CN114891508B - Water-soluble InP core-shell quantum dot with high brightness and stability and synthesis method and application thereof - Google Patents
Water-soluble InP core-shell quantum dot with high brightness and stability and synthesis method and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of detection, and particularly relates to a water-soluble InP core-shell quantum dot with high brightness and stability, and a synthesis method and application thereof. The present invention combines thermodynamic and kinetic growth processes. First, fluorescent quantum dots with a thin ZnS layer were obtained by thermodynamic growth at high temperature. Then, the ZnS shell layer is induced to be dynamically grown at low temperature by a photochemical treatment process, so that the problem of serious fluorescence loss caused by ligand exchange is solved, and the water-soluble InP core-shell quantum dot with the quantum yield of more than 80% is obtained. After the water-soluble InP quantum coupled alpha fetoprotein antibody is adopted, the antibody can be used for sensitively detecting AFP antigen based on a fluorescence immunoassay technology, the detection range is 1-1000 ng/mL, the detection limit is as low as 0.58 ng/mL, the antibody can be used for targeting marking liver cancer cells and liver cancer tumors in mice, and the early diagnosis effect on liver cancer can be improved by combining external analysis and in-vitro and in-vivo imaging data.
Description
Technical Field
The invention belongs to the technical field of quantum dot preparation, and particularly relates to a water-soluble InP core-shell quantum dot with high brightness and stability, a synthetic method thereof and application of the water-soluble InP core-shell quantum dot as a fluorescent probe.
Background
Quantum Dots (QDs) are powerful tools for in vitro diagnosis and biological imaging as fluorescent probes due to unique optical advantages, and play an important role in connecting modern nano-medicine and biological applications. Particularly, with the rapid development of heavy metal-free quantum dots represented by InP, the worry that the traditional CdSe quantum dots seriously affect the health of organisms is reduced. In biological applications, one key issue with quantum dots is maintaining high fluorescence and photochemical stability. However, at present, the high-quality InP core-shell quantum dots are obtained by high-temperature reaction in an organic solvent, so that how to efficiently transfer the hydrophobic InP core-shell quantum dots to an aqueous solution, and maintain good fluorescence and stability is a difficult problem.
Ligand exchange (commonly used ligands such as mercaptopropionic acid, thioglycolic acid, etc.) is the most widely used one of the aqueous phase transfer strategies because it is able to perfectly maintain the small particle size and monodispersity of quantum dots, which is crucial for in vitro or in vivo bioimaging applications. However, it is well known that ligand exchange using mercaptopropionic acid results in loss of fluorescence, probably due to etching of the quantum dot surface, and the prepared quantum dots can aggregate and precipitate in a short time, which is unacceptable in biological applications. In addition, because InP core-shell quantum dots are highly sensitive to water and oxygen, surface modification can severely affect the recombination of internal excitons. To date, there has been little research on the water phase transfer and stability of InP core-shell quantum dots. Therefore, it is difficult to obtain water-soluble InP core-shell quantum dots with high fluorescence and stability by a simple ligand exchange strategy.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the water-soluble InP core-shell quantum dot with high brightness and stability.
The invention also provides a synthesis method of the water-soluble InP core-shell quantum dot, a quantum dot-antibody fluorescent probe manufactured by using the water-soluble InP core-shell quantum dot and application of the water-soluble InP core-shell quantum dot. The fluorescent probe can realize targeted imaging of liver cancer cells and tumor parts and is used for detecting tumors or other markers.
In order to achieve the above object, the present invention provides the following technical solutions:
a synthetic method of a water-soluble InP core-shell quantum dot with high brightness and stability comprises the following steps:
s1: obtaining oil-soluble InP core-shell quantum dots; the oil-soluble InP core-shell quantum dot can be one or more of InP/ZnSe, inP/ZnS, inP/ZnSe/ZnSeSSZnS, inP/Gap/ZnS and the like, can be prepared by adopting a conventional method in the field, and can also be directly purchased from common commercial products;
s2: mixing and reacting the water-soluble sulfydryl type coating with the oil-soluble InP core-shell quantum dots in a water phase to obtain an aqueous solution of the InP core-shell quantum dots;
s3: and adding a Zn-mercaptopropionic acid precursor solution into the aqueous solution of the InP core-shell quantum dot, and then carrying out ultraviolet irradiation (for exciting kinetic growth under the assistance of the ultraviolet irradiation so as to grow a thicker ZnS layer), thereby obtaining the water-soluble InP core-shell quantum dot with high brightness and stability.
