Detection reagent for biological analysis, kit and application

Abstract

The invention relates to the technical field of biological detection, in particular to a detection reagent for biological analysis, a kit and application, the detection reagent comprises a regulating medium, and the regulating medium comprises a buffer system. Through innovative component combination and formula optimization, non-specific adsorption is reduced, the signal-to-noise ratio is increased, the detection sensitivity is improved, particularly, a specific amino acid buffer solution is introduced, the specific amino acid buffer solution can interact with a hydrophobic region on the surface of protein, non-specific adsorption components are reduced, and the biological analysis and detection performance can be remarkably improved.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a detection reagent, a kit and an application thereof for biological analysis. Background Art

[0002] As a fast, simple and economical detection method, bioanalysis technology is widely used in many fields such as clinical diagnosis, food safety, and environmental monitoring. These technologies include but are not limited to immunochromatography, enzyme-linked immunosorbent assay (ELISA), fluorescence analysis, etc. They rely on the specific interaction of biological molecules (such as antigens and antibodies, enzymes and substrates) and achieve detection through various markers (such as chemiluminescence, fluorescence, enzyme catalysis, etc.). However, despite the many advantages of bioanalysis technology, there are still some significant problems in the prior art that affect its detection performance and application effect.

[0003] Nonspecific adsorption is one of the main problems in existing bioanalytical techniques. Nonspecific adsorption refers to the unintended interaction of labeled molecules or other sample components with non-target areas or non-target molecules in the detection system during the detection process. This phenomenon is common in various bioanalytical techniques, including immunochromatography, ELISA and other methods. For example, in immunochromatography, nonspecific adsorption may occur outside the specific area of ​​the test strip, while in ELISA, it may occur at nonspecific binding sites on the microplate.

[0004] Nonspecific adsorption can bring about a series of adverse effects. First, it can significantly increase the background signal. During the detection process, the interfering substances in the labeled molecules or samples bind to nonspecific positions in the detection system, resulting in an uncontrollable increase in the background signal. This background signal is mixed with the signal generated by specific binding, increasing the uncertainty of the test results. Secondly, the reduction in signal-to-noise ratio is a direct consequence of nonspecific adsorption. A significant increase in background signal will reduce the ratio of specific signal to background signal, that is, the signal-to-noise ratio decreases. Under conditions of low signal-to-noise ratio, it is difficult for the detection system to distinguish between the true signal and background noise, thus affecting the accuracy and reliability of the test results.

[0005] In addition, detection sensitivity is also negatively affected by nonspecific adsorption. Sensitivity refers to the ability of the detection system to identify and measure low concentrations of target substances. Due to the increase in background signals and the decrease in signal-to-noise ratio, the signals generated by low concentrations of target substances may be masked, resulting in decreased detection sensitivity. This not only limits the application of bioanalytical technology in high-sensitivity detection needs, but also affects the overall performance of various bioanalytical methods, including the accuracy of quantitative analysis and the reliability of qualitative analysis.

[0006] In the existing technology, researchers have tried to mitigate the impact of nonspecific adsorption through various methods. For example, optimizing the use of blocking agents can reduce nonspecific binding, but the effect is limited and may increase operational complexity and cost. Improving markers has also been widely studied, such as developing new fluorescent markers or optimizing the surface modification of nanoparticles, but in practical applications, the effects of these methods are not satisfactory and often require a trade-off between sensitivity and background signal.

[0007] Although these methods can mitigate the impact of nonspecific adsorption to a certain extent, they often have problems such as limited effect, complex operation, increased cost or difficulty in promotion. Therefore, the development of a new bioanalytical detection reagent that can effectively reduce nonspecific adsorption and improve detection performance remains an important direction of current research. This new detection reagent not only needs to solve the problem of nonspecific adsorption, but also has a wide range of applicability and can play a role in various bioanalytical techniques, thereby improving the overall detection performance and reliability. Summary of the invention

[0008] Based on the above needs, the present invention proposes a new type of bioanalytical detection reagent, which aims to reduce nonspecific adsorption, improve the signal-to-noise ratio and detection sensitivity through innovative component combination and formula optimization. In particular, the introduction of specific amino acid buffers can interact with the hydrophobic areas on the protein surface and reduce nonspecific adsorption components, which can significantly improve the performance of immunochromatographic detection.

[0009] In a first aspect of the present invention, a biological analysis detection reagent is provided, wherein the detection reagent comprises a regulating medium, the regulating medium comprises a buffer system, and the buffer system comprises an amino acid buffer.

[0010] Introducing amino acids into the conditioning medium can reduce nonspecific adsorption. Because in immunochromatographic testing, the protective layer formed by amino acids on the surface of the test paper can prevent non-target proteins from directly contacting the matrix material, thereby reducing nonspecific adsorption. This protective layer can effectively isolate the nonspecific interaction between proteins and matrix materials, and improve the specificity of the test.

