Real-time detection method for fibrinolytic enzyme activity in whole blood

Abstract

The invention discloses a real-time detection method for the activity of fibrinolytic enzyme in whole blood, which comprises the following steps: preparing a fibrinolytic response interference layer film with fibrinolytic enzyme response, and fixing to obtain a microfluidic sample cell; preparing a closed plasmin response and plasmin response interference layer film; analyzing the anti-interference capability of the fibrinolysis response interference layer film on interference protein in whole blood; analyzing the specificity of the fibrinolytic response interference layer film for detecting fibrinolytic enzyme in whole blood; analyzing the sensitivity of the fibrinolytic response interference layer film on the detection of fibrinolytic enzyme in whole blood; diluting the whole blood sample by different multiples with normal saline, introducing the diluted whole blood sample and the diluted whole blood sample with labeled fibrinolytic enzyme into a sample pool formed by the closed fibrinolytic response interference layer film, and detecting the fibrinolytic enzyme activity in the whole blood sample. The method can overcome the interference of colored, complex and viscous whole blood samples, successfully detects the activity of the fibrinolytic enzyme in the whole blood samples, and is stable in detection.

Description

Technical Field

[0001] The invention belongs to the field of biosensor detection, and in particular relates to a real-time detection method for fibrinolytic enzyme activity in whole blood. Background Art

[0002] Fibrin plays an important role in hemostasis, being both the main product of the coagulation cascade to form the blood clot scaffold and the final substrate of fibrinolysis during thrombolysis. Under normal physiological conditions, a constant dynamic balance is maintained between blood coagulation and fibrinolysis. However, the disorder of fibrinolysis in whole blood may lead to some serious diseases. Excessive fibrinolytic activity may trigger and cause life-threatening blood loss, which is common in patients suffering from severe trauma and after surgery. In contrast, low fibrinolytic activity may cause thromboembolism, which is one of the main causes of cardiovascular disease. Thrombosis is also considered to be the "culprit" that hinders blood flow to organs, often leading to serious diseases such as pulmonary embolism, acute myocardial infarction and ischemic stroke. Monitoring fibrinolytic activity is considered to be an emerging science for managing surgical bleeding and preventing thromboembolism. Fibrinolysis is directly caused by the enzymatic action of plasmin. Therefore, the determination of plasmin activity in the blood is crucial for the clinical treatment of cardiovascular diseases.

[0003] According to different mechanisms of action, plasmin can be divided into two types. One is plasmin-like plasmin, such as plasmin, nattokinase and lumbrokinase, which can directly degrade fibrin. The other type is plasminogen activators, such as urokinase and streptokinase, which activate plasminogen (Plg) to convert into active plasmin to degrade fibrin. Some methods have been developed to evaluate and quantify plasmin activity in vitro, but they all have certain defects. For example, the fibrin plate method and its derivative methods that measure the end point of fibrin lysis are not only time-consuming and unstable, but also can obtain very little data information. The turbidity lysis method can be used to evaluate fibrinolytic activity by measuring the turbidity of plasma clots, such as euglobulin clot lysis time. The disadvantage of this method is low sensitivity. Fibrinolysis under flow conditions can most truly reflect the events that occur during fibrinolysis. However, there are few methods available for measuring plasmin activity under flow conditions. There are two main difficulties: (1) Under normal circumstances, fibrin takes hours or even days to dissolve in healthy blood, and (2) the key reaction of fibrinolysis occurs at the heterogeneous solid-liquid interface, to which the classical homogeneous enzymology concepts and analytical methods may not be applicable, which increases the difficulty of detecting plasmin activity under flow conditions. Summary of the invention

[0004] Purpose of the invention: In order to overcome the deficiencies in the prior art, the purpose of the present invention is to provide a real-time detection method for fibrinolytic enzyme activity in whole blood which has strong anti-interference ability, good stability and can perform real-time dynamic quantitative analysis.

[0005] Technical solution: The real-time detection method of fibrinolytic enzyme activity in whole blood of the present invention comprises the following steps:

[0006] S1. Fixing a silica colloidal crystal film on a silica pad to prepare a reaction pool, first injecting fibrinogen into the reaction pool, letting it stand, then dripping thrombin into the reaction pool, removing the silica pad after the cross-linking reaction, and obtaining a fibrinolytic responsive interference layer film that is responsive to plasmin;

[0007] S2, fixing a fibrinolytic responsive interference layer film responsive to plasmin between a glass slide and a silicone pad to obtain a microfluidic sample pool;

[0008] S3, pumping ovalbumin into the microfluidic sample pool for blocking, and then blocking with surfactant S9, and washing with PBS solution after the blocking, to obtain a blocked plasmin-responsive fibrinolytic-responsive interference layer film;

[0009] S4, passing PBS solutions containing excess blood proteins into the sample pools formed by the sealed fibrinolysis response interference layer film, and rinsing with PBS solution after completion, and analyzing the anti-interference ability of the fibrinolysis response interference layer film to the interfering proteins in the whole blood by ordered porous layer interferometry;

