Covalent Immobilization Research Articles

Efficient [Fe-Imidazole@SiO2] Nanohybrids for Catalytic H2 Production from Formic Acid.

Three imidazole-based hybrid materials, coded as IGOPS, IPS and impyridine@SiO2 nanohybrids, were prepared via the covalent immobilization of N-ligands onto a mesoporous nano-SiO2 matrix for H2 generation from formic acid (FA). BET and HRTEM demonstrated that the immobilization of the imidazole derivative onto SiO2 has a significant effect on the SSA, average pore volume, and particle size distribution. In the context of FA dehydrogenation, their catalytic activity (TONs, TOFs), stability, and reusability were assessed. Additionally, the homologous homogeneous counterparts were evaluated for comparison purposes. Mapping the redox potential of solution Eh vs. SHE revealed that poly-phosphine PP3 plays an essential role in FA dehydrogenation. On the basis of performance and stability, [Fe2+/IGOPS/PP3] demonstrated superior activity compared to other heterogeneous catalysts, producing 9.82 L of gases (VH2 + CO2) with TONs = 31,778, albeit with low recyclability. In contrast, [Fe2+/IPS/PP3] showed the highest stability, retaining considerable performance after three consecutive uses. With VH2 + CO2 = 7.8 L, [Fe2+/impyridine@SiO2/PP3] activity decreased, and it was no longer recyclable. However, the homogeneous equivalent of [Fe2+/impyridine/PP3] was completely inactive. Raman, FT/IR, and UV/Vis spectroscopy demonstrated that the reduced recyclability of [Fe2+/IGOPS/PP3] and [Fe2+/impyridine@SiO2/PP3] nanohybrids is due to the reductive cleavage of their C-O-C bonds during catalysis. An alternative grafting procedure is proposed, applying here to the grafting of IPS, resulting in its higher stability. The accumulation of water derived from substrate's feeding causes the inhibition of catalysis. In the case of [Fe2+-imidazole@SiO2] nanohybrids, simple washing and drying result in their re-activation, overcoming the water inhibition. Thus, the low-cost imidazole-based nanohybrids IGOPS and IPS are capable of forming [Fe2+/IGOPS/PP3] and [Fe2+/IPS/PP3] heterogeneous catalytic systems with high stability and performance for FA dehydrogenation.

Influence of Immobilization Strategies on the Antibacterial Properties of Antimicrobial Peptide-Chitosan Coatings.

It is key to fight bacterial adhesion to prevent biofilm establishment on biomaterials. Surface immobilization of antimicrobial peptides (AMP) is a promising strategy to avoid bacterial colonization. This work aimed to investigate whether the direct surface immobilization of Dhvar5, an AMP with head-to-tail amphipathicity, would improve the antimicrobial activity of chitosan ultrathin coatings. The peptide was grafted by copper-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry by either its C- or N- terminus to assess the influence of peptide orientation on surface properties and antimicrobial activity. These features were compared with those of coatings fabricated using previously described Dhvar5-chitosan conjugates (immobilized in bulk). The peptide was chemoselectively immobilized onto the coating by both termini. Moreover, the covalent immobilization of Dhvar5 by either terminus enhanced the antimicrobial effect of the chitosan coating by decreasing colonization by both Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa) bacteria. Relevantly, the antimicrobial performance of the surface on Gram-positive bacteria depended on how Dhvar5-chitosan coatings were produced. An antiadhesive effect was observed when the peptide was grafted onto prefabricated chitosan coatings (film), and a bactericidal effect was exhibited when coatings were prepared from Dhvar5-chitosan conjugates (bulk). This antiadhesive effect was not due to changes in surface wettability or protein adsorption but rather depended on variations in peptide concentration, exposure, and surface roughness. Results reported in this study show that the antibacterial potency and effect of immobilized AMP vary greatly with the immobilization procedure. Overall, independently of the fabrication protocol and mechanism of action, Dhvar5-chitosan coatings are a promising strategy for the development of antimicrobial medical devices, either as an antiadhesive or contact-killing surface.

Reagentless Electrochemical Detection of Tumor Biomarker Based on Stable Confinement of Electrochemical Probe in Bipolar Silica Nanochannel Film.

