CN118858262A - Highly sensitive single-component chemiluminescent substrate, long-term storage container and application thereof - Google Patents

Abstract

The present invention provides a highly sensitive single-component chemiluminescent substrate, a long-term storage container and its application. In order to improve the luminescent signal intensity of HRP chemiluminescent detection, the inventors focus on single-component substrate solutions, improving chemiluminescent intensity and improving long-term storage stability. In the present invention, a novel single-component HRP chemiluminescent substrate solution (and kit) is disclosed for the first time, which has good luminescent performance and a long shelf life.

Description

Highly sensitive single-component chemiluminescent substrate, long-term storage container and application thereof

Technical Field

The invention belongs to the field of chemical engineering, and more specifically, relates to a highly sensitive single-component chemiluminescent substrate, a long-acting storage container and applications thereof.

Background Art

Chemiluminescent immunoassay (CLIA) is the most widely used quantitative immunoassay method in the clinical field. It mainly uses the luminol chemiluminescent system catalyzed by horseradish peroxidase (HRP). In addition to being used in enzyme-linked immunosorbent assay methods, this system is also widely used in blotting tests for protein and nucleic acid analysis.

The reaction rate of luminol and hydrogen peroxide (H ₂ O ₂ ) is very slow. HRP is added to the system as a reaction catalyst to increase the reaction rate, so that the luminol luminescence can be quantitatively measured, and the luminescence intensity is proportional to the concentration of the enzyme; however, HRP itself is very inefficient as a catalyst for the chemiluminescent oxidation of luminol, so an enhancer is usually added to the luminescence system to make HRP catalyze the luminol chemiluminescence faster and more efficient. In this system, luminol and the enhancer are oxidized at the same time, and the enhancer is an HRP substrate with higher activity than luminol. The mechanism of the enhancer oxidation reaction is as follows:

(1) HRP+H ₂ O ₂ →HRP-I

(2) HRP-I+E→HRP-II+ ^E

(3) HRP-II+E→HRP+ ^E

Where E is the enhancer, E ^· is the free radical product of the enhancer oxidation, HRP, HRP-I and HRP-II are horseradish peroxidase and its two oxidation intermediate states. Therefore, the luminol molecule not only reacts with the oxidation intermediate of HRP, but also reacts with the free radical product of the enhancer to generate the luminol free radical L ^·- :

(4) HRP-I+LH ^- →HRP-II+L ^·-

(5) HRP-II+LH ^- →HRP+L ^·-

(6)E ^· +LH ^- →E+L ^·-

(7)2L ^·- →LH ^- +L

(8) L+H ₂ O ₂ →LO ₂ ^2- →(AP ^2- )*+N ₂

(9) (AP ^2- )*→AP ^2- +hν

Among them, two luminol free radicals are disproportionated to generate luminol anion (LH ^- ) and diazaquinone (L), diazaquinone (L) reacts with hydrogen peroxide to generate luminol peroxide (LO ₂ ^2- ), and then further generates excited 3-aminophthalate (3-aminophthalate, (AP ^2- )*), and emits photons (hν) in the process of returning to the ground state (AP ^2- ), producing detectable chemiluminescence. The intensity of chemiluminescence is related to the generation rate of luminol free radicals (L ^·- ), and the presence of enhancers greatly accelerates the generation of luminol free radicals (L ^·- ). In addition, in the absence of enhancers, the rate-limiting step of the entire process is the reduction of HRP's oxidized intermediate HRP-II to HRP (Formula (5)); after the addition of enhancers, the rate of the reduction reaction is improved, and the degree of improvement is also related to the selected enhancer (Formula (3)).

At present, many compounds have been reported as enhancers of HRP-catalyzed luminol luminescence. For example, luciferin used in early reports; various phenol enhancers, such as p-iodophenol; biphenyl compounds, such as p-hydroxybiphenyl and 4-hydroxy-4'-iodobiphenyl; triphenylimidazole derivatives; in addition to organic compounds, some metal ions or their oxides can also play a role in enhancing chemiluminescence. The best enhancer reported so far is N-substituted phenothiazines, among which the most used is 10H-phenothiazine-10-propane sulfonic acid sodium salt (SPTZ).

On this basis, adding a co-enhancer to the system containing the SPTZ enhancer can further promote the reaction and increase the intensity of chemiluminescence. At present, some co-enhancers have been used, such as 4-morpholinopyridine, 4-dimethylaminopyridine (DMAP) and 4-(1-pyrrolidinyl)pyridine (MORP); imidazole (IM), etc., which provide a certain luminescence enhancement effect, but this enhancement effect needs to be further optimized.

The widely used HRP chemiluminescent substrates are mostly two-component formulas, consisting of two pre-prepared solutions, one containing luminol and an enhancer, and the other containing peroxide, which are mixed to obtain a working solution when used. Each of the two solutions can be stored separately for a long time, but the mixed working solution has poor stability, and the usable window time only ranges from ten minutes to a few days. Currently, there are very limited reports on single-component HRP chemiluminescent substrate solutions, especially single-component solutions with long-term storage stability.

Based on the above existing research in this field, there is still a lot of room for improvement in the substrate solution of HRP chemiluminescent detection.

Summary of the invention

The purpose of the present invention is to provide a highly sensitive single-component chemiluminescent substrate, a long-acting storage container and applications thereof.

