A diamine oxidase detection probe and kit

Abstract

The application provides a diamine oxidase detection probe and a kit. The diamine oxidase detection probe with the chemical structure shown in formula (I) can realize fluorescence color development, has an absorption peak value in the range of 592-602 nm, and the absorption peak value is near 596 nm. The diamine oxidase can oxidize the diamine oxidase detection probe shown in formula (I), so that the light absorption in the range of 592-602 nm disappears. The diamine oxidase detection probe with the chemical structure shown in formula (I) is stable in structure and is beneficial to storage. The diamine oxidase detection probe with the chemical structure shown in formula (I) can be applied to diamine oxidase detection. The application provides a detection method for taking the serum diamine oxidase (DAO) level as an early diagnosis index of intestinal mucosal injury.

Description

Technical Field

[0001] This invention relates to the field of fluorescence analysis and detection, specifically to a diamine oxidase detection probe and kit. Background Technology

[0002] Gut microbiota dysbiosis and impaired intestinal barrier function can trigger acute or chronic diseases. Gut microbiota dysbiosis can induce intestinal barrier dysfunction, and conversely, intestinal barrier dysfunction can exacerbate gut microbiota dysbiosis. If not addressed promptly, it can further induce enterogenic infections and even systemic inflammatory responses.

[0003] Intestinal ischemia / reperfusion injury (I / R) is commonly seen in various clinical situations, including severe trauma, shock, infection, strangulated intestinal obstruction, acute mesenteric ischemia, and cardiopulmonary bypass. With early treatment, acute intestinal ischemia can usually be reversed; however, delayed diagnosis can lead to multiple organ dysfunction or even failure, with an extremely high mortality rate. Studies have reported that serum diamine oxidase (DAO) levels have good clinical value as an early diagnostic indicator of intestinal mucosal injury. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a diamine oxidase detection probe and kit.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a diamine oxidase detection probe, characterized in that the chemical structure of the diamine oxidase detection probe is as shown in formula (Ⅰ);

[0006]

[0007] The diamine oxidase detection probe with the chemical structure shown in Formula (I) exhibits fluorescence and has an absorption peak in the range of 592–602 nm, with the peak value near 596 nm. Furthermore, diamine oxidase can oxidize the diamine oxidase detection probe shown in Formula (I), causing its absorption in the 592–602 nm range to disappear. The diamine oxidase detection probe with the chemical structure shown in Formula (I) is structurally stable and easy to store.

[0008] A diamine oxidase detection probe with a chemical structure as shown in formula (I) can be applied to the detection of diamine oxidase. This provides a detection method for serum diamine oxidase (DAO) levels as a diagnostic indicator of early intestinal mucosal damage.

[0009] A method for preparing a diamine oxidase detection probe with a chemical structure as shown in formula (Ⅰ) includes the following steps:

[0010] (1) Rhodamine B and 1,2-diaminonaphthalene were condensed to give the compound shown in formula (II).

[0011]

[0012] (2) The compound shown in (Ⅱ) is subjected to a reduction reaction.

[0013] Preferably, the reducing agent for reducing the compound shown in (II) is lithium aluminum hydride (LiAlH4).

[0014] Preferably, the solvent in the reaction environment of step (2) is tetrahydrofuran (THF).

[0015] Preferably, in step (1), Rhodamine B and 1,2-diaminonaphthalene are subjected to a BOP (benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate) condensation reaction in dichloroethane solvent to obtain the compound shown in formula (II).

[0016] The preparation of the diamine oxidase detection probe with the chemical structure shown in formula (I) is shown in formula (III):

[0017]

[0018] The present invention also provides a diamine oxidase detection kit, the diamine oxidase detection kit comprising the following components: diamine oxidase, diamine oxidase terminator, diamine oxidase detection probe as shown in formula (I) as substrate, buffer, and diamine oxidase detection probe as shown in formula (I) as standard.

[0019]

[0020] In the diamine oxidase assay kit, diamine oxidase is in powder form, while the other reagents are pre-prepared to specific concentrations using buffer solutions.