Specifically, in step S2, the water-soluble mercapto coating includes mercaptopropionic acid, thioglycolic acid, or the like.
Specifically, in step S3, the zinc source in the Zn-mercaptopropionic acid precursor liquid includes zinc chloride, zinc acetate, zinc perchlorate, or the like. And the steps S2 and S3 are not required to be carried out under the protection of nitrogen.
Further, S2 specifically is: dispersing the purified oil-soluble InP core-shell quantum dots in n-hexane or octane, adding concentrated ammonia water and a water-soluble mercapto-group coating, and stirring in a water bath at 50-70 ℃ to react for 1-3 h; wherein the volume ratio of the concentrated ammonia water to the water-soluble sulfydryl coating is 3-7:1; the adding mass ratio of the oil-soluble InP core-shell quantum dots to the water-soluble mercapto-based coating is 1: 30-50. The concentrated ammonia refers to an aqueous solution containing 25 to 28 percent of ammonia, and is a common commercial product.
Further, the S3 specifically is: and (3) purifying the aqueous solution of the InP core-shell quantum dots obtained in the step S2 by using acetonitrile, dispersing the aqueous solution of the InP core-shell quantum dots in 3-4 mL ultrapure water (the concentration is 8-15 mg/mL), adding 3-4 mLZn-mercaptopropionic acid precursor liquid, placing the solution in a water bath at the temperature of 60-80 ℃, and performing ultraviolet irradiation for 30-60 min under stirring.
In particular, the followingThe preparation method of the Zn-mercaptopropionic acid precursor liquid in the S3 comprises the following steps: stirring and dissolving a zinc source, mercaptopropionic acid and ultrapure water, wherein the mass ratio of the zinc source to the mercaptopropionic acid to the ultrapure water is 1:8-10:25-36. Preferably, the water is mixed after adding ultrapure water with corresponding volume, so that ZnCl in the Zn-mercaptopropionic acid precursor liquid S3 is obtained 2 The concentration of (A) is 0.2-0.4 mmol/mL, and the concentration of mercaptopropionic acid is 0.2-0.4 mL/mL.
The invention also provides the water-soluble InP core-shell quantum dot which is synthesized by the method and has high brightness and stability.
The invention makes the water-soluble quantum dot with high brightness and stability as a biological fluorescent probe, such as a coupled antibody, and then is used for detecting tumors or other markers, such as C-reactive protein (CRP), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA) and the like, based on a fluorescence immunoassay technology. Specifically, the invention also provides a method for preparing a quantum dot-antibody fluorescent probe by using the water-soluble InP core-shell quantum dot, which comprises the following steps:
1) Dispersing water-soluble InP core-shell quantum dots in a sodium borate buffer solution, adding N-hydroxy thiosuccinimide and carbodiimide, carrying out an activation reaction for 8-20 min under ultrasonic vibration at the temperature of 2-6 ℃, removing supernatant, and centrifuging to remove unreacted reagents;
2) Dissolving the product obtained in step 1) in sodium borate buffer solution, adding coupling antibody (such as one or more of AFP, CEA and CRP), and incubating in 37 + -2 deg.C incubator for 2-4 h; adding confining liquid, confining for 20-40 min, and terminating reaction with ethanolamine.
The invention also provides the quantum dot-antibody fluorescent probe prepared by the method.
The invention also provides the quantum dot-antibody fluorescent probe as a tracer for in vitro diagnosis or quantitative detection of target labeled cells or related markers, or as an imaging agent for targeted imaging of tumors in vivo. The cell types comprise liver cancer cells such as HepG2, SMMC-7721 and the like.
