Preparation method and application of quantum dot composite fluorescent microsphere solution

Abstract

The invention provides a preparation method and application of a quantum dot composite fluorescent microsphere solution, and the preparation method comprises the following steps: dissolving sodium selenite and sodium sulfite in purified water, stirring, heating and refluxing to obtain a Na2SeSO3 solution; dissolving cadmium acetate in purified water to adjust the pH value, and injecting a stabilizer and a Na2SeSO3 solution to obtain a CdSe quantum dot solution; znSO4. 7H2O is dissolved in purified water, MPA is added, the pH value is adjusted, NaHS powder is added, and a ZnS shell layer precursor solution is prepared; adding the ZnS shell layer precursor solution into the CdSe quantum dot solution, performing heating reflux, and performing centrifugal drying to obtain CdSe / ZnS quantum dots; the preparation method comprises the following steps: coating CdSe / ZnS quantum dots in carboxylated polystyrene microspheres, and carrying out oscillation, swelling equilibrium, centrifugation, washing and drying to obtain a CdSe / ZnS (at) PS-COOH quantum dot fluorescent microsphere solution. The stability and fluorescence efficiency of a quantum dot solution can be effectively improved, and meanwhile, the dog and cat helicobacter pylori antigen detection test strip prepared from the quantum dot fluorescent microspheres combines the advantages of quick reading detection and high sensitivity, and is suitable for on-site quick screening or quantitative detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary in vitro diagnostics, and particularly relates to a preparation method and application of a quantum dot composite fluorescent microsphere solution. Background Art

[0002] Helicobacter pylori is a Gram-negative bacterium that specifically colonizes the surface of gastric mucosal epithelial cells. Although humans are the only hosts of Helicobacter pylori, with the deepening of research, Helicobacter pylori has also been isolated and observed in more and more animals. Research shows that the gene sequence similarity between Helicobacter pylori in humans and pets exceeds 99%. Due to the close contact between dogs and cats and humans, they have become one of the potential infection sources of human Helicobacter pylori infection. Therefore, developing rapid and accurate detection methods is of great significance for preventing and controlling Helicobacter pylori infection in dogs and cats.

[0003] In recent years, with the development of nanotechnology, quantum dot technology has been widely used in the biomedical field, especially in in vitro diagnostics, due to its excellent properties such as high fluorescence efficiency and adjustable emission wavelength. Quantum dot composite fluorescent microspheres can significantly improve the stability and detection sensitivity of quantum dots by embedding quantum dots inside inorganic materials or using methods such as silica coating. Quantum dot immunochromatographic test strips, as a rapid and simple detection tool, can achieve quantitative detection of target molecules. These test strips have the advantages of simple operation, fast detection speed, high sensitivity, and strong specificity, and are very suitable for on-site rapid screening or quantitative detection of Helicobacter pylori antigen in dog and cat fecal samples.

[0004] Currently, the rapid detection test strips on the market are mainly colloidal gold detection test strips. Although they have a certain detection effect, problems such as inaccurate detection results are likely to occur. Therefore, the present invention provides a preparation method and application of a quantum dot composite fluorescent microsphere solution, which can achieve accurate detection of Helicobacter pylori antigen in dog and cat fecal samples. Summary of the Invention

[0005] The object of the present invention is to address the problems such as inaccurate detection results that are prone to occur in current colloidal gold detection test strips, and to propose a preparation method and application of a quantum dot composite fluorescent microsphere solution.

[0006] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0007] In the first aspect, the present invention proposes a preparation method of a quantum dot composite fluorescent microsphere solution, and the preparation method includes the following steps:

[0008] S1. Dissolve sodium selenite and sodium sulfite in purified water, stir until completely dissolved, and then heat to reflux to obtain a Na 2 SeSO 3 solution;

[0009] Dissolve cadmium acetate in purified water, adjust the pH, and inject stabilizer and Na 2 SeSO 3 The solution is stirred and then heated to react to obtain a CdSe quantum dot solution;

[0010] S2. Put ZnSO 4 7H 2 O was dissolved in purified water, MPA was added and the pH value was adjusted, and NaHS powder was added to prepare the ZnS shell precursor solution;

[0011] Adding ZnS shell precursor solution to CdSe quantum dot solution, heating to reflux, and centrifugally drying to obtain CdSe / ZnS quantum dots;

[0012] S3. Encapsulate CdSe / ZnS quantum dots in carboxylated polystyrene microspheres, and obtain CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution after shaking, swelling equilibrium, centrifugation, washing and drying.