The development of simple and probe-integrated aptamer sensors for the electrochemical detection of tumor biomarkers is of great significance for the diagnosis of tumors and evaluation of prognosis. In this work, a probe-integrated aptamer sensor is demonstrated based on the stable confinement of an electrochemical probe in a bipolar nanochannel film, which can realize the reagentless electrochemical detection of the tumor biomarker carcinoembryonic antigen (CEA). To realize the stable immobilization of a large amount of the cationic electrochemical probe methylene blue (MB), a two-layer silica nanochannel array (SNF) with asymmetric charge was grown on the supporting electrode from bipolar SNF (bp-SNF). The inner SNF is negatively charged (n-SNF), and the outer-layer SNF is positively charged (p-SNF). The dual electrostatic interaction including the electrostatic adsorption from n-SNF and the electrostatic repulsion from p-SNF achieve the stable confinement of MB in bp-SNF. The recognitive interface is fabricated by the covalent immobilization of the CEA aptamer on the outer surface of bp-SNF, followed by the blocking of non-specific binding sites. Owing to the stable and abundant immobilized probes and highly specific aptamer interface, the developed aptamer sensor enables the sensitive detection of CEA in the range of 1 pg/mL to 1 μg/mL with a low limit of detection (LOD, 0.22 pg/mL, S/N = 3). Owing to the high selectivity and stability of the developed biosensor, reagentless electrochemical detection of CEA in serum was realized.

Fabrication of a bioconjugated dual-functional SERS probe for facile compound screening and detection

Surface-enhanced Raman spectroscopy (SERS) is an ultrasensitive technique for both detection and structural characterizations. To further exploit these advantages, we designed and fabricated a dual-functional SERS probe for specific capture and fast detection of small molecule ligands binding to target protein from a mixture of compounds such as extracts of natural products. As a proof of concept, we synthesized SiO2@Ag nanoclusters that are coated with 6-chlorohexanoic acid for covalent immobilization of serotonin transporter (5-HTT) fused with a Halo-tag through enzyme-substrate recognition. As such, we fabricated a bioconjugated SERS probe, and the synthesis, coating, protein immobilization, and affinity-based ligand binding have been characterized and verified by transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), and elemental mapping. By applying this probe to analyze Gardenia jasminoides extract, we have successfully identified crocin I as a compound binding to 5-HTT, which was further proved by using mass spectrometry (MS) and nuclear magnetic resonance (NMR). Taken together, we have developed a novel SERS probe by integrating the inherent strength of SERS in molecular analysis with an extended functionality of affinity-guided molecular capture, which has demonstrated the potential in drug screening of challenging systems.

Sustainable synthesis of L-phenylalanine derivatives in continuous flow by immobilized phenylalanine ammonia lyase

The application of phenylalanine ammonia lyases (PALs) for the amination of a variety of cinnamic acids has been shown to be a cost-efficient method to produce a variety of phenylalanine analogues. Nonetheless, as many other biocatalytic tools, the process intensification, especially due to the high equivalents of ammonia needed, and the cost-efficiency of the catalyst production and use have been key points to further prove their usefulness. Here, we investigated the use of previously characterized PALs (AvPAL and PbPAL) for the amination of a series of substituted cinnamic acids. To enhance the process scalability and the reusability of the catalyst, we investigated the use of covalent immobilization onto commercially available supports, creating a heterogeneous catalyst with good recovered activity (50%) and excellent stability. The immobilized enzyme was also incorporated in continuous flow for the synthesis of 3-methoxy-phenyl alanine and 4-nitro-phenylalanine, which allowed for shorter reaction times (20 min of contact time) and excellent conversions (88% ± 4% and 89% ± 5%) respectively, which could be maintained over extended period of time, up to 24 h. This work exemplifies the advantages that the combination of enzyme catalysis with flow technologies can have not only in the reaction kinetics, but also in the productivity, catalyst reusability and downstream processing.

