Microfluidics-based rapid measurement of nitrite in human blood plasma.

Nitric oxide (NO) is one of the vital gasotransmitters that takes part in many biological pathways such as infection, inflammation and ischemia, immune response, neurotransmission, and cardiovascular systems. Nitrite is one of the primary metabolites of NO and is considered to be a circulating storage pool for NO. Here, we report direct and rapid measurement of nitrite in human blood plasma using a fluorescence-based microfluidic method. The study revealed the factors that affect the endogenous concentration of nitrite in blood plasma, mainly the presence of blood cells, hemoglobin, and soluble proteins. We find that separation of blood plasma immediately after sample collection and subsequent dilution of plasma with buffer at a ratio of 1 : 4 eliminates the interference from cells and proteins, providing reliable measurements. The proposed method can measure plasma nitrite in the concentration range of 0-20 µM with a limit of detection of 60 nM and a sensitivity of 5.64 µM-1 within 10 min of sample collection. By spiking nitrite into plasma, a linear correlation between the nitrite concentration and FL intensity is obtained, which is utilized further to measure the endogenous concentration of nitrite present in the plasma of healthy volunteers and patients. The study revealed that the endogenous nitrite concentration in the blood plasma of healthy humans falls in the range of 0.4-1.2 µM. Furthermore, the study with blood samples obtained from patients showed that nitrite levels are inversely correlated with the total cholesterol and low-density lipoproteins levels, which is in good agreement with the literature.

Direct and rapid measurement of hydrogen peroxide in human blood using a microfluidic device.

The levels of hydrogen peroxide ([Formula: see text]) in human blood is of great relevance as it has emerged as an important signalling molecule in a variety of disease states. Fast and reliable measurement of [Formula: see text] levels in the blood, however, continues to remain a challenge. Herein we report an automated method employing a microfluidic device for direct and rapid measurement of [Formula: see text] in human blood based on laser-induced fluorescence measurement. Our study delineates the critical factors that affect measurement accuracy-we found blood cells and soluble proteins significantly alter the native [Formula: see text] levels in the time interval between sample withdrawal and detection. We show that separation of blood cells and subsequent dilution of the plasma with a buffer at a ratio of 1:6 inhibits the above effect, leading to reliable measurements. We demonstrate rapid measurement of [Formula: see text] in plasma in the concentration range of 0-49 µM, offering a limit of detection of 0.05 µM, a sensitivity of 0.60 µM-1, and detection time of 15 min; the device is amenable to the real-time measurement of [Formula: see text] in the patient's blood. Using the linear correlation obtained with known quantities of [Formula: see text], the endogenous [Formula: see text] concentration in the blood of healthy individuals is found to be in the range of 0.8-6 µM. The availability of this device at the point of care will have relevance in understanding the role of [Formula: see text] in health and disease.

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

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.