Acridinium ester enhanced luminescence system

Abstract

The invention discloses an acridinium ester enhanced luminescence system, comprising a pre-excitation liquid, an excitation liquid and an acridinium ester connected to a test object, wherein the disclosed pre-excitation liquid is a mixed solution of a hydrogen peroxide solution and a nitric acid solution, the excitation liquid is a sodium hydroxide solution containing 0.5% to 2.0% of an enhancer, and the enhancer is any one of sodium lauryl sulfoacetate or Zetasperse 179; the invention has simple components, does not need to use other substances such as polyols and metal chelating agents as stabilizers, is easy to prepare, and is suitable for mass production; the invention can enhance the relative luminescence intensity by more than 250%, and can also prolong the luminescence time of the acridinium ester, thereby improving the operability and accuracy of the test.

Description

Technical Field

[0001] The invention belongs to the field of chemiluminescent immunoassay, and in particular relates to an acridinium ester enhanced luminescence system. Background Art

[0002] Chemiluminescence analysis is an analytical method that uses the luminescence phenomenon of chemiluminescent reactions to measure the light signal emitted when chemiluminescent substances transition from an excited state to a ground state. Chemiluminescent immunoassay technology uses the free energy released by chemical reactions to excite intermediates, causing them to return from an excited state to a ground state. When the intermediates return from an excited state to a ground state, they release photons of equal energy levels, and then measure the photons for quantitative analysis. The luminescence types of chemiluminescent reactions are usually divided into two types: flash type and glow type. Among them, the flash type has a very short luminescence time, only a few tenths of a second to a few seconds.

[0003] Acridinium ester is a third-generation chemiluminescent substance, and its luminescent reaction type belongs to the flash type. Antibodies (or antigens) are directly labeled with acridinium esters, and after an immune reaction with the corresponding antigens (or antibodies) in the test specimen, a solid-phase coated antibody-test antigen-acridinium ester labeled antibody complex is formed, and then when oxidized by H2O2 under alkaline conditions, light with a wavelength of 470nm is emitted, which has the advantages of high luminescence efficiency, low background interference, and high detection sensitivity. However, acridinium esters are expensive, so the use of raw materials can be reduced by enhancing the luminescence of acridinium value, thereby reducing the purpose of reducing costs.

[0004] At present, the enhanced luminescence technology of acridinium ester has been studied. For example, the Chinese invention patent application "A luminescence system of acridinium ester substrate" with document number 202211316032.3 discloses an acridinium ester substrate luminescence system including a pre-excitation liquid and an excitation liquid. The pre-excitation liquid is an aqueous solution containing 0.65% to 1.65% hydrogen peroxide and 0.315% nitric acid, and the excitation liquid is a sodium hydroxide solution containing 0.095% to 2% luminescence enhancer TritonX-100 and 0.25% to 5% luminescence enhancer hexadecyltrimethylammonium chloride. The excitation liquid also contains ethanol. This document improves the signal intensity by reducing the blank control luminescence value, thereby improving the luminescence efficiency and stability of the acridinium ester substrate luminescence system. However, this document does not solve the problem that acridinium ester has a short duration as a flash-type luminescent substance.

[0005] Therefore, it is necessary to further develop an acridinium ester luminescence system that can both enhance luminescence and prolong luminescence time. Summary of the invention

[0006] The purpose of the present invention is to study an acridinium ester enhanced luminescence system to enhance the luminescence intensity of acridinium ester and extend the luminescence duration, in view of the problems that the pre-excitation liquid and the excitation liquid used in the prior art have no significant effect on prolonging the luminescence time of acridinium ester and the luminescence intensity is weak.

[0007] The technical solution adopted by the present invention provides an acridinium ester enhanced luminescence system, comprising a pre-excitation liquid, an excitation liquid and an acridinium ester for connecting with a test object, wherein the pre-excitation liquid is a mixed solution of a hydrogen peroxide solution and a nitric acid solution; the excitation liquid is a sodium hydroxide solution containing 0.5% to 2.0% of an enhancer, and the enhancer is any one of sodium lauryl sulfoacetate or Zetasperse179.

