High-sensitivity and high-stability acridinium ester luminescent substrate liquid, and preparation method and application thereof

Abstract

The present application relates to the technical field of chemiluminescence analysis, and discloses a high-sensitivity and high-stability acridinium ester luminous substrate liquid as well as a preparation method and application thereof.The acridinium ester luminous substrate liquid comprises substrate liquid A and substrate liquid B, the volume ratio of the substrate liquid A to the substrate liquid B is (0.5-1):(0.5-1), the substrate liquid A comprises an oxidizing agent, an inorganic acid and a surfactant, and the substrate liquid B comprises a composite enhancer and an inorganic alkali.The stability of the substrate liquid is greatly improved through screening and optimization of the acids, alkalis, surfactants and enhancers contained in the substrate liquid, the substrate liquid can be stably stored at room temperature of 2-30 DEG C for 2 years, low-temperature transportation and refrigeration are not needed, the substrate liquid after being opened still has good stability within 70 days, the storage, transportation and use costs are greatly reduced, the formula is simple, the substrate liquid can be transported and stored at room temperature, the cost is controllable, and the substrate liquid is suitable for industrial production and clinical detection.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescence analysis technology, specifically to a highly sensitive and stable acridinium ester luminescent substrate solution, its preparation method, and its application. Background Technology

[0002] Acridinium ester chemiluminescence systems are widely used in clinical immunodiagnostics and biological detection due to their high luminescence efficiency, rapid reaction, and low background signal. The chemiluminescence of acridinium esters is direct chemiluminescence, characterized by the absence of additional enzyme catalysis; it emits intense light rapidly in an alkaline hydrogen peroxide environment. Under alkaline conditions, the hydrogen peroxide anion in the substrate solution exhibits strong nucleophilicity, attacking the acridinium ring in the acridinium ester molecule to form an unstable intermediate. This intermediate rapidly undergoes intramolecular rearrangement, the acridinium ring quickly opens, and a CO2 molecule is instantaneously removed to form N-methylacridone. This product is in an excited singlet state (high-energy state). When it returns from the excited state to the stable ground state, the excess energy is released as photons, with the emission wavelength peak typically between 425 and 430 nm.

[0003] Existing acridil ester luminescent substrate solutions generally suffer from the problem of balancing luminescence signal intensity and background signal. Insufficient addition of the enhancer results in low sensitivity, while excessive addition increases the background signal and decreases the signal-to-noise ratio. Poor pH control of the substrate solution system can easily lead to unstable luminescence reactions and poor signal reproducibility. Most reported acridil ester luminescent substrate solutions require storage at 2-8°C, which results in poor long-term stability. Transporting and storing at 2-8°C significantly increases transportation and storage costs for users. Summary of the Invention

[0004] The present invention aims to provide a highly sensitive and stable acridine ester luminescent substrate liquid, its preparation method and application, in order to solve the problems of insufficient stability, difficulty in balancing luminescence sensitivity and luminescence intensity, and harsh storage conditions of existing acridine ester luminescent substrate liquids.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a highly sensitive and highly stable acrid ester luminescent substrate liquid, comprising substrate liquid A and substrate liquid B, wherein the volume ratio of substrate liquid A to substrate liquid B is (0.5~1):(0.5~1), substrate liquid A comprises an oxidant, an inorganic acid and a surfactant; substrate liquid B comprises a composite reinforcing agent and an inorganic base.

[0006] Preferably, as an improvement, the volume ratio of substrate liquid A to substrate liquid B is 1:1.

[0007] Preferably, as an improvement, the oxidant is hydrogen peroxide, and the molar concentration of hydrogen peroxide is in the range of 0.1 mol / L to 1 mol / L.

[0008] Preferably, as an improvement, the inorganic acid is nitric acid, and the molar concentration of nitric acid is 15 mmol / L.

[0009] Preferably, as an improvement, the surfactant is at least one of Pluronic F-127, Pluronic F-68, and ethyl phenyl polyethylene glycol, and the mass concentration of the surfactant is 0.02 g / L to 0.5 g / L.

[0010] Preferably, as an improvement, the inorganic base is sodium hydroxide, and the molar concentration of sodium hydroxide is 0.25 mol / L.

[0011] Preferably, as an improvement, the composite reinforcing agent comprises any two or three combinations of tetradecyltrimethylammonium chloride or hexadecyltrimethylammonium chloride with Tween-20 and Triton X-100.

