Electrochemical detection in a microfluidic device of oxidative stress generated by macrophage cells

The release of reactive oxygen species (ROS) or reactive nitrogen species (RNS), i.e., the initial phase of oxidative stress, by macrophage cells has been studied by electrochemistry within a microfluidic device. Macrophages were first cultured into a detection chamber containing the three electrodes system and were subsequently stimulated by the microinjection of a calcium ionophore (A23187). Their production of ROS and RNS was then measured by amperometry at the surface of a platinized microelectrode. The fabricated microfluidic device provides an accurate measurement of oxidative release kinetics with an excellent reproducibility. We believe that such a method is simple and versatile for a number of advanced applications based on the detection of biological processes of secretion by a few or even a single living cell.     

Real‐Time Intracellular Measurements of ROS and RNS in Living Cells with Single Core–Shell Nanowire Electrodes

Nanoelectrodes allow precise and quantitative measurements of important biological processes at the single living‐cell level in real time. Cylindrical nanowire electrodes (NWEs) required for intracellular measurements create a great challenge for achieving excellent electrochemical and mechanical performances. Herein, we present a facile and robust solution to this problem based on a unique SiC‐core–shell design to produce cylindrical NWEs with superior mechanical toughness provided by the SiC nano‐core and an excellent electrochemical performance provided by the ultrathin carbon shell that can be used as such or platinized. The use of such NWEs for biological applications is illustrated by the first quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages. As the shell material can be varied to meet any specific detection purpose, this work opens up new opportunities to monitor quantitatively biological functions occurring inside cells and their organelles.     

Electrochemical Monitoring of ROS/RNS Homeostasis Within Individual Phagolysosomes Inside Single Macrophages

The existence of a homeostatic mechanism regulating reactive oxygen/nitrogen species (ROS/RNS) amounts inside phagolysosomes has been invoked to account for the efficiency of this process but could not be unambiguously documented. Now, intracellular electrochemical analysis with platinized nanowire electrodes (Pt‐NWEs) allowed monitoring ROS/RNS effluxes with sub‐millisecond resolution from individual phagolysosomes impacting onto the electrode inserted inside a living macrophage. This shows for the first time that the consumption of ROS/RNS by their oxidation at the nanoelectrode surface stimulates the production of significant ROS/RNS amounts inside phagolysosomes. These results establish the existence of the long‐postulated ROS/RNS homeostasis and allows its kinetics and efficiency to be quantified. ROS/RNS concentrations may then be maintained at sufficiently high levels for sustaining proper pathogen digestion rates without endangering the macrophage internal structures.     

Fabrication, characterization, and chemical etching of Ag nanoelectrodes

The previously developed methodologies for fabricating flat, polished nanoelectrodes were extended to produce silver electrodes with the radii from 50 nm to micrometers. The prepared electrodes were characterized by steady-state voltammetry, scanning electrochemical microscopy (SECM), and atomic force microscopy. The protocol was developed for controlled chemical etching of silver in ammonia solutions to produce recessed nanoelectrodes. Voltammograms and SECM approach curves were obtained to evaluate the recess depth and other geometric parameters of the etched electrodes.     

Fluorescent and luminescent probes for detection of reactive oxygen and nitrogen species

Oxidative and nitrosative stress induced by ROS/RNS play crucial roles in a wide range of physiological processes and are also implicated in various diseases, including cancer and neurodegenerative disorders. Sensitive and selective methods for the detection of ROS/RNS based on fluorescent and luminescent probes are of great use in monitoring the in vivo production of these species and elucidating their biological functions. This critical review highlights recent advances that have been made in the development of fluorescent and luminescent probes employed to monitor various ROS/RNS (132 references).     

