Secondary Structure in Enzyme-Inspired Polymer Catalysts Impacts Water Oxidation Efficiency.

Protein structure plays an essential role on their stability, functionality, and catalytic activity. In this work, the interplay between the ß-sheet structure and its catalytic implications to the design of enzyme-inspired materials is investigated. Here, inspiration is drawn from the active sites and ß-sheet rich structure of the highly efficient multicopper oxidase (MCO) to engineer a bio-inspired electrocatalyst for water oxidation utilizing the abundant metal, copper. Copper ions are coordinated to poly-histidine (polyCuHis), as they are in MCO active sites. The resultant polyCuHis material effectively promotes water oxidation with low overpotentials (0.15 V) in alkaline systems. This activity is due to the 3D structure of the poly-histidine backbone. By increasing the prevalence of ß-sheet structure and decreasing the random coil nature of the polyCuHis secondary structures, this study is able to modulates the electrocatalytic activity of this material is modulated, shifting it toward water oxidation. These results highlight the crucial role of the local environment at catalytic sites for efficient, energy-relevant transformations. Moreover, this work highlights the importance of conformational structure in the design of scaffolds for high-performance electrocatalysts.

Hydrogen bioelectrogeneration with pH-resilient and oxygen-tolerant cobalt apoenzyme-saccharide.

Hydrogenases are enzymes that catalyze the reversible conversion of protons to hydrogen gas, using earth-abundant metals such as nickel and/or iron. This characteristic makes them promising for sustainable energy applications, particularly in clean hydrogen production. However, their widespread use faces challenges, including a limited pH range and susceptibility to oxygen. In response to these issues, SacCoMyo is introduced as an artificial enzyme. SacCoMyo is designed by replacing the native metal in the myoglobin (Myo) scaffold with a hydroxocobalamin (Co) porphyrin core and complemented by a protective heteropolysaccharide-linked (Sac) shell. This engineered protein proves to be resilient, maintaining robust functionality even in acidic environments and preventing denaturation in a pH 1 electrolyte. The cobalt porphyrin core of SacCoMyo reduces the activation overpotential for hydrogen generation. A high turnover frequency of about 2400 H2 s-1 is demonstrated in the presence of molecular oxygen, showcasing its potential in biohydrogen production and its ability to overcome the limitations associated with natural hydrogenases.

Expanding the application of graphene vertical devices to dual femtomolar detection of SARS-CoV-2 receptor binding domain in serum and saliva.

The emergence of the graphene-based hybrid electrical-electrochemical vertical device (EEVD) has introduced a promising nanostructured biosensor tailored for point-of-care applications. In this study, we present an innovative EEVD capable of simultaneously detecting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in both serum and saliva. The foundation of the EEVD lies in a poly-neutral red-graphene heterojunction, which has been enhanced with a bioconjugate of gold nanoparticles and antibodies. The biodevice demonstrates a remarkable limit of detection, registering at the femtomolar scale (2.86 fmol L-1 or 0.1 pg mL-1). Its sensitivity is characterized by a 6.1 mV/decade response, and its operational range spans 10-12 to 10-7 g mL-1 in both serum and saliva samples. With a 20.0 µL of biological samples and a rapid processing time of under 10 min, the EEVD achieves the feat of dual antigen detection. The tests achieved 100.0% specificity, accuracy, and sensitivity in saliva, and 100.0% specificity, 88.9% accuracy, and 80.0% sensitivity in serum. This study highlights the EEVD as a low-cost solution of rapid viral detection during the crucial initial phases of COVID-19 infections.

Low-dimensionality carbon-based biosensors: the new era of emerging technologies in bioanalytical chemistry.

Since the last decade, carbon nanomaterials have had a notable impact on different fields such as bioimaging, drug delivery, artificial tissue engineering, and biosensors. This is due to their good compatibility toward a wide range of chemical to biological molecules, low toxicity, and tunable properties. Especially for biosensor technology, the characteristic features of each dimensionality of carbon-based materials may influence the performance and viability of their use. Surface area, porous network, hybridization, functionalization, synthesis route, the combination of dimensionalities, purity levels, and the mechanisms underlying carbon nanomaterial interactions influence their applications in bioanalytical chemistry. Efforts are being made to fully understand how nanomaterials can influence biological interactions, to develop commercially viable biosensors, and to gain knowledge on the biomolecular processes associated with carbon. Here, we present a comprehensive review highlighting the characteristic features of the dimensionality of carbon-based materials in biosensing.

