WO2021227336A1 - 一种包封石墨烯量子点的纳米脂质体及制备以及其在生物酶活性检测中的应用 - Google Patents

一种包封石墨烯量子点的纳米脂质体及制备以及其在生物酶活性检测中的应用 Download PDF

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WO2021227336A1
WO2021227336A1 PCT/CN2020/117568 CN2020117568W WO2021227336A1 WO 2021227336 A1 WO2021227336 A1 WO 2021227336A1 CN 2020117568 W CN2020117568 W CN 2020117568W WO 2021227336 A1 WO2021227336 A1 WO 2021227336A1
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graphene quantum
quantum dots
solution
liposome
phospholipase
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French (fr)
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李楠
査勇超
张美莹
薛巍
崔鑫
王宇
周平
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暨南大学
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"

Definitions

  • the invention belongs to the field of organic/inorganic composite optical probe materials and disease-related marker detection, and in particular relates to a nano liposome encapsulating graphene quantum dots, preparation and application thereof in biological enzyme activity detection.
  • Phospholipase (phospholipase) is widely distributed in the human body and plays an important role in physiological processes such as phospholipid renewal, cell membrane remodeling, exocytosis and information transmission.
  • Phospholipases mainly include phospholipases A1, A2, B1, B2, C and D, which specifically act on the ester bonds within the phospholipid molecule to form different products.
  • phospholipase A2 (Phospholipase A 2 , PLA 2 ) can hydrolyze phospholipid sn-2 ester bonds to produce free fatty acids and lysophospholipids, which play a key role in pathophysiological processes such as information transmission and membrane channel activation during inflammation and tissue damage. .
  • Acute pancreatitis is an inflammatory reaction of pancreatic tissue self-digestion, edema and even necrosis caused by the activation of pancreatin.
  • phospholipase A2 will be prematurely activated and excessively released when acute pancreatitis occurs, which can directly participate in and affect the pathogenesis of acute pancreatitis. Therefore, detecting the biological activity of phospholipase A2 in serum has important clinical significance for the diagnosis and treatment of acute pancreatitis.
  • the developed methods for determining phospholipase A2 mainly include: 1) spectrophotometry, 2) electrochemical method, 3) radioimmunoassay, 4) enzyme-linked immunoassay, 5) chromatographic mass spectrometry and other methods.
  • the Chinese patent application "Lipoprotein-related Phospholipase A2 Quantitative Detection Kit and its preparation and operation method with publication number CN 103698535 B combines immunomagnetic bead particles with chemiluminescence enzyme immunoassay technology to achieve sensitive detection and obtain Better performance parameters.
  • Another example is the Chinese patent application "A kit for detecting lipoprotein-related phospholipase A2 and its preparation and application” with the publication number CN 104634970 A.
  • the antibody is coated with a magnetic ball and a tracer marker to label the antibody. Combined, it can accurately and sensitively detect lipoprotein-associated phospholipase A2.
  • some of these methods involve expensive antigens and antibodies, which make the detection cost high, and some are limited by the complicated and time-consuming detection process.
  • Graphene quantum dot a flat carbon material with a thickness of only a few atoms and a width of nanometers, is a new member of the zero-dimensional nanomaterial family. GQD exhibits excellent fluorescence performance due to the quantum effect brought about by its small size. Compared with traditional organic fluorescent probes and semiconductor quantum dots, it has excellent photobleaching resistance, luminescence stability, biocompatibility, and easy surface functionalization. It has become the most promising photoluminescent material one. At present, graphene quantum dots have a wide range of applications in biological imaging, labeling and tracing, and energy storage and conversion.
  • Liposomes are vesicle structures formed by self-assembly of phospholipid molecules and cholesterol molecules dispersed in water, which have a bilayer of cell membrane structure. Such a hollow double-layer vesicle structure has a special ability-both the inner cavity and the bilayer can carry drugs, enzymes, fluorophores or other signaling molecules. Liposome vesicles have been used in drug carriers, tumor imaging, biosensing and other fields. Especially in the field of biosensing detection, probes using liposome vesicles have obvious advantages in signal amplification compared with traditional small molecule probes, and show the characteristics of rapid response and convenient use.
  • the development and application of liposome vesicle probes is gradually becoming a hot field of biosensor research.
  • the Chinese patent "Preparation method of liposome temperature fluorescent probe loaded with gold nanoclusters and anticancer drugs" with the publication number CN 103599070 B uses supercritical carbon dioxide method to mix gold nanocluster suspension with anticancer drugs The solution is encapsulated in the water-phase inner cavity of the liposome, and the prepared liposome has photo-thermosensitivity and can be a new type of temperature fluorescent probe.
  • the added surfactant for the rupture of phospholipid vesicles is not easy to remove from the reaction system, and its introduction not only brings impurities to the system and complicates the reaction process, but even has an adverse effect on biological substances.
  • the method of using liposomes for enzyme-linked immunoassay has the advantage of strong affinity, it still has defects such as poor stability of antigen and antibody and high cost. These also limit the further development of liposomes in the field of biosensors.
  • the primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing nano liposomes encapsulating graphene quantum dots.
  • Another object of the present invention is to provide nano liposomes encapsulating graphene quantum dots prepared by the method.
  • Another object of the present invention is to provide the application of the nano liposome encapsulating graphene quantum dots in the detection of biological enzyme activity.
  • a method for preparing nano liposomes encapsulating graphene quantum dots including the following steps:
  • the graphene quantum dot solution is added to the liposome film, and the ice bath is ultrasonically dispersed to obtain a mixed solution I; then the mixed solution I is repeatedly squeezed through a polycarbonate membrane to obtain a mixed solution II; The solution II was dialyzed to obtain nano liposomes encapsulating graphene quantum dots.
  • the molar ratio of lecithin to cholesterol in step (1) is 1 to 5:1; preferably 5:1.
  • the amount of chloroform mentioned in step (1) is calculated based on 1ml chloroform per 1.8mmol of cholesterol (or 1ml chloroform per 10.8mmol of lecithin and cholesterol).
  • the ultrasound conditions described in step (1) are: 100W ultrasound for 5-10 minutes; preferably, 100W ultrasound for 5 minutes.
  • the conditions of the rotary steaming described in step (1) are: 40°C rotary steaming for 15-60 minutes; preferably 40°C rotary steaming for 60 minutes.
  • the total mass ratio of the graphene quantum dots to the lecithin and cholesterol in step (2) is 0.02-0.4:30; preferably 0.2:30.
  • the graphene quantum dot solution described in step (2) is an aqueous solution of graphene quantum dots, or a solution obtained by dissolving graphene quantum dots in a phosphate buffer solution; its concentration is 0.01 to 0.2 mg/mL; preferably 0.1 mg/mL mL.
  • the phosphate buffer solution is a mixed solution of disodium hydrogen phosphate and sodium dihydrogen phosphate, and the pH is adjusted to 7.0.
  • the graphene quantum dots described in step (2) are preferably prepared by the following method:
  • the carbon black described in step S1 is preferably carbot vulcan XC-72 carbon black.
  • the concentration of the concentrated nitric acid solution in step S1 is 5-8 mol/L; preferably 6 mol/L.
  • step S1 The reflux reaction described in step S1 is preferably carried out in an oil bath.
  • the reflux reaction time described in step S1 is preferably 24 hours.