[0012] Preferably, as an improvement, a method for preparing a highly sensitive and highly stable acridine ester luminescent substrate solution includes the following steps: Step 1, Preparation of substrate solution A: Add nitric acid, hydrogen peroxide, Pluronic F-127 or Pluronic F-68 or ethyl phenyl polyethylene glycol to pure water, stir, adjust the pH to <2.0 with nitric acid and seal for storage. Step 2, Preparation of substrate solution B: Add sodium hydroxide and composite enhancer to pure water, stir, adjust the pH to 12.5~13.5 with sodium hydroxide and store; Step 3, Mixing: When using, mix substrate solution A and substrate solution B at a volume ratio of 1:1.

[0013] Preferably, as an improvement, substrate solution A and substrate solution B are stored at 2~30℃ in a sealed container.

[0014] Preferably, as an improvement, the application of a highly sensitive and stable acridine ester luminescent substrate solution in the preparation of industrial and / or clinical diagnostic reagents.

[0015] The principle and advantages of this scheme are as follows: In practical applications, this technical scheme addresses the problems of insufficient stability of acridine ester luminescent substrate solutions, difficulty in balancing luminescence sensitivity and intensity, and stringent storage conditions in existing technologies. It comprehensively optimizes and upgrades the formulation and preparation process of the luminescent substrate solution. In terms of formulation composition, substrate solution A in this technical scheme is primarily an oxidation system, composed of an oxidant, an inorganic acid, and a nonionic surfactant. Hydrogen peroxide is easily decomposed and inactivated in neutral and alkaline environments. Nitric acid adjusts the pH of substrate solution A to a strongly acidic environment below 2.0, significantly inhibiting hydrogen peroxide decomposition and ensuring the long-term stable retention of the oxidant, laying the foundation for long-term storage of the substrate solution at room temperature. The combination of these two components forms a stable acidic oxidation solution. When mixed with the strongly alkaline substrate solution B, a weakly alkaline reaction environment suitable for acridine ester luminescence can be quickly constructed, ensuring a uniform and stable reaction rate. Regarding surfactant optimization, this technical solution adds Pluronic F-127 and other surfactants to substrate solution A. Existing technologies generally consider that such high molecular weight block polyether surfactants will hydrolyze, precipitate, and emulsify in a high-concentration nitric acid environment, resulting in unstable dissolution. Therefore, traditional substrate solution A only adds small-molecule co-solvents, without introducing high molecular weight block polyethers. Furthermore, it is generally believed that adding surfactants increases stray fluorescence, non-specific luminescence, raises background noise, and reduces signal-to-noise ratio; therefore, only a small amount of low-molecular-weight surfactant is added to alkaline solution B, while acidic oxidant solutions are completely avoided. However, this technical solution breaks away from existing biases by introducing block polyether surfactants into substrate solution A for the first time. The surfactant can encapsulate and disperse acridine ester molecules, reducing energy loss and fluorescence quenching during luminescence, directly improving luminescence efficiency. It also possesses defoaming properties, eliminating bubbles generated by the subsequent introduction of reinforcing agents, avoiding bubble interference with detection, and improving result repeatability. This surfactant has good compatibility with hydrogen peroxide and nitric acid and will not damage the stability of the oxidation system.

[0016] The core of substrate solution B is an alkaline enhancement system, composed of an inorganic base and a composite luminescence enhancer. Sodium hydroxide is chosen as the inorganic base, providing a strongly alkaline environment for the overall reaction and promoting the conversion of hydrogen peroxide into highly reactive hydrogen peroxide anions. The composite enhancer is a complex system formed by tetradecyltrimethylammonium chloride (a cationic surfactant), Tween-20, and Triton X-100 (a nonionic surfactant), unlike a single enhancer. The cationic surfactant alters the interfacial properties of the solution, enhancing the contact efficiency between the acridinium ester and the oxidant, thus amplifying the luminescence signal. The two nonionic surfactants further optimize the system's solubility and molecular dispersion, assisting in improving luminescence intensity. With precise proportions, the luminescence signal is significantly enhanced without causing a spike in background signal due to excessive surfactant, achieving a perfect balance between luminescence intensity and background value. Replacing it with hexadecyltrimethylammonium chloride, or using any two components in combination, can also form an effective enhancement system suitable for different application scenarios.

[0017] When in use, components A and B are mixed at a volume ratio of (0.5~1):(0.5~1), especially (1:1). The acidic oxidizing solution is neutralized with the alkaline solution to precisely form the reaction microenvironment required for acridine ester luminescence: under alkaline conditions, hydrogen peroxide generates highly active hydrogen peroxide anions, which quickly initiate the acridine ester luminescence reaction; various surfactants and enhancers in A and B work together to continuously play the roles of dispersion, anti-adsorption, brightening, and foam suppression, ensuring the smooth progress of the luminescence reaction and the uniform and stable signal throughout the process.

