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

The levels of hydrogen peroxide (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}H2O2 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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}H2O2 in the patient’s blood. Using the linear correlation obtained with known quantities of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}H2O2, the endogenous \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}H2O2 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 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{H}}_{2}{\mathrm{O}}_{2}$$\end{document}H2O2 in health and disease.


Table of contents-
 Acoustics-based for blood-plasma separation.  Flow rates of the buffer, H 2 O 2 stock, plasma, and probe for on-chip mixing and reaction and detection of exogenous H 2 O 2 in the buffer and blood plasma, centrifugation and on-chip, at different concentrations for 1:6 dilution.  Optical transmission of machined, chloroform exposed, and heat-treated PMMA channel.  Fluorescence imaging inside microchannel and in Eppendorf tubes.  Schematic of the experimental setup, Blood plasma separation-images and absorbance measurement, FL signal from H2O2+probe mixture in Eppendorf tube.  Variation of FL intensity with H 2 O 2 concentration in buffer measured using the microfluidic device with onchip and off-chip mixing/incubation.  Flow rates of the buffer, H 2 O 2 stock, plasma, and probe for on-chip mixing and incubation and detection of exogenous H 2 O 2 in the centrifuged blood plasma at different concentrations and for 1:1, 1:3, and 1:10 dilutions.  Limit of detection and sensitivity.

S1: Acoustics-based for blood-plasma separation
The blood plasma separation device works on the principle of acoustophoresis. The blood sample flowing through the microchannel is exposed to bulk acoustic standing waves generated using a PZT resonator. The width of the microchannel is 300 µm and by operating the PZT at a frequency of 1.91 MHz, standing half-waves are produced with the node at the center of the microchannel and anti-nodes at the side walls. Micron-sized objects exposed to standing bulk acoustic wave can experience primary acoustic radiation force given by 25 F p = 4πa 3 E ac k sin(2kz)ϕ, with ϕ = ρ p + , 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 is the velocity of sound in the medium and particle respectively, and the acoustic-pressure amplitude is P a . The blood cells exposed to the standing waves experience the acoustic radiation force due to a higher acoustic impedance compared to the suspending medium or the plasma, consequently a positive contrast factor and therefore migrate towards the nodal plane. The blood cells get focused at the center of the microchannel and exit through the center outlet whereas the cellfree plasma enters into the mixing and incubation module through the side outlets. In the present study, the flow rate of the whole blood sample is kept fixed at 20 µL/min and acoustic energy density is also kept fixed 14.9 J/m 3 to obtain cellfree plasma at a flow rate of 1.0 µL/min."

S2: The details of flow rates for different H2O2 concentrations in various experiments
Here, the H2O2 stock of 10 μM concentration, used as a working solution, is filled in one of the syringes, and the other syringes are separately filled with the probe, buffer, and plasma. The flow rates of probe, buffer, and plasma are adjusted to achieve a total flow rate of 4 μL/min. All the flow rates, such as the H2O2 stock, buffer, probe, and plasma, are adjusted considering the final flow rate of 4 μL/min. For example, in experiments with buffer (see Table S1), to obtain the final H2O2 concentration of 1.0 μM, the flow rates are 3.4 μL/min buffer, 0.4 μL/min H2O2 stock, and 0.2μL/min probe, which gives a total flow rate of 4 μL/min. By molarity calculation, say per minute (or any fixed time duration), we get the concentration of 1.0 μM in a final volume of 4.0 μL by adding 0.4 μL from the stock of 10 μM. The process is continuous and the other concentration is achieved in the same way. Similarly, in the experiments with plasma (see Table S1), a similar procedure is followed; the only difference is that the fixed 1:6 plasma dilution (which is required to overcome the interference from plasma proteins) is achieved by fixing the plasma volume and total (buffer + stock) volume. The individual buffer and stock volumes are varied to achieve a given concentration.
To avoid cross-contamination, the channels are flushed with the buffer between the measurements at different concentrations. During this periodic cleaning step, the setup remains fixed and does not require any manual intervention; only the other syringe infusion pumps are turned off leaving the buffer infusion pump running.

S3: Optical transmission of machined, chloroform exposed and heat-treated PMMA channel
The microchannel was machined in PMMA using a CNC micro-milling machine (Minitech machinery, USA) and then exposed to chloroform vapour for 2 min before sealing it with a planar PMMA substrate and then heating the bonded device at 65°C for 30 min. We have measured the roughness of a microchannel machined and exposed to a heat cycle at the same conditions using a surface profiler (Wyko NT1100, Vecco, USA), which was found to be ~30 nm. This is in agreement with the literature which suggests that micro-milled PMMA after exposure to chloroform and a heating cycle (at 60°C) yields optical quality devices with reduced roughness 2 . We have also compared the transmittance of the PMMA microchannel prepared above with that of unprocessed PMMA and glass. The transmittance measurements were performed using a spectrometer (Flame-T, Ocean Optics, Germany) and a light source (DH-2000-BAL, Ocean Optics, Germany). We observed a negligible difference between the transmittance values obtained in the three different cases indicating that the fabrication process does not affect the optical quality of the PMMA considerably (See Fig. S1).

Fig. S1
Percentage of optical transmissions at different frequencies for different surfaces such as glass, PMMA, and machined PMMA with chloroform exposure and heat cycle of 65°C for 30min.

S4: Fluorescence imaging inside microchannel and in Eppendorf tubes
The images in Figure 1c and S2b are the fluorescence images of the mixture of H 2 O 2 and the chemical probe upon reaction, for different concentrations of H 2 O 2 , captured in the microchannels and Eppendorf tubes. The images shown for the microchannel case are captured by passing a laser beam through the channel in the detection module in the dark field and using a 60X lens of an inverted microscope and a high-speed/resolution colour camera. The images for the Eppendorf tube case are captured with an 18MP phone camera, placing the tubes on a UV illuminator. The RGB values for the pictures after subtracting the background are shown in the table below.       Table S2.