Chemiluminescent substrate solution, chemiluminescent detection kit, and detection method

Abstract

The application 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 chemiluminescence 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 absorber capable of absorbing blue light with a wavelength of 400-500 nm; and the substrate solution 2 comprises an inorganic acid and hydrogen peroxide. The blue light absorber is added in the chemiluminescence substrate solution, can absorb the blue light emitted by acridinium ester, reduce the light emission signal, in particular reduce the light emission value of the background caused by non-specific adsorption, and thus improve the signal-to-noise ratio.

Description

Technical Field

[0001] This application relates to the fields of immunoassay and immunodiagnostic technologies, and in particular to a chemiluminescent substrate solution, a chemiluminescent detection kit, and a detection method. Background Technology

[0002] Chemiluminescent immunoassay (CLIA) has seen rapid development in recent years due to its high sensitivity, wide linear detection range, fast analysis speed, and simple operation. Commonly used chemiluminescent substrates include acridinium esters, alkaline phosphatase, and luminol. Acridinium esters exhibit flash-type luminescence, characterized by rapid, concentrated, and high-intensity emission, resulting in fast detection and high sensitivity and precision, making it an excellent chemiluminescent substrate.

[0003] Acridine esters commonly used for antibody and antigen labeling include NSP-DMAE-NHS and NSP-SA-NHS. Acridine esters, or those labeled on antibodies or antigens, do not emit light themselves; they only emit light through a chemical reaction in the presence of a substrate solution. The substrate solution consists of two independent components: hydrogen peroxide containing an inorganic acid (mainly nitric acid) and an inorganic base (mainly sodium hydroxide). The mechanism of acridine ester luminescence involves the attack of hydrogen peroxide ions on the acridine ester in sodium hydroxide solution, generating an unstable cyclic intermediate. This cyclic intermediate decomposes into carbon dioxide and an excited-state N-methylacridone. When the excited-state N-methylacridone returns to its ground state, it emits blue light with a maximum emission wavelength of 430 nm.

[0004] Chemiluminescent substrate solutions often contain surfactants to enhance the luminescence signal. Commonly used surfactants include Triton X-100, CTAC (hexadecyltrimethylammonium chloride), and Tween-20. CN117769588A introduces benzyldimethyldodecylammonium chloride with an aromatic head group into CTAC, which enhances the signal even 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 improve the quantum yield of chemiluminescent compounds, thereby enhancing the signal.

[0005] Traditional chemiluminescent substrates use surfactants to enhance the signal, but this also amplifies the luminescence value caused by nonspecific adsorption background, often without significantly changing the signal-to-noise ratio. However, at excessively high surfactant concentrations, the background caused by nonspecific adsorption increases faster than the signal, leading to a decrease in signal-to-noise ratio and detection sensitivity. Summary of the Invention

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

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

[0008] The substrate solution 1 includes 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 this application, the substrate solution 1 comprises 0.1 mg / L to 100 mg / L of the blue light absorber. Optionally, the substrate solution 1 comprises 1 mg / L to 30 mg / L of the blue light absorber.

[0011] In some embodiments of this 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 this application, the surfactant includes one or more of Triton X-100, CTAC, and Tween-20.

[0013] In some embodiments of this application, the substrate liquid 1 comprises 0.1 wt% to 3 wt% of the surfactant.

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

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

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

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

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

[0019] In some embodiments of this application, the substrate solution 2 comprises 0.1 wt% to 1.5 wt% of the hydrogen peroxide.

[0020] One or more embodiments of this application provide a chemiluminescence detection kit, the chemiluminescence detection kit comprising the aforementioned chemiluminescence substrate solution.

[0021] In some embodiments of this application, the chemiluminescence detection kit further includes one or more of acridinium esters and antibodies.

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

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

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

[0025] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Detailed Implementation

[0026] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present 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 commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0028] the term

[0029] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0030] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of 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 connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: 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, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0031] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0032] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0033] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0034] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that achieve better results, and should be understood not to limit the scope of protection of this application.