  • the filtering described in step S2 is filtering with a filter paper and a needle filter in sequence.
  • the pore size of the needle filter is 0.22 ⁇ m.
  • step S2 The conditions for centrifugation in step S2 are all: 8000 rpm centrifugation for 10 minutes.
  • the pore size of the ultrafiltration centrifuge tube described in step S2 is 3000 Da.
  • the dialysis described in step S2 is dialysis using a dialysis bag with a molecular weight cut-off of 100 to 500 Da.
  • the conditions of the dialysis described in step S2 are: dialysis with deionized water as the dialysate for 24 hours.
  • the temperature of the extrusion described in step (2) is preferably 40 ⁇ 2°C.
  • step (2) The extrusion described in step (2) is carried out in a liposome extruder.
  • the pore size of the polycarbonate membrane described in step (2) is 200 nm.
  • the number of extrusions described in step (2) is 21 times or more.
  • the dialysis described in step (2) uses a dialysis membrane with a molecular weight cut-off of 8000 Da for dialysis.
  • the dialysis time described in step (2) is 24 hours.
  • the ultrasound conditions described in step (2) are: 100W ultrasound for 40-60 minutes; preferably, 100W ultrasound for 50 minutes.
  • a nano liposome encapsulating graphene quantum dots is prepared by the method described in any one of the above.
  • nano liposomes encapsulating graphene quantum dots in the preparation of fluorescent probes or pancreatitis marker detection sensors.
  • the fluorescent probe is a fluorescent probe for detecting phospholipase A2 (pancreatitis marker phospholipase A2).
  • a method for measuring the content of phospholipase A2 for non-disease diagnosis purposes using the nanoliposomes encapsulating graphene quantum dots including the following steps:
  • step (b) According to the maximum intensity of the fluorescence spectrum obtained in step (a) and the concentration of the phospholipase A2 aqueous solution, draw a standard curve to obtain a linear equation;
  • step (c) Mix the sample to be tested with the nano liposomes encapsulating graphene quantum dots and react in a water bath, and then measure the fluorescence spectrum to obtain the maximum intensity of the fluorescence spectrum of the sample to be tested; and then obtain the result according to step (b)
  • the linear equation obtains the content of the sample to be tested.
  • the amount of the phospholipase A2 aqueous solution in step (a) is added according to its final concentration in the reaction system of 0-20 U/L; preferably according to its final concentration in the reaction system of 0, 2, 5, 10 and 20U/L add.
  • the dosage of the nano liposome encapsulating graphene quantum dots in step (a) is added according to its final concentration in the reaction system of 0.26 ⁇ 0.28 mg/mL; preferably according to its concentration in the reaction system The final concentration of 0.265 ⁇ 0.275mg/mL is added.
  • the maximum intensity of the fluorescence spectrum in steps (a) and (c) is the intensity of the fluorescence spectrum measured at an excitation wavelength of 530 nm.
  • the conditions of the water bath reaction in steps (a) and (c) are: 37°C water bath for 1 hour.
  • the present invention has the following advantages and effects:
  • the present invention uses lecithin and cholesterol as synthetic materials, and uses a thin film dispersion method to prepare liposomes encapsulating graphene quantum dots. Then use the phospholipase A2 enzymatic reaction substrate—phospholipid molecules to self-assemble to form liposomes encapsulating graphene quantum dots. In the presence of phospholipase A2, the liposomes are ruptured and the fluorescent signal molecules encapsulated in them are released. This can effectively avoid the addition of third-party reagents (such as Triton-100) as a liposome disrupting agent. In addition, the use of expensive biological reagents such as antigens and antibodies is avoided, so that the cost of detection is greatly reduced, and it has broad application prospects in biological detection and clinical diagnosis.
  • third-party reagents such as Triton-100
  • the nano liposomes encapsulating graphene quantum dots in the present invention have excellent biocompatibility and long-lasting light stability, high biological safety, long-lasting and stable luminescence, and good biological application potential.
  • the present invention utilizes the unique loading capacity of liposomes to wrap graphene quantum dot fluorescent nanoprobes to provide optical signal amplification strategies for biochemical detection and sensing.
  • the nanoprobe can be used as a disease marker detection sensor.
  • the present invention utilizes the hydrolytic degradation of phospholipase A2 to break down liposomes to release graphene quantum dot fluorescent probes, and the fluorescence intensity change caused by the release degree has a certain quantitative relationship with the activity of phospholipase A2, which is established for use in diseases ( Such as acute pancreatitis) a new fluorescent detection sensor method for the marker phospholipase A2, that is, this method can quantitatively detect phospholipase A2: add a gradient concentration of phospholipase A2 into liposomes to burst and release graphene quantum Spot a fluorescent probe, and then detect its fluorescence intensity at different phospholipase A2 concentrations to create a standard curve that correlates the change in fluorescence intensity with the concentration of phospholipase A2. Finally, add a sample of phospholipase A2 of unknown concentration and detect its fluorescence intensity. The standard curve gives the test result.
  • the present invention adopts the method of directly using the test substance as the liposome rupture, breaking the traditional scheme of using surfactants and other substances to rupture the liposome.
  • the test substance phospholipase A2 is directly used as the rupture of the phospholipid vesicles.
  • the stimulus factors can realize the sensitive detection of pancreatitis marker phospholipase A2, and the method also has the advantages of low cost, convenient and quick detection, and provides for the design of intelligent bionic microvesicles in response to environmental stimuli and the construction of new intelligent bionic systems New ideas.
  • Fig. 1 is a schematic diagram of the principle of the nanoprobe encapsulating graphene quantum dot liposomes in the fluorescence detection of phospholipase A2 of the present invention.
  • Figure 2 is a characterization diagram of graphene quantum dots; among them, A is a scanning electron microscope photo of graphene quantum dots; B is an atomic force microscope photo of graphene quantum dots.
  • Figure 3 is the graphene quantum dot emission spectra under different excitation wavelengths (the inset is the image of the graphene quantum dot solution when irradiated with white light and 365nm ultraviolet light).
  • Figure 4 is a graph of the concentration-induced fluorescence changes of graphene quantum dot aqueous solutions; where A is the emission spectra of graphene quantum dot aqueous solutions at different concentrations at an excitation wavelength of 465 nm; B is the fluorescence intensity of graphene quantum dot aqueous solutions at 530 nm. The standard curve of its concentration change.
  • Figure 5 is a characterization diagram of liposomes; where A is a scanning electron micrograph of liposomes; B is a particle size distribution of liposomes (the inset is an image of liposome solution under white light irradiation).
  • Figure 6 is a graph of the particle size distribution of liposomes encapsulating graphene quantum dots prepared at a molar ratio of lecithin and cholesterol of 1:1; where A is the scanning electron micrograph of the liposome; B is the liposome The particle size distribution chart.
  • Figure 7 is a graph of the particle size distribution of liposomes encapsulating graphene quantum dots prepared at a molar ratio of lecithin and cholesterol of 5:2; where A is a scanning electron micrograph of the liposome; B is a liposome The particle size distribution chart.
  • Figure 8 shows the synthesis of liposomes encapsulating different concentrations of graphene quantum dot solutions (0.01mg/ml, 0.05mg/ml, 0.1mg/ml, 0.2mg/ml) ruptured under the action of phospholipase A2 to release fluorescent probes Graph of the resulting spectral changes.