[0011] In an embodiment of the present invention, the amino acid buffer is at least one selected from the group consisting of glycine buffer, arginine buffer, lysine buffer, histidine buffer, glutamate buffer, aspartic acid buffer and proline buffer.

[0012] By introducing at least one of glycine buffer, arginine buffer, lysine buffer, histidine buffer, glutamic acid buffer, aspartic acid buffer and proline buffer as a regulating medium in an embodiment of the present invention, for example, arginine and lysine are positively charged basic amino acids, which can electrostatically interact with the negatively charged region of the protein surface in the buffer to form a stable charge complex. Glutamic acid and aspartic acid are negatively charged acidic amino acids, which can interact with the positively charged region of the protein surface. These charge interactions help to reduce the nonspecific adsorption of protein on the matrix material. Proline and glycine etc. have hydrophobic side chains, which can interact with the hydrophobic region of the protein surface to form a protective layer to prevent non-target proteins from nonspecifically binding to the matrix material.

[0013] In an embodiment of the present invention, the regulating medium further comprises at least one of a luminescent ligand and an amphiphilic molecule.

[0014] Luminescent ligands such as fluorescent molecules, enzyme markers, etc. can specifically bind to target molecules and emit detectable signals under specific conditions. The introduction of luminescent ligands can directly enhance the intensity of the detection signal, making the detection of target molecules more sensitive and clear. This signal enhancement effect is particularly suitable for the detection of low-concentration target molecules. Amphiphilic molecules (such as surfactants) have a structure with both hydrophilic and hydrophobic parts. This structure can form micromicelles in the sample, improve the solubility and distribution uniformity of the target molecule, and thus optimize the detection signal.

[0015] In an embodiment of the present invention, when the regulating medium includes both luminescent ligands and amphiphilic molecules, the concentration ratio of the luminescent ligands to the amphiphilic molecules is 0 to 2. Luminescent ligands can provide strong detection signals at appropriate concentrations. However, excessive luminescent ligand concentrations may lead to fluorescence quenching or signal saturation, thereby reducing the detection signal. Therefore, by controlling the concentration of the luminescent ligand within a reasonable range, the signal intensity can be maximized. An appropriate amount of luminescent ligands can provide a strong detection signal, making the signal of a low-concentration target molecule more obvious and improving the detection sensitivity. However, an excessive amount of luminescent ligands may cause an increase in background noise and reduce the signal-to-noise ratio. Amphiphilic molecules can form stable micro-micelles at appropriate concentrations, optimize the distribution and solubility of the target molecule, and thus enhance the detection signal intensity. Excessive amphiphilic molecule concentrations may lead to excessive formation of micro-micelles, which in turn inhibits the effect of luminescent ligands. By precisely controlling the concentration ratio of luminescent ligands to amphiphilic molecules to 0 to 2 in the regulating medium, the novel bioanalysis detection reagent of the present invention significantly optimizes the detection signal intensity, improves the detection sensitivity, and enhances the signal stability. The realization of these technical effects benefits from the reasonable ratio of luminescent ligands and amphiphilic molecules, which maximizes their synergistic effect, reduces nonspecific interference, and maintains high signal intensity and stability.

[0016] In an embodiment of the present invention, the concentration of the amphiphilic molecule is 0% to 1.0%. Within the concentration range of 0% to 1.0%, the amphiphilic molecule can effectively reduce signal drift and variation, ensuring that the detection signal remains stable over a period of time, thereby improving the reliability and reproducibility of the detection.

[0017] In an embodiment of the present invention, the luminescent ligand is selected from at least one of the group consisting of β-diketone ligands, carboxylic acid ligands, multidentate nitrogen ligands, crown ether ligands, polyphenol ligands, phosphate ligands, oxygen-containing heterocyclic ligands, macrocyclic ligands, polymer ligands and heteropolyacid ligands.

[0018] In an embodiment of the present invention, by selecting different types of ligands to coordinate with Eu (europium) ions, it is possible to significantly improve luminescence performance, enhance stability and optimize detection sensitivity. Specifically, the selected ligand type includes β-diketone ligands, carboxylic acid ligands, multidentate aza ligands, crown ether ligands, polyphenol ligands, phosphate ligands, oxygen-containing heterocyclic ligands, macrocyclic ligands, polymer ligands and heteropolyacid ligands, and the coordination of each ligand and Eu has unique chemical properties and technical effects. For example, β-diketone ligands can effectively transfer the absorbed energy to Eu ions, thereby significantly enhancing the luminescence intensity of Eu, and Eu-β-diketone complexes usually have a long luminescence life, which helps to improve the sensitivity and signal stability of detection, and can maintain excellent luminescence performance in a variety of detection environments. The complex formed by carboxylic acid ligands and Eu ions has higher stability, can remain stable under different pH values ​​and temperature conditions, provides stable detection signals, and is insensitive to environmental changes. Multidentate aza ligands can selectively coordinate with Eu ions to reduce nonspecific interference. Crown ether ligands have high affinity and can effectively coordinate with Eu ions to improve the luminescence properties of the complex. The complex has good fluidity in the solution, which is conducive to uniform distribution and detection.

