Acridinium ester luminescent substrate and application thereof

Abstract

The invention belongs to the technical field of chemiluminescence immunoassay, and particularly relates to an acridinium ester luminescent substrate and application thereof.The acridinium ester luminescent substrate comprises a pre-excitation solution and an excitation solution, the pre-excitation solution is a hydrogen peroxide acid solution containing 0.05-0.2% of polyvinylpyrrolidone and / or 0.1-0.3% of polyethylene glycol, and the excitation solution is an acridinium ester luminescent substrate. The excitation liquid is an alkali solution containing 0.5 to 2 percent of triton. The polyvinylpyrrolidone and / or polyethylene glycol in the pre-excitation liquid and triton in the excitation liquid are matched, so that the reaction intensity of a substrate and acridinium ester molecules can be enhanced, the light production efficiency of acridinium ester in unit time is improved, and the method has good effects on background or background control, signal-to-noise ratio improvement and the like.

Description

Technical Field

[0001] The invention relates to the technical field of chemiluminescent immunoassay, and in particular to an acridinium ester luminescent substrate and application thereof. Background Art

[0002] Chemiluminescence immunoassay (CLIA) technology is a detection and analysis technology that combines chemiluminescence with antigen-antibody reaction and is used for various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins and drugs. It has been widely used in many fields such as biomedicine, clinical diagnosis, environmental and health testing, forensic identification, food safety, etc. Its basic principle is to use chemiluminescent reagents as markers, directly mark them on analytes such as antigens or antibodies, and form antigen-antibody immune complexes through the reaction of antigens and antibodies. After that, luminescent substrates such as catalysts and oxidants are added. After the chemiluminescent reagents are oxidized, an excited intermediate is formed. The intermediate emits photons to release energy and returns to a stable ground state. The content of the analyte can be quantitatively detected by measuring the luminescence intensity.

[0003] Acridinium esters and acridinium salt luminescent agents (NSP-SA-NHS) are commonly used chemiluminescent markers in CLIA. In an alkaline environment, hydrogen peroxide anions are added to the 9th position of the acridinium ring to generate a strained and unstable dioxyethane, which is then rapidly decomposed into carbon dioxide and electronically excited N-methylacridone. When acridinone returns to the ground state, it emits photons with a maximum absorption wavelength of 470nm. In this process, no additional energy input is required, the luminescence efficiency is high, and the reaction system is relatively simple, and no complex catalyst is required. At the same time, the luminescence of this type of compound is flash-type. After adding the luminescence start reagent, the luminescence intensity reaches the maximum in about 0.4s, and the half-life is about 0.9s, which is very time-saving. Based on the above characteristics, acridinium esters have significant advantages in the field of CLIA rapid detection. However, there are also some shortcomings. The stability of acridinium esters is relatively poor, and they are easily hydrolyzed or photodegraded, resulting in a weakened or even lost luminescence effect.

[0004] In order to improve the stability and chemiluminescence intensity of acridinium esters, surfactants such as Triton X-100 and Tween-20 are usually added to the luminescence initiation reagent to provide a relatively simple and stable condition for the chemical reaction of acridinium ester molecules. However, this will lead to a decrease in the photon output per unit time, affecting the luminescence efficiency and detection sensitivity. Summary of the invention

[0005] In view of the above technical problems in the prior art, the present invention provides an acridinium ester luminescent substrate, which can enhance the reaction intensity between the substrate and the acridinium ester molecule, increase the light production efficiency of the acridinium ester per unit time, and has good effects on background or background control, improvement of signal-to-noise ratio, etc. Therefore, the present invention further provides the use of the aforementioned acridinium ester luminescent substrate in the preparation of a chemiluminescent immunoassay kit.

[0006] To achieve the above object, the present invention is specifically implemented through the following technical solutions:

[0007] The first aspect of the present invention provides an acridinium ester luminescent substrate, comprising a pre-excitation solution and an excitation solution, wherein the pre-excitation solution is a hydrogen peroxide acid solution comprising 0.05-0.2% polyvinyl pyrrolidone and / or 0.1-0.3% polyethylene glycol, and the excitation solution is an alkaline solution comprising 0.5-2% Triton.

