Preparation method of blood separation sensor membrane for dynamic monitoring of perioperative liver function

Abstract

The present invention combines membrane separation technology with biosensor technology to develop a blood separation sensor membrane that can simultaneously achieve blood separation and physiological indicator detection. A membrane tube with an Fe3O4 sensing layer is immersed in a sulfuric acid mixture containing aniline and ammonium persulfate for low-temperature oxidative polymerization, followed by washing and vacuum drying. A lactate oxidase solution and a glutamate oxidase solution are sequentially deposited on the inner surface and pores of the blood separation sensor membrane via vacuum filtration to obtain a blood separation sensor membrane. The separation membrane layer accurately intercepts blood cells while allowing serum to pass through without loss. The octahedral Fe3O4 sensing nanocrystals with high catalytic activity in the inner pores specifically respond to liver function indicators, generating current signals. This enables real-time screening and dynamic monitoring of the biological components of liver function indicators. The preparation process of this separation sensor membrane is simple and controllable, making it easy to implement large-scale and commercial production, thus resolving the current technical bottleneck in real-time dynamic monitoring of liver function in clinical practice.

Description

Technical Field

[0001] The present invention belongs to the intersection of membrane separation technology and electrochemical sensing technology, and relates to a method for preparing a blood separation sensor membrane for dynamic monitoring of perioperative liver function. Background Art

[0002] During the perioperative period, especially in high-risk surgeries such as complex hepatobiliary surgery, liver tumor resection, and liver transplantation, timely and dynamic understanding of liver function status and the extent of liver damage is crucial for clinical decision-making. Similarly, in non-surgical settings, such as patients with critical shock, severe infection, and sepsis, accurate assessment of liver function and prediction of prognosis are particularly necessary. However, real-time and dynamic monitoring of liver function has long been a technical challenge. Real-time dynamic monitoring, like physiological indicators such as electrocardiograms, heart rate, continuous arterial pressure, pulse oxygen saturation, continuous cardiac output measurement, and dynamic lung function monitoring, has not yet been achieved. This has limited the ability of physicians (including anesthesiologists, hepatobiliary surgeons, transplant surgeons, and infectious disease specialists) to accurately judge and effectively manage the liver function status of high-risk patients.

[0003] Currently, there are two main methods for liver examination in clinical practice. One involves direct biopsy to obtain partial liver tissue for pathological analysis. While this method is highly accurate, it is invasive and requires high surgical skill and can be quite painful for the patient. The other method involves blood tests for liver function markers (such as alanine aminotransferase (ALT), glutamate, lactate, and aspartate aminotransferase) to assess the functional status of the liver. This method requires centrifugation to obtain serum before testing. Both techniques are independent and time-consuming, resulting in low efficiency and a significant lag in results. They cannot reflect the dynamic changes in a patient's liver function in real time, making them unsuitable for surgical procedures and organ failure. Furthermore, they require large equipment and specialized personnel, resulting in complex procedures and high costs.

[0004] Given the crucial role of liver function in maintaining life, its dynamic monitoring should be considered as important as the dynamic monitoring of other physiological indicators. Therefore, developing membrane materials that combine separation and sensing properties to simultaneously achieve blood separation and dynamic monitoring of physiological markers of liver function will help improve the clinical diagnosis and treatment of liver diseases, optimize treatment plans, and improve patient outcomes. Summary of the Invention

[0005] In response to the current clinical bottleneck of being unable to realize real-time dynamic detection of liver function in crisis situations such as perioperative period and emergency, the present invention proposes a method for preparing a blood separation sensor membrane that can simultaneously realize blood separation and detection of liver function physiological indicators.

[0006] In order to achieve the above object, the present invention is implemented by adopting the following technical solutions:

[0007] A method for preparing a blood separation sensor membrane for dynamic monitoring of perioperative liver function comprises the following steps.

[0008] (1) Preparation of Fe3O4 sensing layer

[0009] Sodium hydroxide and polyethylene glycol powder are added to an ethylene glycol solvent and heated and stirred until the solution is completely dissolved. Ferric chloride powder is then added to the solution and heated and stirred to produce a deposition solution. A hollow fiber zirconia (YSZ) support is sealed with silicone sealant at one end and connected to a vacuum pump at the other end. The support is immersed in the deposition solution and vacuum-filtered until the deposition solution fills the membrane pores. The support and the deposition solution are then transferred to a hydrothermal reactor and allowed to react hydrothermally for a specified period of time. Finally, the membrane is removed and rinsed multiple times with deionized water. The pores are then flushed with deionized water by filtration to remove any unreacted deposition solution. The cleaned membrane is then vacuum-dried to produce a membrane tube with an Fe3O4 sensing layer.