[0019] In an embodiment of the present invention, the detection reagent further comprises a rare earth compound and a porous carrier, wherein the rare earth compound is coated in the porous carrier. In the porous carrier, the rare earth compound may undergo an exchange reaction with a ligand in a solvent, which may form a more soluble complex, thereby increasing solubility. Certain porous carriers may provide stable complexation sites, allowing the rare earth compound to exist in a stable complexed form, thereby increasing its solubility in the solvent.

[0020] In an embodiment of the present invention, the rare earth compound is europium trioxide, and the porous carrier is dendritic mesoporous silica or its functionalized derivatives. In the field of immunochromatography, the rare earth compound is preferably europium trioxide, because europium trioxide (Eu2O3) has a strong red fluorescence emission characteristic, and can emit bright red fluorescence under ultraviolet or near-ultraviolet light excitation. In addition, the fluorescence emission spectrum peak of europium trioxide is narrow, and the background interference is small, which is conducive to improving the sensitivity and signal-to-noise ratio of detection. At the same time, dendritic mesoporous silica (Dendritic Mesoporous Silica, DMS) has a highly ordered mesoporous structure, providing extremely high specific surface area and large pore volume. This makes it possible to effectively load a large amount of rare earth compounds and improve the loading efficiency of markers.

[0021] In an embodiment of the present invention, the functionalized derivative comprises at least one functional group selected from hydroxyl, carboxyl, amino, thiol, aldehyde and amide. The surface silanol of DMS makes it easy to carry out chemical modification and functionalization, such as the introduction of various functional groups such as amino, carboxyl, sulfhydryl, etc., thereby enhancing its binding ability with biomolecules, increasing specificity and stability. Through functional modification, DMS can be firmly combined with antibodies, antigens or other biomolecules, improving the specificity and sensitivity of immunochromatographic detection.

[0022] In a second aspect of the present invention, a kit for biological analysis is provided, the kit comprising the aforementioned detection reagent.

[0023] In the third aspect of the present invention, the aforementioned detection reagent and kit are used in the preparation of reagents for the following detections: (1) immunochromatographic detection; (2) enzyme-linked immunosorbent assay; or (3) a combined detection comprising both immunochromatographic detection and enzyme-linked immunosorbent assay. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the XRD characterization result of Eu2O3 nanoparticles in Example 1;

[0025] Figure 2 is an electron microscope image of the dendritic mesoporous silica (DMSNs) in Example 2;

[0026] Figure 3 It is the effect of common buffer and amino acid buffer on the coordination-enhanced fluorescence of Eu2O3 nanoparticles;

[0027] Figure 4 This is a diagram of the luminescence effect of Comparative Example 1 and Example 6 on the immunochromatographic test strip;

[0028] Figure 5 is the effect of pH of glycine buffer on the coordination-enhanced fluorescence of Eu2O3 nanoparticles;

[0029] Figure 6 is the effect of the concentration of amphiphilic molecules on the coordination-enhanced fluorescence of Eu2O3 nanoparticles;

[0030] Figure 7 This is the result of PCT detection using Eu2O3@DMSN-PCT CE-TRFIA immunochromatographic test strip. DETAILED DESCRIPTION

[0031] The present invention is further described below by way of examples, but is not intended to be limiting of the present invention. Any person skilled in the art may utilize the above disclosed technical contents to make changes or modifications to equivalent embodiments of equivalent changes. However, any modification, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the technical solution of the present invention still fall within the protection scope of the technical solution of the present invention.

[0032] The test instruments and test methods used in the embodiments of the present application are as follows:

[0033] TEM, HRTEM and EDS Mapping characterization: The prepared Eu2O3 was diluted with n-hexane or ethanol and ultrasonicated, then dropped onto the carbon support film copper grid. After the solvent was completely evaporated, electron microscopy testing was performed.

[0034] XRD characterization: The prepared sample powder was tested using an X'pert PRO diffractometer at a scanning speed of 1° / min (Cu-Kα radiation, tube voltage 40 kV).

[0035] ICP analysis: The sample to be tested was fully dissolved in dilute hydrochloric acid and then filtered through a 0.4 μm aqueous filter membrane, and tested using Therm-Fisher's ICP-AES.

[0036] Fluorescence and absorption spectra: The steady-state fluorescence spectra were measured on an Edinburgh FS5 spectrometer, and the UV-visible absorption spectra were measured on a Shimadzu UV-2600 UV-visible spectrophotometer.

[0037] Example 1 Synthesis of Eu2O3

[0038] Dissolve Eu(Ac)3·4H2O in 20mL xylene, add oleylamine, transfer to a three-necked flask after ultrasonication, and stir under nitrogen for 30 minutes. Then heat to 95°C, add deionized water after reaction, and keep warm for 1h. Precipitate Eu2O3 nanoparticles with anhydrous ethanol, wash the precipitate with ethanol and n-hexane after centrifugation, and finally store in n-hexane.

