Degas-Driven Deterministic Lateral Displacement in Poly

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Degas-driven deterministic lateral displacement in polydimethylsiloxane microfluidic devices Naotomo Tottori, and Takasi Nisisako Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05587 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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Analytical Chemistry

Degas-driven deterministic lateral displacement in polydimethylsiloxane microfluidic devices

Naotomo Tottori† and Takasi Nisisako*‡

† Department of Mechanical Engineering, School of Engineering, Tokyo Institute of Technology, Tokyo, Japan ‡ Institute of Innovative Research, Tokyo Institute of Technology, R2-9, 4259 Nagatsuta-cho, Midoriku, Yokohama, Kanagawa, 226-8503, Japan

*to whom correspondence should be addressed: [email protected]

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ABSTRACT In this work, degas-driven microfluidic deterministic lateral displacement devices were fabricated from poly(dimethylsiloxane). Two device configurations were considered: one with a single input for the enrichment of particles and the other one with sheath inputs for the separation of particles based on their sizes. Using the single-input device, the characteristics of the degas-driven fluid through micropillars were investigated, and then, selective enrichment of fluorescent polymer particles with diameters of around 13 m mixed with similar 7 m particles was demonstrated. Using the sheathinput device, the separation of 13 μm and 7 μm beads was achieved (the corresponding purities exceeded 92.62% and 99.98%, respectively). In addition, clusters composed of 7 μm beads (including doublets, triplets, and quadruplets) were fractionated based on their equivalent sizes. Finally, white blood cells could be separated from red blood cells at a relatively high capture efficiency (95.57%) and purity (86.97%).


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INTRODUCTION The separation of target biological particles from a heterogeneous population is a fundamental sample preparation step in many biological and medical assays. For cell separation, standard fluorescence-activated and magnetic-activated cell sorting methods are widely utilized. However, these techniques require the use of expensive benchtop equipment and involve relatively complex operations. In contrast, various active and passive microfluidic particles separators have been designed1–3 as a promising alternative to the currently used separation techniques because of their advantages such as minimized consumption of samples and reagents, simple user operation, and easy integration of multiple components for comprehensive analysis. One of the passive separation techniques, deterministic lateral displacement (DLD), has been recently investigated in detail4. It can separate particles based on their sizes with exceptionally high resolution via continuous flow through periodically arranged pillars5. In DLD, geometrically-predicted boundary in size for separation, which is called critical diameter Dc, is independent of the flow rate when the particles are not very small and their diffusion by Brownian motion is negligible against their sizes. In previous studies, DLD was used for separating various particles such as polystyrene (PS) beads of micro/nanometer sizes5,6, droplets7, biological particles including DNA5, blood cell subtypes8,9, trypanosomes10, circulating tumor cells from blood11–13, epithelial cells from smaller fibroblasts14, fungal spores15, viable and nonviable mammalian cells16, exosomes6, and pathogenic bacteria17. During conventional DLD separation, off-chip bulky pumps are normally utilized for 3 ACS Paragon Plus Environment


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infusing the sample solution despite the existence of gravity18 and capillary-driven19,20 DLD devices. However, such external pumps possess low portability, require large footprints and a power source, and can produce a relatively high dead volume of the analyzed sample. Thus far, many fluid transportation techniques without external pumps have been considered. They are based on the pressure difference between two droplets with different sizes21, capillary flow22–24, evaporation22,25, gravity-driven flow23,26, and the flow driven by squeezing with a finger27. Moreover, liquids have been autonomously infused into a poly(dimethylsiloxane) (PDMS) channel due to the internal negative pressure induced by the high gas solubility of PDMS28,29. When a degassed PDMS device is exposed to atmospheric pressure and a sample is loaded to fill the inlet of the microchannel, the microchannel pressure becomes lower than the atmospheric pressure because of the re-dissolution of air. As a result, the sample is infused into the channel by its internal negative pressure. This pumping does not require any hydrophilic surface treatment (unlike the capillary flow in PDMS devices), humidity/temperature control (unlike evaporation-driven flow), or a complex operational procedure. As these advantages are suitable for point of care (POC) testing, multiple studies have employed this technique29. However, to the best of our knowledge, the described PDMS property has not been used for fluid transport in DLD devices. Therefore, it remains unclear whether and how the degas-driven fluid infusion could be used for the enrichment of particles and their separation through DLD arrays. In this work, two different degas-driven DLD devices were fabricated from PDMS (Fig. 1a). First, using a single-input device, the degas-driven flow from a source droplet through a DLD array was 4 ACS Paragon Plus Environment

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investigated, and selective enrichment of 13 μm fluorescent PS beads from 7 μm PS beads was demonstrated. Next, the separation of 13 μm and 7 μm PS beads was performed at high purity and capture efficiency using a sheath-input DLD device. Finally, the sheath-input device was utilized for separating white blood cells (WBCs) and red blood cells (RBCs).