In a first aspect of the present invention, a method for enhancing the luminescent signal of HRP-catalyzed luminol chemiluminescence is provided, comprising:

(1) providing a chemiluminescent substrate system, comprising a basic chemiluminescent substrate and a co-enhancer; the co-enhancer is selected from: polyimidazole ionic liquids and polypyridine ionic liquids;

(2) contacting the chemiluminescent substrate system of (1) with HRP (e.g., SA-HRP) to generate an enhanced luminescent signal;

Wherein, the polyimidazole ionic liquid includes: an ionic liquid having polyvinyl imidazolium ions (P[VIM]+);

Wherein, the polyimidazole ionic liquid includes: an ionic liquid having polyvinyl-3-ethylimidazolium ion (P[VEIM]+), an ionic liquid having p[ECH-DMAP]+;

Wherein, the luminol includes luminol derivatives.

In one or more embodiments, the polyvinyl imidazolium ion is

In one or more embodiments, the polyvinyl-3-ethylimidazolium ion is

In one or more embodiments, the polyECH-dimethylaminopyridine is

In one or more embodiments, n=10-100 (e.g., 15, 20, 30, 40, 50, 60, 70, 80, 90, 95).

In one or more embodiments, the ionic liquid further comprises anions; preferably, the anions include: [BF ₄ ]-, [Cl]-, [NO ₃ ]-, [CF ₃ CO ₂ ]-.

In one or more embodiments, the derivative of luminol is L-012.

In one or more embodiments, the basic chemiluminescent substrate includes: luminol or its derivatives, phenothiazine-10-yl-propyl sulfonate (preferably SPTZ), and a peroxide oxidant; preferably, the peroxide oxidant includes perborate.

In one or more embodiments, the basic chemiluminescent substrate is present in a buffer, preferably a Tris buffer.

In one or more embodiments, the pH of the chemiluminescent substrate system is 7.2 to 9.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of the luminol derivative L-012 is 0.01 mM to 0.5 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of luminol is 0.5 mM to 20 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of phenothiazine-10-yl-propyl sulfonate (such as SPTZ) is 0.5 mM to 10 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of the co-enhancer is 0.05 mg/mL to 10 mg/mL.

In one or more embodiments, the pH of the chemiluminescent substrate system is 7.4, 7.5, 7.8, 8.0, 8.2, 8.5, or 8.8.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of the luminol derivative L-012 is 0.03, 0.05, 0.08, 0.1, 0.15, 0.2, 0.25, 0.3, or 0.4 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of luminol is 0.8, 1, 2, 3, 4, or 5 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of phenothiazine-10-yl-propyl sulfonate (such as SPTZ) is 0.6, 0.8, 1, 3, 5, 7, or 9 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of the co-enhancer is 0.08, 0.1, 0.5, 1, 2, 3, 5, 7, 8, or 9 mg/mL.

In another aspect of the present invention, a chemiluminescent substrate system is provided, comprising a basic chemiluminescent substrate and a co-enhancer; the co-enhancer is selected from: imidazole ionic liquids or polymers thereof, pyridine ionic liquids or polymers thereof; wherein the polyimidazole ionic liquids include: ionic liquids having polyvinyl imidazolium ions (P[VIM]+); wherein the polyimidazole ionic liquids include: ionic liquids having polyvinyl-3-ethylimidazolium ions (P[VEIM]+), ionic liquids having p[ECH-DMAP]+; wherein the luminol includes luminol derivatives.

In one or more embodiments, the ionic liquid further comprises anions; preferably, the anions include: [BF ₄ ]-, [Cl]-, [NO ₃ ]-, [CF ₃ CO ₂ ]-.

In one or more embodiments, the luminol derivative is L-012.

In one or more embodiments, the basic chemiluminescent substrate includes: luminol or its derivatives, phenothiazine-10-yl-propyl sulfonate (preferably SPTZ), and a peroxide oxidant; more preferably, the peroxide oxidant includes perborate.

In one or more embodiments, the pH of the chemiluminescent substrate system is 7.2 to 9.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of the luminol derivative L-012 is 0.01 mM to 0.5 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of luminol is 0.5 mM to 20 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of phenothiazine-10-yl-propyl sulfonate (such as SPTZ) is 0.5 mM to 10 mM.

In one or more embodiments, in the chemiluminescent substrate system, the concentration of the co-enhancer is 0.05 mg/mL to 10 mg/mL.

In one or more embodiments, the chemiluminescent substrate system is stable, wherein the polyionic liquid enhances the stability of the substrate system.

In another aspect of the present invention, a chemiluminescent substrate device that maintains long-lasting activity is provided, comprising: (i) a container whose inner surface is coated with a metal-polyphenol network (MPN) nanocoating; the nanocoating comprises: (tannic acid layer-metal layer) _m arranged in sequence, wherein m is a positive integer of 5 to 50; thereby using metal ions (such as Mg ²⁺ ) as metal centers to construct a metal-polyphenol network together with tannic acid; (ii) any of the chemiluminescent substrate systems described above, which is placed in the container of (i).

In one or more embodiments, in the chemiluminescent substrate system, in the chemiluminescent substrate device, the tannic acid is used for spraying at a concentration of 2 to 50 mg/mL (such as 5, 8, 10, 15, 20, 25, 30, 35, 40 mg/mL), and more preferably at a concentration of 6 to 25 mg/mL;

In one or more embodiments, the metal includes Mg, Ca, Al.

In one or more embodiments, a Mg ²⁺ solution (preferably a MgCl ₂ solution) is prepared for spraying.

In one or more embodiments, the Mg ²⁺ solution is used for spraying at a concentration of 1 to 30 mg/mL (e.g., 2, 5, 6, 8, 10, 15, 20, 25 mg/mL), and more preferably at a concentration of 3 to 12 mg/mL.

