A near-infrared afterglow nanoprobes, and a preparation method and application thereof

Abstract

The application discloses a kind of near-infrared afterglow nano probe and its preparation method and application, wherein the near-infrared long afterglow nano probe is near-infrared long afterglow nano probe, particle size is 100nm, and its composition is: ZnGaSnO4:Cr, Y, wherein, the ratio of Zn, Ga, Sn, Cr, Y is 1.3:1.4:0.3:0.005:0.003, the near-infrared long afterglow nano probe of the application has higher luminous intensity and excellent fluorescent performance, is a new type of luminescent material, can emit light for more than several hours under external environment, has high luminous intensity, long life and good chemical stability and other characteristics, has the advantages of low background, strong fluorescent signal, high signal-to-noise ratio and the like, label is connected with oxidized low density lipoprotein monoclonal antibody by covalent bond, and the labeled product is stable, has the characteristics of high sensitivity, high accuracy, rapid and simple detection and the like, early diagnosis, curative effect monitoring and prognosis evaluation of coronary heart disease and atherosclerosis can quickly help clinical diagnosis.

Description

TECHNICAL FIELD

[0001] The present application relates to a near-infrared long afterglow nanoprobes and its preparation method and application. BACKGROUND

[0002] Oxidized Low Density Lipoprotein (OxLDL) is formed by the oxidation modification of Low Density Lipoprotein (LDL) in the body under the action of various complex factors, which mainly occurs in the intima layer of the artery. The oxidation process mainly includes two processes: the oxidation of polyunsaturated fatty acid lipid molecules to form lipid oxidation products and the cross-linking of lipid oxidation products with amino acids on LDL. The transformation of LDL into OxLDL is a key physiological link in the occurrence and promotion of atherosclerosis (AS). After LDL is oxidatively modified into OxLDL, it is mainly phagocytosed by the scavenger receptor on the surface of macrophages, leading to lipid accumulation in macrophages and the formation of foam cells. Foam cells accumulate to form lipid streaks and even lipid plaques. When the lipid plaque ruptures, platelets adhere and aggregate to form thrombus, leading to unstable angina pectoris, acute myocardial infarction and sudden death. Numerous studies have shown that the level of OxLDL in the blood is positively correlated with the severity of Coronary Atherosclerotic Heart Disease (CHD), so detecting the level of OxLDL in the blood is of great significance for the early diagnosis, efficacy monitoring and prognosis evaluation of coronary heart disease.

[0003] The trap states existing in the crystal structure of the infrared long afterglow nanoprobes can trap charge carriers during photoexcitation. The luminescence caused by thermal excitation will gradually de-trap due to the recombination of electron-hole pairs. Long-lifetime long afterglow nanoparticles can effectively eliminate the fluorescence background and improve the sensitivity by reducing the interference of short non-specific fluorescent substances. They have higher light stability and longer fluorescence lifetime than Eu nanoparticles, and can possibly replace lanthanide labels for in vitro diagnosis.

[0004] Near-infrared long afterglow nanomaterials are not affected by external light sources or fluorescence resonance energy transfer. They can effectively maintain fluorescence for a long time after excitation by LED 254 nm light source, eliminate the interference of background fluorescence through time resolution, effectively exclude the interference of excitation light, and thus avoid the influence of the environment during the biological labeling process, effectively improving the analysis sensitivity.

[0005] At present, there is no on-site detection method with high sensitivity, high specificity and high portability in the field of oxidized low-density lipoprotein detection, so it is of great significance to develop a near-infrared long afterglow nano probe kit and method with high sensitivity, rapid determination, high accuracy of oxidized low-density lipoprotein detection in serum, plasma and whole blood samples. SUMMARY

[0006] The purpose of the present application is to provide a near-infrared long afterglow nano probe preparation method and its probe, and apply it to oxidized low-density lipoprotein immunization test paper and its detection box to solve the above technical problems.

[0007] The technical solution to achieve the purpose of the present application is:

[0008] The preparation method of the near-infrared long afterglow nano probe provided by the present application comprises the following steps:

[0009] S1: Preparation of mesoporous silica: triethanolamine, cetyltrimethylammonium bromide are stirred and dissolved in water, and then oil bath stirring is carried out under heating conditions. Then, ethyl silicate and cyclohexane are mixed, stirring is continued after the addition of the mixture into the flask with a pipette, and then the collected reaction solution is centrifuged and washed in a mixture of water and ethanol. The centrifuged product is dried, ground and calcined at high temperature.

[0010] S2: Preparation of metal salt solution: zinc acetate is ultrasonically dissolved in water, and then gallium nitrate hydrate, yttrium nitrate solution and chromium acetate solution are added and stirred. Then, stannic tetrachloride is added and ultrasonically dissolved to obtain a metal salt solution.

