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.

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.

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.

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.

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.

Multiplex Infrared Spectroscopy Imaging for Monitoring Spatially Resolved Redox Chemistry.

IR spectroscopy is an excellent method for understanding surface redox chemistry. However, obtaining sufficient spatial resolution to analyze in situ surface redox reactions is difficult because the aqueous sampling environments provide some challenges for IR spectroscopy. These challenges arise because of the vibrational contribution of water. In this letter, we demonstrate a solution to this problem, where the key development enabling the coupling of spectromicroscopy with electrochemical measurements is a CaF2/electrolyte/Au sandwich IR-sensitive sample holder that acts as an electrochemical cell. In this system, there is a very thin layer of aqueous electrolyte (∼10 µm), and it is possible to monitor, in real time, the vibrational maps and changes to the Au surface modified with iron(II, III) hexacyanoferrate(II, III) by varying the electrochemical potential. By selecting specific vibrational modes with a focal plane array detector, which allows the simultaneous collection of IR spectra from 4096 microscopic regions, chemical maps showing the surface changes were obtained and analyzed using color, providing new insights into how the charge transfer processes affect the chemical composition in specific 2D spatially resolved regions.

Supramolecular Arrangement of Iron Phthalocyanine in Langmuir-Schaefer and Electrodeposited Thin Films.

The supramolecular arrangement in thin film technology has been explored through different deposition techniques aiming to control the film properties at the molecular level. We report on the formation of iron phthalocyanine (FePc) films using both Langmuir-Schaefer (LS) and electrodeposition methods. The multilayer formation was monitored with ultraviolet-visible absorption spectroscopy (UV-vis) and electrochemical measurements. According to Raman spectroscopy, atomic force microscopy (AFM), and scanning electron microscopy (SEM), the surface morphology of electrodeposited films is more homogeneous than LS films at micro and nanometer scales. From FTIR spectroscopy, the FePc molecules in the electrodeposited films are oriented preferentially with the macrocycle parallel to the substrate surface (flat-on), while a slight tilt is suggested in LS films, being both films crystalline. Therefore, the use of different deposition techniques allowed the fabrication of thin films from FePc with distinct supramolecular arrangements, leading to distinct electrochemical properties. For instance, the electrodeposited films show higher surface coverage, suggesting a more compact structure, which favors the charge transfer and smaller energy gap. The possibility of tuning some properties according to deposition technique for the same material can help the development of technological applications such as electronic or sensing devices.

Advances in enzyme bioelectrochemistry.

Bioelectrochemistry can be defined as a branch of Chemical Science concerned with electron-proton transfer and transport involving biomolecules, as well as electrode reactions of redox enzymes. The bioelectrochemical reactions and system have direct impact in biotechnological development, in medical devices designing, in the behavior of DNA-protein complexes, in green-energy and bioenergy concepts, and make it possible an understanding of metabolism of all living organisms (e.g. humans) where biomolecules are integral to health and proper functioning. In the last years, many researchers have dedicated itself to study different redox enzymes by using electrochemistry, aiming to understand their mechanisms and to develop promising bioanodes and biocathodes for biofuel cells as well as to develop biosensors and implantable bioelectronics devices. Inside this scope, this review try to introduce and contemplate some relevant topics for enzyme bioelectrochemistry, such as the immobilization of the enzymes at electrode surfaces, the electron transfer, the bioelectrocatalysis, and new techniques conjugated with electrochemistry vising understand the kinetics and thermodynamics of redox proteins. Furthermore, examples of recent approaches in designing biosensors and biofuel developed are presented.

Electrochemistry of the [4Fe4S] Cluster in Base Excision Repair Proteins: Tuning the Redox Potential with DNA.

Escherichia coli endonuclease III (EndoIII) and MutY are DNA glycosylases that contain [4Fe4S] clusters and that serve to maintain the integrity of the genome after oxidative stress. Electrochemical studies on highly oriented pyrolytic graphite (HOPG) revealed that DNA binding by EndoIII leads to a large negative shift in the midpoint potential of the cluster, consistent with stabilization of the oxidized [4Fe4S]3+ form. However, the smooth, hydrophobic HOPG surface is nonideal for working with proteins in the absence of DNA. In this work, we use thin film voltammetry on a pyrolytic graphite edge electrode to overcome these limitations. Improved adsorption leads to substantial signals for both EndoIII and MutY in the absence of DNA, and a large negative potential shift is retained with DNA present. In contrast, the EndoIII mutants E200K, Y205H, and K208E, which provide electrostatic perturbations in the vicinity of the cluster, all show DNA-free potentials within error of wild type; similarly, the presence of negatively charged poly-l-glutamate does not lead to a significant potential shift. Overall, binding to the DNA polyanion is the dominant effect in tuning the redox potential of the [4Fe4S] cluster, helping to explain why all DNA-binding proteins with [4Fe4S] clusters studied to date have similar DNA-bound potentials.

