Electrochemical Detection Platform Based on RGO Functionalized with Diazonium Salt for DNA Hybridization

In this paper, we propose an improved electrochemical platform based on graphene for the detection of DNA hybridization. Commercial screen-printed carbon electrodes (SPCEs) were used for this purpose due to their ease of functionalization and miniaturization opportunities. SPCEs were modified with reduced graphene oxide (RGO), offering a suitable surface for further functionalization. Therefore, aryl-carboxyl groups were integrated onto RGO-modified electrodes by electrochemical reduction of the corresponding diazonium salt to provide enough reaction sites for the covalent immobilization of amino-modified DNA probes. Our final goal was to determine the optimum conditions needed to fabricate a simple, label-free RGO-based electrochemical platform to detect the hybridization between two complementary single-stranded DNA molecules. Each modification step in the fabrication process was monitored by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) using [Fe(CN)6]3−/4− as a redox reporter. Although, the diazonium electrografted layer displayed the expected blocking effect of the charge transfer, the next steps in the modification procedure resulted in enhanced electron transfer properties of the electrode interface. We suggest that the improvement in the charge transfer after the DNA hybridization process could be exploited as a prospective sensing feature. The morphological and structural characterization of the modified electrodes performed by scanning electron microscopy (SEM) and Raman spectroscopy, respectively, were used to validate different modification steps in the platform fabrication process.

Covalent Immobilization of Antibodies through Tetrazine-TCO Reaction to Improve Sensitivity of ELISA Technique

Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.

Electrospun Magnetic Nanocellulose–Polyethersulfone-Conjugated Aspergillus oryzae Lipase for Synthesis of Ethyl Valerate

A novel greener MNC/PES membrane was developed through an electrospinning technique for lipase immobilization to catalyze the synthesis of ethyl valerate (EV). In this study, the covalent immobilization of Aspergillus oryzae lipase (AOL) onto an electrospun nanofibrous membrane consisting of magnetic nanocellulose (MNC) and polyethersulfone (PES) to produce EV was statistically optimized. Raman spectroscopy, Fourier-transform infrared spectroscopy: attenuated total reflection, field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, thermal gravimetric analysis (TGA), and differential thermal gravimetric (DTG) of MNC/PES-AOL demonstrated that AOL was successfully immobilized onto the fibers. The Taguchi design-assisted immobilization of AOL onto MNC/PES fibers identified that 1.10 mg/mL protein loading, 4 mL reaction volume, 250 rpm stirring rate, and 50 °C were optimal to yield 72.09% of EV in 24 h. The thermal stability of MNC/PES-AOL was improved by ≈20% over the free AOL, with reusability for up to five consecutive esterification cycles while demonstrating an exceptional half-life of 120 h. Briefly, the electrospun MNC/PES fibers that immobilized AOL showed promising applicability in yielding relatively good EV levels. This study suggests that using MNC as fillers in a PES to improve AOL activity and durability for a longer catalytic process could be a viable option.

Hybrid Supercapacitor Electrode Materials via Molecular Imprinting of Nitrogenous Ligands and Iron Complexes into Carbon Support

Novel hybrid supercapacitor materials were made by the covalent immobilization of nitrogenous ligands onto the surface of commercial carbon support (Vulcan XC-72), then coordinated to iron. The covalent attachment of the nitrogenous ligands allows for the controlled deposition of nitrogen functionalities on the surface of the carbon. The supercapacitor tests in acidic media showed significant growth of the capacitance as a result of the nitrogenous ligands on the support. Notably, the increase of the capacitance values directly correlates with the molecular loading on the surface. Following coordination of the iron to the ligands on the surface further elevated the capacitance via Faradaic reactions of the metal center. Remarkably, the overall capacitance of materials significantly increased after the course of long-term cycling tests (ca. 110% or higher). At the beginning of durability studies, a small decline in capacitance was observed, due to some extent of molecular decomposition on the surface of the electrode. However, the intense cycling further propagates a steady growth of the overall capacitance. This could be attributed to the process of polymerization of physisorbed molecules/ radicals that result in the formation of a 3D network structure that eventually boosts the overall capacitance and charge storage of the electrode.