[0010] (2) Preparation of blood separation sensing membrane-PANI separation layer

[0011] A conductive polymer, PANI, was synthesized in situ on the YSZ support using a low-temperature chemical oxidation polymerization process. The other open end of the membrane tube with the Fe3O4 sensing layer obtained in step (1) was also sealed with silicone adhesive and then immersed in a sulfuric acid mixture containing aniline and ammonium persulfate for low-temperature oxidation polymerization. After the reaction, it was washed with deionized water and vacuum-dried to obtain a PANI / Fe3O4-YSZ blood separation sensor membrane.

[0012] (3) Lactate oxidase solution and glutamate oxidase solution were sequentially deposited on the inner surface and pores of the PANI / Fe3O4-YSZ blood separation sensor membrane by vacuum filtration to obtain a blood separation sensor membrane, which was then stored at low temperature.

[0013] Preferably, in step (1), the molar concentration of sodium hydroxide in the deposition solution is 0.2-1.5 M, the concentration of polyethylene glycol is 20-30 g / L, the mass ratio of polyethylene glycol to ferric chloride is 1:(1-2), the heating and stirring temperature is 90-120° C.; the temperature of the hydrothermal synthesis reaction is 180-220° C., and the time is 16-24 h; the vacuum degree of vacuum drying is 0.1 MPa, the vacuum drying temperature is 60° C., and the vacuum drying time is at least 12 h.

[0014] Preferably, the molar concentrations of aniline and ammonium persulfate in the sulfuric acid solution of aniline and ammonium persulfate in step (2) are both 0.5-2 M; the temperature of the low-temperature oxidative polymerization reaction is 2° C., and the reaction time is 1-8 h; the vacuum degree of vacuum drying is 0.1 MPa, the vacuum drying temperature is 60° C., and the vacuum drying time is at least 6 h.

[0015] Preferably, the biological recognition element (enzyme) in step (3) is lactate oxidase or glutamate oxidase, the concentration of both enzyme solutions is 0.5-2 U / mL, and the low-temperature storage temperature is 4°C.

[0016] Based on membrane separation and biosensing technologies, this invention designs a novel dual-functional separation and sensing membrane that can simultaneously separate blood and dynamically monitor liver function physiological indicators (alanine aminotransferase (ALT), glutamate, and lactate). In terms of membrane structure, the present invention employs a hollow fiber membrane with heterogeneous nanostructured channels, combining separation and sensing functions. A relatively dense polyaniline (PANI)-modified separation layer is constructed on the support surface. The biocompatible nanopores of the PANI separation membrane precisely retain blood cells while allowing serum to pass through without loss. A porous Fe₃O₄ electrocatalytic sensing layer is formed within the support and within the pores, specifically responding to physiological indicators and generating a current signal. The heterogeneous nanostructured channels enable real-time screening and dynamic monitoring of biological components. This separation and sensing membrane demonstrates excellent detection performance and anti-interference capabilities in the detection of lactate, glutamate, and ALT. It also demonstrates extremely high accuracy in the detection of lactate, glutamate, and ALT in real blood samples, enabling dynamic separation and real-time analysis of liver function indicators in real human blood samples. The designed separation sensing membrane combines blood separation technology with a biosensor mechanism for long-term, dynamic monitoring of liver function during perioperative periods, breaking through the bottleneck of traditional liver function testing methods that only provide offline data. It significantly shortens testing time, reduces intraoperative risks, and provides a new approach for real-time monitoring of liver function status.

[0017] Compared with the prior art, the advantages and positive effects of the present invention are:

[0018] 1. This invention integrates membrane separation and biosensing theory and technology. Based on precise control of the membrane material's nanostructure, it combines blood component screening and detection functions into one, proposing the fabrication of a blood separation sensor membrane for dynamic perioperative liver function monitoring. This overcomes the current technical bottleneck of dynamic liver function monitoring in clinical practice, providing a new approach for real-time monitoring of patient status during clinical surgery, and boasts unique design.