A primary battery-on-a-chip using monolayer graphene.

We present here a bottom-up approach for realizing on-chip on-demand batteries starting out with chemical vapor deposition-grown graphene. Single graphene monolayers contacted by electrode lines on a silicon chip serve as electrodes. The anode and cathode are realized by electrodeposition of zinc and copper respectively onto graphene, leading to the realization of a miniature graphene-based Daniell cell on a chip. The electrolyte is housed partly in a gel and partly in liquid form in an on-chip enclosure molded using a 3d printer or made out of poly(dimethylsiloxane). The realized batteries provide a stable voltage (∼1.1 V) for many hours and exhibit capacities as high as 15 µAh, providing enough power to operate a pocket calculator. The realized batteries show promise for deployment as on-chip power sources for autonomous systems in lab-on-a-chip or biomedical applications.

Magnetically controlled single-nanoparticle detection via particle-electrode collisions.

This paper reports the magnetic control of nanoparticle collisions on gold ultramicroelectrode surface. Magnetite nanoparticles with diameters of 10 nm and modified with Prussian blue (Fe3O4-NPs-PB) were directed by gravitational force on the electrode surface, and spikes in current-time transients were observed. By modulating a magnetic field parallel to the electrode surface, the number of nanoparticle collisions and the nanoparticle positions could be controlled.

Evidence of short-range electron transfer of a redox enzyme on graphene oxide electrodes.

Direct electron transfer (DET) between redox enzymes and electrode surfaces is of growing interest and an important strategy in the development of biofuel cells and biosensors. Among the nanomaterials utilized at electrode/enzyme interfaces to enhance the electronic communication, graphene oxide (GO) has been identified as a highly promising candidate. It is postulated that GO layers decrease the distance between the flavin cofactor (FAD/FADH2) of the glucose oxidase enzyme (GOx) and the electrode surface, though experimental evidence concerning the distance dependence of the rate constant for heterogeneous electron-transfer (k(het)) has not yet been observed. In this work, we report the experimentally observed DET of the GOx enzyme adsorbed on flexible carbon fiber (FCF) electrodes modified with GO (FCF-GO), where the k(het) between GO and electroactive GOx has been measured at a structurally well-defined interface. The curves obtained from the Marcus theory were used to obtain k(het), by using the model proposed by Chidsey. In agreement with experimental data, this model proved to be useful to systematically probe the dependence of electron transfer rates on distance, in order to provide an empirical basis to understand the origin of interfacial DET between GO and GOx. We also demonstrate that the presence of GO at the enzyme/electrode interface diminishes the activation energy by decreasing the distance between the electrode surface and FAD/FADH2.

Enhanced label-free detection of proteins on Au nanoparticle micropatterns for surface-enhanced infrared absorption spectroscopy.

A label-free one-step lithographically masked deposition technique was implemented for the fabrication of gold nanoparticle (Au NP) micropatterns. These micropatterns serve as active substrates for surface-enhanced infrared absorption spectroscopy (SEIRAS) and exhibit a substantial increase in the IR signal upon adsorption of multiple proteins compared to untreated surfaces. Micro-FTIR chemical imaging was conducted to evaluate the efficacy of Au NP micropatterns as singular enhancers for SEIRAS across diverse IR-active substrates demonstrating a promising application for the detection of proteins at low concentrations within biological fluids.

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.

Flexible carbon cloth electrode modified by hollow core-mesoporous shell carbon as a novel efficient bio-anode for biofuel cell.

A new approach is described to produce an efficient electrode material for biofuel cells using flexible carbon cloth (FCC) and hollow core-mesoporous shell carbon (HCMSC) nanospheres as bio-anode materials. The bio-electrochemical activity of glucose oxidase (GOx) enzyme adsorbed on this bio-anode was evaluated, with the maximum anodic current density varying from 80 microA cm(-2) to 180 microA cm-2 for glucose concentrations up to 5.0 mmol L(-1) for the FCC modified electrode with HCMSCs. The open circuit cell voltage was E(0) = 380 mV, and the catalytic electro-oxidation current of glucose reached 0.1 mA cm(-2) at 0.0 V versus Ag/AgCl. This new system employing HCMSC-based FCC is promising toward novel bio-anodes for biofuel cells using glucose as a fuel.

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.