A functionalized nanoporous membrane and its application in sialic acid molecule recognition

Abstract

This invention provides a functionalized nanoporous membrane and its application in sialic acid molecule recognition. First, a smart polymer is synthesized, and then this polymer is grafted into nanopores. This smart polymer can specifically interact with sialic acid molecules. The interaction force drives the polymer to swell to varying degrees, compressing the effective space within the nanopores to different degrees. This allows for the detection of different changes in transmembrane ion current using electrochemical detection methods, thereby enabling the detection of different sialic acid molecules. This nanoporous membrane for detecting sialic acid molecules has advantages such as low detection limit, high sensitivity, simple operation, low cost, and label-free operation. It can be integrated into a lightweight, portable, simple, rapid, and economical detection device. It provides a valuable tool for the distinguishing detection of sialic acid sugar molecules in glycobiology.

Description

Technical Field

[0001] The present invention relates to the synthesis of smart polymers and their modification on the inner wall of nanopores, as well as the application of smart polymer-modified nanopore membranes in the recognition and detection of different sialic acid molecules, and specifically to a functionalized nanopore membrane and its application in sialic acid molecule recognition. Background Art

[0002] Glycosylation is a type of post-translational modification (PTM) of proteins and lipids that regulates or mediates many biological processes, such as molecular recognition, signal transduction, immune response, and cell-cell interaction. The occurrence and development of many diseases are closely related to changes in cell surface glycosylation. In cancer, changes in glycosylation play an important role in promoting tumor growth, metastasis, evading immune surveillance, escaping apoptosis pathways, leading to cancer cell survival and resistance to treatment. For example, the surface of tumor cells is covered with sugar chains, which can adhere to blood cells and then achieve long-distance migration; HIV-1 disguises itself through sugar chain shielding to evade recognition by the host immune system; and the COVID-19 novel coronavirus has been confirmed to have a large number of sugar chains on its surface.

[0003] Sialylation is a crucial component of glycosylation, and sialylated sugar chains have high biological significance. Terminal sialylated sugar chains account for a very high proportion in human serum. Furthermore, sialic acid levels in serum or plasma have been shown to be a strong predictor of cardiovascular mortality, with elevated levels in the blood of patients with cerebrovascular disease and diabetes. Furthermore, sialic acid is a key component of gangliosides, which are closely linked to the nervous system. The highest concentration of gangliosides is found in the human cerebral cortex, where they promote neuronal adhesion and migration. Furthermore, the sialic acid content in neuronal cell membranes is 20 times higher than in other cell membrane types, indicating a clear role for sialic acid in neural structures. Furthermore, major glycoproteins, glycolipids, and major oligosaccharides associated with Alzheimer's disease are all associated with sialic acid. Therefore, highly sensitive detection of sialic acid is of great significance.

[0004] Sialic acid is a derivative of 9-carbon monosaccharides and a general term for a family of substances, but it mainly includes three important forms: Neu5Ac, KDN, and Neu5Gc. Their structures are highly similar, differing only in one group. Current methods such as mass spectrometry, nuclear magnetic resonance, and high-performance liquid chromatography can achieve differentiation, but they have disadvantages such as complex pretreatment or sufficient purity and quantity of samples. Accurately distinguishing them in a simple, inexpensive, and label-free manner remains a challenge. Therefore, a simple and efficient method is needed to supplement mass spectrometry / nuclear magnetic resonance technology.

[0005] The broken symmetry and internal surface charge of the nanopore make it ion-selective, thus exhibiting a rectification effect. There are exposed carboxyl groups in the conical pore, which can be grafted with molecules such as polymers. At the same time, the response molecules can be introduced into the pore to change the physical and chemical properties of the pore. After the substance to be detected interacts with the response molecules on the polymer, it causes the polymer chain to relax, agglomerate or change the surface charge, which will change the transmembrane current value. Based on this, the detection of sialic acid molecules can be achieved. Moreover, the pore size of the small end of the nanopore is only tens of nanometers, or even more than ten nanometers. The smaller the pore size, the stronger the ability to control the charge conduction properties of the pore, the more obvious the rectification effect, and a small change can trigger a significant change in the current value, thereby enabling the detection of extremely low concentrations of the substance to be detected.

