Chemiluminescence substrate solution, chemiluminescence detection kit and detection method

Abstract

The invention relates to the technical field of immunodetection and immunodiagnosis, in particular to a chemiluminescence substrate solution, a chemiluminescence detection kit and a detection method. The chemiluminescent substrate solution comprises a substrate solution 1, or comprises the substrate solution 1 and a substrate solution 2, the substrate solution 1 comprises sodium hydroxide, a surfactant and a blue light absorbent for absorbing blue light with the wavelength of 400-500 nm; the substrate solution 2 comprises inorganic acid and hydrogen peroxide. According to the application, the blue light absorbent is added into the chemiluminescent substrate liquid, so that blue light emitted by acridinium ester can be absorbed, a luminescent signal is reduced, and particularly, the luminescent value of a background caused by non-specific adsorption is reduced, so that the signal-to-noise ratio is increased.

Description

Technical Field

[0001] The present application relates to the technical field of immunoassay and immunodiagnosis, and in particular to a chemiluminescent substrate solution, a chemiluminescent detection kit and a detection method. Background Art

[0002] Chemiluminescent immunoassay (CLIA) has developed rapidly in recent years due to its high sensitivity, wide linear detection range, fast analysis speed, and simple operation. Commonly used chemiluminescent substrates include acridinium ester, alkaline phosphatase, luminol, etc. Acridinium ester luminescence is flash type, fast, concentrated and strong, fast detection, high sensitivity and precision, and is a very good chemiluminescent substrate.

[0003] Commonly used acridinium esters for antibody and antigen labeling include NSP-DMAE-NHS and NSP-SA-NHS. Acridinium esters or acridinium esters labeled on antibodies and antigens do not emit light by themselves, and only emit light when a chemical reaction occurs under the action of a substrate solution. The substrate solution consists of two independent components: hydrogen peroxide containing inorganic acid (mainly nitric acid) and inorganic base (mainly sodium hydroxide). The mechanism of acridinium ester luminescence is that in sodium hydroxide solution, acridinium ester is attacked by hydrogen peroxide ions to generate unstable cyclic intermediates, which decompose into carbon dioxide and excited N-methylacridone. When the excited N-methylacridone returns to the ground state, it emits blue light with a maximum emission wavelength of 430nm.

[0004] Chemiluminescent substrate solutions often add some surfactants to enhance the luminescent signal. Commonly used surfactants include Triton X-100, CTAC (hexadecyltrimethylammonium chloride), Tween-20, etc. CN117769588A also introduces benzyldimethyldodecylammonium chloride with an aromatic head group into CTAC, which enhances the signal more than CTAC. In addition, the use of fluorescent agents with high fluorescence quantum yields such as sodium fluorescein, rhodamine B and rhodamine 6G can also increase the quantum yield of chemiluminescent compounds, thereby enhancing the signal.

[0005] Traditional chemiluminescent substrates use surfactants to enhance the signal, but also enhance the background luminescence value caused by nonspecific adsorption, and the signal-to-noise ratio often does not change much. When the surfactant concentration is too high, the background caused by nonspecific adsorption increases faster than the signal, causing the signal-to-noise ratio to decrease and the detection sensitivity to decrease. Summary of the invention

[0006] Based on this, one or more embodiments of the present application provide a chemiluminescent substrate solution, a chemiluminescent detection kit and a detection method, including the following technical solutions:

[0007] One or more embodiments of the present application provide a chemiluminescent substrate solution, wherein the chemiluminescent substrate solution includes substrate solution 1, or includes substrate solution 1 and substrate solution 2;

[0008] The substrate solution 1 comprises sodium hydroxide, a surfactant and a blue light absorber that absorbs blue light with a wavelength of 400nm-500nm;

[0009] The substrate solution 2 includes inorganic acid and hydrogen peroxide.

[0010] In some embodiments of the present application, the substrate solution 1 includes 0.1 mg / L-100 mg / L of the blue light absorber. Optionally, the substrate solution 1 includes 1 mg / L-30 mg / L of the blue light absorber.

[0011] In some embodiments of the present application, the blue light absorber includes one or more of auramine O, golden orange G, thiazole orange and sunset yellow.

[0012] In some embodiments of the present application, the surfactant includes one or more of Triton X-100, CTAC and Tween-20.

[0013] In some embodiments of the present application, the substrate solution 1 includes 0.1 wt%-3 wt% of the surfactant.

[0014] In some embodiments of the present application, the inorganic base includes one or more of sodium hydroxide and potassium hydroxide.

