CN115825442A - Application of perovskite nanocrystalline in preparation of probe for tumor diagnosis or treatment - Google Patents
Application of perovskite nanocrystalline in preparation of probe for tumor diagnosis or treatment Download PDFInfo
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- CN115825442A CN115825442A CN202211379981.6A CN202211379981A CN115825442A CN 115825442 A CN115825442 A CN 115825442A CN 202211379981 A CN202211379981 A CN 202211379981A CN 115825442 A CN115825442 A CN 115825442A
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Images
Abstract
The invention provides an application of perovskite nanocrystalline in preparing a probe for tumor diagnosis or treatment. The perovskite nanocrystalline in the application comprises perovskite quantum dots and liposome materials coated on the surfaces of the perovskite quantum dots. The tumor diagnosis method under the application has the characteristics of high sensitivity, rapid detection, simplicity, convenience, intuition and the like, greatly improves the efficiency of detecting tumor cells or tissues, and has guiding significance in assisting tumor resection while clinical operation.
Description
Technical Field
The invention belongs to the technical field of immunodetection, relates to application of perovskite nanocrystalline in preparation of probes for tumor diagnosis or treatment, and particularly relates to a rapid pathological diagnosis means of brain glioma.
Background
Brain glioma is one of common diseases in the nervous system, and has the characteristics of easy invasion, easy metastasis and diffusion, high recurrence rate and high lethality rate, and multi-modal imaging combining various modes such as MRI imaging, near infrared fluorescence imaging, ultrasound and the like in the operation is a common method for guiding tumor resection. MRI imaging can provide good resolution and accuracy, but does not provide real-time continuous guidance and is expensive; ultrasound is portable and low cost, but has limited intraoperative image contrast and resolution, making accurate real-time detection difficult. Therefore, fluorescence imaging is extremely advantageous in detecting tumors in surgical operations, not only can be imaged in real time, but also has high resolution and high accuracy, and can provide real-time and direct visual guidance for tumor cells.
With the increasing demand of tumor cell detection in rapid real-time surgery, the requirement of fluorescent biological detection probes is higher and higher. Although many studies have used traditional fluorescent substances to guide the excision of glioma, the detection requirements of high fluorescence efficiency, narrow emission, low interference and the like cannot be met due to poor fluorescence stability and low fluorescence efficiency, so that the development of a novel nano biological detection probe with high fluorescence intensity, narrow emission and good biocompatibility is of great significance.
The perovskite nanocrystal material becomes a fluorescent labeling material with the highest quantum yield in the existing material due to the size effect and quantum confinement of the material. The perovskite nanocrystal material also has the advantages of narrow emission peak (< 20 nm), controllable emission peak position, wide excitation wavelength range, easy synthesis and the like. However, perovskite quantum dots are not resistant to water and oxygen, so that the application and development of the perovskite quantum dots in various fields such as bioscience and medicine are severely limited. Therefore, how to modify perovskite quantum dots to construct a nano biological probe which can fully exert the advantages of materials and has good biocompatibility is an urgent need to be solved. Here, we propose a preparation method of water-soluble terminal carboxyl polylactic acid-glycolic acid copolymer coated perovskite quantum dots, which maintains the water and oxygen resistance (> 200 days) while maintaining high quantum yield (PLQY = 70%). The water-soluble perovskite nanocrystal can be coupled with a chlorotoxin antibody specifically combined with a receptor on a brain glioma cell membrane through electrostatic interaction to form a water-phase high-fluorescence-intensity perovskite nanocrystal probe. Because the glioma cell can be specifically identified, the tumor tissue, the tissue beside the cancer and the normal tissue can be distinguished, and the real-time and direct visual guidance of tumor excision can be realized.
Brain gliomas are the most direct neurosurgical disease with the greatest impact on nerve function. Brain glioma has the characteristics of higher invasiveness and adjacent important nerve functional structures, and is difficult to accurately cut off in the operation; even if treatment measures such as operation, radiotherapy, chemotherapy and the like are adopted, the prognosis is still poor, and most of patients with brain glioma still easily relapse after receiving comprehensive treatment. Therefore, how to realize quick operation and assist the surgeon in realizing accurate excision of the brain glioma becomes an important problem.
At present, intraoperative auxiliary brain glioma imaging mainly depends on imaging technologies such as MRI, CT, fluorescence and the like. The fluorescence imaging can not only image in real time, has high resolution and high precision, but also provide real-time and direct visual guidance for tumor cells. In recent years, with the development of nanotechnology, different kinds of nanomaterials are used as probes for fluorescence imaging. By means of the unique advantages of the functionalized nano materials, fluorescence imaging shows great advantages in accurate diagnosis and treatment of brain glioma, and shows wide clinical application prospects.
With the increasing demand of biological detection, the nano composite probe presents a diversified development situation. The nano fluorescent probe can realize the rapid identification of the target substance, so the nano fluorescent probe is very suitable for the field real-time detection of the target substance. The first generation of fluorescent labeling materials such as fluorochrome, fluorescein isothiocyanate and rhodamine 6G have higher photoluminescence quantum yield, but the emission peak width of the fluorescent labeling materials exceeds 50nm, so that the application of the fluorescent labeling materials in real-time detection of multi-target detection objects is severely limited. The second generation fluorescent labeling material is quantum dot, and compared with the traditional fluorescent molecule, the quantum dot has the advantages of symmetrical and narrow emission peak, large Stokes shift, high quantum yield, size-dependent emission spectrum and the like. And under the influence of quantum confinement, the emission peak position of the quantum dot can be adjusted to a near infrared light (NIR) region by changing the size and the element composition of the quantum dot. Based on the excellent performance, the quantum dots have incomparable effects compared with other traditional fluorescent labeling materials in the biological application field. Meanwhile, with the continuous development and progress of quantum dot synthesis methods, quantum dots can be combined with various nano materials to further reduce the biotoxicity of the quantum dots, so that a biological small molecule detection method based on the quantum dots is widely concerned, and particularly, the quantum dots based on heavy metal Cd. However, it is still difficult to obtain quantum dots having a peak width of 30nm or less by the above method.
The perovskite nanocrystal material becomes a fluorescent labeling material with the highest quantum yield in the existing material due to the size effect and quantum confinement of the material. The perovskite nanocrystal material also has the advantages of narrow emission peak (< 20 nm), controllable emission peak position, wide excitation wavelength range, easy synthesis and the like. However, perovskite quantum dots are not resistant to water and oxygen, so that the application and development of the perovskite quantum dots in various fields such as bioscience and medicine are severely limited.
With the development of the fields of chemistry, materials science, biology and the like, the research and application of nano biotechnology are receiving more and more attention of researchers, especially in the field of biological analysis. The nanometer material has controllable appearance and size, and the surface functional group has modifiable property and unique optical property, so that the nanometer material occupies an important position in the field of biological small molecule analysis. Compared with the traditional detection probe, the nano biological composite probe has the advantages of multifunction composite, multiple detection channels, easy signal amplification, simple and convenient preparation and the like. Of particular interest, many nanoprobes have excellent optical properties, and can be used for biological detection with conventional optical devices, and even for naked eye detection.
The fluorescence immunoassay technology is characterized in that fluorescent substances are used for marking antibodies or antigen molecules, and after the antibodies or the antigen molecules are specifically combined with an analyte, fluorescent signals are reported, so that qualitative or quantitative detection of the target analyte is realized. The nano fluorescent probe has the advantages of high sensitivity, high specificity, simple and convenient detection instrument and low cost, and is widely researched in the fields of biological detection, sensing, drug molecule identification and the like. With the increasing demand for multi-target detection substances, the requirements for biological detection probes are also increasing. The traditional nano biological probe can not meet the detection requirements of high fluorescence efficiency, narrow emission, low interference and the like, so that the development of a novel nano biological detection probe with high fluorescence intensity, narrow emission and good biocompatibility has great significance.
Compared with the traditional fluorescent material, the perovskite quantum dot material has the advantages of high quantum yield, narrow emission peak, large Stokes shift, easy surface modification and the like, and becomes a hotspot research object. However, perovskite quantum dots are not resistant to water and oxygen, so that the application of the perovskite quantum dots in the research fields of bioscience, medicine and the like is limited. Therefore, how to modify perovskite quantum dots to construct a nano biological probe which can fully exert the advantages of materials and has good biocompatibility is an urgent need to be solved.
Disclosure of Invention
The invention aims to provide application of perovskite nanocrystalline in preparing a probe for tumor diagnosis or treatment.
The perovskite nanocrystalline in the application comprises perovskite quantum dots and liposome materials coated on the surfaces of the perovskite quantum dots.
The liposome coating material is preferably DPPC, DPPE, DSPC and derivatives thereof.
The liposome coating material is preferably DSPC-PEG, DPPC-PEG or DPPE-PEG.
The liposome coating material is most preferably DSPC-PEG-COOH.
The perovskite quantum dots are preferably CsPbBr3 perovskite quantum dots.
The probe in the application is a biological material marked by perovskite nanocrystals; the biological material is one, two or more of antibody, aptamer and polypeptide.
The tumor in the above application is preferably brain glioma.
The biomaterial in the above application is preferably an antibody, aptamer or polypeptide that specifically recognizes brain glioma tissue.
The biological material is further preferably at least one of chlorotoxin, IDH isocitrate dehydrogenase I, glial fibrillary acidic protein antibody, anti-ATRX and rabbit Anti-human H3K27Me3 polyclonal antibody.
The spectrum range of the water-soluble perovskite nanocrystal in the application is preferably 470-650 nm.
The particle size of the water-soluble perovskite nanocrystal in the application is preferably 100-200nm.
The perovskite nanocrystalline in the application is obtained by heating, stirring and reacting a liposome material and perovskite quantum dots.
The invention aims to provide a preparation method of a water-soluble perovskite nanocrystalline probe and application of the water-soluble perovskite nanocrystalline probe in brain glioma cell imaging.
The water-soluble perovskite quantum dot nano material provided by the invention comprises perovskite quantum dots and terminal carboxyl polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) coated on the surfaces of the perovskite quantum dots.
According to an embodiment of the invention, the water-soluble nanomaterial is a nanocrystal.
According to an embodiment of the invention, the perovskite quantum dots have an average particle size of 5 to 20nm, such as 10 to 15nm; illustratively, the quantum dots have an average particle size of 12nm.
According to an embodiment of the invention, the average particle size of the water-soluble perovskite nanomaterial is larger than the average particle size of the perovskite quantum dots, for example 5 to 100nm, preferably 10 to 80nm, exemplarily 10nm, 15nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm.
According to a particular embodiment of the invention, the water-soluble perovskite nanomaterial has an average particle size of 70nm.
According to an embodiment of the invention, the coating may be a full coating.
According to an embodiment of the invention, the water-soluble perovskite nanocrystals are CsPbBr coated with a carboxyl-terminated polylactic-co-glycolic acid (OH-PLGA-COOH) 3 And preparing the surface of the perovskite quantum dot.
According to an embodiment of the present invention, the quantum dot may be a perovskite quantum dot, a carbon quantum dot, a cadmium quantum dot, a zinc sulfide quantum dot, a sulfur quantum dot, or the like, and is preferably CsPbBr 3 Perovskite quantum dots.
According to an embodiment of the invention, the CsPbBr 3 Perovskite quantum dots are yellow in visible light and green in ultraviolet light (e.g., 365nm excitation).
According to an embodiment of the present invention, the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) has a number average molecular weight of 100 to 200000, preferably 110000. Preferably, the carboxyl-terminated polylactic acid-glycolic acid copolymer is synthesized by randomly copolymerizing racemic lactide (DLLA) and Glycolide (GA), and the ratio of the racemic lactide (DLLA) to the Glycolide (GA) is (50-90) to (10-50), preferably 90.
According to an exemplary embodiment of the present invention, the water-soluble quantum dot nanomaterial is the water-soluble perovskite nanomaterial, denoted as P-PQDs, which comprises CsPbBr 3 Perovskite quantum dot and coating CsPbBr 3 Terminal carboxyl polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) on the surface of the perovskite quantum dot.
According to an embodiment of the present invention, the water-soluble quantum dot nanomaterial has almost the same optical properties as quantum dots; for example, P-PQDs have the same chemical formula as CsPbBr 3 Perovskite quantum dots have almost the same optical properties.
In order to overcome the defects of complexity and long period of the existing fluorescence imaging technology, the invention provides a rapid pathological diagnosis means for rapidly detecting brain glioma cells, tissue slices and living tissues, and the diagnosis method has the characteristics of high sensitivity, rapid detection, simplicity, convenience, intuition and the like. The diagnosis method adopts the water-soluble perovskite nanocrystalline material as a fluorescent label, quickly reacts and identifies the tumor region (including cells, tissue slices and living tissues), judges the detection result under the irradiation of a simple handheld ultraviolet lamp, greatly improves the efficiency of detecting the tumor cells or tissues, and has guiding significance in assisting tumor resection during clinical operation.
The technical scheme of the invention is as follows:
the diagnosis method is characterized in that a water-soluble perovskite nanocrystalline material is used as a fluorescent label and coupled with a biomaterial targeting brain glioma to form a perovskite-based probe, and the perovskite-based probe is used for rapid 10min imaging on a cell layer, a tissue slice and a living tissue layer of the brain glioma, thereby providing a rapid pathological diagnosis means for assisting tumor resection in clinical operation.
The perovskite nanocrystalline material is an all-inorganic perovskite quantum dot (CsPbBr) 3 )。
Wherein, the CsPbBr 3 The perovskite quantum dots are light yellow under visible light and green under ultraviolet light (for example, 365nm excitation).
The water-soluble perovskite nanocrystal comprises all-inorganic perovskite quantum dots (CsPbBr) 3 ) And a liposome material coated on the surface of the perovskite quantum dot;
the biological material is selected from one, two or more of antibody, aptamer and polypeptide targeting brain glioma. For example: at least one of Chlorotoxin (CTX), IDH isocitrate dehydrogenase I (IDH-1), glial fibrillary acidic protein antibody (GFAP), anti-ATRX, rabbit Anti-human H3K27Me3 polyclonal antibody, etc., preferably Chlorotoxin (CTX).
The liposome coating material is DPPC (dipalmitoyl phosphatidylcholine), DPPE (dipalmitoyl phosphatidylethanolamine), DSPC (distearoyl phosphatidylcholine) and other substances and derivatives thereofAn agent; for example: DSPC-PEG (distearoyl phosphatidylcholine polyethylene glycol), DPPC-PEG (dipalmitoyl phosphatidylcholine polyethylene glycol), DPPE-PEG (dipalmitoyl phosphatidylethanolamine polyethylene glycol), preferably, with-COOH, -NH 2 And DSPC-PEG-COOH (distearoyl phosphatidyl choline polyethylene glycol with a terminal group modifying carboxyl) as-SH.