From research to market: correlation between publications, patent filings, and investments in development and production of technological innovations in biosensors.

As the global population grows and science and technology development evolve, fulfilling basic human needs has been even more linked to technological solutions. In this review, we present an overview of the biosensor market and discuss the factors that make certain countries more competitive than others in terms of technology and innovation and how this is reflected in the trends in publication and patent filling. Additionally, we expose briefly how the COVID-19 pandemic acts as a catalyst for the integration of research and development, business, and innovation sectors to bring solutions and ideas that have been predicted as tendencies for the future.

Graphene-based hybrid electrical-electrochemical point-of-care device for serologic COVID-19 diagnosis.

The outbreak of COVID-19 pandemics highlighted the need of sensitive, selective, and easy-to-handle biosensing devices. In the contemporary scenario, point-of-care devices for mass testing and infection mapping within a population have proven themselves as of primordial importance. Here, we introduce a graphene-based Electrical-Electrochemical Vertical Device (EEVD) point-of-care biosensor, strategically engineered for serologic COVID-19 diagnosis. EEVD uses serologic IgG quantifications on SARS-CoV-2 Receptor Binding Domain (RBD) bioconjugate immobilized onto device surface. EEVD combines graphene basal plane with high charge carrier mobility, high conductivity, low intrinsic resistance, and interfacial sensitivity to capacitance alterations. EEVD application was carried out in real human serum samples. Since EEVD is a miniaturized device, it requires just 40 µL of sample for a point-of-care COVID-19 infections detection. When compared to serologic assays such ELISA and other immunochromatographic methods, EEVD presents some advantages such as time of analyses (15 min), sample preparation, and a LOD of 1.0 pg mL-1. We glimpse that EEVD meets the principles of robustness and accuracy, desirable analytic parameters for assays destined to pandemics control strategies.

Non-destructive molecular FTIR spectromicroscopy for real time assessment of redox metallodrugs.

Recent emergence of FTIR spectromicroscopy (micro-FTIR) as a dynamic spectroscopy for imaging to study biological chemistry has opened new possibilities for investigating in situ drug release, redox chemistry effects on biological molecules, DNA and drug interactions, membrane dynamics, and redox reactions with proteins at the single cell level. Micro-FTIR applied to metallodrugs has been playing an important role since the last decade because of its great potential to achieve more robust and controlled pharmacological effects against several diseases, including cancer. An important aspect in the development of these drugs is to understand their cellular properties, such as uptake, accumulation, activity, and toxicity. In this review, we present the potential application of micro-FTIR and its importance for studying metal-based drugs, highlighting the perspectives of chemistry of living cells. We also emphasise bioimaging, which is of high importance to localize the cellular processes, for a proper understanding of the mechanism of action.

On the Weak Binding and Spectroscopic Signature of SARS-CoV-2 nsp14 Interaction with RNA.

The SARS-CoV-2 non-structural protein 14 (nsp14), known as exoribonuclease is encoded from the large polyprotein of viral genome and is a major constituent of the transcription replication complex (TRC) machinery of the viral RNA synthesis. This protein is highly conserved among the coronaviruses and is a potential target for the development of a therapeutic drug. Here, we report the SARS-CoV-2 nsp14 expression, show its structural characterization, and ss-RNA exonuclease activity through vibrational and electronic spectroscopies. The deconvolution of amide-I band in the FTIR spectrum of the protein revealed a composition of 35 % α-helix and 25 % ß-sheets. The binding between protein and RNA is evidenced from the spectral changes in the amide-I region of the nsp14, showing protein conformational changes during the binding process. A value of 20.60±3.81 mol L-1 of the binding constant (KD ) is obtained for nsp14/RNA complex. The findings reported here can motivate further studies to develop structural models for better understanding the mechanism of exonuclease enzymes for correcting the viral genome and can help in the development of drugs against SARS-CoV-2.

Nanosized non-proteinaceous complexes III and IV mimicking electron transfer of mitochondrial respiratory chain.

