Alkaline phosphatase luminescent substrate, composition thereof, kit, preparation method and application

Abstract

The invention discloses an alkaline phosphatase luminescent substrate as well as a composition, a kit, a preparation method and application thereof. The structural formula of the alkaline phosphatase luminescent substrate is as follows: # imgabs0 #. The alkaline phosphatase luminescent substrate is stable in structure and suitable for being preserved in an environment of about 4 DEG C, and compared with APS-5 which must be preserved at-20 DEG C, the alkaline phosphatase luminescent substrate has the advantages that the preservation conditions are milder, and convenience is provided for use.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical detection, and in particular to an alkaline phosphatase luminescent substrate, a composition and a kit comprising the substrate, and a preparation method and application of the alkaline phosphatase luminescent substrate. Background Art

[0002] Chemiluminescence is the phenomenon of emitting visible light during a chemical reaction (mainly a redox reaction). As early as the 1870s, Radzisewski et al. (1877) discovered that when luminol was oxidized and reduced with hydrogen peroxide in an alkaline medium, photons were generated (green light was emitted). Albrecht (1928) proved that luminol has a luminescent effect in an alkaline medium. Glue and Petsh (1935) were the first to report that lucigenin reacted with hydrogen peroxide under alkaline conditions to produce chemiluminescence.

[0003] Chemiluminescence reaction consists of two key steps: excitation and emission. Many chemical reactions can release enough free energy to excite one of the substances involved in the reaction to an electronic excited state that can emit light, generating an excited state product (c). When it returns to the ground state, the remaining energy is converted into photon energy (h) to produce luminescence, that is, A+B-c, then c-C+h, where c is in a singlet excited state. The spectrum of chemiluminescence is consistent with the fluorescence of c.

[0004] With the advancement of chemiluminescent material synthesis technology, chemiluminescence is increasingly used in biomedicine and other fields. The chemiluminescence immunoassay and chemiluminescence labeling established by combining chemiluminescence with immune response are the latest detection technologies developed after the three major labeling technologies of fluorescent labeling, radionuclide labeling, and enzyme labeling. Chemiluminescence and chemiluminescence immunoassay can detect a variety of substances, and their detection limit reaches 10 -19 mol.

[0005] Alkaline phosphatase luminescent system is a widely used type. Alkaline phosphatase is an enzyme that can dephosphorylate the corresponding substrate, that is, it removes the phosphate group on the substrate molecule by hydrolyzing the phosphate monoester and generates phosphate ions and free hydroxyl groups. The process of removing the phosphate group is called dephosphorylation or dephosphorylation. Alkaline phosphatase luminescent substrate can quickly release photons under the action of alkaline phosphatase. Within a specific alkaline phosphatase concentration range, the number of photons released is proportional to the concentration of alkaline phosphatase in the solution.

[0006] (4-chlorophenylmercapto) (10-methyl-9,10-dihydroacridinium methylene) phosphate disodium salt, referred to as APS-5, is a commonly used alkaline phosphatase luminescent substrate. For example, CN117186878 A discloses an alkaline phosphatase enzymatic chemiluminescent substrate solution and its preparation method and application, the enzymatic chemiluminescent substrate solution includes sodium tridecyl sulfate, zinc chloride, sodium thiosulfate, 9-(4-chlorophenylmercapto) (10-methyl-9,10-dihydroacridinium methylene) phosphate disodium salt and luscin. CN111077140A discloses an enzymatic chemiluminescent substrate solution preparation method and filling device, wherein the enzymatic chemiluminescent substrate solution includes the following components: luminescent substrate, luminescent enhancer, background inhibitor, surfactant, enzyme stabilizer, preservative and buffer matrix; the luminescent substrate is (4-chlorophenylmercapto) (10-methyl-9,10-dihydroacridinium methylene) phosphate disodium salt. CN109490281A discloses a chemiluminescent substrate solution, which includes the following components: 150 mg / L of (4-chlorophenylmercapto) (10-methyl-9,10-dihydroacridinium methylene) phosphate disodium salt, 10 mmol / L-500 mmol / L of buffer matrix, 0.1 mg / L-20 mg / L of fluorescence enhancing substance, 10 mg / L-5000 mg / L of background luminescence suppressing substance and 0.1 g / L-10 g / L of surfactant. APS-5 has the advantages of high detection sensitivity and short reaction time. However, APS-5 is unstable and needs to be stored at -20°C, which brings inconvenience to transportation and use. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the present invention provides an alkaline phosphatase luminescent substrate, which integrates two APS-5 in the same structure, and the obtained new compound has a stable structure and can increase the luminescence intensity. To this end, the present invention also provides a composition including the alkaline phosphatase luminescent substrate and a chemiluminescent detection kit containing the substrate; in addition, the present invention also provides a preparation method of the alkaline phosphatase luminescent substrate.

