Unusual dark-field hyperspectral and confocal Raman microscopy features of a nanoporous gold electrode coated with porphyrazine complex.

Nanoporous gold electrodes are of great interest in electroanalytical chemistry, because of their unusual activity and large surface area. The electrochemical activity can be further improved by coating with molecular catalysts such as the tetraruthenated cobalt-tetrapyridylporphyrazines investigated in this work. The plasmonic enhancement of the scattered light at the nanoholes and borders modifies the electrode's optical characteristics, improving the transmission through the surface-enhanced Raman scattering (SERS) effect. When monitored by hyperspectral dark-field and confocal Raman microscopy, this effect allows probing of the porphyrazine species at the plasmonic nanholes, improving the understanding of the chemically modified gold electrodes.

Patterning (Electro)chemical Treatment-Free Electrodes with a 3D Printing Pen.

A simple fabrication method to make electrochemical sensors is reported. The electrodes were fabricated with a commercial filament based on polylactic acid and carbon black (PLA/CB). They were engineered with a three-dimensional (3D) printing pen and poly(methyl methacrylate) template. The optimization parameters included the thickness and diameters of the electrodes. The electrode diameter was restricted by the 3D printing pen's nozzle dimension, and larger diameters generated small cracks on the electrode surface, compromising their analytical signal. The electrode thickness can increase the electrical resistance, affecting their electrochemical response. The fabrication showed reproducibility (RSD = 4%). The electrode surface was easily renewed by sanding the electrodes, making them reusable. Additionally, the proposed sensor provided comparable electrochemical responses over traditional glassy carbon electrodes. Moreover, no (electro)chemical surface treatment was required for sensing applications due to the compromise between the thickness and diameters of the electrodes, effectively translating the filaments' electrical properties to resulting materials. The electrodes' analytical performance was shown for organic and inorganic species, including paraquat, Pb2+, and caffeic acid. As proof of concept, the analytical applicability was demonstrated for total polyphenolic quantification in tea samples. Therefore, this work provides an alternative to fabricating miniaturized electrodes, bringing valuable insights into PLA/CB 3D-printed sensors and opening possibilities for designing electrode arrays. Moreover, the proposed electrodes are promising platforms for paper-based microfluidic systems.

Multiple Pulse Amperometry-An Antifouling Approach for Nitrite Determination Using Carbon Fiber Microelectrodes.

Nitrite is a ubiquitous pollutant in modern society. Developing new strategies for its determination is very important, and electroanalytical methods present outstanding performance on this task. However, the use of bare electrodes is not recommended because of their predisposition to poisoning and passivation. We herein report a procedure to overcome these limitations on carbon fiber microelectrodes through pulsed amperometry. A three-pulse amperometry approach was used to reduce the current decay from 47% (after 20 min under constant potential) to virtually 0%. Repeatability and reproducibility were found to have an RSD lower than 0.5% and 7%, respectively. Tap water and synthetic inorganic saliva samples were fortified with nitrite, and the results obtained with the proposed sensor were in good agreement with the amount added.

Sandwich-Type Electrochemical Paper-Based Immunosensor for Claudin 7 and CD81 Dual Determination on Extracellular Vesicles from Breast Cancer Patients.

This study is focused on identifying novel epithelial markers in circulating extracellular vesicles (EVs) through the development of a dual sandwich-type electrochemical paper-based immunosensor for Claudin 7 and CD81 determination, as well as its validation in breast cancer (BC) patients. This immunosensor allows for rapid, sensitive, and label-free detection of these two relevant BC biomarkers. Under optimum conditions, the limit of detection for Claudin 7 was 0.4 pg mL-1, with a wide linear range of 2 to 1000 pg mL-1, while for CD81, the limit of detection was 3 pg mL-1, with a wide linear range of 0.01 to 10 ng mL-1. Finally, we validated Claudin 7 and CD81 determination in EVs from 60 BC patients and 20 healthy volunteers, reporting higher diagnostic accuracy than the one observed with classical diagnostic markers. This analysis provides a low-cost, specific, versatile, and user-friendly strategy as a robust and reliable tool for early BC diagnosis.