[0021] Preferably, the diamine oxidase is a dry powder, the diamine oxidase terminator is aminoguanidine or aminoguanidine hydrochloride, the concentration of the diamine oxidase terminator is 5-50 mmol / L, the pH of the buffer solution is 6.0-7.0, the concentration of the buffer solution is 10-50 mmol / L, and the concentration of the standard is 50-500 mmol / L.

[0022] Preferably, the concentration of the diamine oxidase terminator is 10–20 mmol / L, the pH of the buffer solution is 6.3–6.8, and the concentration of the buffer solution is 20–50 mmol / L.

[0023] This invention also provides a detection method for a diamine oxidase detection kit, the method comprising the following steps:

[0024] (1) Prepare 6–10 gradient standard solutions with concentrations of 0–2.5 mmol / L using buffer solution, and include solvent blank samples. Measure the absorbance (D0) of the gradient standard solutions containing solvent blank samples at 592–602 nm; measure the absorbance (D0) of the solvent blank sample (i.e., the 0 mmol / L gradient standard solution) at 592–602 nm. 空白 ;

[0025] (2) Mix 15-30 μL of gradient standard solution containing solvent blank sample with diamine oxidase in 150-500 μL of buffer system with pH 6.0-7.0 and incubate at 32-38℃ for 5-20 minutes.

[0026] (3) After incubation, add 5-20 μL of diamine oxidase terminator, which is prepared with 20-50 mmol / L buffer solution, and test the absorbance value D1 at 592-602 nm respectively.

[0027] (4) Function fitting of gradient standard solution concentration difference and absorbance value change before and after enzyme incubation reaction D0-D1-D 空白 The functional relationship between them;

[0028] (5) After performing steps (1), (2), and (3) on the sample to be tested, measure the absorbance value D of the sample before and after incubation. x0 D x1 According to Dx0-Dx1-D 空白 The functional relationship with step (3) is used to calculate the concentration difference of the sample solution before and after enzyme incubation; the diamine oxidase activity is obtained by balancing the concentration difference of the sample solution before and after enzyme incubation.

[0029] The diamine oxidase detection probe shown in formula (Ⅰ) is used as a standard.

[0030]

[0031] In the above method, diamine oxidase activity is expressed as the amount of substrate reduction per unit volume per unit time.

[0032] Preferably, the diamine oxidase terminator is aminoguanidine or aminoguanidine hydrochloride, the pH of the buffer solution is 6.0 to 7.0, the concentration of the buffer solution is 10 to 50 mmol / L, and the concentration of the diamine oxidase terminator is 5 to 50 mmol / L.

[0033] The beneficial effects of this invention are as follows: This invention provides a diamine oxidase detection probe and kit. The diamine oxidase detection probe, with a chemical structure as shown in Formula (I), is capable of fluorescence and has an absorption peak in the range of 592–602 nm, with the absorption peak near 596 nm. Furthermore, diamine oxidase can oxidize the diamine oxidase detection probe as shown in Formula (I), causing its absorbance in the 592–602 nm range to disappear. The diamine oxidase detection probe with a chemical structure as shown in Formula (I) has a stable structure, which is beneficial for storage. The diamine oxidase detection probe with a chemical structure as shown in Formula (I) can be used for diamine oxidase detection. This provides a detection method for serum diamine oxidase (DAO) levels as a diagnostic indicator for early intestinal mucosal damage. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the chemical structure of the diamine oxidase detection probe of the present invention.

[0035] Figure 2 This is a schematic diagram of the preparation process of the diamine oxidase detection probe of the present invention.

[0036] Figure 3 This is the absorption spectrum of the diamine oxidase detection probe of the present invention. Detailed Implementation

[0037] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0038] Example 1

[0039] As the diamine oxidase detection probe of Example 1 of the present invention, the chemical structure of the diamine oxidase detection probe is shown in Formula (I);

[0040]

[0041] The preparation method of the diamine oxidase detection probe with the chemical structure shown in formula (Ⅰ) includes the following steps:

[0042] (1) Rhodamine B (2 mmol) and 1,2-diaminonaphthalene (6 mmol) were condensed in dichloroethane solvent (50 mL) at a molar ratio of 1:3 via BOP (benzotriazole-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate) reaction. The reaction was stirred at 30 °C for 10 hours to obtain the compound shown in formula (II).