[0006] In this invention, we innovatively utilize nanochannels for the identification and differentiation of sialic acid molecules, and have developed a highly sensitive sialic acid detection device. By grafting recognition units onto polymers to create smart polymers, which are then modified onto the inner walls of nanopores, we leverage the specific interaction between the recognition units and sialic acid molecules to drive the smart polymers to swell to varying degrees, compressing the effective space within the pores. This, in turn, causes varying degrees of variation in the ionic current flowing through the nanopores, thereby enabling the identification and detection of different sialic acid molecules. This invention provides a means for the identification and detection of sialic acid, offering a valuable tool for the differential detection of sialic acid sugar molecules in glycobiology. Summary of the Invention

[0007] The present invention aims to provide a smart polymer that specifically interacts with sialic acid and chemically grafts it into nanopores to produce a smart polymer-functionalized nanoporous membrane. This functionalized nanoporous membrane can be used to identify and detect sialic acid molecules and accurately distinguish different sialic acid molecules from monosaccharide molecules. Compared with traditional mass spectrometry, nuclear magnetic resonance, and electrochemical detection methods, this functionalized nanoporous membrane has the advantages of low detection limit, high sensitivity, simple operation, low cost, and no labeling required. It is suitable for the identification and detection of sialic acid molecules.

[0008] The technical solution adopted by the present invention is:

[0009] A functionalized nanoporous membrane is used for sialic acid molecule recognition detection by an electrochemical detection method.

[0010] The nanoporous membrane is characterized in that: the nanoporous membrane is an organic membrane; the nanoporous membrane is track-etched on the organic membrane, and the pore density is 1-1.0×10 8 pieces / cm 2The pore size range is 10-1000nm. The method for preparing the functionalized nanoporous membrane is to first graft a functional recognition monomer—acetylated arginine (Ac-Arg) molecule—on the polymer chain, and then modify the smart polymer grafted with the recognition monomer to the inner wall of the nanopore. The specific steps are as follows:

[0011] (1) 0.757 g of acetylated arginine, 0.7-0.9 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and 0.5-0.6 g of N-hydroxysuccinimide (NHS) were dissolved in 5-30 ml of 0.01-1 M 2-morpholinoethanesulfonic acid (MES) buffer solution (pH = 4-6) and stirred at room temperature for 2-3 hours;

[0012] Dissolve 0.5-2.0 g of polyethyleneimine (PEI) in MES solution, then add the above solution, and stir the mixed solution at room temperature for 8-36 hours. After the reaction, the solution is placed in a dialysis bag, dialyzed with water for 2-5 days, and freeze-dried to obtain the desired smart polymer.

[0013] (2) Activation and modification of nanopores: The nanoporous membrane was placed between the two chambers of the electrochemical cell, and a deionized water solution of 5-30 mg / mol EDC and 2-15 mg / mol NHS was added to the two chambers to ensure that both sides of the nanoporous membrane were immersed in the solution. The membrane was activated at room temperature for 0.5-3 hours. The solution was then removed and washed with deionized water. A 1-20 mg / ml polymer solution was added to ensure that both sides of the nanoporous membrane were immersed in the solution. The reaction was allowed to react overnight (6-12 hours) at room temperature to obtain a smart polymer-modified nanoporous membrane.

[0014] The functionalized nanoporous membrane comprises a nanoporous membrane and a smart polymer grafted onto the inner surface of the pores of the nanoporous membrane. The molecular structure of the polymer is schematically shown as follows:

[0015]

[0016] The grafting amount is 1 to 99% (based on the number of primary amino groups); n is 10 to 100

[0017] The organic film is one of a polyethylene terephthalate film, a polyimide film, and a polycarbonate film.