[0039] Results Reference Figure 1 , Figure 1XRD characterization of Eu2O3 nanoparticles. XRD characterization found that the particles are amorphous. This is because they are wrapped by oleic acid molecules after rapid nucleation in alkaline solution at a lower temperature, which does not meet the crystallization conditions. Compared with crystals, the interaction between atoms on the surface and inside of amorphous particles is weaker, which helps to achieve faster coordination.

[0040] Example 2 Preparation of dendritic mesoporous silica (DMSNs)

[0041] Dendritic mesoporous silica (DMSNs) was prepared in one step using the CTAB template method. Take triethanolamine and add it to 25mL of water and stir it magnetically. Add sodium salicylate and CTAB, and continue stirring for 1h. Under stirring at 300rpm, add 4mL of LTEOS dropwise, keep stirring at 300rpm for 2h, then stop stirring and let stand at 80℃ for 6 hours. Collect the product by centrifugation and wash it with water and ethanol several times. Finally, use a mixture of hydrochloric acid and ethanol, stir at 80℃ for 2 hours and wash it 3 times to remove CTAB.

[0042] Results Figure 2 TEM images show that the prepared silica (DMSNs) are uniformly spherical and have abundant dendritic mesoporous channels. The rich pore structure of MSNs lays the foundation for the large-scale loading of ultra-small nanoparticles.

[0043] Example 3 Preparation of Eu2O3@DMSNs microspheres

[0044] Considering that it is difficult to couple antibodies on the surface of Eu2O3 nanoparticles, and that ultra-small particles are easily adsorbed by the chromatographic NC membrane, resulting in an increase in background signals, it is designed to load a large number of ultra-small Eu2O3 nanoparticles into mesoporous silica microspheres, which can not only utilize the abundant silanol groups on the SiO2 surface to achieve surface carboxyl modification for stable antibody coupling, but also utilize the enriched Eu to achieve signal magnitude amplification, and can smoothly climb the membrane to reduce background interference.

[0045] In this example, Eu2O3@DMSNs microspheres were prepared by in-situ growth method. The specific experimental steps are as follows:

[0046] Take the mesoporous silica nanoparticle DMSNs solution (8 mL) prepared in Example 2, centrifuge to remove the supernatant, add 10 mL of xylene and 0.1 mL of oleylamine, and ultrasonically dissolve it to make it uniformly dispersed. At the same time, 0.5 mmol of Eu(CH3CO2)3.xH2O was dissolved in 20 mL of xylene and 3.5 g of oleylamine and ultrasonically dissolved. After that, the solution was added dropwise to the DMSNs solution under magnetic stirring, heated to 60 ° C and stirred for 60 minutes, then cooled to room temperature, and centrifuged to remove the Eu precursor not adsorbed in DMSN. Add 20 mL of xylene and 3.5 g of oleylamine to the precipitate, ultrasonically re-disperse it, heat it to 95 ° C under magnetic stirring, add H2O, and keep it warm for reaction. Rapidly cool to room temperature, add ethanol, centrifuge to separate the product, continue to centrifuge and wash twice with ethanol and n-hexane, and the final product Eu2O3@DMSNs is dispersed in toluene for storage.

[0047] Using the dendritic mesoporous DMSNs prepared above as a template and europium acetate as a precursor, amorphous ultra-small Eu2O3 nanoparticles were grown in situ in the pores, and a Eu2O3@DMSNs nanocomposite material with a large amount of Eu2O3 loaded in the mesoporous pores was successfully prepared. The average statistical particle size was about 282.3nm, which was slightly increased compared to the particle size of pure DMSNs. A large amount of Eu2O3 was evenly distributed in the DMSNs. The electron microscopy results showed that there were no obvious lattice fringes and the atoms were irregularly distributed, indicating an amorphous structure, proving that the composite material was amorphous. This has the same amorphous characteristics as the Eu2O3 synthesized separately, which is conducive to the consistency of the dissociation enhanced fluorescence properties.

[0048] Example 4 Preparation of Eu2O3@DMSNs microspheres

[0049] This example uses a coating method to prepare Eu2O3@DMSNs microspheres. The following are the specific experimental steps:

[0050] The n-hexane solution of Eu2O3 nanoparticles prepared in Example 1 was centrifuged and then ultrasonically re-dissolved with 10 mL of toluene. The dendritic mesoporous silica nanoparticles (DMSNs) in Example 2 were centrifuged and added with 10 mL of toluene and 100 uL of oleylamine, and magnetically stirred after ultrasonication. Ethanol was then added, and the nanoparticles were centrifuged and washed twice with ethanol and n-hexane. Finally, the product Eu2O3@MSNs was dispersed in toluene for storage.