[0043]

[0044] (2) The solvent of the reagent after the reaction in step (1) was removed by vacuum evaporation. The compound (0.3 g) shown in (II) was reduced in 40 mL of tetrahydrofuran (THF) solvent with lithium aluminum hydride (0.2 g) as the reducing agent. The reaction mixture was extracted twice with 100 mL of dichloromethane. The organic phase was collected and dried with anhydrous magnesium sulfate. The solvent was removed by vacuum distillation. The crude product was obtained by column chromatography with CH2Cl2 / CH3CH2OH eluent at a volume ratio of 6:1 to 2:1. The chemical structure of the diamine oxidase detection probe is shown in formula (I), m / z (M+H + =569.3.

[0045] The preparation of the diamine oxidase detection probe with the chemical structure shown in formula (I) is shown in formula (III):

[0046]

[0047] Experimental methods

[0048] I. Absorption Spectroscopy Detection

[0049] The absorption spectrum of the diamine oxidase detection probe with a chemical structure as shown in formula (Ⅰ) is as follows: Figure 3 As shown, it can exhibit fluorescence and color development, with an absorption peak in the range of 592–602 nm, and the absorption peak is near 596 nm.

[0050] II. Diamine oxidase activity assay

[0051] DAO enzyme activity in the sample: One activity unit is defined as the DAO enzyme activity required per minute to hydrolyze 1 μmol of substrate per liter of the test sample at 37°C.

[0052] 1. Testing Method

[0053] The pH of the phosphate buffer is 6.5, and the concentration of the buffer is 25 mmol / L.

[0054] Includes the following steps:

[0055] (1) Prepare gradient standard solutions with concentrations of 0, 0.2, 0.5, 0.8, 1.2, 1.5, and 2.5 mmol / L using phosphate buffer, and measure the absorbance value D0 at 597 nm for each solution; the absorbance value D0 of the solvent blank sample (i.e., the gradient standard solution with a concentration of 0 mmol / L) at 597 nm is also measured. 空白 ;

[0056] (2) Mix 20 μL of gradient standard solution containing solvent blank sample with 30 μL of diamine oxidase solution and 250 μL of phosphate buffer with pH 6.5, and incubate at 37°C for 10 minutes; the concentration of diamine oxidase solution is 10 mg / mL;

[0057] (3) After incubation, add 10 μL of diamine oxidase terminator, which is prepared by phosphate buffer, and test the absorbance value D1 at 597 nm.

[0058] (4) Function fitting of gradient standard solution enzyme concentration difference and absorbance value change before and after enzyme incubation reaction D0-D1-D 空白 The functional relationship between them;

[0059] (5) After performing steps (1), (2), and (3) on the sample to be tested, measure the absorbance values ​​Dx0 and Dx1 of the sample before and after incubation. Then, calculate the absorbance values ​​based on Dx0-Dx1-Dx0. 空白 The concentration difference of the sample solution before and after enzyme incubation is calculated based on the functional relationship with step (3); the diamine oxidase activity is obtained by balancing the concentration difference of the sample solution before and after enzyme incubation; the diamine oxidase detection probe is used as a standard as shown in formula (I).

[0060] Fitting curve for standard sample: y = ax + b

[0061] x: The concentration difference of the known standard

[0062] a: Slope of the standard curve

[0063] b: Intercept of the standard curve

[0064] y: The absorbance difference before and after incubation of the corresponding standard (Dx0-Dx1-D blank), that is, the absorbance value before incubation minus the absorbance value after incubation, with solvent blank correction removed.

[0065] According to the standard curve experiment, y = 0.2389x - 0.0169. R 2 =0.9916.

[0066] It is known that diamine oxidase can oxidize the diamine oxidase detection probe shown in formula (I), causing it to lose absorbance in the 592–602 nm range, thus enabling its application in diamine oxidase detection. This provides a detection method for serum diamine oxidase (DAO) levels as a diagnostic indicator for early intestinal mucosal damage.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A diaminooxidase detection probe, characterized in that, The chemical structure of the diamine oxidase detection probe is shown in formula (I); Formula (I).

