A single-component acridinium ester luminescent substrate solution and a preparation method thereof

Abstract

The application belongs to the technical field of luminescent substrate liquid preparation, and particularly relates to a single-component acridinium ester luminescent substrate liquid 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 hazardous components, and is safer to use. The luminescent substrate liquid has the advantages of high luminescent intensity, low background signal, good stability and thermal stability, low cost, convenient use and the like. The preparation method of the luminescent substrate liquid is simple, and the luminescent substrate liquid can be used in all types of magnetic microparticle chemiluminescence acridinium ester projects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of preparation of luminescent substrate solution, and particularly relates to a single-component acridinium ester luminescent substrate solution and a preparation method thereof. BACKGROUND

[0002] The magnetic particle chemiluminescence acridinium ester system is widely used in the fields of human external diagnosis reagent, veterinary animal epidemic diagnosis reagent, etc. due to its high luminescent intensity, good stability, convenient use, high detection accuracy and wide linear range.

[0003] The acridinium ester substrate solution as a general component of the kit affects the sensitivity and stability of the kit. The acridinium ester luminescent substrate solution used in the market at present is double-component, which is composed of pre-excitation liquid and excitation liquid.

[0004] The application No. 202410084061.4 discloses a zinc transporter 8 antibody detection kit and a preparation method thereof. The detection kit comprises a zinc transporter 8 antigen coated magnetic particle solution, a goat anti-human IgG antibody fluorescent marker solution, a sample diluent, a calibration solution and a chemiluminescent substrate solution. The chemiluminescent substrate solution comprises a pre-excitation liquid and an excitation liquid; the pre-excitation liquid is a NaOH solution with a concentration of 0.05-0.25 mol / L, and the excitation liquid is an H2O2 solution with a concentration of 0.05-0.2 mol / L.

[0005] The application No. 202410147811.8 discloses a functionalized magnetic microsphere, a preparation method and a tyrosine phosphatase antibody determination kit. The preparation method of the functionalized magnetic microsphere is as follows: the silicate, active blocking agent and magnetic bead body are added into a solvent and stirred to be uniformly mixed, the temperature is raised to 60-90 DEG C under the protection of inert atmosphere, then the acid catalyst solution is added dropwise for stirring reaction, and after the reaction is completed, the functionalized magnetic microsphere is obtained through filtration, washing and drying. The tyrosine phosphatase antibody determination kit comprises a tyrosine phosphatase antigen coated functionalized magnetic microsphere solution, a goat anti-human IgG antibody fluorescent marker solution, a sample diluent, a calibration solution and a chemiluminescent substrate solution. The chemiluminescent substrate solution comprises 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] The application number 202411563857.4 discloses a chemical luminescence substrate liquid, a chemical luminescence detection kit and a detection method. The chemical luminescence substrate liquid comprises substrate liquid 1 or substrate liquid 1 and substrate liquid 2; the substrate liquid 1 comprises sodium hydroxide, a surfactant and a blue light absorber capable of absorbing blue light with a wavelength of 400-500 nm; and the substrate liquid 2 comprises inorganic acid and hydrogen peroxide. The blue light absorber added in the chemical luminescence substrate liquid can absorb the blue light emitted by acridinium ester, reduce the luminescence signal, especially reduce the luminescence value of the background caused by non-specific adsorption, and thus improve the signal-to-noise ratio.

[0007] In addition, the existing luminescent substrate liquid formula contains many hazardous components, such as nitric acid, and the hydrogen peroxide used has poor stability and is easy to degrade. SUMMARY

[0008] Based on the above technical background, the main purpose of the present application is to provide a single-component acridinium ester luminescent substrate liquid and a preparation method thereof to overcome the deficiencies in the prior art.

[0009] To achieve the above-mentioned purposes, the technical scheme adopted by the present application comprises:

[0010] The first aspect of the present application is to provide a single-component acridinium ester luminescent substrate liquid, which comprises the following raw materials: sodium hydroxide, peroxylurea, N-methyl pyrrolidone, a surfactant, sodium percarbonate, a stabilizer, triton 405 and a catalyst.

[0011] Preferably, the single-component acridinium ester luminescent substrate liquid comprises the following raw materials with the following mass concentrations: sodium hydroxide 0.4-2 g / L, peroxylurea 1-5 g / L, N-methyl pyrrolidone 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.

[0012] More preferably, the single-component acridinium ester luminescent substrate liquid comprises the following raw materials with the following mass concentrations: sodium hydroxide 1 g / L, peroxylurea 2 g / L, N-methyl pyrrolidone 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.

