The use of microelectrodes to investigate compartmentation and the transport of metabolized inorganic ions in plants

Microelectrode measurements can be used to investigate both the intracellular pools of ions and membrane transport processes of single living cells. Microelectrodes can report these processes in the surface layers of root and leaf cells of intact plants. By careful manipulation of the plant, a minimum of disruption is produced and therefore the information obtained from these measurements most probably represents the 'in vivo' situation. Microelectrodes can be used to assay for the activity of particular transport systems in the plasma membrane of cells. Compartmental concentrations of inorganic metabolite ions have been measured by several different methods and the results obtained for the cytosol are compared. Ion-selective microelectrodes have been used to measure the activities of ions in the apoplast, cytosol and vacuole of single cells. New sensors for these microelectrodes are being produced which offer lower detection limits and the opportunity to measure other previously unmeasured ions. Measurements can be used to determine the intracellular steady-state activities or report the response of cells to environmental changes.     

Biochips and other microtechnologies for physiomics

This paper presents a review of microtechnologies relevant to applications in cellular physiology, including biochips, electrochemical sensors and optrodic sensing techniques. Microelectrodes have been the main tools for measuring cellular electrophysiology, oxygen, nitric oxide, neurotransmitters, pH and various ions. Optical fiber sensing methods, such as indicator-based optrodes, with fluorescence lifetime measurement, are now emerging as viable alternatives to electroanalytical chemistry. These new optrode techniques are possible because of recent advances in the optoelectronics industry and are comparably easier to miniaturize, have faster response times, do not consume the analyte and have lower operational costs. This review serves as a summary and predicts future trends for both electrochemical and optical luminescence lifetime sensing as components in lab-on-a-chip devices for physiological sensing.     

Nanostructured material-based optical and electrochemical detection of amoxicillin antibiotic

The penicillin derivative amoxicillin (AMX) plays an important role in treating various types of infections caused by bacteria. However, excessive use of AMX may have negative health effects. Therefore, it is of utmost importance to detect and quantify the AMX in pharmaceutical drugs, biological fluids, and environmental samples with high sensitivity. Therefore, this review article provides valuable and up-to-date information on nanostructured material-based optical and electrochemical sensors to detect AMX in various biological and chemical samples. The role of using different nanostructured materials on the performance of important optical sensors such as colorimetric sensors, fluorescence sensors, surface-enhanced Raman scattering sensors, chemiluminescence/electroluminescence sensors, optical immunosensors, optical fibre-based sensors, and several important electrochemical sensors based on different electrode types have been discussed. Moreover, nanocomposites, polymer, and MXenes-based electrochemical sensors have also been discussed, in which such materials are being used to further enhance the sensitivity of these sensors. Furthermore, nanocomposite-based photo-electrochemical sensors and the market availability of biosensors including AMX have also been discussed briefly. Finally, the conclusion, challenges, and future perspectives of the above-mentioned sensing techniques for AMX detection are presented.     

An improved Na+-selective microelectrode for intracellular measurements in plant cells.

The high background K+ concentration in plant cells is a problem for intracellular measurements of Na+ using ion-selective microelectrodes. The discrimination between Na+ and K+ of the microelectrode ionophore molecule limits the usefulness of this technique. A new Na+-selective microelectrode with an ionophore incorporating a tetramethoxyethyl ester derivative of p-t-butyl calix[4]arene has been developed. Microelectrodes made with this new sensor have superior selectivity for Na+ over K+ resulting in a lower limit of detection when compared with microelectrodes made using a commercially available ionophore (ETH227). Both types of microelectrodes were insensitive to changes in ionic strength and physiological ranges of pH, but only the calixarene-based electrodes showed no protein interference. To test the suitability of the calixarene-based microelectrodes for measurements in plants, they were used to measure Na+ in epidermal cells in the zone 10-20 mm from the root apex of barley (Hordeum vulgare L.). Seedlings were grown in a nutrient solution containing 200 mM NaCl for 1-6 d. The range of intracellular Na+ activity (a(Na)) measured varied from < or =0.1 mM (limit of detection) to over 100 mM, and these values increased significantly with time. The membrane potential (E(m)) of these cells was variable, but the values became significantly more negative with time, although there was no significant correlation between E(m) and a(Na). These intracellular measurements could not be separated into distinct populations that might be representative of subcellular compartments.     