Copolymer Coatings for DNA Biosensors: Effect of Charges and Immobilization Chemistries on Yield, Strength and Kinetics of Hybridization

The physical–chemical properties of the surface of DNA microarrays and biosensors play a fundamental role in their performance, affecting the signal’s amplitude and the strength and kinetics of binding. We studied how the interaction parameters vary for hybridization of complementary 23-mer DNA, when the probe strands are immobilized on different copolymers, which coat the surface of an optical, label-free biosensor. Copolymers of N, N-dimethylacrylamide bringing either a different type or density of sites for covalent immobilization of DNA probes, or different backbone charges, were used to functionalize the surface of a Reflective Phantom Interface multispot biosensor made of a glass prism with a silicon dioxide antireflective layer. By analyzing the kinetic hybridization curves at different probe surface densities and target concentrations in solution, we found that all the tested coatings displayed a common association kinetics of about 9 × 104 M−1·s−1 at small probe density, decreasing by one order of magnitude close to the surface saturation of probes. In contrast, both the yield of hybridization and the dissociation kinetics, and hence the equilibrium constant, depend on the type of copolymer coating. Nearly doubled signal amplitudes, although equilibrium dissociation constant was as large as 4 nM, were obtained by immobilizing the probe via click chemistry, whereas amine-based immobilization combined with passivation with diamine carrying positive charges granted much slower dissociation kinetics, yielding an equilibrium dissociation constant as low as 0.5 nM. These results offer quantitative criteria for an optimal selection of surface copolymer coatings, depending on the application.

Monolithic Papain-Immobilized Enzyme Reactors for Automated Structural Characterization of Monoclonal Antibodies

The characterization of monoclonal antibodies (mAbs) requires laborious and time-consuming sample preparation steps before the liquid chromatography–mass spectrometry (LC-MS) analysis. Middle-up approaches entailing the use of specific proteases (papain, IdeS, etc.) emerged as practical and informative methods for mAb characterization. This work reports the development of immobilized enzyme reactors (IMERs) based on papain able to support mAb analytical characterization. Two monolithic IMERs were prepared by the covalent immobilization of papain on different supports, both functionalized via epoxy groups: a Chromolith® WP 300 Epoxy silica column from Merck KGaA and a polymerized high internal phase emulsion (polyHIPE) material synthesized by our research group. The two bioreactors were included in an in-flow system and characterized in terms of immobilization yield, kinetics, activity, and stability using Nα-benzoyl-L-arginine ethyl ester (BAEE) as a standard substrate. Moreover, the two bioreactors were tested toward a standard mAb, namely, rituximab (RTX). An on-line platform for mAb sample preparation and analysis with minimal operator manipulation was developed with both IMERs, allowing to reduce enzyme consumption and to improve repeatability compared to in-batch reactions. The site-specificity of papain was maintained after its immobilization on silica and polyHIPE monolithic supports, and the two IMERs were successfully applied to RTX digestion for its structural characterization by LC-MS. The main pros and cons of the two supports for the present application were described.

Advances in Colorimetric Assay Based on AuNPs Modified by Proteins and Nucleic Acid Aptamers

This review is focused on the biosensing assay based on AuNPs (AuNPs) modified by proteins, peptides and nucleic acid aptamers. The unique physical properties of AuNPs allow their modification by proteins, peptides or nucleic acid aptamers by chemisorption as well as other methods including physical adsorption and covalent immobilization using carbodiimide chemistry or based on strong binding of biotinylated receptors on neutravidin, streptavidin or avidin. The methods of AuNPs preparation, their chemical modification and application in several biosensing assays are presented with focus on application of nucleic acid aptamers for colorimetry assay for determination of antibiotics and bacteria in food samples.

Preparation and characterization of stable Core/Shell Fe3O4@Au ‎decorated with amine group for immobilization of lipase by covalent ‎attachment

The self-assembly approach was used for amine decoration of core/shell Fe3O4@Au with 4-aminothiphenol. This structure was used for covalent immobilization of lipase using a Ugi 4-component reaction. The amine group on the structure and carboxylic group from lipase can react in the Ugi reaction and a firm and stable covalent bond creates between enzyme and support. The synthesized structure was fully characterized and its activity was explored in different situations. The results displayed the pH and temperature stability of immobilized lipase compared to free lipase in a wide range of pH and temperature. Also after 60 days, it showed excellent activity while residual activity for the free enzyme was only 10%. The synthesized structure was conveniently separated using an external magnetic field and reused 6 times without losing the activity of the immobilized enzyme.