Search Results (42)

A new approach in amperometric enzyme electrodes production based on all-electrochemically assisted procedures will be described. Enzyme (glucose oxidase) immobilization was performed by in situ co-crosslinking of enzyme molecules through electrophoretic protein deposition, assuring enzyme immobilization exclusively onto the transducer surface (Pt electrode). Analogously, the poor selectivity of the transducer was dramatically improved by the electrosynthesis of non-conducting polymers with built-in permselectivity, permitting the formation of a thin permselective film onto the transducer surface, able to reject common interferents usually found in real samples. Since both approaches required a proper and distinct electrochemical perturbation (a pulsed current sequence for electrophoretic protein deposition and cyclic voltammetry for the electrosynthesis of non-conducting polymers), an appropriate coupling of the two all-electrochemical approaches was assured by a thorough study of the likely combinations of the electrosynthesis of permselective polymers with enzyme immobilization by electrophoretic protein deposition and by the use of several electrosynthesized polymers. For each investigated combination and for each polymer, the analytical performances and the rejection capabilities of the resulting biosensor were acquired so to gain information about their sensing abilities eventually in real sample analysis. This study shows that the proper coupling of the two all-electrochemical approaches and the appropriate choice of the electrosynthesized, permselective polymer permits the easy fabrication of novel glucose oxidase biosensors with good analytical performance and low bias in glucose measurement from typical interferent in serum. This novel approach, resembling classical electroplating procedures, is expected to allow all the advantages expected from such procedures like an easy preparation biosensor, a bi-dimensional control of enzyme immobilization and thickness, interferent- and fouling-free transduction of the electrodic sensor and, last but not the least, possibility of miniaturization of the biosensing device. Full article

This study describes the development of catalytic surface immobilizing dopamine via cross-linking or tyrosinase through covalent bonds on an electrografted screen-printed carbon electrode with a 4-nitrobenzenediazonium ion. A simple electrochemical reduction approach was used to covalently graft aryldiazonium ions onto the surface of commercial electrodes. After functionalization with aminophenyl groups, dopamine, an important neurotransmitter, was immobilized by imine bond formation using glutaraldehyde as a bifunctional cross-linking molecule. The presence of immobilized dopamine was confirmed by cyclic voltammetry following the electrochemical response of the hydroquinone/quinone redox process from catechol functionalities on the surface, which are responsible for the catalytic activity. In addition, the surface was also characterized by cyclic voltammetry using the redox probe, [Fe(CN)₆]^3−/4−, obtaining a signal approximately 14 times higher than that of a bare electrode, achieving a dynamic concentration range spanning three orders of magnitude. Remarkable sensitivity was also obtained by combining the electrografting, in situ diazotation, to generate grafted aryl diazonium ions on the surface, and coupling reaction to anchor the tyrosinase enzyme to the electrode surface. The response of the TYR-biosensor towards catechol, using the redox probe as mediator, was 10 times higher than that obtained with the dopamine modified catalytic surface. These modified surfaces offer promising alternatives for the voltammetric quantification of catechol in environmental fields. Full article

The immobilization of enzymes in polyamide-based polymeric materials through covalent bonding is an established technique to stabilize and reuse biocatalysts in industrial processes. Traditionally, enzymes are immobilized using crosslinking agents that activate functional groups on both the support and the enzyme, creating strong bonds that securely anchor the enzyme to the surface. While effective for maintaining enzyme activity over multiple cycles, this method can reduce catalytic efficiency due to rigid binding and involves complex activation steps. Recently, in situ immobilization approaches have emerged as promising alternatives. In this method, enzymes are directly entrapped within the polymer matrix during the synthesis of the polyamide support, such as nylon, simplifying the process and offering enhanced control over enzyme distribution. For instance, studies have demonstrated that in situ immobilization can improve enzyme stability by protecting it within the polymeric network, while reducing production costs and waste. This review explores the ability of polyamide as a support material for immobilization of enzymes, analyzing key techniques, performance across applications, and future strategies to optimize polymer-enzyme interactions for industrial use. Full article

