Rapid measurement of hydrogen sulphide in human blood plasma using a microfluidic method

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.

Figure 1

A schematic of the proposed microfluidic device, the mixing and incubation module and the detection module are shown.

Figure 2

(a) Effect of mixing and incubation time on the FL intensity, sulfide concentration 100 µM in 1.0 mL of PBS, in one of the samples, probe and sulphide thoroughly mixed externally using a vortex mixer for ~30 s and in the other the probe just added to sulphide but not mixed, (b) Effect of pH on the change in fluorescence intensity at different sulphide concentrations one with a pH of 7.1 and other with a pH of 5.6, (c) Variation of fluorescence spectrum with concentration of sulphide in PBS, (d) Effect of sulphide concentration in PBS on the FL intensity measured using spectrometer, off-chip mixing and on-chip detection, and on-chip mixing and detection, probe concentration 200 μM in all cases, the inset shows the change in fluorescence intensity for the range of 0 µM to 500 µM of sulfide measured in spectrofluorometer. For each data point, the error bars represent the standard deviation from three different readings.

Figure 3

(a) Effect of dilution of plasma (containing endogenous sulphide) without the addition of exogenous sulphide in plasma on the fluorescence intensity, probe concentration 200 µM, inset shows the change in FL intensity with addition of sulfide in undiluted plasma, (b) Variation of FL intensity with exogenous sulphide in diluted plasma of different dilution factors (2.0 and 3.3), (c) Decay of FL intensity measured at different time points after exogenous sulphide added to three-fold diluted plasma, inset shows the corresponding sulphide concentration with time, (d) Change in the FL intensity of the endogenous sulfide in three-fold diluted plasma with time, probes added to diluted plasma at different time points, mixed thoroughly and detected immediately, probe concentration 200 µM in all cases. For each data point, the error bars represent the standard deviation from three different readings.

Figure 4

(a) Fluorescence spectrum obtained for different exogenous concentrations (0 to 100 µM) in plasma, (b) Linear response of the probe from three different schemes of detection in plasma, (b) Effect of exogenous sulphide concentration in plasma on the normalized FL intensity measured using spectrometer, off-chip mixing but on-chip detection, and on-chip mixing and detection, probe concentration 200 μM, sulphide and probe thoroughly mixed, pH = 7.1. For each data point, the error bars represent the standard deviation from three different readings.

Figure 5

(a) Optical images of the fluorescence signal in the detection zone for different concentrations of exogenous sulfide (0 to 100 µM) in plasma obtained using a FL microscope, the scale bar represents 150 μm, (b) Variation of FL intensity with exogenous sulphide concentration for the plasma samples of different individuals, characteristic curves are linear (with R² ≥ 0.92) in all cases with a slope of 0.0374 ± 0.0013 μM⁻¹, inset shows concentration of sulfide measured from the linear fit to the exogenous sulphide added in the three plasma samples. For each data point, the error bars represent the standard deviation from three different readings.