DEVICE DESIGN AND MECHANISM The single-input device (Fig. 1b) can easily enrich particles without further dilution, although smaller particles cannot be removed perfectly from a larger particle population11. In contrast, the sheath-input device (Fig. 1c) can separate particles with high purity and capture efficiency although the injected sample solution is diluted by the sheath fluid5. Both degas-driven DLD devices consist of a PDMS chip on a glass slide, which contains DLD pillars between the inlet and outlet reservoirs with diameters, heights, internal volumes, and surface areas equal to 4 mm, 2.5 mm, 31.4 μL, and 31.4 mm2, respectively. The height of the channel is 40 μm, and the outlet reservoirs are closed using adhesive tape. Unlike previous devices containing dead-end channels30,31, the internal volume of the outlet reservoir significantly exceeds that of the channel to continuously infuse liquid after filling the microchannel28. In both the single-input and sheath-input devices, particles larger than the critical diameter Dc of the DLD array flow at a small angle toward the sidewall (displacement mode), while particles with diameters smaller than Dc flow along the fluid streamline and retain their original positions in the 5 ACS Paragon Plus Environment


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vertical direction with respect to the global fluid stream (zigzag mode). This size-based difference in the migration modes allows the selective enrichment of particles larger than Dc by the single-input device and their separation from smaller particles using the sheath-input device. We used the following equation, which is based on streamlines model32, as a guidance to determine the desired Dc values for the enrichment or separation of particles: 0.48

𝐷c = 1.4 × 𝑑 × (∆𝜆 𝜆)

(1)

where d is the gap between the posts, Δλ is the shift of the posts, and λ is the center-to-center distance between the neighboring posts (Fig. 1d). Meanwhile, it was reported previously that the DLD model based on streamlines might not be valid for particles that are large compared to the gap between obstacles and that the Dc value calculated using Eq. (1) was slightly smaller than the experimentally determined magnitude10,16,33–35. Therefore, the corresponding DLD parameters were adjusted to obtain desired Dc values16 (see Supporting Information Section S-1). In addition, the gaps near the sidewalls of the DLD array were adjusted to reduce their effect on the particle separation process36. The single-input device contains a DLD array (width: 1.5 mm, length: 40 mm) between the single inlet and outlet reservoirs. The volume of the channel is equal to 1.5 μL, and the utilized DLD parameters are listed in Table S-1. In this device, the infused particles enter the DLD array from the broadly distributed positions (corresponding to gap numbers (GNs) ranging from 1 to 19), after which the particles with sizes larger than Dc flow toward the sidewall in the displacement mode, while the other particles (with diameters smaller than Dc) flow in the zigzag mode, resulting in the presence of 6 ACS Paragon Plus Environment

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highly enriched larger particles along the sidewall (GN = 19; see the inset of Fig. 1b). For particle and blood separations, two sheath-input devices containing DLD arrays (width: 2.0 mm, length: 29 mm) between the three inlet and two outlet reservoirs were fabricated (Table S-1). The inner volumes of the channels for particle and blood separation were 3.0 μL and 2.9 μL, respectively. In order to separate the WBCs from RBCs20, the Dc of the DLD array for blood separation was set to 4.99 μm, while the Dc for separating PS particles was equal to 6.65 μm. The infused particles entered the DLD array near the sidewall because the sample input channel was sandwiched between the asymmetric sheath-flow channels. By introducing the particle suspension near the sidewall, the entire DLD array could be used more effectively. After the infused particles enter the DLD array at the specific places, only the particles with diameters larger than Dc flow toward the sidewall in the displacement mode from the original input band stream containing smaller particles, resulting in the separation of the larger particles at a relatively high purity and capture efficiency.

EXPERIMENTAL Device microfabrication. The PDMS devices were fabricated via standard soft lithography (Fig. S-1)7. The volume of the poured precursor was adjusted to obtain a final thickness of 2.5 mm, which ensured sufficient degas-driven flow in the microchannel31. Following the peel-off of the cured PDMS replica from the mold, holes for the inlet and outlet reservoirs with diameters of around 4 mm were produced using a punching tool (Harris Uni-Core, Ted Pella, CA, USA). The replicated microgrooves 7 ACS Paragon Plus Environment


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of the PDMS part were sealed with a planar glass slide (size: 76 mm × 26 mm, thickness: 0.9–1.2 mm) using the self-sticking property of PDMS.

