A method for detecting the density and shedding rate of heparin-based coating based on a sleeve

Abstract

The application provides a heparin coating density and shedding rate detection method based on a package, and relates to the technical field of heparin quantitative detection. The heparin coating density and shedding rate detection method based on the package comprises the following steps: S1, drawing a heparin concentration-delta absorbance standard curve by using the toluidine blue method; S2, measuring the dynamic circulation and shedding amount of the heparin coating in the package; S3, measuring the residual amount of the heparin coating in the package; and S4, calculating the heparin coating density p and the coating shedding rate F at each time point in the package n The application can more truly simulate the fluid environment in the extracorporeal circulation process, realizes accurate measurement, can deeply study the coating stability under the dynamic scouring environment, and provides more accurate and effective evaluation basis for the long-term stability and service life of the long package.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of heparin quantitative detection, and particularly relates to a heparin coating density and shedding rate detection method based on a package. BACKGROUND

[0002] The long-acting disposable membrane oxygenator package (hereinafter referred to as a package) can realize long-term cardiopulmonary support, greatly expand the treatment window, and has high durability, better biocompatibility and anticoagulation performance. Therefore, in addition to using more durable biocompatibility materials, an anticoagulant coating technology needs to be introduced. As an anticoagulant, heparin is widely used in anticoagulant coating technology, which can effectively reduce thrombosis and reduce inflammation. Since the long-acting package can provide effective cardiopulmonary support for several days to several weeks during use, coating shedding in the clinic not only can cause the anticoagulant effect to decrease, but also can cause thrombosis and inflammation, which can adversely affect the treatment of patients. Therefore, the long-term stability of heparin coating technology applied to the long-acting package needs to be focused on. Developing an accurate and reliable method for detecting the heparin coating density and shedding rate of the package is of great significance to evaluate the service life of the long-acting package and ensure its safety.

[0003] The existing research does not disclose a method for testing the heparin coating density and shedding rate of the package as a whole. Only a quantitative and qualitative test method for the shedding rate of a single heparin coating is disclosed. Even if the coating is applied to the package as a whole, the coating density and shedding rate are tested by disassembling the parts. Therefore, the heparin coating stability state of the package in the use state cannot be dynamically simulated, and the evaluation effect of the detection results is not good. The prior art CN118252986A discloses a test method for the long-term stability of the coating. The method is to immerse the coating in PBS buffer for 7 days, then test the heparin content before and after immersion using the toluidine blue colorimetric method, and calculate the coating shedding rate. This method can quantitatively determine the shedding rate of the heparin coating. However, the immersion process cannot simulate the dynamic shear force when the blood is in contact, and 2 coating samples are needed before and after the test, which is easy to produce errors. CN119868670A proposes a method for coating heparin on the package. The method uses the toluidine blue method to quantitatively test and test the shedding rate of heparin on different parts removed from the package. However, since different materials in the package have different adhesion abilities for the heparin coating, the quantitative evaluation of the package as a whole cannot be accurately realized. At the same time, in this method, the use of non-coated products for background deduction and dynamic shedding rate detection during the flushing process requires the use of multiple samples, which has a high requirement for the amount of samples and is easy to produce errors. SUMMARY

[0004] This invention addresses the shortcomings of current methods for detecting the density and peeling rate of heparin coatings on protective sleeves. These methods cannot accurately simulate the dynamic usage state of the sleeves and require the removal of different components for testing, thus failing to provide an accurate quantitative assessment of the overall sleeve. The invention provides a method for detecting the density and peeling rate of the heparin coating on the sleeve in real time during dynamic simulation of use. This method does not require the removal of different components and can accurately and dynamically quantitatively assess the overall density and peeling rate of the heparin coating on the sleeve, enabling a true and accurate evaluation of the service life of long-lasting protective sleeves.

