A single-component luminol chemiluminescent substrate solution and a preparation method thereof

Abstract

The application belongs to the field of detection reagents, and particularly relates to a single-component luminol chemiluminescence substrate liquid and a preparation method thereof. The single-component luminol chemiluminescence substrate liquid comprises the following components: sodium acetate, sodium perborate, N-N dimethylacetamide, beta-cyclodextrin, luminol, 4-morpholinopyridine, p-acetylaminophenol, 4-hydroxyphenylacetic acid, phenothiazine-10-yl-propyl sulfonic acid sodium salt and isothiazolinone. The purpose of the application is to solve the technical defects that the luminol chemiluminescence substrate liquid in the prior art must be packaged in two components due to the instability of the luminous agent and the oxidizing agent in the liquid phase, leading to inconvenient use and easy introduction of operation errors, so as to provide a single-component luminol chemiluminescence substrate liquid and a preparation method thereof, which have high storage stability, high luminous performance and convenient use.

Description

Technical Field

[0001] This invention belongs to the field of detection reagents, specifically relating to a single-component luminol chemiluminescent substrate solution and its preparation method. Background Technology

[0002] Magnetic microparticle chemiluminescence technology has been widely used in in vitro diagnostics and animal disease detection due to its advantages such as high sensitivity, wide linear range, and good stability. In luminol-based chemiluminescence systems, the substrate solution is the key reagent providing the signal, and its performance directly affects the sensitivity and stability of the detection.

[0003] Traditional luminol chemiluminescent substrate solutions are typically in a two-component form, with the luminescent agent (such as luminol) and the oxidant (such as hydrogen peroxide or its stable precursor) placed in separate solutions A and B. This is because the luminescent agent and oxidant are chemically unstable in a single liquid phase environment, and a slow reaction occurs after mixing, leading to attenuation of the luminescence signal and an increase in background value during storage, failing to meet the requirements for long-term storage. Therefore, users must mix solutions A and B in a specific ratio before detection. This operation not only adds steps and causes inconvenience but is also prone to introducing errors due to inaccurate or uneven mixing ratios, affecting the accuracy and repeatability of the detection results.

[0004] To address the inconvenience of using two-component reagents, some technical solutions attempt to achieve single-sample addition through physical isolation. For example, solid luminescent agents and solid oxidants (such as sodium perborate) are immobilized in different areas of an immunochromatographic strip, and then dissolved and mixed as the sample solution flows through. However, this solid-support solution cannot meet the needs of large-scale automated, high-throughput magnetic particle chemiluminescence analysis platforms, which typically require stable, ready-to-use liquid reagents.

[0005] Therefore, developing a single-component liquid substrate that can stably coexist with luminescent agents and oxidants in a single liquid phase and has long-term storage stability remains a technical challenge that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to address the technical shortcomings of existing luminol chemiluminescent substrate solutions, which require two-component packaging due to the instability of the luminescent agent and oxidant in the liquid phase, leading to inconvenience and easy introduction of operational errors. This invention provides a single-component luminol chemiluminescent substrate solution and its preparation method that combine high storage stability, high luminescent performance, and ease of use.

[0007] To address the problems existing in the prior art, the present invention provides a single-component luminol chemiluminescent substrate solution, comprising the following components: sodium acetate, sodium perborate, N,N dimethylacetamide, β-cyclodextrin, luminol, 4-morpholinopyridine, acetaminophen, 4-hydroxyphenylacetic acid, sodium phenthiazide-10-yl-propylsulfonate, and isothiazolinone.

[0008] To facilitate understanding of this invention, the principles of this invention will be explained as follows:

[0009] Ten components work together to form a synergistic and stable system. Sodium acetate provides a stable, weakly alkaline buffer environment, which is crucial for maintaining the chemical stability of luminol and the efficiency of the subsequent chemiluminescence reaction. Sodium perborate, as a solid peroxide precursor, slowly releases hydrogen peroxide in aqueous solution. Compared to using hydrogen peroxide directly, it exhibits better stability and is the preferred oxidant in this system. Luminol is the core substrate for the chemiluminescence reaction.