Determination of [Mg2+]i – an update on the use of Mg2+-selective electrodes

Since their invention, ion-selective microelectrodes have become an indispensable tool for investigations of intracellular ion regulation and transport. While highly selective sensors for all major intracellular monovalent ions have been available for decades, the development of sensors for divalent cations seems to have presented more difficulties. As ion-selective microelectrodes typically have time-constants in the range of 0.5 to several seconds they turned out to be inapt for the investigation of intracellular Ca²⁺. The development of sensors for Mg²⁺-selective electrodes has made its most striking progress only over the past few years. While the first Mg²⁺ sensor, ETH 1117, was barely able to detect physiological Mg²⁺ concentrations in the presence of other mono- and divalent cations, the newest sensors allow measurements in the micromolar range. When used in macroelectrodes, the most recent developments, ETH 5506 and ETH 5504, have even been reported to do so in the presence of millimolar Ca²⁺ concentrations. Although there is still room for improvement to make these sensors applicable in microelectrodes, some preliminary data look extremely promising and indicate that a new era for Mg²⁺-selective microelectrodes is about to start.     

Microelectrode measurement of oxygen tensions in collagen-hepatocyte gels

Summary Microelectrodes have been used for the measurement of oxygen tension in various biological systems.(Silver, 1987) Although not previously reported, microelectrodes allow the direct measurement of oxygen tension profiles within collagen gels containing entrapped hepatocytes (collagcn-hepatocyte gels). These oxygen tension profiles, along with hepatocyte oxygen consumption data, allowed the estimation of a diffusion coefficient for oxygen in collagen-hepatocyte gels, D _g = 2.99 × 10^–5 cm²/s.     

Rube Goldberg goes (ribo)nuclear? Molecular switches and sensors made from RNA

Switches and sensors play important roles in our everyday lives. The chemical properties of RNA make it amenable for use as a switch or sensor, both artificially and in nature. This review focuses on recent advances in artificial RNA switches and sensors. Researchers have been applying classical biochemical principles such as allostery in elegant ways that are influencing the development of biosensors and other applications. Particular attention is given here to allosteric ribozymes (aptazymes) that are regulated by small organic molecules, by proteins, or by oligonucleotides. Also discussed are ribozymes whose activities are controlled by various nonallosteric strategies.     

Ca2+-selective microelectrodes

Ca2+-selective microelectrodes based on the synthetic neutral carrier ETH 1001 can be used for quantitative intracellular measurements of resting Ca2+-activities and of slowly changing Ca2+-levels (response time in the order of seconds). Microelectrodes with tip diameters greater than 0.3 micron show selectivities that yield a detection limit between 10(-8) and 10(-7) M Ca2+ in an intracellular background. The Ca2+-activity is obtained together with electrical parameters of the cell (e.g. cell membrane potential and membrane resistance or conductivity). Simultaneous monitoring of other ion-activities is accessible (double- or multi-barrelled microelectrodes). The Ca2+-determination is extremely local, i.e. it probably does not indicate an averaged cytosolic activity in every situation (e.g. localized transients).     

