A sensor with antioxidant and antifouling properties and a preparation method and application thereof

Abstract

The application provides a sensor with antioxidant and antifouling properties and a preparation method and application thereof, and belongs to the technical field of marine chemistry. The sensor takes a glassy carbon electrode (GC) as a substrate, adopts polyaniline as a solid contact layer, and takes a calcium ion selective membrane (ISM) as a detection core. The innovation of the sensor is that a fucoidan-based functional polypeptide (PFp) is modified on the ISM to construct an antifouling layer. The PFp layer forms a triple synergistic defense mechanism of "anti-adhesion-sterilization-antioxidation". Experiments prove that the sensor can effectively inhibit the adhesion and colonization of bacteria such as escherichia coli and staphylococcus aureus and chlorella, and significantly slow down the oxidative decay of the sensing membrane. In addition, when the sensor is used for calcium ion detection in actual seawater, it exhibits excellent long-term stability and high precision, and provides an innovative strategy for continuous in-situ monitoring in complex media.

Description

Technical Field

[0001] This invention belongs to the field of marine chemistry technology, and in particular relates to a sensor with antioxidant and antifouling properties, its preparation method and application. Background Technology

[0002] Biofouling is a core bottleneck restricting the long-term in-situ monitoring of ion-selective electrodes (ISEs) in complex media such as seawater, wastewater, and body fluids. The adhesion and colonization of biological components such as microorganisms, proteins, and algae on the surface of ion-selective membranes significantly inhibits the transmembrane diffusion of target ions, leading to electrode potential drift, prolonged response time, and decreased sensitivity and selectivity. Ultimately, this causes the sensor to lose its reliable detection capability within a few days, severely limiting its practical deployment in fields such as marine environmental monitoring, implantable diagnostics, and industrial process control.

[0003] Existing antifouling strategies are mainly divided into two categories: bactericidal and anti-adhesion. Bactericidal strategies, represented by silver nanoparticles, quaternary ammonium salts, and antimicrobial peptides, inhibit bacterial growth by disrupting bacterial cell membranes or inducing reactive oxygen species (ROS) bursts. However, dead bacterial debris remains on the electrode surface, forming a non-specific adsorption layer that continuously blocks ion channels, leading to sluggish response and irreversible drift. Anti-adhesion strategies construct strong hydration layers using polyethylene glycol (PEG), zwitterionic polymers, or dopamine, forming spatial repulsion and hydration barriers at the interface, effectively reducing initial adhesion. However, their ability to remove already adhered microorganisms is limited, and under high fouling loads (such as in marine environments), the hydration layer is susceptible to competitive damage from salt ions, polyvalent cations, and proteins. Therefore, achieving a multi-layered, synergistic antifouling interface combining hydrophilic hydration, antioxidant properties, and mild antibacterial activity is a problem that needs to be solved. Summary of the Invention

[0004] The purpose of this invention is to provide a sensor with antioxidant and antifouling properties, its preparation method and application, thereby enabling accurate detection of calcium ions in seawater.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] First, this invention provides a method for preparing an antifouling electrochemical sensor based on a functional bactericidal peptide of fucoidan, wherein the antifouling electrochemical sensor is GC / PANI / Ca 2+ -ISM / PFp sensor;

[0007] The method for preparing the anti-fouling electrochemical sensor includes the following steps:

[0008] (1) After polishing the bare glassy carbon electrode GC with alumina powder, the electrode was thoroughly cleaned to obtain a polished glassy carbon electrode GC.

[0009] (2) The polished glassy carbon electrode GC was placed in an aniline solution and deposited using a chronoamperometry method to obtain a GC / PANI electrode;

[0010] (3) Dissolve calcium ion carrier, sodium [3,5-bis(trifluoromethyl)phenyl]borate, polyvinyl chloride, and 2-nitrophenyl n-octyl ether in tetrahydrofuran and mix ultrasonically to prepare Ca 2+ Selective membrane mixed solution;

[0011] (4) The Ca 2+ The selective membrane mixture solution was dropped onto the GC / PANI electrode and dried overnight to obtain GC / PANI / Ca. 2+ -ISM electrode;

[0012] (5) In the GC / PANI / Ca 2+ - The anti-fouling electrochemical sensor was obtained by incubating a fucoidan-functional bactericidal peptide solution on an ISM electrode for 12-24 hours.

[0013] Preferably, in step (5), the fucoidan-functional bactericidal peptide solution corresponding to each 5-10 mg of fucoidan is prepared by the following method:

[0014] (a) Add 5–10 mg of fucoidan, 3 mg of dopamine, and 2–5 mg of functional bactericidal peptide to 5–10 mL of 0.1 mol·L⁻¹. -1 In the MES buffer solution, shake or stir thoroughly until completely dissolved to form a mixed solution a;

[0015] (b) Add 5-10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the mixed solution a, mix thoroughly to obtain mixed solution b;

[0016] (c) Transfer the mixed solution b to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and administer at 0.005 mol·L⁻¹ -1 The HCl solution was used as the external dialysis solution, and the solution was dialyzed at 4°C for 24-48 hours to obtain the fucoidan-functional bactericidal peptide solution.

[0017] Preferably, the amino acid sequence of the functional bactericidal peptide is KFKFKFKF;

[0018] The fucoidan is Fucus vesiculosus fucoidan, and the CAS number of the Fucus vesiculosus fucoidan is 9072-19-9.

