Thrombin fluorescent substrate applied to whole blood, its preparation method and application

Abstract

The application relates to the technical field of thrombin detection, in particular to a thrombin fluorescent substrate applied to whole blood, a preparation method and application thereof. The thrombin fluorescent substrate comprises a specific recognition peptide chain D-Phe (4-MeSO2)-Pro-Gly-Arg and a fluorescent group. The thrombin fluorescent substrate has high affinity with thrombin, high catalytic efficiency and strong anti-interference capability, and can be widely applied to thrombin detection technology.

Description

Technical Field

[0001] This invention relates to the field of thrombin detection technology, specifically to a thrombin fluorescent substrate for whole blood, its preparation method, and its application. Background Technology

[0002] Thrombin, a key enzyme in the coagulation cascade, plays a crucial regulatory role in physiological hemostasis and pathological thrombosis by catalyzing the conversion of fibrinogen into fibrin clots. Abnormal thrombin activity is directly associated with the development of hemorrhagic diseases (such as hemophilia) or thrombotic diseases (such as deep vein thrombosis and myocardial infarction). Therefore, accurate detection of thrombin activity is a vital aspect of clinical diagnosis, treatment monitoring, and basic research. Fluorescent substrate technology, with its high sensitivity, real-time detection capability, and ease of operation, has gradually replaced traditional colorimetric methods and become the mainstream approach for thrombin activity analysis. This technology utilizes the design of molecules coupled with specific peptide sequences and fluorescent groups, leveraging the enzymatic hydrolysis of the substrate by thrombin to release a fluorescent signal, thereby achieving quantitative detection of enzyme activity.

[0003] However, existing thrombin fluorescent substrates have poor specificity for thrombin, and blood samples contain a wide variety of proteases, such as plasmin, kallikrein, and factor Xa. These proteases can also recognize and cleave thrombin substrates, leading to false positives when testing plasma samples.

[0004] Furthermore, the fluorophore of existing thrombin fluorescent substrates is AMC, and components such as hemoglobin and bilirubin in whole blood quench the blue fluorescence emitted by AMC (emission wavelength approximately 460 nm), affecting the detection of thrombin activity. Additionally, hemoglobin has a very strong absorption peak near 405 nm; to ensure detection accuracy, whole blood must usually be centrifuged to separate plasma, but this destroys the integrity of the whole blood and loses crucial information about the role of blood cells (especially platelets and red blood cells) in physiological coagulation. Therefore, existing thrombin fluorescent substrates cannot effectively detect whole blood samples.

[0005] There is an urgent need in the market for a fluorescent substrate for thrombin that can accurately detect complex samples such as plasma and whole blood. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a novel fluorescent substrate for detecting thrombin activity, the details of which are as follows:

[0007] In a first aspect, the present invention provides a thrombin fluorescent substrate for whole blood, the thrombin fluorescent substrate comprising a specific recognition peptide chain D-Phe(4-MeSO2)-Pro-Gly-Arg and a fluorescent group, wherein the specific recognition sequence of the thrombin fluorescent substrate of the present invention, from the N-terminus to the C-terminus, is 4-(methanesulfonyl)-D-phenylalanine (D-Phe(4-MeSO2)), proline, glycine, and arginine, wherein 4-(methanesulfonyl)-D-phenylalanine is obtained by attaching a methanesulfonyl group at the 4th position (i.e., the para position) of the benzene ring of D-type phenylalanine.

[0008] Furthermore, the thrombin fluorescent substrate contains a specific recognition peptide chain D-Phe(4-MeSO2)-Pro-Gly-Arg and a fluorescent group linked by chemical bonds or non-chemical bonds.

[0009] Optionally, the thrombin fluorescent substrate contains a specific recognition peptide chain D-Phe(4-MeSO2)-Pro-Gly-Arg and a fluorescent group linked by a chemical bond.

[0010] Furthermore, the fluorescent group includes at least one of 7-amino-4-methylcoumarin (AMC), 7-amino-4-trifluoromethylcoumarin (AFC), rhodamine and its derivatives, and near-infrared (NIR) fluorescent groups.

[0011] Preferably, the fluorescent group is a rhodamine derivative, more preferably rhodamine 110 (Rh110).

