Covalent Cross-linking Method Research Articles

Microfluidic Assembly of Degradable, Stereocomplexed Hydrogel Microparticles.

Hydrogel microparticles (HMPs) have been investigated widely for their use in tissue engineering and drug delivery applications. However, translation of these highly tunable systems has been hindered by covalent cross-linking methods within microparticles. Stereocomplexation, a stereospecific form of physical cross-linking, provides a robust yet degradable alternative for creating translationally relevant HMPs. Herein, 4-arm polyethylene glycol (PEG) stars were used as macromolecular initiators from which oligomeric poly(l-lactic acid) (PLLA) was polymerized with a degree of polymerization (DPn) of 20 on each arm. Similarly, complementary propargyl-containing ABA cross-linkers with enantiomeric poly(d-lactic acid) (PDLA) segments (DPn = 20) on each arm. Droplets of these gel precursors were formed via a microfluidic organic-in-oil-in-water system where microparticles self-assembled via stereocomplexation and were stabilized after precipitation in deionized water. By varying the flow rate of the dispersed phase, well-defined microparticles with diameters of 33.7 ± 0.5, 62.4 ± 0.6, and 105.7 ± 0.8 μm were fabricated. Gelation due to stereocomplexation was confirmed via wide-angle X-ray scattering in which HMPs exhibited the signature diffraction pattern of stereocomplexed PLA at 2θ = 12.2, 21.2, 24.2°. Differential scanning calorimetry also confirmed stereocomplexation by the appearance of a crystallization exotherm (Tc = 37 °C) and a high-temperature endotherm (Tm = 159 °C) that does not appear in the homocrystallization of PLLA or the hydrogel precursors. Additionally, the propargyl handle present on the cross-linker allows for pre- or post-assembly thiol-yne "click" functionalization as demonstrated by the addition of thiol-containing fluorophores to the HMPs.

Effects of polyphenol and gelatin types on the physicochemical properties and emulsion stabilization of polyphenol-crosslinked gelatin conjugates

Herein, six types of polyphenol-crosslinked gelatin conjugates (PGCs) with ≥ two gelatin molecules were prepared using a covalent crosslinking method with two types of polyphenols (tannic acid and caffeic acid) and three types of gelatins (bovine bone gelatin, cold water fish skin gelatin, and porcine skin gelatin) for the emulsion stabilization. The structural and functional properties of the PGCs were dependent on both polyphenol and gelatin types. The storage stability of the conjugate-stabilized emulsions was dependent on the polyphenol crosslinking, NaCl addition, and heating pretreatment. In particular, NaCl addition promoted the liquid-gel transition of the emulsions: 0.2 mol/L > 0.1 mol/L > 0.0 mol/L. Moreover, NaCl addition also increased the creaming stability of the emulsions stabilized by PGCs except tannic acid-crosslinked bovine bone gelatin conjugate. All the results provided useful knowledge on the effects of molecular modification and physical processing on the properties of gelatins.

Multifunctional Suture Coating for Combating Surgical Site Infections and Mitigating Associated Complications.

Despite advancements in preventive measures and hospital protocols, surgical site infections (SSIs) remain a significant concern following surgeries. Sutures, commonly used for wound closure, can serve as a platform for microbial adherence and contamination, leading to extensive debridement and recurrent antibiotic therapy. The emergence of drug resistance and the formation of biofilms on sutures have further complicated the management of SSIs. Drug-eluting sutures incorporating biocides like triclosan have limitations due to uncontrolled release and associated toxicity. Therefore, there is a need for alternative approaches to impart antimicrobial properties to sutures. In this study, we present a one-step covalent cross-linking method to coat surgical sutures with an antimicrobial small molecule, quaternary benzophenone-based antimicrobial (QSM). Additionally, the sutures are dip-coated with ibuprofen, a nonsteroidal anti-inflammatory drug with analgesic properties. The coated sutures maintained their morphological and tensile properties after in vivo implantation. The antimicrobial coating demonstrated efficacy against a broad-spectrum pathogens, including drug-resistant bacteria and fungi. The optimized formulation retained its biodegradability in vivo. Furthermore, the coated sutures exhibited ∼3 log reduction in methicillin-resistant Staphylococcus aureus (MRSA) burden in a subcutaneous implantation mouse model. Overall, this multifunctional coating provides antimicrobial properties to surgical sutures while preserving their mechanical integrity and biodegradability. These coated sutures have the potential to address the challenge of SSIs and contribute to improved surgical outcomes.

