Diamine oxidase detection probe and kit

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of fluorescence analysis and detection, and in particular to a diamine oxidase detection probe and a kit. Background Art

[0002] Intestinal flora imbalance, barrier function damage and other reasons can induce the occurrence of acute or chronic diseases. Intestinal flora imbalance can induce intestinal barrier function damage, and intestinal barrier function damage can also aggravate intestinal flora imbalance. If not intervened in time, it will further induce enterogenic infection and even systemic inflammatory response.

[0003] Intestinal ischemia / reperfusion (I / R) is common in a variety of clinical situations, including severe trauma, shock, infection, strangulated intestinal obstruction, acute mesenteric ischemia, and extracorporeal circulation. If treated early, acute intestinal ischemia can usually be reversed, but delayed diagnosis can lead to multiple organ dysfunction or even failure, with a very 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 the present invention is to overcome the shortcomings of the prior art and provide a diamine oxidase detection probe and a kit.

[0005] To achieve the above object, the technical solution adopted by the present invention is: 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 a chemical structure as shown in formula (I) can be fluorescent, has an absorption peak in the range of 592 to 602 nm, and the absorption peak is near 596 nm. Diamine oxidase can oxidize the diamine oxidase detection probe as shown in formula (I) to make its light absorption in the range of 592 to 602 nm disappear. The diamine oxidase detection probe with a chemical structure as shown in formula (I) has a stable structure and is easy to store.

[0008] The diamine oxidase detection probe with a chemical structure as shown in formula (I) can be applied to diamine oxidase detection, providing a detection method for using serum diamine oxidase (DAO) levels as an early diagnostic indicator for intestinal mucosal damage.

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

[0010] (1) condensing rhodamine B and 1,2-diaminonaphthalene to obtain a compound as shown in formula (II),

[0011]

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

[0013] Preferably, the reducing agent for the reduction reaction of the compound represented by (II) is lithium aluminum hydride (LiAlH4).

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

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

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

[0017]

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

[0019]

[0020] The diamine oxidase in the diamine oxidase detection kit is a powder, and the other reagents are pre-mixed with a buffer solution to a specific concentration.

[0021] Preferably, 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 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.

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

[0023] The present invention also provides a detection method of a diamine oxidase detection kit, the method comprising the following steps:

[0024] (1) Prepare 6 to 10 gradient standard solutions with concentrations of 0 to 2.5 mmol / L using a buffer solution and include a solvent blank sample. Test the absorbance values ​​D0 at 592 to 602 nm of the gradient standard solutions including the solvent blank sample. The absorbance value D0 at 592 to 602 nm of the solvent blank sample, i.e., the gradient standard solution with a concentration of 0 mmol / L, is 空白 ;

[0025] (2) respectively mixing 15 to 30 μL of the gradient standard solution including the solvent blank sample and diamine oxidase in 150 μL to 500 μL of a buffer system having a pH of 6.0 to 7.0, and incubating at 32 to 38° C. for 5 to 20 minutes;

[0026] (3) After incubation, 5 to 20 μL of diamine oxidase terminator is added, and the diamine oxidase terminator is prepared by a buffer solution with a concentration of 20 to 50 mmoL / L, and the absorbance values ​​D1 at 592 to 602 nm are measured respectively;

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

[0028] (5) The sample to be tested is operated according to steps (1), (2) and (3) and the absorbance value D of the sample to be tested before and after incubation is measured. 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 the enzyme incubation reaction; the diamine oxidase activity is obtained by balancing the concentration difference of the sample solution before and after the enzyme incubation reaction;

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

[0030]

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

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

[0033] The beneficial effects of the present invention are as follows: the present invention provides a diamine oxidase detection probe and a kit. The diamine oxidase detection probe with a chemical structure as shown in formula (I) can be fluorescent, has an absorption peak in the range of 592 to 602 nm, and the absorption peak is near 596 nm. And diamine oxidase can oxidize the diamine oxidase detection probe as shown in formula (I) to make its absorbance in the range of 592 to 602 nm disappear. The diamine oxidase detection probe with a chemical structure as shown in formula (I) has a stable structure and is easy to store. The diamine oxidase detection probe with a chemical structure as shown in formula (I) can be used for diamine oxidase detection. A detection method is provided for serum diamine oxidase (DAO) levels as an early diagnostic indicator for intestinal mucosal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The chemical structure diagram of the diamine oxidase detection probe of the present invention is shown in FIG.

[0035] Figure 2 The figure 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 DESCRIPTION

[0037] In order to better illustrate the purpose, technical solutions 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 having a chemical structure as shown in formula (I) comprises the following steps:

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

[0043]

[0044] (2) The reagents after the reaction in step (1) are evaporated in vacuo to remove the solvent, and the compound (II) (0.3 g) is added with a reducing agent, namely, lithium aluminum hydride (0.2 g) in a tetrahydrofuran (THF) solvent (40 mL) for reduction reaction. The reaction mixture is extracted twice with 100 mL of dichloromethane, and the organic phase is collected and dried over anhydrous magnesium sulfate. The solvent is removed by reduced pressure distillation, and the crude product is subjected to column chromatography using a CH2Cl2 / CH3CH2OH eluent with a volume ratio of 6:1 to 2:1 to obtain a diamine oxidase detection probe having a chemical structure as shown in formula (I), m / z (M+H + )=569.3.

