CN113030036A - Antibody probe for tracing under near-infrared condition and preparation method and application thereof - Google Patents
Antibody probe for tracing under near-infrared condition and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an antibody probe for tracing under a near infrared condition, which is characterized in that the probe is a NaYF4 Yb, Ho @ NaGdF4-SCC antibody probe, a target head SCC antibody and NaYF4 Yb, Ho @ NaGdF4 crystals are jointed through EDC-NHS reaction by using rare earth down-conversion NaYF4 Yb, Ho @ NaGdF4 crystals, and the characteristic data of the probe is as follows: an emission peak at 1150nm and a particle size at 75 nm. The invention also discloses a preparation method and application thereof. The novel probe prepared by the invention can identify the squamous cell lung carcinoma cells by a non-invasive method, and has low biological toxicity. A rare earth down-conversion material is modified by a squamous cell carcinoma antibody target head to construct a NaYF4: Yb, Ho @ NaGdF4-SCC antibody probe, tracing is carried out under the near infrared condition, and identification is obtained through wavelength.
Description
Technical Field
The invention relates to the technical field of squamous cell lung carcinoma and nano rare earth materials, in particular to an antibody probe for tracing under the near infrared condition, a preparation method and application thereof.
Background
Near-infrared fluorescence NIRF (near-infrared fluorescence) is currently being studied more extensively in optical imaging, with wavelengths in the range of 700 and 1000 nm. In biological tissue, the wavelength range has low absorption and the tissue has little spontaneous fluorescence, so the penetration depth on the biological tissue is deep and the background interference can be reduced with little, thereby providing high signal-to-noise ratio. Compared with conventional tomography imaging technologies such as CT (computed tomography), PET-CT (position emission tomography), mri (magnetic resonance imaging), and the like, the optical imaging technology based on photoluminescence has the advantages of high feedback speed, high imaging resolution, high sensitivity, low cost, no radiation, and the like. The application of NIRF in bioluminescence imaging is mainly focused on the conventional near infrared window (NIR-I; lambda: 700 and 900nm), and research is expanded to the second near infrared window (NIR-II; lambda: 1000 and 1700 nm).
It further reduces tissue absorption, autofluorescence and scattering, and is particularly suitable for human applications. Due to its broad wavelength region, it can maximize the number of investigatable fluorescent probes. Nanoparticles containing NIRF dyes and anti-cancer agents are useful for the synergistic treatment of cancer, which can be integrated into imaging and therapeutic functions. Therefore, the current NIRF has wide application prospect in the biological field.
NIRF fluorescent dyes are divided into organic and inorganic dyes. The organic dye comprises cyanine dyes, phthalocyanines, squaraines, rhodamines, porphyrin derivatives, boron dipalmorphyrin methyl derivatives and the like, has certain chemical and light stability, wherein the cyanine dyes and the porphyrin derivatives are most widely applied, the fluorescence intensity of the organic dye is sharply reduced along with the increase of the irradiation time of an excitation light source, and the imaging time is short.
Inorganic dyes include heavy metal molecules such as selenium and cadmium, and such particles are highly cytotoxic and cannot be applied to biological tissues. And after the reaction with corresponding articles, the emission peak position and intensity of the inorganic nano rare earth materials are not influenced under the excitation of near infrared light, so that special species can be identified. Indocyanine green (ICG, Ind ℃green) is the only near-infrared optical imaging contrast enhancer approved by the U.S. Food and Drug Administration (FDA) for clinical use.
The nano-encapsulated ICG can show good tumor-specific imaging, but the chemical properties of ICG easily cause the disadvantages of high biological toxicity and difficult excretion.
The rare earth near-infrared luminescent material is a new material in the near-infrared fluorescence application field, has the advantages of good light stability, low self-fluorescence background and strong tissue penetration, becomes one of detection means with high selectivity and high sensitivity due to the advantages, and is expected to replace organic NIRF fluorescent dye and quantum dot material.