The growing demand for sustainable chemical production has spurred significant interest in biocatalysis. This study is framed within the biocatalytic production of 1,3-dihydroxyacetone (DHA) from glycerol, a byproduct of biodiesel manufacturing. The main goal of this study is to address the challenge of identifying the optimal operating conditions. To achieve this, catalytic potential, a lumped parameter that considers both the activity and stability of immobilized biocatalysts, was used to guide the design of a multi-enzymatic system. The multi-enzymatic system comprises glycerol dehydrogenase (GlyDH) and NADH oxidase (NOX). The enzymatic oxidation of glycerol to DHA catalyzed by GlyDH requires the cofactor NAD+. The integration of NOX into a one-pot reactor allows for the in situ regeneration of NAD+, enhancing the overall efficiency of the process. Furthermore, immobilization on Ni⁺² agarose chelated supports, combined with post-immobilization modifications (glutaraldehyde crosslinking for GlyDH), significantly improved the stability and activity of both enzymes. The catalytic potential enabled the identification of the optimal operating conditions, which were 30 °C and pH 7.5, favoring NOX stability. This work establishes a framework for the rational design and optimization of multi-enzymatic systems. It highlights the crucial interplay between individual enzyme properties and process conditions to achieve efficient and sustainable biocatalytic transformations. Full article

New carriers for protein immobilization are objects of interest in various fields of biomedicine. Immobilization is a technique used to stabilize and provide physical support for biological micro- and macromolecules and whole cells. Special efforts have been made to develop new materials for protein immobilization that are non-toxic to both the body and the environment, inexpensive, readily available, and easy to modify. Currently, biodegradable and non-toxic polymers, including cellulose, are widely used for protein immobilization. Bacterial cellulose (BC) is a natural polymer with excellent biocompatibility, purity, high porosity, high water uptake capacity, non-immunogenicity, and ease of production and modification. BC is composed of glucose units and does not contain lignin or hemicellulose, which is an advantage allowing the avoidance of the chemical purification step before use. Recently, BC–protein composites have been developed as wound dressings, tissue engineering scaffolds, three-dimensional (3D) cell culture systems, drug delivery systems, and enzyme immobilization matrices. Proteins or peptides are often added to polymeric scaffolds to improve their biocompatibility and biological, physical–chemical, and mechanical properties. To broaden BC applications, various ex situ and in situ modifications of native BC are used to improve its properties for a specific application. In vivo studies showed that several BC–protein composites exhibited excellent biocompatibility, demonstrated prolonged treatment time, and increased the survival of animals. Today, there are several patents and commercial BC-based composites for wounds and vascular grafts. Therefore, further research on BC–protein composites has great prospects. This review focuses on the major advances in protein immobilization on BC for biomedical applications. Full article

Detection and enumeration of coliform bacteria using traditional methods and current molecular techniques against E. coli usually involve long processes with less sensitivity and specificity to distinguish between viable and non-viable bacteria for microbiological water analysis. This approach involves developing and validating an immunosensor comprising ring resonators functionalized with specific antibodies surrounded by a network of microchannels as an alternative method for detecting and indirectly enumerating Escherichia coli in samples of water for consumption. Different ELISA assays were conducted to characterize monoclonal and polyclonal antibodies selected as detection probes for specific B-galactosidase enzymes and membrane LPS antigens of E. coli. An immobilization control study was performed on silicon nitride surfaces used in the immunosensor, immobilized with the selected antibodies from the ELISA assays. The specificity of this method was confirmed by detecting as few as 10 CFU/mL of E. coli from viable and non-viable target bacteria after applying various disinfection methods to water samples intended for human consumption. The 100% detection rate and a 100 CFU/mL Limit of Quantification of the proposed method were validated through a comprehensive assessment of the immunosensor-coupled microfluidic system, involving at least 50 replicates with a concentration range of 10 to 10⁶ CFU/mL of the target bacteria and 50 real samples contaminated with and without disinfection treatment. The correlation coefficient of around one calculated for each calibration curve obtained from the results demonstrated sensitive and rapid detection capabilities suitable for application in water resources intended for human consumption within the food industry. The biosensor was shown to provide results in less than 4 h, allowing for rapid identification of microbial contamination crucial for ensuring water monitoring related to food safety or environmental diagnosis and allowing for timely interventions to mitigate contamination risks. Indeed, the achieved setup facilitates the in situ execution of laboratory processes, allowing for the detection of both viable and non-viable bacteria, and it implies future developments of simultaneous detection of pathogens in the same contaminated sample. Full article