How Solid Surfaces Control Stability and Interactions of Supported Cationic CuI(dppf) Complexes─A Solid-State NMR Study

Organometallic complexes are frequently deposited on solid surfaces, but little is known about how the resulting complex-solid interactions alter their properties. Here, a series of complexes of the type Cu(dppf)(Lx)+ (dppf = 1,1'-bis(diphenylphosphino)ferrocene, Lx = mono- and bidentate ligands) were synthesized, physisorbed, ion-exchanged, or covalently immobilized on solid surfaces and investigated by 31P MAS NMR spectroscopy. Complexes adsorbed on silica interacted weakly and were stable, while adsorption on acidic γ-Al2O3 resulted in slow complex decomposition. Ion exchange into mesoporous Na-[Al]SBA-15 resulted in magnetic inequivalence of 31P nuclei verified by 31P-31P RFDR and 1H-31P FSLG HETCOR. DFT calculations verified that a MeCN ligand dissociates upon ion exchange. Covalent immobilization via organic linkers as well as ion exchange with bidentate ligands both lead to rigidly bound complexes that cause broad 31P CSA tensors. We thus demonstrate how the interactions between complexes and functional surfaces determine and alter the stability of complexes. The applied Cu(dppf)(Lx)+ complex family members are identified as suitable solid-state NMR probes for investigating the influence of support surfaces on deposited inorganic complexes.

Incorporation of metal-organic framework MOFs-5 into the polymer monolith via the surface covalent immobilization method for enhanced capillary liquid chromatographic separation of benzene homologues

In this study, metal organic framework material MOFs-5 was immobilized covalently on the surface of polymer monolith to improve the separation efficiency of small molecular compounds in the mode of capillary liquid chromatography (cLC) firstly. Scanning electron microscopy, fourier transform infrared spectroscopy and flow-through property test were applied to characterize the novel [email protected] monolith, confirming that MOFs-5 was effectively immobilized on the surface of polymer monolith via covalent reactions. More importantly, Benzene homologues (benzene and phenol) which could be separated by the commercialized C18 packed chromatographic column have been well separated via the [email protected] monolith. By contrast, there wasn't any separation between benzene and phenol via the original polymer monolith. Moreover, the relative standard deviation was less than 2.2 % for run-to-run. These results showed that the [email protected] monolith is a kind of potential commercialized material for small molecular compound separation in the mode of cLC.

Development of albumin monitoring system with hepatic hypoxia-on-a-chip

Hypoxia plays an essential role in the pathogenesis of various liver diseases, and albumin is one of the important biomarkers secreted by the liver. In this study, we developed an albumin monitoring system composed of hepatic hypoxia-on-a-chip and an albumin sensor to study liver function change due to hypoxia. In hepatic hypoxia-on-a-chip, we vertically stack an oxygen-scavenging channel on a liver on a chip with a thin gas-permeable membrane in the middle. This unique design of the hepatic hypoxia-on-a-chip can help to induce hypoxia quickly, attaining <5% within 10 min. An electrochemical albumin sensor was fabricated based on the covalent immobilization of antibodies on the Au electrode to monitor albumin secreting function on the hepatic hypoxia-on-a-chip. Standard albumin samples spiked in PBS, and culture media were measured by the electrochemical impedance spectroscopy using the fabricated immunosensor. The LOD was calculated to be 10 ag/mL in both cases. Using the electrochemical albumin sensor, we measured albumin secretion in normoxia and hypoxia in the chips. The albumin concentration decreased to 27% after 24 h in hypoxia compared to normoxia. This response was consistent with physiological studies. With technical refinements, the present albumin monitoring system can be a powerful tool in studying hepatic hypoxia with real-time liver function monitoring.

A novel microbial-derived family 19 endochitinase with exochitinase activity and its immobilization

A novel chitinase gene of 888bp from Streptomyces bacillaris was cloned and expressed in Escherichia coli BL21. The purified recombinant enzyme (SbChiAJ103) was identified as the first microbial-derived family 19 endochitinase that showed exochitinase activity. SbChiAJ103 exhibited the substrate preference for N-acetylchitooligosaccharides with even degrees of polymerization and the capability to specifically hydrolyze colloidal chitin into (GlcNAc)2. Mono-methyl adipate was employed as a novel linker for the efficient covalent immobilization of chitinase on magnetic nanoparticles (MNPs). The immobilized SbChiAJ103, SbChiAJ103@MNPs, exhibited superior pH tolerance, temperature stability, and storage stability than free SbChiAJ103. Even after incubation at 45°C for 24h, SbChiAJ103@MNPs could retain more than 60.0% initial activity. As a result, the enzymatic hydrolysis yield of SbChiAJ103@MNPs increased to 1.58 times that of free SbChiAJ103. Moreover, SbChiAJ103@MNPs could be reused by convenient magnetic separation. After 10 recycles, SbChiAJ103@MNPs could retain almost 80.0% of its initial activity. The immobilization of the novel chitinase SbChiAJ103 paves the way to the efficient and eco-friendly commercial production of (GlcNAc)2. KEY POINTS: • The first microbial GH19 endochitinase with exochitinase activity was reported. • Mono-methyl adipate was first employed to immobilize chitinase. • SbChiAJ103@MNPs showed excellent pH stability, thermal stability, and reusability.

