Silk fibroin molecular imprinting preparation method, silk fibroin molecular imprint prepared by the method and application thereof

Abstract

This invention discloses a method for preparing silk fibroin molecular imprints, the prepared silk fibroin molecular imprints, and their applications, belonging to the field of molecular imprinting technology. The molecular imprints are prepared by the following method: silk fibroin is dissolved in formic acid and stirred until the solution is clear, obtaining a silk fibroin solution; a template molecule solution is added to the silk fibroin solution, and the mixture is stirred to react; after the reaction is complete, the mixture is eluted, centrifuged, the supernatant is removed, and the precipitate is retained to obtain the silk fibroin molecular imprints. This invention uses silk fibroin simultaneously as a functional monomer and a cross-linking agent, achieving the construction of novel molecular imprints in a green and harmless manner. It fundamentally solves the problems of poor biocompatibility, potential toxicity, and limited in vivo application of traditional MIPs due to the use of organic chemical reagents. Its application prospects are broad, and it has significant application value in fields such as biomedical detection, drug delivery, and tissue engineering.

Description

Technical Field

[0001] This invention belongs to the field of molecular imprinting technology, specifically relating to a method for preparing silk fibroin molecular imprints, the prepared silk fibroin molecular imprints, and their applications. Background Technology

[0002] Molecular imprinting technology utilizes molecularly imprinted polymers (MIPs) to mimic the interactions between enzymes and substrates or antibodies and antigens, enabling the specific recognition of template molecules. Its core lies in the preparation of molecularly imprinted polymers, which mainly involves mixing template molecules with suitable functional monomers (usually small molecule compounds) and cross-linking agents to induce their interaction and polymerize them. The template molecules are then removed, resulting in the molecularly imprinted polymer.

[0003] Current methods for preparing molecular imprints mainly include surface imprinting and traditional bulk polymerization. The components that constitute the imprint cavity are mostly chemical reagents. During preparation, an initiator (such as azobisisobutyronitrile, AIBN) is often needed to initiate a crosslinking reaction between functional monomers (such as MAA methacrylate, AM acrylamide, etc.) and crosslinking agents (such as ethylene glycol dimethacrylate, EDMA, TRIM, etc.). These monomers interact with the template molecules to form a prepolymer complex. Finally, the hydrogen bonds and other forces are broken by a highly destructive solvent such as acetic acid, allowing the template molecules to be removed and the final product obtained.

[0004] Current molecular imprinting has certain limitations; for example, its recognition efficiency in aqueous phase is relatively low; traditional functional monomers (such as MAA) mainly recognize through hydrophobic interactions or hydrogen bonds, which are weak in aqueous environments, resulting in poor selectivity for interfering substances; imprinted sites are buried too deeply; traditional bulk imprinted polymers tend to form thick layers, causing a large number of recognition sites to be buried deep inside the polymer, making it difficult for template molecules to access them, resulting in low binding capacity and slow kinetics; and it has poor biocompatibility, as molecular imprinting is mostly polymerized in organic solvents, which may lead to rejection reactions or sensitization when used in vivo or on the body surface.

[0005] Silk fibroin (SF), also known as silk protein, is a natural polymer material that has attracted widespread attention due to its excellent mechanical properties, good biocompatibility, and controllable degradation characteristics. Silk fibroin is also an ideal template and reducing agent for nanostructure growth, containing abundant functional groups (such as tyrosine and serine). Its rich amino acid residues can bind to template molecules through various interactions (hydrogen bonds, hydrophobic interactions, π-π stacking), thus providing a good structural basis for constructing highly selective molecularly imprinted sites. Using silk fibroin as a monomer for molecular imprinting is a green, biocompatible, and efficient imprinting method that can overcome the limitations of traditional molecularly imprinted (MIP) technology in biomedical and environmental applications. Therefore, providing a simple method for preparing silk fibroin-based molecularly imprinted sites is of great significance. Summary of the Invention

[0006] This invention provides a method for preparing silk fibroin molecular imprints, the steps of which are as follows: Silk fibroin was dissolved in formic acid and stirred until the solution became clear to obtain a silk fibroin solution. The template molecule solution was added to the silk fibroin solution and stirred to react. After the reaction was completed, the mixture was eluted, centrifuged, and the supernatant was removed while the precipitate was retained to obtain a silk fibroin molecular imprint.