[0012] In a second aspect, the present invention provides a kit for detecting thrombin activity, the kit comprising the aforementioned thrombin fluorescent substrate.

[0013] Furthermore, the kit also includes additives or matrices acceptable in the biological and / or chemical fields.

[0014] Furthermore, the kit also includes at least one of reaction buffer, thrombin standard, and sample diluent.

[0015] In a third aspect, the present invention provides a method for detecting thrombin activity, the method comprising the step of adding the thrombin fluorescent substrate to a sample to be tested.

[0016] Furthermore, the method includes the following steps:

[0017] Thrombin standards were diluted into a series of concentration gradients using dilution buffer. Substrate was added to thrombin, and the fluorescence signal intensity after hydrolyzing the thrombin fluorescent substrate with different concentrations of thrombin was recorded. A standard curve of thrombin concentration-fluorescence intensity was plotted.

[0018] Obtain the fluorescence signal intensity of the sample to be tested, and calculate the thrombin concentration of the sample to be tested based on the standard curve.

[0019] Optionally, the sample to be tested is a biological sample, preferably blood or blood products.

[0020] In a fourth aspect, the present invention provides the application of the thrombin fluorescent substrate or the kit described herein in the preparation of products for detecting thrombin.

[0021] Furthermore, the product is suitable for biological samples.

[0022] Furthermore, the biological sample includes at least one of blood or blood products, cells, tissues, and body fluids.

[0023] Furthermore, the biological sample is at least one of whole blood, plasma, and serum.

[0024] In a fifth aspect, the present invention provides a method for preparing the thrombin fluorescent substrate, the method comprising at least one of chemical synthesis and enzymatic / biosynthesis.

[0025] Preferably, the method is a chemical synthesis method. More preferably, the chemical synthesis method includes at least one of liquid-phase synthesis, solid-liquid phase mixed synthesis, and solid-phase synthesis.

[0026] In one specific embodiment of the present invention, the method is a solid-phase synthesis method.

[0027] The beneficial effects of the present invention include, but are not limited to:

[0028] This invention provides a novel thrombin fluorescent substrate that exhibits a low Km value, indicating high affinity between the substrate and thrombin. A high kcat value indicates high catalytic efficiency once the substrate binds. An extremely high kcat / Km ratio further demonstrates superior performance as a specific probe for thrombin.

[0029] The thrombin fluorescent substrate of this invention maintains an extremely high reaction rate in whole blood samples, exhibiting strong anti-interference capabilities. For the target enzyme, the reaction rate of the target substrate is 1.9 times that of the universal substrate, indicating superior sequence design. For FXa, the background signal of the target substrate is only 3% of that of the universal substrate (a 97% reduction); for plasmin, the background signal is only 1.7% of that of the universal substrate (a 98.3% reduction); for kallikrein, the background signal is only 6.7% of that of the universal substrate (a 93.3% reduction). Under the most complex interference, the background signal of the target substrate is only 3.1% of that of the universal substrate (a 96.9% reduction), demonstrating outstanding anti-interference capabilities. In thrombin and mixed interfering enzymes, even under the strongest interference background, the detection accuracy of the target substrate for thrombin remains unaffected (recovery rate ≈ 100%), proving its excellent anti-interference and detection reliability. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0031] Figure 1 This is a schematic diagram of the chemical structure of the D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110 fluorescent substrate in an embodiment of the present invention.

[0032] Figure 2 These are the mass spectrometry chromatogram and 1H NMR spectrum of the D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110 fluorescent substrate in the embodiments of the present invention, wherein... Figure 2 a) Its chromatogram; Figure 2 b) Its mass spectrum; Figure 2 c) is the hydrogen NMR spectrum.

[0033] Figure 3 This is the Michaelis-Menten curve fitted in this embodiment of the invention, with V0 as the vertical axis (Y) and the corresponding substrate concentration [S] as the horizontal axis (X).

[0034] Figure 4 The graph shows the maximum initial rate (V0) of fluorescence growth at each thrombin concentration in the embodiments of the present invention. The thrombin concentration-initial reaction rate result is obtained by plotting V0 against thrombin concentration and performing linear regression fitting.

[0035] Figure 5 This is the standard curve of free Rh110 fluorescence value in an embodiment of the present invention.

