Abstract
Hydrogen sulfide (H2S) is emerging as an important gasotransmitter in both physiological and pathological states. Rapid measurement of H2S remains a challenge. We report a microfluidic method for rapid measurement of sulphide in blood plasma using Dansyl-Azide, a fluorescence (FL) based probe. We have measured known quantities of externally added (exogenous) H2S to both buffer and human blood plasma. Surprisingly, a decrease in FL intensity with increase in exogenous sulphide concentration in plasma was observed which is attributed to the interaction between the proteins and sulphide present in plasma underpinning our observation. The effects of mixing and incubation time, pH, and dilution of plasma on the FL intensity is studied which revealed that the FL assay required a mixing time of 2 min, incubation time of 5 min, a pH of 7.1 and performing the test within 10 min of sampling; these together constitute the optimal parameters at room temperature. A linear correlation (with R2 ≥ 0.95) and an excellent match was obtained when a comparison was done between the proposed microfluidic and conventional spectrofluorometric methods for known concentrations of H2S (range 0–100 µM). We have measured the baseline level of endogenous H2S in healthy volunteers which was found to lie in the range of 70 μM – 125 μM. The proposed microfluidic device with DNS-Az probe enables rapid and accurate estimation of a key gasotransmitter H2S in plasma in conditions closely mimicking real time clinical setting. The availability of this device as at the point of care, will help in understanding the role of H2S in health and disease.
Highlights
Gasotransmitters, nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are emerging as potentially key signalling molecules and may have important roles in physiology[1,2,3]
The probe dansyl azide (DNS-Az) that is compatible with plasma and has a smaller reaction time when mixed with sulphide and on reaction sulfide forms dansyl amine, which is a fluorescent compound with excitation at 360 nm and emission peak at 530 nm
The results showed a linear increase in FL intensity for sulphide in the range 0 to 200 μM beyond which the linearity was lost which could be attributed to the fact that the stoichiometric ratio of the probe with sulfide is 1:1 since the probe concentration was fixed at 200 μM
Summary
Gasotransmitters, nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) are emerging as potentially key signalling molecules and may have important roles in physiology[1,2,3]. In one of the techniques, dissolved H2S present in an aqueous medium is permeated through a silicone membrane to reach the working electrode (where current is generated) immersed in an electrolyte solution[20] This method detects only the H2S in the solution and not the HS− ions which forms the 80% composition in the pH range 7.0 to 8.2 and solely depends on the membrane permeability and the sensor dimensions[20]. There have been attempts to measure and monitor hydrogen sulfide in biosamples such as, microdialysis effluents[30] and artificial cerebrospinal fluids[31], albeit without miniaturisation This was done by nanoparticle conjugation with a fluorophore, incubated with the sample in droplets in a PTFE tube and detected off chip by a commercially available real-time PCR instrument. To the best of our knowledge, there has been no attempt to develop a miniaturized method for detection of hydrogen sulphide in blood plasma
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