Directional extraction and purification method of giant spiny frog skin bradykinin

Abstract

The invention relates to the technical field of biological separation, and discloses a directional extraction and purification method of giant spiny frog skin bradykinin, which comprises the following steps: S1, preparing an intelligent integrated molecularly imprinted hydrogel membrane; s2, enabling the intelligent integrated molecular imprinting hydrogel film to be in contact with skin tissues of giant spiny frogs; s3, applying specific external stimulation to the intelligent integrated molecularly imprinted hydrogel film; and S4, collecting the giant spiny frog skin bradykinin dissociated from the intelligent integrated molecularly imprinted hydrogel film. By means of the antibody-level recognition capacity of molecular imprinting sites, high-specificity capture of the giant spiny frog skin bradykinin is achieved, co-extraction of non-target molecules is effectively avoided, active driving force is provided for the main target peptide through application of an external electric field, release of the main target peptide from tissue and migration of the main target peptide into a hydrogel film are accelerated, and the hydrogel film has a good application prospect. The efficiency and pertinence of the extraction process are greatly improved from the source, and a high-purity foundation is laid for subsequent purification work.

Description

Technical Field

[0001] This invention relates to the field of bioseparation technology, specifically a method for the targeted extraction and purification of bradykinin from the skin of the spiny-breasted frog. Background Technology

[0002] Natural bioactive peptides have broad application prospects in the biomedical field due to their unique biological functions and pharmacological activities. Obtaining these bioactive peptides from natural biological resources is fundamental to their research and application. Currently, the extraction and purification of bioactive peptides from biological tissues typically relies on physicochemical methods.

[0003] In existing technologies, common methods for extracting bioactive peptides from biological tissues include tissue homogenization or enzymatic hydrolysis, followed by extraction using organic solvents or acidic aqueous solutions. The resulting crude extract then undergoes a series of separation and purification steps, including but not limited to solid-phase extraction, membrane filtration, and multi-step tandem high-performance liquid chromatography (HPLC), such as ion-exchange chromatography, size exclusion chromatography, and reversed-phase HPLC, to progressively separate and enrich the target bioactive peptides.

[0004] However, existing technologies have significant drawbacks in efficiently extracting trace amounts of target bioactive peptides from complex biological matrices. These methods typically suffer from low extraction selectivity, resulting in the initial extract containing a large number of non-target proteins, peptides, and other biomacromolecules. To separate the target bioactive peptide from this highly complex mixture, a lengthy and time-consuming multi-step chromatographic purification process must be employed. This not only increases the complexity of the operation, reagent consumption, and operating costs, but more importantly, the target bioactive peptide may inevitably suffer loss in each purification step, leading to a low overall yield of the final product. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a method for the targeted extraction and purification of bradykinin from the skin of the spiny-breasted frog, solving the problems of insufficient selectivity and lengthy processes in the extraction and purification of natural active peptides.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for the targeted extraction and purification of bradykinin from the skin of the spiny-breasted frog, comprising the following steps: S1. Prepare an intelligent integrated molecularly imprinted hydrogel membrane, wherein the polymer network of the intelligent integrated molecularly imprinted hydrogel membrane contains molecularly imprinted sites that match the bradykinin molecules of the spiny-breasted frog skin, and the polymer network is also covalently grafted with competing molecules through cleavable chemical bonds. S2. The intelligent integrated molecularly imprinted hydrogel membrane is brought into contact with the skin tissue of the spiny-breasted frog, and an external physical field is applied to induce the spiny-breasted frog skin bradykinin to migrate from the skin tissue and be selectively captured by the molecularly imprinted sites. S3. Apply a specific external stimulus to the intelligent integrated molecularly imprinted hydrogel membrane to induce the breaking of the breakable chemical bonds, release the competitive molecule in situ inside the intelligent integrated molecularly imprinted hydrogel membrane, and dissociate the captured bradykinin of the spiny-breasted frog skin through competitive displacement. S4. Collect the bradykinin from the spiny-breasted frog skin, which is dissociated from the intelligent integrated molecularly imprinted hydrogel membrane.