[0013] The stabilizer is selected from one or more of diethylene triamine pentaacetic acid, zinc stearate, dibutyl ene dilaurate, dibutyl tin dimaleate and antimony mercaptan.

[0014] Preferably, the stabilizer is diethylene triamine pentaacetic acid.

[0015] 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.

[0016] The surfactant is selected from one or more of quaternary ammonium salts and quaternary ammonium alkali salts.

[0017] Preferably, the surfactant is hexadecyltrimethylammonium bromide.

[0018] 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.

[0019] The catalyst is selected from one or two of molybdenum tetrachloride and nickel chloride.

[0020] Preferably, the catalyst is molybdenum tetrachloride.

[0021] The use of molybdenum tetrachloride as a catalyst can improve reaction efficiency and make the detection efficiency of the luminescent substrate solution higher.

[0022] By adding the above-mentioned raw materials, the luminescent substrate liquid system can basically solve various problems of the entire magnetic microparticle chemiluminescence process, with low cost and better social adaptability.

[0023] The optimal pH range for the buffer solution used with the luminescent substrate solution is 10–12.

[0024] 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:

[0025] 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.

[0026] 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.

[0027] The steps described above are described in detail below.

[0028] 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.

[0029] Preferably, the mixture is stirred evenly at room temperature and a stirring speed of 200 rpm. Even mixing is achieved at room temperature.

[0030] Add stabilizer, urea peroxide, sodium percarbonate and surfactant, and stir until homogeneous at 20-30°C and 200-500 rpm.

[0031] Preferably, the mixture is stirred evenly at 25°C and 300 rpm.

[0032] 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.

[0033] Preferably, the mixture is stirred evenly at 25°C and 500 rpm.

[0034] The beneficial effects of this invention are as follows:

[0035] (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.

[0036] (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.

[0037] (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.

[0038] (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

[0039] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

[0040] Example

[0041] 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.

[0042] Example 1

[0043] 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.

[0044] Example 2

[0045] 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.

[0046] Example 3

[0047] 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.

[0048] Example 4

[0049] 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.

[0050] Example 5

[0051] 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.

[0052] Experimental Example

[0053] Experiment Example 1: Luminescence Value Test

[0054] 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.

[0055] Table 1

[0056]

[0057] 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.

[0058] Experiment Example 2 Stability Test

[0059] 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.

[0060] Table 2

[0061]

[0062] 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.

[0063] Experiment Example 3 Thermal Stability Test

[0064] 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.

[0065] Table 3

[0066]

[0067] 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.

[0068] 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 acridinium ester luminous substrate solution, characterized in that, the single-component acridinium ester luminous substrate solution comprises the following raw materials in the following mass concentrations: sodium hydroxide 0.4-2 g / L, urea peroxide 1-5 g / L, N-methyl pyrrolidone 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. The stabilizer is selected from one or more of diethylene triamine pentaacetic acid, zinc stearate, dibutyl ene dilaurate, dibutyl tin dimaleate, and antimony mercaptide. The surfactant is selected from one or more of quaternary ammonium salt and quaternary ammonium base salt. The catalyst is selected from one or both of molybdenum tetrachloride oxide and nickel chloride. 2.The single-component acridinium ester luminous substrate solution of claim 1, characterized in that, the single-component acridinium ester luminous substrate solution comprises the following raw materials in the following mass concentrations: sodium hydroxide 1 g / L, urea peroxide 2 g / L, N-methyl pyrrolidone 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. 3.A preparation method of the single-component acridinium ester luminous substrate solution of claim 1 or 2, the preparation method comprising the following steps: Step 1: adding sodium hydroxide to water, stirring until uniform, then adding stabilizer, urea peroxide, sodium percarbonate, and surfactant, stirring until uniform to obtain an intermediate substrate solution; Step 2: adding N-methyl pyrrolidone, triton 405, and catalyst to the intermediate substrate solution, stirring until uniform to obtain the single-component acridinium ester luminous substrate solution. In step 1, Sodium hydroxide is added to water, and stirring is performed at a stirring speed of 100-300 rpm at room temperature until uniform.

4. The production method according to claim 3, characterized by, In step 1, The stabilizer, urea peroxide, sodium percarbonate, and surfactant are added, and stirring is performed at a stirring speed of 200-500 rpm at 20-30 ℃ until uniform.

5. The preparation method according to claim 3, characterized in that, In step 2, N-methyl pyrrolidone, triton 405, and catalyst are added to the intermediate substrate solution, and stirring is performed at a stirring speed of 400-700 rpm at 20-30 ℃ until uniform.

6. The preparation method according to claim 3, characterized in that, ​ ​

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