[0019] 2. Detection of multiple liver function indicators. By switching the separation sensing membrane with corresponding functions and the corresponding signal processing fitting parameters, it can meet the needs of in-situ online long-term dynamic detection of multiple liver function indicators.

[0020] 3. Building on the separation, purification, and mass transfer capabilities of the YSZ hollow fiber support, a sensing module has been cleverly integrated. The hollow channels of the fiber membrane serve as a container for separated serum, providing a reaction microenvironment for sensing and detection, and simultaneously establishing pathways for electron and signal transmission. This provides a novel design approach for the integration of membrane and sensor technologies.

[0021] 4. This method further simplifies the preparation process of blood separation sensor membranes, resulting in low cost, good separation effect, and promising large-scale production. The prepared PANI / Fe3O4-YSZ blood separation sensor membrane has a 100% retention rate for blood cells and a maximum sensitivity of 182.6μA / mM for lactate, 85.6μA / mM for glutamate, and 12.6μA / mM for ALU. -1 . BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a structural schematic diagram of the separation sensing membrane assembly detection device.

[0023] Figure 2 This is an electron microscope image of the sensing layer of the PANI / Fe3O4-YSZ blood separation sensor membrane.

[0024] Figure 3 This is an electron microscope image of the separation layer of the PANI / Fe3O4-YSZ blood separation sensor membrane.

[0025] Figure 4 This is a scanning electron micrograph of the cross section of the PANI / Fe3O4-YSZ blood separation sensor membrane.

[0026] The reference numerals of the figures are: 1 blood transfusion channel, 2 peristaltic pump, 3 whole blood storage container, 4 reference electrode, 5 counter electrode, 6 separation sensor membrane, 7 signal processing module, 8 signal display module. DETAILED DESCRIPTION

[0027] In order to more clearly understand the above-mentioned objects, features and advantages of the present invention, the present invention is further described below in conjunction with specific embodiments. It should be noted that, in the absence of conflict, the embodiments of the present application and the features therein can be combined with each other.

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways than those described herein. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0029] Example 1

[0030] This embodiment provides a preparation method and detection process of a blood separation sensor membrane for dynamic monitoring of perioperative liver function, and the steps are as follows.

[0031] (1) Preparation of blood separation sensor membrane-Fe3O4 sensing layer: 0.96g sodium hydroxide and 1g polyethylene glycol powder were added to 40mL ethylene glycol solvent and heated at 100℃ with stirring until the solution was completely dissolved. 1.3g ferric chloride powder was added to the above solution and heated at 100℃ with stirring until uniformity was obtained to obtain a deposition solution. One end of a hollow fiber zirconia support (YSZ, length 6cm, wall thickness 1mm, tube diameter 2.5mm, average pore size 400nm) was sealed with silicone glue and the other end was connected to a vacuum pump. The support was immersed in the deposition solution and vacuum filtered to fill the membrane pores with the deposition solution. Then, it was transferred to a hydrothermal reactor with the deposition solution and hydrothermally reacted at 200℃ for 20h. Finally, it was removed and the membrane surface was rinsed with deionized water several times, and the pores were rinsed with deionized water by filtration to remove the unreacted deposition solution. Finally, the cleaned hollow fiber membrane was vacuum dried at a vacuum degree of 0.1 MPa and a temperature of 60°C for 12 h to obtain a Fe3O4 sensing layer (Fe3O4-YSZ).

[0032] (2) Prepare two sets of 1M sulfuric acid solutions, 10 mL each, as solvents, add aniline and ammonium persulfate respectively, so that the concentrations of aniline and ammonium persulfate in the two sets of solvents reach 1M, then take 5 mL of each and mix them evenly to obtain a mixed solution. Preparation of blood separation sensor membrane-PANI separation layer: The other end of the membrane tube (Fe3O4-YSZ) prepared in step (1) is also sealed with silicone glue and immersed in a sulfuric acid mixed solution containing aniline and ammonium persulfate at a low temperature of 2°C for 2 hours. After the reaction is completed, wash with running deionized water 3 times, each time for 5-20 seconds, and place in a vacuum drying oven at 60°C for dehydration and drying for 10 hours, with a vacuum degree of 0.1MPa. The PANI / Fe3O4-YSZ blood separation sensor membrane is obtained.