[0015] In some embodiments of the present application, the substrate solution 1 includes 0.2M-0.6M of the inorganic base.

[0016] In some embodiments of the present application, the substrate solution 1 includes 0.2M-0.6M of sodium hydroxide.

[0017] In some embodiments of the present application, the substrate solution 2 includes 0.02M-0.2M of the inorganic acid.

[0018] In some embodiments of the present application, the inorganic acid includes one or more of nitric acid, hydrochloric acid and sulfuric acid.

[0019] In some embodiments of the present application, the substrate solution 2 includes 0.1 wt%-1.5 wt% of the hydrogen peroxide.

[0020] One or more embodiments of the present application provide a chemiluminescent detection kit, which includes the chemiluminescent substrate solution.

[0021] In some embodiments of the present application, the chemiluminescent detection kit further comprises one or more of an acridinium ester and an antibody.

[0022] In some embodiments of the present application, the acridinium ester includes one or more of NSP-DMAE-NHS, NSP-SA-NHS and NSP-DMAE-HEG-Glu-NHS.

[0023] One or more embodiments of the present application provide a method for detecting a target substance using chemiluminescence, wherein the chemiluminescent substrate solution or the chemiluminescent detection kit is used in the process of detecting the target substance.

[0024] In some embodiments of the present application, the volume ratio of the substrate solution 1 to the substrate solution 2 is 1:(0.5-2.5).

[0025] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. DETAILED DESCRIPTION

[0026] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0028] the term

[0029] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0030] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0031] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0032] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0033] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0034] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0035] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0036] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0037] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0038] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0039] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0040] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0041] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0042] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0043] In a first aspect of the embodiments of the present application, a chemiluminescent substrate solution is provided, wherein the chemiluminescent substrate solution includes substrate solution 1, or includes substrate solution 1 and substrate solution 2;

[0044] The substrate solution 1 comprises sodium hydroxide, a surfactant and a blue light absorber that absorbs blue light with a wavelength of 400nm-500nm;

[0045] The substrate solution 2 includes inorganic acid and hydrogen peroxide.

[0046] In the embodiment of the present application, a blue light absorber is added to the chemiluminescent substrate solution, which can absorb the blue light emitted by the acridinium ester, reduce the luminescent signal, and especially reduce the background luminescent value caused by nonspecific adsorption, thereby improving the signal-to-noise ratio.

[0047] In the present application, the chemiluminescent substrate solution refers to its working state, i.e., the use state. In fact, it can be ready-to-use or ready-to-mix.

[0048] In some examples of the present application, the substrate solution 1 includes 0.1 mg / L-100 mg / L of the blue light absorber, and the concentration of the blue light absorber is, for example, 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / L. Optionally, the substrate solution 1 includes 1 mg / L-30 mg / L of the blue light absorber.

[0049] The present application does not particularly limit the type of blue light absorber, as long as it can absorb blue light. In some examples of the present application, the blue light absorber includes one or more of auramine O, golden orange G, thiazole orange and sunset yellow.

[0050] The present application does not specifically limit the type of surfactant, as long as it can enhance the light signal. In some examples of the present application, the surfactant includes one or more of Triton X-100, CTAC and Tween-20.

[0051] The present application does not particularly limit the amount of surfactant used, as long as it can meet the detection requirements. In some examples of the present application, the substrate solution 1 includes 0.1wt%-3wt% of the surfactant, for example, 0.1wt%, 0.2wt%, 0.4wt%, 0.6wt%, 0.8wt%, 1wt%, 1.2wt%, 1.4wt%, 1.6wt%, 1.8wt%, 2wt%, 2.2wt%, 2.4wt%, 2.6wt%, 2.8wt%, and 3wt%.

[0052] The present application does not specifically limit the type of inorganic base, as long as it can meet the detection requirements. The inorganic base can be sodium hydroxide or potassium hydroxide. In some examples of the present application, the substrate solution 1 includes 0.2M-0.6M of sodium hydroxide, and the concentration of sodium hydroxide is, for example, 0.2, 0.3, 0.4, 0.5, or 0.6M.

[0053] The present application does not specifically limit the type and concentration of the inorganic acid in the substrate solution 2, as long as it can meet the detection requirements. In some examples of the present application, the substrate solution 2 includes 0.02M-0.2M of the inorganic acid, and the concentration of the inorganic acid is, for example, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2 M. In some examples of the present application, the inorganic acid includes one or more of nitric acid, hydrochloric acid and sulfuric acid.