[0010] The composite stabilizer consists of four components: N,N-dimethylacetamide, β-cyclodextrin, acetaminophen, and isothiazolinone. These four components work synergistically through different mechanisms to effectively inhibit the premature non-specific reaction between luminol (as a luminescent agent) and sodium perborate (as an oxidant) during long-term storage. Specifically, N,N-dimethylacetamide, as an aprotic polar solvent, can alter the microscopic solvent environment of the solution by adding an appropriate amount, reducing the activity of water molecules and thus slowing down the hydrolysis and mutual reaction rates of luminol and sodium perborate. β-cyclodextrin has a unique cyclic structure with a hydrophobic inner cavity and a hydrophilic outer wall. Through host-guest interactions, it can encapsulate luminol molecules or their easily oxidized portions within its inner cavity, forming inclusion complexes. This physically shields luminol from direct contact with the oxidant, greatly improving its chemical stability. Acetaminophen, as an effective free radical scavenger, can capture free radicals in the system that may be generated by trace reactions or external factors (such as light and temperature fluctuations), blocking reaction pathways that may trigger the chain degradation of luminol. Isothiazolinone, in addition to acting as a broad-spectrum preservative to prevent microbial growth and system deterioration, also contributes to the stability of the chemical system. This combination of four stabilizers constructs a comprehensive protection system from multiple dimensions, including solvent environment, physical isolation, and chemical quenching, which is key to achieving long-term stable coexistence of single-component liquids.

[0011] Furthermore, the composite catalyst consists of three components: 4-morpholinopyridine, 4-hydroxyphenylacetic acid, and sodium phenothiazine-10-yl-propylsulfonate. In practical detection applications, when the substrate solution comes into contact with a labeled substance containing horseradish peroxidase (HRP), these three catalysts work synergistically to significantly improve the quantum yield and luminescence kinetics of the luminol luminescence reaction. 4-Hydroxyphenylacetic acid is a horseradish peroxidase luminescence catalyst; it can be oxidized by the horseradish peroxidase-hydrogen peroxide complex to form phenoxy radicals, which then act as electron transfer mediators, efficiently oxidizing luminol to induce luminescence. 4-morpholinopyridine and sodium phenothiazine-10-yl-propylsulfonate act as even more efficient electron transfer catalysts, further accelerating the electron transfer process between horseradish peroxidase, the catalyst, and luminol. This not only significantly enhances the peak intensity of the luminescence signal but, more importantly, stabilizes the luminescence process, forming a sustained signal plateau. The design of this composite catalyst ensures that the substrate liquid can "instantly start" when a reaction is needed, generating a strong and persistent signal, thereby guaranteeing high detection sensitivity and a wide linear range.

[0012] Preferably, the single-component luminol chemiluminescent substrate solution comprises the following components at the following concentrations: sodium acetate 10-20 g / L, sodium perborate 5-10 g / L, NN dimethylacetamide 2-6 vol%, β-cyclodextrin 4-8 g / L, luminol 0.1-0.5 g / L, 4-morpholinopyridine 0.05-0.2 g / L, acetaminophen 0.1-0.5 g / L, 4-hydroxyphenylacetic acid 0.1-0.5 g / L, sodium phenothiazine-10-yl-propylsulfonate 0.05-0.7 g / L, and isothiazolinone 0.1-0.4 g / L.

[0013] Preferably, the single-component luminol chemiluminescent substrate solution comprises the following components at the following concentrations: sodium acetate 15 g / L, sodium perborate 6 g / L, NN dimethylacetamide 5 vol%, β-cyclodextrin 5 g / L, luminol 0.4 g / L, 4-morpholinopyridine 0.06 g / L, acetaminophen 0.2 g / L, 4-hydroxyphenylacetic acid 0.2 g / L, phenothiazine-10-yl-propylsulfonate sodium salt 0.3 g / L, and isothiazolinone 0.2 g / L.

[0014] Preferably, the pH of the single-component luminol chemiluminescent substrate solution is 8-9.

[0015] Based on the same technical concept, the present invention further provides a method for preparing the single-component luminol chemiluminescent substrate solution, characterized in that the preparation method includes the following steps:

[0016] (I) Sodium acetate, N,N dimethylacetamide, β-cyclodextrin, acetaminophen, sodium perborate, luminol, 4-morpholinopyridine, sodium phenthiazide-10-yl-propylsulfonate, 4-hydroxyphenylacetic acid and isothiazolinone were added to water in sequence to obtain a mixed solution;

[0017] (II) The mixed solution is brought to a target volume to obtain the single-component luminol chemiluminescent substrate solution.