[0033] (3) Before loading the enzyme, remove the silicone sealant on one end of the PANI / Fe3O4-YSZ blood separation sensor membrane, while keeping the silicone sealant on the other end. Then prepare 10 mL of a 1U / mL lactate oxidase (LOX) solution, and deposit LOX on the inner surface and pores of the PANI / Fe3O4-YSZ blood separation sensor membrane by vacuum filtration. Store it at low temperature to obtain the LOX-PANI / Fe3O4 blood separation sensor membrane. Then prepare 10 mL of a 1U / mL glutamate oxidase (GOX) solution, and also use vacuum filtration to deposit GOX on the inner surface and pores of the PANI / Fe3O4-YSZ blood separation sensor membrane. Store it at 4°C to obtain the GOX-PANI / Fe3O4 blood separation sensor membrane.

[0034] The PANI / Fe3O4-YSZ blood separation sensor membrane obtained in step (2) was characterized by scanning electron microscopy. Figure 2 , 3 and Figure 4 As shown. Figure 2 As can be seen, by adjusting the molar concentration of sodium hydroxide, octahedral Fe3O4 sensing crystals with high catalytic activity were successfully prepared. The octahedral structure of Fe3O4 has a superior catalytic reaction ability towards hydrogen peroxide, strengthening the electron transport channel and improving the detection performance of liver function markers. Figure 3 The separation layer of the prepared PANI / Fe3O4-YSZ blood separation sensor membrane is a network porous nanostructure. Thanks to the excellent biocompatibility, hydrophilicity and electronegativity of PANI, it can ensure that the serum passes through the separation layer intact when separating the whole blood solution, avoiding irreversible interference of blood cell rupture on the detection. Figure 4 This is a scanning electron micrograph of a cross-section of the PANI / Fe3O4-YSZ blood separation sensor membrane, showing that the prepared membrane is a heterogeneous nanostructure, which enables the prepared separation sensor membrane to separate real blood samples and detect liver function markers (blood lactate, glutamate, ALT) simultaneously.

[0035] The prepared separation sensing membrane is placed in the designed component (such as Figure 1), the separation sensor membrane assembly includes a blood transfusion channel 1, a peristaltic pump 2, a whole blood storage container 3, a reference electrode 4, a counter electrode 5, a separation sensor membrane 6, a signal processing module 7, and a signal display module 8. The separation sensor membrane is placed in the whole blood storage container 3, with the separation sensor membrane 6 itself serving as the working electrode. A platinum wire counter electrode 5 and an Ag / AgCl wire reference electrode 4 are also integrated within the assembly, ensuring that the lower ends of the reference electrode 4 and counter electrode 5 are immersed in the whole blood in the whole blood storage container 3. A vacuum pressure differential is used to drive the whole blood through the separation sensor membrane 6, achieving serum separation and delivery. The separation sensor membrane 6 can convert the concentration signals of liver function markers (blood lactate, glutamate, ALT) in the blood into electrochemical signals through biochemical reactions. Three signal transmission wires extend from the signal processing module 7 and are connected to the counter electrode 5, the separation sensor membrane 6, and the reference electrode 4, respectively. The detected electrical signals are transmitted to the signal processing module 7 for analysis and conversion, and the detection results are displayed on the signal display module 8, enabling in situ dynamic monitoring of liver function markers in the human body. The prepared blood separation sensor membrane 6 achieves a 100% retention rate for blood cells, achieving a sensitivity of 182.6 μA / mM for blood lactate and 85.6 μA / mM for glutamate. Monosodium glutamate can also be considered an intermediate product produced by the reaction of α-ketoglutarate and L-alanine in the presence of another enzyme, ALT. Therefore, the prepared GOX-PANI / Fe3O4 blood separation sensor membrane can also be used to detect the level of ALT, a liver function marker. Before ALT detection, a certain amount of the reactants α-ketoglutarate and L-alanine must be pre-added to the whole blood storage container. Since α-ketoglutarate has the effect of inhibiting GOX activity, adding 10 mM α-ketoglutarate and 100 mM L-alanine to 20 mL of whole blood is the best ratio after investigation. The prepared GOX-PANI / Fe3O4 blood separation sensor membrane can achieve a detection sensitivity of 12.6 μA / UL for ALU. -1 After the test was completed, the separation sensing membrane was stored in PBS buffer solution at 4°C for 30 days, and its response signal was 96% of the initial signal, indicating that the separation sensing membrane has excellent stability.

