An immunochromatographic detection reagent and a preparation method of a test strip thereof

Abstract

The application relates to the technical field of detection, in particular to an immunochromatographic detection reagent and a preparation method of a test strip thereof. The immunochromatographic 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 a signal in a luminescence adjusting medium containing a luminescence ligand, a buffer and a surfactant. The technical scheme of the application not only simplifies a detection process, reduces a detection cost and improves detection sensitivity, but also provides powerful technical support for various rapid detections, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to an immunochromatographic detection reagent and a preparation method of a test strip thereof. BACKGROUND

[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 visually present the detection results through a visual means. Traditionally, this visual means often involves rare earth compounds, especially rare earth nanoparticles, as a marker. Rare earth elements, such as lanthanum, cerium and neodymium, are widely used in luminescent labeling due to their unique optical and electrochemical properties, to achieve signal amplification and improve detection sensitivity.

[0003] However, although the application of rare earth elements in immunochromatographic detection brings many advantages, there are obvious problems in practical application. First, the abundance of rare earth elements is low and unevenly distributed, mainly concentrated in specific geographical regions. This uneven distribution leads to high cost of mining and purification of rare earth resources, which in turn makes the price of rare earth-based detection materials relatively expensive. Second, the mining and processing of rare earth elements often cause serious environmental problems, including the generation of radioactive waste and the emission of toxic by-products, which pose potential threats to the ecological environment and human health. In addition, the market price of rare earth elements has high volatility, which may adversely affect the cost of health care 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 detection, the cost and environmental problems brought by these rare earth compounds, as well as the uncertainty of resource supply, pose challenges to the widespread application and sustainable development of this technology. In view of this, developing a lower-cost, less environmentally damaging and stably supplied alternative marker material has become an important issue for the development of immunochromatographic detection technology. SUMMARY

[0005] In order to solve the problems existing in the prior art, the present application provides an immunochromatographic detection reagent and a preparation method of a test strip thereof. By introducing mesoporous microspheres coated with metal oxides into the immunochromatographic detection reagent, transition metal oxides and main group metal oxides are used to replace rare earth elements in the prior art. By introducing a luminescence adjusting medium, the luminescence properties are improved. The strong coordination ability of the luminescence adjusting medium and the metal coordination dissociation enhance the luminescence characteristics. Through a secondary linkage fluorescence signal amplification strategy, the detection sensitivity is improved.

[0006] The immunochromatographic detection reagent of the present application comprises mesoporous microspheres and metal oxides, the metal oxides are distributed in the mesoporous microspheres, and the detection reagent is used for enhancing the signal in a luminescence adjusting medium containing a luminescent ligand, a buffer and a surfactant.

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

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

[0009] By further preferably the metal oxide is at least one of aluminum oxide (Al2O3), magnesium oxide (MgO) and zinc oxide (ZnO), firstly, aluminum oxide (Al2O3) is well 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 enables the stability of the detection reagent to be maintained in different detection environments. In addition, it itself has certain light absorption performance, which can play a role in filtering stray light in some detection applications, thereby improving the accuracy of detection. Zinc oxide (ZnO) is a commonly used semiconductor material with significant photoelectric properties, especially under ultraviolet excitation. This property can be utilized to further enhance the detection signal, especially in situations requiring an ultraviolet excitation source. By taking aluminum oxide, magnesium oxide and zinc oxide as representatives of metal oxides, the immunochromatographic detection reagent of the present application 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, and their abundant sources, low cost characteristics, and wide application in industrial production ensure the sustainable production and wide application of the detection reagent. The resulting technical effects provide a new, cost-effective and environmentally friendly signal enhancement approach for the field of immunochromatographic detection.

[0010] The luminescent ligand structure is as follows:

[0011]

[0012] wherein 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 any of R1 to R6 is not connected to any group.

[0013] The above structure can form stable complexes with many metal ions, and these complexes usually have good fluorescence properties. The selection of R groups can not only maintain or enhance the luminescent properties of the above structure after coordination with metal ions, but also does not destroy the chelating sites. In addition, the selection of R groups in the above structure can maintain the overall planarity of the molecule, which is beneficial to the π-π interaction with metal ions, and helps to form stable complexes and enhance the luminescent properties of the complexes.