[0005] This invention provides a method for detecting the density and peeling rate of heparin-based coatings based on a packaging system, comprising the following steps:

[0006] S1. A standard curve of heparin concentration versus absorbance was plotted using the toluidine blue method;

[0007] S2. Determination of heparin coating shedding in the sample pack: Simulated blood was injected into the sample pack circuit and dynamically circulated to flush the pack. Simulated blood samples were collected at different flushing times, and the absorbance value (A) was measured using the toluidine blue method. n Substituting the values ​​into the heparin concentration-Δabsorbance standard curve described in S1, the heparin concentration C is calculated. n The amount of heparin coating detachment (N) from samples with different rinsing times was determined using the following formula. n= C n ×V1, where V1 is the simulated blood volume injected into the sample sleeve circuit;

[0008] S3. Determination of residual heparin coating in the package: The sample package from S2, after reaching the set dynamic cyclic rinsing time, was stained with toluidine blue until saturated, and the absorbance value was measured as A. m The blank control kit was stained with toluidine blue until saturated, and the absorbance value was measured as A0. A0 was then... m Substituting A0 and A0 into the heparin concentration-Δabsorbance standard curve described in S1, the heparin concentration C is calculated. m And C0, the amount of heparin coating residue N in the package after rinsing S= (C) m -C0)×V2, where V2 is the volume of toluidine blue injected into the encapsulation circuit;

[0009] S4. Calculate the heparin coating density ρ and the coating peeling rate F at each time point in the package. n .

[0010] According to the present invention, a method for detecting the density and peeling rate of heparin-based coatings based on a package is preferably used in step S3, wherein the absorbance value A is obtained. m The sample kit after measurement and removal of residual toluidine blue serves as a blank control kit.

[0011] According to the heparin coating density and shedding rate detection method based on the package provided by the application, preferably, the removing residual toluene blue adopts the following operation: adopting 2-3LPM water flushing to complete the absorbance value A m The sample is determined for 20-30min.

[0012] According to the heparin coating density and shedding rate detection method based on the package provided by the application, preferably, the flow of the dynamic circulation flushing in S2 is less than or equal to 7LPM, and the post-membrane pressure is greater than or equal to 150mmHg.

[0013] According to the heparin coating density and shedding rate detection method based on the package provided by the application, preferably, the simulated blood in S2 is physiological saline or a mixture of physiological saline and glycerol.

[0014] According to the heparin coating density and shedding rate detection method based on the package provided by the application, preferably, the viscosity range of the mixture of physiological saline and glycerol is 2.5*10 -3 Pa·s-3.5*10 -3 Pa·s.

[0015] According to the heparin coating density and shedding rate detection method based on the package provided by the application, preferably, the different flushing times in S2 are 0h, 6h, 24h, 2d, 3d, 5d, 7d, 14d and 30d respectively.

[0016] According to the heparin coating density and shedding rate detection method based on the package provided by the application, preferably, the specific operation of toluene blue staining to saturation in S3 is that the same concentration of toluene blue solution in S1 is used to circulate and perfuse the package circuit, and the circulation and perfusion time is greater than or equal to 30min.

[0017] According to the heparin coating density and shedding rate detection method based on the package provided by the application, preferably, the absorbance value is the absorbance value determined at 630nm wavelength.

[0018] According to the heparin coating density and shedding rate detection method based on the package provided by the application, preferably, the heparin concentration-△absorbance standard curve in S1 takes the heparin concentration as the x-axis and the △absorbance as the y-axis, the heparin concentration range is 0-20μg / mL, and the concentration gradient is set to 2μg / mL.

[0019] Beneficial effects:

[0020] The heparin coating density and shedding rate detection method based on a casing provided by the application adopts the maximum flow dynamic flushing of the casing in extracorporeal circulation to measure the shedding rate of the coating on the surface of the casing, can more truly simulate the fluid environment in the extracorporeal circulation process, realizes accurate measurement, and only uses one set of casing to realize the initial coverage density measurement of the heparin coating on the surface of the casing, and can further study the coating stability of the casing in the dynamic flushing environment and quantitatively evaluate the shedding rate, thereby providing a more accurate and effective evaluation basis for the long-term stability and service life of the long-acting disposable membrane oxygenator casing. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings.

[0022] Figure 1 A heparin concentration-△absorbance standard curve diagram of the application.