[0036] Example 2

[0037] The conditions in this embodiment and subsequent embodiments not specifically described are consistent with those in Example 1. This embodiment provides a method for preparing a blood separation sensor membrane for dynamic monitoring of perioperative liver function, comprising the following steps:

[0038] (1) Preparation of blood separation sensor membrane-Fe3O4 sensing layer: 0.32g sodium hydroxide and 1g polyethylene glycol powder were added to 40mL ethylene glycol solvent and heated and stirred at 90℃ until the solution was completely dissolved. Then 1g ferric chloride powder was added to the above solution and heated and stirred at 90℃ until uniform deposition liquid was obtained. One end of the hollow fiber zirconia support (YSZ) was sealed with silicone glue and the other end was connected to a vacuum pump. The support was immersed in the deposition liquid and vacuum filtered to fill the membrane pores. Then it was transferred to a hydrothermal reactor with the deposition liquid and hydrothermally reacted at 180℃ for 16 hours. Finally, it was taken out and the membrane surface was rinsed with deionized water several times, and the pores were rinsed with deionized water by filtration to remove the unreacted deposition liquid. Finally, the cleaned hollow fiber membrane was vacuum dried at a vacuum degree of 0.1MPa and a temperature of 60℃ for 16 hours to obtain the Fe3O4 sensing layer (Fe3O4-YSZ).

[0039] (2) Preparation of blood separation sensor membrane-PANI separation layer: The other end of the membrane tube (Fe3O4-YSZ) prepared above was also sealed with silicone glue and reacted in a sulfuric acid solution containing aniline and ammonium persulfate at a low temperature of 2°C for 1 hour. The mixed solution of aniline and ammonium persulfate was obtained by mixing 5 mL of 0.5M aniline solution with 5 mL of 0.5M ammonium persulfate solution (0.5M sulfuric acid as solvent). After the reaction, it was washed three times with running deionized water and placed in a vacuum drying oven at 60°C for 10 hours with a vacuum degree of 0.1 MPa. The PANI / Fe3O4-YSZ blood separation sensor membrane was obtained.

[0040] (3) Prepare 10 mL of 0.5 U / mL lactate oxidase (LOX) solution, and deposit LOX on the inner surface and pores of the PANI / Fe3O4-YSZ blood separation sensor membrane by vacuum filtration. Store it at low temperature to obtain the LOX-PANI / Fe3O4 blood separation sensor membrane. Prepare another 10 mL of 0.5 U / mL glutamate oxidase (GOX) solution, and also use vacuum filtration to deposit GOX on the inner surface and pores of the PANI / Fe3O4-YSZ blood separation sensor membrane. Store it at low temperature to obtain the GOX-PANI / Fe3O4 blood separation sensor membrane. After testing, the sensor membrane prepared in this embodiment achieved a sensitivity of 164.5 μA / mM in the detection of blood lactate and a sensitivity of 72.6 μA / mM in the detection of glutamate. The detection sensitivity of ALU can reach 11.2 μA / UL -1 After completing the standard curve test, the separation sensing membrane was stored in PBS buffer solution at 4°C for 30 days, and its response signal was 93% of the initial signal, indicating that the separation sensing membrane has excellent stability.

[0041] Example 3

[0042] (1) Preparation of blood separation sensor membrane-Fe3O4 sensing layer: 2.4g sodium hydroxide and 1g polyethylene glycol powder were added to 40mL ethylene glycol solvent, heated and stirred at 120℃ until the solution was completely dissolved. Then 1.6g ferric chloride powder was added to the above solution, heated and stirred at 120℃ until uniform deposition liquid was obtained. One end of the hollow fiber zirconia support (YSZ) was sealed with silicone glue, and the other end was connected to a vacuum pump. The support was immersed in the deposition liquid, and vacuum filtered to fill the membrane pores with the deposition liquid. Then, it was transferred to a hydrothermal reactor with the deposition liquid and hydrothermally reacted at 220℃ for 24 hours. Finally, it was taken out and the membrane surface was rinsed with deionized water several times, and the pores were rinsed with deionized water by filtration to remove the unreacted deposition liquid. Finally, the cleaned hollow fiber membrane was vacuum dried at a vacuum degree of 0.1MPa and a temperature of 60℃ for 14 hours to obtain the Fe3O4 sensing layer (Fe3O4-YSZ).

