Mediating Millisecond Reaction Time around Particles and Cells

Jan 7, 2014 - Mediating Millisecond Reaction Time around Particles and Cells. Jaideep S. Dudani,. †,⊥. Derek E. Go,. †. Daniel R. Gossett,. †,...
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Mediating Millisecond Reaction Time around Particles and Cells Jaideep S. Dudani,†,⊥ Derek E. Go,† Daniel R. Gossett,†,‡,¶ Andrew P. Tan,† and Dino Di Carlo*,†,‡,§ †

Department of Bioengineering, University of California Los Angeles, 420 Westwood Plaza, 5121 Engineering V, Box 951600, Los Angeles, California 90095, United States ‡ California NanoSystems Institute, Los Angeles, California 90095, United States § Jonsson Comprehensive Cancer Center, Los Angeles, California 90095, United States S Supporting Information *

ABSTRACT: Precise spatiotemporal control of how particles and cells interact with reagents is critical for numerous laboratory and industrial processes. Novel tools for exerting this control at shorter time scales will enable development of new chemical processes and biomedical assays. Previously, we have developed a generalized approach to manipulate cells and particles across fluid streams termed rapid inertial solution exchange (RInSE), which utilizes inertial lift forces at finite Reynolds number and high Peclet number to transfer particles from an initial solution to another within a millisecond. Here, we apply these principles toward developing a continuous flow microfluidic platform that enables transient chemical treatments of cells and particles (on the order of 1 ms). We also demonstrate how the reactant stream can be employed as a diffusion barrier, preventing adverse reactions between coflowing solutions. In order to demonstrate the utility of the method, we applied it to various operations in molecular biology and automated cell staining including cell permeabilization, fluorescent staining, and molecular delivery to viable cells. We expect this method will enable previously unexplored studies of the dynamics of molecular events, improve uniformity of reactions carried on the surface of beads, and increase uniformity in cell-based assays through automation.

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low complexity and integration in fabrication, (iii) independence from molecular labels, (iv) and control of reaction initiation and termination for every cell or particle. A method that meets these demands will enable automation of a wide variety of laboratory operations and novel techniques for molecular and cellular biology. One operation that will benefit from rapid solution exchange is chemical poration for molecular delivery, as lack of temporal control of poration agents can lead to either low efficiency or high levels of cell death, making alternate methods of delivery a stronger option.21 Viral methods of molecular delivery are limited due to the potential for immune responses and toxicity.22 Electroporation lacks precise control and uniformity. Despite advances in microfluidic electroporation that promise increased uniformity, throughput and the ability to continuously operate are still limited.3,23−28 An ideal tool for poration should operate continuously and work inline with other cell preparation methods for robust processing and operation. As such, a chemical method inline with cell preparation will provide a valuable alternative to current poration methods by enabling operations to be coupled with any number of inline systems.

emporal regulation is an important aspect of biological and chemical reactions. Miniaturized systems enable precise temporal reaction control, with methods such as creation of droplet microreactors1 or through chaotic or diffusive mixing of reagents.2 In biology, these systems have been devised to perform chemical treatments of cells for molecular delivery,3 sample preparation,4−9 and study of molecular events.10,11 However, current approaches for chemical treatments of cells and chemical reactions possess several limitations. They typically operate on slow time scales (∼seconds to minutes),12,13 limiting the realm of fast molecular events that can be studied. Specifically, in the realm of cellular biology, many processes occur at time scales not accessible by existing methods. Automation with robotics for fluid and particle handling is not universally available, and these tools are also limited in temporal resolution. A divergent approach has been to develop miniaturized systems for precise particle and fluid control. For example, external force fields (dielectric, acoustic, or magnetic) have been used to transfer particles between solutions at high Peclet number (when convection dominates over diffusion)14−17 or across fluids separated by an immiscible layer.18−20 These methods not only mix particles into a new solution but also allow complete and repeatable solution exchange. However, to our knowledge, not one of these methods provides all of the key features for robust, uniform, rapid, and commercially scalable treatment of cells for timesensitive operations: (i) treatments on the scale of 1 ms, (ii) © 2014 American Chemical Society

Received: September 12, 2013 Accepted: January 7, 2014 Published: January 7, 2014 1502

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Figure 1. Method for inertial lift-mediated rapid solution exchange and short exposures of cells and particles to reactants. (A) The microchannel design is drawn in black with three inlets and two outlets. (B) The main transfer channel is a low aspect ratio wide rectangular channel, which results in a blunted velocity profile in the xy plane. This confines the shear gradient lift force to near-wall regions and results in stable equilibrium positions in the center of the wide face of the channel. (C) Device operation is depicted. Cells rapidly transfer across Solution 2 enabling extremely fast incubations. An input code describes the solutions and the flow conditions within the channel. The relative width of each flowing stream is approximately proportional to the relative flow rate. Inset green box: In the transfer channels, particles migrate more quickly in the z direction to one of the two stable z equilibrium heights and then more slowly laterally at that z height to the channel centerline. Inset orange box: This results in two equilibrium positions in the center of the channel. (D) High-speed image stacks of device operation depict a single microparticle transferring from Solution 1 to Solution 3. Boxes around each micrograph correspond to colored boxes in (A). Dotted lines indicate Solution 2. Scale bars = 500 μm.

demonstrate the application of this strategy in several areas including chemical poration, segregation of reactive reagents, and labeling or staining cells for analysis, and we elucidate mechanisms for controlling incubation time.

