Marker for improving chemiluminescence signal as well as preparation method and application thereof

Abstract

The invention discloses a marker for improving a chemiluminescence signal as well as a preparation method and application of the marker. The marker comprises horse radish peroxidase and a phenothiazine group coupled to the horse radish peroxidase. The marker can significantly enhance the signal intensity of a horse radish peroxidase catalytic chemiluminescence substrate and effectively improve the chemiluminescence efficiency, and the kit prepared based on the marker can significantly improve the detection sensitivity when detecting a target substance.

Description

Technical Field

[0001] The present application relates to the field of in vitro detection technology, and specifically to a marker for enhancing chemiluminescence signals, a preparation method thereof, and an application thereof. Background Art

[0002] In the field of in vitro diagnostic reagents, HRP (horseradish peroxidase) is the most commonly used tracer label and has the widest application. Two of the most important applications are in the preparation of enzyme-linked immunosorbent assay kits and chemiluminescence kits. HRP catalyzes the reaction between luminol and peroxide, producing strong chemiluminescence (emission wavelength 420-430 nm). While many factors influence kit performance, the chemiluminescence catalytic activity of the HRP enzyme clearly has a direct impact on kit sensitivity. Improving the chemiluminescence catalytic activity of HRP has always been a key issue in the field of in vitro diagnostic reagents. Because HRP is extracted from the plant horseradish, the potential for signal enhancement during production is very limited. Before HRP is labeled with antibodies or other proteins, if the chemiluminescence catalytic activity of HRP can be enhanced through structural modification, the signal level of the chemiluminescence kit system can be significantly improved. In the field of chemiluminescent substrates, phenothiazines are often used as "enhancers," significantly boosting the signal level of chemiluminescent substrates. With the help of the enhancement effect of phenothiazine compounds, the detection sensitivity of HRP enzyme-labeled chemiluminescence reagents has been significantly improved, but there is still a certain gap compared with the currently widely used electrochemiluminescence and acridinium ester direct luminescence kits.

[0003] Therefore, how to improve the chemiluminescence catalytic ability of HRP enzyme and enhance the chemiluminescence signal is the difficulty in improving the sensitivity of chemiluminescence detection. Summary of the Invention

[0004] Based on this, it is necessary to provide a marker for enhancing chemiluminescence signal and its preparation method and application.

[0005] A first aspect of the present application provides a label for enhancing a chemiluminescent signal, wherein the label comprises horseradish peroxidase and a phenothiazine group coupled to the horseradish peroxidase.

[0006] In some embodiments, the phenothiazine group is coupled to the amino position of a lysine residue in the horseradish peroxidase via an amide bond.

[0007] The second aspect of the present application provides a method for preparing the marker described in the first aspect of the present application, comprising the step of coupling the phenothiazine group to the horseradish peroxidase to prepare the marker.

[0008] In some embodiments, the method for preparing the marker comprises the following steps:

[0009] The raw material containing the phenothiazine group is mixed with the horseradish peroxidase to carry out coupling reaction, and the obtained coupling product is quenched and dialyzed to prepare the label.

[0010] In some embodiments, the raw material containing a phenothiazine group includes one or more of phenothiazine-10-propionic acid and phenothiazine-10-carbonyl chloride.

[0011] In some embodiments, the raw material containing phenothiazine groups is dissolved in dimethyl sulfoxide at a concentration of 1 w / v% to 2 w / v%.

[0012] In some embodiments, the horseradish peroxidase is dissolved in a phosphate buffer at a concentration of 10 mg / mL to 15 mg / mL; optionally, the concentration of the phosphate buffer is 0.1 M to 0.2 M, and the pH of the phosphate buffer is 7.4 to 7.6.

[0013] In some embodiments, the coupling reaction time is 4 h to 6 h.

[0014] In some embodiments, the coupling reaction temperature is 35°C to 40°C.

[0015] In some embodiments, the quenching is performed by adding an aqueous solution of glycine.