According to an embodiment of the invention, the water-soluble perovskite nanocrystals have a spectral range of 470 to 650nm, e.g. 480nm, 520nm, 580nm, 650nm, preferably the spectral position of the water-soluble perovskite nanocrystals is 520nm.
According to an embodiment of the invention, the water-soluble perovskite nanocrystals have a particle size of 100-200nm, e.g. 100nm, 150nm or 200nm, preferably the water-soluble perovskite nanocrystals have a particle size of 100nm.
According to an embodiment of the invention, the perovskite quantum dots have an average particle size of 5 to 20nm, for example 10 to 15nm; illustratively, the perovskite quantum dots have an average particle size of 5nm, 10nm, 12nm, 15nm.
According to an embodiment of the invention, the mass ratio (mg: mg) of the perovskite quantum dots to the liposome material is 116 (25-50); for example, 116 (25 to 40) may be used. For example, can be 116.
According to an embodiment of the invention, the coating is a full coating. For example, complete coating can be achieved when the mass ratio (mg: mg) of the perovskite quantum dots to DSPC-PEG-COOH (end-group modified carboxyl distearoyl phosphatidylcholine polyethylene glycol) is at least 116.
According to an embodiment of the invention, the weight average molecular weight of the DSPC-PEG-COOH (end group modified carboxyl group distearoyl phosphatidylcholine polyethylene glycol) is 1000-20000, such as 3400-20000, further such as 3400.
According to an embodiment of the invention, the water-soluble perovskite nanocrystal comprises CsPbBr 3 Perovskite quantum dot and coating CsPbBr 3 DSPC-PEG-COOH (distearoyl phosphatidylcholine polyethylene glycol with carboxyl modified at the end group) on the surface of the perovskite quantum dot is marked as D-PNCs.
According to an embodiment of the invention, the water-soluble perovskite nanocrystals have almost the same optical properties as perovskite quantum dots; for example, D-PNCs have a chemical affinity with CsPbBr 3 Perovskite quantum dots have almost the same optical properties.
According to an embodiment of the present invention, the method for preparing the water-soluble perovskite nanocrystal comprises the steps of: heating and stirring the distearoyl phosphatidylcholine polyethylene glycol with the terminal group modified carboxyl and the perovskite quantum dots for reaction to obtain the water-soluble perovskite nanocrystal.
According to the embodiment of the invention, the preparation method of the water-soluble perovskite nanocrystal specifically comprises the following steps:
(A1) Mixing and dissolving a raw material for preparing the perovskite quantum dots and distearoyl phosphatidylcholine polyethylene glycol with terminal group modified carboxyl in a solvent, and adding an organic ligand to form a stable solution;
(A2) And (2) adding the stable solution obtained in the step (A1) into an anti-solvent, heating for reaction, and precipitating water-soluble perovskite nanocrystalline by using an anti-solvent supersaturation method to prepare the water-soluble perovskite nanocrystalline.
According to an embodiment of the invention, the perovskite quantum dot is CsPbBr 3 In the case of perovskite quantum dots, the raw materials for preparing the perovskite quantum dots are, for example, csBr and PbBr 2 。
Wherein, the CsPbBr 3 The perovskite quantum dots can be prepared by methods known in the art.
According to an embodiment of the present invention, in the step (A1), the mixing order of the raw material for preparing the perovskite quantum dot and the distearoyl phosphatidylcholine polyethylene glycol with the terminal group modified carboxyl group is not limited, for example, the raw material for preparing the perovskite quantum dot and the distearoyl phosphatidylcholine polyethylene glycol with the terminal group modified carboxyl group may be added to the solvent at the same time, or the raw material for preparing the perovskite quantum dot may be added to the solvent first, and then the distearoyl phosphatidylcholine polyethylene glycol with the terminal group modified carboxyl group is added to the solvent.
According to an embodiment of the invention, in the step (A1), the ratio (mg: mg) of the sum of the masses of the raw materials for preparing the perovskite quantum dots to the mass of the distearoyl phosphatidylcholine polyethylene glycol (DSPC-PEG-COOH) with the terminal group modified carboxyl is 116 (25-50); for example, 116 (25 to 40) may be used.
According to an embodiment of the present invention, in step (A1), the mass-to-volume ratio of the end-group-modified carboxyl group distearoylphosphatidylcholine polyethylene glycol to the solvent is (10 to 30) mg:1mL, for example, 10mg 1ml, 15mg, 18mg, 120mg, 25mg.
According to an embodiment of the present invention, in the step (A1), the solvent may be selected from one or two of N, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO).
According to an embodiment of the present invention, in the step (A1), the organic ligand may be at least one selected from oleic acid, oleylamine, octylamine, and caprylic acid, for example, a mixture of oleic acid and oleylamine in any ratio.
Preferably, the volume ratio of the organic ligand to the solvent is (0.5 to 5): 10, such as (1 to 3): 10, exemplary 0.5.
According to an embodiment of the invention, step (A1) is synthesized in an all air process.
According to an embodiment of the present invention, in the step (A2), the antisolvent is at least one selected from the group consisting of toluene, chlorobenzene, and n-hexane.
According to an embodiment of the present invention, step (A2) comprises: dripping the stable solution in the step (A1) into an anti-solvent to obtain water-soluble perovskite nanocrystal; heating for reaction, collecting precipitate in a high-speed centrifugation mode after the reaction is finished, and performing ultrasonic dispersion in water to obtain the water-soluble perovskite nanocrystal.
Preferably, the volume ratio of the stabilizing solution to the anti-solvent is (0.1-5): 10, for example (0.5-3): 10.
Preferably, the dropping is slowly dropping dropwise, for example, at a rate of 3 to 10. Mu.L/s, illustratively 3. Mu.L/s, 5. Mu.L/s, 10. Mu.L/s.
Preferably, the dropwise addition is carried out under vigorous stirring of the anti-solvent. For example, the stirring speed is 800 to 1000r, illustratively 800r, 850r, 900r, 950r, 1000r.
Preferably, the water-soluble perovskite nanocrystal solution is added into an excessive antisolvent, heated and stirred for reaction, and the water-soluble perovskite nanocrystal is obtained through precipitation.
For example, the stirring time is 2-5h, such as 2h, 3h, 4h, 5h. For example, the temperature of the reaction is 40 to 60 ℃, e.g., 40 to 50 ℃, such as 30, 40 ℃, 45 ℃, 50 ℃, 60 ℃.
According to an embodiment of the present invention, the test water-soluble perovskite nanocrystalline material can be excited by a hand-held ultraviolet lamp and exhibits green fluorescence.
Further, the wavelength range of the ultraviolet lamp is 320-450 nm, preferably 365nm.
Further, the power of the ultraviolet lamp is 10-50W, preferably 10W.
According to the embodiment of the invention, the preparation method of the probe formed by coupling the water-soluble perovskite nano-crystal fluorescent material and the biomaterial targeting brain glioma comprises the following steps:
(1) Dispersing water-soluble perovskite nanocrystalline (D-PNCs) powder into ultrapure water (pH = 6.2-6.8), and uniformly dispersing by ultrasonic.
(2) And (2) adding the biological material in the step (1), mixing, and collecting the part with fluorescence to prepare a probe solution.
According to the embodiment of the invention, the detection probe is formed by combining the perovskite nanocrystalline material with strong positive charges and the biological material with negative charges targeting brain glioma through electrostatic interaction by regulating and controlling the pH of the solution.
According to an embodiment of the invention, the probe is stored in a diluent; the diluent is ultrapure water containing 0.01 to 0.1 weight percent of triton X-100. Illustratively, the pH of the ultrapure water is from 6.2 to 6.8.
According to an embodiment of the present invention, the obtained test solution may also be stored under refrigeration. For example, the temperature for cold storage is 1 to 5 ℃, for example, 1 ℃,2 ℃,3 ℃,4 ℃ or 5 ℃.
According to an embodiment of the invention, in step (1), the ultrasound is performed for 5-10min, such as 1min, 2min, 3min, 4min, 5min, 8min, 10min.
As a preferred embodiment of the present invention, the method for preparing the probe comprises the steps of:
A. dissolving D-PNCs (such as D-PNCs with wavelength of 520 nm) in ultrapure water, and subjecting to ultrasonic treatment for 5min to uniformly disperse the D-PNCs in the solvent.
B. Adding antibody (such as chlorotoxin CTX), and reacting at 37 deg.C for 10min;
C. after the reaction is finished, concentrating the reaction product to 30-100 ul by using an ultrafiltration centrifugal tube with the molecular cut-off of 30-100 KDa, purifying the concentrated solution by adopting a gel size exclusion method, collecting the part with fluorescence, concentrating the concentrated solution by using the ultrafiltration centrifugal tube, storing the concentrated solution in a diluent (containing 0.05 percent of triton X-100), and storing the concentrated solution at 4 ℃ for later use to obtain the probe solution.
The invention also provides application of the rapid brain glioma diagnosis means in the fields of medical detection, medical diagnosis and treatment and the like, and the rapid brain glioma diagnosis means is preferably used for detecting brain glioma cells, tissue slices and living tissue imaging.
The invention also provides a specific step of the diagnosis means, which comprises the following steps:
and (3) dropwise adding the probe solution onto a substance to be detected for reaction, and observing whether fluorescence appears on the target substance to be detected by adopting a handheld ultraviolet lamp for irradiation.
According to an embodiment of the present invention, the probe is added in an amount of 100 to 300. Mu.L.
According to an embodiment of the present invention, the temperature of the reaction is room temperature; the reaction time is 5 to 15min, and illustratively, the reaction time is 10min.
According to the embodiment of the invention, after the reaction is completed, the target substance to be detected can be washed by using a washing liquid; the flushing liquid is ultrapure water or normal saline.
According to an embodiment of the present invention, the wavelength of the ultraviolet light is 320 to 450nm.
As a preferred embodiment of the present invention, the use method is, for example: and (3) dripping 200 mu L of the detection solution on a target substance to be detected, reacting at room temperature for 10min, flushing with flushing liquid for 5-6 times after reaction, and carrying out imaging observation by using an ultraviolet lamp with hands.
According to an embodiment of the present invention, the target detection substance may be a glioma cell, a frozen section in glioma sectioning, a paraffin section, and a excised living tissue in glioma sectioning.
The evaluation criteria of the diagnostic method of the invention are: irradiating a tissue area by using a common handheld ultraviolet lamp with a light source range of exciting water-soluble perovskite nanocrystalline being 320-450 nm, dripping detection liquid into the tissue area when a target substance to be detected contains related antigens of a glioma tissue area according to an antibody antigen specificity identification principle, flushing the detection liquid for 5-6 times by using flushing liquid after reacting for 10min, wherein the tumor area is subjected to fluorescence under the excitation and irradiation of the handheld ultraviolet lamp, and the detection result is the glioma tumor area; on the contrary, the target substance to be detected is normal tissue, namely the target substance does not contain the related antigen of the glioma region, the tumor region does not have fluorescence under the excitation and irradiation of the handheld ultraviolet lamp, and the detection result is the normal tissue region, namely the non-tumor region.
The higher the fluorescence intensity is, the higher the canceration degree of the glioma contained in the tissue section to be detected is, whereas the lower the fluorescence intensity is, the lower the canceration degree is.
The invention also provides a preparation method of the water-soluble perovskite quantum dot nano material, which comprises the following steps: and coating the surface of the perovskite quantum dot with a carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) to obtain the water-soluble perovskite quantum dot nano material.
According to the embodiment of the invention, the preparation method of the water-soluble perovskite quantum dot nano material comprises the following steps:
(A1) Mixing a preparation raw material of the quantum dot and a carboxyl-terminated polylactic acid-glycolic acid copolymer in a solvent, and adding a ligand material after the raw material and the carboxyl-terminated polylactic acid-glycolic acid copolymer are completely dissolved to form a stable solution;
(A2) And adding the stable solution into an anti-solvent, separating out the water-soluble perovskite nanocrystalline material by using an anti-solvent supersaturation method, collecting precipitates in a high-speed centrifugation mode, and performing ultrasonic dispersion in water to obtain the water-soluble perovskite nanocrystalline material.
According to an embodiment of the invention, in step (A1), the perovskite quantum dots have the meaning as described above. The preparation raw material may be selected according to the perovskite quantum dot used. For example, csPbBr 3 The perovskite quantum dots are prepared from CsBr and PbBr 2 。
Wherein, the perovskite quantum dot can be prepared by adopting a method known in the field.
According to an embodiment of the present invention, in the step (A1), the mixing order of the raw material for preparing the quantum dots and the terminal carboxy polylactic acid-glycolic acid copolymer is not limited, for example, the raw material for preparing the quantum dots and the terminal carboxy polylactic acid-glycolic acid copolymer may be added to the solvent at the same time, or the raw material for preparing the quantum dots may be added to the solvent first, and then the terminal carboxy polylactic acid-glycolic acid copolymer may be added to the solvent.
According to an embodiment of the invention, in the step (A1), the molar ratio or molar mass ratio of the perovskite quantum dots and the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is 1mol (300-600) mg, for example 0.2mol.
According to an embodiment of the present invention, in step (A1), the mass-to-volume ratio of the terminal carboxyl polylactic acid-glycolic acid copolymer to the solvent is (5 to 30) mg:1mL, for example, 90mg.
According to an embodiment of the present invention, in the step (A1), the solvent may be one or two selected from N, N-Dimethyldiamide (DMF), dimethyl sulfoxide (DMSO).
According to an embodiment of the present invention, in step (A1), the ligand material may be selected from oleic acid, oleylamine and/or octylamine, preferably oleic acid and oleylamine.
Preferably, the ligand material is added in a ratio of (0.5-5) to solvent volume of 10, for example (1-3) to 10, illustratively 0.75.
According to an embodiment of the invention, step (A1) is carried out under anhydrous and anaerobic conditions. Preferably in an inert atmosphere, such as nitrogen.
According to an embodiment of the present invention, in the step (A2), the antisolvent is at least one selected from the group consisting of toluene, chlorobenzene, and n-hexane.
According to an embodiment of the present invention, step (A2) comprises: firstly, dropwise adding the stable solution into an anti-solvent to obtain a water-soluble perovskite quantum dot nano material solution; and collecting the precipitate in a high-speed centrifugation mode, and performing ultrasonic dispersion in water to obtain the water-soluble perovskite nanocrystalline material. Preferably, the volume ratio of the stabilizing solution to the anti-solvent is (0.1-5): 10, for example (0.5-3): 10.
Preferably, the dropping is slowly dropping dropwise.
Preferably, the dropwise addition is carried out under vigorous stirring of the anti-solvent.
Preferably, the volume ratio of the water-soluble perovskite quantum dot nano material solution to the anti-solvent is (0.5-3): 15, such as (1-2.5): 15.