[0013] Preferably, in step S1, sodium selenite and sodium sulfite are dissolved in purified water, stirred until completely dissolved, and then heated to 101° C. and refluxed overnight to obtain Na 2 SeSO 3 Solution;

[0014] Dissolve cadmium acetate in purified water, adjust the pH value, and quickly inject stabilizer and Na 2 SeSO 3 The solution was stirred at 25°C for 1 h, then heated to 63°C and stirred for one day and one night to obtain a CdSe quantum dot solution.

[0015] Preferably, in step S2, ZnSO 4 7H 2 O was dissolved in purified water, MPA was added and the pH value was adjusted, and NaHS powder was added after sufficient stirring to prepare a ZnS shell precursor solution;

[0016] Adding ZnS shell precursor solution to CdSe quantum dot solution, heating to 63°C and reflux overnight to obtain CdSe / ZnS core-shell quantum dot solution;

[0017] A cleaning solution was added to the CdSe / ZnS core-shell quantum dot solution and then placed in a centrifuge. After high-speed centrifugation, the supernatant was removed and the precipitate was placed in a 37° C. constant temperature oven for drying to finally obtain CdSe / ZnS quantum dots.

[0018] Preferably, in step S3, CdSe / ZnS quantum dots are coated in carboxylated polystyrene microspheres. After shaking, swelling equilibrium, centrifugation, washing, and drying, a CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution is prepared, which specifically includes the following sub-steps:

[0019] S31: Add carboxylated polystyrene microspheres and a mixed solution of n-butanol and chloroform into a centrifuge tube to obtain a first mixture.

[0020] S32: Mix the first mixture thoroughly until a uniformly dispersed system is obtained.

[0021] S33: Add the CdSe / ZnS quantum dots obtained in step S2 into the uniformly dispersed system to obtain a second mixture.

[0022] S34: Under room temperature conditions, vigorously shake the second mixture for 12 h to fully incorporate the CdSe / ZnS quantum dots into the carboxylated polystyrene microspheres. Let it stand for one day to wait for the swelling to reach the equilibrium state. After high-speed centrifugation, remove the supernatant, and wash repeatedly for multiple times until the supernatant becomes colorless to obtain CdSe / ZnS@PS-COOH quantum dot fluorescent microspheres.

[0023] S35: Dissolve the quantum dot fluorescent microspheres in purified water to prepare a CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution.

[0024] In a second aspect, the present invention provides a quantum dot composite fluorescent microsphere solution prepared by the preparation method as described in any one of the above.

[0025] In a third aspect, the present invention provides an application of the quantum dot composite fluorescent microsphere solution in quantum dot markers. Disperse the CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution in an activation buffer solution, vortex and mix well, add EDC and NHS solutions, mix well by shaking, and place it in a constant temperature shaker at 37 °C for activation to obtain activated quantum dot fluorescent microspheres.

[0026] Divide the activated quantum dot fluorescent microspheres into two equal parts, perform centrifugation treatment, remove the supernatant, and then add T-coupling reaction solution and C-coupling reaction solution respectively, mix evenly. Disperse the HP-labeled antibody in the T-coupling reaction solution to obtain a T-antibody solution, and disperse the sheep IgG-labeled antibody in the C-coupling reaction solution to obtain a C-antibody solution.

[0027] Place the T-antibody solution and the C-antibody solution in a constant temperature shaker at 37 °C for incubation and coupling to obtain a coupling product.

[0028] Add BSA solution to the conjugate product for blocking. After pipetting and mixing evenly, perform the blocking. After centrifuging the blocked conjugate product, remove the free antibody. After removing the supernatant, add the preservation solution respectively for preservation to obtain the quantum dot label.

[0029] Preferably, disperse the CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution in the activation buffer, vortex and mix evenly, add the EDC and NHS solutions, mix evenly by oscillation, place it in a constant temperature shaker at 37 °C, and activate for 30 min at 200 rpm to obtain the activated quantum dot fluorescent microspheres.