[0007] Preferably, the intelligent integrated molecularly imprinted hydrogel membrane is polymerized from a prepolymer solution, the prepolymer solution comprising bradykinin from the skin of the spiny-breasted frog as a template molecule, a host functional monomer for forming the molecularly imprinted sites, an electroresponsive monomer that imparts charge properties to the polymer network, a covalently grafted competitive elution monomer containing cleavable bonds, which is composed of polymerizable groups, cleavable chemical bonds, and the competing molecules, a crosslinking agent, and an initiator.

[0008] Preferably, the external physical field in step S2 is a direct current electric field; the direct current electric field is applied by placing the spiny-breasted frog skin tissue and the intelligent integrated molecularly imprinted hydrogel membrane between the anode and the cathode, so as to drive the spiny-breasted frog skin bradykinin to migrate directionally to the intelligent integrated molecularly imprinted hydrogel membrane by electrophoresis.

[0009] Preferably, the specific external stimulus in step S3 is light irradiation of a specific wavelength; the breakable chemical bond is a photosensitive breakable chemical bond.

[0010] Preferably, the photosensitive breakable chemical bond comprises an ortho-nitrobenzyl structure.

[0011] Preferably, the competing molecule is Nα-benzoyl-L-arginine ethyl ester.

[0012] Preferably, the electroresponsive monomer is 2-acrylamido-2-methylpropanesulfonic acid.

[0013] Preferably, the specific external stimulus in step S3 is light irradiation of a specific wavelength, and the breakable chemical bond is a photosensitive breakable chemical bond.

[0014] Preferably, step S1 further includes, after the polymerization reaction, removing the spiny-breasted frog skin bradykinin, which serves as a template molecule, by solvent elution to form the molecular imprint site.

[0015] Preferably, the main functional monomer comprises methacrylic acid providing a carboxyl group and N-isopropylacrylamide providing a hydrophobic interaction, for non-covalent interaction with the spiny-breasted frog skin bradykinin, which serves as the template molecule, through ionic bonds and hydrophobic interactions.

[0016] This invention provides a method for the targeted extraction and purification of bradykinin from the skin of the spiny-breasted frog. It has the following beneficial effects: 1. This invention achieves highly specific capture of bradykinin from the skin of the spiny-breasted frog by utilizing the antibody-level recognition capability of molecularly imprinted sites, effectively avoiding the co-extraction of non-target molecules. Furthermore, the application of an external electric field provides an active driving force for the main target peptide, accelerating its release from the tissue and migration into the hydrogel membrane. This significantly improves the efficiency and targeting of the extraction process from the source, laying a high-purity foundation for subsequent purification work.

[0017] 2. This invention combines extraction and highly selective enrichment by directly applying molecular imprinting to the surface of intact biological tissues. Furthermore, it operates under mild conditions, avoiding the potential damage to the bradykinin structure and bioactivity of spiny-breasted frog skin caused by high temperatures, strong acids and alkalis, or violent mechanical shearing, thus helping to maintain its natural activity.

[0018] 3. This invention achieves the in-situ, high-concentration instantaneous release of these competitive molecules inside the membrane through external stimulation. This self-excited competitive displacement enables bradykinin from the skin of the spiny-breasted frog to be efficiently and rapidly dissociated from the imprinted site and diffused into a very small amount of the collection solution, which significantly improves the elution efficiency, reduces solvent consumption, and further increases the concentration of the product in the collection solution. Attached Figure Description

[0019] Figure 1 This is a flowchart of the present invention. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Please see the appendix Figure 1 This invention provides a method for the targeted extraction and purification of bradykinin from the skin of the spiny-breasted frog, specifically including the following steps: S1. Preparation of intelligent integrated molecularly imprinted hydrogel membrane. The polymer network of the intelligent integrated molecularly imprinted hydrogel membrane contains molecularly imprinted sites that match the bradykinin molecules of the spiny-breasted frog skin, and the polymer network also contains competing molecules covalently grafted through cleavable chemical bonds. This step aims to prepare the core functional material for subsequent extraction and purification operations. The membrane is characterized by the pre-constructed molecular imprinting sites in its polymer network structure, which have the ability to specifically recognize the target substance, bradykinin from the skin of the spiny-breasted frog, and simultaneously covalently immobilizes the competing molecules in the unexcited state.

