Detection reagents, kits and applications for bioanalysis

Abstract

This application relates to the field of bioassay technology, and more particularly to a detection reagent, kit, and application for bioanalysis. The detection reagent includes a conditioning medium, which comprises a buffer system. Through innovative component combinations and formulation optimization, it aims to reduce non-specific adsorption, improve the signal-to-noise ratio and detection sensitivity. In particular, the introduction of specific amino acid-based buffers can interact with hydrophobic regions on protein surfaces, reducing non-specific adsorption and significantly improving the performance of bioanalytical detection.

Description

Technical Field

[0001] This application relates to the field of biological detection technology, and in particular to a detection reagent, kit, and application for biological analysis. Background Technology

[0002] Bioanalytical technologies, as rapid, simple, and economical detection methods, are widely used in various fields such as clinical diagnosis, food safety, and environmental monitoring. These technologies include, but are not limited to, immunochromatography, enzyme-linked immunosorbent assay (ELISA), and fluorescence analysis. They rely on the specific interactions of biomolecules (such as antigens and antibodies, enzymes and substrates) and achieve detection through various labels (such as chemiluminescence, fluorescence, and enzyme catalysis). However, despite the many advantages of bioanalytical technologies, some significant problems still exist in existing technologies, affecting their detection performance and application effectiveness.

[0003] Non-specific adsorption is one of the major problems in existing bioanalytical techniques. Non-specific adsorption refers to the unexpected interactions that occur between labeled molecules or other sample components and non-target regions or molecules within the detection system during the detection process. This phenomenon is prevalent in various bioanalytical techniques, including immunochromatography and ELISA. For example, in immunochromatography, non-specific adsorption may occur outside the specific areas of the test strip, while in ELISA, it may occur at non-specific binding sites on the microplate.

[0004] Non-specific adsorption has a series of adverse effects. First, it significantly increases the background signal. During detection, the labeled molecules or interfering substances in the sample bind to non-specific sites in the detection system, leading to an uncontrollable increase in the background signal. This background signal mixes with the signal generated by specific binding, increasing the uncertainty of the detection results. Second, a decrease in the signal-to-noise ratio (SNR) is a direct consequence of non-specific adsorption. The significant increase in the background signal reduces the proportion of the specific signal relative to the background signal, i.e., the SNR decreases. Under low SNR conditions, the detection system struggles to distinguish between the true signal and background noise, thus affecting the accuracy and reliability of the detection results.

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

[0006] In existing technologies, researchers have attempted to mitigate the effects 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 labels has also been extensively studied, such as developing novel fluorescent labels or optimizing the surface modification of nanoparticles; however, in practical applications, these methods are not entirely satisfactory and often require a trade-off between sensitivity and background signal.

[0007] While these methods can mitigate the effects of nonspecific adsorption to some extent, they often suffer from limited effectiveness, operational complexity, increased costs, or difficulty in widespread adoption. Therefore, developing novel bioanalytical reagents that can effectively reduce nonspecific adsorption and improve detection performance remains a crucial research direction. Such novel reagents not only need to address the nonspecific adsorption problem but should also possess broad applicability, functioning across various bioanalytical techniques to enhance overall detection performance and reliability. Summary of the Invention

[0008] Based on the above-mentioned needs, this invention proposes a novel bioanalytical detection reagent. Through innovative component combination and formulation optimization, it aims to reduce non-specific adsorption, improve signal-to-noise ratio and detection sensitivity. In particular, the introduction of specific amino acid buffers can interact with hydrophobic regions on the protein surface, reduce non-specific adsorption components, and significantly improve the performance of immunochromatographic detection.

[0009] In a first aspect of the invention, a bioanalytical detection reagent is provided. The detection reagent includes a conditioning medium, the conditioning medium comprising a buffer system, the buffer system comprising an amino acid-based buffer solution.

[0010] Introducing amino acids into the conditioning medium can reduce non-specific adsorption. In immunochromatographic assays, the protective layer formed by amino acids on the test strip surface prevents direct contact between non-target proteins and the matrix material, thus reducing non-specific adsorption. This protective layer effectively isolates non-specific interactions between proteins and the matrix material, improving the specificity of the detection.