[0008] Furthermore, the pre-stimulation solution is a hydrogen peroxide acid solution comprising 0.05-0.2% polyvinyl pyrrolidone and 0.1-0.3% polyethylene glycol.

[0009] Furthermore, the pre-stimulation solution is a hydrogen peroxide acid solution including 0.1% polyvinyl pyrrolidone and 0.2% polyethylene glycol.

[0010] Furthermore, the hydrogen peroxide acid solution is an aqueous solution comprising 0.2 mol / L HCl and 58.8 mmol / L H2O2.

[0011] Furthermore, the pre-stimulation solution consists of an aqueous solution of 0.05-0.2% polyvinyl pyrrolidone, 0.1-0.3% polyethylene glycol, 0.2 mol / L HCl and 58.8 mmol / L H2O2.

[0012] Furthermore, the stimulating solution is an alkaline solution including 0.5% Triton.

[0013] Furthermore, the alkaline solution is an aqueous solution including 0.5 mol / L NaOH.

[0014] Furthermore, the stimulating solution is composed of an aqueous solution of 0.5-2% Triton and 0.5 mol / L NaOH.

[0015] Furthermore, the polyvinyl pyrrolidone is selected from PVP-10, the polyethylene glycol is selected from PEG-6000, and the Triton is selected from Triton X-100.

[0016] Furthermore, the volume ratio of the pre-excitation liquid to the excitation liquid is 1:1.

[0017] The second aspect of the present invention provides the use of the acridinium ester luminescent substrate as described above in the preparation of a chemiluminescent immunoassay kit.

[0018] The advantages and positive effects of the present invention are:

[0019] The present invention improves the components and ratios of the pre-excitation solution and the excitation solution, and can enhance the reaction intensity of the substrate and the acridinium ester molecule by combining polyvinyl pyrrolidone (PVP) and / or polyethylene glycol (PEG) in the pre-excitation solution and Triton in the excitation solution, thereby increasing the light production efficiency of the acridinium ester per unit time, and has good effects on background or base control, improvement of the signal-to-noise ratio, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0021] Figure 1 The curve showing the change of the luminescence intensity of the acridinium ester marker detected by the luminescent substrate in the experimental group A of the embodiment of the present invention as a function of the reaction time;

[0022] Figure 2 The curve showing the change of the luminescence intensity of the acridinium ester marker with the luminescent substrate in the experimental group B of the embodiment of the present invention as a function of the reaction time;

[0023] Figure 3 The curve showing the change of the luminescence intensity of the acridinium ester marker with the luminescent substrate in the experimental group C of the embodiment of the present invention as a function of the reaction time;

[0024] Figure 4 The curve showing the change of the luminescence intensity of the acridinium ester marker with the reaction time when the luminescent substrate is used to detect the acridinium ester marker in the experimental group D of the embodiment of the present invention;

[0025] Figure 5 This is a curve showing the change in luminescence intensity of the acridinium ester marker detected by the luminescent substrate in experimental group E of the embodiment of the present invention as a function of reaction time. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0027] Based on the information contained in the present invention, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, because these embodiments and other descriptions are only for the purpose of illustrating specific aspects of the present invention. In fact, various changes that a person skilled in the art or related fields can obviously make to the embodiments of the present invention are covered within the scope of the appended claims.

[0028] In order to better understand the present invention but not to limit the scope of the present invention, all the numbers and other numerical values ​​used in the present invention to express the amount, percentage, etc. should be understood as modified by the word "approximately" in all cases. The term "approximately" has its usual meaning, which is used to indicate that a value includes the inherent variation of the error of the device or method used to determine the value, or contains a value close to the value, such as within 10% of the value (or range of values). Therefore, unless otherwise specified, the numerical parameters listed in the specification and the attached claims are approximate values, which may be changed according to the different ideal properties attempted to be obtained. In addition, it should be noted that the meaning of the terms "including", "comprising", "containing", "having" and the like is non-restrictive, that is, other steps and other ingredients that do not affect the results can be added.