[0007] In the above-mentioned method for preparing silk fibroin molecular imprints, the concentration of the silk fibroin solution is 10~50 mg / mL.

[0008] In the above method for preparing silk fibroin molecular imprints, the concentration of the template molecule solution is 0.5~3 M.

[0009] In the above-mentioned method for preparing silk fibroin molecular imprints, the template molecule is any substance that can enable silk fibroin to form a complementary cavity in the template molecule structure; for example, pesticide pollutants (acetamiprid, etc.), human health markers (cortisol, etc.), and organic dye environmental pollutants (bisphenol A, methylene blue, etc.).

[0010] In the above method for preparing silk fibroin molecular imprints, the stirring reaction conditions are: stirring at 200-600 rpm for 100-200 min at 50-70℃.

[0011] In the above method for preparing silk fibroin molecular imprints, the elution solution used is a methanol / acetic acid mixed solution; wherein the volume ratio of methanol to acetic acid is 9:1.

[0012] In the above-mentioned method for preparing silk fibroin molecular imprinting, the silk fibroin can be prepared by the following method: Silkworm cocoons are cut into thin slices, boiled in NaHCO3 solution to remove sericin, rinsed and dried to obtain degummed silk. The degummed silk is then dissolved in a ternary solution and reacted at 90℃ for 2-3 h. After dialysis, small molecule impurities and salts in the fibroin solution are removed. The dialyzed solution is then freeze-dried to obtain fibroin protein.

[0013] In the above method for preparing silk fibroin, the ternary solution is a mixed solution composed of calcium chloride, ethanol and water in a molar ratio of 1:2:8.

[0014] This invention provides a silk fibroin molecular imprint prepared by the above method.

[0015] Since the above-mentioned silk fibroin molecular imprinting can achieve the recognition and loading of template molecules, based on this, the present invention provides applications of the above-mentioned silk fibroin molecular imprinting, including at least one of the following: (1) Applications in separation and purification; for example, for enantiomer resolution of chiral drugs; (2) Applications in sensing and detection; for example, in constructing chemical sensors to achieve trace detection of environmental pollutants; (3) Applications in catalytic system design; for example, for designing molecularly imprinted catalysts with enzyme-mimicking catalytic activity; (4) Applications in drug delivery; for example, for developing targeted drug carriers to achieve precise drug release from lesion tissues.

[0016] The beneficial effects of this invention are as follows: This invention uses silk fibroin as both a functional monomer and a cross-linking agent (without requiring the addition of other chemical cross-linking agents), completely replacing chemically synthesized monomers and cross-linking agents such as methacrylic acid and ethylene glycol dimethacrylate in traditional molecularly imprinted polymers. It achieves the construction of novel molecular imprints in a green and harmless manner, fundamentally solving the problems of poor biocompatibility, potential toxicity, and limited in vivo application caused by the use of organic chemical reagents in traditional MIPs. Its application prospects are broad, and it has important application value in fields such as biomedical detection, drug delivery, and tissue engineering.

[0017] This invention breaks through the traditional imprinting paradigm that uses synthetic monomers and cross-linking agents. Through controlled depolymerization, it simultaneously provides functional monomers (short peptide chains) and cross-linking agents (long peptide chains). Utilizing the "self-linking effect" of silk fibroin, it completes compounding, immobilization, and cavity formation in one step, eliminating the need for external molecules and simplifying the traditional imprinting process. The short peptide chains are responsible for recognition, while the long peptide chains are responsible for three-dimensional network cross-linking. This natural division of labor gives the imprinted structure precise recognition sites. Furthermore, the synergistic effect of multiple forces (β-sheet, hydrogen bonding, carbonyl groups, and hydrophobic interactions) enables diverse and stable interactions with the template molecules, thereby improving the affinity and selectivity of the imprinted cavity.

[0018] Silk fibroin, derived from natural silk, is renewable, biodegradable, and biocompatible, making it suitable for applications in biomedicine, in vivo detection, and environmentally friendly sensors. It provides a novel natural protein-based solution for molecular imprinting technology, propelling the technology towards a greener, simpler, and more biocompatible direction. It is expected to expand the application scope of molecular imprinting, particularly in high-end fields such as flexible sensing, controlled drug release, bioseparation, and tissue engineering, achieving a balance between high performance and sustainability.