[0035] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0036] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

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

[0038] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0039] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0040] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0041] In this 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 references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0043] A first aspect of the embodiments of this application provides a chemiluminescent substrate liquid, the chemiluminescent substrate liquid comprising substrate liquid 1, or comprising substrate liquid 1 and substrate liquid 2;

[0044] The substrate solution 1 includes 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 this embodiment, a blue light absorber is added to the chemiluminescent substrate solution, which can absorb the blue light emitted by acridine ester, reduce the luminescence signal, especially reduce the background luminescence value caused by non-specific adsorption, thereby improving the signal-to-noise ratio.

[0047] In this application, the chemiluminescent substrate liquid refers to its working state, i.e., its use state. In reality, it can be either ready-to-use or ready-to-prepare.

[0048] In some examples of this application, the substrate solution 1 comprises 0.1 mg / L to 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, or 100 mg / L. Optionally, the substrate solution 1 comprises 1 mg / L to 30 mg / L of the blue light absorber.

[0049] This application does not impose any particular limitation on the type of blue light absorber, as long as it can absorb blue light. In some examples of this application, the blue light absorber includes one or more of auramine O, golden orange G, thiazole orange, and sunset yellow.

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

[0051] This application does not impose a particular limitation on the amount of surfactant used, as long as it meets the detection requirements. In some examples of this 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] This application does not impose any particular limitation on the type of inorganic base, as long as it meets the detection requirements. The inorganic base can be sodium hydroxide or potassium hydroxide. In some examples of this 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] This application does not impose any particular limitation on the type and concentration of the inorganic acid in the substrate solution 2, as long as it meets the detection requirements. In some examples of this 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, or 0.2M. In some examples of this application, the inorganic acid includes one or more of nitric acid, hydrochloric acid, and sulfuric acid.

[0054] This application does not impose any particular limitation on the concentration of hydrogen peroxide in substrate solution 2, as long as it meets the detection requirements. In some examples of this application, the substrate solution 2 includes 0.1wt%-1.5wt% of the hydrogen peroxide, and the concentration of 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 this application, the substrate solution 1 comprises 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 comprises 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 this application, the substrate solution 2 comprises 0.06M-0.2M of the inorganic acid and 0.9wt%-1.5wt% of the hydrogen peroxide.

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

[0058] A second aspect of this application provides a chemiluminescence detection kit, the chemiluminescence detection kit comprising the aforementioned chemiluminescence substrate solution.

[0059] The embodiments of this application may also include other chemiluminescent detection reagents, including but not limited to acridine esters and antibodies. For example, they may include a first antibody and a second antibody targeting a specific antigen, and either the first antibody or the second antibody being labeled with an acridine ester.

[0060] This application does not specifically limit the types of acridine esters, including but not limited to NSP-DMAE-NHS, NSP-SA-NHS and NSP-DMAE-HEG-Glu-NHS.

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

[0062] This application does not specifically limit the target substance, which can be an antigen. When the target substance is an antigen, the method of this application can be a double-antibody sandwich method.

[0063] During the testing process, this application does not impose any particular limitation on the volume ratio of substrate liquid 1 to substrate liquid 2, as long as the testing requirements are met. In some examples of this application, the volume ratio of substrate liquid 1 to 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, 1:1.2, 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, and 1:2.5.

[0064] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0065] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0066] Example 1

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

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

[0069] Table 1

[0070]

[0071] Configuration process:

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

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

[0074] Auramine O (maximum absorption wavelength 436 nm) has the following structural formula:

[0075] Example 2

[0076] This embodiment provides an acridinium ester chemiluminescent substrate solution, which is prepared according to the formulation in the table:

[0077] Table 2

[0078]

[0079] The configuration process is the same as in 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, which is prepared according to the formulation in the table:

[0084] Table 3

[0085]

[0086]

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

[0088] The structural formula of thiazole orange (maximum absorption wavelength 475 nm) is as follows:

[0089]

[0090] Example 4

[0091] This embodiment provides an acridinium ester chemiluminescent substrate solution, which is prepared according to the formulation in the table:

[0092] Table 4

[0093]

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

[0095] The structural formula of Sunset Yellow (maximum absorption wavelength 482nm) is as follows:

[0096]

[0097] Example 5

[0098] This embodiment provides an acridinium ester chemiluminescent substrate solution, which is prepared according to the formulation in the table:

[0099] Table 5

[0100]

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

[0102] Example 6

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

[0104] Comparative Example 1

[0105] Compared with the formulation of Example 1, the chemiluminescent substrate solution of Comparative Example 1 did not contain auramine O, and the preparation process was the same as in Example 1.