Preferably, the water-soluble perovskite quantum dot nano material solution is added into an excessive antisolvent, heated, stirred and reacted, and the water-soluble perovskite quantum dot nano material is obtained through precipitation. For example, the reaction time is 5-10h, such as 5h, 7h, 8h, 10h. For example, the temperature of the stirring reaction is 30 to 60 ℃, e.g., 40 to 50 ℃, such as 30, 40 ℃, 42 ℃, 45 ℃,48 ℃, 50 ℃, 60 ℃.
According to the embodiment of the invention, the preparation method of the water-soluble perovskite quantum dot nanometer material further comprises the step (A3) of separating the precipitated water-soluble perovskite quantum dot nanometer material from a solution system and drying to obtain the solid water-soluble perovskite nanometer crystal material.
According to an embodiment of the present invention, the preparation method of the water-soluble perovskite quantum dot nanomaterial further comprises a step (A4) of dispersing the solid water-soluble perovskite quantum dot nanomaterial obtained in the step (A3) in water to obtain a water-soluble perovskite nanocrystal solution.
The invention also provides the water-soluble perovskite nanocrystalline solution prepared by the method.
The invention also provides application of the water-soluble quantum dot perovskite nano material in a medical diagnosis probe or a kit. For example, the medical diagnostic probe may be a fluorescent biological detection probe or a cellular imaging probe.
The invention also provides a nano probe which comprises the water-soluble quantum dot nano material.
The invention belongs to the field of materials science and biomedicine, relates to a water-soluble perovskite nanocrystalline probe, a preparation method thereof and brain glioma cell imaging application, and particularly relates to a perovskite nanocrystalline probe stably existing in a water system for a long time, a preparation method thereof and brain glioma cell imaging application.
According to the embodiment of the invention, the nano probe is a biological material marked by the water-soluble quantum dot nano material, and is formed by coupling the water-soluble quantum dot perovskite nano material with the biological material.
According to an embodiment of the present invention, the biological material may be selected from one, two or more of antibodies, aptamers, polypeptides and the like, preferably polypeptides. Illustratively, the biological material is a polypeptide; such as chlorotoxin polypeptides.
According to the embodiment of the invention, the nanoprobe can generate strong fluorescence in the range of 500-540 nm and generate strongest emission at 515 +/-5 nm under the excitation of 365 +/-5 nm.
According to an exemplary embodiment of the invention, the nanoprobe is a chlorotoxin polypeptide labeled by a water-soluble perovskite nano material P-PQDs, and is formed by electrostatic interaction of the P-PQDs and the chlorotoxin polypeptide.
According to the embodiment of the invention, the mass ratio of the water-soluble quantum dot nano material to the biological material is (10-50): 1, and preferably 20.
According to an embodiment of the present invention, the average particle diameter of the nanoprobe is in the range of 20 to 100nm, preferably 50nm.
The invention also provides a preparation method of the nano probe, which comprises the step of coupling the water-soluble perovskite nano crystal and the biological material to form the nano probe.
According to an embodiment of the invention, the preparation method comprises the steps of: directly mixing a water-soluble perovskite nanocrystalline material with a biological material, and obtaining the nanoprobe through strong charge difference electrostatic interaction;
the water-soluble perovskite nanocrystalline material and the biomaterial have the meaning as described above.
According to an embodiment of the present invention, the solvent in the crosslinking reagent liquid is an ultrapure water solution (e.g., pH = 6.42).
According to the embodiment of the invention, the mass ratio of the biological material to the water-soluble quantum dot perovskite nano material is 1 (10-50), such as 1.
According to an embodiment of the present invention, the step (B2) further comprises cryopreserving the obtained nanoprobe. For example, the temperature for cold storage is 1 to 5 ℃, for example, 1 ℃,2 ℃,3 ℃,4 ℃, 5 ℃.
The invention also provides the nano probe prepared by the method.
The invention also provides a kit, and the kit comprises the nanoprobe.
The invention also provides application of the water-soluble perovskite quantum dot nano material, the nano probe or the kit in the fields of medical detection, medical diagnosis and treatment and the like. Preferably, the medical test may be a cellular imaging or biological test.
According to an embodiment of the present invention, the nanoprobe or kit can specifically recognize the following target analytes: antibodies, aptamers, polypeptides, antigens, target molecules, proteases, and the like. Such as polypeptides, e.g., chlorotoxin polypeptides, and the like.
The invention also provides a method for specifically recognizing the target analyte by using the nano probe or the kit, which comprises the following steps: and contacting the nano probe with the target analyte, and identifying through fluorescence detection.
A rapid brain glioma detection diagnosis means comprises a detection probe, wherein the detection liquid contains a nanocrystalline probe which is a biological material marked by water-soluble perovskite nanocrystals;
the water-soluble perovskite nanocrystal comprises perovskite quantum dots and DSPC-PEG-COOH (distearoyl phosphatidylcholine polyethylene glycol with terminal group modified carboxyl) coated on the surfaces of the perovskite quantum dots;
the biological material is selected from one, two or more of antibodies, aptamers and polypeptides.
The spectrum range of the water-soluble perovskite nanocrystal of the nanoprobe is 470-650 nm.
Preferably, the particle size of the water-soluble perovskite nanocrystal is 100-200nm.
The fluorescent marker is characterized in that the perovskite quantum dot is CsPbBr 3 Perovskite quantum dots.
Preferably, the weight average molecular weight of the DSPC-PEG-COOH (distearoyl phosphatidyl choline polyethylene glycol with terminal group modified carboxyl) is 1000-20000.
The detection probe, the water-soluble perovskite nanocrystal comprises CsPbBr 3 Perovskite quantum dot and coating CsPbBr 3 DSPC-PEG-COOH (distearoyl phosphatidyl choline polyethylene glycol with carboxyl modified at the end group) on the surface of the perovskite quantum dot.
The preparation method of the water-soluble perovskite nanocrystal comprises the following steps: and (3) heating and stirring DSPC-PEG-COOH (distearoyl phosphatidylcholine polyethylene glycol with terminal group modified carboxyl) and perovskite quantum dots for reaction to obtain the water-soluble perovskite nanocrystal.
The preparation method of the nanoprobe and the water-soluble perovskite nanocrystal specifically comprises the following steps:
(A1) Mixing and dissolving a raw material for preparing perovskite quantum dots and DSPC-PEG-COOH (distearoyl phosphatidylcholine polyethylene glycol with terminal group modified carboxyl) in a solvent, and adding an organic ligand to form a stable solution;
(A2) And (2) adding the stable solution obtained in the step (A1) into an anti-solvent, heating for reaction, and precipitating water-soluble perovskite nanocrystalline by using an anti-solvent supersaturation method to prepare the water-soluble perovskite nanocrystalline.
The visual portable observation equipment, portable equipment is handheld ultraviolet lamp.
Preferably, the wavelength range of the ultraviolet lamp is 320-450 nm.
Preferably, the power of the ultraviolet lamp is 10-50W.
Preferably, the biological material is an antibody, aptamer or polypeptide that specifically recognizes brain glioma tissue; is at least one of Chlorotoxin (CTX), IDH isocitrate dehydrogenase I (IDH-1), glial fibrillary acidic protein antibody (GFAP), anti-ATRX and rabbit Anti-human H3K27Me3 polyclonal antibody.
The nanometer probe combines the perovskite nanometer crystal material with strong positive charge and the biological material with negative charge targeting brain glioma through electrostatic interaction by regulating and controlling the PH of the solution, and the preparation method of the nanometer probe comprises the following steps:
(1) Dispersing water-soluble perovskite nanocrystalline (D-PNCs) powder into ultrapure water (pH = 6.2-6.8), and uniformly dispersing by ultrasonic.
(2) And (2) adding the biological material in the step (1), mixing, and collecting the part with fluorescence to prepare a probe solution.
A rapid diagnosis means for brain glioma is applied to rapid 10min imaging of cell layer, tissue slice and living tissue of brain glioma.
The invention has the beneficial effects that:
1. the invention utilizes polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) to CsPbBr 3 And the perovskite quantum dots are packaged to prepare the water-soluble perovskite nanocrystalline material, and the optical properties of the perovskite quantum dots are almost completely reserved. (ii) a
2. The product of the invention uses degradable polylactic acid to coat the quantum dot material to construct the biological nano probe, thereby not only keeping the high fluorescence intensity and narrow emission of the quantum dot, but also endowing the quantum dot with the characteristics of good biocompatibility, no toxicity, environmental protection and the like. (ii) a
3. Because the perovskite quantum dot has the characteristic of narrow emission peak (< 20 nm), the water-soluble quantum dot nanocrystalline biological nanoprobe can be used for simultaneous detection of multiple analytes in the medical field, can avoid false negative results caused by fluorescence emission peak crossing, improves the detection reliability, has universality, can realize simultaneous detection of multiple targets, greatly meets the requirements of modern biotechnology and medical detection, and has important significance in the medical diagnosis and treatment field. (ii) a
4. The water-soluble perovskite nanocrystalline can be simply and quickly connected with the biological material by realizing the electrostatic interaction through simple strong potential difference, so that the complex coupling reaction and the use of a coupling kit are avoided, the cost is low, the operation is simple, and the commercialization is easy to realize. (ii) a
5. The novel biological nano probe is constructed on the basis of the water-soluble perovskite quantum dot material. The chlorotoxin nanoprobe is taken as an example, glioma cells can be specifically identified, so that tumor tissues, tissues beside cancer and normal tissues can be distinguished, and real-time and direct visual guidance of tumor excision can be realized.
The invention is based on the characteristic of high quantum yield of the water-soluble perovskite-based nano probe, realizes rapid and high-sensitivity 10min imaging on the cell level, the tissue slice and the living tissue level of the brain glioma, and can finish visual observation by holding an ultraviolet lamp with hands.
The rapid brain glioma diagnosis means combines the antibody and the antigen specifically and can be used for high-sensitivity rapid detection of brain glioma tissues or cells.
The invention has the advantages of good labeling stability (for example, covalent bonding of biomolecules and perovskite nanocrystals), high speed and high sensitivity (for example, the perovskite nanocrystals have high fluorescence intensity and can realize the detection of low-concentration target molecules), simple and quick operation, short detection time, easy result interpretation, qualitative and quantitative detection and the like. Is particularly suitable for clinical application guided by the resection of brain glioma by doctors.
In order to improve the technical problem, the invention provides a hydrophobic perovskite nanocrystal which comprises a perovskite quantum dot and a polymer outer layer coated on the surface of the perovskite quantum dot, wherein a polymer in the polymer outer layer is a hydrophobic polymer containing terminal carboxyl.
According to an embodiment of the present invention, the hydrophobic polymer having a terminal carboxyl group includes at least one of a lactic acid-glycolic acid copolymer having a terminal carboxyl group (OH-PLGA-COOH); preferably, in the lactic-glycolic acid copolymer, the ratio of racemic lactide (DLLA) to Glycolide (GA) can be (50 to 90) (10 to 50), exemplary being 90; for example, the ratio of racemic lactide DLLA to glycolide GA is 50.
According to an embodiment of the invention, the hydrophobic carboxyl end group containing polymer has a number average molecular weight of 1000 to 100000, preferably 5000 to 80000, exemplarily 1000, 2000, 5000, 10000, 30000, 50000, 80000, 100000.
According to an embodiment of the invention, the perovskite quantum dots have the chemical formula ABX 3 (ii) a Wherein:
a represents at least one of methylamine cation, formamidine cation, lithium ion, sodium ion, potassium ion, rubidium (Rb) ion and cesium (Cs) ion; preferably Cs ions;
b represents at least one of lead (Pb) ions, tin (Sn) ions, cadmium (Cd) ions, manganese (Mn) ions, zinc (Zn) ions, and nickel (Ni) ions; preferably a Pb ion;
x represents a monovalent anion; for example, a halogen ion, illustratively at least one selected from the group consisting of F ion, cl ion, br ion, and I ion; br ions are preferred.
According to an embodiment of the invention, the particle size of the perovskite quantum dots is 10 to 20nm, exemplary 10nm, 15nm, 20nm.
According to an embodiment of the present invention, the perovskite quantum dot has the chemical formula CsPbX 3 (ii) a Wherein, X represents at least one of F, cl, br and I; br is preferred.
According to an embodiment of the present invention, the mass ratio of the perovskite quantum dots to the hydrophobic polymer containing terminal carboxyl groups is (40-70): (30-60), preferably (50-60): (40-50), exemplary is 40.
According to an embodiment of the invention, the particle size of the hydrophobic perovskite nanocrystal is 40 to 90nm, preferably 40 to 70nm, exemplary 40nm, 50nm, 60nm, 70nm, 80nm.
The invention also provides a preparation method of the hydrophobic perovskite nanocrystalline, which comprises the step of coating the hydrophobic polymer containing terminal carboxyl on the surface of the perovskite quantum dot to prepare the hydrophobic perovskite nanocrystalline.
According to an embodiment of the present invention, the preparation method specifically comprises:
preparing perovskite quantum dots by reacting raw materials comprising an A source and a B source, and then mixing a hydrophobic polymer containing terminal carboxyl with the perovskite quantum dots to prepare the hydrophobic perovskite nanocrystals;
or mixing the hydrophobic polymer containing the terminal carboxyl with raw materials comprising an A source and a B source, and reacting to obtain the hydrophobic perovskite nanocrystal.
According to an embodiment of the present invention, the preparation method specifically comprises the steps of:
(1) Sequentially dissolving a source A, a source B and a hydrophobic polymer containing terminal carboxyl in a first solvent, and then adding a stabilizer into the mixed solution;
(2) And adding the mixed solution into a second solvent (such as toluene, chlorobenzene, cyclohexane and n-hexane) to obtain the hydrophobic perovskite nanocrystal solution.
According to an embodiment of the invention, the a source is provided by a compound containing a selected from at least one of methylamine cation, formamidine cation, lithium ion, sodium ion, potassium ion, rubidium (Rb) ion and cesium (Cs) ion.
Preferably, the compound containing a may be at least one of bromide, chloride, fluoride and iodide containing a. More preferably a-containing bromide.
According to an embodiment of the present invention, the B source is provided by a compound containing B selected from at least one of lead (Pb) ions, tin (Sn) ions, cadmium (Cd) ions, manganese (Mn) ions, zinc (Zn) ions, and nickel (Ni) ions.
Preferably, the compound containing B may be at least one of bromide, chloride, fluoride and iodide containing B. More preferably a bromide containing B.
According to an embodiment of the invention, the reaction is carried out in the presence of a source a, a source B, a stabilizer and a first solvent.
According to an embodiment of the invention, the ratio of the amounts of the a source, B source and hydrophobic polymer containing terminal carboxyl groups is (0.1-0.5) mmol to (0.1-0.5) mmol, preferably (0.2-0.4) mmol to 90mg, exemplary is.
According to an embodiment of the present invention, the amount ratio of the hydrophobic polymer containing a terminal carboxyl group and the stabilizer is 90mg (0.2 to 1.5) mL, preferably 90mg (0.2 to 1) mL, exemplified by 90mg.