Synthetic biology pursues the understanding of biological processes and their possible mimicry with artificial bioinspired materials. A number of materials have already been used to mimic the active site of simple redox proteins, including nanosized iron oxides due to their redox properties. However, the mimicry of membrane redox protein complexes is still a challenge. Herein, magnetic iron oxide nanoparticles (NPs), incorporated as non-proteinaceous complexes III and IV in a mitochondrial model membrane, catalyze electron transfer (ET) similarly to the natural complexes towards cytochrome c. The associated molecular mechanism is experimentally proven in solution and in a Langmuir-Blodgett film. A direct and entropy-driven ET, with rate constant of 2.63 ± 0.05Lmol-1 at 25 °C, occurs between the iron sites of the NPs and the cytochrome c heme group, not affecting the protein secondary and tertiary structures. This process requires an activation energy of 40.2 ± 1.5 kJ mol-1 resulting in an overall Gibbs free energy of -55.3 kJ mol-1. Furthermore, the protein-NP system is governed by electrostatic and non-polar forces that contribute to an associative mechanism in the transition state. Finally, the incorporated NPs in a model membrane were able to catalyze ET, such as the natural complexes in respiratory chain. This work presents an experimental approach demonstrating that inorganic nanostructured systems may behave as embedded proteins in the eukaryotic cells membrane, opening the way for more sophisticated and robust mimicry of membrane protein complexes.

Highly sensitive interfaces of graphene electrical-electrochemical vertical devices for on drop atto-molar DNA detection.

The development of novel high-sensitivity DNA-based biosensors is beneficial, as these devices have applications in the identification of genetic risk factors, medical diagnostics, and environmental monitoring. Herein, we report on the first robust device capable of detecting DNA on a microliter drop with a zepto-molar (10-21) concentration. To accomplish this, we engineered an electrical-electrochemical vertical device (EEVD) that comprises a novel drain and source terminal in a short-circuited configuration, paired with an ideal non-polarizable reference electrode. Vertical electron transfer occurs perpendicularly to the graphene plane, while the electronic current flows through the graphene van der Waals (vdW) heterojunctions. Ferrocene adsorbed on graphene was strategically chosen as the vdW heterojunction redox component. Charge carrier insertion into the graphene makes the EEVD 10 times more sensitive than traditional graphene field-effect transistors. Interfacial potential changes were measured for single-stranded DNA detection with an unprecedented zepto-molar limit of detection.

On the Challenges for the Diagnosis of SARS-CoV-2 Based on a Review of Current Methodologies.

Diagnosis of COVID-19 has been challenging owing to the need for mass testing and for combining distinct types of detection to cover the different stages of the infection. In this review, we have surveyed the most used methodologies for diagnosis of COVID-19, which can be basically categorized into genetic-material detection and immunoassays. Detection of genetic material with real-time polymerase chain reaction (RT-PCR) and similar techniques has been achieved with high accuracy, but these methods are expensive and require time-consuming protocols which are not widely available, especially in less developed countries. Immunoassays for detecting a few antibodies, on the other hand, have been used for rapid, less expensive tests, but their accuracy in diagnosing infected individuals has been limited. We have therefore discussed the strengths and limitations of all of these methodologies, particularly in light of the required combination of tests owing to the long incubation periods. We identified the bottlenecks that prevented mass testing in many countries, and proposed strategies for further action, which are mostly associated with materials science and chemistry. Of special relevance are the methodologies which can be integrated into point-of-care (POC) devices and the use of artificial intelligence that do not require products from a well-developed biotech industry.

Problems of Interpreting Diagnostic Tests for SARS-CoV-2: Analytical Chemistry Concerns.

The COVID-19 pandemic outbreak made the development of reliable, sensitive, and reproducible testing methods crucial throughout the world. Without proper analytical validation, testing results can be misinterpreted, leading to a certain degree of misinformation in the clinical area. To accurately assess the methods, the determination of an analytical linear range of response of the assay is fundamental. Based on this curve, the evaluation of some parameters as sensitivity, limit of detection, and limit of quantification can be done, as well as the establishment of cut-off values. Statistical treatments of the collected data can be performed for reproducibility and reliability evaluations. In this context, there is a wide range of analytical concerns that should be in-depth discussed in medical, biomedical and chemical areas. This letter aims to briefly clarify some analytical chemistry concepts, as sensitivity, cut-off and limit of detection, and their application towards clinical diagnosis.