[0010] S5, passing a PBS solution containing an excess of interfering protease into a sample pool formed by a closed fibrinolysis response interference layer film, washing with a PBS solution after the reaction is completed, and analyzing the specificity of the fibrinolysis response interference layer film for the detection of plasmin in whole blood by an ordered porous interferometry system;

[0011] S6, passing PBS solutions containing different concentrations of fibrinolytic enzymes into the sample pool formed by the closed fibrinolytic response interference layer film, flushing the system with PBS solution after the reaction is completed, and analyzing the sensitivity of the fibrinolytic response interference layer film to the detection of fibrinolytic enzymes in whole blood by an ordered porous interferometry system;

[0012] S7. Dilute the whole blood sample with physiological saline at different multiples, and pass the diluted whole blood sample and the diluted whole blood sample spiked with plasmin into the sample pool formed by the closed fibrinolysis response interference layer film. After the reaction is completed, rinse the system with PBS solution, and detect the plasmin activity in the whole blood sample by the ordered porous interferometry measurement system.

[0013] A fibrinolysis-responsive interference layer film was constructed using silica colloidal crystal film as a support, and a sensing system that can monitor the activity of fibrinolytic enzymes in different fibrinolytic pathways in whole blood in real time was established based on the ordered porous layer interferometry technique. This method can monitor the entire process of fibrinolytic enzymes degrading fibrin in real time, which provides more possibilities for real-time dynamic quantitative analysis of fibrinolytic enzyme activity in clinical practice.

[0014] Furthermore, in step S1, the concentration of fibrinogen is 1-20 mg / mL, the activity of thrombin is 10-150 U / mL, and the ratio of the injection amount of fibrinogen to the injection amount of thrombin is 20-80:2-10. Preferably, fibrinogen is replaced by a mixture of fibrinogen and plasminogen, and the activity of the plasminogen is 0.1-1 U / mL. The cross-linking reaction time is 1-6 hours.

[0015] Furthermore, in step S3, the volume percentage of ovalbumin is 1 to 3 vol%, the mass percentage of surfactant S9 is 0.1 to 0.3 wt%, and the blocking time is 1 to 5 hours.

[0016] Furthermore, in step S4, the blood protein is one or more of human serum albumin and human immunoglobulin G.

[0017] Furthermore, in step S5, the interfering protease is one or more of alkaline phosphatase, thrombin, lipase, lactate dehydrogenase, and alanine aminotransferase.

[0018] Furthermore, in step S6, the fibrinolytic enzyme is plasmin or streptokinase, the concentration of the plasmin is 0.001 to 0.5 U / mL, and the concentration of the streptokinase is 10 to 2000 U / mL.

[0019] Furthermore, in step S7, the volume percentage of the whole blood sample after dilution is 5-50 vol%.

[0020] Furthermore, in steps S4 to S7, the pH of the PBS solution is 7.2 to 7.6, and the concentration is 0.01 to 0.1 M. Sodium chloride with a concentration of 0.01 to 0.1 M and a surfactant S9 with a concentration of 0.02 to 0.15 wt % are added to the PBS solution.

[0021] Detection principle: By loading fibrin or a mixture of fibrin and plasminogen into a highly ordered silica colloidal crystal film scaffold, and then dripping thrombin, a fibrinolysis-responsive interference layer film is constructed. Using the fibrinolysis-responsive interference layer film as the interference substrate, a sensing system for real-time, label-free detection of plasmin activity in whole blood is developed in combination with ordered porous interferometry. Fibrinolysis triggered by plasmin causes the interference fringes of the interference layer to blue shift, which can be manifested as a change in optical thickness.

[0022] This method can monitor the fibrinolytic process of different fibrinolytic enzymes: (1) plasmin directly dissolves fibrin; (2) streptokinase activates plasminogen in the interference layer and then begins to dissolve fibrin. The fibrinolytic response interference sensing layer film can shield nonspecific adsorption in complex environments after being sealed with ovalbumin and surfactant S9, so it can be used for the analysis of complex whole blood systems. When the whole blood sample flows into the interference layer, the fibrin therein will be specifically cut by the plasmin, and when it is rinsed with buffer, the influence of the solvent effect is eliminated and the free and weakly bound interference molecules are eluted. This entire process can be recorded in real time, and the final optical thickness change can be obtained. According to the relationship curve between the optical thickness change and the activity of the plasmin in the buffer system, the activity of the plasmin in the whole blood can be calculated.