The unique electronic structure of rare earth makes it have unique spectral property, low self light absorption capacity and very small molar extinction coefficient. The rare earth NIRF probe has deep spatial resolution for detecting the distribution of tumor cells, and can realize high-sensitivity noninvasive visualization. Such as a NIRF probe based on gadolinium, loaded with nanoparticles, due to their unique structure, have unique optical properties that allow for accurate detection and treatment of cancer.
The monodisperse spherical nanoparticles formed by self-assembly of gold nanoclusters mediated by gadolinium ions show remarkable enhancement of luminous intensity in NIRF imaging, higher X-ray attenuation in CT imaging and reasonable R1 relaxivity in MR imaging, and have great application potential for polymorphic clinical diagnosis of tumors. The degree of anastomoses of NIRF in tissue applications such as arthritis at the lesion site is highly consistent with that at the site of pathologically confirmed diagnosis.
NIRF imaging has been less studied in lung cancer, but since lung cancer is always the first in new worldwide cases every year, it is particularly necessary to explore the use of NIRF in the field of lung cancer, particularly in cancer cell localization, assisted surgery, etc. because of its advantages, it is worth further study.
Cohen identified lung cancer cell lines with high endogenous delta expression using the binding affinity of the lanthanide time-resolved fluorescence competitive binding assay delta, showing that NIRF has good selectivity for cancer cells. The NIRF is combined with nanoparticles of an anti-epidermal growth factor receptor (anti-EGFR), has a targeting induction function on the EGFR, and has strong targeting detection capability. In a more studied in vivo in situ growth model of Lewis lung cancer cell mice, the NIRF probe can track the tumor progression in a targeted manner. In some cases of targeted drug resistance, the near-infrared fluorescent photosensitizer is combined with a molecular targeted drug, the targeted drug can be induced to enter tumor cells to generate strong fluorescence, and then the targeted therapy and the photodynamic therapy are combined to overcome the drug resistance.
At present, the imaging diagnosis technology based on the rare earth nanoparticle carrier is not reported in the lung cancer. The lung cancer is classified into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC), among which squamous cell carcinoma is a common type.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention utilizes rare earth down-conversion nanocrystals in combination with SCC antibody target heads to prepare down-conversion nanocrystal complexes, so as to identify squamous cell lung cancer by constructing NaYF4: Yb, Ho @ NaGdF4-SCC antibody probes.
In order to realize the purpose of the invention, the adopted technical scheme is as follows:
an antibody probe for tracing under the near infrared condition is disclosed, which is
NaYF4 Yb, Ho @ NaGdF4-SCC antibody probe by down-conversion using rare earth
Reaction of NaYF4 Yb, Ho @ NaGdF4 crystals in EDC-NHS
The probe is obtained by crystal bonding of NaYF4, Ho @ NaGdF4, and the characterization data are as follows: an emission peak at 1150nm and a particle size at 75 nm.
A preparation method of an antibody probe for tracing under the near infrared condition comprises the following steps:
the method comprises the following steps:
firstly, respectively preparing a Gd-OA precursor and a Na-TFA-OA precursor for standby,
the Gd-OA precursor is a gadolinium-oleic acid precursor of 0.1M;
the Na-TFA-OA precursor is a sodium trifluoroacetate-oleic acid precursor.
Step two:
preparing a core-shell structure efficient anti-quenching holmium-doped down-conversion fluorescent nanocrystal by taking the Gd-OA precursor and the Na-TFA-OA precursor as shell layers;
step three:
performing surface modification on the down-conversion fluorescent nano crystal to obtain a water-soluble efficient anti-quenching down-conversion near-infrared fluorescent crystal, wherein the water-soluble efficient anti-quenching down-conversion near-infrared fluorescent crystal is a rare earth down-conversion NaYF4: Yb, Ho @ NaGdF4 crystal;
step four:
and (3) jointing the target SCC antibody with NaYF4: Yb, Ho @ NaGdF4 crystals through EDC-NHS reaction to obtain a NaYF4: Yb, Ho @ NaGdF4-SCC antibody probe.
