Observation of nonspherical particle behaviors for continuous shape-based separation using hydrodynamic filtration

Abstract

Selection of particles or cells of specific shapes from a complex mixture is an essential procedure for various biological and industrial applications, including synchronization of the cell cycle, classification of environmental bacteria, and elimination of aggregates from synthesized particles. Here, we investigate the separation behaviors of nonspherical and spherical particles∕cells in the hydrodynamic filtration (HDF) scheme, which was previously developed for continuous size-dependent particle∕cell separation. Nonspherical particle models were prepared by coating the hemisphere of spherical polymer particles with a thin Au layer and by bonding the Janus particles to form twins and triplets resembling dividing and aggregating cells, respectively. High-speed imaging revealed a difference in the separation behaviors of spherical and nonspherical particles at a branch point; nonspherical particles showed rotation behavior and did not enter the branch channel even when their minor axis was smaller than the virtual width of the flow region entering the branch channel, w(1). The confocal-laser high-speed particle intensity velocimetry system visualized the flow profile inside the HDF microchannel, demonstrating that the steep flow-velocity distribution at the branch point is the main factor causing the rotation behavior of nonspherical particles. As applications, we successfully separated spherical and nonspherical particles with various major∕minor lengths and also demonstrated the selection of budding∕single cells from a yeast cell mixture. We therefore conclude that the HDF scheme can be used for continuous shape-based particle∕cell separation.

Figure 1

Schematic diagrams showing the separation behaviors of particles in the HDF scheme. (a) Size-dependent separation of spherical particles and [(b)–(d)] possible rotation behaviors of spherical (b) and nonspherical [(c) and (d)] particles at the branch point.

Figure 2

(a) Fabrication process of nonspherical particle models. Janus particles are initially prepared and then bonded to form nonspherical twin and triplet particles. (b) Micrographs of the fabricated single, twin, and triplet particles. Scale bar: 10 μm.

Figure 3

Schematic illustrations of the designs of HDF microchannels. (a) Microdevice A for particle separation and (b) microdevice B for sorting budding yeast cells. Details of these microchannels are shown in the supplemental information (Ref. 43). Drawing is not to scale.

Figure 4

Visualization of the virtual regions entering into branch channels connected to (a) outlet 2 and (b) outlet 6. The widths of the flow region entering the branch channels, w₁, were (a) 9.0 and (b) 5.7 μm, respectively. Scale bar: 10 μm.

Figure 5

Behaviors of single and twin particles at a branch point connected to outlet 6 in microdevice A. [(a) and (b)] Micrographs of (a) a spherical particle and (b) a nonspherical twin particle at every 1 ms. Scale bar: 20 μm. [(c) and (d)] Schematic illustrations of positions of (c) spherical and (d) nonspherical twin particle micrographs. The numbers correspond to the times (ms) shown in (a) and (b) (enhanced online).

Figure 6

Visualized flow-velocity distribution at a branch point connected to outlet 6 in microdevice A when the inlet flow rate was 2.0 μl∕min. (a) Image showing the flow-velocity distribution. Scale bar: 30 μm. (b) Definition of x- and y-axes at the branch point. The location of a nonspherical twin particle corresponds to that shown in Fig. 5a at 4 ms. [(c) and (d)] The distributions of flow velocities (c) v_x and (d) v_y along the x=1.91 μm line. (e) Schematic image showing the flow forces in the x- and y-directions on the twin particle. The particle positions correspond to those at 4 and 8 ms in Fig. 5b.

Figure 7

Separation results for spherical and nonspherical particles. (a) Micrographs and schematic diagrams of spherical and nonspherical particles used for separation. Scale bar: 10 μm. The sizes of the triplets varied depending on the bonding angles, which were 60° and 110° for triplets 1 and 2. (b) Micrographs of particles before separation and after recovery from outlets 1–6. Scale bar: 20 μm. (c) Distribution ratios of particles at each outlet. Each value shows the mean±SD from three independent experiments.

Figure 8

Sorting results for budding yeast cells. (a) Micrographs of cells before separation and after recovery from outlets 1–5. Scale bar: 10 μm. (b) The ratios of cell types in the fractions recovered from each outlet and (c) the distribution ratios of cells according to cell type. Each value shows the mean±SD from three independent experiments.