Immunochromatography detection reagent and preparation method of test strip thereof

Abstract

The invention relates to the technical field of detection, in particular to an immunochromatography detection reagent and a preparation method of a test strip thereof. The immunochromatography detection reagent comprises mesoporous microspheres and metal oxides, the metal oxides are distributed in the mesoporous microspheres, and the detection reagent is used for enhancing signals in a luminescence regulation medium containing a luminescent ligand, a buffer solution and a surfactant. According to the technical scheme, the detection process is simplified, the detection cost is reduced, the detection sensitivity is improved, powerful technical support is provided for various rapid detections, and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a preparation method of an immunochromatographic detection reagent and a test strip thereof. Background Art

[0002] Immunochromatographic detection technology is a portable, rapid and cost-effective detection method, which is widely used in medical diagnosis, food safety inspection, environmental monitoring and scientific research. The core of this technology is to use the specific binding of antigen and antibody to intuitively present the test results through a visualization method. Traditionally, this visualization method often involves rare earth compounds, especially rare earth nanoparticles, as labeling substances. Rare earth elements, such as lanthanum, cerium and neodymium, are widely used in the field of luminescent labeling due to their unique optical and electrochemical properties to achieve detection signal amplification and improve detection sensitivity.

[0003] However, despite the many advantages of using rare earth elements in immunochromatographic testing, there are obvious problems in its practical application. First, the abundance of rare earth elements is low and their distribution is uneven, mainly concentrated in specific geographical areas. This uneven distribution leads to high costs for mining and purification of rare earth resources, which in turn makes the price of rare earth-based detection materials relatively expensive. Secondly, the mining and processing of rare earth elements usually brings serious environmental problems, including the generation of radioactive waste and the emission of toxic byproducts, posing a potential threat to the ecological environment and human health. In addition, the market price of rare earth elements is highly volatile, which may have an adverse impact on health care costs and the availability of detection products.

[0004] Therefore, although the current immunochromatographic detection technology relies on rare earth compounds to achieve high sensitivity and specificity, the cost and environmental issues brought by these rare earth compounds, as well as the uncertainty of resource supply, have brought challenges to the widespread application and sustainable development of this technology. In view of this, the development of an alternative labeling material with lower cost, less environmental impact and stable supply has become an important issue in the development of immunochromatographic detection technology. Summary of the invention

[0005] In order to solve the problems existing in the above-mentioned prior art, the present invention provides an immunochromatographic detection reagent and a method for preparing a test strip thereof, by introducing mesoporous microspheres coated with metal oxides into the immunochromatographic detection reagent, replacing the rare earth elements in the prior art with transition metal oxides and main group metal oxides, and by introducing a luminescence regulating medium, the luminescence properties are improved, relying on the strong coordination ability of the luminescence regulating medium and the metal coordination dissociation to enhance the luminescence characteristics, and through a secondary linkage fluorescence signal amplification strategy, the detection sensitivity is improved.

[0006] The immunochromatographic detection reagent of the present invention comprises mesoporous microspheres and metal oxides, wherein the metal oxides are distributed in the mesoporous microspheres, and the detection reagent is used for enhancing signals in a luminescent regulating medium containing a luminescent ligand, a buffer solution and a surfactant.

[0007] The immunochromatographic detection reagent provided by the present invention adopts a combination of uniquely structured mesoporous microspheres and metal oxides, which effectively overcomes the limitations of traditional rare earth compound markers in terms of cost, environmental impact, and supply chain stability. The mesoporous microspheres have a highly ordered nanopore structure, which not only provides a larger specific surface area, but also achieves a better loading capacity. The metal oxides are distributed in the mesoporous microspheres, and due to their inherent physical and chemical stability, they can maintain structural integrity and functional durability during the detection process.

[0008] In the present invention, the selection of metal oxides covers transition metal oxides and main group metal oxides, which are not only abundant in source and low in cost, but also have little environmental impact. The role they play in detection is to enhance the detection signal by acting on the luminescent ligand in the luminescence regulating medium. The luminescence regulating medium is composed of luminescent ligands, buffer and surfactant, which not only provides a suitable reaction environment for metal oxides, but also can optimize the detection signal and improve the sensitivity and specificity of detection. More importantly, the distribution of metal oxides in mesoporous microspheres provides more active sites, which can greatly amplify the detection signal when acting with luminescent ligands. At the same time, this unique structural design is also conducive to the stable transmission of signals, avoiding the decrease in sensitivity caused by signal loss. Therefore, the technical solution of the present invention not only simplifies the detection process, reduces the detection cost, and improves the detection sensitivity, but also provides strong technical support for various rapid detections and has broad application prospects.