In one or more embodiments, the molar ratio of the tannic acid to the Mg ²⁺ solution is 1:(3-30); preferably 1:(5-20).

In one or more embodiments, the molar ratio of the tannic acid to the Mg ²⁺ solution may be, for example, 1:6, 1:8, 1:10, 1:15, 1:20, 1:25.

In one or more embodiments, after the spraying, drying is performed; preferably, the drying is performed at 70-90°C, such as 80°C.

In one or more embodiments, the pH value of the tannic acid is 7.0.

In another aspect of the present invention, there is provided a use of any of the aforementioned methods, any of the aforementioned chemiluminescent substrate systems or any of the aforementioned chemiluminescent substrate devices for HRP-catalyzed luminol chemiluminescent reaction, wherein the luminol reaction has an enhanced luminescent signal; wherein the luminol includes a luminol derivative.

In another aspect of the present invention, a kit for HRP-catalyzed luminol chemiluminescent reaction is provided, which comprises any of the above-mentioned chemiluminescent substrate systems, or any of the above-mentioned chemiluminescent substrate devices, wherein the luminol comprises a luminol derivative.

In one or more embodiments, the kit further includes: a reaction buffer.

Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Structural formulas of luminol, L-012 and enhancer SPTZ.

Figure 2, co-enhancer and synthesis route;

(a) Structural formulas of ionic liquids [MIM][Cl] and [MIM][BF ₄ ];

(b) Synthesis route of polyionic liquid P[VEIM][BF ₄ ];

(c) Synthesis route of polyionic liquid P[VIM][BF4];

(d) Synthesis route of ionic liquid [B-DMAP][BF ₄ ];

(e) Synthesis route of polyionic liquid P[ECH-DMAP][BF ₄ ].

FIG3 . Bar graphs of chemiluminescent signals of unenhanced and SPTZ-enhanced luminol and L-012 substrates in the presence of various alternative co-enhancers.

Figure 4. Effect of pH on the signal enhancement of chemiluminescent substrates by polyionic liquid co-enhancers.

Figure 5. Effect of L-012 concentration on luminescence signal intensity.

Fig. 6. Effect of SPTZ concentration on luminescence signal intensity.

Figure 7. Effect of the concentration of each polyionic liquid co-enhancer on the signal enhancement effect.

FIG8 is a graph showing the chemiluminescent signals of L-012/P[VIM][BF ₄ ] and luminol/MORP substrates as a function of SA-HRP concentration.

Figure 9. Signal decline trend of chemiluminescent substrate formulations of luminol/MORP and L-012 after storage at 37°C.

Fig. 10. Contact angle measurement of (a) PP material before and (b) after spraying; (c) MPN film thickness after 10 spraying cycles and 20 spraying cycles.

Figure 11. Signal decline trends of the two storage methods at (a) 37°C and (b) 4°C at different time points. The signals were normalized to the signals of the freshly prepared solutions.

DETAILED DESCRIPTION

In order to improve the luminescent signal intensity of HRP chemiluminescent detection, the inventors focused on single-component substrate solutions, improving chemiluminescent intensity and improving long-term storage stability. After in-depth research, screening and optimization, a new single-component HRP chemiluminescent substrate solution (and kit) was revealed for the first time, which has good luminescent performance and long shelf life.

As used in the present invention, the term “contains,” “includes,” or “has” includes “comprises,” “mainly consists of,” “substantially consists of,” and “consisting of”; “mainly consists of,” “substantially consists of,” and “consisting of” are subordinate concepts of “contains,” “has,” or “includes.”

As used in the present invention, "about", "approximately", "roughly" or "substantially" generally refers to a specific value or range such as within 20%, preferably within 10%, and more preferably within 5%. The numerical values used herein are approximate values, which means that if not explicitly stated, the words "about", "approximately", "roughly" or "substantially" can be inferred to apply.

As used in the present invention, the "main ingredient (component)" or "main active ingredient" or "active ingredient" refers to the necessary ingredient that plays a role in maintaining protein stability, and the present invention mainly includes the following components: amphiphilic block polymers, ionic liquids and antimicrobial peptides, preferably also including sucrose; or consisting of them.

In the present invention, the chemiluminescent substrate system comprises a basic chemiluminescent substrate and a co-enhancer; the co-enhancer is selected from: imidazole ionic liquids or polymers thereof, pyridine ionic liquids or polymers thereof.

In the chemiluminescent substrate system, the basic chemiluminescent substrate can be some reagents known in the art that are required for HRP-based chemiluminescent reactions. The reagents may include: luminol or its derivatives, phenothiazine-10-yl-propyl sulfonate (preferably SPTZ), and a peroxide oxidant. For example, the peroxide oxidant includes perborate.

The inventors have found that the derivative of luminol, L-012, is more suitable for use in the technical solution of the present invention than luminol itself. As a preferred luminescent substrate, the derivative of luminol, L-012, is suitable for physiological pH conditions and matches the appropriate reaction conditions of HRP.

In a specific embodiment of the present invention, pyridine ionic liquids and polymers thereof, and imidazole ionic liquids and polymers thereof were evaluated as co-enhancers. The results showed that ionic liquids having polyvinyl imidazolium ions (P[VIM]+), ionic liquids having polyvinyl-3-ethylimidazolium ions (P[VEIM]+), and ionic liquids having p[ECH-DMAP]+ were extremely effective as co-enhancers, and were significantly more synergistic than existing enhancers.

