Fibrinogen concentration testing method based on A280 absorbance and application

Abstract

The invention belongs to a protein concentration detection method, and particularly relates to a fibrinogen concentration detection method based on A280 absorbance and application. The detection method comprises the following steps: (1) detecting A280 absorbance of multiple groups of fibrinogen solutions with known concentrations; (2) constructing a relational expression between the fibrinogen solution concentration and the A280 absorbance according to the multiple groups of A280 absorbance; and (3) detecting A280 absorbance of a to-be-detected sample, and obtaining the fibrinogen concentration in the to-be-detected sample according to the relational expression. The detection method is high in accuracy, and the concentration of the fibrinogen can be obtained according to the relational expression by testing the absorbance of A280, so that the detection experiment process is optimized, and the test reagent and the test cost are saved.

Description

Technical Field

[0001] The invention belongs to a protein concentration detection method, and specifically relates to a fibrinogen concentration detection method based on A280 absorbance and an application thereof. Background Art

[0002] The EVAHEARTⅠ system is a third-generation centrifugal left ventricular assist system that uses hydraulic suspension technology. It allows the sliding bearings and mechanical sealing systems in the artificial heart's blood pump to be suspended in water, isolating the blood and bearings, thereby reducing damage to blood cells and preventing strokes. Among them, the pure water sealing system is an important component of the EVAHEARTⅠ system. Its function is to drive the pure water sealing liquid to circulate between the blood pump and the controller, cool the blood pump seal, and lubricate the sliding bearings and cool the motor coils.

[0003] In addition, a small amount of plasma protein components will overflow from the sealing part. Fibrinogen in plasma protein is one of the "central" proteins in the coagulation system. It is the final substrate for the successive activation of coagulation factors during the coagulation process and has a hemostatic function. In addition to directly participating in the later stages of the coagulation process, it also mediates platelet aggregation and affects blood viscosity. Therefore, the pure water sealing system also has the function of cleaning and taking away a small amount of plasma protein.

[0004] The filter is the main component for cleaning plasma proteins in the EVAHEARTⅠ system. Existing filter products need to meet the specifications of a fibrinogen residual rate of less than 10% and a fibrinogen removal rate of 90%. Therefore, the evaluation of the filter's fibrinogen filtration efficiency is important for product quality control. Summary of the invention

[0005] The present invention has developed a method for detecting fibrinogen concentration, which is a detection method based on A280 absorbance. The detection method has high accuracy, does not require multiple tests, and has low reagent costs. The evaluation of the filtration efficiency of filter fibrinogen in the present invention is based on the detection method.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides a method for detecting fibrinogen concentration based on A280 absorbance, comprising: (1) detecting the A280 absorbance of multiple groups of fibrinogen solutions with known concentrations; (2) constructing a relationship between the concentration of the fibrinogen solution and the A280 absorbance according to the multiple groups of A280 absorbances; and (3) detecting the A280 absorbance of a sample to be tested, and obtaining the fibrinogen concentration in the sample to be tested according to the relationship.

[0008] Preferably, in the above step (1), the fibrinogen solution is prepared using a phosphate buffer solution, and the pH of the phosphate buffer solution is 7.0-7.4.

[0009] Preferably, a UV-visible spectrophotometer is used to detect the A280 absorbance of multiple groups of fibrinogen solutions with known concentrations, and the UV-visible spectrophotometer is a Pulse T6-1650F.

[0010] Preferably, in the above step (2), the relationship is: fibrinogen concentration of the sample to be tested = X*A280±Y, wherein A280 is the A280 absorbance, X is 600-3000, and Y is 0-1; fibrinogen purity is ≥70%.

[0011] Preferably, when the fibrinogen purity is 70%≤fibrinogen purity<85%, the relationship is fibrinogen concentration (μg / mL)=(1750-3000)*A280±(0-1.0); or when the fibrinogen purity is 85%≤fibrinogen purity<95%, the relationship is fibrinogen concentration (μg / mL)=(950-1800)*A280±(0-1.0; or when the fibrinogen purity is 95%≤fibrinogen purity≤100%, the relationship is=(659-950)*A280±(0-1.0).