The structural and functional properties of hemp protein isolate-epigallocatechin-3-gallate biopolymer covalent complex during heating.

It is well known that hemp proteins have the disadvantages of poor solubility and poor emulsification. To improve these shortcomings, an alkali covalent cross-linking method was used to prepare hemp protein isolate-epigallocatechin-3-gallate biopolymer (HPI-EGCG) and the effects of different heat treatment conditions on the structure and emulsifying properties of the HPI-EGCG covalent complex were studied. The secondary and tertiary structures, solubility, and emulsification ability of the HPI-EGCG complexes were evaluated using particle size, zeta potential, circular dichroism (CD), and fluorescence spectroscopy indices. The results showed that the absolute value of zeta potential of HPI-EGCG covalent complex was the largest, 18.6 mV, and the maximum binding amount of HPI to EGCG was 29.18 μmol g-1 . Under heat treatment at 25-35 °C, the α-helix content was reduced from 1.87% to 0%, and the β-helix content was reduced from 82.79% to 0% after the covalent binding of HPI and EGCG. The solubility and emulsification properties of the HPI-EGCG covalent complexes were improved significantly, and the emulsification activity index (EAI) and emulsion stability index (ESI) were increased by 2.77-fold and 1.21-fold, respectively. A new HPI-EGCG covalent complex was developed in this study to provide a theoretical basis for the application of HPI-EGCG in food industry. © 2023 Society of Chemical Industry.

Genipin versus Ferric Chloride cross-linked unmodified Gum Arabic/Chitosan/nano-Hydroxyapatite nanocomposite hydrogels as potential scaffolds for bone regeneration

Ferric chloride (FeCl3) and Genipin were utilized as cross-linkers to create two types of nanocomposite hydrogels through physical and covalent cross-linking methods, respectively. The hydrogels were composed of unmodified Gum Arabic (GA), Chitosan (Ch), and natural nano-Hydroxyapatite (nHA) using an acrylic acid solvent. Both the natural nHA and the FeCl3 vs. genipin cross-linked GA/Ch/nHA nano-composite hydrogels were prepared and characterized using various in vitro and in vivo analysis techniques. The use of FeCl3 and genipin cross-linkers resulted in the formation of novel hydrogels with compressive strengths of (15.43–22.20 MPa), which are comparable to those of natural cortical bone. In vivo evaluation was conducted by creating calvarial defects (6 mm) in Sprague–Dawley male rats. The results showed the formation of new, full-thickness bone at the implantation sites in all groups, as evidenced by digital planar tomography and histological staining with Hematoxylin and Eosin stain (H & E). Additionally, the use of genipin as a cross-linker positively affected the hydrogel's hydrophilicity and porosity. These findings justify further investigation into the potential of these nanocomposite hydrogels for bone regeneration applications.

A Skin-Inspired Multifunctional Conductive Hydrogel with High Stretchable, Adhesive, Healable, and Decomposable Properties for Highly Sensitive Dual-Sensing of Temperature and Strain.

Developing smart hydrogels with excellent physicochemical properties and multi-sensing capabilities for various simulation of human skin's functions still remains a great challenge. Here, based on simple and convenient one-step covalent cross-linking method enhanced by dynamic RS-Ag interactions, a skin-inspired multifunctional conductive hydrogel with desirable physicochemical properties (including high stretchability, self-adhesion, self-healing, decomposition and removability) is developed for highly sensitive dual-sensing of temperature and strain. Benefiting from the synergistic action of multiple hydrogen bonds, RS-Ag bonds and S-S bonds, the gel exhibited a novel thermosensitive mechanism. The prepared hydrogels exhibited extremely high mechanical properties (maximum tensile strength of 0.35MPa, elongation at break nearly 1800%, compressive stress over 4.43MPa), excellent self-healing (96.82% (stress), 88.45% (temperature), 73.89% (mechanical property)), decomposition (the molecular weight after decomposition is below 700) and self-adhesion (enhanced contact with the material interface). In addition, this conductive hydrogel could also simultaneously achieve highly sensitive temperature-sensing (TCR: 10.89) and stress-sensing (GF: 1.469). As a proof-to-concept, the hydrogel displayed superior capability for simulation of human skin to perception of touch, pressure and ambient temperature simultaneously, indicating promising applications in the fields of wearable devices, personal health care, and human-machine interfaces.