Recent progress on performances and mechanisms of carbon dots for gas sensing

Carbon dots (CDs), as an attractive zero-dimensional carbon nanomaterial with unique photoluminescent merits, have recently exhibited significant application potential in gas sensing as a result of their excellent optical/electronic characteristics, high chemical/thermal stability, and tunable surface states. CDs exhibit strong light absorption in the ultraviolet range and tunable photoluminescence characteristics in the visible range, which makes CDs an effective tool for optical sensing applications. Optical gas sensor based on CDs have been investigated, which generally responds to the target gas by corresponding changes in optical absorption or fluorescence. Moreover, electrical gas sensor and quartz crystal microbalance sensor whose sensing layer involves CDs have also been designed. Electrical gas sensor exhibits an increase or a decrease in electrical current, capacitance, or conductance once exposed to the target gas. Quartz crystal microbalance sensor responds to the target gas with a frequency shift. CDs greatly promote the absorption of the target gas and improve the sensitivity of both sensors. In this review, we aim to summarize different types of gas sensors involving CDs, and sensing performances of these sensors for monitoring diverse gases or vapors, as well as the mechanisms of CDs in different types of sensors. Moreover, this review provides the prospect of the potential development of CDs based gas sensors.     

Coupled Effects of Mass Transfer and Uptake Kinetics on In Vivo Microdialysis of Dopamine

Abstract: Voltammetric microelectrodes and microdialysis probes were used simultaneously to monitor extracellular dopamine in rat striatum during electrical stimulation of the medial forebrain bundle. Microelectrodes were placed far away (1 mm) from, immediately adjacent to, and at the outlet of microdialysis probes. In drug-naive rats, electrical stimulation (45 Hz, 25 s) evoked a robust response at microelectrodes far away from the probes, but there was no response at microelectrodes adjacent to and at the outlet of the probes. After nomifensine administration (20 mg/kg i.p.), stimulation evoked robust responses at all three microelectrode placements. These results demonstrate first that evoked release in tissue adjacent to microdialysis probes is suppressed in comparison with evoked release in tissue far away from the probes and second that equilibration of the dopamine concentration in the extracellular fluid adjacent to and far away from the probes is prevented by the high-affinity dopamine transporter. Hence, models of microdialysis, which assume the properties of tissue to be spatially uniform, require modification to account for the distance that separates viable sites of evoked dopamine release from the probe. We introduce new mass transfer resistance parameters that qualitatively explain the observed effects of uptake inhibition on stimulation responses recorded with microdialysis and voltammetry.     

Procedures for manufacturing double-barrelled ion-sensitive microelectrodes employing liquid sensors

Liquid ion-sensitive/selective sensors are available for most inorganic ions of physiological and biochemical importance. In order to measure intracellular ionic activities in relatively small cells, it is advisable to manufacture and use double-barrelled microelectrodes. Procedures for making two types of double-barrelled ion-sensitive microelectrode are described in detail. Such microelectrodes have been used successfully to measure intracellular K+, Cl- and Na+ activities in retinal horizontal cells of fish and body-wall muscles of insect larvae.     

Fixed charges of the heart muscle interstitium.

The interstitium of the heart muscle is primarily composed of ground substance. The glycoproteins and proteoglycans that formed the ground substance bore negative charges at neutral pH like the glycosaminoglycans and proteoglycans of the water-rich phase of the interstitium. Microelectrodes were used to look for the existence of an electrical potential between the interstitium of the rabbit papillary muscle and an ambient medium. Evidence is presented for the existence of such an electrical potential. When the ambient solution was a Krebs solution, this potential was evaluated at -5.7 mV. This electrical potential is dependent on the filling solution of the microelectrodes and on the ambient medium; in rabbit serum, the electrical potential diminished to -0.6 mV. Assuming that this potential is a measure of the Donnan potential, the Cl and Na activities in the interstitium were evaluated to 76 and 135 mM when the rabbit papillary muscle was superfused with a Kreb's solution.     