[0023] Beneficial effects: Compared with the prior art, the present invention has the following significant features:

[0024] 1. It can overcome the interference of colored, complex and viscous whole blood samples and successfully detect the fibrinolytic enzyme activity in whole blood samples;

[0025] 2. Successfully overcome the difficulty of detecting enzymatic reactions occurring at the solid-liquid interface of a heterogeneous phase, and can monitor the degradation of fibrin by fibrinolytic enzymes under flow conditions;

[0026] 3. It not only directly records the process of fibrinolytic enzymes degrading fibrin, but also records the effect of fibrinolytic enzymes at each time point during the fibrinolysis process, and can provide real-time fibrinolysis data. Compared with the traditional endpoint method, it records more meaningful data;

[0027] 4. Ovalbumin can be used to block the adsorption sites on the film that are not occupied by fibrin, and the surfactant S9 shields the hydrophobic groups on the surface of fibrin to prevent interfering molecules from binding to fibrin through hydrophobic interactions. This completely shields nonspecific adsorption in the whole blood environment, making the test more stable and less subject to interference;

[0028] 5. The three-dimensional fibrin network is loaded on the reaction interface, which truly simulates the interaction process between plasmin and fibrin under flow state. Compared with the traditional closed detection system, it is more in line with the biological characteristics of the fibrinolytic process, which is crucial for the clinical detection of plasmin activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the detection principle diagram of the present invention;

[0030] Figure 2 1 is a scanning electron microscope image of a silica colloidal crystal film (A), a film loaded with fibrin (B), and a film loaded with a complex of fibrin and plasminogen (C) in Example 1 of the present invention;

[0031] Figure 3 Schematic diagram of the whole blood fibrinolytic enzyme activity detection method of the present invention, (A) real-time flow unit positioned by a microscope, (B) fibrinolysis response interference layer constructed by silica colloidal crystal film, (C) mechanism of real-time monitoring of fibrinolytic enzyme activity by the fibrinolysis response interference layer, (D) reflection interference spectrum diagram, (E) real-time change diagram of optical thickness;

[0032] Figure 4 It is a real-time change diagram of the optical thickness of the fibrinolysis-responsive interference layer film during the whole process of detecting a PBS solution containing a fibrinolytic enzyme in Example 1 (A) and Example 2 (B) of the present invention;

[0033] Figure 5 The real-time optical thickness curves of the fibrinolysis-responsive interference layer film in Example 1 (A) and Example 2 (B) of the present invention are obtained by passing a PBS solution spiked with excess interfering proteins (2 mg / mL HSA and IgG) and then washing with a PBS solution;

[0034] Figure 6 Graphs showing changes in optical thickness caused by the introduction of 10 μg / mL of different interfering proteases and fibrinolytic enzymes into the fibrinolytic response interference layer films in Examples 1 (A) and 2 (B) of the present invention;

[0035] Figure 7 is the optical thickness change curve caused by different concentrations of plasmin in the present invention;

[0036] Figure 8 is the plasmin activity detection calibration curve of the present invention;

[0037] Fig. 9 is a real-time optical thickness curve of the same whole blood sample of the present invention at different dilutions when pumped into the fibrinolysis response interference layer film in Example 1;

[0038] Fig.10The fibrinolytic response curve of the fibrinolytic response interference layer film with different plasminogen contents when 2000 U / mL of streptokinase is applied to the film;

[0039] Fig.11 The optical thickness change curve (A) caused by different concentrations of streptokinase (10, 20, 100, 200, 1000, 2000 U / mL) and the streptokinase activity detection calibration curve (B) of the present invention;

[0040] Fig.12 The real-time optical thickness curves (A) of the same whole blood sample of the present invention at different dilutions pumped into the fibrinolysis-responsive interference layer film in Example 2, and the real-time optical thickness curves (B) of the same whole blood sample spiked with 1000 U / mL streptokinase pumped into the fibrinolysis-responsive interference layer film in Example 2 at different dilutions;

[0041] Fig.13 The dissolution circle after plasmin acts on the fibrin plate in Comparative Example 1 (A), the calibration curve (B), and the dissolution circle after the whole blood sample spiked with 1000U / mL plasmin acts on the fibrin plate (C);

[0042] Fig.14 The dissolution circle (A) and calibration curve (B) after the medium chain kinase acts on the fibrin plate in Comparative Example 2;

[0043] Fig.15 This is a graph showing the relationship between the absorbance value obtained using a commercial ELISA kit and the plasmin concentration in Comparative Example 3. DETAILED DESCRIPTION

[0044] The experimental methods described in the examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0045] Example 1

[0046] like Figure 1 , a real-time detection method for fibrinolytic enzyme activity in whole blood, comprising the following steps:

[0047] S1. Soak the slide in piranha solution overnight and wash it with ultrapure water. Use the vertical deposition method to vertically insert the slide into the purified silica sol (about 1wt%), control the ambient temperature to 25℃ and the humidity to 10%, and after ethanol evaporation for 7 days, a silica colloidal crystal film with a thickness of 5 microns is obtained.