[0043] (2) Preparation of blood separation sensor membrane-PANI separation layer: The other end of the membrane tube (Fe3O4-YSZ) prepared above was also sealed with silicone glue and reacted in a sulfuric acid solution containing aniline and ammonium persulfate at a low temperature of 2°C for 8 hours. The mixed solution of aniline and ammonium persulfate was obtained by mixing 5 mL of a 2M aniline solution with 5 mL of a 2M ammonium persulfate solution (2M sulfuric acid as a solvent). After the reaction, it was washed three times with running deionized water and placed in a vacuum drying oven at 60°C for 12 hours with a vacuum degree of 0.1 MPa. The PANI / Fe3O4-YSZ blood separation sensor membrane was obtained.

[0044] (3) Prepare 10 mL of a 2 U / mL lactate oxidase (LOX) solution and deposit LOX on the inner surface and pores of the PANI / Fe3O4-YSZ blood separation sensor membrane by vacuum filtration. Store the membrane at low temperature to obtain a LOX-PANI / Fe3O4 blood separation sensor membrane. Prepare another 10 mL of a 2 U / mL glutamate oxidase (GOX) solution and deposit GOX on the inner surface and pores of the PANI / Fe3O4-YSZ blood separation sensor membrane by vacuum filtration. Store the membrane at low temperature to obtain a GOX-PANI / Fe3O4 blood separation sensor membrane.

[0045] The results show that the sensor membrane prepared in this example has a sensitivity of 156.4 μA / mM in the detection of blood lactate and 58.6 μA / mM in the detection of glutamate. The detection sensitivity of ALU can reach 10.3 μA / mM. -1 After completing the standard curve test, the separated sensor membrane was stored in PBS buffer solution at 4°C for 30 days, and its response signal was 90% of the initial signal.

[0046] The separation and sensing membranes prepared in Examples 1-3 were used to separate and detect liver function markers (blood lactate, glutamate, and ALT) from different real blood samples (the real blood samples were from Nanjing Drum Tower Hospital). The test results are shown in Table 1.

[0047] Table 1 Real blood sample test results

[0048]

[0049] Table 1 shows the detection results of lactate, glutamate, and ALT in real blood samples using the separation sensor membranes prepared in Examples 1-3. The reference data (actual values) are from a Mindray fully automatic biochemical analyzer. Testing showed that the plasma separated by the blood separation sensor membranes prepared in Examples 1-3 was free of blood cells, and the blood cell retention rate reached 100%. The results in Table 1 demonstrate that the separation sensor membranes prepared in Examples 1-3 can accurately detect liver function markers (blood lactate, glutamate, and ALT) in real blood samples, with low detection limits and a wide linear range, demonstrating excellent stability, anti-interference ability, and detection accuracy.

[0050] Comparative Example 1

[0051] This comparative example differs from Example 1 in that the conductive polymer PANI was synthesized in situ on the Fe3O4-YSZ surface using a low-temperature chemical oxidative polymerization process. Hydrochloric acid was used as the solvent for the mixed solution of aniline and ammonium persulfate, while all other conditions remained unchanged. The resulting PANI / Fe3O4-YSZ blood separation sensor membrane destroyed blood cells when tested in real blood samples, making it unsuitable for detecting liver function markers in real blood samples.

[0052] Comparative Example 2

[0053] This comparative example differs from Example 1 in that the prepared Fe3O4-YSZ membrane tube was not subjected to further low-temperature oxidative polymerization to deposit polyaniline on its surface. Instead, the Fe3O4-YSZ membrane was used alone to prepare the blood sample separation sensor membrane, with all other conditions remaining unchanged. When testing real blood samples, this separation sensor membrane failed to completely retain blood cells and caused them to rupture, failing to meet actual testing requirements.

[0054] Comparative Example 3

[0055] This comparative example differs from Example 1 in that Fe₃O₄ is not deposited in the pores and inner surface of the support. Instead, PANI is directly oxidatively polymerized on the YSZ surface at low temperature to produce a PANI-YSZ separation sensing membrane. All other conditions remain unchanged. Experimental verification shows that this PANI-YSZ separation sensing membrane exhibits little response to liver function markers (blood lactate, glutamate, and ALT), failing to meet practical detection requirements. This demonstrates the crucial role of Fe₃O₄ nanosensing crystals in the detection of physiological components in blood.