[0009] By further preferably selecting the metal oxide as at least one of aluminum oxide (Al2O3), magnesium oxide (MgO) and zinc oxide (ZnO), firstly, aluminum oxide (Al2O3) is known for its excellent chemical stability, high specific surface area and good biocompatibility, and its high specific surface area can provide more active sites, thereby enhancing the interaction with the luminescent ligand, making the signal enhancement effect more significant. Magnesium oxide (MgO) has high thermal stability and good corrosion resistance, which makes it possible to maintain the stability of the detection reagent under different detection environments. In addition, it itself has certain light absorption properties, which can play a role in filtering stray light in certain detection applications, thereby improving the accuracy of detection. Zinc oxide (ZnO) is a commonly used semiconductor material with significant photoelectric properties, especially luminescence characteristics under ultraviolet light excitation. This property can be used to further enhance the detection signal, especially in situations where an ultraviolet excitation source is required. By using aluminum oxide, magnesium oxide and zinc oxide as representatives of metal oxides, the immunochromatographic detection reagent of the present invention has significant advantages in improving the stability and sensitivity of the detection signal. At the same time, the above metal oxides are all environmentally friendly materials. Their abundant sources, low-cost characteristics, and wide application in industrial production ensure the sustainable production and wide application of detection reagents. The resulting technical effect provides a new, cost-effective and environmentally friendly signal enhancement method for the field of immunochromatographic detection.

[0010] The structure of the luminescent ligand is as follows:

[0011]

[0012] Among them, R1 is selected from one of alkyl, halogen, methoxy, amino, and thioether; R2 is selected from one of C1-C5 alkyl, halogen, nitro, alkoxy, and amino; R3 to R5 are each independently selected from one of alkyl, halogen, nitro, alkoxy, and amino; R6 is selected from one of alkyl, halogen, and C1-C5 alkoxy; or no group is connected to any group among R1 to R6.

[0013] The above structure can form stable complexes with many metal ions, and these complexes usually have good fluorescence properties. The selection of the R group can not only maintain or enhance the luminescent properties of the above structure after coordination with the metal ion, but also does not destroy the chelating site. At the same time, the space occupied by the group is small, which will not affect the approach and coordination of the above structure with the metal ion. In addition, the selection of the R group in the above structure can maintain the overall planarity of the entire molecule, which is beneficial to its π-π interaction with the metal ion, helps to form a stable complex and enhance the luminescent properties of the complex.

[0014] Mesoporous microspheres are preferably mesoporous silica nanoparticles. Mesoporous microspheres are preferably mesoporous silica nanoparticles (MSNs) to bring multiple technical effects and advantages to the immunochromatographic detection reagent of the present invention. Mesoporous silica nanoparticles have extremely high specific surface area and large pore volume, which enables them to load a large amount of metal oxides, thereby increasing the active sites acting with the luminescent ligand, and significantly improving the intensity of the generated signal. The chemical stability of silicon dioxide is excellent, not only can it stably exist under various pH conditions, but also shows good compatibility to a variety of chemical reagents and biomolecules, ensuring the reliability of the detection reagent under different environments. The transparency of silicon dioxide itself ensures that it will not interfere with the detection of signals in optical detection, which is particularly important for immunochromatographic detection reagents based on optical principles. Therefore, using mesoporous silica nanoparticles as the material of mesoporous microspheres not only improves the loading capacity and signal intensity of the detection reagent, but also ensures the chemical stability, biocompatibility and operability of the detection reagent, and provides a kind of efficient, stable and safe detection reagent selection for clinical detection and laboratory analysis.