[0048] S2. Fix the film obtained in step S1 on a square groove silicone pad to prepare a reaction pool, first inject 60 μL of 10 mg / mL fibrinogen into the reaction pool, let it stand for 20 minutes, then add 8 μL of thrombin with an enzyme activity of 80 U / mL into the reaction pool, and perform cross-linking reaction at 37°C for 5 hours. After the reaction, remove the silicone pad to obtain a plasmin-responsive fibrinolytic interference layer film.

[0049] S3, fix the film obtained in S2 on a plate with a needle area of ​​0.2 cm 2 The circular grooved silicone pad was placed underneath and a glass slide was covered on top of the silicone pad to obtain a microfluidic sample pool.

[0050] S4. Use a peristaltic pump to pump 2 vol% ovalbumin into the sample pool obtained in S3 at a flow rate of 0.45 mL / min for sealing for 2 hours, and then continue to seal with 0.25 wt% surfactant S9 for 1 hour. After sealing, rinse with PBS solution (containing 0.08 wt% surfactant S9, concentration of 0.05 M, pH 7.4) for 15 minutes to obtain a sealed plasmin-responsive fibrinolytic-responsive interference layer film.

[0051] S5. Pass 2 mg / mL PBS solutions of human serum albumin and human immunoglobulin G into the sample pool formed by the closed fibrinolysis response interference layer film respectively. After completion, rinse the system with PBS solution (containing 0.8 M sodium chloride and 0.08 wt % surfactant S9, with a concentration of 0.05 M and a pH of 7.4). Analyze the anti-interference ability of the fibrinolysis response interference layer film to interfering proteins in whole blood by ordered porous layer interferometry (OPLI).

[0052] S6. Pass 10 μg / mL PBS solutions of alkaline phosphatase, thrombin, lipase, lactate dehydrogenase, alanine aminotransferase and plasmin into the sample pool formed by the closed fibrinolysis response interference layer film. After the reaction, rinse the system with PBS solution (containing 0.8 M sodium chloride and 0.08 wt % surfactant S9, with a concentration of 0.05 M and a pH of 7.4). Analyze the specificity of the fibrinolysis response interference layer film for the detection of plasmin in whole blood by the ordered porous interferometry measurement system.

[0053] S7. PBS solutions containing different concentrations of plasmin (0.001, 0.005, 0.01, 0.05, 0.1, and 0.5 U / mL) were passed into the sample pool formed by the closed fibrinolysis response interference layer film. After the reaction, the system was flushed with PBS solution (containing 0.8 M sodium chloride and 0.08 wt % surfactant S9, with a concentration of 0.05 M and a pH of 7.4). The sensitivity of the fibrinolysis response interference layer film to the detection of plasmin in whole blood was analyzed by an ordered porous interferometry measurement system.

[0054] S8. Dilute the whole blood sample to 5, 10, 20, and 50% with 0.9% saline, and pass the diluted whole blood sample and the diluted whole blood sample spiked with different concentrations (0.01, 0.05, and 0.1U / mL) of plasmin into the sample pool formed by the closed fibrinolysis response interference layer film. After the reaction, rinse the system with PBS solution (containing 0.8M sodium chloride and 0.08wt% surfactant S9, with a concentration of 0.05M and a pH of 7.4), and detect the plasmin activity in the whole blood sample by an ordered porous interferometry system. The real whole blood sample contains 0.8M sodium chloride and 0.08wt% surfactant S9.

[0055] like Figure 2 A and Figure 2 B. Silica is tightly packed in a large area on the glass slide, showing a typical colloidal crystal structure. The surface of the film loaded with fibrin is covered with a dense fibrin network, and the fibrin fibers are evenly filled in the gaps between the silica balls. In addition, the loading of fibrin does not destroy the regularity of the structure of the silica colloidal crystal film, which is conducive to its use as a scaffold for interferometric sensing measurement.

[0056] Figure 3 The principle of the present invention for real-time, online and in-situ detection of fibrinolytic enzyme activity is shown. As shown in 3B, since the interference fringes are collected from the back of the sensing film element, and since the nanoporous structure of the film can block blood cells outside the film, turbid and colored samples will not affect the collection of interference signals. Figure 3 D. Figure 3 E, plasmin-like fibrinolytic enzymes respond to fibrinolysis. The degradation of fibrin in the interference layer reduces the mass of the interference layer, resulting in a blue shift in the interference spectrum, which is manifested as a decrease in the optical thickness value. This method can monitor the fibrinolytic process of different fibrinolytic enzymes: (1) plasmin-like fibrinolytic enzymes directly dissolve fibrin; (2) plasminogen in the interference layer is activated and begins to dissolve fibrin. Figure 3 C, Real-time in situ monitoring of fibrinolytic enzyme activity by collecting optical thickness through the OPLI system.

[0057] Figure 4 It is divided into 4 stages: (i) ovalbumin blocking, (ii) surfactant S9 blocking, (iii) PBS flushing system, and (iv) detection of PBS solution containing plasmin or streptokinase. Figure 4 As shown in A, after the plasmin-responsive fibrinolytic interference layer film is blocked by ovalbumin and surfactant S9, the optical thickness drops sharply when the plasmin solution flows through it, indicating that the film is responsive to plasmin.