[0036] Figure 6These are the thrombin activity-time curves of the D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110 fluorescent substrate and the positive control substrate in the embodiments of the present invention. Detailed Implementation

[0037] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0038] 1. Preparation of reagents and materials

[0039] Resin: Rink Amide MBHA resin (0.6 mmol / g, 100-200 mesh).

[0040] Protected amino acids:

[0041] Fmoc-D-Phe(4-MeSO2)-OH (ordered from Sigma-Aldrich);

[0042] Fmoc-Pro-OH;

[0043] Fmoc-Gly-OH;

[0044] Fmoc-Arg(Pbf)-OH (Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl).

[0045] Condensing agent: HBTU / HOBt / DIPEA (1:1:2).

[0046] Cutting reagent: TFA / TIS / H2O (95:2.5:2.5).

[0047] Others: DMF, DCM, piperidine, diethyl ether, acetonitrile.

[0048] Example 1 Preparation of thrombin fluorescent substrate

[0049] First, the specific peptide sequence of the fluorescent substrate was constructed based on the specific recognition sequence of thrombin. The obtained sequence is: D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110, with the following structural formula: Figure 1 As shown.

[0050] The fluorescent substrate was synthesized using a solid-phase peptide synthesis method, and the specific steps are as follows:

[0051] Place 1.0 g of Wang resin in a reaction column and swell it with 20 mL of DMF for 30 minutes. Dry the column under vacuum, wash three times with DCM (10 mL each time), add 20 mL of 20% piperidine / DMF solution, stir at room temperature for 20 minutes, and dry under vacuum. Wash five times with DMF (10 mL each time) and three times with DCM to remove the initial Fmoc protecting groups on the resin and expose the free amino groups.

[0052] Dissolve 1 mmol Fmoc-Arg(Pbf)-OH, 1 mmol HBTU, and 1 mmol HOBt in 15 mL DMF. Add 2 mmol DIPEA and activate for 5 minutes. Add to the reaction column and stir at room temperature for 4 hours. Use ninhydrin to determine if the reaction is complete. Dry the column, wash 5 times with DMF, and then wash 3 times with DCM. Treat the resin with a DMF solution containing acetic anhydride and DIPEA for 10 minutes to block unreacted hydroxyl groups. A negative ninhydrin test (colorless / yellow) indicates successful loading.

[0053] The coupling was performed sequentially in the order of Arg→Gly→Pro→D-Phe(4-MeSO2), with ninhydrin detection performed after each coupling step, until the coupling of Fmoc-D-Phe(4-MeSO2)-OH was completed. At this point, the resin contained a fully protected tetrapeptide: Fmoc-D-Phe(4-MeSO2)-Pro-Gly-Arg(Pbf)-Wang Resin.

[0054] The resin was treated with 20% piperidine / DMF for 20 minutes to remove the N-terminal Fmoc group, yielding HD-Phe(4-MeSO2)-Pro-Gly-Arg(Pbf)-Wang Resin. The resin was then thoroughly washed sequentially with DMF, DCM, and methanol, and dried under vacuum to obtain the fully protected peptide resin.

[0055] Place the dried peptide resin in a centrifuge tube and add 10 mL of a TFA / DCM mixture of 1:99 (volume ratio). Gently stir at room temperature for 30–60 minutes. Filter and wash the resin with a small amount of DCM. Combine the filtrates and gently concentrate to dryness at ≤30°C using nitrogen or a rotary evaporator to obtain an oily or foamy fully protected peptide acid, HD-Phe(4-MeSO2)-Pro-Gly-Arg(Pbf)-OH.

[0056] Dissolve the above fully protected peptide acid in 10 mL of anhydrous DMF. Add 1.2 mmol HBTU and 1.2 mmol HOBt, then add 2.4 mmol DIPEA, and activate in an ice bath for 10 minutes.

[0057] Weigh 1 mmol Rh110, dissolve it in 5 mL DMF, and add 1.1 mmol DIPEA to dissolve it as a salt. Add the Rh110 solution dropwise to the activated fully protected peptide acid HD-Phe(4-MeSO2)-Pro-Gly-Arg(Pbf)-OH solution.