The ionic liquid optimized by the present invention has high ionic conductivity and polarizability, and can act as a promoter to accelerate the transfer of electrons during the reaction, thereby further improving the catalytic activity; polyionic liquids are generated by the polymerization of ionic liquid monomers, and have anionic and cationic groups on the repeating units, achieving a higher density and more ordered charge arrangement, thereby enhancing the catalytic properties of ionic liquid electron transfer.

Since the selection of the co-enhancer (i.e., ionic liquid) in the present invention is based on electron transfer and ionic bond interactions, and also based on the presence of a matching primary enhancer SPTZ in the system, rather than adjusting the system to a suitable pH to form micelles to promote the reaction, the ionic liquids optimized in the present invention are all carbon chain-free or methyl-only substituted imidazole ionic liquids, and pyridine ionic liquids with a structure similar to dimethylaminopyridine.

The co-enhancer effect of the ionic liquid in the present invention is mainly determined by the cation, while its water solubility is determined by the anion. Common hydrophilic anions include [Cl], [BF ₄ ], etc., and the hydrophobic anion is mainly [PF ₆ ]. Although [BF ₄ ] is preferably used as the anion in the embodiments of the present invention, other types of anions can also be included in the scope of the present invention.

Due to its advantages in stability, the chemiluminescent substrate system of the present invention can be a stable composition that can be prepared in advance. The components in the composition are compatible with each other in appropriate amounts and act synergistically, so that the chemiluminescent signal in the HRP reaction is significantly increased and the storage stability of the substrate is significantly improved.

The present invention also includes compounds, chemicals, analogs and/or salts, hydrates or precursors equivalent to the above-mentioned main active ingredients. For high molecular polymers, the present invention includes polymers with a certain degree of polymerization or polymers within a certain molecular weight range. Preferred specific polymers are provided in the embodiments and effect tests have been carried out, but it should be understood that polymers with a degree of polymerization floating within a certain range are also included in the present invention.

It should be understood that the chemiluminescent substrate system (composition) of the present invention can be concentrated or diluted. The content of active ingredients in concentrated compositions is relatively high, while the content of active ingredients in diluted compositions and compositions actually used may be relatively low. In addition, other suitable chemicals, synergists, trace elements, stabilizers, dispersants, solvents and other commonly used components may also be included, which generally do not change the active functions of the main active ingredients.

The chemiluminescent substrate system (composition) of the present invention can also be packaged in containers, packaged, and made into a kit for the convenience of use by those skilled in the art. In addition, the package or kit can also include instructions for use.

In a preferred embodiment of the present invention, the container is a container that helps to improve the stability/storage time of the chemiluminescent substrate system (composition). The inner surface of the container is coated with a metal-polyphenol network (MPN) nanocoating; the nanocoating includes: (tannic acid layer-metal layer) _m arranged in sequence; so that a metal ion (such as Mg ²⁺ ) can be used as a metal center to construct a metal-polyphenol network together with tannic acid. After the chemiluminescent substrate system of the present invention is placed in the container, it can be stored for a long time.

In the metal-polyphenol network (MPN), tannin, as a polyphenol, contains a large number of phenolic hydroxyl structures, wherein the ortho-phenolic hydroxyl groups are easily oxidized to quinone structures, thereby consuming oxygen in the environment, and also having a strong ability to capture free radicals such as active oxygen, so it has antioxidant properties and the ability to scavenge free radicals. At the same time, the phenolic hydroxyl groups in the polyphenols can act as ligands to react with metal ions to form stable chelates.

In a preferred embodiment, Fe ³⁺ is used as the metal center in MPN. The presence of Fe ³⁺ is not conducive to the long-term storage of luminol (and its derivatives) chemiluminescent solution because it will catalyze itself in the substrate solution. Similarly, some common metal center ions, such as Cu ²⁺ , Co ³⁺ and Cr ³⁺ , will also have similar problems. The container coated with the metal-polyphenol network (MPN) nanocoating established by the present invention effectively solves this problem.

The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

Example 1. Selection and preparation of enhancing agents

1. Enhanced single-component chemiluminescent substrate

A new co-enhancer is used to increase the chemiluminescence intensity, thereby improving the response sensitivity.

The luminescent agents used in the present invention include luminol and L-012.

The enhancer includes SPTZ (sodium phenothiazine-10-yl-propylsulfonate), and their structural formula is shown in FIG1 .

2. Selection and synthesis of co-enhancers

(A) Synthesis of Imidazole Ionic Liquids

The imidazolium ionic liquids used are all water-soluble ionic liquids, specifically 1-methylimidazolium chloride ([MIM][Cl]) and 1-methylimidazolium tetrafluoroborate ([MIM][BF ₄ ]), as shown in Figure 2a. While performing the functional test, the effects of different anions on the intensity of the luminescent signal can also be preliminarily compared.

(B) Synthesis of polyimidazole ionic liquids

(a) Polyvinyl-3-ethylimidazolium tetrafluoroborate ionic liquid (P[VEIM][BF ₄ ]):

The preparation is carried out by free radical polymerization. 3g of 1-vinyl-3-ethylimidazole tetrafluoroborate is dissolved in 30mL of chloroform, and the solution is protected by _N2 . 0.06g of azobisisobutyronitrile (AIBN) is added to the system, heated to 60°C under _N2 protection, and the reaction is carried out for 5 hours. At this time, a milky white solid is generated in the solution, which is cooled to room temperature. After filtering, the obtained solid is washed with chloroform several times to remove the unreacted monomers and initiators, and then dried in a vacuum oven at 40°C for 24 hours to obtain a white solid product P[VEIM][ _BF4 ] (Figure 2b) (n=10-100, n is 36 in subsequent examples).