Investigation of adsorption and biosorption features of bio-functionalized poly(GMA-Co-EGDMA) polymer beads in the treatment of nicotine from tobacco industry

The investigation of multifunctional materials for modern enzyme immobilization is an attractive subject in advanced adsorption and biosorption applications. In the present study, the feasibility of immobilization of Lipozyme TL 100L (LPZM) on 3-aminopropyltriethoxysilane (APTES) modified poly-(GMA-co-EGDMA) (PEGDMA) was investigated for adsorption and biosorption of nicotine from aqueous solution. Characterization tests confirmed successful immobilization of lipozyme which significantly altered thermal behavior, surface characteristics, and surface morphology of PEGDMA and PEGDMA/APTES. In addition, the immobilization yields were calculated as 85.0% and 72.0% onto PEGDMA/APTES using physical adsorption and covalent immobilization methods, respectively. The nicotine removal efficiencies were calculated to be 66.4%, 79.0%, 98.9%, and 85.7%, using raw PEGDMA, PEGDMA/APTES, PEGDMA/APTES@LPZM, and PEGDMA/APTES/GU@LPZM, respectively. For the raw PEGDMA, the Langmuir isotherm was best fitted to the adsorption data, while Langmuir-Freundich model described well the adsorption process on PEGDMA/APTES and PEGDMA/APTES@LPZM. The maximum adsorption capacities of Langmuir-Freundlich model increased from 8.118 to 17.32mg/g after enzyme immobilization. The negative enthalpy value, ΔH° (- 10.37kJ/mol), revealed that the nicotine adsorption on PEGDMA/APTES@LPZM was exothermic in nature, which was corroborated by the decrease observed in the number of adsorbed molecules with increasing temperature. In the kinetic experiments, the adsorption on PEGDMA and PEGDMA/APTES@LPZM reached equilibrium with the removal percentages as 66.4% and 98.9% at the end of 3h, respectively. The nicotine adsorption performances in real water matrices were also investigated, and PEGDMA/APTES@LPZM showed satisfactory reusability with removal percentage decreased from 98.9% (1st cycle) to 83.0% (6th cycle).

Magnetic Amine-Functionalized ZIF-8 with Hierarchical Pores for Efficient Covalent Immobilization of α-Amylase

Magnetic Amine-Functionalized ZIF-8 with Hierarchical Pores for Efficient Covalent Immobilization of α-Amylase

Hierarchical Emulsion-Templated Monoliths (polyHIPEs) as Scaffolds for Covalent Immobilization of P. acidilactici.

The immobilized cell fermentation technique (IMCF) has gained immense popularity in recent years due to its capacity to enhance metabolic efficiency, cell stability, and product separation during fermentation. Porous carriers used as cell immobilization facilitate mass transfer and isolate the cells from an adverse external environment, thus accelerating cell growth and metabolism. However, creating a cell-immobilized porous carrier that guarantees both mechanical strength and cell stability remains challenging. Herein, templated by water-in-oil (w/o) high internal phase emulsions (HIPE), we established a tunable open-cell polymeric P(St-co-GMA) monolith as a scaffold for the efficient immobilization of Pediococcus acidilactici (P. acidilactici). The porous framework's mechanical property was substantially improved by incorporating the styrene monomer and cross-linker divinylbenzene (DVB) in the HIPE's external phase, while the epoxy groups on glycidyl methacrylate (GMA) supply anchoring sites for P. acidilactici, securing the immobilization to the inner wall surface of the void. For the fermentation of immobilized P. acidilactici, the polyHIPEs permit efficient mass transfer, which increases along with increased interconnectivity of the monolith, resulting in higher L-lactic acid yield compared to that of suspended cells with an increase of 17%. The relative L-lactic acid production is constantly maintained above 92.9% of their initial relative production after 10 cycles, exhibiting both its great cycling stability and the durability of the material structure. Furthermore, the procedure during recycle batch also simplifies downstream separation operations.