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

[0012] In embodiments of the present invention, at least one of glycine buffer, arginine buffer, lysine buffer, histidine buffer, glutamate buffer, aspartic acid buffer, and proline buffer is introduced as a conditioning medium. For example, arginine and lysine are positively charged basic amino acids that can electrostatically interact with negatively charged regions on the protein surface in the buffer to form stable charge complexes. Glutamate and aspartic acid are negatively charged acidic amino acids that can interact with positively charged regions on the protein surface. These charge interactions help reduce non-specific adsorption of proteins onto the matrix material. Proline and glycine, among others, have hydrophobic side chains, and these amino acids can interact with hydrophobic regions on the protein surface to form a protective layer, preventing non-target proteins from non-specifically binding to the matrix material.

[0013] In embodiments of the present invention, the modulation medium further includes at least one of a luminescent ligand and an amphiphilic molecule.

[0014] Luminescent ligands, such as fluorescent molecules and enzyme labels, can specifically bind to target molecules and emit detectable signals under specific conditions. Introducing luminescent ligands can directly enhance the intensity of the detection signal, making the detection of target molecules more sensitive and precise. This signal enhancement effect is particularly suitable for the detection of low concentrations of target molecules. Amphiphilic molecules (such as surfactants) have structures with both hydrophilic and hydrophobic parts. This structure can form micelles in the sample, improving the solubility and uniform distribution of target molecules, thereby optimizing the detection signal.

[0015] In embodiments of the present invention, when the conditioning medium simultaneously comprises a luminescent ligand and an amphiphilic molecule, the concentration ratio of the luminescent ligand to the amphiphilic molecule is 0–2. The luminescent ligand, at an appropriate concentration, can provide a strong detection signal. However, excessively high concentrations of the luminescent ligand 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 ligand can provide a strong detection signal, making the signal of low-concentration target molecules more prominent and improving detection sensitivity. However, excessive luminescent ligand may lead to increased background noise and reduced signal-to-noise ratio. At an appropriate concentration, the amphiphilic molecule can form stable micelles, optimizing the distribution and solubility of the target molecule, thereby enhancing the detection signal intensity. Excessively high concentrations of the amphiphilic molecule may lead to excessive micelle formation, which in turn inhibits the effect of the luminescent ligand. By precisely controlling the concentration ratio of the luminescent ligand to the amphiphilic molecule in the conditioning medium to be 0–2, the novel bioanalytical reagent of the present invention significantly optimizes the detection signal intensity, improves detection sensitivity, and enhances signal stability. These technical effects are achieved through a reasonable ratio of luminescent ligands and amphiphilic molecules, which maximizes their synergistic effect, reduces non-specific interference, and maintains high signal strength and stability.

[0016] In embodiments 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 embodiments of the present invention, the luminescent ligand is selected from at least one group consisting of β-diketone ligands, carboxylic acid ligands, polydentate aza ligands, crown ether ligands, polyphenol ligands, phosphate ligands, oxygen-containing heterocyclic ligands, macrocyclic ligands, polymer ligands, and heteropolyacid ligands.

[0018] In embodiments of the present invention, by selecting different types of ligands to coordinate with Eu (eu) ions, luminescence performance, stability, and detection sensitivity can be significantly improved. Specifically, the selected ligand types include β-diketone ligands, carboxylic acid ligands, polydentate aza ligands, crown ether ligands, polyphenol ligands, phosphate ligands, oxygen-containing heterocyclic ligands, macrocyclic ligands, polymer ligands, and heteropolyacid ligands. Each ligand has unique chemical properties and technical effects when coordinated with Eu. For example, β-diketone ligands can effectively transfer absorbed energy to Eu ions, thereby significantly enhancing the luminescence intensity of Eu. Eu-β-diketone complexes typically have a long luminescence lifetime, which helps improve detection sensitivity and signal stability, and can maintain excellent luminescence performance in various detection environments. The complexes formed by carboxylic acid ligands and Eu ions have high stability, remaining stable under different pH and temperature conditions, providing a stable detection signal, and are insensitive to environmental changes. Polydentate aza ligands can selectively coordinate with Eu ions, reducing nonspecific interference. Crown ether ligands have high affinity and can effectively coordinate with Eu ions, improving the luminescence performance of the complexes. The complexes also have good fluidity in solution, which is beneficial for uniform distribution and detection.