¹ H NMR (d-DMSO, 400MHz) 9.6-9.41 (1H, N CH N); 8.2-7.92 (1H, CHN CH CHN); 7.81-7.71 (1H, CHNCH CH N); 4.76 (1H, CH CH ₂ ); 4.20 (2H,N CH ₂ CH ₃ ); 2.60-2.54 (2H, CH CH ₂ ); 1.48 (3H, NCH ₂ CH ₃ )

(b) Polyvinyl imidazolium tetrafluoroborate ionic liquid (P[VIM][BF ₄ ]):

Preparation of monomer: 10g 1-vinylimidazole was dissolved in 30mL deionized water. 17mL tetrafluoroboric acid solution (48wt.%) was placed in an ice bath and stirred, and vinyl imidazole aqueous solution was added dropwise thereto. The resulting solution was stirred and reacted at 0°C (ice bath) for 12 hours, then transferred to a rotary vacuum apparatus and heated to 40°C, and water in the system was removed under reduced pressure. The resulting solid was washed twice with 300mL ether to remove unreacted raw materials, and then placed in a vacuum oven and dried at room temperature for 24 hours to obtain a monomer 1-vinylimidazole tetrafluoroborate ionic liquid.

¹ H NMR (d-DMSO, 400MHz) 9.33 (1H, N CH N); 8.18 (1H, CHN CH CHN); 7.82 (1H, CH CH ₂ ); 7.25-7.32 (1H, CHNCH CH N); 5.4- 5.95(2H,CH CH ₂ ).

Preparation of polyionic liquid: 3 g of 1-vinyl imidazolium tetrafluoroboric acid was dissolved in 30 mL of ethanol, and the solution was protected by N _2. 0.15 g of AIBN was added to the system, and the system was heated to 70°C under N ₂ protection, and the reaction was carried out for 3 hours. The reaction solution was distilled under reduced pressure at 60°C on a rotary vacuum apparatus to remove ethanol to obtain a white solid, which was then transferred to a vacuum oven and dried at 60°C for 24 hours to obtain the product P[VIM][BF ₄ ] (Figure 2c) (n=10-100, and n is 21 in subsequent examples).

¹ H NMR (d-DMSO, 400MHz) 9.7-9.44 (1H, N CH NH); 8.18-7.96 (1H, N CH CHNH); 7.85-7.75 (1H, NCH CH NH); 3.83 (1H, CH CH ₂ ); 2.2-2.13(2H,CH CH ₂ ).

Afterwards, the pyridine ionic liquid and the polyionic liquid are synthesized. The selected ionic liquid is a 4-dimethylaminopyridine (DMAP) ionic liquid.

(c) [B-DMAP] [BF ₄ ]

12.2g DMAP (0.1mol) was dissolved in 30mL ethyl acetate, and 13.7g n-butyl bromide (0.1mol) was added dropwise under stirring at room temperature. After heating to 70℃ and stirring for 48 hours, a precipitate was generated in the system. The precipitate was filtered, washed with ethyl acetate three times, and dried in a vacuum oven at 70℃ for 10 hours to obtain a white solid intermediate product [B-DMAP][Br]. 2g [B-DMAP][Br] was dissolved in 60mL anhydrous acetone, and 1.3g NaBF ₄ was added thereto under stirring. The solution was stirred at room temperature for 48 hours. The generated white precipitate was filtered off, and the reaction solution was washed twice with saturated sodium bicarbonate solution, and anhydrous sodium sulfate was added to remove water. The sodium sulfate was filtered off, and the induction system was dried in a vacuum oven at 40℃ to obtain a white solid [B-DMAP][BF ₄ ] (Figure 2d).

¹ H NMR (d-DMSO, 400MHz) 8.28 (2H, N CH CHC); 7.02 (2H, NC CH CH); 4.15 (2H, N CH ₂ CH ₂ ); 3.16 (6H, N CH ₃ ); 1.75- 1.66(2H,NCH ₂ CH ₂ CH ₂ ); 1.26-1.18(2H,CH ₂ CH ₂ CH ₂ CH ₃ ); 0.90(3H,CH ₂ CH ₂ CH ₃ ).

(d) Polyionic liquid (P[ECH-DMAP][BF ₄ ])

4g of polyepichlorohydrin was dissolved in 30mL of toluene, 10g of DMAP was added thereto under stirring, the system was heated to 80°C and stirred for 12 hours, and an oily solid was generated in the solution. After cooling to room temperature, 30mL of ether was added thereto, transferred to a rotary evaporator, and distilled under reduced pressure at 90°C to obtain a crude product. The crude product was washed with a small amount of acetone, and then placed in a vacuum oven and dried at room temperature for 24 hours to obtain an intermediate product P[ECH-DMAP][Cl]. 2g of P[ECH-DMAP][Cl] was dissolved in 18g of methanol to prepare a 10wt% solution. Similarly, 2.4g of LiBF ₄ was dissolved in 21.6g of methanol to prepare a 10wt% solution. The above two solutions were mixed and stirred for reaction at room temperature for 30 minutes. The resulting product was an oily substance that was immiscible with the reaction system. After the reaction, the oily product was separated and dried in a vacuum oven at 80° C. for at least 24 hours. The obtained white solid was P[ECH-DMAP][BF ₄ ] ( FIG. 2 e ) (n=10-100, and n was 90 in the subsequent examples).

¹ H NMR (d-DMSO, 400MHz) 8.76 (2H, N CH CHC); 7.48 (2H, NC CH CH); 5.30-5.02 (2H, N CH ₂ CH); 3.62-3.35 (backbone, O CHCH ₂ ) ;3.05(6H,N CH ₃ ).

