Covalent Immobilization Research Articles

Covalent Immobilization of Cellulase Enzyme on Chitosan and Eudragit S-100 Biopolymers for Recovery and Reusability in Denim Fading Application

Covalent Immobilization of Cellulase Enzyme on Chitosan and Eudragit S-100 Biopolymers for Recovery and Reusability in Denim Fading Application

Synthesis of Ferrocene‐Containing Schiff Bases Based on 3‐Aminopropyltriethoxysilane Oligomers for Covalent Modification of Glass Surfaces and Creation of Soft Magnetic Materials

ABSTRACTA ferrocene‐containing oligoorganosiloxane with a number‐average degree of polymerization of 8.8 and a number‐average molecular weight of 3200 is synthesized by hydrolytic condensation of 3‐aminopropyltriethoxysilane followed by chemical modification of the resulting oligomer with acetylferrocene. Its structure is characterized by MALDI‐TOF, NMR and IR spectroscopy. 1H NMR spectroscopy shows the predominance of more thermodynamically stable units containing anti‐configuration Schiff bases. Using IR spectroscopy as well as experimental determination of surface energy and its polar (acid–base) and dispersion components, the covalent immobilization of ferrocene‐containing oligoorganosiloxane on a glass surface after heating at 110°C is shown. The formed coating provides hydrophobization, acid–base indifference of the glass surface and serves as a precursor for the formation of magnetically soft materials after pyrolysis in argon already at 350°C. The main stages of ferrocene‐containing oligoorganosiloxane thermal destruction in an inert atmosphere and the formation of a mixture of iron and silicon oxides during its thermal oxidative destruction are established by combination of TGA/DTA, IR spectroscopy and elemental analysis. The proposed approach opens up new possibilities for functionalizing silicates surfaces, creating magnetic glasses, as well as regulating surface energy and its components.

Antimicrobial Peptide SAAP-148-Functionalized Hydrogels from Photocrosslinkable Polymers with Broad Antibacterial Activity.

Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics for treating skin wound infections. Nonetheless, their short half-life in biological environments restricts clinical applicability. Covalent immobilization of AMPs onto suitable substrates offers a comprehensive solution, creating contact-killing surfaces with long-term functionality. Here, a copolymer of poly[(hydroxy ethyl acrylamide)-co-(4-benzophenone acrylamide)-co-(pentafluorophenyl acrylate)-co-(ECOSURF EH-3 acrylate)], in short poly(HEAAm-co-BPAAm-co-PFPA-co-EH3A), is synthesized by free radical polymerization. Subsequent modification of active ester groups with the amine groups of SAAP-148, results in a copolymer, that is non-cytotoxic to human lung fibroblasts. UV photocrosslinking of the benzophenone units yields a polymer network that forms a hydrogel after swelling with aqueous medium. Both the SAAP-148-modified polymer in solution and the photocrosslinked hydrogels show good antimicrobial activity against strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, including multidrug-resistant strains, frequently found in wound infections. The covalent attachment of SAAP-148 prevents leaching, ensuring sustained antimicrobial activity for at least 48 h in diluted human blood plasma and 14 days in PBS. This prolonged retention of antimicrobial activity in human blood plasma significantly enhances its clinical potential. Overall, this study shows the potential of the AMP-functionalized photocrosslinkable polymer as antimicrobial wound dressings, providing an effective alternative to antibiotics.

Immobilization of laccase on Fe3O4@SiO2 core@shell magnetic nanoparticles for methylene blue biodegradation

