Single-component acridinium ester luminescent substrate solution and preparation method thereof

Abstract

The invention belongs to the technical field of preparation of luminescent substrate solutions, and particularly relates to a single-component acridinium ester luminescent substrate solution and a preparation method thereof. The single-component acridinium ester luminescent substrate liquid comprises the following raw materials: sodium hydroxide, urea peroxide, N-methyl pyrrolidone, a surfactant, sodium percarbonate, a stabilizer, triton 405 and a catalyst, does not contain dangerous components, and is safer to use. The luminous substrate solution has the advantages of high luminous intensity, low background signal, good stability and thermal stability, low cost, convenience in use and the like. The preparation method of the luminescent substrate solution is simple and can be used for all types of magnetic particle chemiluminescence acridinium ester items.

Description

Technical Field

[0001] This invention belongs to the field of luminescent substrate preparation technology, specifically relating to a single-component acridine ester luminescent substrate and its preparation method. Background Technology

[0002] Magnetic microparticle chemiluminescent acridine ester systems have been widely used in human in vitro diagnostic reagents, veterinary animal disease diagnostic reagents, and other fields due to their high luminescence intensity, good stability, ease of use, high detection accuracy, and wide linear range.

[0003] Acridinium ester substrate solutions are a common component of reagent kits, affecting the kit's sensitivity and stability. Currently, commercially available acridinium ester luminescent substrate solutions are all two-component, consisting of a pre-excitation solution and an excitation solution.

[0004] For example, application number 202410084061.4 discloses a zinc transporter 8 antibody detection kit and its preparation method. The detection kit includes a zinc transporter 8 antigen-coated magnetic microparticle solution, a goat anti-human IgG antibody fluorescent labeling solution, a sample diluent, a calibrator solution, and a chemiluminescent substrate solution. The chemiluminescent substrate solution includes a pre-excitation solution and an excitation solution; the pre-excitation solution is a NaOH solution with a concentration of 0.05–0.25 mol / L, and the excitation solution is an H2O2 solution with a concentration of 0.05–0.2 mol / L.

[0005] Application No. 202410147811.8 discloses a functionalized magnetic microsphere, its preparation method, and a tyrosine phosphatase antibody assay kit. The preparation method of the functionalized magnetic microspheres involves adding silicate ester, an active capping agent, and the magnetic bead body to a solvent and stirring until homogeneous. Under an inert atmosphere, the mixture is heated to 60-90°C, and then an acid catalyst solution is added dropwise while stirring. After the reaction is complete, the mixture is filtered, washed, and dried to obtain the functionalized magnetic microspheres. The tyrosine phosphatase antibody assay kit includes a tyrosine phosphatase antigen-coated functionalized magnetic microsphere solution, a goat anti-human IgG antibody fluorescent label solution, a sample diluent, a calibrator solution, and a chemiluminescent substrate solution. The chemiluminescent substrate solution contains a NaOH solution with a concentration of 0.05-0.25 mol / L and an H₂O₂ solution with a concentration of 0.05-0.2 mol / L.

[0006] Application No. 202411563857.4 discloses a chemiluminescent substrate solution, a chemiluminescent detection kit, and a detection method. The chemiluminescent substrate solution comprises substrate solution 1, or substrate solution 1 and substrate solution 2; substrate solution 1 comprises sodium hydroxide, a surfactant, and a blue light absorber that absorbs blue light at wavelengths of 400nm-500nm; substrate solution 2 comprises an inorganic acid and hydrogen peroxide. This application adds a blue light absorber to the chemiluminescent substrate solution, which can absorb the blue light emitted by acridine esters, reducing the luminescence signal, especially reducing the background luminescence value caused by non-specific adsorption, thereby improving the signal-to-noise ratio.

[0007] In addition, existing luminescent substrate liquid formulations contain many hazardous components, such as nitric acid, and the hydrogen peroxide used is unstable and easily degraded. Summary of the Invention

[0008] Based on the above technical background, the main objective of this invention is to provide a single-component acridine ester luminescent substrate liquid and its preparation method, so as to overcome the shortcomings of the prior art.