Recognizing conductive islands in polymeric redox surfaces using electrochemical-coupled vibrational spectromicroscopy.

We introduce a set up by coupling multiplex FTIR microscopy to electrochemistry through a home-made spectroelectrochemical cell to observe real time changes in the electronic states of polymeric islands by monitoring the oxidation states of polyaniline (PANI). The resultant technique, called electrochemical-coupled vibrational spectromicroscopy (EVSM), enables the measurement of structural changes in the conductive islands of PANI with the spatial resolution as high as 2.5 µm. Unique 2D and 3D chemical maps obtained by the integration of the spectral bands in the subtractively normalized interfacial infrared (SNIFTIR) spectra reveal electrochemical heterogeneity, showing promising topological properties control for conducting polymer-based electronic devices.

A combined Far-FTIR, FTIR Spectromicroscopy, and DFT Study of the Effect of DNA Binding on the [4Fe4S] Cluster Site in EndoIII.

Endonuclease III (EndoIII) is a DNA glycosylase that contains the [4Fe4S] cluster, which is essential for the protein to bind to damaged DNA in a process called base excision repair (BER). Here we propose that the change in the covalency of Fe-S bonds of the [4Fe4S] cluster caused by double-stranded (ds)-DNA binding is accompanied by a change in their strength, which is due to alterations of the electronic structure of the cluster. Micro-FTIR spectroscopy in the mid-IR region and FTIR spectroscopy in the far IR (450 and 300 cm-1) were used independently to study the structural changes in EndoIII and the behavior of the [4Fe4S] cluster it contains, in the native form and upon its binding to ds-DNA. Structural changes in the DNA itself were also examined. The characteristics vibrational modes, corresponding to Fe-S (sulfide) and Fe-S (thiolate) bonds were identified in the cluster through far IR spectroscopy as well through quantum chemistry calculations. Based on the experimental results, these vibrational modes shift in their spectral positions caused by negatively charged DNA in the vicinity of the cluster. Modifications of the Fe-S bond lengths upon DNA binding, both of the Fe-S (sulfide) and Fe-S (thiolate) bonds in the [4Fe4S] cluster of EndoIII are responsible for the stabilization of the cluster towards higher oxidation state (3+), and hence its redox communication along the ds-DNA helix.

Real-time redox monitoring of a nitrosyl ruthenium complex acting as NO-donor agent in a single A549 cancer cell with multiplex Fourier-transform infrared microscopy.

Multiplex Fourier-transform infrared microscopy (µFT-IR) helped to monitor trans-[Ru(NO) (NH3)4 (isn)]3+(I), uptake by A549 lung carcinoma cell, as well as the generation of its product, nitric oxide (NO), inside the cell. Chronoamperometry with NO-sensor and µFT-IR showed that exogenous NADH and the A549 cell induced the NO release redox mechanism. Chemical imaging confirmed that (I) was taken up by the cell, and that its localization coincided with its consumption in the cellular environment within 15 min of exposure. The Ru-NO absorption band in the IR spectrum shifted from 1932 cm-1, when NO was coordinated to Ru as {RuII-NO+}3+, to 1876 cm-1, due the formation of reduced species {RuII-NO0}2+, a precursor of NO release. Futhermore, the µFT-IR spectral profile demonstrated that, as a result of the NO action on the target, NO interacted with nucleic acids, which provided a biochemical response that is detectable in living cells.

Assessing electron transfer reactions and catalysis in multicopper oxidases with operando X-ray absorption spectroscopy.

Here we propose an experimental setup based on operando X-ray absorption spectroscopy (XAS) to understand why copper-containing oxidoreductase enzymes show exceptional performance as catalysts for the oxygen reduction reaction (ORR). An electrode based on carbon nanoparticles organized in mesoporous structures with bilirubin oxidase (BOD) was developed to be used in a home-made operando XAS electrochemical cell, and we probed the electron transfer under ORR regime. In the presence of molecular oxygen, the BOD cofactor containing 4 copper ions require an overpotential about 150 mV to be reduced as compared to that in the absence of oxygen. A second electron transfer step, which occurs faster than the cofactor reduction, suggests that the cooper ions act as a tridimensional redox active electronic bridges for the electron transfer reaction.