Here we report the preparation and characterization of novel enzyme supports based on silica-coated Fe3O4 magnetic nanoparticles. These nanomaterials were modified at their outer silica surface with isocyanate, trimethylammonium and β-cyclodextrin moieties to immobilize laccase from Trametes versicolor through covalent, electrostatic and supramolecular interactions, respectively, with protein immobilization yields ranging from 21.7% to 53.5%. The effect of the immobilization approach on the activity, optimal working conditions, stability and reusability of the resulting biocatalysts were studied. Best results were achieved for native and adamantane-modified laccase supramolecularly immobilized on β-cyclodextrin bearing supports in terms of their catalytic properties, showing 18.0 U and 14.0 U of immobilized laccase activity per gram of support. However, high thermal stability was observed for the enzyme covalently immobilized on isocyanate-modified nanoparticles, with 14.8-fold increase in the half-life time at 65ºC in comparison with native laccase. Best reusability properties were also achieved by covalent immobilization, retaining over 88% of the initial catalytic activity after 13 cycles of magnetic reuses. All enzyme derivatives were evaluated for the catalytic degradation of methylene blue as pollutant model, showing significant reduction of the dye. In special, a 68-fold increase in the removal efficacy was observed for covalently immobilized enzyme compared to the free laccase. These results suggest high potential application of these biocatalysts in wastewater treatment.

Micro-fluidic covalent immobilization of multi-gradient RGD peptides on a gelatin surface for studying endothelial cell migration.

Collective endothelial migration is a hallmark of wound healing, which is regulated by spatial concentration gradients of extracellular biochemical factors. Arginine-glycine-aspartate (RGD) peptides play a vital role in regulating cell migration through specific binding to integrins. In this study, a micro-fluidic technology combined with a photopolymerization technique is developed to create gelatin methacryloyl (GelMA)-based substrates with various concentration gradients of RGD peptides. The capability of generating linear and nonlinear RGD concentration gradients was quantitatively verified through numerical simulation and immunohistochemical quantitative experiments. The results of the concentration gradients show a strong concurrence between the immunohistochemical quantification experiments and numerical simulations. Furthermore, endothelial migration experiments were conducted with various concentration gradients of RGD peptides. We have observed that endothelial cells on the surface of gels with a linear concentration gradient exhibit a larger cell area, a longer cell perimeter, and more stress fiber density. Furthermore, the cells demonstrate directional alignment and migration towards regions with a higher RGD concentration. High concentration gradients significantly enhance endothelial cell migration, consistent with observations on surfaces of gels with nonlinear concentration gradients. In brief, we proposed a simple and effective micro-fluidic photopolymerization technique capable of generating diverse concentration gradients of RGD and probing their effects on cell migration. The results suggest that regulating the RGD peptide concentration gradients can alter the migration of endothelial cells, showing potential for promoting wound healing.

A bioinspired porous and electroactive reduced graphene oxide hydrogel based biosensing platform for efficient detection of tumor necrosis factor-α.

Oral cancer is one of the leading cancer types, which is frequently diagnosed at an advanced stage, giving patients a poor prognosis and fewer therapeutic choices. To address this gap, exploiting biosensors utilizing anti-biofouling hydrogels for early-stage oral cancer detection in non-invasive body fluids is gaining utter importance. Herein, we have demonstrated the fabrication of an innovative electrochemical immunosensor for the rapid, label-free, non-invasive, and affordable detection of tumor necrosis factor-α (TNF-α), a biomarker associated with oral cancer progression in artificial saliva samples. The gold screen-printed electrodes (gSPEs) are modified with a green synthesized porous and electroactive reduced graphene oxide (rGO) hydrogel utilizing L-cystine (L-cys) as both in situ reducing and surface functionalization agent, followed by covalent immobilization of anti-TNF-α and blocking of residual sites with bovine serum albumin (BSA) to fabricate the BSA/anti-TNF-α/L-cys_rGO hydrogel/gSPE immunosensing platform. The fabricated platform demonstrates excellent performance, with a low limit of detection of 1.20 pg mL-1, a broad linear range from 1 to 200 pg mL-1, and a high sensitivity of 2.10 μA pg-1 mL cm-2 carried out with differential pulse voltammetry (DPV) technique. Moreover, it exhibits specificity towards TNF-α, even in the presence of potential interferents and other cancer biomarkers. Besides, the biosensor showed good reproducibility and repeatability with a relative standard deviation (%RSD) of 5.11% and 1.85%, respectively. Thus, integrating the L-cys_rGO hydrogel in the immunosensor design offers enhanced performance, paving the way for its application in early-stage oral cancer diagnosis.