[0009] To achieve the aforementioned objectives, the technical solution adopted by this invention includes: The first aspect of the present invention is to provide a one-component acridine ester luminescent substrate liquid, the one-component acridine ester luminescent substrate liquid comprising the following raw materials: sodium hydroxide, urea peroxide, N-methylpyrrolidone, surfactant, sodium percarbonate, stabilizer, Triton 405 and catalyst.

[0010] Preferably, the single-component acridine ester luminescent substrate solution comprises the following raw materials at the following mass concentrations: sodium hydroxide 0.4–2 g / L, urea peroxide 1–5 g / L, N-methylpyrrolidone 10–50 g / L, surfactant 0.2–0.8 g / L, sodium percarbonate 0.4–0.7 g / L, stabilizer 0.1–0.3 g / L, Triton 405 1–5 g / L, and catalyst 0.1–0.5 g / L.

[0011] More preferably, the single-component acridine ester luminescent substrate solution comprises the following raw materials at the following mass concentrations: sodium hydroxide 1 g / L, urea peroxide 2 g / L, N-methylpyrrolidone 20 g / L, surfactant 0.5 g / L, sodium percarbonate 0.5 g / L, stabilizer 0.2 g / L, Triton 405 2 g / L, and catalyst 0.3 g / L.

[0012] The stabilizer is selected from one or more of diethylenetriaminepentaacetic acid, zinc stearate, dibutylene dilaurate, dibutyltin dimaleate, and antimony thiolate.

[0013] Preferably, the stabilizer is diethylenetriaminepentaacetic acid.

[0014] The sodium percarbonate and urea peroxide used in the substrate solution of this invention have good stability. The use of diethylenetriaminepentaacetic acid as a stabilizer can further enhance the stability of sodium percarbonate and urea peroxide. The substrate solution has no decrease in performance after being stored at 2-8°C for 5 years.

[0015] The surfactant is selected from one or more of quaternary ammonium salts and quaternary ammonium alkali salts.

[0016] Preferably, the surfactant is hexadecyltrimethylammonium bromide.

[0017] The addition of N-methylpyrrolidone, hexadecyltrimethylammonium bromide, and Triton 405 to the substrate solution can increase the stability and solubility of the nonpolar acridine ester intermediate.

[0018] The catalyst is selected from one or two of molybdenum tetrachloride and nickel chloride.

[0019] Preferably, the catalyst is molybdenum tetrachloride.

[0020] The use of molybdenum tetrachloride as a catalyst can improve reaction efficiency and make the detection efficiency of the luminescent substrate solution higher.

[0021] By adding the above-mentioned raw materials, the luminescent substrate liquid system can basically solve all the problems of chemiluminescence of magnetic particles, with low cost and better social adaptability.

[0022] The optimal pH range for the buffer solution used with the luminescent substrate solution is 10–12.

[0023] A second aspect of the present invention is to provide a method for preparing the single-component acridine ester luminescent substrate liquid described in the first aspect of the present invention, the preparation method comprising the following steps: Step 1: Add sodium hydroxide to water and stir well. Then add stabilizer, urea peroxide, sodium percarbonate and surfactant, and stir well to obtain intermediate substrate solution. Step 2: Add N-methylpyrrolidone, Triton 405 and catalyst to the intermediate substrate solution, stir well to obtain a single-component acridine ester luminescent substrate solution.

[0024] The steps described above are described in detail below.

[0025] In step 1, sodium hydroxide is added to the water and stirred until homogeneous at room temperature and a stirring speed of 100-300 rpm.

[0026] Preferably, the mixture is stirred evenly at room temperature and a stirring speed of 200 rpm. Even mixing is achieved at room temperature.

[0027] Add stabilizer, urea peroxide, sodium percarbonate and surfactant, and stir until homogeneous at 20-30°C and 200-500 rpm.

[0028] Preferably, the mixture is stirred evenly at 25°C and 300 rpm.