[0012] On the other hand, the present invention provides a method for testing the fibrinogen filtration efficiency of an artificial heart pure water sealed system filter, which may include: (i) circulating a fibrinogen standard solution in an artificial heart pure water sealed system filter to obtain a circulating fluid; and (ii) testing the fibrinogen concentration of the circulating fluid using the fibrinogen concentration testing method of the present invention, thereby determining the fibrinogen filtration efficiency of the artificial heart pure water sealed system filter.

[0013] Preferably, the above step (i) comprises:

[0014] (a) using phosphate buffer to prepare a fibrinogen standard solution; using the phosphate buffer to circulate in an artificial heart; (b) after the phosphate buffer is circulated, the fibrinogen standard solution is dripped into it and circulated to obtain a circulating fluid.

[0015] Preferably, the concentration of the fibrinogen standard solution may be 4 mg / ml.

[0016] Preferably, the circulation rate may be 130 ml / min.

[0017] Preferably, in the above step (b), dripping the fibrinogen standard solution for circulation comprises: dripping 1 ml of the fibrinogen standard solution for 3 minutes; recording the pressure value 3 minutes after dripping; repeating the operation until 10 ml of the fibrinogen standard solution is dripped; after dripping the 10th ml of the fibrinogen standard solution; after 3 minutes, taking 6 ml of the circulating liquid; after taking the liquid, continuing the circulation for up to 3 hours, and taking the circulating liquid again at the end.

[0018] Preferably, the above-mentioned artificial heart may be EVAHEARTⅠ.

[0019] The beneficial effects of the present invention include at least:

[0020] (1) Correlation R between the detection value of the fibrinogen concentration detection method based on A280 absorbance in the present invention and the theoretical value of the standard solution 2 is 1, indicating that the detection method of the present invention has high accuracy; and the test results of the fibrinogen filtration efficiency of the artificial heart filter based on the detection method have no significant difference with the test results of the BCA test method in the prior art CN114062220B, further indicating that the detection method of the present invention has high accuracy.

[0021] (2) In the present invention, a relationship between the concentration of fibrinogen and the A280 absorbance is constructed. By testing the A280 absorbance, the concentration of fibrinogen can be obtained according to the relationship, thereby optimizing the detection experiment process and saving test reagents and test costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of some components of the EVAHEARTⅠ system;

[0023] Figure 2 is a filter schematic diagram;

[0024] Figure 3 is the relationship between fibrinogen concentration and absorbance in the method of the present invention;

[0025] Figure 4 The correlation between the prepared fibrinogen concentration and the concentration calculated according to formula 1 based on the absorbance;

[0026] Figure 5 It is the schematic diagram of the test device structure;

[0027] Figure 6 This is the absorbance of the first group of CS310 filter samples;

[0028] Figure 7 This is the absorbance of the second group of CS310 filter samples;

[0029] In the figure, 1: filter; 2: pure water sealing component; 3: pump cable; 4: blood pump; 5: diaphragm pump; 6: solution bottle; 7: pipette; 8: water bath; 9: CS tube; 10: flow meter; 11: pressure gauge. DETAILED DESCRIPTION

[0030] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0031] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0032] The embodiment of the present invention provides a method for detecting fibrinogen concentration based on A280 absorbance, comprising: (1) detecting the A280 absorbance of multiple groups of fibrinogen solutions with known concentrations; (2) constructing a relationship between the concentration of the fibrinogen solution and the A280 absorbance according to the multiple groups of A280 absorbances; and (3) detecting the A280 absorbance of a sample to be tested, and obtaining the fibrinogen concentration in the sample to be tested according to the relationship.

[0033] It should be noted that the present invention has found that a relationship can be established between the value of A280 absorbance and the concentration of fibrinogen solution. The established relationship can be used to quickly detect the fibrinogen concentration in sample detection, and the correlation R between the detected concentration value and the actual value is 2 is 1, indicating that the detection method of the present invention has high accuracy.

[0034] It should also be noted that the method of the present invention is established when the fibrinogen concentration is in the range of 0.5 μg / mL-1000 μg / mL, and the circulating water of the entire system is about 300 mL. When the concentration is lower than 10 μg / mL, the accuracy of the A280 method is not as good as when the fibrinogen concentration is above 10 μg / mL.