[0030] Divide the activated quantum dot fluorescent microspheres into 2 equal parts on average, centrifuge in a centrifuge at 15000 rpm for 10 min, remove the supernatant, and then add the T coupling reaction solution and the C coupling reaction solution respectively, mix evenly. Disperse the HP-labeled antibody in the T coupling reaction solution to obtain the T antibody solution, and disperse the sheep IgG-labeled antibody in the C coupling reaction solution to obtain the C antibody solution.

[0031] Place the T antibody solution and the C antibody solution in a constant temperature shaker at 37 °C, incubate at 200 rpm for 300 min for coupling to obtain the conjugate product.

[0032] Add a BSA solution with a final concentration of 1% to the conjugate product for blocking. After pipetting and mixing evenly, place it at 2 - 8 °C for blocking for 15 - 30 min. After centrifuging the blocked conjugate product in a centrifuge at 15000 rpm for 10 min, remove the free antibody. After removing the supernatant, add the preservation solution respectively, disperse evenly by ultrasonic treatment, and then place it at 2 - 8 °C for dark storage to obtain the quantum dot label.

[0033] Fourthly, the present invention proposes a preparation method of a quantum dot fluorescence immunochromatographic test strip, and the test strip contains the quantum dot label.

[0034] Preferably, the preparation method of the quantum dot fluorescence immunochromatographic test strip specifically includes the following steps:

[0035] After diluting the HP-coated antibody and the mouse anti-sheep IgG antibody respectively, coat them on the nitrocellulose membrane to form a T line and a C line, and paste the nitrocellulose membrane on the PVC board with absorbent paper attached.

[0036] After diluting the quantum dot label, spray it on the pretreated glass fiber, and dry it by suction to obtain the labeled conjugate pad.

[0037] Soak the glass fiber evenly in the glass fiber treatment solution, take it out after drying by suction overnight to obtain the sample pad.

[0038] Assemble the labeled conjugate pad and the sample pad on the PVC board with the absorbent paper and the nitrocellulose membrane pasted in sequence, and cut it into a preset width to obtain the quantum dot fluorescence immunochromatographic test strip.

[0039] Preferably, the quantum dot label is sprayed on the pretreated glass fiber and transferred to a freeze dryer to be dried for 1 - 12 h to obtain a label binding pad.

[0040] In summary, compared with the prior art, a preparation method and application of a quantum dot composite fluorescent microsphere solution provided by the present invention have the following beneficial effects:

[0041] (1) Improving detection sensitivity: The present invention uses CdSe / ZnS core-shell quantum dots and encapsulates them inside carboxylated polystyrene microspheres by a swelling method to form CdSe / ZnS@PS-COOH quantum dot fluorescent microspheres. This structure not only improves the stability and biocompatibility of quantum dots but also significantly enhances the fluorescence efficiency.

[0042] (2) Optimizing the preparation process: In the preparation process of quantum dots and quantum dot fluorescent microspheres, the present invention strictly controls the reaction conditions and parameters to ensure the quality and performance of the obtained products. At the same time, in the preparation process of quantum dot labels, through fine activation, coupling, blocking, and preservation steps, the performance of the "microsphere-antibody" composite product is effectively improved, making the test strip show better stability in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Other features, objects, and advantages of the present application will become more apparent by reading the detailed description of the non-limiting embodiments with reference to the following drawings:

[0044] Figure 1 is a flowchart of the preparation method of the quantum dot composite fluorescent microsphere solution according to the present invention;

[0045] Figure 2 is a schematic diagram of the relationship between the T / C mean value and the concentration of the corresponding standard product. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0046] The present application will be further described in detail below with reference to the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the related invention and do not limit the invention. In addition, it should be noted that for the convenience of description, only parts related to the relevant invention are shown in the drawings.

[0047] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The present application will be described in detail below with reference to the drawings and embodiments.