[0019] In embodiments of the present invention, the detection reagent further includes a rare earth compound and a porous support, wherein the rare earth compound is coated within the porous support. Within the porous support, the rare earth compound may undergo an exchange reaction with ligands in the solvent, which can form more soluble complexes, thereby increasing solubility. Some porous supports can provide stable complexation sites, allowing the rare earth compound to exist in a stable complexed form, thus improving its solubility in the solvent.

[0020] In embodiments of the present invention, the rare earth compound is europium trioxide (Eu₂O₃), and the porous support is dendritic mesoporous silica or its functionalized derivatives. In the field of immunochromatography, europium trioxide is preferred because it exhibits strong red fluorescence emission characteristics, emitting bright red fluorescence under ultraviolet or near-ultraviolet light excitation. Furthermore, europium trioxide has a narrow fluorescence emission peak and less background interference, which is beneficial for improving detection sensitivity and signal-to-noise ratio. Simultaneously, dendritic mesoporous silica (DMS) possesses a highly ordered mesoporous structure, providing extremely high specific surface area and large pore volume. This allows it to effectively load large amounts of rare earth compounds, improving the loading efficiency of the labeled substances.

[0021] In embodiments of the present invention, the functionalized derivative comprises at least one functional group selected from hydroxyl, carboxyl, amino, thiol, aldehyde, and amide groups. The surface silanol groups of DMS facilitate chemical modification and functionalization, such as the introduction of various functional groups like amino, carboxyl, and thiol groups, thereby enhancing its binding affinity to biomolecules and increasing its specificity and stability. Through functionalization, DMS can bind firmly to antibodies, antigens, or other biomolecules, improving the specificity and sensitivity of immunochromatographic detection.

[0022] In a second aspect of the invention, a kit for bioanalysis is provided, the kit comprising the aforementioned detection reagents.

[0023] In a third aspect of the invention, the aforementioned detection reagents and kits are used in the preparation of reagents for the following detections: (1) immunochromatographic detection; (2) enzyme-linked immunosorbent assay (ELISA); or (3) a combined detection comprising both immunochromatographic detection and ELISA. Attached Figure Description

[0024] Figure 1 These are the XRD characterization results of the Eu2O3 nanoparticles in Example 1;

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

[0026] Figure 3 The effects of ordinary buffer and amino acid buffer on the coordination-enhanced fluorescence of Eu2O3 nanoparticles;

[0027] Figure 4 These are images showing the luminescence effects of Comparative Example 1 and Example 6 on immunochromatographic test strips;

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

[0029] Figure 6 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 the Eu2O3@DMSN-PCT CE-TRFIA immunochromatographic test strip. Detailed Implementation

[0031] The following embodiments further illustrate the present invention, but are not intended to limit the invention. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

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

[0033] TEM, HRTEM and EDS Mapping characterization: The prepared Eu2O3 was diluted with n-hexane or ethanol and sonicated, then dropped onto a copper grid with a carbon support film. After the solvent had completely evaporated, electron microscopy 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 40kV).

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

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

[0037] Example 1: Synthesis of Eu2O3

[0038] Eu(Ac)3·4H2O was dissolved in 20 mL of xylene, oleylamine was added, and the mixture was sonicated and then transferred to a three-necked flask. Nitrogen gas was purged and the mixture was stirred for 30 minutes. The temperature was then raised to 95 °C, and after the reaction, deionized water was added and the mixture was kept at this temperature for 1 hour. Eu2O3 nanoparticles were precipitated with anhydrous ethanol, centrifuged, and then washed with ethanol and n-hexane. The precipitate was finally stored in n-hexane.