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

[0046]

[0047] Experimental methods

[0048] 1. Absorption spectrum 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 show fluorescence, has an absorption peak in the range of 592-602nm, and has an absorption peak near 596nm.

[0050] 2. Diamine oxidase activity detection

[0051] DAO enzyme activity in the sample: Under 37°C conditions, the DAO enzyme activity required to hydrolyze 1 μmol of substrate per minute per liter of the sample to be tested is one activity unit.

[0052] 1. Test methods

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

[0054] The following steps are involved:

[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 in phosphate buffer and test the absorbance values ​​D0 at 597 nm respectively; the absorbance value of the solvent blank sample, i.e., the gradient standard solution with a concentration of 0 mmol / L, at 597 nm is D 空白 ;

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

[0057] (3) After incubation, 10 μL of diamine oxidase terminator was added, the diamine oxidase terminator was prepared by phosphate buffer, and the absorbance value D1 at 597 nm was measured respectively;

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

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

[0060] Standard product fitting curve: y = ax + b

[0061] x: known concentration difference of the standard

[0062] a: slope of the curve

[0063] b: intercept of the curve

[0064] y: corresponds to the absorbance difference before and after incubation of the standard (Dx0-Dx1-Dblank), that is, the absorbance value before incubation minus the absorbance value after incubation, excluding the solvent blank correction.

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

[0066] It can be seen that diamine oxidase can oxidize the diamine oxidase detection probe shown in formula (I), causing its absorbance in the range of 592-602 nm to disappear, and can be applied to diamine oxidase detection. This provides a detection method for using serum diamine oxidase (DAO) levels as an early diagnostic indicator for intestinal mucosal damage.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A diamine oxidase detection probe, characterized in that: The chemical structure of the diamine oxidase detection probe is shown in formula (I); 2. Use of the diamine oxidase detection probe as claimed in claim 1 in diamine oxidase detection.

3. The method for preparing a diamine oxidase detection probe according to claim 1, characterized in that: The preparation method comprises the following steps: (1) condensing rhodamine B and 1,2-diaminonaphthalene to obtain a compound as shown in formula (II), (2) The compound represented by (II) is subjected to a reduction reaction.

4. The method for preparing a diamine oxidase detection probe according to claim 3, characterized in that: The reducing agent for the reduction reaction of the compound represented by (II) is lithium aluminum hydride.

5. The method for preparing a diamine oxidase detection probe according to claim 3, characterized in that: The reaction environment solvent in step (2) is tetrahydrofuran.

6. The method for preparing a diamine oxidase detection probe according to claim 3, characterized in that: In step (1), rhodamine B and 1,2-diaminonaphthalene are subjected to a BOP condensation reaction in a dichloroethane solvent to obtain a compound as shown in formula (II).

7. A diamine oxidase detection kit, characterized in that: The diamine oxidase detection kit comprises the following components: diamine oxidase, a diamine oxidase terminator, a diamine oxidase detection probe as shown in formula (I) as a substrate, a buffer, and a diamine oxidase detection probe as shown in formula (I) as a standard; 8. The diamine oxidase detection kit according to claim 7, characterized in that: 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 to 50 mmol / L, 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 standard is 50 to 500 mmoL / L.

9. A diamine oxidase detection kit detection method, characterized in that: The method comprises the following steps: (1) Prepare 6 to 10 gradient standard solutions with concentrations of 0 to 2.5 mmol / L using a buffer solution and include a solvent blank sample. Test the absorbance values ​​D0 at 592 to 602 nm of the gradient standard solutions including the solvent blank sample. The absorbance value D0 at 592 to 602 nm of the solvent blank sample, i.e., the gradient standard solution with a concentration of 0 mmol / L, is 空白 ; (2) respectively mixing 15 to 30 μL of the gradient standard solution including the solvent blank sample and diamine oxidase in 150 μL to 500 μL of a buffer system having a pH of 6.0 to 7.0, and incubating at 32 to 38° C. for 5 to 20 minutes; (3) After incubation, 5 to 20 μL of diamine oxidase terminator is added, and the diamine oxidase terminator is prepared by a buffer solution with a concentration of 20 to 50 mmoL / L, and the absorbance values ​​D1 at 592 to 602 nm are measured respectively; (4) Function fitting of the concentration difference of the gradient standard solution and the change in absorbance value before and after the enzyme incubation reaction D0-D1-D 空白 The functional relationship between (5) The sample to be tested is operated according to steps (1), (2) and (3) and the absorbance value D of the sample to be tested before and after incubation is measured. 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 the enzyme incubation reaction; the diamine oxidase activity is obtained by balancing the concentration difference of the sample solution before and after the enzyme incubation reaction; The diamine oxidase detection probe shown in formula (I) is used as a standard; 10. The detection method of the diamine oxidase detection kit according to claim 9, characterized in that: The diamine oxidase terminator is aminoguanidine or aminoguanidine hydrochloride, 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 diamine oxidase terminator is 5-50 mmol / L.

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