Molecular mechanisms associated with acidification and alkalization along the larval midgut of Musca domestica.

To disclose the molecular mechanisms involved in luminal midgut buffering of M. domestica, we used RNA-seq analyses from triplicate samples of seven sections along the midgut to evaluate the expression levels of genes coding for selected manually curated protein sequences. Channels, pumps and transporters were confirmed as being apical by proteomics of purified microvillar membranes. Midgut pH determinations with a microsensor and a pH indicator were carried out in larvae in different diets with or without added compounds to evaluate the role of proteins in buffering. The data suggested that acidification occurs at middle midgut by the action of H+ V-ATPase with protons produced by carbonic anhydrase, followed by chloride ions transported by a K+Cl- symporter. K+ ions are recovered by an apical K+ channel and K+ homeostasis maintained by a basolateral Na+/K+-ATPase. Acidification is also affected by a Na+/H+ exchanger and a multidrug resistance protein. Posterior midgut alkalization results from the action of a NH3 transporter and H+-coupled peptide transporter, mainly in a diet rich in free peptides. A working model was proposed for the midgut luminal acidification and alkalization, as well as for mucosal protection against acid by a neutralized mucus layer.

Scanning Bipolar Electrochemical Microscopy.

Electrochemical techniques offer high temporal resolution for studying the dynamics of electroactive species at samples of interest. To monitor fastest concentration changes, a micro- or nanoelectrode is accurately positioned in the vicinity of a sample surface. Using a microelectrode array, it is even possible to investigate several sites simultaneously and to obtain an instantaneous image of local dynamics. However, the spatial resolution is limited by the minimal electrode size required in order to contact the electrodes. To provide a remedy, we introduce the concept of scanning bipolar electrochemical microscopy and the corresponding experimental system. This technique allows precise positioning of a wireless scanning bipolar electrode to convert spatially heterogeneous concentrations of the analyte of interest into an electrochemiluminescence map of the sample reactivity. After elucidating the working principle by recording bipolar line and array scans, a bipolar electrode array is positioned at the site of interest to record an electrochemical image of the localized release of analyte molecules.

Monitoring H2O2 inside Aspergillus fumigatus with an Integrated Microelectrode: The Role of Peroxiredoxin Protein Prx1.

Peroxiredoxins (Prx) are important proteins involved in hydroperoxide degradation and are related to virulence in several pathogens, including Aspergillus fumigatus. In this work, in vivo studies on the degradation of hydrogen peroxide (H2O2) in the microenvironment of A. fumigatus fungus were performed by using an integrated Pt microelectrode. Three A. fumigatus strains were used to confirm the role of the cytosolic protein Prx1 in the defense mechanism of this microorganism: a wild-type strain, capable to expressing the protein Prx1; a Δprx strain, whose gene prx1 was removed; and a genetically complemented Δprx1::prx1+ strain generated from the Δprx1 and in which the gene prx1 was reintroduced. The fabricated microelectrode was shown to be a reliable inert probe tip for in situ and real-time measurements of H2O2 in such microenvironments, with potential applications in investigations involving the mechanism of oxidative stress.

Electrochemical dopamine sensor using a nanoporous gold microelectrode: a proof-of-concept study for the detection of dopamine release by scanning electrochemical microscopy.

Nanoporous gold (NPG) structures were prepared on the surface of a gold microelectrode (Au-µE) by an anodization-reduction method. Cyclic voltammetry and field emission scanning electron microscopy were used to study the electrochemical properties and the morphology of the nanostructured film. Voltammetry showed an improved sensitivity for dopamine (DA) oxidation at this microelectrode when compared to a bare gold microelectrode, with a peak near 0.2 V (vs. Ag/AgCl) at a scan rate of 0.1 V s-1. This is due to the increased surface area and roughness. Square wave voltammetry shows a response that is linear in the 0.1-10 µmol L-1 DA concentration range, with a 30 nmol L-1 detection limit and a sensitivity of 1.18 mA (µmol L-1)-1 cm-2. The sensor is not interfered by ascorbic acid. The reproducibility, repeatability, long-term stability and real sample analysis (spiked urine) were assessed, and acceptable performance was achieved. The "proof-of-concept" detection of dopamine release was demonstrated by using scanning electrochemical microscopy (SECM) with the aim of future applications for single cell analysis. Graphical abstract A reproducible electrochemical approach was proposed to fabricate an NPG-microelectrode for DA detection, with enhanced sensitivity and selectivity. Besides, a proof-of-concept detection of DA release was also demonstrated by using SECM.