[0014] The mesoporous microspheres are preferably mesoporous silica nanoparticles. The mesoporous microspheres, preferably mesoporous silica nanoparticles (MSNs), bring multiple technical effects and advantages to the immunochromatographic detection reagent of the application. The mesoporous silica nanoparticles have a very high specific surface area and a large pore volume, which enables them to load a large amount of metal oxides, thereby increasing the active sites for interaction with luminescent ligands and significantly improving the intensity of the generated signal. The chemical stability of silica is excellent, and it can exist stably under various pH conditions and shows good compatibility with various chemical reagents and biological molecules, ensuring the reliability of the detection reagent under different environments. The transparency of silica itself ensures that it does 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, providing an 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, glycine buffer. The selection of the above buffer solution can effectively maintain the stability under different pH conditions, ensuring the activity of biological molecules and the accuracy of detection reaction. The specific buffer solution can resist the influence of external factors (such as temperature change, ion strength change, etc.) on pH, which is particularly important for immunochromatographic detection in complex samples. In addition, the above-mentioned buffer solution is compatible with biological systems and will not damage or affect the activity of biological molecules, which is crucial for maintaining the 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 dodecyl benzene sulfonate, cetyltrimethylammonium bromide, cocamide propyl betaine, and dodecyl sulfobetaine. The above surfactant can reduce non-specific protein adsorption and reduce background noise, thereby improving the specificity of detection. In addition, the surfactant can adjust the affinity of proteins with ligands or antibodies by changing the surface properties of proteins.

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

[0018] A blank nitric acid cellulose membrane (NC membrane) is attached to a polyvinyl chloride (PVC) base plate; a predetermined concentration of PCT detection antibody and goat anti-chicken IgY are sprayed onto the NC membrane at the predetermined test line (T line) and control line (C line) positions using a membrane marker, and placed in an oven for drying; a solution containing a labeled probe biological conjugate is uniformly sprayed on the 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 biological conjugate is obtained after dilution with a solidification solution containing 25 mM glycine with a pH of 8.0; the sample pad is treated by soaking in a 25 mM glycine buffer (pH 7.4, containing 0.5% sodium caseinate and 0.3% Tween-20), and then dried and stored in a drying oven at 37°C for future use; the PVC base plate with the dried NC membrane attached is removed, and the prepared sample pad, binding pad, and absorbent paper are attached to the base plate in order, ensuring that each part overlaps at the connection by about 2 mm; the assembled test strip is cut along the predetermined width (about 0.4 mm) to obtain multiple immunochromatographic test strips. In this embodiment, the labeled probe biological conjugate contains an immunochromatographic detection reagent as described above.

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

[0020] After the immunoreaction is completed, a luminescence adjusting medium is applied above the NC membrane, and the adjusting medium contains a luminescence ligand, a buffer system, and a surfactant.

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

[0022] By applying a luminescence adjusting medium on the test strip, the detection signal can be improved, the detection sensitivity can be enhanced, and low-concentration antigens can be detected. Using a predetermined concentration of PCT detection antibody and goat anti-chicken IgY as the capture and control lines can specifically bind to the target antigen and the label, reduce non-specific signals, and improve the accuracy of the results. The design of the above-mentioned immunochromatographic test strip aims to provide a rapid, simple, sensitive, specific, stable, and intuitive detection method, which is suitable for a wide range of applications, including but not limited to clinical diagnosis, on-site rapid detection, food safety detection, and other fields. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure for the luminescence performance of ZnO@DMSNs nanoparticles in different buffers in the embodiments of the present application;

[0024] Figure 2 Figure for the luminescence performance of MgO@DMSNs nanoparticles in different buffers in the embodiments of the present application;

[0025] Figure 3 Figure of luminescence performance of Al2O3@DMSNs nanoparticles in different buffers in the embodiment of the present application;

[0026] Figure 4 Figure of luminescence performance of ZnO@DMSNs nanoparticles in different amino acid buffers in the embodiment of the present application;

[0027] Figure 5 Figure of correlation between CLEFIA test strip and Beckman Coulter scatter turbidimetry test results of CRP in the embodiment of the present application. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0029] Embodiment 1

[0030] The embodiment provides a preparation method of ZnO nanoparticles.

[0031] Zinc acetate was dissolved in 20 mL of methanol, and 100 μL of oleic acid was added after heating and stirring. The mixture was continuously stirred for 10 min, and then gradually heated to 95 ℃ to form a mixed solution A. Tetramethylammonium hydroxide (TMAH) was dissolved in 5 mL of methanol solution to form a mixed solution B. The mixed solution A and the mixed solution B were reacted for 10 min. After the reaction was completed, the white precipitate was cooled. The product was collected by centrifugal separation and washed with ethanol and n-hexane alternately for 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 aggregation, and the particle size distribution was between 2.5 nm and 6 nm. The XRD diffraction peak position and intensity were consistent with the standard card.