[0015] The buffer solution is selected from at least one of the buffer systems selected from glutamine, glutamic acid, arginine, cysteine, lysine, valine, and glycine buffers. The selection of the above buffer solutions can effectively maintain stability under different pH conditions, ensuring the activity of biological molecules and the accuracy of detection reactions. Specific buffer solutions can resist the effects of external factors (such as temperature changes, ionic strength changes, etc.) on pH, which is particularly important for immunochromatographic detection in complex samples. In addition, the above buffer solutions are compatible with biological systems and will not damage biological molecules or affect their activity, which is crucial to maintaining good reactivity between antigens and antibodies.

[0016] The surfactant is selected from at least one of nonylphenol polyoxyethylene ether, Tween 20, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, hexadecyl trimethyl ammonium bromide, cocamidopropyl betaine, and dodecyl sulfobetaine. The above surfactant can reduce nonspecific protein adsorption and background noise, thereby improving the specificity of detection. In addition, the surfactant can adjust the affinity of the protein with the ligand or antibody by changing the surface properties of the protein.

[0017] The present application provides a method for preparing an immunochromatographic test strip, and the preparation steps are as follows:

[0018] A blank nitrocellulose membrane (NC membrane) is pasted onto a polyvinyl chloride (PVC) bottom plate; a predetermined concentration of PCT detection antibody and goat anti-chicken IgY are sprayed onto the preset test line (T line) and control line (C line) positions on the NC membrane using a film scribing instrument, respectively, and then placed in an oven for drying; a solution containing a labeled probe bioconjugate is evenly sprayed onto a sample binding pad using a gold spraying instrument, and the sprayed sample binding pad is placed in an oven for drying, wherein the labeled probe bioconjugate is obtained by diluting a solidifying solution containing 25 mM glycine at a pH of 8.0; the sample pad is soaked in a 25 mM glycine buffer (pH 7.4, containing 0.5% casein sodium salt and 0.3% Tween-20), then dried in a drying oven at 37°C and stored for later use; take out the PVC base plate with the dried NC film attached, and paste the prepared sample pad, conjugate pad and absorbent paper to the base plate in sequence, ensuring that each part has an overlap of about 2 mm at the connection; cut the above-assembled test strip along a predetermined width (about 0.4 mm) to prepare multiple immunochromatographic test strips. In this embodiment, the labeled probe-containing bioconjugate comprises the immunochromatographic detection reagent as described above.

[0019] The concentration of the labeled probe bioconjugate solution preferably ranges from 10 μg / mL to 70 μg / mL.

[0020] After the immune reaction is completed, the immunochromatographic test strip applies a luminescent regulating medium on the NC membrane, wherein the regulating medium contains a luminescent ligand, a buffer system and a surfactant.

[0021] The immune response time is preferably 2 min to 20 min.

[0022] By applying a luminescent regulating medium to the test strip, the detection signal can be improved, the detection sensitivity can be enhanced, and it is helpful to detect low concentrations of antigens. Using a predetermined concentration of PCT detection antibody and sheep anti-chicken IgY as the capture and control lines, it can specifically bind to the target antigen and marker, reduce non-specific signals, and improve the accuracy of the results. The above-mentioned immunochromatographic test strip is designed to provide a fast, simple, sensitive, specific, stable and intuitive detection method, which is suitable for a wide range of application scenarios, including but not limited to clinical diagnosis, on-site rapid detection, food safety testing and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a graph showing the luminescence performance of ZnO@DMSNs nanoparticles in different buffer solutions in the examples of this application;

[0024] Figure 2 This is a graph showing the luminescence performance of MgO@DMSNs nanoparticles in different buffer solutions in the examples of this application;

[0025] Figure 3 This is a graph showing the luminescence performance of Al2O3@DMSNs nanoparticles in different buffer solutions in the examples of this application;

[0026] Figure 4 This is a graph showing the luminescence performance of ZnO@DMSNs nanoparticles in different amino acid buffers in the examples of this application;

[0027] Figure 5 This is a correlation diagram of the CRP test results of the CELFIA test strip and the Beckman Coulter nephelometry method in the examples of this application. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] This embodiment provides a method for preparing ZnO nanoparticles.

