Application of perovskite nanocrystalline probe in preparation of cancer detection reagent

Abstract

The invention provides an application of a perovskite nanocrystalline probe in preparation of a cancer detection reagent. The perovskite nanocrystalline probe is formed by a biological material targeting a Survivin gene and a water-soluble perovskite nanocrystalline through electrostatic interaction or covalent coupling. The water-soluble perovskite nanocrystal is formed by in-situ coating and packaging of perovskite quantum dots and a polymer packaging material. The high-molecular packaging material comprises a lipidosome for modifying a functional group; the liposome is selected from one or more than two of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylcholine and distearoyl phosphatidylethanolamine; the functional group is selected from carboxyl, amino and sulfydryl; and the perovskite quantum dots are selected from CsPbBr3. The universal perovskite nanocrystalline probe provided by the invention can be used for high-sensitivity rapid detection of various cancer tissues or cells.

Description

Technical Field

[0001] The present invention relates to the technical field of luminescent materials, and in particular to an application of a perovskite nanocrystal probe in the preparation of a cancer detection reagent. Background Art

[0002] There are many ways to diagnose tumors, including clinical diagnosis, imaging diagnosis, surgical diagnosis, and biochemical diagnosis, etc., but histopathological diagnosis is the most ideal and reliable basis for diagnosis, and is the gold standard for diagnosing cancer. However, the diagnostic targets of tumor pathology in each part are different. Therefore, it is of great clinical significance to discover a universal target for multiple cancers.

[0003] Fluorescence immunopathology diagnosis can assist clinical diagnosis of cancerous areas or the degree of malignancy. Existing fluorescent dyes have relatively low fluorescence luminescence efficiency and weak resistance to photobleaching. Using fluorescent materials to form a universal pathology diagnosis probe to achieve rapid screening and diagnosis in pathology is what those skilled in the art are pursuing. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides a perovskite nanocrystal probe and its application in the preparation of cancer detection reagents, thereby realizing rapid screening and diagnosis in the pathology department.

[0005] In a first aspect, the present invention provides a perovskite nanocrystal probe, which is formed by electrostatic interaction or covalent coupling between a biomaterial targeting the Survivin gene and a water-soluble perovskite nanocrystal.

[0006] According to an embodiment of the present invention, the water-soluble perovskite nanocrystals include perovskite quantum dots and polymer encapsulation materials.

[0007] According to an embodiment of the present invention, the water-soluble perovskite nanocrystals are formed by in-situ coating and encapsulation of perovskite quantum dots and polymer encapsulation materials.

[0008] According to an embodiment of the present invention, the polymer encapsulation material is a liposome with modified functional groups. The liposome is, for example, selected from one or more of DPPC (dipalmitoylphosphatidylcholine), DPPE (dipalmitoylphosphatidylethanolamine), DSPC (distearoylphosphatidylcholine), DSPE (distearoylphosphatidylethanolamine), etc.

[0009] According to an embodiment of the present invention, the modified functional group may be a carboxyl group, an amino group, a thiol group, or the like.

[0010] According to an embodiment of the present invention, the perovskite quantum dots may be selected from CsPbBr3.

[0011] According to an embodiment of the present invention, the spectral range of the water-soluble perovskite nanocrystal is 510-530 nm, for example, 515 nm, 520 nm, 525 nm, preferably, the spectral position of the water-soluble perovskite nanocrystal is 520 nm.

[0012] According to an embodiment of the present invention, the particle size of the water-soluble perovskite nanocrystal is 20 to 90 nm, for example, 20 nm, 30 nm, 40 nm, 50 nm or 80 nm. Preferably, the particle size of the water-soluble perovskite nanocrystal is 40 nm.

[0013] According to an embodiment of the present invention, the average particle size of the perovskite quantum dots is 5 to 20 nm, for example, 10 to 15 nm; illustratively, the average particle size of the perovskite quantum dots is 5 nm, 8 nm, 10 nm, 12 nm, 15 nm or 20 nm.