[0035] In some specific examples, in the above step (1), the fibrinogen solution is prepared using a phosphate buffer solution, and the pH of the phosphate buffer solution is 7.0-7.4.

[0036] It should be noted that the fibrinogen solution in the present invention can be prepared using a phosphate buffer, which is well known in the art, preferably a phosphate buffer with a pH of 7.0-7.4.

[0037] It should be noted that phosphate buffer has a wide pH buffer range, thanks to the secondary dissociation characteristics of its components such as Na2HPO4 and KH2PO4, which enables phosphate buffer to effectively maintain the pH stability of the solution when the acid-base environment changes, providing a suitable acid-base environment for biological molecules; in addition, in addition to phosphates, PBS also contains salts such as NaCl and KCl. The main function of these salts is to increase the salt ion concentration of the solution, which helps to maintain the stability and activity of biological molecules; in addition, phosphate buffer has a good dissolving and protective effect on biological molecules (such as proteins, DNA, RNA, etc.); it will not destroy the structure and biological characteristics of these biological molecules, thereby ensuring the accuracy and reliability of biological experiments.

[0038] In some specific examples, a UV-visible spectrophotometer is used to detect the A280 absorbance of multiple groups of fibrinogen solutions with known concentrations, and the UV-visible spectrophotometer is a Pulse T6-1650F.

[0039] It should be noted that A280 in the present invention can be detected by a UV-visible spectrophotometer known in the art, preferably a UV-visible spectrophotometer T6-1650F, which is more accurate in testing the concentration of the fibrinogen solution.

[0040] In some specific examples, in the above step (2), the relationship is: fibrinogen concentration of the sample to be tested = X*A280±Y, wherein A280 is the A280 absorbance, X is 600-3000, and Y is 0-1; fibrinogen purity is ≥70%.

[0041] In some specific examples, when the fibrinogen purity is 70% ≤ fibrinogen purity < 85%, the relationship is fibrinogen concentration (μg / mL) = (1750-3000) * A280 ± (0-1.0); or when the fibrinogen purity is 85% ≤ fibrinogen purity < 95%, the relationship is fibrinogen concentration (μg / mL) = (950-1800) * A280 ± (0-1.0; or when the fibrinogen purity is 95% ≤ fibrinogen purity ≤ 100%, the relationship is = (659-950) * A280 ± (0-1.0).

[0042] An embodiment of the present invention also provides a method for testing the fibrinogen filtration efficiency of an artificial heart pure water sealed system filter, which may include: (i) using a fibrinogen standard solution to circulate in the artificial heart pure water sealed system filter to obtain a circulating fluid; (ii) using the fibrinogen concentration testing method of the present invention to test the fibrinogen concentration of the circulating fluid, thereby determining the fibrinogen filtration efficiency of the artificial heart pure water sealed system filter.

[0043] It should be noted that the artificial hearts in the present invention are all artificial hearts known in the art, that is, the fibrinogen filtration efficiency test method in the present invention is applicable to all artificial hearts.

[0044] In some specific examples, the above step (i) includes:

[0045] (a) preparing a fibrinogen standard solution using a phosphate buffer solution; and circulating the phosphate buffer solution in an artificial heart;

[0046] (b) After the phosphate buffer is circulated, a fibrinogen standard solution is dropped and circulated to obtain a circulating solution.

[0047] In some specific examples, the concentration of the fibrinogen standard solution may be 4 mg / ml.

[0048] It should be noted that 4 mg / mL is calculated based on the maximum value of the fibrinogen concentration in normal human plasma; the fibrinogen content refers to the concentration of fibrin in plasma, and the normal value is 2 mg / mL-4 mg / mL; so 4 mg / mL is preferred in the present invention.

[0049] In some specific examples, the circulation rate may be 130 ml / min.

[0050] It should be noted that when the CSU is operating normally, the flow rate is 130 ml / min, so the circulation rate can be set to 130 ml / min.

[0051] In some specific examples, in the above step (b), dripping the fibrinogen standard solution for circulation includes: dripping 1 ml of the fibrinogen standard solution for 3 minutes; recording the pressure value 3 minutes after dripping; repeating the operation until 10 ml of the fibrinogen standard solution is dripped; after dripping the 10th ml of the fibrinogen standard solution; after 3 minutes, taking 6 ml of the circulating liquid; after taking the liquid, the circulation is continued for up to 3 hours, and the circulating liquid is taken again at the end.