[0008] In a first aspect of the present invention, there is provided an alkaline phosphatase luminescent substrate, the structural formula of which is as follows:

[0009] Wherein, n1 and n2 are selected from integers of 1-10.

[0010] The second aspect of the present invention provides an alkaline phosphatase luminescent substrate composition, which comprises the above-mentioned alkaline phosphatase luminescent substrate.

[0011] The third aspect of the present invention provides an alkaline phosphatase luminescent substrate composition, comprising 0.01mol / L-0.5mol / L tris buffer, 0.05%-1% Tween 20, 10mg / L-100mg / L sodium sulfite, 5mg / L-10mg / L lucigenin and 100mg / L-300mg / L of the above-mentioned alkaline phosphatase luminescent substrate, and the above-mentioned percentages are mass percentages.

[0012] As a preferred embodiment, the alkaline phosphatase luminescent substrate composition includes 0.2 mol / L tris buffer, 0.1% Tween 20, 10 mg / L sodium sulfite, 5 mg / L lucigenin and 100 mg / L of the above alkaline phosphatase luminescent substrate, and the above percentages are mass percentages.

[0013] The fourth aspect of the present invention provides a chemiluminescent detection kit, comprising the above-mentioned alkaline phosphatase luminescent substrate or the above-mentioned alkaline phosphatase luminescent substrate combination.

[0014] The fifth aspect of the present invention provides a method for preparing the above-mentioned alkaline phosphatase luminescent substrate, comprising the following steps:

[0015] S1. Compound A reacts with succinimide trifluoroacetic acid under the action of a catalyst to generate compound B; S2, dissolving compound C in a solvent and reacting with compound D to generate compound E; S3, reacting compound B with compound E under nitrogen protection to generate compound F; S4, adding sodium hydroxide to compound F to obtain compound G; Wherein, n1 and n2 are selected from integers of 1-10.

[0016] As a preferred embodiment, the above step S1 satisfies one or more of the following conditions: (1) The molar ratio of compound A to succinimide trifluoroacetic acid is 1:1.2 to 1:1.5; (2) The catalyst is selected from pyridine; (3) The molar ratio of compound A to catalyst is 1:0.1 to 1:0.2.

[0017] As a preferred embodiment, the above step S2 satisfies one or more of the following conditions: (1) The molar ratio of compound C to compound D is 1:1.5 to 1:2; (2) The solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or tetrahydrofuran; (3) The reaction temperature is -10~-5℃.

[0018] As a preferred embodiment, the molar ratio of compound B to compound E in the above step S3 is 1:2.5~1:3.

[0019] The sixth aspect of the present invention provides the use of the above-mentioned alkaline phosphatase luminescent substrate or alkaline phosphatase luminescent substrate composition or chemiluminescent detection kit in the preparation of products for detecting thyroid stimulating hormone content, carcinoembryonic antigen content, and alpha-fetoprotein content.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The alkaline phosphatase luminescent substrate of the present invention has a stable structure and can be stored at about 4°C. Compared with APS-5 which must be stored at -20°C, the storage conditions are milder, which provides convenience for transportation and use.

[0021] (2) The alkaline phosphatase luminescent substrate of the present invention can improve the efficiency of generating photons, has the effect of enhancing chemiluminescent signals, and improves the accuracy and sensitivity of detection.