[0039] Results Reference Figure 1 , Figure 1XRD characterization of Eu₂O₃ nanoparticles revealed that they are amorphous. This is because after rapid nucleation in an alkaline solution at a lower temperature, the particles are encapsulated by oleic acid molecules, preventing the formation of crystals. Compared to crystalline particles, the amorphous particles exhibit weaker interatomic forces on their surface and within, facilitating faster coordination.

[0040] Example 2: Preparation of Dendritic Mesoporous Silica (DMSNs)

[0041] Dendritic mesoporous silica (DMSNs) were prepared in one step using the CTAB template method. Triethanolamine was added to 25 mL of water and magnetically stirred. Sodium salicylate and CTAB were added, and stirring continued for 1 h. 4 mL of LTEOS was added dropwise while stirring at 300 rpm for 2 h, then stirring was stopped and the mixture was allowed to stand at 80 °C for 6 h. The product was collected by centrifugation and washed several times with water and ethanol. Finally, it was washed three times with a mixture of hydrochloric acid and ethanol at 80 °C with stirring for 2 hours to remove CTAB.

[0042] See results Figure 2 TEM images show that the prepared silica (DMSNs) are uniform spheres with abundant dendritic mesoporous channels. The rich pore structure of MSNs lays the foundation for the large-scale loading of ultra-small diameter nanoparticles.

[0043] Example 3: Preparation of Eu2O3@DMSNs microspheres

[0044] Considering the difficulty of antibody conjugation on the surface of Eu2O3 nanoparticles and the ease with which ultra-small particles are adsorbed onto NC chromatography membranes, leading to increased background signal, a design was developed to load a large number of ultra-small Eu2O3 nanoparticles into mesoporous silica microspheres. This approach leverages the abundant silanol groups on the SiO2 surface for carboxyl modification, enabling stable antibody conjugation, while also utilizing the enriched Eu to amplify the signal by orders of magnitude. Furthermore, the nanoparticles can successfully climb the membrane, reducing background interference.

[0045] This embodiment uses an in-situ growth method to prepare Eu2O3@DMSNs microspheres. The specific experimental steps are as follows:

[0046] Take 8 mL of the mesoporous silica nanoparticle (DMSNs) solution prepared in Example 2, centrifuge to remove the supernatant, add 10 mL of xylene and 0.1 mL of oleylamine, and sonicate to redissolve and uniformly disperse. Simultaneously, dissolve 0.5 mmol of Eu(CH3CO2)3·xH2O in 20 mL of xylene and 3.5 g of oleylamine, and sonicate to dissolve. Then, add the solution dropwise to the DMSNs solution under magnetic stirring, heat to 60 °C and stir continuously for 60 minutes, then cool to room temperature, and centrifuge to remove the Eu precursor not adsorbed in the DMSNs. Add 20 mL of xylene and 3.5 g of oleylamine to the precipitate, sonicate to redisperse, heat to 95 °C under magnetic stirring, add H2O, and maintain the reaction temperature. Rapidly cool to room temperature, add ethanol, centrifuge to separate the product, and wash twice more with ethanol and n-hexane. The final product, Eu2O3@DMSNs, is dispersed in toluene and stored.

[0047] Using the dendritic mesoporous DMSNs prepared above as a template and europium acetate as a precursor, amorphous ultrafine Eu₂O₃ nanoparticles were grown in situ within the pores, successfully preparing Eu₂O₃@DMSNs nanocomposites with a large amount of Eu₂O₃ loaded within the mesoporous channels. The average statistical particle size was approximately 282.3 nm, slightly larger than that of pure DMSNs. A large amount of Eu₂O₃ was uniformly distributed within the DMSNs. Electron microscopy results showed no obvious lattice fringes and irregular atomic distribution, indicating an amorphous structure and confirming that the composite material is amorphous. This consistent amorphous characteristic with separately synthesized Eu₂O₃ is beneficial for maintaining the consistency of dissociation-enhanced fluorescence properties.