[0023] Figure 2 A casing sampling schematic diagram of Comparative Example 2. DETAILED DESCRIPTION

[0024] The following examples are used to illustrate the application, but are not used to limit the scope of the application. If the specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product instruction is used. If the reagent or instrument is not specified by the manufacturer, it is a conventional product that can be purchased through a regular channel.

[0025] In the detailed description, the application provides a heparin coating density and shedding rate detection method based on a casing, which comprises the following steps:

[0026] S1. A heparin concentration-△absorbance standard curve is drawn by using the toluidine blue method.

[0027] S2. Heparin coating shedding amount determination in the casing: simulate blood is injected into the sample casing circuit to dynamically circulate and flush the casing, and the absorbance value A n of the simulate blood is measured at different flushing times by using the toluidine blue method. n The heparin concentration C n= is calculated by substituting into the heparin concentration-△absorbance standard curve in S1. n The heparin coating shedding amount N m of the sample casing at different flushing times is measured according to the following formula: C n ×V1, wherein V1 is the volume of the simulate blood injected into the sample casing circuit.

[0028] S3. Heparin coating residual amount determination in the casing: the sample casing reaching the set dynamic circulation flushing time in S2 is dyed with toluidine blue until saturation, and the absorbance value A mThe blank control kit was stained with toluidine blue until saturated, and the absorbance value was measured as A0. A0 was then... m Substituting A0 and A0 into the heparin concentration-Δabsorbance standard curve described in S1, the heparin concentration C is calculated. m And C0, the amount of heparin coating residue N in the package after rinsing S= (C) m -C0)×V2, where V2 is the volume of toluidine blue injected into the encapsulation circuit;

[0029] S4. Heparin coating density ρ and coating peeling rate F at each time point in the package. n .

[0030] In a specific embodiment, the density ρ of the heparin coating in the package mentioned in this invention can be calculated using the following method: =(Nn+N S ) / S, where S is the coating area of ​​the heparin-based coating, and Nn is the amount of heparin-based coating detached from the sample at different rinsing times, N S This represents the amount of heparin-based coating residue remaining in the package after rinsing.

[0031] In a specific embodiment, the coating peeling rate F of the heparin-based coating in the package mentioned in this invention at various time points is... n It can be calculated using any of the following methods:

[0032] One method: Fn = (N n / (N) n +N S ))×100%, where the amount of heparin coating detached from the sample at different rinsing times, N n The amount of heparin coating detached from the sample package at different rinsing times, N S This represents the amount of heparin-based coating residue remaining in the package after rinsing.

[0033] Method Two: Fn=N n / S ×100%, where the amount of heparin coating detached from the sample at different rinsing times is N. n ρ is the density of the heparin coating in the package, and S is the coating area of ​​the heparin coating in the package.

[0034] The total peeling rate of the heparin coating in the package can be calculated as follows:

[0035] F=C m / (C m +C1)×100%, in this calculation V1=V2.

[0036] It should be noted that:

[0037] The method for detecting the density and peeling rate of heparin coatings based on a package provided by this invention is based on the dynamic shear-scouring process simulating blood contact, and measures the density ρ of the heparin coating in the package and the peeling rate F at each time point. n The quantitative detection method can simulate dynamic usage conditions, providing accurate and reliable quantitative results. Furthermore, it can measure the overall dynamic heparin coating density ρ and coating peeling rate F at various time points. n The quantitative detection method eliminates the need to disassemble and test different components in the kit, enabling a more accurate quantitative assessment of the overall stability of the heparin coating in the kit. This avoids the differences in adhesion of different component materials to the heparin coating and the errors caused by using multiple samples for testing.

[0038] This invention uses a single package to simultaneously test the density and dynamic shedding rate of the heparin coating on the package surface. During the rinsing test, the amount of heparin shed can be sampled and tested multiple times to achieve dynamic detection of the coating shedding rate. The method has good repeatability, high accuracy, and high sample utilization.