[0022] (3) The method for preparing the alkaline phosphatase luminescent substrate of the present invention has readily available raw materials and does not require harsh reaction conditions, thereby reducing production costs. DETAILED DESCRIPTION

[0023] In order to make the technical means, creative features, objectives and effects of the invention easier to understand, the present invention is further described. However, the present invention is not limited to the following implementation cases.

[0024] It should be noted that the structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology, and are not used to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any structural modification, change in proportion or adjustment of size should still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0025] The structural formula of APS-5 is as follows: As a luminescent substrate for alkaline phosphatase, APS-5 has the advantages of high sensitivity and short reaction time. However, APS-5 is unstable and needs to be stored at a low temperature of -20°C, which brings inconvenience to transportation, storage and use. The inventors of the present application tried to integrate two APS-5 into the same structure to increase the stability of APS-5.

[0026] The present invention provides an alkaline phosphatase luminescent substrate, the structural formula of which is as follows:

[0027] Wherein, n1 and n2 are selected from integers of 1-10.

[0028] The present invention also provides an alkaline phosphatase luminescent substrate composition, which comprises the alkaline phosphatase luminescent substrate mentioned above.

[0029] The above alkaline phosphatase luminescent substrate is combined with other commonly used auxiliary agents, such as buffer, surfactant, catalyst, etc. to prepare a composition, which will emit fluorescence in the environment where alkaline phosphatase is located, and can be used to determine the content of thyroid stimulating hormone, carcinoembryonic antigen, alpha-fetoprotein, etc. In this application, the alkaline phosphatase luminescent substrate can be used instead of APS-5, and the selection of other auxiliary agents can refer to APS-5.

[0030] The present invention also provides an alkaline phosphatase luminescent substrate composition, comprising 0.01mol / L-0.5mol / L tris buffer, 0.05%-1% Tween 20, 10mg / L-100mg / L sodium sulfite, 5mg / L-10mg / L lucigenin and 100mg / L-300mg / L of the above alkaline phosphatase luminescent substrate, and the above percentages are mass percentages.

[0031] As a preferred embodiment, the alkaline phosphatase luminescent substrate composition includes 0.2 mol / L tris buffer, 0.1% Tween 20, 10 mg / L sodium sulfite, 5 mg / L lucigenin and 100 mg / L of the above alkaline phosphatase luminescent substrate, and the above percentages are mass percentages.

[0032] The present invention provides a chemiluminescent detection kit, comprising the above alkaline phosphatase luminescent substrate or the above alkaline phosphatase luminescent substrate composition.

[0033] The present invention also provides a method for preparing the alkaline phosphatase luminescent substrate, comprising the following steps:

[0034] S1. Compound A reacts with succinimide trifluoroacetic acid under the action of a catalyst to generate compound B; S2, dissolving compound C in a solvent and reacting with compound D to generate compound E; S3, reacting compound B with compound E under nitrogen protection to generate compound F; S4, adding sodium hydroxide to compound F to obtain compound G; Wherein, n1 and n2 are selected from integers of 1-10.

[0035] As a preferred embodiment, in step S1, the molar ratio of compound A to succinimide trifluoroacetic acid is 1:1.2 to 1:1.5.

[0036] As a preferred embodiment, in step S1, the catalyst is selected from pyridine.

[0037] As a preferred embodiment, the molar ratio of compound A to catalyst in step S1 is 1:0.1 to 1:0.2.

[0038] As a preferred embodiment, the molar ratio of compound C to compound D in step S2 is 1:1.5~1:2.

[0039] As a preferred embodiment, in step S2, the solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or tetrahydrofuran.

[0040] As a preferred embodiment, the reaction temperature in step S2 is -10~-5°C.