  • Figure 9 is a graph of the fluorescence detection results of liposomes encapsulating graphene quantum dots for detecting the activity of phospholipase A2; where A is the trend graph of the fluorescence intensity of the released graphene quantum dots as a function of phospholipase A2 activity (its The activity concentration varies from 0 to 250 U/L); B is the standard curve of the fluorescence intensity of the released graphene quantum dots with the change of phospholipase A2 activity from 0 to 20 U/L.
  • Figure 10 is a graph showing the experimental results of the selectivity of liposomes encapsulating graphene quantum dots to phospholipase A2.
  • FIG. 11 is a comparison diagram of the biological safety of the encapsulated graphene quantum dot liposome of the present invention as a novel fluorescent probe for biological detection.
  • Fig. 12 is a comparison diagram of the photostability of the encapsulated graphene quantum dot liposome of the present invention as a novel fluorescent probe for biological detection.
  • the present invention will be further described in detail below in conjunction with examples, but the implementation of the present invention is not limited thereto.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the test methods that do not indicate specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions recommended by the manufacturer.
  • the reagents and raw materials used in the present invention are all commercially available.
  • This embodiment provides a method for synthesizing graphene quantum dots.
  • the solution 2 was filtered again with a 0.22 ⁇ m syringe filter to obtain a solution 3.
  • the solution 3 was centrifuged at 8000 rpm for 10 minutes, and then the supernatant was pipetted into an ultrafiltration centrifuge tube (with a pore size of 3000 Da) to obtain a solution 4.
  • Centrifuge the solution 4 at 8000 rpm (10 minutes) to basically separate all the clear liquid from the precipitate, and finally put the separated clear liquid into a dialysis bag with a molecular weight cut-off of 100 to 500 Da.
  • the solution is added to the centrifuge tube, and the graphene quantum dots are formed after freeze-drying treatment.
  • the scanning electron microscope photo of the graphene quantum dots is shown in Figure 2A, and the atomic force microscope photo is shown in Figure 2B.
  • the emission spectra of graphene quantum dots at different excitation wavelengths fluorescence spectrophotometer, 405, 425, 445, 465, 485, 505, 525nm
  • images of graphene quantum dot solutions under white light and 365nm ultraviolet light irradiation As shown in Figure 3.
  • Fig. 4A shows the emission spectra of graphene quantum dot aqueous solutions at different concentrations at an excitation wavelength of 465 nm
  • Fig. 4B is a standard curve of the fluorescence intensity of graphene quantum dot aqueous solutions at 530 nm as a function of its concentration.
  • This embodiment provides a method for synthesizing liposomes.
  • Lecithin and cholesterol were mixed at a ratio of 5:1 (molar ratio, 43.2mmol, 30mg), dissolved in 4ml of chloroform, and sonicated (power 100W) for 5 minutes to make the dispersion uniform. Subsequently, the organic solvent was removed by rotary evaporator at 40° C. under reduced pressure for 1 hour, and a transparent film was uniformly formed on the bottom of the flask. At this time, add 2mL 0.1mg/ml graphene quantum dot solution (dissolve the graphene quantum dots prepared in Example 1 in a phosphate buffer solution (pH 7.0)), and ultrasound in an ice bath (power 100W) for 50 minutes to obtain a milky white Turbid liquid.
  • 2mL 0.1mg/ml graphene quantum dot solution dissolve the graphene quantum dots prepared in Example 1 in a phosphate buffer solution (pH 7.0)
  • ultrasound in an ice bath power 100W
  • the obtained liposome solution was dialyzed against a dialysis membrane (with a molecular weight cut-off of less than 8000D), using deionized water as the dialysate, dialyzed for 24 hours to remove the unencapsulated graphene quantum dots, and store the obtained liposome solution At 4°C.
  • Fig. 5A The scanning electron microscope results of the liposomes are shown in Fig. 5A, and the particle size distribution is shown in Fig. 5B (the inset is the image of the liposome solution under white light irradiation). It can be seen from the particle size distribution and scanning electron microscopy results that the liposome vesicles prepared in this example have uniform size and good dispersibility.
  • the synthesis method is the same as that of Example 2, except that lecithin and cholesterol are mixed at a ratio of 1:1 (molar ratio, 52.37 mmol, 30 mg in total) or 5:2 (molar ratio, 45.99 mmol, 30 mg in total).
  • the synthesis method is the same as in Example 2, except that the concentration of the graphene quantum dot solution (the graphene quantum dots prepared in Example 1 are dissolved in a phosphate buffer solution (pH 7.0)) are 0.01 mg/ml and 0.05 mg/ml. ml, 0.1mg/mL, 0.2mg/ml.
  • Figure 8 shows the changes in fluorescence intensity caused by the rupture of liposomes encapsulating graphene quantum dot solutions at concentrations of 0.01 mg/ml, 0.05 mg/ml, 0.1 mg/mL, and 0.2 mg/ml under the action of a certain phospholipase A2 concentration.
  • the graphene quantum dot solution is 0.2mg/ml
  • the detected fluorescence intensity is not significantly changed compared to 0.1mg/ml, indicating that it is limited by the size and volume of the liposome cavity.
  • the graphene quantum dot solution of 0.1 mg/ml is preferred to prepare the liposome probe.
  • This embodiment provides a method for sensitive and specific detection of a disease marker phospholipase A2 using a fluorescent probe of nanoliposomes encapsulating graphene quantum dots. The principle is shown in FIG. 1.
  • Figure 9 is the result of fluorescence detection of phospholipase A2 activity using liposomes encapsulating graphene quantum dots; among them, Figure 9A shows the fluorescence intensity of the released graphene quantum dots as a function of phospholipase A2 activity Change trend graph (the activity concentration change range is 0 ⁇ 250U/L); Fig. 9B is the standard curve of the fluorescence intensity of the released graphene quantum dots with the change of phospholipase A2 activity from 0 ⁇ 20U/L. The results show that the fluorescence intensity at 530nm increases with the increase of the concentration of phospholipase A2, and there is a good linear relationship between 0 and 20 U/L.
  • This experiment uses different types of fluorescent probes with different mass concentrations, such as emerging nano fluorescent probes (including graphene quantum dots, water-soluble CdSe semiconductor quantum dots) and traditional organic molecular fluorescent probes (including rhodamine B and 1-naphthalene).
  • Phenol is an example of infecting Hela cells, and their respective biological safety assessments are carried out by detecting the survival rate of the cells.
  • the materials involved are as follows: Hela cell strain (Hela) was purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.; the preparation of graphene quantum dots was the same as in Example 1; CdSe semiconductor quantum dots were purchased from Guangzhou Lige Technology Co., Ltd.
  • Rhodamine B and 1-Naphthol were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity greater than 99%; DMEM high-sugar medium was purchased from Guangzhou Hengyan Biotechnology Co., Ltd.; Trypsin (0.25%, containing EDTA) and Fetal bovine serum was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; MTT detection kit was purchased from Guangzhou Lucheng Biochemical Technology Co., Ltd.
  • Hela cell culture medium 500mL of DMEM high glucose medium, 50mL of fetal bovine serum, to obtain DMEM high glucose medium containing 10% fetal bovine serum, store at 4°C for later use;
  • Hela cells (cell seeding density is 1 ⁇ 10 4 /cm 2 ) are cultured in DMEM high glucose medium containing 10% fetal bovine serum, culture conditions are 5% (v/v) CO 2 , 37° C., saturated humidity .
