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A diagnostic probe for thrombotic thrombocytopenic purpura and its preparation method and application

A technology for platelet and purpura disease, applied in the field of biological analysis, can solve the problems of high technical requirements, expensive kits, complicated operation, etc., and achieve the effect of good biocompatibility, mild conditions and high resolution

Active Publication Date: 2021-09-28
苏州智影特生物医药技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most of these detection methods are complicated to operate, take a long time, the kits are expensive, and require high technical conditions.

Method used

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  • A diagnostic probe for thrombotic thrombocytopenic purpura and its preparation method and application
  • A diagnostic probe for thrombotic thrombocytopenic purpura and its preparation method and application
  • A diagnostic probe for thrombotic thrombocytopenic purpura and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Synthesis and characterization of the molecular probe probe-3 for the diagnosis of thrombotic thrombocytopenic purpura and the comparison probes probe-1 and probe-2

[0060] (1) Add compound 8 (10 mg, 0.0086 mmol, structural formula below) into a 10 mL round bottom flask, dissolve it with 4 mL of N,N-dimethylformamide, and then add DABCYL (3.8 mg, 0.01 mmol) and diisopropylethylamine (1.5 mg, 0.017 mmol), stirred at room temperature for 2 h. After the reaction, it was separated and purified by HPLC to obtain intermediate 1 (its structure is as follows) (9.7 mg, yield: 80%). MS (MALDI-TOF) Calc'd for C 68 h 95 N 15 o 16 S Na[M+Na] + , 1432.680,; found, 1432.454.

[0061]

[0062]

[0063] Add Intermediate 1 (9 mg, 0.0064 mmol) and 5-fluorescein isothiocyanate (FITC-N 3 ) (3.75 mg, 0.010 mmol), and dissolved in DMSO (1 mL), stirred well. At the same time, sodium ascorbate (0.5 mg, 0.0026 mmol) and anhydrous copper sulfate (0.2 mg, 0.0013 mmol) wer...

Embodiment 2

[0072] The DMSO mother solution of the thrombotic thrombocytopenic purpura diagnostic molecular probe probe-3 prepared in Example 1 was dissolved in a conventional buffer solution (5 mmol / L Bis-Tris, 25 mmol / L CaCl 2 , 0.005% Tween20, pH 6.0), and then add normal human plasma, 37 o C. Incubate for 2 h under shaking at 500rpm, and then use a fluorescence spectrometer to detect the change of the fluorescence signal of the solution: when no plasma is added, the solution containing probe-3 cannot detect fluorescence, after adding plasma for incubation, the energy between FITC and DABCYL The resonance shift becomes weaker and the fluorescence recovers, indicating that probe-3 can be used as a detection probe for von Willebrand factor cleavage protease (ADAMTS13).

Embodiment 3

[0073] Example 3 HPLC purity characterization and high-resolution mass spectrometry characterization of molecular probes probe-1, probe-2, and probe-3

[0074] After diluting the diagnostic molecular probes probe-1, probe-2 and probe-3 for thrombotic thrombocytopenic purpura prepared in Example 1 with solvent methanol to a concentration of 5 μM, the molecular weight of the probes was determined by high-resolution mass spectrometry identified and analyzed for purity using high performance liquid chromatography.

[0075] Such as image 3 As shown in a, using Agilent 1260 high performance liquid chromatography to analyze the sample, the retention time of probe-1, the diagnostic molecular probe for thrombotic thrombocytopenic purpura, was 8.163 minutes, and the peak area was further integrated to calculate the The probe concentration was as high as 98%. image 3 b shows the theoretical m / z of probe-1, a molecular probe for the diagnosis of thrombotic thrombocytopenic purpura: 19...

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Abstract

The invention discloses a diagnostic probe for thrombotic thrombocytopenic purpura, a preparation method and application thereof. The probe of the present invention utilizes the highly efficient click reaction between 1,2-aminothiol and 2-cyanobenzothiazole (CBT) for the first time to link the two compounds together, which can greatly reduce the synthesis cost, and the probe has good effect on blood vessels. Hemophilia factor cleavage protease (ADAMTS13) has good specificity and high detection sensitivity, and can be applied to the detection of ADAMTS13 activity, so as to diagnose thrombotic thrombocytopenic purpura.

Description

technical field [0001] The invention belongs to the field of biological analysis, and specifically relates to a diagnostic probe for thrombotic thrombocytopenic purpura based on fluorescence resonance energy transfer, a preparation method and an application. Background technique [0002] Von Willebrand factor cleavage protease (ADAMTS13), as an important metalloprotease in the body, can act on the A2 region of von Willebrand factor and cleave the ultra-large molecule VWF (UL-VWF) with high adhesion in plasma It is a low-adhesive small molecule VWF, which prevents platelets from excessively adhering to cause aggregation and resulting in microthrombus formation. The lack of plasma ADAMTS13 activity will lead to the occurrence of thrombotic thrombocytopenic purpura (TTP), so the detection of ADAMTS13 activity is of great significance for the diagnosis of TTP. At present, the methods for detecting ADAMTS13 activity in plasma mainly include residual collagen binding test, ELISA ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/06C09K11/06C12Q1/527
CPCC07K7/06C09K11/06C09K2211/1037C09K2211/1088C12Q1/527G01N2800/224
Inventor 史海斌王安娜史沛洋
Owner 苏州智影特生物医药技术有限公司
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