According to an embodiment of the invention, the ratio of the amount of the hydrophobic polymer containing terminal carboxyl groups to the first solvent is 90mg (2 to 15) mL, preferably 90mg (3 to 10) mL, exemplified by 90mg (2ml), 90mg (3ml), 90mg (5ml), 90mg (10ml).
Preferably, the stabilizer is selected from oleic acid and/or oleylamine. More preferably, when the stabilizer is selected from two of oleic acid and oleylamine, the volume ratio of oleic acid to oleylamine is 1 (0.1 to 1), exemplary is 1.
Preferably, the first solvent is at least one of N, N-Dimethyldiamide (DMF) and Dimethylsulfoxide (DMSO), preferably N, N-Dimethyldiamide (DMF).
Preferably, in the step (2), the mixed solution is added to the second solvent in a dropwise manner. Preferably, the volume ratio of the mixed solution to the second solvent is 1 (5-30), and exemplarily comprises 1.
Preferably, the preparation method further comprises:
(3) And adding the hydrophobic perovskite nanocrystal solution into a second solvent (such as toluene, chlorobenzene, cyclohexane and n-hexane) again. Preferably, the volume ratio of the hydrophobic perovskite nanocrystal solution to the second solvent is 1 (5-25), exemplarily 1.
Preferably, the preparation process is carried out under stirring conditions. For example, the stirring time is not more than 50 hours, preferably 4 to 48 hours, and exemplarily 4 hours, 12 hours, 16 hours, 24 hours, 30 hours, 36 hours, 40 hours, and 48 hours.
Preferably, the preparation method further comprises:
(4) And carrying out solid-liquid separation on the hydrophobic perovskite nanocrystal solution. For example, the solid-liquid separation may be by means known in the art, such as centrifugation. Preferably, the rotation speed of the centrifugation is 6000 to 12000rpm, such as 7000 to 10000rpm, exemplary 6000rpm, 7000rpm, 8000rpm, 9000rpm, 10000rpm. Further, the time of the centrifugation is 3 to 10min, such as 5 to 8min, exemplary 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min.
According to an embodiment of the present invention, the step (4) further comprises: and drying the reaction product obtained by solid-liquid separation. For example, the drying temperature is 60 to 90 ℃, preferably 70 to 80 ℃, and exemplary 60 ℃, 70 ℃, 80 ℃, 90 ℃. Further, the drying time is 1 to 12 hours, preferably 1 to 10 hours, and exemplarily 1 hour, 4 hours, 8 hours, 10 hours, 12 hours.
According to an embodiment of the invention, the preparation method further comprises:
(5) And dispersing the dried product in water to obtain the perovskite nano crystal water dispersion liquid. Preferably, the method further comprises the step of subjecting the perovskite nanocrystalline water dispersion to ultrasonic treatment. For example, the time of the ultrasonic treatment is 1 to 10min, preferably 2 to 8min, and exemplified by 1min, 2min, 5min, 8min, and 10min.
According to an exemplary embodiment of the present invention, the method for preparing the hydrophobic perovskite nanocrystal comprises the steps of:
(1) Reacting CsBr and PbBr 2 And polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) containing terminal carboxyl is dissolved in N, N-dimethyl Diamide (DMF) solvent, and oleic acid and oleylamine are added for stabilizing the solution after the copolymer is completely dissolved;
(2) Under the condition of stirring, the solution is taken and dripped into a toluene solution to obtain CsPbBr 3 -QDs @ PLGA perovskite nanocrystalline solution；
(3) Adding 1mL of the solution obtained in the step (2) into the toluene solution, and stirring for reaction to completely coat the solution to obtain CsPbBr 3 -qds @ plga perovskite nanocrystals;
(4) CsPbBr prepared in the step (3) 3 Centrifuging and drying the-QDs @ PLGA perovskite nanocrystalline solution;
(5) Dispersing the product prepared in the step (4) in water, and performing ultrasonic treatment to obtain uniformly dispersed CsPbBr 3 -qds @ plga perovskite nanocrystalline water dispersion.
The invention also provides application of the hydrophobic perovskite nanocrystal in fluorescence biological detection. For example, in the construction of aqueous phase fluorescent biological detection probes.
The invention also provides a fluorescent biological detection probe which comprises the hydrophobic perovskite nanocrystal.
The invention has the beneficial effects that:
(1) The invention utilizes a hydrophobic polymer containing terminal carboxyl (such as polylactic-co-glycolic acid (PLGA-COOH)) for the first time to realize perovskite quantum dots (such as CsPbBr) in a simple and green anti-solvent one-step method 3 Perovskite quantum dots), realizes the stable existence of the perovskite quantum dots in a water phase, and almost completely retains the optical properties of the perovskite quantum dots.
(2) The hydrophobic perovskite nanocrystalline product prepared by the invention can prepare fluorescent groups with different emission wavelength ranges by adjusting the element composition of perovskite quantum dots.
(3) The product of the invention uses degradable polymers (such as polylactic acid polymers) as coating materials, is green and environment-friendly, has good biocompatibility, simple operation and low cost, and can be used for the mass preparation of perovskite nanocrystals.
(4) Specifically, the invention utilizes polylactic acid-glycolic acid copolymer (PLGA-COOH) to CsPbBr 3 The perovskite quantum dots are coated, and the polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) containing terminal carboxyl is a drug delivery carrier with great potential, and has the advantages of high drug encapsulation rate, strong modifiability, good biological safety and the likeAdvantageously, it can therefore be used for the protection of biological materials.
(5) The coating layer of the hydrophobic perovskite nanocrystalline material prepared by the invention can expose a large amount of active functional groups such as-COOH and the like, can provide reactive active sites for the construction of a biological probe by a later-stage coupling biological material, and can react with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to realize the coupling labeling of perovskite quantum dots and the biological material through simple and green coupling reaction of carboxyl and amino so as to construct a novel biological fluorescence detection probe.
The hydrophobic perovskite nanocrystal comprises a perovskite quantum dot and a polymer outer layer coated on the surface of the perovskite quantum dot, wherein a polymer in the polymer outer layer is a hydrophobic polymer containing terminal carboxyl.
The hydrophobic perovskite nanocrystal comprises at least one of carboxyl-terminated lactic acid-glycolic acid copolymers;
preferably, in the lactic-glycolic acid copolymer, the ratio of racemic lactide (DLLA) to Glycolide (GA) can be (50 to 90) (10 to 50), exemplary being 90.
Preferably, the hydrophobic carboxyl end group-containing polymer has a number average molecular weight of 1000 to 100000, preferably 5000 to 80000.
The chemical formula of the hydrophobic perovskite nano crystal is ABX 3 (ii) a Wherein:
a represents at least one of methylamine cation, formamidine cation, lithium ion, sodium ion, potassium ion, rubidium (Rb) ion and cesium (Cs) ion; preferably Cs ions;
b represents at least one of lead (Pb) ions, tin (Sn) ions, cadmium (Cd) ions, manganese (Mn) ions, zinc (Zn) ions, and nickel (Ni) ions; preferably a Pb ion;
x represents a monovalent anion; for example, a halogen ion, illustratively at least one selected from the group consisting of F ion, cl ion, br ion, and I ion; br ions are preferred.
Preferably, the particle size of the perovskite quantum dot is 10-20 nm.
Preferably, the perovskite quantum dot has a chemical formula of CsPbX 3 (ii) a Wherein, X represents at least one of F, cl, br and I; br is preferred.
The hydrophobic perovskite nanocrystal is characterized in that the mass ratio of the perovskite quantum dot to the hydrophobic polymer containing the terminal carboxyl is (40-70) to (30-60), preferably (50-60) to (40-50).
Preferably, the particle size of the hydrophobic perovskite nanocrystal is 40 to 90nm, preferably 40 to 70nm.
The preparation method of the hydrophobic perovskite nanocrystal comprises the step of coating a hydrophobic polymer containing terminal carboxyl on the surface of a perovskite quantum dot to prepare the hydrophobic perovskite nanocrystal.
Preferably, the preparation method specifically comprises:
preparing perovskite quantum dots by reacting raw materials comprising an A source and a B source, and then mixing a hydrophobic polymer containing terminal carboxyl with the perovskite quantum dots to prepare the hydrophobic perovskite nanocrystals;
or mixing the hydrophobic polymer containing the terminal carboxyl with raw materials comprising an A source and a B source, and reacting to obtain the hydrophobic perovskite nanocrystal.
The preparation method comprises the following steps:
(1) Sequentially dissolving a source A, a source B and a hydrophobic polymer containing terminal carboxyl in a first solvent, and then adding a stabilizer into the mixed solution;
(2) And adding the mixed solution into a second solvent (such as toluene, chlorobenzene, cyclohexane and n-hexane) to obtain the hydrophobic perovskite nanocrystal solution.
Preferably, the a source is provided by a compound containing a selected from at least one of methylamine cations, formamidine cations, lithium ions, sodium ions, potassium ions, rubidium (Rb) ions and cesium (Cs) ions.
Preferably, the compound containing a may be at least one of bromide, chloride, fluoride and iodide containing a. More preferably a-containing bromide.
Preferably, the B source is provided by a compound containing B selected from at least one of lead (Pb) ions, tin (Sn) ions, cadmium (Cd) ions, manganese (Mn) ions, zinc (Zn) ions, and nickel (Ni) ions.
Preferably, the compound containing B may be at least one of bromide, chloride, fluoride and iodide containing B. More preferably a bromide containing B.
The preparation method is characterized in that the reaction is carried out in the presence of a source A, a source B, a stabilizer and a first solvent.
Preferably, the amount ratio of the A source, the B source and the hydrophobic polymer containing terminal carboxyl groups is (0.1-0.5) mmol, (0.1-0.5) mmol:90mg, preferably (0.2-0.4) mmol, (0.2-0.4) mmol:90mg.
Preferably, the amount ratio of the hydrophobic polymer containing terminal carboxyl groups to the stabilizer is 90mg (0.2-1.5) mL, preferably 90mg (0.2-1) mL.
Preferably, the dosage ratio of the hydrophobic polymer containing terminal carboxyl groups to the first solvent is 90mg (2-15) mL, preferably 90mg (3-10) mL.
Preferably, the stabilizer is selected from oleic acid and/or oleylamine. More preferably, when the stabilizing agent is selected from two of oleic acid and oleylamine, the volume ratio of the oleic acid to the oleylamine is 1 (0.1-1).
Preferably, the first solvent is at least one of N, N-Dimethyldiamide (DMF) and Dimethylsulfoxide (DMSO), preferably N, N-Dimethyldiamide (DMF).
Preferably, in the step (2), the mixed solution is added to the second solvent in a dropwise manner. Preferably, the volume ratio of the mixed solution to the second solvent is 1 (5-30).
Preferably, the preparation method further comprises:
(3) And adding the hydrophobic perovskite nanocrystal solution into a second solvent (such as toluene, chlorobenzene, cyclohexane and n-hexane) again. Preferably, the volume ratio of the hydrophobic perovskite nanocrystal solution to the second solvent is 1 (5-25).
Preferably, the preparation process is carried out under stirring conditions. For example, the stirring time is not more than 50 hours, preferably 4 to 48 hours.
Preferably, the preparation method further comprises:
(4) And carrying out solid-liquid separation on the hydrophobic perovskite nanocrystal solution. For example, the solid-liquid separation may be by means known in the art, such as centrifugation. Preferably, the centrifugation is performed at a speed of 6000 to 12000rpm, such as 7000 to 10000rpm. Further, the time of the centrifugation is 3 to 10min, for example 5 to 8min.
Preferably, the step (4) further comprises: and drying the reaction product obtained by solid-liquid separation. For example, the drying temperature is 60 to 90 ℃, preferably 70 to 80 ℃. Further, the drying time is 1 to 12 hours, preferably 1 to 10 hours.
Preferably, the preparation method further comprises
(5) And dispersing the dried product in water to obtain the perovskite nano crystal water dispersion liquid. Preferably, the method further comprises the step of subjecting the perovskite nanocrystalline water dispersion to ultrasonic treatment. For example, the time for the ultrasonic treatment is 1 to 10min, preferably 2 to 8min.
The preparation method of the hydrophobic perovskite nanocrystal comprises the following steps:
(1) Reacting CsBr and PbBr 2 And polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) containing terminal carboxyl is dissolved in N, N-dimethyl Diamide (DMF) solvent, and oleic acid and oleylamine are added for stabilizing the solution after the copolymer is completely dissolved;
(2) Under the condition of stirring, the solution is taken and dripped into a toluene solution to obtain CsPbBr 3 -qds @ plga perovskite nanocrystalline solution;
(3) Adding 1mL of the solution obtained in the step (2) into the toluene solution, and stirring for reaction to ensure that the coating is complete to obtain CsPbBr 3 -qds @ plga perovskite nanocrystals;
(4) CsPbBr prepared in the step (3) 3 Centrifuging and drying the-QDs @ PLGA perovskite nanocrystalline solution;
(5) Dispersing the product prepared in the step (4) in water, and performing ultrasonic treatment to obtain uniformly dispersed CsPbBr 3 -qds @ plga perovskite nanocrystalline dispersion.
The hydrophobic perovskite nanocrystalline and/or the hydrophobic perovskite nanocrystalline prepared by the preparation method are applied to fluorescence biological detection. For example, in the construction of aqueous phase fluorescent biological detection probes.
A fluorescence biological detection probe comprises the hydrophobic perovskite nanocrystal and/or the hydrophobic perovskite nanocrystal prepared by the preparation method.
The invention provides a hydrophobic quantum dot nano material, a nano probe of the nano material, and preparation methods and applications of the hydrophobic quantum dot nano material and the nano probe.
The hydrophobic quantum dot nano material provided by the invention comprises quantum dots and a carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) coated on the surfaces of the quantum dots.
According to an embodiment of the invention, the hydrophobic quantum dot nanomaterial is a nanocrystal.
According to an embodiment of the invention, the quantum dots have an average particle size of 5 to 20nm, such as 10 to 15nm; illustratively, the quantum dots have an average particle size of 12nm.
According to an embodiment of the invention, the hydrophobic quantum dot nanomaterial has an average particle size larger than the average particle size of the quantum dots, e.g. 5 to 100nm, preferably 10 to 80nm, exemplarily 10nm, 15nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm.
According to a specific embodiment of the present invention, the hydrophobic quantum dot nanomaterial has an average particle size of 80nm.
According to an embodiment of the invention, the coating is a full coating.
According to the embodiment of the invention, the hydrophobic quantum dot nano material is prepared by coating carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) on the surface of a quantum dot.
According to an embodiment of the invention, the quantum dots may be selected from modified or unmodifiedThe following quantum dots: perovskite quantum dots, carbon quantum dots, cadmium quantum dots, sulfur quantum dots, or the like, preferably modified or unmodified perovskite quantum dots. Wherein, the modification means that the quantum dot can be modified by adopting a fluorescent group pair (such as sulfydrylyl). Illustratively, the quantum dot may be CsPbBr 3 Perovskite quantum dots or Sulfydryl-PQDs perovskite quantum dots.