Materials. Various mixtures of fluorescence polystyrene beads with diameters of around 13 μm (excitation wavelength: 542 nm, emission wavelength: 612 nm, 36-4, Thermo Fisher Scientific, MA, USA) and 7 μm (excitation wavelength: 480 nm, emission wavelength: 520 nm, FS06F, Bangs Laboratories, IN, USA) in an aqueous solution of 0.1 vol.% Tween 20 reagent (Sigma-Aldrich, MO, USA) were prepared. The total concentration of the beads in solution was adjusted to 105106 particles/mL. As the sheath-input solution without particles, an aqueous solution of 0.1 vol.% Tween 20 was used. For the separation of blood cells, a 40 μL droplet of peripheral blood sample was extracted from a healthy volunteer via finger pricking (Nipro, Osaka, Japan) based on the protocol approved by an institutional review board. To prevent the coagulation of the blood sample and fluorescently label the WBCs, ethylenediaminetetraacetic acid (EDTA; Dojindo, Kumamoto, Japan) and Hoechst 33342 reagent (Dojindo, Kumamoto, Japan) were added to the whole blood sample at final concentrations of 5.0 mM and 29.7 μM, respectively. The final volume of the blood sample solution was 42.7 μL. We measure the concentrations of WBCs and RBCs contained in the sample solution by using a hemocytometer (Neubauer Improved, ISOLAB, Germany). As the sheath fluid, a phosphate-buffered saline (PBS) solution containing 5.0 mM EDTA and 2% (w/v) bovine serum albumin (BSA; Wako 8 ACS Paragon Plus Environment

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Pure Chemical Industries, Osaka, Japan) was used.

Infusion of sample solution. Before experiments, the PDMS device with a sealed outlet reservoir was evacuated inside a vacuum chamber (BP-1; Samco, Tokyo, Japan) at a background pressure of ~5 Pa. The degas time was set to 1 h unless specified otherwise. Within 50 s after the exposure of the degassed PDMS device to the atmospheric pressure, a droplet of the sample solution (droplets of the sheath-fluid solution) was (were) injected into the inlet reservoir(s) to introduce the solution into the microchannel by its negative internal pressure.

Measurement and image analysis procedures. The degas-driven flow through the DLD arrays was monitored and recorded using either an upright fluorescence optical microscope (BX51; Olympus, Tokyo, Japan) or an inverted optical microscope (CKX41; Olympus, Tokyo, Japan) equipped with a digital video camera (HC-750M; Panasonic, Osaka, Japan). Image processing was conducted using the ImageJ software (NIH, NY, USA) to superimpose the recorded photomicrographs for the visualization of particle trajectories and measure the particle velocities (the mean velocity values were calculated by averaging the velocities of all particles observed in the DLD array for 20 s). The number of particles flowing through each DLD gap was counted from the recorded video to determine their distribution in the DLD array.



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RESULTS AND DISCUSSION Degas-driven flow through the DLD array. First, the degas-driven fluid infusion into the singleinput device was investigated. After 1 h of degassing, a droplet of 20 μL aqueous solution without particles was injected into the inlet. Within 10 s (7.3±0.5 s, n = 3) of injection, the aqueous solution appeared at the entrance of the DLD region, and the water-air interface started to gradually move through the arrays toward the outlet because of the internal negative pressure caused by air absorption in the PDMS channels. In particular, faster interface movement was consistently observed near the sidewall in the direction of the displacement (Fig. S-2), which might be caused by the low flow resistance of the channel in this location due to the larger gap value36. The mean velocity of the interface was 0.66 mm/s, and the end of the DLD array was reached in about 1 min (67.7±6.6 s, n = 3). After the aqueous solution passed through the entire DLD array, several remaining void areas (bubbles) were detected, which were subsequently absorbed and completely removed from the system in less than 5 min (284.7±16.2 s, n = 3). To optimize the degas time, it was varied between 10 min, 1 h, 3 h, and 10 h at a pressure of ~5 Pa followed by measurements of the infusion time, which corresponded to the time elapsed until the DLD array was filled with the flow (Fig. S-3). When the degas time was 10 min, the infusion time was 394.7±12.2 s. In contrast, when the degas time was 1 h or longer, the infusion time was around 300 s or smaller, indicating that its value did not decrease significantly at larger degas times. Next, the prepared particle suspension was infused into the channel, and particle image velocimetry 10 ACS Paragon Plus Environment