In a preferred embodiment of the invention, the Gd-OA precursor is prepared by the following method:
2.5mmol of GdCl are initially introduced into the vessel3·6H2O (gadolinium trichloride hexahydrate), 10mL oleic acid, and 15mL octadecene;
followed by heating to 140 ℃ and holding for at least 1 hour under vacuum with magnetic stirring, then a clear and transparent 0.1M Gd-OA precursor was obtained.
In a preferred embodiment of the present invention, the Na-TFA-OA precursor is prepared by the following method:
4mmol of sodium trifluoroacetate and 10mL of oleic acid are added into a container in sequence, the room temperature is kept, and the components are dissolved under the conditions of vacuum environment and stirring until a clear, colorless and transparent Na-TFA-OA precursor solution is finally obtained.
In a preferred embodiment of the invention, the holmium-doped down-conversion fluorescent nanocrystal with a core-shell structure and high efficiency anti-quenching performance is prepared by the following steps:
0.93mmol YCl was first added to the vessel3·6H2O (yttrium trichloride hexahydrate) and 0.05mmol of YbCl3 & 6H2O (ytterbium trichloride hexahydrate) and 0.02mmol of HoCl3·6H2O (holmium chloride hexahydrate), then 6mL of oleic acid and 15mL of octadecene are added, stirred and heated to 140 ℃, and a transparent mixed solution is obtained after dehydration and deoxidation treatment in a vacuum environment;
cooling the mixed solution to room temperature, adding a 10mL methanol mixed solution dissolved with 2.5mmol of NaOH and 4mmol of NH4F into the mixed solution, and stirring for continuous reaction;
then heating to 80 ℃ and removing methanol under vacuum condition;
under the protection of high-purity argon gas, the temperature is increased to 285 ℃ at the speed of 10 min-1 to react until the reaction is finished, the temperature is reduced to 50 ℃, ethanol solution is added to precipitate out a product, and then the product is centrifuged,
repeatedly washing the product by absolute ethyl alcohol, and dispersing the product in a cyclohexane solution for later use, wherein the concentration of the initial product of the cyclohexane solution is about 0.5 mmol;
adding 5mL of the prepared cyclohexane solution into 8mL of oleic acid and 12mL of octadecene solution, mixing, heating to 70 ℃, and removing cyclohexane and oxygen in a reaction system in a vacuum environment;
then heating the reaction system to 280 ℃ at the speed of 20 ℃ min < -1 >;
then, 1mL of Gd-OA precursor and 0.5mL of Na-TFA-OA precursor are alternately added into the reactants, and the intermittent time of each adding is 15 min;
and adding 4 rounds of shell layer precursors to obtain the holmium-doped down-conversion fluorescent nanocrystal with the core-shell structure and high-efficiency anti-quenching performance.
In a preferred embodiment of the present invention, the surface modification is specifically:
adding 1mL of cyclohexane solution containing the holmium-doped down-conversion fluorescent nanocrystal with efficient anti-quenching core-shell structure and 1mL of DSPE-PEG-COOH (1, 2-distearoyl-SN-glycerol-3-phosphorylethanolamine-N-carboxyl-polyethylene glycol) chloroform solution into a container, wherein the concentration of the solution is 12.5mg mL-1, uniformly mixing, and volatilizing the solvent;
then adding 1mL of ultrapure water to dissolve the product in water by thick ultrasonic treatment, stirring at 75 ℃, transferring the solution into a centrifuge tube, centrifuging at the speed of 14500rpm to remove the precipitate so as to remove the agglomerate, and obtaining the water-soluble high-efficiency anti-quenching down-conversion near-infrared fluorescent crystal.
In a preferred embodiment of the invention, the EDC-NHS reaction is:
dissolving the SCC antibody in 10mL MES (2-Morpholinoethanesulfonic Acid ) buffer at a concentration of 0.1M and a pH of 5;
then adding 8mg of EDC, uniformly mixing, then adding 8mg of NHS, and reacting for 30 min;
meanwhile, 1mmol of down-conversion fluorescent nanocrystals were dispersed in 5mL of MES buffer, and the above target solution was added rapidly, and reacted at room temperature with rapid stirring for 2 h. After the reaction is finished, centrifuging and washing for 2 times by using MES buffer solution;
finally, preparing the NaYF4 Yb, Ho @ NaGdF4-SCC antibody probe.
