Probing the intracellular glutathione redox potential by in-cell NMR spectroscopy.

Non-invasive and real-time analysis of cellular redox processes has been greatly hampered by lack of suitable measurement techniques. Here we describe an in-cell nuclear magnetic resonance (NMR) based method for measuring the intracellular glutathione redox potential by direct and quantitative measurement of isotopically labeled glutathione introduced exogenously into living yeast. By using this approach, perturbations in the cellular glutathione redox homeostasis were also monitored as yeast cells were subjected to oxidative stress.

Quantitative measurements of glutathione in yeast cell lysate using 1H NMR.

Methods for quantifying the level of glutathione (GSH) in yeast cell lysate are described using (1)H NMR analysis. For quantification purposes, the (1)H resonances corresponding to the Cys βCH2 of GSH were identified as having the fewest overlapping spectral interferences from lysate matrix components using GSH spiked yeast lysate samples. Two methods, standard addition based on peak integration and a spectral subtraction approach, were evaluated for quantifying GSH in lysate samples.

Automated spectral smoothing with spatially adaptive penalized least squares.

A variety of data smoothing techniques exist to address the issue of noise in spectroscopic data. The vast majority, however, require parameter specification by a knowledgeable user, which is typically accomplished by trial and error. In most situations, optimized parameters represent a compromise between noise reduction and signal preservation.

Centrifugal microfluidics with integrated sensing microdome optodes for multiion detection.

An array of four sensing microdome optodes (potassium, sodium, calcium, and chloride) was incorporated into a centrifugal microfluidics platform to obtain a multiion analysis system. The behavior of each sensing microdome was in good agreement with a theoretical model describing the response. The selectivity of each optode over common interfering ions was established and was used to identify calibrant solutions that can be employed for the simultaneous calibration of all four optodes without significant cross-interference.

Decyl methacrylate-based microspot optodes.

Optode sensing membranes employing decyl methacrylate cross-linked with 1,6-hexanediol dimethacrylate as the polymer support were fabricated by a direct microspotting method on several surfaces. Photopolymerization was used to attach the microspots to the substrate. Using this method, diameters in the micrometer domain were obtained.