  • Cell survival rate is a commonly used indicator in cytotoxicity experiments.
  • MTT detection kit was used to determine cell survival rate by colorimetry. Adjust the density of Hela cells to 1 ⁇ 10 5 cells/mL, inoculate them in a 96-well plate, 100 ⁇ L per well, after cell culture for 24 hours, aspirate and discard the original culture medium, add a final mass concentration of 0, 50 to each well each time , 100, 150, 200 ⁇ g/mL graphene quantum dot solution, each mass concentration is set to 3 replicates, with water-soluble CdSe semiconductor quantum dots, rhodamine B and 1-naphthol as comparisons, the method of adding each experiment is the same as above.
  • the graphene quantum dots used in the present invention as a new type of fluorescent probes have significantly superior biocompatibility compared to other organic fluorescent molecules and semiconductor quantum dots. It has good biological application potential.
  • This experiment compares the fluorescence luminescence stability of different types of fluorescent probes including graphene quantum dots, water-soluble CdSe semiconductor quantum dots and 1-naphthol, and evaluates their long-term performance.
  • the materials involved are as follows: the preparation of graphene quantum dots is the same as in Example 1, CdSe semiconductor quantum dots were purchased from Guangzhou Lige Technology Co., Ltd.; 1-naphthol was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity greater than 99%. Specific steps are as follows:
  • the three sample solutions prepared above were respectively irradiated with a xenon lamp (Beijing Zhuoli Hanguang Instrument Co., Ltd., Zolix LSB-500W, power 500W) for 120 minutes.
  • Fluorescence spectrophotometer (Hitachi, F-4500) detects the fluorescence intensity changes of the three sample solutions before and after irradiation (A0 is before irradiation, A is the fluorescence intensity at 500nm wavelength after irradiation for different time).
  • the quantum dot fluorescent probe used in the existing method in the control group exhibits a slow luminescence intensity decay under long-term strong light excitation and irradiation
  • the luminescence of CdSe quantum dots attenuates by 35%
  • 1-naphthol as an organic light-emitting molecule
  • its molecular chemical structure is significantly damaged under long-term high-intensity laser irradiation, showing obvious The photobleaching properties of the luminous intensity attenuate 75% after 120 minutes of irradiation. Therefore, the graphene quantum dots used in the present invention have excellent long-lasting and stable luminescence advantages compared with other quantum dots and organic molecular fluorescent probes in bioanalytical sensing and detection applications.

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Abstract

本发明公开了一种包封石墨烯量子点的纳米脂质体及制备以及其在生物酶活性检测中的应用。该方法包括如下步骤：(1)将卵磷脂与胆固醇加入到氯仿中，超声使其分散均匀，然后旋蒸除去氯仿，得到脂质体薄膜；(2)将石墨烯量子点溶液加入到脂质体薄膜中，冰浴超声分散均匀，得到混合溶液I；然后将混合溶液通过聚碳酸酯膜反复挤压，得到混合溶液II；再将混合溶液II进行透析，得到包封石墨烯量子点的纳米脂质体。本发明中制备的纳米脂质体生物安全性高且发光长效稳定，在磷脂酶A2存在下使得该脂质体破裂，释放包裹在其中的荧光信号分子，因此可将其用于胰腺炎标志物磷脂酶A2的活性检测，在生物检测和临床诊断等方面具有广泛应用前景。