Covalent Lysozyme Immobilization on Enzymatic Cellulose Nanocrystals.

Nanostructured materials represent promising substrates for biocatalyst immobilization and activation. Cellulose nanocrystals (CNCs), accessible from waste and/or renewable sources, are sustainable and biodegradable, show high specific surface area for anchoring a high number of enzymatic units, and high thermal and mechanical stability. In this work, we present a holistic enzyme-based approach to functional antibacterial materials by bioconjugation between the lysozyme from chicken egg white and enzymatic cellulose nanocrystals. The neutral CNCs were prepared by endoglucanase hydrolysis from Avicel. We explore the covalent immobilization of lysozyme on enzymatic CNCs and on their TEMPO oxidized derivatives (TO-CNCs), comparing immobilization yields, material properties, and enzymatic activities. The materials were characterized by X-ray diffractometry (XRD), attenuated total reflectance Fourier Transform infrared spectroscopy (ATR-FTIR), bicinchoninic acid (BCA) assay, field-emission scanning electron microscopy (FE-SEM) and dynamic light scattering (DLS). We demonstrate the higher overall efficiency of the immobilization process carried out on TO-CNCs, based on the success of covalent bonding and on the stability of the isolated bioconjugates.

Enhanced Electrochemiluminescence of Luminol and-Dissolved Oxygen by Nanochannel-Confined Au Nanomaterials for Sensitive Immunoassay of Carcinoembryonic Antigen.

Simple development of an electrochemiluminescence (ECL) immunosensor for convenient detection of tumor biomarker is of great significance for early cancer diagnosis, treatment evaluation, and improving patient survival rates and quality of life. In this work, an immunosensor is demonstrated based on an enhanced ECL signal boosted by nanochannel-confined Au nanomaterial, which enables sensitive detection of the tumor biomarker-carcinoembryonic antigen (CEA). Vertically-ordered mesoporous silica film (VMSF) with a nanochannel array and amine groups was rapidly grown on a simple and low-cost indium tin oxide (ITO) electrode using the electrochemically assisted self-assembly (EASA) method. Au nanomaterials were confined in situ on the VMSF through electrodeposition, which catalyzed both the conversion of dissolved oxygen (O2) to reactive oxygen species (ROS) and the oxidation of a luminol emitter and improved the electrode active surface. The ECL signal was enhanced fivefold after Au nanomaterial deposition. The recognitive interface was fabricated by covalent immobilization of the CEA antibody on the outer surface of the VMSF, followed with the blocking of non-specific binding sites. In the presence of CEA, the formed immunocomplex reduced the diffusion of the luminol emitter, resulting in the reduction of the ECL signal. Based on this mechanism, the constructed immunosensor was able to provide sensitive detection of CEA ranging from 1 pg·mL-1 to 100 ng·mL-1 with a low limit of detection (LOD, 0.37 pg·mL-1, S/N = 3). The developed immunosensor exhibited high selectivity and good stability. ECL determination of CEA in fetal bovine serum was achieved.

Precise Immobilization Strategy Combined with Rational Design to Improve β-Agarase Stability.

Recently, the orientational immobilization of enzymes has attracted extensive attention. In this study, we report the development of a strategy combined with rational design to achieve precise site-specific covalent immobilization of β-agarase. We first rationally screened six surface sites that can be mutated to cysteine by combining molecular dynamics simulation and energy calculation. Site-specific immobilization was successfully achieved by Michael addition reaction of mutant enzymes and maleimide-modified magnetic nanoparticles (MAL-MNPs). The enzyme activity retention rate of R66C-MAL-MNPs and K588C-MAL-MNPs was greater than 96%. The thermal deactivation kinetics study revealed that the site-specific immobilization strategy significantly improved the thermal stability of Aga50D, resulting in a substantial increase in its antidenaturation activity at elevated temperatures, and the highest t1/2 of the immobilized mutant enzymes was increased by an impressive 21.25-fold at 40 °C. The immobilized mutant enzymes also showed significantly enhanced tolerance to metal ions and organic reagents. For instance, all of the immobilized enzymes maintained over 90% of their enzymatic activity in the 50% (v/v) acetone/water solution. The present work may pave the way for the design of precisely immobilized enzymes, which can help promote green manufacturing.

