CN111153962B - Thrombotic thrombocytopenic purpura disease diagnosis probe and preparation method and application thereof - Google Patents

Thrombotic thrombocytopenic purpura disease diagnosis probe and preparation method and application thereof Download PDF

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CN111153962B
CN111153962B CN202010044420.5A CN202010044420A CN111153962B CN 111153962 B CN111153962 B CN 111153962B CN 202010044420 A CN202010044420 A CN 202010044420A CN 111153962 B CN111153962 B CN 111153962B
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史海斌
王安娜
史沛洋
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Suzhou zhiyingte Biomedical Technology Co.,Ltd.
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Abstract

The invention discloses a thrombotic thrombocytopenic purpura diagnostic probe and a preparation method and application thereof. The probe provided by the invention has the advantages that the 1, 2-amino thiol and the 2-Cyanobenzothiazole (CBT) are subjected to efficient click reaction for the first time, the two compounds are connected together, the synthesis cost can be greatly reduced, the probe has better specificity and higher detection sensitivity for von Willebrand factor lyase (ADAMTS 13), and the probe can be applied to activity detection of ADAMTS13, so that diagnosis of thrombotic thrombocytopenic purpura is performed.

Description

Thrombotic thrombocytopenic purpura disease diagnosis probe and preparation method and application thereof
Technical Field
The invention belongs to the field of biological analysis, and particularly relates to a thrombotic thrombocytopenic purpura disease diagnosis probe based on fluorescence resonance energy transfer, a preparation method and application.
Background
The von willebrand factor lyase (ADAMTS 13) is an important in vivo metalloprotease, and can act on the A2 region of von willebrand factor to cleave the ultra-large molecular VWF (UL-VWF) with high adhesiveness in plasma into small molecular VWF with low adhesiveness, so as to prevent the platelet from being excessively adhered to cause aggregation to cause microthrombosis. Lack of plasma ADAMTS13 activity may lead to the development of Thrombotic Thrombocytopenic Purpura (TTP), so detection of ADAMTS13 activity is of great importance for the diagnosis of TTP. Currently, the methods for detecting the activity of ADAMTS13 in plasma mainly include residual collagen binding assay, ELISA method, guanidine-hydrochloric acid (or urea) denaturation, and the like. However, most of the detection methods are complex in operation, long in time consumption, expensive in kit price and high in requirements on technical conditions. Therefore, in order to overcome the above defects, it is necessary to develop a rapid, simple, accurate and noninvasive method for detecting the activity of ADAMTS 13.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention constructs a molecular probe specifically identified by ADAMTS13 based on fluorescence resonance energy transfer. The method has the advantages of high energy resonance transfer resolution, high sensitivity, good reproducibility and strong specificity, and can detect the activity of ADAMTS13 more simply and rapidly, thereby realizing the diagnosis of thrombotic thrombocytopenic purpura, and having wide application prospects in experimental research and clinical diagnosis.
The invention adopts the following technical scheme:
a diagnostic probe for thrombotic thrombocytopenic purpura disease has the following chemical structural formula:
Figure 907803DEST_PATH_IMAGE002
the thrombotic thrombocytopenic purpura disease diagnostic probe is applied to the preparation of thrombotic thrombocytopenic purpura disease diagnostic reagents, is applied as a thrombotic thrombocytopenic purpura disease diagnostic probe, or is applied to the preparation of von willebrand factor lyase activity detection reagents, and is applied as an von willebrand factor lyase activity detection probe.
The preparation method of the diagnostic probe for thrombotic thrombocytopenic purpura comprises the following steps:
(1) carrying out amide condensation reaction on the compound 1 and 2-amino-6-cyanobenzothiazole to obtain a compound 2; removing a protecting group from the compound 2 to obtain a compound 3;
(2) reacting the compound 3 with 5-fluorescein isothiocyanate to obtain a compound 4; removing a protecting group from the compound 4 to obtain a compound 5;
(3) reacting the compound 5 with DABCYL to obtain a compound 6;
(4) and carrying out click condensation reaction on the compound 6 and the compound 7 to obtain the thrombotic thrombocytopenic purpura disease probe.