[0018] The shape of the nanopore is one or more of cylindrical, asymmetric conical, cigar-shaped, funnel-shaped, hourglass-shaped, and dumbbell-shaped;

[0019] Preferably, asymmetric conical pores are track-etched on the organic film with a pore density of 1-1.0×10 8 pieces / cm 2The pore size on one side of the membrane surface is 500-1000 nm, and the pore size on the other side of the membrane surface is 10-100 nm. The pore ports on one side of the membrane surface are larger than the pore ports on the other side of the membrane surface.

[0020] The functionalized nanoporous membrane is applied to electrochemical detection and identification of sialic acid molecules.

[0021] The functionalized nanoporous membrane is used for detecting sialic acid molecules. The specific steps for detecting different sialic acid molecules using an electrochemical detection method are as follows:

[0022] Step 1: Sandwich the nanoporous membrane modified with a smart polymer between the two chambers of an electrochemical cell. Add electrolyte to both sides of the membrane. Connect electrodes to the two chambers of the electrochemical cell and apply a transmembrane step voltage difference of -2V to +2V. The step voltage is 0.05 to 0.4V, and the step time is 0.1 to 4 seconds. During the test, the voltage starts at -2V and ends at +2V. Use a picoammeter to measure the transmembrane ion current every 1-5 minutes. After 6-10 runs, record the data.

[0023] Step 2: Remove the electrolyte added in Step 1 and add the electrolyte of the substance to be tested. Connect electrodes to the two chambers of the electrochemical cell and apply a transmembrane step voltage difference of -2V to +2V. The step voltage is 0.05 to 0.4V and the step time is 0.1 to 4 seconds. During the test, the voltage starts at -2V and ends at +2V. Use a picoammeter to measure the transmembrane current every 1-5 minutes. After 6-10 runs, record the data.

[0024] The nanoporous membrane has different degrees of current changes for different sialic acids, thereby achieving the distinction between different sialic acids.

[0025] The electrolyte is a solution of one or more of sodium chloride, potassium chloride, calcium chloride or magnesium chloride at a concentration of 0.001 to 1.0 mol / L. The pH of the electrolyte is 4 to 11. The solvent is deionized water, ultrapure water or distilled water.

[0026] The electrode is one or more of a platinum wire electrode, a glassy carbon electrode, a graphite electrode, a stainless steel electrode, a silver-silver chloride electrode or a mercury-mercury chloride electrode.

[0027] The technical advantages of the present invention are:

[0028] 1. The polymer used in the smart polymer-modified nanoporous membrane prepared by the present invention is made of commercial polymer side chain coupled recognition units, which is simple to prepare, low in cost and has strong scalability;

[0029] 2. The functionalized nanoporous membrane prepared by the present invention has different responsiveness to different sialic acid molecules when identifying and detecting sialic acid molecules, and can achieve accurate identification and differentiation of different types of sialic acid;

[0030] 3. The functionalized nanoporous membrane prepared by the present invention occupies very little space and can be integrated into a lightweight, portable, simple, fast and economical detection device. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1 Smart polymer-modified nanopores and polymer structures;

[0033] Figure 2 XPS images of the nanoporous membrane before (a) and after (b) polymer modification.

[0034] Figure 3 Contact angle diagrams of nanoporous membranes before (a) and after (b) polymer modification.

[0035] Figure 4 .Schematic diagram of the electrochemical test structure of the nanoporous membrane;

[0036] Figure 5 .Voltage-current curves of nanoporous membranes before and after modification with smart polymers;

[0037] Figure 6 The voltage-current curve changes caused by the addition of different concentrations of Neu5Ac sialic acid molecules to the smart polymer-modified nanoporous membrane;

[0038] Figure 7 . Current changes of smart polymer-modified nanoporous membranes with different sialic acid concentrations and +2V voltage;

[0039] Figure 8 .Chart of current changes when different monosaccharides were added to the smart polymer-modified nanoporous membrane at different concentrations and +2V voltage;

[0040] Figure 9 .Chart of current changes of smart polymer modified nanoporous membrane with different weak acids added at different concentrations and +2V voltage;

[0041] Figure 10. Current changes of smart polymer modified nanoporous membrane, unmodified nanoporous membrane, and polymer modified nanoporous membrane without grafted recognition unit when Neu5Ac was added at different concentrations and +2V voltage. DETAILED DESCRIPTION

[0042] In order to make the content, technical solutions and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments and drawings. These embodiments are only used to illustrate the present invention, and the present invention is not limited to the following embodiments.