Tyrosinase (TYR) emerges as a key enzyme that exerts a regulatory influence on the synthesis of melanin, thereby assuming the role of a critical biomarker for the detection of melanoma. Detecting the authentic concentration of TYR in the skin remains a primary challenge. Distinguished from ex vivo detection methods, this study introduces a novel sensor platform that integrates a microneedle (MN) biosensor with surface-enhanced Raman spectroscopy (SERS) technology for the in situ detection of TYR in human skin. The platform utilized dopamine (DA)-functionalized gold nanoparticles (Au NPs) as the capturing substrate and 4-mercaptophenylboronic acid (4-MPBA)-modified silver nanoparticles (Ag NPs) acting as the SERS probe. Here, the Au NPs were functionalized with mercaptosuccinic acid (MSA) for DA capture. In the presence of TYR, DA immobilized on the MN is preferentially oxidized to dopamine quinone (DQ), a process that results in a decreased density of SERS probes on the platform. TYR concentration was detected through variations in the signal intensity emitted by the phenylboronic acid. The detection system was able to evaluate TYR concentrations within a linear range of 0.05 U/mL to 200 U/mL and showed robust anti-interference capabilities. The proposed platform, integrating MN-based in situ sensing, SERS technology, and TYR responsiveness, holds significant importance for diagnosing cutaneous melanoma. Full article

Nowadays, mycotoxin contamination in cereals and wastewater exposes a safety hazard to consumer health. This work describes the design of a simple, low-cost, and sensitive origami microfluidic paper-based device using electrochemical detection for zearalenone determination. The microfluidic immunosensor was designed on a paper platform by a wax printing process. The graphitized carbon working electrode modified with carbon nanohorns-decorated nanoporous gold showed a higher surface area, sensitivity, and adequate analytical performance. Electrodes were characterized by scanning electron microscopy, energy-dispersive spectroscopy, and cyclic voltammetry. The determination of zearalenone was carried out through a competitive immunoassay using specific antibodies immobilized by a covalent bond on the electrode surface. In the presence of HRP-labeled enzyme conjugate, substrate, and catechol, zearalenone was detected employing the developed immunosensor by applying −0.1 V to the working electrode vs silver as a pseudo-reference electrode. A calibration curve with a linear range between 10 and 1000 µg Kg⁻¹ (R² = 0.998) was obtained, and the limit of detection and quantification for the electrochemical immunosensor were 4.40 and 14.90 µg Kg⁻¹, respectively. The coefficient of variation for intra- and inter-day assays was less than 5%. The selectivity and specificity of the sensor were evaluated, comparing the response against zearalenone metabolites and other mycotoxins that could affect the corn samples. Therefore, origami is a promising approach for paper-based electrochemical microfluidic sensors coupled to smartphones as a rapid and portable tool for in situ mycotoxins detection in real samples. Full article

Electrophoretic deposition is a powerful tool for depositing materials onto a substrate by using an electric field; its application in biotechnological areas, namely, electrophoretic protein deposition (EPD), is the most promising for, e.g., fabricating novel amperometric biosensors. Unfortunately, EPD suffers from several drawbacks due to coupled parasite electrochemical processes damaging the deposit; moreover, the nature of the deposition process, the deposit, and its stability are still controversial and unknown. The present research presents a deep investigation of the EPD processes conducted by using several electroanalytical techniques and an electrochemical quartz crystal microbalance (EQCM); notably, EPD was used here as a novel tool for performing an electrophoretically assisted, classical enzyme immobilization technique like co-crosslinking, thus permitting the immobilization of the desired protein in situ, i.e., exclusively onto the deposition electrode. An electrochemical study permitted the acquisition of useful insights about electrophoresis processes as well as solvent discharge and gas evolution at the deposition electrode; further, the use of appropriate current or potential pulse sequences, as investigated and improved in this study, together with fine-tuned chemical conditions, allowed the optimization of this novel EPD approach. Moreover, an EQCM study gave useful insights into the kinetics of the process, permitting a quantitative estimate of the deposit. Full article