Local electron transfer rate measurements on modified and unmodified glassy carbon electrodes

A natural and artificial distribution of electron transfer activity on glassy carbon electrodes can be observed and quantified by the use of scanning electrochemical microscopy (SECM). A large (sevenfold) spread in rate constant is found for randomly sampled sites on polished, untreated glassy carbon surfaces. Direct-mode oxidation with the SECM tip was used to produce small regions of oxidized carbon on a polished surface. A large increase in electron transfer rate for the Fe(II/III) ion is observed on the locally oxidized carbon surface in comparison to the unoxidized region. Rate constant measurements made along a line profiles the transition from unoxidized to oxidized surfaces. SECM images of defect sites show reaction–rate variations. Rate constants measured at several locations of the defective surface allows discrimination between the kinetic and topographic components of the SECM image. Dedicated to the 80th birthday of Keith B. Oldham     

Detection of Reactive Oxygen and Nitrogen Species by Upconversion Nanoparticle-Based Near-Infrared Nanoprobes: Recent Progress and Perspectives

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are essential oxidative metabolites of organisms, which are closely related to physiological, pathological and pharmacological processes. The accurate detection of ROS/RNS is important for the understanding of biological processes, monitoring of pharmacological effects, and predicting the course of disease. The recently developed NIR nanoprobes based on upconversion nanoparticles (UCNPs) hold great prospects in sensitive and deep-tissue detection of ROS/RNS, and considerable progress has been achieved so far. In this review, we systematically summarize the up-to-date advances of UCNPs-based near-infrared (NIR) probes for ROS/RNS sensing, and the potential challenges and perspectives for further research are also highlighted. We envision that such a research field will have a bright future for modern biomedical applications.     

Biological applications of scanning electrochemical microscopy: chemical imaging of single living cells and beyond

Recent applications of scanning electrochemical microscopy (SECM) to studies of single biological cells are reviewed. This scanning probe microscopic technique allows the imaging of an individual cell on the basis of not only its surface topography but also such cellular activities as photosynthesis, respiration, electron transfer, single vesicular exocytosis and membrane transport. The operational principles of SECM are also introduced in the context of these biological applications. Recent progress in techniques for high-resolution SECM imaging are also reviewed. Future directions, such as single-channel detection by SECM, high-resolution imaging with nanometer-sized probes, and combined SECM techniques for multidimensional imaging are also discussed.     

Nanotechnology for Electroanalytical Biosensors of Reactive Oxygen and Nitrogen Species

Over the past several decades, nanotechnology has contributed to the progress of biomedicine, biomarker discovery, and the development of highly sensitive electroanalytical / electrochemical biosensors for in vitro and in vivo monitoring, and quantification of oxidative and nitrosative stress markers like reactive oxygen species (ROS) and reactive nitrogen species (RNS). A major source of ROS and RNS is oxidative stress in cells, which can cause many human diseases, including cancer. Therefore, the detection of local concentrations of ROS (e. g. superoxide anion radical; O₂^•−) and RNS (e. g. nitric oxide radical; NO^• and its metabolites) released from biological systems is increasingly important and needs a sophisticated detection strategy to monitor ROS and RNS in vitro and in vivo. In this review, we discuss the nanomaterials‐based ROS and RNS biosensors utilizing electrochemical techniques with emphasis on their biomedical applications.     

Electrochemical sensors for oxidative stress monitoring

Electrochemical sensors are ideally suited for the detection of reactive oxygen and nitrogen species (ROS and RNS) generated during biological processes. This review discusses the latest work in the development of electrochemical microsensors for ROS/RNS and their applications for monitoring oxidative stress in biological systems. The performance of recent designs of microelectrodes and electrode materials is discussed along with their functionality in preclinical models of drug efficacy, mitochondrial distress, and endothelial dysfunction. Challenges and opportunities in translating this methodology to study the pathophysiology associated with various diseases are discussed.     

A comparison of electrochemically pre-treated and spark-platinized carbon fiber microelectrode. Measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in human urine and plasma

A novel method of carbon fiber microelectrode activation using spark discharge was demonstrated and compared to conventional electrochemical pretreatment by potential cycling. The spark discharge was performed at 800 V between the microelectrode connected to positive pole of the power supply and platinum counter electrode. Spark discharge led both to trimming of the fiber tip into conical shape and to the modification of carbon fiber microelectrode with platinum, as proven by scanning electron microscopy and electron dispersive X-ray spectroscopy. After the characterization of electrochemical properties using ferricyanide voltammetry, the activated electrodes were used for electrochemical analysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine, an oxidative stress marker. Subnanomolar detection limits (0.55 nmol L⁻¹) in high-performance liquid chromatography were achieved for spark platinized electrodes incorporated into the flow detection cell.     