Description

一种包封石墨烯量子点的纳米脂质体及制备以及其在生物酶活性检测中的应用 技术领域
本发明属于有机/无机复合光学探针材料及疾病相关标志物检测领域，特别涉及一种包封石墨烯量子点的纳米脂质体及制备以及其在生物酶活性检测中的应用。
背景技术
磷脂酶(phospholipase)广泛分布于人体，在磷脂更新、细胞膜重构、胞吐和信息传递等生理过程中发挥重要作用。磷脂酶主要包含有磷脂酶A1、A2、B1、B2、C和D，它们会特异的作用于磷脂分子内部的各个酯键，形成不同的产物。其中磷脂酶A2(Phospholipase A 2，PLA 2)能够水解磷脂sn-2酯键产生游离脂肪酸和溶血磷脂，其在炎症和组织损伤时对于信息传递和膜通道活化等病理生理过程中起关键性作用。急性胰腺炎(Acute pancreatitis，AP)是胰酶激活后引起的胰腺组织自身消化、水肿甚至坏死的炎症反应。研究发现，磷脂酶A2在急性胰腺炎发生时会出现过早激活和过度释放，可直接参与并影响急性胰腺炎的发病过程。因此，检测血清中磷脂酶A2的生物活性对于急性胰腺炎的诊断和治疗来说具有重要临床意义。目前，已开发的磷脂酶A2测定方法主要有：1)分光光度法、2)电化学法、3)放射性免疫法、4)酶联免疫法、5)色谱质谱联用等方法。如公开号为CN 103698535 B的中国专利申请“脂蛋白相关磷脂酶A2定量检测试剂盒及制备、操作方法中以免疫磁珠微粒与化学发光酶免疫分析技术相结合，实现了灵敏检测并获得了较佳的性能参数。又如公开号为CN 104634970 A的中国专利申请“用于检测脂蛋白相关磷脂酶A2的试剂盒及其制备和应用”以抗体包被磁球和示踪标记物标记抗体结合，能够准确灵敏检测脂蛋白相关磷脂酶A2。然而，这些方法中有的由于涉及到昂贵的抗原抗体使得检测成本较高，而有的受限于检测流程繁杂耗时。
近些年来，新兴纳米材料在生物传感领域的潜在应用价值日益突显。石墨烯量子点(Graphene quantum dot,GQD)，是一种只有几个原子厚度和纳米级别的宽度的平面碳材料，属于零维纳米材料家族中的新成员。GQD由于其小尺寸所带来的量子效应而表现出优异的荧光性能。相比传统的有机荧光探针和半导体 量子点，其具有优异的抗光漂白性、发光稳定性、生物相容性和易于表面功能化等优点，已成为了最具应用潜力的光致发光材料之一。目前，石墨烯量子点在生物成像、标记与示踪、能量的储存与转换等具有广泛的应用。
脂质体(Liposomes)是由磷脂分子和胆固醇分子分散在水中自组装形成的囊泡结构，其具有类细胞膜结构的双分子层。这样的中空双层囊泡结构具有特殊能力—内腔和双分子层都可以负载药物，酶，荧光团或其他信号分子。脂质体囊泡已应用于药物载体、肿瘤成像、生物传感等领域。尤其在生物传感检测领域，使用脂质体囊泡的探针相比于传统小分子探针，具有明显的信号放大优势，并显现响应快速和使用方便等特点。因此，近年来脂质体囊泡探针的开发应用正逐渐成为生物传感器研究的一个热门领域。如公开号为CN 103599070 B的中国专利“负载金纳米簇和抗癌药物的脂质体温度荧光探针的制备方法”，利用超临界二氧化碳法将金纳米簇悬浊液和抗癌药物的混合溶液包封与脂质体的水相内腔中，制备得到的脂质体具有光致热敏性，可成为新型温度荧光探针。再如专利公开号为CN 107966568A的中国专利“基于铁(Ⅱ)与邻菲罗啉体系和脂质体包裹葡萄糖的免疫分析方法”，通过脂质体包裹葡萄糖用于信号放大，并且利用亚铁离子和邻菲罗啉指示剂作为显色基团，可用于链霉素的检测。再如专利公开号为CN 107674884A的中国专利“一种用于检测胰腺癌标记物REG1A的生物素化脂质体及其制备方法和应用”，利用免疫LAMP反应与脂质体纳米囊泡结合，实现了胰腺癌标志物REG1A的灵敏检测，并且该方法具有成本低、特异性好以及稳定性高等优点。值得注意的是，这些方法中脂质体破裂以释放包封在内的信号分子手段都是通过加入曲拉通-100(Triton X-100)或其他表面活性剂的方式。然而，加入的用于磷脂囊泡破裂的表面活性剂不容易从反应体系中除去，其引入不仅给体系带来杂质使得反应过程变得复杂，甚至对生物物质存在不良影响。虽然利用脂质体进行酶联免疫测定的方法具有亲和力强的优点，但是仍存在着抗原抗体的稳定性差以及成本高昂等缺陷。这些也限制脂质体在生物传感器领域进一步发展。
发明内容
本发明的首要目的在于克服现有技术的缺点与不足，提供一种包封石墨烯量子点的纳米脂质体的制备方法。
本发明的另一目的在于提供所述方法制备得到的包封石墨烯量子点的纳米脂质体。
本发明的再一目的在于提供所述包封石墨烯量子点的纳米脂质体在生物酶活性检测中的应用。
本发明的目的通过下述技术方案实现：一种包封石墨烯量子点的纳米脂质体的制备方法，包括如下步骤：
(1)将卵磷脂与胆固醇加入到氯仿中，超声使其分散均匀，然后旋蒸除去氯仿，得到脂质体薄膜；
(2)将石墨烯量子点溶液加入到脂质体薄膜中，冰浴超声分散均匀，得到混合溶液I；然后将混合溶液I通过聚碳酸酯膜反复挤压，得到混合溶液II；再将混合溶液II进行透析，得到包封石墨烯量子点的纳米脂质体。
步骤(1)中所述的卵磷脂与胆固醇的摩尔比为1～5：1；优选为5：1。
步骤(1)中所述的氯仿的用量为按每1.8mmol胆固醇配比1ml氯仿计算(或每10.8mmol卵磷脂和胆固醇配比1ml氯仿计算)。
步骤(1)中所述的超声的条件为：100W超声5～10min；优选为：100W超声5min。
步骤(1)中所述的旋蒸的条件为：40℃旋蒸15～60分钟；优选为40℃旋蒸60分钟。
步骤(2)中所述的石墨烯量子点与所述卵磷脂和胆固醇的总质量比为0.02～0.4:30；优选为0.2:30。
步骤(2)中所述的石墨烯量子点溶液为石墨烯量子点水溶液，或将石墨烯量子点溶于磷酸缓冲溶液得到的溶液；其浓度为0.01～0.2mg/mL；优选为0.1mg/mL。
所述的磷酸缓冲溶液为磷酸氢二钠和磷酸二氢钠的混合溶液，调节pH为7.0。
步骤(2)中所述的石墨烯量子点优选为通过如下方法制备得到：
S1、将碳黑加入到浓硝酸溶液中，于130℃条件下搅拌回流反应，待反应结束后冷却至室温，吸取上清液，加热除酸至pH为5～7，得到溶液A；
S2、将溶液A过滤，取滤液；然后将滤液进行离心，取上清液；再将上清液加入到超滤离心管中，离心，取清液；最后将清液进行透析，待透析结束后，冷冻干燥，得到石墨烯量子点。
步骤S1中所述的碳黑优选为carbot vulcan XC-72碳黑。
步骤S1中所述的浓硝酸溶液的浓度5～8mol/L；优选为6mol/L。
步骤S1中所述的回流反应优选为在油浴下进行回流反应。
步骤S1中所述的回流反应的时间优选为24小时。
步骤S2中所述的过滤为依次用滤纸和针式过滤器进行过滤。
所述的针式过滤器的孔径大小为0.22μm。
步骤S2中所述的离心的条件均为：8000rpm离心10分钟。
步骤S2中所述的超滤离心管的孔径大小为3000Da。
步骤S2中所述的透析为采用截留分子量为100～500Da的透析袋进行透析。
步骤S2中所述的透析的条件为：以去离子水为透析液透析24h。
步骤(2)中所述的挤压的温度优选为40±2℃。
步骤(2)中所述的挤压为在脂质体挤出仪中进行。
步骤(2)中所述的聚碳酸酯膜的孔径大小为200nm。
步骤(2)中所述的挤出的次数为21次以上。