Efficient decolorization of melanoidin in raw molasses wastewater by thermophilic esterase in actual extreme conditions

This work was developed to explore the versatility of thermophilic esterase for decolorizing raw molasses wastewater at high temperature and acidic pH. Combining covalent crosslinking method with deep eutectic solvent, a thermophilic esterase from Pyrobaculum calidifontis was immobilized on chitosan/macroporous resin composite carrier. The application of this immobilized thermophilic esterase eliminated 92.35% of colorants in raw molasses wastewater, achieving maximal decolorization efficiency across all the enzymes tested. Strikingly, this immobilized thermophilic esterase was capable of engaging in continuous activity for a 5-day period while removing 76.23% of pigments from samples. It effectively and continuously eliminated BOD5 and COD, effectively and directly facilitating raw molasses wastewater decolorization under extreme conditions more readily than control group. In addition, this thermophilic esterase was believed to achieve decolorization through an addition reaction that disrupted conjugated system of melanoidins. Together, these results highlight an efficient and practical means of achieving enzyme-based molasses wastewater decolorization.

Photocrosslinkable natural polymers in tissue engineering.

Natural polymers have been widely used in scaffolds for tissue engineering due to their superior biocompatibility, biodegradability, and low cytotoxicity compared to synthetic polymers. Despite these advantages, there remain drawbacks such as unsatisfying mechanical properties or low processability, which hinder natural tissue substitution. Several non-covalent or covalent crosslinking methods induced by chemicals, temperatures, pH, or light sources have been suggested to overcome these limitations. Among them, light-assisted crosslinking has been considered as a promising strategy for fabricating microstructures of scaffolds. This is due to the merits of non-invasiveness, relatively high crosslinking efficiency via light penetration, and easily controllable parameters, including light intensity or exposure time. This review focuses on photo-reactive moieties and their reaction mechanisms, which are widely exploited along with natural polymer and its tissue engineering applications.

Fabrication of hydrogel microspheres via microfluidics using inverse electron demand Diels-Alder click chemistry-based tetrazine-norbornene for drug delivery and cell encapsulation applications.

Microfluidic on-chip production of polymeric hydrogel microspheres (MPs) can be designed for the loading of different biologically active cargos and living cells. Among different gelation strategies, ionically crosslinked microspheres generally show limited mechanical properties, meanwhile covalently crosslinked microspheres often require the use of crosslinking agents or initiators with limited biocompatibility. Inverse electron demand Diels Alder (iEDDA) click chemistry is a promising covalent crosslinking method with fast kinetics, high chemoselectivity, high efficiency and no cross-reactivity. Herein, in situ gellable iEDDA-crosslinked polymeric hydrogel microspheres are developed via water-in-oil emulsification (W/O) glass microfluidics. The microspheres are composed of two polyethylene glycol precursors modified with either tetrazine or norbornene as functional moieties. Using a single co-flow glass microfluidic platform, homogenous MPs of sizes 200-600 μm are developed and crosslinked within 2 minutes. The rheological properties of iEDDA crosslinked bulk hydrogels are maintained with a low swelling degree and a slow degradation behaviour under physiological conditions. Moreover, a high-protein loading capacity can be achieved, and the encapsulation of mammalian cells is possible. Overall, this work provides the possibility of developing microfluidics-produced iEDDA-crosslinked MPs as a potential drug vehicle and cell encapsulation system in the biomedical field.

Applications of Designed Alpha‐Helical Metallopeptides

A variety of strategies have been employed to generate alpha‐helical peptides to mimic native protein‐protein interactions. Here, we present applications of a method that induces secondary structure in short peptides using engineered metal‐binding sites. We propose that the incorporation of metal ions to create helical peptides provides additional benefits that the more widely utilized covalent crosslinking methods lack. We have created a peptide system designed to bind ribose 5‐phosphate isomerase B (RpiB), a protein target in developing treatments for Chagas disease caused by Trypanosoma cruzi. In this system, we have compared peptide structure and stability when using our metal‐crosslinking system to hydrocarbon, lactam, and thioether staples across 1‐ or 2‐turns of an alpha‐helix. We have also measured the ability of each of these peptide constructs to bind RpiB and prevent homodimerization, which is required for parasitic activity. Further, we have studied whether metal‐bound peptides are better able to cross cellular membranes and enter the cytoplasm of mammalian cells compared to metal‐free peptides, as has been previously suggested in the literature, and deduced the impact of metal ion charge, identity, and secondary structure in enhanced permeability.