[0011] S3: Adsorption of metal ions: mesoporous silica in S1 is added into the metal salt solution in S2, and then stirred and ultrasonically dispersed. After drying and grinding, high-temperature calcination is carried out.

[0012] The above preparation method is simple, easy to operate and easy to realize industrialization.

[0013] Further, the oil bath stirring temperature in step S1 is 60℃, the stirring time after adding into the flask is 18 hours, the water: ethanol ratio in the centrifugation washing process is 2:1, the drying temperature of the centrifuged product is 60℃, the high-temperature calcination temperature after grinding is 550℃, the time is 5h, and the heating rate is 5℃ / min.

[0014] In step S3, the amount of mesoporous silica is 100mg, the amount of metal salt solution is 500μL, the drying temperature is 60℃, the drying time is 8 hours, the high-temperature calcination temperature is 950℃, and the calcination time is 3h.

[0015] In actual operation, different parameters in the synthesis process can be changed to obtain near-infrared long afterglow nano probes with different properties. The synthesis temperature can be adjusted as needed.

[0016] A near-infrared long afterglow nano probe, characterized in that the near-infrared long afterglow nano probe is composed of Zn1.3Ga1.4Sn0.3O4:Cr0.005:Y0.003, wherein the ratio of Zn, Ga, Sn, Cr and Y is 1.3:1.4:0.3:0.005:0.003; the colon ":" represents chromium and yttrium doping.

[0017] The above-mentioned near-infrared long afterglow nano probe has the advantages of low background, long light-emitting life, strong fluorescence signal, high signal-to-noise ratio and the like, and has higher sensitivity than existing probes, is not affected by external light source stimulation or fluorescence resonance energy transfer, and has a long light-emitting life, so that more accurate and reliable detection results can be obtained. After excitation by an LED 254 nm light source, there is a long afterglow light emission, thereby excluding the interference of excitation light, eliminating the background fluorescence interference through time resolution, and the environment is not easily affected in the biological labeling process.

[0018] The application also provides an oxidized low-density lipoprotein immunization test paper prepared based on the above-mentioned near-infrared long afterglow nano probe,

[0019] The test paper comprises a bottom lining, a coating film arranged inside the surface of the bottom lining, a sample pad lapped on one end of the upper surface of the coating film, and a water absorption paper lapped and arranged on the other end of the upper surface of the coating film.

[0020] The sample pad is sprayed with a microsphere line, the microsphere line is an oxidized low-density lipoprotein monoclonal antibody 1 labeled by the activated near-infrared long afterglow nano probe; the coating film is sequentially provided with a detection line and a quality control line, and the detection line is close to the sample pad; the detection line is coated with an oxidized low-density lipoprotein monoclonal antibody 2, and the quality control line is coated with a goat anti-rabbit IgG antibody.

[0021] In the above-mentioned oxidized low-density lipoprotein immunization test paper, the probe is connected with the antibody through a covalent bond, the labeled product is stable, has the characteristics of wide detection range, high sensitivity (the minimum detection limit is 3 U / L), high accuracy, rapid and simple detection, and the like, and can be used for rapid detection.

[0022] Further, the content of the oxidized low-density lipoprotein monoclonal antibody labeled by the near-infrared long afterglow nano probe sprayed on the sample pad is 100-300 µg antibody / 200 µl fluorescent microspheres.

[0023] During the preparation process, the antibody concentration can be adjusted according to the color development requirement, and the higher the antibody concentration, the greater the color development intensity.

[0024] Further, the oxidized low density lipoprotein monoclonal antibody 2 in the detection line is coated at a concentration of 0.1-2 mg / ml, and the amount of coating liquid per cm of membrane is 0.5-1.5 μl.

[0025] According to the above step, the antibody coating concentration can be adjusted according to the color development requirement.

[0026] Further, the fluorescent microspheres are diluted 4-20 times by the microsphere diluent, and the amount of liquid per cm of sample pad is 2-4 μl; the microsphere diluent is 20 mM Tris-HCl buffer solution containing 0.5% BSA, 5% trehalose, and 20% sucrose, and the pH is 8.0.