[0014] According to an embodiment of the present invention, the average particle size of the water-soluble perovskite nanocrystals is greater than the average particle size of the perovskite quantum dots, for example, greater than 5 nm and less than or equal to 60 nm, preferably 15 to 50 nm, and can be 8 nm, 10 nm, 15 nm, 20 nm, 30 nm, 40 nm, or 50 nm.

[0015] According to an embodiment of the present invention, the mass ratio (mg:mg) of the perovskite quantum dots to the polymer encapsulation material is 116:(10-50); for example, it can be 116:10, 116:20, 116:30, 116:40, or 116:50.

[0016] According to an embodiment of the present invention, the encapsulation is complete encapsulation. In one embodiment, complete encapsulation can be achieved when the mass ratio (mg:mg) of perovskite quantum dots to liposomes is at least 116:10.

[0017] Among them, CsPbBr3 perovskite quantum dots appear yellow under visible light and green under ultraviolet light (such as 365nm excitation).

[0018] According to an embodiment of the present invention, the method for preparing the water-soluble perovskite nanocrystals comprises the following steps: heating liposomes and perovskite quantum dots to react to obtain the water-soluble perovskite nanocrystals.

[0019] According to an embodiment of the present invention, the water-soluble perovskite nanocrystal is prepared by the following method:

[0020] (1) mixing cesium bromide, lead bromide, liposomes and an organic solvent to form a stable solution, adding oleylamine and oleic acid to react;

[0021] Preferably, the reaction temperature is 50-70°C, for example 60°C;

[0022] Preferably, the organic solvent includes any one of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), γ-butyrolactone (GBL), and N,N-dimethylacetamide (DMA);

[0023] (2) adding the solution of step (1) into an anti-solvent to react and precipitate water-soluble perovskite nanocrystals;

[0024] Preferably, the anti-solvent is selected from at least one of toluene, chlorobenzene and n-hexane;

[0025] Preferably, the reaction temperature is 35-50°C, such as 45°C.

[0026] According to an embodiment of the present invention, the method for preparing water-soluble perovskite nanocrystals specifically comprises the following steps:

[0027] (A1) 0.4 mmol cesium bromide (CsBr), 0.4 mmol lead bromide (PbBr2) and liposomes were added to 10 mL N,N-dimethylformamide (DMF) to form a stable solution, and 0.5 mL oleylamine and 1 mL oleic acid were added as a ligand stabilizing solution. The entire reaction was carried out at 60 °C.

[0028] (A2) adding 0.75 mL of the stabilizing solution described in step (A1) to 15 mL of toluene as an anti-solvent, heating the mixture at 45° C. to precipitate water-soluble perovskite nanocrystals using an anti-solvent supersaturation method, thereby preparing water-soluble perovskite nanocrystals;

[0029] (A3) separating the water-soluble perovskite nanocrystals in step (A2), drying the precipitate, and obtaining solid water-soluble perovskite nanocrystals;

[0030] (A4) dispersing the solid water-soluble perovskite nanocrystals obtained in step (A3) in water to obtain a water-soluble perovskite nanocrystal solution.

[0031] The electrical property of the water-soluble perovskite nanocrystal is positive charge.

[0032] According to an embodiment of the present invention, the biological material targeting the Survivin gene includes antibodies, peptides, aptamers, enzymes and other biological materials. Preferably, the antibody is Anti-BIRC5.

[0033] In a second aspect, the present invention provides a method for preparing a perovskite nanocrystal probe, comprising covalently coupling a water-soluble perovskite nanocrystal with a biomaterial targeting a Survivin gene to obtain a perovskite nanocrystal probe;

[0034] Preferably, the perovskite nanocrystal probe is the probe mentioned above.

[0035] According to an embodiment of the present invention, the covalent coupling comprises activating the perovskite nanocrystal and then performing a coupling reaction with a biomaterial targeting the Survivin gene.