[0058] like Figure 5As shown in A, after the fibrinolytic response interference layer film is blocked by ovalbumin and surfactant S9, it has no response to the interfering proteins (serum albumin and human immunoglobulin G) in the excess blood, and the optical thickness value returns to the initial value. This proves that the blocking process of Example 1 can completely shield the interference of nonspecific adsorption. In addition, Figure 6 The fibrinolysis-responsive interference layer film shown in A has an excellent specific response to plasmin. The film has basically no reaction to 10 μg / mL (excess) of alkaline phosphatase, thrombin, lipase, lactate dehydrogenase, and alanine aminotransferase pumped in. The final optical thickness value is similar to that of the control group, indicating that this fibrinolysis model provides excellent selectivity for plasmin, making the detection more targeted.

[0059] The fibrinolytic process of different concentrations of plasmin Figure 7 As shown. When plasmin flows into the sample pool, the optical thickness value continues to decrease, and the fibrinolysis rate increases with the increase of enzyme concentration, which is consistent with the nature of the enzymatic reaction. Since the optical thickness value did not change during the final PBS rinse stage, it was selected as the reaction endpoint to calculate the fibrinolysis rate. Subsequently, a linear regression analysis was performed with the logarithm of the plasmin concentration as the horizontal axis and the final optical thickness value as the vertical axis. The calibration curve of plasmin dissolving fibrin is shown in Figure 8 As shown, when the enzyme concentration is in the range of 0.001-0.5U / mL, lg(C 纤溶酶 ) is linearly related to the optical thickness value with a linearity of 0.95.

[0060] Fig. 9 The real-time optical thickness changes of the interference layer in response to pumping fibrinolysis are shown at different dilution levels of whole blood samples. Figure 8 By substituting the final optical thickness change into the standard curve, the concentration of plasmin can be calculated as 0.0104U / mL (5%), 0.0105U / mL (10%), 0.0114U / mL (20%) and 0.0071U / mL (50%). When the concentration of whole blood sample is lower than 20%, the measured plasmin activity is consistent with the concentration of plasmin in the same whole blood sample detected by the fibrin plate method and the commercial ELISA kit (0.0101U / mL, 0.0109U / mL) ( Fig.13 C and Fig.15 ). To further verify the reliability of this method for determining the activity of plasmin in whole blood samples, the same whole blood sample was spiked with plasmins of different activities and the activity of plasmin was detected after 5-fold dilution. As can be seen from Table 1, the recovery rates of plasmin were 97%-110%, and the relative standard deviation was less than 7%. In summary, the method of this embodiment is feasible for the detection of plasmin activity in whole blood samples.

[0061] Table 1 Recovery of plasmin spiked with whole blood samples after 5-fold dilution

[0062] Spiked amount (U / mL) Detection value (U / mL) Recovery rate (%) Relative standard deviation (%) 0.01 0.011 110 6.8 0.05 0.052 104 4.2 0.1 0.097 97 3.6

[0063] Example 2

[0064] A real-time detection method for fibrinolytic enzyme activity in whole blood comprises the following steps:

[0065] S1. Soak the slide in piranha solution overnight and wash it with ultrapure water. Use the vertical deposition method to vertically insert the slide into the purified silica sol (about 1wt%), control the ambient temperature to 25℃ and the humidity to 10%, and after ethanol evaporation for 7 days, a silica colloidal crystal film with a thickness of 5 microns is obtained.

[0066] S2. Fix the film obtained in step A1 on a square groove silicone pad to prepare a reaction pool. First, inject 50 μL of a mixture containing 10 mg / mL fibrinogen and 0.5 U / mL plasminogen into the reaction pool. After standing for 20 minutes, add 6 μL of thrombin with an enzyme activity of 100 U / mL into the reaction pool. Cross-link the reaction at 37°C for 5 hours. After the reaction, remove the silicone pad to obtain a streptokinase-responsive fibrinolytic interference layer film.

[0067] S3, fix the film obtained in A2 on a plate with a needle area of ​​0.2 cm 2 The circular grooved silicone pad was placed underneath and a glass slide was covered on top of the silicone pad to obtain a microfluidic sample pool.

[0068] S4. Use a peristaltic pump to pump 1.5 vol% ovalbumin into the sample pool obtained in A3 at a flow rate of 0.5 mL / min for blocking for 2 hours, and then continue to block with 0.2 vol% surfactant S9 for 1 hour. After blocking, rinse with PBS solution (containing 0.05 wt% surfactant S9, concentration of 0.05 M, pH 7.4) for 15 minutes to obtain a blocked streptokinase-responsive fibrinolysis-responsive interference layer film.