[0106] Comparative Example 2

[0107] Compared with the formulation of Example 2, the chemiluminescent substrate solution of Comparative Example 2 did not contain Golden Orange G, and the preparation process was 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 did not contain thiazole orange, and the preparation process was the same as in Example 3.

[0110] Performance Test 1: NSP-DMAE-NHS Direct Light Emission Test

[0111] The DMSO solution (10 g / L) of NSP-DMAE-NHS 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 assay.

[0112] The tests were performed using an iFlash3000 chemiluminescence immunoassay analyzer. 30 μL of NSP-DMAE-NHS solutions of different concentrations were pipetted into reaction vessels, 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. The luminescence values ​​were then measured. Three parallel measurements were performed, and the average value was taken (see 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] As can be seen from the table above:

[0116] Examples 1, 2, and 3, in which blue light absorbers were added, all showed lower luminescence values ​​compared to Comparative Examples 1, 2, and 3, in which no blue light absorbers were added. This is because the blue light emitted by acridine ester was absorbed by the blue light absorber, resulting in less detected light. For the three concentrations of NSP-DMAE-NHS (1 ng / mL, 10 ng / mL, and 100 ng / mL), the decrease in luminescence value was basically the same for all three concentrations of acridine ester in Examples 1, 2, and 3. However, for the two concentrations of acridine ester with very low luminescence values ​​(0.01 ng / mL and 0.1 ng / mL), the decrease in luminescence value was greater, indicating that at these two concentrations, the luminescence value of acridine ester was very low, and a larger proportion of the light was absorbed by the blue light absorber.

[0117] Compared to Example 1, Example 6 had the same substrate formulation except for the amount of Orange O. The concentration of Orange O in Example 6 was 5 mg / mL, while in Example 1 it was 1 mg / mL. The luminescence values ​​of Example 6 (acridinium ester concentrations of 1, 10, and 100 ng / mL) were only 0.37 times that of Comparative Example 1 (without Orange O), while Example 1 was 0.84 times. At very low acridine ester concentrations (0.01 and 0.1 ng / mL), the luminescence values ​​of Example 6 decreased by a greater proportion than those of Example 1 (0.25 times vs. 0.69 times, 0.18 times vs. 0.52 times). This shows that with the addition of a blue light absorber, the luminescence values ​​of different concentrations of acridine ester decreased by varying proportions. The higher the amount of blue light absorber, the greater the decrease in luminescence value. For higher concentrations of acridine ester, the decrease in luminescence value was basically the same for the same amount of blue light absorber. However, for acridine esters with lower concentrations, the lower the concentration of acridine ester, the greater the decrease in luminescence value when the same amount of blue light absorber is used. Increasing the amount of blue light absorber can help to further reduce the proportion of luminescence value decrease in low-concentration acridine esters.

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

[0119] Replace NSP-DMAE-NHS with NSP-DMAE-HEG-Glu-NHS, and keep the other test conditions the same as in performance test one. 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 replacing acridine ester with NSP-DMAE-NHS results in the blue light absorber having a similar effect.

[0123] Performance Test 3: Project Luminescence Test

[0124] NSP-DMAE-NHS was labeled onto the PIVKA-II antibody, and another paired antibody was coated onto magnetic beads. The double-antibody sandwich method was used for testing.