[0052] In some specific examples, the artificial heart may be EVAHEARTⅠ.

[0053] It should be noted that, as mentioned above, the artificial heart in the present invention is any artificial heart known in the art, and the method of the present invention is particularly suitable for the EVAHEARTⅠ artificial heart, whose structure is shown in FIG. Figure 1 As shown; in addition, the filters are as follows Figure 2 shown.

[0054] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0055] In the following examples, the UV-visible spectrophotometer is 752N; the UV-visible spectrophotometer is Puxi T6-1650F purchased from Beijing Puxi General Instrument Co., Ltd.; the ultrasonic flow sensor is purchased from Enshi, FD-XS8; the BCA microprotein assay kit is from Pierce TM BCA Protein Assay Kits were purchased from Thermofisher, catalog number: 23235, containing Pierce TM Bovine serum albumin BSA standard solution, 2 mg / mL (Cat. No.: 23209); fibrinogen purchased from Solarbio, model: F8051); phosphate buffer (1X PBS PH7.0-7.4) purchased from XiGene, model: XG3650.

[0056] Use BSA to verify the UV-visible spectrophotometer. Protein molecules often contain benzene ring structures such as tyrosine, tryptophan, and phenylalanine. They have a maximum absorption peak at the ultraviolet wavelength of 280nm. The light absorption value is proportional to the protein concentration. Therefore, the absorption value at 280nm wavelength can be used to determine the protein content. According to literature records, bovine serum albumin (BSA) c = A280 / ε280, ε280 = 0.660 ± 0.008, indicating that at a wavelength of 280nm, the absorbance of 1mg / mL of bovine serum albumin is 0.660 ± 0.008; that is, at 280nm, when the absorbance (A280) is 1, the concentration of BSA is 1.497mg / ml-1.533mg / ml. That is, the relationship between the concentration of BSA and A280 can be calculated according to the following formula 1, which is as follows:

[0057] Formula 1: BSA concentration (mg / mL) = (1.497-1.533)*A280.

[0058] To verify the feasibility of the method, BSA was prepared into BSA standard solutions with BSA concentrations of 0.5 mg / ml, 1.0 mg / ml, 1.5 mg / ml and 2.0 mg / ml using PBS phosphate buffer (1*PBS, pH 7.0-7.4, 2.7 mM KCL, 137 mM NaCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4), and their A280 were measured using a UV-visible spectrophotometer T6-1650F. The results are shown in Table 1.

[0059] Table 1 A280 values ​​of BSA standard solutions with different concentrations

[0060] concentration A280 k 0.5mg / ml 0.326 1.5337 1.0mg / ml 0.651 1.5361 1.5mg / ml 0.986 1.5213 2.0mg / ml 1.301 1.5373

[0061] It can be seen from Table 1 above that the k value of the relationship between the BSA concentration and A280 tested at different concentrations is about 1.532, which is consistent with the existing BSA concentration (mg / mL) = (1.497-1.533)*A280; this shows that the accuracy of the UV-visible spectrophotometer T6-1650F in the present invention is reliable, the operating error is small, and the PBS phosphate buffer does not affect the detection of protein.

[0062] 2. Establishment of the relationship between fibrinogen solution concentration and A280

[0063] Fibrinogen (purity 93%) was prepared into fibrinogen standard solutions of different concentrations using PBS phosphate buffer (1*PBS pH 7.0-7.4), and the A280 was measured using a UV-visible spectrophotometer T6-1650F. According to the prepared fibrinogen concentration and A280 (see Table 2), the relationship between the fibrinogen concentration and A280 was obtained, as shown in Figure 3 shown.

[0064] Table 2 Different concentrations of fibrinogen and A280

[0065] A280 concentration 0.002 0.5 μg / mL 0.003 1.0 μg / mL 0.006 2.0 μg / mL 0.007 5.0 μg / mL 0.011 10 μg / mL 0.016 15 μg / mL 0.021 20 μg / mL 0.043 50 μg / mL 0.09 100 μg / mL 0.239 250 μg / mL 0.488 500 μg / mL 0.985 1000 μg / mL

[0066] The relationship between fibrinogen concentration and A280 is shown in Formula 2:

[0067] Formula 2: Fibrinogen concentration (μg / mL) = 1018.1*A280+0.6586.