Oriental covalent immobilization of N-glycan binding protein via N-terminal selective modification

Lectin affinity chromatography is one of powerful tools for the study of protein glycosylation. Different lectin proteins can recognize different structures of monosaccharides or oligosaccharide units, allowing for the selective separation of glycopeptides or glycoproteins containing different polysaccharide structures. However, the N-glycans were only partially captured by most of common lectins, reducing the coverage rate of identifying N-glycoconjugates. Recently, it has been reported that the engineering variant of glycan binding protein Fbs1 has a high affinity for innermost Man3GlcNAc2 structure and is able to bind diverse types of N-glycans, which can be suitable for the analysis of protein N-glycosylation. However, efficient immobilization of protein to separation matrix is particularly challenging as it requires the functionality and integrity of the protein to be preserved. Herein, we describe a simple and robust strategy for oriental covalent immobilization of proteins on magnetic nanoparticles by N-terminal selective labeling techniques. We inserted the enterokinase cleavage site to produce the specific N- terminal glycine of protein. Under physiological conditions, the protein was immobilized on the surface of the MNPs by this glycine tag, and the enrichment process could be completed within 30 min. A whole enrichment and purification of glycan and glycopeptides were completed and analyzed by MALDI TOF-MS. The functional materials achieved stable enrichment of glycan structure in different enzyme digestion systems or complex samples, showing excellent anti-interference and applicability.

Inhibition of bacteria adhesion and biofilm formation using a precisely structured nitric oxide-releasing coating with repeatedly renewing antimicrobial and antifouling ability

Bacterial adhesion and colonization usually causes the formation of biofilm that cannot be easily eradicated by common antibiotics and disinfectants. Surface covalent immobilization of nitric oxide (NO)-releasing polymer is an effective surface modification strategy to combat the threat from bacteria. Although surface covalent strategy avoided the leaching of toxic NO donors, the existence of hydrophobic NO donors on topsurface could inevitably accelerate the microorganism adhesion and accumulation, and the NO-releasing period of time was still very limited. In this work, we prepared an antimicrobial and antibiofilm surface via tethering a precision-structured NO-releasing copolymer brush to an organic-inorganic hybrid hard coating (> 5H pencil hardness) on one end. The precision-structured diblock copolymer brush was composed of a surface antifouling block and a subsurface antimicrobial block of NO-releasing units. A typical effect of “kill two birds with one stone” was observed, the NO releasing subsurface segment within a polymeric matrix prolongs NO release while also preventing surface fouling caused by hydrophobic NO donors on top surface. The impacts of polymer architecture on bioactivity was detailed investigated, and the block copolymer brush exhibited the optimal inhibitory effects against bacteria colonization and biofilm formation. The developed bioactive diblock copolymer brush was grafted from a hard hybrid persistent coating that embeds considerable initiation sites for surface modification. Thanks to the presence of initiator throughout the hybrid network, the initiation layer could initiate polymerization repeatedly after the polymer brushes are worn off at high pressure. Thus, the coating exhibited repeatedly renewing antimicrobial and antifouling ability after multiple abrasion cycles. This work not only developed a novel strategy for regulating NO releasing via modulating the polymer architecture, but provided a feasible route for obtaining a robust initiator layer for synthesizing polymer brush for antimicrobial and antifouling applications.