[0027] Further, the preparation method of the oxidized low density lipoprotein immunization test paper comprises the following steps:

[0028] I. Material modification: add PEG in purified water, ultrasonic dissolution, then add ZnGaSnO4:Cr, Y for ultrasonic dispersion, and stand at room temperature;

[0029] II. Activate the near-infrared long-afterglow nanoprobes to prepare activated near-infrared long-afterglow nanoprobes;

[0030] III. Add oxidized low density lipoprotein monoclonal antibody 1 to the activated near-infrared long-afterglow nanoprobes at 100-300 μg / 200 μl, block with blocking solution after blocking, centrifuge at high speed, wash with storage solution and resuspend, and store at 0-10°C in the dark;

[0031] IV. Prepare a coated membrane: take a nitrocellulose membrane, and coat the oxidized low density lipoprotein monoclonal antibody 2 and the goat anti-rabbit IgG antibody as the detection line and the quality control line on the nitrocellulose membrane respectively, dry, and prepare a coated membrane;

[0032] V. Prepare a sample pad: use a sample pad treatment solution containing 1% NaCl, 0.5% S17, 0.5% casein, 2 mg / ml anti-RBC antibody, 20 mM Tris-HCl, and pH 8.0 to evenly spray two lines on the side close to the sample pad at 37°C, dry overnight, and spray a line of oxidized low density lipoprotein monoclonal antibody 1 labeled with near-infrared long-afterglow nanoprobes on the sample pad close to the NC membrane, and dry;

[0033] VI. Paste the sample pad, coated membrane, and absorbent paper on the backing in sequence and overlap each other to obtain a test paper board, and cut to obtain the oxidized low density lipoprotein immunization test paper.

[0034] The application further provides an oxidized low-density lipoprotein detection card prepared by using the oxidized low-density lipoprotein immunization test paper.

[0035] The detection card prepared by using the oxidized low-density lipoprotein immunization test paper is prepared by using ZnGaSnO4:Cr,Y near-infrared long afterglow nano probes to form biological tracer particles, and is applied to an in vitro diagnostic reagent, has high luminous intensity and excellent fluorescence performance, is a new type of luminescent material, can stably emit light for more than several hours under an external environment, has high luminous intensity, long life and good chemical stability, and has the advantages of low background, strong fluorescence signal, high signal-to-noise ratio and the like, and can be used for rapid detection.

[0036] By adopting the technical scheme, the application has the following beneficial effects:

[0037] Compared with existing reagents, the tracer particles prepared by using the near-infrared long afterglow nano probes have better stability under an external environment, higher luminous intensity and longer life, and better chemical stability, have low background, strong fluorescence signal and high signal-to-noise ratio, and have better sensitivity when used for rapid detection. BRIEF DESCRIPTION OF DRAWINGS

[0038] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments and in combination with the drawings, in which

[0039] Figure 1 FIG. 1 is a structural schematic diagram of an oxidized low-density lipoprotein detection card of the application.

[0040] Figure 2 FIG. 4 is a standard curve diagram drawn by an oxidized low-density lipoprotein quality control product concentration and sample signal T / C average value of the application.

[0041] Figure 3 FIG. 6 is a comparison curve diagram of detection results of the same sample by an oxidized low-density lipoprotein test kit of the application and an oxidized low-density lipoprotein detection kit (magnetic particle immunochromatography method) kit of Xi'an Jinmagnetic.

[0042] The reference signs are: bottom lining 1, sample pad 2, microsphere line 21, coated membrane 3, detection line 31, quality control line 32, and water absorption paper 4. DETAILED DESCRIPTION

[0043] In order to better understand the above technical scheme, the above technical scheme will be described in detail below in combination with the drawings of the specification and specific embodiments.

[0044] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown in the drawings can be arranged and designed in various different configurations.

[0045] Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed present application, but only represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative labor are within the scope of protection of the present application.

[0046] It should be noted that: similar reference numerals and letters represent similar items in the following drawings, therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0047] In the description of the embodiments of the present application, it should be understood that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship commonly understood by those skilled in the art, and are only for the purpose of facilitating the description of the present application and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application.

[0048] In the description of the embodiments of the present application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "arrangement", "installation", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be directly connected, or indirectly connected through an intermediate medium, or it can be the communication inside two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances. The present application will be further described below with reference to the drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present application, and cannot limit the protection scope of the present application.

[0049] (Embodiment 1)

[0050] The preparation method of the near-infrared long-persistence nanoprobes of the present embodiment is as follows:

[0051] Step one: Preparation of mesoporous silica: In a 100 mL round bottom flask, add 0.18 g of triethanolamine, 24 mL of cetyltrimethylammonium chloride (25% wt) and 36 mL of water, stir under 60 °C oil bath for 1 hour, then mix 4 mL of TEOS and 16 mL of cyclohexane, stop stirring, then use a pipette to add dropwise into the flask (control in 15~20 min, along the wall, the feeding speed will affect the product diameter), continue stirring for 18 hours. Then centrifuge the collected reaction solution and wash it three times, the washing solution is prepared according to water: ethanol = 2:1. After pouring off the last washing solution, it is placed in a 60 °C oven for drying. After drying, the dried product is ground into powder with a mortar and transferred to a ceramic crucible and placed in a box furnace for high temperature calcination (550 °C, 5h, 5 °C / min).

