CN114755403A - Use of indole derivatives as chemiluminescence enhancer and kit containing enhancer - Google Patents

Abstract

The invention belongs to the field of chemiluminescent reagents, and discloses an application of indole derivatives as a chemiluminescent enhancer and a kit containing the enhancer. In the application, the indole derivative is used as a second reinforcing agent and is combined with a phenothiazine compound as a first reinforcing agent; further, the phenothiazine compound is selected from one of 3- (10-phenothiazinyl) propane-1-sodium sulfonate, 10- (3-dimethylaminopropyl) phenothiazine and derivatives thereof; further, the indole derivatives are selected from one or more of tryptophan, 5-hydroxytryptamine, auxin and indole-3-acetic acid. The invention utilizes the combination of phenothiazine compounds and indole derivatives to exert synergistic effect, thereby not only improving the brightness of the luminescent reagent, but also prolonging the luminescent time due to the mutual transmission of electrons.

Description

Use of indole derivatives as chemiluminescence enhancer and kit containing enhancer

Technical Field

The invention belongs to the field of chemiluminescent reagents, and discloses an application of indole derivatives as a chemiluminescent enhancer and a kit containing the enhancer.

Background

Horse Radish Peroxidase (HRP) catalyzed luminol and peroxy compound chemiluminescence reaction is widely applied to detection and analysis of antigens and antibodies, especially enzyme-linked immunosorbent assay (ELISA) and immunoblotting assay. Horseradish peroxidase was conjugated to an antigen or antibody, and the amount of antigen or antibody was analyzed indirectly by measuring the amount of HRP.

Luminol as a classical chemiluminescent reaction, a number of compounds have been successfully used to enhance HRP-induced chemiluminescence, including: p-iodophenol, p-coumarin and other phenolic enhancers, and acetanilides and phenothiazines besides the phenolic enhancers, wherein the phenothiazines belong to the more newly discovered enhancers. Phenothiazines have a stronger luminescence intensity compared to the remaining luminescence enhancers, especially a stronger luminescence intensity and luminescence stability when an enhancer such as imidazole, 4-morpholinopyridine is added as a second enhancer, i.e., a co-enhancer.

However, the current phenothiazine-type enhancers and the reported second enhancers have a large increase in brightness, but poor stability. Therefore, on the basis of the theory of new enhancers and second enhancers, it is still necessary to develop new enhancer combinations to achieve improved stability of chemiluminescence without reducing the sensitivity of chemiluminescence too much, ensuring that the luminescence intensity decays less than 10% within two hours.

Disclosure of Invention

In view of the above situation, the present invention discloses the use of indole derivatives as a chemiluminescence enhancer, and a kit comprising the enhancer.

The technical scheme of the invention is as follows:

use of an indole derivative as a second enhancer in combination with a phenothiazine compound as a first enhancer as a chemiluminescence enhancer.

In the above use, the phenothiazine compound is selected from one of sodium 3- (10-phenothiazinyl) propane-1-sulfonate, 10- (3-dimethylaminopropyl) phenothiazine and a derivative thereof.

Further, in the above use, the indole derivative is selected from one or more of tryptophan, 5-hydroxytryptamine, auxin and indole-3-acetic acid.

Further, in the above use, the final concentration of the indole derivative in the chemiluminescent reagent is between 1 and 30 mM.

Further, a chemiluminescent reagent comprising a phenothiazine compound as a first enhancer; indole derivatives as second enhancers; luminol, isoluminol or a luminol-related derivative as a luminescent substrate.

Further, a chemiluminescence kit comprises the chemiluminescence reagent.

Furthermore, the chemiluminescence kit comprises a solution A and a solution B,

the solution A comprises a chemiluminescent reagent, a buffering agent A and a humectant;

the liquid B comprises a hyperoxidant, a stabilizer and a buffer B.

Furthermore, the chemiluminescence kit can be used for preparing a fluorescent material,

the liquid A comprises:

1) and a luminescent substrate: selected from luminol, isoluminol or a luminol-related derivative;

2) and a first enhancer: one selected from 3- (10-phenothiazinyl) propane-1-sodium sulfonate, 10- (3-dimethylaminopropyl) phenothiazine and derivatives thereof;

3) and a second enhancer: one or more selected from tryptophan, 5-hydroxytryptamine, auxin, and indole-3-acetic acid;

4) and a buffer A: a buffer at a pH between 8 and 9.5;

the liquid B comprises:

1) and a peroxide: one selected from hydrogen peroxide, urea hydrogen peroxide or perborate;

2) and a stabilizer: one selected from ethylenediamine tetraacetic acid, cyclohexane ethylenediaminetetraacetic acid, ethylene glycol tetraacetic acid and diethyl triaminepentaacetic acid;

3) and a buffer B: the pH value is 2-6, and is selected from acetic acid, benzoic acid, phosphoric acid, and citric acid.