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analysis was performed (Fig. 2a) to evaluate the effect of the degas time on the device operational time and time dependence of the flow velocity. First, after degassing for 1 h, 20 L of the particle aqueous solution was injected into the input, and the resulting flow velocity was measured as a function of time (Fig. 2b). The flow velocity was maximum when the particles appeared at the DLD entrance for the first time, and then decreased dramatically within the next 5 min. After that, the flow velocity decreased gradually and almost reached zero in 30 min. From the maximum flow velocity v ~ 626 μm/s, fluid density ρ = 998.2 kg/m3, the characteristic length of the DLD array (i.e. gap between the posts) L = 20 μm, fluid viscosity μ = 1.002 mPa s), the corresponding Reynolds number (Re = ρvL/μ) was about 0.01, suggesting that the flow was laminar. When the device was degassed for more than 1 h, the flow velocity variation with time and operational time did not change significantly. Meanwhile, at shorter degas times (such as 10 min), the operational time decreased by a factor of 0.6 as compared with those measured at degas times over 1 h (Fig. 2b). The degassing progress inside the PDMS slab can be characterized by the dimensionless index m of a one-dimensional model, which is defined by the amount of remaining air molecules in the PDMS system at a certain point of time divided by the amount of air to be ultimately degassed as follows:28 𝑙

𝑚=

∫ ―𝑙(𝐶 ― 𝐶1)𝑑𝑥 𝑙

∫ ―𝑙(𝐶0 ― 𝐶1)𝑑𝑥

≈

(

)

8 𝑡 𝜋2𝐷 ― 𝑡 exp = exp ― 4 𝑙2 𝜏 𝜋2 𝜋2 8

( )

(2)

where D is the diffusion coefficient of air in PDMS, C(x, t) is the air concentration inside the PDMS slab as a function of position (x) and time (t), C1 is the boundary air concentration, C0 = C(x, 0) is the initial concentration of air molecules inside the PDMS slab, and l is the thickness of the PDMS slab. 11 ACS Paragon Plus Environment


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At sufficiently large t values (> 4.3 min), the approximation in eqn. (2) remains valid because of the negligible error of m (< 1%) in our system28. Using eqn. (2), the time constant  = (4/)(l2/D) equal to 12.4 min was calculated by substituting the experimental values l = 2.5 mm and D = 3.4×105 cm2s1 (Ref.37). Its magnitude corresponds to m ~ 0.0064, indicating that more than 99% of the degassing process was completed within 1 h. This prediction agrees with our experimental results stating that the driving time and time dependence of the flow velocity were similar when the degas time was 1 h or greater. In contrast, the degas time of 10 min resulted in m ~ 0.362, which suggested that 36.2% of air molecules remained in the PDMS slab. Again, this result is in good agreement with the experimental data obtained at shorter and longer degas times. In addition, the rate of re-dissolution of air from the atmosphere into the evacuated PDMS slab was estimated using a one-dimensional model that compared the velocity attenuation factor with the theoretical parameter. The air flux F through the interface can be determined via the following equation:28 ∂𝐶(𝑥,𝑡) 𝐹=𝐷 ∂𝑥

|

≈

2𝐷(𝐶1 ― 𝐶0) 𝑙

𝑥=𝑙

(

exp ―

𝜋2𝐷 4𝑙

)

𝑡

2

(3)

For sufficiently large t (> 7.4 min), the approximation in eqn. (3) remains valid because of the negligible error of F (< 1%). At the time constant  = 12.4 min, the activity decreases by a factor of 0.44 after 10 min, whereas the experimental flow velocity decreases by a factor of 0.32 after the same period (between 10 min and 20 min; see Fig. 2b). Thus, the theoretical prediction was consistent with the experimental results. 12 ACS Paragon Plus Environment

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Furthermore, in order to evaluate how the volume and surface area of the outlet reservoir affect the flow velocity over time, the diameter and number of the outlet reservoir(s) and the thickness of the PDMS were varied (Table S-2). As the volume and surface area of the outlet reservoir(s) increased, the flow velocity also increased (Figs. S-4 and S-5). Similarly, as the thickness of the PDMS increased, the flow velocity also increased (Fig. S-6).

Bead enrichment in the single-input DLD device. Selective enrichment of 13 m beads in the population containing 7 m beads in the degas-driven single-input device was demonstrated. After 1 h of degassing, a 20 L droplet of the sample solution was injected into the device inlet, and the resulting liquid infusion process and trajectories of the 13 μm and 7 μm beads flowing through the DLD array were monitored. When the air-water interface advanced through the DLD array, some air bubbles were trapped between the DLD pillars (Fig. S-7a), which disturbed the bead trajectories (Fig. S-7b). However, the particles eventually circumvented these bubbles and returned onto their original paths. Moreover, we estimate that the flow field near the progressing air-water interface is not fully developed as Hagen-Poiseuille flow. Therefore, the conventional DLD models based on uniform flow might be inappropriate before the solution fills the device. After the complete removal of the trapped bubbles, the trajectories of the 13 μm and 7 μm beads flowing through the DLD pillars were observed (Fig. 3a), and their spatial distribution was determined (Fig. 3b). For better visualization, videos with various durations were recorded at different points of 13 ACS Paragon Plus Environment


