CN114075434A - Up-conversion luminescence nanocrystalline material and application thereof in specific immunoassay - Google Patents

Up-conversion luminescence nanocrystalline material and application thereof in specific immunoassay Download PDF

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CN114075434A
CN114075434A CN202010823006.4A CN202010823006A CN114075434A CN 114075434 A CN114075434 A CN 114075434A CN 202010823006 A CN202010823006 A CN 202010823006A CN 114075434 A CN114075434 A CN 114075434A
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王从相
王清清
王双兰
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Wenzhou Guangli Biomedical Technology Co ltd
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Abstract

The application relates to the field of a novel nanocrystalline material and instant detection thereof, in particular to an up-conversion luminescence nanocrystalline material, a preparation method thereof and application thereof in specific immunodetection of chronic obstructive pulmonary disease. An up-conversion luminescence nanocrystal material, the molecular formula of the nanocrystal is CaClF: Bi/Er @ SiO2. The surface of the nanocrystalline is coated with SiO2Obtaining a large number of hydrophilic hydroxyl groups which can be covalently coupled with antibody proteins to formThe fluorescence labeling antibody is used for specifically binding and detecting a target antigen and has good biocompatibility.

Description

Up-conversion luminescence nanocrystalline material and application thereof in specific immunoassay
Technical Field
The application relates to the field of a novel nanocrystalline material and instant detection thereof, in particular to an up-conversion luminescence nanocrystalline material, a preparation method thereof and application thereof in specific immunodetection of chronic obstructive pulmonary disease.
Background
Rare earth fluorides serve as an excellent down-and up-conversion matrix material. Rare earth ion doped fluorides have broad prospects in biological applications and photonic devices, particularly in bioluminescent labeling, surgical equipment and medical analysis involving optical elements. The advantages of low autofluorescence background, large anti-Stokes shift, sharp emission bandwidth, high photobleaching resistance, high penetration depth and time resolution of the rare earth fluoride nano-crystal are excellent substitutes for organic fluorophores and quantum dots, but the morphology of the rare earth fluoride nano-crystal is influenced by various factors, and in addition, although the rare earth doped fluoride nano-crystal has wide application in the aspect of applying biological fluorescence labeling, the application in the immune test paper for instant detection is still to be further researched.
The point-of-care test (POC) test has the characteristics of rapidness, portability, easy use, low cost, good field performance and the like, and in various paper-based POC tests, the specific immune test paper (LFS) is simpler, quicker and cheaper, can detect a plurality of targets within 20 minutes, and can obtain results under simple step operation. Therefore, the specific immunity test paper is a promising household detection platform. Among the numerous specific immunoassay strip fluorescent markers, LFS based on gold nanoparticles (AuNPs) has been successfully converted into a large number of commercial products due to its low cost and visual readability of the detection results. In addition, the technology can also detect multiple analytes by designing multiple test lines or an integration of multiple strips. However, gold nanoparticles (AuNPs) have limited LFS sensitivity and can only achieve qualitative or semi-quantitative detection, thus limiting their application in disease and quantitative prediction. In recent years, various fluorescent markers have been used instead of AuNPs to improve the sensitivity and quantitative detection capability of the test strip. However, fluorescent labels, such as fluorescent dyes and Quantum Dots (QDs), are now used which are excited by ultraviolet light, but they produce a strong background signal. In addition, fluorescent dyes are associated with photobleaching and are not stable on storage at room temperature. In contrast, upconversion nanoparticles (UCNPs) emit visible light, rather than ultraviolet light, under excitation by Near Infrared (NIR) light. The unique optical characteristics minimize background fluorescence, provide high photostability, improve signal-to-noise ratio, and allow sensitive detection in complex biological samples. Therefore, LFS based on UCNPs have received increasing attention in recent years, and their wide application includes detection of fish infections, various antibiotics, parasitic infections, zoonotic pathogens, and the like. However, no test strip for detecting diseases such as chronic obstructive pulmonary disease has been studied. Chronic obstructive pulmonary disease is a fatal chronic disease that requires long-term monitoring of a patient's health. However, the lack of effective monitoring methods for the immediate detection of chronic obstructive pulmonary disease has greatly limited their use in immediate detection.