步骤(2)中所述的透析为采用截留分子量为8000Da的透析膜进行透析。
步骤(2)中所述的透析的时间为24小时。
步骤(2)中所述的超声的条件为：100W超声40～60min；优选为：100W超声50min。
一种包封石墨烯量子点的纳米脂质体，通过上述任一项所述的方法制备得到。
所述的包封石墨烯量子点的纳米脂质体在制备荧光探针或胰腺炎标志物检测传感器中的应用。
所述的荧光探针为用于检测磷脂酶A2(胰腺炎标记物磷脂酶A2)的荧光探针。
一种利用所述包封石墨烯量子点的纳米脂质体进行非疾病诊断目的的测定磷脂酶A2含量的方法，包括如下步骤：
(a)将至少五个浓度梯度的磷脂酶A2水溶液分别与所述的包封石墨烯量子点的纳米脂质体混合后水浴反应，然后分别测量荧光光谱，得到荧光光谱最大强度；
(b)根据步骤(a)中得到的荧光光谱最大强度与磷脂酶A2水溶液的浓度，绘制标准曲线，得到线性方程；
(c)将待测样品与所述的包封石墨烯量子点的纳米脂质体混合后水浴反应，然后测量荧光光谱，得到待测样品的荧光光谱最大强度；再根据步骤(b)得到的线性方程获得待测样品的含量。
步骤(a)中所述的磷脂酶A2水溶液的用量为按其在所述反应体系的终浓度为0～20U/L添加；优选为按其在所述反应体系的终浓度为0、2、5、10和20U/L添加。
步骤(a)中所述的包封石墨烯量子点的纳米脂质体的用量为按其在所述反应体系的终浓度为0.26～0.28mg/mL添加；优选为按其在所述反应体系的终浓度为0.265～0.275mg/mL添加。
步骤(a)和(c)中所述的荧光光谱最大强度为激发波长为530nm下测定的荧光光谱强度。
步骤(a)和(c)中所述的水浴反应的条件为：37℃水浴1小时。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明以卵磷脂和胆固醇为合成材料，使用薄膜分散法制备包裹石墨烯量子点的脂质体。然后使用磷脂酶A2的酶促反应底物—磷脂分子自组装形成包裹了石墨烯量子点的脂质体，在磷脂酶A2存在下使得该脂质体破裂，释放包裹在其中的荧光信号分子，这样可有效避免第三方试剂(如曲拉通-100)作为脂质体破裂剂的加入。此外，避免了昂贵抗原抗体等生物试剂的使用，使得检测成本大大降低，在生物检测和临床诊断等方面具有广泛应用前景。
(2)本发明中的包封石墨烯量子点的纳米脂质体具有优异的生物相容性和长效光稳定性，其生物安全性高、发光长效稳定，具有良好的生物应用潜力。
(3)本发明利用脂质体独特的负载能力包裹石墨烯量子点荧光纳米探针，提供生化检测传感的光学信号放大策略，该纳米探针可作为疾病标志物检测传感器。
(4)本发明利用磷脂酶A2水解降解使得脂质体分解破裂释放石墨烯量子点荧光探针，其释放程度所引起的荧光强度变化与磷脂酶A2活性存在一定定量关系，建立用于疾病(如急性胰腺炎)标志物磷脂酶A2的荧光检测传感新方法，即该方法可对磷脂酶A2进行定量检测：将梯度浓度的磷脂酶A2加入脂质体，使其破裂释放出石墨烯量子点荧光探针，然后检测其在不同磷脂酶A2浓度下的荧光强度，制作出荧光强度变化与磷脂酶A2浓度相关的标准曲线，最后加入未知浓度的磷脂酶A2样品并检测其荧光强度，根据标准曲线得出检测结果。
(5)本发明采用将待测物直接作为脂质体破裂的方法，打破传统利用表面活性剂等物质破裂脂质体的方案，而是将待测物磷脂酶A2直接作为引起磷脂囊泡破裂的刺激因素，能够实现胰腺炎标志物磷脂酶A2的灵敏检测，并且该方法还具有成本低，检测方便快捷的优点，为环境刺激响应的智能仿生微囊泡的设 计和构建新型智能仿生系统提供新思路。
附图说明
图1是本发明包封石墨烯量子点脂质体的纳米探针用于磷脂酶A2荧光检测的原理示意图。
图2是石墨烯量子点的表征图；其中，A为石墨烯量子点的扫描电镜照片；B为石墨烯量子点的原子力显微镜照片。
图3是在不同激发波长下的石墨烯量子点发射光谱图(插图为白光和365nm紫外光照射时石墨烯量子点溶液的图像)。
图4是石墨烯量子点水溶液的浓度诱导荧光变化图；其中，A为不同浓度的石墨烯量子水溶液在465nm激发波长下的发射光谱图；B是石墨烯量子点水溶液在530nm处发射荧光强度随其浓度变化的标准曲线。
图5是脂质体的表征图；其中，A为脂质体的扫描电镜照片；B为脂质体的粒径分布情况(插图为白光照射时脂质体溶液图像)。
图6是在卵磷脂和胆固醇的摩尔比1:1下制备的包封石墨烯量子点的脂质体的粒径分布图；其中，A为脂质体的扫描电镜照片；B为脂质体的粒径分布图。
图7是在卵磷脂和胆固醇的摩尔比5:2下制备的包封石墨烯量子点的脂质体的粒径分布图；其中，A为脂质体的扫描电镜照片；B为脂质体的粒径分布图。
图8是包封不同浓度的石墨烯量子点溶液(0.01mg/ml、0.05mg/ml、0.1mg/ml、0.2mg/ml)合成的脂质体在磷脂酶A2作用下破裂释放荧光探针引起的光谱变化图。
图9是包封石墨烯量子点的脂质体用于检测磷脂酶A2活性的荧光检测结果图；其中，A为释放的石墨烯量子点的荧光强度随磷脂酶A2活性变化的趋势图(其活性浓度变化范围为0～250U/L)；B为释放的石墨烯量子点的荧光强度随磷脂酶A2活性变化从0～20U/L的标准曲线。
图10是包封石墨烯量子点的脂质体对磷脂酶A2的选择性实验结果图。
图11是本发明包封石墨烯量子点脂质体作为新型荧光探针用于生物检测的生物安全性比较图。
图12是本发明包封石墨烯量子点脂质体作为新型荧光探针用于生物检测的光稳定性比较图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述，但本发明的实施方式不限于此。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。除非特别说明，本发明所用试剂和原材料均可通过市售获得。
实施例1
本实施例提供一种石墨烯量子点合成方法。
称取0.4g carbot vulcan XC-72碳黑(品牌：麦考林，购于广州普智生物科技有限公司)，加入到100mL 6mol/L的HNO 3中，130℃(油浴)条件下搅拌回流反应24小时。然后将反应后的溶液冷却至室温，吸取上清液，加热除酸至pH为5～7，最终溶液体积为50mL，命名为溶液1。将得到的溶液1用滤纸(中速定性滤纸(速率102)，孔径为30～50微米，品牌：Biorad,北京百诺威生物科技有限公司)过滤两次，得到溶液2。再将溶液2用0.22μm的针式过滤器进行再次过滤，得到溶液3。将溶液3在8000rpm下离心10分钟，然后将上清液吸取到超滤离心管(孔径大小为3000Da)中，得到溶液4。将溶液4在8000rpm下离心(10分钟)，基本将所有清液与沉淀分离，最后将分离得到的清液放入截留分子量为100～500Da的透析袋中，以去离子水为透析液，透析24h。透析结束后，将溶液加到离心管内，冻干处理后即为石墨烯量子点。
石墨烯量子点的扫描电镜照片如图2A所示，原子力显微镜照片如图2B所示。石墨烯量子点在不同激发波长(荧光分光光度计，405、425、445、465、485、505、525nm)下的发射光谱，以及在白光和365nm紫外光照射下的石墨烯量子点溶液的图像如图3所示。