[0016] In some embodiments, the concentration of glycine in the glycine aqueous solution is 1M~2M.

[0017] In some embodiments, the amount of the glycine aqueous solution added is 0.1 mL to 0.3 mL.

[0018] In some embodiments, before mixing the raw material containing phenothiazine groups with the horseradish peroxidase, the process further comprises: mixing the raw material containing phenothiazine groups with N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide for activation.

[0019] The third aspect of the present application provides a chemiluminescent detection kit, which includes the marker described in the first aspect of the present application.

[0020] In some embodiments, the chemiluminescent detection kit further comprises a luminescent substrate.

[0021] In some embodiments, the luminescent substrate comprises luminol.

[0022] The fourth aspect of the present application provides a labeled antibody, which includes a detection antibody and the marker described in the first aspect of the present application marked on the detection antibody.

[0023] The fifth aspect of the present application provides an immunoassay kit, which includes the labeled antibody described in the fourth aspect of the present application.

[0024] In some embodiments, the immunoassay kit further comprises a luminescent substrate.

[0025] In some embodiments, the luminescent substrate comprises luminol.

[0026] The marker provided above can significantly enhance the signal intensity of the horseradish peroxidase-catalyzed chemiluminescent substrate and effectively improve the chemiluminescence efficiency. The kit prepared based on the marker can significantly improve the detection sensitivity when detecting the target substance. DETAILED DESCRIPTION

[0027] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference thereto. Preferred embodiments of the present application are provided. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, the purpose of providing these embodiments is to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0029] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0030] In this application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this application.

[0031] As used herein, the terms "having," "containing," "including," and "comprising" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0032] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0033] In this application, when referring to the unit of a data range, if the unit is only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0034] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0035] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0036] In this application, the terms "first," "second," and "third," etc., in "the first aspect," "the second aspect," "the third aspect," etc., are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," and "third," etc., are only used for non-exhaustive enumeration and description purposes and should be understood not to constitute closed-ended limitations on quantity.

[0037] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​within the numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. "Numerical interval" allows for a broad range of numerical interval types including percentage intervals, ratio intervals, and ratio intervals.

[0038] In the field of chemiluminescent substrates, phenothiazines are frequently used as "enhancers," significantly boosting the signal level of chemiluminescent substrates. While this enhancement effect significantly improves the sensitivity of HRP-labeled chemiluminescent reagents, it still lags behind widely used kits such as electrochemiluminescence and acridinium ester direct luminescence.

[0039] Based on this, the embodiments of the present application at least provide a marker for enhancing chemiluminescence signals, and a preparation method and application thereof.

[0040] In a first aspect of the present application, a label for enhancing chemiluminescence signal is provided. The label comprises horseradish peroxidase and a phenothiazine group coupled to the horseradish peroxidase.

[0041] In the present application, the phenothiazine group refers to the remaining portion after hydrogen atoms or atomic groups in a phenothiazine or phenothiazine derivative are replaced by other atoms or atomic groups.

[0042] In some embodiments, the phenothiazine group is coupled to the amino position of a lysine residue in horseradish peroxidase via an amide bond.

[0043] The present application discovered that although adding a phenothiazine enhancer to a chemiluminescent substrate sensitive to horseradish peroxidase can enhance the luminescence signal, combining horseradish peroxidase and the phenothiazine group into one molecule allows the phenothiazine to be accurately positioned in the environment where horseradish peroxidase catalyzes the reaction between luminol and peroxide, exerting a more significant signal enhancement effect and successfully increasing the luminescence signal by 5-8 times.

[0044] The marker provided above can significantly enhance the signal intensity of the chemiluminescent substrate catalyzed by horseradish peroxidase, effectively improving the chemiluminescence efficiency.