[0029] In step 2, N-methylpyrrolidone, Triton 405, and the catalyst are added to the intermediate substrate solution, and the mixture is stirred until homogeneous at 20–30°C and 400–700 rpm. Homogenization can be achieved at low temperatures.

[0030] Preferably, the mixture is stirred evenly at 25°C and 500 rpm.

[0031] The beneficial effects of this invention are as follows: (1) The luminescent substrate liquid of the present invention does not contain any hazardous components and has the advantages of high luminescence intensity, low background signal, good stability and thermal stability. In addition, the single-component acridine ester luminescent substrate liquid of the present invention can be stored for 5 years after real-time stability testing. It has a long service life, low cost, more convenient use and high luminescence intensity. It can be used in all types of magnetic microparticle chemiluminescent acridine ester projects. (2) The luminescent substrate solution described in this invention uses diethylenetriaminepentaacetic acid as a stabilizer, which can greatly enhance the stability of the luminescent substrate solution. Its performance has not decreased after being placed at 2-8℃ for 5 years. The N-methylpyrrolidone, hexadecylamine bromide and Triton 405 added to the luminescent substrate solution can increase the stability and solubility of non-polar acridine ester intermediates. Molybdenum tetrachloride is used as a catalyst, which makes the luminescent substrate solution have higher and faster reaction efficiency and high detection efficiency.

[0032] (3) The preparation method of the single-component acridine ester luminescent substrate liquid of the present invention is simple and can be prepared by stirring at low temperature. It is non-toxic and harmless, and the preparation process and use are highly safe.

[0033] (4) The single-component luminescent substrate liquid described in this invention can basically solve various problems of chemiluminescence of magnetic microparticles, and has the advantages of low cost and strong industrial adaptability. Detailed Implementation

[0034] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

[0035] Example The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All raw materials used in the embodiments of the present invention were commercially available.

[0036] Example 1 Measure 900 ml of water, add 1 g of sodium hydroxide, and stir at 200 rpm at room temperature until completely dissolved. Then, add 0.2 g of diethylenetriaminepentaacetic acid, 2 g of urea peroxide, 0.5 g of sodium percarbonate, and 0.5 g of hexadecyltrimethylammonium bromide in sequence, and stir at 25°C and 300 rpm until completely dissolved to obtain an intermediate substrate solution. Then, add 20 mL of N-methylpyrrolidone, 2 mL of Triton 405, and 0.3 g of molybdenum tetrachloride, and stir at 25°C and 500 rpm until completely dissolved. Finally, add water to a final volume of 1 L to obtain a single-component acridine ester luminescent substrate solution, and store at 4°C for later use.

[0037] Example 2 Measure 900 ml of water, add 0.4 g of sodium hydroxide, and stir at 200 rpm at room temperature until completely dissolved. Then, add 0.1 g of diethylenetriaminepentaacetic acid, 1 g of urea peroxide, 0.4 g of sodium percarbonate, and 0.2 g of hexadecyltrimethylammonium bromide in sequence, and stir at 25°C and 300 rpm until completely dissolved to obtain an intermediate substrate solution. Then, add 10 mL of N-methylpyrrolidone, 1 mL of Triton 405, and 0.1 g of molybdenum tetrachloride, and stir at 25°C and 500 rpm until completely dissolved. Finally, add water to a final volume of 1 L to obtain a single-component acridinium ester luminescent substrate solution, and store at 4°C for later use.