[0039] Toluidine blue can electrostatically bind with heparin to form a purple complex, resulting in a decrease in absorbance. A heparin-coated package is perfused with a toluidine blue solution of a specific concentration. The absorbance change after the complexation reaction is measured to calculate the heparin coating content. Considering that the substrate material is also stained by the toluidine blue solution, causing a slight change in absorbance, a blank package control is required. In some specific embodiments, to mitigate the control error caused by the use of an uncoated product for background subtraction blank control in existing technologies, the present invention, in S3, preferably uses a complete absorbance value A. m The sample kit after measurement and removal of residual toluidine blue serves as a blank control kit. In this invention, the stained kit is washed to remove excess dye before further staining to account for absorbance changes caused by the substrate. Using the same kit as both the test and control samples further avoids errors caused by differences in kit materials and saves on the amount of kits used in the test. All tests can be completed with just one kit, resulting in better economic efficiency.

[0040] This method uses a dyeing saturation and then washing away the excess dye to replace the heparin-free coating saturation ...

[0041] In some specific embodiments, the present invention does not specify how to completely remove the absorbance value A. mThe way of determining the residual toluene blue of the sample package after the determination is defined, and any implementation that can achieve the effect of complete removal of residual toluene blue can be used, for example, the removal of residual toluene blue mentioned in S2 of the present application can be performed as follows: using 2-3 LPM flowing water to flush to complete the absorbance value A m The sample package is determined for 20-30 min.

[0042] In some embodiments, in order to more truly and accurately simulate the blood flow state of the long-acting disposable membrane oxygenator package in actual use, and simulate the dynamic shear force when the blood contacts, the flow rate of the dynamic circulation flushing in S2 is preferably controlled to be ≥7 LPM, and the post-membrane pressure is ≥150 mmHg.

[0043] In some embodiments, it is more preferred to use the rated maximum flow rate of 7 LPM or more of the package for blood dynamic circulation flushing simulation. The shedding rate under the extreme dynamic circulation flushing flow rate is closer to the maximum shedding rate, and the evaluation results of the package use stability and service life are more conducive to avoiding the risk of actual application of the package.

[0044] In some embodiments, the simulation blood of the present application can use the conventional physiological saline and other commonly used simulation liquids in the art. In order to more truly and accurately simulate the blood viscosity and other properties of the long-acting disposable membrane oxygenator package in actual use, the simulation blood can also be preferably a mixture of physiological saline and glycerol, and more preferably, the viscosity range of the mixture of physiological saline and glycerol is 2.5×10 -3 Pa·s~3.5×10 -3 Pa·s. In order to control the viscosity of the above mixture, for example, the mass ratio of physiological saline to glycerol in the mixture of physiological saline and glycerol can be controlled to be 3:2.

[0045] In some embodiments, in order to more truly simulate the dynamic shedding state of the heparin coating of the long-acting disposable membrane oxygenator package in actual use, the different flushing times in S2 of the present application are preferably 0 h, 6 h, 24 h, 2 d, 3 d, 5 d, 7 d, 14 d and 30 d, respectively.

[0046] In some embodiments, in order to ensure that the dynamic circulation flushing is not affected after taking simulation blood at different flushing times in S2, it is preferred that after taking simulation blood at different flushing times in S2, an equal amount of fresh simulation blood is immediately supplemented into the sample package circuit, for example, a sampling amount of 3 mL can be taken, and at the same time, 3 mL of fresh simulation blood is supplemented into the sample package circuit in time after each sampling.

[0047] In some embodiments, in order to determine the determination of the residual amount of heparin coating of the set after the dynamic circulation flushing is completed, the sample set in S2 reaching the set dynamic circulation flushing time needs to be stained with toluidine blue to saturation, and then the absorbance value is determined, wherein the specific operation of staining with toluidine blue to saturation in S3 can be: using the same concentration of toluidine blue solution in S1 to circulate and perfuse the set circuit, and the circulation and perfusion time is greater than or equal to 30 minutes.

[0048] In specific embodiments, all the absorbance values mentioned in the present application are absorbance values measured at a wavelength of 630 nm.

[0049] Further, in order to obtain a heparin concentration-△absorbance standard curve with better fitting effect and more accurately reflect the quantitative relationship between heparin concentration and absorbance value, in some specific embodiments, the heparin concentration range when drawing the heparin concentration-△absorbance standard curve is preferably 0-20 μg / mL, and the concentration gradient is set to 2 μg / mL.