[0045] In a second aspect of the present application, a method for preparing the above-mentioned marker is provided, comprising the step of coupling a phenothiazine group to horseradish peroxidase to prepare the marker; further, the preparation method comprises the following steps:

[0046] A raw material containing a phenothiazine group is mixed with horseradish peroxidase to carry out a coupling reaction, and the obtained coupling product is quenched and dialyzed to prepare a label.

[0047] In some embodiments, the raw material containing a phenothiazine group includes one or more of phenothiazine-10-propionic acid and phenothiazine-10-carbonyl chloride. It should be noted that other derivatives containing a phenothiazine group may also be included, such as, but not limited to, nitro or halogen substituents on the phenothiazine ring.

[0048] In some embodiments, the raw material containing phenothiazine groups is dissolved in dimethyl sulfoxide at a concentration of 1 w / v% to 2 w / v%. Without limitation, the concentration of the raw material solution containing phenothiazine groups can be, but is not limited to, 1 w / v%, 1.2 w / v%, 1.4 w / v%, 1.6 w / v%, 1.8 w / v%, 2 w / v%, or a value or range between any two of the foregoing values.

[0049] In some embodiments, horseradish peroxidase is dissolved in phosphate buffer at a concentration of 10 mg / mL to 15 mg / mL. In a non-limiting manner, the concentration of the horseradish peroxidase solution can be, but is not limited to, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, or a value or range between any two of the above values.

[0050] In some embodiments, the coupling reaction time is 4 h to 6 h. In a non-limiting manner, the coupling reaction time can be, but is not limited to, 4 h, 5 h, 6 h, or a value or range between any two of the above values.

[0051] In some embodiments, the coupling reaction temperature is 35° C. to 40° C. Without limitation, the coupling reaction temperature can be, but is not limited to, 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., or a value or range between any two of the above values.

[0052] In some embodiments, the resulting coupled product is quenched by the addition of aqueous glycine solution.

[0053] In some embodiments, the concentration of glycine in the glycine aqueous solution is 1 M to 2 M. Without limitation, the concentration of glycine in the glycine aqueous solution can be, but is not limited to, 1 M, 1.2 M, 1.4 M, 1.6 M, 1.8 M, 2 M, or a value or range between any two of the above values.

[0054] In some embodiments, the amount of the glycine aqueous solution added is 0.1 mL to 0.3 mL. In a non-limiting manner, the amount of the glycine aqueous solution added can be, but is not limited to, 0.1 mL, 0.2 mL, 0.3 mL, or a value or range between any two of the above values.

[0055] In some embodiments, before mixing the raw material containing a phenothiazine group with horseradish peroxidase, the process further includes: mixing the raw material containing a phenothiazine group with N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide for activation. It should be noted that for carboxylic acid-based raw materials containing phenothiazine groups, such as but not limited to phenothiazine-10-propionic acid, activation is required before mixing with horseradish peroxidase to achieve coupling with the horseradish peroxidase; for acyl chloride-based raw materials containing phenothiazine groups, such as but not limited to phenothiazine-10-carbonyl chloride, direct coupling is possible without activation.

[0056] In some embodiments, the mass ratio of the raw material containing phenothiazine groups to N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide is (2-3):(1-2):(2-3). Without limitation, the mass ratio of the raw material containing phenothiazine groups to N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide can be, but is not limited to, 2:1:2, 3:1:2, 2:2:3, 3:2:2, 3:2:3, or a ratio or range between any two of the above ratios.

[0057] The third aspect of the present application provides a chemiluminescent detection kit, which includes the above-mentioned marker.

[0058] In some embodiments, the chemiluminescent detection kit further comprises a luminescent substrate. Exemplarily, the luminescent substrate comprises luminol luminescent solution A and luminol luminescent solution B; luminol luminescent solution A comprises luminol, Tris buffer, and a certain amount of enhancer (phenothiazine); and luminol luminescent solution B comprises a peroxide component. Furthermore, the mixture ratio of luminol luminescent solution A to luminol luminescent solution B is 1:1.