Further, in the above chemiluminescence kit, the solution a further comprises: 5) and (3) a humectant: one or more selected from glycerol, hyaluronic acid and propylene glycol.

Further, the chemiluminescence kit preferably comprises the following components:

the liquid A and the liquid B are mixed before use, and the mixing proportion is determined according to the final concentration and is not limited to a specific mixing proportion.

Wherein the solution A comprises the following components:

1) and a luminescent substrate comprising luminol, isoluminol and luminol-related derivatives, wherein the dosage is as follows: between 0.5 and 10mM

2) And a first enhancer: belongs to phenothiazine compounds, one of 3- (10-phenothiazinyl) propane-1-sodium sulfonate, 10- (3-dimethylaminopropyl) phenothiazine and derivatives thereof, and the dosage is as follows: between 1 and 20 mM.

3) And a second enhancer: belongs to indole derivatives, mainly comprisesTryptophan5-hydroxytryptamine, auxin, and indole-3-acetic acid in an amount of 1-30 mM.

4) And a buffer A: mainly selected from common buffer solutions with the buffering range between pH8-9.5, including Tris-HCl, borate and the like, and the dosage is as follows: 10-200 mM.

5) And (3) a humectant: comprises glycerol, hyaluronic acid and propylene glycol, and the dosage is between 500 and 2000 mM.

Wherein the liquid B comprises the following components:

1) the peroxidating agent comprises one of hydrogen peroxide, urea hydrogen peroxide and perborate, and the dosage of the peroxidating agent is between 0.01mM and 10 mM.

2) The stabilizer is also a chelating agent and is one of ethylenediamine tetraacetic acid, cyclohexane ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid and diethyl triaminepentaacetic acid, and the dosage is 0.001mM-0.1 mM.

3) And a buffer B with a pH value ranging from 2 to 6, wherein the buffer B is one selected from acetic acid, benzoic acid, phosphoric acid and citric acid and is used in an amount of 10mM to 100 mM.

Further, the chemiluminescence reagent or chemiluminescence kit is used for preparing a kit for an immunoblotting experiment or an enzyme-linked immunosorbent assay.

The invention has the following beneficial effects:

in the prior art, the single luminol reacts with the peroxide compound, so that the quantum yield of the luminescence reaction is low and the reaction speed is high. The addition of the enhancer makes the chemiluminescence oxidation faster, brighter and more stable. According to the existing chemiluminescence principle, the mechanism can be called as 'ping-pong' mechanism, horseradish peroxidase is activated into an oxidation intermediate through hydrogen peroxide in the luminescence process, an enhancer is oxidized into a free radical product through the oxidation intermediate of the enzyme, the product can be in reversible reaction with luminol molecules, finally, after the luminol is subjected to multi-step basic reaction, the excited state of 3-aminophthalate is generated along with the generation of molecular nitrogen, and blue light with the wavelength of 425nm is released when the luminol returns to the ground state. The principle of the synergistic reaction is shown in the attached FIG. 1, and can be referred to in the literature (Development of a high purity biochemical enzyme immunoassay enhanced luminescence as substrate.)

The invention provides a sensitive chemiluminescence reagent with stable luminescence, which is used for detecting horseradish peroxidase in a sample. The invention highlights the fact that by proposing a new second enhancer, in combination with the already reported first enhancer, the light emitted by the system is stabilized, ensuring that the sensitivity is not reduced too much. The new second enhancer is DL-tryptophan or L-tryptophan, and can be extended to indole derivatives such as 5-hydroxytryptamine, auxin, indole-3-acetic acid, etc.

On the basis of the formula, glycerol, hyaluronic acid, propylene glycol and the like are added into the reagent formula, so that the density of the substances is higher than that of pure water, and the deep layer bonding of the surface of the reagent solid phase carrier is increased in actual use; meanwhile, the moisture absorption and moisture retention function is achieved, so that long-time detection can be facilitated, and the problem of reagent volatilization is not worried about.