[0048] Example 4: Preparation of Eu2O3@DMSNs microspheres

[0049] This embodiment uses a coating method to prepare Eu2O3@DMSNs microspheres. The specific experimental steps are as follows:

[0050] The Eu₂O₃ nanoparticles prepared in Example 1 were centrifuged in hexane and then reconstituted with 10 mL of toluene by sonication. Dendritic mesoporous silica nanoparticles (DMSNs) from Example 2 were centrifuged, and then 10 mL of toluene and 100 μL of oleylamine were added. The mixture was sonicated and magnetically stirred. Ethanol was then added, and the nanoparticles were separated by centrifugation. The nanoparticles were washed twice with ethanol and hexane by centrifugation. The final product, Eu₂O₃@MSNs, was dispersed in toluene and stored.

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

[0052] Preparation of Eu2O3@MSNs-COOH: A certain amount of Eu2O3@DMSNs nanoparticles were centrifuged to remove the supernatant. 20 mL of toluene was added, and the mixture was reconstituted by sonication. A 0.1 M Silane-PEG2000-COOH toluene solution was added dropwise under magnetic stirring, with continuous stirring. Then, a 0.1 M TMAH methanol solution was added, and the mixture was heated to 85 °C for reaction. After cooling to room temperature, hexane was added for centrifugation. The nanoparticles were washed three times with hexane, followed by two more washes with ethanol. Finally, pure water was added, and the mixture was sonicated to obtain the product Eu2O3@DMSNs-COOH.

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

[0054] A 50 mM glycine buffer solution with the same pH was prepared. The amphiphilic component was 0.1% Triton X-100, and the luminescent ligand was β-NTA (150 μM) + TOPO (100 μM), where TOPO refers to topoisomerase. All components were mixed and sonicated for 5 min. Then, according to a reaction system with a molar ratio of Eu:β-NTA:TOPO = 1:3:2, the nanoparticle solution was mixed with the prepared adjustment medium solution in a cuvette. The effect of different surfactants on the luminescent properties of the four materials was tested, including luminescence kinetic curve testing, fluorescence spectroscopy testing, and imaging under a 365 nm UV lamp.

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

[0056] The experimental procedure for this embodiment is the same as that in Example 6, except that the glycine buffer is replaced with arginine buffer.

[0057] Example 8: Performance study of Eu2O3@DMSNs microspheres in lysine buffer.

[0058] The experimental procedure for this embodiment is the same as in Example 6, except that the glycine buffer is replaced with a lysine buffer.

[0059] Example 9: Performance study of Eu2O3@DMSNs microspheres in histidine buffer.

[0060] The experimental procedure for this embodiment is the same as in Example 6, except that the glycine buffer is replaced with histidine buffer.

[0061] Example 10: Performance study of Eu2O3@DMSNs microspheres in glutamate buffer.

[0062] The experimental procedure for this embodiment is the same as that in Example 6, except that the glycine buffer is replaced with glutamate buffer.

[0063] Example 11: Performance study of Eu2O3@DMSNs microspheres in aspartic acid buffer.

[0064] The experimental procedure for this embodiment is the same as that in Example 6, except that the glycine buffer is replaced with aspartic acid buffer.

[0065] Example 12: Performance study of Eu2O3@DMSNs microspheres in proline buffer

[0066] The experimental procedure for this embodiment is the same as in Example 6, except that the glycine buffer is replaced with a proline buffer.

[0067] Example 13: Performance study of Eu2O3@DMSNs microspheres in citrate-sodium citrate buffer solution

[0068] The experimental procedure for this comparative example is described in Example 6, except that the glycine buffer was replaced with a citrate-sodium citrate buffer.

[0069] Comparative Example 1

[0070] The experimental procedure for this comparative example is described in Example 6, except that the glycine buffer was replaced with deionized water.

[0071] The fluorescence performance results of Examples 6-10 and Comparative Examples 1 and 2 are as follows: Figure 3 .

[0072] Experimental results show that the performance of the luminescent system is closely related to the selection and use of the buffer solution. The luminescence performance was poor with the introduction of a standard buffer solution, but the introduction of an amino acid buffer significantly enhanced the luminescence intensity, exhibiting superior performance. This phenomenon may be attributed to the fact that the amino acid buffer, while maintaining a suitable pH value, also provides additional stability and optimizes the reaction environment for the luminescent reaction. Figure 4 The results shown are those of Comparative Example 1 and Example 6 on immunochromatographic test strips.