[0018] Preferably, step (I) includes the following specific operations:

[0019] (i) Dissolve sodium acetate in water and stir to form a buffer solution;

[0020] (ii) Then, N,N dimethylacetamide, β-cyclodextrin and p-acetaminophen are added sequentially to the buffer solution and stirred until dissolved;

[0021] (iii) Continue adding sodium perborate and luminol while stirring to dissolve;

[0022] (iv) Finally, add 4-morpholinopyridine, sodium phenthiazide-10-yl-propylsulfonate, 4-hydroxyphenylacetic acid and isothiazolinone and stir to dissolve to obtain a mixed solution.

[0023] Preferably, the stirring speed is 80-100 r / min and the stirring time is 3-5 min.

[0024] Preferably, in step (II), after volume adjustment, the pH is adjusted to 8-9.

[0025] The beneficial effects of this invention are as follows:

[0026] 1. The single-component luminol chemiluminescent substrate liquid of the present invention has extremely high storage stability. Through a specific composite stabilizer and composite enhancer formulation, the long-term stable coexistence of the luminescent agent and oxidant in a single liquid is successfully achieved, overcoming the technical problem of instability of the two in the liquid phase in the prior art. Real-time stability testing shows that it can last for up to 5 years.

[0027] 2. The single-component luminol chemiluminescent substrate solution described in this invention is a ready-to-use single-component reagent, which is convenient to use and does not require users to perform mixing operations before use. This simplifies the experimental process, avoids errors introduced by improper manual mixing, and improves the reliability of the detection results.

[0028] 3. The single-component luminol chemiluminescent substrate solution described in this invention exhibits excellent luminescence performance. Benefiting from the synergistic effect of the composite enhancer, it rapidly reaches the luminescence plateau within 2 minutes after reaction triggering, and its catalytic efficiency remains stable without decline. Overall, it features high luminescence intensity and a long luminescence period. After reaching the luminescence peak, it continues to emit light for 60 minutes without decreasing luminescence intensity. The luminescence intensity remains unchanged after 5 years of storage at 2–8°C, ensuring high detection sensitivity and a wide linear range.

[0029] 4. The formulation components of this application are readily available, the preparation process is simple, and it has good cost-effectiveness. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0031] Example 1

[0032] This embodiment provides a composition and concentration formulation for a single-component luminol chemiluminescent substrate solution (the substrate solution is a clear, homogeneous, ready-to-use liquid system) (taking the preparation of 1000 mL of substrate solution as an example), specifically:

[0033] Sodium acetate: 15g, as a buffer. Sodium perborate: 6g, as an oxidizing agent. Luminol (3-aminophenyl hydrazide): 0.4g, as a luminescent agent. N,N-dimethylacetamide: 50mL, as a component of the composite stabilizer. β-Cyclodextrin: 5g, as a component of the composite stabilizer. Acetaminophen: 0.2g, as a component of the composite stabilizer. Isothiazolinone: 0.2g, as a component of the composite stabilizer and also has antiseptic properties. 4-Morphyrin: 0.06g, as a component of the composite catalyst. 4-Hydroxyphenylacetic acid: 0.2g, as a component of the composite catalyst. Phenothiazine-10-yl-propylsulfonate sodium salt: 0.3g, as a component of the composite catalyst. The remainder is deionized water.

[0034] The preparation method of the single-component luminol chemiluminescent substrate solution includes the following steps:

[0035] (1) At room temperature, accurately weigh 15g of sodium acetate solid and add it to a clean beaker. Then add approximately 900mL of high-purity deionized water to the beaker. Start a magnetic stirrer and stir at medium speed (80r / min, 5min) until the sodium acetate solid is completely dissolved, forming a clear and transparent buffer solution. This step aims to first establish the basic buffer environment of the system.

[0036] (2) While maintaining stirring, add portions of the composite stabilizer to the buffer solution in a predetermined order. First, pipette 50 mL of N,N-dimethylacetamide and slowly add it to the solution. Then, weigh 5 g of β-cyclodextrin and 0.2 g of p-acetaminophen and add them sequentially. Continue stirring to ensure that all added stabilizer components are completely dissolved and the solution remains clear. This specific order of addition helps the β-cyclodextrin and other components to be fully dispersed and dissolved before being introduced into the core reactants, preparing for subsequent stabilization.