[0052] Step two: Preparation of metal salt solution: First, add 0.5707 g of zinc acetate in 1.8 mL of water solution, ultrasonic to dissolve, then add 0.7161 g of gallium nitrate hydrate, 600 μL of yttrium nitrate solution (0.01M), 40 μL of chromium acetate solution (0.25M) and 560 μL of water, finally add 0.2104 g of crystalline tin tetrachloride, ultrasonic to completely dissolve the product.

[0053] Step three: Adsorption of metal ions: Take 100 mg of mesoporous silica in step one, add 500 μL of metal salt solution in step two, stir and open, dry in a vacuum drying oven at 60 °C for more than 8 hours, then grind and transfer to a small crucible for high temperature calcination (950 °C, 3h), collect the powder.

[0054] The oil bath stirring temperature in step S1 is 60 °C, the stirring time after adding the flask is 18 hours, the water: ethanol = 2:1 during the centrifugal washing process, the drying temperature of the centrifugal product is 60 °C, the high temperature calcination temperature after grinding is 550 °C, the time is 5h, and the heating rate is 5 °C / min;

[0055] The amount of mesoporous silica in step S3 is 100 mg, the amount of metal salt solution is 500 μL, the drying temperature is 60 °C, the drying time is 8 hours, the high temperature calcination temperature is 950 °C, and the calcination time is 3h.

[0056] The composition of the near-infrared long afterglow nanoprobes in this embodiment is Zn1.3Ga1.4Sn0.3O4:Cr0.005:Y0.003, wherein the ratio of Zn, Ga, Sn, Cr and Y is 1.3:1.4:0.3:0.005:0.003; the colon “:” represents chromium and yttrium doping.

[0057] As Figure 1The above near-infrared long afterglow nano probe is prepared into an oxidized low-density lipoprotein immunization test paper, which comprises: a bottom lining 1; a substrate for the oxidized low-density lipoprotein detection card;

[0058] A coating film 2 is arranged on the upper surface of the middle part of the bottom lining 1;

[0059] A sample pad 3 is arranged on one end of the upper surface of the coating film 3

[0060] An absorbent paper 4 is arranged on the other end of the upper surface of the coating film 3;

[0061] The sample pad 2 is sprayed with a microsphere line 21, the microsphere line 21 is the near-infrared long afterglow nano probe labeled oxidized low-density lipoprotein monoclonal antibody 1 provided in Embodiment 1; the coating film 3 is provided with a detection line 31 and a quality control line 32, the detection line 31 is close to the sample pad 2, and the detection line 31 and the quality control line 32 are parallel to each other with a spacing distance of 3-5 mm; the detection line 31 is coated with oxidized low-density lipoprotein monoclonal antibody 2, and the quality control line 32 is coated with goat anti-rabbit IgG antibody.

[0062] The content of the near-infrared long afterglow nano probe labeled oxidized low-density lipoprotein monoclonal antibody 1 sprayed on the sample pad 3 is 100-300 µg antibody / 200 µl fluorescent microspheres.

[0063] The coating concentration of the oxidized low-density lipoprotein monoclonal antibody 2 in the detection line 31 is 0.1-2 mg / ml, and the amount of coating liquid per cm membrane is 0.5-1.5 µl; the coating concentration of the goat anti-rabbit IgG antibody in the quality control line 32 is 0.5-2 mg / ml, and the amount of coating liquid per cm membrane is 0.5-1.5 µl.

[0064] The near-infrared long afterglow nano probe labeled oxidized low-density lipoprotein monoclonal antibody is diluted 4-20 times by a microsphere diluent, and the amount of liquid per cm sample pad is 2-4 µl; the microsphere diluent is a 20 mM, pH 8.0 Tris-HCl buffer solution containing 0.5% BSA, 5% trehalose, and 20% sucrose.

[0065] After the preparation is completed, it is fixed on a plastic bottom card matched with the size of the detection card, the surface of the detection card is pressed by a card surface, and sample holes and observation windows are respectively reserved in the parts corresponding to the sample pad 2 and the coating film 3.

[0066] A preparation method of an oxidized low-density lipoprotein immunization test paper comprises the following steps:

[0067] Step one: prepare near-infrared long afterglow nanoprobes according to the method provided in Example 1 and perform surface modification and activation; add 60 mg of PEG to 10 ml of purified water, ultrasonically dissolve, then add 30 mg of ZnGaSnO4:Cr, Y and ultrasonically disperse, stand at room temperature for 24 hours at 600 rpm. Then ultrasonically treat and centrifuge the material, wash the precipitate with 10-100 mM of MES solution with a pH of 5.0-7.0; add carbodiimide, N-hydroxysuccinimide, mix well, then high-speed centrifuge, wash the precipitate with a pH of 5.0-7.0 of MES solution, and obtain the activated near-infrared long afterglow nanoprobes.

