Biological applications of microchip electrophoresis with amperometric detection: in vivo monitoring and cell analysis.

Microchip electrophoresis with amperometric detection (ME-EC) is a useful tool for the determination of redox active compounds in complex biological samples. In this review, a brief background on the principles of ME-EC is provided, including substrate types, electrode materials, and electrode configurations. Several different detection approaches are described, including dual-channel systems for dual-electrode detection and electrochemistry coupled with fluorescence and chemiluminescence. The application of ME-EC to the determination of catecholamines, adenosine and its metabolites, and reactive nitrogen and oxygen species in microdialysis samples and cell lysates is also detailed. Lastly, approaches for coupling of ME-EC with microdialysis sampling to create separation-based sensors that can be used for near real-time monitoring of drug metabolism and neurotransmitters in freely roaming animals are provided. Graphical abstract.

Evaluation of dual electrode configurations for microchip electrophoresis used for voltammetric characterization of electroactive species.

Microchip electrophoresis coupled with amperometric detection is more popular than voltammetric detection due to the lower limits of detection that can be achieved. However, voltammetry provides additional information about the redox properties of the analyte that can be used for peak identification. In this paper, two dual electrode configurations for microchip electrophoresis are described and evaluated for obtaining voltammetric information using amperometry. The dual-series electrode configuration was first evaluated to generate current ratios in a single run by applying two different potentials to the working electrodes placed perpendicular to the separation channel. However, it was found that it is difficult to obtain realistic current ratios with this configuration, primarily due to the relative placement of electrodes with respect to the channel end of the simple-t microchip. Correction factors were needed to obtain current ratios similar to those that would be obtained for sequential injections at two different potentials using a single electrode. A second approach using a dual-channel chip with two parallel electrodes was then developed and evaluated for obtaining voltammetric identification. The newly developed microchip permitted the injection of same amount of sample into two unique separation channels, each with an electrode at a different detection potential. Migration times and current ratios for several biologically important molecules and potential interferences including nitrite, tyrosine, hydrogen peroxide, and azide were obtained and compared to the responses obtained for analytes found in macrophage cell lysates.

Optimization of a microchip electrophoresis method with electrochemical detection for the determination of nitrite in macrophage cells as an indicator of nitric oxide production.

Nitric oxide (NO) is involved in many biological functions, including blood pressure regulation, the immune response, and neurotransmission. However, excess production of NO can lead to the generation of reactive nitrogen species and nitrosative stress and has been linked to several neurodegenerative diseases and cardiovascular disorders. Because NO is short-lived and generally difficult to detect, its primary stable degradation product, nitrite, is frequently monitored in its place. In this paper, an improved method using microchip electrophoresis with electrochemical detection (ME-EC) was developed for the separation and detection of nitrite in cell lysates. A separation of nitrite from several electroactive cell constituents and interferences was optimized, and the effect of sample and buffer conductivity on peak efficiency was explored. It was found that the addition of 10 mM NaCl to the run buffer caused stacking of the nitrite peak and improved limits of detection. A platinum black working electrode was also evaluated for the detection of nitrite and other electroactive cellular species after electrophoretic separation. The use of a modified platinum working electrode resulted in 2.5-, 1.7-, and 7.2-fold signal enhancement for nitrite, ascorbic acid, and hydrogen peroxide, respectively, and increased the sensitivity of the method for nitrite twofold. The optimized ME-EC method was used to compare nitrite production by native and lipopolysaccharide-stimulated RAW 264.7 macrophage cells.

Non-toxic engineered carbon nanodiamond concentrations induce oxidative/nitrosative stress, imbalance of energy metabolism, and mitochondrial dysfunction in microglial and alveolar basal epithelial cells.