The water-soluble InP core-shell quantum dot with high brightness and stability comprises a mercaptopropionic acid ligand exchange layer and a thicker ZnS layer grown under the assistance of ultraviolet radiation; after the InP core-shell quantum dot is subjected to ultraviolet irradiation to assist in growing the ZnS layer, the overall size is increased by 1-2 nm, the thicker ZnS layer passivates surface defects caused by ligand exchange, electrons and holes can be further limited in the core, the fluorescence of the quantum dot is increased, the quantity of mercaptopropionic acid on the surface of the quantum dot is increased, and the stability of the quantum dot is improved.
The invention also provides a fluorescence immunoassay technology for quantitatively detecting a liver cancer marker Alpha Fetoprotein (AFP), the water-soluble InP core-shell quantum dot with high brightness and stability has high fluorescence, the sensitivity of a detection signal is improved, and the higher ligand density on the surface is beneficial to coupling more AFP antibodies while the good stability of the quantum dot is ensured. When the water-soluble InP quantum dot with high brightness and stability is used for detecting AFP antigen based on the fluorescence immunoassay technology, the detection range is 1-1000 ng/mL, and the lowest detection limit is 0.58 ng/mL, so that the detection range and the detection limit are remarkably improved compared with the water-soluble InP quantum dot obtained by simple mercaptopropionic acid ligand exchange.
The invention also provides a method for targeting labeling liver cancer cells in vitro and targeting tumor imaging in vivo. The quantum dot-Antibody (AFP) fluorescent probe designed based on the water-soluble InP core-shell quantum dot with high brightness and stability has an obvious targeting marking effect on liver cancer cells, and can also target tumors after tail intravenous injection into tumor-bearing mice.
The present invention combines thermodynamic and kinetic growth processes. First, fluorescent quantum dots with a thin ZnS layer were obtained by thermodynamic growth at high temperature. Then, the ZnS shell layer is induced to be dynamically grown at low temperature by a photochemical treatment process, so that the problem of serious fluorescence loss caused by ligand exchange is solved, and the water-soluble InP core-shell quantum dot with the quantum yield of more than 80% is obtained. After the water-soluble InP quantum coupled alpha-fetoprotein antibody is adopted, the antibody can be used for sensitively detecting AFP antigen based on a fluorescence immunoassay technology, the detection range is 1-1000 ng/mL, the detection limit is as low as 0.58 ng/mL, the antibody can be used for targeting markers of liver cancer cells and liver cancer tumors in mice, and the early diagnosis effect on liver cancer can be improved by combining in-vitro analysis and in-vivo and in-vitro imaging data.
The water-soluble InP quantum dot provided by the invention combines the advantages of high-temperature reaction in an organic solvent, keeps good crystallinity and luminescence property, and reduces the influence of a water transfer process on optical performance by further growing a thicker ZnS layer in an aqueous solution; the finally obtained water-soluble quantum dots have high quantum yield and good stability. The invention has the innovativeness that: the method solves the problems in the prior art, develops a method for synthesizing InP quantum dots with high brightness and stability in aqueous solution, and combines the thermodynamic growth process and the kinetic growth process. Firstly, obtaining fluorescent quantum dots with a thin ZnS layer through thermodynamic growth at high temperature; subsequently introducing it into an aqueous solution containing a zinc source and 3-mercaptopropionic acid (3-MPA), and exciting S cleaved from 3-MPA by ultraviolet irradiation 2- Then with Zn in solution 2fa A chemical reaction occurs that promotes the kinetic growth of the ZnS shell at low temperature. In this process, the thermal motion of the ions in the quantum dots is suppressed, which determines that no additional lattice defects are generated during the growth of the thicker ZnS shell. The thick shell structure effectively reduces the influence of surface modification on exciton recombination, thereby successfully improving the yield of the fluorescence quantum. It is worth noting that the surplus 3-MPA in the solution further improves the dispersibility of the quantum dots in the aqueous solution during the growth of the kinetic shell. Moreover, the strategy increases the ligand density on the surface of the InP core-shell quantum dot, which is important for maintaining the stability in aqueous solution and simultaneously improves the coupling efficiency with the antibody. The oil-water two-phase method not only ensures the advantages of synthesis in organic solvents, ensures good crystallinity and optical performance of quantum dots, but also avoids fluorescence loss caused by ligand exchange. The prepared InP core-shell quantum dots can be stably dispersed in an aqueous solution for a long time and have bright fluorescence. Compared with the prior art, the invention has the following beneficial effects:
1) The prepared water-soluble InP core-shell quantum dots have ultrahigh brightness and stability by combining the advantages of high-temperature oil phase thermodynamic growth and low-temperature water phase kinetic growth;
2) The method realizes the sensitive detection of the AFP antigen based on the quantum dot fluorescence immunoassay technology, and has ultra-low detection limit and larger detection range;
3) Studies with cellular and in vivo imaging showed that: the quantum dot-antibody fluorescent probe prepared based on the InP core-shell quantum dot has good liver cancer targeting property, and the sensitivity of early screening of liver cancer can be improved by combining the results of in vitro diagnosis and in vitro and in vivo imaging.