[0068] Step two: prepare near-infrared long afterglow nanoprobes labeled oxidized low-density lipoprotein monoclonal antibody 1: after ultrasonic treatment of the activated near-infrared long afterglow nanoprobes in step one for 1-2 min, add 100-300 µg of oxidized low-density lipoprotein monoclonal antibody 1 per 200 µl, mix well for 3 hours, block with 0.5% BSA in 10-50 mM of Tris-HCl with a pH of 7.5-8.5 for 0.5-1 hour, then high-speed centrifuge at 12000-14000 rpm for 5-15 min, wash and resuspend with 0.5% NaCl, 0.5% BSA, 0.2% Tween-20 in 10-50 mM of Tris-HCl with a pH of 7.5-8.5, and store at 4°C in the dark;

[0069] Step three: prepare a coating film: use oxidized low-density lipoprotein monoclonal antibody 2 and goat anti-rabbit IgG antibody as the detection line and quality control line respectively, parallelly draw on the nitrocellulose membrane for coating, and dry in an oven; adjust the concentration of the oxidized low-density lipoprotein monoclonal antibody 2 and goat anti-rabbit IgG antibody to 0.5-2 mg / ml respectively with coating buffer, use an amount of 0.5-1.5 µl of coating solution per cm of membrane, parallelly draw on the nitrocellulose membrane for coating as the detection line and quality control line respectively, with a spacing of 3-7 mm between the quality control line and the detection line, and dry in an oven at 45°C overnight;

[0070] Step four: prepare a sample pad: use a sample pad treatment solution containing 1% NaCl, 0.5% S17, 0.5% casein, 2 mg / ml of anti-RBC antibody in 20 mM of Tris-HCl with a pH of 8.0 to parallelly and evenly spray two lines near the sample pad side, dry at 37 degrees overnight, and dilute the near-infrared long afterglow nanoprobes labeled oxidized low-density lipoprotein monoclonal antibody 1 with microsphere diluent 4-20 times on the sample pad near the NC membrane, use an amount of 2-4 µl of liquid per cm of sample pad, and dry in an oven at 37°C overnight; the microsphere diluent is 20 mM of Tris-HCl buffer containing 0.5% BSA, 5% trehalose, and 20% sucrose with a pH of 8.0.

[0071] Step five: sequentially paste sample pad, sample pad, coating film and water absorption paper on the bottom liner to obtain a test paper board, and cut to obtain the oxidized low density lipoprotein detection card.

[0072] The above oxidized low density lipoprotein immunization test paper can be prepared into an oxidized low density lipoprotein detection card to obtain an oxidized low density lipoprotein detection card.

[0073] The oxidized low density lipoprotein detection card of the present application is used in a plastic shell formed by buckling a plastic upper shell and a plastic lower shell, the plastic upper shell is provided with two openings, which are a sample adding hole and an observation window, the sample adding hole corresponds to the sample pad 2, the plastic lower shell is provided with a cartridge for fixing the test paper strip, and the result observation window corresponds to the detection line 31 and the quality control line 32, and the oxidized low density lipoprotein detection card can be taken out of the plastic shell.

[0074] The above oxidized low density lipoprotein detection card is used for data verification.

[0075] Specifically, the following steps are included:

[0076] Step one: using serum / plasma / whole blood sample as the detection sample;

[0077] Step two: accurately pipette 20 µL of the blood sample to be tested into the sample diluent, and mix well;

[0078] Step three: pipette 80 µL of the mixed sample into the sample adding hole of the detection card;

[0079] Step four: after 15 min, the test paper strip is irradiated with 659 nm LED for 5 min, and then immediately placed in the photographing area of the IVIS Lumina II imaging system, and then exposed for 5 min for photographing, then the gray value of the detection line and the quality control line is read by ImageJ software, and the gray value ratio of the detection line and the quality control line is calculated for quantitative detection.