Pencil it in: pencil drawn electrochemical sensing platforms.

Inspired by recent reports concerning the utilisation of hand drawn pencil macroelectrodes (PDEs), we report the fabrication, characterisation (physicochemical and electrochemical) and implementation (electrochemical sensing) of various PDEs drawn upon a flexible polyester substrate. Electrochemical characterisation reveals that there are no quantifiable electrochemical responses upon utilising these PDEs with an electroactive analyte that requires an electrochemical oxidation step first, therefore the PDEs have been examined towards the electroactive redox probes hexaammineruthenium(iii) chloride, potassium ferricyanide and ammonium iron(ii) sulfate. For the first time, characterisation of the number of drawn pencil layers and the grade of pencil are examined; these parameters are commonly overlooked when utilising PDEs. It is demonstrated that a PDE drawn ten times with a 6B pencil presented the most advantageous electrochemical platform, in terms of electrochemical reversibility and peak height/analytical signal. In consideration of the aforementioned limitation, analytes requiring an electrochemical reduction as the first process were solely analysed. We demonstrate the beneficial electroanalytical capabilities of these PDEs towards p-benzoquinone and the simultaneous detection of heavy metals, namely lead(ii) and cadmium(ii), all of which are explored for the first time utilising PDEs. Initially, the detection limits of this system were higher than desired for electroanalytical platforms, however upon implementation of the PDEs in a back-to-back configuration (in which two PDEs are placed back-to-back sharing a single connection to the potentiostat), the detection limits for lead(ii) and cadmium(ii) correspond to 10 µg L(-1) and 98 µg L(-1) respectively within model aqueous (0.1 M HCl) solutions.

Chemical Characterization of Urate Hydroperoxide, A Pro-oxidant Intermediate Generated by Urate Oxidation in Inflammatory and Photoinduced Processes.

Urate hydroperoxide is a strong oxidant generated by the combination of urate free radical and superoxide. The formation of urate hydroperoxide as an intermediate in urate oxidation is potentially responsible for the pro-oxidant effects of urate in inflammatory disorders, protein degradation, and food decomposition. To understand the molecular mechanisms that sustain the harmful effects of urate in inflammatory and oxidative stress related conditions, we report a detailed structural characterization and reactivity of urate hydroperoxide toward biomolecules. Urate hydroperoxide was synthesized by photo-oxidation and by a myeloperoxidase/hydrogen peroxide/superoxide system. Multiple reaction monitoring (MRM) and MS(3) ion fragmentation revealed that urate hydroperoxide from both sources has the same chemical structure. Urate hydroperoxide has a maximum absorption at 308 nm, ε308nm = 6.54 ± 0.38 × 10(3) M(-1) cm(-1). This peroxide decays spontaneously with a rate constant of k = 2.80 ± 0.18 × 10(-4) s(-1) and a half-life of 41 min at 22 °C. Urate hydroperoxide undergoes electrochemical reduction at potential values less negative than -0.5 V (versus Ag/AgCl). When incubated with taurine, histidine, tryptophan, lysine, methionine, cysteine, or glutathione, urate hydroperoxide reacted only with methionine, cysteine, and glutathione. The oxidation of these molecules occurred by a two-electron mechanism, generating the alcohol, hydroxyisourate. No adduct between cysteine or glutathione and urate hydroperoxide was detected. The second-order rate constant for the oxidation of glutathione by urate hydroperoxide was 13.7 ± 0.8 M(-1) s(-1). In conclusion, the oxidation of sulfur-containing biomolecules by urate hydroperoxide is likely to be a mechanism by which the pro-oxidant and damaging effects of urate are mediated in inflammatory and photo-oxidizing processes.