Generation and detection of single metal nanoparticles using scanning electrochemical microscopy techniques

Different pathways towards the generation and detection of a single metal nanoparticle (MNP) on a conductive carbon support for testing as an electrocatalyst are described. Various approaches were investigated including interparticle distance enhancement, electrochemical and mechanical tip-substrate MNP transfer onto macroscopic surfaces, scanning electrochemical microscopy (SECM)-controlled electrodeposition, and the use of selective binding monolayers on carbon fiber electrodes (CFEs) for solution-phase-selective adsorption. A novel SECM technique for electrodepositing MNPs on CFE tips immersed 100-200 nm below the electrolyte level was developed and used to generate single Pt and Ni nanoparticles. Following their generation, we demonstrate electrocatalytic detection of Fe3+ on individual Pt particles with the CFE in a Fe3+/H2SO4 solution. We also describe an approach of attaching MNPs to CFEs by controlling the composition of monolayers bonded to the CFE. By employing a monolayer with a low ratio of binding (e.g., 4-aminopyridine) to nonbinding molecules (e.g., aniline) and controlling the position of the CFE in a colloidal Pt solution with a SECM, we attached a single 15 nm radius Pt nanoparticle to the CFE. Such chemisorbed Pt particles exhibited a stronger adhesion on surface-modified CFEs and better mechanical stability during proton reduction than MNPs electrodeposited directly on the CFE.     

Scanning electrochemical microscopy studies of glutathione-modified surfaces. An erasable and sensitive-to-reactive oxygen species surface

A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the -SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O(2) to superoxide, O(2)(?-), near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O(2)(?-) and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O(2)(?-) at the tip probe electrode.     

Substituent Effects in BODIPY in Live Cell Imaging

Small‐molecule organoselenium‐based fluorescent probes possess great capacity in understanding biological processes through the detection of various analytes such as reactive oxygen/nitrogen species (ROS/RNS), biothiols (cysteine, homocysteine and glutathione), lipid droplets, etc. Herein, we present how substituents on the BODIPY system play a significant part in the detection of biologically important analytes for in vitro conditions and live cell imaging studies. The fluorescence of the probe was quenched by 2‐chloro and 6‐phenyl selenium groups; the probe shows high selectivity with NaOCl among other ROS/RNS, and gives a turn‐on response. The maximum fluorescence intensity is attained within ≈1–2 min with a low detection limit (19.6 nm ), and shows a ≈110‐fold fluorescence enhancement compared to signals generated for other ROS/RNS. Surprisingly, in live cell experiments, the probe specifically located and accumulated in lipid droplets, and showed a fluorescence turn‐on response. We believe this turn‐on response occurred because of aggregation‐induced emission (AIE), which surprisingly occurred only by introducing one lipophilic mesityl group at the meso position of the BODIPY.     

In situ formation and scanning electrochemical microscopy assisted positioning of NO-sensors above human umbilical vein endothelial cells for the detection of nitric oxide release

Pt microelectrodes (50 μm diameter) were positioned by means of scanning electrochemical microscopy assisted z-approach curves and in situ modified with nickel tetrasulfonated phthalocyanine tetrasodium salt as electrocatalytic layer for the specific oxidative detection of nitric oxide. The thus modified electrodes were then moved over a layer of adherently growing human umbilical vein endothelial cells (HUVEC) in order to amperometrically detect nitric oxide (NO) released from the cells upon stimulation with bradykinin. This approach actually takes advantage of the use of SECM to define a sequential procedure that enables the in situ functionalisation of the SECM tip thus allowing to accurately control the separation between the functionalised SECM tip and the cell population.     