[0080] (Example 2)

[0081] The structure of the oxidized low density lipoprotein detection card in this embodiment is consistent with that in Embodiment 1, the difference is that: the microsphere line on the sample pad uses specific excitation light (254 nm) / emission light (696 nm) wavelength near-infrared long afterglow nanoprobes (Zn1.3Ga1.4Sn0.3O4:Cr0.005, Y0.003) (diameter about 100 nm) to label oxidized low density lipoprotein monoclonal antibody 1 (200 μg antibody / 200 μl nanoprobes) on the sample pad, the detection line is coated with oxidized low density lipoprotein monoclonal antibody 2 (1 mg / ml), and the quality control line is coated with 1 mg / ml goat anti-rabbit IgG antibody (wherein the oxidized low density lipoprotein monoclonal antibody is purchased from Nanjing Baikang Biological Technology Co., Ltd., and the goat anti-rabbit IgG antibody is from Changsha Boyou Biological Technology Co., Ltd.). The amount of coating solution used for the microsphere line is 4 μl / cm sample pad, and the amount of coating solution used for the detection line and the quality control line is 1 μl / cm membrane.

[0082] The preparation method of the near-infrared long afterglow nanoprobes of the embodiment, characterized by comprising the following steps:

[0083] (1) Preparation of mesoporous silica:

[0084] In a 100 mL round-bottom flask, 0.18 g of triethanolamine, 24 mL of cetyltrimethylammonium chloride (25% wt), and 36 mL of water were added, and the reaction was stirred at 60°C for 1 hour. Then 4 mL of TEOS and 16 mL of cyclohexane were mixed, and after stopping the stirring, the mixture was added dropwise to the flask with a pipette, and the stirring was continued for 18 hours. After that, the collected reaction solution was centrifuged and washed three times, and the washing solution was prepared according to the ratio of water: ethanol = 2:1. After pouring out the last washing solution, it was placed in a 60°C oven for drying. After drying, the dried product was ground into powder with a mortar, transferred to a ceramic crucible, and placed in a box furnace for high-temperature calcination (550°C, 5h, 5°C / min).

[0085] (2) Preparation of metal salt solution:

[0086] First, 0.5707 g of zinc acetate was added to 1.8 mL of water solution, and ultrasonic was used to dissolve it. Then 0.7161 g of gallium nitrate hydrate, 600 μL of yttrium nitrate solution (0.01 M), 40 μL of chromium acetate solution (0.25 M), and 560 μL of water were added. Finally, 0.2104 g of crystalline tin tetrachloride was added, and ultrasonic was used to completely dissolve the product.

[0087] (3) Adsorption of metal ions:

[0088] Take 100 mg of mesoporous silica in step one, add 500 μL of metal salt solution in step two, stir and open, and dry in a vacuum drying oven at 60°C for more than 8 hours. Then grind and transfer to a small crucible for high-temperature calcination (950°C, 3h), and collect the powder.

[0089] The preparation method of the oxidized low-density lipoprotein immunization test paper comprises the following steps:

[0090] (1) Modification and activation of near-infrared long afterglow nano probes:

[0091] The near-infrared long afterglow nano probes prepared by the method provided in step (3) are surface-modified, 60 mg of PEG is added to 10 ml of purified water and ultrasonically dissolved, 30 mg of ZnGaSnO4:Cr, Y is added and ultrasonically dispersed, and the material is left to stand at room temperature at 600 rpm for 24 hours, after which the material is ultrasonically treated and centrifuged, and the precipitate is washed with a 10-100 mM MES solution with a pH of 5.0-7.0; carbodiimide and N-hydroxysulfosuccinimide are added, the mixture is uniformly mixed and high-speed centrifuged, and the precipitate is washed with a pH 5.0-7.0 MES solution to obtain activated near-infrared long afterglow nano probes.

[0092] (2) Preparation of near-infrared long afterglow nano probe labeled oxidized low-density lipoprotein monoclonal antibody 1:

[0093] After ultrasonic treatment of the above-mentioned activated fluorescent microspheres for 2 minutes, 200 µg / 200 µl of oxidized low-density lipoprotein monoclonal antibody 1 is added, the mixture is uniformly mixed for 2 hours, and after blocking for 1 hour with a 0.5% BSA-containing 50 mM, pH 8.0 Tris-HCl blocking solution, the mixture is high-speed centrifuged at 14000 rpm for 15 minutes, washed twice with a 0.5% (w / w) Nacl, 0.5% (w / w) BSA, 0.2% (w / w) Tween-20-containing 50 mM, pH 8.0 Tris-HCl storage buffer, and ultrasonically treated and resuspended to 200 µl at 4°C in the dark.