[0019] Preferably, in step (2), the concentration of the aniline solution is 1-3 mmol·L⁻¹. -1 ;

[0020] The potential range of the chronoamperometry method is -2V to 2V, and the deposition time of the chronoamperometry method is 30 minutes.

[0021] Preferably, in step (3), the mass-to-volume ratio of the calcium ion carrier, sodium [3,5-bis(trifluoromethyl)phenyl]borate, polyvinyl chloride, 2-nitrophenyl n-octyl ether and tetrahydrofuran is 2.5 mg:1.2 mg:66 mg:135 mg:1.5 mL.

[0022] Preferably, in step (4), the Ca 2+ The selective membrane mixture solution was added in a volume of 90-100 μL.

[0023] The GC / PANI / Ca 2+ The amount of fucoidan-functional bactericidal peptide solution used for incubation on the ISM electrode is 10-50 μL.

[0024] Preferably, the antifouling electrochemical sensor is an antifouling electrochemical sensor that inhibits the adhesion of Escherichia coli and Staphylococcus aureus to the sensor surface, promotes the colonization of Chlorella, and provides antioxidant properties.

[0025] The anti-fouling electrochemical sensor is used for the detection of calcium ions in seawater.

[0026] Secondly, this invention provides an antifouling electrochemical sensor based on fucoidan-functional bactericidal peptides. The electrochemical sensor is characterized by using a glassy carbon electrode as a substrate, polyaniline as a solid conductive layer, a calcium ion-selective membrane as a detection layer, and a self-designed fucoidan-functional bactericidal peptide as an antifouling layer (GC / PANI / Ca). 2+ -ISM / PFp sensor;

[0027] The anti-fouling electrochemical sensor was prepared by the above-described preparation method.

[0028] Preferably, the antifouling electrochemical sensor is an antifouling electrochemical sensor that inhibits the adhesion of Escherichia coli and Staphylococcus aureus, colonization of Chlorella, and antioxidant activity.

[0029] The anti-fouling electrochemical sensor is used for the detection of calcium ions in seawater.

[0030] Finally, this invention provides the application of the above-described anti-fouling electrochemical sensor in the detection of calcium ions in seawater.

[0031] The beneficial effects of this invention are as follows:

[0032] 1) This invention develops an all-solid-state calcium ion selective electrochemical sensor based on fucoidan-functionalized peptides as an antifouling layer for long-term accurate monitoring of calcium ions in the ocean.

[0033] 2) This invention utilizes a self-designed fucoidan-functionalized peptide as an antifouling layer, achieving a triple defense of antifouling, sterilization, and antioxidation at the interface. Fucoidan forms a hydration layer with its sulfated polysaccharide backbone to resist non-specific adsorption; its antioxidant activity scavenges reactive oxygen species at the interface, thereby slowing down the aging of the ion-selective membrane, inhibiting biofilm formation, and delaying the inactivation of antimicrobial peptides. The bactericidal peptides achieve rapid sterilization by electrostatically disrupting bacterial membranes. The functional bactericidal peptides based on fucoidan are firmly anchored on the sensor surface via dopamine.

[0034] 3) The surface modification method using fucoidan-functionalized peptides in this invention significantly reduces bacterial cell adhesion and inhibits the formation of closed bacterial communities in attached bacterial cells and extracellular polymers. Its antioxidant activity can scavenge reactive oxygen species at the interface, thereby slowing down the aging of ion-selective membranes, inhibiting biofilm formation, and delaying the inactivation of antimicrobial peptides. Furthermore, this sensor maintains good selectivity, excellent stability, and a low detection limit even after modification with fucoidan-functionalized peptides.

[0035] In summary, this invention has developed a pollution-resistant all-solid-state calcium ion selective electrochemical sensor, which has broad potential for monitoring the marine environment. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0037] Figure 1 Molecular dynamics simulations and mechanism diagrams of PFp;

[0038] Among them, (A) representative snapshots of the simulated system at different time intervals (dopamine is represented in yellow, fucoidan in blue, and peptide in red); (B) the change of the solvent contact surface area (SASA) of the system over time; (C) the change of the root mean square deviation (RMSD) of the system over time; (D) the change of the radius of gyration (Rg) of the system over time; (E) the change of the interaction energy between fucoidan and dopamine over time; (F) the change of the interaction energy between fucoidan and peptide over time; (G) the change of the interaction energy between peptide and dopamine over time; (H) the change of the number of hydrogen bonds in the system over time; (I) the free energy distribution of the system; and (J) the electrostatic potential distribution of the system at 100 ns.

[0039] Figure 2The graph shows the results of the characteristic analysis of PFp.

[0040] Among them, (A) transmission electron microscopy image of fucoidan-functionalized peptides; (B) infrared spectra of dopamine, polypeptides, fucoidan, and fucoidan-functionalized peptides; (C) ultraviolet absorption spectra of dopamine, polypeptides, fucoidan, and fucoidan-functionalized peptides; (D) Ca 2+ -ISM and Ca 2+ -ISM / PFp contact angle;

[0041] Figure 3 For Ca 2+ -Graph showing the antibacterial effect test results of ISM / PFp;