[0022] In one specific embodiment, the preparation process includes the following steps: First, a prepolymer solution is prepared by dissolving bradykinin from the spiny-breasted frog skin as the template molecule, the main functional monomer, the electroresponsive monomer, the covalently grafted competitive eluting monomer containing a cleavable bond, the crosslinking agent, and the photoinitiator in a specific solvent system.

[0023] In a preferred embodiment, in the prepolymer solution, the molar ratio of the spiny-breasted frog skin bradykinin (as template molecule), the host functional monomer, the electroresponsive monomer, the covalently grafted competitive eluting monomer containing cleavable bonds, and the crosslinking agent ranges from 1:(4-12):(1-5):(0.5-3):(10-30). The solvent system is a mixture of dimethyl sulfoxide and water in a volume ratio of 7:3 to 9:1.

[0024] The bradykinin from the skin of the spiny-breasted frog, serving as a template molecule, provides the basis for the three-dimensional structure and chemical information for the subsequent formation of molecular imprinting sites.

[0025] The selection of the main functional monomer is based on the fact that its functional group can undergo reversible non-covalent interaction with the functional group of the template molecule.

[0026] In this embodiment, the main functional monomer is a combination of methacrylic acid and N-isopropylacrylamide. The carboxyl group of methacrylic acid can form ionic bonds with basic groups such as arginine residues in bradykinin from the skin of the spiny-breasted frog under certain pH conditions. N-isopropylacrylamide provides hydrophobic segments that can interact with hydrophobic amino acid residues in bradykinin from the skin of the spiny-breasted frog. This recognition site, which is formed through multiple non-covalent interactions, has higher specificity and affinity.

[0027] The role of electroresponsive monomers is to impart fixed charge properties to the entire hydrogel membrane, thereby generating electroosmotic or electrophoretic enrichment effects in subsequent electric field-assisted extraction steps.

[0028] In this embodiment, 2-acrylamido-2-methylpropanesulfonic acid was selected. Its sulfonic acid group is a strong acid group, which makes the polymer network stably carry a negative charge over a wide pH range.

[0029] Covalently grafted competitive eluting monomers containing cleavable bonds are key components for achieving in-situ self-eluting. These monomers consist of three parts: a group that can participate in the polymerization reaction, a chemical bond that can be broken by external stimuli, and a competitive molecule that can bind to the molecular imprinting site.

[0030] In this embodiment, the cleavable chemical bond is a photosensitive cleavable chemical bond, specifically adopting a structure containing an ortho-nitrobenzyl group. The competitive molecule is Nα-benzoyl-L-arginine ethyl ester. This molecular structure can effectively mimic the key binding group of bradykinin in the skin of the spiny-breasted frog, thereby competing with the target molecule for binding molecular imprinting sites in subsequent steps.

[0031] N,N'-methylenebisacrylamide was selected as the crosslinking agent to construct the three-dimensional network framework of the hydrogel. A photoinitiator was selected as the initiator to initiate free radical polymerization under ultraviolet light irradiation.

[0032] The resulting intelligent integrated molecularly imprinted hydrogel membrane has an internal structure of a three-dimensional polymer network. Within this network, three-dimensional vacancies, i.e., molecularly imprinted sites, are distributed through a pre-polymerization template method. Simultaneously, competing molecules are connected to the polymer network segments via photosensitive breakable chemical bonds.

[0033] Then, polymerization and film formation are carried out. The prepared prepolymer solution is poured into a mold and irradiated under ultraviolet light of a specific wavelength to initiate the polymerization reaction. After the reaction is completed, a hydrogel film with a specific thickness and flexibility is formed. The ultraviolet polymerization is carried out under the conditions of a wavelength of 365nm and a light intensity of 10-25mW / cm², and the irradiation time is 15-45 minutes.

[0034] Finally, template molecules are eluted by placing the polymerized hydrogel membrane in an acidic organic solvent for thorough washing to disrupt the non-covalent interactions between the functional monomers and the template molecules, thus completely eluting the template molecules from the polymer network. After this step, a large number of vacant three-dimensional molecular imprint sites remain in the polymer network, which are highly matched with the bradykinin of the spiny-breasted frog skin in terms of spatial configuration and interaction sites. Meanwhile, the covalently grafted competitive eluting monomers containing cleavable bonds remain in the membrane due to covalent bonding. After elution, the membrane is washed with ultrapure water until neutral and set aside. The acidic organic solvent is a mixed solution of methanol and acetic acid with a volume ratio of 9:1. The elution process lasts for 24 hours, during which the eluent is replaced with fresh eluent every 4 hours.