[0125] Samples containing different concentrations of PIVKA-II were prepared. 50 μL of each sample was added to 100 μL of magnetic beads coated with PIVKA-II antibody (concentration 0.2 mg / mL), followed by 50 μL of acridine-labeled PIVKA-II antibody (200 ng / mL). The mixture was incubated at 37°C for 10 min. Then, magnetic separation was performed, followed by washing three times. Finally, 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, and the luminescence value was measured. Three parallel measurements were performed, and the average value was taken. The results are shown in Table 8.

[0126] Table 8

[0127]

[0128] As shown in Table 8, the luminescence values ​​of Examples 1, 2, and 3, which had blue light absorbers added, were all lower than those of Comparative Examples 1, 2, and 3, which did not have blue light absorbers added. Since the luminescence value is also high (equivalent to CAL4 and CAL3) when the acridinium ester concentration is high, the decrease in luminescence value for samples with high luminescence values ​​is basically the same for both. Therefore, the CAL4 / CAL3 values ​​of Examples 1-6 (with added blue light absorbers) and Comparative Examples 1-3 (without added blue light absorbers) are both around 6, indicating that the blue light absorber cannot change the signal-to-noise ratio of medium-to-high value samples.

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

[0130] Using substrate solutions with different formulations, there are components that increase luminescence value as well as components that decrease luminescence value. By optimizing and adjusting the amount of each component, it is possible to obtain results with a high signal-to-noise ratio while improving luminescence value.

[0131] Performance Test 4: Project Luminescence Test

[0132] NSP-SA-NHS was labeled onto a PIC antibody, and another paired antibody was coated onto a magnetic bead. The test was performed using a double-antibody sandwich methodology.

[0133] Samples containing different concentrations of PIC were prepared. 20 μL of each sample was added to 50 μL of magnetic beads coated with PIC antibody (concentration 0.2 mg / mL), followed by 50 μL of acridine-labeled PIC antibody (400 ng / mL). The mixture was incubated at 37°C for 10 min. Then, magnetic separation was performed, followed by washing three times. Finally, 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, and the luminescence value was measured. Three parallel measurements were performed, and the average value was taken. 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 when acridine ester is replaced with NSP-SA-NHS for different test items, the blue light absorber plays a similar role.

[0137] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments 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 embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

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

3. The chemiluminescent substrate solution for acridine esters according to claim 2, characterized in that, The substrate solution 1 comprises 1 mg / L to 30 mg / L of the blue light absorber.

4. The chemiluminescent substrate solution for acridine esters according to claim 1, characterized in that, The surfactant includes one or more of Triton X-100, CTAC, and Tween-20; and / or the substrate liquid 1 includes 0.1 wt% to 3 wt% of the surfactant.

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

6. The chemiluminescent substrate solution for acridine esters according to any one of claims 1 to 4, characterized in that, The substrate solution 2 comprises 0.02M-0.2M of the inorganic acid; and / or the inorganic acid comprises one or more of nitric acid, hydrochloric acid and sulfuric acid.

7. The chemiluminescent substrate solution for acridine esters according to any one of claims 1 to 4, characterized in that, The substrate solution 2 comprises 0.1wt%-1.5wt% of the hydrogen peroxide.

8. A chemiluminescence detection kit, characterized in that, The chemiluminescence detection kit includes the chemiluminescent substrate solution for acridine esters as described in any one of claims 1 to 7.

9. The chemiluminescence detection kit according to claim 8, characterized in that, The chemiluminescence detection kit also includes one or more of acridinium esters and antibodies.

10. The chemiluminescence detection kit according to claim 9, characterized in that, The acridine ester includes one or more of NSP-DMAE-NHS, NSP-SA-NHS, and NSP-DMAE-HEG-Glu-NHS.

11. A method for detecting a target substance using chemiluminescence immunoassay, characterized in that, The chemiluminescent substrate solution for acridine esters as described in any one of claims 1 to 7 or the chemiluminescent detection kit as described in any one of claims 8 to 10 is used in the detection of the target substance.

12. The method for detecting a target substance using chemiluminescence immunoassay according to claim 11, characterized in that, The volume ratio of substrate liquid 1 to substrate liquid 2 is 1:(0.5-2.5).

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