A method for detecting von willebrand factor lyase activity for non-disease diagnosis and treatment purposes, comprising the steps of:
(1) carrying out amide condensation reaction on the compound 1 and 2-amino-6-cyanobenzothiazole to obtain a compound 2; removing a protecting group from the compound 2 to obtain a compound 3;
(2) reacting the compound 3 with 5-fluorescein isothiocyanate to obtain a compound 4; removing a protecting group from the compound 4 to obtain a compound 5;
(3) reacting the compound 5 with DABCYL to obtain a compound 6;
(4) carrying out click condensation reaction on the compound 6 and the compound 7 to obtain the thrombotic thrombocytopenic purpura disease probe;
(5) and incubating the thrombotic thrombocytopenic purpura probe and the blood plasma to be detected, and then carrying out fluorescence detection to finish the activity detection of von willebrand factor lyase.
In the invention, plasma to be detected is thawed at 37 ℃ and then mixed with buffer solution (5 mmol/L Bis-Tris, 25 mmol/L CaCl) of molecular probe20.005% Tween20, pH 6.0), reacting at 37 ℃ with shaking at 500rpm for 2 hours, and detecting the activity of ADAMTS13 by fluorescence detection after the reaction is finished.
In the invention, the molar ratio of the compound 1 to the 2-amino-6-Cyanobenzothiazole (CBT) is 1: 1.2; the amide condensation reaction is carried out in the presence of IBCF, NMM and THF; the time of amide condensation reaction is 12-18 hours; removing protective groups of the compound 2 in a dichloromethane/trifluoroacetic acid mixed solvent; the volume ratio of the dichloromethane to the trifluoroacetic acid is 1: 4.
In the invention, the reaction of the compound 3 and the 5-fluorescein isothiocyanate is carried out in the presence of diisopropylethylamine; the mol ratio of the compound 3 to the 5-fluorescein isothiocyanate to the diisopropylethylamine is 1:1.2: 2; removing protective groups of the compound 4 in a mixed solvent of N, N-dimethylformamide and piperidine; the volume ratio of the N, N-dimethylformamide to the piperidine is 4: 1.
In the invention, the molar ratio of the compound 5 to the quencher DABCYL is 1: 1.2; the click condensation reaction is carried out in PBS (phosphate buffered saline, pH = 7.2-7.4); the molar ratio of compound 6 to compound 7 was 1: 1.2.
Specifically, the method provided by the invention comprises the following steps:
according to the designed synthesis steps: first, compound 1 was amide-condensed with 2-amino-6-Cyanobenzothiazole (CBT), followed by 80% trifluoroacetic acid (dichloromethane: trifluoroacetic acid = 1:4, v/v); removing protecting groups of Boc, t-Bu and Trt of intermediate compounds; followed by reaction with fluorescein 5-isothiocyanate and deprotection of the Fmoc group of the resulting intermediate compound with 20% piperidine (N, N-dimethylformamide: piperidine = 4:1, v/v) followed by amide condensation with DABCYL, a FITC quencher which has been activated with NHS, to give compound 6 of the formula:
Figure 293785DEST_PATH_IMAGE003
the compound 6 further reacts with the compound 7 in a PBS (pH = 7.2-7.4) buffer solution to generate a click chemical reaction, so that the product molecular probe is obtained.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
(1) according to the invention, a plurality of compounds are connected together by utilizing efficient click reaction between 1, 2-aminothiol and 2-Cyanobenzothiazole (CBT) for the first time, and the condensation reaction has the advantages of mild condition, high speed, good biocompatibility and the like;
(2) the fluorescence resonance energy transfer detection method used in the invention has the advantages of high resolution, high sensitivity, good reproducibility, strong specificity and the like.