[0038] Example 3 Measure 900 ml of water, add 2 g of sodium hydroxide, and stir at 200 rpm at room temperature until completely dissolved. Then, add 0.3 g of diethylenetriaminepentaacetic acid, 5 g of urea peroxide, 0.7 g of sodium percarbonate, and 0.8 g of hexadecyltrimethylammonium bromide in sequence, and stir at 25°C and 300 rpm until completely dissolved to obtain an intermediate substrate solution. Then, add 50 mL of N-methylpyrrolidone, 5 mL of Triton 405, and 0.5 g of molybdenum tetrachloride, and stir at 25°C and 500 rpm until completely dissolved. Finally, add water to a final volume of 1 L to obtain a single-component acridine ester luminescent substrate solution, and store at 4°C for later use. Example 4 Measure 900 ml of water, add 1 g of sodium hydroxide, and stir at 100 rpm at room temperature until completely dissolved. Then, add 0.2 g of diethylenetriaminepentaacetic acid, 2 g of urea peroxide, 0.5 g of sodium percarbonate, and 0.5 g of hexadecyltrimethylammonium bromide in sequence, and stir at 20°C and 500 rpm until completely dissolved to obtain an intermediate substrate solution. Then, add 20 mL of N-methylpyrrolidone, 2 mL of Triton 405, and 0.3 g of molybdenum tetrachloride, and stir at 20°C and 700 rpm until completely dissolved. Finally, add water to a final volume of 1 L to obtain a single-component acridine ester luminescent substrate solution, and store at 4°C for later use. Example 5 Measure 900 ml of water, add 1 g of sodium hydroxide, and stir at 300 rpm at room temperature until completely dissolved. Then, add 0.2 g of diethylenetriaminepentaacetic acid, 2 g of urea peroxide, 0.5 g of sodium percarbonate, and 0.5 g of hexadecyltrimethylammonium bromide in sequence, and stir at 30°C and 200 rpm until completely dissolved to obtain an intermediate substrate solution. Then, add 20 mL of N-methylpyrrolidone, 2 mL of Triton 405, and 0.3 g of molybdenum tetrachloride, and stir at 30°C and 400 rpm until completely dissolved. Finally, add water to a final volume of 1 L to obtain a single-component acridine ester luminescent substrate solution, and store at 4°C for later use.

[0039] Experimental Example Experiment Example 1: Luminescence Value Test 1. The luminescence values ​​of the acridinium ester luminescent substrate solution prepared in Example 1 and the imported two-component acridinium ester luminescent substrate solution (Abbott) were detected using a magnetic particle chemiluminescence analyzer. Mouse monoclonal antibody conjugated with magnetic beads and goat anti-mouse secondary antibody conjugated with acridinium ester were added according to a pre-programmed machine sequence, and the luminescence values ​​were read after 10 minutes. A comparison of the luminescence values ​​of the single-component acridinium ester luminescent substrate solution prepared in Example 1 and the imported two-component acridinium ester luminescent substrate solution is shown in Table 1. Table 1

[0040] As shown in Table 1, when the concentrations of acridine ester goat anti-mouse agent were 100 ng / mL, 10 ng / mL, 1 ng / mL, and 0.1 ng / mL, the luminescence values ​​of the luminescent substrate solution prepared in Example 1 of this invention were all higher than those of the two-component acridine ester luminescent substrate solution. This indicates that the acridine ester luminescent substrate solution of this invention has higher luminescence values ​​and luminescence intensity than the two-component acridine ester luminescent substrate solution, suggesting that the acridine ester luminescent substrate solution of this invention has superior detection performance compared to the two-component acridine ester luminescent substrate solution.

[0041] Experiment Example 2: Stability Test The luminescence values ​​of the acridinium ester luminescent substrate solution prepared in Example 1 were tested after storage for 0 days, 6 months, 12 months, 24 months, 36 months, 48 ​​months, and 60 months. The luminescence value testing process was the same as that in Experiment 1. The test results are shown in Table 2.

[0042] Table 2

[0043] As shown in Table 1, the luminescence value of the acridine ester luminescent substrate solution prepared in Example 1 was 11,732,295. After 60 months of storage, the luminescence value of the acridine ester luminescent substrate solution was 11,741,572. The comparison shows that the luminescence value of the acridine ester luminescent substrate solution after 60 months of storage is similar to that of the freshly prepared acridine ester luminescent substrate solution, indicating that the acridine ester luminescent substrate solution described in this invention still has a high luminescence value after 60 months of storage. These results demonstrate that the acridine ester luminescent substrate solution described in this invention has good stability and still exhibits a high luminescence value after 60 months of storage.