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the elapsed time in the upstream, midstream, and downstream DLD regions (Fig. S-8). After the entrance of the 13 μm and 7 μm beads into the DLD array from the broadly distributed positions, the 13 μm beads flowed in the displacement mode and gradually drifted toward the sidewall, while the 7 μm beads flowed in the zigzag mode and retained their original positions with respect to the DLD array width, resulting in the enrichment of the 13 μm beads near the sidewall. Despite the gradual decrease in the flow velocity with time (Fig. S-5), both the 13 μm and 7 μm beads retained their trajectories in the displacement and zigzag modes, respectively. This observation is consistent with the fact that DLD separation is independent of the flow speed when the diffusion rate is negligibly small5. To evaluate the enrichment process during the entire operation, the total number of beads flowing through each DLD gap at the end of the DLD array was counted. It was found that more than 95% of the 13 μm beads were collected at GN = 19 (Figs. 4 and S-9, and Table S-3). The mean purity ± standard deviation (s.d.) calculated for the 13 µm beads at GN = 19 was 95.6±1.8% (n = 3) (Table S3). In this experiment, the purity of the 13 µm beads in the initial sample solution was around 70%, indicating that they were enriched by a factor of 1.37. A similar enrichment rate was obtained at a degas time of 10 min (Fig. S-10), although the corresponding operation time was shorter, and the processed volume was lower than that of the device outgassed for longer periods (such as 1 h). The processed sample volume was roughly determined by calculating the ratio of the number of particles in the originally loaded sample solution to the total number of the collected particles in the outlet reservoir. The number of particles in the original 20 µL droplet was approximately 4000, which 14 ACS Paragon Plus Environment

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was calculated from the concentration of the prepared sample solution (~2×105 particles/mL), and the total number of particles at the outlet was 780±80 (n = 3) (Figs. 4 and S-9). Hence, the processed sample volume was estimated to be about 3.9±0.4 µL.

Bead separation in the sheath-input DLD device. Separation experiments in the sheath-input device were performed by injecting the sample and sheath-fluid droplets into each input within 50 s after 1 h of degassing. Regardless of the loading order, the sample and sheath solutions did not start to flow until all of the three inlets were filled. A particle-containing 20 μL droplet was injected into the middle input, whereas two sheath-fluid droplets with volumes of 22 μL and 20 μL were infused into the side inlets connected to the wider and narrower channels, respectively, to balance the overall fluid consumption. After all inlets were filled, the injected solutions started to enter the input channels. The sheath solution flowing through the wider channel reached the DLD entrance much faster than the two other solutions because of its lower flow resistance. During this process, the sheath fluid interface spread throughout the DLD array and approached the two other input reservoirs. The void area between the sheath fluid and the two other solutions disappeared in about 3 min, after which the particles started to enter the DLD array as a sheath-focused stream. In the three experiments with the same device, the initial average inflow rates of all particles were 182±82 μm/s, 172±82 μm/s, and 140±71 μm/s. The large deviation in each experiment was presumably due to velocity profile in Hagen–Poiseuille flow. 15 ACS Paragon Plus Environment


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In addition, unlike the single-input device, the DLD array in the sheath-input device was already filled with the sheath fluid before the first particles entered it, which enabled their uninterrupted flow through the DLD array without taking a detour around trapped bubbles. Figure 5 shows the trajectories of the beads and their spatial distributions throughout the DLD array. After entering the array as a sheath-focused stream (GN = 6–12), the 13 μm beads started to shift toward one sidewall in the displacement mode, while the 7 μm beads continued flowing in the zigzag mode as a stream with a limited width (GN = 8–18). Thus, in the downstream region, both the 13 μm and 7 μm beads flowed toward two separate outlets, although a small number of green beads flowing together with the 13 m ones were observed, which could possibly consist of 7 m beads (Fig. S-11). While the flow rate gradually decreased with time, the trajectories of the beads did not change until the flow finally stopped in about 40 min. After the flow stopped, the numbers of the 13 μm and 7 μm beads collected at each outlet were counted by their colors (Fig. 6a). The capture efficiencies of the beads collected at outlet-L and outlet-S were 99.97±0.04% and 94.52±1.59% (n = 3) (Figs. 6b, c and Table S-4), whereas their corresponding purities were 92.62±2.46% and 99.98±0.03% (n = 3), respectively (Figs. 6d, S-12 and Table S-4). Next, it was found that the 7 μm beads collected at outlet-L represented various clusters such as doublets, triplets, and over-quadruplets (Figs. 7a, b). In order to estimate the trajectories of these clusters inside the DLD array, spatial distributions of their constituent beads were obtained at each outlet (Figs. 7c–f). At outlet-L, 7 μm clusters were distributed in different positions based on their 16 ACS Paragon Plus Environment