Disclosure of Invention
In order to solve the above technical problems, an object of the present application is to provide an upconversion luminescent nanocrystal material, wherein the nanocrystal has a molecular formula of CaClF: Bi/Er @ SiO2By wrapping a layer of SiO outside the nanocrystal2So that a large number of hydrophilic hydroxyls are obtained, the fluorescent conjugated antibody can be covalently coupled with antibody protein to form a fluorescent labeled antibody for specifically binding and detecting a target antigen, and the fluorescent labeled antibody has good biocompatibility. The application also provides a preparation method and application of the nanocrystal, and the formed nanocrystal fluorescent marker is used for preparing specific immune test paper, and has the advantages of simplicity, rapidness and accuracy in the instant detection of certain diseases such as chronic obstructive pulmonary disease.
In order to achieve the above object, the present application adopts the following technical solutions:
an up-conversion luminescence nanocrystal material, the molecular formula of the nanocrystal is CaClF: Bi/Er @ SiO2。
Preferably, the molecular formula of the nanocrystal is CaClF: 10Bi/20Er @ SiO2。
Preferably, the nanocrystal is a water-soluble nanocrystal.
Further, the present application provides a method for preparing an upconversion luminescent nanocrystalline material, comprising the following steps:
1) adding bismuth acetate and erbium acetate with required doping amounts, 0.7 mmol of calcium acetate, 2 ml of trifluoroacetic acid, 2 ml of trichloroacetic acid, 10 ml of oleic acid and 25 ml of octadecene into a three-neck flask, stirring at 100 ℃ under the condition of nitrogen protection atmosphere, keeping the temperature for 90 minutes, then rapidly heating to 180 ℃, and keeping the temperature for 1 hour;
2) and after the solution is cooled to room temperature, washing for 3-5 times by using a mixed solution of ethanol and deionized water to obtain oily fluorochloride nanocrystalline CaClF: Bi/Er;
3) 0.1 ml of CO-520, 6 ml of cyclohexane and 4 ml (0.01M) of CaClF: adding the Bi/Er nano-particles into a beaker, stirring for 10 minutes, adding 0.4 ml of CO-520 and 0.08 ml of ammonia water, sealing the beaker, and performing ultrasonic treatment for 20 minutes until the solution becomes clear from milky white;
4) then 0.04 ml TEOS is added into the solution, the beaker is sealed, the reaction is carried out for 2 days at the rotating speed of 600 rpm, after the reaction is finished, acetone is added into the mixed solution for precipitation, and the mixed solution is dispersed in deionized water after being washed for 2 times by ethanol/deionized water (1: 1 v/v).
Preferably, the method further comprises a step of surface treatment of the nanocrystals in step 2): and (3) mixing the prepared 10-100mg of nanocrystalline CaClF: dispersing Bi/Er in a mixed solution of 1-3mL of ethanol and 0.5-1mL of HCl with the concentration of 0.2mol/L, carrying out ultrasonic treatment on the mixed solution for 5-10min, and then adding ethanol for washing, centrifuging, drying and the like to obtain the water-based nanocrystal.
Further, the application provides an application of the nanocrystalline material in preparation of specific immunity test paper.
Further, the application provides an application of the nanocrystalline material in specific immunoassay of chronic obstructive pulmonary disease.
Further, the application provides a specific immunoassay kit comprising the nanocrystalline material.
Further, the application provides a specific immunoassay kit for chronic obstructive pulmonary disease, which comprises the nanocrystalline material.
The present application prepares CaClF by a solvothermal process: and (3) treating the Bi/Er nanocrystalline by using an HCl solution to remove an oleic acid ligand on the surface of the nanocrystalline, wherein the nanocrystalline can be well dispersed in an aqueous solution. And wrapping SiO on the surface2Obtaining the CaClF: Bi/Er @ SiO2To obtain a largeThe hydrophilic hydroxyl group can be covalently coupled with antibody protein to form a fluorescence labeling antibody for specifically combining and detecting a target antigen, and has good biocompatibility.