[0036] Preferably, the activators used are EDC and NHS.

[0037] In a further embodiment of the present invention, the method for preparing the water-soluble perovskite nanocrystal probe specifically comprises the following steps:

[0038] (A1) 0.4 mmol cesium bromide (CsBr), 0.4 mmol lead bromide (PbBr2) and liposomes were added to 10 mL N,N-dimethylformamide (DMF) to form a stable solution, and 0.5 mL oleylamine and 1 mL oleic acid were added as a ligand stabilizing solution. The entire reaction was carried out at 60 °C.

[0039] (A2) adding 0.75 mL of the stabilizing solution described in step (A1) to 15 mL of toluene as an anti-solvent, heating the mixture at 45° C. to precipitate water-soluble perovskite nanocrystals using an anti-solvent supersaturation method, thereby preparing water-soluble perovskite nanocrystals;

[0040] (A3) separating the water-soluble perovskite nanocrystals in step (A2), drying the precipitate, and obtaining solid water-soluble perovskite nanocrystals;

[0041] (A4) dispersing the solid water-soluble perovskite nanocrystals obtained in step (A3) in water to obtain a water-soluble perovskite nanocrystal solution;

[0042] (A5) coupling the water-soluble perovskite nanocrystal solution of step (A4) with a biomaterial targeting the Survivin gene.

[0043] In a third aspect, the present invention provides a kit comprising the above-mentioned water-soluble perovskite nanocrystal probe.

[0044] In one embodiment of the present invention, the kit further comprises a handheld ultraviolet lamp, a flushing solution, a pipette and a storage medium.

[0045] Preferably, the storage medium is an RFID tag, an IC chip, a magnetic code or a bar code.

[0046] Preferably, the flushing liquid is water, buffer solution or the like.

[0047] In a fourth aspect, the present invention provides the use of the water-soluble perovskite nanocrystal probe or kit in detecting the Survivin gene.

[0048] In a fifth aspect, the present invention provides the use of the above-mentioned water-soluble perovskite nanocrystal probe or kit in the preparation of cancer detection reagents.

[0049] According to an embodiment of the present invention, the cancer may be any one of liver cancer, lung cancer, intestinal cancer, breast cancer or gastric cancer.

[0050] The present invention has the following beneficial effects:

[0051] (1) According to the analysis of the cancer gene atlas TCGA database, the inventors found that the Survivin gene is highly expressed in almost all malignant tumors, while the corresponding normal tissues are lowly expressed or not expressed. This discovery provides us with a new target for pan-cancer detection. Combining perovskite quantum dots with the Survivin gene target to form a universal pathological diagnostic probe has important medical significance for rapid screening and diagnosis in pathology. A universal perovskite nanocrystal probe of the present invention can be used for high-sensitivity rapid detection of a variety of cancer tissues or cells.

[0052] (2) The labeling method of the present invention has the advantages of good stability (for example, electrostatic binding of biological molecules to perovskite nanocrystals), rapidity and high sensitivity (for example, the fluorescence intensity of perovskite nanocrystals is high, which can realize the detection of low-concentration target molecules), simple and rapid operation, short detection time, easy result interpretation, and both qualitative and quantitative analysis are possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 It is a transmission electron microscope image of CsPbBr3 perovskite quantum dots in Example 1;

[0054] Figure 2 is a transmission electron microscope image of the water-soluble perovskite nanocrystal in Example 1;

[0055] Figure 3 The S element spectrum in XPS of CsPbBr3 perovskite quantum dots and water-soluble perovskite nanocrystals in Example 1;

[0056] Figure 4 This is a universal perovskite probe kit for fluorescence imaging Confocal images on liver, lung, intestine, breast and stomach tissue sections. The Confocal laser excitation wavelengths are 405nm and 488nm. DETAILED DESCRIPTION

[0057] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0058] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0059] Example 1 Preparation of water-soluble perovskite nanocrystals

[0060] 1. Dissolve 85 mg CsBr, 146.8 mg PbBr2 and 10 mg DSPE-PEG-SH in 10 mL N,N-dimethylformamide (DMF) solvent. After complete dissolution, add 1 mL oleic acid and 0.5 mL oleylamine to form a stable precursor solution.