Metallic modified (bismuth, antimony, tin and combinations thereof) film carbon electrodes.

In this paper in situ bismuth, antimony, tin modified electrodes and combinations thereof are explored towards the model target analytes cadmium(II) and lead(II), chosen since they are the most widely studied, to explore the role of the underlying electrode substrate with respect to boron-doped diamond, glassy carbon, and screen-printed graphite electrodes. It is found that differing electrochemical responses are observed, dependent upon the underlying electrode substrate. The electrochemical response using the available range of metallic modifications is only ever observed when the underlying electrode substrate exhibits relatively slow electron transfer properties; in the case of fast electron transfer properties, no significant advantages are evident. Furthermore these bismuth modified systems which commonly employ a pH 4 acetate buffer, reported to ensure the bismuth(III) stability upon the electrode surface can create create a problem when sensing at low concentrations of heavy metals due to its high background current. It is demonstrated that a simple change of pH can allow the detection of the target analytes (cadmium(II) and lead(II)) at levels below that set by the World Health Organisation (WHO) using bare graphite screen-printed electrodes.

Screen-printed back-to-back electroanalytical sensors: heavy metal ion sensing.

Screen-printed back-to-back microband electroanalytical sensors are applied to the quantification of lead(II) ions for the first time. In this configuration the electrodes are positioned back-to-back with a common electrical connection to the two working electrodes with the counter and reference electrodes for each connected in the same manner as a normal "traditional" screen-printed sensor. Proof-of-concept is demonstrated for the electroanalytical sensing of lead(II) ions utilising square-wave anodic stripping voltammetry where an increase in the electroanalytical sensitivity is observed by a factor of 5 with the back-to-back microband configuration at a fixed lead(II) ion concentration of 5 µg L(-1) utilising a deposition potential and time of -1.2 V and 30 seconds respectively, compared to a conventional (single) microband electrode. The back-to-back microband configuration allows for the sensing of lead(II) ions with a linear range from 5 to 110 µg L(-1) with a limit of detection (based on 3σ) corresponding to 3.7 µg L(-1). The back-to-back microband configuration is demonstrated to quantify the levels of lead(II) ions within drinking water corresponding to a level of 2.8 (±0.3) µg L(-1). Independent validation was performed using ICP-OES with the levels of lead(II) ions found to correspond to 2.5 (±0.1) µg L(-1); the excellent agreement between the two methods validates the electroanalytical procedure for the quantification of lead(II) ions in drinking water. This back-to-back configuration exhibits an excellent validated analytical performance for the determination of lead(II) ions within drinking water at World Health Organisation levels (limited to 10 µg L(-1) within drinking water).

A facile electrochemical approach to fabricate a nanoporous gold film electrode and its electrocatalytic activity towards dissolved oxygen reduction.

A nanoporous gold film (NPGF) was prepared onto a gold electrode surface using a facile and green electrochemical approach via anodization at 2.0 V in 0.5 mol L(-1) H2SO4. The as-synthesized NPGF electrode showed superior electrocatalytic activity towards dissolved oxygen reduction at a low overpotential (-0.045 V) along with 2-fold enhanced sensitivity.

A home-made nanoporous gold microsensor for lead(II) detection in seawater with high sensitivity and anti-interference properties.