Use of an antibody probe for tracking under near infrared conditions, wherein the use is in identifying squamous cell lung carcinoma under near infrared fluorescent NIRF conditions.
The invention has the beneficial effects that:
the novel probe prepared by the invention can identify the squamous cell lung carcinoma cells by a non-invasive method, and has low biological toxicity.
A rare earth down-conversion material is modified by a Squamous Cell Carcinoma (SCC) antibody target head to construct a NaYF4: Yb, Ho @ NaGdF4-SCC antibody probe, tracing is carried out under the near infrared condition, and identification is obtained through wavelength.
Drawings
Fig. 1 is a visible light luminescence diagram of holmium-doped down-conversion fluorescent nanocrystals (in the figure, a white aperture is a luminescent region).
Fig. 2 is a near-infrared spectrum of a holmium-doped down-conversion fluorescent nanocrystal.
FIG. 3 is an electron micrograph of NaYF4: Yb, Ho @ NaGdF4-SCC antibody probe (200 nm).
Fig. 4 is a graph of near-infrared peak values of holmium-doped down-conversion fluorescent nanocrystals.
FIG. 5 is a bright field image of lung squamous carcinoma cells.
FIG. 6 is a DAPI map of squamous cell lung carcinoma cells.
FIG. 7 is a NIRF map of squamous cell lung carcinoma cells.
FIG. 8 shows the brightfield, DAPI, and NIRF fusion of lung squamous carcinoma cells.
Detailed Description
1. Probe preparation
1.1 preparation of Shell precursor Material
Preparation of Gd-OA (Gadolinium-Oleic acid, 0.1M) precursor:
in a 50mL 3-neck round-bottom flask, 2.5mmol of GdCl was first added3·6H2O (Gadolinium (III) chloride hexahydrate, gadolinium trichloride hexahydrate), 10mL oleic acid, 15mL octadecene.
Subsequently, the mixture was heated to 140 ℃ under vacuum and magnetic stirring and kept for 1 hour, and then a clear and transparent Gd-OA (0.1M) precursor was obtained.
Preparation of Na-TFA-OA (Sodium-trifluoracetic-Oleic acid, Sodium trifluoroacetate-Oleic acid) precursor:
in a 25mL 3-neck round-bottom flask, 4mmol of sodium trifluoroacetate and 10mL of oleic acid are added in sequence, kept at room temperature and dissolved under vacuum and stirring conditions until a clear, colorless and transparent Na-TFA-OA precursor solution is finally obtained.
1.2. Synthesis of holmium-doped down-conversion fluorescent nanocrystal
In a 50mL 3-neck round-bottom flask, 0.93mmol of YCl was first added3·6H2O (yttrium trichloride hexahydrate) and 0.05mmol of YbCl3 & 6H2O (ytterbium trichloride hexahydrate) and 0.02mmol HoCl3·6H2O (holmium chloride hexahydrate), followed by 6mL of oleic acid, 15mL of octadecene. And stirring and heating the mixture of the samples to 140 ℃, dehydrating and deoxidizing for 60min under a vacuum environment to obtain a transparent mixed solution. After the transparent mixed solution is cooled to room temperature, a 10mL methanol mixed solution containing 2.5mmol of NaOH and 4mmol of NH4F is added into the obtained mixed solution for continuous reaction. Continuously stirring for 20min, heating to 80 deg.C, removing methanol in vacuum environment for 60min, heating the reactant to 285 deg.C at 10 deg.C min-1 under the protection of high purity argon gas, and reacting for 100 min. After the reaction is finished, cooling the reaction liquid to 50 ℃, adding an ethanol solution to precipitate and separate out a corresponding product from the solution, then centrifuging, repeatedly washing by absolute ethanol for 2 times to obtain a product, and dispersing the product in 10mL of cyclohexane solution for later use.