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morphologies (Fig. 7d). The triplet and over-quadruplet clusters were present together with 13 μm beads, while the doublet 7 μm beads were clearly separated from the 13 μm bead population (Figs. 7c, d). A few single 7 μm beads were also observed at outlet-L. Doublets of 7 μm beads were also detected at outlet-S, which were separated from the single 7 μm bead population (Figs. 7a, e, and f). The effective diameters of the triplet and over-quadruplet clusters (>15 m) were larger than the actual Dc values (8 μm < Dc < 10 μm), suggesting that these clusters migrated in the displacement mode and were collected at outlet-L near the sidewall. In contrast, the doublets of the 7 μm beads had a longer (~14 μm) and a shorter (~7 μm) axes rotating through the DLD array, which could cause their migration in the intermediate mode between the displacement and zigzag modes17, thus explaining their entry near the center of the bifurcating junction and final locations at the two outlets. The single 7 m beads collected at outlet-L were likely present due to the breakup of 7 m clusters or clusters containing both 7 m and 13 m beads. To increase the purity of the separation process, the formation of clusters must be prevented in advance. As this formation may be caused by the hydrophobic interactions of PS beads, optimal parameters of the surfactant (such as type and concentration) and particle suspensions with lower concentrations should be used. On the other hand, the observed separation of clusters with different morphologies suggests that the degas-driven DLD device is suitable for the fractionation of fragile aggregates because of its low shear stream. Similar to the single-input device, the volume of the processed sample solution was estimated. The 17 ACS Paragon Plus Environment


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20 L droplet of the sample with a concentration of around 2×106 particles/mL injected into the inlet contained 4×104 particles, while the total number of particles at the outlets was 2505±408 (n = 3) (Figs. 7 and S-9). The processed sample volume estimated from these values was equal to 1.3±0.2 µL. Further, the processed volume of the sheath fluid was determined by calculating the ratio of the bandwidths of the sheath-flow and sample streams. At the DLD entrance (Fig. 5a), this ratio was about 5.9, and the corresponding processed volume of the sheath fluid was 7.7±1.2 µL, which suggested that the total processed volume was 9.0 ± 1.4 µL. This magnitude was about twice greater than that obtained for the single-input device because the sheath-input device contained two outlets that promoted the aspiration of fluids with larger volumes.

Blood separation. The degas-driven DLD separation of WBCs from RBCs was demonstrated. After 1 h of degassing, 28 μL and 10 μL droplets of the sheath solution were loaded into the inlets connected to the wider and narrower channels, respectively. In addition, the 10 μL blood sample (Table S-5) was injected into the central inlet. After all inlets were filled, the solutions started to enter the channels. As was observed in the previous bead separation experiments, the sheath solution flowing through the wider input channel rapidly filled the DLD array, and after about 6 min, blood cells started to enter the DLD array while the voids remaining in the other two input channels finally disappeared. The initial inflow rate of the blood cells was 162±87 μm/s. Because the DLD region was pre-filled with the sheath fluid, blood cells could flow through the array without taking a detour around trapped bubbles. 18 ACS Paragon Plus Environment

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The blood components entered the DLD array in the form of a focused stream (Fig. 8). After their entrance, the blood components with sizes smaller than Dc started to move in the zigzag mode and nearly retained their original stream position despite the observed increase in the stream width (Fig. 8a). This expansion of the blood stream might be due to non-deterministic particle collision in the nondiluted sample stream38. Fluorescent microscopy observations showed that WBCs shifted their positions towards the sidewall in the displacement mode (Fig. 8b). Unlike previous bead studies, it was found that some WBCs were not fully displaced through the DLD pillars because the WBCs with sizes close to Dc flowed through the DLD array in a mixed motion17. Thus, WBCs were separated continuously for about 40 min until the degas-driven flow finally stopped. After the flow stopped, the numbers of WBCs collected at each outlet were counted by their fluorescent label, and the numbers of RBCs collected at outlet-L were counted according to their shape (Fig. 9a). The purity and capture efficiency of the WBCs collected at outlet-L were 86.97±6.62% and 95.57±1.65%, respectively (Figs. 9b, c and Table S-6). Thus, the purity of WBCs was increased by approximately 681 times as compared to that of the original blood sample. Two possible reasons for the contamination of the separated WBCs by RBCs exist: 1) a small number of RBCs flowing near the bifurcating junction were collected at outlet-L; and 2) the aggregated RBCs with diameters larger than Dc, flowed in the displacement mode and were collected at outlet-L. Both these issues can be solved by using a diluted blood sample. Meanwhile, we found a large variation in the number of processed WBCs between the three experiments with the same device (Table S-6). This difference might be 19 ACS Paragon Plus Environment