[0041] As a preferred embodiment, in step S3, the molar ratio of compound B to compound E is 1:2.5 to 1:3; The present invention also provides the use of the above-mentioned alkaline phosphatase luminescent substrate or alkaline phosphatase luminescent substrate composition or chemiluminescent detection kit in the preparation of products for detecting thyroid stimulating hormone content, carcinoembryonic antigen content, and alpha-fetoprotein content. Example 1

[0042] 1. Synthesis of Compound B

[0043] 100 mg of compound A was dissolved in 2 mL of dry N,N-dimethylformamide, and then 0.5 mL of dry pyridine and 80 mg of succinimide trifluoroacetate were added. The mixture was stirred at room temperature for 5 hours, filtered and dried to obtain 90 mg of compound B.

[0044] NMR data of compound B: 1 HNMR (400MHz, DMSO-d6) δ8.5~8.6(m,4H), 8.35~8.45(m,4H), 7.9~8.25(m,8H), 5.9~6.05(s,4H), 2.7~3.0(m,8H).

[0045] 2. Synthesis of Compound E

[0046] 200 mg of compound C was dissolved in 3 mL of dry pyridine, cooled to -10~-5°C, 3 mL of dry pyridine solution (containing 150 mg of phosphorus oxychloride) was slowly added with a syringe, the temperature was maintained and stirred for 6 hours, compound D was added, and the reaction was stirred overnight until the reaction was complete. The reaction solution was poured into an ice-water mixture, extracted with ethyl acetate, and the organic phase was washed with ammonium chloride solution. After concentration, rapid column chromatography was performed to obtain 150 mg of compound E.

[0047] NMR data of compound E: 1 HNMR(400MHz, DMSO-d6) δ7.8~7.9(m,2H), 7.45~7.55(m,2H), 7.15~7.35(m,4H), 4.0~4.2(m,6H), 3.27~3.35(m,2H), 3.15~3.25(s,3H), 1.2~1.4(m,6H).

[0048] 3. Synthesis of Compound F

[0049] 40 mg of compound B was added to 3 ml of dry N,N-dimethylformamide, and 90 mg of compound E was added under nitrogen protection. The mixture was stirred overnight. 10 ml of ethyl acetate was added to the reaction solution, and the mixture was stirred at room temperature for half an hour to precipitate the product. The product was filtered and washed with ethyl acetate to obtain 120 mg of compound F.

[0050] NMR data of compound F: 1 HNMR (400MHz, DMSO-d6) δ8.5~8.6(m,4H), 8.35~8.45(m,4H), 7.9~8.25(m,8H), 7.5~7.65(m,1H), 7.0~7.45(m,12H), 5.25~5.35 (d,4H),4.15~4.35(m,4H),3.4~3.5(m,5H),3.25~3.5(s,6H),3.1~3.2(s,6H),1.2~1.4(m,12H).

[0051] 4. Synthesis of Compound G

[0052] 100 mg of compound F was added to a mixed solution of 3 ml of acetone and 2 ml of pure water, and 30 mg of sodium hydroxide was added. The mixture was stirred for 12 hours in the dark, and filtered to obtain 100 mg of compound G as a dark brown powder.

[0053] NMR data of compound G: 1HNMR (400MHz, D2O) δ8.6~8.7(m,4H), 8.35~8.45(m,4H),8.1~8.35(m,8H), 7.6~7.7(m,1H),7.0~7.7(m,12H), 5.2~5.3(d,4H), 4.2~4.5(m,4H), 3.4~3.5(m,5H), 3.2~3.3(s,6H). Example 2

[0054] The influence of different values ​​of n1 and n2 of compound G on the test results is investigated.

[0055] Alkaline phosphatase luminescent substrate composition: pH 9.0 0.2 mol / L tris, 0.1% Tween 20, 100 mg / L compound G (different n2 values, n1=n2), 10 mg / L sodium sulfite, 5 mg / L lucigenin.