[0043] Polymer synthesis:

[0044] Example 1

[0045] Polymer structure such as Figure 1 As shown in the structural schematic on the right, 0.757 g of acetylated arginine (Ac-Arg), 0.805 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.524 g of N-hydroxysuccinimide (NHS) were dissolved in 10 ml of 0.1 M 2-morpholineethanesulfonic acid (MES) buffer solution (pH = 5) and stirred at room temperature for 2.5 hours.

[0046] 1.0 g of branched polyethyleneimine (PEI, molecular weight Mw = 10,000 g / mol, Alfa Aesar, branched, 99%) was dissolved in 5 ml of MES solution, then added to the above solution. The mixed solution was stirred at room temperature for 24 hours. After the reaction, the solution was placed in a dialysis bag (molecular weight cut-off 3,500 g / mol) and dialyzed against deionized water for 4 days. After freeze-drying, the desired smart polymer (PEI-Ac-Arg) was obtained. The grafting amount was 22.1% (based on the number of primary amino groups).

[0047] Preparation of polymer-modified nanoporous membranes, i.e., preparation of functionalized nanoporous membranes:

[0048] Example 2

[0049] (1) Polymer modified nanoporous membrane (Schematic diagram of the pores obtained by track etching in the nanoporous membrane is shown in Figure 1 As shown); taking the polyethylene terephthalate (PET) conical porous membrane obtained by track etching as an example.

[0050] First, each side of a heavy metal ion-irradiated PET film (GSI, Darmstadt) was irradiated with ultraviolet light (365 nm) for 30 minutes to sensitize the damage track. Afterwards, the UV-irradiated PET film was installed in an electrochemical cell, which was divided into two mutually separated chambers by a nanoporous membrane. One chamber was filled with a 9M NaOH solution (etching solution), and the other chamber was filled with a 1M HCOOH and 1M KCl solution (blocking solution). Etching was carried out at 45°C, and electrodes were inserted into the two chambers of the electrochemical cell. A picoammeter was used to apply a transmembrane voltage of 1 V to monitor the etching process. When the ion current reached 3.0×10 -5 At A, the etching solution was removed by rinsing and filling both chambers with a blocking solution to stop etching. Afterwards, the blocking solution was replaced by rinsing and filling both chambers with water. Finally, the prepared nanoporous membrane was preserved by soaking in water to remove residual salt and acid. The resulting nanoporous membrane had asymmetric conical pores with a pore density of 1.0×10 6 The average pore size on one side of the membrane is 660 nm, and the average pore size on the other side of the membrane is 30 nm. The pore ports on one side of the membrane are larger than those on the other side of the membrane.

[0051] The nanoporous membrane was sandwiched within an electrochemical cell, which was then divided into two separate chambers by the nanoporous membrane. A deionized water solution of 15 mg / mol EDC and 6 mg / mol NHS was added to the two chambers, ensuring that both surfaces of the nanoporous membrane were immersed in the solution. The solution was activated at room temperature for 1 hour. The solution was then removed and rinsed with deionized water. A 5 mg / ml solution of the polymer from Example 1 was then added, ensuring that both surfaces of the nanoporous membrane were immersed in the solution. The reaction was allowed to proceed overnight (12 hours) at room temperature. After the reaction was completed, the nanoporous membrane was removed, rinsed with deionized water, and then dried with nitrogen to obtain a smart polymer-modified nanoporous membrane.

[0052] (2) X-ray photoelectron spectroscopy (XPS) analysis of nanoporous membranes before (a) and after (b) polymer modification Figure 2 As shown, it can be seen that the N1s peak increases significantly after polymer modification, and the nitrogen element signal mainly comes from the polymer, indicating that the polymer is successfully modified on the nanoporous membrane.

