Measurement of Alcohol-Dependent Physiological Changes in Red Blood Cells Using Resistive Pulse Sensing

Abstract

Alcohol exposure has been postulated to adversely affect the physiology and function of the red blood cells (RBCs). The global pervasiveness of alcohol abuse, causing health issues and social problems, makes it imperative to resolve the physiological effects of alcohol on RBC physiology. Alcohol consumed recreationally or otherwise almost immediately alters cell physiology in ways that is subtle and still unresolved. In this paper, we introduce a high-resolution device for quantitative electrofluidic measurement of changes in RBC volume upon alcohol exposure. We present an exhaustive calibration of our device using model cells to measure and resolve volume changes down to 0.6 fL. We find an RBC shrinkage of 5.3% at 0.125% ethanol (the legal limit in the United States) and a shrinkage of 18.5% at 0.5% ethanol (the lethal limit) exposure. Further, we also measure the time dependence of cell volume shrinkage (upon alcohol exposure) and then recovery (upon alcohol removal) to quantify shrinkage and recovery of RBC volumes. This work presents the first direct quantification of temporal and concentration-dependent changes in red blood cell volume upon ethanol exposure. Our device presents a universally applicable highresolution and high-throughput platform to measure changes in cell physiology under native and diseased conditions. KEYWORDS: size of red blood cells, alcohol-induced shrinkage of biological cells, resistive pulse technique, micropores, electrofluidic devices ■ SIGNIFICANCE STATEMENTThe physiological changes in red blood cells caused by alcohol are not well understood, specifically the changes in cell volume that directly affect the oxygen carrying function of RBCs. Most of these changes are subtle and difficult to quantify. In the last decade, multiple methods have been used to measure this subtle change in cell size caused by alcohol exposure. The focus of this work is an electrofluidic sensing approach that accurately measures changes in cell volume of a cell population at single-cell resolution. The approach has potential to rapidly provide changes in cell volumes in cases like chemical exposure, malaria infection, large population sickle-cell disease screening, and quantifying cell lysis in a label-free, low-cost, and microscopy-independent way.

Customized low-cost high-throughput amplifier for electro-fluidic detection of cell volume changes in point-of-care applications

“…The bar plot in this figure shows comparison of measured ΔG values of 24.1 ± 0.7, 26.7 ± 0.3 and 33.9 ± 0.9 nS respectively, for beads of diameter 1.90 ± 0.04, 2.00 ± 0.04, and 2.1 ± 0.3 μm translocating through a 3.0 μm micropore device. In our previous work, we have shown similar contrast between particles using commercial amplifiers [ 48 ], Fig 5D demonstrates that in terms of resolutions, our custom made lab-amplifier compares very well to the commercial amplifiers.…”

“…We next show cell volume detection capabilities using model cells measured with our electro-fluidic device and lab-amplifiers. Our devices make electrical measurements of cell volumes, with single-cell resolution, as individual cells translocate through the micropore under an applied flow and electrical potential [ 48 ]. Fig 4A shows 1.5 sec long time series of translocation events caused by 4.98 μm beads translocating through a 6.8 μm micropore device as measured using the L1A (blue), L10A (green) and D001-1 (red) amplifiers.…”

“…As the model cells translocate through the micropore, they block the pore conductance (ΔG (nS)) for the duration of translocation (dwell time, Δt (ms)). An electrical conductance drop (ΔG) signals the translocation of a single cell and is directly proportional to the cell volume [ 48 , 51 , 52 , 54 ]. In each dataset, we collect electrical conductance blockage events for 500 cells or more that translocate through the pore to measure population average cell volumes and changes in it, if any.…”

“…Borosilicate glass capillaries (Part # B100-50-10, Sutter Instruments) were pulled using a micropipette puller (P-2000, Shutter Instrument) and then polished using a flame polisher (MF-900, Micro Forge, Narishige) to fabricate the desired micropores (electro-fluidic device). The detailed information on the fabrication of the micropores (electro-fluidic device), sample preparation, and electro-fluidic measurements are presented in our previous work [ 48 ]. The schematic and brief details on how the micropore devices are prepared is included in the supplementary information file (see S2 Fig in S1 File ).…”

“…Lab amplifier with higher bandwidth (L1 series) works better for higher throughput but at the cost of slightly higher noise than the L10 series. L1 series amplifiers are more suitable for larger cells with large resistive pulse signals [ 48 ], whereas the L10 series amplifiers are more suitable to measure subtle changes in cell volumes with higher resolution. All the electrical characterization of the lab amplifier is compared with two commercial amplifiers under identical experimental conditions.…”