[0042] Among them, (A)Ca 2+ -ISM, Ca 2+ -ISM / pep and Ca 2+ -ISM / PFp in E. coli suspension (10 6 CFU·mL -1 (A) CLSM (laser confocal microscopy) image after immersion in Ca for 12 h; (B) Ca 2+ -ISM, Ca 2+ -ISM / pep and Ca 2+ -ISM / PFp in Staphylococcus aureus suspension (10 6 CFU·mL -1 CLSM images after immersion in 12 h: live bacteria stained with SYTO 9 show green fluorescence, while dead bacteria stained with PI (propidium iodide) show red fluorescence; (C)Ca 2+ -ISM / pep and Ca 2+ -ISM / pep in bacterial suspension (10 6 CFU·mL -1 (D) Scanning electron microscopy image after soaking in LB agar for 12 h; (D) Colony count on LB agar plate: positive control (10 6 CFU·mL -1 ), from Ca 2+ -ISM, Ca 2+ -ISM / pep, Ca 2+ -ISM / PFp bacterial washing solution, and blank (negative control); (E) in Ca 2+ -ISM / pep and Ca 2+ Typical bacterial growth curves recorded by a non-contact conductivity sensor on an ISM / PFp: left graph for Escherichia coli, right graph for Staphylococcus aureus; (F)Ca 2+ -ISM / pep and Ca 2+-Microscopic images of ISM / PFp after soaking in Chlorella suspension for 1 week;

[0043] Figure 4 The UV spectra of DPPH, PFp, and vitamin C are shown.

[0044] Figure 5 For Ca 2+ -Electrochemical measurement results of ISM / PFp;

[0045] Among them, (A)Ca 2+ -ISM / pep and Ca 2+ -ISM / PFp in CaCl2 solutions of different concentrations (10 -7 – 10 -1 mol·L -1 The background solution was 0.5 mol. -1 (A) Potential-time response curves in NaCl; (B) GC / PANI / Ca 2+ -ISM / pep and GC / PANI / Ca 2+ -ISM / PFp calibration curve;

[0046] Figure 6 For GC / PANI / Ca 2+ -ISM and GC / PANI / Ca 2+ - The changes in the potential response slope of the ISM / PFp electrode after immersion in seawater, artificial saliva, and artificial urine are shown, where the error bar represents the standard deviation of three independent measurements. Detailed Implementation

[0047] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0048] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; other experimental methods and technical means not specifically mentioned herein refer to experimental methods and technical means commonly used by one of ordinary skill in the art.

[0049] The materials and reagents used in this invention are as follows:

[0050] The designed peptide (pep, KFKFKFKF, purity >95%) was synthesized by Hefei Guotai Biotechnology Co., Ltd. Dopamine hydrochloride (98%), Fucus vesiculosus fucoidan (>95%, CAS: 9072-19-9), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, >99%), sodium [3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), polyvinyl chloride (PVC), 2-nitrophenyl n-octyl ether (o-NPOE), and calcium ion carrier IV were all purchased from Sigma-Aldridge (Shanghai). Calcium chloride, tetrahydrofuran (THF), glycerol, ethanol, and glutaraldehyde were all purchased from Sinopharm Group (Shanghai). Phosphate-buffered saline (PBS) was purchased from Sangon Biotech Co., Ltd. (Shanghai). LB medium and nutrient agar were purchased from Qingdao Haibo Biotechnology Co., Ltd. (Qingdao High-tech Industrial Park). Ultrapure water (resistivity ≤18 MΩ) was used throughout the experiment. All other chemical reagents were of analytical grade and could be used without further purification.

[0051] The entire experiment of this invention uses ultrapure water (resistivity ≤ 18 MΩ cm). -1 All chemicals are analytical grade and are used without further purification.

[0052] The instruments used in this invention are as follows:

[0053] All electrochemical measurements were performed at room temperature using a CHI 660E electrochemical workstation. The morphology of various modified surfaces was characterized using a Hitachi S-4800 scanning electron microscope (SEM) (Hitachi Group, Japan). The binding state of PFp was verified using a Fourier transform infrared (FTIR) spectrometer (Thermo Nicolet iS5) and a UV spectrometer. The water contact angle of different modified interfaces was measured using a JC2000D1 contact angle meter (Shanghai Zhongchen Digital Technology Equipment Co., Ltd.). The fluorescence of bacteria attached to the samples was observed using a TCS-SP5 confocal laser scanning microscope (Leica, Germany). An eight-channel capacitively coupled non-contact conductivity detector (manufactured by eDAQ Pty Ltd, Sydney, Australia) and TERA TERM software were used to monitor and test the conductivity changes of the water medium in a glass tube (outer diameter 5.0 mm, inner diameter 4.0 mm) online. The Ca in actual ocean water was measured using the BIOSYS intelligent biosensor system (Shenzhen Ruihua Intelligent Technology Co., Ltd., China). 2+ .

[0054] Example 1

[0055] Preparation of a pollution-resistant electrochemical sensor for calcium ions (GC / PANI / Ca) 2+-ISM / PFp)

[0056] (1) Polish the bare glassy carbon electrode (GC) with alumina powder and thoroughly clean the electrode;

[0057] (2) Place the glassy carbon electrode GC treated in step (1) in a 1 mmol·L⁻¹ solution. -1 In an aniline solution, a chronoamperometry method was used to deposit GC / PANI electrodes for 30 minutes within a potential range of -2 V to 2 V.