Drawings
For convenience of description, inP core-shell quantum dots dispersed in organic solvent n-hexane are referred to as "Original", quantum dots subjected to simple ligand exchange are referred to as "QDs-1", and quantum dots subjected to photochemical treatment are referred to as "QDs-2";
FIG. 1 is a TEM image of example 1 after organic solvent (a), ligand exchange (b), and photochemical treatment (c), respectively;
FIG. 2 is a fluorescence spectrum of example 1 after organic solvent, ligand exchange, and photochemical treatment, respectively;
FIG. 3 is a graph showing fluorescence lifetimes of the samples of example 1 after organic solvent, ligand exchange, and photochemical treatment, respectively; wherein Original is dispersed in n-hexane, and QDs-1 and QDs-2 are dispersed in water;
FIG. 4 is a chart of surface ligand density analysis (thermogravimetric analysis) after ligand exchange (a, QDs-1) and photochemical treatment (b, QDs-2), respectively, for example 1: the testing temperature range is 30-500 ℃, and the heating rate is 5 ℃/min;
FIG. 5 is a standard graph of the quantum dot-antibody probe prepared in example 1 for AFP antigen detection by fluorescence immunoassay; (a) the change of fluorescence intensity of the QDs-1 fluorescent probe along with the antigen concentration, (b) the change of fluorescence intensity of the QDs-2 fluorescent probe along with the antigen concentration, (c) a standard curve for AFP antigen detection, wherein the detection range of the QDs-1 is 5-500 ng/mL, the detection range of the QDs-2 is 1-1000 ng/mL, and (d) the standard curve of the probe within the range of 1-100 ng/mL;
FIG. 6 is a diagram of the quantum dot probe prepared in example 1 for targeting and labeling hepatoma cells and tumor sites; (a) In vitro imaging of cancer cells with the QDs-1-AFP-Ab probe, (b) in vitro imaging of cancer cells with the QDs-2-AFP-Ab probe, wherein nuclei are stained blue with Hoechst 33342, and the scale bar is 8 μm; (c) Fluorescence images of QDs-1-AFP-Ab probe at different times in each organ of the mouse; (d) Fluorescence images of QDs-2-AFP-Ab probes at various times in various organs of mice. The sample injection concentration of the probe is 5 mg/kg, and the sample injection amount is 200 mu L.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
The names and models of the experimental instruments are as follows:
a Perkin-Elmer Lambda-850 UV spectrophotometer;
Perkin-Elmer Ls55 spectrofluorometer;
JEOL JEM-200CX transmission electron microscope;
METTLER TOLEDO thermogravimetric analyzer;
a SpectraMax i3x multifunctional microplate reader;
leica SP5 confocal fluorescence microscope.
In the following examples, all the raw materials were common commercial products which were directly available.
The oil-soluble InP core-shell Quantum Dots (InP/ZnSe/ZnS) used in example 1 were prepared by reference to the literature (Choi S-W, kim H-M, yoon S-Y, et al, amino phosphor-Derived, high-Quality Red-Emissive Inp quant Dots by the Use of an inorganic in semiconductor Chemistry [ J ]. Journal of Materials Chemistry C, 2022, 10 (6): 2213-2222).