2. The diamine oxidase detection probe for use in the diagnosis and treatment of non-diseases according to claim 1.

3. The method of claim 1, wherein the diamine oxidase detection probe is prepared by the steps of: (a) synthesizing a diamine oxidase detection probe comprising a diamine oxidase substrate and a diamine oxidase inhibitor; and (b) purifying the diamine oxidase detection probe. The preparation method comprises the following steps: (1) condensation of rhodamine B and 1,2-diaminonaphthalene to obtain a compound shown in formula (II), Formula (II); (2) reduction of the compound shown in formula (II).

4. The method of claim 3, wherein the diamine oxidase detection probe is prepared by the steps of: (a) synthesizing a diamine oxidase detection probe comprising a diamine oxidase substrate and a diamine oxidase inhibitor; and (b) purifying the diamine oxidase detection probe. The reducing agent for the reduction of the compound shown in formula (II) is lithium aluminum hydride.

5. The method of claim 3, wherein the diamine oxidase detection probe is prepared by the steps of: (a) synthesizing a diamine oxidase detection probe comprising a diamine oxidase substrate and a diamine oxidase inhibitor; and (b) purifying the diamine oxidase detection probe. The reaction environment solvent in step (2) is tetrahydrofuran.

6. The method of claim 3, wherein the diamine oxidase detection probe is prepared by the steps of: (a) synthesizing a diamine oxidase detection probe comprising a diamine oxidase substrate and a diamine oxidase inhibitor; and (b) purifying the diamine oxidase detection probe. In step (1), rhodamine B and 1,2-diaminonaphthalene are subjected to BOP condensation reaction in dichloroethane solvent to obtain a compound shown in formula (II).

7. A diamine oxidase test kit characterized in that, The diamine oxidase detection kit comprises the following components: diamine oxidase, diamine oxidase terminator, diamine oxidase detection probe shown in formula (I) as a substrate, buffer, and diamine oxidase detection probe shown in formula (I) as a standard; Formula (I).

8. The diamine oxidase test kit according to claim 7, characterized in that, The diamine oxidase is in the form of dry powder, the diamine oxidase terminator is aminoguanidine or aminoguanidine hydrochloride, the concentration of the diamine oxidase terminator is 5-50 mmol / L, the pH of the buffer is 6.0-7.0, the concentration of the buffer is 10-50 mmol / L, and the concentration of the standard is 50-500 mmol / L.

9. A method of detecting diamine oxidase by a kit for detecting diamine oxidase, the method of detection being a method for diagnosis and treatment of disease, characterized by, The method comprises the following steps: (1) Prepare 6-10 gradient standard solutions with concentrations of 0-2.5 mmol / L using buffer solution, and include solvent blank samples. Measure the absorbance value D0 of the gradient standard solutions containing solvent blank samples at 592-602 nm; the absorbance value D0 of the solvent blank sample (i.e., the 0 mmol / L gradient standard solution) at 592-602 nm is... 空白 ; (2) 15-30 μL of the gradient standard solution in the solvent blank sample is mixed with diamine oxidase in 150-500 μL of a buffer system with a pH of 6.0-7.0, and incubated at 32-38 °C for 5-20 minutes; (3) after incubation, 5-20 μL of diamine oxidase terminator is added, the diamine oxidase terminator is prepared from a buffer with a concentration of 20-50 mmol / L, and the absorbance value D1 at 592-602 nm is tested respectively; (4) Function fitting the function relationship between the concentration difference of the gradient standard solution and the absorbance value change D0-D1-D before and after the enzyme incubation reaction 空白 ; (5) The absorbance values D of the sample before and after incubation are measured according to steps (1), (2) and (3) x0 , D x1 x0-Dx1-D 空白 x2, the concentration difference of the sample solution before and after the enzyme incubation reaction is calculated according to the functional relationship of step (3); and the diamine oxidase activity is calculated according to the concentration difference of the sample solution before and after the enzyme incubation reaction. The diamine oxidase detection probe shown in formula (I) as a standard; Formula (I).

10. The method of claim 9, wherein the diamine oxidase test kit is a Diazyme® kit. The diamine oxidase terminator is aminoguanidine or aminoguanidine hydrochloride, the pH of the buffer is 6.0-7.0, the concentration of the buffer is 10-50 mmol / L, and the concentration of the diamine oxidase terminator is 5-50 mmol / L.

Images