According to an embodiment of the invention, the CsPbBr 3 The perovskite quantum dots are light yellow under visible light and green under ultraviolet light (for example, 365nm excitation).
According to an exemplary embodiment of the present invention, the Sulfydryl-PQDs perovskite quantum dots have a cyan color under visible light and a blue color under ultraviolet light (e.g., 365nm excitation).
According to an embodiment of the present invention, the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) has a number average molecular weight of 100 to 100000, preferably 30000. Preferably, the terminal carboxyl polylactic acid-glycolic acid copolymer is randomly copolymerized from racemic lactide (DLLA) and Glycolide (GA), and the percentage of the racemic lactide (DLLA) to the Glycolide (GA) is (50-90), (10-50), preferably 90.
According to an exemplary embodiment of the present invention, the hydrophobic quantum dot nanomaterial is a hydrophobic perovskite nanomaterial, denoted as pqds @ plga, comprising CsPbBr 3 Perovskite quantum dot and coating CsPbBr 3 And the terminal carboxyl polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) on the surface of the perovskite quantum dot.
According to an embodiment of the present invention, the hydrophobic quantum dot nanomaterial has almost the same optical properties as quantum dots; for example, PQDs @ PLGA has CsPbBr 3 Perovskite quantum dots have almost the same optical properties.
The invention also provides a preparation method of the hydrophobic quantum dot nano material, which comprises the following steps: and coating the surface of the quantum dot with a carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) to obtain the hydrophobic quantum dot nano material.
According to an embodiment of the present invention, the preparation method of the hydrophobic quantum dot nanomaterial comprises the following steps:
(A1) Mixing a preparation raw material of the quantum dot with a terminal carboxyl polylactic acid-glycolic acid copolymer in a solvent, and adding a ligand material after the quantum dot is completely dissolved to form a stable solution;
(A2) And adding the stable solution into an anti-solvent to form a hydrophobic quantum dot nano material solution, and then separating out the hydrophobic quantum dot nano material by using an anti-solvent supersaturation method.
According to an embodiment of the invention, in step (A1), the quantum dots have the meaning as described above. The preparation raw material may be selected according to the quantum dot used. For example, csPbBr 3 The perovskite quantum dot is prepared from CsBr and PbBr 2 。
The quantum dots can be prepared by adopting a method known in the field.
According to the embodiment of the present invention, in the step (A1), the mixing order of the raw materials for preparing the quantum dots and the carboxyl-terminated polylactic acid-glycolic acid copolymer is not limited, for example, the raw materials for preparing the quantum dots and the carboxyl-terminated polylactic acid-glycolic acid copolymer may be added to the solvent at the same time, or the raw materials for preparing the quantum dots may be added to the solvent first, and then the carboxyl-terminated polylactic acid-glycolic acid copolymer may be added to the solvent.
According to an embodiment of the present invention, in the step (A1), the molar mass ratio of the quantum dot to the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is 1mmol (300-600) mg, for example 0.2mmol.
According to an embodiment of the present invention, in step (A1), the mass-to-volume ratio of the terminal carboxyl polylactic acid-glycolic acid copolymer to the solvent is (5 to 30) mg:1mL, for example, 90mg.
According to an embodiment of the present invention, in the step (A1), the solvent may be one or two selected from N, N-Dimethyldiamide (DMF), dimethyl sulfoxide (DMSO).
According to an embodiment of the present invention, in step (A1), the ligand material may be selected from oleic acid, oleylamine and/or mercaptoundecanoic acid (Sulfydryl), preferably oleic acid and oleylamine, or oleic acid, oleylamine and mercaptoundecanoic acid.
Preferably, the ligand material is added in a ratio to the volume of solvent of (0.5-5): 10, e.g. (1-3): 10, exemplary 0.75.
According to an embodiment of the invention, step (A1) is carried out under anhydrous and anaerobic conditions. Preferably in an inert atmosphere, such as nitrogen.
According to an embodiment of the present invention, in the step (A2), the antisolvent is at least one selected from the group consisting of toluene, chlorobenzene, and n-hexane.
According to an embodiment of the present invention, step (A2) comprises: firstly, dropwise adding the stable solution into an anti-solvent to obtain a hydrophobic quantum dot nano material solution; and then adding the hydrophobic quantum dot nano material solution into an excessive anti-solvent to separate out the hydrophobic quantum dot nano material.
Preferably, the volume ratio of the stabilizing solution to the anti-solvent is (0.1-5): 10, for example (0.5-3): 10.
Preferably, the dropping is slow dropping.
Preferably, the dropwise addition is carried out under vigorous stirring of the anti-solvent.
Preferably, the volume ratio of the hydrophobic quantum dot nano material solution to the anti-solvent is (0.5-3): 15, such as (1-2.5): 15.
Preferably, the hydrophobic quantum dot nano-material solution is added into an excessive antisolvent, heated and stirred for reaction, and the hydrophobic quantum dot nano-material is obtained through precipitation. For example, the reaction time is 20 to 60 hours, such as 30 hours, 40 hours, 48 hours, 50 hours. For example, the temperature of the stirring reaction is 30 to 60 ℃, e.g., 40 to 50 ℃, such as 30 ℃, 40 ℃, 42 ℃, 45 ℃,48 ℃, 50 ℃, 60 ℃.
According to the embodiment of the invention, the preparation method of the hydrophobic quantum dot nanomaterial further comprises a step (A3) of separating the precipitated hydrophobic quantum dot nanomaterial from a solution system and drying the separated hydrophobic quantum dot nanomaterial to obtain a solid hydrophobic quantum dot nanomaterial.
According to an embodiment of the present invention, the method for preparing a hydrophobic quantum dot nanomaterial further comprises a step (A4) of dispersing the solid hydrophobic quantum dot nanomaterial obtained in the step (A3) in water to obtain an aqueous dispersion of the hydrophobic quantum dot nanomaterial.
The invention also provides the hydrophobic quantum dot nano material prepared by the method.
The invention also provides application of the hydrophobic quantum dot nano material in a medical diagnosis probe or a kit. For example, the medical diagnostic probe may be a fluorescent biological detection probe or a cellular imaging probe.
The invention also provides a nano probe which comprises the hydrophobic quantum dot nano material.
According to the embodiment of the invention, the nano probe is a biological material marked by the hydrophobic quantum dot nano material, and is formed by coupling the hydrophobic quantum dot nano material and the biological material.
According to an embodiment of the present invention, the biological material may be selected from one, two or more of antibodies, aptamers, polypeptides and the like, preferably antibodies. Illustratively, the antibody is an IgG antibody; such as human IgG antibodies.
According to the embodiment of the invention, the nanoprobe can generate strong fluorescence in the range of 500-540 nm and generate strongest emission at 515 +/-5 nm under the excitation of 365 +/-5 nm.
According to an exemplary scheme of the invention, the nanoprobe is a hydrophobic perovskite nano material PQDs @ PLGA labeled human IgG antibody, and is formed by coupling PQDs @ PLGA and the human IgG antibody.
According to the embodiment of the invention, the mass ratio of the hydrophobic quantum dot nano material to the biological material is (10-50): 1, and preferably 20.
According to an embodiment of the present invention, the average particle diameter of the nanoprobe is 100 to 500nm, preferably 200nm.
The invention also provides a preparation method of the nano probe, which comprises the step of coupling the hydrophobic quantum dot nano material and the biological material to form the nano probe.
According to an embodiment of the invention, the preparation method comprises the steps of: dispersing a hydrophobic quantum dot nano material in a crosslinking reagent solution, and adding a biological material and a surfactant into the crosslinking reagent solution to obtain the nano probe;
the hydrophobic quantum dot nanomaterials and biomaterials have the meaning as described above.
According to an embodiment of the present invention, the method for preparing the nanoprobe includes the steps of:
(B1) Dispersing a hydrophobic quantum dot nano material in a crosslinking reactant solution, stirring, and activating carboxyl on the surface of the hydrophobic quantum dot nano material;
(B2) And (B) adding a biological material and a surfactant into the solution obtained in the step (B1) to obtain the nano probe.
According to an embodiment of the invention, the crosslinking reagents are 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); preferably, the mass ratio of EDC to NHS is 1 (3-10), such as 1.
According to an embodiment of the invention, the solvent in the crosslinking reagent solution is PBS buffer (e.g. PBS buffer with pH = 7.3).
According to a specific embodiment of the present invention, the crosslinking reagent solution is a mixed solution of EDC and NHS; for example, a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution and an N-hydroxysuccinimide (NHS) solution may be prepared separately and mixed to obtain a mixed solution of EDC and NHS. Preferably, the use method of the mixed solution of EDC and NHS is in-situ preparation.
According to a specific embodiment of the present invention, the mass concentration of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) solution is 6mg/mL; the mass concentration of the N-hydroxysuccinimide (NHS) solution was 30mg/mL. Preferably, the volume ratio of the EDC solution to the NHS solution when the two solutions are mixed is 1.
According to an embodiment of the invention, the mass ratio of the biomaterial to the crosslinking reagent is (5-15) 1, such as 15.
According to the embodiment of the invention, in order to fully activate the carboxyl on the surface of the hydrophobic quantum dot nano material, magnetic stirring is carried out at room temperature during dispersion. For example, the stirring time is 10 to 60min, for example, 10min, 20min, 30min, 40min, 50min, 60min.
According to the embodiment of the invention, the surfactant is added to avoid agglomeration of the hydrophobic quantum dot nanomaterial. Preferably, the surfactant is a nonionic surfactant, such as one, two or three of triton X-100, alkyl polyglycoside APG and fatty alcohol polyoxyethylene ether AEO.
According to an embodiment of the invention, the mass to volume ratio of the biological material to the surfactant is 1mg (0.1 to 1) μ L.
According to the embodiment of the invention, the mass ratio of the biological material to the hydrophobic quantum dot nano material is 1 (10-50), such as 1.
According to an embodiment of the present invention, in the step (B2), when the biomaterial and the surfactant are added to the solution obtained in the step (B1), a rocking bed mild reaction is performed; preferably, the time of the mild reaction of the shaking table is 10-60 min, such as 10min, 20min, 30min, 40min, 50min, 60min.
According to an embodiment of the present invention, the step (B2) further comprises cryopreserving the obtained nanoprobe. For example, the temperature for cold storage is 1 to 5 ℃, for example, 1 ℃,2 ℃,3 ℃,4 ℃, 5 ℃.
The invention also provides the nano probe prepared by the method.
The invention also provides a kit, and the kit comprises the nanoprobe.
The invention also provides application of the hydrophobic quantum dot nano material, the nano probe or the kit in the fields of medical detection, medical diagnosis and treatment and the like. Preferably, the medical test may be a cellular imaging or biological test.
According to an embodiment of the present invention, the nanoprobe or kit can specifically recognize the following target analytes: antibodies, aptamers, polypeptides, antigens, target molecules, proteases, and the like. Examples of the antibody include antibodies such as human IgG antibodies. Examples thereof include antigens such as goat anti-human IgG, rabbit anti-human IgG, and mouse anti-human IgG.
The invention also provides a method for specifically recognizing the target analyte by using the nano probe or the kit, which comprises the following steps: and contacting the nano probe with the target analyte, and identifying through fluorescence detection.
According to an embodiment of the invention, the method comprises the steps of:
(S1) coating the well plate with a target analyte or an element-labeled target analyte, and washing the well plate coated with the target analyte or the element-labeled target analyte after incubation;
(S2) sealing and cleaning the coated pore plate obtained in the step (S1);
and (S3) adding the nanoprobe into the closed coated pore plate obtained in the step (S2), contacting with the target analyte, cleaning, and identifying the target analyte through fluorescence detection.
According to an embodiment of the invention, the target analyte has the meaning as described above. Illustratively, the target analyte is an antigen, for example Eu-labeled anti-human IgG.
According to an embodiment of the invention, the cleaning is performed by: washing was performed with PBST (PBS buffer, pH =7.3, containing five parts per million volume concentration of Tween-20). Preferably, the number of washing is 3 to 5.
According to an embodiment of the present invention, in step (S1), the element labeling the target analyte is Eu.
According to an embodiment of the invention, the well plate is a 96 well plate.
According to an embodiment of the invention, in step (S1), the incubation is performed in a well plate, e.g. a 96 well plate.
According to an embodiment of the invention, the incubation is a low temperature overnight incubation. For example, the incubation temperature is 1 to 5 ℃, such as 1 ℃,2 ℃,3 ℃,4 ℃, 5 ℃. For example, the incubation time is 8 to 18 hours, such as 8 to 14 hours, 10 to 12 hours.
According to an embodiment of the invention, the sealing treatment comprises: and (3) sealing the coated pore plate obtained in the step (S2) by using a skimmed milk powder solution. For example, the skim milk powder may be skim milk powder. For example, the mass fraction of the skim milk powder solution is 5%.
According to an embodiment of the invention, the sealing treatment is carried out for a time of 0.5 to 2 hours at a temperature of 30 to 45 ℃. According to a particular embodiment of the invention, the blocking treatment is carried out for a period of 1h and at a temperature of 37 ℃.
According to an embodiment of the present invention, in the step (S3), the contacting is performed for 0.5 to 2 hours at a temperature of 30 to 45 ℃. Illustratively, the contact time is 1h and the temperature is 37 ℃.
The invention has the beneficial effects that:
1. the invention utilizes polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) to encapsulate the quantum dots, realizes the stable existence of the quantum dot material in the water phase, and almost completely retains the optical performance of the quantum dots;
2. the product of the invention can obtain fluorescent groups with different emission wavelength ranges by adjusting the components of the quantum dots;
3. the degradable polylactic acid is used for coating the quantum dot material to construct the biological nano probe, so that the characteristics of good biocompatibility, no toxicity, environmental protection and the like are endowed while the high fluorescence intensity and narrow emission of the quantum dot are maintained;
4. as the perovskite quantum dot has the characteristic of narrow emission peak (< 20 nm), the hydrophobic quantum dot nanocrystal provided by the invention can be used as a biological nanoprobe for simultaneous detection of multiple analytes in the medical field, can avoid false negative results caused by the cross of emission peaks of fluorescent substances, improves the detection reliability, has universality, greatly meets the requirements of modern biotechnology and medical detection, and has important significance in the medical diagnosis and treatment field;
5. the novel biological nano probe constructed based on the stable quantum dot material in the water phase realizes biological detection with high sensitivity and high fluorescence intensity.
The hydrophobic quantum dot nano material comprises quantum dots and a carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) coated on the surfaces of the quantum dots.
The hydrophobic quantum dot nano material is characterized in that the quantum dot is selected from modified or unmodified quantum dots shown as follows: perovskite quantum dots, carbon quantum dots, cadmium quantum dots or sulfur quantum dots;
preferably, the hydrophobic quantum dot nano material is prepared by coating a carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) on the surface of a quantum dot.