[0035] S2. The intelligent integrated molecularly imprinted hydrogel membrane is brought into contact with the skin tissue of the spiny-breasted frog, and an external physical field is applied to induce the bradykinin of the spiny-breasted frog skin to migrate from the skin tissue and be selectively captured by the molecularly imprinted sites. Before performing this step, the spiny-breasted frog skin tissue needs to be pretreated. Take fresh or frozen and thawed spiny-breasted frog skin, wash it with physiological saline to remove surface impurities, and use the inner surface of the entire skin tissue, i.e. the dermal layer side, as the contact surface with the intelligent integrated molecularly imprinted hydrogel membrane to ensure good electrical contact and mass transfer efficiency. Before contact, the surface is wetted with the buffer solution used in the operation.

[0036] This step combines the active driving force of the electric field with the passive selective capture of molecular imprinted sites to achieve efficient and targeted enrichment of target peptides in the skin tissue of the spiny-breasted frog.

[0037] The directional extraction device is constructed as an electrochemical cell structure. During operation, the intelligent integrated molecularly imprinted hydrogel membrane is directly applied to the surface of the skin tissue of the spiny-breasted frog, and the cathode and anode are respectively set on the outside of the membrane and the skin tissue to form a complete electrical circuit.

[0038] A DC electric field is applied to both ends of the device. In a preferred embodiment, the electric field strength ranges from 0.5 to 5 V / cm. The entire extraction process lasts for 10 to 60 minutes. The buffer solution used is a phosphate buffer with a pH of 7.0 to 8.0 and an ionic strength of 10 to 50 mM. Because the bradykinin molecules of the spiny-breasted frog skin carry a net positive charge in the set buffer pH environment, the electric field force drives them to actively detach from the skin tissue matrix and migrate electrophoretically towards the cathode. When they migrate into the intelligent integrated device with a fixed negative charge... When the target peptide is absorbed into the molecularly imprinted hydrogel membrane, it is first electrostatically enriched by the negatively charged environment inside the membrane. Subsequently, it encounters a large number of molecularly imprinted sites within the membrane. Due to the high degree of matching between the spatial configuration and the chemical action sites, bradykinin molecules from the spiny-breasted frog skin are preferentially and firmly captured by these molecularly imprinted sites. Other impurity molecules in the tissue that are mismatched in size, shape, or charge properties have difficulty entering the imprinted sites, are directly repelled by the electric field, or pass through the membrane without being effectively enriched. Through the synergistic effect of electrophoretic migration and specific molecular recognition, selective capture of the target peptide is achieved.

[0039] S3. Apply specific external stimulation to the intelligent integrated molecularly imprinted hydrogel membrane to induce the breaking of breakable chemical bonds, release competitive molecules in situ inside the intelligent integrated molecularly imprinted hydrogel membrane, and dissociate the captured bradykinin of the spiny-breasted frog skin through competitive displacement. This step aims to efficiently dissociate the captured target peptide from the membrane by applying external stimulation.

[0040] After the extraction process is completed, the intelligent integrated molecularly imprinted hydrogel membrane enriched with bradykinin from the skin of the spiny-breasted frog is taken out and irradiated with light of a specific wavelength. In this embodiment, it is ultraviolet light with a wavelength of 365nm. The light intensity of the irradiation is 20-50mW / cm², and the irradiation time is 5-20 minutes.

[0041] At the microscopic level, light energy triggers a photolysis reaction of photosensitive, cleavable chemical bonds covalently grafted onto the polymer network. Specifically, the o-nitrobenzyl structure absorbs photons, rearranges, and breaks, causing the competing molecules attached to it to detach from the polymer network and become free molecules. Since all competing molecules are released simultaneously throughout the entire membrane volume, their local concentration around the molecular imprinting site instantaneously reaches an extremely high level. These high-concentration free competing molecules immediately engage in a fierce competitive substitution with the captured spiny-breasted frog skin bradykinin molecules, using their numerical advantage to displace the spiny-breasted frog skin bradykinin molecules from the imprinting site and restore them to a free state.