A nanoporous gold microelectrode (NPG-µE) was fabricated and used for Pb(II) detection in seawater samples via square wave anodic stripping voltammetry (SWASV). The Au microelectrode (Au-µE) was fabricated by embedding a gold microfiber into a Pasteur pipette, and its surface was further modified by an anodization-electrochemical reduction (A-ECR) method, yielding the NPG-µE. The fabricated electrodes were characterized by cyclic voltammetry (CV) and field emission scanning electron microscopy (FE-SEM) for electrochemical and structural morphological investigations. SWASV results show a Pb(II) stripping peak at around -0.05 V vs. Ag/AgCl, sat. KCl, which is unusual for common Pb(II) detection (typically occurring at around -0.40 V) in anodic stripping voltammetry (ASV) analysis. The Pb(II) detection at less negative stripping potential is more beneficial. Hence, it exhibited anti-interference properties with Cd(II), which is attributed to the preferential deposition and stripping of the target analyte on the low-indexed crystal planes of the NPG structure. The calibration plot obtained by SWASV was linear in the concentration range of 0.1-10 µM, and the detection limit was found to be 57 nM (correlation coefficient of 0.9974). The NPG microsensor presented a 15-fold enhanced current response compared to Au-µE, with excellent sensitivity (27.2 µA µM-1 cm-2). The application of the NPG microsensor was examined by detecting Pb(II) in seawater samples, and a satisfactory performance was obtained.

Electrochemical microfluidic immunosensor with graphene-decorated gold nanoporous for T-2 mycotoxin detection.

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at -0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 µg kg-1 and 10 µg kg-1, and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples.

Caffeine Release from Magneto-Responsive Hydrogels Controlled by External Magnetic Field and Calcium Ions and Its Effect on the Viability of Neuronal Cells.

Caffeine (CAF) is a psychostimulant present in many beverages and with rapid bioabsorption. For this reason, matrices that effectuate the sustained release of a low amount of CAF would help reduce the intake frequency and side effects caused by high doses of this stimulant. Thus, in this study, CAF was loaded into magnetic gelatin/alginate (Gel/Alg/MNP) hydrogels at 18.5 mg/ghydrogel. The in vitro release of CAF was evaluated in the absence and presence of an external magnetic field (EMF) and Ca2+. In all cases, the presence of Ca2+ (0.002 M) retarded the release of CAF due to favorable interactions between them. Remarkably, the release of CAF from Gel/Alg/MNP in PBS/CaCl2 (0.002 M) at 37 °C under an EMF was more sustained due to synergic effects. In PBS/CaCl2 (0.002 M) and at 37 °C, the amounts of CAF released after 45 min from Gel/Alg and Gel/Alg/MNP/EMF were 8.3 ± 0.2 mg/ghydrogel and 6.1 ± 0.8 mg/ghydrogel, respectively. The concentration of CAF released from Gel/Alg and Gel/Alg/MNP hydrogels amounted to ~0.35 mM, thereby promoting an increase in cell viability for 48 h. Gel/Alg and Gel/Alg/MNP hydrogels can be applied as reservoirs to release CAF at suitable concentrations, thus forestalling possible side effects and improving the viability of SH-SY5Y cells.

Highly sensitive and selective nanostructured microbiosensors for glucose and lactate simultaneous measurements in blood serum and in vivo in brain tissue.

Highly sensitive and selective nanostructured lactate and glucose microbiosensors for their in vivo simultaneous determination in rat brain were developed based on carbon fiber microelectrodes (CFM) modified with nanoporous gold (NPG) using the Dynamic Hydrogen Bubble Template (DHBT) method. Electrodeposition of platinum nanoparticles (PtNP) onto the NPG film enhances the sensitivity and the electrocatalytic properties towards H2O2 detection. The nanostructured microelectrode platform was modified by glucose oxidase (GOx) and lactate oxidase (LOx) enzyme immobilization. High selective measurements were achieved by covering with a perm-selective layer of electropolymerized m-phenylenediamine, deposition of a Nafion® film and by using a null sensor. The morphological characteristics and electroanalytical performance of the microbiosensors were assessed, by scanning electron microscopy and electrochemical techniques, respectively. The PtNP/NPG/CFM shows a high sensitivity to H2O2 (5.96 A M-1 cm-2) at 0.36 V vs. Ag/AgCl, with a linear range from 0.2 to 200 µM, and an LOD of 10 nM. The microbiosensors were applied to the simultaneous determination of lactate and glucose in blood serum samples. Moreover, the basal extracellular concentrations of lactate and glucose were measured in vivo in four different rat brain structures. These results support the potential of the microbiosensor to be used as a valuable tool to investigate brain neurochemicals in vivo.