[0058] (3) Dissolve 2 mg of calcium ion carrier (ETH 5234, N,N-dicyclohexyl-N′,N′-bis(octadecyl-3-oxapranyl)amide), 1 mg of sodium [3,5-bis(trifluoromethyl)phenyl]borate NaTFPB, 66 mg of polyvinyl chloride (PVC), and 132 mg of 2-nitrophenyl n-octyl ether o-NPOE in 1.6 mL of tetrahydrofuran (THF) and sonicate for 1 h to prepare Ca 2+ Selective membrane mixed solution;

[0059] (4) Take 100 μL of Ca 2+ The selective membrane mixture solution was dropped onto the pre-deposited electrode and dried overnight to obtain GC / PANI / Ca. 2+ -ISM electrode;

[0060] (5) In Ca 2+ A fouling-resistant electrochemical sensor (GC / PANI / Ca) based on cyclic peptides was prepared by incubating 10 μL of 3 mg / mL fucoidan-functionalized peptide solution on an ion-selective electrode for 12 hours. 2+ -ISM / PFp).

[0061] The preparation method for the fucoidan-functionalized peptide solution is as follows:

[0062] (a) Completely dissolve fucoidan (10 mg), dopamine (3 mg), and peptide (5 mg) in 5 mL of MES buffer (0.1 mol·L⁻¹). -1 In (pH 6.0);

[0063] (b) Add EDC (10 mg) to the solution, mix thoroughly, and then transfer to a dialysis membrane (MWCO = 3.5 kDa) using acidified water (0.005 mol·L⁻¹). -1 Dialysis was performed using an HCl solution.

[0064] (c) After freeze-drying, store in a refrigerator for later use and label it as PFp.

[0065] In subsequent experiments, a 3 mg / mL fucoidan-functionalized peptide aqueous solution was prepared as needed.

[0066] In subsequent experiments, the GC / PANI / Ca were detected. 2+ -ISM / PFp are both GC / PANI / Ca prepared according to the method in Example 1. 2+ -ISM / PFp.

[0067] Example 2

[0068] Preparation of a pollution-resistant electrochemical sensor for calcium ions (GC / PANI / Ca) 2+ -ISM / PFp)

[0069] (1) Polish the bare glassy carbon electrode (GC) with alumina powder and thoroughly clean the electrode;

[0070] (2) Place the glassy carbon electrode GC treated in step (1) in a 3 mmol·L⁻¹ solution. -1 In an aniline solution, a chronoamperometry method was used to deposit GC / PANI electrodes for 45 minutes within a potential range of -2 V to 2 V.

[0071] (3) Dissolve 2.5 mg of calcium ion carrier (ETH 5234, N,N-dicyclohexyl-N′,N′-bis(octadecyl-3-oxapranyl)amide), 1.2 mg of sodium [3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), 66 mg of polyvinyl chloride (PVC), and 135 mg of 2-nitrophenyl n-octyl ether (o-NPOE) in 1.5 mL of tetrahydrofuran (THF) and sonicate for 1 h to prepare Ca 2+ Selective membrane mixed solution;

[0072] (4) Take 90 μL of Ca 2+ The selective membrane mixture solution was dropped onto the pre-deposited electrode and dried overnight to obtain GC / PANI / Ca. 2+ -ISM electrode;

[0073] (5) In Ca 2+ A fouling-resistant electrochemical sensor (GC / PANI / Ca) based on cyclic peptides was prepared by incubating 50 μL of a 3 mg / mL fucoidan-functionalized peptide solution on an ion-selective electrode for 24 hours. 2+ -ISM / PFp).

[0074] The preparation method for the fucoidan-functionalized peptide solution is as follows:

[0075] (a) Completely dissolve fucoidan (10 mg), dopamine (3 mg), and peptide (5 mg) in 5 mL of MES buffer (0.1 mol / L).-1 In (pH 6.0);

[0076] (b) Add EDC (10 mg) to the solution, mix thoroughly, and then transfer to a dialysis membrane (MWCO = 3.5 kDa) using acidified water (0.005 mol·L⁻¹). -1 Dialysis was performed using an HCl solution.

[0077] (c) After freeze-drying, store in a refrigerator for later use and label it as PFp.

[0078] In subsequent experiments, a 3 mg / mL fucoidan-functionalized peptide aqueous solution was prepared as needed.

[0079] Comparative Example 1

[0080] Preparation of GC / PANI / Ca 2+ -ISM electrode

[0081] (1) Polish the bare glassy carbon electrode (GC) with alumina powder and thoroughly clean the electrode;

[0082] (2) Place the glassy carbon electrode GC treated in step (1) in a 1 mmol L -1 In an aniline solution, a chronoamperometry method was used to deposit GC / PANI electrodes for 30 minutes within a potential range of -2 V to 2 V.

[0083] (3) Dissolve 2 mg of calcium ion carrier (ETH 5234, N,N-dicyclohexyl-N′,N′-bis(octadecyl-3-oxapranyl)amide), 1 mg of sodium [3,5-bis(trifluoromethyl)phenyl]borate NaTFPB, 66 mg of polyvinyl chloride (PVC), and 132 mg of 2-nitrophenyl n-octyl ether o-NPOE in 1.6 mL of tetrahydrofuran (THF) and sonicate to prepare Ca 2+ Selective membrane mixed solution;

[0084] (4) Take Ca 2+ The selective membrane mixture solution was dropped onto the pre-deposited electrode and dried overnight to obtain GC / PANI / Ca. 2+ -ISM electrode.