[0068] In addition, the fibrinogen standard with a purity of 84.4% was used to construct a relationship as shown in Formula 3 using the above method:

[0069] Formula 3: Fibrinogen concentration (μg / mL) = 1887.5*A280-0.1632.

[0070] 3. Correlation between measured and theoretical values ​​of fibrinogen solution concentration

[0071] PBS phosphate buffer (1*PBS PH 7.0-7.4) was used to prepare the absorbance (A280) of fibrinogen solutions of different concentrations (10 μg / mL, 16.5 μg / mL, 23.5 μg / mL, 44.0 μg / mL, 117.5 μg / mL, 256.5 μg / mL and 317.5 μg / mL). The obtained value was introduced into Formula 2 to compare the calculated concentration and the prepared concentration. The comparison is shown in Table 3.

[0072] Table 3 Comparison of prepared fibrinogen solution concentration and calculated concentration

[0073]

[0074] In addition, the prepared fibrinogen concentration was plotted on the horizontal axis and the calculated concentration on the vertical axis, and the correlation between the prepared concentration and the concentration calculated by formula 2 was confirmed. The results are as follows Figure 4 As shown, the results show that R 2 =1(see Figure 4 ), indicating that within the range of 10 μg / mL-319.5 μg / mL, the fibrinogen solution concentration calculated using the relationship between the fibrinogen solution concentration and A280 in the present invention has a very high correlation, indicating that the fibrinogen solution concentration calculated using the relationship between the fibrinogen solution concentration and A280 in the present invention is relatively accurate.

[0075] In addition, by testing using Formula 3 and Formula 4 according to the above method, it was found that the fibrinogen solution concentration calculated using the relationship between the fibrinogen solution concentration and A280 in the present invention also had a very high correlation.

[0076] 4. Filter filtration effect test

[0077] (I) Method of the present invention

[0078] The filtration performance of the CS310 filter sample was tested using the method of the present invention, as follows:

[0079] (1) Preparation of fibrinogen solution

[0080] 0.16 g of fibrinogen reagent was weighed using an electronic balance and dissolved in 40 mL (concentration of 4 mg / mL) of PBS phosphate buffer (1*PBS PH 7.0-7.4, 2.7 mM KCL, 137 mM NaCl, 10 mM Na2HPO4·12H2O, 1.8 mM KH2PO4) as the test solution.

[0081] (2) Start water injection

[0082] Start the water injection test to see if the device can operate normally and remove the bubbles in the circulation line. The details are as follows: Use a 50mL syringe to fill the filter with phosphate buffer; then connect the test device (refer to Figure 5 ), circulate at a flow rate of 130 mL / min for 10 min; in addition, take about 10 mL of the circulating liquid as a blank liquid;

[0083] (3) Fibrinogen dripping

[0084] Use a micropipette to drip 1 mL of the prepared 40 mL test solution of 4 mg / mL at 3-minute intervals (wait 3 minutes after dripping before dripping again, the same below); record the pressure value (In side pressure value) 3 minutes after dripping; repeat the operation until the maximum dripping is 10 mL (fibrinogen amount 40 mg); 3 minutes after the 10th mL is dripped, take 6 mL of circulating liquid for measuring A280; after taking the liquid, continue to circulate for up to 3 hours, and at the end, take 6 mL of circulating liquid again for measuring A280;

[0085] (4) Circulating fluid A280 test

[0086] The A280 absorbance value of the circulating liquid obtained each time was measured and substituted into Formula 1 to obtain the concentration of the circulating liquid. A total of 12 sets of values ​​were used to calculate the residual rate. The results are shown in Table 4 below.

[0087] Table 4 A280 absorbance values ​​of different circulating fluids

[0088]

[0089] (II) Existing technical methods

[0090] Two CS310 filter samples were tested with reference to the BCA test method in the authorized patent CN114062220B, as follows:

[0091] (1) In the anhydrous state, weigh the pure water liquid tube, liquid storage box and filter and record it as g1.