Characterization of electrodes modified with nanocomposites of cobalt tetraaminophenoxyphthalocyanine,reduced graphene and multi-walled carbon nanotubes

Abstract

Glassy carbon electrodes or plates were modified with nanocomposites consisting of cobalt tetraaminophenoxyphthalocyanine (CoTAPhPc), reduced graphene oxide nanosheets (rGONs) and multi-walled carbon nanotubes (MWCNTs). The modified electrodes were characterized using cyclic voltammetry, scanning electrochemical microscopy (SECM) and time-of-flight-secondary ion mass spectrometer (TOF-SIMS). The electrocatalytic activity of the modified electrode was tested for detection of L-cysteine. The presence of CoTAPhPc on sequential layers of MWCNT and rGONs resulted in improved detection currents compared to CoTAPhPc alone or when MWCNT/rGONs are mixed in CoTAPhPc–MWCNT/rGONs (mix)–glassy carbon electrode (GCE). CoTAPhPc–MWCNT–GCE (without rGONS) showed higher sensitivity toward l-cysteine as compared to the probes incorporating rGONs with a catalytic rate constant of 4.62?×?10⁴ M^?1s^?1 and a detection limit of 30?nM. The presence of rGONs improved the stability of the electrode.     

Multiscale electrochemistry of hydrogels embedding conductive nanotubes

The local functionalities of biocompatible objects can be characterized under conditions similar to the operating ones, using scanning electrochemical microscopy (SECM). In the case of alginate beads entrapping carbon nanotubes (CNTs), SECM allows evidencing of the local conductivity, organization, and communication between the CNTs. It shows that the CNT network is active enough to allow long range charge evacuation, enabling the use of alginate/CNT beads as soft 3D electrodes. Direct connection or local interrogation by a microelectrode allows visualization of their communication as a network and eventually the study of them individually at the nanoscale.     

Photoreduction of catalytic platinum particles using immobilized multilayers of Photosystem I

Using the abundance of available electrons generated by immobilized multilayers of the photoactive protein complex Photosystem I (PSI), we have photoreduced platinum particles that are catalytically active for the H(2)/H(+) redox couple. The resulting platinized PSI films were optimized using electrochemical measurements and then characterized using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and scanning electrochemical microscopy (SECM). These results demonstrate a novel method for generating immobilized platinum catalysts that are readily available on the surface of a photoactive PSI multilayer.     

Fabricating and imaging carbon-fiber immobilized enzyme ultramicroelectrodes with scanning electrochemical microscopy.

The scanning electrochemical microscope (SECM) is used to image the activity of enzymes immobilized on the surfaces of disk-shaped carbon-fiber electrodes. SECM was used to map the concentration of enzymatically produced hydroquinone or hydrogen peroxide at the surface of a 33-microm diameter disk-shaped carbon-fiber electrode modified by an immobilized glucose-oxidase layer. Sub-monolayer coverage of the enzyme at the electrode surface could be detected with micrometer resolution. The SECM was also employed as a surface modification tool to produce microscopic regions of enzyme activity by using a variety of methods. One method is a gold-masking process in which microscopic gold patterns act as mask for producing patterns of chemical modification. The gold masks allow operation in both a positive or negative process for patterning enzyme activity. A second method uses the direct mode of the SECM to produce covalently attached amine groups on the carbon surface. The amine groups are anchors for attachment of glucose oxidase by use of a biotin/avidin process. The effect of non-uniform enzyme activity was investigated by using the SECM tip to temporarily damage an immobilized enzyme surface. SECM imaging can observe the spatial extent and time-course of the enzyme recovery process.     

Quantitative characterization of shear force regulation for scanning electrochemical microscopy

The quest for higher spatial resolution in scanning electrochemical microscopy (SECM) calls for the application of smaller probe electrodes. When electrodes are to be used in the feedback mode, smaller electrodes require higher intrinsic kinetics at the sample. The fabrication of nanoelectrodes, as well as their use as SECM probes at constant distance, are reported. The properties of shear force regulation system are characterized quantitatively. Simultaneous topography and reactivity imaging were demonstrated using gold microstructures on a glass substrate.