Example 2: Preliminary test of the signal enhancement effect of various co-enhancers

Prepare the basic chemiluminescent substrate solution (Group I) according to the proportions in Table 1 below:

Table 1

Add various reagents to be analyzed respectively, and the amounts are as follows;

DMAP, MORP, IM, [MIM][Cl], [MIM][BF ₄ ], [B-DMAP][BF ₄ ]: 1mM

Each polyionic liquid (P[VEIM][BF ₄ ], P[VIM][BF ₄ ], P[ECH-DMAP][BF ₄ ]): 0.4 mg/mL

Prepare the basic chemiluminescent substrate solution (Group II) according to the proportions in Table 2 below:

Table 2

Various reagents to be analyzed were added thereto, and the amounts thereof were as follows:

DMAP, MORP, [B-DMAP][BF ₄ ]: 2mM;

IM, [MIM][Cl], [MIM][BF ₄ ]: 10mM;

Each polyionic liquid (P[VEIM][BF ₄ ], P[VIM][BF ₄ ], P[ECH-DMAP][BF ₄ ]): 1.5 mg/mL.

100 μL of each luminescent solution was added to a 96-well plate, and 1 ng/mL of streptavidin-cross-linked horseradish peroxidase (SA-HRP) was added thereto, mixed well, and then waited for 30 seconds for chemiluminescence detection. The results are shown in FIG3 .

It can be seen from the figure that (1) as expected, the luminescence efficiency of L-012 is 10 times or more that of luminol under the same conditions; (2) in the case of the same cation, when the anion is [BF ₄ ] in [MIM][Cl] and [MIM][BF ₄ ], the luminescence signal is relatively strong; (3) the presence of a co-enhancer can greatly promote the chemiluminescence intensity of luminol and L-012.

Among them, the L-012 system with the addition of the co-enhancer P[VIM][BF ₄ ] has the highest sensitivity, followed by the other two polyionic liquids. The main structural difference between P[VIM][BF ₄ ] and P[VEIM][BF ₄ ] is that P[VEIM][BF ₄ ] has an additional ethyl substitution, and the signal is reduced compared with the unsubstituted P[VIM][BF ₄ ], which is consistent with the inventors' consideration of the imidazole cationic structure when selecting ionic liquid co-enhancers.

Example 3: Effect of pH on the signal enhancement effect of co-enhancer

In this example, the effect of different pH conditions on the signal enhancement of the co-enhancer was analyzed. The reaction system was set up as shown in Table 3 below, and the pH value in the system was changed (basically the same as the above-mentioned group I, but the pH value was changed):

Table 3

Each polyionic liquid (P[VEIM][BF ₄ ], P[VIM][BF ₄ ], P[ECH-DMAP][BF ₄ ]) was added thereto at 0.4 mg/mL.

The detection method is the same as before, and the results are shown in Figure 4. As can be seen from the figure, pH 7.4 to 8.5 can achieve a good enhancement effect. Among them, for the polyionic liquids P[VEIM][BF ₄ ] and P[VIM][BF ₄ ], the most suitable pH is 7.8; for the polyionic liquid P[ECH-DMAP][BF ₄ ], the most suitable pH is 7.6.

According to FIG. 4 , the signal enhancement effect of P[VIM][BF ₄ ] is relatively the highest.

Example 4: Effect of L-012 concentration on chemiluminescent signal intensity

In this example, the effect of different concentrations of L-012 on the chemiluminescent signal intensity was analyzed. The reaction system was set as shown in Table 4 below:

Table 4

L-012 was added thereto at a concentration of 0.01 mM to 0.5 mM. The detection method was the same as before.

The results are shown in FIG5 , which shows that L-012 can achieve good enhancement effects at relatively low concentrations, preferably 0.06 mM to 0.5 mM, and preferably higher than 0.1 mM.

Example 5: Effect of enhancer concentration on chemiluminescent signal

In this example, the effect of different concentrations of the analysis enhancer (SPTZ) on the chemiluminescent signal was analyzed. The reaction system was set up as shown in Table 5 below:

Table 5

SPTZ was added thereto at a concentration of 0.5 mM to 10 mM. The detection method was the same as before.

The regulatory effect of SPTZ concentration on the intensity of luminescent signal is shown in FIG6 . It can be seen that SPTZ concentrations of 0.3 mM to 5 mM can have a good enhancement effect, among which the enhancement effect is more significant at 1 mM to 3 mM, and the highest is about 2 mM.

Example 6: Effect of polyionic liquid co-enhancer concentration on chemiluminescent signal

In this example, the effect of different concentrations of polyionic liquid co-enhancer on chemiluminescent signal was analyzed. The reaction system was set as shown in Table 6 below:

Table 6

The co-enhancer (P[VEIM][BF ₄ ], P[VIM][BF ₄ ], P[ECH-DMAP][BF ₄ ]) was added thereto at a concentration of 0.05 mg/mL to 2 mg/mL. The detection method was the same as above.

The regulatory effect of each polyionic liquid co-enhancer concentration on the signal enhancement effect is shown in FIG7 . It can be seen that 0.2 mg/mL to 2 mg/mL can have a good enhancement effect, and the enhancement effect is more significant at 0.4 mg/mL to 1 mg/mL.

According to FIG. 7 , the signal enhancement effect of P[VIM][BF ₄ ] is relatively the highest.

Example 7. Comparison of optimized composition and luminol signal

In this example, the optimized composition was compared with luminol for signal.

Prepare the luminol/MORP chemiluminescent substrate solution according to the exemplary concentration of Group II in Example 2.