[0094] (3) Preparation of a coating film:

[0095] Oxidized low-density lipoprotein monoclonal antibody 2 and goat anti-rabbit IgG antibody are respectively adjusted to a concentration of 1 mg / ml with a coating buffer (20 mM Tris-HCl buffer containing 2.5% (w / w) trehalose, pH 8.0), and the amount of coating solution used is 1 µl / cm membrane, and the two are respectively coated on the nitrocellulose membrane as a test line and a quality control line, with a 4 mm interval between the test line and the quality control line, and the membrane is dried in an oven at a humidity of <30% and a temperature of 45°C overnight, and then sealed and stored for use;

[0096] (4) Preparation of a sample pad:

[0097] The sample pad is treated with a solution containing 1% NaCl, 0.5% S17, 0.5% casein, 2 mg / ml anti-RBC antibody in 20 mM, pH 8.0 Tris-HCl. Two parallel lines are evenly sprayed on one side of the sample pad, and the sample pad is dried at 37°C overnight. The near-infrared long afterglow nano probe labeled oxidized low density lipoprotein monoclonal antibody 1 is diluted 20 times with microsphere diluent (20 mM, pH 8.0 Tris-HCl buffer containing 0.5% (w / w) BSA, 5% (w / w) trehalose, 20% (w / w) sucrose) and evenly sprayed on the sample pad near the NC membrane, with a volume of 4 μl / cm sample pad. Place in an oven and dry at 37°C overnight.

[0098] (5) Test card assembly:

[0099] The sample pad (size 31*300mm, glass fiber cotton material), the coating membrane (size 25*300mm, nitrocellulose material) and the water absorption paper (size 28*300mm) are sequentially and mutually overlapped on the backing (size 80*300mm) to obtain a test strip, which is cut into a test strip with a width of 4mm according to the requirements.

[0100] The antigen calibrators are diluted with negative clinical samples to obtain samples with concentrations of 150, 120, 80, 60, 45, 10, and 3 U / L. The oxidized low density lipoprotein test card obtained in Example 1 and Example 2 is tested three times, and the average T / C value is taken to establish a standard curve with the calibrator concentration, as shown in Table 1:

[0101]

[0102] From the above test results, it can be seen that:

[0103] A standard curve is drawn with the antigen concentration and the average sample signal T / C value. The curve data is shown in Table 1, and the standard curve is shown in Figure 2 The R value of oxidized low density lipoprotein is 0.9984, respectively. The standard curve is used to quantitatively determine the concentration of oxidized low density lipoprotein in the sample. The performance of the oxidized low density lipoprotein test card is tested as follows:

[0104] (1) The lowest detection limit: the zero value sample is repeatedly measured for 20 times, and the average value M and the standard deviation SD of the 20 results are calculated. The detection limit of the method is reported as the blank average value plus twice the standard deviation (M+2SD), and the result of oxidized low density lipoprotein is 2.57 U / L, which meets the sensitivity standard of 3 U / L.

[0105] (2) Linear range: take 3~150 U / L of oxidized low density lipoprotein 7 concentration values respectively, each concentration is repeated three times, the average value of the measured concentration is linearly analyzed with the theoretical concentration, the linear equation of oxidized low density lipoprotein y == 0.9948x + 0.4842, r=0.9986, which indicates that the oxidized low density lipoprotein test kit has good correlation in the linear range.

[0106] (3) Precision: take three batches of reagent kits of the embodiment, respectively detect three batches of repeatability quality control samples, each batch of reagent kit is detected by parallel detection of repeatability quality control samples for 10 times, wherein the three batches of batch CV of 80 U / L of oxidized low density lipoprotein are 8.94%, 8.82%, 7.97% respectively, the batch CV is 8.33%, the three batches of batch CV of 45 U / L are 6.76%, 6.45%, 6.33% respectively, and the batch CV is 6.35%, all of which are less than 10%.

[0107] The sample in the application is compared with the magnetic microparticle immunochromatography method for detecting oxidized low density lipoprotein reagent kit of Xi'an Jinmagnetic Nanobiotechnology Co., Ltd., and the test results are shown in Table 1. Figure 3

[0108] The linear analysis of the detection results is shown in Table 2. Figure 3

[0109] It can be seen that the kit of the application realizes high sensitivity, accurate quantification, simple and rapid quantitative detection of oxidized low density lipoprotein antibody by using the sensitivity of rare earth nanometer fluorescence immunochromatography technology and combining with a dry-type immunofluorescence analyzer.