Example 1
A synthetic method of a water-soluble InP core-shell quantum dot with high brightness and stability comprises the following steps:
1) Simple ligand exchange:
after purifying oil-soluble InP core-shell (InP/ZnSe/ZnS) quantum dots (quantum dots purified using toluene-ethanol, the volume ratio of the quantum dots to toluene and ethanol is 1. Putting a 4 mL quantum dot solution into a single-mouth bottle without the protection of nitrogen or argon, adding 4 mL concentrated ammonia water and 1 mL mercaptopropionic acid (the corresponding pH is about 11), and stirring in a water bath at 60 ℃ to react for 1 h to obtain an InP core-shell quantum dot aqueous solution, which is marked as 'QDs-1';
2) Photochemical treatment to grow thicker ZnS layer:
the preparation of the Zn-mercaptopropionic acid precursor liquid comprises the following specific steps: taking ZnCl 2 4 mmol of mercaptopropionic acid 4 mL, adding ultrapure water 16 mL, stirring and fully dissolving;
after the InP core-shell quantum dot aqueous solution obtained in the above way is centrifugally purified by acetonitrile (the volume ratio of the quantum dot aqueous solution to the acetonitrile is 1:5), the InP core-shell quantum dot aqueous solution is re-dispersed in ultrapure water of 4 mL (the concentration is 10 mg/mL), zn-mercaptopropionic acid precursor liquid 4 mL is added, the obtained InP core-shell quantum dot aqueous solution is placed in a water bath at 75 ℃, stirred and assisted with ultraviolet irradiation (the wavelength is 365 nm) to grow a thicker ZnS layer, and the ultraviolet irradiation time is 40 min. After the ultraviolet irradiation is finished, acetonitrile is used for centrifugal purification twice, and the acetonitrile is dispersed in pure water again, so that the water-soluble InP core-shell quantum dots with high brightness and stability are obtained, and are marked as 'QDs-2'.
The water-soluble InP core-shell quantum dots prepared in example 1, which have both high brightness and stability, are subjected to TEM morphology characterization after organic solvent, ligand exchange and photochemical treatment, and the results are shown in fig. 1. As can be seen from a in fig. 1, the average size of the oil-soluble InP core-shell quantum dots is 10.08 nm; as can be seen in FIG. 1 b, the average size after ligand exchange was 9.76 nm, which resulted in a reduction in size relative to that of nm in organic solvent due to the etching effect of mercaptopropionic acid. As can be seen from c in fig. 1: the average size of the quantum dots increased to 11.83 nm after photochemical treatment, which was about 2 nm increased relative to after ligand exchange. TEM data indicate that a thicker ZnS layer is grown on the surface of InP quantum dots by the photochemical treatment strategy.
Fig. 2 is a fluorescence spectrum of the water-soluble InP core-shell quantum dot prepared in example 1 after organic solvent, ligand exchange and photochemical treatment. As can be seen from fig. 2, the fluorescence peak after ligand exchange has a relatively significant red shift, which is presumed to be due to poor dispersibility and forster fluorescence energy resonance transfer occurring between particles, and the red shift amplitude decreases after photochemical treatment.
FIG. 3 is a graph showing fluorescence lifetimes of the samples of example 1 after organic solvent, ligand exchange, and photochemical treatment, respectively. As can be seen from fig. 3, the fluorescence lifetime sharply decayed after ligand exchange, presumably due to increased surface defects caused by the etching of the quantum dot surface by mercaptopropionic acid, while the newly grown ZnS layer eliminated surface defects after photochemical treatment, exhibiting fluorescence decay similar to that in organic solvent n-hexane.