Drawings
FIG. 1 is a scheme showing the synthesis of probe molecule probe-1 for diagnosing thrombotic thrombocytopenic purpura in example 1;
FIG. 2 is a synthesis scheme of molecular probes probe-2 and probe-3 for diagnosing thrombotic thrombocytopenic purpura in example 1;
FIG. 3 shows HPLC purity characterization (a) and high-resolution mass spectrometry characterization (b) of the molecular probe-1 in example 3;
FIG. 4 is a graph showing the change in the UV absorption spectrum of the molecular probe-2 after the click condensation reaction with cysteine (Cys) and cysteine (CR-59) on polypeptide substrate fragment 2 (CR-59) specifically recognized by von Willebrand factor cleavage protease (ADAMTS 13) in example 4;
FIG. 5 is a graph showing the change in fluorescence intensity of bacterial reactions in which ADAMTS13 enzyme was expressed by varying concentration gradients of probe-1 in example 5;
FIG. 6 shows the fluorescence intensity changes of probe-1 and ADAMTS 13-expressing bacteria and inhibitor competition experiment and the fluorescence intensity changes of the probe and different biological enzymes in example 6;
FIG. 7 is a graph showing the change in fluorescence intensity of the molecular probes probe-1, probe-2, and probe-3 in example 7 in response to different concentration gradients of normal human plasma or plasma of von Willebrand disease patients;
FIG. 8 is a graph showing the fluorescence intensity of the molecular probe for diagnosing thrombotic thrombocytopenic purpura in example 8 reacting with normal human plasma of different concentration gradients and the change with concentration gradient (a), a straight line (b) fitted, and the change in fluorescence intensity of the probe after reacting with normal human plasma in the presence or absence of an inhibitor (c).
Detailed Description
The experimental method according to the embodiment of the present invention is as follows.
Detection of ADAMTS 13-specific recognition molecular probes probe-1, probe-2 and probe-3 based on fluorescence resonance energy transfer on ADAMTS13 activity in plasma:
the obtained molecular probe-1,DMSO mother solutions of probe-2 and probe-3 were dissolved in buffer solution (5 mmol/L Bis-Tris, 25 mmol/L CaCl)20.005% Tween20, pH 6.0) at a final concentration of 2. mu.M, 0, 1,2, 3, 4, 5 uL of normal human plasma or von Willebrand's plasma was mixed with the above buffer, 37oC, reacting for 2 hours, and carrying out fluorescence detection after the reaction is finished.
Study on the concentration dependence and specificity of the molecular probe-3 for diagnosing thrombotic thrombocytopenic purpura on ADAMTS13 in plasma of normal human:
dissolving the DMSO mother liquor of the molecular probe-3 for diagnosing the thrombotic thrombocytopenic purpura in a buffer solution (5 mmol/L Bis-Tris, 25 mmol/L CaCl)20.005% Tween20, pH 6.0) at a final concentration of 2. mu.M, 0, 1,2, 3, 4, 5, 6 uL of normal human plasma was mixed with the above buffer, 37oC, reacting for 2 hours, and carrying out fluorescence detection after the reaction is finished. And the molecular probe-3 and 6 uL of normal human plasma were mixed at 37 deg.C in the presence/absence of inhibitor EDTA (20 mM)oAnd C, respectively incubating for 2 h, and carrying out fluorescence detection after the reaction is finished.
In the invention, the molecular probe-2 and the compound 7 (CR-59) are subjected to click condensation reaction, and are separated and purified by using semi-preparative high performance liquid chromatography to obtain the thrombotic thrombocytopenic purpura diagnostic probe-3, and the product is light yellow solid powder. The embodiment relates to a high performance liquid chromatography separation method, which comprises the following steps: c18 column, 3.5 μm, 4.6X 100 mm; mobile phase: a is trifluoroacetic acid to water = 1: 1000; b is trifluoroacetic acid acetonitrile = 1: 1000; flow rate: 1 mL/min; linear gradient elution procedure: 0 min, A: B = 95: 5; 10 min, A: B = 0: 100.
The invention will be further elucidated with reference to the drawings and specific embodiments. It should be understood that these examples are only for explaining and illustrating the technical solutions of the present invention, and are not intended to limit the scope of the present invention. In addition, unless otherwise specified, materials, reagents, instruments and the like used in the following examples are commercially available.
All plasma originated from the tin-free civilian hospital.