[0008] Specifically, the mass concentration of the hydrogen peroxide solution is 1% to 2%, and the molar concentration of the nitric acid solution is 0.4 mol / L to 0.8 mol / L.

[0009] More specifically, the volume ratio of the hydrogen peroxide solution to the nitric acid solution is 1:9-10.

[0010] Furthermore, the molar concentration of the sodium hydroxide solution is 0.4 mol / L to 0.8 mol / L.

[0011] Preferably, the molar concentration of the nitric acid solution is 0.5 mol / L, and the molar concentration of the sodium hydroxide solution is 0.5 mol / L.

[0012] Preferably, the above-mentioned stimulating solution is a sodium hydroxide solution containing 1% of an enhancer.

[0013] Preferably, the concentration of the acridinium ester used to connect with the analyte in the sample to be tested is 3×10 - 8 mmol / L~5×10 -8 mmol / L.

[0014] Furthermore, the mass ratio of the pre-excitation liquid to the excitation liquid in the acridinium ester enhanced luminescence system is 1:1.5-2.5.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] In the enhanced luminescence system of the present invention, only low-concentration hydrogen peroxide is used as an enhancer in the pre-excitation liquid, and low-concentration nitric acid solution is used as a stabilizer. The two are mixed to form a pre-excitation liquid, in which no enhancer or other stabilizers are needed; the excitation liquid is a low-concentration sodium hydroxide solution with simple ingredients, in which the content of the enhancer is not high, only 0.5% to 2.0%, and no other organic solvents are needed. Regardless of the pre-excitation liquid or the excitation liquid of the present application, the components are very simple, and no other substances such as polyols and metal chelating agents are needed as stabilizers. It is easy to prepare and suitable for mass production.

[0017] The present invention uses only surfactants as enhancers in the excitation solution, which simplifies the enhanced luminescence process of the system. The enhancers selected by the present invention are two surfactants (sodium lauryl sulfoacetate or Zetasperse 179). When the enhancer content is 0.5% to 2.0%, the effect of enhancing the relative luminescence intensity by more than 250% can be achieved.

[0018] Among them, sodium lauryl sulfoacetate is also known as houttuynia cordata, houttuynia cordata sodium or neohouttuynia cordata sodium. Although it has the properties of a surfactant, this substance is a commonly used antibacterial and anti-inflammatory drug, and is widely used clinically to treat various diseases such as upper respiratory tract infection, chronic bronchitis, pneumonia and adnexitis. This substance can improve the solubility of the intermediate in the excitation system, but it is not easy to produce bubbles when used in low concentrations. Zetasperse179 is a novel alcohol ethoxylate substance, which is currently often used as a dispersant for coatings. In the present invention, because Zetasperse179 has a strong steric stabilization function, it can better improve the wettability of the substrate and improve the stability of the system.

[0019] Moreover, the present invention can also prolong the luminescence time of acridinium ester, reduce the requirements for instrument precision, and reduce the probability of failure. In addition, the present invention can also show a good luminescence enhancement effect under the condition of low acridinium ester content, so the present invention can improve the luminescence efficiency of the acridinium ester substrate luminescence system, and achieve the purpose of reducing the amount of acridinium ester used and reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a luminescence kinetics curve diagram of the present invention. DETAILED DESCRIPTION

[0021] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] If no specific conditions are specified in the examples, the experiments can be carried out under conventional conditions; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0023] Embodiment 1

[0024] 0.1 L of a 1% hydrogen peroxide solution and 0.9 L of a 0.5 mol / L nitric acid solution were mixed to prepare a pre-excitation solution sample 1;

[0025] 13.0 g of sodium lauryl sulfoacetate was added as an enhancer to 1 L of a sodium hydroxide solution with a molar concentration of 0.6 mol / L (density of about 1.3 g / mL) to prepare an exciting solution sample 1.