[0058] The reaction was carried out with slow stirring at room temperature for 4-12 hours. After the reaction was completed, the reaction solution was poured into ice water, and a precipitate was formed. The precipitate was extracted with ethyl acetate, and the organic phase was washed successively with dilute citric acid aqueous solution, saturated sodium bicarbonate solution, and brine. The phase was dried over anhydrous sodium sulfate and concentrated to obtain the crude product (a fluorescent substrate with a protecting group).

[0059] Add the crude product to 20 mL of cutting reagent (volume ratio of TFA / TIS / H2O = 95:2.5:2.5), stir at room temperature for 2 hours, shaking once every 30 minutes during the process. Filter using a sintered glass funnel, collect the filtrate, wash the resin with 5 mL of TFA, and combine the filtrates.

[0060] Slowly add the filtrate to 400 mL of pre-cooled diethyl ether, stir, and let stand at 4°C for 2 hours until the white precipitate has completely precipitated. Centrifuge at 3000 rpm for 10 minutes and discard the supernatant. Wash the precipitate three times with diethyl ether and dry under vacuum.

[0061] Example 2 Identification of thrombin fluorescent substrate

[0062] Preparative reversed-phase HPLC purification:

[0063] Column: C18 column (250×20mm, 5μm);

[0064] Mobile phase: A = 0.1% TFA aqueous solution, B = 0.1% TFA acetonitrile solution;

[0065] Elution gradient: 0-30 min 10%-40%B, 30-40 min 40%-80%B, 40-50 min 80%-10%B;

[0066] Flow rate: 10 mL / min, detection wavelength: 380 nm;

[0067] The target peak was collected, and most of the acetonitrile was removed by rotary evaporation. The product was freeze-dried for 48 hours to obtain a white powder. The pure product should be protected from light, kept dry, and stored at -80°C for extended periods.

[0068] Structural characterization such as Figure 2 As shown, Figure 2 The chromatogram in a) shows that a peak appeared at 14.5 min. Figure 2b) The mass-to-charge ratio is 776.0, indicating that the obtained substance is the thrombin fluorescent substrate D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110. Figure 2 c) It was further confirmed that the obtained substance was the thrombin fluorescent substrate D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110.

[0069] Example 3: Activity identification of thrombin fluorescent substrate

[0070] Experimental system:

[0071] Buffer: 50 mM Tris-HCl, 150 mM NaCl, pH 7.4 (containing 0.1% BSA to reduce nonspecific adsorption).

[0072] Enzyme: Purified human α-thrombin (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S10117ku, specific activity 2000 U / mg).

[0073] Substrate: Prepare a high-concentration stock solution (10 mM) of the validated substrate using DMSO, and dilute with buffer before use.

[0074] Experimental steps:

[0075] Add 80 μL of buffer and 10 μL of thrombin substrate working solution at different concentrations (0, 0.8, 1.6, 2.4, 3.2, 4.8, 6.4, 9.6, 16, 32, 64 μM) to each well of a black 96-well plate, with three replicates for each concentration. Set up a background control with only buffer and substrate, excluding the enzyme. Set the microplate reader temperature to 37°C and incubate the plate for 5–10 minutes. Using the microplate reader's autosampler, quickly add 10 μL of enzyme working solution to each well (except the background control wells). Start reading immediately. Immediately begin monitoring fluorescence intensity (excitation / emission: 496 nm / 520 nm), taking readings every 10–30 seconds for 10–20 minutes.

[0076] Calculate the initial reaction rate (V0): Subtract the average value of the background control wells from the fluorescence readings. Convert the fluorescence intensity values ​​to product concentration (μM) using the Rh110 standard curve. Select the initial linear portion of the reaction progress curve (product formation < 5% of total substrate) and calculate the slope to obtain the initial reaction rate V0 (unit: μM / s).

[0077] A Michaelis-Menten curve was fitted with V0 as the ordinate (Y) and the corresponding substrate concentration [S] as the abscissa (X). kcat was calculated.

[0078] Formula: kcat = Vmax / [E]0;

[0079] Where [E]0 is the total molar concentration of thrombin in the reaction system.

[0080] The results are as follows:

[0081] Km = 3.25 ± 0.2 μM;

[0082] Vmax = 0.152 ± 0.008 μM / s;

[0083] kcat = 122 s - ¹ ;

[0084] kcat / Km = 3.75 × 10 7 M - ¹s - ¹.