Biocompatible epoxysilane substituted polymer-based nano biosensing platform for label-free detection of cancer biomarker SP17 in patient serum samples

Herein, we report the results of the studies relating to developing a simple, sensitive, cost-effective, and disposable electrochemical-based label-free immunosensor for real-time detection of a new cancer biomarker, sperm protein-17 (SP17), in complex serum samples. An indium tin oxide (ITO) coated glass substrate modified with self-assembled monolayers (SAMs) of 3-glycidoxypropyltrimethoxysilane (GPTMS) was functionalized via covalent immobilization of monoclonal anti-SP17 antibodies using EDC(1-(3-(dimethylamine)-propyl)-3-ethylcarbodiimide hydrochloride) - NHS (N-hydroxy succinimide) chemistry. The developed immunosensor platform (BSA/anti-SP17/GPTMS@SAMs/ITO) was characterized via scanning electron microscopy (SEM), atomic force microscopy (AFM), contact angle (CA), Fourier transform infrared (FT-IR) spectroscopic, and electrochemical techniques such as cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) techniques. The fabricated BSA/anti-SP17/GPTMS@SAMs/ITO immunoelectrode platform was used to measure changes in the magnitude of the current of the electrodes through an electrochemical CV and DPV technique. A calibration curve between current and SP17 concentrations exhibited a broad linear detection range of (100–6000 & 50–5500 pg mL−1), with enhanced sensitivity (0.047 & 0.024 μA pg mL−1 cm−2), limit of detection (LOD) and limit of quantification (LOQ) of 47.57 & 142.9 pg mL−1 and 158.58 & 476.3 pg mL−1, by CV and DPV technique, respectively with a rapid response time of 15 min. It possessed exceptional repeatability, outstanding reproducibility, five-time reusability, and high stability. The biosensor's performance was evaluated in human serum samples, giving satisfactory findings obtained via the commercially available enzyme-linked immunosorbent assay (ELISA) technique, proving the clinical applicability for early diagnosis of cancer patients. Moreover, various in vitro studies in murine fibroblast cell line L929 have been performed to assess the cytotoxicity of GPTMS. The results demonstrated that GPTMS has excellent biocompatibility and can be used for biosensor fabrication.

Covalent immobilization of phosphotungstic acid and amino acid on metal-organic frameworks with different structures: Acid-base bifunctional heterogeneous catalyst for the production of biodiesel from insect lipid

To provide more acid-base active sites for the conversion of the insect lipid into biodiesel, three bifunctional heterogeneous catalysts (amino acid-PTA/ZIF-8) with different structures were designed and compared by modifying ZIF-8 with amino acid and phosphotungstic acid (PTA) as active species in the current research. The as-prepared amino acid-PTA/ZIF-8 composite catalyst features a large surface area and an increased acidity and basicity, thus combining the merits of acid and base with the heterogeneous microenvironment of ZIF-8. Meanwhile, various methods were used to investigate the physicochemical properties of the synthesized composite catalyst. Furthermore, the effects of reaction conditions on the esterification and transesterification of the acid model lipids were used to assess the best catalytic activity process. The optimum conditions (molar ratio of Arginine/PTA = 2:1, weight ratio of Arg2PTA/ZIF-8 = 3:1, reaction temperature = 60 °C, catalyst loading = 3 wt%, methanol/lipid molar ratio = 9:1, and stirring rate = 500 rpm) resulted in a maximum lipid conversion to biodiesel of 94.15%. In addition, the stability and reusability of the prepared catalysts with different structures during four reaction cycles were also demonstrated. Finally, the physicochemical properties of the biodiesel studied were measured and compared to the ASTM standard. Hence, this work provides great potential for acid-base functionalization of porous materials for future biodiesel production.