[0018] In addition, during the research and development phase of the technical solution, we tried to optimize the surfactant by using a surfactant compound system. However, the results showed that it is sufficient to choose only one surfactant, and the compound surfactant has no obvious advantage in terms of effect, and is even slightly inferior.

[0019] In summary, the beneficial effects of this technical solution are as follows: 1. In traditional technologies, it is difficult to control the amount of enhancer added. Insufficient addition results in low sensitivity, while excessive addition leads to increased background and decreased signal-to-noise ratio. This solution employs a multi-stage compound system of Pluronic F-127 + cationic surfactant + two nonionic surfactants: The first layer utilizes Pluronic F-127 to achieve dispersion, anti-adsorption, and defoaming functions, reducing the basic background; the second layer uses three different types of surfactants to form a composite enhancer, which directionally improves the luminescence intensity through the synergistic effect between components, while suppressing background rise by limiting the concentration range, creatively solving the problem of the inability to simultaneously achieve both luminescence sensitivity and background signal.

[0020] 2. This technical solution has been experimentally verified to have: high luminescence intensity, low background signal, and a signal-to-noise ratio significantly superior to commercially available products; good detection repeatability with low coefficient of variation (CV) across batches; improved detection sensitivity, with a blank limit far lower than existing commercially available substrate solutions when applied to clinical TSH and other immunoassay tests, resulting in higher detection accuracy; excellent long-term stability at room temperature, capable of being sealed and stored for 2 years at 2-30℃ with a luminescence signal attenuation of less than 7% and no significant increase in background; good acceleration stability, with no significant performance degradation after 14 days of acceleration at 55℃, and tolerance to temperature fluctuations during transportation and storage; stable performance after opening, maintaining good luminescence performance even after 70 days of room temperature on the instrument, eliminating the need for frequent substrate solution replacements, making it suitable for continuous clinical testing scenarios.

[0021] 3. This technical solution can prevent non-specific adsorption of acridine esters and eliminate bubble interference, and is compatible with various reaction containers, pipelines and detection equipment; the formulation system has strong versatility and can be widely used in various acridine ester chemiluminescence scenarios such as clinical immunodiagnosis and biological detection, and is suitable for large-scale industrial production and batch clinical testing. Detailed Implementation

[0022] The following detailed description provides further details on specific embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art; the experimental methods used are all conventional methods; and the materials and reagents used are all commercially available.

[0023] Overview of the plan: A highly sensitive and stable acridinium ester luminescent substrate liquid includes substrate liquid A and substrate liquid B, with a volume ratio of substrate liquid A to substrate liquid B of (0.5~1):(0.5~1). Substrate liquid A includes an oxidant, an inorganic acid, and a surfactant; substrate liquid B includes a composite reinforcing agent and an inorganic base.

[0024] In substrate solution A: The oxidant is hydrogen peroxide, and the molar concentration of hydrogen peroxide ranges from 0.1 mol / L to 1 mol / L; the preferred optimal molar concentration is 0.4 mol / L. The inorganic acid is nitric acid, and the molar concentration of nitric acid is 15 mmol / L; The surfactant is at least one of Pluronic F-127, Pluronic F-68, and ethyl phenyl polyethylene glycol, and the mass concentration of the surfactant is 0.02 g / L to 0.5 g / L; the preferred optimal concentration is 0.1 g / L.

[0025] In substrate solution B: The inorganic base is sodium hydroxide, and the molar concentration of sodium hydroxide is 0.25 mol / L; The composite reinforcing agent includes any two or three combinations of tetradecyltrimethylammonium chloride or hexadecyltrimethylammonium chloride with Tween-20 and Triton X-100; the optimal combination of composite reinforcing agents is tetradecyltrimethylammonium chloride, Tween-20 and Triton X-100, wherein the mass concentration of tetradecyltrimethylammonium chloride ranges from 0.5 g / L to 10 g / L, the concentration range of Tween-20 ranges from 0.01% to 1%, and the concentration range of Triton X-100 ranges from 0.5% to 4%.

[0026] A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Preparation of substrate solution A: Add nitric acid, hydrogen peroxide, Pluronic F-127 or Pluronic F-68 or ethyl phenyl polyethylene glycol to pure water, stir and mix at room temperature, and after complete dissolution and mixing, add pure water to make up the volume. Adjust the pH to <2.0 with nitric acid, and store in a sealed container at 2~30℃.

[0027] Step 2, Preparation of Substrate Solution B: Add sodium hydroxide and composite enhancer to pure water, stir and mix at room temperature. After complete dissolution and mixing, add pure water to make up the volume. Adjust the pH to 12.5~13.5 with sodium hydroxide, and store in a sealed container at 2~30℃.