[0059] The fourth aspect of the present application provides a labeled antibody, which includes a detection antibody and the above-mentioned marker labeled on the detection antibody.

[0060] The fifth aspect of the present application provides an immunoassay kit, which includes the above-mentioned labeled antibody.

[0061] In some embodiments, the immunoassay kit further includes a luminescent substrate. The luminescent substrate includes luminol luminescent solution A and luminol luminescent solution B. Luminol luminescent solution A includes luminol, Tris buffer, and a certain amount of enhancer (phenothiazine); luminol luminescent solution B includes a peroxide component. Furthermore, the mixture ratio of luminol luminescent solution A to luminol luminescent solution B is 1:1.

[0062] In some embodiments, the immunoassay kit is used to perform a double antibody sandwich method to detect the target antigen. The immunoassay kit also includes at least one of avidin-coated magnetic beads, a biotinylated capture antibody, and a buffer. Both the detection antibody and the biotinylated capture antibody can specifically bind to the target antigen, and the binding sites are different.

[0063] The sixth aspect of the present application provides a method for enhancing the luminescent signal of luminol chemiluminescence catalyzed by HRP enzyme, comprising: adding the above-provided marker to the chemiluminescent substrate system.

[0064] In some embodiments, the concentration of the marker is 8 ng / mL to 12 ng / mL. Without limitation, the concentration of the marker can be, but is not limited to, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, or a value or range between any two of the foregoing values.

[0065] Some examples are provided below.

[0066] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0067] In the following examples, 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 testing accuracy or operating accuracy are allowed.

[0068] In the following examples, all reagents used were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Phenothiazine-10-propionic acid and phenothiazine-10-carbonyl chloride were purchased from Beijing Bailingwei Technology Co., Ltd.

[0069] Example 1

[0070] 1. Synthesis of Phenothiazine-10-Propionic Acid NHS Ester

[0071] To 2.7 g (10 mmol) of phenothiazine-10-propionic acid, add 1.2 g (10.4 mmol) of N-hydroxysuccinimide (NHS) and 2.2 g (10.7 mmol) of N,N'-dicyclohexylcarbodiimide (DCC). Dissolve the product in 200 ml of anhydrous DMSO (dimethyl sulfoxide). Incubate at 50°C for 5 hours. Clearly crystalline insoluble material will form. The resulting solution is a 50 µmol / mL solution of phenothiazine-10-propionic acid NHS ester. This solution can be directly used in the next reaction or frozen for later use.

[0072] 2. Modification of HRP enzyme with phenothiazine-10-propionic acid NHS ester

[0073] Weigh 0.1 g of HRP enzyme and dissolve it in 10 ml of 0.1 M PB buffer (pH 7.5). Add 0.2 ml of the phenothiazine-10-propionic acid NHS ester solution prepared in step 1 to the solution. After shaking to mix, react in a 37°C water bath for 5 hours. After the reaction, quench the solution by adding 0.2 ml of 1 M aqueous glycine. Subsequently, dialyze the resulting solution against 0.1 M PB (pH 7.5) to remove unbound phenothiazines.

[0074] Example 2 Modification of HRP enzyme with phenothiazine-10-carbonyl chloride

[0075] Weigh 0.1 g of HRP enzyme and dissolve it in 10 ml of 0.1 M PB buffer (pH 7.5). Add 0.1 ml of a 2% phenothiazine-10-carbonyl chloride solution in anhydrous DMSO to the solution. After shaking, react in a 37°C water bath for 5 hours. After the reaction, quench the solution with 0.2 ml of 1 M glycine solution. The resulting solution is then dialyzed against 0.1 M PB (pH 7.5) to remove unbound phenothiazine compounds.