Drawings
Fig. 1 (a) and (b) are CaClF: XRD pattern and transmission electron microscopy pattern of 10Bi/20Er nanocrystalline, (c) CaClF: 10Bi/20Er @ SiO2Transmission electron microscopy of nanocrystals.
Fig. 2 (a) CaClF: the up-conversion emission spectrum of 10Bi/20Er nanocrystalline under the excitation condition of 1530nm near-infrared laser, (b) the up-conversion emission intensity is along with Er3+The variation curve of ion doping concentration, (c) the physical schematic diagram of up-conversion luminescence machine, (d) the luminous intensity of the product along with the doping ion Bi3+The variation of (2).
FIG. 3 is an assessment of the specificity of LFS for FIB antigen and different hormones. Three interference biomarkers of GC, TRH and TSH are selected, the concentration of the three interference biomarkers is 0.5IU/ml (the concentration of the three interference biomarkers is 10 times of the clinical critical value), and the selectivity of LFS is verified. I is the fluorescence intensity of the test article, I0Background fluorescence intensity.
FIG. 4 shows fluorescence spectra of test strip detection bands (T line) at different concentrations (100 mIU/ml, 500 mIU/ml, 1 IU/ml, 5IU/ml, 10 IU/ml and 25 IU/ml) of standard samples added dropwise.
FIG. 5 shows the relationship b) between the fluorescence intensity a) and the concentration of FIB antigen in the detection zone of the test strip, which is obtained by quantifying the fluorescence intensity of the detection zone.
Detailed Description
1. Experimental part
1.1 main raw materials and reagents:
bismuth acetate (99.0%), trichloroacetic acid (99.0%), trifluoroacetic acid (99.0%), calcium acetate (99.0%), erbium acetate (99.9%), alkylphenol ethoxylate (Igepal CO-520), oleic acid (90.0%) and octadecene (90.0%) were purchased from Sigma-Aldrich, goat anti-mouse IgG, specific antibody protein 1, specific antibody protein 2 from glerin biotechnology limited, and anhydrous ethanol, tetraethyl orthosilicate (TEOS) and concentrated hydrochloric acid from national drug group chemical agents limited.
1.2 CaClF：Bi/Er@SiO2Preparing a nanocrystal:
the method comprises the following steps of mixing the components of CaClF: taking 10Bi/20Er nanocrystals as an example, 0.1 mmol of bismuth acetate, 0.7 mmol of calcium acetate, 0.2 mmol of erbium acetate, 2 ml of trifluoroacetic acid, 2 ml of trichloroacetic acid, 10 ml of oleic acid and 25 ml of octadecene are added into a three-neck flask, stirred and kept at 100 ℃ for 90 minutes under the condition of nitrogen protection atmosphere, and then rapidly heated to 180 DEG C
Figure DEST_PATH_IMAGE001
And incubated for 1 hour. And after the solution is cooled to room temperature, washing for 3-5 times by using a mixed solution of ethanol and deionized water to obtain oily fluorochloride nanocrystalline CaClF: 10Bi/20 Er; 0.1 ml of CO-520, 6 ml of cyclohexane and 4 ml (0.01M) of CaClF: adding 10Bi/20Er nano-particles into a beaker, stirring for 10 minutes, adding 0.4 ml of CO-520 and 0.08 ml of ammonia water, sealing the beaker, and performing ultrasonic treatment for 20 minutes until the solution becomes clear from milky white; and adding 0.04 ml of TEOS into the solution, sealing the beaker, reacting at the rotating speed of 600 rpm for 2 days, adding acetone into the mixed solution after the reaction is finished, precipitating, washing for 2 times by using ethanol/deionized water (1: 1 v/v), and dispersing in the deionized water to obtain the CaClF: 10Bi/20Er @ SiO2And (4) nanocrystals.