[0028] Step 3, Mixing: Substrate solution A and substrate solution B are packaged and stored separately; when using, substrate solution A and substrate solution B are mixed at a volume ratio of (0.5~1):(0.5~1).

[0029] Example 1 A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Prepare substrate solution A: 15 mmol / L nitric acid + hydrogen peroxide of different concentrations. Adjust the pH to <2.0 with nitric acid and store in a sealed container at 2~30℃ for later use.

[0030] Step 2: Prepare substrate solution B: 0.25 mol / L sodium hydroxide + 3 g / L tetradecyltrimethylammonium chloride + 0.1% Tween-20 + 2% Triton X-100. Adjust the pH to 12.5~13.5 with sodium hydroxide and store in a sealed container at 2~30℃ for later use.

[0031] Purified water was used as a blank sample for background testing; a 1 nmol / L acridinium ester solution prepared with purified water was used as the acridinium ester sample for luminescence intensity testing. On a fully automated chemiluminescence immunoassay analyzer, 10 μL of blank sample or acridinium ester sample was added to 100 μL of substrate solution A and 100 μL of substrate solution B, reacted for 2 min, and then detected. Each sample was tested 10 times.

[0032] The effects of different concentrations of hydrogen peroxide in substrate solution A on the background signal are shown in Table 1, and the effects of different concentrations of hydrogen peroxide in substrate solution A on the luminescence intensity are shown in Table 2.

[0033] Table 1. Effects of different hydrogen peroxide concentrations on background signal.

[0034] Table 2 Effect of different hydrogen peroxide concentrations on luminescence intensity

[0035] The results in Tables 1 and 2 show that there is no significant difference in background signal values ​​when the hydrogen peroxide concentration is 0.4 mol / L and 0.1 mol / L. However, when the hydrogen peroxide concentration is 1 mol / L, the background signal is significantly higher than that at 0.4 mol / L and 0.1 mol / L. The luminescence intensity is lowest at a hydrogen peroxide concentration of 0.1 mol / L and highest at 1 mol / L, but the background is also highest, resulting in a worse signal-to-noise ratio (S / N) than at a 0.4 mol / L concentration. Therefore, a 0.4 mol / L hydrogen peroxide concentration provides the lowest background, the highest luminescence intensity, and the highest S / N.

[0036] Example 2 A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Prepare substrate solution A: 15 mmol / L nitric acid + 0.4 mol / L hydrogen peroxide + different concentrations of Pluronic F-127 or Pluronic F-68 or ethyl phenyl polyethylene glycol. Adjust the pH to <2.0 with nitric acid and store in a sealed container at 2~30℃ for later use.

[0037] Step 2: Prepare substrate solution B: 0.25 mol / L sodium hydroxide + 3 g / L tetradecyltrimethylammonium chloride + 0.1% Tween-20 + 2% Triton X-100. Adjust the pH to 12.5~13.5 with sodium hydroxide and store in a sealed container at 2~30℃ for later use.

[0038] Purified water was used as a blank sample for background testing; a 1 nmol / L acridinium ester solution prepared with purified water was used as the acridinium ester sample for luminescence intensity testing. On a fully automated chemiluminescence immunoassay analyzer, 10 μL of either the blank sample or the acridinium ester sample was added to 100 μL of substrate solution A and 100 μL of substrate solution B. After reacting for 2 min, the sample was detected. Each sample was tested 10 times, and the average value and repeatability (CV) were calculated. The effects of different surfactants in substrate solution A on the background signal are shown in Table 3, and the effects of different surfactants in substrate solution A on luminescence intensity and repeatability are shown in Table 4.

[0039] Table 3. Effects of different surfactants in substrate solution A on background signal.

[0040] Table 4. Effect of different surfactants in substrate solution A on luminescence intensity

[0041] The results in Tables 3 and 4 show that when the concentration of Pluronic F-127 added is 0.1 g / L, the signal value of the blank sample (i.e., the background signal) is the lowest, and the repeatability of the 10-well replicate is the best. When the concentration of Pluronic F-127 added is 0.1 g / L, the signal value of the acridinium ester sample has reached its highest value, and the repeatability of the 10-well replicate is the best. Further increasing the concentration of Pluronic F-127 will not further increase the luminescence intensity or improve repeatability.

[0042] Example 3 A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Prepare substrate solution A: 15 mmol / L nitric acid + 0.4 mol / L hydrogen peroxide + 0.1 g / L Pluronic F-127. Adjust the pH to <2.0 with nitric acid and store in a sealed container at 2~30℃ for later use.