Page 20 of 34

caused by the settling of WBCs in inlet reservoir or the variation of the surface affinity and fluidic resistance in the PDMS device. The degas-driven DLD has some advantages over its counterparts. Unlike capillary-driven DLD19,20, the channels used for the infusion of aqueous solution do not need to be hydrophilic. Similarly, unlike gravity-driven DLD18, this method does not require the use of a complex setup, and the particle density should not be necessarily higher than that of the medium. Thus, the degas-driven DLD can be potentially used in various POC testing procedures such counting CD4+ T-cells to determine the current stage of human immune deficiency virus39 and separation of plasma from blood cells for the enhancement of sensitivity and reliability of blood plasma analysis24,30. Furthermore, a portable degasdriven DLD device can be prepared by storing a degassed device in an airtight package, which is suitable for on-site POC diagnostics and environmental monitoring without a bulky pump.

CONCLUSION In this work, PDMS-based degas-driven DLD microfluidic devices were fabricated. When a droplet of the sample solution was injected into the inlet of the pre-degassed device, the negative pressure induced by air absorption into the PDMS walls made the solution flow into the confined DLD channel. After that, the solution particles were continuously enriched and separated based on their sizes inside the DLD array until the air pressure reached equilibrium and the fluid flow finally stopped. In addition to the fluorescent PS beads, separation of WBCs from RBCs at a high capture efficiency and purity 20 ACS Paragon Plus Environment

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was demonstrated. We believe that the fabricated portable and pump-free DLD devices have many potential applications including POC testing and on-site environmental monitoring.

Supporting Information. Adjustment of DLD parameters. Effect of PDMS thickness, and the volume and surface area of the outlet reservoirs on flow velocity. Geometry and parameters of the DLD devices. Summarized results of enrichment and separation experiments.

ACKNOWLEDGEMENTS This work was supported by JSPS KAKENHI Grant Numbers 17J10441.



Page 22 of 34

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Page 25 of 34

(a) Liquid Degassed PDMS Air absorption DLD array

(b) Inlet

Liquid flow Outlet

(i)

Lid (d)

19

λ

2.5 mm 4 mm

40 μm

Glass Δλ

1

(c)

PDMS

Dp λ d

1 mm

(i)

Gap number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Sheath fluid

Outlet

DLD array

Sample

Sheath fluid

13 μm (displacement mode) 7 μm (zigzag mode)

1 mm

L

Lid

S

Lid

Fig. 1. Degas-driven microfluidic DLD devices. Schematic illustrations of the (a) device concept and the (b) device for enrichment of particles. The dashed rectangle denotes a magnified view, and each DLD gap is numbered from 1 to 19. (c) Schematic illustration of the device for the separation of particles. (d) A rhombic unit cell of the DLD array with the post diameter Dp, center-to-center distance between the neighboring posts λ, specific distance Δλ shifted for every row, and gap width d = λ Dp.



(a)

FLOW

Velocity [µm/s] (b) 0 2032 1000

100 μm

Velocity [µm/s]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 34

10 min 1h 3h 10 h

500 0 0

10 20 Time [min]

30

Fig. 2. Effect of the degas time on the flow velocity in the DLD array. (a) Particle tracking performed during flow velocity measurements. The degas time was 1 h, and the degas pressure was around 5 Pa. (b) Variations of flow velocity with time observed at degas times of 10 min, 1 h, 3 h, and 10 h.


Page 27 of 34

(a)

Upstream

FLOW

Midstream

Downstream

13 μm 7 μm

100 μm

10 1 0

19

13 μm (n = 14) 7 μm (n = 28)

10

50 Fraction [%]

100

1 0

50 Fraction [%]

19

Gap number

13 μm (n = 182) 7 μm (n = 75)

Gap number

(b) 19

Gap number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


100

10 1 0

13 μm (n = 79) 7 μm (n = 64)

50 Fraction [%]

100

Fig. 3. Degas-driven enrichment of particles flowing through the DLD array. (a) Trajectories of 13 µm (yellow) and 7 µm (green) beads flowing in the upstream, midstream, and downstream DLD regions, which demonstrate selective enrichment of the 13 µm beads near the sidewall. The white dashed lines denote the sidewalls. (b) Spatial distributions of the flowing beads.