[0053] (3) Analysis of water contact angle of nanoporous membrane before (a) and after (b) polymer modification Figure 3 As shown (at room temperature), it can be seen that the contact angle is significantly reduced after polymer modification, and the polymer has better hydrophilicity than the PET film itself, indicating that the polymer is successfully modified on the nanoporous membrane.

[0054] Application of polymer-modified nanoporous membranes in the detection of sialic acid and monosaccharide molecules:

[0055] Example 3

[0056] (1) Schematic diagram of the electrochemical test structure of the nanoporous membrane Figure 4 As shown, the nanoporous membrane modified with the smart polymer of Example 2 (taking PEI-Ac-Arg modified PET nanoporous membrane as an example) and the nanoporous membrane before modification are respectively clamped in the electrochemical cell, and the inside of the electrochemical cell is divided into two mutually spaced chambers by the nanoporous membrane; electrolyte (taking 0.01M sodium chloride as an example) is added to the two chambers respectively to ensure that the surfaces on both sides of the nanoporous membrane are immersed in the solution, and electrodes (taking platinum wire electrodes as an example) are inserted into the two chambers respectively, and a picoammeter is used to apply a transmembrane step voltage difference of -2V to +2V on the two electrodes, with a step voltage of 0.2V and a step time of 1s. During the measurement of the transmembrane ion current, the voltage starts from -2V and ends at +2V. The measurement is performed every 3 minutes until the data is stable (usually 6-8 times, 7 times here). The changes in transmembrane current before and after polymer modification are shown in FIG. Figure 5 As shown, it can be clearly seen that after polymer modification, the change in the charge on the inner wall of the nanopore causes the current-voltage curve to flip.

[0057] (2) Using 0.01M sodium chloride solution as solvent, different concentrations (1×10 -12 M, 1×10 -11 M, 1×10 -10 M, 1×10 -9 M, 1×10 -8 M, 1×10 -7 M) of sialic acid solution (Neu5Ac). The lowest concentration (1×10 - 12 M) sialic acid solution, and measure the transmembrane ion current every 3 minutes until the data is stable. Then, change to a high concentration (1×10 -11 M, 1×10 -10 M, 1×10 -9 M, 1×10 -8 M, 1×10 -7 M) sialic acid solution. The current-voltage curve is as follows Figure 6 As shown in the figure, it is obvious that as the concentration of sialic acid increases, the effective pore size of the nanopore decreases, resulting in a decrease in current. Therefore, this characteristic of current change can be used to detect sialic acid at different concentrations.

[0058] Example 5

[0059] Using 0.01M sodium chloride solution as solvent, different concentrations (1×10 -12 M, 1×10 -11 M, 1×10 - 10 M, 1×10 -9 M, 1×10-8 M, 1×10 -7 For each sialic acid molecule, a new smart polymer-modified nanoporous membrane was placed inside an electrochemical cell. The nanoporous membrane separated the interior of the electrochemical cell into two separate chambers. Sialic acid solutions were added sequentially from low to high concentrations. The transmembrane ion current was measured every 3 minutes at each concentration until the data stabilized. The current changes at different concentrations and a voltage of +2V were plotted, as shown in Figure 2. Figure 7 As shown, it can be seen that the reduction in nanopore ion current caused by different sialic acid molecules is significantly different. By utilizing this different current change characteristic, the smart polymer-modified nanopore can realize the recognition and detection of different sialic acid molecules.

[0060] Example 6

[0061] Using 0.01M sodium chloride solution as solvent, different concentrations (1×10 -12 M, 1×10 -11 M, 1×10 -10 M, 1×10 -9 M, 1×10 -8 M, 1×10 -7 M) different monosaccharide solutions (fucose, glucose, galactose) and Neu5Ac solutions. For each monosaccharide molecule and Neu5Ac molecule, a new smart polymer-modified nanoporous membrane was sandwiched between the two chambers of the electrochemical cell. Monosaccharide solutions and Neu5Ac solutions were added sequentially from low to high concentrations. At each concentration, the transmembrane ion current was measured every 3 minutes until the data stabilized. The current changes at different concentrations and a voltage of +2V were plotted, as shown in Figure 2. Figure 8 As shown, it can be seen that monosaccharide molecules basically do not cause changes in transmembrane ion current, while the changes caused by Neu5Ac molecules are very significant. By utilizing this different current change characteristic, the polymer-modified nanopore can realize the distinction and detection of sialic acid molecules and monosaccharide molecules.