[0048] Figure 1 is a flowchart of the preparation method of the quantum dot composite fluorescent microsphere solution according to the present invention, as Figure 1As shown in the figure, the present invention provides a method for preparing a quantum dot composite fluorescent microsphere solution, which includes the following steps:

[0049] I. Synthesize CdSe core quantum dots

[0050] First, take 2 g of sodium selenite and 5 g of sodium sulfite, add purified water to make up to 100 mL, continuously stir with a rotor until these two substances are fully dissolved, heat up to 101 °C and reflux overnight to obtain Na 2 SeSO 3 solution. Then, take 1.74 g of cadmium acetate, dissolve it in 1000 mL of purified water, adjust the pH value of the solution with NaOH solution and HCl solution after stirring and dissolving. After completing the pH adjustment, add a certain amount of stabilizer and Na 2 SeSO 3 solution. At this time, it can be observed that the solution gradually changes from the original white to a transparent state. Under normal temperature environment, continuously stir the mixed solution for 1 h, and then quickly heat up to 63 °C and stir for one day and one night. At this time, CdSe quantum dots are obtained.

[0051] II. Add ZnS shell

[0052] Take 2.93 g of ZnSO 4 ·7H 2 0, dissolve it in 1000 mL of purified water, add 3.6 mL of MPA (mercapto propionic acid) and stir well to make it evenly mixed. Then use NaOH solution and HCl solution to adjust the pH value of the solution. During this process, the originally transparent solution will first gradually turn white and then return to the transparent state again. After completing the pH adjustment, add an appropriate amount of NaHS powder and mix well to obtain the ZnS shell precursor solution; add an appropriate amount of the above ZnS shell precursor solution to the CdSe quantum dot solution, heat up to 63 °C and reflux overnight, so that CdSe / ZnS core-shell quantum dots can be obtained. Wash the CdSe / ZnS core-shell quantum dot solution, add the cleaning solution and centrifuge at high speed. After removing the supernatant, place the precipitate in a constant temperature oven at 37 °C to dry, and finally obtain CdSe / ZnS quantum dots.

[0053] III. Coating of surface polystyrene (PS)

[0054] The present invention prepares CdSe / ZnS@PS-COOH quantum dot fluorescent microspheres by the swelling method, and the specific operation process is as follows: In the first step, take a centrifuge tube, add 0.5 mL of carboxylated polystyrene microspheres and a mixed solution of n-butanol and chloroform, and mix well. In the second step, add 100 mg of the CdSe / ZnS quantum dots obtained in the second step, and perform violent shaking for 12 h to allow the quantum dots to fully integrate into the carboxylated polystyrene microspheres. In the third step, leave it for one day to wait for the swelling to reach the equilibrium state, remove the supernatant after high-speed centrifugation, and wash it repeatedly for many times until the supernatant becomes colorless. After completion, use a freeze dryer to dry it. Finally, weigh 0.25 mg of the quantum dot fluorescent microspheres and dissolve them in 1 mL of purified water to prepare a quantum dot fluorescent microsphere solution with a solid content of 0.25%.

[0055] In the second aspect, the present invention provides a quantum dot composite fluorescent microsphere solution prepared by the above preparation method.

[0056] In the third aspect, the present invention provides an application of the quantum dot composite fluorescent microsphere solution in quantum dot markers. The preparation method of the quantum dot markers is as follows:

[0057] 1) Activation: Take 200 μl of the quantum dot fluorescent microsphere solution and disperse it in 200 μl of the activation buffer solution. Mix well by vortex oscillation. Observe under a microscope that there is no aggregation. Add an appropriate amount of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) solutions prepared with the activation diluent to a concentration of 0.02%, mix well by oscillation, and place it in a constant temperature shaker at 37 °C at 200 rpm for 30 min for activation.

[0058] 2) Coupling: Divide the activated quantum dot fluorescent microspheres into two equal parts on average, place them in a centrifuge and centrifuge at 15000 rpm for 10 min. After removing the supernatant, add appropriate 200 μl of coupling reaction solutions respectively, mix well. Take an appropriate amount of HP-labeled antibody and disperse it in 200 μl of the T coupling reaction solution, and take an appropriate amount of sheep IgG-labeled antibody and disperse it in 200 μL of the C coupling reaction solution to form a C antibody solution; place it in a constant temperature shaker at 37 °C at 200 rpm and incubate for 300 min for coupling.