Plasma surface functionalization: A comprehensive review of advances in the quest for bioinstructive materials and interfaces

Surface biofunctionalization aims to create cell-instructive surfaces that control the behavior of cells and modulate cellular interactions by incorporating cell signaling moieties at the materials–biosystem interface. Despite advances in developing bioinert and biocompatible materials, blood clotting, inflammation, and cell death continue to be observed upon the contact of foreign materials with living tissues leading to the materials' rejection. Specific examples include the application of foreign materials in implantable devices (e.g., bone implants, antimicrobial surfaces, and cardiovascular stents), biosensors, drug delivery, and 3D-bioprinting. Biofunctionalization of materials to date has been predominantly realized using wet chemical approaches. However, the complexity of wet chemistry, toxicity of reactants, waste disposal issues, reaction time, poor reproducibility, and scalability drive a need for a paradigm shift from wet chemical approaches to dry methods of surface biofunctionalization. Plasma-based technologies that enable covalent surface immobilization of biomolecules have emerged as dry, reagent-free, and single-step alternatives for surface biofunctionalization. This review commences by highlighting the need for bioinstructive surfaces and coatings for various biomedical applications such as bone implants, antimicrobial surfaces, biosensors, and 3D-bioprinted structures, followed by a brief review of wet chemical approaches for developing biofunctionalized surfaces and biomimetic devices. We then provide a comprehensive review of the development of plasma-based technologies for biofunctionalization, highlighting the plasma–surface interactions and underpinning mechanisms of biomolecule immobilization.

Nonmigrating Active Antibacterial Packaging: Antimicrobial Mechanism against Staphylococcus Aureus and Its Application in Large Yellow Croaker

In this study, chitosan (CS) was grafted onto the surface of polylactic acid (PLA) film by covalent immobilization to prepare four kinds of CS-grafted PLA (CS-g-PLA) films with different molecular weights. The properties of the films were characterized, and their antibacterial effect and mechanism of action against Staphylococcus aureus (S. aureus) were explored. After being treated with CS-g-PLA films, the cell morphology was destroyed, the permeability of the cell membrane changed, and malate dehydrogenase (MDH) activity decreased. Furthermore, the large yellow croaker fillets were packaged by the films mentioned above and stored at 4 °C, the physicochemical changes and microbial counts were measured. The results showed that the CS-g-PLA films could effectively delay the decay of fish fillets for 1–4 days. Overall, this study demonstrated that grafted films showed great application potential in aquatic product packaging and preservation.

Electrodeposited Carbonyl Functional Polymers as Suitable Supports for Preparation of the First-Generation Biosensors.

The aim of this electrochemical study was to ascertain which type of electrochemically deposited carbonyl functionalized polymer represents the most suitable electrode substrate for direct covalent immobilization of biological catalysts (enzymes). For this purpose, a triad of amperometric biosensors differing in the type of conductive polymers (poly-vanillin, poly-trans-cinnamaldehyde, and poly-4-hydroxybenzaldehyde) and in the functioning of selected enzymes (tyrosinase and alkaline phosphatase) has been compared for the biosensing of neurotransmitters (dopamine, epinephrine, norepinephrine, and serotonin) and phenyl phosphates (p-aminophenyl phosphate and hydroquinone diphosphate). The individual layers of the polymers were electrochemically deposited onto commercially available screen-printed carbon electrodes (type C110) using repetitive potential cycling in the linear voltammetric mode. Their characterization was subsequently performed by SEM imaging and attenuated total reflectance FTIR spectroscopy. Molecules of enzymes were covalently bonded to the free carbonyl groups in polymers via the Schiff base formation, in some cases even with the use of special cross-linkers. The as-prepared biosensors have been examined using cyclic voltammetry and amperometric detection. In this way, the role of the carbonyl groups embedded in the polymeric structure was defined with respect to the efficiency of binding enzymes, and consequently, via the final (electro)analytical performance.

Continuous fixed-bed column studies to remove polycyclic aromatic hydrocarbons by degrading enzymes immobilized on polyimide aerogels