Visualizing circuits and systems using transgenic reporters of neural activity

Genetically encoded sensors of neural activity enable visualization of circuit-level function in the central nervous system. Although our understanding of the molecular events that regulate neuronal firing, synaptic function, and plasticity has expanded rapidly over the past 15 years, an appreciation for how cellular changes are functionally integrated at the circuit level has lagged. A new generation of tools that employ fluorescent sensors of neural activity promises unique opportunities to bridge the gap between cellular level and system level analysis. This review will focus on genetically encoded sensors. A primary advantage of these indicators is that they can be nonselectively introduced to large populations of cells using either transgenic-mediated or viral-mediated approaches. This ability removes the nontrivial obstacles of how to get chemical indicators into cells of interest, a problem that has dogged investigators who have been interested in mapping neural function in the intact CNS. Five different types of approaches and their relative utility will be reviewed here: first, reporters of immediate-early gene (IEG) activation using promoters such as c-fos and arc; second, voltage-based sensors, such as GFP-coupled Na+ and K+ channels; third, Cl*-based sensors; fourth, Ca2+-based sensors, such as Camgaroo and the troponin-based TN-L15; and fifth, pH-based sensors, which have been particularly useful for examining synaptic activity of highly convergent afferents in sensory systems in vivo. Particular attention will be paid to reporters of IEG expression, because these tools employ the built-in threshold function that occurs with activation of gene expression, provoking new experimental questions by expanding the timescale of analysis for circuit-level and system-level functional mapping.     

Microelectrode Measurements of the Activity Distribution in Nitrifying Bacterial Aggregates

Microelectrodes for ammonium, oxygen, nitrate, and pH were used to study nitrifying aggregates grown in a fluidized-bed reactor. Local reactant fluxes and distribution of microbial activity could be determined from the microprofiles. The interfacial fluxes of the reactants closely reflected the stoichiometry of bacterial nitrification. Both ammonium consumption and nitrate production were localized in the outer shells, with a thickness of approximately 100 to 120 μm, of the aggregates. Under conditions in which ammonium and oxygen penetrated the whole aggregate, nitrification was restricted to this zone; oxygen was consumed in the central parts of the aggregates as well, probably because of oxidation of dead biomass. A sudden increase of the oxygen concentration to saturation (pure oxygen) was inhibitory to nitrification. The pH profiles showed acidification in the aggregates, but not to an inhibitory level. The distribution of activity was determined by the penetration depth of oxygen during aggregate development in the reactor. Mass transfer was significantly limited by the boundary layer surrounding the aggregates. Microelectrode measurements showed that the thickness of this layer was correlated with the diffusion coefficient of the species. Determination of the distribution of nitrifying activity required the use of ammonium or nitrate microelectrodes, whereas the use of oxygen microelectrodes alone would lead to erroneous results.     

Qualitative measurements of the entry of L-lactate into single surface fibres of frog skeletal muscle using a lactate-sensitive microelectrode

The present results demonstrate the sensitivity of the Corning chloride liquid ion exchanger 477913 to L-lactate. Microelectrodes filled with this exchanger showed responses to changes in L-lactate concentration in chloride-free solutions. In these experiments L-lactate replaced gluconate in equimolar amounts. Microelectrodes filled with this exchanger were used to qualitatively detect changes in intracellular anion in chloride-depleted frog sartorius muscle fibres during exposure to extracellular concentrations of L-lactate. The increase in intracellular anion concentration is consistent with the movement of L-lactate into the cell. This microelectrode enables one to qualitatively monitor changes in intracellular L-lactate in chloride-free experiments without incorporating selectivity coefficients.     