5mL of the prepared cyclohexane solution (the concentration of the dispersion in the cyclohexane solution is about 0.5mmol) is added into 8mL of oleic acid and 12mL of octadecene solution, mixed and heated to 70 ℃, and kept for 30min under a vacuum environment to remove cyclohexane and oxygen in the reaction system. The reaction system was then heated to 280 ℃ by a rate of 20 ℃ min-1. Then, 1mL of Gd-OA precursor and 0.5mL of Na-TFA-OA precursor were alternately added to the above reaction flask. The intermittent time of each filling is 15 min. After 4 rounds of shell layer precursors are added, the holmium-doped down-conversion fluorescent nanocrystal with a core-shell structure and high-efficiency anti-quenching performance can be obtained.
1.3. Surface modification of down-converting fluorescent nanocrystals
The surface modification of the efficient anti-quenching doped down-conversion near-infrared fluorescent crystal is carried out by taking a 10mL single-neck flask as a reaction container, adding 1mL cyclohexane solution and 1mL of a chloroform solution (12.5mg mL-1) of DSPE-PEG-COOH (1, 2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-carboxyl-polyethylene glycol) and uniformly mixing. The flask was opened to the air and stirred slowly for 2 days to evaporate the solvent, and the flask was placed in a 75 ℃ oven for 5min to ensure complete removal of the solvent. 1mL of ultrapure water was added to the reaction flask, the product was dissolved in water by sonication, and stirred vigorously for 10min at 75 ℃ with an open mouth. And transferring the solution into a centrifuge tube, centrifuging at high speed (14500rpm for 10min), discarding the precipitate to remove the agglomerates, and obtaining the water-soluble high-efficiency anti-quenching down-conversion near-infrared fluorescent crystal. The concentration of the nanocrystals in the solution needs to be quantitatively determined by subsequent detection.
By EDC-NHS
(1-Ethyl-3- (3' -methylenepropyl) carbodiimide-N-Hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride-N-Hydroxysuccinimide) reaction the targeting SCC antibody was grafted onto the down-converting nanocrystals by the following procedure:
SCC antibody was dissolved in 10mL MES (2-Morpholinoethanesulfonic Acid ) buffer (concentration of buffer 0.1M, pH 5), followed by addition of 8mg EDC, stirring for 5min, addition of 8mg NHS, and reaction for 30 min. Meanwhile, 1mmol of down-conversion fluorescent nanocrystals were dispersed in 5mL of MES buffer, and the above target solution was added rapidly, and reacted at room temperature with rapid stirring for 2 h.
After the reaction was completed, it was centrifuged and washed 2 times with MES buffer. Finally, preparing the NaYF4 Yb, Ho @ NaGdF4-SCC antibody probe.
2. Testing and characterization of probes
Characterization of Transmission Electron Microscope (TEM), High Resolution Transmission Electron Microscope (HRTEM), Selective Area Electron Diffraction (SAED), high angle annular dark field-scanning transmission electron microscope (HAADF-STEM), X-ray energy spectrum (EDS) was done by JEM-2100F type transmission electron microscope (acceleration voltage 200 kV).
Preparation of a sample:
the sample was dispersed in cyclohexane or aqueous solution, 10uL was pipetted onto a carbon film-loaded copper mesh using a pipette, dried and used directly for observation. Characterization by X-ray powder diffraction (XRD) measured by X-ray powder diffractometer model D8 (CuK alpha, wavelength: X)
)。
The absorption spectrum was measured by a Lambda35 model UV-VIS-NIR spectrophotometer. The near infrared fluorescence spectrum is measured by an FLS 980 type fluorescence spectrometer which can be externally connected with a 808nm or 980nm continuous laser (the maximum power is 2W).
The concentration of the nanocrystals in the solution was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES) model IRISAdvantageDuoER/S. Near infrared imaging was measured by a nirvana ccd camera.