[0073] Example 14

[0074] A glycine buffer system with a pH of 3.0 was prepared as the luminescence modulation medium, in which 0.1 wt% TX-100 was selected as the amphiphilic molecule, and 150 μM β-NTA and 100 μM TOPO were selected as ligands. The mixture was sonicated for 5 min before use. Subsequently, the luminescence properties of Eu2O3@DMSNs microspheres were tested according to the aforementioned method.

[0075] Example 15

[0076] The process of this embodiment is the same as that in Embodiment 14, except that pH 3.0 is replaced with pH = 4.0.

[0077] Example 16

[0078] The process of this embodiment is the same as that in Embodiment 14, except that pH 3.0 is replaced with pH = 5.0.

[0079] Example 17

[0080] The process of this embodiment is the same as that in Embodiment 14, except that pH 3.0 is replaced with pH = 6.0.

[0081] Example 18

[0082] The process of this embodiment is the same as that in Example 14, except that pH 3.0 is replaced with pH = 7.5.

[0083] Comparative Example 2

[0084] The process for this comparative example is the same as in Example 14, except that pH 3.0 is replaced with pH = 2.0.

[0085] Comparative Example 3

[0086] The process for this comparative example is described in Example 14, except that pH 3.0 is replaced with pH = 8.0.

[0087] Comparative Example 4

[0088] The process for this comparative example is the same as in Example 14, except that pH 3.0 is replaced with pH = 9.0.

[0089] The experimental results of Examples 14-18 and Comparative Examples 2-4 above are as follows: Figure 5 As shown in the figure, analysis of the experimental data clearly reveals the significant impact of pH on the reaction kinetics. Under the same conditions of ligand, amphiphilic molecule, and buffer solution, the optimal pH range is clearly between 3 and 7.5. Within this range, Eu₂O₃ exhibits excellent performance under both weakly acidic and neutral conditions, demonstrating both strong signal intensity and a generally rapid reaction rate. Particularly at pH 6, the reaction still reaches equilibrium within 3 minutes; even in a neutral environment (pH 7), although the reaction rate is slightly slower than in a weakly acidic environment, equilibrium is still reached within 5 minutes. In contrast, the results of Comparative Examples 2-4 show a significant decrease in signal intensity and a markedly slower reaction rate under strongly acidic conditions at pH 2 (Comparative Example 2), making it difficult to reach equilibrium within a reasonable time. Similarly, in alkaline environments, the experimental results at pH 8 (Comparative Example 3) and pH 9 (Comparative Example 4) both show a sharp decrease in signal intensity and a significant slowdown in reaction kinetics. These comparative experiments clearly demonstrate that the performance of the reaction system deteriorates significantly under conditions where the pH is below 3 or above 7.5, failing to meet the requirements of practical applications.

[0090] Example 19

[0091] This embodiment uses 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] Prepare luminescence modulation media containing gradient concentrations of TX-100 surfactant: concentrations of 0%, 0.05%, 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, and 1.0%; other components are the same: the buffer system is a glycine buffer solution with pH=4, and the ligands (150 μM β-NTA and 100 μM TOPO) are sonicated for 5 min before use.

[0093] The luminescence properties of Eu2O3@DMSNs in different concentrations of the amphiphilic molecule TX-100 were tested according to the aforementioned method. (See [link to relevant documentation]). Figure 6 The results showed that as the concentration of TX-100 increased from 0 to 0.1%, the fluorescence intensity gradually increased, but when the concentration exceeded 0.2%, Eu... 3+ The luminescence intensity at 613 nm decreased rapidly, indicating that amphiphilic molecules facilitate coordination reactions, but excessively high concentrations would weaken coordination ability, resulting in reduced luminescence.

