe and preparation method thereof

[0079] (1) A protective gas is passed through an octadecene solvent, and then 0.2 mmol of silver acetate and 700 μL of octanethiol are added and heated to 160° C. to obtain a mixed solution; 0.2 mmol of ODE-Se precursor is injected into the mixed solution and the temperature is rapidly reduced to 130° C. for reaction to obtain near-infrared hydrophobic Ag2Se quantum dots;

[0080] (2) 20 mg of the hydrophobic Ag2Se quantum dots were coated and modified with 50 mg of octylamine-grafted polyacrylic acid OPA in a chloroform solution, and then the excess OPA was removed by centrifugation, and the Ag2Se-OPA quantum dots with a concentration of 0.4 μM and hydrophilic surface and carboxyl groups were obtained by dispersion in PBS;

[0081] (3) 500 μL of the Ag2Se-OPA quantum dots and 50 μL (100 μM) of a PD-L1 aptamer having an amino group at one end were subjected to an amidation reaction at 25° C. to obtain a FSAP detection probe.

[0082] 2. Used to distinguish healthy volunteers from patients with malignant tumors

[0083] The detection probe prepared above was incubated with plasma samples of healthy volunteers and malignant tumor patients diluted 70 times in advance, and placed in a fluorescence instrument cuvette for detection. According to the properties of the synthesized near-infrared second-zone Ag2Se quantum dots, the excitation wavelength of the fluorescence instrument was set to 450nm, the emission wavelength was set to 1020nm, and the horizontal and vertical polarizers and horizontal and vertical analyzers of the instrument were selected. By analyzing the level of extracellular vesicle PD-L1, it is possible to distinguish healthy volunteers from malignant tumor patients.

[0084] The results are as follows Figure 6 As shown, the change in fluorescence anisotropy value of plasma samples of healthy volunteers is lower than that of plasma samples of patients with malignant tumors, and thus the level of extracellular vesicle PD-L1 is lower than that of plasma samples of patients with malignant tumors. This indicates that by analyzing the level of extracellular vesicle PD-L1, it is possible to distinguish healthy volunteers from patients with malignant tumors.

[0085] Experimental Example 1: Performance measurement of FSAP detection probe and kit

[0086] 1. Sensitivity detection

[0087] The samples with different gradient concentrations were detected by FSAP probe, and the detection limit could reach 375 particles / μL, indicating that the probe and its kit had high detection sensitivity.

[0088] 2. No interference from free PD-L1 in complex matrix

[0089] The fluorescence anisotropy values ​​of the samples after FSAP, FSAP incubated with free PD-L1, FSAP incubated with extracellular vesicle PD-L1, and FSAP co-incubated with free PD-L1 and extracellular vesicle PD-L1 were measured. Figure 2 As shown in Figure 2, it is shown that the detection is not interfered by the free form of PD-L1 in the complex matrix (see Appendix Figure 2 ,FP: free form PD-L1,sEV: extracellular vesicle PD-L1).

[0090] 3. Stability test

[0091] We investigated the photostability and detection ability of FSAP for 21 days, testing the fluorescence intensity of FSAP and its ability to detect extracellular vesicle PD-L1 every 3 days during the 21 days.

[0092] The results are as follows Figure 3 As shown, its fluorescence intensity and ability to bind to extracellular vesicle PD-L1 remain stable, indicating that the FSAP probe used for detection is labeled with near-infrared second region Ag2Se quantum dots and has good photostability.

[0093] 4. Specificity detection

[0094] We used random sequence aptamers (Random Sequence) to construct detection probes for comparison with FSAP for detection.

[0095] The results are attached Figure 4 As shown, it was found that only the FSAP constructed by the PD-L1 aptamer of the present application could bind to the extracellular vesicle PD-L1, and the detection probe constructed by the random sequence aptamer did not produce a change in the fluorescence anisotropy value after co-incubation with the extracellular vesicle PD-L1. This shows that FSAP can achieve specific detection of extracellular vesicle PD-L1.