[0056] Take 20 μL of the sample containing AFP, add 50 μL of the magnetic bead solution labeled with the corresponding antibody and 50 μL of the alkaline phosphatase mixed solution labeled with the corresponding antibody (the alkaline phosphatase mixed solution includes 0.01 mol / L PBS, 1% bovine serum albumin by mass, 1% alkaline phosphatase by mass, and 0.1% biological preservative by mass), react for 15 minutes, separate and wash the magnetic beads, add the magnetic beads to 100 μL of the alkaline phosphatase luminescent substrate composition, and use a chemiluminescent immunoassay to detect. The measurement results are shown in Tables 1 and 2: Table 1

[0057] Table 2

[0058] It can be seen from Tables 1 and 2 that when the n2 value of G is 1-10, the maximum value of the 1000 / 0 ratio is 11793 and the minimum value is 10019, and the signal change is within 20%; when the n value is greater than 10, the 1000 / 0 value changes greatly, so the optimal n2 value is in the range of 1-10, and when the n2 value is 2, the optimal signal-to-noise ratio can be obtained. Example 3

[0059] Formulation 1: pH 10.0 0.2 mol / L tris, 0.1% Tween 20, 100 mg / L compound G, 10 mg / L sodium sulfite, 5 mg / L lucigenin.

[0060] Formula 2: pH 10.0 0.2 mol / L tris, 0.1% Tween 20, 100 mg / L patent US5772926A compound, 10 mg / L sodium sulfite, 5 mg / L lucigenin.

[0061] Formula 3: pH 10.0 0.2 mol / L tris, 0.1% Tween 20, 100 mg / L patented CN1180349A compound, 10 mg / L sodium sulfite, 5 mg / L lucite.

[0062] Formula 4: pH 10.0 0.2 mol / L tris, 0.1% Tween 20, 100 mg / L patent CN115433229A compound, 10 mg / L sodium sulfite, 5 mg / L lustrous essence.

[0063] Formula 5: pH 10.0 0.2 mol / L tris, 0.1% Tween 20, 100 mg / L patent CN104418887A compound, 10 mg / L sodium sulfite, 5 mg / L lucite.

[0064] Formulas 1-5 were used to measure the levels of TSH (thyroid stimulating hormone), CEA (carcinoembryonic antigen), and AFP (alpha-fetoprotein).

[0065] Test method: Take 20 μL of sample (such as TSH), add 50 μL of magnetic bead solution labeled with corresponding antibody and 50 μL of alkaline phosphatase mixed solution labeled with corresponding antibody (the alkaline phosphatase mixed solution includes 0.01 mol / L PBS, 1% bovine serum albumin, 1% alkaline phosphatase, and 0.1% biological preservative), react for 15 minutes, separate the magnetic beads, wash them 3 times with 0.01 mol / L PBS solution, add the magnetic beads to 100 μL of liquid of formula 1-5, and detect them with chemiluminescence immunoassay. The test results are shown in Table 3: Table 3

[0066] In Table 3, 1000 / 0 and 100 / 0 respectively represent the photon signal of 1000 mg / L divided by the photon signal of 0 mg / L and the photon signal of 100 mg / L divided by the photon signal of 0 mg / L, which reflects the signal-to-noise ratio. The higher the value, the smaller the noise impact.

[0067] As can be seen from Table 3, when testing the AFP (alpha-fetoprotein) content, the corresponding signal-to-noise ratios of Formula 1-Formula 5 were 14494, 4351, 6563, 4380, and 6614, respectively; when testing the CEA (carcinoembryonic antigen) content, the corresponding signal-to-noise ratios of Formula 1-Formula 5 were 14049, 4586, 6484, 4623, and 5895, respectively; when testing the TSH (thyroid stimulating hormone) content, the corresponding signal-to-noise ratios of Formula 1-Formula 5 were 14268, 4558, 5885, 4760, and 6237, respectively. It can be seen that when Formula 1 is testing the TSH (thyroid stimulating hormone), CEA (carcinoembryonic antigen), and AFP (alpha-fetoprotein) content, the noise effect is minimal, and the test results are more stable and reliable. Example 4

[0068] The stability of the alkaline phosphatase luminescent substrate G prepared in the present application (prepared in Example 1) was compared with that of APS-5.

[0069] Formulation 1: pH 10.0 0.2 mol / L tris, 0.1% Tween 20, 100 mg / L compound G, 10 mg / L sodium sulfite, 5 mg / L lucigenin.