[0031] Zinc acetate was dissolved in 20 mL of methanol, and 100 μL of oleic acid was added after heating and stirring. Stirring was continued for 10 min, and then the temperature was gradually raised to 95 ° C to form a mixed solution A. Tetramethylammonium hydroxide (TMAH) was then dissolved in 5 mL of methanol solution to form a mixed solution B. Mixed solution A and mixed solution B were reacted for 10 min. After the reaction was completed, it was cooled and a white precipitate appeared. The product was collected by centrifugation and washed alternately with ethanol and n-hexane several times. The final product was dispersed in n-hexane to obtain a clear solution. The results showed that the ZnO nanoparticles were uniformly dispersed without agglomeration, the particle size distribution was between 2.5 nm and 6 nm, and the XRD diffraction peak position and intensity were consistent with the standard card.

[0032] Example 2

[0033] This embodiment provides a method for preparing MgO nanoparticles.

[0034] Dissolve magnesium acetate in 20 mL of methanol and place it in a flask. After heating and stirring, add 100 μL of oleic acid, continue stirring for 10 minutes, and then gradually heat to 95°C. Dissolve TMAH in 5 mL of methanol solution, quickly add it to the flask, and react for 30 minutes. After the reaction is completed, cool it and a white precipitate will appear. Collect the product by centrifugation and wash it alternately with ethanol and n-hexane several times. The final product is dispersed in n-hexane for storage. The results show that the MgO nanoparticles are evenly dispersed, most of them are triangular, regularly arranged, and there is no agglomeration phenomenon. The particle size distribution of the nanoparticles is between 3 and 10 nm. The XRD diffraction peak position and intensity are consistent with the standard card PDF#97-018-8324, indicating that the synthesized substance is cubic MgO nanoparticles.

[0035] Example 3

[0036] This embodiment provides a method for preparing Al2O3 nanoparticles.

[0037] AlCl3·6H2O was dissolved in xylene, oleic acid was added, and stirred at about 80°C for a period of time to obtain a light yellow transparent solution, which was transferred to a three-necked flask. Subsequently, octadecene and a small amount of oleylamine were added and magnetically stirred under the protection of inert gas. The temperature was slowly raised to about 300°C and refluxed, and the temperature was gradually lowered after several hours of reaction. The product was separated by centrifugation using n-hexane and ethanol, and washed several times. The final product was dispersed in n-hexane for storage. The results showed that the particles were short rod-shaped and relatively uniformly dispersed. The particle size statistics showed that the average particle size was about 5.2nm. The XRD diffraction peak was consistent with the standard card PDF#97-006-6559, which was cubic Al2O3.

[0038] Example 4

[0039] This embodiment provides a method for preparing ZnO@DMSNs.

[0040] Take a certain amount of the prepared ultra-small ZnO nanoparticles in Example 1 and mix them with dendritic mesoporous silica (DMSNs) nanoparticles, add 20mL toluene solution, then add 100μL oleylamine, ultrasonicate for 10min, and magnetically stir at room temperature for 10h. Then add ethanol, centrifuge to separate the nanoparticles, wash them by centrifugation with ethanol and n-hexane for 3 times, and finally disperse the product ZnO@DMSNs in toluene for storage. The results show that a large number of ultra-small ZnO particles are uniformly enriched in the pores of mesoporous silica. The diffraction peaks contain hexagonal ZnO (PDF#97-029-0968) and SiO2 with characteristic bulge peaks near 22°, indicating that the ZnO phase in the ZnO@DMSNs particles is consistent with the individual ultra-small ZnO nanoparticles.

[0041] Example 5

[0042] This embodiment provides a surface modification method of ZnO@DMSNs.

[0043] Take a certain amount of ZnO@DMSNs nanoparticles, centrifuge and remove the supernatant, add 20mL toluene for ultrasonic re-dissolution, magnetic stirring, drop 2mL Silane-PEG2000-COOH toluene solution (0.1M), stir for 5 minutes, add 2mL TMAH methanol solution (0.1M), heat to 85℃ and react for 45 minutes. After cooling, add ethanol and centrifuge, wash with n-hexane for 3 times, wash with ethanol for 2 times, add pure water, and disperse with ultrasound to obtain the product ZnO@DMSNs-COOH.

[0044] Example 6

[0045] This embodiment provides a method for preparing MgO@DMSNs. The preparation process of MgO@DMSNs is similar to that of embodiment 4, except that ZnO nanoparticles are replaced with MgO nanoparticles.

[0046] Example 7

[0047] This embodiment provides a method for preparing Al2O3@DMSNs. The preparation process of Al2O3@DMSNs is similar to that of Example 4, except that ZnO nanoparticles are replaced with Al2O3 nanoparticles.