[0062] Example 7

[0063] Using 0.01M sodium chloride solution as solvent, different concentrations (1×10 -12 M, 1×10 -11 M, 1×10 -10 M, 1×10 -9 M, 1×10 -8 M, 1×10 -7M) different weak acid solutions (tartaric acid, gluconic acid, ascorbic acid) and Neu5Ac solutions. For each weak acid molecule and Neu5Ac molecule, a new smart polymer-modified nanoporous membrane was placed inside an electrochemical cell. The nanoporous membrane separated the interior of the electrochemical cell into two separate chambers. Weak acid solutions and Neu5Ac solutions were added sequentially from low to high concentrations. At each concentration, the transmembrane ion current was measured every 3 minutes until the data stabilized. The current changes at different concentrations and a voltage of +2V were plotted, as shown in Figure 2. Figure 9 As shown, it can be seen that weak acid molecules basically do not cause changes in transmembrane ion current, while Neu5Ac molecules cause very significant changes. Using this different current change characteristic, it can be proved that the polymer-modified nanopore is specific for recognizing and detecting sialic acid molecules, and it is not weak acidity that causes the change in current value.

[0064] Example 8

[0065] Using 0.01M sodium chloride solution as solvent, different concentrations (1×10 -12 M, 1×10 -11 M, 1×10 -10 M, 1×10 -9 M, 1×10 -8 M, 1×10 -7 M) Neu5Ac solution. The polymer without grafted recognition unit was modified onto the nanoporous membrane in the same way. Then the nanoporous membrane modified with smart polymer, the unmodified nanoporous membrane, and the nanoporous membrane modified with polymer without grafted recognition unit were sandwiched in the middle of the homemade electrochemical cell, and Neu5Ac solution from low to high concentration was added in sequence. At each concentration, the transmembrane ion current was measured every 3 minutes until the data stabilized. The current changes at different concentrations and +2V voltage were plotted, as shown in Figure 2. Figure 10 As shown, it can be seen that the transmembrane ion current of the unmodified nanopore membrane and the polymer-modified nanopore membrane without grafted recognition units has basically no change, while the change of the nanopore membrane modified with the smart polymer is very significant. Using this different current change characteristic, the important role of the recognition unit in the smart polymer can be demonstrated.

[0066] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. Application of a functionalized nanoporous membrane in electrochemical detection and identification of sialic acid molecules, characterized in that: The nanoporous membrane is an organic membrane; the nanoporous membrane is track-etched on the organic membrane, and the pore density is 1-1.0×10 8 Pieces / cm 2 ; The pore size range is 10-1000nm; The specific steps of the preparation method of the functionalized nanoporous membrane are as follows: (1) Dissolve 0.757 g of acetylated arginine, 0.7-0.9 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 0.5-0.6 g of N-hydroxysuccinimide (NHS) in 5-30 ml of 0.01-1 M 2-morpholineethanesulfonic acid (MES) buffer solution, pH = 4-6, and stir at room temperature for 2-3 hours; Dissolve 0.5-2.0g polyethyleneimine (PEI) in MES solution, then add the above solution, stir the mixed solution at room temperature for 8-36 hours, put the solution into a dialysis bag after the reaction, dialyze with water for 2-5 days, and obtain the desired smart polymer after freeze-drying; (2) Activation and modification of nanopores: Place the nanoporous membrane in an electrochemical cell, and separate the interior of the electrochemical cell into two mutually spaced chambers through the nanoporous membrane. Add an aqueous solution containing 5-30 mg / mol EDC and 2-15 mg / mol NHS to the two chambers, ensuring that both sides of the nanoporous membrane are immersed in the solution, and activate at room temperature for 0.5-3 hours; then remove the solution and wash with water, add 1-20 mg / ml smart polymer solution, ensuring that both sides of the nanoporous membrane are immersed in the solution, and react at room temperature for 6-12 hours to obtain a smart polymer modified nanoporous membrane; The organic film is a polyethylene terephthalate film, The application is not suitable for diagnosis and treatment of diseases.

