Craig A Aspinwall

PMID: 22911376;PMCID: PMC3716455;Abstract:

Photolytic optical gating (POG) facilitates rapid, on-line and highly sensitive analyses, though POG utilizes UV lasers for sample injection. We present a low-cost, more portable alternative, employing an ultraviolet light-emitting diode (UV-LED) array to inject caged fluorescent dyes via photolysis. Utilizing the UV-LED array, labeled amino acids were injected with nanomolar limits of detection (270 ± 30 nM and 250 ± 30 nM for arginine and citrulline, respectively). When normalized for the difference in light intensity, the UV-LED array provides comparable sensitivity to POG utilizing UV lasers. Additionally, the UV-LED array yielded sufficient beam quality and stability to facilitate coupling with a Hadamard transform, resulting in increased sensitivity. This work shows, for the first time, the use of an UV-LED for online POG with comparable sensitivity to conventional laser sources but at a lower cost. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Abstract:

A ruthenium-oxide-type catalytic film (RuOx) was produced on carbon fiber microelectrodes by cycling the electrode potential between 0.65 and -0.85V vs. SSCE at 100 V s^-1 in an air-equilibrated acidic solution of RuCl3. The film catalyzes oxidation of insulin in a saline buffer at pH7.4. The minimum number of electrons transferred during the insulin oxidation at 0.65 V is 6.7. The analytical performance of the modified electrode as an amperometric detector for insulin was characterized using flow injection analysis. Linear least squares calibration curves over the range 0.10 to 1.0 μM (five points) had slopes of 72 ± 2 pA μM^-1 and correlation coefficients of 0.999 or greater. The detection limit, calculated as the concentration that would yield a signal equal to three times the root mean square noise, was 23 nM and response time (t90%) was 40ms or less. The electrode response to 0.2 μM insulin was stable for 3 days. The modified electrode was used for amperometric detection of exocytosis from individual pancreatic β-cells.

Nitroxyl (HNO) donors exhibit promising pharmacological characteristics for treatment of cardiovascular disorders, cancer, and alcoholism. However, whether HNO also serves as an endogenous signaling agent is currently unknown, largely because of the inability to selectively and sensitively detect HNO in a cellular environment. Although a number of methods to detect HNO have been developed recently, sensitivity and selectivity against other nitrogen oxides or biological reductants remain problematic. To improve selectivity, the electrophilic nature of HNO has been harnessed to generate modifications of thiols and phosphines that are unique to HNO, especially compared to nitric oxide (NO). Given high bioavailability, glutathione (GSH) is expected to be a major target of HNO. As a result, the putative selective product glutathione sulfinamide (GS(O)NH2) may serve as a high-yield biomarker of HNO production. In this work, the formation of GS(O)NH2 after exposure to HNO donors was investigated. Fluorescent labeling followed by separation and detection using capillary zone electrophoresis with laser-induced fluorescence allowed quantitation of GS(O)NH2 with nanomolar sensitivity, even in the presence of GSH and derivatives. Formation of GS(O)NH2 was found to occur exclusively upon exposure of GSH to HNO donors, thus confirming selectivity. GS(O)NH2 was detected in the lysate of cells treated with low-micromolar concentrations of HNO donors, verifying that this species has sufficient stability to server as a biomarker of HNO. Additionally, the concentration-dependent formation of GS(O)NH2 in cells treated with an HNO donor suggests that the concentration of GS(O)NH2 can be correlated to intracellular levels of HNO.

We report an online, optical injection interface for capillary zone electrophoresis (CZE) based upon photophysical activation of a caged, fluorogenic label covalently attached to the target analyte. This injection interface allows online analysis of biomolecular systems with high temporal resolution and high sensitivity. Samples are injected onto the separation capillary by photolysis of a caged-fluorescein label using the 351-364 nm irradiation of an Ar+ laser. Following injection, the sample is separated and detected via laser-induced fluorescence detection at 488 nm. Detection limits for online analysis of arginine, glutamic acid, and aspartic acid were less than 1 nM with separation times less than 5 s and separation efficiencies exceeding 1,000,000 plates/m. Rapid injection of proteins was demonstrated with migration times less than 500 ms and 0.5 nM detection limits. Online monitoring was performed with response times less than 20 s, suggesting the feasibility of this approach for online, in vivo analysis for a range of biologically relevant analytes.

PMID: 10362508;Abstract:

A novel oxygen microsensor was used to measure oxygen levels in single mouse islets as a function of glucose concentration. Oxygen consumption of individual islets was 5.99 ± 1.17, 9.21 ± 2.15, and 12.22 ± 2.16 pmol/min at 3, 10, and 20 mM glucose, respectively (mean ± SEM, n = 10). Consumption of oxygen was islet-size dependent as larger islets consumed more oxygen than smaller islets but smaller islets consumed more oxygen per unit volume than larger islets. Elevating glucose levels from 3 to 10 mM induced pronounced fast oscillations in oxygen level (period of 12.1 ± 1.7 s, n = 6) superimposed on top of large slow oscillations (period of 3.3 ± 0.6 min, n = 6). The fast oscillations could be completely abolished by treatment with the L-type Ca²⁺-channel blocker nifedipine (40 μM) with a lesser effect on slow oscillations. Slow oscillations were almost completely dependent upon extracellular Ca²⁺. The oxygen patterns closely mimic those that have previously been reported for intracellular Ca²⁺ levels and are suggestive of an important role for Ca²⁺ in amplifying metabolic oscillations.