[0044] Experiment Example 3 Thermal Stability Test The acridinium ester luminescent substrate solution prepared in Example 1 was stored at 37°C for 28 days. The luminescence values ​​of the acridinium ester luminescent substrate solutions stored for 0, 7, 14, 21 and 28 days were tested, and the test results are shown in Table 3.

[0045] Table 3

[0046] As shown in Table 3, the luminescence value of the acridine ester luminescent substrate solution prepared in Example 1 was 11,738,652. After storage at 37°C for 28 days, the luminescence value of the acridine ester luminescent substrate solution was 11,725,693. This comparison shows that the luminescence value of the acridine ester luminescent substrate solution after storage at 37°C for 28 days is similar to that of the freshly prepared acridine ester luminescent substrate solution. This indicates that the acridine ester luminescent substrate solution described in this invention has good thermal stability and can still be used after storage at 37°C for 28 days, maintaining good performance even after 28 days of storage.

[0047] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A single-component acridine ester luminescent substrate solution, characterized in that, The single-component acridine ester luminescent substrate solution comprises the following raw materials: sodium hydroxide, urea peroxide, N-methylpyrrolidone, surfactant, sodium percarbonate, stabilizer, Triton 405, and catalyst.

2. The single-component acridine ester luminescent substrate solution according to claim 1, characterized in that, The single-component acridine ester luminescent substrate solution comprises the following raw materials at the following mass concentrations: sodium hydroxide 0.4–2 g / L, urea peroxide 1–5 g / L, N-methylpyrrolidone 10–50 g / L, surfactant 0.2–0.8 g / L, sodium percarbonate 0.4–0.7 g / L, stabilizer 0.1–0.3 g / L, Triton 405 1–5 g / L, and catalyst 0.1–0.5 g / L.

3. The single-component acridine ester luminescent substrate solution according to claim 2, characterized in that, The single-component acridine ester luminescent substrate solution comprises the following raw materials at the following mass concentrations: sodium hydroxide 1 g / L, urea peroxide 2 g / L, N-methylpyrrolidone 20 g / L, surfactant 0.5 g / L, sodium percarbonate 0.5 g / L, stabilizer 0.2 g / L, Triton 405 2 g / L, and catalyst 0.3 g / L.

4. The single-component acridine ester luminescent substrate solution according to claim 1, characterized in that, The stabilizer is selected from one or more of diethylenetriaminepentaacetic acid, zinc stearate, dibutylene dilaurate, dibutyltin dimaleate, and antimony thiolate.

5. The single-component acridine ester luminescent substrate solution according to claim 1, characterized in that, The surfactant is selected from one or more of quaternary ammonium salts and quaternary ammonium alkali salts.

6. The single-component acridine ester luminescent substrate solution according to claim 1, characterized in that, The catalyst is selected from one or two of molybdenum tetrachloride and nickel chloride.

7. A method for preparing a single-component acridine ester luminescent substrate solution according to any one of claims 1 to 6, the preparation method comprising the following steps: Step 1: Add sodium hydroxide to water and stir well. Then add stabilizer, urea peroxide, sodium percarbonate and surfactant, and stir well to obtain intermediate substrate solution. Step 2: Add N-methylpyrrolidone, Triton 405 and catalyst to the intermediate substrate solution, stir well to obtain a single-component acridine ester luminescent substrate solution.

8. The preparation method according to claim 7, characterized in that, In step 1, Add sodium hydroxide to the water and stir until homogeneous at room temperature and a stirring speed of 100–300 rpm.

9. The preparation method according to claim 7, characterized in that, In step 1, Add stabilizer, urea peroxide, sodium percarbonate and surfactant, and stir until homogeneous at 20-30°C and 200-500 rpm.

10. The preparation method according to claim 7, characterized in that, In step 2, Add N-methylpyrrolidone, Triton 405 and catalyst to the intermediate substrate solution, and stir until homogeneous at 20-30°C and 400-700 rpm.

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