[0026] Embodiment 2

[0027] Pre-excitation solution sample 2 was prepared by mixing 0.1 L of a 1.5% mass concentration hydrogen peroxide solution and 0.9 L of a 0.4 mol / L molar concentration nitric acid solution;

[0028] 26 g of Zetasperse 179 was added as an enhancer to 1 L of 0.4 mol / L sodium hydroxide solution (density about 1.3 g / mL) to prepare the excitation solution sample 2.

[0029] Embodiment 3

[0030] 0.1 L of a 1% hydrogen peroxide solution and 0.9 L of a 0.5 mol / L nitric acid solution were mixed to prepare a pre-excitation solution sample 3;

[0031] 13 g of Zetasperse 179 was added as an enhancer to 1 L of 0.5 mol / L sodium hydroxide solution (density about 1.3 g / mL) to prepare the excitation solution sample 3.

[0032] Embodiment 4

[0033] 0.1 L of a 2% hydrogen peroxide solution and 1 L of a 0.8 mol / L nitric acid solution were mixed to prepare a pre-excitation solution sample 4;

[0034] 19.5 g of Zetasperse 179 was added as an enhancer to 1 L of 0.6 mol / L sodium hydroxide solution (density about 1.3 g / mL) to prepare the excitation solution sample 4.

[0035] Embodiment 5

[0036] 0.1 L of a 2% hydrogen peroxide solution and 1 L of a 0.4 mol / L nitric acid solution were mixed to prepare a pre-excitation solution sample 5;

[0037] 6.5 g of sodium lauryl sulfoacetate was added as an enhancer to 1 L of a sodium hydroxide solution with a molar concentration of 0.5 mol / L (density of about 1.3 g / mL) to prepare an exciting solution sample 5.

[0038] Embodiment 6

[0039] 0.1 L of a 1.5% mass concentration hydrogen peroxide solution and 1 L of a 0.6 mol / L molar concentration nitric acid solution were mixed to prepare a pre-excitation solution sample 6;

[0040] 9.8 g of sodium lauryl sulfoacetate was added as an enhancer to 1 L of a sodium hydroxide solution with a molar concentration of 0.8 mol / L (density of about 1.3 g / mL) to prepare an exciting solution sample 6.

[0041] Comparative Example 1

[0042] The comparative example is the same as Example 1, except that when preparing the stimulating solution, sodium lauryl sulfoacetate or Zetasperse 179 is not used as an enhancer, but 10g, 15g and 20g of Triton X-100 are used respectively to prepare the stimulating solution reference substances 1-1, 1-2 and 1-3.

[0043] Comparative Example 2

[0044] Comparative Example 2 is the same as Example 1, except that when preparing the stimulating solution, sodium lauryl sulfoacetate or Zetasperse 179 is not used as an enhancer, but 20 g, 30 g and 40 g of hexadecyltrimethylammonium chloride are used respectively to prepare the stimulating solution reference substances 2-1, 2-2 and 2-3.

[0045] Comparative Example 3

[0046] Comparative Example 3 is the same as Example 1, except that when preparing the stimulating solution, sodium lauryl sulfoacetate or Zetasperse 179 is not used as an enhancer, but 10 g of Triton X-100 and 40 g of hexadecyl trimethyl ammonium chloride, 15 g of Triton X-100 and 30 g of hexadecyl trimethyl ammonium chloride, and 20 g of Triton X-100 and 20 g of hexadecyl trimethyl ammonium chloride are used respectively to prepare stimulating solution reference substances 3-1, 3-2 and 3-3.

[0047] Blank control (basic luminescence system)

[0048] The blank control was the same as Example 1, except that when preparing the stimulating solution, neither sodium lauryl sulfoacetate nor Zetasperse 179 was used as an enhancer, nor any other type of surfactant was used as an enhancer.

[0049] Analysis and testing

[0050] 1. Experiment on the effect of different formulas of pre-excitation solution and excitation solution on the relative luminescence intensity of acridinium ester

[0051] Add 10 μL of diluted acridinium ester to the reaction cup to label anti-AFP antibody, thyroglobulin, and troponin, respectively. The acridinium ester content is 3×10 -8 mmol / L, 4×10 -8 mmol / L and 5×10 -8 mmol / L.