[0085] Example 3

[0086] In this embodiment, we used Materials Studio to construct fucoidan, dopamine, and H3O. +The model was constructed using density functional theory calculations with Gaussian 16 software. All calculations employed B3LYP functionals combined with D3BJ dispersion correction. For geometry optimization, def2tzvp basis sets were used for all atmospheres. TIP3P5 force fields were applied to water molecules. The restrained electrostatic potential (RESP) charge was then fitted using Multiwfn. Other bonding and non-bonding parameters were obtained using AuToFF software. The KFKFKFKF peptide sequence was constructed using AlphaFold37. The OPLS-AA force field parameters of the peptide were obtained using the pdb2gmx command. Molecular dynamics (MD) simulations were performed for 100 ns, and the system was characterized using various parameters. The results are shown below. Figure 1 As shown.

[0087] like Figure 1 As shown in Figure A, in the initial stage of the simulation, the three types of molecules are randomly dispersed; as the simulation progresses, the molecules gradually aggregate into clusters, eventually evolving into a relatively compact, irregular spherical structure. This process intuitively reflects the spontaneous self-assembly behavior of the system driven by intermolecular forces. The solvent-accessible surface area (SASA) is used to assess the compactness of the system, and its value represents the degree of exposure of the molecular surface to the solvent.

[0088] Figure 1 B shows that SASA decreased significantly over time and tended to stabilize, indicating that the system gradually changed from a dispersed state to a compact aggregated state, and the number of atoms exposed to the solvent decreased.

[0089] like Figure 1 As shown in Figure C, the root mean square deviation (RMSD) curve reflects the overall structural stability of the system. Significant fluctuations in RMSD occur between 0 and 36 ns, indicating that the system is in a conformational adjustment phase; thereafter, the RMSD stabilizes, indicating that the system has formed a relatively stable assembly. Simultaneously, the trend of the system's radius of gyration (Rg) is consistent with that of the RMSD. Figure 1 D): The value gradually decreases in the first 36 ns and then remains stable, indicating that the system shrinks from a loose structure to a dense aggregate.

[0090] In addition, to explore the driving force of self-assembly, this study analyzed the interaction energies between three pairs of molecules: fucoidan-dopamine (FD), fucoidan-peptide (Fp), and peptide-dopamine (Dp). Figure 1 E–G).

[0091] The results showed that the system's stability mainly stemmed from electrostatic interactions. The electrostatic interaction energy between fucoidan and polypeptide was significantly higher than that between other pairings, indicating that these two were the core factors driving the system's self-assembly, while dopamine played only a supporting role. This trend was further validated by hydrogen bond analysis.

[0092] Figure 1 The H-values ​​show that the number of hydrogen bonds in the system increases over time and tends to stabilize in the later stages, indicating that a hydrogen bond network gradually forms during the self-assembly process. Notably, the number of hydrogen bonds between fucoidan and polypeptide is much higher than that between other molecular pairs, indicating a stronger mutual recognition and binding ability, thus further confirming their dominant role in self-assembly. This is mainly attributed to the sulfate ester groups (–SO3) on the fucoidan backbone. - The high complementarity between the amino side chain and the peptide molecule provides more binding sites and stronger binding energy.

[0093] Figure 1 Figure 1 illustrates the two-dimensional and three-dimensional free energy landscape of the system. The two-dimensional model shows multiple local energy valleys, indicating that the system may possess multiple self-assembly paths and conformational diversity. As the simulation progresses, the molecules gradually tend towards a major energy minimum valley, indicating that the system eventually reaches a stable configuration. The three-dimensional free energy surface more intuitively displays the distribution of energy valleys and barriers: deep valleys correspond to stable assembled states, while energy barriers represent the energy obstacles that the system must overcome during the transition from a dispersed state to an assembled state. This "barrier-valley" model reveals the dynamic evolution path of the system—molecules overcome energy barriers under the combined drive of electrostatic and hydrogen bonding interactions, ultimately reaching a stable aggregated state.

[0094] Figure 1 J shows the electrostatic potential distribution of the system at 100 ns (red represents negative charge, blue represents positive charge, and the color intensity corresponds to the surface charge strength). Binding site analysis indicates that fucoidan is mainly distributed in the blue region, due to its sulfate (–SO3) ions. - Significant electrostatic interactions occur between dopamine and peptides. The charge distribution of dopamine is relatively localized, thus playing a supporting rather than decisive role in the assembly process. In summary, MD simulations systematically reveal the self-assembly mechanism of the three-component system: the system can spontaneously evolve from a dispersed state into stable spherical aggregates, and its stability mainly originates from the strong electrostatic interactions and hydrogen bond network formed between fucoidan and peptides.

[0095] Example 4

[0096] This embodiment employs a transmission electron microscope, infrared and ultraviolet spectrometers, and an antennal angle measuring instrument. For the transmission electron microscope, an aqueous solution of PFp is dropped onto a copper grid for imaging. For the infrared spectrometer, the freeze-dried particles are ground with potassium bromide, compressed into tablets, and then vacuum-sealed for detection. For the ultraviolet spectrometer, the particles are prepared in 3 mg / mL solutions. -1 The aqueous solution was measured, and the background signal of water was subtracted before measurement. GC / PANI / Ca 2+ The characteristics of -ISM / PFp were analyzed, and the results are as follows: Figure 2 As shown.

[0097] Figure 2 Image A presents a transmission electron microscope (TEM) image of the PFp complex. The TEM image shows a cross-linked network structure. The darker regions with high cross-linking density can be attributed to the formation of amide bonds mediated by EDC, which covalently couples the fucoidan chains with dopamine and peptides; while the lighter outer regions are likely dominated by non-covalent interactions, such as hydrogen bonds, electrostatic interactions, and π–π stacking between catechol groups and aromatic residues of peptides.