In the invention, the chemical structural formulas of the compounds are respectively as follows:
Figure 401418DEST_PATH_IMAGE004
Figure 265469DEST_PATH_IMAGE005
Figure 345420DEST_PATH_IMAGE006
Figure 512091DEST_PATH_IMAGE007
Figure 247965DEST_PATH_IMAGE008
Figure 774762DEST_PATH_IMAGE009
the chemical structural formula of the 5-fluorescein isothiocyanate is as follows:
Figure 912482DEST_PATH_IMAGE010
the compound 7 is amino-cysteine-proline-alanine-serine-aspartic acid-glutamic acid-isoleucine-lysine-arginine-leucine-proline-glycine-aspartic acid-isoleucine-glutamine-valine-proline-isoleucine-glycine-valine-glycine-proline-asparagine-alanine-asparagine-valine-glutamine-glutamic acid-leucine-glutamic acid-arginine-isoleucine-glycine-proline-asparagine-alanine-glutamine-alanine-isoleucine-tryptophan-proline-asparagine-alanine-proline-isoleucine-leucine-isoleucine-glutamine-asparagine Acid-phenylalanine-glutamic acid-threonine-leucine-proline-arginine-glutamic acid-alanine-proline-aspartic acid-leucine-valine-leucine-glutamine-arginine-carboxyl, with three letters being represented as:
Figure 250054DEST_PATH_IMAGE011
the chemical structure of DABCYL is as follows:
Figure 269962DEST_PATH_IMAGE012
example 1: synthesis and characterization of molecular probe-3 and contrast probes probe-1 and probe-2 for diagnosing thrombotic thrombocytopenic purpura
(1) A10 mL round bottom flask was charged with Compound 8 (10 mg, 0.0086 mmol, formula shown below), dissolved in 4 mL of N, N-dimethylformamide, followed by DABCYL (3.8 mg, 0.01 mmol) and diisopropylethylamine (1.5 mg, 0.017 mmol), and the reaction stirred at room temperature for 2 h. After completion of the reaction, separation and purification by HPLC were carried out to obtain intermediate 1 (the structure is shown below) (9.7 mg, yield: 80%). MS (MALDI-TOF) Calc'd for C68H95N15O16S Na[M+Na]+，1432.680,；found，1432.454。
Figure 475816DEST_PATH_IMAGE013
Figure 327097DEST_PATH_IMAGE014
A5 mL round bottom flask was charged with intermediate 1 (9 mg, 0.0064 mmol) and fluorescein 5-isothiocyanate (FITC-N)3) (3.75 mg, 0.010 mmol) and dissolved in DMSO (1 mL) and stirred well. Sodium ascorbate (0.5 mg, 0.0026 mmol) and anhydrous copper sulfate (0.2 mg, 0)0013 mmol) was dissolved in 1 mL of deionized water, and the mixture was added to the reaction flask and stirred at room temperature for 8 h. After the completion of the reaction, it was separated and purified by semi-preparative high performance liquid chromatography to obtain probe-1 (8.5 mg, yield: 70%) as a comparative molecular probe. MS (MALDI-TOF) Calc'd for C92H114N20O21S2Na[M+Na]+，1921.791；found，1921.876。
Figure 225783DEST_PATH_IMAGE015
(2) Compound 1 (20 mg, 0.01 mmol) was added to a 10 mL round-bottom flask, dissolved in anhydrous tetrahydrofuran, and N-methylmorphine (1.5 mg, 0.015 mmol) was added, and the round-bottom flask was placed in a salt-ice bath and cooled to 0oC, then isobutyl chloroformate (2.05 mg, 0.015 mmol) was added dropwise, and after activation for half an hour, 2-amino-6-cyanobenzothiazole (2.15 mg, 0.012 mmol) dissolved in dry tetrahydrofuran was added thereto, and the solution was kept at 0oC was reacted for 1 hour, then stirred at room temperature overnight. After the reaction was completed, separation and purification by HPLC were carried out, and fractions having an absorption spectrum at 350 nm were collected to obtain Compound 2 (the structure of which is shown as Compound 2 in FIG. 1) as a pale yellow powder (12.1 mg, yield: 60%). MS (MALDI-TOF) Calc'd for C109H140N16O18S2Na[M+Na]+，2048.997；found，2048.777。