Fabrication of heat-treated soybean protein isolate-EGCG complex nanoparticle as a functional carrier for curcumin

In this study, soybean protein isolate (SPI) was first heat-treated to yield modified SPI (HSPI). Then SPI- and HSPI-epigallocatechin-3-gallate (EGCG) biopolymers (SPI-E and HSPI-E) were fabricated using an alkali covalent crosslinking method and their abilities to function as novel delivery carriers to load curcumin (Cur) were investigated. The particle size of loaded-Cur SPI-E and HSPI-E complex nanoparticles (SPI-E-C and HSPI-E-C) were all reduced and the zeta-potentials were all negative. FTIR and fluorescence spectra analysis indicated the presence of hydrogen bondings, hydrophobic interactions, and electrostatic interactions were the driving force for the formation of the carrier-Cur complex. Moreover, compared with SPI-E, HSPI-E had a high loading rate of Cur, improved bioaccessibility and scavenging capacity, smaller size, thinner sheet shape morphology, and stronger binding affinity. In its protective capacity as a delivery carrier, HSPI-E significantly improved the thermal stability, acid stability and controlled release characteristics of Cur, thereby overcoming the instability of a single protein carrier and possessing superior properties. These findings indicate that the HSPI-E polymer is a favorable carrier for delivering Cur and suit for development as a delivery polymer carrier to protect hydrophobic nutraceuticals and drugs with high-loading rate-performance and health-related functions without the use of crosslinkers.

Tannic acid: a crosslinker leading to versatile functional polymeric networks: a review.

With the thriving of mussel-inspired polyphenol chemistry as well as the demand for low-cost analogues to polydopamine in adhesive design, tannic acid has gradually become a research focus because of its wide availability, health benefits and special chemical properties. As a natural building block, tannic acid could be used as a crosslinker either supramolecularly or chemically, ensuring versatile functional polymeric networks for various applications. Up to now, a systematic summary on tannic-acid-based networks has still been waiting for an update and outlook. In this review, the common features of tannic acid are summarized in detail, followed by the introduction of covalent and non-covalent crosslinking methods leading to various tannic-acid-based materials. Moreover, recent progress in the application of tannic acid composites is also summarized, including bone regeneration, skin adhesives, wound dressings, drug loading and photothermal conversion. Above all, we also provide further prospects concerning tannic-acid-crosslinked materials.

Development and characterization of enzymatic biosensor based on lipase from porcine pancreas for propylparaben detection

PurposeThe purpose of this paper is to study the immobilization of porcine pancreatic lipase (PPL), in an organic matrix by a covalent cross-linking method to sense propylparaben (PP) present in aqueous solution.Design/methodology/approachPPL immobilization was performed by the covalent cross-linking method, using bovine serum albumin (BSA) in the presence of saturated glutaraldehyde vapor (GA). The preparation of the enzymatic membrane involves the incorporation of porcine pancreatic lipase (PPL), bovine serum albumin (BSA) and glycerol into a phosphate buffer solution (PBS). Characterization of this sensor was performed by impedance spectroscopy (EIS) and scanning electron microscope (SEM). The effect of experimental conditions such as PPL activity, potential, scan rate, PP concentration, pH and presence of interfering elements were studied by cyclic voltammetry.FindingsUnder the optimal experimental conditions, a number of significant factors were optimized. The method exhibited good linearity in the range of 10–14to 10–9 mol/L with a good correlation coefficient of 0.957, detection limit (LOD) of 3.66 × 10–15 mol/L and high sensitivity of 1.086 mA mol−1L. The authors also obtained a very good coverage rate of the surface equal to 91.44%, and hydrolytic activity of lipase is evaluated to 26.64 mmol min−1. The stability and the interference were also evaluated. The equivalent circuit used to explain the electrochemical behavior of modified electrode is a Randle circuit.Practical implicationsThe main application of biosensors is the detection of biomolecules that are either indicators of a disease. For example, electrochemical biosensing techniques can be used as clinical tools to detect breast tumors, because these compounds (PP) were found in breast tumors.Originality/valueThe result registered in this paper indicates that the developed sensor is an efficient, fast, simple and inexpensive analytical tool that can be used for the analysis of water containing PP.

Dual Cross-Linked Chitosan/PVA Hydrogels Containing Silver Nanoparticles with Antimicrobial Properties.