Non-invasive self-referencing electrochemical sensors for quantifying real-time biofilm analyte flux

Current techniques for characterizing biofilm physiology lack the signal filtering capability required for quantifying signals associated with real time biologically active transport. Though a great deal was learned from previous investigations, no results have been reported on the characterization of in vivo, real time biofilm flux using non-invasive (non-destructive) techniques. This article introduces the self-referencing technique for applications in biofilm physiology. Self-referencing is a non-invasive sensing modality which is capable of sensing changes in biologically active analyte flux as small as 10 fmol cm(-2) s(-1). Studies directly characterizing flux, as opposed to concentration, have the advantage of quantifying real time changes in biologically active transport which are otherwise lost to background noise. The use of this modality for characterizing biofilm physiology is validated with a reversible enzyme inhibition study. The experiment used self-referencing potentiometric sensors for quantifying real time ammonium and nitrite flux. Amperometric and optical sensing methods, though not presented herein, are also powerful sensing tools which benefit from operation in self-referencing mode. Reversible ammonia monooxygenase inhibition by a copper chelator (thiourea), and subsequent relief by excess copper addition was successfully demonstrated using self-referencing ion-selective microelectrodes for a mature Nitrosomonas europaea biofilm.     

Are biological sensors modulated by their structural scaffolds? The role of the structural muscle proteins α-actinin-2 and α-actinin-3 as modulators of biological sensors

Biological sensors and their ability to detect and respond to change in the cellular environment can be modulated by protein scaffolds acting within their interaction network. The skeletal muscle α-actinins have been considered as primarily structural scaffold proteins. However, deficiency of α-actinin-3 due to a common null polymorphism results in predominantly metabolic changes in skeletal muscle function. In this review, we explore the range of phenotypes associated with α-actinin-3 deficiency, and draw supporting evidence from known interaction partners for its role as a scaffold which acts to modulate biological sensors that result in changes in muscle metabolism and structure.     

Nanopore sensing: A physical-chemical approach

Protein nanopores have emerged as an important class of sensors for the understanding of biophysical processes, such as molecular transport across membranes, and for the detection and characterization of biopolymers. Here, we trace the development of these sensors from the Coulter counter and squid axon studies to the modern applications including exquisite detection of small volume changes and molecular reactions at the single molecule (or reactant) scale. This review focuses on the chemistry of biological pores, and how that influences the physical chemistry of molecular detection.     

Automated biomonitors — first line of defense

Automated biomonitors operate on a real-time basis and utilize living organisms as the sensors. Traditionally, chemical monitors have been used to assess water quality. However, biological monitors respond to a greater number of toxic conditions. An overview of the various automated biomonitors, assessed by the types of biological sensors employed, is presented. The sensors used include bacteria, algae, invertebrates, and fish. Of all the systems, those monitoring the ventilatory behavior of fish have evolved the furthest with respect to their research, development, commercial availability, and field testing.     

Electrochemical properties of Na+- and K+-selective glass microelectrodes.

Electrochemical properties of Na+-selective glass microelectrodes were studied and compared with those of K+-selective glass microelectrodes. The selectivity of Na+-selective glass microelectrodes depended on the ion concentration of test solutions. With aging, resistance of Na+-selective microelectrodes increased and their selectivity for Na over K decreased. Na+-selective microelectrodes potential measured in NaCl solution remained constant with aging, while the potential measured in KCl solution decreased and became more positive. The changes in resistance and potential of Na+-selective microelectrodes may be due to the effects of the less mobile cation, i.e., H+ or K+ on the Na ion exchange in the Na-sensing region. The results indicate that Na+-selective microelectrodes must be used as soon after filling as possible. The selectivity of Na+-selective microelectrodes increased with increase of the sensitive exposed-tip length, whereas their response time became slow due to a large recessed volume, indicating requirement of an optimum exposed-tip length for intracellular applications. The changes in the properties of Na+-selective glass microelectrodes with aging contrasted with those of K+-selective glass microelectrodes in which resistance decreased and K+-selectivity increased. The K+-selective microelectrodes required aging before use for a high selectivity and low resistance. The K+-selective microelectrodes with low resistance after sufficient aging can be used without insulation to measure K+ and Na+ activities in aqueous solutions. The different properties between Na+- and K+-selective microelectrodes are understandable, because hydration of N+-selective glass is much less extensive than that of K+-selective glass.