According to the invention, the phenothiazine compound and the indole derivative are used in combination, and the synergistic effect is exerted by combining the two reinforcing agents according to the 'ping-pong theory', so that the brightness is improved, and the luminescence time is prolonged due to the mutual transmission of electrons. Furthermore, the invention can also add humectant at the same time, which ensures the stability of the incubation of the reagent on the peroxidase, and simultaneously can prevent inaccurate luminescence detection result caused by excessive residual liquid of the carrier due to the increase of the density, and the improvement effect is most obvious particularly in an immunoblotting experiment. Therefore, the luminescent reagent can be suitable for various enzyme-linked immunosorbent assays and immunoblotting assays.

Drawings

FIG. 1 is a principle of a synergistic reaction;

fig. 2 is a luminescence detection diagram in embodiment 3, and the sample size on each diagram is respectively 100ug, 50ug, 25ug, 12.5ug, 6.25ug and 3.125ug from left to right.

Detailed Description

The test methods used in the following examples are all conventional methods unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention relates to a high-sensitivity signal-stable chemiluminescent reagent, which comprises a reagent A liquid and a reagent B liquid;

formulation I

Solution A:

luminol 5mM

Sodium 3- (10-phenothiazinyl) propane-1-sulfonate 10mM

5-hydroxytryptamine 10mM

Tris 100mM

The pH was adjusted to between 8.5 and 8.8 using 6M hydrochloric acid.

And B, liquid B:

0.01mM hydrogen peroxide

EDTA 0.002mM

Glycerol 300mM

Hyaluronic acid 0.1mM

Acetic acid-sodium acetate buffer 50mM pH 5.0-5.5

Formulation II

Solution A:

luminol 2mM

Sodium 3- (10-phenothiazinyl) propane-1-sulfonate 10mM

Tryptophan 15mM

Tris 100mM

The pH was adjusted to between 8.5 and 8.8 using 6M hydrochloric acid.

And B, liquid B:

0.01mM hydrogen peroxide

EDTA 0.002mM

Glycerol 300mM

Sodium benzoate solution 50mM pH 2-4

The formula III is as follows:

solution A:

luminol 10mM

Sodium 3- (10-phenothiazinyl) propane-1-sulfonate 10mM

Tryptophan 5mM

Indole-3-acetic acid 5mM

Tris 100mM

The pH was adjusted to between 8.5 and 8.8 using 6M hydrochloric acid.

And B, liquid B:

0.01mM hydrogen peroxide

EDTA 0.002mM

Propylene glycol 20mM

Sodium benzoate solution 50mM pH 2-4

The above formula is representative of one, and the compounds listed and corresponding concentration ranges can be combined into a formula.

Example 1

In this example, antigen-antibody detection was performed using formulation one.

The kit can be used for detecting various in-vitro antibody antigens, the antigen antibodies are coated in a white or black opaque polystyrene microporous plate, and then the content of the antigen or the antibody is detected through a coupled HRP marker, taking common alpha-fetoprotein (AFP) as an example.

The reference substance is a commercial alpha fetoprotein quantitative detection kit (chemiluminescence immunoassay (panoramic life)), a reagent for detecting and establishing a standard curve in a calibrator is used, the reagent is coated in a white polystyrene micropore plate, the concentration range is 0-500ng/ml, the reagent is incubated by using a corresponding antibody coupled with HRP, the luminous intensity of the reagent is detected by using a BioTek Synergy H1 full-function micropore plate detector, micropores with the concentration of 250ng/ml are selected, and the luminous value is detected once every 5min, and the measurement is carried out for 2 hours.

Results table 1 shows the concentration of alpha-fetoprotein (AFP) and the corresponding luminescence intensity,

TABLE 1 concentration of alpha-fetoprotein (AFP) and corresponding luminescence intensity

Concentration of calibrator (ng/ml)	Example formulation 1	Reference substance
			0	749	1325
15.625	28346	29259
			31.25	61263	51918
62.5	136642	106476
			125	270878	241821
250	537853	538905
			500	1352011	1015941

According to the above luminescence results, in the example formula I and the reference substance, the washing related to the linear fitting of the protein concentration and the luminescence value data is more than 0.95. However, the luminous value of the same concentration is larger than that of the contrast, and the standard curve has larger slope, which indicates that the detection result of the formula has higher accuracy and sensitivity.