3. Cellular imaging of probes
Cell imaging was performed by incubating the synthesized down-converted fluorescent nanocrystals with cells for 2 hours, centrifuging to wash free nanocrystals, and then placing the enriched cells in a confocal petri dish for infrared imaging using a microscope (Olympus IX71) coupled with a NIR CCD camera (Princeton Instruments Inc.). An 808nm continuous laser, a 980 laser, was used as the excitation source and equipped with two long-wavelength pass filters (1000LP, Chroma Corp and 1200LP, thorlabs).
4. Probe NIRF coloration
According to the invention, for the down-conversion material, the pure holmium-doped down-conversion near-infrared fluorescent crystal with the core-shell structure and high-efficiency anti-quenching performance is prepared, the composite solution is transferred to a centrifugal tube by a chemical method to be centrifuged at high speed, and the precipitate is discarded to remove the agglomerate, so that the water-soluble high-efficiency anti-quenching down-conversion near-infrared fluorescent crystal is obtained.
The visible luminescence diagram is shown below (see fig. 1), and the near infrared light image is shown below (see fig. 2).
The rare earth down-conversion NaYF4 is carried out on Yb, Ho @ NaGdF4 crystals, a targeting SCC antibody is combined with NaYF4: Yb, Ho @ NaGdF4 crystals through EDC-NHS reaction to obtain a NaYF4: Yb, Ho @ NaGdF4-SCC antibody probe, and an electron microscope picture is shown as below (see figure 3). The near infrared wavelengths are shown below (see FIG. 4).
Then, the obtained lung squamous carcinoma cells are cultured in a culture medium in advance, and DAPI (4' 6-diamidino-2-phenylindole) staining and NIRF color development are carried out on the lung squamous carcinoma cells at the cell level, so that the NaYF4: Yb, Ho @ NaGdF4-SCC antibody probe can be accurately identified in the lung squamous carcinoma cells (see the figure 5-8).
Claims (8)
1. The probe is an NaYF4: Yb, Ho @ NaGdF4-SCC antibody probe, and is obtained by utilizing rare earth down-conversion NaYF4: Yb, Ho @ NaGdF4 crystals to perform EDC-NHS reaction to join a target head SCC antibody and NaYF4: Yb, Ho @ NaGdF4 crystals, wherein the characterization data of the probe are as follows: an emission peak at 1150nm and a particle size at 75 nm.
2. The method for preparing an antibody probe for tracking under near infrared conditions according to claim 1, comprising the steps of:
the method comprises the following steps:
firstly, respectively preparing a Gd-OA precursor and a Na-TFA-OA precursor for standby,
the Gd-OA precursor is a gadolinium-oleic acid precursor of 0.1M;
the Na-TFA-OA precursor is a sodium trifluoroacetate-oleic acid precursor.
Step two:
preparing a core-shell structure efficient anti-quenching holmium-doped down-conversion fluorescent nanocrystal by taking the Gd-OA precursor and the Na-TFA-OA precursor as shell layers;
step three:
performing surface modification on the down-conversion fluorescent nano crystal to obtain a water-soluble efficient anti-quenching down-conversion near-infrared fluorescent crystal, wherein the water-soluble efficient anti-quenching down-conversion near-infrared fluorescent crystal is a rare earth down-conversion NaYF4: Yb, Ho @ NaGdF4 crystal;
step four:
and (3) jointing the target SCC antibody with NaYF4: Yb, Ho @ NaGdF4 crystals through EDC-NHS reaction to obtain a NaYF4: Yb, Ho @ NaGdF4-SCC antibody probe.
3. The method of claim 2, wherein the Gd-OA precursor is prepared by the following steps:
2.5mmol of GdCl are initially introduced into the vessel3·6H2O, 10mL oleic acid, and 15mL octadecene;
followed by heating to 140 ℃ and holding for at least 1 hour under vacuum with magnetic stirring, then a clear and transparent 0.1M Gd-OA precursor was obtained.
