Direct and Rapid Measurement of Hydrogen Peroxide in Human Blood Using a Microfluidic Device

Abstract

The levels of hydrogen peroxide (H2O2) 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 H2O2 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 H2O2 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 H2O2 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 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 H2O2 in the patient’s blood. Using the linear correlation obtained with known quantities of H2O2, the endogenous H2O2 concentration in the blood of healthy individuals is found to be in the range 2 – 6 µM. The availability of this device at the point of care will have relevance in understanding the role of H2O2 in health and disease.

Microfluidics-based rapid measurement of nitrite in human blood plasma

“…We observed a fall in the FL intensity with an increase in the externally added nitrite concentration at smaller nitrite concentrations. The same effect was observed in our previous work, 45 where we demonstrated the detection of hydrogen peroxide in blood plasma. This reduction in the FL intensity could be attributed to the interference of the soluble proteins and platelets with the hydrogen peroxide and nitrite.…”

“…Half-wavelength standing waves are generated inside the channel using a PZT transducer having a resonant frequency of 1.90 MHz. Micron-sized objects such as particles or cells flowing through the channel experience primary acoustic radiation force because of the standing bulk acoustic wave, and force expression is given by 45,47 F p = 4π a 3 E ac k sin(2 kz ) ϕ , with , , , , where a is the particle size, E ac is the acoustic energy density, k is the wavenumber, z is the distance from the wall, ϕ is the contrast factor, ρ p and ρ 0 are the density of the particles and the density of the medium, respectively, β p and β 0 are the compressibility of the particles and the medium, respectively. c 0 and c p are the velocity of sound in the medium and particle, respectively and the acoustic-pressure amplitude is P a .…”

“…The first module (module I) is the blood plasma separation module, wherein the cell-free plasma is obtained from whole blood by separating blood cells using acoustophoresis. 44,45 The second module (module II) is the mixing and incubation module, wherein the plasma obtained from module-I interacts with reagents such as externally added nitrite, buffer, DAN reagent, and NaOH solution. Under acidic conditions, nitrite reacts with DAN reagent to form a fluorescent compound 2,3-naphthotriazole (NAT), and the fluorescence is further enhanced by adding NaOH solution 46 (see Fig.…”

“…Module I is an acoustics-based blood plasma separation device fabricated on a silicon substrate. 44,45 A microchannel width of 400 µm supports a half-wave frequency of 1.91 MHz, having a pressure node at the center of the channel and antinodes at the channel walls. Half-wavelength standing waves are generated inside the channel using a PZT transducer having a resonant frequency of 1.90 MHz.…”

“…Half-wavelength standing waves are generated inside the channel using a PZT transducer having a resonant frequency of 1.90 MHz. Micron-sized objects such as particles or cells flowing through the channel experience primary acoustic radiation force because of the standing bulk acoustic wave, and force expression is given by 45,47…”