[0059] 3) Blocking: Add a 1% BSA solution with a final concentration to the coupling product for blocking, blow and mix well, and place it at 2-8 °C for blocking for 15-30 min.

[0060] 4) Collection and storage of the markers: Centrifuge the blocked coupling product at 15000 rpm for 10 min to remove the free antibody. After removing the supernatant, add 100 μl of the storage solution respectively, disperse it evenly by ultrasonic wave, and place it at 2-8 °C in the dark for storage for standby, and thus obtain the quantum dot markers.

[0061] Fourthly, the present invention provides a method for preparing a quantum dot fluorescence immunochromatographic test strip, which test strip contains a quantum dot label. The method for preparing the quantum dot fluorescence immunochromatographic test strip specifically includes the following steps:

[0062] 1) Coating: Dilute the HP-coated antibody and the mouse anti-sheep IgG antibody with a coating buffer to obtain a T-line coating solution and a C-line coating solution respectively; paste the NC membrane (nitrocellulose membrane) on the PVC large plate with absorbent paper attached, and coat the prepared T-line coating solution and C-line coating solution on the NC membrane at a coating amount of 1 μL / cm, and transfer it to an oven to dry at 37 °C for 16 - 30 h.

[0063] 2) Preparation of the marker conjugate pad: Uniformly spray the working solution of the pretreatment pad on a glass fiber membrane with a specification of 105 * 300 mm at 8 μL / cm, take it out after drying by suction, cut it into a pretreatment pad with a width of 17 mm for standby. Then uniformly spray the quantum dot label on the pretreatment pad at a spraying amount of 4 μL / cm, take it out after drying by suction, and thus obtain the marker conjugate pad.

[0064] 3) Preparation of the sample pad

[0065] Soak a glass fiber membrane with a specification of 200 * 300 mm uniformly in the glass fiber treatment solution, take it out after drying by suction overnight, and cut it into a sample pad with a width of 14 mm.

[0066] 4) Assembly of the test strip

[0067] Assemble the marker conjugate pad and the sample pad on the PVC plate that has been coated with the NC membrane in sequence, laminate them with each other and then cut them into a test strip with a width of 3.5 mm, put it into a plastic card bottom and cover it, and fasten and compact it to obtain the quantum dot fluorescence immunochromatographic test strip.

[0068] It should be understood that the preparation principle of the fluorescence immunochromatographic test strip provided by the present invention is: adopting the double antibody sandwich method, pre-coating the antibody of Helicobacter pylori labeled with quantum dots on the glass fiber membrane, and coating the antibody of Helicobacter pylori and the secondary antibody at the test line (T-line) and the quality control line (C-line) on the nitrocellulose membrane respectively. When detecting a positive sample, the Helicobacter pylori antigen in the sample binds to the antibody of Helicobacter pylori labeled with quantum dots on the glass fiber membrane to form a complex. Due to the chromatography effect, the complex moves forward along the test strip and is captured by the pre-coated antibody of Helicobacter pylori when passing through the test line, and a fluorescence signal is detected. For a negative specimen, a fluorescence signal is only detected at the quality control line.

[0069] Example 1 (Sensitivity analysis)

[0070] The Helicobacter pylori antigen standard was diluted into a total of 8 concentration gradients as the sensitivity reference, with 0 pg / ml as the blank control. 75 μl of each of the 8 sensitivity references and the blank control were respectively added dropwise into the sample addition wells of the quantum dot fluorescence immunochromatographic test strip prepared above. After 15 minutes, it was inserted into the fluorescence immunoassay analyzer for reading, and each reference was detected 3 times. The mean signal values of the quantum dot fluorescence immunochromatographic test strip on the T line and the C line were obtained, and the mean T / C value was calculated, as shown in Table 1. The standard curve regression equation was obtained by calculating the relationship between the mean T / C and the concentration of the corresponding standard, as shown in Figure 2 .