[0085] result( Figure 3 The thrombin fluorescent substrate described in this invention exhibits a low Km value, indicating a high affinity between the substrate and thrombin. A high kcat value indicates high catalytic efficiency once the substrate binds. An extremely high kcat / Km ratio is the gold standard for measuring "catalytic efficiency" or "specificity constant." The higher this value, the better the substrate's performance as a specific probe for thrombin.

[0086] Example 4: Simulated Plasma Experiment

[0087] 1. Calibration curve and sensitivity determination in simulated plasma

[0088] Target fluorescent substrate: D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110, prepared as a 10 mM stock solution with DMSO, stored at -20℃ protected from light.

[0089] The simulated plasma buffer was prepared with 3 mg / mL BSA (simulated plasma protein background), 1 mg / mL trypsin-hydrolyzed casein, 50 mM Tris-HCl, 150 mM NaCl, and pH 7.4.

[0090] Human α-thrombin: purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S10117ku, with a specific activity of 2000 U / mg.

[0091] Experimental steps:

[0092] Add 80 µL of simulated plasma buffer to each well of a black 96-well plate. Add 10 µL of thrombin working solution at different concentrations (diluted with simulated plasma buffer to 0, 0.1, 0.5, 1, 2, 5, 10, 20 pM) to each well, with three replicates for each concentration. Set up a zero-enzyme well (add an equal volume of buffer) as background. Incubate the plate in a microplate reader at 37°C for 5 minutes. Quickly add 10 µL of target substrate working solution (diluted with simulated plasma buffer to a final concentration of 10 µM) to each well. Begin measurements immediately. Excitation / emission wavelengths: 496 nm / 520 nm, read fluorescence intensity every 30 seconds for 60–90 minutes, or until the signal reaches plateau.

[0093] Plotting calibration curves and calculating sensitivity: Calculate the maximum initial rate of fluorescence growth (V0) at each thrombin concentration, plot V0 against thrombin concentration, and perform linear regression fitting.

[0094] The results are as follows Figure 4 As shown, the fitted curve is y = 0.2714x + 0.0175, R0 2 = 0.9997.

[0095] The lowest detection limit is calculated based on the fitted curve using the formula: LOD = (3 × SD_blank) / a;

[0096] Where a is the slope of the calibration curve, and SD_blank is the standard deviation of the blank sample V0.

[0097] Six independent replicate experiments were performed on the blank sample (0 pM), and the V0 of each blank sample was calculated. The results were: 0.002 μM / min, -0.001 μM / min, 0.003 μM / min, 0.001 μM / min, 0.002 μM / min, and 0.000 μM / min.

[0098] The standard deviation was calculated, and the result was SD_blank = 0.00147 μM / min.

[0099] The lowest detection limit was obtained as 0.0163 pM.

[0100] 2. Specificity verification experiment

[0101] Interfering proteases: Factor Xa (Sigma-Aldrich, F9302-25UG), plasmin (Sigma-Aldrich, P1867-1MG), and kallikrein (Sigma-Aldrich, K1879-100UG). Each was prepared using simulated plasma buffer.

[0102] Positive control substrate: Commercially available universal fluorescent substrate (Z-Gly-Gly-Arg-AMC) (Sigma-Aldrich, C9521-1MG).

[0103] Set up reaction groups: Set up the following groups in the orifice plate, with 3 replicates per group:

[0104] A. Experimental group: simulated plasma + target fluorescent substrate + thrombin (1 pM);

[0105] B. Interference control group 1: simulated plasma + target fluorescent substrate + factor Xa (FXa, 1 nM);

[0106] C. Interference control group 2: simulated plasma + target fluorescent substrate + plasmin (Plasmin, 10 nM);

[0107] D. Interference control group 3: simulated plasma + target fluorescent substrate + kallikrein (500pM);

[0108] E. Interference control group 4: Simulated plasma + target fluorescent substrate + mixed interfering enzyme: FXa (1 nM) + Plasmin (10 nM) + Kallikrein (500 pM);

[0109] F. Interference control group 5: Simulated plasma + target fluorescent substrate + thrombin (1 pM) + mixed interfering enzyme: FXa (1 nM) + Plasmin (10 nM) + Kallikrein (500 pM);

[0110] G. Cross-reactive group 1: Simulated plasma + positive control substrate + thrombin;