[0056] When the prepared separation sensing membrane of the present invention is used to detect liver function markers (blood lactate, glutamate, ALT) in real blood samples, the blood sample is first separated by a PANI-modified separation layer. Since PANI has good biocompatibility, hydrophilicity and electronegativity, it can effectively inhibit the adhesion of a large number of negatively charged proteins in whole blood when separating blood. At the same time, the surface of red blood cells has a negative charge, and electrostatic repulsion can prevent cell surface damage and the occurrence of hemolysis. Therefore, it can penetrate serum without loss. Lactate in the serum reacts with lactate oxidase (LOX) in the pores and inner surface of the separation sensor membrane to produce H2O2. Similarly, glutamate in the serum reacts with glutamate oxidase (LOX) in the pores and inner surface of the separation sensor membrane to produce H2O2. Alanine aminotransferase (ALT) in the blood sample first reacts with α-ketoglutarate and L-alanine, the reactants pre-added to the whole blood storage container, to produce glutamate. The serum is then separated by the PANI-modified separation layer. Glutamate in the serum reacts with glutamate oxidase (LOX) in the pores and inner surface of the separation sensor membrane to produce H2O2. Because Fe3O4 is a natural catalase, it reduces H2O2 in the presence of O2 with 100% selectivity. This process results in the transfer of two electrons, generating a corresponding response current feedback signal. By utilizing the regular, highly catalytically active octahedral Fe3O4 evenly distributed in the pores and inner surface of the separation sensing membrane, the concentration signals of liver function markers (blood lactate, glutamate, ALT) in the blood can be converted into electrochemical signals through biochemical reactions, thereby realizing online monitoring of liver function.

[0057] The concentration of sodium hydroxide plays a key role in the morphology of the prepared Fe3O4. A reasonable sodium hydroxide concentration allows the preparation of Fe3O4 with a regular octahedral structure. Fe3O4 with a regular octahedral structure exhibits superior detection performance compared to Fe3O4 with an irregular morphology. This is due to the excellent catalytic activity and enhanced electron transport of Fe3O4 with a regular octahedral structure.

[0058] In Comparative Example 1, when the conductive polymer PANI was in situ synthesized on the Fe3O4-YSZ surface to construct a separation layer, 1M hydrochloric acid was selected as the solvent for the mixed solution of aniline and ammonium persulfate. The morphology of the PANI synthesized under this condition was not as Figure 3When the PANI / Fe3O4-YSZ blood separation sensor membrane is used to detect real blood samples, the needle-shaped PANI will destroy blood cells, causing a large amount of interfering substances such as hemoglobin to enter the blood environment, causing serious signal interference. Therefore, the separation sensor membrane cannot be used to detect liver function markers in real blood samples.

[0059] The Fe3O4-YSZ membrane prepared in Comparative Example 2 was not further subjected to low-temperature oxidative polymerization to deposit polyaniline on its surface. Instead, the Fe3O4-YSZ membrane alone was used to prepare the blood separation sensor membrane. Because the pore size of the YSZ support is larger than that of blood cells, it is unable to completely retain blood cells and separate serum. More importantly, YSZ has extremely poor biocompatibility and hydrophilicity, and lacks electronegativity. Therefore, during blood separation, a large amount of protein in whole blood adheres to the membrane surface, destroying red blood cells and causing hemolysis. Therefore, this separation sensor membrane cannot be used to detect liver function markers in real blood samples.

[0060] In Comparative Example 3, Fe₃O₄ was not deposited in the pores and inner surface of the support. Instead, PANI was directly oxidatively polymerized on the YSZ surface at low temperature to produce a PANI-YSZ separation sensing membrane. All other conditions remained unchanged. However, due to the lack of catalytically active sensing nanomaterials, the H₂O₂ product of the reaction between the biorecognition element (enzyme) and liver function markers (blood lactate, glutamate, and ALT) could not be recognized. Consequently, the concentration signals of these liver function markers (blood lactate, glutamate, and ALT) in the blood could not be converted into electrochemical signals through biochemical reactions, making online, real-time monitoring of liver function impossible.