将石墨烯量子点配制成0.01，0.02，0.04，0.06，0.08，0.1mg/ml的石墨烯量子点水溶液，观察石墨烯量子点水溶液的浓度诱导荧光变化性质。结果如图4所示：图4A为不同浓度的石墨烯量子水溶液在465nm激发波长下的发射光谱图；图4B为石墨烯量子点水溶液在530nm处发射荧光强度随其浓度变化的标准曲线。
实施例2
本实施例提供一种脂质体的合成方法。
卵磷脂和胆固醇以5：1(摩尔比，共43.2mmol，30mg)的比例混合，溶 于4ml氯仿中，超声(功率100W)5分钟使其分散均匀。随后通过旋转蒸发器在40℃下、减压旋蒸1小时以除去有机溶剂，烧瓶底部均匀的形成一层透明薄膜。此时加入2mL 0.1mg/ml的石墨烯量子点溶液(将实施例1制备石墨烯量子点溶于磷酸缓冲溶液(pH7.0)中)，冰浴超声(功率100W)50分钟，得到乳白色的浑浊液体。将其通过200nm的聚碳酸酯膜(即脂质体挤出仪使用的滤膜孔径为200nm)，反复挤压21次(40℃)。最后将得到的脂质体溶液用透析膜(截留分子量小于8000D)透析，以去离子水为透析液，透析24小时，移除未包封的石墨烯量子点，将获得的脂质体溶液储存在4℃下。
脂质体的扫描电镜结果如图5A所示，粒径分布情况如图5B所示(插图为白光照射时脂质体溶液图像)。从粒径分布和扫描电镜结果可知，本实施例制备的脂质体囊泡尺寸均匀，分散性好。
实施例3
不同比例的卵磷脂与胆固醇合成的脂质体
合成方法同实施例2，不同之处在于：卵磷脂和胆固醇以1:1(摩尔比，共52.37mmol，30mg)或5:2(摩尔比，共45.99mmol，30mg)的比例混合。
其扫描电镜结果和粒径分布如图6和7所示。
实施例4
不同浓度的石墨烯量子点溶液合成的脂质体
合成方法同实施例2，不同之处在于：石墨烯量子点溶液(将实施例1制备石墨烯量子点溶于磷酸缓冲溶液(pH7.0)中)的浓度为0.01mg/ml、0.05mg/ml、0.1mg/mL、0.2mg/ml。
分别取4uL上述不同浓度的石墨烯量子点包封所制备的脂质体溶液(浓度均为13.6mg/mL)用水稀释50倍，然后分别与磷脂酶A2(品牌：源叶，上海源叶生物科技有限公司)混合，混合体系中磷脂酶A2的终浓度均为10U/L，37℃水浴反应1小时，然后测量荧光强度。
图8是包封石墨烯量子点溶液浓度为0.01mg/ml、0.05mg/ml、0.1mg/mL、0.2mg/ml的脂质体在一定磷脂酶A2浓度作用下破裂引起荧光强度变化情况。如图8所示，当采用的石墨烯量子点溶液为0.2mg/ml时所检测的荧光强度比起0.1mg/ml的变化不明显，说明受限于脂质体空腔大小容积的限制，制备包封石墨烯量子点脂质体时加入更大浓度的石墨烯量子点也包封不进去了，所以本发 明优选为0.1mg/ml的石墨烯量子点溶液来制备脂质体探针。
实施例5
本实施例提供一种包封石墨烯量子点的纳米脂质体的荧光探针用于疾病标志磷脂酶A2的灵敏特异检测方法，其原理如图1所示。
(1)将4uL 13.6mg/mL的脂质体溶液(即实施例2制备的脂质体)用水稀释50倍，加入5uL不同浓度的磷脂酶A2(品牌：源叶，上海源叶生物科技有限公司)在37℃水浴1小时后(磷脂酶A2的终浓度分别为0、2、5、10、20、50、100、150、200、300U/L)，530nm处观察荧光强度。
结果如图9所示：图9是包封石墨烯量子点的脂质体用于磷脂酶A2活性的荧光检测结果；其中，图9A为释放的石墨烯量子点的荧光强度随磷脂酶A2活性变化的趋势图(其活性浓度变化范围为0～250U/L)；图9B为释放的石墨烯量子点的荧光强度随磷脂酶A2活性变化从0～20U/L的标准曲线。结果表明：530nm处的荧光强度随着磷脂酶A2的浓度增加而增加，其中0到20U/L之间存在良好线性关系。
(2)为验证该方法对磷脂酶A2的特异性检测，用10uL 50U/L的磷脂酶C(PLC)，磷脂酶D(PLD)(磷脂酶C和磷脂酶D，品牌：源叶生物，均购自广州齐云生物科技有限公司)以及磷脂酶A2(PL A2)溶液分别与50倍稀释后的脂质体(即实施例2制备的脂质体)溶液400uL混合水浴1小时(37℃)，以不加入磷脂酶为空白对照(同样含量的脂质体，但没有任何酶加入时，磷脂囊泡不会自己破裂释放石墨烯量子点荧光探针)(BLANK)。
结果如图10所示，与50U/L磷脂酶C，磷脂酶D溶液混合水浴后的脂质体溶液，荧光强度并没有明显变化。而与磷脂酶A2溶液混合水浴后的脂质体，荧光强度显著升高，表明该方法对磷脂酶A2具有良好选择性。
实施例6
1.生物安全性实验
本实验以不同质量浓度的不同类型的荧光探针，如新兴纳米荧光探针(包括石墨烯量子点、水溶性CdSe半导体量子点)和传统有机分子荧光探针(包括罗丹明B和1-萘酚)为例染毒Hela细胞，通过检测细胞存活率进行其各自的生物安全性评估。其中，所涉及的材料如下：海拉细胞株(Hela)购于生工生物工 程(上海)股份有限公司；石墨烯量子点的制备同实施例1；CdSe半导体量子点购于广州锂阁科技有限公司；罗丹明B和1-萘酚购于上海阿拉丁生化科技有限公司，纯度大于99％；DMEM高糖培养基购于广州恒研生物科技有限公司；胰蛋白酶(0.25％，含EDTA)和胎牛血清购于上海麦克林生化科技有限公司；MTT检测试剂盒购于广州鲁诚生化科技有限公司。
以不同质量浓度(0、50、100、150和200μg/mL)的四种荧光探针(石墨烯量子点、水溶性CdSe半导体量子点、罗丹明B和1-萘酚)分别染毒Hela细胞，采用MTT法(常用于细胞毒性/存活率的分析方法)检测细胞存活率，具体步骤如下：
(1)配制荧光探针溶液和细胞培养液
①石墨烯量子点、水溶性CdSe半导体量子点、罗丹明B和1-萘酚的母液配制：分别取1mg的石墨烯量子点、水溶性CdSe半导体量子点、罗丹明B和1-萘酚的粉末分散溶解于1mL磷酸缓冲溶液PBS(pH7.0)，得到终浓度均为1mg/mL的溶液；
②Hela细胞培养液配制：用DMEM高糖培养基500mL，加胎牛血清50mL，得到含l0％胎牛血清的DMEM高糖培养基，4℃保存备用；
(2)细胞培养
Hela细胞(细胞的接种密度为1×10 4/cm 2)在含l0％胎牛血清的DMEM高糖培养基中培养，培养条件为5％(v/v)CO 2，37℃，饱和湿度。
(3)细胞存活率检测
方法：细胞存活率是细胞毒性实验常用指标，本实验采用MTT检测试剂盒以比色法测定细胞存活率。调整Hela细胞密度为1×10 5cells/mL，接种于96孔板中，每孔100μL，细胞培养24小时后，吸弃原培养液，每次分别在每孔加入终质量浓度为0、50、100、150、200μg/mL的石墨烯量子点溶液，每个质量浓度设3个重复，以水溶性CdSe半导体量子点，罗丹明B和1-萘酚为对比，每次实验加入方法同上。分别作用24h后，每孔加入20μL 5mg/mL MTT试剂，继续孵育2h后，选择490nm波长，在酶标仪上测定各孔吸光度(OD)值。细胞相对存活率计算公式为：
Figure PCTCN2020117568-appb-000001
结果如图11所示：由图11的实验结果可知，Hela细胞经过与不同质量浓度的不同类型的荧光探针共培养24小时后，随着加入的不同探针的质量浓度升 高，石墨烯量子点未引起Hela细胞存活率的明显变化，即使浓度为200μg/m时，Hela的细胞存活率还保持96.8％，说明石墨烯量子点具有优异的生物安全性，适合生物分析应用；有机荧光分子如罗丹明B和1-萘酚也都表现出一定的细胞毒性，随着浓度的不断增加，Hela细胞存活率大大降低分别至(45.4％和38.6％)；相比之下，水溶性CdSe半导体量子点具有明显毒性，导致Hela细胞存活率显著降低(18.4％)。