[0050] The specific steps for drawing the heparin concentration-△absorbance standard curve can be referred to as follows:

[0051] Prepare 0.005% toluidine blue solution and prepare heparin solutions with different concentrations of 0-20 μg / mL, and the concentration gradient is set to 2 μg / mL.

[0052] Take 3 mL of each concentration of heparin solution and 0.005% toluidine blue solution, mix well, and place the mixed solution in a dark environment for 3-4 minutes to allow the complexation reaction to occur fully. Add 5 mL of n-hexane and shake well to extract the complex into the organic layer, and remove the complex organic layer. Extract multiple times until no purple complex appears, and obtain the water phase solution for absorbance determination.

[0053] Take 200 μL of the water phase solution and place it in a 96-well plate, inject at least 3 wells per sample, and use an enzyme marker to measure the absorbance at a wavelength of 630 nm.

[0054] Take 200 μL of the water phase solution and place it in a 96-well plate, inject at least 3 wells per sample, and use an enzyme marker to measure the absorbance at a wavelength of 630 nm.

[0055] The fitting equation of the heparin concentration-△absorbance standard curve provided by the present application is y=0.06613x-0.0442, the R 2 of the fitting equation is 0.99723, and the equation fitting degree is high.

[0056] Example 1

[0057] A set-based heparin coating density and shedding rate detection method, comprising the following steps:

[0058] S1. Draw the heparin concentration-△absorbance standard curve using toluidine blue method:

[0059] Prepare a 0.005% toluidine blue solution, and prepare heparin solutions with different concentrations of 0~20μg / mL with a concentration gradient set at 2μg / mL. Take 3mL of each concentration of heparin solution and 0.005% toluidine blue solution, and mix them using a vortex mixer for 30s to obtain a mixed solution;

[0060] Place the mixed solution in a dark environment for 5min to allow the complexation reaction to occur fully, then add 5mL of extractant n-hexane to the mixed solution, shake it thoroughly, and extract the complex in the mixed solution into the organic layer. Remove the complex organic layer with a dropper, and extract the mixed solution multiple times until no purple complex appears. Obtain the aqueous solution;

[0061] Use a pipette to take 200μL of the aqueous solution and place it in a 96-well plate, with at least 3 wells per sample. Use an enzyme marker to measure the absorbance at 630nm. Draw the heparin concentration-△absorbance standard curve with heparin concentration (μg / mL) as the x-axis and △absorbance as the y-axis, as shown in Figure 1 ;

[0062] S2. Measurement of heparin coating shedding amount in the set:

[0063] Prepare simulated blood with a physiological saline:glycerol ratio of 3:2 (mass ratio) to simulate the viscosity of blood, and the mixture has a viscosity of 3.0×10 -3 Pa·s;

[0064] Fill the simulated blood into the set circuit, record the filling volume V1, set the flow rate to the maximum rated flow rate of the set, and set the blood channel flow rate to the rated maximum flow rate of 7LPM (liters / minute) of a common set, with a post-membrane pressure ≥150mmHg. Start timing from the time when the flushing begins, and at 0h, 6h, 24h, 2d, 3d, 5d, 7d, 14d, and 30d, take 3mL of sample solution from the circuit and place it in a centrifuge tube, then add 3mL of simulated blood to the circuit;

[0065] Use the method in S1 to add toluidine blue solution to the simulated blood extracted at different times to complex and extract it, and test its absorbance value A n . Substitute it into the heparin concentration-△absorbance standard curve described in S1 to calculate the heparin concentration C n . According to the following formula, determine the heparin coating shedding amount N n of the sample set at different flushing times, Nn=C n ×V1, where V1 is the volume of simulated blood injected into the sample set circuit;

[0066] S3. Determination of residual heparin coating in the set:

[0067] The set was taken out after flushing for 30 days in S2, dried, and the set circuit was perfused with a 0.005% concentration of toluidine blue solution, and the perfusion volume was recorded as V2. The circuit was opened to drive circulation, and the reaction was circulated for 30 min at a flow rate of 1 LPM to ensure that the set was saturated with dye;