[0071] Table 1 Relationship between mean T / C and concentration of corresponding standard

[0072] Concentration pg / ml T mean value C mean value T / C mean value 0 100 12467 0.0080 50 165 12074 0.0137 100 146 11733 0.0124 250 358 12968 0.0276 500 540 13138 0.0411 1000 1149 10441 0.1100 2500 2614 11142 0.2346 5000 4142 11679 0.3547 10000 7125 9530 0.7476

[0073] As can be seen from Table 1, when the antigen concentration was 50 pg / mL, the mean T / C was significantly different from the mean T / C of the blank control (0 pg / mL). Therefore, the detection limit of this quantum dot fluorescence immunochromatographic test strip was determined to be 50 pg / mL, which means that this quantum dot fluorescence immunochromatographic test strip can detect at least 50 pg / mL of Helicobacter pylori antigen. From the data in Table 1 and Figure 2 the curve therein, it can be seen that this quantum dot fluorescence immunochromatographic test strip showed a stable signal response at different antigen concentrations, and there was a good linear relationship between the T / C value and the antigen concentration. This indicates that this quantum dot fluorescence immunochromatographic test strip has high sensitivity and accuracy and is suitable for the quantitative detection of Helicobacter pylori antigen.

[0074] Example 2 (Specificity analysis)

[0075] 1) Preparation of control samples

[0076] The following interfering substance solvents were respectively added to the Hp antigen-negative fecal samples and weakly positive fecal samples to prepare negative control samples and positive control samples.

[0077] Hemoglobin control sample: 900 μL of fecal sample + 100 μL of 10 mM PBS (pH 7.4)

[0078] Omeprazole control sample: 950 μL of fecal sample + 50 μL of 0.1 M sodium hydroxide

[0079] Domperidone (Motilium) control sample: 950 μL of fecal sample + 50 μL of isopropanol

[0080] Hydrotalcite control sample: 950 μL of fecal sample + 50 μL of 2 M hydrochloric acid

[0081] Clarithromycin control sample: 950 μL of fecal sample + 50 μL of methanol

[0082] Amoxicillin control sample: 950 μL of fecal sample + 50 μL of 4.6% sodium bicarbonate

[0083] Metronidazole control sample: 950 μL of fecal sample + 50 μL of 2M hydrochloric acid

[0084] Azithromycin control sample: 950 μL of fecal sample + 50 μL of 20% ethanol

[0085] 2) Preparation of interference samples

[0086] Add the following interferents to Hp antigen-negative fecal samples and weakly positive fecal samples respectively to prepare corresponding negative interference samples and positive interference samples.

[0087] Hemoglobin interference sample: 900 μL of fecal sample + 100 μL of 100 mg / mL hemoglobin

[0088] Omeprazole interference sample: 950 μL of fecal sample + 50 μL of 0.5 mg / mL omeprazole

[0089] Domperidone (Motilium) interference sample: 950 μL of fecal sample + 50 μL of 0.125 mg / mL domperidone

[0090] Hydrotalcite interference sample: 950 μL of fecal sample + 50 μL of 12.5 mg / mL hydrotalcite

[0091] Clarithromycin interference sample: 950 μL of fecal sample + 50 μL of 6.25 mg / mL clarithromycin

[0092] Amoxicillin interference sample: 950 μL of fecal sample + 50 μL of 6.25 mg / mL amoxicillin

[0093] Metronidazole interference sample: 950 μL of fecal sample + 50 μL of 5 mg / mL metronidazole

[0094] Azithromycin interference sample: 950 μL of fecal sample + 50 μL of 6.25 mg / mL azithromycin

[0095] Take three batches of quantum dot fluorescence immunochromatographic test strips, and detect the above-prepared negative interference samples, positive interference samples, negative control samples and positive control samples respectively. Add 75 μl of each sample to the sample addition hole of the quantum dot fluorescence immunochromatographic test strip, and insert it into the fluorescence immunoassay analyzer to read the value after 15 minutes. Each sample is detected 3 times.

[0096] It was verified that the test results of the negative interference samples and the negative control samples were both negative, and the test results of the positive interference samples and the positive control samples were both positive, indicating that there was no interference phenomenon under the experimental concentration conditions.

[0097] Example 3 (Stability Experiment)

[0098] Randomly select 300 copies from each of the 3 batches of quantum dot fluorescence immunochromatographic test strips obtained from the above preparation, store them sealed and dry at room temperature (4 - 30 °C), and place them for 26 months. Take them out at the 6th, 9th, 12th, 15th, 18th, 20th, 22nd, 24th, and 26th months respectively, and add samples of blank control, sensitivity reference product, and repeatability reference product for testing to detect the minimum detection limit, standard curve, and repeatability of the reagent.

