(F-218) Simultaneously measuring electrophoretic mobility and size of single particles using a novel microfluidic device based on AC electrophoresis

Introduction:: The ability to precisely measure the charge and size of single particles is essential in understanding particle adhesion and interactions with their environment. The charge of cancer cells determines their state of aggregation and adhesion, and polarization of platelets promotes binding. A particle’s surface charge is often defined by the zeta potential that largely impacts the electrophoretic mobility (μ_ep). Several electrophoretic and size measurement methods are used to measure particles’ μ_ep and size. However, precise measurement of a single particle’s physical property remains challenging. Thus, developing an improved multi-parameter measurement method is expected to benefit many biological applications. A few reports have utilized particle tracking velocimetry (PTV) with DC electrophoresis to measure the particles’ size and electrophoretic mobility. However, relatively low electric fields are utilized, and large noise reduces the precision of the μ_ep measurements in these systems. We introduce transverse AC electrophoresis (TrACE), which provides improved measurements of m_ep by applying an oscillating field across the microchannel width. The cross-channel field eliminates longitudinal EOF. TrACE devices are anticipated to be especially amenable as a point-of-care tool since the low-frequency and low-power waveforms for operations are easily generated using a smartphone. Many oscillations can be observed for a single particle, and the small inter-electrode distance creates high electric fields. Here, we develop a quasi-equilibrium model and characterize the ability of TrACE to simultaneously measure the μ_ep and size of mammalian cells and bacteria from PTV analysis of their recorded trajectories.




Materials and Methods:: The TrACE devices were fabricated from two main components: a glass slide with micropatterned electrodes and a polydimethylsiloxane (PDMS) capping layer with a single microchannel and an inlet and outlet reservoir. The silicon wafer mold for a PDMS microchannel was fabricated using a standard photolithography method, and the electrodes were photopatterned onto the glass substrate via a standard wet etching process. An electric field wave, either a square or sine wave, was applied between an electrode pair using a function generator that created a transverse electric field to the bulk flow along the microchannel. The TrACE device was placed on an inverted fluorescent microscope and imaged at 1000 fps at 20 or 40-X magnification. A sequence of 1000 images was processed utilizing a PTV algorithm, and the particle trajectories were analyzed to calculate each particle’s μ_ep and size. Samples were prepared in 0.1 mM PB solution (pH 7.4). Polystyrene particles with 0.53, 0.84, 1, and 2 µm in diameter are tested in the studies. Upon sample injection into the inlet reservoir, particles travel through the microchannel via hydrodynamic flow created by the height difference between the inlet and outlet reservoirs. For the calculation of μ_ep, the maximum y-velocity was calculated for every half-cycle in a trajectory oscillation and was divided by the known electric field. For the calculation of a diameter, spatial fluctuations from Brownian motion were used to determine a diffusion coefficient or via image analysis.

Results, Conclusions, and Discussions:: We describe a new microfluidic TrACE system that measures the electrophoretic mobility and size of single particles using particle trajectories. A simple quasi-equilibrium model is developed to understand the basic particle response to the externally applied sine or square wave. A model shows that the particle oscillates at the same frequency as the wave that drives its motion. Additionally, the amplitude of the particle trajectory is proportional to the frequency of the applied potential because the longer excursions at lower frequencies increase the trajectory amplitude. Changing the amplitude of the electric potential also increases the trajectory amplitude because of the increase in driving electric force. Individual particles are measured in the TrACE system using a square or sine wave at 10, 50, and 100 Hz and 0.75, 0.9, 1, 1.1, 1.25, and 1.5 V amplitudes. Recorded trajectories closely agree with the predicted particle trajectory. Electrophoretic mobility of 0.53, 0.84, 1, and 2 µm particles are measured using the particle trajectories recorded while applying a 1 V amplitude square wave at 50 Hz. 50 Hz better utilizes the residence time of the particles within the detection window by forcing particles to oscillate more quickly and allowing for ~100 measurements. The precision of the measured electrophoretic mobility values is represented with a standard error of the mean value as close as ~2% to the true mean. Additionally, a large ratio ( > 100) between electrophoresis and Brownian motion shows that electrophoresis is a primary force driving the particle motion. While the size of larger particles, ≥2 µm, can be measured directly from imaging, size measurements of smaller particles are more precise when calculated from Brownian motion in the x-dimension. Multiplexing via simultaneous tracking of multiple particle trajectories in the detection window alleviates low particle throughput. Additionally, a low bulk flow velocity and long detection window provide a longer observation time and increase the number of measurements for improved precision. We anticipate that the measurement precision and capabilities of the TrACE system will grow as our understanding of particle response improves.




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