[0019] Using the method of this invention, a variety of substances can be used as template molecules to prepare novel molecular imprints, thereby opening up new functional material systems.

[0020] Silk fibroin molecular imprinting can specifically recognize template molecules, and the cavity formed can fix the target material in it through forces such as hydrogen bonds. Based on this characteristic and function, it can be applied to multiple fields.

[0021] In the fields of biomedicine and pharmacy, silk fibroin molecular imprinting can be used as a drug carrier. By loading drug molecules into the imprinted cavity, targeted recognition and controlled release at lesions can be achieved; furthermore, the excellent biocompatibility of silk fibroin ensures drug safety. Silk fibroin molecular imprinting can also be used as a loading layer to fabricate recognition elements for the detection of certain disease biomarkers, improving the selectivity and sensitivity of detection.

[0022] It can also be used as a means of identification for purification and separation. By imprinting silk fibroin molecules with solidification technology to form a separation layer, and by fixing it in a purification column, the target substance can be selectively separated from a complex mixture by utilizing the specific recognition function of the cavity.

[0023] In addition, the specific recognition capabilities of molecular imprinting can be applied to the detection of various targets, including environmental monitoring, to develop silk fibroin imprinted sensors or adsorbent materials targeting specific environmental pollutants (such as pesticide residues, antibiotics, and heavy metal ions) for the monitoring and treatment of environmental water bodies. It can also be applied to food safety testing, detecting trace amounts of harmful substances such as pesticide residues, veterinary drug residues, and biotoxins in food, thus ensuring food safety. Attached Figure Description

[0024] Figure 1 Electrochemical impedance spectroscopy for molecular imprinting of acetamiprid fibroin protein; where A represents molecular imprinting and B represents non-molecular imprinting.

[0025] Figure 2 The image shows a scanning electron microscope image of the molecular imprint of acetamiprid serine protein; where A represents the non-molecular imprint and B represents the molecular imprint.

[0026] Figure 3Affinity and adsorption specificity tests were performed on the molecular imprinted acetamiprid fibroin protein; where A represents the affinity test and B represents the specific adsorption.

[0027] Figure 4 To conduct enzyme activity experiments using molecularly imprinted gels containing acetamiprid nanofiber protein.

[0028] Figure 5 This study aims to verify the feasibility of cortisol-based silk fibroin molecular imprinting and methylene blue-based silk fibroin molecular imprinting; where A, B, and C represent cortisol molecular imprinting, and D, E, and F represent methylene blue molecular imprinting.

[0029] Figure 6 Selectivity testing for wearable pesticide sensors for plants.

[0030] Figure 7 The standard curve for acetamiprid is shown below. A represents the open-circuit voltage signal of the wearable plant pesticide sensor at different concentrations of acetamiprid, from top to bottom: 0 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL; B represents the relationship between the open-circuit voltage signal of the wearable plant pesticide sensor and the concentration of acetamiprid.

[0031] Figure 8 The actual sample test results for the wearable pesticide sensor for plants are shown; where A represents the open-circuit voltage test results for twelve consecutive days; and B represents the pesticide degradation simulation curve for twelve consecutive days.

[0032] Figure 9 These experiments validated the effects of different molecular weight peptide chains; A, B, and C were validation experiments on the treatment time of peptide chains with different formic acid concentrations; D, E, and F were validation experiments on the degree of peptide chain treatment with different concentrations of formic acid. Detailed Implementation

[0033] In this invention, silk fibroin can be obtained through commercial channels or by self-production.

[0034] This invention provides a method for preparing silk fibroin, comprising the following steps: Take 20 g of silkworm cocoons and cut them into 2 cm pieces. 2Thin slices of silkworm cocoons were boiled in 2 L of 1% NaHCO3 solution for 60 min to remove sericin. The cocoons were then rinsed with warm water, and this process was repeated 5 times. The cocoons were then dried at 70 °C to obtain degummed silk. 150 g of a ternary solution containing calcium chloride, ethanol, and deionized water (molar ratio 1:2:8) was taken, and 5 g of degummed silk was dissolved in the solution. The reaction was carried out at 90 °C for 3 h. Using traditional dialysis techniques, a dialysis bag (MWCO 3500) was used with 3000 mL of deionized water as the medium for 24 h of dialysis purification. Small molecule impurities and salts in the silk fibroin solution were removed. The dialyzed solution was then freeze-dried to obtain silk fibroin protein.