[0094] Example 20

[0095] The conjugation of Eu2O3@DMSNs with PCT antibodies employs a covalent conjugation method. Specifically, the surface carboxyl groups of Eu2O3@DMSNs are activated by EDC / NHS before being bioconjugated with PCT antibodies. The specific steps are as follows:

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

[0097] 2) Labeling: Add 0.2 mg of 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 to the labeled microsphere solution as a blocking agent, and the mixture was rotated and mixed at room temperature for 2 hours. After centrifugation and washing, BSA-blocked nanoparticles were obtained.

[0099] 4) Storage: Disperse the above nanoparticles in BBS buffer (0.05M BBS, pH=7.0, 0.5% BSA and 0.05% proclin300) and store at 4°C for later use.

[0100] The method for conjugating chicken IgY antibodies to Eu2O3@DMSNs is the same as above.

[0101] Example 21

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

[0103] 1) NC membrane coating: After cutting a blank NC membrane and pasting it onto a PVC base plate, spray 0.8 mg / mL of PCT detection antibody and 1 mg / mL of goat anti-chicken IgY onto the T and C lines of the NC membrane using the capillary tube of the membrane coating instrument nozzle, and dry it overnight in an oven at 37°C.

[0104] 2) Binding pad preparation: The labeled probe bioconjugate complex (Eu2O3@DMSNs-AbPCT, 0.1 mg / mL) was uniformly sprayed onto the sample binding pad using a gold sprayer and dried overnight in an oven.

[0105] The labeled probe bioconjugate complex was diluted with a curing buffer (25 mM glycine buffer, 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% sodium caseinate and 0.3% Tween-20) and dried in a 37℃ drying oven for 2 hours before storage.

[0107] 4) Plate preparation: Take out the dry plastic base plate with the NC film coating, and attach the prepared sample pad, conjugate pad, and absorbent paper to the base plate in sequence.

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

[0109] Example 22

[0110] The immunochromatographic test strip consists of a support base (PVC), a sample pad, a conjugate pad, an NC membrane, and an absorbent pad. Based on the immunochromatographic double-antibody sandwich method, after the sample solution is added to the sample well, it flows through the sample pad to the conjugate pad under capillary action, releasing the signal probes on the conjugate pad. These probes then flow through the NC membrane to the absorbent paper side. If the sample does not contain the target analyte PCT, all signal probes flow directly across the T-line without binding to the detection antibody (PCT secondary antibody) immobilized on the T-line. However, 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 cross the NC membrane with the detection antibody on the T-line to form a "sandwich" double-antibody sandwich immune complex, thus causing the signal probe to remain 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 goat anti-chicken IgY. After the immunochromatographic reaction was completed, a conditioning medium was added directly to the NC membrane, and the luminescence signal was read using a fluorescence analyzer.

[0111] The results show that, see Figure 7 The NC membrane without the added luminescence modulator only showed strong blue-blue fluorescence, while the test strip with the added luminescence modulator Eu2O3@DMSNs-Ab IgY emitted a brighter red afterglow. This not only verifies that Eu2O3@DMSNs and the luminescence modulator can achieve luminescence enhancement on a solid-phase NC membrane, but also demonstrates that Eu2O3@DMSNs and the luminescence modulator can utilize the fluorescence enhancement technology. 3+ It features a long lifespan and utilizes time-resolved immunoassay analyzer readings to remove interference from background fluorescence signals on the membrane surface, thereby further improving detection sensitivity. Furthermore, the image at 365nm shows the CELFIA immunochromatographic test strip detecting PCT under UV light. The linear range for detecting procalcitonin (PCT) in clinical serum is 0.05 ng / mL to 20 ng / mL, with a limit of detection (LoD) of 0.04 ng / mL.

[0112] Comparative Example 5

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

[0114] In comparison, the detection limit of the immunochromatographic assay reagent in Example 22 is ten times higher than that of existing technologies. This is mainly based on the fact that, in a system with added buffer, the novel assay reagent allows the ligands in the buffer to rapidly react with rare earth Eu. 3+Coordination compounds with long-afterglow luminescence properties were formed, and a time-resolved coordination-enhanced luminescence immunochromatographic assay reagent was constructed. This effectively reduced background fluorescence interference while using a strategy of rapid coordination dissociation to enhance luminescence and amplify the signal, thereby further improving the sensitivity of immunochromatographic detection.