[0042] S4. Collect bradykinin from the skin of the spiny-breasted frog, which is dissociated from the intelligent integrated molecularly imprinted hydrogel membrane; The displaced bradykinin molecules from the spiny-breasted frog skin diffuse out of the intelligent integrated molecularly imprinted hydrogel membrane due to the concentration gradient. By placing the membrane in a very small amount of buffer solution, in a preferred embodiment, the volume ratio of the very small amount of buffer solution to the volume of the intelligent integrated molecularly imprinted hydrogel membrane is 0.5:1 to 2:1, a high-concentration, preliminarily purified bradykinin solution from the spiny-breasted frog skin can be collected. This solution can be directly used for subsequent activity testing or further fine purification.

[0043] This invention constructs a complete integrated method from in-situ extraction of complex biological samples to collection of high-purity target products by organically combining the above steps. This method not only significantly simplifies the traditional multi-step separation and purification process, but also ensures the structural integrity and biological activity of the target bioactive molecules through mild electrochemical and photochemical conditions.

[0044] To verify the beneficial effects of the present invention, the following examples and comparative examples are provided.

[0045] Example 1: Targeted extraction and purification method of the present invention Accurately weigh 5.0 grams of pretreated fresh spiny-breasted frog skin tissue and proceed with the operation according to the method described in detail in the specific embodiments of the present invention above: The smart integrated molecularly imprinted hydrogel membrane was prepared using the preferred ratio (molar ratio 1:8:2:1:20).

[0046] Extraction was performed for 30 minutes under a DC electric field with an electric field strength of 3 V / cm.

[0047] Irradiation with 365nm ultraviolet light at an intensity of 30 mW / cm² for 10 minutes triggered the release of competitive molecules.

[0048] Collection was performed using PBS buffer (pH 7.4) at a volume equal to one membrane volume.

[0049] The collected solution was filtered through a 0.22 μm filter membrane, and the extracted products were qualitatively and quantitatively analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0050] Comparative Example 1: Using the traditional tissue homogenization-chromatographic separation method Accurately weigh 5.0 grams of skin tissue from the same batch of spiny-breasted frogs and process it according to the conventional methods described in the background art: Skin tissue was mixed with pre-cooled 0.1 M acetic acid solution at a ratio of 1:5 (w / v) and mechanically homogenized in an ice-water bath.

[0051] The homogenate was centrifuged at 4°C and 12,000 rpm for 20 minutes, and the supernatant was collected as the crude extract.

[0052] The crude extract was successively desalted by solid-phase extraction column and preliminarily separated by cation exchange chromatography.

[0053] Finally, preparative reversed-phase high-performance liquid chromatography was used to collect the target peak for subsequent analysis.

[0054] Comparison and Analysis of Results The final products obtained in Example 1 and Comparative Example 1 were tested respectively, and the key performance indicators are compared in Table 1: Table 1: Comparison of the effects of the method of the present invention and the traditional method on the extraction and purification of bradykinin from the skin of the spiny-breasted frog. As shown in Table 1, compared with the traditional tissue homogenization-multi-step chromatographic separation method (Comparative Example 1), the directional extraction and purification method provided by the present invention (Example 1) exhibits significant advantages: Extremely efficient: This invention combines extraction and preliminary purification into one process, significantly reducing the total operation time from over 24 hours to approximately 1.5 hours.

[0055] Significantly improved recovery rate: By avoiding sample loss in multi-step processing, the recovery rate of the target peptide increased from 58.3% to 92.5%, which is crucial for obtaining rare natural bioactive peptides.

[0056] Higher purity: Thanks to the highly specific capture of molecular imprinted sites, the purity of the final product reaches 95.8%, which is better than the 90.1% of the traditional method.

[0057] Green and environmentally friendly: This invention significantly reduces the amount of organic solvents used, which is more in line with the requirements of green chemistry.

[0058] Simplified process: This invention does not require a complex multi-step chromatography system, is easy to operate, and is easy to scale up.