[0051] Example 5 Surface modification of Eu2O3@DMSNs microspheres

[0052] Preparation of Eu2O3@MSNs-COOH: Take a certain amount of Eu2O3@DMSNs nanoparticles, centrifuge and remove the supernatant, add 20mL toluene and ultrasonically dissolve, add 0.1M Silane-PEG2000-COOH toluene solution dropwise under magnetic stirring, and continue stirring. Then, add 0.1M TMAH methanol solution and heat to 85℃ for reaction. After cooling to room temperature, add n-hexane for centrifugal separation, wash with n-hexane by centrifugation 3 times, and then wash with ethanol by centrifugation 2 times. Finally, add pure water and ultrasonically disperse to obtain the product Eu2O3@DMSNs-COOH.

[0053] Example 6 Performance study of Eu2O3@DMSNs microspheres in glycine buffer

[0054] Prepare a glycine buffer with the same pH and a concentration of 50mM, the amphiphilic component is 0.1% Triton X-100, and the luminescent ligand is β-NTA (150μM) + TOPO (100μM), where TOPO refers to topoisomerase. After mixing the components, ultrasonicate for 5 minutes for use. Afterwards, according to the reaction system of the molar ratio of Eu:β-NTA:TOPO=1:3:2, the nanoparticle solution is mixed with the above-prepared regulating medium solution in a cuvette, and the effects of different surfactant types on the luminescent properties of the four materials are tested, including luminescence kinetic curve test, fluorescence spectrum test and photography under 365nm ultraviolet light.

[0055] Example 7 Performance study of Eu2O3@DMSNs microspheres in arginine buffer

[0056] The experimental process of this embodiment is similar to that of embodiment 6, except that the glycine buffer is replaced by an arginine buffer.

[0057] Example 8 Study on the performance of Eu2O3@DMSNs microspheres in lysine buffer

[0058] The experimental process of this embodiment refers to that of embodiment 6, except that the glycine buffer is replaced by a lysine buffer.

[0059] Example 9 Study on the performance of Eu2O3@DMSNs microspheres in histidine buffer

[0060] The experimental process of this example is similar to that of Example 6, except that the glycine buffer is replaced by a histidine buffer.

[0061] Example 10 Study on the performance of Eu2O3@DMSNs microspheres in glutamate buffer

[0062] The experimental process of this embodiment is similar to that of embodiment 6, except that the glycine buffer is replaced by a glutamate buffer.

[0063] Example 11 Study on the performance of Eu2O3@DMSNs microspheres in aspartic acid buffer

[0064] The experimental process of this embodiment refers to that of embodiment 6, except that the glycine buffer is replaced by aspartic acid buffer.

[0065] Example 12 Study on the performance of Eu2O3@DMSNs microspheres in proline buffer

[0066] The experimental process of this example is similar to that of Example 6, except that the glycine buffer is replaced by a proline buffer.

[0067] Example 13 Study on the performance of Eu2O3@DMSNs microspheres in citric acid-sodium citrate buffer

[0068] The experimental process of this comparative example is referred to Example 6, except that the glycine buffer is replaced by a citric acid-sodium citrate buffer.

[0069] Comparative Example 1

[0070] The experimental process of this comparative example is similar to that of Example 6, except that the glycine buffer solution is replaced with deionized water.

[0071] The fluorescence performance results of Example 6-Example 10 and Comparative Example 1 and Comparative Example 2 refer to Figure 3 .

[0072] The experimental results show that the performance of the luminescence system is closely related to the selection and use of the buffer. The introduction of the ordinary buffer system resulted in poor luminescence performance, but the introduction of the amino acid buffer significantly enhanced the luminescence intensity, showing superior performance. This phenomenon may be attributed to the fact that the amino acid buffer can provide additional stability and reaction environment optimization for the luminescence reaction while maintaining a suitable pH value. Figure 4 Shown are the results of Comparative Example 1 and Example 6 presented on the immunochromatographic test strips.

[0073] Embodiment 14

[0074] A glycine buffer system luminescence regulating medium with a pH of 3.0 was prepared, wherein the amphiphilic molecule was 0.1wt% TX-100, the ligand was 150μM β-NTA and 100μM TOPO, and ultrasonication was performed for 5 minutes. Afterwards, the luminescence performance of Eu2O3@DMSNs microspheres was tested according to the above method.

[0075] Embodiment 15

[0076] The process of this embodiment is similar to that of Embodiment 14, except that pH=3.0 is replaced by pH=4.0.

[0077] Example 16

[0078] The process of this embodiment is similar to that of Embodiment 14, except that pH=3.0 is replaced by pH=5.0.

[0079] Embodiment 17

[0080] The process of this embodiment is similar to that of Embodiment 14, except that pH 3.0 is replaced by pH=6.0.

[0081] Embodiment 18

[0082] The process of this example is similar to that of Example 14, except that pH 3.0 is replaced by pH = 7.5.

[0083] Comparative Example 2

[0084] The process of this comparative example is similar to that of Example 14, except that pH 3.0 is replaced by pH=2.0.

[0085] Comparative Example 3

[0086] The process of this comparative example is similar to that of Example 14, except that pH 3.0 is replaced by pH=8.0.