FIG. 4 is a graph of surface ligand density analysis (thermogravimetric analysis) after ligand exchange and photochemical treatment, respectively, for example 1. As can be seen from fig. 4: after photochemical treatment, QDs-2 loses more weight between 100 and 500 ℃, which indicates that the density of mercaptopropionic acid ligand on the surface is higher, and the densities of ligand exchange and the surface ligand after photochemical treatment are respectively 1.57 and 5.36 and are calculated as units/nm 2 。
Example 2
A method for preparing a quantum dot-antibody fluorescent probe by using the water-soluble InP core-shell quantum dot comprises the following steps:
1) Firstly, 300 mu L of the water-soluble InP core-shell quantum dots (10 mg/mL, QDs-2, and using QDs-1 as a contrast) prepared in the example 1 are dispersed in 750 mu L of sodium borate buffer (5 mM, pH = 7.2) and placed in a 1.5 mL centrifuge tube; subsequently, 50. Mu.L of 0.226M N-hydroxythiosuccinimide (sulfo-NHS) and 50. Mu.L of 0.09M carbodiimide (EDC) were added to the reaction solution, and the reaction was activated for 10 min under ultrasonic vibration at 4 ℃. Discarding the supernatant, and centrifuging at low temperature (4 ℃) to remove unreacted reagents;
2) The product from step 1) was redissolved in 400 μ L of sodium borate buffer (5 mM, pH = 8.0). Then, 20 μ L of AFP-conjugated antibody (finland Medix, available from shanghai youning vitamin science and technology ltd., product model 5107, concentration 5 mg/mL) was added to the solution, and incubated in 37 ℃ incubator 3 h;
3) Finally, 25. Mu.L of a blocking solution (0.01 mol/L phosphate buffer solution containing 0.5% by mass of bovine serum albumin and 5% by mass of sucrose, pH = 7.4) was added to perform blocking at 37 ℃ for 30 min, and the reaction was stopped with 12. Mu.L of ethanolamine for 30 min. After the whole antibody coupling experiment process is finished, the quantum dot-antibody fluorescent probe is stored in 50 μ L of preservation solution (0.005 mol/L borax buffer solution containing bovine serum albumin with the mass ratio of 0.5%, pH = 8.0) for subsequent testing.
The quantum dot-antibody fluorescent probe prepared in example 2 was subjected to the following test experiment.
A quantum dot based fluorescence immunoassay (QDs-FLISA) assay comprising the steps of:
1) Before the detection, AFP standard antigen was first diluted with a sample diluent (0.01 mol/L phosphate buffer containing 10% by volume of human negative serum, pH = 7.4) at a series of concentrations (here, 10 concentrations of 1, 2, 5, 10, 20, 50, 100, 200, 500, and 1000 ng/mL were diluted, respectively) to prepare standard solutions. 100. Mu.L of each of the series of standard solutions with different concentrations was added to the wells of the microplate and incubated in a constant temperature shaker at 37 ℃ for 30 min. Excess standard solution was then removed and blotted dry on absorbent paper, and 200. Mu.L of wash solution (1X 10 containing 0.05% Tween 20 by volume) was added to each well -5 mmol/mL phosphate buffer, pH 7.4) for 5 washes and patting dry again;
2) Next, the probe diluent (1X 10 containing 30% by volume of newborn calf serum) for the InP quantum dot-antibody fluorescent probe prepared in example 2 above was added -5 mmol/mL phosphate buffer solution with pH value of 7.4), adding 100 μ L into each well of the enzyme label plate, incubating in a constant temperature shaking table at 37 ℃ for 30 min, washing with washing solution for 5 times, and patting to dry;
3) The excitation wavelength was set at 450 nm and the fluorescence intensity of each well in the microplate was read automatically using SpectraMax-i 3. The collection wavelength range is 550-750 nm.
FIG. 5 is a graph of fluorescence change and standard curve of AFP antigen concentration measured by QDs-FLISA technology, in example 1, after ligand exchange (QDs-1) and photochemical treatment (QDs-2) are respectively coupled with fluorescent probes made of AFP. As can be seen from fig. 5: the effective detection range of the QDs-1-AFP-Ab probe is 5-100 ng/mL. In contrast, the fluorescence intensity of the QDs-2-AFP-Ab probe is highly linearly related to the AFP antigen concentration in the range of 1-1000 ng/mL, and a larger detection range is shown. Meanwhile, the calculation shows that the detection limit of the QDs-1-AFP-Ab antibody probe is 3.38 ng/mL, while the detection sensitivity of the QDs-2-AFP-Ab antibody probe is improved by about 5.8 times, and the detection limit is as low as 0.58 ng/mL. The quantum dot probe designed based on QDs-2 obtains a wider detection range and higher sensitivity, and is enough to meet the requirements of clinic for AFP antigen detection (in the serum of a normal person, the content of AFP is lower than 25 ng/mL, clinically, 400 ng/mL is generally taken as a standard, and the possibility of liver cancer should be considered above the value).