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0030] The basic principle of acridinium ester chemiluminescence includes two steps: (1) the formation of excited intermediates: under alkaline conditions, acridinium ester reacts with hydrogen peroxide to generate an unstable dioxyethane intermediate, which decomposes rapidly in a very short time to produce carbon dioxide (CO2) and an N-methylacridone molecule in an electronically excited state. During this process, energy is stored in the excited N-methylacridone molecule; (2) the release of photons: when the excited N-methylacridone molecule returns to the ground state, its stored energy is released in the form of photons to form visible light. The luminescent substrate that plays an exciting role in the acridinium ester luminescence process is a key component in the entire reaction system. Acridinium ester is connected with antigens, antibodies and other analytes to form acridinium ester markers. The acridinium ester markers form unstable excited intermediates under the combined action of the pre-excitation solution and the excitation solution, resulting in a luminescent reaction. The luminescence intensity of the reaction can be detected using a chemiluminometer to qualitatively and quantitatively analyze the concentration of the analyte. The pre-excitation solution is usually an acidic solution containing hydrogen peroxide, which is used in conjunction with the immunoassay analyzer to provide a reaction environment for the chemiluminescent reaction and maintain the stability of hydrogen peroxide. The excitation solution is usually an alkaline solution, which is used to provide an alkaline environment and excite the chemiluminescent reaction.

[0031] The embodiment of the present invention provides an acridinium ester luminescent substrate, including a pre-excitation solution and an excitation solution, wherein the pre-excitation solution is a hydrogen peroxide acid solution including 0.05-0.2% polyvinyl pyrrolidone (PVP) and / or 0.1-0.3% polyethylene glycol (PEG), and the excitation solution is an alkaline solution including 0.5-2% Triton. The above mass proportions are all mass percentages of the solvent in the solution, and generally speaking, the solvent can be water.

[0032] The present invention uses only hydrogen peroxide (hydrogen peroxide, H2O2) as an oxidant in the pre-excitation solution, uses low-concentration hydrochloric acid as a stabilizer, and simultaneously adds polyvinyl pyrrolidone (PVP) and / or 0.1-0.3% polyethylene glycol (PEG) as a luminescence enhancer, and mixes to form a pre-excitation solution, and cooperates with an alkaline solution containing low-concentration Triton as an excitation solution to form a luminescent substrate. Wherein, polyethylene glycol (PEG) and polyvinyl pyrrolidone (PVP) are both non-ionic compounds with excellent dispersibility and biocompatibility. Polyethylene glycol (PEG) can be used as a protein protector and signal amplifier of the buffer to improve the stability and sensitivity of the reagent, and reduce nonspecific binding and background signals. Polyvinyl pyrrolidone (PVP) is a high molecular homopolymer that can be dissolved in both water and most organic solvents, has good solubility, film-forming properties and binding ability, is relatively stable to salt, acid and heat, and plays a role in stabilizing the reaction system during the light-promoted reaction of acridinium esters. Triton is a nonionic surfactant and emulsifier, which can improve the hydrophilicity of acridinium ester, make the whole reaction have lower background, and thus improve the signal-to-noise ratio. The above three substances cooperate with each other to enhance the reaction intensity between substrate and acridinium ester molecules, increase the light production efficiency of acridinium ester per unit time, and have good effects on background or base control, signal-to-noise ratio improvement, etc.

[0033] The present invention experiments found that the combination of PVP and PEG is more conducive to improving the luminescence efficiency of acridinium ester and improving the signal-to-noise ratio. Therefore, preferably, the pre-excitation solution is a hydrogen peroxide acid solution comprising 0.05-0.2% PVP and 0.1-0.3% PEG. More preferably, the pre-excitation solution is a hydrogen peroxide acid solution comprising 0.1% PVP and 0.2% PEG.

[0034] Optionally, the hydrogen peroxide acid solution includes 0.2moL / L HCl and 58.8mmoL / L H2O2, which can be prepared by adding 0.2moL HCl and 58.8mmoL H2O2 to 1000mL water. At this time, the pre-excitation solution includes 0.05-0.2% polyvinyl pyrrolidone (PVP), 0.1-0.3% polyethylene glycol (PEG), 0.2moL / L HCl and 58.8mmoL / L H2O2. In a preferred embodiment, the pre-excitation solution consists of an aqueous solution of 0.05-0.2% PVP, 0.1-0.3% PEG, 0.2moL / L HCl and 58.8mmoL / L H2O2.