A dialdehyde pullulan cross-linking strategy for immobilizing protamine onto silk fiber surfaces to achieve durable antibacterial function

Covalent immobilization of antibacterial peptides (APs) onto silk protein-based materials has always been a challenge due to the lack of green and efficient macromolecular cross-linkers. Here, we proposed a dialdehyde polysaccharide cross-linker oxidized from pullulan for grafting a natural AP protamine (PM) onto silk fiber surface through a simple cold pad-batch process. The oxidized pullulan (OP) was linked to silk fiber surface through Schiff reaction and used for mediated cross-linking of PM also via Schiff base linkages. This modification introduced abundant PM guanidine groups on the fiber leading to much-desired antibacterial activity, and considerable improvement in the moisture transfer properties and shade depth. FTIR, XPS, SEM studies confirmed the presence of PM and the cross-linking structure between the polysaccharide and peptides on the fiber surfaces. The antibacterial activity imparted by this process was retained even after 20 washing and 50 rubbing cycles proving versatility and durability. Further, the process did not affect other critical silk properties such as appearance, tensile strength, biological safety, etc. Immobilization of PM onto silk fibers through this novel green polysaccharide cross-linker makes silk more appealing and usable and could also enlighten the attempts of cross-linking other protein materials.

Enhancing rhamnolipid production via immobilized Pseudomonas stutzeri lipase: A comparative study

Rhamnolipids (RLs) are widely studied biosurfactants with significant industrial potential in cosmetics, pharmaceuticals, and bioremediation due to their excellent surface activity, emulsifying properties and bioactive characteristics. However, high production costs impede their mass production. This study investigates the immobilization of Pseudomonas stutzeri lipase (PSL) on various supports to enhance RL synthesis efficiency, focusing on yield and regioselectivity in the enzymatic synthesis of 4-O-lauroylrhamnose by the transesterification of rhamnose with vinyl laurate. Three immobilization methods were compared: covalent binding, adsorption on Celite, and adsorption on hydrophobic supports. The immobilization efficiency varied depending on the method used, with the lowest observed for adsorption on Celite (56 %), followed by covalent immobilization on Sepabeads (EC-EP/S 78 % and EC-EP/L 70 %), and the highest for adsorption on hydrophobic supports (83–97 %, with EC-OD being the best at 97 %). For the enzymatic synthesis of 4-O-lauroylrhamnose, covalent immobilization on Sepabeads™ EC-EP yielded low conversions due to restricted conformational freedom of the enzyme. Celite® 545 adsorption resulted in moderate conversion rates, limited by the electrostatic interactions restricting enzyme activity. The most promising results were obtained with hydrophobic supports, particularly Purolite® ECR8806F, achieving nearly complete conversion and maintaining high regioselectivity at the 4-position of rhamnose in both THF and the green solvent 2-methyltetrahydrofuran (2-MeTHF). The study highlights the critical role of support hydrophobicity and active surface area in the immobilized enzyme performance. PSL immobilized on Purolite® ECR8806F demonstrated significant potential for sustainable RLs production, showing excellent reusability, stability and productivity across multiple reaction cycles. This study presents a significant advancement in RLs production by optimizing PSL immobilization and reaction conditions, facilitating the way for more cost-effective and sustainable industrial applications.

Fast Production of Covalent Organic Frameworks for Covalent Enzyme Immobilization with Boosted Enzymatic Catalysis by Solar‐Driven Photothermal Effect

Developing new enzyme‐immobilization systems to stabilize their dynamic structures and meanwhile enhance their catalytic activity is of great significance but very challenging. Herein, we design and fabricate a class of robust mesoporous covalent organic frameworks (COFs) via Michael addition‐elimination reaction. It is found that highly crystalline COFs can be produced in 10 min, which is attributed to the promoting effect of the intramolecular hydrogen bond activation. The COFs rich in hydroxyl groups can be facilely post‐modified by epibromohydrin to covalently immobilize enzymes with both high loading and activity. Furthermore, we create a solar‐driven photothermal‐promoted strategy by introducing photoactive azo groups to COF carriers, which can boost the enzyme catalytic performance (lipase) with much higher conversion of various racemic substrates and chiral resolution upon solar light irradiation. The heterogeneous biocatalysts also demonstrate exceptional reusability and stability. This work provides a green and energy‐efficient approach to facilitate the scale application of enzyme‐immobilized biocatalysts.