Example 2

The second formula is used for comparison of the chemiluminescence reagent thermal stability experiment

Preparing reagent according to formula II, placing the obtained reagent at 37 deg.C for 4 days, 8 days, and 12 days, respectively, comparing with the luminous reagent prepared on the same day, directly using horse radish peroxidase dissolved in physiological saline, and diluting to 1X10^-15And (3) taking 100ul of the luminescent liquid A and the luminescent liquid B which are different in standing days respectively for standby application, mixing, adding the mixture into a white polystyrene micropore plate, and then adding the diluted horseradish peroxidase into each hole. The luminescence value was measured using a BioTek Synergy H1 full-function microplate detector. The results are shown in Table 2.

TABLE 2 chemiluminescence reagent thermal stability experiment

From the above results, it was found that the luminescent reagent is stable in properties and can be stored for a long period of time.

Example 3

In the embodiment, the third formula is used, and the detection effect of the western blot detection is realized

The specific operation process is as follows:

selecting mouse liver tissue, extracting protein with RIPA lysate, detecting protein concentration, and calculating protein adding amount of 100ug, 50ug, 25ug, 12.5ug, 6.25ug and 3.125ug for each well according to the detected protein concentration. Protein electrophoresis and membrane transfer were performed according to the conventional procedure using NC membrane, which was closed with skim milk powder and incubated with Histone H3 primary antibody overnight, followed by HRP-labeled secondary antibody and luminescence detection using a luminescence reagent. The selected control products were imported good-selling products: SuperSignal West Dura chemiluminescent substrate. In order to ensure that the result is always consistent, the same NC membrane is used in detection, and different luminous liquids are repeatedly used for detection and comparison.

As shown in FIG. 2, the exposure time was 30s, and the stability of the product of the present invention was slightly higher than that of the control.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

Claims (10)

1. Use of an indole derivative as a chemiluminescent enhancer characterized in that the indole derivative is used as a second enhancer in combination with a phenothiazine compound as a first enhancer.

2. The use according to claim 1, wherein the phenothiazine-like compound is selected from the group consisting of sodium 3- (10-phenothiazinyl) propane-1-sulfonate, 10- (3-dimethylaminopropyl) phenothiazine, and derivatives thereof.

3. The use according to claim 1, wherein the indole derivative is selected from one or more of tryptophan, 5-hydroxytryptamine, auxin or indole-3-acetic acid.

4. Use according to claim 1, wherein the final concentration of the indole derivative in the chemiluminescent reagent is between 1 and 30 mM.

5. A chemiluminescent reagent comprising a phenothiazine compound as a first enhancer; indole derivatives as second enhancers; luminol, isoluminol or a luminol-related derivative as a luminescent substrate.

6. A chemiluminescent kit comprising the chemiluminescent reagent of claim 5.

7. A chemiluminescent kit according to claim 6 comprises solution A and solution B, wherein the solution A comprises a chemiluminescent reagent, a buffer A and a humectant;

the liquid B comprises a hyperoxidant, a stabilizer and a buffer B.

8. A chemiluminescent kit according to claim 7,

the liquid A comprises:

1) and a luminescent substrate: selected from luminol, isoluminol or a luminol-related derivative;

2) and a first enhancer: one selected from 3- (10-phenothiazinyl) propane-1-sodium sulfonate, 10- (3-dimethylaminopropyl) phenothiazine and derivatives thereof;

3) and a second enhancer: one or more selected from tryptophan, 5-hydroxytryptamine, auxin, and indole-3-acetic acid;

4) and a buffer A: a buffer at a pH between 8 and 9.5;

the liquid B comprises:

1) and a peroxide: one selected from hydrogen peroxide, urea hydrogen peroxide or perborate;

2) and a stabilizer: one selected from ethylenediamine tetraacetic acid, cyclohexane ethylenediamine tetraacetic acid, ethylene glycol tetraacetic acid, diethyl triamine pentaacetic acid;

3) and a buffer B: the pH value is 2-6, and is selected from acetic acid, benzoic acid, phosphoric acid, and citric acid.

9. The chemiluminescent kit according to claim 8, wherein the solution A further comprises: 5) and a humectant: one or more selected from glycerol, hyaluronic acid and propylene glycol.

10. Use of a chemiluminescent reagent or chemiluminescent kit of any one of claims 5 to 9 in the manufacture of a kit for immunoblotting or enzyme-linked immunosorbent assays.

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