[0054] The present application does not particularly limit the concentration of the hydrogen peroxide in the substrate liquid 2, as long as it can meet the detection requirements. In some examples of the present application, the substrate liquid 2 includes 0.1wt%-1.5wt% of the hydrogen peroxide, and the concentration of the hydrogen peroxide is, for example, 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%, 0.6wt%, 0.7wt%, 0.8wt%, 0.9wt%, 1wt%, 1.1wt%, 1.2wt%, 1.3wt%, 1.4wt%, and 1.5wt%.

[0055] In some examples of the present application, the substrate solution 1 includes 0.2M-0.6M of the sodium hydroxide, 0.1wt%-3wt% of the surfactant, and 0.1mg / L-100mg / L of the blue light absorber. Optionally, the substrate solution 1 includes 0.3M-0.6M of the sodium hydroxide, 0.5wt%-3wt% of the surfactant, and 1mg / L-30mg / L of the blue light absorber.

[0056] In some examples of the present application, the substrate solution 2 includes 0.06M-0.2M of the inorganic acid and 0.9wt%-1.5wt% of the hydrogen peroxide.

[0057] In some examples of the present application, the chemiluminescent substrate solution includes substrate solution 1 and substrate solution 2; the substrate solution 1 includes 0.2M-0.6M sodium hydroxide, 0.1wt%-3wt% surfactant, and 0.1mg / L-100mg / L blue light absorber. Optionally, the substrate solution 1 includes 0.3M-0.6M sodium hydroxide, 0.5wt%-3wt% surfactant, and 1mg / L-30mg / L blue light absorber; the substrate solution 2 includes 0.06M-0.2M inorganic acid and 0.9wt%-1.5wt% hydrogen peroxide.

[0058] A second aspect of the embodiments of the present application provides a chemiluminescent detection kit, which includes the chemiluminescent substrate solution.

[0059] The present embodiment may also include other chemiluminescent detection reagents, including but not limited to acridinium esters and antibodies, for example, a first antibody and a second antibody targeting a specific antigen, and either of the first antibody and the second antibody is labeled with an acridinium ester.

[0060] The present application does not particularly limit the types of acridinium esters, including but not limited to NSP-DMAE-NHS, NSP-SA-NHS and NSP-DMAE-HEG-Glu-NHS.

[0061] A third aspect of the embodiments of the present application provides a method for detecting a target substance using chemiluminescence, wherein the chemiluminescent substrate solution or the chemiluminescent detection kit is used in the process of detecting the target substance.

[0062] The target substance is not particularly limited in the present application, and may be an antigen. In the case where the target substance is an antigen, the method of the present application may be a double antibody sandwich method.

[0063] During the detection process, the present application does not specifically limit the usage ratio of the substrate liquid 1 and the substrate liquid 2, as long as the detection requirements are met. In some examples of the present application, the volume ratio of the substrate liquid 1 and the substrate liquid 2 is 1:(0.5-2.5), for example, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1., 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2.0, 1:2.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5.

[0064] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0065] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0066] Example 1

[0067] This embodiment provides an acridinium ester chemiluminescent substrate solution, and the preparation method thereof is as follows:

[0068] Prepare the acridinium ester chemiluminescent substrate solution according to the formula in the table:

[0069] Table 1

[0070]

[0071] Configuration process:

[0072] (1) Weigh sodium hydroxide, CTAC, and auramine O and add them to 500 mL of water. Stir until dissolved and then add purified water to make up to 1 L.

[0073] (2) Use a measuring cylinder to measure nitric acid (16 M), add it to 500 mL of water, add 30% hydrogen peroxide, and finally add water to make up to 1 L.

[0074] Auramine O (maximum absorption wavelength 436nm), the structural formula is as follows:

[0075]

[0076] This embodiment provides an acridinium ester chemiluminescent substrate solution. The acridinium ester chemiluminescent substrate solution is prepared according to the formula in the table:

[0077] Table 2

[0078]

[0079] The configuration process is the same as that of Example 1.

[0080] The structural formula of Golden Orange G (maximum absorption wavelength 478nm) is as follows:

[0081]

[0082] Example 3

[0083] This embodiment provides an acridinium ester chemiluminescent substrate solution. The acridinium ester chemiluminescent substrate solution is prepared according to the formula in the table:

[0084] Table 3

[0085]

[0086]

[0087] The configuration process is the same as that of Example 1.