[0115] Furthermore, it should be noted that traditional immunochromatographic test strips typically immobilize luminescent materials (such as rare earth complexes) directly onto the strip. When the sample to be tested is added, if the target analyte is present, a specific immune response is 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 the target analyte, the luminescent material immobilized on the test strip itself will produce a certain degree of background luminescence. This persistent background signal may interfere with the detection results, especially when detecting low concentrations of the target analyte, reducing the sensitivity and accuracy of the detection. The embodiments of this invention employ a stepwise activation strategy, the core of which lies in dividing the luminescence process into two independent steps. First, only the cationic component of the luminescent material (such as Eu) is immobilized on the immunochromatographic test strip. 3+ These ions themselves do not possess luminescence capabilities, therefore no background signal is generated at this stage. After the sample is added and the immunoassay is complete, a conditioning 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. Stepwise activation allows for better control of the luminescence process and reduces interference from non-specific reactions. This method enables multiplex detection; for example, by using different metal ions and corresponding ligands, multiple targets can be detected simultaneously on the same test strip.

[0116] Example 23

[0117] This embodiment demonstrates the application of the detection reagent Eu2O3@DMSNs and luminescence modulation medium of the present invention in 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 overnight at 4°C. Discard the coating solution, add 200 μL of 1% BSA to each well, and incubate at room temperature for 2 hours. Add 100 μL of serially 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 modulation medium (buffer system: glycine buffer at pH = 4, ligands (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 with citrate-sodium citrate buffer. See Example 23 for other steps.

[0121] The limit of detection (LOD) for PSA detection using the Eu2O3@DMSNs detection reagent and luminescence modulation medium in a TR-ELISA method was 0.01 ng / mL, while the LOD of the comparative TR-ELISA method using conventional citrate-sodium citrate buffer was 0.1 ng / mL. This indicates that the detection reagent of the present invention significantly improves the detection sensitivity of TR-ELISA, approximately 10 times that of conventional buffers. In the detection of low-concentration PSA (0.05 ng / mL), the signal-to-noise ratio (S / N) using the detection reagent of the present invention was 15.3, while the S / N of the comparative example was only 4.7. This indicates that the detection reagent of the present invention significantly improves signal intensity, reduces background signal, and improves detection reliability.

[0122] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. The above preparation methods can be stepwise preparation or one-step preparation. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A detection reagent for bioanalysis, characterized by, The detection reagent comprises a conditioning medium; the conditioning medium comprises a buffer system, the buffer system comprises an amino acid buffer, and the conditioning medium further comprises a luminescent ligand and an amphiphilic molecule; the pH range of the conditioning medium is 3 to 7.5; The detection reagent for biological analysis further comprises a rare earth compound and a porous carrier, the rare earth compound is coated in the porous carrier; the rare earth compound is di-europium trioxide, and the porous carrier is dendritic mesoporous silica or a functional derivative thereof.

2. The detection reagent for biological analysis according to claim 1, characterized by, The amino acid buffer is at least one selected from the group consisting of glycine buffer, arginine buffer, lysine buffer, histidine buffer, glutamic acid buffer, aspartic acid buffer, and proline buffer.

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

4. The detection reagent for biological analysis according to claim 3, characterized by, The luminescent ligand is at least one selected from β-naphthyl trifluoroacetyl propionone and trioctylphosphine oxide.

5. The detection reagent for biological analysis according to claim 1, wherein The functional derivative comprises at least one functional group selected from hydroxyl, carboxyl, amino, thiol, aldehyde, and amide.

6. A kit for biological analysis, characterized by, The kit comprises the detection reagent according to any one of claims 1-5.

7. Use of the detection reagent according to any one of claims 1-5 or the kit according to claim 6 in the preparation of a reagent for the detection of: (1) an immunochromatographic detection; (2) an enzyme-linked immunoassay; or (3) a combined detection comprising both an immunochromatographic detection and an enzyme-linked immunoassay.

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