[0037] (3) After the stabilizer system is constructed, continue stirring. Accurately weigh 6g of sodium perborate and 0.4g of luminol, and add these two solid powders to the solution sequentially. Since these two are the core reactants, adding them to the solution already containing the stabilizer can immediately protect them and minimize their potential reactions and losses during the dissolution and mixing process. Continue stirring until both solids are completely dissolved.

[0038] (4) After luminol and sodium perborate have completely dissolved, accurately weigh 0.06 g of 4-morpholinopyridine, 0.3 g of sodium phenthiazide-10-yl-propylsulfonate, 0.2 g of 4-hydroxyphenylacetic acid and 0.2 g of isothiazolinone, and add them to the solution in sequence, stirring thoroughly until completely dissolved.

[0039] (5) Transfer the thoroughly mixed solution to a 1000 mL volumetric flask. Rinse the beaker several times with a small amount of deionized water, transferring the washings into the flask to ensure no loss of components. Then, carefully dilute the solution to the 1000 mL mark with deionized water. Stopper the flask and invert it several times to mix. Measure the pH of the solution using a calibrated pH meter and fine-tune it using dilute hydrochloric acid or dilute sodium hydroxide solution until the pH is precisely stable at 8.5. Precise pH control is crucial for ensuring the long-term stability and optimal luminescence performance of the system.

[0040] (6) Filter the final prepared substrate solution through a 0.22-micron filter membrane for sterilization, then dispense it into clean, light-proof reagent bottles and seal the bottle openings. Store the dispensed reagent bottles in a refrigerator at 2-8℃ for long-term storage until use.

[0041] Example 2

[0042] This embodiment provides a formulation and concentration of a single-component luminol chemiluminescent substrate solution (taking the preparation of 1000 mL of substrate solution as an example), with the concentration at the lower limit of the range, specifically:

[0043] Sodium acetate: 10g, as a buffer. Sodium perborate: 5g, as an oxidizing agent. Luminol (3-aminophenyl hydrazide): 0.1g, as a luminescent agent. N,N-dimethylacetamide: 20mL, as a component of the composite stabilizer. β-Cyclodextrin: 4g, as a component of the composite stabilizer. Acetaminophen: 0.1g, as a component of the composite stabilizer. Isothiazolinone: 0.1g, as a component of the composite stabilizer and also has antiseptic properties. 4-Morpholinpyridine: 0.05g, as a component of the composite catalyst. 4-Hydroxyphenylacetic acid: 0.1g, as a component of the composite catalyst. Phenothiazine-10-yl-propylsulfonate sodium salt: 0.05g, as a component of the composite catalyst. The remainder is deionized water.

[0044] In this embodiment, the concentrations of each component correspond to the lower limit of the preferred concentration range claimed. The preparation method is exactly the same as in Example 1, namely: first, sodium acetate is dissolved, then N,N dimethylacetamide, β-cyclodextrin, and acetaminophen are added, followed by sodium perborate and luminol, and finally 4-morpholinopyridine, phenothiazine-10-yl-propylsulfonate sodium salt, 4-hydroxyphenylacetic acid, and isothiazolinone are added. The solution is then brought to a final volume of 1000 mL, and the pH is adjusted to 8.0 using dilute hydrochloric acid or dilute sodium hydroxide solution. The finished product is then dispensed and stored at 2-8°C for later use.

[0045] Example 3

[0046] This embodiment provides a formulation and concentration of a single-component luminol chemiluminescent substrate solution (taking the preparation of 1000 mL of substrate solution as an example). The concentration is at the upper limit of the range, aiming to achieve effectiveness in application scenarios that pursue extreme luminescence intensity and the highest detection sensitivity. Specifically:

[0047] Sodium acetate: 20g, as a buffer. Sodium perborate: 10g, as an oxidizing agent. Luminol (3-aminophenyl hydrazide): 0.5g, as a luminescent agent. NN dimethylacetamide: 60mL, as a component of the composite stabilizer. β-Cyclodextrin: 8g, as a component of the composite stabilizer. Acetaminophen: 0.5g, as a component of the composite stabilizer. Isothiazolinone: 0.4g, as a component of the composite stabilizer and also has antiseptic properties. 4-Morphyrin pyridine: 0.2g, as a component of the composite catalyst. 4-Hydroxyphenylacetic acid: 0.5g, as a component of the composite catalyst. Phenothiazine-10-yl-propylsulfonate sodium salt: 0.7g, as a component of the composite catalyst. The remainder is deionized water.