[0099] The test results showed that when the quantum dot fluorescence immunochromatographic test strips were placed sealed and dry at room temperature (4 - 30 °C) for 26 months, the test results at different time periods did not change significantly. During the storage period, the kit could achieve a detection sensitivity of 50 pg / ml and had good repeatability.

[0100] In summary, a preparation method of a quantum dot solution and a test strip provided by the present invention can effectively improve the stability and fluorescence efficiency of the quantum dot solution. At the same time, the test strip labeled with quantum dots combines the advantages of rapid detection and high sensitivity, makes up for the deficiencies of the prior art, and is suitable for on-site rapid screening or quantitative detection of Helicobacter pylori in dogs and cats.

[0101] The above description is only a preferred embodiment of the present application and an explanation of the applied technical principle. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solution formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the above inventive concept. For example, the technical solutions formed by mutually replacing the above features with the (but not limited to) technical features with similar functions disclosed in the present application.

Claims

1. A method for preparing a quantum dot composite fluorescent microsphere solution, characterized in that: The preparation method comprises the following steps: S1, dissolving sodium selenite and sodium sulfite in purified water, stirring until completely dissolved, and heating and refluxing to obtain a Na2SeSO3 solution; Dissolve cadmium acetate in purified water, adjust the pH value, inject a stabilizer and the Na2SeSO3 solution, stir and heat to react, and obtain a CdSe quantum dot solution; S2, dissolving ZnSO4·7H2O in purified water, adding MPA and adjusting the pH value, and adding NaHS powder to prepare a ZnS shell precursor solution; Adding the ZnS shell precursor solution to the CdSe quantum dot solution, heating to reflux, and centrifugally drying to obtain CdSe / ZnS quantum dots; S3, encapsulating the CdSe / ZnS quantum dots in carboxylated polystyrene microspheres, and obtaining a CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution after shaking, swelling equilibrium, centrifugation, washing and drying.

2. The method for preparing the quantum dot composite fluorescent microsphere solution according to claim 1, characterized in that: In step S1, sodium selenite and sodium sulfite are dissolved in purified water, stirred until completely dissolved, and then heated to 101° C. and refluxed overnight to obtain a Na2SeSO3 solution; Dissolve cadmium acetate in purified water, adjust the pH value, quickly inject the stabilizer and the Na2SeSO3 solution, stir at 25°C for 1 hour, then heat to 63°C and stir for one day and one night to obtain a CdSe quantum dot solution.

3. The method for preparing the quantum dot composite fluorescent microsphere solution according to claim 1, characterized in that: In step S2, ZnSO4·7H2O is dissolved in purified water, MPA is added and the pH value is adjusted, and NaHS powder is added after sufficient stirring to prepare a ZnS shell precursor solution; Adding the ZnS shell precursor solution to the CdSe quantum dot solution, heating to 63° C. and reflux overnight to obtain a CdSe / ZnS core-shell quantum dot solution; A cleaning solution was added to the CdSe / ZnS core-shell quantum dot solution and then placed in a centrifuge. After high-speed centrifugation, the supernatant was removed and the precipitate was placed in a 37° C. constant temperature oven for drying to finally obtain CdSe / ZnS quantum dots.

4. The method for preparing the quantum dot composite fluorescent microsphere solution according to claim 1, characterized in that: In step S3, the CdSe / ZnS quantum dots are coated in carboxylated polystyrene microspheres, and after shaking, swelling equilibrium, centrifugation, washing and drying, a CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution is obtained, which specifically includes the following sub-steps: S31, adding carboxylated polystyrene microspheres and a mixed solution of n-butanol and chloroform into a centrifuge tube to obtain a first mixture; S32, fully mixing the first mixture until a uniformly dispersed system is obtained; S33, adding the CdSe / ZnS quantum dots obtained in step S2 to the uniformly dispersed system to obtain a second mixture; S34, at room temperature, the second mixture was vigorously shaken for 12 hours to allow the CdSe / ZnS quantum dots to fully integrate into the carboxylated polystyrene microspheres, and then left for one day to wait for the swelling to reach equilibrium, and the supernatant was removed after high-speed centrifugation, and the supernatant was repeatedly washed until the supernatant became colorless to obtain CdSe / ZnS@PS-COOH quantum dot fluorescent microspheres; S35, dissolving the quantum dot fluorescent microspheres in purified water to obtain the CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution.