[0088] Thiazole orange (maximum absorption wavelength 475nm) has the following structural formula:

[0089]

[0090] Example 4

[0091] This embodiment provides an acridinium ester chemiluminescent substrate solution. The acridinium ester chemiluminescent substrate solution is prepared according to the formula in the table:

[0092] Table 4

[0093]

[0094] The configuration process is the same as that of Example 1.

[0095] Sunset Yellow (maximum absorption wavelength 482nm) has the following structural formula:

[0096]

[0097] Example 5

[0098] This embodiment provides an acridinium ester chemiluminescent substrate solution. The acridinium ester chemiluminescent substrate solution is prepared according to the formula in the table:

[0099] Table 5

[0100]

[0101] The configuration process is the same as that of Example 1.

[0102] Example 6

[0103] Compared with the formula of Example 1, the amount of auramine O in the chemiluminescent substrate solution of Example 6 is changed from 1 mg / L to 5 mg / L, and the preparation process is referred to Example 1.

[0104] Comparative Example 1

[0105] Compared with the formula of Example 1, the chemiluminescent substrate solution of Comparative Example 1 does not contain auramine O, and the preparation process is the same as that of Example 1.

[0106] Comparative Example 2

[0107] Compared with the formula of Example 2, the chemiluminescent substrate solution of Comparative Example 2 does not contain Golden Orange G, and the preparation process is the same as that of Example 2.

[0108] Comparative Example 3

[0109] Compared with the formulation of Example 3, the chemiluminescent substrate solution of Comparative Example 3 does not contain thiazole orange, and the preparation process is the same as that of Example 3.

[0110] Performance test 1: NSP-DMAE-NHS direct luminescence test

[0111] The DMSO solution of NSP-DMAE-NHS (10 g / L) was diluted with 50 mM PBS (pH 7.0) to 0.01 ng / L, 0.1 ng / L, 1 ng / L, 10 ng / L and 100 ng / L for direct luminescence test.

[0112] The test was completed in an iFlash3000 chemiluminescent immunoassay analyzer. 30 μL of NSP-DMAE-NHS solution of different concentrations was drawn into a reaction cup, and 100 μL of sodium hydroxide solution and 100 μL of hydrogen peroxide solution prepared in Examples 1-6 and Comparative Examples 1-3 were added respectively to measure the luminescence value. Three measurements were performed in parallel, and the average value was taken, as shown in Table 6.

[0113] Table 6

[0114] NSP-DMAE-NHS concentration (ng / mL) 0.01 0.1 1 10 100 Example 1 812 8989 84319 939810 9247621 Example 2 312 2818 34617 340036 3613571 Example 3 232 2509 29839 310769 3248440 Example 4 181 1814 16132 176895 1770137 Example 5 438 6188 72274 796033 7918812 Example 6 282 3223 37141 409072 4069390 Comparative Example 1 1564 12892 100380 1105601 10998350 Comparative Example 2 1269 7251 68114 679712 7171180 Comparative Example 3 861 5835 48916 501240 5239421 Example 1 / Comparative Example 1 0.52 0.69 0.84 0.85 0.84 Example 6 / Comparative Example 1 0.18 0.25 0.37 0.37 0.37 Example 2 / Comparative Example 2 0.25 0.39 0.51 0.50 0.50 Example 3 / Comparative Example 3 0.27 0.43 0.61 0.62 0.62

[0115] From the table above we can see that:

[0116] The luminescence values ​​of Examples 1, 2 and 3 to which a blue light absorber was added were lower than those of Comparative Examples 1, 2 and 3 to which a blue light absorber was not added, respectively. This is because the blue light emitted by the acridinium ester was absorbed by the blue light absorber, and less light was detected. For NSP-DMAE-NHS at three concentrations of 1 ng / mL, 10 ng / mL and 100 ng / mL, the magnitude of the reduction in the luminescence values ​​of the acridinium esters at these three concentrations in Examples 1, 2 and 3 was basically the same, but for the acridinium esters at two concentrations of 0.01 ng / mL and 0.1 ng / mL, which had very low luminescence values, the magnitude of the reduction in luminescence values ​​was greater, indicating that at these two concentrations, the luminescence values ​​of the acridinium esters were very low, and the proportion of light absorbed by the blue light absorber was greater.