A10 mL round bottom flask was charged with 1 mL of methylene chloride and 4 mL of trifluoroacetic acid and stirred well. Intermediate compound 2 (12.1 mg, 0.006 mmol) was then added to the reaction flask and the reaction stirred at room temperature for 1 h. After the reaction was completed, the reaction solution was removed by rotary evaporation, separation and purification was performed by HPLC, and a fraction having an absorption spectrum at 350 nm was collected to obtain Compound 3 (the structure is shown as Compound 3 in FIG. 2) as a pale yellow powder (8.0 mg, yield: 85%). MS (MALDI-TOF) Calc'd for C77H102N16O16S2Na[M+Na]+，1593.710；found，1594.127。
Compound 3 (8.0 mg, 0.0051 mmol) was charged into a 10 mL round-bottomed flask, and dissolved in 4 mL of N, N-dimethylformamide, followed by addition of fluorescein 5-isothiocyanate (2.4 mg, 0.0061 mmol) and diisopropylethylamine (1.3 mg, 0.01 mmol), and the reaction was stirred at room temperature for 1 h. After the completion of the reaction, separation and purification by HPLC were carried out to obtain Compound 4 (the structure of which is shown as Compound 4 in FIG. 2) (8.0 mg, yield: 80%). MS (MALDI-TOF) Calc'd for C98H113N17O21S3Na[M+Na]+，1982.746；found，1982.746。
Compound 4 (8.0 mg, 0.0041 mmol) was added to a 10 mL round-bottomed flask, which was then dissolved in 3.6 mL of N, N-dimethylformamide, and the round-bottomed flask was placed in a salt-ice bath and cooled to 0oC, then adding 400 uL piperidine dropwise, keeping 0oC for 10 minutes, and after completion of the reaction, separation and purification by HPLC were carried out to obtain Compound 5 (the structure of which is shown as Compound 5 in FIG. 2) (6.1 mg, yield: 85%). MS (MALDI-TOF) Calc'd for C83H103N17O19S3Na[M+Na]+，1760.678；found，1760.836。
A10 mL round-bottom flask was charged with Compound 5 (6.1 mg, 0.0035 mmol), dissolved in 2 mL of N, N-dimethylformamide, followed by DABCYL (1.54 mg, 0.0042 mmol) and diisopropylethylamine (0.91 mg, 0.007 mmol), and the reaction was stirred at room temperature for 2 h. After the completion of the reaction, separation and purification were carried out by HPLC to obtain the probe-2 of comparative molecule, i.e., Compound 6 (the structure of which is shown by Compound probe-2 in FIG. 2) (5.9 mg, yield: 85%). MS (MALDI-TOF) Calc'd for C98H116N20O20S3Na[M+Na]+，2011.784；found，2011.956。
A10 mL round bottom flask was charged with probe-2 (1 mg, 5X 10)-4mmol), dissolved in 1 mL of PBS (pH = 7.2-7.4) buffer solution, and then compound 7 (4.02 mg, 6.1 × 10) is added-4 mmol），37oC, reacting for 4 hours. After the reaction is finished, separating and purifying by HPLC to obtain the product molecular probe-3 (1) of the invention8 mg, yield: 43%), one letter in the chemical structural formula is the abbreviation for the compound 7 three-letter amino acid. MS (MALDI-TOF) Calc'd for C388H582N99O107S4Na6[M+6Na+2H]8+/8，1076.461；found，1076.540。
Example 2
DMSO stock solution of diagnostic molecular probe for thrombotic thrombocytopenic purpura-3 prepared in example 1 was dissolved in a conventional buffer solution (5 mmol/L Bis-Tris, 25 mmol/L CaCl)20.005% Tween20, pH 6.0), and then normal human plasma, 37oC. Incubating for 2 h under shaking at 500rpm, and detecting the change of the fluorescence signal of the solution by using a fluorescence spectrometer: when no plasma was added, the solution containing probe-3 showed no fluorescence detection, and after incubation with plasma, the energy resonance transfer between FITC and DABCYL became weak and fluorescence recovered, indicating that probe-3 can be used as a detection probe for von Willebrand factor cleaving protease (ADAMTS 13).
EXAMPLE 3 high performance liquid chromatography purity characterization and high resolution Mass Spectrometry characterization of molecular probes Probe-1, Probe-2, Probe-3
The molecular probes probe-1, probe-2 and probe-3 for diagnosing thrombotic thrombocytopenic purpura prepared in example 1 were diluted with a solvent methanol to a concentration of 5 μ M, and then molecular weight of the probes was determined by high resolution mass spectrometry and purity analysis was performed using a high performance liquid chromatograph.