[0048] In this embodiment, the concentrations of each component correspond to the upper limit of the preferred concentration range claimed. The preparation method is exactly the same as in Example 1, namely: first, dissolve sodium acetate, then add N,N-dimethylacetamide, β-cyclodextrin, and acetaminophen, followed by sodium perborate and luminol, and finally add 4-morpholinopyridine, phenothiazine-10-yl-propylsulfonate sodium salt, 4-hydroxyphenylacetic acid, and isothiazolinone. The solution is then brought to a final volume of 1000 mL, and the pH is adjusted to 9.0 using dilute hydrochloric acid or dilute sodium hydroxide solution. The finished product is then dispensed and stored at 2-8°C for later use.

[0049] Examples 4 and 5

[0050] Prepare a concentrated mother liquor of approximately 950 mL without pH adjustment, following the component types and concentration ratios described in Example 1. After thoroughly mixing the mother liquor, divide it into two equal portions and place each portion in a separate beaker.

[0051] For the first solution, transfer it to a 500 mL volumetric flask and dilute to the mark with deionized water. Then, using a high-precision pH meter, add diluted hydrochloric acid solution dropwise while stirring continuously until the pH of the solution is precisely stable at 8.0 (this is Example 4).

[0052] For the second solution, it was also transferred to another 500 mL volumetric flask and brought to volume. Then, using a high-precision pH meter, diluted sodium hydroxide solution was added dropwise while stirring continuously until the pH of the solution was precisely stabilized at 9.0 (this is Example 5).

[0053] The resulting two batches of single-component luminol chemiluminescent substrate solutions (Examples 4 and 5) had identical component concentrations but pH values ​​of 8.0 and 9.0, respectively. These two batches of substrate solutions were filtered, dispensed, and sealed for storage at 2-8°C for later use.

[0054] Verification Example

[0055] The single-component luminol chemiluminescent substrate solution obtained in Example 1 of this invention was compared with a commercially available two-component luminol luminescent substrate solution (comparative example: Thermo Scientific chemiluminescence SuperSignal). TM West Atto hypersensitive substrates were detected by a magnetic microparticle chemiluminescence analyzer. Magnetic beads-conjugated mouse monoclonal antibody and goat anti-mouse-HRP were added according to a pre-programmed machine procedure, and the luminescence values ​​were read after 10 minutes (as shown in Table 1).

[0056] The testing process (hereinafter the same) is as follows:

[0057] (1) Information entry: Open the fully automated chemiluminescence immunoassay analyzer program setting software, set the program as required, generate a QR code, scan the QR code generated by the matching program, and enter the detection method into the fully automated chemiluminescence immunoassay analyzer.

[0058] (2) Verification of detection method: Verify reaction system: 50 μl Ra (i.e. mouse IgG labeled magnetic beads) + 50 μl Rb (i.e. goat anti-mouse-HRP), react for 15 minutes; add 100 μL of excitation solution A, 100 μL of excitation solution B (comparative example) or 200 μL of single-component substrate solution, and measure the luminescence value.

[0059] (3) Reagent preparation: Before use, gently rotate or shake the reagent to mix it. On the "Reagent" page, insert the reagent tray of the instrument and start the test.

[0060] Table 1

[0061] sheep anti-mouse HRP concentration Example 1: Luminous value (U) Comparative luminescence value (U) 100ng / mL 16174398 15968688 10 ng / mL 4106889 3989886 1ng / mL 461907 458277 0.1 ng / mL 89592 88071

[0062] As shown in Table 1, the substrate solution prepared in Example 1 exhibited excellent beneficial effects after performance testing. When detecting analytes at concentrations of 100, 10, 1, and 0.1 ng / mL, the luminescence values ​​produced by the single-component substrate solution of Example 1 were significantly higher than those of the comparative two-component substrate solution of the prior art, indicating that it has higher luminescence efficiency and detection sensitivity.

[0063] The duration of the reaction plateau phase in Example 1 was retested and is shown in Table 2.