[0069] S5. Pass 2 mg / mL human serum albumin and human immunoglobulin G PBS solutions into the sample pool formed by the closed fibrinolysis response interference layer film respectively. After completion, rinse the system with PBS solution (containing 0.8 M sodium chloride and 0.05 wt % surfactant S9, with a concentration of 0.05 M and a pH of 7.4). Analyze the anti-interference ability of the fibrinolysis response interference layer film to interfering proteins in whole blood by ordered porous layer interferometry (OPLI).

[0070] S6. 10 μg / mL PBS solutions of alkaline phosphatase, thrombin, lipase, lactate dehydrogenase, alanine aminotransferase and plasmin are respectively passed into the sample pool formed by the closed fibrinolysis response interference layer film. After the reaction, the system is rinsed with PBS solution (containing 0.8 M sodium chloride and 0.05 wt % surfactant S9, with a concentration of 0.05 M and a pH of 7.4). The specificity of the fibrinolysis response interference layer film for the detection of chain kinase in whole blood is analyzed by an ordered porous interferometry measurement system.

[0071] S7. PBS solutions containing different concentrations (10, 20, 100, 200, 1000, 2000 U / mL) of streptokinase were passed into the sample pool formed by the closed fibrinolysis response interference layer film. After the reaction, the system was rinsed with PBS solution (containing 0.8 M sodium chloride and 0.05 wt % surfactant S9, with a concentration of 0.05 M and a pH of 7.4). The sensitivity of the fibrinolysis response interference layer film to the detection of streptokinase in whole blood was analyzed by an ordered porous interferometry measurement system.

[0072] S8. Dilute the whole blood sample to 5, 10, 20, and 50% with 0.9% physiological saline, and pass the diluted whole blood sample and the diluted whole blood sample spiked with streptokinase at different concentrations (50, 500, and 1000 U / mL) into the sample pool formed by the closed fibrinolysis response interference layer film. After the reaction is completed, rinse the system with PBS solution (containing 0.8M sodium chloride and 0.05wt% surfactant S9, with a concentration of 0.05M and a pH of 7.4), and detect the streptokinase activity in the whole blood sample by an ordered multi-hole interferometry system. The real whole blood sample contains 0.8M sodium chloride and 0.05wt% surfactant S9.

[0073] like Figure 2 As shown in the SEM image of C, the streptokinase-responsive fibrinolytic interference layer film has a similar microstructure. The fibrin network covers and inlays the SCC film, and the network structure seems to be more dense, which may be related to the additional Plg loading. When streptokinase flows through the sample pool, it activates plasminogen in the interference layer and begins to dissolve fibrin ( Figure 3 C) Figure 4 As shown in B, the streptokinase-responsive fibrinolytic interference layer film is responsive to streptokinase after being blocked with ovalbumin and surfactant S9.

[0074] like Figure 5 As shown in B, after being blocked, the fibrinolysis-responsive interference layer film of streptokinase response can completely shield the nonspecific adsorption interference of serum albumin and human immunoglobulin G. Figure 6 The film was shown to have an excellent specific response to streptokinase. Fig.10The optical thickness change of the fibrinolysis-responsive interference layer film in response to streptokinase with different Plg loadings is shown after 2000U / mL of streptokinase is pumped in. When the Plg loading is less than 0.5U / mL, the optical thickness value increases with the increase of Plg loading, and maintains a relative stability in the range of 0.5-0.6U / mL. However, as the loading amount increases further, the decrease in optical thickness will decrease. This may be attributed to the fact that low concentrations of Plg loading produce less plasmin, while excessive Plg loading will occupy more adsorption sites and is not conducive to the mass transfer of streptokinase in the sensing layer, all of which are not conducive to the fibrinolytic reaction. Therefore, for the sake of system response sensitivity and cost considerations, the Plg loading in the film was determined to be 0.5U / mL. The fibrinolysis process of different concentrations of streptokinase is shown in Figure 2. Fig.11 As shown in A, after streptokinase flows into the sample pool, the optical density decreases as the enzyme concentration increases. The calibration curve of streptokinase dissolving fibrin is shown in Fig. 9 As shown in B, when the enzyme concentration is in the range of 10-2000U / mL, lg(C 链激酶 ) is linearly related to the optical thickness value with a linearity of 0.97.