As shown in FIG. 3a, when the sample was analyzed by Agilent 1260 high performance liquid chromatography, the retention time of the molecular probe-1 for diagnosing thrombotic thrombocytopenic purpura was 8.163 minutes, and the peak area was further integrated to calculate the probe concentration in the sample as high as 98%. FIG. 3b shows the theoretical m/z of the molecular probe for diagnosing thrombotic thrombocytopenic purpura-1: 1921.791, actually obtaining m/z in a high-resolution mass spectrum: 1921.876, the two are identical, thus obtaining the desired compound.
As shown in FIG. 4c, when the sample was analyzed by Agilent 1260 high performance liquid chromatography, the retention time of the molecular probe-2 for diagnosing thrombotic thrombocytopenic purpura was 9.41 minutes, and the peak area was further integrated to calculate the probe concentration in the sample as high as 93.8%. FIG. 4d shows the theoretical m/z of the molecular probe for diagnosis of thrombotic thrombocytopenic purpura-2: 2011.784, actually obtaining m/z in a high-resolution mass spectrum: 2011.956, the two are identical, thus obtaining the desired compound.
As shown in FIG. 5e, when the sample was analyzed by Agilent 1260 high performance liquid chromatography, the retention time of the molecular probe-3 for diagnosing thrombotic thrombocytopenic purpura was 7.184 minutes, and the peak area was further integrated to calculate the probe concentration in the sample as high as 90%. FIG. 5f shows the theoretical m/z of the molecular probe for diagnosis of thrombotic thrombocytopenic purpura-3: 1076.461, actually obtaining m/z in a high-resolution mass spectrum: 1076.540, the two are identical, thus obtaining the desired compound.
Example 4: change in UV absorption Spectrum after click condensation reaction of Probe-2 with cysteine (Cys) (FIG. 6 a) and change in UV absorption Spectrum after click condensation reaction of Probe-2 with Compound 7 (CR-59) (FIG. 6 b)
Probe-2 prepared in example 1 was reacted with cysteine and Compound 7 (reaction System: PBS, pH = 7.2-7.4), respectively, 37oC, reacting for 4 hours. After the reaction is finished, the ultraviolet absorption spectrum of the product at about 320 nm is compared, and it can be further confirmed that probe-2 and cysteine on compound 7 have performed click condensation reaction, and the result is shown in fig. 6.
Example 5: graph of fluorescence intensity changes of molecular probes Probe-1, Probe-2, Probe-3 in response to different concentration gradients of normal human plasma or plasma of von Willebrand patients
The molecular probe-1DMSO stock solution prepared in example 1 (2 uL of the probe-1DMSO stock solution was taken and diluted with a buffer solution so that the final concentration of the probe was 2 uM (2 uM) and the concentration was the same as that of the probe in 37oAt C with different volumes of normal human plasma or von Willebrand patient plasma (0, 1,2, 3, 4, 5 uL) in 200uL of buffer solution (5 mmol/L Bis-Tris, 25 mmol/L CaCl)20.005% Tween20, pH 6.0) at 37 deg.C, 5Incubate for 2 h with shaking at 00rpm, and then detect the change in the fluorescence signal of the solution with a fluorescence spectrometer. As shown in FIG. 7, the molecular probe-1 at the same concentration showed no significant concentration dependence in the reaction with normal human plasma (FIG. 7 a) or plasma of von Willebrand disease patients (FIG. 7 b), and could not be detected as ADAMTS13 activity.
Molecular probe-2 DMSO stock (2 uM) prepared in example 1 was added at 37oAt C with different volumes of normal human plasma or von Willebrand patient plasma (0, 1,2, 3, 4, 5 uL) in 200uL of buffer solution (5 mmol/L Bis-Tris, 25 mmol/L CaCl)20.005% Tween20, pH 6.0) at 37 ℃ for 2 h with shaking at 500rpm, and then detecting the change in the fluorescence signal of the solution with a fluorescence spectrometer. As shown in FIG. 7, the molecular probe-2 at the same concentration showed no significant concentration dependence in the reaction with normal human plasma (FIG. 7 c) or plasma of von Willebrand disease patients (FIG. 7 d), and could not be detected as ADAMTS13 activity.