[0064] Table 2

[0065] time Example 1: Luminous value (U) 10min 26397669 20min 26390142 30min 26403066 60min 26457123

[0066] As shown in Table 2, after the reaction is triggered, the luminescence signal reaches its peak in a very short time, and then remains relatively stable with minimal fluctuations for the next 60 minutes, forming a broad signal plateau. This provides the automated instrument with ample reading time window, greatly improving the stability and reliability of the detection.

[0067] Example 1 was retested, and its shelf life data is shown in Table 3.

[0068] Table 3

[0069] Time (month) Example 1: Luminous value (U) 0 17598447 6 17593428 12 17602044 24 17638083 36 17617827 48 17607234 60 17612358

[0070] As shown in Table 3, the substrate solution was sealed and stored at 2-8℃, and its performance was tested periodically. The results showed that even after storage for up to 60 months (5 years), the luminescence value decreased by a very low rate compared to the initial value, and there was no significant difference in performance.

[0071] Example 1 was retested, and its thermal stability (37°C) data are shown in Table 4.

[0072] Time (days) Example 1: Luminous value (U) 0 17607978 7 17588886 14 17604276 21 17603292 28 17618541

[0073] As shown in Table 4, even at a relatively high temperature of 37°C, the luminescence value showed a very low attenuation rate compared to the initial value, and the performance was not significantly different.

[0074] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A single-component luminol chemiluminescent substrate solution, characterized in that, It includes the following components: sodium acetate, sodium perborate, N,N-dimethylacetamide, β-cyclodextrin, luminol, 4-morpholinopyridine, acetaminophen, 4-hydroxyphenylacetic acid, sodium phenthiazide-10-yl-propylsulfonate and isothiazolinone; The pH of the single-component luminol chemiluminescent substrate solution is 8-9.

2. The single-component luminol chemiluminescent substrate solution according to claim 1, characterized in that, The components include the following concentrations: sodium acetate 10-20 g / L, sodium perborate 5-10 g / L, N,N-dimethylacetamide 2-6 vol%, β-cyclodextrin 4-8 g / L, luminol 0.1-0.5 g / L, 4-morpholinopyridine 0.05-0.2 g / L, acetaminophen 0.1-0.5 g / L, 4-hydroxyphenylacetic acid 0.1-0.5 g / L, sodium phenthiazide-10-yl-propylsulfonate 0.05-0.7 g / L, and isothiazolinone 0.1-0.4 g / L.

3. The single-component luminol chemiluminescent substrate solution according to claim 1 or 2, characterized in that, The components include the following concentrations: sodium acetate 15 g / L, sodium perborate 6 g / L, N,N-dimethylacetamide 5 vol%, β-cyclodextrin 5 g / L, luminol 0.4 g / L, 4-morpholinopyridine 0.06 g / L, acetaminophen 0.2 g / L, 4-hydroxyphenylacetic acid 0.2 g / L, phenothiazine-10-yl-propylsulfonate sodium salt 0.3 g / L, and isothiazolinone 0.2 g / L.

4. The method for preparing the single-component luminol chemiluminescent substrate liquid according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: (I) Sodium acetate, N,N-dimethylacetamide, β-cyclodextrin, acetaminophen, sodium perborate, luminol, 4-morpholinopyridine, sodium phenthiazide-10-yl-propylsulfonate, 4-hydroxyphenylacetic acid and isothiazolinone were added to water in sequence to obtain a mixed solution; (II) The mixed solution is brought to the target volume to obtain the single-component luminol chemiluminescent substrate solution.

5. The preparation method according to claim 4, characterized in that, (I) Includes the following steps: (i) Dissolve sodium acetate in water and stir to form a buffer solution; (ii) Then N,N-dimethylacetamide, β-cyclodextrin and p-acetaminophen are added sequentially to the buffer solution and stirred to dissolve; (iii) Continue adding sodium perborate and luminol while stirring to dissolve; (iv) Finally, add 4-morpholinopyridine, sodium phenthiazide-10-yl-propylsulfonate, 4-hydroxyphenylacetic acid and isothiazolinone and stir to dissolve to obtain a mixed solution.

6. The preparation method according to claim 4, characterized in that, The stirring speed for dissolving is 80~100 r / min, and the stirring time is 3~5 min.

7. The preparation method according to claim 4, characterized in that, In step (II), after making up the volume, adjust the pH to 8-9.