[0117] Compared with Example 1, Example 6 has the same composition except that the amount of Golden Orange O is different in the substrate solution formula. The concentration of Golden Orange O in Example 6 is 5 mg / mL, while the concentration of Golden Orange O in Example 1 is 1 mg / mL. The luminescence value of Example 6 (acridinium ester concentrations of 1, 10 and 100 ng / mL) is only 0.37 times that of Comparative Example 1 without the addition of Golden Orange O, while that of Example 1 is 0.84 times. When the concentration of acridinium ester is very low (0.01 and 0.1 ng / mL), the luminescence value of Example 6 decreases more than that of Example 1 (0.25 times VS 0.69 times, 0.18 times VS 0.52 times). It can be seen that with the addition of blue light absorbers, the luminescence values ​​of acridinium esters of different concentrations all show a decrease in different proportions. The higher the amount of blue light absorber, the greater the amplitude of the decrease in luminescence value. For acridinium esters with higher concentrations, the amplitude of the decrease in luminescence value is basically the same for the same amount of blue light absorber. However, for acridinium esters with lower concentrations, with the same amount of blue light absorber, the lower the acridinium ester concentration, the greater the decrease in luminescence value; increasing the amount of blue light absorber can help further reduce the proportion of the decrease in luminescence value of low-concentration acridinium esters.

[0118] Performance Test 2: NSP-DMAE-HEG-Glu-NHS Direct Luminescence Test

[0119] NSP-DMAE-NHS was replaced with NSP-DMAE-HEG-Glu-NHS. Other test conditions were the same as those in performance test 1. The test results are shown in Table 7.

[0120] Table 7

[0121] NSP-DMAE-HEG-Glu-NHS concentration (ng / mL) 0.01 0.1 1 10 100 Example 1 717 8230 75632 856710 8474722 Example 2 324 2782 34311 341692 3644573 Example 3 298 3192 36997 414246 4230172 Example 4 244 2618 23752 273681 2710171 Example 5 408 5662 65038 716712 7126881 Example 6 244 2640 31819 338702 3462750 Comparative Example 1 1433 11477 90912 996183 9893571 Comparative Example 2 1299 7447 66882 683132 7180031 Comparative Example 3 1206 7776 65287 651348 6811252 Example 1 / Comparative Example 1 0.50 0.72 0.83 0.86 0.86 Example 6 / Comparative Example 1 0.17 0.23 0.35 0.34 0.35 Example 2 / Comparative Example 2 0.25 0.37 0.51 0.50 0.51 Example 3 / Comparative Example 3 0.25 0.41 0.57 0.64 0.62

[0122] The test results in Table 7 are similar to those in Table 6, indicating that when the acridinium ester is replaced by NSP-DMAE-NHS with NSP-DMAE-HEG-Glu-NHS, the blue light absorber plays a similar role.

[0123] Performance test 3. Project luminescence test

[0124] NSP-DMAE-NHS was labeled on the PIVKA-II antibody, and another paired antibody was coated on magnetic beads, and the test was performed using the double antibody sandwich methodology.

[0125] Prepare samples containing different concentrations of PIVKA-II, take 50 μL of each sample, add to 100 μL of magnetic beads coated with PIVKA-II antibody (concentration of 0.2 mg / mL), add 50 μL of acridine marker (200 ng / mL) of PIVKA-II antibody, and incubate at 37°C for 10 min. Then magnetically separate, wash 3 times, and finally add 100 μL of sodium hydroxide solution and 100 μL of hydrogen peroxide solution prepared in Example 1-6 and Comparative Example 1-3, respectively, and measure the luminescence value. Perform 3 measurements in parallel, take the average value, and the results are shown in Table 8.

[0126] Table 8

[0127]

[0128] As can be seen from Table 8, the luminescence values ​​of Examples 1, 2 and 3 to which the blue light absorber is added are lower than those of Comparative Examples 1, 2 and 3 to which the blue light absorber is not added. Since the luminescence value of samples with high luminescence values ​​decreases with the blue light absorber at a high concentration (the luminescence value is also high, equivalent to CAL4 and CAL3), the magnitude of the decrease in the luminescence value of samples with high luminescence values ​​is basically the same, so the CAL4 / CAL3 values ​​of Examples 1-6 to which the blue light absorber is added and the CAL4 / CAL3 values ​​of Comparative Examples 1-3 to which the blue light absorber is not added are both around 6, indicating that the blue light absorber cannot change the signal-to-noise ratio of the medium and high value samples.