The target labeling experiment of the InP quantum dot-antibody fluorescent probe on the HepG2 cell comprises the following steps:
1) HepG2 cells in the logarithmic growth phase were taken and digested with pancreatin, then collected and made into single cell suspensions (5-10X 10) 4 one/mL), then 500. Mu.L of the InP quantum dot-antibody fluorescent probe is inoculated into a laser confocal dish, the laser confocal dish is placed into an incubator, the InP quantum dot-antibody fluorescent probe prepared in the example 2 is added into the dish after the cells are completely attached to the wall, and the InP quantum dot-antibody fluorescent probe is incubated in the incubator at 37 ℃ for 6 h. Then removing the redundant culture medium and washing off the unbound quantum dot-antibody fluorescent probe by soft washing for 3 times;
2) Adding Hoechst 33342 to perform cell nucleus staining;
3) Finally, the confocal laser microscope was placed under a confocal laser microscope for observation, and the results are shown in FIG. 6.
3. In vitro imaging of major organs of tumor-bearing mice comprising the steps of:
1) Taking out H22 murine liver cancer cells in liquid nitrogen, resuscitating in water bath at 37 ℃, centrifuging and removing supernatant. The cells were washed repeatedly with physiological saline three to four times until the supernatant was colorless. Then by physiologySaline resuspended cells at 2X 10 5 One/μ L, inoculated to the abdominal cavity of a mouse with a 1 mL syringe;
2) On the 7 th day after the mouse liver cancer H22 cells are planted in the abdominal cavity, the mouse is placed into 75% alcohol for soaking for 1-2 min, the mouse is taken into a super clean bench under the aseptic condition, a 5 mL injector is used for extracting ascites, the mouse is placed into a centrifugal tube, the cell is washed by normal saline, a liquid gun is fully and uniformly blown when the cell is washed, the washing frequency is that the centrifugal supernatant is colorless, the mouse is centrifuged for 5 min at the speed of 1000 r/min, the supernatant is poured out, and then the mouse is diluted by normal saline according to a certain proportion (the density is 1-2 × 10) 7 One/μ L of cells) was counted with a cell counting plate to prepare a tumor cell suspension, and 200 ten thousand cells, 0.2 mL/mouse, were inoculated to each mouse;
3) Female mice weighing 18-20 g Balb/c were selected and inoculated in the right forelimb axilla in a volume of 0.2 mL (about 200 ten thousand cells). During inoculation, the needle head is inserted into the subcutaneous space to a depth of about 1 cm, and is rotated to withdraw slowly, and the middle and back parts of the armpit are 100 mm after 3-5 days of inoculation 3 The experiment can be started by the lumps with the sizes of the soybean grains;
4) To assess the biodistribution of these probes in vivo, quantum dot-antibody fluorescent probes (i.e., QDs-1-AFP-Ab or QDs-2-AFP-Ab) after coupling with AFP antibody were injected into mice via the tail vein (injected at 5 mg/kg), 1, 2, 4, 6, 8, 12 h after injection, and the mice were dissected to obtain hearts (Heart), livers (Liver), spleens (spleens), lungs (Lung), kidneys (Kidney), and tumors (tumours), and fluorescence imaging was performed using the small animal in vivo imaging system (IVIS luminea XRMS Series III), with the results shown in fig. 6.
All procedures for animal studies were performed according to the guidelines for resource care and use of laboratory animals.
FIG. 6 is a graph showing in vitro imaging of cancer cells and imaging of various organs in mice of fluorescent probes prepared based on QDs-1 and QDs-2 in example 2.In fig. 6, a and b can be seen: the QDs-2 probe has obvious imaging effect in the liver cancer cells; in fig. 6 c, d can be seen: the strong and bright fluorescence signals are mainly gathered at the liver and tumor parts, and no obvious fluorescence signals exist in organs such as heart, spleen, lung and the like, which shows that the quantum dot probe has good effect of specifically targeting the liver cancer. After 1 h acts in a mouse body, the tumor part can be obviously gathered, the intensity of a fluorescence signal reaches a peak value after 6 h is detected, the fluorescence signal is gradually weakened after 12 h, and the metabolism is supposed to occur in the mouse body. The image of the corresponding 6 h clearly shows the imaging of the QDs-2 quantum dot probe on the whole tumor site, which indicates that the QDs-2-AFP-Ab probe has better tumor targeting. Based on this, the present invention is expected to provide accurate guidance for the physician to find and excise HCC lesions in the future.