[0098] from Figure 2 The infrared spectrum of B indicates that PFp is in the range of 3000–3100 cm⁻¹. -1 Several weak peaks appear in the range, which are attributed to the stretching vibration of the unsaturated –CH (i.e., benzene ring –CH), originating from the benzene ring structure of PDA and PEP; PFp is in the range of 3500–3300 cm⁻¹. -1 It still shows a bimodal interval of about 140 cm -1 This characteristic is typical of amide A bands (N–H stretching vibrations). Secondly, at 1650 and 1520 cm⁻¹... -1 Two characteristic peaks appear at 1270 cm⁻¹, corresponding to amide I and II bands respectively (a combination of C=O-dominated composite vibrations and the superposition of C–N stretching and N–H in-plane bending). -1 The infrared characteristic peak at this location is attributed to the S=O stretching vibration; this peak appears in PFp and originates from the sulfonic acid group (or sulfate ester) introduced by the fucoidan itself. Meanwhile, at approximately 1050 cm⁻¹... -1 The presence of C–O–C infrared characteristic peaks indicates the presence of ether bonds in the PFp product, which is also attributed to the introduction of fucoidan.

[0099] from Figure 2 A comparison of the UV spectra of C shows that: the spectrum of fucoidan is relatively flat; after the addition of PEP, two peaks appear at 215 nm and 261 nm, which are attributed to the UV absorption of the amide and benzene ring, respectively; after further introduction of PAD, the UV absorption is significantly enhanced, indicating the introduction of more benzene rings and amide structures. In summary, it can be inferred that PEP and PDA have been successfully introduced into fucoidan to form PFp.

[0100] from Figure 2 As can be seen from D, the calcium ion-selective membrane (Ca) modified with PFp... 2+ The contact angle of PFp decreased from 94.13° to 23.28°, indicating that PFp has good hydrophilicity and further proving that PFp has been successfully modified onto Ca. 2+ -ISM surface.

[0101] In summary, PFp was successfully constructed into a cross-linked network structure, which combines excellent hydrophilicity, structural stability and good compatibility with calcium ion transport, providing a promising solution for antifouling strategies of membrane surface modification.

[0102] Example 5

[0103] Testing the antifouling performance of ISM surfaces modified with different antifouling materials

[0104] To evaluate the antifouling performance of PFp, *Escherichia coli* and *Staphylococcus aureus* were used as model strains. A combination of confocal laser scanning microscopy (CLSM), plate counting, scanning electron microscopy (SEM), and a multi-channel non-contact conductivity sensor (CCS) was employed to assess microbial attachment and growth kinetics. In short, Ca... 2+ -ISM / PFp and control samples were immersed in bacterial suspension (10 6 CFU mL -1 *Escherichia coli* or *Staphylococcus aureus* were cultured for 24 hours. After culture, samples were washed with PBS to remove loosely attached bacteria. Live / dead bacterial cells were observed using CLSM stained with SYTO 9 / PI. To assess bacterial adhesion, the membrane was immersed in vials containing 5 mL of PBS solution and sonicated for 5 minutes to separate surface-bound bacteria. The resulting bacterial suspension was diluted 1000-fold, and 10 μL aliquots were spread on LB agar and incubated overnight at 37°C for colony counting. For SEM imaging, samples were fixed with 2.5% glutaraldehyde and dehydrated with fractionated ethanol. For CCS analysis, the washed membrane was placed in 2 mL NMR tubes containing growth medium to monitor bacterial proliferation on different surfaces. The antifouling effect against *Chlorella* was also evaluated. 2+ -ISM / PFp and control samples were incubated overnight in Chlorella cultures, rinsed with deionized water, and examined under an optical microscope to assess algal adhesion. The results are as follows: Figure 3 As shown.

[0105] like Figure 3 As shown in A and 3B, in Ca 2+ A large number of live bacteria, including Escherichia coli and Staphylococcus aureus, were observed adhering to the surface of the ISM. In contrast, on the Ca... 2+ On the -ISM / pep-modified surface, the number of viable bacteria was significantly reduced, indicating that the introduced pep exerted effective bactericidal activity by killing adhered bacteria. However, dead bacteria residue was still visible on the surface, indicating a problem of residue accumulation after bacterial death. It is noteworthy that Ca... 2+The number of both viable and dead bacteria on the surface modified with -ISM / PFp was significantly reduced, indicating that PFp materials possess excellent antifouling properties. This performance improvement can be attributed to the triple defense effect of the PFp interface: antifouling, bactericidal, and antioxidant. Fucoidan is rich in hydroxyl and sulfate groups, which not only endows it with excellent hydrophilicity but also inhibits early bacterial adhesion and biofilm formation. The bactericidal properties of PFp were further confirmed by scanning electron microscopy (SEM).

[0106] like Figure 3 As shown in C, adhering to unmodified Ca 2+ The bacteria on the ISM retained their typical rod-shaped and spherical morphologies (Escherichia coli and Staphylococcus aureus), respectively. In contrast, Ca... 2+ Bacterial cells on the ISM / pep surface exhibited significant morphological damage, including cell membrane rupture and cell structure deformation, clearly demonstrating the bactericidal effect of PEP. Plate counting was performed to further quantify its antifouling properties.

[0107] like Figure 3 As shown in D, Ca 2+ The -ISM / PFp electrode exhibits the lowest number of colony-forming units (CFU), confirming its excellent antifouling ability.