[0032] Embodiment 2

[0033] The embodiment provides a preparation method of MgO nanoparticles.

[0034] Magnesium acetate was dissolved in 20 mL of methanol in a flask, heated and stirred, and then 100 μL of oleic acid was added, and stirring was continued for 10 min, and then the temperature was gradually increased to 95°C. TMAH was dissolved in 5 mL of methanol solution, and was quickly added to the flask, and the reaction was carried out for 30 min. After the reaction was completed, the white precipitate was obtained. The product was collected by centrifugation, and was washed several times with ethanol and n-hexane. The final product was dispersed in n-hexane for storage. The results show that the MgO nanoparticles are uniformly dispersed, most of which are triangular, regularly arranged, and there is no agglomeration phenomenon. The particle size distribution of the nanoparticles is between 3-10 nm. The XRD diffraction peak position and intensity are consistent with the standard card PDF#97-018-8324, indicating that the synthesized substance is MgO nanoparticles in a cubic crystal system.

[0035] Example 3

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

[0037] AlCl3·6H2O was dissolved in xylene, and 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. Then, octadecene and a small amount of oleylamine were added, and magnetic stirring was carried out under inert gas protection. The temperature was slowly increased to about 300°C to reflux, and the reaction was carried out for several hours, and then the temperature was gradually decreased. The product was separated by centrifugation using n-hexane and ethanol, and was washed several times, and the final product was dispersed in n-hexane for storage. The results show that the particles are short rods, and are uniformly dispersed. The particle size statistics show that the average particle size is about 5.2 nm. The XRD diffraction peak is consistent with the standard card PDF#97-006-6559, which is cubic crystal system Al2O3.

[0038] Example 4

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

[0040] A certain amount of the prepared ultra-small ZnO nanoparticles in Example 1 was mixed with dendritic mesoporous silica (DMSNs) nanoparticles, 20 mL of toluene solution was added, and then 100 μL of oleylamine was added, and ultrasonic treatment was carried out for 10 min, and then magnetic stirring was carried out at room temperature for 10 h. Then, ethanol was added, and the nanoparticles were separated by centrifugation, and were washed with ethanol and n-hexane for 3 times, and finally the product ZnO@DMSNs was dispersed in toluene for storage. The results show that a large amount of ultra-small particle size ZnO particles are uniformly enriched in the pore channels of mesoporous silica. There are diffraction peaks of hexagonal ZnO (PDF#97-029-0968) and characteristic bump peaks of SiO2 near 22°, indicating that the ZnO phase in the ZnO@DMSNs particles is consistent with the ultra-small ZnO nanoparticles alone.

[0041] Example 5

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

[0043] A certain amount of ZnO@DMSNs nanoparticles is centrifuged to remove supernatant, 20 mL of toluene is added for ultrasonic redissolution, magnetic stirring is performed, 2 mL of Silane-PEG2000-COOH toluene solution (0.1 M) is added dropwise, stirring is performed for 5 minutes, 2 mL of TMAH methanol solution (0.1 M) is added, and the temperature is increased to 85 DEG C for reaction for 45 minutes. After cooling, ethanol is added for centrifugal separation, n-hexane is used for centrifugal washing for 3 times, ethanol is used for centrifugal washing for 2 times, pure water is added, ultrasonic dispersion is performed, and the product ZnO@DMSNs-COOH is obtained.

[0044] Example 6

[0045] The embodiment provides a preparation method of MgO@DMSNs. The preparation process of MgO@DMSNs is referred to the embodiment 4, and the difference is that ZnO nanoparticles are replaced by MgO nanoparticles.

[0046] Example 7

[0047] The embodiment provides a preparation method of Al2O3@DMSNs. The preparation process of Al2O3@DMSNs is referred to the embodiment 4, and the difference is that ZnO nanoparticles are replaced by Al2O3 nanoparticles.

[0048] Example 8

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

[0050] The adjusting medium component in the embodiment is 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 adjusting medium is added in a transparent cuvette, then ZnO@DMSNs nanoparticles prepared in the embodiment 4 are added, rapid mixing is performed, and then the reaction kinetics curve and the fluorescence emission spectrum (λex=365 nm, λem=520 nm) of 10 minutes are measured on an FS5 spectrometer.