Claims (9)
1. A synthetic method of a water-soluble InP core-shell quantum dot with high brightness and stability is characterized by comprising the following steps:
s1: obtaining oil-soluble InP core-shell quantum dots; the oil-soluble InP core-shell quantum dots are one or more of InP/ZnSe, inP/ZnS, inP/ZnSe/ZnSeSSN and InP/GaP/ZnS;
s2: mixing and reacting the water-soluble sulfydryl type coating with the oil-soluble InP core-shell quantum dots in a water phase to obtain an aqueous solution of the InP core-shell quantum dots; the S2 specifically comprises the following steps: dispersing oil-soluble InP core-shell quantum dots in n-hexane or octane, adding concentrated ammonia water and a water-soluble mercapto-group coating, and stirring in a water bath at 50-70 ℃ to react for 1-3 h; wherein the volume ratio of the concentrated ammonia water to the water-soluble sulfydryl coating is 3-7:1; the addition mass ratio of the oil-soluble InP core-shell quantum dots to the water-soluble mercapto coating is 1-50;
s3: and adding Zn-mercaptopropionic acid precursor liquid into the aqueous solution of the InP core-shell quantum dots, and then carrying out ultraviolet irradiation to obtain the InP core-shell quantum dots.
2. The method for synthesizing the water-soluble InP core-shell quantum dot with high brightness and stability as claimed in claim 1, wherein the water-soluble mercapto-based coating comprises mercaptopropionic acid or thioglycolic acid.
3. The method for synthesizing the water-soluble InP core-shell quantum dot with high brightness and stability as claimed in claim 1, wherein the zinc source in the Zn-mercaptopropionic acid precursor solution comprises zinc chloride, zinc acetate or zinc perchlorate.
4. The method for synthesizing the water-soluble InP core-shell quantum dot with high brightness and stability as claimed in claim 3, wherein S3 is specifically: and (3) purifying the aqueous solution of the InP core-shell quantum dots obtained in the step (S2) by using acetonitrile, dispersing the aqueous solution in ultrapure water again, adding Zn-mercaptopropionic acid precursor liquid, placing the solution in a water bath at the temperature of 60-80 ℃, and carrying out ultraviolet irradiation for 30-60 min under stirring.
5. The method for synthesizing the water-soluble InP core-shell quantum dot with high brightness and stability as claimed in claim 4, wherein the Zn-mercaptopropionic acid precursor solution in S3 is prepared as follows: stirring and dissolving a zinc source, mercaptopropionic acid and ultrapure water, wherein the mass ratio of the zinc source to the mercaptopropionic acid is 1:8-10.
6. The water-soluble InP core-shell quantum dot which is synthesized by the method of any one of claims 1 to 5 and has high brightness and stability.
7. A method for manufacturing a quantum dot-antibody fluorescent probe by using the water-soluble InP core-shell quantum dot as claimed in claim 6, which is characterized by comprising the following steps:
1) Dispersing water-soluble InP core-shell quantum dots in a sodium borate buffer solution, adding N-hydroxy thiosuccinimide and carbodiimide, carrying out an activation reaction for 8-20 min under ultrasonic vibration at the temperature of 2-6 ℃, removing supernatant, and centrifuging to remove unreacted reagents;
2) Dissolving the product obtained in the step 1) in a sodium borate buffer solution, adding a coupling antibody, and incubating 2-4 h in a 37 +/-2 ℃ thermostat; adding confining liquid, confining for 20-40 min, and terminating reaction with ethanolamine;
the conjugated antibody is one or more of AFP, CEA and CRP.
8. The quantum dot-antibody fluorescent probe prepared by the method of claim 7.
9. Use of the quantum dot-antibody fluorescent probe of claim 8 in the preparation of a tracer or an imaging agent.
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