Stable chitosan/PVA-based hydrogels were obtained by combining covalent and physical cross-linking methods. As covalent cross-linkers, epoxy agents with different chain lengths were used, while freeze–thaw cycles were applied for additional physical cross-linking. The chemical structure of the hydrogel was examined by FTIR spectroscopy whereas the morphology was analyzed by SEM, showing well-defined pores with dimensions of around 50 μm in diameter. It was proved that gel fraction and the network morphology were deeply influenced by the synthesis conditions. Chitosan/PVA hydrogel showed a relative high swelling rate, reaching equilibrium in the first hour. The values obtained for the elastic modulus were relatively low (3–30 kPa); as a result, these hydrogels are soft and very flexible, and are ideal candidates for medical applications as wound or oral dressings. In addition, the natural antimicrobial activity of chitosan was enhanced by in situ generation of silver nanoparticles (AgNPs) under UV irradiation. The total amount of Ag from hydrogel was determined by elemental analyses and its crystalline state was confirmed by XRD. The CS/PVA hydrogels entrapped with AgNPs exhibited high inhibitory activity against S. aureus and K. pneumonia. The vitality tests confirmed the lack of cytotoxicity of CS/PVA hydrogels without and with AgNPs.

Crosslinking Strategies for the Microfluidic Production of Microgels.

This article provides a systematic review of the crosslinking strategies used to produce microgel particles in microfluidic chips. Various ionic crosslinking methods for the gelation of charged polymers are discussed, including external gelation via crosslinkers dissolved or dispersed in the oil phase; internal gelation methods using crosslinkers added to the dispersed phase in their non-active forms, such as chelating agents, photo-acid generators, sparingly soluble or slowly hydrolyzing compounds, and methods involving competitive ligand exchange; rapid mixing of polymer and crosslinking streams; and merging polymer and crosslinker droplets. Covalent crosslinking methods using enzymatic oxidation of modified biopolymers, photo-polymerization of crosslinkable monomers or polymers, and thiol-ene “click” reactions are also discussed, as well as methods based on the sol−gel transitions of stimuli responsive polymers triggered by pH or temperature change. In addition to homogeneous microgel particles, the production of structurally heterogeneous particles such as composite hydrogel particles entrapping droplet interface bilayers, core−shell particles, organoids, and Janus particles are also discussed. Microfluidics offers the ability to precisely tune the chemical composition, size, shape, surface morphology, and internal structure of microgels by bringing multiple fluid streams in contact in a highly controlled fashion using versatile channel geometries and flow configurations, and allowing for controlled crosslinking.

Enzymatic and Nonenzymatic Conjugates of Lactoferrin and (-)-Epigallocatechin Gallate: Formation, Structure, Functionality, and Allergenicity.

The impact of covalent attachment of (-)-epigallocatechin gallate (EGCG) to lactoferrin (LF) on the structure, morphology, functionality, and allergenicity of the protein was studied. These polyphenol-protein conjugates were formed using various enzymatic (laccase- and tyrosinase-catalyzed oxidation) and nonenzymatic (free radical grafting and alkali treatment) methods. The preparation conditions for forming the enzymatic conjugates were optimized by exploring the influence of order-of-addition effects: protein, polyphenols, and enzymes. The total phenol content of the LF-EGCG conjugates was quantified using the Folin-Ciocalteu method. The nature of the conjugates formed was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Fourier transform infrared (FTIR) spectroscopy analyses. These studies showed that enzymatic cross-linking was a highly effective means of forming LF-EGCG conjugates. Analysis of these conjugates using various spectroscopic methods showed that conjugation to EGCG changed the molecular structure of LF. Atomic force microscopy showed that the four covalent cross-linking methods affected the size and morphology of these LF-EGCG conjugates formed. The antioxidant activity and emulsifying stability of LF were significantly improved by conjugation to EGCG. Finally, an enzyme-linked immunosorbent assay (ELISA) and a western blot assay indicated that conjugation of EGCG reduced the binding capacity of LF to immunoglobulin E (IgE) and immunoglobulin G (IgG), which is consistent with a decrease in allergenicity. Overall, this study suggests that LF-EGCG conjugates formed using enzymatic or nonenzymatic methods have potential applications as functional ingredients in foods.