2. The use of the functionalized nanoporous membrane according to claim 1 in electrochemical detection and identification of sialic acid molecules, characterized in that: The functionalized nanoporous membrane comprises a nanoporous membrane and a smart polymer grafted to the inner surface of the pores of the nanoporous membrane. The molecular structure of the smart polymer is schematically shown as follows: ， The grafting amount is 1-99% based on the number of primary amino groups; n is 10-100.

3. The use of the functionalized nanoporous membrane according to claim 1 in electrochemical detection and identification of sialic acid molecules, characterized in that: The shape of the nanopore is one or more of a cylindrical shape, an asymmetric cone shape, a cigar shape, a funnel shape, an hourglass shape, and a dumbbell shape.

4. The use of the functionalized nanoporous membrane according to claim 1 in electrochemical detection and identification of sialic acid molecules, characterized in that: Track etching of asymmetric conical pores in organic membranes with a pore density of 1-1.0×10 8 Pieces / cm 2 The pore size on one side of the membrane is 500-1000nm, and the pore size on the other side of the membrane is 10-50nm. The pore ports on one side of the membrane are larger than those on the other side of the membrane.

5. The use of the functionalized nanoporous membrane according to claim 1 in electrochemical detection and identification of sialic acid molecules, characterized in that: The functionalized nanoporous membrane is used for the detection of sialic acid molecules. The specific steps of realizing the detection of different sialic acid molecules by electrochemical detection method are as follows: Step 1, clamp the nanoporous membrane modified with the smart polymer in the electrochemical cell, divide the inside of the electrochemical cell into two mutually spaced chambers through the nanoporous membrane, add electrolyte to both sides of the membrane to ensure that the surfaces on both sides of the nanopores are immersed in the solution, insert electrodes into the two chambers of the electrochemical cell, apply a transmembrane step voltage difference of -2V~+2V to the two electrodes on both sides of the membrane, the step voltage is 0.05~0.4V, and the step time is 0.1~4s; during the test, the voltage starts from -2V and ends at +2V, and the transmembrane ion current is measured every 1-5 minutes with a picoammeter, and the data is recorded after running 6-10 times; Step 2, remove the electrolyte added in step 1 by suction, and add the electrolyte of the substance to be detected respectively, ensuring that the surfaces on both sides of the nanopore are immersed in the solution; connect electrodes to the two chambers of the electrochemical cell, apply a transmembrane step voltage difference of -2V~+2V on the two electrodes, the step voltage is 0.05~0.4V, and the step time is 0.1~4s. During the test, the voltage starts from -2V and ends at +2V. Use a picoammeter to measure the transmembrane current every 1-5 minutes. After running 6-10 times, record the data; The nanoporous membrane has different degrees of current changes for different sialic acids, thereby achieving the distinction between different sialic acids.

6. The use of the functionalized nanoporous membrane according to claim 5 in electrochemical detection and identification of sialic acid molecules, characterized in that: The electrolyte is a solution of one or more of sodium chloride, potassium chloride, calcium chloride or magnesium chloride at a concentration of 0.001-1.0 mol / L, the pH of the electrolyte is 4-11, and the solvent is deionized water, ultrapure water or distilled water.

7. The use of the functionalized nanoporous membrane according to claim 5 in electrochemical detection and identification of sialic acid molecules, characterized in that: The electrode is one or more of a platinum wire electrode, a glassy carbon electrode, a graphite electrode, a stainless steel electrode, a silver-silver chloride electrode or a mercury-mercury chloride electrode.

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