[0035] In this invention, silk fibroin itself has excellent biocompatibility, degradability and good mechanical properties, making the prepared molecular imprint more suitable for the biomedical field (such as drug delivery, in vivo sensing).

[0036] Other materials used in this invention, unless otherwise stated, are commercially available. Other terms used in this invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and not intended to limit the scope of the invention in any way. Example 1

[0037] The steps for preparing acetamiprid serine protein molecular imprints are as follows: 200 mg of silk fibroin was dissolved in 10 mL of formic acid and stirred at 60 °C for 2 h until the solution became clear, yielding a silk fibroin solution (20 mg / mL). 1 mL of acetamiprid solution (1 M) was added to 5 mL of the silk fibroin solution, and the mixture was stirred at 400 rpm for 120 min at 60 °C to allow for sufficient covalent bonding between the silk fibroin and the template molecules. After the reaction, the resulting MIP was placed in a methanol:acetic acid (9:1 v / v) solution and centrifuged at 8000 rpm for 5 min to remove the template molecules. The supernatant was removed, and the precipitate was retained to obtain the acetamiprid-imprinted silk fibroin molecular imprint.

[0038] I. Electrochemical Impedance Test A non-molecularly imprinted (NIP) sample without the addition of a template molecule (acetamiprid) was prepared using the same steps as in Example 1 above as a control experiment. The molecularly imprinted and non-molecularly imprinted samples were verified using electrochemical impedance spectroscopy.

[0039] The molecularly imprinted acetamipridin protein (MIP gel) was subjected to electrochemical impedance spectroscopy using an electrochemical workstation. Cyclic voltammetry was performed in the three-electrode system of the electrochemical workstation, using Pt wire as the counter electrode, Ag / AgCl as the reference electrode, and the MIP gel as the working electrode in a 500 mmol / L iron standard solution.

[0040] The test results are as follows Figure 1 As shown: As shown in Figure A, the impedance value of the standalone molecularly imprinted (MIP) is relatively small. When the template molecule acetamiprid is added, a significant increase in impedance is observed during retesting, proving that the template molecule is bound to the cavity of the molecularly imprinted membrane. Eluting the MIP+ATM with a methanol-acetic acid solution disrupts the forces within the imprint, removing acetamiprid, and a subsequent test shows that its resistance returns to its original level. In the non-molecularly imprinted control group, as shown in Figure B, the impedance values ​​of the non-molecularly imprinted (NIP) and the NIP+ATM with the added template molecule are almost identical, demonstrating that the non-molecularly imprinted membrane has no recognition effect on the template molecule.

[0041] II. Scanning Electron Microscope The morphologies of molecularly imprinted (MIP) and non-molecularly imprinted (NIP) structures were characterized using scanning electron microscopy, and the results are as follows: Figure 2 As shown: Figure A shows the morphology of NIP, and Figure B shows the morphology of MIP. At 500 nm magnification, it can be seen that the surface of NIP is smooth and flat; while in the magnified image of MIP, a porous structure is observed after the formation of the imprinted cavity, which is significantly different from NIP.

[0042] III. Affinity test and adsorption specificity test A gradient was set for the imprinting polymerization time of silk fibroin and template molecules. The UV absorption peak of the target extract was considered by testing. The optimal recognition ability of the molecular imprint was determined by studying the imprinting polymerization time of the formed molecular imprint.

[0043] The imprinting time of molecular imprinting was optimized by controlling the molecular imprinting under different conditions to extract acetamiprid from solution, and the imprinting factor was studied through experimental parameters. To further verify its specific recognition ability, different solutions were specifically extracted using MIP gels, and the absorbance values ​​of various interferences were measured by UV light.

[0044] The test results are as follows Figure 3 As shown: As shown in Figure A, the concentration of ATM recognized by the MIP gradually increases with increasing imprinting time, reaching a maximum at 150 min with an absorbance of approximately 1.5. The absorption efficiency slightly decreases at 180 min. Combined with the non-specific adsorption of NIP, the imprinting factor (IF value) was calculated, revealing that the IF value was optimal at an imprinting time of 120 min, approximately 5.3. Therefore, this time is considered the best imprinting time. Simultaneously, the specific recognition of the target by MIP and NIP was tested. As shown in Figure B, NIP exhibits adsorption for a variety of substrates without specificity, while MIP shows specific selectivity for ATM absorption, thus confirming the feasibility of the molecular imprinting method prepared in this invention.