[0043] Step 2: Prepare substrate solution B: 0.25 mol / L sodium hydroxide + 3 g / L tetradecyltrimethylammonium chloride or 3 g / L hexadecyltrimethylammonium chloride, and any combination of two or three of 0.1% Tween-20 and 2% Triton X-100. Adjust the pH to 12.5~13.5 with sodium hydroxide, and store in a sealed container at 2~30℃ for later use.

[0044] Meanwhile, prepare the control group substrate solution B0: 0.25 mol / L sodium hydroxide, without any enhancer. Adjust the pH to 12.5~13.5 with sodium hydroxide and store it in a sealed container at 2~30℃ for later use.

[0045] Purified water was used as a blank sample for background testing; a 1 nmol / L acridinium ester solution prepared with purified water was used as the acridinium ester sample for luminescence intensity testing. On a fully automated chemiluminescence immunoassay analyzer, 10 μL of either the blank sample or the acridinium ester sample was added to 100 μL of substrate solution A and 100 μL of substrate solution B. After reacting for 2 min, the sample was detected. Each sample was tested 10 times, and the average value and repeatability (CV) were calculated. The effects of different enhancer combinations in substrate solution B on the background signal are shown in Table 5, the effects of different enhancer combinations in substrate solution B on the luminescence intensity are shown in Table 6, and the effects of different enhancer combinations in substrate solution B on the signal-to-noise ratio are shown in Table 7.

[0046] Table 5. Effects of different enhancer combinations in substrate solution B on background signal. Blank sample test results (luminescence value) Tetradecyltrimethylammonium chloride + Tween-20 Tetradecyltrimethylammonium chloride + Triton X-100 Hexadecyltrimethylammonium chloride + Tween-20 Hexadecyltrimethylammonium chloride + Triton X-100 Tween-20+TritonX-100 Tetradecyltrimethylammonium chloride + Tween-20 + Triton X-100 Hexadecyltrimethylammonium chloride + Tween-20 + Triton X-100 control group Repeat 1 106 135 106 148 87 97 113 72 Repeat 2 100 151 92 148 95 113 106 88 Repeat 3 96 116 112 129 97 92 112 76 Repeat 4 93 134 92 153 85 101 101 70 Repeat 5 110 139 113 148 81 101 118 66 Repeat 6 107 126 104 121 69 106 119 69 Repeat 7 98 146 97 138 71 103 115 72 Repeat 8 85 118 106 155 70 95 130 72 Repeat 9 105 140 100 161 74 92 102 63 Repeat 10 91 129 113 153 89 90 121 74 mean 99 133 104 145 82 99 114 72 CV 8.1% 8.5% 7.7% 8.6% 12.7% 7.3% 7.9% 9.3% Table 6. Effect of different enhancer combinations in substrate solution B on luminescence intensity Acridinium ester sample detection results (luminescence value) Tetradecyltrimethylammonium chloride + Tween-20 Tetradecyltrimethylammonium chloride + Triton X-100 Hexadecyltrimethylammonium chloride + Tween-20 Hexadecyltrimethylammonium chloride + Triton X-100 Tween-20+TritonX-100 Tetradecyltrimethylammonium chloride + Tween-20 + Triton X-100 Hexadecyltrimethylammonium chloride + Tween-20 + Triton X-100 control group Repeat 1 6572343 8071237 6477844 7780620 6249467 8552852 8390023 3621119 Repeat 2 6481099 7939494 6514154 8062907 6316752 8584532 8428046 3508055 Repeat 3 6551199 7954617 6552300 8096805 6122413 8494182 8469503 3597239 Repeat 4 6482856 8057141 6464729 8050250 6135623 8454505 8414004 3517720 Repeat 5 6412503 7977511 6490974 8009418 6175745 8413929 8548424 3755867 Repeat 6 6680775 7984913 6550716 7991886 6196461 8501227 8612556 3575630 Repeat 7 6555222 8118932 6488361 7762222 6080126 8493886 8449081 3902298 Repeat 8 6573581 8089612 6253132 7786722 6194722 8544685 8457337 3610273 Repeat 9 6565936 8220593 6438516 8000123 6156361 8580923 8494317 3514514 Repeat 10 6536373 7746239 6482330 7876667 6228181 8591206 8524217 3616020 mean 6541189 8016029 6471306 7941762 6185585 8521193 8478751 3621874 CV 1.1% 1.6% 1.3% 1.6% 1.1% 0.7% 0.8% 3.4% Table 7. Effect of different enhancer combinations on signal-to-noise ratio in substrate solution B Group Enhancer combination Signal-to-noise ratio (S / N) 1 Tetradecyltrimethylammonium chloride + Tween-20 66073 2 Tetradecyltrimethylammonium chloride + Triton X-100 60271 3 Hexadecyltrimethylammonium chloride + Tween-20 62224 4 Hexadecyltrimethylammonium chloride + Triton X-100 54771 5 Tween-20 + Triton X-100 75434 6 Tetradecyltrimethylammonium chloride + Tween-20 + Triton X-100 86073 7 Hexadecyltrimethylammonium chloride + Tween-20 + Triton X-100 74375 8 control group 50304 The results in Tables 5, 6, and 7 show that the background signal value is the lowest when no enhancer is added to substrate solution B, but the luminescence intensity and signal-to-noise ratio are also the lowest. When Tween-20 and Triton X-100 are added to substrate solution B, the background signal value is also low, but the luminescence intensity is also low. Only when tetradecyltrimethylammonium chloride, Tween-20, and Triton X-100 are added to substrate solution B as a composite enhancer, the background signal value is low, the luminescence intensity is the highest, the signal-to-noise ratio is the highest, and the CV of 10 replicates is the best. It is worth noting that the optimal combination for substrate solution B is 3 g / L tetradecyltrimethylammonium chloride, 0.1% Tween-20, and 2% Triton X-100. However, any combination of tetradecyltrimethylammonium chloride or hexadecyltrimethylammonium chloride with any two or three of Tween-20 and Triton X-100 can achieve a relatively ideal low background signal and high luminescence intensity. The concentration range of tetradecyltrimethylammonium chloride or hexadecyltrimethylammonium chloride is 0.5 g / L to 10 g / L, the concentration range of Tween-20 is 0.01% to 1%, and the concentration range of Triton X-100 is 0.5% to 4%.