Fast Production of Covalent Organic Frameworks for Covalent Enzyme Immobilization with Boosted Enzymatic Catalysis by Solar-Driven Photothermal Effect.

Developing new enzyme-immobilization systems to stabilize their dynamic structures and meanwhile enhance their catalytic activity is of great significance but very challenging. Herein, we design and fabricate a class of robust mesoporous covalent organic frameworks (COFs) via Michael addition-elimination reaction. It is found that highly crystalline COFs can be produced in 10 min, which is attributed to the promoting effect of the intramolecular hydrogen bond activation. The COFs rich in hydroxyl groups can be facilely post-modified by epibromohydrin to covalently immobilize enzymes with both high loading and activity. Furthermore, we create a solar-driven photothermal-promoted strategy by introducing photoactive azo groups to COF carriers, which can boost the enzyme catalytic performance (lipase) with much higher conversion of various racemic substrates and chiral resolution upon solar light irradiation. The heterogeneous biocatalysts also demonstrate exceptional reusability and stability. This work provides a green and energy-efficient approach to facilitate the scale application of enzyme-immobilized biocatalysts.

Non-enzymatic cholesterol biosensor: Electrochemical sensing based on peptide-polylactic acid thin film

Cholesterol is a fundamental lipid prevalent in eukaryotic cell membranes and circulating in the bloodstream bound to lipoproteins. It serves as a precursor to steroid hormones and is regarded as a biomarker for cardiovascular disease and other metabolic disorders. Numerous cholesterol detection methods predominantly rely on enzymes, which suffer from instability, leading to non-cost-effective biosensors with low sensitivity and poor reusability. Therefore, monitoring cholesterol levels with a feasible, rapid, and stable biosensor is critical for diagnosing and treating various disorders. This study aimed to develop a non-enzymatic cholesterol biosensor based on a selected cholesterol recognition peptide as the detection element. Screen-printed carbon electrodes (SPEs) modified with biocompatible poly-L-lactic acid (PLLA) porous nanomembranes (NMs) were utilized as support for the covalent immobilization of the peptide. Data obtained from electrochemical impedance spectroscopy (EIS) demonstrated the peptide's effective binding affinity towards cholesterol, paving the way for its implementation. The determination of cholesterol with the proposed biosensor exhibited a low limit of detection of 6.31 μM with linear responses ranging from 2–15 μM and 20–40 μM. These findings present an alternative method for cholesterol sensing by integrating novel peptides as biorecognition motifs with biocompatible polymeric materials, potentially useful as biocompatible and future point-of-care sensors.

Nanoarchitectonics of Biofunctionalized Hydrogen‐Terminated 2D‐Germanane Heterostructures as Highly Sensitive Biorecognition Transducers: The Case Study of Cocaine Drug

Hydrogen‐terminated 2D‐germanane (2D‐GeH), as one inorganic 2D material akin to graphene, is attracting widespread interest owing to its predicted (opto)electronic properties. Nonetheless, the chemical reactivity of 2D‐GeH requires further exploration to expand its real implementation. Herein, a simple and straightforward bottom‐up biofunctionalization approach is reported aiming at providing the bases toward the robust design of 2D‐GeH‐based biorecognition systems with electrical readout. For this goal, 2D‐GeH has been firstly functionalized with gold nanoparticles (Au‐NPs) via an organometallic approach, followed by the covalent immobilization of a thiolated single‐stranded DNA (ssDNA) aptamer via AuS bond interactions. After an accurate material characterization, the resulting ssDNA/Au@GeH heterostructure is drop‐casted on a fluorine‐doped tin oxide (FTO) electrode for impedimetrically monitoring cocaine as a model drug. Interestingly, the aptamer–cocaine interactions hinder the interfacial electron‐transfer process of the benchmark [Fe(CN)6]3−/4− redox marker with increasing concentration of the cocaine target, leading to a detection limit as low as 4.9 ± 0.1 aM, the lowest one reported in literature by far. Overall, the ssDNA/Au@GeH electrochemical biosensor exhibits outstanding selectivity, specificity, and reproducibility, demonstrating the potential use of 2D‐GeH as an emerging highly sensitive transducer for biosensing applications. The reported method is general and might be simply customized by tailoring the biorecognition component.