Unveiling the contribution of the reproductive system of individual Caenorhabditis elegans on oxygen consumption by single-point scanning electrochemical microscopy measurements.

Metabolic analysis in animals is usually either evaluated as whole-body measurements or in isolated tissue samples. To reveal tissue specificities in vivo, this study uses scanning electrochemical microscopy (SECM) to provide localized oxygen consumption rates (OCRs) in different regions of single adult Caenorhabditis elegans individuals. This is achieved by measuring the oxygen reduction current at the SECM tip electrode and using a finite element method model of the experiment that defines oxygen concentration and flux at the surface of the organism. SECM mapping measurements uncover a marked heterogeneity of OCR along the worm, with high respiration rates at the reproductive system region. To enable sensitive and quantitative measurements, a self-referencing approach is adopted, whereby the oxygen reduction current at the SECM tip is measured at a selected point on the worm and in bulk solution (calibration). Using genetic and pharmacological approaches, our SECM measurements indicate that viable eggs in the reproductive system are the main contributors in the total oxygen consumption of adult Caenorhabditis elegans. The finding that large regional differences in OCR exist within the animal provides a new understanding of oxygen consumption and metabolic measurements, paving the way for tissue-specific metabolic analyses and toxicity evaluation within single organisms.

Fabrication of dendritic nanoporous gold via a two-step amperometric approach: Application for electrochemical detection of methyl parathion in river water samples.

In this work, nanoporous gold (NPG) was prepared according to three different approaches, such as (i) anodization-electrochemical reduction (A-ECR, NPGA), (ii) dynamic hydrogen bubble template (DHBT, NPGB), and (iii) the combination of both methods (NPGA+B). Field-emission scanning electron microscopy (FE-SEM) and cyclic voltammetry (CV) were used to investigate the structural morphology and the electrochemical behavior of the fabricated materials. The NPGA+B electrode showed a large amount of surface defects and/or edges, greater electrochemical surface area (2.5 cm2), and increased roughness factor (35.4). Such outstanding features of the NPGA+B platform were demonstrated by the sensitive detection of methyl parathion (MP) in river water samples. CV results indicated nearly 25-fold, 6-fold, and 2.5-fold higher sensitivity for NPGA+B compared to that of bare Au, NPGA, and NPGB, respectively. Differential pulse voltammetry (DPV) results show a linear behavior in the MP concentration range of 5-50 ng mL-1 with a limit of detection (LOD) of 0.6 ng mL-1 and limit of quantification (LOQ) of 2.0 ng mL-1. Besides, the NPGA+B sensor also revealed excellent selectivity towards MP detection in the presence of other interfering molecules or ions, reproducibility, and repeatability.

Ultrasensitive microfluidic electrochemical immunosensor based on electrodeposited nanoporous gold for SOX-2 determination.

An ultrasensitive and portable microfluidic electrochemical immunosensor for SOX-2 cancer biomarker determination was developed. The selectivity and sensitivity of the sensor were improved by modifying the microfluidic channel. This was accomplished through a physical-chemical treatment to produce a hydrophilic surface, with an increased surface to volume/ratio, where the anti-SOX-2 antibodies can be covalently immobilized. A sputtered gold electrode was used as detector and its surface was activated by using a dynamic hydrogen bubble template method. As a result, a gold nanoporous structure (NPAu) with outstanding properties, like high specific surface area, large pore volume, uniform nanostructure, good conductivity, and excellent electrochemical activity was obtained. SOX-2 present in the sample was bound to the anti-SOX-2 immobilized in the microfluidic channel, and then was labeled with a second antibody marked with horseradish peroxidase (HRP-anti-SOX-2) like a sandwich immunoassay. Finally, an H2O2 + catechol solution was added, and the enzymatic product (quinone) was reduced on the NPAu electrode at +0.1 V (vs. Ag). The current obtained was directly proportional to the SOX-2 concentration in the sample. The detection limit achieved was 30 pg mL-1, and the coefficient of variation was less than 4.75%. Therefore, the microfluidic electrochemical immunosensor is a suitable clinical device for in situ SOX-2 determination in real samples.