The main sources of polycyclic aromatic hydrocarbons (PAHs), entering aquatic environments are industrial discharges, petroleum spills, combustion of fossil fuels, urban runoff, and atmospheric deposition. For the biodegradation of PAHs, the use of biocatalysts, such as enzymes, is an environmentally friendly method. Despite this, it is necessary to immobilize the enzymes to facilitate their recovery and reusability, as well as to prevent their loss. Using covalent bonding, (PAHs) degrading enzymes including naphthalene and catechol 2,3 dioxygenase enzymes were immobilized on modified polyimide aerogels. Covalent immobilization of enzymes on surface-modified polyimide aerogels resulted in around 9- and 6-fold lower enzyme leaching for naphthalene and catechol 2,3 dioxygenase enzymes compared to enzyme immobilization using adsorption. The Fourier Transform Infrared Spectrum (FT-IR) confirmed the enzyme immobilization and aerogel modification. SEM images indicated completely different textures for surface-modified aerogel attributed to the presence of functional groups after surface modification that enhanced enzyme loading. The removal capacity (∼20mganthracenegaerogel) of enzyme loaded aerogel was determined to select an appropriate range for column tests. Finally, the effects of flow rate, size of aerogels and inlet concentration of anthracene on the removal efficiency of pollutants were examined. Using the derived model as a basis for prediction, in terms of removal efficiency, the highest result was achieved to be 84.01% at a flow rate of 22 mL min−1, initial concentration of 34 mg L−1and aerogel size of 2 cm while under these conditions, the removal efficiency was experimentally measured to be 87.14 %. A fixed-bed column packed with enzyme-loaded aerogel for the removal of PAHs provides novel insight into the application of aerogel base materials for water treatment and PAH removal with possibilities of scaling up for larger applications.

Nanostructured poly(thiophene acetic acid)/Au/poly(methylene blue) interface for electrochemical immunosensing of p53 protein.

A poly(thiophene acetic acid)/Au/poly(methylene blue) nanostructured interface was electrochemically assembled step-by-step on screen-printed carbon electrodes (SPCE) for label-free detection of p53 protein. The initial electrical conductive properties of the polymeric interface were increased with an additional layer of poly(methylene blue) electropolymerized in the presence of gold nanoparticles. The nano-immunosensing architecture was prepared by covalent immobilization of anti-p53 antibodies as bioreceptors through the poly(thiophene acetic acid) moieties. The nano-immunosensor assembly was extensively characterized by ultraviolet-visible spectrophotometry, dynamic and electrophoretic light scattering, scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, atomic force microscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Under optimal conditions, p53 was specifically and selectively detected by square wave voltammetry in a linear range between 1 and 100ngmL-1 with a limit ofdetection of 0.65ngmL-1. In addition, the electrochemical nano-immunosensor detected p53 in spiked human serum samples and colorectal cancer cell lysates, and the results were validated with a standard spectrophotometric method using a paired samples t test, which did not exhibit significant differences between both methods. The resultant p53 nano-immunosensor is simple to assemble, robust, and has the potential for point-of-care biomarker detection applications.

Glutaraldehyde-copper gelled chitosan beads: Characterization and utilization as covalent immobilizers

The robust chitosan (CHS)–Cu2+ interactions were utilized to prepare CHS beads. Glutaraldehyde (GA) was also added to the gelling medium to provide the functional entities needed for β-d-galactosidase (βG) covalent immobilization. The preparation of the GA-Cu-CHS beads was honed with respect to the concentrations of the utilized CuSO4, CHS, and GA solutions. A CHS concentration of 2% was favored. The optimal gelling medium comprised 0.2% CuSO4-1.12% GA. The beads were characterized via SEM and EDX. The beads enhanced mechanical traits were verified. The GA-Cu-CHS beads immobilized βG, and the immobilization efficiency reached 47.67% when 2.56 Ug−1 βG loading activity was adopted. The immobilized βG (iβG) experienced an acidic shift in its optimal pH range from 4.4 to 5.5 to 3.6–4.4. Moreover, its optimal temperature was slightly shifted following immobilization from 55-60 °C–60 °C. The Km and Vmax values of βG were reduced from 84.4 mM to 19.0 μmol/min mg enzyme to 64.9 mM and 5.6 μmol/min mg enzyme following immobilization. The operational stability of iβG was also studied while performing the 15 min lactose hydrolytic reaction at 37 °C, and 81.79% activity was obtained throughout the 15th hydrolytic cycle. Moreover, the iβG provided 97.30% activity after 77 days of storage in distilled water in the fridge. Finally, the iβG was utilized to degrade lactose in cheese whey and also in buffered lactose solution (BLS) at 45 °C and at pH 4.4. Each degradation cycle lasted 24 h and 78.52% of the inceptive released glucose was released during the BLS 5th degradation cycle.