Designing mixed cationic/anionic supports to covalently immobilize/stabilize enzymes with high isoelectric point by enzyme adsorption and support-enzyme glutaraldehyde crosslinking

Ficin fully immobilized on Asp-agarose beads at pH 7 but not on an aminated support. This made enzyme adsorption plus glutaraldehyde modification non-viable for this enzyme. Modifying glyoxyl-agarose beads with mixtures of Asp and 1,6-hexamethylenediamine (HA) at different ratios, mixed anion/cation exchanger supports were built. Only if HA greatly exceed Asp in the support, immobilization did not work. While only using the Asp-agarose support immobilized enzyme molecules were only ionically adsorbed after glutaraldehyde treatment (visualized in SDS-PAGE analysis), the mixed supports gave covalent immobilization. The glutaraldehyde modification of these biocatalysts permitted to establish covalent bonds with the support, and this was more effective when using higher amounts of HA in the support. When around 60 % of the groups in the support were HA, the treatment with glutaraldehyde fully suppressed enzyme release from the support after boiling in SDS. The glutaraldehyde treated biocatalysts were more stable than just the adsorbed enzymes or the enzyme adsorbed only on Asp supports and then treated with glutaraldehyde (the optimal biocatalyst retained 90 % of the initial activity while the just adsorbed ficin retained 50 % of the initial activity). This strategy can be utilized to immobilize other proteins with high isoelectric points following this immobilization strategy.

Functionalization of Si‐ and Al‐Based Materials with Phosphorus Dendrimers and their Properties

AbstractThe interactions of dendrimers having a phosphorus atom at each branching point, of type poly(phosphorhydrazone) (PPH), with different types of materials based on silicon and aluminum are reviewed. These dendrimers can be used for the functionalization of solid surfaces at the nanoscale, either by covalent immobilization, or by electrostatic interactions, especially for constructing perfectly controlled multi‐layers by layer‐by‐layer (LbL) deposit. The properties of such hybrid materials as chemical sensors, as very sensitive biological sensors, as well as substrates for the growing of cells are described. The PPH dendrimers can also be embedded inside materials. The resulting hybrid materials have been used for the capture of pollutants, in particular CO2 in air and chromate and methylene blue in waste water.

A novel Affi-Cova magnetic nanoparticles for one-step covalent immobilization of His-tagged enzyme directly from crude cell lysate

Owing to the rapid advancement of in vitro synthetic biology, functional carriers capable of covalently binding target proteins from crude lysates under mild conditions have garnered escalating attention. Herein, a magnetic nanoparticle with affinity/covalent bifunction (MNP@Affi-Cova) was developed for the direct covalent immobilization of the recombinant enzyme of His-tagged birA (r-birA) from crude cell lysates in a single step. This innovative approach is attributed to the presence of chelated Ni2+ ions and epoxy groups on the surface of the beads. The fabricated magnetic nanoparticles were characterized by SEM, FT-IR spectrum, and zeta potential. The application conditions and stability of the MNP@Affi-Cova beads were systematically evaluated. Notably, the MNP@Affi-Cova beads exhibited a covalent capture efficiency of 91.25 μg r-birA/mg beads from a cell lysate supernatant containing 2.62 mg/mL crude protein. The immobilized r-birA exhibited significantly enhanced pH and thermal stability compared to the free counterpart. Additionally, the reusability of the immobilized r-birA on MNP@Affi-Cova demonstrated the retention of 76.1 % of its initial activity over ten cycles. These results suggest that the MNP@Affi-Cova presents considerable potential as a support for the covalent immobilization of recombinant His-tagged enzymes directly from crude lysates, thereby circumventing the labor-intensive purification process typically required before enzyme immobilization.