[0092] (2) Inject pure water into the pure water liquid tube, liquid storage box and filter. The injection of pure water is mainly carried out through a syringe to ensure that pure water can be injected and air can be discharged. Here, the pure water sealed tube with liquid inside, the liquid storage box and the filter are weighed together to obtain the total weight of the liquid plus the pure water sealed tube, the liquid storage box and the filter. The mass of the injected pure water can be calculated using the formula g2-g1.

[0093] (3) Collection of pure water solution (blank)

[0094] Collect Vml of pure water through the liquid storage box and use this as blank data.

[0095] (4) Inject the fibrinogen solution into the pure water pipe of the pure water seal component

[0096] The fibrinogen concentration may be 40 mg fibrinogen: 40 mL 1*PBS phosphate buffer = 1 mg / mL.

[0097] Use a syringe to inject V ml of fibrinogen solution from the Shure connector; repeatedly push and pull the piston of the syringe to mix the injection water and fibrinogen solution in the reservoir box, and use this as the supply concentration of fibrinogen to calculate according to Formula 4.

[0098] Formula 4: Fibrinogen supply concentration = fibrinogen V*1 / pure water weight (g2-g1);

[0099] For convenience of calculation, the fibrinogen supply concentration can be assumed to be 133.3 μg / mL to calculate V.

[0100] (5) Collection of pure water (sample)

[0101] Turn on the diaphragm pump, and collect 10 ml of pure water from the outlet pipe after 1 hour and 4 hours as described above.

[0102] (6) Preparation of standard solution series for calibration curve

[0103] A series of standard fibrinogen solutions (0, 0.25 μg / mL, 0.50 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 10.0 μg / mL, 20.0 μg / mL) were prepared using fibrinogen and phosphate buffer. A calibration curve (the relationship between concentration and absorbance) was created using this series of standard solutions to determine the concentration of an unknown sample.

[0104] (7)Micro BCA TM Protein Assay Kit Reaction

[0105] Mix 3mL of fibrinogen solution in the standard solution series and 3ml of Micro BCA TM Protein Assay Kit (BCA protein concentration determination kit) preparation solution; then 3ml of pure water collected in a 15ml sterile centrifuge tube and 3ml of MicroBCA TM Mix the prepared solutions of the Protein Assay Kit; place the mixed solutions in a thermostatic bath and react at 60°C for 1 hour.

[0106] (8) Determination of absorbance

[0107] The absorbance of the reaction solution after reacting for 1 hour was measured using a UV-visible spectrophotometer T6-1650F (measurement wavelength: 562 mm).

[0108] (9) Creation of calibration curve

[0109] A calibration curve was prepared based on the concentration and absorbance of a series of standard solutions of fibrinogen solution.

[0110] (10) Determination of fibrinogen concentration in pure water (sample)

[0111] The fibrinogen concentration of the pure water solution (sample) was calculated from the calibration curve and this was taken as the fibrinogen filtrate concentration. The fibrinogen rejection rate (%) was calculated according to Formula 3.

[0112] Formula 3: Fibrinogen rejection rate (%) = 1-filtrate concentration / supply concentration × 100.

[0113] The absorbance of the first group of CS310 filter samples is shown in Table 5 below, and the graph is as follows Figure 6 shown.

[0114] Table 5 Absorbance of the first group of CS310 filter samples

[0115]

[0116]

[0117] In addition, the rejection rate (filtration rate) test results of the first group of CS310 filter samples are shown in Table 6 below.

[0118] Table 6 Rejection rate of the first group of CS310 filter samples

[0119]

[0120] Combining the test results of Table 6 and Table 4 above, it can be seen that the filtration rate (100%-residual rate) of the filter used to detect the filtration effect of the filter using the test method of the present invention is not significantly different from the filtration rate of the BCA test method used in the existing patent CN114062220A, further illustrating the accuracy of the test method of the present invention.

[0121] The absorbance of the second group of CS310 filter samples is shown in Table 7 below, and the graph is as follows Figure 7 shown.

[0122] Table 7 Absorbance of the first group of CS310 filter samples

[0123] concentration A1(Absorbance) A2(Absorbance) Average 0 0.119 0.122 0.121 0.25 0.124 0.126 0.125 0.5 0.126 0.127 0.127 1 0.136 0.137 0.137 2 0.151 0.153 0.152 5 0.208 0.209 0.209 10 0.342 0.344 0.343 20 0.650 0.637 0.644

[0124] In addition, the rejection rate (filtration rate) test results of the second group of CS310 filter samples are shown in Table 8 below.