[0068] 3 mL of the perfusate was taken out, 3 mL of purified water was added, and the absorbance at a wavelength of 630 nm was measured. The average value of the absorbance was recorded as A m , the absorbance value of the toluidine blue solution after combining with the set with heparin coating;

[0069] The set that had been quantitatively tested was washed, and 2 LPM of water was used to flush for 30 min to wash away the residual toluidine blue liquid in the set, and the set was dried. The same test method was used to test the set that had been saturated with dye, and the average value of the absorbance was recorded as A0, the absorbance value of the toluidine blue solution after combining with the base material.

[0070] The absorbance values A m and A0 were substituted into the heparin concentration-△absorbance standard curve for calculation, and the heparin concentrations were recorded as C m and C0. The residual amount of heparin coating in the set after flushing was N S= (C m -C0) x V2, where V2 was the volume of toluidine blue injected into the set circuit.

[0071] S4. Heparin coating density p in the set and coating shedding rate F at each time point n The coating shedding rate F at each time point was calculated by the following formula:

[0072] = (Nn+N S ) / S, where S was the coating area of the heparin coating in the set;

[0073] Fn= (N n / (N n +N S )) x 100%.

[0074] As shown in Table 1 below, it can be seen that the heparin shedding amount density B (N n ) and the residual heparin content density C (N SThe sum of the additive and the total content density (A) of the package circuit heparin is basically equal. It is shown that the density and shedding rate detection method of the package-based heparin coating of the application can accurately reflect the dynamic and real shedding of the package-based heparin coating in use, can accurately realize dynamic quantitative evaluation of the density and shedding rate of the package-based heparin coating, and can accurately evaluate the service life of the long-acting package.

[0075] Table 1.

[0076]

[0077] Example 2

[0078] A density and shedding rate detection method of a package-based heparin coating comprises the following steps:

[0079] S1. Draw a heparin concentration-△absorbance standard curve by using toluidine blue method, same as example 1;

[0080] S2. Heparin coating shedding amount determination in package: same as example 1;

[0081] S3. Heparin coating residual amount determination in package:

[0082] After 30 days of flushing, the package is taken out, dried, and perfused with a 0.005% toluidine blue solution, and the perfusion volume is recorded as V. The driving circuit is opened for circulation, and the circulation reaction is carried out at a flow rate of 1LPM for 30 minutes to ensure that the staining in the package reaches a saturated state.

[0083] Take out 3mL perfusion liquid, add 3mL purified water, and record the average value of the absorbance at 630nm wavelength, which is tested in parallel for 3 times, as A m , the absorbance value of toluidine blue solution combined with heparin coating package after staining;

[0084] A heparin-free coating package is used as a blank control sample. The same test method as described above is used to test the blank control sample after staining, and the average value of the absorbance is recorded as A0, which is the absorbance value of the blank material combined with the toluidine blue solution after staining;

[0085] The above absorbance values A m and A0 are substituted into the heparin concentration-△absorbance standard curve for calculation, and the heparin concentration is recorded as C m and C0, the residual amount of heparin coating in the package after flushing N S= (C m -C0)×V, wherein V2 is the volume of toluidine blue injected into the package circuit;

[0086] S4. Heparin coating density ρ and coating shedding rate F at each time point in packagen The calculation is made by the following formula:

[0087] = (Nn + N S ) / S, wherein S is the coating area of the sheath package heparin coating;

[0088] Fn= (N n / (N n + N S )) / 100%.