The components of L-012 chemiluminescent substrate solution are as follows in Table 7:

Table 7

The prepared chemiluminescent substrate solution was added to each well of a 96-well plate, 100 μL, and 20 μL SA-HRP solution was added thereto to make the final concentration as shown in the figure, spanning from 0 to 1 ng/mL. The blank was a solution without any SA-HRP added.

The curves of the chemiluminescent signals of L-012/P[VIM][BF ₄ ] and luminol/MORP substrates changing with SA-HRP concentration are shown in FIG8 . It can be seen that the regression slopes of the two differ by more than 10 times, and the optimized composition of the present invention exhibits extremely remarkable excellent performance.

Example 8, Stability Analysis after Seven Days of Storage

According to the example in Example 7, luminol/MORP and L-012 chemiluminescent substrate solutions were prepared respectively, stored at 37°C in the dark, and tested once every 24 hours. During the test, the prepared chemiluminescent substrate solutions were added to 96-well plates, 100 μL per well, and 20 μL of SA-HRP solution was added thereto to make the final concentration 1 ng/mL. After mixing, wait for 30 seconds for chemiluminescent detection.

The signal decline trend of the chemiluminescent substrate formula of Minol/MORP and L-012 after storage at 37°C is shown in Figure 9. As time goes by, the signal declines significantly. It can also be seen that after the addition of polyionic liquid P[VIM][BF ₄ ], it has a significant maintenance effect on the stability of the formula. Although the principle is unknown, based on previous studies, the main reason for the poor long-term stability of single-component chemiluminescent substrates with added enhancers and co-enhancers is that the enhancer is rapidly oxidized and decomposed, rather than the substrate (luminol) or peroxide being consumed during storage.

Therefore, it is suggested that the polyionic liquid has a stabilizing effect on the enhancer SPTZ, thereby improving the stability of the chemiluminescent substrate solution as a whole, while the signal of the substrate solution using the traditional co-enhancer drops rapidly.

Example 9: Stability analysis of long-term storage

The inventor's previous research and the above stability test results show that the stability of the enhancer has a strong correlation with the overall stability of the chemiluminescent substrate signal. Existing schemes attempt to use enhancers with better stability, but this method requires screening and testing of compounds with similar structures, and there is a certain probability that no suitable alternatives can be found, and it does not have the universality that can be extended to various substrate solutions of peroxidase to prevent component oxidation.

Different from what is disclosed in the patent, the inventors intend to adopt a different solution, namely, to establish an antioxidant solution environment, which can be successfully used for the long-term storage of the single-component chemiluminescent substrate in the present invention, and can also be subsequently extended to the long-term preservation of various single-component substrate solutions of single-component peroxidase.

After repeated analysis and experiments, the inventors adopted a method of constructing a metal-polyphenol network (MPN) nanocoating on the inner surface of the container in use by layer-by-layer assembly to achieve the purpose of preventing oxidation of components in the solution.

The inventors used Mg ²⁺ as the metal center and constructed MPN together with tannic acid (TA) to eliminate this problem; in addition, the uncomplexed phenolic hydroxyl group can also capture a small amount of Fe ³⁺ in the solution to prevent the catalytic consumption of luminol in the substrate.

1. MPN Construction

After washing the polypropylene (PP) sheet with acetone, dry it in an oven to constant weight. Prepare a 10 mg/mL tannic acid (TA) ethanol solution and add a small amount of sodium hydroxide to adjust the solution pH to 7.0. Prepare a 6 mg/mL MgCl ₂ ethanol solution so that the molar ratio of TA:Mg ²⁺ is 1:10 at the same volume. Put the two solutions into containers with the same specifications and nozzles. When preparing self-assembled membranes, first spray the TA solution, press the nozzle once, and the solution covers the PP material; wait for 20 seconds and then immediately spray the MgCl ₂ solution, which is consistent with the method of spraying the TA solution, and wait for 20 seconds. The above process is one spray cycle, and a total of 20 spray cycles are required. After spraying, wash the sheet with ethanol and dry it in an oven at 80°C.

The contact angle of the PP material before and after spraying was measured to characterize its surface modification; the film thickness after spraying was tested by ellipsometry, and the results are shown in Figure 10.

According to Figure 10, the hydrophobic PP material before spraying becomes extremely hydrophilic after MPN coating. The thickness of the MPN film after 20 sprayings is about 48 nm.

Furthermore, the PP container was cleaned with acetone, dried in an oven to constant weight, and a TA-Mg self-assembled film was prepared on the inner wall thereof using the same method as above.

2. Long-term preservation evaluation

The L-012/P[VIM][BF4] (the optimized composition ratio in Example 7) chemiluminescent substrate was prepared and placed in a common container and a container with MPN coating, respectively, and stored away from light.

The test conditions are set as follows:

The chemiluminescent substrates of the two storage conditions were placed at 4°C and 37°C, respectively. For 4°C, the samples were taken out for detection at 30, 90, 120 and 150 days; for 37°C, the samples were taken out for detection at 1, 3, 7 and 14 days. During the detection, 100 μL of each luminescent liquid was added to a 96-well plate, and 1 ng/mL streptavidin-cross-linked horseradish peroxidase (SA-HRP) was added thereto. After mixing, wait for 30 seconds for chemiluminescent detection.

The results are shown in Figure 11. It can be seen that the chemiluminescent base solution stored in the container without the protective coating has a significant signal reduction due to the decomposition of the enhancer and cannot be stored for a long time as a single component solution. In the container with the protective coating, the oxygen free radicals are effectively captured, the solution is in an antioxidant environment, and the stability of each component can be maintained for a long time.