Molecular probe-3 DMSO stock solution (2 uM) prepared in example 1 was added to the reaction mixture at 37oAt C with different volumes of normal human plasma or von Willebrand patient plasma (0, 1,2, 3, 4, 5 uL) in 200uL of buffer solution (5 mmol/L Bis-Tris, 25 mmol/L CaCl)20.005% Tween20, pH 6.0) at 37 ℃ for 2 h with shaking at 500rpm, and then detecting the change in the fluorescence signal of the solution with a fluorescence spectrometer. As shown in FIG. 7, the fluorescence intensity of the molecular probe-3 at the same concentration increased with the plasma dose of normal persons (FIG. 7 e), but did not increase with the plasma dose of von Willebrand patients (FIG. 7 f). This shows that the molecular probe-3 has better responsiveness to ADAMTS13 in the plasma of normal people, and the plasma of von Willebrand patients has no influence on the molecular probe-3, and can be applied to activity detection of ADAMTS13, thereby diagnosing thrombotic thrombocytopenic purpura.
Example 8: study on concentration dependence and specificity of molecular probe-3 for diagnosing thrombotic thrombocytopenic purpura in plasma of normal human ADAMTS13
2uL of probe-prepared in example 1 was taken3DMSO stock solution, diluted in buffer to a final concentration of 2 uM with different volumes of normal human plasma (0, 1,2, 3, 4, 5, 6 uL) in 200uL buffer (5 mmol/L Bis-Tris, 25 mmol/L CaCl) at 37 deg.C20.005% Tween20, pH 6.0) for 2 h under the shaking of 500rpm, detecting the change of a fluorescence signal of the solution by using a fluorescence spectrometer (the excitation wavelength is 495nm, the emission range is 500-600 nm, the same as the embodiment 7), and fitting a straight line graph of the fluorescence intensity of the plasma of normal persons with different concentration gradients and the change along with the concentration gradients. As shown in FIGS. 8a and 8b, the fluorescence intensity of the molecular probe-3 at the same concentration increases with the plasma dosage of normal human, and increases linearly.
The DMSO stock solution of the molecular probe-3 prepared in example 1 was dissolved in a conventional buffer solution (5 mmol/L Bis-Tris, 25 mmol/L CaCl)20.005% Tween20, pH 6.0), to a final concentration of 2 uM, and preparing seven identical portions; under the same conditions, one part of the solution was used as a blank, Caspase-3, MMP-2, MMP-9, Furin, BSA, and ADAMTS13 (final concentrations were 640 ng/mL, enzymes were all commercially available) were added to the six parts of the solution, respectively, at 37oC, incubating for 2 h, and carrying out fluorescence detection after the reaction is finished, wherein the solution has stronger fluorescence signal increase at 520 nm and almost no enhancement of the fluorescence of other enzymes (compared with a blank control, the fluorescence intensity change is small) after the probe-3 only acts with ADAMTS 13; this demonstrates that the probes of the invention have good specificity and selectivity for von Willebrand factor cleaving protease (ADAMTS 13).
To further study the specificity of the probes, probe-3 DMSO stock solution (2 uM) and normal human plasma (6 uL) were incubated in the presence/absence of inhibitor EDTA (20 mM) at 37 ℃ for 2 h with shaking at 500rpm, and the fluorescence intensity of the corresponding solutions was finally measured by fluorescence spectroscopy. As shown in FIG. 8c, when the probe reacts with normal human plasma, the fluorescence intensity of the solution at 520 nm is significantly enhanced compared with the background of the probe, while the fluorescence intensity of the solution added with the inhibitor in advance is significantly inhibited, which proves that the probe-3 has certain specificity to von Willebrand factor lyase (ADAMTS 13) in normal human plasma. The experimental results show that the probe-3 has good specificity and high detection sensitivity on von Willebrand factor lyase (ADAMTS 13), and can be applied to activity detection of ADAMTS13 so as to diagnose thrombotic thrombocytopenic purpura.

Claims (3)

1. A thrombotic thrombocytopenic purpura probe, characterized in that it has the following chemical structural formula:
Figure DEST_PATH_IMAGE002
。
2. use of the thrombotic thrombocytopenic purpura probe in claim 1 for preparing reagents for detecting von willebrand factor cleaving protease activity.
3. Use of the thrombotic thrombocytopenic purpura probe according to claim 1 for the preparation of a diagnostic reagent for thrombotic thrombocytopenic purpura.
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