4. The method of claim 2, wherein the Na-TFA-OA precursor is prepared by the following steps:
4mmol of sodium trifluoroacetate and 10mL of oleic acid are added into a container in sequence, the room temperature is kept, and the components are dissolved under the conditions of vacuum environment and stirring until a clear, colorless and transparent Na-TFA-OA precursor solution is finally obtained.
5. The method for preparing an antibody probe for tracking under the near-infrared condition as claimed in claim 2, wherein the core-shell structure efficient anti-quenching holmium-doped down-conversion fluorescent nanocrystal is prepared by the following steps:
0.93mmol YCl was first added to the vessel3·6H2O, 0.05mmol of YbCl3 & 6H2O and 0.02mmol HoCl3·6H2O, then adding 6mL of oleic acid and 15mL of octadecene, stirring, heating to 140 ℃, dehydrating and deoxidizing in a vacuum environment to obtain a transparent mixed solution;
cooling the mixed solution to room temperature, adding a 10mL methanol mixed solution dissolved with 2.5mmol of NaOH and 4mmol of NH4F into the mixed solution, and stirring for continuous reaction;
then heating to 80 ℃ and removing methanol under vacuum condition;
under the protection of high-purity argon gas, the temperature is increased to 285 ℃ at the speed of 10 min-1 to react until the reaction is finished, the temperature is reduced to 50 ℃, ethanol solution is added to precipitate out a product, and then the product is centrifuged,
repeatedly washing the product by absolute ethyl alcohol, and dispersing the product in a cyclohexane solution for later use, wherein the concentration of the initial product of the cyclohexane solution is about 0.5 mmol;
adding 5mL of the prepared cyclohexane solution into 8mL of oleic acid and 12mL of octadecene solution, mixing, heating to 70 ℃, and removing cyclohexane and oxygen in a reaction system in a vacuum environment;
then heating the reaction system to 280 ℃ at the speed of 20 ℃ min < -1 >;
then, 1mL of Gd-OA precursor and 0.5mL of Na-TFA-OA precursor are alternately added into the reactants, and the intermittent time of each adding is 15 min;
and adding 4 rounds of shell layer precursors to obtain the holmium-doped down-conversion fluorescent nanocrystal with the core-shell structure and high-efficiency anti-quenching performance.
6. The method for preparing an antibody probe for tracking under near infrared conditions as claimed in claim 2 or 5, wherein the surface modification is specifically:
adding 1mL of cyclohexane solution containing the holmium-doped down-conversion fluorescent nanocrystal with efficient anti-quenching core-shell structure and 1mL of DSPE-PEG-COOH (1, 2-distearoyl-SN-glycerol-3-phosphorylethanolamine-N-carboxyl-polyethylene glycol) chloroform solution into a container, wherein the concentration of the solution is 12.5mg mL-1, uniformly mixing, and volatilizing the solvent;
then adding 1mL of ultrapure water, dissolving the product in water by ultrasonic treatment, stirring at 75 ℃, transferring the solution into a centrifuge tube, centrifuging at 14500rpm to remove precipitates and obtain the water-soluble high-efficiency anti-quenching down-conversion near-infrared fluorescent crystal.
7. The method of claim 2, wherein the EDC-NHS reaction is:
dissolving the SCC antibody in 10mL MES buffer solution, wherein the concentration of the buffer solution is 0.1M, and the pH value is 5;
then adding 8mg of EDC, uniformly mixing, then adding 8mg of NHS, and reacting for 30 min;
meanwhile, 1mmol of down-conversion fluorescent nanocrystals were dispersed in 5mL of MES buffer, and the above target solution was added rapidly, and reacted at room temperature with rapid stirring for 2 h. After the reaction is finished, centrifuging and washing for 2 times by using MES buffer solution;
finally, preparing the NaYF4 Yb, Ho @ NaGdF4-SCC antibody probe.
8. Use of an antibody probe for tracking under near infrared conditions according to any one of claims 1 to 8 for identifying squamous cell lung carcinoma under near infrared fluorescent NIRF conditions.
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