[0051] Comparative Example 1

[0052] The ligand and the surfactant in the adjusting medium used in the comparative example have the same component and concentration as those in the embodiment 8, and the difference is 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 adjusting medium used in this comparative example are the same as those in Example 8, except that the glycine-HCl buffer is replaced by a phthalic acid-NaOH buffer (PA).

[0055] Comparative Example 3

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

[0057] Comparative Example 4

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

[0059] The results of Example 8 and Comparative Examples 1 to 4 are shown in Table 1. Figure 1 The experimental data show that, under the same conditions of ligand and surfactant, the ZnO@DMSNs nanoparticles exhibit significantly different luminescence properties in different buffer systems. Specifically, the effect of each buffer system on the luminescence enhancement of the ZnO@DMSNs nanoparticles, from strong to weak, is 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 test of the luminescence effect of MgO@DMSNs nanoparticles in a glycine buffer.

[0062] The experimental process is as described in Example 8, except that the ZnO@DMSNs nanoparticles are replaced by MgO@DMSNs nanoparticles.

[0063] Comparative Example 5

[0064] The components and concentrations of the ligand and surfactant in the adjusting 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 adjusting medium used in this comparative example are the same as those in Example 9, except that the glycine-HCl buffer is replaced by a phthalic acid-NaOH buffer (PA).

[0067] Comparative Example 7

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

[0069] Comparative Example 8

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

[0071] The results of Example 9 and Comparative Examples 5 to 8 are shown in Table 1. Figure 2 The data analysis shows that there is a significant difference in the luminescence performance of MgO@DMSNs nanoparticles in different buffer systems. Specifically, the luminescence intensity of MgO@DMSNs nanoparticles in glycine buffer is about 5 times that in other buffer systems. In contrast, the nanoparticles exhibit weaker luminescence intensity in phosphoric acid 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 test of the luminescence effect of Al2O3@DMSNs nanoparticles in glycine buffer.

[0074] The experimental process is as described in Example 8, except that ZnO@DMSNs nanoparticles are replaced by Al2O3@DMSNs nanoparticles.

[0075] Comparative Example 9

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

[0077] Comparative Example 10

[0078] The components and concentrations of the ligand and surfactant in the adjusting 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 adjusting 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 adjusting 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).

[0083] The results of Example 10 and Comparative Examples 9 to 12 are shown in Table 1. Figure 3 The experimental data show that, under the same conditions of ligand and surfactant, the Al2O3@DMSNs nanoparticles exhibit significantly different luminescence properties in different buffer systems. Specifically, the effect of each buffer system on the luminescence enhancement of the Al2O3@DMSNs nanoparticles, from strong to weak, is as follows: glycine-HCl buffer (Gly) > phosphoric acid 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 exhibits the most excellent performance. Compared with other buffer systems, the Gly buffer exhibits a significant fluorescence enhancement effect on the three metal compounds ZnO, MgO and Al2O3. This result indicates that the Gly buffer system can effectively promote the coordination reaction between the metal compounds and 8-hydroxyquinoline (8-HQ). In contrast, the citric acid buffer system performs the worst. After adding the three metal compounds in this system, the fluorescence signal is very weak. This phenomenon indicates that the citric acid system significantly inhibits the coordination ability of the metal compounds with 8-HQ, and is 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 exhibit a moderate degree of fluorescence enhancement effect. Although these three buffer solutions can promote the coordination reaction between ZnO and Al2O3 and 8-HQ, their fluorescence enhancement effect is not as good as that of the glycine system. It is worth noting that the PA buffer solution has solubility problems, which limits its application in the preparation and preservation of adjusting media. Due to the unique chemical structure of Gly, which has both amino and carboxylate ions, it becomes a typical bidentate ligand. This structural feature endows Gly with the ability to form stable chelates with various metal ions, including rare earth ions. Based on this feature, in the Gly buffer system, the metal ions on the surfaces of the ZnO, MgO and Al2O3 nanoparticles may undergo complexation reactions with the large number of Gly molecules present in the solution. This process promotes the coordination of the metal ions (Zn 2+ , Mg2+ and Al 3+ ) dissociation from the surface of the nanoparticles. Subsequently, these dissociated metal ions will further coordinate with 8-hydroxyquinoline (8-HQ) in the solution. As a ligand with strong complexing ability, 8-HQ can form new coordination compounds, namely ZnQ2, MgQ2and AlQ3, with these metal ions. It is these newly formed coordination compounds that exhibit a significant improvement in luminescent performance.