Preferably, the number average molecular weight of the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is 100 to 100000;
preferably, the hydrophobic quantum dot nanomaterial is a nanocrystal;
preferably, the coating is a full coating;
preferably, the average particle size of the quantum dots is 5-20 nm; the average particle size of the hydrophobic quantum dot nano material is larger than that of the quantum dot;
preferably, the modification refers to the modification of the quantum dot with a fluorescent group, such as mercaptoundecanoic acid (Sulfydryl).
The preparation method of the hydrophobic quantum dot nano material comprises the following steps: and coating the surface of the quantum dot with a carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) to obtain the hydrophobic quantum dot nano material.
The preparation method of the hydrophobic quantum dot nano material comprises the following specific steps:
(A1) Mixing a preparation raw material of the quantum dot with a terminal carboxyl polylactic acid-glycolic acid copolymer in a solvent, and adding a ligand material after the quantum dot is completely dissolved to form a stable solution;
(A2) Adding the stable solution into an anti-solvent to form a hydrophobic quantum dot nano material solution, and then separating out the hydrophobic quantum dot nano material by using an anti-solvent supersaturation method;
preferably, in the step (A1), the molar mass ratio of the quantum dots to the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is 1mmol (300-600) mg;
preferably, in the step (A1), the mass-to-volume ratio of the terminal carboxyl polylactic acid-glycolic acid copolymer to the solvent is (5-30) mg:1mL;
preferably, in the step (A1), the solvent may be one or two selected from N, N-Dimethyldiamide (DMF), dimethylsulfoxide (DMSO);
preferably, in step (A1), the ligand material may be selected from oleic acid, oleylamine and/or mercaptoundecanoic acid (Sulfydryl);
preferably, the ratio of the adding amount of the ligand material to the volume of the solvent is (0.5-5): 10;
preferably, step (A1) is carried out under anhydrous and anaerobic conditions;
preferably, in the step (A2), the antisolvent is at least one selected from toluene, chlorobenzene and n-hexane;
preferably, the volume ratio of the stabilizing solution to the anti-solvent is (0.1-5): 10;
preferably, the volume ratio of the hydrophobic quantum dot nano material solution to the anti-solvent is (0.5-3): 15;
preferably, the preparation method of the hydrophobic quantum dot nano material further comprises a step (A3) of separating the precipitated hydrophobic quantum dot nano material from a solution system, and drying to obtain a solid hydrophobic quantum dot nano material;
preferably, the preparation method of the hydrophobic quantum dot nanomaterial further comprises a step (A4) of dispersing the solid hydrophobic quantum dot nanomaterial obtained in the step (A3) in water to obtain an aqueous dispersion of the hydrophobic quantum dot nanomaterial.
A nanoprobe comprising the hydrophobic quantum dot nanomaterial of claim 1 or 2;
preferably, the nanoprobe is a biological material marked by the hydrophobic quantum dot nanometer material and is formed by coupling the hydrophobic quantum dot nanometer material and the biological material;
preferably, the biological material may be selected from one, two or more of antibodies, aptamers, polypeptides and the like;
preferably, the mass ratio of the hydrophobic quantum dot nano material to the biological material is (10-50): 1, preferably 20;
preferably, the average particle size of the nanoprobe is 100 to 500nm.
The preparation method of the nano probe comprises the steps of coupling the hydrophobic quantum dot nano material and the biological material to form the nano probe;
preferably, the preparation method specifically comprises the following steps: dispersing the hydrophobic quantum dot nano material in a crosslinking reactant solution, and adding a biological material and a surfactant into the crosslinking reactant solution to obtain the nano probe.
The preparation method of the nanoprobe specifically comprises the following steps:
(B1) Dispersing a hydrophobic quantum dot nano material in a crosslinking reactant solution, stirring, and activating carboxyl on the surface of the hydrophobic quantum dot nano material;
(B2) Adding a biological material and a surfactant into the solution obtained in the step (B1) to obtain the nano probe;
preferably, the crosslinking reactants are 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS); preferably, the mass ratio of EDC to NHS is 1 (3-10);
preferably, the solvent in the crosslinking reactant solution is PBS buffer solution;
preferably, the crosslinking reactant solution is a mixed solution of EDC and NHS;
preferably, the mass ratio of the biological material to the crosslinking reactant is (5-15) 1;
preferably, the surfactant is a nonionic surfactant;
preferably, the nonionic surfactant is one, two or three of triton X-100, alkyl polyglycoside APG and fatty alcohol-polyoxyethylene ether AEO.
Preferably, in the step (B2), when the biological material and the surfactant are added to the solution obtained in the step (B1), a rocking bed mild reaction is required;
preferably, the step (B2) further comprises refrigerating the nanoprobe.
A kit comprising said hydrophobic quantum dot nanomaterial or said nanoprobe.
The hydrophobic quantum dot nano material, the nano probe or the kit are applied to the fields of medical detection and medical diagnosis and treatment;
preferably, the medical assay is a cellular imaging or biological assay;
preferably, the nanoprobe or kit is capable of specifically recognizing the following target analytes: an antibody, an aptamer, a polypeptide, an antigen, a target molecule, or a protease.
A method of specifically recognizing a target analyte, the method comprising: contacting the nanoprobe with a target analyte, and identifying through fluorescence detection; the target analyte has the aforementioned selection.
Drawings
FIG. 1 is a transmission electron micrograph of the water-soluble perovskite nanocrystal of example 1. As can be seen from the figure, the particle size of the water-soluble perovskite nanocrystal is about 100nm.
FIG. 2 is a PL spectrum of perovskite quantum dots (PQDs-Toluene) dissolved in Toluene and water-soluble perovskite nanocrystals (PNCs-water).
FIG. 3 is a Confocal image of the perovskite-based nanoprobe on brain glioma cells, the Confocal laser excitation wavelength being 405nm and 488nm. FIG. 4 is a Confocal image of the perovskite-based nanoprobe on brain glioma tissue sections, the Confocal laser excitation wavelength being 405nm and 488nm.
Fig. 5 is a real image of the perovskite-based nanoprobe on brain glioma biopsy.
FIG. 6 is a transmission electron micrograph of PQDs quantum dots of preparation example 11, representing the size of the PQDs quantum dots to 12nm.
FIG. 7 is a transmission electron micrograph of the P-PQDs nanocrystal of preparation example 11, representing the size of the P-PQDs quantum dots ranging from 30nm.
FIG. 8 is a graph showing the fluorescence image of P-PQDs perovskite nanocrystals of example 11 under 420-450 nm excitation, which is green.
FIG. 9 is a fluorescence emission spectrum of P-PQDs nanocrystals and Probe-P QDs of preparation example 11.
FIG. 10 is a graph of the Z eta potentials of the P-PQDs quantum dots, CTX and Probe-P QDs of example 21.
FIG. 11 is a Confocal fluorescence imaging chart showing that the biological nanoprobe Probe-PQDs formed by P-PQDs perovskite nanocrystals and CTX of example 31 specifically recognizes glioma 251 cells and the control group hardly recognizes HA cells, and the two-channel excitation is 405nm and 488nm.
FIG. 12 shows CsPbBr prepared in example 12 3 Fluorescence emission and absorption spectra of QDs quantum dots.
FIG. 13 shows CsPbBr obtained in example 12 3 Transmission electron microscopy of QDs quantum dots (left panel) and their lattice fringes (right panel).
FIG. 14 shows CsPbBr obtained in example 22 3 Transmission electron micrograph of-QDs @ PLGA perovskite nanocrystalline, nanocrystalline grain size ~ 80nm.
FIG. 15 shows CsPbBr prepared in example 22 3 The surface topography of-QDs @ PLGA perovskite nanocrystal, the grain size of the nanocrystal is 80nm.
FIG. 16 shows CsPbBr 3 -QDs Quantum dots and CsPbBr 3 -XRD pattern of qds @ plga perovskite nanocrystals.
FIG. 17 shows CsPbBr 3 -QDs Quantum dots and CsPbBr 3 -qds @ plga perovskite nanocrystals quantum yield.
FIG. 18 shows CsPbBr prepared in example 32 3 Zeta potential maps of QDs @ PLGA, human IgG, and probe CsPbBr3-QDs @ PLGA @ IgG in MES buffer solution (pH = 5.15), PBS buffer solution (pH = 7.3), tris buffer solution (pH = 8.17), and the like, respectively.
FIG. 19 is a transmission electron micrograph of PQDs quantum dots of preparation example 13 and lattice fringes thereof; the left image represents a transmission electron microscope image of the quantum dots, and the particle size of the quantum dots is 12nm; the right panel represents the fringe spacing of 0.295nm.
FIG. 20 shows fluorescence emission spectrum and absorption spectrum of PQDs quantum dot of preparation example 13, wherein the emission peak position of the quantum dot is 515nm.
FIG. 21 shows a fluorescence emission spectrum and an absorption spectrum of Sulfydryl-PQDs of preparation example 23, wherein the emission peak of the quantum dot is 461nm.
FIG. 22 is a transmission electron micrograph of PQDs @ PLGA perovskite nanocrystal of example 13.
FIG. 23 is an optical microscopic photograph of PQDs @ PLGA perovskite nanocrystal of example 13, the left image represents the fluorescence imaging image of the nanocrystal under excitation at 420-450 nm, and the right image represents the light field image of the nanocrystal.
FIG. 24 shows fluorescence emission spectrum and absorption spectrum of PQDs @ PLGA perovskite nanocrystal of example 13.
FIG. 25 is the SEM image and EDS spectrum of the biological nano-probe PQDs @ PLGA @ IgG formed by coupling the PQDs @ PLGA perovskite nanocrystal of example 23 with human IgG antibody.
FIG. 26 is a Confocal fluorescence imaging plot of the binding verification of the biological nanoprobes PQDs @ PLGA @ IgG formed by coupling the PQDs @ PLGA perovskite nanocrystals of example 33 with human IgG antibodies and Eu-labeled anti-human IgG, with double channel excitation at 488nm and 561 nm.
FIG. 27 is a schematic diagram of the experiment in which PQDs @ PLGA perovskite nanocrystal of example 33 is coupled with human IgG antibody to form a biological nano probe PQDs @ PLGA @ IgG, and is identified and verified with Eu-labeled anti-human IgG.
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the techniques realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1
The preparation steps of the water-soluble perovskite nanocrystal are as follows:
(1) Under the environment of full room temperature and full air,42.5mg CsBr and 73.4mg PbBr 2 Dissolving 25mg of distearoyl phosphatidylcholine polyethylene glycol (DSPC-PEG-COOH, mw = 3400) with a mercapto group modified at the end group in 5mL of N, N-dimethyl Diamide (DMF) solvent, and adding 0.5mL of oleic acid and 0.25mL of oleylamine after the distearoyl phosphatidylcholine polyethylene glycol and the DSPC-PEG-COOH are completely dissolved to form a stable precursor solution;
(2) Taking 500 mu L of the solution, and slowly dripping the solution into 10mL of toluene solution which is vigorously stirred to obtain a perovskite quantum dot solution; stirring and reacting for 4h to completely coat the perovskite nano crystal to obtain the water-soluble perovskite nano crystal.
(3) Separating and purifying the water-soluble perovskite nanocrystal: centrifuging the toluene solution in (2) at 10000r for 20min, air drying the obtained precipitate in a fume hood to obtain light yellow powder, and making the powder green under irradiation of ultraviolet lamp.
(4) And (3) dispersing the powder in water by ultrasonic for 5min, and storing the water-soluble perovskite nanocrystal solution at room temperature.
Fig. 1 is a transmission electron micrograph of the water-soluble perovskite nanocrystal of example 1. As can be seen from the figure, the particle size of the water-soluble perovskite nanocrystal is about 200nm.
FIG. 2 is a PL spectrum of perovskite quantum dots (PQDs-Toluene) dissolved in Toluene and water-soluble perovskite nanocrystals (PNCs-water); as can be seen from the PL spectrum of fig. 2, the water-soluble perovskite nanocrystals can stably exist in the aqueous phase, almost retain the optical properties of the perovskite quantum dots, and have uniform coating particle sizes.
Example 2
A preparation method of a perovskite nanocrystalline probe for rapidly detecting brain glioma tissue imaging comprises the following specific steps:
(1) Dissolving D-PNCs (such as D-PNCs with wavelength of 520 nm) in ultrapure water, and subjecting to ultrasonic treatment for 5min to uniformly disperse the D-PNCs in the solvent.
(2) Adding antibody (such as chlorotoxin CTX), and reacting at 37 deg.C for 10min;
(3) After the reaction is finished, concentrating the reaction product to 30-100 ul by using an ultrafiltration centrifugal tube with the molecular cut-off of 30-100 KDa, purifying the concentrated solution by adopting a gel size exclusion method, collecting the part with fluorescence, concentrating the concentrated solution by using the ultrafiltration centrifugal tube, storing the concentrated solution in a diluent (containing 0.05 percent of triton X-100), and storing the concentrated solution at 4 ℃ for later use to obtain the probe solution.
Example 3
The invention relates to the concrete steps of simple and rapid brain glioma diagnosis and the evaluation standard of brain glioma canceration
The application range is as follows: the target detection substance can be brain glioma cells, frozen sections in brain glioma sectioning, paraffin sections and cut living tissues in brain glioma sectioning.
The using method comprises the following steps: 200-300 ul of detection liquid is dripped on the slice to be detected, the reaction is carried out for 10min at room temperature, washing liquid (such as ultrapure water) is washed for 5-6 times after the reaction, and the imaging observation is carried out by a hand-held ultraviolet lamp.
Evaluation criteria for brain glioma canceration: irradiating a target object to be detected by using a common handheld ultraviolet lamp with the wavelength of 320-450 nm, dropping a detection liquid into the target object to be detected when the target object to be detected contains related antigens of a glioma area according to an antibody antigen specificity recognition principle, reacting for 10min, washing the detection liquid for 5-6 times by using a washing liquid, and exciting a tumor area by using the handheld ultraviolet lamp to generate fluorescence, wherein the detection result is the glioma tumor area; on the contrary, the target object to be detected is a normal tissue, namely the target object does not contain the related antigen of the glioma region, the tumor region does not have fluorescence under the excitation and irradiation of the handheld ultraviolet lamp, and the detection result is a normal tissue region, namely a non-tumor region; the higher the fluorescence intensity is, the higher the canceration degree of the brain glioma contained in the object to be detected is, and conversely, the lower the fluorescence intensity is, the lower the canceration degree is.
FIG. 3 is a Confocal image of the perovskite-based nanoprobe on brain glioma cells, the Confocal laser excitation wavelength being 405nm and 488nm. 172 brain glioma cells are selected as an experimental group, and green fluorescence can be shown; the HA cells are normal brain glial cells and have no fluorescence; the targeting functionality of the perovskite nanocrystal probe is successfully verified.