[0047] Example 4 A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Prepare substrate solution A: 15 mmol / L nitric acid + 0.4 mol / L hydrogen peroxide + 0.1 g / L Pluronic F-127. Adjust the pH to <2.0 with nitric acid and store in a sealed container at 2~30℃ for later use.

[0048] Step 2: Prepare substrate solution B: 0.25 mol / L sodium hydroxide + 3 g / L tetradecyltrimethylammonium chloride + 0.1% Tween-20 + 2% Triton X-100. Adjust the pH to 12.5~13.5 with sodium hydroxide and store in a sealed container at 2~30℃ for later use.

[0049] A portion of each of the prepared substrate solutions A and B was placed at 55℃ for 14 days for acceleration. The accelerated substrate solutions and the substrate solutions stored at 2-30℃ were used to test blank samples and acrid ester samples, respectively. Each sample was tested 10 times, and the average value and repeatability (CV) were calculated. The changes in background signal and luminescence intensity of the accelerated substrate solutions are shown in Table 8.

[0050] Table 8. Results of accelerated stability of substrate liquid

[0051] The results in Table 8 show that after 14 days of acceleration at 55℃, the background signal values ​​of substrate solutions A and B did not change significantly and remained low. The luminescence intensity signal values ​​did not decrease significantly, with a decrease of less than 10%, and the CV remained good. Therefore, substrate solutions A and B exhibit good acceleration stability and remained stable after 14 days of acceleration at 55℃.

[0052] Example 5 A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Prepare substrate solution A: 15 mmol / L nitric acid + 0.4 mol / L hydrogen peroxide + 0.1 g / L Pluronic F-127. Adjust the pH to <2.0 with nitric acid and store in a sealed container at 2~30℃ for later use.

[0053] Step 2: Prepare substrate solution B: 0.25 mol / L sodium hydroxide + 3 g / L tetradecyltrimethylammonium chloride + 0.1% Tween-20 + 2% Triton X-100. Adjust the pH to 12.5~13.5 with sodium hydroxide and store in a sealed container at 2~30℃ for later use.

[0054] After opening substrate solutions A and B, they were loaded onto a fully automated chemiluminescence immunoassay analyzer and stored at room temperature (25±5℃) for 0, 10, 20, 40, and 70 days. Blank samples and acridil ester samples were then tested, with each sample tested 10 times. The mean and repeatability (CV) were calculated. The results of the open-bottle stability of the substrate solutions are shown in Tables 9 and 10.

[0055] Table 9. Background signal results of substrate solution at different days after opening.

[0056] Table 10 Results of luminescence intensity signal values ​​of substrate solution at different days after opening.

[0057] The results in Tables 9 and 10 show that after the substrate solution was first opened and placed on the instrument, the background signal was still low after 70 days, and the luminescence intensity signal value only decreased by about 10% compared with day 0. This indicates that the substrate solution has good stability after opening and can be stored in the instrument for at least 70 days after opening.