[0035] Preferably, the stimulating solution is an alkaline solution including 0.5% Triton.

[0036] Optionally, the alkaline solution includes 0.5 mol / L NaOH, which can be prepared by adding 0.5 mol / L NaOH to 1000 mL water. In this case, the stimulating solution includes 0.5-2% Triton and 0.5 mol / L NaOH. In a preferred embodiment, the stimulating solution is composed of an aqueous solution of 0.5-2% Triton and 0.5 mol / L NaOH.

[0037] In a preferred embodiment, the polyvinyl pyrrolidone (PVP) is selected from PVP-10, the polyethylene glycol (PEG) is selected from PEG-6000, and the Triton is selected from Triton X-100.

[0038] In actual detection, the volume ratio of the pre-excitation liquid to the excitation liquid is 1:1.

[0039] Another embodiment of the present invention provides the use of the acridinium ester luminescent substrate as described above in the preparation of a chemiluminescent immunoassay kit.

[0040] The advantages of using the acridinium ester luminescent substrate in preparing a chemiluminescent immunoassay kit are the same as the advantages of the acridinium ester luminescent substrate over the prior art as described above, and will not be repeated here.

[0041] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0042] Example

[0043] 1. Preparation of acridinium ester luminescent substrate

[0044] In this embodiment, the luminescent substrate includes a pre-excitation solution and an excitation solution. An aqueous solution containing 0.2 mol / L HCl and 58.8 mmol / L H2O2 is used as the pre-excitation solution, and an aqueous solution containing 0.5 mol / L NaOH is used as the excitation solution to form a basic luminescent system. In addition, different concentrations of Tween 20 (Tween-20), betaine, Triton X-100, polyethylene glycol 6000 (PEG-6000), PVP-10 and other surfactants are added to the pre-excitation solution and the excitation solution to prepare different luminescent substrates. The specific groups and formulas of the luminescent substrates are shown in Table 1. It should be noted that, in the pre-excitation solution and the excitation solution, unless otherwise specified, the percentages are weight percentages, that is, the ratio of the mass of the solute in the solution to the mass of the solvent, wherein the solvent is water. For example, the pre-excitation solution contains 0.1% PEG-6000, which means that 0.1g of PEG-6000 is contained in every 100g of water, and the excitation solution contains 0.5% Triton X-100, which means that 0.5g of Triton X-100 is contained in every 100g of water.

[0045] Table 1 Formulas of different acridinium ester luminescent substrates in the embodiments of the present invention

[0046]

[0047]

[0048] 2. Effect of acridinium ester luminescent substrate on the luminescent properties of acridinium ester

[0049] Luminescence detection: initialize the chemiluminometer (purchased from Coleus, catalog number Venus100S), confirm that the instrument is operating normally, and record the power-on time; take out the test samples human interleukin 6 (IL-6) recombinant protein (purchased from Abotek, catalog number RP00201), human interleukin 6 (IL-6) monoclonal antibody, excitation solution and pre-excitation solution from the refrigerator and place them for a period of time to restore their temperature to room temperature; add pre-excitation solution and excitation solution to the samples to be tested in sequence, wherein the test samples S1-S5 are phosphate buffer containing different concentrations of human interleukin 6 (IL-6) recombinant protein, S0 is phosphate buffer without any recombinant protein, and the protein concentrations of S1-S5 are 61.7 pg / mL, 185.2 pg / mL, 555.6 pg / mL, 1667 pg / mL and 5000 pg / mL, respectively.