[0061] In summary, the novel PANI / Fe3O4-YSZ blood separation sensor membrane with heterogeneous nanostructured channels prepared in this invention can simultaneously achieve non-destructive blood separation and dynamic monitoring of liver function markers (blood lactate, glutamate, and ALT). This overcomes the current technical bottleneck that prevents real-time and dynamic liver function monitoring in clinical practice. By designing and constructing a membrane channel that simultaneously performs separation and electrocatalysis, it simultaneously facilitates serum transfer, electron transport, and signal transmission. The membrane's micro-nanostructure enables precise control of the transfer rate and pathways of different serum components. Electrocatalytically active substances (such as enzymes and Fe3O4) are embedded within the membrane. These substances act as electron transfer media, facilitating electron transfer between serum components and electrodes, thereby capturing and amplifying electrochemical signals. The sensors integrated into the membrane channels can monitor changes in liver function marker concentrations in real time, converting these changes into electrical signals for recording and analysis. This real-time capability significantly improves diagnostic accuracy and timeliness.

[0062] The clinical application of this technology, particularly for high-risk patients in departments such as anesthesiology, hepatobiliary medicine, transplantation, and infectious diseases, enables immediate and accurate assessment of liver function status, providing an important basis for physicians to formulate personalized treatment plans. By continuously monitoring liver function indicators, physicians can adjust treatment plans in a timely manner, avoid complications caused by overdose or underdose, optimize treatment effects, and improve patient prognosis. This innovative design not only promotes the deep integration of membrane and sensing technologies, but also provides new research directions for multiple disciplines such as biomedical engineering and clinical medicine.

[0063] The above description is merely a preferred embodiment of the present invention and does not constitute any other form of limitation to the present invention. Any person skilled in the art may utilize the technical contents disclosed above to change or modify them into equivalent embodiments with equivalent changes for application in other fields. However, any simple modification, equivalent change, and modification of the above embodiments made in accordance with the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a blood separation sensor membrane for dynamic monitoring of perioperative liver function, characterized in that: Here are the steps: (1) Preparation of Fe3O4 sensing layer Sodium hydroxide and polyethylene glycol powder are added to ethylene glycol, heated and stirred until completely dissolved, and ferric chloride powder is added, heated and stirred to obtain a deposition liquid. One end of a hollow fiber zirconia support is sealed with silicone glue, and the other end is connected to a vacuum pump and immersed in the deposition liquid. Vacuum filtration is performed to ensure that the deposition liquid fills the pores of the support. The support and the deposition liquid are then transferred to a hydrothermal kettle for a hydrothermal reaction. After the reaction, the surface and interior of the support are rinsed with deionized water. The membrane tube with the Fe3O4 sensing layer is then vacuum dried. (2) Preparation of PANI separation layer The other open end of the membrane tube having the Fe3O4 sensing layer obtained in step (1) is also sealed with silicone glue, and then immersed in a sulfuric acid mixed solution containing aniline and ammonium persulfate for low-temperature oxidative polymerization reaction. After the reaction, it is washed with deionized water and vacuum dried to obtain a PANI / Fe3O4-YSZ blood separation sensor membrane; (3) Lactate oxidase solution and glutamate oxidase solution were sequentially deposited on the inner surface and pores of the PANI / Fe3O4-YSZ blood separation sensor membrane by vacuum filtration to obtain a blood separation sensor membrane.

2. The method for preparing a blood separation sensor membrane for dynamic monitoring of perioperative liver function according to claim 1, characterized in that: In step (1), the molar concentration of sodium hydroxide in the sedimentation liquid is 0.2-1.5M, the concentration of polyethylene glycol is 20-30g / L, the mass ratio of polyethylene glycol to ferric chloride is 1:(1-2), the heating and stirring temperature is 90-120°C; the temperature of the hydrothermal synthesis reaction is 180-220°C, and the time is 16-24h; the vacuum degree of vacuum drying is 0.1MPa, the vacuum drying temperature is 60°C, and the vacuum drying time is at least 12h.

3. The method for preparing a blood separation sensor membrane for dynamic monitoring of perioperative liver function according to claim 1, characterized in that: In step (2), the molar concentrations of aniline and ammonium persulfate in the sulfuric acid mixed solution are both 0.5-2 M, the temperature of the low-temperature oxidative polymerization reaction is 2° C., and the reaction time is 1-8 h.

4. The method for preparing a blood separation sensor membrane for dynamic monitoring of perioperative liver function according to claim 1, characterized in that: In step (3), the concentrations of the lactate oxidase solution and the glutamate oxidase solution are both 0.5-2 U / mL; after the blood separation sensor membrane is prepared, it can be used directly or stored at a low temperature for future use, and the low temperature storage temperature is 4°C.

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