[0089] Table 2

[0090]

[0091] Comparative Example 1

[0092] A sheath package-based heparin coating density detection method includes the following steps:

[0093] S1. Draw a heparin concentration-△absorbance standard curve using the toluidine blue method:

[0094] Prepare a 0.005% toluidine blue solution, and prepare heparin solutions of different concentrations with a concentration gradient set to 5 μg / mL of 0~20 μg / mL. Take 3 mL of each concentration of heparin solution and 0.005% toluidine blue solution, and mix them using a vortex mixer for 30 s to obtain a mixed solution;

[0095] Place the mixed solution in a dark environment for 5 min to allow it to fully undergo complexation to obtain a complex. Then add 5 mL of extractant n-hexane to the mixed solution, shake it thoroughly, and extract the complex in the mixed solution into the organic layer. Remove the complex organic layer with a dropper, and extract the mixed solution multiple times until no purple complex appears. Obtain the aqueous solution;

[0096] Use a pipette to take 200 μL of the aqueous solution and place it in a 96-well plate, with at least 3 wells per sample. Use an enzyme marker to measure the absorbance at 630 nm. Draw a heparin concentration-△absorbance standard curve with heparin concentration (μg / mL) as the x-axis and △absorbance as the y-axis;

[0097] S2. Test the sheath package heparin coating density. The specific detection method is also toluidine blue quantitative detection, and the material is coated and directly measured:

[0098] Contact different material samples with a known surface area with 0.0025% toluidine blue for 30 min, extract the remaining liquid, and test the absorbance. According to the absorbance change value and the above standard curve, calculate the heparin content density per unit area.

[0099] The heparin content of the surface of the sheath package of different materials was measured. The specific test results are shown in Table 3.

[0100] Table 3

[0101]

[0102] As can be seen from Table 3, for different materials, the coating adhesion ability of heparin coating when different substrates are coated is different. The method of sampling and detecting the residual amount and shedding of the coating after flushing in the prior art is not reliable, because the sheath package is made of a plurality of different materials, the coating adhesion ability of heparin coating of different sampling positions is different, and it cannot represent the adhesion and shedding of the heparin coating of the whole sheath package, and there is obvious detection error.

[0103] Comparative Example 2

[0104] A heparin coating density detection method based on a sheath package includes the following steps:

[0105] S1. Draw a heparin concentration-△absorbance standard curve using toluidine blue method:

[0106] Prepare a 0.005% toluidine blue solution, and prepare heparin solutions with different concentrations of 0~20μg / mL with a concentration gradient set to 5μg / mL. Take 3mL of each concentration of heparin solution and 0.005% toluidine blue solution, and mix them well using a vortex mixer for 30s to obtain a mixed solution;

[0107] Place the above mixed solution in a dark environment for 5min to allow it to fully complex and obtain a complex. Then add 5mL of extractant n-hexane to the mixed solution, shake it well, and extract the complex in the mixed solution to the organic layer. Remove the complex organic layer with a dropper, and mix the solution and extract it multiple times until no purple complex appears. Obtain the aqueous solution;

[0108] Use a pipette to take 200μL of the aqueous solution and place it in a 96-well plate, with at least 3 wells per sample. Use an enzyme marker to measure the absorbance at 630nm wavelength. Draw a heparin concentration-△absorbance standard curve with heparin concentration (μg / mL) as the x-axis and △absorbance as the y-axis;

[0109] S2. Test the heparin coating density of the sheath package:

[0110] The heparin content of the surface of the sheath package at different positions is measured:

[0111] Draw a heparin concentration-△absorbance standard curve as above, and take samples from different positions of the sheath package with known surface area (such as the inner wall of the sheath package, the outer wall of the sheath package, the inner wall of the sheath package, and the outer wall of the sheath package) to measure the absorbance at 630nm wavelength. Figure 2As shown, the samples are contacted with 0.0025% toluidine blue for 30 min, and the remaining liquid is extracted for testing the absorbance. According to the absorbance change value and the standard curve, the heparin content density per unit area is calculated.

[0112] The specific detection results are shown in Table 4.

[0113] Table 4

[0114]

[0115] As shown in Table 4, the heparin coating density is determined by sampling at different positions of the sheath, and the detection values also have large differences, which indicates that the heparin coating at different positions of the sheath has certain differences in the process of use. The method of sampling and detecting the coating residual amount and the shedding condition after flushing from different positions of the sheath in the prior art is unreliable, the shedding rates of the heparin coating at different sampling positions are not the same, and cannot represent the adhesion and shedding condition of the heparin coating of the whole sheath, and there is obvious detection error.