[0058] Example 6 A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Prepare substrate solution A: 15 mmol / L nitric acid + 0.4 mol / L hydrogen peroxide + 0.1 g / L Pluronic F-127. Adjust the pH to <2.0 with nitric acid and store in a sealed container at 2~30℃ for later use.

[0059] Step 2: Prepare substrate solution B: 0.25 mol / L sodium hydroxide + 3 g / L tetradecyltrimethylammonium chloride + 0.1% Tween-20 + 2% Triton X-100. Adjust the pH to 12.5~13.5 with sodium hydroxide and store in a sealed container at 2~30℃ for later use.

[0060] The prepared substrate solutions A and B were stored at room temperature (25±5℃) for 2 years. During this period, blank samples and acrid ester samples were tested at 0, 6, 12, and 24 months, with each sample tested 10 times. The mean and repeatability (CV) were calculated. The results of the room temperature storage stability of the substrate solutions are shown in Tables 11 and 12.

[0061] Table 11 Background signal results of substrate solution stored at room temperature

[0062] Table 12 Results of luminescence intensity signals of substrate solutions stored at room temperature

[0063] The results in Tables 11 and 12 show that after 24 months of storage at room temperature (25±5℃), the background signal value of the prepared substrate solution remained low. Furthermore, the luminescence intensity signal value of the acridinium ester sample showed no significant difference from the value at month 0, with a signal decrease of less than 10%. This indicates that the substrate solution provided by this invention has good stability and can be stored at room temperature for at least 2 years.

[0064] Example 7 A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Prepare substrate solution A: 15 mmol / L nitric acid + 0.4 mol / L hydrogen peroxide + 0.1 g / L Pluronic F-127. Adjust the pH to <2.0 with nitric acid and store in a sealed container at 2~30℃ for later use.

[0065] Step 2: Prepare substrate solution B: 0.25 mol / L sodium hydroxide + 3 g / L tetradecyltrimethylammonium chloride + 0.1% Tween-20 + 2% Triton X-100. Adjust the pH to 12.5~13.5 with sodium hydroxide and store in a sealed container at 2~30℃ for later use.

[0066] Using a self-prepared substrate solution as the experimental group and a commercially available substrate solution as the control group, the blank limit and repeatability of the thyroid-stimulating hormone (TSH) test were compared. Blank limit detection method: Zero-concentration calibrator and calibrator S2 (concentration 0.08 mIU / mL) were used as test samples. The zero-concentration calibrator was measured 20 times, and the luminescence values ​​of the 20 measurements were obtained. The mean (M) and standard deviation (SD) of the 20 measurements were calculated to obtain M+2SD. Calibrator S2 was measured 3 times, and the mean was calculated. A two-point regression was performed based on the concentration-luminescence value results between the zero-concentration calibrator and calibrator S2 to obtain a linear equation. The luminescence value of the zero-concentration calibrator (M+2SD) was substituted into the above equation to obtain the corresponding concentration value, which is the blank limit. Repeatability detection method: The TSH sample (concentration 100 mIU / mL) was measured 10 times, and the CV value was calculated. The blank limit test results of the substrate solutions of the experimental group and the control group for the TSH test are shown in Table 13, and the repeatability test results of the TSH test are shown in Table 14.

[0067] Table 13. Blank limit detection results of substrate solutions in the experimental and control groups for TSH.

[0068] Table 14 Repeatability test results of substrate solutions for TSH in experimental and control groups Repeatability Experimental group (self-prepared substrate solution) Control group (commercially available substrate solution) Repeat 1 7326531 5646961 Repeat 2 7029789 5594353 Repeat 3 7115301 5261856 Repeat 4 7198856 5557510 Repeat 5 7079523 5489321 Repeat 6 7239797 5609919 Repeat 7 7042692 5465965 Repeat 8 7072515 5560847 Repeat 9 7304040 5540762 Repeat 10 7178365 5704040 mean 7158741 5543153 CV 1.5% 2.2% The results in Tables 13 and 14 show that the substrate solution provided by this invention can effectively improve the blank limit of the TSH test, approximately three times that of commercially available substrate solutions. Furthermore, the substrate solution provided by this invention also improves the reproducibility of the TSH test, with a coefficient of variation (CV) superior to that of commercially available substrate solutions.

[0069] Example 8 A method for preparing a highly sensitive and stable acridine ester luminescent substrate solution includes the following steps: Step 1: Prepare substrate solution A: 15 mmol / L nitric acid + 0.4 mol / L hydrogen peroxide + 0.1 g / L Pluronic F-127, and adjust the pH to <2.0 with nitric acid.