As the largest group of synthetic dyes, azo dyes can pose various health and environmental risks due to their widespread use and challenging degradation. Laccases are efficient green biocatalysts for the degradation of organic pollutants. Herein, we report the in situ packaging of laccase in copper-doped zeolitic imidazolate framework-8 (ZIF-8) for the decolorization of reactive black 5, which is a model azo dye. The immobilization support (Cu5/mZIF-8) was obtained via lowering the precursor ratio of ZIF-8 and incorporating copper ions during the synthesis process. Cu5/mZIF-8 were found to be nanospheres with an average diameter of around 150 nm. Laccase encapsulated in Cu5/mZIF-8 showed an activity recovery of 75.6%, which was 2.2 times higher than that of the laccase embedded in ZIF-8. Meanwhile, the immobilized laccase (Lac@Cu5/mZIF-8) showed a higher catalytic activity in organic solvents than that of the free enzyme. In the presence of a mediator, Lac@Cu5/mZIF-8 could remove 95.7% of reactive black 5 in 40 min. After four consecutive cycles, the dye decolorization efficiency declined to 28%. About four transformation products of reactive black 5 were identified via LC-MS analysis, and the potential decolorization mechanism was proposed. The results indicated that the immobilized laccase could be used as an efficient biocatalyst in dye decolorization. Full article

The solubilization capacity of a series of sustainable phenylalanine-derived surface-active ionic liquids (SAILs) was evaluated towards polycyclic aromatic hydrocarbons—naphthalene, anthracene and pyrene. The key physico-chemical parameters of the studied systems (critical micelle concentration, spectral properties, solubilization parameters) were determined, analyzed and compared with conventional cationic surfactant, CTABr. For all studied PAH solubilization capacity increases with extension of alkyl chain length of PyPheOC_n SAILs reaching the values comparable to CTABr for SAILs with n = 10–12. A remarkable advantage of the phenylalanine-derived SAILs PyPheOC_n and PyPheNHC_n is a possibility to cleave enzymatically ester and/or amide bonds under mild conditions, to separate polycyclic aromatic hydrocarbons in situ. A series of immobilized enzymes was tested to determine the most suitable candidates for tunable decomposition of SAILs. The decomposition pathway could be adjusted depending on the choice of the enzyme system, reaction conditions, and selection of SAILs type. The evaluated systems can provide selective cleavage of the ester and amide bond and help to choose the optimal decomposition method of SAILs for enzymatic recycling of SAILs transformation products or as a pretreatment towards biological mineralization. The concept of a possible practical application of studied systems for PAHs solubilization/separation was also discussed focusing on sustainability and a green chemistry approach. Full article

The development and characterization of a microfluidic electrochemical glucose biosensor are presented herein. The transducer part is based on thin-film metal electrodes on a glass substrate. The biological recognition element of the biosensor is the pyrroloquinoline quinone–glucose dehydrogenase (PQQ-GdhB) enzyme, selectively in situ immobilized via microcontact printing of a mixed self-assembling monolayer (SAM) on a gold working electrode, while the microfluidic part of the device comprises microchannel and microfluidic connections formed in a polydimethylsiloxane (PDMS) elastomer. The electrode properties throughout all steps of biosensor construction and the biosensor response to glucose concentration and analyte flow rate were characterized by cyclic voltammetry and chronoamperometry. A measurement range of up to 10 mM in glucose concentration with a linear range up to 200 μM was determined. A detection limit of 30 µM in glucose concentration was obtained. Respective biosensor sensitivities of 0.79 nA/µM/mm² and 0.61 nA/µM/mm² were estimated with and without a flow at 20 µL/min. The developed approach of in situ enzyme immobilization can find a wide number of applications in the development of microfluidic biosensors, offering a path towards continuous and time-independent detection. Full article