And doping the sample with ions of different concentrations or species by changing the corresponding ion concentration or species in the precursor solution.
And (3) surface treatment of the nanocrystalline: 10-100mg of oily fluorochloride nanocrystalline CaClF: dispersing 10Bi/20Er in a mixed solution of 1-3mL ethanol and HCl (0.2 mol/L, 0.5-1 mL), carrying out ultrasonic treatment on the mixed solution for 5-10min, and then adding ethanol for washing, centrifuging, drying and the like to obtain the water-based nanocrystal.
1.3 characterization Instrument
X-ray diffraction patterns (bruker d8Advance, Cu-K α (λ =1.5405 a)), transmission electron microscope (TEM, feitecnag 2F 20), spectrometer (FLUROHUB-B, horiba jobinyvon), ultraviolet lamp power 50W, 1530nm near infrared laser (power range 0-1W).
Preparation of X-ray diffraction samples: paving the dried nanocrystalline in the groove of the sample support;
preparation of transmission electron microscope samples: dissolving all the nanocrystals synthesized in each time in 4 ml of ethanol solution, and dropping 3-6 drops of liquid on the ultrathin carbon film after ultrasonic treatment for 5 minutes.
CaClF: the X-ray diffraction pattern of the 10Bi/20Er nanocrystal is shown in figure 1a, all diffraction peaks correspond to JCPDS24-0185 number of standard PDF card one to one, and no redundant diffraction peaks exist, so that the product obtained by the method is a pure hexagonal phase. The transmission electron microscopy analysis results are shown in FIG. 1b, which shows that the product is in a uniform block form and has good dispersibility. It should be noted that the crystal structure of the nanocrystals was not changed after the hydrochloric acid treatment. When the outside is coated with SiO2Referring to FIG. 1c, it can be seen that each nanocrystal is uniformly coated with a layer of SiO2And the thickness is between 6 and 8 nm.
1.4 development of upconversion fluorescence immunochromatographic test strip
1.4.1 coupling of upconversion luminescent nanocrystals with antibody proteins
1) Up-conversion luminescent nanocrystal activation
1mg of the upconverting luminescent nanocrystals were added to an EP tube, centrifuged for 20 minutes (15000 rpm) and the supernatant removed. 1ml of buffer (borax solution: pH = 8.5) was added and dispersed by sonication (2 sonications in ice bath, 1 s/time, 5s intervals each). Then adding 1mg of EDAC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), 1mg of NHS (N-hydroxysuccinimide) and 200 mul of deionized water, uniformly dispersing, adding the up-conversion luminescence nanocrystal, placing the up-conversion luminescence nanocrystal mixed solution on a rotary push wheel, and incubating for 1 h at room temperature. After centrifugation for 20 minutes (15000 rpm), 1ml of buffer was added after removing the supernatant and the mixture was dispersed by sonication for further use.
2) Coupling of
The up-conversion luminescence nanocrystals were mixed with protein a antibody 1 at a mass ratio of 8:1 (protein a antibody 1: 1). The mixture was then placed on a rotating wheel, incubated at room temperature for 4 hours, centrifuged for 20 minutes (15000 rpm), the supernatant removed and 1ml buffer added, and 200. mu.l ethanolamine added after ultrasonic dispersion. And placing the up-conversion luminescence nanocrystal-protein antibody 1 mixed solution on a rotary push wheel, and incubating for 1 hour at room temperature. Centrifuge for 20 minutes (15000 rpm) and remove the supernatant. Then adding 1ml of confining liquid (borax-Casin) and carrying out ultrasonic dispersion. And finally, placing the up-conversion luminescence nanocrystal-protein antibody 1 mixed solution on a rotary push wheel, and incubating for 12 hours at room temperature. Centrifuge for 20 min (15000 rpm) and remove supernatant. 1ml of buffer was added and dispersed by sonication.