[0087] Comparative Example 4

[0088] The process of this comparative example is similar to that of Example 14, except that pH 3.0 is replaced by pH = 9.0.

[0089] The experimental results of the above examples 14-18 and comparative examples 2-4 are as follows Figure 5 As shown. By analyzing the experimental data, it can be clearly observed that the acidity and alkalinity of the reaction system have a significant effect on the reaction kinetics. Under the same conditions of keeping the ligand, amphiphilic molecules and buffer, the preferred range of pH value is clearly between 3 and 7.5. Within this range, Eu2O3 exhibits excellent performance under both weak acid and neutral conditions, that is, not only the signal intensity is strong, but also the overall reaction rate is fast. Especially at pH = 6, the reaction can still reach equilibrium within 3 minutes; even in a neutral environment (pH 7), although the reaction rate is slightly slower than that in a weak acid environment, it can still reach equilibrium within 5 minutes. In contrast, the results of Comparative Examples 2-4 show that under the strongly acidic conditions of pH = 2 (Comparative Example 2), it is observed that the signal intensity is significantly reduced, the reaction rate is significantly slowed down, and it is difficult to reach equilibrium within a reasonable time. Similarly, in an alkaline environment, the experimental results of pH = 8 (Comparative Example 3) and pH = 9 (Comparative Example 4) both show a sharp drop in signal intensity and a significant delay in the reaction kinetics. These comparative experiments clearly show that under conditions where the pH is lower than 3 or higher than 7.5, the performance of the reaction system is greatly reduced and cannot meet the needs of practical applications.

[0090] Embodiment 19

[0091] This example takes the amphiphilic molecule of TX-100 as an example to illustrate the effect of the concentration of the amphiphilic molecule on the luminescence enhancement of Eu2O3@DMSNs.

[0092] A luminescence regulating medium containing gradient concentrations of TX-100 surfactant was prepared: 0%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%; other components were the same: the buffer system was glycine buffer at pH = 4, ligand (150 μM β-NTA and 100 μM TOPO), and it was set aside after ultrasonication for 5 minutes.

[0093] The luminescence performance of Eu2O3@DMSNs in the presence of different concentrations of the amphiphilic molecule TX-100 was tested according to the aforementioned method. Figure 6 The results show that as the concentration of TX-100 increases from 0 to 0.1%, the fluorescence intensity gradually increases, but when the concentration exceeds 0.2%, the fluorescence intensity of Eu 3+ The luminescence intensity at 613 nm drops rapidly, indicating that the amphiphilic molecules contribute to the coordination reaction, but too high a concentration will weaken the coordination ability, resulting in weakened luminescence.

[0094] Embodiment 20

[0095] The coupling of Eu2O3@DMSNs and PCT antibody adopts the covalent coupling method, that is, the surface carboxyl groups of Eu2O3@DMSNs are activated by EDC / NHS and then biocoupled with PCT antibody. The specific steps are as follows:

[0096] 1) Activation: Carboxylated Eu2O3@DMSNs were added to MES (25 mM, pH = 6.0) buffer, followed by 20 μL of freshly prepared EDC (10 mg / mL) and NHS (10 mg / mL) solutions, mixed quickly and activated at room temperature. After centrifugation and washing, the samples were ultrasonically dispersed in BBS (pH = 7.4) buffer.

[0097] 2) Labeling: Add 0.2 mg PCT labeled antibody to the activated microsphere solution, rotate and mix at room temperature, and incubate for 3 hours.

[0098] 3) Blocking: 10 wt % BSA was added as a blocking agent to the labeled microsphere solution, and after rotating and mixing at room temperature for 2 hours, the solution was centrifuged and washed to obtain BSA-blocked nanoparticles.

[0099] 4) Storage: The above nanoparticles were dispersed in BBS buffer (0.05 M BBS, pH=7.0, 0.5% BSA and 0.05% proclin300), and stored in a refrigerator at 4° C. for future use.

[0100] The method of coupling Eu2O3@DMSNs to chicken IgY antibody is the same as above.

[0101] Embodiment 21

[0102] The preparation steps of Eu2O3@DMSNs immunochromatographic test strips are as follows:

[0103] 1) NC membrane coating: Cut a blank NC membrane and stick it to the PVC base plate, then spray 0.8 mg / mL PCT detection antibody and 1 mg / mL goat anti-chicken IgY on the T and C lines of the NC membrane through the capillary of the membrane scribing instrument nozzle, and dry it in a 37°C oven overnight.

[0104] 2) Preparation of conjugate pad: The labeled probe bioconjugate complex (Eu2O3@DMSNs-AbPCT, 0.1 mg / mL) was evenly sprayed on the sample conjugate pad using a gold spraying apparatus and dried in an oven overnight.

[0105] The labeled probe bioconjugate complex was diluted with a solidification solution (25 mM glycine buffer at pH 8.0, containing 3% NaCl, 3% BSA, and 7.5% sucrose).