Microwires of Au-Ag Nanocages Patterned via Magnetic Nanoadhesives for Investigating Proteins using Surface Enhanced Infrared Absorption Spectroscopy.

The controlled assembly of metal nanoparticles into ordered structures interacting with biological molecules is an emerging concept in surface science. Here, bare magnetite nanoparticles (Fe3O4-NPs) were employed as nanoadhesives to capture hollow metallic nanostructures (Au-Ag nanocages) from aqueous suspensions, and these coupled nanostructures were patterned onto various types of substrate via magnetolithography. Microwires of Au-Ag nanocages patterned onto an Au substrate behaved as optical antennas, providing a plasmonic enhancement exploited in surface-enhanced infrared absorption spectroscopy (SEIRAS) to investigate the proteins cytochrome c, bilirubin oxidase, alcohol dehydrogenase, bovine serum albumin, and glucose oxidase. Chemical maps containing more than 4000 spectra, acquired within only 2 min with a focal plane array detector, indicate that proteins were adsorbed along the microwires with their secondary structure preserved according to the spatial distribution of their amide groups. We believe there are significant practical aspects of the methodology proposed here to develop an alternative label-free assay for investigating biological molecules.

The structure-property relationship in LAPONITE® materials: from Wigner glasses to strong self-healing hydrogels formed by non-covalent interactions.

Rheology, small-angle X-ray scattering (SAXS), and dynamic light scattering (DLS) analysis, zeta potential measurement, scanning electron microscopy (SEM), and micro-FTIR and absorbance spectroscopy were used to enlighten the controversial literature about LAPONITE® materials. Our data suggest that pristine LAPONITE® in water does not form hydrogels induced by the so-called "house of cards" assembly, but rather forms Wigner glasses governed by repulsive forces. Ionic interactions between anisotropic LAPONITE® nanodiscs, sodium polyacrylate and inorganic salts afforded hydrogels that were transparent, self-standing, moldable, strong, and biocompatible with shear-thinning and self-healing behavior. An extensive study on the role of salts in the gelification process dictates a trend that relates the valence of cations with the viscoelastic properties of the bulk material (G' values follow the trend, monovalent < divalent < trivalent). These hydrogels present G' values up to 5.1 × 104 Pa, which are considered high values for non-covalent hydrogels. Hydrogels crosslinked with sodium phosphate salts are biocompatible, and might be valid candidates for injectable drug delivery systems due to their shear-thinning behavior with rapid self-healing after injection.

Nano-flocks of a bimetallic organic framework for efficient hydrogen evolution electrocatalysis.

Nano-flocks of a bimetallic organic framework (NiCo-MOF) were grown on a nickel mesh and covered with a graphene oxide aerosol skeleton by employing a nebulizer air compressor. The resulting material induces a high accessible active surface area and shows enhanced electrocatalytic activity towards the hydrogen evolution reaction with excellent stability in alkaline medium.

Interplay of non-uniform charge distribution on the electrochemical modification of graphene.

Graphene is considered a model material for surfaces because it is stable despite being composed of a single layer of carbon atoms. Although the thermal and electronic properties of graphene are well reported, the behavior of graphene sheets with the addition of charges to the structure is not well understood. Combining infrared spectroscopy, electrochemical analysis, and computational simulations, we report the effect of an electrochemically induced covalent anchoring of 4-carboxyphenyl (4-CP) units on the optical and electronic properties of graphene. Charges in graphene become concentrated at specific sites of the sheet when electrochemically perturbed and the functionalization occurs inhomogeneously along the graphene sheet. We observed that, when graphene is covalently functionalized, the resistance to heterogeneous electron transfer is increased by a factor of 1.4. Furthermore, scattering-type scanning near-field optical microscopy and atomic force microscopy show that the covalent functionalization affects drastically the optical and physical properties of the graphene/SiO2 system, especially the plasmon-phonon coupling after the functionalization. In addition, from these we infer that a comparatively higher degree of functionalization occurs near the electrode edges. These results are supported by computational simulations, which show that the covalent anchoring of 4-CP units weakens electron transfer because the charges are retained on the sp3-hybridized carbon atoms generated upon functionalization, suggesting that graphene properties are deeply influenced by the way the molecules are immobilized on its structure.