FIG. 4 is a Confocal image of the perovskite-based nanoprobe on brain glioma tissue sections, the Confocal laser excitation wavelength being 405nm and 488nm.
The upper panel of fig. 4 is a negative control group, and the components of the detection solution are perovskite quantum dots only, and the results in the figure show no fluorescence, indicating no specific recognition.
The lower graph of fig. 4 is an experimental group, the components of the detection solution are water-soluble perovskite nano-crystals or water-soluble perovskite quantum dot probes, and from the results in the graph, strong green fluorescence can be seen, which indicates that the specific recognition of the antibody and the antigen occurs due to the existence of the specific recognition antibody, and the specific recognition function of the detection solution is verified.
Fig. 5 is a real image of the perovskite-based nanoprobe on brain glioma biopsy. A hand-held ultraviolet lamp is used for imaging at 10W and 365nm. The figure shows the staining condition of the living tissue of a glioma mouse, and the living tissue is irradiated by a handheld ultraviolet lamp to show green fluorescence so as to verify the targeting functionality of the nano probe on the living tissue.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
The carboxyl-terminated polylactic-co-glycolic acid (OH-PLGA-COOH) used in the following examples was purchased from dendri handle bio-engineering ltd with a number average molecular weight of 110000,dlla as a percentage of 50.
Preparation example 11
The preparation steps of the PQDs perovskite quantum dot are as follows:
(1) 42.5mg CsBr and 73.4mg PbBr 2 Dissolving in 5mL of N, N-dimethyl Diamide (DMF) solvent, and adding 0.5mL of oleic acid and 0.25mL of oleylamine for stabilizing the solution after complete dissolution;
(2) Taking 500 mu L of the solution, placing the solution in a glove box, and adding N 2 Slowly dropwise adding the solution into 10mL of toluene solution which is vigorously stirred under the protection of atmosphere to obtain the PQDs quantum dot solution.
(3) Centrifuging the prepared PQDs perovskite quantum dot at 10000r/min for 20min, and separating to obtain a supernatant, namely obtaining a purified PQDs perovskite quantum dot solution which is bright green.
FIG. 6 is a transmission electron microscope image of the prepared PQDs perovskite quantum, from which the particle size of quantum dots can be seen to be 12nm, which is in accordance with CsPbBr 3 Quantum dot size.
Example 11
P-PQDs water-soluble perovskite nanocrystal
In order to achieve the hydrophobic effect, on the basis of preparation example 11, a hydrophobic carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is coated on the surface of a high-fluorescence perovskite quantum dot to form a water-soluble perovskite nanocrystal, which comprises the following steps:
(1) 42.5mg CsBr and 73.4mg PbBr 2 Dissolving 90mg of terminal carboxyl polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) in 5mL of N, N-dimethyl Diamide (DMF) solvent, and after completely dissolving, adding 0.5mL of oleic acid and 0.25mL of oleylamine for stabilizing the solution;
(2) Taking 750 mu L of the solution, and dropwise and slowly adding the solution into 15mL of toluene solution which is stirred violently to obtain P-PQDs perovskite nanocrystalline solution; stirring and reacting for 8h to ensure that the coating is complete, and obtaining the water-soluble P-PQDs perovskite nanocrystal.
(3) Separating and purifying P-PQDs perovskite nanocrystalline, centrifuging at 10000r/min for 20min, drying the obtained precipitate in an oven at 60 ℃ for 1h to obtain dark yellow powder, and making the powder green under the irradiation of a 365nm ultraviolet lamp.
(4) Dispersing the powder in water, and storing the water-soluble P-PQDs perovskite nano crystal at room temperature.
FIG. 7 is a transmission electron micrograph of the water-soluble P-PQDs perovskite nanocrystal prepared in example 11. As can be seen from FIG. 21, the average particle size of the P-PQDs perovskite nanocrystal is 50nm, which is larger than that of preparation example 11, and thus the terminal carboxyl polylactic acid-glycolic acid copolymer coats a plurality of PQDs quantum dots into an integral shape, and the coating effect is good.
FIG. 8 is a graph showing the fluorescence images of P-PQDs perovskite nanocrystals of example 11 under 420-450 nm excitation.
Example 21
Water-soluble perovskite nanocrystalline biological nanoprobe
In order to further couple the hydrophobic perovskite nanocrystal with the biomaterial to construct a novel nano biological probe, on the basis of the embodiment 1, the high positive charge performance of the surface of the perovskite quantum dot can be utilized, and the hydrophobic perovskite nanocrystal is connected with the biomaterial with negative charges through the positive and negative charge electrostatic interaction to construct a water-soluble perovskite nanocrystal probe, which comprises the following specific implementation steps:
(1) The chlorotoxin polypeptide was dissolved in ultrapure water (PH = 6.42), and the solution concentration was set to 10mg/mL.
(2) Mixing 1mL of P-PQDs solution with the concentration of 0.1mg/mL and 10ul of chlorotoxin polypeptide with the concentration of 10mg/mL, vortexing for 30s to mix the solution uniformly, and storing the prepared Probe-PQDs Probe solution at 4 ℃.
FIG. 9 shows fluorescence emission spectra of P-PQDs and nanoprobe-PQDs in example 21. As can be seen from FIG. 9, the P-PQDs linked biomaterials undergo a slight blue shift because the size of the P-PQDs is changed by the linked biomaterials, resulting in a slight change in their corresponding spectra; meanwhile, the Probe-PQDs of the nanoprobe almost completely keep the optical performance of the original P-PQDs.
Example 31
Nano probe for identifying 251 cells of brain glioma
And after the construction of the biological nano probe is completed, carrying out identification function verification on the probe. On the basis of example 2, the probe recognition function was verified by a conventional experimental method in a biological experiment. In this example, the nanoprobes P-PQDs prepared in example 21 were used to label chlorotoxin polypeptides, and target analytes were brain glioma U251 cells and HA cells.
(1) U251 cells and HA cells were plated at 1X 10 per well 5 Density of individual cells seeded in a confocal dish at 37 ℃ CO 2 Culturing in an incubator.
(2) The confocal dish was removed and the cells were washed 3 times with PBS (0.01M, pH = 7.3).
(3) After fixation for 15min with 2mL of 4% paraformaldehyde, the cells were washed again 3 times with PBS (0.01M, pH = 7.3).
(4) 2mL goat serum working solution was added for blocking for 30min, and the cells were washed again 3 times with PBS (0.01M, pH = 7.3).
(5) The prepared 1mL of 0.1mg/mL Probe-PQDs Probe was added thereto, and the mixture was subjected to room temperature discrimination for 1 hour.
(6) The probe solution was recovered, and the cells were washed again with PBS (0.01M, pH = 7.3) 4 to 6 times.
(7) Nuclei were counterstained with DAPI (1 mg/ml), incubated for 15min in the dark, and the cells were washed 4 to 6 times with PBS (0.01M, pH = 7.3).
(8) Confocal microscopy was observed. Two-channel excitation at 405nm and 488nm.
FIG. 11 is a Confocal fluorescent image of the Probe-PQDs Probe of example 31, and the identification of U251 cells and HA cells, with two-channel excitation at 405nm and 488nm. The 405nm channel corresponds to the DAPI channel; the 488nm channel corresponds to Probe-PQDs. From the figure, it can be seen that Probe-PQDs specifically recognize brain glioma U251 cells, but hardly recognize HA cells which are hardly expressed, which accords with the theoretical basis.
The biomaterial in the above embodiment may also be replaced with an aptamer or an antibody to obtain a nanoprobe in which the perovskite nanocrystal is coupled with the aptamer or the antibody, and the obtained nanoprobe also has a specific recognition function.
Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 12
CsPbBr 3 The synthesis method of the-QDs perovskite quantum dot comprises the following steps:
(1) 0.2mmol CsBr and 0.2mmol PbBr 2 Dissolving in 5mL of N, N-dimethyl Diamide (DMF) solvent, and adding 0.5mL of oleic acid and 0.25mL of oleylamine for stabilizing the solution after complete dissolution;
(2) 0.5mL of the solution is taken and slowly dripped into 10mL of toluene solution which is vigorously stirred, and CsPbBr is obtained 3 QDs quantum dot solutions.
(3) The prepared CsPbBr 3 Centrifuging the-QDs perovskite quantum dots at 10000r for 10min, separating to obtain supernatant, namely obtaining purified CsPbBr 3 -QDs perovskite quantum dot solution.
FIG. 12 shows CsPbBr prepared in example 12 3 Fluorescence emission and absorption spectra of QDs perovskite quantum dots. As can be seen from the figure, csPbBr prepared in this example 3 The maximum emission peak of the-QDs perovskite quantum dot is 514nm, and CsPbBr 3 The half-peak width of the-QDs perovskite quantum dot is 18nm. This indicates that CsPbBr prepared in this example 3 The QDs perovskite quantum dots have narrow fluorescence emission half-peak width, and the absorption edge of the quantum dots corresponds to the maximum emission peak 514nm.
FIG. 13 shows CsPbBr prepared in example 12 3 Transmission electron microscopy (left) and lattice fringe pattern (right) of QDs perovskite quantum dots. As can be seen from the transmission electron micrograph (left image) in FIG. 13, csPbBr prepared in this example 3 The particle size of the-QDs perovskite quantum dots is about 12nm. As can be seen from the lattice fringe pattern (right) in FIG. 13, csPbBr produced in this example 3 The stripe spacing of-QDs perovskite quantum dots is 0.295nm. This indicates that CsPbBr prepared in this example 3 the-QDs perovskite quantum dots have good quality and uniform particle size.
Example 22
To further achieve the hydrophobic effect, on the basis of example 12, a hydrophobic polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) containing terminal carboxyl groups is coated on the surface of the high-fluorescence perovskite quantum dot to form a hydrophobic perovskite nanocrystal, which comprises the following steps:
(1) 0.2mmol CsBr and 0.2mmol PbBr 2 And 90mg of polylactic acid-glycolic acid copolymer containing terminal carboxyl (OH-PLGA-COOH) was dissolved in 5mL of N, N-Dimethyldiamide (DMF) solvent, and after complete dissolution, 0.5mL of oleic acid and 0.25mL of oleylamine were added for stabilization of the solution;
(2) 0.5mL of the mixed solution prepared in the step (1) is taken and added into 10mL of toluene solution which is vigorously stirred, and CsPbBr is obtained 3 -qds @ plga quantum dot solution;
(3) Adding 1mL of the solution obtained in the step (2) into 15mL of toluene solution, and stirring for reaction for 48 hours to completely coat the solution to obtain CsPbBr 3 -qds @ plga perovskite nanocrystal mixed solution;
(4) CsPbBr prepared in the step (3) 3 Centrifuging the mixed solution of-QDs @ PLGA perovskite nanocrystals at 10000r for 10min, and drying the obtained precipitate in an oven at 60 ℃ for 1h;
(5) To the dried product in step (4)Adding 2-4 mL of water, and carrying out ultrasonic treatment for 2min to obtain the uniformly dispersed CsPbBr 3 -qds @ plga perovskite nanocrystalline dispersion. FIG. 14 shows CsPbBr prepared in example 22 3 Transmission electron micrograph of-qds @ plga perovskite nanocrystals. As can be seen from the figure, csPbBr prepared by the present embodiment 3 The grain size of-QDs @ PLGA perovskite nanocrystal is about 80nm, which is larger than that of CsPbBr prepared in example 12 3 The particle size of the-QDs perovskite quantum dot is increased, thereby indicating that the terminal carboxyl polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is successfully coated on CsPbBr 3 -QDs perovskite quantum dots. Therefore, the method is expected to be applied to various fields such as biological detection.
FIG. 15 shows CsPbBr prepared in example 22 3 -QDs @ PLGA perovskite nanocrystals scanning electron microscopy. As can be seen from the figure, csPbBr prepared by the present embodiment 3 The grain size of-QDs @ PLGA perovskite nanocrystal is about 80nm, and CsPbBr 3 the-QDs @ PLGA perovskite nano crystal particles are uniformly dispersed and consistent with the characterization result of a transmission electron microscope.
FIG. 16 shows CsPbBr prepared in example 12 3 -QDs perovskite nanocrystals (bottom) and CsPbBr prepared in example 22 3 XRD pattern of (on) QDs @ PLGA perovskite nanocrystals. As can be seen from the results in the figure, csPbBr prepared in example 12 of the present invention 3 the-QDs perovskite quantum dot has a strong and sharp diffraction peak, is a pure phase structure and has good quality. And CsPbBr formed after being coated by carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) 3 The diffraction peak intensity of-QDs @ PLGA perovskite nanocrystal is slightly reduced. Thus, the success of the polymer coating of the terminal carboxyl polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is further shown.
FIG. 17 shows CsPbBr prepared in example 12 3 -QDs perovskite nanocrystals and CsPbBr prepared in example 22 3 -qds @ plga perovskite nanocrystals quantum yield histogram. From the figure it follows that: csPbBr prepared by the invention 3 The quantum yield of-QDs perovskite quantum dots is as high as 83.10%, which indicates that the-QDs perovskite quantum dots have high fluorescence intensity. Hydrophobic CsPbBr formed after carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) coating 3 Quantum yield of-QDs @ PLGA perovskite nanocrystalsStill as high as 70.07%, thereby indicating that the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is coated on CsPbBr 3 After the-QDs perovskite nanocrystalline is arranged, the perovskite quantum dots can stably exist in the water phase. Almost completely reserves the optical property of the perovskite quantum dot (basically has no influence on the fluorescence intensity), and the hydrophobic CsPbBr is formed after the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is coated 3 the-QDs @ PLGA perovskite nano crystal still has higher fluorescence intensity.
Example 32
The hydrophobic perovskite nanocrystal is used for aqueous phase fluorescence detection application:
the hydrophobic CsPbBr3-QDs @ PLGA perovskite nanocrystal formed after being coated by the carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) has more-COOH functional groups exposed on the surface, and the electric property of the nanocrystal in solution is negative. The essence of the antibody is a protein which is an ampholyte, and when the pH value of an external solution is greater than the isoelectric point pI value of zwitterions, the zwitterions release protons with negative charges; when the pH value of the external solution is less than the pI value of the zwitterion, the zwitterion is protonated and positively charged. Therefore, the electric property of the antibody can be regulated by adjusting the pH value of the buffer solution. By electrostatic charge interaction, we can convert hydrophobic CsPbBr 3 Coupling the-QDs @ PLGA perovskite nano crystal with an antibody (such as IgG, igM, igA and the like) to form a novel perovskite nano crystal biological probe, and specifically comprising the following steps:
(1) Several buffer solutions common in biology were prepared: MES buffer (pH = 5.15), PBS buffer (pH = 7.3), tris buffer (pH = 8.17).