1.4.2 preparation of up-conversion fluorescence immunochromatographic test strip
Using a CaClF: Bi/Er @ SiO2The nanocrystalline is used as a fluorescent marker of the immunochromatography test strip, emits an optical signal of visible light under the irradiation of infrared light, and can be interpreted by an instrument, so that qualitative and quantitative detection of an object to be detected is realized. The immunochromatographic test paper mainly comprises the following components: sample pad, conjugate pad, analytical membrane, absorbent pad, adhesive backing and plastic housing. The debugging of the up-conversion luminescence test paper mainly comprises the following steps: mucosa, spot membrane, label, spot marker, processing, assembly, detection.
1) Mucosa: firstly, adhering a proper nitrocellulose membrane (sartorius membrane) on a plastic big card;
2) film dotting: preparing an antibody (antigen or other substances) for detection into a solution with a certain concentration, uniformly spraying the solution on the stuck nitrocellulose membrane through a machine, and drying in an oven;
raw materials of wire C: goat anti-mouse IgG, concentration used: 2 mg/ml, spray volume: 1.0 ul/cm, the film drying temperature is 37 ℃, and the film drying time is 24 hours;
t line raw materials: specific antibody protein 2, using concentrations: 1 mg/ml, spray amount: 1.0 ul/cm, film drying temperature: film drying time at 37 ℃: for 24 hours.
3) Marking: additional specific antibody protein 1 and up-converting luminescent nanocrystals were coupled together.
4) Point markers: the coupled labels were formulated into a solution at 1 mg/ml, which was dispensed by machine onto the polyester film of the treated conjugate pad at a dose of 2.0 ul/cm. The polyester film treatment formula comprises: (Tween 200.5%, Sodium Azide 0.002%, T-Casein 0.5%), and oven drying. The film drying temperature is 37 ℃, and the film drying time is 24 hours;
5) and (3) treatment: according to the formula of the treating fluid: tris (0.5-1%), Tween 20 (0.5-1%), Sodium Azide (< 0.02%) Polyvinylpyrrolidone (0.5-1%) Bovine Sodium album (0.5-1%) to prepare a solution, and treating a certain amount of solution on glass fiber, drying at the drying temperature of 37 ℃ for 24 hours;
6) assembling: assembling the processed sample pad, the bonding pad and the cellulose membrane together according to a certain position, adding absorbent paper, cutting into pieces with width of 4 mm, and packaging in corresponding fittings;
7) and (3) detection: and (3) detecting the sample by using the assembled test paper, wherein the detection method comprises the following steps: 75 ul of the sample to be tested was added and the timer was started and the test read after 15 minutes.
1.5 practical detection of Fibrinogen (FIB) specific immunological test paper
In order to better prepare specific immune test paper (LFS) for detecting FIB, the FIB is utilized to research the adaptability experiment of the prepared up-conversion luminescence nanocrystals and the specific immune chromatography test paper.
The detection principle is as follows: we select the FIB which is a biomarker sensitive to chronic obstructive pulmonary disease and develop a specific immune test paper. The up-conversion luminescence nanocrystalline probe on the test paper sample pad is responsible for detecting the biomarker, and goat anti-mouse immunoglobulin G (IgG) in the quality control band can capture all fluorescent markers. When the sample to be detected is dripped on the LFS, if the antigen to be detected FIB exists in the sample to be detected, the sample to be detected is firstly specifically combined with the protein 1 probe forming the up-conversion luminescence nanocrystal-FIB antibody. The conjugate flows through the nitrocellulose membrane driven by capillary forces and is then captured by FIB antibody protein 2 immobilized in the test strip, and the excess analyte-unbound upconverting luminescent nanocrystal probe will react with IgG and be captured in the quality control strip. If FIB is not present in the sample, all of the FIB antibody protein 1-bearing UCNPs probes will flow directly through the detection line without reaction and eventually be captured on the control band. After the reaction, the fluorescence signal at the LFS was visually observed after 15 minutes. And the fluorescence signal of the test paper has a linear rule along with the change of the concentration, and the antigen in the sample can be quantitatively detected by detecting the test paper signal through a spectrometer.