[0085] Example 11

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

[0087] The experimental process of this example is as in 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). The fluorescence intensity of Zn 2+ -8-HQ complex in different amino acid buffer systems is compared. The results are shown in Figure 4 The amino acid buffer systems all significantly promote the coordination reaction of metal ions with 8-HQ, showing a clear fluorescence enhancement effect. The experimental results confirm the universal effectiveness of amino acid buffer systems in enhancing the fluorescence performance of the coordination reaction of metal ions with 8-HQ.

[0088] Example 12

[0089] Gly buffer system 8-HQ luminescence adjusting medium (containing 0.8 mM 8-HQ, 0.1% TX-100) with different pH (2.0-9.0) was prepared by ultrasonic dispersion. Then, the luminescence enhancement effect of 8-HQ luminescence adjusting medium and three kinds of metal oxide nanoparticle coordination was tested. Glycine buffer system was used to prepare 8-HQ luminescence adjusting medium (containing 0.8 mM 8-HQ, 0.1% TX-100) with different pH (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0), and the coordination reaction of three kinds of metal compounds (ZnO, MgO and Al2O3) with 8-HQ was tested by fluorescence spectrum. The effect of different pH on luminescence performance was observed. The results show that when the pH of the solution is increased to 3.0, the fluorescence signal is very small when the three kinds of metal oxides are added; when the pH is 4.0, different brightness of fluorescence signal begins to appear, and the fluorescence signal further increases with the increase of pH; the fluorescence intensity of ZnO is the strongest at pH 7.0, but the fluorescence intensity of ZnO decreases with the further increase of pH, while the luminescence intensity of the other two metal oxides MgO and Al2O3 gradually increases with the increase of pH, and the fluorescence intensity is the highest at pH 9.0, but the absolute intensity is lower than that of ZnO.

[0090] Example 13

[0091] The CRP labeled antibody was modified on the surface of carboxylated ZnO@DMSNs by EDC and NHS chemical crosslinking method, and was 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 then NHS (10 mg / mL) and EDC (10 mg / mL) were added in sequence. After activation, centrifugal washing was performed to remove excess crosslinking reagents, and then ultrasonic dispersion was performed to resuspend in BBS (0.01 M, pH = 7.4) buffer.

[0093] 2) Labeling: CRP labeled antibody was added to the above activated microsphere solution, and incubation was performed at room temperature.

[0094] 3) Blocking: BSA solution was added to the labeled solution for blocking. After reaction, centrifugal washing was performed to remove the supernatant, and then the centrifugal product was added to BBS stock solution (0.01 M, pH = 7.4) and ultrasonic resuspension was performed.

[0095] 4) Preservation: the above coupled microspheres were preserved in the stock solution and stored in a refrigerator at 4°C for standby use. The conjugate was named as: ZnO@DMSNs-Ab CRP.

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

[0097] Example 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 onto the NC membrane at the predetermined test line (T line) and control line (C line) positions, respectively, and placed in an oven for drying; a solution containing a labeled probe bioconjugate is uniformly sprayed on the sample binding pad, and the sprayed sample binding pad is placed in an oven for drying, wherein the ZnO@DMSNs conjugate is obtained after dilution with a solidification solution containing 25 mM glycine at pH 8.0; the sample pad is treated by soaking in 25 mM glycine buffer, then dried in a drying oven and stored for use; the polyvinyl chloride base plate with the dried NC membrane pasted is removed, and the prepared sample pad, binding pad and absorbent paper are pasted to the base plate in order, ensuring that each part has an overlap of about 2 mm at the connection; the assembled test strip is cut along the predetermined width to obtain multiple immunochromatographic test strips. Among them, the CRP detection antibody and goat anti-chicken IgY antibody on the NC membrane are diluted to 0.8 mg / mL and 1 mg / mL respectively for standby, and the final concentration of ZnO@DMSNs-Ab conjugate (labeled probe) is selected as 0.05 mg / mL.

[0099] The feasibility of coordination-enhanced luminescence strategy for immunochromatography was verified by adding an optimized 8-HQ luminescence adjusting medium on the NC membrane of the test strip. Different luminescence intensities of 8-hydroxyquinoline zinc (ZnQ2) complex micelles were formed on the NC membrane, and after the coordination-enhanced luminescence immunochromatography was immunoreacted, the luminescence adjusting medium was added dropwise on the NC membrane of the card for 2 min, and then the fluorescence signals of the T line and the C line were read by the immunization analyzer, thereby realizing quantitative detection.