[0108] Furthermore, this invention also evaluated the anti-algae properties of PFp. The growth kinetics of bacteria at different modified interfaces were analyzed using a non-contact conductivity sensor (CCS). Bacteria metabolize uncharged or weakly charged substrates (e.g., yeast extracts, peptones, and sugars) into highly charged products, including amino acids, aldehydes, ketones, acids, and other metabolites. This leads to an increase in the conductivity of the aqueous solution, allowing for indirect monitoring of bacterial growth. The x-axis represents culture time, and the y-axis represents the normalized apparent conductivity (NACV) value, expressed as voltage. Figure 3 As shown in E, Ca 2+ -ISM / PFp's NACV value is significantly lower than that of Ca. 2+ -ISM indicates extremely low bacterial adhesion and demonstrates the excellent antifouling properties of the PFp-modified electrode.

[0109] Figure 3F shows that, compared with unmodified Ca... 2+ Compared to -ISM, Chlorella has Ca 2+ The significantly reduced adhesion of the -ISM / PFp surface indicates that it has strong algicidal activity.

[0110] Example 6

[0111] Verification of GC / PANI / Ca using DPPH radical scavenging assay 2+ Oxidation properties of ISM / PFp materials

[0112] The specific steps are as follows:

[0113] Different concentrations of PFp (1-5 mg·mL) were compared. -1 ) and 2 mL 0.1 mmol·L -1 The DPPH ethanol solution was mixed and incubated in the dark at room temperature for 30 minutes. The absorbance was then recorded at 517 nm using a UV spectrophotometer. An ethanol solution of vitamin C was used as a positive control, and the results are as follows. Figure 5 As shown.

[0114] from Figure 4 The results show that as the PFp concentration (1-5 mg·mL) increases... -1 With increasing concentrations of PFp, the DPPH free radical scavenging rate showed a significant dose-dependent increase. More importantly, the scavenging capacity of PFp at the same concentration was highly similar to that of vitamin C, a recognized strong antioxidant; their curves almost completely overlapped, demonstrating excellent antioxidant capabilities.

[0115] Example 7

[0116] Electrochemical performance measurements were performed on the GC / PANI / Ca2+-ISM / PFp material. Electromotive force (EMF) measurements were conducted at room temperature using an electrochemical workstation employing a dual-electrode system. 2+ -ISM or GC / PANI / Ca 2+ The ISM / PFp electrode is used as the working electrode, and the Ag / AgCl electrode is used as the reference electrode. In the detection of Ca... 2+ Previously, all electrodes were pretreated at 1 mmol·L⁻¹ -1 Soak in calcium chloride solution for 12 hours.

[0117] First, to assess whether surface modification significantly reduces the analytical performance of the electrode, we systematically evaluated the effect of the PFp coating on the sensor response, and the results are as follows: Figure 5 As shown.

[0118] Figure 5 The results from A show that, under simulated seawater conditions (0.5 mol·L⁻¹), -1 Under NaCl conditions, we measured the GC / PANI / Ca ratio. 2+ -ISM and GC / PANI / Ca 2+ -ISM / PFp electrode at a concentration range of 10 -7 Up to 10 -1 mol L -1 The potential behavior in CaCl2 solution.

[0119] Figure 5The results of B show that the two electrodes at 10 -5 Up to 10 -1 mol·L -1 It exhibited a linear response across the entire concentration range, with a detection limit of 10 for the uncoated electrode. -5.10 mol L -1 The detection limit of the PFp-coated electrode is 10. -1 mol·L -5.09 This indicates that the introduction of the PFp antifouling layer has minimal impact on the sensor's sensitivity. This can be attributed to Ca... 2+ Ion recognition and transfer within selective membranes are controlled by thermodynamic equilibrium.

[0120] Example 8

[0121] By comparing bacterial growth curves at different modified interfaces, the antifouling ability of different interfaces can be detected.

[0122] This embodiment uses the GC / PANI / Ca prepared according to the present invention. 2+ -ISM / PFp electrode and GC / PANI / Ca 2+ The -ISM ​​electrode was immersed in seawater, artificial saliva, and artificial urine for up to 60 days. EMF measurements were performed at room temperature using an electrochemical workstation employing a dual-electrode system, consisting of GC / PANI / Ca. 2+ -ISM or GC / PANI / Ca 2+ The ISM / PFp electrode was used as the working electrode, and the Ag / AgCl electrode was used as the reference electrode. Their potential responses were systematically evaluated, and the results are as follows: Figure 6 As shown.

[0123] from Figure 6 It can be seen that, under the same conditions, GC / PANI / Ca 2+ -After immersion for 25 days, the potential response slope of the ISM electrode decreased significantly, from 28.52 ± 0.25, 28.21 ± 0.11, and 28.30 ± 0.21 mV dec in seawater, artificial saliva, and artificial urine, respectively. -1 Decreased to 19.20 ± 0.29, 19.60 ± 0.12 and 15.90 ± 0.37 mV dec -1 In contrast, GC / PANI / Ca 2+ The -ISM / PFp electrode exhibited excellent stability, maintaining 25.28 ± 0.47, 25.53 ± 0.28, and 26.01 ± 0.39 mV dec after immersion for 55 days. -1 The response slope, with initial values ​​of 27.91 ± 0.12, 27.90 ± 0.38, and 27.76 ± 0.35 mV dec.-1 .