(2) 200mg of CsPbBr prepared in example 22 was added 3 -qds @ plga perovskite nanocrystals were added to 2mL MES buffer (pH = 5.15), PBS buffer (pH = 7.3), tris buffer (pH = 8.17) respectively, magnetically stirred at room temperature 300r,30min;
(3) Adding 10 μ L human IgG antibody (1 mg/mL) and 4 μ L triton X-100 into the buffer solution in step (2), and reacting in a shaker at 37 deg.C for 30min to obtain CsPbBr 3 -QDs @ PLGA perovskite nanocrystal labeled human IgG probe CsPbBr 3 -QDs@PLGA@IgG；
(4) The probe CsPbBr prepared in the step (3) 3 -QDs @ PLGA @ IgG stored refrigerated at 4 ℃.
FIG. 18 shows CsPbBr prepared in example 32 3 Zeta potential maps of QDs @ PLGA, human IgG, and probe CsPbBr3-QDs @ PLGA @ IgG in MES buffer solution (pH = 5.15), PBS buffer solution (pH = 7.3), tris buffer solution (pH = 8.17), and the like, respectively. As can be seen from the figure, csPbBr 3 -qds @ plga has negative electrical properties in acidic, neutral, alkaline solutions of pH =5.15, pH =7.3, pH =8.17, etc.; the electric property of the human IgG antibody is positive under acidic conditions of pH =5.15, and is negative in neutral and alkaline solutions of pH =7.3, pH =8.17, and the like; therefore, the CsPbBr3-QDs @ PLGA perovskite nanocrystal and human IgG are placed in MES (pH = 5.15) buffer solution to realize charge electrostatic interaction so as to construct CsPbBr 3 -qds @ plga novel perovskite nanocrystal probes.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
The carboxyl-terminated polylactic-co-glycolic acid (OH-PLGA-COOH) used in the following examples was purchased from dendri handle bio-engineering ltd with a number average molecular weight of 30000 and a percentage of dlla of 75.
Preparation example 13
The preparation steps of the PQDs perovskite quantum dot are as follows:
(1) 42.5mg CsBr and 73.4mg PbBr 2 Dissolving in 5mL of N, N-dimethyl Diamide (DMF) solvent, and adding 0.5mL of oleic acid and 0.25mL of oleylamine for stabilizing the solution after complete dissolution;
(2) Taking 500 mu L of the solution, placing the solution in a glove box, and adding N 2 Slowly dropwise adding the solution into 10mL of toluene solution which is stirred violently under the protection of atmosphere to obtain the PQDs quantum dot solution.
(3) Centrifuging the prepared PQDs perovskite quantum dot at 10000r/min for 20min, and separating to obtain a supernatant, namely obtaining a purified PQDs perovskite quantum dot solution which is bright green.
FIG. 19 is a photograph of a photograph obtained byThe transmission electron microscope picture of PQDs perovskite quantum and the lattice stripe thereof, the left picture represents the transmission electron microscope picture, the particle size of the quantum dot is 12nm, which accords with CsPbBr 3 The size of the quantum dots; the right panel represents the stripe spacing of 0.295nm. The quantum dots synthesized by the method have good quality and uniform particle size.
FIG. 20 shows fluorescence emission spectrum and absorption spectrum of the prepared PQDs perovskite quantum. As can be seen from FIG. 20, the PQDs perovskite quantum dot can generate strong fluorescence under 365nm excitation, the emission peak position is 515nm, the half-peak width is 18nm, which indicates that the quantum dot has narrow fluorescence emission and the absorption edge corresponds to the emission peak of 515nm.
Preparation example 23 preparation of Sulfydryl-PQDs perovskite Quantum dots
(1) 42.5mg CsBr and 73.4mg PbBr 2 Dissolving in 5mL of N, N-Dimethyldiamide (DMF) solvent, and after complete dissolution, adding 0.5mL of oleic acid, 0.25mL of oleylamine, and 0.2mL of mercaptoundecanoic acid (Sulfydryl) for stabilizing the solution;
(2) Taking 500 mu L of the solution, placing the solution in a glove box, and adding N 2 And slowly dripping the solution into 10mL of toluene solution which is stirred violently under the protection of atmosphere to obtain the PQDs quantum dot solution.
(3) Centrifuging the prepared Sulfydryl-PQDs perovskite quantum dots at 10000r/min for 20min, and separating to obtain supernatant, namely obtaining purified PQDs perovskite quantum dot solution, wherein the solution is bright cyan; under the irradiation of an ultraviolet lamp (365 nm), the solution is blue.
FIG. 21 shows fluorescence emission spectrum and absorption spectrum of Sulfydryl-PQDs perovskite quantum prepared. As can be seen from FIG. 21, sulfydryl-PQDs perovskite quantum dots can generate strong fluorescence under 365nm excitation, and the emission peak position is 461nm. Therefore, by adjusting the perovskite quantum dot composition, fluorophores with different emission wavelength ranges can be obtained.
Example 13PQDs @ PLGA hydrophobic perovskite nanocrystal
In order to achieve the hydrophobic effect, on the basis of preparation example 13, a hydrophobic carboxyl-terminated polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) is coated on the surface of a high-fluorescence perovskite quantum dot to form a hydrophobic perovskite nanocrystal, and the steps are as follows:
(1) 42.5mg CsBr and 73.4mg PbBr 2 Dissolving 90mg of terminal carboxyl polylactic acid-glycolic acid copolymer (OH-PLGA-COOH) in 5mL of N, N-dimethyl Diamide (DMF) solvent, and adding 0.5mL of oleic acid and 0.25mL of oleylamine for stabilizing the solution after completely dissolving;
(2) Taking 500 mu L of the solution, and slowly dripping the solution into 10mL of toluene solution which is vigorously stirred to obtain PQDs @ PLGA quantum dot solution; and adding 1mL of the obtained solution into 15mL of toluene solution, and stirring for reaction for 48 hours to completely coat the solution to obtain the hydrophobic PQDs @ PLGA perovskite nanocrystal.
(3) Separating and purifying PQDs @ PLGA perovskite nanocrystal, centrifuging the nanocrystal at 10000r/min for 20min, drying the obtained precipitate in an oven at 60 ℃ for 1h to obtain light yellow powder, and enabling the powder to be green under the irradiation of an ultraviolet lamp.
(4) Dispersing the powder in water, and storing the hydrophobic PQDs @ PLGA perovskite nano crystal at room temperature.
FIG. 22 is a transmission electron micrograph of the hydrophobic PQDs @ PLGA perovskite nanocrystal prepared in example 13. As can be seen from FIG. 22, the average particle size of the PQDs @ PLGA perovskite nanocrystal is 80nm, which is larger than that of preparation example 13, and shows that the terminal carboxyl polylactic acid-glycolic acid copolymer coats a plurality of PQDs quantum dots into an integral appearance, and the coating effect is good.
FIG. 23 is an optical microscopic photograph of PQDs @ PLGA perovskite nanocrystals of example 13. Wherein, the left graph represents the fluorescence imaging graph of the nanocrystal under the excitation of 420-450 nm, and the right graph represents the bright field graph of the nanocrystal.
FIG. 24 shows fluorescence emission spectrum and absorption spectrum of PQDs @ PLGA perovskite nanocrystal placed in water for at least 90 days. As can be seen from fig. 24, the PL spectrum of the PQDs @/plga perovskite nanocrystal left for at least 90 days is almost consistent with the emission peak position and half-peak width of the pure perovskite quantum dot PQDs, and although the absorption spectrum intensity is reduced due to the coating of a layer of polymer on the outer layer, the PQDs @/plga perovskite nanocrystal can stably exist in the aqueous phase, almost retains the optical properties of the PQDs quantum dot, and the coated particle size is uniform.
Example 23 preparation of hydrophobic perovskite nanocrystalline biological nanoprobes by coupling method
In order to further couple the hydrophobic perovskite nanocrystal with the biomaterial to construct a novel nano-biological probe, on the basis of the embodiment 1, the surface group of the polylactic acid of the packaging coating material is utilized, EDC/NHS is used as a cross-linking agent, and the novel nano-biological probe is coupled with the biomaterial to construct a novel fluorescence detection probe. The specific implementation steps are as follows:
(1) 6mg/mL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 30mg/mL of N-hydroxysuccinimide (NHS) were prepared, and a volume ratio of 1.
(2) Adding 10mg PQDs @ PLGA perovskite nano crystal into 2mL mixed solution of EDC and NHS, and magnetically stirring at room temperature for 300r/min and 30min to fully activate-COOH on the surface of the PQDs @ PLGA perovskite nano crystal.
(3) Adding 10 μ L human IgG antibody (1 mg/mL) and 4 μ L triton X-100 into the solution (2), and reacting with mild shaking bed for 30min to obtain PQDs @ PLGA labeled human IgG probe PQDs @ PLGA @ IgG.
FIG. 25 is the SEM and EDS spectra of the biological nano-probe PQDs @ PLGA @ IgG formed by coupling the PQDs @ PLGA perovskite nanocrystal in example 23 with human IgG antibody. As can be seen from the figure, the average particle size of the biological nanoprobe PQDs @ PLGA @ IgG is 200nm; the perovskite quantum dots encapsulated by the polymer are successfully coupled with antibody protein, and the presence of main elements Pb, cs and Br forming the perovskite quantum dots is further proved by an EDS energy spectrum.
Example 33 Nanoprobe recognition of antigens
And after the construction of the biological nano probe is completed, carrying out identification function verification on the probe. On the basis of example 23, the probe recognition function was verified by a conventional experimental method in a biological experiment. In this example, the nanoprobe PQDs @ PLGA labeled human IgG prepared in example 23 was used as an antibody, and the target analyte was Eu-labeled anti-human IgG as an antigen. FIG. 27 is a schematic diagram of the experiment for identifying and verifying the biological nanoprobes PQDs @ PLGA @ IgG and Eu-labeled anti-human IgG in this example.
(1) Coating the bottom of the plate by using a 96-well plate, coating the plate with Eu-labeled anti-human IgG (10 mu g/mL) by using a dosage of 50 mu L/well to set the plate as an experimental group, and incubating overnight at 4 ℃; at this time, a control experiment group was set, and bovine serum albumin BSA was coated on the plate bottom with other substances to incubate the control group and a blank group of a full blank control.
The coated 96-well plate was removed, and the incubation well plates of the experimental group, the control group, and the blank group were washed 3 to 5 times with PBST (PBS buffer, pH =7.3, containing Tween-20 at a concentration of five ten thousandths of volume), and excess uncoated Eu-labeled anti-human IgG or BSA was washed away.
(2) Sealing the coated pore plate, preparing 5% of defatted milk powder by mass fraction, sealing and incubating the pore plate with the dosage of 200 mu L/well, and occupying the site at the bottom of the plate as the coated antibody at 37 ℃ for 1h to reduce non-specific adsorption; the closed well plate was washed 3-5 times with PBST (PBS buffer, pH =7.3, containing five parts per million by volume Tween-20) and the excess skim milk powder solution was washed away.
(3) Adding 100 μ L of the biological nanoprobe PQDs @ PLGA @ IgG prepared in example 33 to a 96-well plate coated with Eu-labeled anti-human IgG and bovine serum albumin BSA after blocking, and keeping the temperature at 37 ℃ for 1h; the recognition well plate was washed 3 to 5 times with PBST (PBS buffer, pH =7.3, containing five parts per million volume concentration of Tween-20) and excess unrecognized PQDs @ PLGA @ IgG was washed away.
FIG. 26 is a Confocal fluorescence imaging graph of the coupling of PQDs @ PLGA perovskite nanocrystals in example 33 with human IgG antibodies to form biological nanoprobes PQDs @ PLGA @ IgG, and the recognition of Eu-labeled anti-human IgG, with double channel excitation at 488nm and 561 nm.
The first line is an experimental group, the porous plate is coated with Eu-labeled anti-human IgG, the number of a corresponding picture is 1-3, PQDs @ PLGA-labeled human IgG is used for identification, red fluorescence (namely, the picture with the number 3) is presented in an excitation channel at 561nm, and green fluorescence (namely, the picture with the number 2) is presented in an excitation channel at 488 nm; merge stands for 488nm and 561nm two-channel excitation, and the superimposed yellow fluorescence imaging of red and green fluorescence is presented (i.e. picture No. 1). A second behavior bovine serum albumin control group, wherein bovine serum albumin is adopted to coat the pore plate, PQDs @ PLGA is added to mark human IgG for identification, and the number of a corresponding picture is 4-6; the third row is Blank group (Blank), the pore plate without antigen coating is added with PQDs @ PLGA labeled human IgG for recognition, and the corresponding pictures are numbered as 7-9.
As can be seen from the fluorescence imaging graph in FIG. 26, the experiment group showed a red fluorescence image under 561nm excitation light due to Eu-labeled anti-human IgG coated on the bottom of the plate, and the identified human IgG showed green fluorescence imaging under 488nm excitation condition with PQDs @ PLGA label, which can prove the success of antibody-antigen recognition; meanwhile, the control group uses antigens which can not be mutually identified to replace and blank groups, so that a red-free fluorescence image and a green fluorescence image are respectively presented, and the biological nano probe is further proved to be not only successfully coupled, but also retain the original identification function.
The biomaterial in the above embodiment may also be replaced with an aptamer or a polypeptide to obtain a nanoprobe in which the perovskite nanocrystal is coupled with the aptamer or the polypeptide, and the obtained nanoprobe also has a specific recognition function.
Claims (10)
1. The application of the perovskite nanocrystal in preparing a probe for tumor diagnosis or treatment is characterized in that the perovskite nanocrystal comprises perovskite quantum dots and liposome materials coated on the surfaces of the perovskite quantum dots.
2. The use of claim 1, wherein the liposome coating material is DPPC, DPPE, DSPC and derivatives thereof.
3. Use according to claim 2, wherein the liposome coating material is DSPC-PEG, DPPC-PEG, DPPE-PEG.
4. Use according to claim 3, wherein the liposome coating material is DSPC-PEG-COOH.
5. The use of claim 1, 2, 3 or 4, wherein the perovskite quantum dots are CsPbBr 3 Perovskite quantum dots.
6. The use of claim 1, 2, 3, 4 or 5, wherein the probe is a perovskite nanocrystal labeled biological material; the biological material is one, two or more of an antibody, an aptamer and a polypeptide; and/or
The tumor is brain glioma.
7. The use according to claim 6, wherein the biological material is an antibody, aptamer or polypeptide that specifically recognizes brain glioma tissue.
8. The use according to claim 7, wherein said biological material is at least one of chlorotoxin, IDH isocitrate dehydrogenase I, glial fibrillary acidic protein antibody, anti-ATRX, rabbit Anti-human H3K27Me3 polyclonal antibody.
9. The use according to any one of claims 1 to 8, wherein the water-soluble perovskite nanocrystals have a spectral range of 470 to 650nm; and/or
The particle size of the water-soluble perovskite nanocrystal is 100-200nm.
10. The use according to any one of claims 1 to 9, wherein the perovskite nanocrystals are obtained by heating and stirring a liposome material and perovskite quantum dots to react.
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