[0050] The order of sample testing is S0, S1, S2, S3, S4, S5, and the sample volume used is 50 μL. Two human interleukin 6 monoclonal antibodies (purchased from Ibotek, catalog number RM17650 and RM17651) were used to detect the above-mentioned human interleukin 6 (IL-6) recombinant protein using the double antibody sandwich method, one of which was coupled to magnetic beads as a capture antibody, and the other was labeled with acridinium ester (purchased from Meiket, catalog number 203126) as a detection antibody. When switching different pre-excitation solutions and excitation solutions, pay attention to emptying the residual liquid in the pipeline; record the luminescence value of each excitation solution and pre-excitation solution test sample, and organize the data.

[0051] Taking the basic luminescence system as the control group, the optimal substrate formula was determined by the luminescence signal value (RLU) of different experimental groups, the light production efficiency and the signal-to-noise ratio (the ratio of S1-S5 to S0), and the corresponding data are summarized in Table 2. Furthermore, by testing the curve of the change of luminescence intensity in the sample to be tested with the reaction time, the reaction results of the experimental group AE are shown in Table 2. Figure 1-5 , where the horizontal axis represents time and the vertical axis represents luminous intensity, which is calculated by dividing the actual amount of product produced by the theoretical amount.

[0052] Table 2 Luminescence performance test of different acridinium ester luminescent substrates in the present invention

[0053]

[0054] The above results show that the addition of conventional surfactants such as betaine and Tween-20 has little effect on improving the light-generating performance of acridinium esters. However, the present invention adds PVP-10 and / or PEG-6000 to the pre-excitation solution and Triton X-100 to the excitation solution, which can enhance the reaction intensity between the substrate and acridinium ester molecules, increase the light-generating efficiency of acridinium esters per unit time, and help reduce or maintain the background value unchanged, thereby achieving the purpose of improving the signal-to-noise ratio. Among them, the experimental group E1 in which 0.1% PVP-10 and 0.2% PEG-6000 are added to the pre-excitation solution and 0.5% Triton X-100 is added to the excitation solution has the shortest reaction time, and the light signal can reach a peak in about 0.1s, the chemical reaction efficiency is the highest, the number of photons produced per unit time increases by about 50%, and the signal-to-noise ratio result is the best.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An acridinium ester luminescent substrate, characterized in that The invention comprises a pre-stimulating solution and a stimulating solution, wherein the pre-stimulating solution is a hydrogen peroxide acid solution comprising 0.05-0.2% polyvinyl pyrrolidone and / or 0.1-0.3% polyethylene glycol, and the stimulating solution is an alkaline solution comprising 0.5-2% Triton.

2. The acridinium ester luminescent substrate according to claim 1, characterized in that The pre-stimulation solution is a hydrogen peroxide acid solution comprising 0.05-0.2% polyvinyl pyrrolidone and 0.1-0.3% polyethylene glycol.

3. The acridinium ester luminescent substrate according to claim 2, characterized in that The pre-stimulation solution is a hydrogen peroxide acid solution including 0.1% polyvinyl pyrrolidone and 0.2% polyethylene glycol.

4. The acridinium ester luminescent substrate according to claim 1, characterized in that The hydrogen peroxide acid solution is an aqueous solution including 0.2 mol / L HCl and 58.8 mmol / L H2O2.

5. The acridinium ester luminescent substrate according to claim 4, characterized in that The pre-stimulation solution consists of an aqueous solution of 0.05-0.2% polyvinyl pyrrolidone, 0.1-0.3% polyethylene glycol, 0.2 mol / L HCl and 58.8 mmol / L H2O2.

6. The acridinium ester luminescent substrate according to claim 1, characterized in that The provocative solution is an alkaline solution containing 0.5% Triton.

7. The acridinium ester luminescent substrate according to claim 1, characterized in that The alkaline solution is an aqueous solution including 0.5 mol / L NaOH.

8. The acridinium ester luminescent substrate according to claim 7, characterized in that The stimulating solution is composed of an aqueous solution of 0.5-2% Triton and 0.5 mol / L NaOH.

9. The acridinium ester luminescent substrate according to claim 1, characterized in that The polyvinyl pyrrolidone is selected from PVP-10, the polyethylene glycol is selected from PEG-6000, and the Triton is selected from Triton X-100.

10. Use of the acridinium ester luminescent substrate according to any one of claims 1 to 9 in the preparation of a chemiluminescent immunoassay kit.

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