[0045] IV. Acetamiprid nanofiber fibroin molecularly imprinted gel enzyme activity experiment The feasibility of using nanozymes to imprint silk fibroin molecules was verified. Platinum nanoparticles were prepared in situ on the fiber using the reducing power of silk fibroin. Furthermore, the molecular imprinting method provided by this invention was used to simultaneously prepare silk fibroin molecular imprints and silk fibroin nanozymes with laccase-like activity to obtain a composite material.

[0046] Hydrogels were constructed by combining molecular imprinting with nanozymes. The hydrogels were incubated in a 2,4-DP, 4-AP system. Experimental groups were set up: the gel after acetamiprid absorption and the gel after acetamiprid elution, to verify the effect of molecular imprinting on nanozyme activity.

[0047] The test results are as follows Figure 4 As shown: No absorption peak was observed in the 2,4-DP+4-AP control group. The 2,4-DP+4-AP+MIP group exhibited the strongest absorption, approximately 0.58. After the addition of the template molecule ATM, the absorption peak decreased due to the restriction of substrate-nanozyme binding by MIP adsorption, leading to reduced activity. However, elution of ATM with methanol-acetic acid solution restored the activity, with an absorption peak of approximately 0.4. Example 2

[0048] The steps for preparing cortisol silk fibroin molecular imprints are as follows: 200 mg of silk fibroin was dissolved in 10 mL of formic acid and stirred at 60 °C for 2 h until the solution became clear, yielding a silk fibroin solution (20 mg / mL). 1 mL of cortisol solution (1 M) was added to 5 mL of the silk fibroin solution, and the mixture was stirred at 400 rpm for 120 min at 60 °C to allow for sufficient covalent bonding between the silk fibroin and the template molecules. After the reaction, the resulting MIP was placed in a methanol:acetic acid (9:1 v / v) solution and centrifuged at 8000 rpm for 5 min to remove the template molecules. The supernatant was removed, and the precipitate was retained to obtain the cortisol-imprinted silk fibroin molecular pattern. Example 3

[0049] The steps for preparing methylene blue silk fibroin molecular imprints are as follows: 200 mg of silk fibroin was dissolved in 10 mL of formic acid and stirred at 60 °C for 2 h until the solution became clear, yielding a silk fibroin solution (20 mg / mL). 1 mL of methylene blue solution (1 M) was added to 5 mL of the silk fibroin solution, and the mixture was stirred at 400 rpm for 120 min at 60 °C to allow for sufficient covalent bonding between the silk fibroin and the template molecules. After the reaction, the resulting MIP was placed in a methanol:acetic acid (9:1 v / v) solution and centrifuged at 8000 rpm for 5 min to remove the template molecules. The supernatant was removed, and the precipitate was retained to obtain the methylene blue silk fibroin molecular imprint.

[0050] V. Feasibility Verification of Molecular Imprinting The constructed cortisol / methylene blue molecularly imprinted polymer was tested using electrochemical impedance spectroscopy, adsorption specificity, and affinity assays. The experimental results are as follows: Figure 5 As shown.

[0051] Figures A and D show that the blank MIP has a low impedance value; when the template molecules cortisol or methylene blue are added, its impedance increases significantly; after elution, its resistance returns to the original level.

[0052] By studying the imprinting polymerization time of molecularly imprinted molecules, the optimal recognition ability of the molecular imprints was determined. As shown in Figures B and E, by setting a gradient in the imprinting polymerization time of silk fibroin and template molecules, the absorption peak of the target analyte extraction was tested. It was found that the optimal imprinting time for cortisol imprinting was 120 min, with an imprinting factor of approximately 10; and the optimal imprinting time for methylene blue imprinting was 120 min, with an imprinting factor of approximately 4.

[0053] The specific recognition of the target by MIP and NIP was tested. As shown in Figures C and D, the prepared NIP exhibited adsorption for a variety of substrates but lacked specificity, while the MIP showed specific selectivity for the absorption of cortisol or methylene blue, confirming the feasibility of the molecular imprinting prepared in this invention.