[0048] Example 8

[0049] This example provides a luminescence effect test of ZnO@DMSNs nanoparticles in glycine buffer.

[0050] The conditioning medium components in this embodiment are 8-hydroxyquinoline (8-HQ) as a ligand, 0.1% TX-100 as a surfactant, and glycine-HCl buffer (Gly) as a buffer system. The test method is as follows: the conditioning medium is added to a transparent cuvette, and then the ZnO@DMSNs nanoparticles prepared in Example 4 are added, and after rapid mixing, the reaction kinetic curve and fluorescence emission spectrum (λex=365nm, λem=520nm) are measured on an FS5 spectrometer for 10 minutes.

[0051] Comparative Example 1

[0052] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 8, except that the glycine-HCl buffer is replaced by an acetic acid-sodium acetate buffer (Ac-NaAc).

[0053] Comparative Example 2

[0054] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 8, except that the glycine-HCl buffer is replaced by phthalic acid-NaOH buffer (PA).

[0055] Comparative Example 3

[0056] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 8, except that the glycine-HCl buffer is replaced by citric acid-sodium citrate (CA).

[0057] Comparative Example 4

[0058] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 8, except that the glycine-HCl buffer is replaced by phosphate buffer (PBS).

[0059] The results of Example 8 and Comparative Examples 1 to 4 are as follows Figure 1 As shown. The experimental data show that under the same ligand and surfactant conditions, ZnO@DMSNs nanoparticles exhibit significantly different luminescence properties in different buffer systems. Specifically, the effects of each buffer system on the luminescence enhancement of ZnO@DMSNs nanoparticles are as follows: glycine-HCl buffer (Gly) > phosphate buffer (PBS) > acetic acid-sodium acetate buffer (Ac-NaAc) > phthalic acid-NaOH buffer (PA) > citric acid-sodium citrate buffer (CA).

[0060] Example 9

[0061] This example provides a luminescence effect test of MgO@DMSNs nanoparticles in glycine buffer.

[0062] The experimental process is similar to that of Example 8, except that the ZnO@DMSNs nanoparticles are replaced with MgO@DMSNs nanoparticles.

[0063] Comparative Example 5

[0064] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 9, except that the glycine-HCl buffer is replaced by an acetic acid-sodium acetate buffer (Ac-NaAc).

[0065] Comparative Example 6

[0066] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 9, except that the glycine-HCl buffer is replaced by phthalic acid-NaOH buffer (PA).

[0067] Comparative Example 7

[0068] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 9, except that the glycine-HCl buffer is replaced with citric acid-sodium citrate (CA).

[0069] Comparative Example 8

[0070] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 9, except that the glycine-HCl buffer is replaced with phosphate buffered saline (PBS).

[0071] The results of Example 9 and Comparative Examples 5 to 8 are as follows Figure 2 As shown. Data analysis shows that there are significant differences in the luminescence performance of MgO@DMSNs nanoparticles in different buffer systems. Specifically, in glycine buffer, the luminescence intensity of MgO@DMSNs nanoparticles is about 5 times that of other buffer systems. In contrast, the nanoparticles showed weaker luminescence intensity in phosphate buffer (PBS), acetic acid-sodium acetate buffer (Ac-NaAc), phthalic acid-NaOH buffer (PA), and citric acid-sodium citrate buffer (CA).

[0072] Example 10

[0073] This example provides a luminescence effect test of Al2O3@DMSNs nanoparticles in glycine buffer.

[0074] The experimental process is similar to that of Example 8, except that the ZnO@DMSNs nanoparticles are replaced with Al2O3@DMSNs nanoparticles.

[0075] Comparative Example 9

[0076] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 10, except that the glycine-HCl buffer is replaced by an acetic acid-sodium acetate buffer (Ac-NaAc).

[0077] Comparative Example 10

[0078] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 10, except that the glycine-HCl buffer is replaced by phthalic acid-NaOH buffer (PA).

[0079] Comparative Example 11

[0080] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 10, except that the glycine-HCl buffer is replaced by citric acid-sodium citrate (CA).

[0081] Comparative Example 12

[0082] The components and concentrations of the ligand and surfactant in the regulating medium used in this comparative example are the same as those in Example 10, except that the glycine-HCl buffer is replaced with phosphate buffered saline (PBS).