[0075] On this basis, the activity of streptokinase in whole blood was detected. Complex whole blood samples contain fibrinolytic substances. In order to reduce the influence of the fibrinolytic ability of whole blood samples on the analysis results, a set of control data was prepared under the same conditions using the same spiked whole blood sample. The effective optical thickness change value after deducting the optical thickness change in the control data was used for the quantitative analysis of streptokinase fibrinolytic activity. Fig.12 A shows the real-time optical thickness changes of the same whole blood sample spiked with streptokinase (1000 U / mL) pumped into the fibrinolytic response interference layer at different dilution levels. Fig.12 B shows control data obtained under the same conditions using unspiked whole blood samples. Fig.11The standard curve in B, by substituting the effective optical thickness change value of the same whole blood sample at different dilution multiples, the concentration of streptokinase can be calculated as 926.0U / mL (5%), 962.8U / mL (10%), 968.4U / mL (20%) and 409.2U / mL (50%). It can be seen that when the concentration of the whole blood sample is lower than 20%, the measured streptokinase activity is closer to the spiked value. When the whole blood sample is diluted to 50%, the measured value of its activity is significantly lower than the spiked value. This may be due to the fact that the excessive complex proteins in the whole blood will still adsorb on the sensing layer or interfere with the degradation of fibrin by streptokinase. Therefore, in order to minimize the impact of the overly complex whole blood system on the analysis results, the whole blood sample should be diluted more than 5 times for testing. In addition, the activity of the same whole blood sample spiked with different active streptokinase was measured. It can be seen from Table 2 that the recovery rate of streptokinase is 92.9% to 107.6%, respectively, and the relative standard deviation is less than 6%. This further validates the reliability of this method for detecting streptokinase activity in whole blood samples.

[0076] Table 2 Recovery of spiked streptokinase in whole blood samples diluted 5 times

[0077] Spiked amount (U / mL) Detection value (U / mL) Recovery rate (%) Relative standard deviation (%) 50 47 94.0 3.85 500 538 107.6 5.21 1000 929 92.9 4.65

[0078] Comparative Example 1

[0079] Fibrin plate method to detect plasmin activity in whole blood:

[0080] First, dissolve agarose in PBS solution (concentration of 0.05M, pH 7.4) to prepare 20mL 1wt% agarose solution, heat it to completely melt, wait until it cools to about 40°C, add 200μL containing 10mg / mL fibrinogen, and then quickly add 20μL 80U / mL thrombin, stir evenly, and immediately put it in a glass culture dish. After the agar is completely solidified, make several small holes with a diameter of 2mm. Add 8μL of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5U / mL streptokinase PBS solution, whole blood sample and whole blood sample spiked with plasmin to the small holes, respectively, and place the above fiber plate in a 37°C incubator for 18h. Stain with protein staining solution until a clear dissolution circle can be seen.

[0081] Fig.13 A is a photo of the dissolution zone of the fibrin plate with different concentrations of plasmin in the fibrin plate method. The area of ​​the dissolution zone decreases with the decrease of the plasmin concentration. The linear relationship between the dissolution zone area and the inverse of the plasmin concentration is as follows: Fig.13As shown in B, it is linearly correlated in the range of 0.005 to 0.5U / mL, with a correlation coefficient of 0.96. And the measured plasmin activity of the whole blood sample spiked with 0.1U / mL plasmin was 0.089U / mL. Unlike the OPLI method, 0.001U / mL plasmin cannot be detected by the fibrin plate method, and the accuracy is low. The method of this embodiment can provide real-time fibrinolysis data, greatly shorten the fibrinolysis detection time, and simulate the real fibrinolysis process of plasmin in human blood vessels. This is something that traditional fibrinolysis detection methods cannot do. This plays an important role in the treatment of acute thrombolytic diseases.

[0082] Comparative Example 2

[0083] Fibrin plate method to detect the activity of streptokinase in whole blood:

[0084] First, dissolve agarose in PBS solution (concentration of 0.05M, pH 7.4) to make 20mL 1wt% agarose solution, heat it to completely melt, wait until it cools to about 40°C, add 200μL of a mixture containing 10mg / mL fibrinogen and 0.5U / mL plasminogen, and then quickly add 20μL 100U / mL thrombin, stir evenly, and immediately put it in a glass culture dish. After the agar is completely solidified, make several small holes with a diameter of 2mm. Add 8μL of 10, 20, 100, 200, 1000, 2000U / mL streptokinase PBS solution, whole blood sample and whole blood sample spiked with streptokinase to the small holes, respectively, and place the above fiber plate in a 37°C incubator for 18h. Stain with protein staining solution until a clear dissolution circle can be seen.

[0085] Fig.14 A is a photo of the dissolution zone of the fibrin plate dissolved by different concentrations of streptokinase. The dissolution zone area also decreases with the decrease of streptokinase concentration, and 10U / mL streptokinase cannot be detected. The linear relationship between the dissolution zone area and the inverse of the streptokinase concentration is as follows: Fig.14 As shown in B, there is a linear correlation in the range of 20 to 1000 U / mL, with a correlation coefficient of 0.96. The measured plasmin activity of the whole blood sample spiked with 1000 U / mL streptokinase is 1126 U / mL.