[0054] Application examples To verify the feasibility of using silk fibroin to construct novel molecular imprinting technologies, acetamiprid (ATM) molecular imprinting was used as an example to apply silk fibroin molecular imprinting to pesticide monitoring. By combining silk fibroin molecular imprinting with flexible sensors, a highly selective and stable wearable sensing device was constructed.

[0055] The specific method for constructing a wearable pesticide monitoring device for plants (acetamiprid molecular imprinted sensor) is as follows: Platinum nanoparticles were prepared in situ on the fiber surface using the reducing power of silk fibroin. Further, using the molecular imprinting method provided by this invention, silk fibroin molecular imprints and silk fibroin nanozymes with laccase-like activity were simultaneously prepared. After crosslinking with acrylamide, a platinum nanofiber silk fibroin molecularly imprinted gel was successfully obtained. A plant wearable sensor was then constructed using an anode with grape oxidase-like activity.

[0056] 1. Selective testing Neonicotinoid and non-neonicotinoid pesticides were used as interfering agents and sprayed together with acetamiprid onto the surface of tomato leaves. Phosphate, thiamethoxam, thiamethoxam, methyl parathion, fipronil, and imidacloprid were selected as interfering agents. The same concentration of pesticide mixtures was used, and detection was performed using a plant wearable sensor, with all procedures maintained consistently.

[0057] Test results are as follows Figure 6 As shown: Only acetamiprid and the mixture suppressed the sensor's output signal, making its open-circuit voltage almost 0 V. Other pesticide interfering substances (imidacloprid, fipronil, etc.) could not be captured by the molecular imprint, so their open-circuit voltage was relatively stable at about 0.225 V.

[0058] 2. Standard Curve Test The constructed sensor was tested using a standard curve for the target pesticide (acetamiprid). Tomato was used as a model crop, and different concentrations of acetamiprid solutions (0 fg / mL, 10 fg / mL, 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 1 μg / mL, 10 μg / mL) were sprayed onto tomato leaves. After complete drying, the wearable sensor was attached to the leaf surface to monitor E. OCV And plot the standard curve.

[0059] Test results are as follows Figure 7 As shown: As shown in Figure A, with increasing pesticide concentration, the silk fibroin molecular imprint captures more template molecules, resulting in a greater inhibitory effect on the sensor and a gradual decrease in open-circuit voltage. As shown in Figure B, the voltage signal exhibits a good linear relationship with the logarithm of pesticide concentration.

[0060] 3. Continuous monitoring test An acetamiprid molecularly imprinted sensor was placed on plant leaves and subjected to long-term continuous monitoring experiments. This demonstrates that wearable plant sensors based on silk fibroin serve as a verifiable application example for green agriculture, and that using silk fibroin to prepare molecular imprints is a method with practical application value.

[0061] Tomato leaves were sprayed with a solution of acetamiprid (1 μg / mL), and the dynamic degradation of the pesticide was monitored using a designed plant wearable sensor. After spraying acetamiprid, the sensor was attached to the leaves on different days to monitor E0.05. OCV Finally, dynamic degradation curves were plotted based on the relevant results.

[0062] The test results are as follows Figure 8 As shown: As shown in Figure A, over time, the amount of pesticide on the plant surface gradually decreases, thus reducing the amount of target material captured by the molecular imprint, and the sensor's output signal gradually increases. After converting the measured signal, the results are shown in Figure B, indicating that the pesticide's degradation pattern is consistent with its half-life variation trend. In summary, the acetamiprid molecularly imprinted sensor prepared in this invention possesses excellent recognition capabilities.

[0063] 4. Validation experiments of peptide chains with different molecular weights First, formic acid was used to treat silk fibroin for different durations. The formic acid unchained the peptide chains in the silk fibroin, breaking them down into a mixture of long and short chains. Based on this, molecular imprints were prepared using silk fibroin with different components, and their extraction activity against acetamiprid was tested. Furthermore, the composition of the silk fibroin solutions treated at different times was verified using SDS-PAGE.

[0064] Meanwhile, different concentrations of formic acid were used to treat silk fibroin. Since the formic acid content directly affects the length distribution of the silk fibroin peptide chain, the resulting molecular imprints were also different.