[0052] The IC2000 fully automatic chemiluminescence instrument of Shenyang Yilu Biotechnology Co., Ltd. was used to measure the relative luminescence intensity of each formula with the test time set to 0.8s. The relative luminescence intensity of the basic luminescence system was taken as 100%, and the relative luminescence intensity and relative activity of each group of formula were calculated respectively. The results are shown in Tables 1, 2 and 3.

[0053] Table 1: Test results of relative luminescence intensity of acridinium esters (anti-alpha-fetoprotein)

[0054]

[0055] Table 2: Test results of relative luminescence intensity of acridinium esters (thyroglobulin)

[0056]

[0057]

[0058] Table 3: Test results of relative luminescence intensity of acridinium ester (troponin)

[0059]

[0060] From the results in Tables 1, 2 and 3, it can be seen that for different labeled substrates, when the acridinium ester content is 3×10 -8 mmol / L, 4×10 -8 mmol / L and 5×10 -8 mmol / L, the enhanced luminescence system of the present invention can increase the relative luminescence intensity to more than 250%. When the acridinium ester content is 5×10 -8mmol / L, the highest enhancement for different proteins can be up to 395.94%, 332.14% and 365.15%. Although the control group has a poor enhancement effect when the acridinium ester content is low, it can also achieve an enhancement effect of more than 250% when the acridinium ester content is high.

[0061] 2. Experiment on the effect of different formulas of pre-excitation solution and excitation solution on the duration of acridinium ester luminescence

[0062] The intensity of acridinium ester was measured over time. The luminescence kinetic curves of sample 1, reference substance 1-3, reference substance 2-3, reference substance 3-2 and blank control were shown in Figure 1 .

[0063] Depend on Figure 1 It can be seen that the present invention can prolong the luminous time, reduce the requirements for the accuracy of the inspection instrument, and thus reduce the probability of instrument failure during the inspection process, improve the inspection efficiency and expand the scope of application.

[0064] 3. Effect of enhanced luminescence systems at different ratios on the relative luminescence intensity of acridinium esters

[0065] Adjust the mass ratio of the pre-stimulation solution and the stimulation solution, add acridinium ester to the reaction cup, and label the anti-AFP antibody, where the acridinium ester content is 4×10 -8 mmol / L; the test time was set to 0.8s to measure the relative luminescence intensity under different ratios. Taking the relative luminescence intensity of the basic luminescence system as 100%, the relative luminescence intensity and relative activity of each group of formulas were calculated respectively. The test results are shown in Table 4.

[0066] Table 4: Effect of different ratios on the relative luminescence intensity of acridinium esters

[0067]

[0068] It can be seen from Table 4 that when the mass ratio of the pre-excitation liquid to the excitation liquid is 1:2, the signal value is optimal and the enhanced luminescence effect is strongest.

Claims

1. An acridinium ester enhanced luminescence system, comprising a pre-excitation liquid, an excitation liquid and an acridinium ester for connecting with a test object, characterized in that: The pre-stimulation solution is a mixed solution of hydrogen peroxide solution and nitric acid solution; the stimulation solution is a sodium hydroxide solution containing 0.5% to 2.0% of an enhancer; the enhancer is Zetasperse 179; The mass concentration of the hydrogen peroxide solution is 1% to 2%; The volume ratio of the hydrogen peroxide solution to the nitric acid solution is 1:9-10; The mass ratio of the pre-excitation liquid to the excitation liquid in the acridinium ester enhanced luminescence system is 1:1.5-2.5; The molar concentration of the nitric acid solution is 0.5 mol / L, and the molar concentration of the sodium hydroxide solution is 0.5 mol / L.

2. The acridinium ester enhanced luminescence system according to claim 1, characterized in that: The exciting solution is a sodium hydroxide solution containing 1% of an enhancer.

3. The acridinium ester enhanced luminescence system according to claim 1, characterized in that: The concentration of the acridinium ester used to connect with the test object in the test sample is 3×10 -8 mmol / L~5×10 -8 mmol / L.

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