[0106] 3) Sample pad pretreatment: The sample pad was immersed in glycine buffer (pH=7.4, containing 0.5% casein sodium salt and 0.3% Tween-20), dried in a 37° C. drying oven for 2 h, and then stored for future use.

[0107] 4) Preparation of large plate: Take out the dry plastic base plate with the NC film, and stick the prepared sample pad, conjugate pad, and absorbent paper to the base plate in sequence.

[0108] 5) Cutting strips: On the PVC base plate, the sample pad, conjugate pad, and absorbent paper prepared above are sequentially pasted to the two ends of the NC membrane (coated with antibodies), with an overlap of about 2 mm at each connection, and then cut into chromatography strips of a certain width (about 0.4 mm).

[0109] Embodiment 22

[0110] The immunochromatographic test paper consists of a supporting bottom plate (PVC), a sample pad, a conjugate pad, a NC membrane, and an absorbent pad. Based on the detection principle of the double antibody sandwich method of immunochromatography, after the sample solution is added to the sample well, it swims to the conjugate pad under capillary action through the sample pad, releases the signal probe on the conjugate pad, and then flows to the side of the absorbent paper through the NC membrane. If there is no target analyte PCT in the sample, all signal probes flow directly through the T line without binding to the detection antibody (PCT secondary antibody) fixed on the T line; when the sample contains the target analyte PCT, the PCT antibody will first bind to the signal probe (Eu2O3@DMSNs-Ab PCT conjugate) on the conjugate pad, and then pass through the NC membrane and the detection antibody on the T line through the target analyte (PCT antigen) to form a "sandwich" double antibody sandwich immune complex, so that the signal probe stays on the T line; regardless of whether the sample contains the target analyte, the Eu2O3@DMSNs-Ab chicken IgY on the C line in the independent quality control system will bind to the sheep anti-chicken IgY. After the immunochromatographic reaction is completed, the conditioning medium is directly added to the NC membrane, and the luminescent signal is read using a fluorescence analyzer.

[0111] The results show that, see Figure 7 The NC film without the addition of the regulating medium only showed a strong blue background fluorescence, while the test strip with the addition of the luminescence regulating medium Eu2O3@DMSNs-Ab chicken IgY emitted a brighter red afterglow. This not only verified that Eu2O3@DMSNs and the luminescence regulating medium can achieve luminescence enhancement on the solid phase carrier NC film, but also can utilize Eu 3+ It has the characteristics of long life, and the interference of the background fluorescence signal on the membrane surface is removed by time-resolved immunoanalyzer reading, thereby further improving the detection sensitivity. In addition, 365nm is a photo of the CELFIA immunochromatographic test strip detecting PCT under ultraviolet light. The linear range of the detection of procalcitonin (PCT) in clinical serum is: 0.05ng / mL~20ng / mL, and the detection limit LoD is 0.04ng / mL.

[0112] Comparative Example 5

[0113] Referring to Example 22, the difference is that the clinical serum samples were tested using the Beckman chemiluminescence reagent in the prior art, and it was found that the detection limit of the Beckman chemiluminescence reagent for PCT was 0.5 ng / mL.

[0114] In contrast, the detection limit of the immunochromatographic detection reagent in Example 22 is ten times higher than that of the prior art. This is mainly based on the fact that the new detection reagent can quickly react with the ligand in the medium in the system where the buffer is added. 3+The coordination compounds with long afterglow luminescence characteristics were formed by coordination, and a time-resolved coordination-enhanced luminescence immunochromatography detection reagent was constructed. While effectively reducing the background fluorescence interference, the strategy of rapid coordination dissociation and enhanced luminescence was used to achieve signal amplification, further improving the sensitivity of immunochromatography detection.

[0115] In addition, it should be noted that traditional immunochromatographic test strips usually fix the luminescent material (such as a rare earth complex) directly on the test strip. When the sample to be tested is added, if the target object is present, a specific immune response will be triggered, causing the luminescent material to aggregate or redistribute, thereby generating a detectable light signal. However, this method has an inherent problem: even in the absence of a target object, the luminescent material fixed on the test strip itself will produce a certain degree of background luminescence. This persistent background signal may interfere with the test results, especially when detecting low-concentration targets, which will reduce the sensitivity and accuracy of the detection. The embodiment of the present invention adopts a step-by-step activation strategy, the core of which is to divide the luminescence process into two independent steps. First, only the cationic component of the luminescent material (such as Eu 2+) is fixed on the immunochromatographic test strip. 3+ ). These ions themselves do not have the ability to emit light, so no background signal is generated at this stage. After the addition of the sample to be tested and the completion of the immune reaction, a regulating medium containing specific ligands is added. These ligands react with the Eu on the test strip. 3+ Ion coordination forms rare earth complexes with luminescent properties. Through step-by-step activation, the luminescence process can be better controlled and the interference caused by non-specific reactions can be reduced. This method provides the possibility of multiple detections. For example, by using different metal ions and corresponding ligands, multiple targets can be detected simultaneously on the same test strip.