1.5.1 investigation of test strip Selectivity to FIB antigen
To test the selectivity of the test strip for FIB antigen, we tested a blank sample (for background subtraction), TRH (thyroid stimulating hormone releasing hormone) 0.5IU/ml, TSH (thyroid stimulating hormone) standard sample 0.5IU/ml, GC (glucocorticoid) standard sample 0.5IU/ml and FIB standard sample 0.5IU/ml with the assembled test strip, and tested the different standard sample test strips with a 980 nm laser in the dark, and it can be seen from fig. 3 that the average visible light intensity of the FIB-tested standard sample is much higher than that of the other standards. The test paper strip is proved to have good selectivity.
1.5.2 stability test of FIB test paper strip
To test the stability of the test strips, the FIB-tested test strips were subjected to stability testing for 60 days, as shown in Table 1. The labeled up-converting fluorescent particles have high value sensitivity reduced after 28 days of accelerated stability experiments at 55 ℃, and have low value sensitivity reduced after 49 days of accelerated stability experiments at 55 ℃.
TABLE 1 color development intensity of test paper under different concentrations of analytes (FIB) at different times
Figure 406765DEST_PATH_IMAGE002
Figure DEST_PATH_IMAGE003
1.5.3 sensitivity test of FIB test paper strip
Standard samples of 5 mIU/ml, 12.5 mIU/ml, 25 mIU/ml and 50 mIU/ml in a series of concentrations of diluted FIB antigen were taken and tested with prepared test strips. After the reaction is finished, an external 980 nm laser is used for excitation after 15 minutes, and a spectrometer is used for detecting luminescence signals. With the continuous reduction of concentration, the optical signal of the test strip is continuously weakened. The sensitivity of the reagent strip can reach 10 mIU/ml, and the requirements of qualitative and quantitative sensitivity are met.
TABLE 2 color development intensity of test strips at different FIB concentrations
Figure 74638DEST_PATH_IMAGE004
Quantitative detection of 1.5.4 FIB test strip
A series of prepared FIB antigen standard samples with concentration gradient, such as 0.1 IU/ml, 0.5IU/ml, 1 IU/ml, 5IU/ml, 10 IU/ml and 25 IU/ml, are used for detection by using prepared corresponding test strips. After the reaction is finished and the reaction product is kept stand for 15 minutes, a 980 nm near-infrared laser is used for exciting the reaction product, and a spectrometer is used for detecting the test strip in a dark place. As can be seen from the spectrum shown in fig. 4, at the detection line (tline) of the analysis film, the luminance of visible light becomes higher as the concentration of the FIB antigen increases. The fluorescence intensity was strongest at 10 IU/ml, and HOOK occurred at an excessively high concentration of 25 IU/ml (the antigen-antibody binding was rather weakened at an excessively high concentration of antigen). For quantitative detection of the concentration of the antigen FIB, FIB solutions of different concentrations and 541 nm fluorescence intensity curves were obtained. And then, taking a logarithmic lgC sample of the concentration of the FIB sample to obtain a working curve graph of y (fluorescence intensity of the T line of the detection line) and the lgC sample, and after linear fitting, obtaining a linear equation of y =22434x (lgC sample) +24963 (R2 = 0.996), so that a good linear response relation between y (fluorescence intensity of the T line of the detection line) and the lgC sample can be obtained, as shown in FIG. 5.