[0061] 2. Take 750 μL of the above solution and slowly add it dropwise into 15 mL of vigorously stirred toluene solution to obtain a perovskite quantum dot solution; stir the reaction for 4 h to ensure complete coating and obtain water-soluble perovskite nanocrystals.

[0062] 3. Separation and purification of perovskite nanocrystals: Centrifuge the toluene solution in (2) at 8000 r for 10 min, and dry the obtained precipitate in a fume hood for 1 h to obtain a light yellow powder, which turns green under ultraviolet light.

[0063] 4. Disperse the powder in water by ultrasonic for 1 min and store the water-soluble perovskite nanocrystals (i.e., PNCs-Water) at room temperature.

[0064] Transmission electron microscopy images of CsPbBr3 perovskite quantum dots Figure 1 As shown in the transmission electron microscope image of perovskite nanocrystals Figure 2 The S element spectrum of CsPbBr3 perovskite quantum dots and water-soluble perovskite nanocrystals in XPS is shown in Figure 3 shown.

[0065] The results show that the particle size of water-soluble perovskite nanocrystals is about 12nm. The particle size of water-soluble perovskite nanocrystals is about 40nm. The SH bond in DSPE-PEG-SH is bonded to the perovskite structure, and S and Pb elements form Pb-S bonds to stabilize the perovskite quantum dot structure and are encapsulated by liposomes.

[0066] Example 2 Preparation of universal perovskite nanocrystal probe

[0067] 10 mg of perovskite nanocrystal powder was dissolved in ultrapure water, reacted with Anti-Survivin (100 uL, 1 mg / mL) at room temperature through EDC (40 mg / mL) and NHS (60 mg / mL) activators for 20 min, and centrifuged through an ultrafiltration centrifuge tube to obtain a universal perovskite nanocrystal probe.

[0068] Example 3 A perovskite nanocrystal kit for rapid detection of multiple cancer tissue imaging

[0069] The test kit comprises a handheld ultraviolet lamp, a detection liquid, a flushing liquid, a pipette and a storage medium, and the storage medium is an RFID tag, an IC chip, a magnetic code or a bar code.

[0070] Usage: Take 200-300 μL of detection solution and add it to the slice to be tested. Let it react at room temperature for 15 minutes. After the reaction, rinse it with a rinse solution (such as ultrapure water) for 5-6 times and observe the image with a handheld UV lamp.

[0071] Evaluation criteria for distinguishing tumors or normal tissues: Use an ordinary handheld ultraviolet lamp with a wavelength of 320 to 450 nm to irradiate the slice area to be tested. According to the principle of antibody-antigen specific recognition, when the relevant antigens of the tumor area in the tissue slice to be tested are detected, the test solution is added to the tissue area, reacted for 15 minutes, and then rinsed 5-6 times with the rinse solution. Fluorescence appears in the tumor area under the excitation of the handheld ultraviolet lamp, and the test result is the tumor area; conversely, if the tissue slice to be tested is normal tissue, that is, it does not contain the relevant antigens of the tumor area, there is no fluorescence in the tumor area under the excitation of the handheld ultraviolet lamp, and the test result is the normal tissue area, that is, the non-tumor area; the higher the fluorescence intensity, the higher the degree of canceration in the tissue slice to be tested, and conversely, the lower the fluorescence intensity, the lower the degree of canceration.

[0072] Figure 4 This is a universal perovskite probe kit for fluorescence imaging Confocal images on liver, lung, intestine, breast and stomach tissue sections. The Confocal laser excitation wavelengths are 405nm and 488nm.