[0111] H. Cross-reactive group 2: simulated plasma + positive control substrate + factor Xa (FXa, 1 nM);

[0112] I. Cross-reactive group 3: simulated plasma + positive control substrate + plasmin (Plasmin, 10 nM);

[0113] J. Cross-reactive group 4: simulated plasma + positive control substrate + kallikrein (500pM);

[0114] K. Cross-reactive group 5: simulated plasma + positive control substrate + mixed interfering enzyme: FXa (1 nM) + Plasmin (10 nM) + Kallikrein (500 pM);

[0115] L. Cross-reactive group 6: Simulated plasma + positive control substrate + thrombin (1 pM) + mixed interfering enzymes: FXa (1 nM) + Plasmin (10 nM) + Kallikrein (500 pM);

[0116] M. Background group: simulated plasma + target fluorescent substrate (without any enzymes).

[0117] Experimental Procedure: Add 80 µL of simulated plasma buffer to each well of a black 96-well plate. Add 10 µL of different working solutions to each well according to the requirements of each reaction group, with three replicates for each concentration. Set up a zero-enzyme well (with an equal volume of buffer added) as background. Incubate the plate in a microplate reader at 37°C for 5 minutes. Quickly add 10 µL of target substrate working solution (dilute the target fluorescent substrate or positive control substrate with simulated plasma buffer to a final concentration of 10 µM) to each well. Begin measurement immediately. Read the fluorescence intensity every 30 seconds for 60-90 minutes, or until the signal reaches the plateau phase.

[0118] Calculate the specificity ratio: Calculate the response rate V0 for the experimental group (A) and each interference control group (BE).

[0119] Specificity ratio = V0 (thrombin) / V0 (interfering protease).

[0120] The results are shown in Table 1. The results indicate that the substrate described in this invention has the following advantages:

[0121] Extremely high specificity: The target substrate produced a strong signal (1500 RFU / min) under the action of 1 pM thrombin. However, when various interfering enzymes (1 nM FXa, 10 nM Plasmin, 500 pM Kallikrein) are present alone or in mixtures at concentrations 100-10000 times higher, the background signal (3-15 RFU / min) is extremely low.

[0122] Quantitative advantages: The specificity ratios are all much greater than 100, with the highest reaching 500. This means that the target substrate reduces the non-specific background signal by more than 99.8% (for factor Xa, the background is only 3 / 1500 = 0.2% of the target signal).

[0123] High signal-to-noise ratio: Even under the most demanding mixed interfering enzyme (E group) challenge, the signal-to-noise ratio SNR = V0(A) / V0(E) is still as high as 100, which fully meets the requirements for complex sample detection.

[0124] Table 1. Response rates of the experimental group (A) and each interference control group (BE).

[0125]

[0126] Table 2 shows the performance comparison between the target substrate and the positive control substrate. The results indicate that the target substrate's reaction rate was 1.9 times that of the universal substrate for the target enzyme, indicating superior sequence design. For FXa, the background signal of the target substrate was only 3% of that of the universal substrate (a 97% reduction); for plasminogen activator, the background signal was only 1.7% of that of the universal substrate (a 98.3% reduction); and for kallikrein, the background signal was only 6.7% of that of the universal substrate (a 93.3% reduction). Under the most complex interference, the background signal of the target substrate was only 3.1% of that of the universal substrate (a 96.9% reduction), demonstrating outstanding anti-interference ability. In thrombin + mixed interfering enzymes, under the strongest interference background, the detection accuracy of the target substrate for thrombin was not affected in any way (recovery rate ≈ 100%), indicating that its specificity and anti-interference performance were better than that of the positive control substrate.

[0127] Table 2 Results of positive control substrate performance assay

[0128]

[0129] Example 5: Thrombin Activity Detection Experiment in Whole Blood Samples

[0130] Target fluorescent substrate: D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110 (10 mM DMSO stock solution). Dilute to working concentration (10 µM) with Hepes buffered saline (HBSS) or physiological saline before use.

[0131] Thrombin activation reagent: Activated with a solution containing 2.5 pM tissue factor (TF) and 4 mM CaCl2.