[0083] The results of Example 10 and Comparative Examples 9 to 12 are as follows Figure 3 As shown. The experimental data show that under the same ligand and surfactant conditions, Al2O3@DMSNs nanoparticles exhibit significantly different luminescence properties in different buffer systems. Specifically, the effects of each buffer system on the luminescence enhancement of Al2O3@DMSNs nanoparticles are as follows: glycine-HCl buffer (Gly) > phosphate buffer (PBS) > phthalic acid-NaOH buffer (PA) > acetic acid-sodium acetate buffer (Ac-NaAc) > citric acid-sodium citrate buffer (CA).

[0084] Therefore, the glycine (Gly) buffer system exhibited the best performance. Compared with other buffer systems, the Gly buffer showed obvious fluorescence enhancement effects on the three metal compounds ZnO, MgO and Al2O3. This result shows that the Gly buffer system can effectively promote the coordination reaction between metal compounds and 8-hydroxyquinoline (8-HQ). On the contrary, the citric acid buffer system performed the worst. After adding the three metal compounds to the system, the fluorescence signals were very weak. This phenomenon shows that the citric acid system significantly inhibited the coordination ability of metal compounds with 8-HQ and was not suitable for use as a buffer medium for fluorescence enhancement. The acetic acid-sodium acetate (Ac-NaAc), phthalic acid (PA) and phosphate (PBS) buffer systems showed moderate fluorescence enhancement effects. Although these three buffers can promote the coordination reaction between ZnO and Al2O3 and 8-HQ, their fluorescence enhancement effects are not as good as those of the glycine system. It is worth noting that the PA buffer has solubility problems, which limits its application in the preparation and preservation of conditioning media. Because the Gly molecule has a unique chemical structure and contains both amino and carboxylate ions, it becomes a typical bidentate ligand. This structural characteristic gives Gly the ability to form stable chelates with various metal ions (including rare earth ions). Based on this characteristic, in the Gly buffer system, the metal ions on the surface of ZnO, MgO and Al2O3 nanoparticles may react with the Gly molecules present in large quantities in the solution. This process promotes the metal ions (Zn 2+ Mg2+ and Al 3+ ) from the surface of the nanoparticles. Subsequently, these dissociated metal ions will undergo further coordination reactions with 8-hydroxyquinoline (8-HQ) in the solution. As a ligand with strong complexing ability, 8-HQ can form new coordination compounds with these metal ions, namely ZnQ2, MgQ2 and AlQ3. It is these newly formed coordination compounds that show a significant improvement in luminescence performance.

[0085] Embodiment 11

[0086] This example further verifies the effect of other amino acids on the improvement of the luminescence performance of the coordination reaction between metal ions and 8-HQ.

[0087] The experimental process of this example is similar to that of Example 8, except that a series of amino acid buffer systems are prepared, including glutamine (Gln), glutamic acid (SGlu), arginine (Arg), cysteine ​​(Cys), lysine (Lys), valine (Val), and glycine (Gly). 2+ -8-HQ complex fluorescence intensity. Results see Figure 4 The amino acid buffer system significantly promoted the coordination reaction between metal ions and 8-HQ, showing a significant fluorescence enhancement effect. The experimental results confirmed the general effectiveness of the amino acid buffer system in enhancing the fluorescence performance of the coordination reaction between metal ions and 8-HQ.

[0088] Example 12

[0089] Gly buffer system 8-HQ luminescence regulating medium (containing 0.8mM 8-HQ, 0.1% TX-100) with different pH (2.0-9.0) was prepared, and ultrasonic dispersion was used for standby. Afterwards, the luminescence enhancement effect of 8-HQ luminescence regulating medium and three metal oxide nanoparticles after coordination was tested. Using glycine buffer system, 8-HQ luminescence regulating medium (containing 0.8mM 8-HQ, 0.1% TX-100 in Gly buffer system) with different pH (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0) was prepared respectively, and fluorescence spectrum tests were carried out on the coordination reaction of three metal compound materials (ZnO, MgO and Al2O3) with 8-HQ to observe the influence of different pH on luminescence performance. The results showed that when the pH of the solution was ≤3.0, the fluorescence signal was negligible when the three metal oxides were added; when pH = 4.0, fluorescence signals of different brightness began to appear, and the fluorescence signal further enhanced with the increase of pH; ZnO had the strongest fluorescence intensity at pH = 7.0, but as the pH value further increased, the fluorescence intensity of ZnO decreased, while the luminescence intensity of the other two metal oxides MgO and Al2O3 gradually increased with the increase of pH, and the fluorescence intensity was highest at pH = 9.0, but in terms of absolute intensity, they were inferior to ZnO.