(a) 19

(b)

Gap number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 34

10

1 0

13 μm (n = 443) 7 μm (n = 226)

50 Fraction [%]

100

50 μm

Fig. 4. Selective enrichment of particles. (a) Spatial distributions showing the enriched 13 m beads near one sidewall (GN =19). (b) Bright-field (BF, left) and fluorescent (FL, right) microscopy images of the particles in the outlet reservoir.


Page 29 of 34

(a)

Upstream

FLOW

Midstream

Downstream

13 μm 7 μm

13 μm 7 μm

100 μm 13 μm (n = 173) 7 μm (n = 166)

14 1 0

40

27

27

13 μm (n = 22) 7 μm (n = 68)

14

25 Fraction [%]

50

1 0

Gap number

27

40

Gap number

(b) 40

Gap number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


25 Fraction [%]

50

14 1 0

13 μm (n = 70) 7 μm (n = 78)

25 Fraction [%]

50

Fig. 5. Degas-driven DLD separation of particles with different sizes. (a) Trajectories illustrating the separation of the 13 µm (yellow) and 7 µm (green) beads, which were obtained by recording 100 s videos in the upstream, downstream, and midstream regions. The white dashed lines denote the sidewalls. (b) Spatial distributions of the flowing beads.



(b)

Outlet-L

Outlet

(a)

L

Outlet

10 μm Outlet-S

S 0

13 μm 7 μm

5.48%

S

(d) L

10 μm

99.97%

S 0.03%

(c) L

Outlet

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 34

94.52% 92.62% 7.38%

13 μm 7 μm

0.02% 99.98%

50 Fraction [%]

100

Fig. 6. Separation of particles with different sizes. (a) BF (left) and FL (right) images of the beads collected at the two outlets. (b) Capture efficiency of the 13 m beads. (c) Capture efficiency of the 7 m beads. (d) Fractions of the 13 m and 7 m beads determined for each outlet. The error bars indicate standard deviations (n = 3).


Page 31 of 34

(a)

Outlet (i)

L 0

(ii) (iii)

x

x

(iii)

0

S (b)

100 μm (ii) Triplet

(i) Quadruplet

(iii) Doublet Outlet-L

Outlet-S 10 μm

x

0 -1

0

x

0 -1

1

0 x [mm]

(f) 0.8

7 μm (n = 985)

S

15

1

7 μm (n = 985) Doublet Single

0.4

(f)

0 -1

Doublet Single

15

0 x [mm]

(e) 30

7 μm (n = 62) > Quadruplet Triplet

Fraction [%]

0

30

Fraction [%]

13 μm (n = 896) 7 μm (n = 62)

L

15

(d)

Channel width

30

Fraction [%]

(c)

Fraction [%]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 x [mm]

0 -1

1

0 x [mm]

1

Fig. 7. Spatial distribution of the aggregated 7 µm beads. (a) BF and FL images of the beads collected at the two outlets. (b) Clusters composed of different numbers of 7 m beads. (c–d) Spatial distributions of the (c) 13 µm and 7 µm beads and (d) clusters containing 7 µm beads at outlet-L. (e– f) Spatial distributions of the (e) 13 µm and 7 µm beads and (f) aggregated 7 µm beads at outlet-S.



(a)

Upstream

Midstream

Page 32 of 34

Downstream

FLOW

RBCs 100 μm

(b)

WBCs

100 μm

Fig. 8. Degas-driven DLD separation of RBCs and WBCs. (a) BF microscopy images of the blood cells flowing through the DLD array. (b) Superimposed FL images showing the trajectories of WBCs (light blue lines) flowing in the displacement mode. The area between the two white dotted lines represents the location where the entire blood sample entered the DLD region as a sheath-focused stream.


Page 33 of 34

(a)

Outlet-L

Outlet-S RBCs

WBCs 10 μm 86.97% 13.03%

0

10 μm (c) L

WBCs RBCs

50 Fraction [%]

Outlet

(b)

Outlet-L

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


S

100

95.57% 4.43%

0

WBCs

50 Fraction [%]

100

Fig. 9. Separation of WBCs and RBCs. (a) BF (left) and FL (right) images of the separated WBCs and RBCs at the two outlets. (b) Fractions of the WBCs and RBCs collected at outlet-L. (c) Fraction of the WBCs collected at outlet-L and outlet-S. The error bars indicate standard deviations (n = 3).



Page 34 of 34

Air absorption Liquid flow

Degas-driven DLD device

Liquid

Degassed PDMS

Upstream FLOW

Midstream

Downstream

13 μm 7 μm

100 μm

Table of contents (TOC)


Degas-Driven Deterministic Lateral Displacement in Poly

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