[0100] Example 16

[0101] Twenty CRP serum samples certified by Beckman Coulter scatter turbidimetry were selected for detection by ZnO@DMSNs TR-CELFIA test strips. The detection results of the two methods were linearly fitted to analyze and judge the correlation of the two methods. The results are shown in Figure 5 , the correlation coefficient r between the two methods is greater than 0.99, and there is no significant difference, indicating that the ZnO@DMSNs coordination-enhanced luminescence immunochromatography has good detection accuracy.

[0102] In summary, the embodiment of the present application coats metal oxide in mesoporous silica nanospheres, uses the strong coordination ability of neutral 8-HQ luminescence adjustment medium and the metal coordination dissociation to enhance the luminescence characteristics, uses the secondary linkage fluorescent signal amplification strategy, improves the detection sensitivity, realizes the rapid and sensitive detection of CRP, the linear range of the serum sample is 1 mg / L-320 mg / L, the detection limit LoD is 0.38 mg / L, which is significantly better than the clinical reference value 10 mg / L, and is beneficial to realize the early diagnosis of diseases. In addition, the comparative detection results of the clinical samples show that the detection results of the method and the commonly used detection method in the clinic have good consistency, indicating that the method has good detection accuracy.

[0103] The above embodiments are only used to illustrate the technical solutions of the present application, rather than limit them, and the above preparation methods can be step-by-step preparation or one-step preparation; although the present application is described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can be modified or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An immunochromatographic test reagent, characterized by, The detection reagent comprises mesoporous microspheres and metal oxides distributed in the mesoporous microspheres, and is used for enhancing a luminescence signal in a luminescence adjusting medium containing a luminescence ligand, a buffer system and a surfactant. The luminescence ligand has the following structure: ； 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 any of R1 to R6 is not connected to any group.

2. The immunochromatographic test reagent according to claim 1, characterized by, The metal oxide is at least one of a transition metal oxide and a main group metal oxide.

3. The immunochromatographic test reagent according to claim 2, characterized by, The metal oxide is at least one of aluminum oxide, magnesium oxide and zinc oxide.

4. The immunochromatographic test reagent according to claim 1, characterized by, The mesoporous microspheres are dendritic mesoporous silica nanomicrospheres.

5. The immunochromatographic test reagent according to claim 1, characterized by, The buffer system is at least one of glutamine, glutamic acid, arginine, cysteine, lysine and valine glycine buffer.

6. The immunochromatographic test reagent according to claim 5, characterized by The pH range of the buffer system is 3-9.

7. The immunochromatographic test reagent according to claim 1, characterized by, The surfactant is at least one of nonylphenol polyoxyethylene ether, Tween 20, sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, cetyltrimethylammonium bromide, cocamidopropyl betaine and dodecyl sulfobetaine.

8. A method for preparing an immunochromatographic test strip, characterized by, The preparation steps are as follows: A nitrocellulose membrane is pasted onto a polyvinyl chloride base plate; PCT detection antibodies and goat anti-chicken IgY are sprayed onto the test line and control line positions on the NC membrane respectively, and are placed in an oven for drying; A solution containing a labeled probe biological conjugate is uniformly sprayed on a sample binding pad, and the sprayed sample binding pad is placed in an oven for drying, wherein the labeled probe biological conjugate is obtained after dilution with a solidification liquid containing glycine; The sample pad is soaked in a glycine buffer solution for treatment, is dried in a drying oven and is stored for standby use; The polyvinyl chloride base plate to which the dried nitrocellulose membrane has been pasted is taken out, and the prepared sample pad, binding pad and absorbent paper are pasted onto the base plate in sequence, so as to ensure that the parts are overlapped at the connection positions; The assembled test strip is cut along a predetermined width to obtain a plurality of immunochromatographic test strips. The labeled probe biological conjugate solution has a concentration range of 10 μg / mL to 70 μg / mL.

9. The method of claim 8, wherein the sample is applied to the sample pad. After the immunoreaction is completed, a luminescence adjusting medium is applied above the nitrocellulose membrane, and the luminescence adjusting medium contains a luminescence ligand, a buffer system and a surfactant.

10. The method of claim 8, wherein the method further comprises the step of drying the test strip. The time of the immunoreaction is 2 min to 20 min.

11. The method of claim 10, wherein the method further comprises the step of applying a sample to the test strip. ​

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