[0090] Example 13

[0091] The CRP-labeled antibody was modified on the surface of carboxylated ZnO@DMSNs by EDC and NHS chemical cross-linking method and named as ZnO@DMSNs-AbCRP. The specific coupling process is as follows:

[0092] 1) Activation: ZnO@DMSNs (10 mg / mL) was added to MES buffer (25 mM, pH = 6.0), and NHS (10 mg / mL) and EDC (10 mg / mL) were added in sequence. After activation, the mixture was centrifuged and washed to remove excess cross-linking reagents, and then ultrasonically dispersed and redissolved in BBS (0.01 M, pH = 7.4) buffer.

[0093] 2) Labeling: Add CRP labeled antibody to the activated microsphere solution and incubate at room temperature.

[0094] 3) Blocking: Add BSA solution to the labeled solution for blocking, centrifuge after the reaction, remove the supernatant, add BBS stock solution (0.01 M, pH = 7.4) to the centrifuged product, and re-dissolve by ultrasonication.

[0095] 4) Storage: The coupled microspheres were stored in the reserve solution and placed in a 4°C refrigerator for future use. The conjugate was named: ZnO@DMSNs-Ab CRP.

[0096] The coupling method of ZnO@DMSNs and chicken IgY antibody was the same as above, named: ZnO@DMSNs-Ab chicken IgY.

[0097] Embodiment 14

[0098] A nitrocellulose membrane (NC membrane) is pasted to a polyvinyl chloride base plate; a predetermined concentration of PCT detection antibody and goat anti-chicken IgY are sprayed to the preset test line (T line) and control line (C line) positions on the NC membrane using a film scribing instrument, and then placed in an oven for drying; a solution containing a labeled probe bioconjugate is evenly sprayed on a sample conjugate pad, and the sprayed sample conjugate pad is placed in an oven for drying, wherein the ZnO@DMSNs conjugate is obtained by diluting a solidifying solution containing 25mM glycine at a pH of 8.0; the sample pad is soaked in a 25mM glycine buffer solution, and then dried in a drying oven and stored for later use; the polyvinyl chloride base plate with the dried NC membrane pasted is taken out, and the prepared sample pad, conjugate pad and absorbent paper are pasted to the base plate in sequence, ensuring that the parts overlap by about 2mm at the connection; the assembled test strips are cut along a predetermined width to obtain multiple immunochromatographic test strips. Among them, the CRP detection antibody and goat anti-chicken IgY antibody on the NC membrane were diluted to 0.8 mg / mL and 1 mg / mL respectively for use, and the final concentration of the ZnO@DMSNs-Ab conjugate (labeled probe) was selected to be 0.05 mg / mL.

[0099] The feasibility of the coordination-enhanced luminescence strategy for immunochromatography was verified by adding an optimized 8-HQ luminescence regulating medium to the NC membrane of the test strip. 8-Hydroxyquinoline zinc (ZnQ2) complex micelles with different luminescence intensities were formed on the NC membrane. After the antigen-antibody immune reaction, the luminescence regulating medium was dripped on the NC membrane of the card strip for 2 minutes, and the fluorescence signals of the T line and C line could be read by the immunoanalyzer, thereby achieving quantitative detection.

[0100] Example 16

[0101] 20 CRP serum samples were selected and measured by Beckman Coulter nephelometry. The ZnO@DMSNs TR-CELFIA test strips were used for testing. The correlation between the two methods was analyzed by linear fitting. Figure 5 The correlation coefficient between the values ​​measured by the two methods was r>0.99, with no significant difference, indicating that the ZnO@DMSNs-based coordination-enhanced luminescence immunochromatography method has good detection accuracy.