As can be seen from the figure, the signal loss of the protected substrate solution is only 8% after being placed at 37°C for 2 weeks. After conversion, it can be concluded that the signal can be kept stable at room temperature for at least two months, and the downward trend curve can be inferred that it can be kept stable at room temperature for a longer time.

The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims (10)

1. A method for enhancing the luminescent signal of HRP-catalyzed luminol chemiluminescence, comprising: (1) providing a chemiluminescent substrate system, comprising a basic chemiluminescent substrate and a co-enhancer; the co-enhancer is selected from: polyimidazole ionic liquids and polypyridine ionic liquids; (2) contacting the chemiluminescent substrate system of (1) with HRP to generate an enhanced luminescent signal; Wherein, the polyimidazole ionic liquid includes: an ionic liquid having polyvinyl imidazolium ions; Wherein, the polyimidazole ionic liquid includes: an ionic liquid having polyvinyl-3-ethylimidazolium ions, an ionic liquid having p[ECH-DMAP]+; Wherein, the luminol includes luminol derivatives. 2. The method according to claim 1, wherein the polyvinyl imidazolium ion is The polyvinyl-3-ethylimidazolium ion is The polyECH-dimethylaminopyridine is Where n=10～100. 3. The method according to claim 1, characterized in that the ionic liquid further comprises anions; preferably, the anions include: [BF ₄ ]-, [Cl]-, [NO ₃ ]-, [CF ₃ CO ₂ ]-; or The luminol derivative is L-012; or The basic chemiluminescent substrate includes: luminol or its derivatives, phenothiazine-10-yl-propyl sulfonate, and a peroxide oxidant; preferably, the peroxide oxidant includes perborate. 4. The method according to claim 3, wherein the pH of the chemiluminescent substrate system is 7.2 to 9; or In the chemiluminescent substrate system, the concentration of the luminol derivative L-012 is 0.01 mM to 0.5 mM; or In the chemiluminescent substrate system, the concentration of luminol is 0.5 mM to 20 mM; or In the chemiluminescent substrate system, the concentration of phenothiazine-10-yl-propyl sulfonate is 0.5 mM to 10 mM; or In the chemiluminescent substrate system, the concentration of the co-enhancer is 0.05 mg/mL to 10 mg/mL. 5. A chemiluminescent substrate system, comprising a basic chemiluminescent substrate and a co-enhancer; the co-enhancer is selected from: imidazole ionic liquids or polymers thereof, pyridine ionic liquids or polymers thereof; Wherein, the polyimidazole ionic liquid includes: an ionic liquid having polyvinyl imidazolium ions; Wherein, the polyimidazole ionic liquid includes: an ionic liquid having polyvinyl-3-ethylimidazolium ions, an ionic liquid having p[ECH-DMAP]+; Wherein, the luminol includes luminol derivatives. 6. The chemiluminescent substrate system according to claim 5, characterized in that the ionic liquid further comprises anions; preferably, the anions include: [BF ₄ ]-, [Cl]-, [NO ₃ ]-, [CF ₃ CO ₂ ]-; or The luminol derivative is L-012; or The basic chemiluminescent substrate includes: luminol or its derivatives, phenothiazine-10-yl-propyl sulfonate, and a peroxide oxidant; more preferably, the peroxide oxidant includes perborate; Preferably, the pH of the chemiluminescent substrate system is 7.2 to 9; Preferably, in the chemiluminescent substrate system, the concentration of the luminol derivative L-012 is 0.01 mM to 0.5 mM; Preferably, in the chemiluminescent substrate system, the concentration of luminol is 0.5 mM to 20 mM; Preferably, in the chemiluminescent substrate system, the concentration of phenothiazine-10-yl-propyl sulfonate is 0.5 mM to 10 mM; Preferably, in the chemiluminescent substrate system, the concentration of the co-enhancer is 0.05 mg/mL to 10 mg/mL. 7. A chemiluminescent substrate device that maintains long-lasting activity, comprising: (i) A container having a metal-polyphenol network nanocoating coated on the inner surface; the nanocoating comprises: (tannic acid layer-metal layer) _m arranged in sequence, wherein m is a positive integer of 5 to 50; thereby, the metal ion is used as the metal center to construct a metal-polyphenol network together with tannic acid; (ii) The chemiluminescent substrate system according to any one of claims 5 to 6, which is placed in the container of (i). 8. The chemiluminescent substrate device according to claim 7, characterized in that the tannic acid is used for spraying at a concentration of 2 to 50 mg/mL, more preferably at a concentration of 6 to 25 mg/mL; The metal includes Mg, Ca, Al; preferably, a Mg ²⁺ solution is prepared for spraying; preferably, the Mg ²⁺ solution is used for spraying at a concentration of 1 to 30 mg/mL, more preferably at a concentration of 3 to 12 mg/mL; Preferably, the molar ratio of the tannic acid to the Mg ²⁺ solution is 1:(3-30); more preferably 1:(5-20). 9. Use of the method according to any one of claims 1 to 4, the chemiluminescent substrate system according to any one of claims 5 to 6, or the chemiluminescent substrate device according to any one of claims 7 to 8 for HRP-catalyzed luminol chemiluminescent reaction, wherein the luminescent reaction has an enhanced luminescent signal; wherein the luminol includes a luminol derivative. 10. A kit for HRP-catalyzed luminol chemiluminescent reaction, comprising the chemiluminescent substrate system according to any one of claims 5 to 6, or the chemiluminescent substrate device according to any one of claims 7 to 8, wherein the luminol comprises a luminol derivative.