Engineered nanoparticles are finding a wide spectrum of biomedical applications, including drug delivery and capacity to trigger cytotoxic phenomena, potentially useful against tumor cells. The full understanding of their biosafety and interactions with cell processes is mandatory. Using microglial (BV-2) and alveolar basal epithelial (A549) cells, in this study we determined the effects of engineered carbon nanodiamonds (ECNs) on cell viability, nitric oxide (NO) and reactive oxygen species (ROS) production, as well as on energy metabolism. Particularly, we initially measured decrease in cell viability as a function of increasing ECNs doses, finding similar cytotoxic ECN effects in the two cell lines. Subsequently, using apparently non-cytotoxic ECN concentrations (2 µg/mL causing decrease in cell number < 5%) we determined NO and ROS production, and measured the concentrations of compounds related to energy metabolism, mitochondrial functions, oxido-reductive reactions, and antioxidant defences. We found that in both cell lines non-cytotoxic ECN concentrations increased NO and ROS production with sustained oxidative/nitrosative stress, and caused energy metabolism imbalance (decrease in high energy phosphates and nicotinic coenzymes) and mitochondrial malfunctioning (decrease in ATP/ADP ratio).These results underline the importance to deeply investigate the molecular and biochemical changes occurring upon the interaction of ECNs (and nanoparticles in general) with living cells, even at apparently non-toxic concentration. Since the use of ECNs in biomedical field is attracting increasing attention the complete evaluation of their biosafety, toxicity and/or possible side effects both in vitro and in vivo is mandatory before these highly promising tools might find the correct application.

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation.

It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O2•-) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O2•- in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O2•- ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O2•- ratios were 0.60 ± 0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08 ± 0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14 ± 0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca2+ (intracellular messenger) on the NO/O2•- ratio was also investigated. Graphical Abstract Simultaneous detection of nitric oxide and superoxide in macrophage cell lysates.

Carnosine modulates nitric oxide in stimulated murine RAW 264.7 macrophages.

Excess nitric oxide (NO) production occurs in several pathological states, including neurodegeneration, ischemia, and inflammation, and is generally accompanied by increased oxidative/nitrosative stress. Carnosine [ß-alanine-histidine (ß-Ala-His)] has been reported to decrease oxidative/nitrosative stress-associated cell damage by reducing the amount of NO produced. In this study, we evaluated the effect of carnosine on NO production by murine RAW 264.7 macrophages stimulated with lipopolysaccharides + interferon-γ. Intracellular NO and intracellular and extracellular nitrite were measured by microchip electrophoresis with laser-induced fluorescence and by the Griess assay, respectively. Results showed that carnosine causes an apparent suppression of total NO production by stimulated macrophages accompanied by an unexpected simultaneous drastic increase in its intracellular low toxicity endproduct, nitrite, with no inhibition of inducible nitric oxide synthase (iNOS). ESI-MS and NMR spectroscopy in a cell-free system showed the formation of multiple adducts (at different ratios) of carnosine-NO and carnosine-nitrite, involving both constituent amino acids (ß-Ala and His) of carnosine, thus providing a possible mechanism for the changes in free NO and nitrite in the presence of carnosine. In stimulated macrophages, the addition of carnosine was also characterized by changes in the expression of macrophage activation markers and a decrease in the release of IL-6, suggesting that carnosine might alter M1/M2 macrophage ratio. These results provide evidence for previously unknown properties of carnosine that modulate the NO/nitrite ratio of stimulated macrophages. This modulation is also accompanied by changes in the release of pro-inflammatory molecules, and does not involve the inhibition of iNOS activity.

Determination of heterogeneous electron transfer and homogeneous comproportionation rate constants of tetracyanoquinodimethane using scanning electrochemical microscopy.

We report the use of scanning electrochemical microscopy (SECM) in determining the heterogeneous electron transfer and homogeneous comproportionation kinetics of tetracyanoquinodimethane (TCNQ) in acetonitrile at Pt tip UMEs (radius 12.5­1 µm). TCNQ undergoes two consecutive one-electron reductions with comproportionation occurring between TCNQ2­, the product of the second reduction, and bulk TCNQ to produce TCNQ­. A standard rate constant, k(1)(0) = 2.9 ± 0.5 cm/s, for the first reduction was determined by tip voltammetry with total positive feedback and a large Pt substrate. A comparatively smaller rate constant, k(2)(0) = 0.44 ± 0.05 cm/s, for the second reduction was determined in the absence of comproportionation by tip voltammetry with the tip shielded from the bulk TCNQ solution by the TCNQ­ diffusion layer of the large Pt substrate. A comproportionation rate constant, k(comp) = 1 × 10(6) M(­1) s(­1), was determined by tip pulse chronoamperometry at coaxially aligned tip and substrate UMEs of the same radius. Diffusion coefficients of the TCNQ species and standard potentials for the reductions were also determined. Experimental results were compared with 2D transient axisymmetric simulations and reported analytical equations.