Nanocellulose aerogel for highly efficient adsorption of uranium (VI) from aqueous solution

Cellulose nanofibers (CNFs) aerogel was prepared via simple covalent crosslinking and freeze-drying method. The porous cellulose aerogel possessed high specific surface area and high metal-chelating capacity, which showed fast adsorption kinetics and high adsorption capacity (440.60 mg g−1) in static uranium adsorption process. In the dynamic filtration system, the maximum adsorption capacity reached 194 mg g−1 with the initial concentration of 10 mg L−1. In addition, the CNFs aerogel possessed excellent selectivity and good regeneration ability for uranium adsorption. The integrated analyses of attenuated total reflection Fourier transform infrared (ATR-FTIR), X-Ray photoelectron spectroscopy (XPS) and extended X-ray absorption fine structure (EXAFS) suggested that the predominant UO22+ species formed inner-sphere surface complexes with two active carboxyl groups in the coordination model. This strategy may provide a sustainable route for development of efficient biomass-based adsorbents for selective uranium removal from aqueous solution.

A mechanically enhanced metal-organic framework/PDMS membrane for CO2/N2 separation

Interface compatibility is crucial to performance and robustness of nanocomposite membranes for molecular separation. In this work, a covalent crosslinking method was exploited to fabricate polydimethylsiloxane/metal-organic-frameworks (PDMS/MOFs) mixed matrix membranes with improved interfacial compatibility. Zirconium MOF particles (UiO-66-NH2) were grafted with methoxysilane groups, which further formed covalent crosslinks with silanol terminated PDMS matrix. The strengthened interfacial compatibility prevented sedimentation and aggregation of MOF particles in PDMS precursor solution during the solvent evaporation process, thus the composites showed good membrane formation ability at high MOFs loading (60 wt%). Tensile strength of the hybrid membrane is 2.8 times improved while retaining good flexibility. Moreover, the CO2/N2 selectivity and CO2 permeability of the PDMS composite membrane incorporated with 40 wt% UiO-66-MS particles are improved by 1.3 times and 0.7 times, respectively.

HKUST-1 modified ultrastability cellulose/chitosan composite aerogel for highly efficient removal of methylene blue

A novel composite HKUST-1/cellulose/chitosan aerogel (HKUST-1/CCSA) as an efficient adsorbent with hierarchical pores was prepared through a facile in situ growth way combining covalent cross-linking, vacuum freeze-drying, and solvothermal methods. By incorporating with cellulose (CE), covalently cross-linked cellulose (CE)/chitosan (CS) composite aerogel exhibits good stability, maintaining fine morphology and structures in acidic solutions under solvothermal conditions. Meantime, a high content of CS is beneficial to enhancing the growth of HKUST-1. Finally, the mass loading ratio of HKUST-1 is as high as 42.54 % in HKUST-1/CCSA. The BET specific surface area of HKUST-1/CCSA reaches 457.75 m2 g−1, which is much larger than that of CCSA (9.74 m2 g−1). HKUST-1/CCSA was applied to remove methylene blue with high adsorption capacity (526.3 mg g−1) and good recycling capability. This strategy can provide an effective and facile pathway to prepare ultra-stable polysaccharide-based composite aerogel with high specific surface area and hierarchical pores, branching out more applications in pollutant treatment fields.

Multi-layered enzyme coating on highly conductive magnetic biochar nanoparticles for bisphenol A sensing in water

This work introduced stable immobilization of tyrosinase (TYR) enzymes onto the highly conductive magnetic biochar nanoparticles with carboxyl functionality to construct sensitive and reusable electrochemical biosensor for bisphenol A (BPA) detection. The newly synthesized biochar nanoparticles (BCNPs) from biomass had outstanding electrochemical activity, after functionalization with carboxylic functional groups and magnetic nanoparticles (Mag-BCNPs-COOH), it was used for multi-layered TYR immobilization with covalent attachment, precipitation and cross-linking method (ML-TYR/Mag-BCNPs-COOH). The BPA sensing system was simply constructed by directly attaching the biocatalyst of ML-TYR/Mag-BCNPs-COOH onto a glassy carbon electrode with magnet force. Owning to great properties of the ML-TYR/Mag-BCNPs-COOH, such as high stability, biocatalytic activity and conductivity, the BPA sensing signal was significantly enhanced. And the lowest detection limit of 2.78 nM with linear ranges from 0.01 to 1.01 μM was achieved. The modified electrode could be reused at least eight cycles without reducing the sensing signal, and could be used for the real environmental water detection.