[0070] Step 2: Prepare substrate solution B: 0.25 mol / L sodium hydroxide + 3 g / L tetradecyltrimethylammonium chloride + 0.1% Tween-20 + 2% Triton X-100, and adjust the pH to 12.5~13.5 with sodium hydroxide.

[0071] Substrate solution A and substrate solution B were used at volume ratios of 1:0.5, 1:1, and 0.5:1, respectively, to detect blank samples and acrid ester samples. Each sample was tested 10 times, and the average value and CV were calculated. The effect of different ratios of substrate solutions A and B on the detection results is shown in Tables 15 and 16.

[0072] Table 15 Effect of different ratios of substrate solutions A and B on background levels

[0073] Table 16 Effect of different ratios of substrate solutions A and B on luminescence intensity

[0074] The results in Tables 15 and 16 show that the optimal volume ratio of substrate liquid A to substrate liquid B provided by this invention is 1:1. When the volume ratio of substrate liquid A to substrate liquid B is 1:0.5 and 0.5:1, the luminescence intensity is slightly worse. However, when the volume ratio of substrate liquid A to substrate liquid B is 1:0.3 and 0.3:1, the luminescence intensity is the worst, and the signal-to-noise ratio is much lower than when the volume ratio of substrate liquid A to substrate liquid B is 1:1.

[0075] In summary, the substrate solution provided by the present invention has the advantages of low background, high luminescence intensity, high signal-to-noise ratio, and high stability. It solves the problem that existing acrid ester luminescent substrate solutions generally have difficulty in balancing luminescence signal intensity and background signal. At the same time, it can be transported and stored at room temperature, which greatly reduces storage, transportation and usage costs, making it suitable for large-scale industrial production and clinical testing.

[0076] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A highly sensitive and highly stable acridine ester luminescent substrate solution, characterized in that: It includes substrate solution A and substrate solution B, with a volume ratio of (0.5~1):(0.5~1). Substrate solution A includes an oxidant, an inorganic acid, and a surfactant; substrate solution B includes a composite reinforcing agent and an inorganic base.

2. The highly sensitive and highly stable acridine ester luminescent substrate liquid according to claim 1, characterized in that: The volume ratio of substrate liquid A to substrate liquid B is 1:

1.

3. The highly sensitive and highly stable acridine ester luminescent substrate solution according to claim 2, characterized in that: The oxidant is hydrogen peroxide, and the molar concentration of hydrogen peroxide is in the range of 0.1 mol / L to 1 mol / L.

4. The highly sensitive and highly stable acridinium ester luminescent substrate solution according to claim 3, characterized in that: The inorganic acid is nitric acid, and the molar concentration of nitric acid is 15 mmol / L.

5. The highly sensitive and highly stable acridinium ester luminescent substrate solution according to claim 4, characterized in that: The surfactant is at least one of Pluronic F-127, Pluronic F-68, and ethyl phenyl polyethylene glycol, and the mass concentration of the surfactant is 0.02 g / L to 0.5 g / L.

6. The highly sensitive and highly stable acridinium ester luminescent substrate liquid according to claim 5, characterized in that: The inorganic base is sodium hydroxide, and the molar concentration of sodium hydroxide is 0.25 mol / L.

7. The highly sensitive and highly stable acridinium ester luminescent substrate liquid according to claim 6, characterized in that: The composite reinforcing agent comprises any two or three combinations of tetradecyltrimethylammonium chloride or hexadecyltrimethylammonium chloride with Tween-20 and Triton X-100.

8. A method for preparing a highly sensitive and highly stable acridine ester luminescent substrate solution according to any one of claims 1 to 7, characterized in that, Includes the following steps: Step 1, Preparation of substrate solution A: Add nitric acid, hydrogen peroxide, Pluronic F-127 or Pluronic F-68 or ethyl phenyl polyethylene glycol to pure water, stir, adjust the pH to <2.0 with nitric acid and seal for storage. Step 2, Preparation of substrate solution B: Add sodium hydroxide and composite enhancer to pure water, stir, adjust the pH to 12.5~13.5 with sodium hydroxide and store; Step 3, Mixing: When using, mix substrate solution A and substrate solution B in a volume ratio of (0.5~1):(0.5~1).

9. The method for preparing a highly sensitive and highly stable acridine ester luminescent substrate solution according to claim 8, characterized in that: Substrate solution A and substrate solution B should be stored in a sealed container at 2~30℃.

10. The application of a highly sensitive and highly stable acridine ester luminescent substrate solution according to any one of claims 1 to 7 in the preparation of industrial and / or clinical diagnostic reagents.

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