[0116] Embodiment 23

[0117] This example demonstrates the application of the detection reagent Eu2O3@DMSNs of the present invention and the luminescence regulating medium in a time-resolved fluorescence enzyme-linked immunosorbent assay (TR-ELISA) to improve the detection sensitivity of prostate-specific antigen (PSA) in human serum.

[0118] Dilute the capture antibody (5 μg / mL) in carbonate buffer (pH = 9.6), add 100 μL to each well, and incubate at 4°C overnight. Discard the coating solution, add 200 μL 1% BSA to each well, and incubate at room temperature for 2 hours. Add 100 μL of gradient diluted PSA standard or serum sample to be tested, and incubate at 37°C for 2 hours. Add 100 μL of biotinylated detection antibody (2 μg / mL) and incubate at 37°C for 1 hour. Add 100 μL of streptavidin-modified Eu2O3@DMSNs (1:1000 dilution) and incubate at room temperature for 30 minutes. Add 100 μL of luminescence adjustment medium (buffer system is glycine buffer with pH = 4, ligand (150 μM β-NTA and 100 μM TOPO)) and incubate at room temperature for 10 minutes. A time-resolved fluorescence detector was used with an excitation wavelength of 340 nm, an emission wavelength of 615 nm, a delay time of 400 μs, and a gate width of 400 μs.

[0119] Comparative Example 6

[0120] The difference is that the glycine buffer is replaced by a citric acid-sodium citrate buffer. For other steps, see Example 23.

[0121] The minimum detection limit (LOD) of PSA detected by the TR-ELISA method using Eu2O3@DMSNs detection reagent and luminescence regulating medium is 0.01ng / mL, while the LOD of the comparative TR-ELISA method using conventional citric acid-sodium citrate buffer is 0.1ng / mL. This shows that the detection reagent of the embodiment of the present invention significantly improves the detection sensitivity of TR-ELISA, which is about 10 times that of conventional buffer. In the detection of low-concentration PSA (0.05ng / mL), the signal-to-noise ratio (S / N) using the detection reagent of the embodiment of the present invention is 15.3, while the S / N of the comparative example is only 4.7. This shows that the detection reagent of the present invention significantly improves the signal intensity, reduces the background signal, and improves the reliability of detection.

[0122] 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. A detection reagent for biological analysis, characterized in that The detection reagent includes a regulating medium, the regulating medium includes a buffer system, and the regulating medium also includes at least one of a luminescent ligand and an amphiphilic molecule.

2. The detection reagent for biological analysis according to claim 1, characterized in that The buffer system includes an amino acid buffer.

3. The detection reagent for biological analysis according to claim 2, characterized in that The amino acid buffer is at least one selected from the group consisting of glycine buffer, arginine buffer, lysine buffer, histidine buffer, glutamate buffer, aspartic acid buffer and proline buffer.

4. The detection reagent for biological analysis according to claim 1, characterized in that The pH range of the conditioning medium is 3 to 7.

5.

5. The detection reagent for biological analysis according to claim 1, characterized in that When the regulating medium includes both the luminescent ligand and the amphiphilic molecule, the concentration ratio of the luminescent ligand to the amphiphilic molecule is 0-2.

6. The detection reagent for biological analysis according to claim 1, characterized in that The concentration of the amphiphilic molecule is between 0% and 1.0%.

7. The detection reagent for biological analysis according to claim 1, characterized in that The luminescent ligand is at least one selected from the group consisting of β-diketone ligands, carboxylic acid ligands, multidentate nitrogen ligands, crown ether ligands, polyphenol ligands, phosphate ligands, oxygen-containing heterocyclic ligands, macrocyclic ligands, polymer ligands and heteropolyacid ligands.

8. The detection reagent for biological analysis according to claim 7, characterized in that The luminescent ligand is selected from at least one of β-naphthoyltrifluoroacetone and trioctylphosphine oxide.

9. The detection reagent for biological analysis according to claim 1, characterized in that The detection reagent for biological analysis also includes a rare earth compound and a porous carrier, and the rare earth compound is coated in the porous carrier.

10. The detection reagent for biological analysis according to claim 9, characterized in that: The rare earth compound is europium trioxide, and the porous carrier is dendritic mesoporous silica or its functionalized derivatives.

11. The detection reagent for biological analysis according to claim 10, characterized in that: The functionalized derivative comprises at least one functional group selected from the group consisting of a hydroxyl group, a carboxyl group, an amino group, a thiol group, an aldehyde group and an amide group.

12. A kit for biological analysis, characterized in that: The kit comprises the detection reagent according to any one of claims 1-11.

13. Use of the detection reagent according to any one of claims 1 to 11 or the kit according to claim 12 in the preparation of a reagent for the following detection: (1) immunochromatographic detection; (2) enzyme-linked immunosorbent assay; or (3) a combined detection comprising both immunochromatographic detection and enzyme-linked immunosorbent assay.

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