[0116] Furthermore, it is also illustrated that the heparin coating density and shedding rate detection method based on the sheath provided by the present application has dynamic reliability, which can not only accurately realize the dynamic quantitative evaluation of the heparin coating density and shedding rate of the whole sheath, but also the detection results will not bring measurement error due to different materials at different positions of the sheath and different sampling areas, and the detection results are more accurate, and the service life of the long-acting sheath can be accurately evaluated.

[0117] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, but not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for detecting the density and shedding rate of heparin-based coating based on a jacketing package, characterized in that, The method comprises the following steps: S1. Draw a heparin concentration-△ absorbance standard curve by toluidine blue method; S2. Determination of heparin shedding amount from the sample set: simulate blood was injected into the sample set circuit to dynamically circulate and flush the sample set, and the simulate blood was taken at different flushing times to determine the absorbance value A by toluidine blue method n , and the heparin concentration C was calculated by substituting the heparin concentration-absorbance standard curve in S1 n , and the heparin shedding amount N of the sample set at different flushing times was determined according to the following formula n= C n ×V1, wherein V1 is the volume of simulate blood injected into the sample set circuit; S3. Determination of residual heparin coating in the package: The sample package from S2, after reaching the set dynamic cyclic rinsing time, was stained with toluidine blue until saturated, and the absorbance value was measured as A. m The blank control kit was stained with toluidine blue until saturated, and the absorbance value was measured as A0. A0 was then... m Substituting A0 and A0 into the heparin concentration-Δabsorbance standard curve described in S1, the heparin concentration C is calculated. m And C0, the amount of heparin coating residue N in the package after rinsing S= (C) m -C0)×V2, where V2 is the volume of toluidine blue injected into the encapsulation circuit; S4. Calculate the density of heparin coating in the set ρ and the coating shedding rate F at each time point n ; The completed absorbance value A is used in S3 m The sample set after determination and removal of residual toluidine blue is the blank control set.

2. The method according to claim 1, wherein the method is a method for detecting the density and the shedding rate of a heparin-based coating by using a kit. The removal of residual toluidine blue is performed by washing with running water at 2-3 LPM until the absorbance value A m The sample is measured for 20-30 min.

3. The method according to claim 1, wherein the method is a method for detecting the density and the shedding rate of a heparin-based coating by using a kit. The flow rate of the dynamic circulation flushing in S2 is ≥7 LPM, and the post-membrane pressure is ≥150 mmHg.

4. The method according to claim 1, wherein the method is a method for detecting the density and the shedding rate of a heparin-based coating by using a kit. The simulated blood in S2 is physiological saline or a mixture of physiological saline and glycerol.

5. The method for detecting the density and peeling rate of heparin-based coatings based on a packaging system according to claim 4, characterized in that, The viscosity range in the mixture of the physiological saline and the glycerol is 2.5 x 10 -3 Pa s~3.5 x 10 -3 Pa s.

6. The method of claim 1, wherein the method is a method of detecting the density and shedding rate of heparin-based coating based on a kit. The different flushing times in S2 are 0 h, 6 h, 24 h, 2 d, 3 d, 5 d, 7 d, 14 d and 30 d, respectively.

7. The method of claim 1, wherein the method is a method of detecting the density and shedding rate of heparin-based coating based on a kit. The specific operation of toluidine blue staining to saturation in S3 is as follows: the same concentration of toluidine blue solution in S1 is used to circulate and perfuse the sleeve package loop, and the circulation and perfusion time is ≥30 min.

8. The method according to any one of claims 1 to 7, wherein the method is a method for detecting the density and the shedding rate of heparin-based coating by using a kit. The absorbance value is the absorbance value measured at 630 nm.

9. The method according to any one of claims 1 to 7, wherein the method is a method for detecting the density and the shedding rate of heparin-based coating by using a kit. The heparin concentration-△ absorbance standard curve in S1 takes heparin concentration as the x-axis and △ absorbance as the y-axis, the heparin concentration range is 0-20 μg / mL, and the concentration gradient is set to 2 μg / mL.

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