因此，通过比较不同类型的荧光探针的生物安全性，本发明所采用的石墨烯量子点作为新型荧光探针较其他的有机荧光分子和半导体量子点等具有明显的优异的生物相容性，具有良好的生物应用潜力。
2.光稳定性实验
本实验比较不同类型的荧光探针包括石墨烯量子点、水溶性CdSe半导体量子点和1-萘酚的荧光发光稳定性，评估其长效应用性能。其中涉及的材料如下：石墨烯量子点的制备同实施例1，CdSe半导体量子点购于广州锂阁科技有限公司；1-萘酚购于上海阿拉丁生化科技有限公司，纯度大于99％。具体步骤如下：
(1)石墨烯量子点、水溶性CdSe半导体量子点和1-萘酚的溶液配制：
分别取200ng石墨烯量子点、水溶性CdSe半导体量子点和1-萘酚的粉末分散溶解于1mL磷酸缓冲溶液PBS(pH7.0)，得到终浓度均为200ng/mL的样品溶液；
(2)荧光发光稳定性实验
将上述配制的三个样品溶液分别用氙灯(北京卓立汉光仪器有限公司，Zolix LSB-500W，功率500瓦)连续照射120分钟。荧光分光光度计(Hitachi,F-4500)检测照射前后三个样品溶液分别照射前后的荧光强度变化(A0为照射前，A为经历不同时间照射后在500nm波长处的荧光强度)。
结果如图12所示：由图12的实验结果可知，经过120分钟的连续强激光照射，本发明中所采用的荧光探针石墨烯量子点在照射前后并未发生明显的光致发光强度变化，说明该材料作为荧光探针具有优异的长效光稳定性；而对照组中现有方法所采用的量子点荧光探针在经历了长时间的强光激发照射下呈现出缓慢的发光强度衰减，120分钟连续照射后，CdSe量子点的发光衰减35％；相比之下，1-萘酚作为有机发光分子，其分子化学结构在长时间高强度激光照射下发生明显的破坏，呈现出明显的光漂白性，其发光强度在120分钟照射后衰减了75％。因此，本发明所采用的石墨烯量子点相比其他量子点和有机分子荧 光探针在生物分析传感检测应用中具有优异的发光长效稳定的优点。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种包封石墨烯量子点的纳米脂质体的制备方法，其特征在于，包括如下步骤：
    (1)将卵磷脂与胆固醇加入到氯仿中，超声使其分散均匀，然后旋蒸除去氯仿，得到脂质体薄膜；
    (2)将石墨烯量子点溶液加入到脂质体薄膜中，冰浴超声分散均匀，得到混合溶液I；然后将混合溶液I通过聚碳酸酯膜反复挤压，得到混合溶液II；再将混合溶液II进行透析，得到包封石墨烯量子点的纳米脂质体。
  2. 根据权利要求1所述的包封石墨烯量子点的纳米脂质体的制备方法，其特征在于：
    步骤(1)中所述的卵磷脂与胆固醇的摩尔比为1～5：1；
    步骤(2)中所述的石墨烯量子点与所述卵磷脂和胆固醇的总质量比为0.02～0.4:30；
    步骤(2)中所述的石墨烯量子点溶液为石墨烯量子点水溶液，或将石墨烯量子点溶于磷酸缓冲溶液得到的溶液；所述的石墨烯量子点溶液的浓度为0.01～0.2mg/mL。
  3. 根据权利要求2所述的包封石墨烯量子点的纳米脂质体的制备方法，其特征在于：
    步骤(1)中所述的卵磷脂与胆固醇的摩尔比为5：1；
    步骤(2)中所述的石墨烯量子点与所述卵磷脂和胆固醇的总质量比为0.2:30；
    步骤(2)中所述的石墨烯量子点溶液的浓度为0.1mg/mL。
  4. 根据权利要求1所述的包封石墨烯量子点的纳米脂质体的制备方法，其特征在于：
    步骤(2)中所述的石墨烯量子点通过如下方法制备得到：
    S1、将碳黑加入到浓硝酸溶液中，于130℃条件下搅拌回流反应，待反应结束后冷却至室温，吸取上清液，加热除酸至pH为5～7，得到溶液A；
    S2、将溶液A过滤，取滤液；然后将滤液进行离心，取上清液；再将上清液加入到超滤离心管中，离心，取清液；最后将清液进行透析，待透析结束后，冷冻干燥，得到石墨烯量子点。
  5. 根据权利要求4所述的包封石墨烯量子点的纳米脂质体的制备方法，其特征在于：
    步骤S1中所述的碳黑为carbot vulcan XC-72碳黑；
    步骤S1中所述的浓硝酸溶液的浓度5～8mol/L；
    步骤S1中所述的回流反应为在油浴下进行回流反应；
    步骤S1中所述的回流反应的时间为24小时；
    步骤S2中所述的过滤为依次用滤纸和针式过滤器进行过滤；
    所述的针式过滤器的孔径大小为0.22μm；
    步骤S2中所述的离心的条件均为：8000rpm离心10分钟；
    步骤S2中所述的超滤离心管的孔径大小为3000Da；
    步骤S2中所述的透析为采用截留分子量为100～500Da的透析袋进行透析；
    步骤S2中所述的透析的条件为：以去离子水为透析液透析24h。
  6. 根据权利要求1所述的包封石墨烯量子点的纳米脂质体的制备方法，其特征在于：
    步骤(1)中所述的超声的条件为：100W超声5～10min；
    步骤(1)中所述的旋蒸的条件为：40℃旋蒸15～60分钟；
    步骤(2)中所述的超声的条件为：100W超声40～60min；
    步骤(2)中所述的挤压的温度为40±2℃；
    步骤(2)中所述的挤压为在脂质体挤出仪中进行；
    步骤(2)中所述的聚碳酸酯膜的孔径大小为200nm；
    步骤(2)中所述的挤出的次数为21次以上；
    步骤(2)中所述的透析为采用截留分子量为8000Da的透析膜进行透析；
    步骤(2)中所述的透析的时间为24小时。
  7. 一种包封石墨烯量子点的纳米脂质体，其特征在于：通过权利要求1～6任一项所述的方法制备得到。
  8. 权利要求7所述的包封石墨烯量子点的纳米脂质体在制备荧光探针或胰腺炎标志物检测传感器中的应用。
  9. 一种利用权利要求7所述包封石墨烯量子点的纳米脂质体进行非疾病诊断目的的测定磷脂酶A2含量的方法，其特征在于，包括如下步骤：
    (a)将至少五个浓度梯度的磷脂酶A2水溶液分别与权利要求7所述的包封石墨烯量子点的纳米脂质体混合后水浴反应，然后分别测量荧光光谱，得到荧光光谱最大强度；
    (b)根据步骤(a)中得到的荧光光谱最大强度与磷脂酶A2水溶液的浓度，绘制标准曲线，得到线性方程；
    (c)将待测样品与包封石墨烯量子点的纳米脂质体混合后水浴反应，然后测量荧光光谱，得到待测样品的荧光光谱最大强度；再根据步骤(b)得到的线性方程获得待测样品的含量。
  10. 根据权利要求9所述的方法，其特征在于：
    步骤(a)中所述的磷脂酶A2水溶液的用量为按其在所述反应体系的终浓度为0～20U/L添加；
    步骤(a)中所述的包封石墨烯量子点的纳米脂质体的用量为按其在所述反应体系的终浓度为0.26～0.28mg/mL添加；
    步骤(a)和(c)中所述的荧光光谱最大强度为激发波长为530nm下测定的荧光光谱强度；
    步骤(a)和(c)中所述的水浴反应的条件为：37℃水浴1小时。
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