[0065] Experimental results are as follows Figure 9 As shown: As shown in Figure A, the specific recognition ability of the molecular imprint for acetamiprid decreased from 60 min onwards as the treatment time of silk fibroin with formic acid increased. SDS gel electrophoresis experiments revealed, as shown in Figures B and C, that at 30 min, silk fibroin consisted entirely of long chains greater than 130 kDa in the black and blue gel films. At 60 min, the peptide chains exhibited a uniform distribution of long and short chains between 17 and 130 kDa, indicating that both long and short peptide chains were functional and the molecular imprint had optimal recognition ability. However, after 90 min, the peptide chains were decomposed, resulting in most peptide chains being too short (less than 17 kDa) and a higher proportion of medium-length peptide chains (40-95 kDa). This imbalance in the crosslinking of the imprint led to a decrease in recognition effectiveness.

[0066] As shown in Figure D, the recognition ability of the prepared molecular imprint for acetamiprid continuously increases with the gradual increase of formic acid content. Combined with the SDS gel electrophoresis results, as shown in Figures E and F, at low concentrations (0%-40%), the peptide chains are mostly 72-130 kDa, and the binding force with acetamiprid is relatively weak; while at concentrations of 60%-80%, the specific recognition effect gradually increases with the gradual increase of peptide chain content; when treated with 100% formic acid, the peptide chain length in the system is uniform, and the prepared molecular imprint has the best recognition effect.

[0067] In summary, under the treatment of formic acid solvent, the long-chain structure of silk fibroin is gradually decomposed into a system of alternating long and short chains. At this point, both long and short chains interact with acetamiprid, with the short chains more readily entraining acetamiprid and forming hydrogen bonds, while the long chains are coupled externally, forming a stable framework structure. When the eluent removes the template molecules, the alternating long and short chain structure is stably maintained, thus yielding a molecularly imprinted cavity with specific recognition capabilities.

[0068] This invention breaks through the traditional imprinting paradigm that uses synthetic monomers and cross-linking agents. Through controlled depolymerization, it simultaneously provides functional monomers (short peptide chains) and cross-linking agents (long peptide chains). Utilizing the "self-linking effect" of silk fibroin, it completes compounding, immobilization, and cavity formation in one step, eliminating the need for external molecules and simplifying the traditional imprinting process. The short peptide chains are responsible for recognition, while the long peptide chains are responsible for three-dimensional network cross-linking. This natural division of labor gives the imprinted structure precise recognition sites. Furthermore, the synergistic effect of multiple forces (β-sheet, hydrogen bonding, carbonyl groups, and hydrophobic interactions) enables diverse and stable interactions with the template molecules, thereby improving the affinity and selectivity of the imprinted cavity.

[0069] In this invention, silk fibroin is used as both a polymer monomer and a crosslinking agent to construct a novel molecular imprinting method. This method is a green, biocompatible, and efficient imprinting method that can overcome the limitations of traditional molecular imprinting (MIP) technology in biomedical and environmental applications.

[0070] First, silk fibroin is a natural water-soluble protein. MIPs constructed using silk fibroin as a functional monomer and cross-linking agent can complete the imprinting in the aqueous phase, making them more suitable for recognition in aqueous solutions or physiological environments. This avoids the problem of a sharp drop in efficiency when traditional MIPs are prepared in organic systems and recognized in aqueous phases.

[0071] Breaking away from the dependence on chemical reagents and complex synthesis in traditional molecular imprinting, silk fibroin, derived from natural silk, is a renewable and biodegradable green material. By utilizing the abundant functional groups within silk fibroin to form interactions with template molecules, molecular imprinting is achieved, significantly reducing or even completely eliminating the use of toxic chemical cross-linking agents, thereby improving the biocompatibility of molecular imprints.

[0072] To address the issues of entrapment and slow mass transfer in imprinted sites, an imprinted layer constructed from silk fibroin adheres to the surface of the recognition region, ensuring that most imprinted cavities are located on the material surface or in a shallow layer. This facilitates the entry of target analytes into the cavities, improving mass transfer efficiency and adsorption rate.

[0073] The amino acid residues in silk fibroin can bind to template molecules through various interactions (hydrogen bonds, hydrophobic interactions, π-π stacking), providing a perfect structural basis for constructing highly selective molecular imprinting sites.

[0074] By using formic acid as a solvent, the network structure of silk fibroin aggregates is opened, reducing its molecular weight and forming sparse peptide chains within. This increases the content of β-sheet structures in the silk fibroin, which become the main framework of the imprinted matrix. This facilitates the formation of various interactions between the silk fibroin and template molecules after the introduction of template molecules, resulting in better molecular imprinting, improved mechanical strength and durability of MIPs in complex environments, and reduced deformation or dissolution during use.