[0059] As shown in Table 1, compared with the traditional tissue homogenization-multi-step chromatographic separation method (Comparative Example 1), the directional extraction and purification method provided by the present invention (Example 1) exhibits significant advantages: Extremely efficient: This invention combines extraction and preliminary purification into one process, significantly reducing the total operation time from over 24 hours to approximately 1.5 hours.

[0060] Significantly improved recovery rate: By avoiding sample loss in multi-step processing, the recovery rate of the target peptide increased from 58.3% to 92.5%, which is crucial for obtaining rare natural bioactive peptides.

[0061] Higher purity: Thanks to the highly specific capture of molecular imprinted sites, the purity of the final product reaches 95.8%, which is better than the 90.1% of the traditional method.

[0062] Green and environmentally friendly: This invention significantly reduces the amount of organic solvents used, which is more in line with the requirements of green chemistry.

[0063] Simplified process: This invention does not require a complex multi-step chromatography system, is easy to operate, and is easy to scale up.

[0064] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for the directional extraction and purification of bradykinin from the skin of Rana temporaria, characterized in that, The method comprises the following steps: S1, preparing an intelligent integrated molecularly imprinted hydrogel film, wherein molecular imprinting sites matching with the skin bradykinin of the cane toad are contained in a polymer network of the intelligent integrated molecularly imprinted hydrogel film, and a competitive molecule is covalently grafted in the polymer network through a breakable chemical bond; S2, contacting the intelligent integrated molecularly imprinted hydrogel film with the skin tissue of the cane toad, and applying an external physical field to promote the skin bradykinin of the cane toad to migrate from the skin tissue of the cane toad and be selectively captured by the molecular imprinting sites; S3, applying a specific external stimulus to the intelligent integrated molecularly imprinted hydrogel film to induce the breakage of the breakable chemical bond, release the competitive molecule in situ in the intelligent integrated molecularly imprinted hydrogel film, and dissociate the captured skin bradykinin of the cane toad through competitive displacement; S4, collecting the skin bradykinin of the cane toad dissociated from the intelligent integrated molecularly imprinted hydrogel film.

2. The method according to claim 1, wherein the method is characterized by, The intelligent integrated molecularly imprinted hydrogel film is polymerized from a prepolymer solution containing the skin bradykinin of the cane toad as a template molecule, a host functional monomer for forming the molecular imprinting sites, an electroresponsive monomer for imparting charge characteristics to the polymer network, a covalently grafted competitive elution monomer containing a breakable bond composed of a polymerizable group, the breakable chemical bond and the competitive molecule, a crosslinking agent and an initiator.

3. The method according to claim 1, wherein the method is characterized by, The external physical field in the step S2 is a direct current electric field, which is applied by placing the skin tissue of the cane toad and the intelligent integrated molecularly imprinted hydrogel film between an anode and a cathode to drive the skin bradykinin of the cane toad to directionally migrate to the intelligent integrated molecularly imprinted hydrogel film through electrophoresis.

4. The method according to claim 1, wherein the method is characterized by, The specific external stimulus in the step S3 is light irradiation of a specific wavelength, and the breakable chemical bond is a photosensitive breakable chemical bond.

5. The method according to claim 4, wherein the skin of Pachina maculata is used as the raw material. The photosensitive breakable chemical bond contains an o-nitrobenzyl structure.

6. The method according to claim 1, wherein the method is characterized by, The competitive molecule is Nα-benzoyl-L-arginine ethyl ester.

7. The method according to claim 2, wherein the method is characterized by, The electroresponsive monomer is 2-acrylamido-2-methylpropanesulfonic acid.

8. The method according to claim 3, wherein the method is characterized by, The specific external stimulus in the step S3 is light irradiation of a specific wavelength, and the breakable chemical bond is a photosensitive breakable chemical bond.

9. The method according to claim 1, wherein the method is characterized by, The step S1 further comprises a step of removing the skin bradykinin of the cane toad as the template molecule by solvent elution after the polymerization reaction to form the molecular imprinting sites.

10. The method according to claim 2, wherein the method is characterized by, The host functional monomer contains methacrylic acid for providing a carboxyl group and N-isopropyl acrylamide for providing a hydrophobic effect, so as to non-covalently interact with the skin bradykinin of the cane toad as the template molecule through ionic bonds and hydrophobic effects.

Images