[0073] In order to verify the location of the Suvivin gene targeting the cell nucleus, DAPI was used to stain the cell nucleus and co-localize with the perovskite probe. As can be seen from the figure, tumors in liver, lung, intestine, breast and stomach tissue sections show strong green fluorescence, while the corresponding normal liver, lung, intestine, breast and stomach tissues have almost no green fluorescence. This is consistent with the theory that the Suvivin gene is highly expressed in tumors and lowly expressed or not expressed in normal tissues.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A perovskite nanocrystal probe, characterized in that: It is formed by electrostatic interaction or covalent coupling between biomaterials targeting the Survivin gene and water-soluble perovskite nanocrystals.

2. A perovskite nanocrystal probe as claimed in claim 1, characterized in that: The water-soluble perovskite nanocrystals include perovskite quantum dots and polymer encapsulation materials; Preferably, the water-soluble perovskite nanocrystals are formed by in-situ encapsulation of perovskite quantum dots and polymer encapsulation materials; Preferably, the polymer encapsulation material comprises liposomes with modified functional groups; the liposomes are selected from one or more of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylcholine, and distearoylphosphatidylethanolamine; the functional groups are selected from carboxyl, amino, and thiol; Preferably, the perovskite quantum dots are selected from CsPbBr3; Preferably, the mass ratio of the perovskite quantum dots to the polymer encapsulation material is 116:(10-50).

3. A perovskite nanocrystal probe as claimed in claim 1, characterized in that: Preferably, the spectral range of the water-soluble perovskite nanocrystals is 510-530 nm; Preferably, the particle size of the water-soluble perovskite nanocrystal is 20 to 90 nm; Preferably, the average particle size of the perovskite quantum dots is 5 to 20 nm; Preferably, the difference between the particle size of the water-soluble perovskite nanocrystals and the average particle size of the perovskite quantum dots is greater than 5 and less than or equal to 60 nm.

4. A perovskite nanocrystal probe as claimed in claim 1, characterized in that: The preparation method of the water-soluble perovskite nanocrystal comprises heating liposomes and perovskite quantum dots to obtain.

5. A perovskite nanocrystal probe as claimed in claim 4, characterized in that: The following steps are involved: (1) mixing cesium bromide, lead bromide, liposomes and an organic solvent to form a stable solution, adding oleylamine and oleic acid to react; Preferably, the reaction temperature is 50-70°C; Preferably, the organic solvent includes any one of dimethylformamide (DMF), dimethyl sulfoxide (DMSO), γ-butyrolactone (GBL), and N,N-dimethylacetamide (DMA); (2) adding the solution of step (1) into an anti-solvent to react and precipitate water-soluble perovskite nanocrystals; Preferably, the anti-solvent is selected from at least one of toluene, chlorobenzene and n-hexane. Preferably, the reaction temperature is 35-50°C.

6. A perovskite nanocrystal probe as claimed in claim 1, characterized in that: The biological material targeting the Survivin gene includes antibodies, polypeptides, aptamers, and enzymes; more preferably, the antibody is Anti-BIRC5.

7. A method for preparing a perovskite nanocrystal probe targeting a cell nucleus according to any one of claims 1 to 6, characterized in that: The method comprises covalently coupling the perovskite nanocrystal with a biomaterial targeting the Survivin gene to obtain a perovskite nanocrystal probe; Preferably, the covalent coupling comprises the perovskite nanocrystal being activated and then undergoing a coupling reaction with a biomaterial targeting the Survivin gene; Preferably, the activators used are EDC and NHS.

8. A kit, characterized in that: A perovskite nanocrystal probe comprising any one of claims 1-6.

9. Use of a perovskite nanocrystal probe targeting a cell nucleus as claimed in any one of claims 1 to 6 and a kit as claimed in claim 8 in detecting Survivin gene.

10. Use of a perovskite nanocrystal probe targeting a cell nucleus according to any one of claims 1 to 6 and a kit according to claim 8 in the preparation of a cancer detection reagent.

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