Abstract
Nitric oxide (NO) is a key regulator of endothelial cell and vascular function. The direct measurement of NO is challenging due to its short half-life, and as such surrogate measurements are typically used to approximate its relative concentrations. Here we demonstrate that ruthenium-based [Ru(bpy)2(dabpy)]2+ is a potent sensor for NO in its irreversible, NO-bound active form, [Ru(bpy)2(T-bpy)]2+. Using spectrophotometry we established the sensor’s ability to detect and measure soluble NO in a concentration-dependent manner in cell-free media. Endothelial cells cultured with acetylcholine or hydrogen peroxide to induce endogenous NO production showed modest increases of 7.3 ± 7.1% and 36.3 ± 25.0% respectively in fluorescence signal from baseline state, while addition of exogenous NO increased their fluorescence by 5.2-fold. The changes in fluorescence signal were proportionate and comparable against conventional NO assays. Rabbit blood samples immediately exposed to [Ru(bpy)2(dabpy)]2+ displayed 8-fold higher mean fluorescence, relative to blood without sensor. Approximately 14% of the observed signal was NO/NO adduct-specific. Optimal readings were obtained when sensor was added to freshly collected blood, remaining stable during subsequent freeze-thaw cycles. Clinical studies are now required to test the utility of [Ru(bpy)2(dabpy)]2+ as a sensor to detect changes in NO from human blood samples in cardiovascular health and disease.
Highlights
Nitric oxide (NO) is a ubiquitous, gaseous molecule that acts as a messenger in numerous regulatory functions of various cells and tissues[1]
As reported previously[23], the increase in luminescence intensity of this sensor is NO-concentration dependent, making it possible to obtain a relative quantification of NO levels
We demonstrate that [Ru(bpy)2(dabpy)]2+ can be used as a reliable, potent, concentration-dependent NO sensor in cell-free media, endothelial cell culture and rabbit plasma
Summary
A mean 27.5% reduction in fluorescence count was observed when Ru(bpy)2(dabpy)]2+ was added to the thawed plasma samples after snap-freezing, in comparison to adding Ru(bpy)2(dabpy)]2+ to blood at collection (p = 0.045, paired t-test) (Fig. 8c) Such differences in fluorescence could be due to metabolism of soluble NO in the samples, effects of temperature changes during processing and variations in the quenching of sensor by different cells in the blood sample that were not included in the analysis. We observed that after initial fluorescence readings were taken from Ru(bpy)2(dabpy)]2+ added samples, the addition of NOC13 still resulted in increased fluorescence counts (mean of 1.8 fold vs Ru(bpy)2(dabpy)]2+-only, p = 0.03, paired t-test), showing that active sensor remained present in the plasma sample throughout the entire process of freeze-thawing (Fig. 8e). Corrections for haemolysis need to be addressed in future blood-based applications of [Ru(bpy)2(dabpy)]2+
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