Conclusion
Due to the CaClF studied in this application: Bi/Er @ SiO2The surface of the nano crystal is treated by hydrochloric acid solution and then coated with a layer of SiO2The nano crystal can be well dispersed in the aqueous solution. At the same time because of SiO2The coating enables a large number of hydrophilic hydroxyl groups to exist on the surface of the nano-crystal, can be coupled with antibody protein so as to be used for specific detection of target antigen, and has certain bioaffinity. Due to the fact thatThe application also applies the nanocrystalline material to the preparation and application of the specific immune test paper, the immune test paper prepared by the nanocrystalline material can carry out sensitive and accurate quantitative detection on disease-related biomarkers, so that the immune test paper can be used for carrying out long-term monitoring on certain diseases such as chronic obstructive pulmonary disease, and the instant detection technology using the nanocrystalline material has the advantages of simplicity, rapidness, sensitivity and accuracy and has good application prospect.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present disclosure, including any person skilled in the art, having the benefit of the present disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art. The general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (9)

1. An up-conversion luminescence nanocrystal material is characterized in that the molecular formula of the nanocrystal is CaClF: Bi/Er @ SiO2。
2. The up-conversion luminescent nanocrystal material of claim 1, wherein the nanocrystal has a molecular formula of CaClF: 10Bi/20Er @ SiO2。
3. The up-conversion luminescent nanocrystal material of claim 1, wherein the nanocrystal is a water-soluble nanocrystal.
4. A method for preparing an upconversion luminescent nanocrystalline material according to claim 1, 2 or 3, characterized in that the method comprises the following steps:
1) adding bismuth acetate and erbium acetate with required doping amounts, 0.7 mmol of calcium acetate, 2 ml of trifluoroacetic acid, 2 ml of trichloroacetic acid, 10 ml of oleic acid and 25 ml of octadecene into a three-neck flask, stirring at 100 ℃ under the condition of nitrogen protection atmosphere, keeping the temperature for 90 minutes, then rapidly heating to 180 ℃, and keeping the temperature for 1 hour;
2) and after the solution is cooled to room temperature, washing for 3-5 times by using a mixed solution of ethanol and deionized water to obtain oily fluorochloride nanocrystalline CaClF: Bi/Er;
3) 0.1 ml of CO-520, 6 ml of cyclohexane and 4 ml (0.01M) of CaClF: adding the Bi/Er nano-particles into a beaker, stirring for 10 minutes, adding 0.4 ml of CO-520 and 0.08 ml of ammonia water, sealing the beaker, and performing ultrasonic treatment for 20 minutes until the solution becomes clear from milky white;
4) then 0.04 ml TEOS is added into the solution, the beaker is sealed, the reaction is carried out for 2 days at the rotating speed of 600 rpm, after the reaction is finished, acetone is added into the mixed solution for precipitation, and the mixed solution is dispersed in deionized water after being washed for 2 times by ethanol/deionized water (1: 1 v/v).
5. The method for preparing a nano material according to claim 4, further comprising a step of treating the surface of the nano crystal in the step 2): and (3) mixing the prepared 10-100mg of nanocrystalline CaClF: dispersing Bi/Er in a mixed solution of 1-3mL of ethanol and 0.5-1mL of HCl with the concentration of 0.2mol/L, carrying out ultrasonic treatment on the mixed solution for 5-10min, and then adding ethanol for washing, centrifuging, drying and the like to obtain the water-based nanocrystal.
6. Use of a nanocrystalline material according to any one of claims 1-3 in the preparation of a specific immune test strip.
7. Use of a nanocrystalline material according to any one of claims 1-3 in a specific immunoassay for chronic obstructive pulmonary disease.
8. A specific immunoassay kit comprising the nanocrystalline material according to any one of claims 1-3.
9. A specific immunoassay kit for chronic obstructive pulmonary disease comprising the nanocrystalline material of any one of claims 1-3.
CN202010823006.4A 2020-08-17 2020-08-17 Up-conversion luminescence nanocrystalline material and application thereof in specific immunoassay Withdrawn CN114075434A (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
CN115505387A (en) * 2022-09-27 2022-12-23 浦江县富盛塑胶新材料有限公司 Toy safety detection material and preparation method thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115505387A (en) * 2022-09-27 2022-12-23 浦江县富盛塑胶新材料有限公司 Toy safety detection material and preparation method thereof
CN115505387B (en) * 2022-09-27 2024-05-28 汕头市启龙玩具有限公司 Toy safety detection material and preparation method thereof

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