[0096] 5. Verification of the ability of FSAP to be applied in complex matrices

[0097] To verify the ability of FSAP to be used in complex matrices, we measured the fluorescence emission spectra of FSAP in PBS and complex matrix SBF (artificial plasma buffer), and performed quantitative detection of extracellular vesicle PD-L1 with known concentrations.

[0098] The results are as follows Figure 5 As shown, the fluorescence emission spectrum of FSAP remains consistent in PBS and SBF, and the change value of fluorescence anisotropy is not significantly affected in the complex matrix SBF, indicating that the FSAP of the present application can avoid the interference of complex matrix autofluorescence to the greatest extent and can be applied to the detection of complex samples.

[0099] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0100] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0101] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a FSAP detection probe, characterized in that: The method comprises: A protective gas is passed through an octadecene solvent, and then silver acetate and octyl mercaptan are added and heated to 160-170° C. to obtain a mixed solution; an ODE-Se precursor is injected into the mixed solution and the temperature is rapidly reduced to 110-130° C. to react, thereby obtaining near-infrared hydrophobic Ag2Se quantum dots; The hydrophobic Ag2Se quantum dots are coated and modified with polyacrylic acid OPA grafted with octylamine in a chloroform solution, and then the excess OPA is removed by centrifugation to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface; The Ag2Se-OPA quantum dots are subjected to an amidation reaction with a PD-L1 aptamer having an amino group at one end to obtain a FSAP detection probe.

2. The method for preparing a FSAP detection probe according to claim 1, characterized in that: The preparation method of the ODE-Se precursor comprises: dissolving selenium powder in octadecene solvent under a protective gas atmosphere, heating to 200-220° C. until a transparent solution is obtained, and then maintaining the temperature for a period of time to obtain the ODE-Se precursor.

3. The method for preparing a FSAP detection probe according to claim 1, characterized in that: The molar ratio of the silver acetate, the ODE-Se precursor and the octanethiol is 1:1:(18-22).

4. The method for preparing a FSAP detection probe according to claim 1, characterized in that: The hydrophobic Ag2Se quantum dots are coated and modified with polyacrylic acid OPA grafted with octylamine in a chloroform solution, and then the excess OPA is removed by centrifugation to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface, including: Obtaining octylamine-grafted polyacrylic acid OPA; Dissolving the hydrophobic Ag2Se quantum dots and the OPA in chloroform respectively to obtain a quantum dot chloroform solution and an OPA chloroform solution; The OPA chloroform solution is added dropwise to the quantum dot chloroform solution to mix well, and then the chloroform is evaporated to dryness and dissolved in phosphate buffer; The excess OPA is removed by ultra-speed density gradient centrifugation to obtain hydrophilic Ag2Se-OPA quantum dots with carboxyl groups on the surface.

5. The method for preparing a FSAP detection probe according to claim 4, characterized in that: The mass ratio of the octylamine-grafted polyacrylic acid OPA to the hydrophobic Ag2Se quantum dots is (2-3):

1.

6. The method for preparing a FSAP detection probe according to claim 4, characterized in that: In the quantum dot chloroform solution, the mass volume ratio of the hydrophobic Ag2Se quantum dots to chloroform is (8-12) mg / ml; in the OPA chloroform solution, the mass volume ratio of OPA to chloroform is (8-12) mg / ml.

7. The method for preparing a FSAP detection probe according to claim 1, characterized in that: The Ag2Se-OPA quantum dots are subjected to an amidation reaction with a PD-L1 aptamer having an amino group at one end to obtain a FSAP detection probe, comprising: Ag2Se-OPA quantum dots were dispersed in PBS buffer, EDC was first added to activate the surface carboxyl groups, and then a PD-L1 aptamer with an amino group at one end was added for amidation reaction. After washing, the FSAP detection probe was obtained.

8. A FSAP detection probe prepared by the method according to any one of claims 1 to 7.

9. A detection kit, characterized in that: The detection kit comprises the FSAP detection probe according to claim 8.

10. Use of the FSAP detection probe according to claim 8 or the detection kit according to claim 9 in preparing a PD-L1 positive extracellular vesicle detection product.

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