[0125] Table 8 Rejection rate of the second group of CS310 filter samples

[0126]

[0127] Combining the test results of Table 8 and Table 4 above, it can be seen that there is no significant difference between the filtration rate (100% - residual rate) of the filter used to detect the filtration effect of the filter using the test method of the present invention and the filtration rate of the BCA test method used in the existing patent CN114062220A, which further illustrates that the test method of the present invention is more accurate.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A method for detecting fibrinogen concentration based on A280 absorbance, characterized in that: include: (1) detecting the A280 absorbance of multiple groups of fibrinogen solutions with known concentrations; (2) constructing a relationship between the concentration of the fibrinogen solution and the A280 absorbance based on the multiple groups of A280 absorbances; (3) detecting the A280 absorbance of the sample to be tested, and obtaining the fibrinogen concentration in the sample to be tested based on the relationship.

2. The method for detecting fibrinogen concentration according to claim 1, characterized in that: In step (1), the fibrinogen solution is prepared using a phosphate buffer solution, and the pH of the phosphate buffer solution is 7.0-7.

4.

3. The method for detecting fibrinogen concentration according to claim 1 or 2, characterized in that: A UV-visible spectrophotometer was used to detect the A280 absorbance of multiple groups of fibrinogen solutions with known concentrations. The UV-visible spectrophotometer was a Puxi T6-1650F.

4. The method for detecting fibrinogen concentration according to claim 1 or 2, characterized in that: In step (2), the relationship is: fibrinogen concentration of the sample to be tested = X*A280±Y, wherein A280 is the A280 absorbance, X is 600-3000, and Y is 0-1; fibrinogen purity is ≥70%.

5. The method for detecting fibrinogen concentration according to claim 4, characterized in that: When fibrinogen purity is 70%≤fibrinogen purity<85%, the relationship is: fibrinogen concentration (μg / mL)=(1750-3000)*A280±(0-1.0); or When the fibrinogen purity is 85%≤fibrinogen purity<95%, the relationship is fibrinogen concentration (μg / mL)=(950-1800)*A280±(0-1.0; or When the fibrinogen purity is 95%≤fibrinogen purity≤100%, the relationship is =(659-950)*A280±(0-1.0).

6. A method for testing the fibrinogen filtration efficiency of a pure water sealing system filter of an artificial heart, characterized in that: include: (i) circulating a fibrinogen standard solution in a pure water sealed system filter of an artificial heart to obtain a circulating fluid; (ii) Using the fibrinogen concentration testing method described in any one of claims 1 to 5 to test the fibrinogen concentration of the circulating fluid, thereby determining the fibrinogen filtration efficiency of the filter of the artificial heart pure water sealing system.

7. The fibrinogen filtration efficiency test method according to claim 6, characterized in that: Step (i) comprises: (a) preparing a fibrinogen standard solution using a phosphate buffer solution; circulating the phosphate buffer solution in a pure water sealing system filter of an artificial heart; (b) After the phosphate buffer is circulated, a fibrinogen standard solution is dropped and circulated to obtain a circulating solution.

8. The fibrinogen filtration efficiency test method according to claim 7, characterized in that: The concentration of the fibrinogen standard solution is 4 mg / ml; and / or the circulation rate is 130 ml / min.

9. The fibrinogen filtration efficiency test method according to claim 7 or 8, characterized in that: In step (b), dripping the fibrinogen standard solution for circulation includes: dripping 1 ml of the fibrinogen standard solution for 3 minutes; recording the pressure value after 3 minutes after dripping; repeating the operation until 10 ml of the fibrinogen standard solution is dripped; after dripping the 10th ml of the fibrinogen standard solution; after 3 minutes, taking 6 ml of the circulating liquid; after taking the liquid, the circulation is continued for a maximum of 3 hours, and the circulating liquid is taken again at the end.

10. The fibrinogen filtration efficiency test method according to claim 7 or 8, characterized in that: The artificial heart is EVAHEARTⅠ.

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