5. A quantum dot composite fluorescent microsphere solution prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the quantum dot composite fluorescent microsphere solution as claimed in claim 5 in quantum dot markers, characterized in that: The CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution is dispersed in an activation buffer solution, vortexed and mixed, EDC and NHS solutions are added, vortexed and mixed, and placed in a 37° C. constant temperature shaker for activation to obtain activated quantum dot fluorescent microspheres; The activated quantum dot fluorescent microspheres are equally divided into two parts, centrifuged, and after removing the supernatant, T coupling reaction solution and C coupling reaction solution are added respectively, mixed evenly, HP labeled antibody is dispersed in T coupling reaction solution to obtain T antibody solution, and goat IgG labeled antibody is dispersed in C coupling reaction solution to obtain C antibody solution; Placing the T antibody solution and the C antibody solution in a 37° C. constant temperature shaker for incubation and coupling to obtain a coupling product; BSA solution is added to the coupling product for blocking, and the product is mixed by pipetting and then blocked. The blocked coupling product is centrifuged to remove free antibodies, and the supernatant is removed and then respectively added with preservation solution for preservation to obtain quantum dot markers.

7. The use of the quantum dot composite fluorescent microsphere solution in a quantum dot marker according to claim 6, characterized in that: The CdSe / ZnS@PS-COOH quantum dot fluorescent microsphere solution is dispersed in an activation buffer solution, vortexed and mixed, EDC and NHS solutions are added, vortexed and mixed, and placed in a 37° C. constant temperature shaker at 200 rpm for activation for 30 min to obtain activated quantum dot fluorescent microspheres; The activated quantum dot fluorescent microspheres were equally divided into two parts, centrifuged at 15000 rpm for 10 min, and after removing the supernatant, T coupling reaction solution and C coupling reaction solution were added respectively, mixed evenly, HP labeled antibody was dispersed in T coupling reaction solution to obtain T antibody solution, and goat IgG labeled antibody was dispersed in C coupling reaction solution to obtain C antibody solution; The T antibody solution and the C antibody solution were placed in a 37° C. constant temperature shaker and incubated at 200 rpm for 300 min for coupling to obtain a coupling product; A BSA solution with a final concentration of 1% was added to the coupling product for blocking. After mixing by blowing, the mixture was placed at 2-8°C for blocking for 15-30 minutes. The blocked coupling product was placed in a centrifuge at 15000 rpm for 10 minutes to remove free antibodies. After removing the supernatant, preservation solution was added respectively. After ultrasonic dispersion, the mixture was placed at 2-8°C and protected from light to obtain a quantum dot marker.

8. A method for preparing a quantum dot fluorescent immunochromatographic test strip, characterized in that: The test strip comprises the quantum dot marker as described in claim 6.

9. The method for preparing the quantum dot fluorescent immunochromatographic test strip according to claim 7, characterized in that: The preparation method comprises the following steps: HP coated antibody and mouse anti-goat IgG antibody were diluted respectively, and then coated on a nitrocellulose membrane to form a T line and a C line, and the nitrocellulose membrane was pasted on a PVC plate with absorbent paper; After diluting the quantum dot marker, spray it on the pretreated glass fiber, and dry it to obtain a marker binding pad; The glass fiber is evenly immersed in the glass fiber treatment solution, and then taken out after being drained overnight to obtain a sample pad; The marker binding pad and the sample pad are sequentially assembled on a PVC plate with absorbent paper and a nitrocellulose membrane, and cut into a preset width to obtain a quantum dot fluorescent immunochromatography test strip.

10. The method for preparing the quantum dot fluorescent immunochromatographic test strip according to claim 9, characterized in that: The quantum dot marker is sprayed onto the pretreated glass fiber, and transferred to a freeze dryer for drying for 1-12 hours to obtain a marker binding pad.

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