[0132] Whole blood samples: Venous blood was collected from healthy volunteers and anticoagulated using 3.2% sodium citrate (1:9 ratio). Gentle handling was maintained throughout the process to avoid platelet activation or hemolysis. The experiment was completed within 2 hours of blood collection, during which time the blood was kept at room temperature and free from shaking.

[0133] Quenching calibration standard: Prepare a standard solution of free Rh110 for calibration.

[0134] Experimental steps:

[0135] Preheat sodium citrate anticoagulated whole blood, CaCl2 solution, substrate working solution, and TF activator in a 37°C water bath for 10 minutes. In each well of the preheated 96-well plate, add 10 µL of the target fluorescent substrate working solution (final concentration 10 µM).

[0136] Quickly add 70 µL of preheated whole blood to the well containing the substrate. Gently blow and mix several times, avoiding the formation of air bubbles.

[0137] Using the autosampler of an ELISA reader or manually, quickly add 20 µL of preheated “TF / CaCl2 mixed activation solution” to each well. Immediately begin fluorescence readings (RFU) (Ex / Em: 490 / 520 nm), taking readings every 15–30 seconds for at least 60 minutes.

[0138] Set up the following groups (3 duplicate holes per group):

[0139] Experimental group: whole blood + target fluorescent substrate + TF / CaCl2 activator.

[0140] Background control group: whole blood + target fluorescent substrate + equal volume of physiological saline.

[0141] Positive control group: whole blood + positive control substrate (Z-Gly-Gly-Arg-AMC) + TF / CaCl2 activator (Ex / Em: 380 / 460 nm).

[0142] Matrix effect group: whole blood + free Rh110 standard (10µM) + TF / CaCl2 activator.

[0143] Measure F_blood (fluorescence of Rh110 standard in whole blood) and F_buffer (fluorescence in buffer solution). Calculate the quenching coefficient Q = F_blood / F_buffer. Correct all raw fluorescence data by dividing by Q.

[0144] A standard curve was prepared using different concentrations (0, 0.1, 0.5, 1, 2, 5, 10 µM) of free Rh110 in buffer solution. Figure 5 The corrected fluorescence values ​​were converted to concentration using the Rh110 standard curve, thus obtaining the thrombin activity-time curve.

[0145] The calculation formulas for each step are as follows:

[0146] Net RFU = Experimental group RFU - Background group RFU;

[0147] Corrected RFU = Net RFU / Q.

[0148] Substitution Figure 5 The standard curve formula was used to plot the thrombin activity-time curve, and the results (e.g.) Figure 6 As shown in the figure, the target fluorescent substrate has a shorter start-up time (delay time), a higher curve position (peak), and a faster rise rate (rate constant), indicating that its sensitivity and signal-to-noise ratio are comprehensively superior to traditional AMC substrates. Figure 6 The parameters of the obtained target fluorescent substrate and positive control substrate are shown in Table 3.

[0149] The formula for calculating the rate constant (taking the linear slope of the steepest interval of the curve):

[0150] .

[0151] Table 3. Parameters of target fluorescent substrate and positive control substrate

[0152]

[0153] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. A thrombin fluorescent substrate for whole blood, characterized in that, The thrombin fluorescent substrate is formed by the covalent binding of a specific recognition peptide chain D-Phe(4-MeSO2)-Pro-Gly-Arg and a fluorescent group Rhodamine 110, with the sequence D-Phe(4-MeSO2)-Pro-Gly-Arg-Rh110.

2. A kit for detecting thrombin activity, characterized in that, The kit includes the thrombin fluorescent substrate as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes additives or matrices acceptable in the biological and / or chemical fields.

4. The reagent kit according to claim 2, characterized in that, The kit also includes at least one of the following: reaction buffer, thrombin standard, and sample diluent.

5. The use of the thrombin fluorescent substrate as described in claim 1 or the kit as described in any one of claims 2-4 in the preparation of products for detecting thrombin.

6. The application according to claim 5, characterized in that, The product is suitable for biological samples.

7. The application according to claim 6, characterized in that, The biological sample includes at least one of cells, tissues, and body fluids.

8. The application according to claim 6, characterized in that, The biological samples include blood.

9. The application according to claim 6, characterized in that, The biological samples include blood products.

10. A method for preparing the thrombin fluorescent substrate of claim 1, characterized in that, The method includes chemical synthesis.

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