[0070] Formula 1-1: pH 10.0 0.2 mol / L tris, 0.1% Tween 20, 100 mg / L APS-5, 10 mg / L sodium sulfite, 5 mg / L lucigenin.

[0071] The solutions of Formula 1 and Formula 1-1 were placed at 37° C. for 0 days, 3 days, 7 days, and 10 days, and the AFP (alpha-fetoprotein) content was tested using the same method as Example 3. The test results after 0 days, 3 days, 7 days, and 10 days are shown in Table 4.

[0072] Table 4

[0073] It can be seen from Table 4 that the signal-to-noise ratios (1000 / 0) of the solutions of Formula 1 were 14104, 14548, 14716, and 14584, respectively, when placed for 0-10 days; while the signal-to-noise ratios (1000 / 0) of the solutions of Formula 1-1 were 9448, 8996, 7851, and 6794, respectively, when placed for 0-10 days, indicating that the solution of Formula 1 has better stability.

[0074] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any person skilled in the art can make a modification and change based on the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art.

Claims

1. An alkaline phosphatase luminescent substrate, characterized in that: The structural formula is as follows: ； Wherein, n1 and n2 are selected from integers of 1-10.

2. An alkaline phosphatase luminescent substrate composition, characterized in that: The invention comprises the alkaline phosphatase luminescent substrate according to claim 1.

3. An alkaline phosphatase luminescent substrate composition, characterized in that: It comprises 0.01mol / L-0.5mol / L tris buffer, 0.05%-1% Tween 20, 10mg / L-100mg / L sodium sulfite, 5mg / L-10mg / L lucigenin and 100mg / L-300mg / L alkaline phosphatase luminescent substrate according to claim 1, and the above percentages are mass percentages.

4. The alkaline phosphatase luminescent substrate composition according to claim 3, characterized in that: The composition comprises 0.2 mol / L tris buffer, 0.1% Tween 20, 10 mg / L sodium sulfite, 5 mg / L lucigenin and 100 mg / L alkaline phosphatase luminescent substrate, and the above percentages are mass percentages.

5. A chemiluminescent detection kit, characterized in that: It comprises the alkaline phosphatase luminescent substrate according to claim 1 or the alkaline phosphatase luminescent substrate composition according to any one of claims 2-4.

6. A method for preparing the alkaline phosphatase luminescent substrate according to claim 1, characterized in that: The steps include: ； S1. Compound A reacts with succinimide trifluoroacetic acid under the action of a catalyst to generate compound B; S2, dissolving compound C in a solvent and reacting with compound D to generate compound E; S3, reacting compound B with compound E under nitrogen protection to generate compound F; S4, adding sodium hydroxide to compound F to obtain compound G; Wherein, n1 and n2 are selected from integers of 1-10.

7. The method for preparing the alkaline phosphatase luminescent substrate according to claim 6, characterized in that: The S1 step satisfies one or more of the following conditions: (1) The molar ratio of compound A to succinimide trifluoroacetic acid is 1:1.2 to 1:1.5; (2) The catalyst is selected from pyridine; (3) The molar ratio of compound A to catalyst is 1:0.1 to 1:0.

2.

8. The method for preparing the alkaline phosphatase luminescent substrate according to claim 6, characterized in that: The S2 step satisfies one or more of the following conditions: (1) The molar ratio of compound C to compound D is 1:1.5 to 1:2; (2) The solvent is selected from N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide or tetrahydrofuran; (3) The reaction temperature is -10~-5℃.

9. The method for preparing the alkaline phosphatase luminescent substrate according to claim 6, characterized in that: In step S3, the molar ratio of compound B to compound E is 1:2.5 to 1:

3.

10. Use of the alkaline phosphatase luminescent substrate according to claim 1, the alkaline phosphatase luminescent substrate composition according to any one of claims 2 to 4, or the chemiluminescent detection kit according to claim 5 in the preparation of products for detecting thyroid stimulating hormone content, carcinoembryonic antigen content, and alpha-fetoprotein content.

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