[0102] In summary, the embodiment of the present invention, by coating metal oxides in mesoporous silica nanospheres, relying on the strong coordination ability of the neutral 8-HQ luminescence regulating medium and the metal coordination dissociation enhanced luminescence characteristics, improves the detection sensitivity through a secondary linkage fluorescence signal amplification strategy, and realizes rapid and sensitive detection of CRP. The detection linear range of serum samples is 1mg / L to 320mg / L, and the detection limit LoD reaches 0.38mg / L, which is significantly better than the clinical reference value of 10mg / L, which is conducive to the early diagnosis of the disease. In addition, the comparative test results of clinical samples show that the test results of this method are very consistent with those of commonly used clinical detection methods, indicating that this method has good detection accuracy.

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. The above preparation method can be a step-by-step preparation or a one-step preparation. Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solutions described in the above embodiments can still be modified, or some of the technical features can be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immunochromatographic detection reagent, characterized in that: The detection reagent comprises mesoporous microspheres and metal oxides, wherein the metal oxides are distributed in the mesoporous microspheres. The detection reagent is used to enhance the luminescent signal in a luminescent regulating medium containing a luminescent ligand, a buffer system and a surfactant.

2. The immunochromatographic detection reagent according to claim 1, characterized in that: The metal oxide is at least one of a transition metal oxide and a main group metal oxide.

3. The immunochromatographic detection reagent according to claim 2, characterized in that: The metal oxide is at least one of aluminum oxide, magnesium oxide and zinc oxide.

4. The immunochromatographic detection reagent according to claim 1, characterized in that: The luminescent ligand structure is as follows: Among them, R1 is selected from one of alkyl, halogen, methoxy, amino, and thioether; R2 is selected from one of C1-C5 alkyl, halogen, nitro, alkoxy, and amino; R3 to R5 are each independently selected from one of alkyl, halogen, nitro, alkoxy, and amino; R6 is selected from one of alkyl, halogen, and C1-C5 alkoxy; or no group is connected to any group among R1 to R6.

5. The immunochromatographic detection reagent according to claim 1, characterized in that: The mesoporous microspheres are dendritic mesoporous silica nanospheres.

6. The immunochromatographic detection reagent according to claim 1, characterized in that: The buffer system is selected from at least one of glutamine, glutamic acid, arginine, cysteine, lysine, valine and glycine buffer.

7. The immunochromatographic detection reagent according to claim 6, characterized in that: The pH range of the buffer system is 3-9.

8. The immunochromatographic detection reagent according to claim 1, characterized in that: The surfactant is selected from at least one of nonylphenol polyoxyethylene ether, Tween 20, sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, hexadecyltrimethylammonium bromide, cocamidopropyl betaine, and dodecyl sulfobetaine.

9. A method for preparing an immunochromatographic test strip, characterized in that: The preparation steps are as follows: Paste the nitrocellulose membrane onto a polyvinyl chloride substrate; Spray PCT detection antibody and goat anti-chicken IgY onto the test line and control line on the NC membrane respectively, and place in an oven for drying; Evenly spraying a solution containing a labeled probe bioconjugate on a sample binding pad, and placing the sprayed sample binding pad in an oven for drying, wherein the labeled probe bioconjugate is obtained by diluting a solidifying solution containing glycine; The sample pad was soaked in glycine buffer, dried in a drying oven and stored for later use; Take out the polyvinyl chloride base plate with the dried nitrocellulose membrane attached, and stick the prepared sample pad, conjugate pad and absorbent paper to the base plate in order, making sure that the parts overlap at the joints; cutting the assembled test strips along a predetermined width to obtain a plurality of immunochromatographic test strips; Wherein, the bioconjugate containing the labeled probe comprises the immunochromatographic detection reagent according to any one of claims 1 to 8.

10. The method for preparing an immunochromatographic test strip according to claim 9, characterized in that: The concentration of the labeled probe bioconjugate solution ranges from 10 μg / mL to 70 μg / mL.

11. The method for preparing an immunochromatographic test strip according to claim 9, characterized in that: After the immune reaction is completed, the immunochromatographic test strip is coated with a luminescent regulating medium on the nitrocellulose membrane, wherein the regulating medium contains a luminescent ligand, a buffer system and a surfactant.

12. The method for preparing an immunochromatographic test strip according to claim 11, characterized in that: The immune response time is 2 min to 20 min.

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