[0110] The above specific embodiments further illustrate the purpose, technical solutions and beneficial effects of the application, and it should be understood that the above description is only a specific embodiment of the application and is not used to limit the application, and any modification, equivalent replacement, improvement, etc. within the spirit and principle of the application should be included in the protection scope of the application.​​

Claims

1. An oxidized low density lipoprotein immunization test paper, characterized in that: it comprises a bottom support, a coating film arranged inside the surface of the bottom support, a sample pad lapped on one end of the upper surface of the coating film, and water-absorbing paper lapped and arranged on the other end of the upper surface of the coating film; the sample pad is sprayed with a microsphere line, which is oxidized low density lipoprotein monoclonal antibody 1 labeled by activated near-infrared long afterglow nano probes; the coating film is sequentially provided with a detection line and a quality control line, the detection line is close to the sample pad; the detection line is coated with oxidized low density lipoprotein monoclonal antibody 2, and the quality control line is coated with goat anti-rabbit IgG antibody; the preparation method of the activated near-infrared long afterglow nano probe comprises the following steps: S1: preparation of mesoporous silica: triethanolamine, cetyltrimethylammonium bromide are stirred and dissolved in water, stirred under heating condition in an oil bath, then ethyl silicate and cyclohexane are mixed, after stopping stirring, a pipette is used to add the mixture into the flask drop by drop along the cup wall within 15-20 min, and the stirring is continued; the collected reaction solution is washed by centrifugation in a mixture of water and ethanol, and then dried, ground and calcined at high temperature; S2: preparation of metal salt solution: zinc acetate is ultrasonically dissolved in water, then gallium nitrate hydrate, yttrium nitrate solution and chromium acetate solution are added, followed by addition of tin tetrachloride, and then ultrasonic dissolution is performed to obtain a metal salt solution; S3: adsorption of metal ions: mesoporous silica in S1 is taken, the metal salt solution in S2 is added, stirring is performed, ultrasonic dispersion is adopted, drying is performed, grinding is performed, and high-temperature calcination is performed; the content of the oxidized low density lipoprotein monoclonal antibody 1 labeled by near-infrared long afterglow nano probes sprayed on the sample pad is 100-300 µg of antibody / 200 µl of fluorescent microspheres; the coating concentration of the oxidized low density lipoprotein monoclonal antibody 2 in the detection line is 0.1-2 mg / ml, and the amount of coating liquid per cm of membrane is 0.5-1.5 µl; the coating concentration of the goat anti-rabbit IgG antibody in the quality control line is 0.5-2 mg / ml, and the amount of coating liquid per cm of membrane is 0.5-1.5 µl; the microsphere line is diluted 4-20 times by using a microsphere diluent to dilute the oxidized low density lipoprotein monoclonal antibody labeled by near-infrared long afterglow nano probes, and the amount of liquid per cm of sample pad is 2-4 µl; the microsphere diluent is a Tris-HCl buffer solution containing 0.5% BSA, 5% trehalose and 20% sucrose, and the pH value is 8.0; it comprises the following steps: I. material modification: PEG is added to purified water, ultrasonic dissolution is performed, ZnGaSnO4:Cr, Y is added for ultrasonic dispersion, and room temperature standing is performed; II. activation of near-infrared long afterglow nano probes to prepare activated near-infrared long afterglow nano probes; III. 100-300 µg of oxidized low density lipoprotein monoclonal antibody 1 is added to 200 µl of activated near-infrared long afterglow nano probes, blocking is performed by using a blocking solution, high-speed centrifugation is performed, washing and resuspension are performed by using a preservation solution, and preservation is performed at 0-10°C in the dark. ​ ​ The near-infrared long afterglow nanoprobe is composed of Zn 1.3 Ga 1.4 Sn 0.3 O4:Cr 0.005 :Y 0.003 , and the particle size is 100 nm; ​ ​ ​ ​ 2. The oxidized low density lipoprotein immunochromatographic test paper according to claim 1, wherein: ​ 3. The oxidized low density lipoprotein immunochromatographic test paper according to claim 1, wherein: ​ 4. The oxidized low density lipoprotein immunochromatographic test paper according to claim 1, wherein: ​ 5. The method for preparing an oxidized low-density lipoprotein immunoassay strip according to claim 1, characterized in that... ​ ​ ​ ​ IV. Preparation of coated membrane: take nitrocellulose membrane, respectively, use oxidized low density lipoprotein monoclonal antibody 2 and goat anti-rabbit IgG antibody as detection line and quality control line parallelly draw on the nitrocellulose membrane for coating, dry, and prepare coated membrane; V. Preparation of sample pad: use sample pad treatment liquid containing 1% NaCl, 0.5% S17, 0.5% casein, 2mg / ml anti-RBC antibody 20mM, pH8.0 Tris-HCl, spray two lines on the side close to the sample pad, dry at 37 degrees overnight, spray a line of near-infrared long afterglow nanoprobes labeled oxidized low density lipoprotein monoclonal antibody 1 on the sample pad close to the NC membrane, and dry; VI. Paste the sample pad, coated membrane and water absorption paper on the backing in sequence and overlap each other to obtain a test paper, and cut to obtain the oxidized low density lipoprotein immunization test paper.

6. An oxidized low density lipoprotein test card, characterized by: The oxidized low density lipoprotein immunization test paper of claim 4 is used for preparation.

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