[0124] Example 9

[0125] Detection of GC / PANI / Ca 2+ -ISM / PFp's actual detection performance in seawater

[0126] To evaluate GC / PANI / Ca 2+ -ISM / PFp electrode for detecting Ca in real samples 2+ To assess the performance, we filtered seawater collected from different areas along the Qingdao coast and performed EMF measurements at room temperature using an electrochemical workstation with a dual-electrode system, in which GC / PANI / Ca... 2+ The ISM / PFp electrode was used as the working electrode, and the Ag / AgCl electrode was used as the reference electrode. The results are shown in Table 1.

[0127] Table 1 GC / PANI / Ca 2+ -ISM / PFp's actual detection performance in seawater

[0128]

[0129] a Statistical parameters were obtained from three separate devices.

[0130] As can be seen from Table 1, the results summarized in Table 1 are in high agreement with the results of inductively coupled plasma atomic emission spectrometry (ICP-AES). This indicates that the sensor of the present invention is suitable for use in seawater containing Ca. 2+ Accurate detection.

Claims

1. A method for preparing an antifouling electrochemical sensor based on functional bactericidal peptides of fucoidan, characterized in that, The anti-fouling electrochemical sensor is GC / PANI / Ca. 2+ -ISM / PFp sensor; The method for preparing the anti-fouling electrochemical sensor includes the following steps: (1) After polishing the bare glassy carbon electrode GC with alumina powder, the electrode was thoroughly cleaned to obtain a polished glassy carbon electrode GC. (2) The polished glassy carbon electrode GC was placed in a polyaniline PANI solution and deposited using a chronoamperometry method to obtain a GC / PANI electrode; (3) Dissolve calcium ion carrier, sodium [3,5-bis(trifluoromethyl)phenyl]borate, polyvinyl chloride, and 2-nitrophenyl n-octyl ether in tetrahydrofuran and mix ultrasonically to prepare Ca 2+ Selective membrane mixed solution; (4) The Ca 2+ The selective membrane mixture solution was dropped onto the GC / PANI electrode and dried overnight to obtain GC / PANI / Ca. 2+ -ISM electrode; (5) In the GC / PANI / Ca 2+ - Incubate the fucoidan-functionalized bactericidal peptide PFp solution on the ISM electrode for 12-24 hours to obtain the antifouling electrochemical sensor GC / PANI / Ca. 2+ -ISM / PFp; In step (5), the fucoidan-functional bactericidal peptide PFp solution is prepared by the following method: (a) Add 5–10 mg of fucoidan, 3 mg of dopamine, and 2–5 mg of functional bactericidal peptide to 5–10 mL of 0.1 mol·L⁻¹. -1 In the MES buffer solution, shake or stir thoroughly until completely dissolved to form a mixed solution a; (b) Add 5-10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the mixed solution a, mix thoroughly to obtain mixed solution b; (c) Transfer the mixed solution b to a dialysis bag with a molecular weight cutoff of 3.5 kDa, and administer at 0.005 mol·L⁻¹ -1 The HCl solution was used as the external dialysis solution, and the solution was dialyzed at 4°C for 24-48 hours to obtain the fucoidan-functional bactericidal peptide PFp solution. The amino acid sequence of the functional bactericidal peptide is KFKFKFKF; The fucoidan is Fucus vesiculosus fucoidan.

2. According to the preparation method of claim 1, in step (2), the concentration of the polyaniline PANI solution is 1-3 mmol·L⁻¹. -1 ; The potential range of the chronoamperometry method is -2V to 2V, and the deposition time of the chronoamperometry method is 30-45 minutes.

3. The preparation method according to claim 2, characterized in that, In step (3), the mass-to-volume ratio of the calcium ion carrier, sodium [3,5-bis(trifluoromethyl)phenyl]borate, polyvinyl chloride, 2-nitrophenyl n-octyl ether and tetrahydrofuran is 2.5 mg: 1.2 mg: 66 mg: 135 mg: 1.5 mL.

4. The preparation method according to claim 3, characterized in that, In step (4), the Ca 2+ The selective membrane mixture solution was added in a volume of 90-100 μL. The GC / PANI / Ca 2+ The volume of fucoidan-functional bactericidal peptide PFp solution used for incubation on the ISM electrode is 10-50 μL.

5. The preparation method according to claim 4, characterized in that, The anti-fouling electrochemical sensor is an anti-fouling electrochemical sensor that inhibits the adhesion of Escherichia coli and Staphylococcus aureus, the colonization of Chlorella, and has antioxidant properties on the sensor surface. The anti-fouling electrochemical sensor is used for the detection of calcium ions in seawater.

6. An antifouling electrochemical sensor based on functional bactericidal peptides of fucoidan, characterized in that, The electrochemical sensor is a GC / PANI / Ca sensor with a glassy carbon electrode as the substrate, polyaniline as the solid conductive layer, a calcium ion selective membrane as the detection layer, and fucoidan-functional bactericidal peptide PFp as the antifouling layer. 2+ -ISM / PFp sensor; The anti-fouling electrochemical sensor is prepared by the preparation method according to any one of claims 1-5.

7. The anti-fouling electrochemical sensor according to claim 6, characterized in that, The anti-fouling electrochemical sensor is an anti-fouling electrochemical sensor that inhibits the adhesion of Escherichia coli and Staphylococcus aureus, the colonization of Chlorella, and has antioxidant properties on the sensor surface. The anti-fouling electrochemical sensor is used for the detection of calcium ions in seawater.

8. The application of the anti-fouling electrochemical sensor as described in claim 6 in the detection of calcium ions in seawater.

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