[0075] Meanwhile, in formic acid solution, due to chain-breaking, silk fibroin forms more nanofiber structures. This greatly increases the specific surface area of ​​the material, providing more binding sites for template molecules and significantly improving the adsorption capacity and mass transfer efficiency of MIPs.

[0076] At the component level, silk fibroin acts as a functional monomer. Its various amino acid residues (such as tyrosine, serine, and aspartic acid) provide abundant hydroxyl, carboxyl, and amino groups, enabling it to form hydrogen bonds, ionic interactions, and hydrophobic interactions with various template molecules. It can perfectly replace the role of chemical functional monomers, forming sufficient affinity and selectivity with template molecules. During the formation of molecular imprints, silk fibroin achieves self-crosslinking through conformational changes, requiring no additional crosslinking agent. Under specific conditions, its secondary structure can spontaneously form β-sheets, with stable crystalline regions acting as crosslinking nodes, promoting the formation of a stable network structure.

[0077] In summary, when formic acid is used for dissolution, it disrupts the peptide chain interactions in SF, transforming the long-chain, disordered silk fibroin network into a structure where short peptide chains and long chains coexist. The β-sheets, hydrogen bonds, and carbonyl groups present in silk fibroin act as functional monomers, aggregating between template molecules and forming various interactions. Meanwhile, the long-chain structures in the system remain free, surrounding the short peptide chain-template molecule, acting as cross-linking agents to fix the complex formed in the previous step, creating a cavity. When hydrogen bonds, hydrophobic interactions, and covalent forces take effect, the resulting complex structure is fixed, thus forming a molecularly imprinted cavity after gel solidification following template elution. Therefore, using short peptide chains as functional monomers and long peptide chains as cross-linking agents to construct molecular imprints with template molecules through self-linking is a feasible novel method for imprinting, which can be used to develop new molecular imprints with a wide range of applications.

[0078] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing silk fibroin molecular imprints, characterized in that, The steps are as follows: Silk fibroin was dissolved in formic acid and stirred until the solution became clear to obtain a silk fibroin solution. The template molecule solution was added to the silk fibroin solution and stirred to react. After the reaction was completed, the mixture was eluted, centrifuged, and the supernatant was removed while the precipitate was retained to obtain a silk fibroin molecular imprint.

2. The method for preparing silk fibroin molecular imprints according to claim 1, characterized in that, The concentration of the silk fibroin solution is 10~50 mg / mL.

3. The method for preparing silk fibroin molecular imprints according to claim 1, characterized in that, The concentration of the template molecule solution is 0.5~3 M.

4. The method for preparing silk fibroin molecular imprints according to claim 1, characterized in that, The template molecule is any substance that can enable silk fibroin to form a complementary cavity in the template molecule structure.

5. The method for preparing silk fibroin molecular imprints according to claim 1, characterized in that, The conditions for the stirring reaction are: stirring at 200-600 rpm for 100-200 min at 50-70℃.

6. The method for preparing silk fibroin molecular imprints according to claim 1, characterized in that, The elution solution used is a methanol / acetic acid mixture; wherein the volume ratio of methanol to acetic acid is 9:

1.

7. The method for preparing silk fibroin molecular imprints according to claim 1, characterized in that, The silk fibroin can be prepared by the following method: Silkworm cocoons are cut into thin slices, boiled in NaHCO3 solution to remove sericin, rinsed and dried to obtain degummed silk. The degummed silk is then dissolved in a ternary solution and reacted at 90℃ for 2-3 hours. After dialysis, small molecule impurities and salts in the fibroin solution are removed. The dialyzed solution is then freeze-dried to obtain fibroin protein.

8. The method for preparing silk fibroin molecular imprints according to claim 7, characterized in that, The ternary solution is a mixture of calcium chloride, ethanol and water in a molar ratio of 1:2:

8.

9. Silk fibroin molecular imprints prepared by the method according to any one of claims 1 to 8.

10. The application of the silk fibroin molecular imprinting according to claim 9, characterized in that, Including at least one of the following applications: (1) Application in separation and purification; (2) Application in sensing and detection; (3) Application in catalytic system design; (4) Application in drug delivery.

Images