The present work describes the development of an easy-to-use portable electrochemical biosensor based on alkaline phosphatase (ALP) as a recognition element, which has been immobilized in acrylamide-based hydrogels prepared through a green protocol over disposable screen-printed electrodes. To carry out the electrochemical transduction, an electroinactive substrate (hydroquinone diphosphate) was used in the presence of the enzyme and then it was hydrolyzed to an electroactive species (hydroquinone). The activity of the protein within the matrix was determined voltammetrically. Due to the adhesive properties of the hydrogel, this was easily deposited on the surface of the electrodes, greatly increasing the sensitivity of the biosensor. The device was optimized to allow the determination of phosphate ion, a competitive inhibitor of ALP, in aqueous media. Our study provides a proof-of-concept demonstrating the potential use of the developed biosensor for in situ, real-time measurement of water pollutants that act as ALP inhibitors. Full article

Mesoporous silica materials have attracted great research interest for various applications ranging from (bio)catalysis and sensing to drug delivery. It remains challenging to prepare hollow mesoporous silica nanoparticles (HMSN) with large center-radial mesopores that could provide a more efficient transport channel through the cell for guest molecules. Here, we propose a novel strategy for the preparation of HMSN with large dendritic mesopores to achieve higher enzyme loading capacity and more efficient bioreactors. The materials were prepared by combining barium sulfate nanoparticles (BaSO₄ NP) as a hard template and the in situ-formed 3-aminophenol/formaldehyde resin as a porogen for directing the dendritic mesopores’ formation. HMSNs with different particle sizes, shell thicknesses, and pore structures have been prepared by choosing BaSO₄ NP of various sizes and adjusting the amount of tetraethyl orthosilicate added in synthesis. The obtained HMSN-1.1 possesses a high pore volume (1.07 cm³ g⁻¹), a large average pore size (10.9 nm), and dendritic mesopores that penetrated through the shell. The advantages of HMSNs are also demonstrated for enzyme (catalase) immobilization and subsequent use of catalase-loaded HMSNs as bioreactors for catalyzing the H₂O₂ degradation reaction. The hollow and dendritic mesoporous shell features of HMSNs provide abundant tunnels for molecular transport and more accessible surfaces for molecular adsorption, showing great promise in developing efficient nanoreactors and drug delivery vehicles. Full article

Bacteria repellent surfaces and antibody-based coatings for bacterial assays have shown a growing demand in the field of biosensors, and have crucial importance in the design of biomedical devices. However, in-depth investigations and comparisons of possible solutions are still missing. The optical waveguide lightmode spectroscopy (OWLS) technique offers label-free, non-invasive, in situ characterization of protein and bacterial adsorption. Moreover, it has excellent flexibility for testing various surface coatings. Here, we describe an OWLS-based method supporting the development of bacteria repellent surfaces and characterize the layer structures and affinities of different antibody-based coatings for bacterial assays. In order to test nonspecific binding blocking agents against bacteria, OWLS chips were coated with bovine serum albumin (BSA), I-block, PAcrAM-g-(PMOXA, NH₂, Si), (PAcrAM-P) and PLL-g-PEG (PP) (with different coating temperatures), and subsequent Escherichia coli adhesion was monitored. We found that the best performing blocking agents could inhibit bacterial adhesion from samples with bacteria concentrations of up to 10⁷ cells/mL. Various immobilization methods were applied to graft a wide range of selected antibodies onto the biosensor’s surface. Simple physisorption, Mix&Go (AnteoBind) (MG) films, covalently immobilized protein A and avidin–biotin based surface chemistries were all fabricated and tested. The surface adsorbed mass densities of deposited antibodies were determined, and the biosensor;s kinetic data were evaluated to divine the possible orientations of the bacteria-capturing antibodies and determine the rate constants and footprints of the binding events. The development of affinity layers was supported by enzyme-linked immunosorbent assay (ELISA) measurements in order to test the bacteria binding capabilities of the antibodies. The best performance in the biosensor measurements was achieved by employing a polyclonal antibody in combination with protein A-based immobilization and PAcrAM-P blocking of nonspecific binding. Using this setting, a surface sensitivity of 70 cells/mm² was demonstrated. Full article