[0129] For samples with low acridinium ester concentration (CAL1 and CAL2), the luminescence values ​​of Examples 1, 2 and 3 with the addition of blue light absorbers all decreased to varying degrees compared to Comparative Examples 1, 2 and 3 without the addition of blue light absorbers. Since the magnitude of the decrease in the luminescence value of CAL2 was greater than that of CAL3, and the magnitude of the decrease in the luminescence value of CAL1 was greater than that of CAL2, CAL3 / CAL2 and CAL2 / CAL1 of Examples 1, 2 and 3 increased to varying degrees compared to Comparative Examples 1, 2 and 3 without the addition of blue light absorbers. This indicates that the addition of blue light absorbers can improve the signal-to-noise ratio of low- and medium-concentration samples.

[0130] Using substrate solutions with different formulations, there are components that increase the luminescence value, as well as components that reduce the luminescence value. By optimizing the dosage of each component, a result with a higher signal-to-noise ratio can be obtained while increasing the luminescence value.

[0131] Performance Test 4: Project Luminescence Test

[0132] NSP-SA-NHS was labeled on the PIC antibody, and the other paired antibody was coated on the magnetic beads, and the double antibody sandwich methodology was used for testing.

[0133] Prepare samples containing different concentrations of PIC, take 20 μL of each sample, add to 50 μL of magnetic beads coated with PIC antibody (concentration of 0.2 mg / mL), add 50 μL of acridine marker of PIC antibody (400 ng / mL), and incubate at 37°C for 10 min. Then magnetic separation, wash 3 times, and finally add 100 μL of sodium hydroxide solution and 100 μL of hydrogen peroxide solution prepared in Example 1-6 and Comparative Example 1-3, and measure the luminescence value. Perform 3 measurements in parallel, take the average value, and the results are shown in Table 9.

[0134] Table 9

[0135]

[0136] The test results in Table 9 are similar to those in Table 8, indicating that in different test items, when the acridinium ester is replaced by NSP-DMAE-NHS into NSP-SA-NHS, the blue light absorber plays a similar role.

[0137] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0138] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description can be used to interpret the content of the claims.

Claims

1. A chemiluminescent substrate solution, characterized in that: The chemiluminescent substrate solution includes substrate solution 1, or includes substrate solution 1 and substrate solution 2; The substrate solution 1 comprises an inorganic base, a surfactant and a blue light absorber that absorbs blue light with a wavelength of 400nm-500nm; The substrate solution 2 includes inorganic acid and hydrogen peroxide.

2. The chemiluminescent substrate solution according to claim 1, characterized in that The substrate solution 1 includes 0.1 mg / L-100 mg / L of the blue light absorber; or / and, the blue light absorber includes one or more of auramine O, golden orange G, thiazole orange and sunset yellow; Optionally, the substrate solution 1 includes 1 mg / L-30 mg / L of the blue light absorber.

3. The chemiluminescent substrate solution according to claim 1, characterized in that The surfactant comprises one or more of TritonX-100, CTAC and Tween-20; or / and, the substrate solution 1 comprises 0.1wt%-3wt% of the surfactant.

4. The chemiluminescent substrate solution according to any one of claims 1 to 3, characterized in that The inorganic base includes one or more of sodium hydroxide and potassium hydroxide; or / and, the substrate solution 1 includes 0.2M-0.6M of the inorganic base.

5. The chemiluminescent substrate solution according to any one of claims 1 to 3, characterized in that The substrate solution 2 includes 0.02M-0.2M of the inorganic acid; or / and the inorganic acid includes one or more of nitric acid, hydrochloric acid and sulfuric acid.

6. The chemiluminescent substrate solution according to any one of claims 1 to 3, characterized in that: The substrate solution 2 includes 0.1wt%-1.5wt% of the hydrogen peroxide solution.

7. A chemiluminescent detection kit, characterized in that: The chemiluminescent detection kit comprises the chemiluminescent substrate solution according to any one of claims 1 to 6.

8. The chemiluminescent detection kit according to claim 7, characterized in that: The chemiluminescent detection kit further comprises one or more of an acridinium ester and an antibody; Optionally, the acridinium ester includes one or more of NSP-DMAE-NHS, NSP-SA-NHS and NSP-DMAE-HEG-Glu-NHS.

9. A method for detecting a target substance using chemiluminescence, characterized in that: In the process of detecting the target substance, the chemiluminescent substrate solution according to any one of claims 1 to 6 or the chemiluminescent detection kit according to any one of claims 7 to 8 is used.

10. The method for detecting a target substance according to claim 9, characterized in that: The volume ratio of the substrate solution 1 to the substrate solution 2 is 1:(0.5-2.5).

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