[0086] Comparative Example 3: Detection of plasmin activity in whole blood samples using a commercial ELISA kit

[0087] First, different active human plasmin standards provided by the commercial ELISA kit (Beijing Ruida Henghui Technology Development Co., Ltd.) were reacted with the chromogenic substrate, and a relationship curve between the plasmin concentration and the absorbance value was established by using an ELISA reader. Further, the diluted whole blood sample was reacted with the chromogenic substrate, and its absorbance value was measured and substituted into the relationship curve to calculate the plasmin concentration of the whole blood sample. The standard curve of the commercial chromogenic substrate for plasmin response is shown in Fig.15 As shown in Figure 2, the concentration of plasmin in the same whole blood sample measured by ELISA was 0.0109 U / mL. The ELISA method is limited by its detection principle and is more suitable for measuring plasmin content rather than activity, because the results measured may include inactive plasmin.

[0088] It can be seen from the above results that, compared with the traditional fibrin plate method and ELISA method, the whole blood fibrinolytic enzyme activity detection method of the present invention overcomes the difficulty of detecting enzymatic reactions occurring at the solid-liquid interface of a heterogeneous phase, and has a lower detection limit and higher stability. More importantly, the method can provide real-time fibrinolysis data and simulate the real fibrinolysis process of fibrinolytic enzyme in human blood vessels under a flow state, which is impossible for traditional fibrinolytic enzyme activity detection methods, and plays an important role in the treatment of acute thrombolytic diseases.

Claims

1. A method for real-time detection of fibrinolytic enzyme activity in whole blood, characterized in that: The following steps are involved: S1. Fixing a silica colloidal crystal film on a silica pad to prepare a reaction pool, first injecting fibrinogen into the reaction pool, letting it stand, then dripping thrombin into the reaction pool, removing the silica pad after the cross-linking reaction, and obtaining a fibrinolytic responsive interference layer film that is responsive to plasmin; S2, fixing a fibrinolytic responsive interference layer film responsive to plasmin between a glass slide and a silicone pad to obtain a microfluidic sample pool; S3, pumping ovalbumin into the microfluidic sample pool for blocking, and then blocking with surfactant S9, and washing with PBS solution after the blocking, to obtain a blocked plasmin-responsive fibrinolytic-responsive interference layer film; S4, passing PBS solutions containing excess blood proteins into the sample pools formed by the sealed fibrinolysis response interference layer film, and rinsing with PBS solution after completion, and analyzing the anti-interference ability of the fibrinolysis response interference layer film to the interfering proteins in the whole blood by ordered porous layer interferometry; S5, passing a PBS solution containing an excess of interfering protease into a sample pool formed by a closed fibrinolysis response interference layer film, washing with a PBS solution after the reaction is completed, and analyzing the specificity of the fibrinolysis response interference layer film for the detection of plasmin in whole blood by an ordered porous interferometry system; S6, passing PBS solutions containing different concentrations of fibrinolytic enzymes into the sample pool formed by the closed fibrinolytic response interference layer film, flushing the system with PBS solution after the reaction is completed, and analyzing the sensitivity of the fibrinolytic response interference layer film to the detection of fibrinolytic enzymes in whole blood by an ordered porous interferometry system; S7. Dilute the whole blood sample with physiological saline at different multiples, and pass the diluted whole blood sample and the diluted whole blood sample spiked with plasmin into the sample pool formed by the closed fibrinolysis response interference layer film. After the reaction is completed, rinse the system with PBS solution, and detect the plasmin activity in the whole blood sample by the ordered porous interferometry measurement system.

2. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 1, characterized in that: In the step S1, the concentration of fibrinogen is 1-20 mg / mL, the activity of thrombin is 10-150 U / mL, and the ratio of the injected amount of fibrinogen to the injected amount of thrombin is 20-80:2-10.

3. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 2, characterized in that: In the step S1, fibrinogen is replaced by a mixture of fibrinogen and plasminogen, and the activity of the plasminogen is 0.1-1 U / mL.

4. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 1, characterized in that: In the step S1, the cross-linking reaction time is 1 to 6 hours.

5. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 1, characterized in that: In the step S3, the volume percentage of ovalbumin is 1-3 vol%, the mass percentage of surfactant S9 is 0.1-0.3 wt%, and the blocking time is 1-5 hours.

6. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 1, characterized in that: In step S4, the blood protein is one or more of human serum albumin and human immunoglobulin G.

7. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 1, characterized in that: In step S5, the interfering protease is one or more of alkaline phosphatase, thrombin, lipase, lactate dehydrogenase, and alanine aminotransferase.

8. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 1, characterized in that: In the step S6, the fibrinolytic enzyme is plasmin or streptokinase, the concentration of the plasmin is 0.001-0.5 U / mL, and the concentration of the streptokinase is 10-2000 U / mL.

9. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 1, characterized in that: In step S7, the volume percentage of the whole blood sample after dilution is 5-50 vol%.

10. The method for real-time detection of fibrinolytic enzyme activity in whole blood according to claim 1, characterized in that: The pH of the PBS solution is 7.2-7.6, and the concentration is 0.01-0.1 M. Sodium chloride with a concentration of 0.01-0.1 M and a surfactant S9 with a concentration of 0.02-0.15 wt % are added to the PBS solution.

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