[0076] Measurement results

[0077] 1. The chemiluminescence level of luminol catalyzed by the HRP enzyme prepared in Examples 1 and 2 was measured. The specific measurement method is described in the patent document (see the measurement protocol in Example 1 of Patent CN105277537A). The measurement results are shown in Table 1. This shows that by coupling phenothiazine with HRP enzyme, the phenothiazine can be precisely positioned within the environment where the HRP enzyme catalyzes the reaction between luminol and peroxide, resulting in a more pronounced signal enhancement effect.

[0078] Table 1

[0079]

[0080] 2. The HRP enzymes prepared in Examples 1 and 2 were used in the preparation of a plate-based chemiluminescent kit (a chemiluminescent assay kit for alpha-fetoprotein (AFP)) using the classic sodium periodate labeling method. The sodium periodate labeling method and the preparation of the plate-based chemiluminescent kit were performed using conventional methods in the art. The resulting kits were used to assay high-value quality control samples and blank samples. The results are shown in Table 2. As can be seen, the chemiluminescent signal level of the chemiluminescent kit prepared using the phenothiazine-conjugated HRP enzyme was significantly increased. This demonstrates that phenothiazine-conjugated HRP enzymes can be successfully used in the preparation of chemiluminescent kits. The HRP enzymes prepared in Examples 1 and 2 can significantly improve the signal level and sensitivity of the kit.

[0081] Table 2

[0082]

[0083] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments 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.

[0084] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A marker for enhancing chemiluminescent signals, characterized in that: The label comprises horseradish peroxidase and a phenothiazine group coupled to the horseradish peroxidase.

2. The marker according to claim 1, wherein The phenothiazine group is coupled to the amino position of the lysine residue in the horseradish peroxidase via an amide bond.

3. A method for preparing a marker according to claim 1 or 2, characterized in that: The method comprises the steps of coupling the phenothiazine group to the horseradish peroxidase to prepare the label; Optionally, the method for preparing the marker comprises the following steps: The raw material containing the phenothiazine group is mixed with the horseradish peroxidase to carry out coupling reaction, and the obtained coupling product is quenched and dialyzed to prepare the label.

4. The method for preparing a marker according to claim 3, wherein: The raw material containing a phenothiazine group includes one or more of phenothiazine-10-propionic acid and phenothiazine-10-carbonyl chloride.

5. The method for preparing a marker according to claim 4, wherein: Meet one or more of the following conditions: The raw material containing phenothiazine groups is dissolved in dimethyl sulfoxide at a concentration of 1 w / v% to 2 w / v%; The horseradish peroxidase is dissolved in a phosphate buffer at a concentration of 10 mg / mL to 15 mg / mL; optionally, the concentration of the phosphate buffer is 0.1 M to 0.2 M, and the pH of the phosphate buffer is 7.4 to 7.6; The coupling reaction time is 4h~6h; The temperature of the coupling reaction is 35°C to 40°C.

6. The method for preparing a marker according to claim 5, wherein: The quenching is performed by adding an aqueous solution of glycine; Optionally, the concentration of glycine in the glycine aqueous solution is 1M~2M; Optionally, the amount of the glycine aqueous solution added is 0.1 mL to 0.3 mL.

7. The method for preparing a marker according to any one of claims 3 to 6, wherein: Before mixing the raw material containing phenothiazine groups with the horseradish peroxidase, the method further comprises: mixing the raw material containing phenothiazine groups with N-hydroxysuccinimide and N,N'-dicyclohexylcarbodiimide for activation.

8. A chemiluminescence detection kit, characterized in that: The chemiluminescent detection kit comprises the marker according to claim 1 or 2; Optionally, the chemiluminescent detection kit further comprises a luminescent substrate; Further optionally, the luminescent substrate comprises luminol.

9. A labeled antibody, characterized in that The labeled antibody comprises a detection antibody and the label according to claim 1 or 2 labeled on the detection antibody.

10. An immunoassay kit, characterized in that: The immunoassay kit comprises the labeled antibody according to claim 9; Optionally, the immunoassay kit further comprises a luminescent substrate; Further optionally, the luminescent substrate comprises luminol.

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