An obstacle to implementing microfluidic strategies for solution exchange is the potential for adverse reactions (e.g., precipitation or crystallization) between solutions in close proximity (e.g., coflows or adjacent reservoirs). A separating or mediating solution can be an important component of a microfluidic coflow, and the high Peclet numbers, easily achievable in microfluidic systems, preclude mixing on the relevant time scale. Recently, we reported the use of inertial lift forces, present in particle-laden high-speed confined flows, to perform submillisecond solution exchange around flowing cells and particles (termed “rapid inertial solution exchange” or RInSE).4 Inertial lift forces are intrinsic to these flows, requiring no integration with external force fields or biochemical labeling, and can be controlled purely by manipulating microchannel geometry and flow conditions within the channel. Typically, for inertial lift forces to substantially affect particle position in centimeter-long microchannels, the ratio of particle diameter to channel dimensions must be approximately 0.07 or larger (as the inertial lift force away from walls scales as FL ∝ a6/H4, where a is particle diameter and H is channel height).29 The high speed of these flows (mL/min and m/s) affords high throughput (>102 cells/s). In this work, we apply this principle to achieve not only solution exchange around particles and cells but also ultrashort exposures of particles and cells to a solution on the scale of a few milliseconds. This platform, termed Dip Chip, consists of three solutions meeting to form a laminar coflow. Inertial lift forces transfer particles from an initial suspension across another stream, in which they reside for several milliseconds and finally into a third solution, which is collected. We

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EXPERIMENTAL SECTION

Numerical Simulation. Finite element method analysis was performed using COMSOL Multiphysics (Comsol, Inc., Burlington, MA, USA) to solve the full Navier−Stokes equations for a segment of the microchannel containing the joining of the three solution inlets. Inlet conditions were normal and inflow velocity of 0.2 m/s for the cell solution (Q1, W = 50 μm, H = 30 μm), 0.1 m/s for the incubating solution flow (Q2, W = 30 μm, H = 30 μm), and 0.8 m/s for the final receiving solution (Q3, W = 50 μm, H = 30 μm). The outlet boundary condition was set to pressure, no viscous stress at 0 Pa. No slip boundary conditions were used for the remaining walls. The simulation was performed in 3D, and velocity heat maps and velocity profiles were generated for the xy plane at z = 15 μm and yz plane at x = 100 μm using postprocessing tools as seen in Supporting Information Figure 1. The transfer channel was 100 μm wide, 300 μm long, and 30 μm tall. The simulation was solved for 15 700 degrees of freedom. The viscosity and density of the fluid are that of water. This yielded a Reynolds number of ∼25 in the main channel. Microfluidic Device Fabrication. Microfluidic devices were fabricated using standard photolithographic and replica molding methods.30 Briefly, the negative photoresist, KMPR (MicroChem Corp., Newton, MA, USA), was cast on mechanical grade silicon wafers. KMPR was exposed through a transparency mask (CAD/Art Services, Inc., Bandon, OR, 1503

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USA) and developed as prescribed. Four mm of polydimethylsiloxane (PDMS; Sylgard 184 Silicone Elastomer Kit; Dow Corning Corp., Midland, MI, USA) was cured on the masters at 65 °C overnight. Cured PDMS was peeled off the wafers, exposed to air plasma, and bonded to glass to create the final device. Through-holes were bored at inlet and outlet positions using a pin vice assembly. Polyetheretherketone (PEEK) tubing (ID 0.02 in., OD 1/32 in.) was inserted into through-holes forming interference fits. Device Operation and Measurement of Performance Metrics. To test for successful particle transfer, 19 μm polystyrene particles (Thermo Fisher Scientific Inc., Waltham, MA, USA) were diluted in phosphate buffered saline (PBS) and pumped into the Dip Chip at Q1 = 30 μL/min, while Trypan Blue (Thermo Fisher Scientific Inc., Waltham, MA, USA) was pumped at Q2 = 15 μL/min and PBS at Q3 = 120 μL/min (Figure 1). All solutions were loaded into 3 mL syringes, which were capped with luer stubs and connected to PEEK tubing via interference fits. Flow rate was controlled via syringe pumps (PHD 2000 syringe pumps, Harvard Apparatus, Holliston, MA, USA). The beads in Solution 1 were transferred across the Trypan Blue coflow into the second coflowing solution of PBS. The purity of the solution receiving the beads post-transfer was measured using a spectrophotometer, taking absorbance readings of the collection stream at λ = 607 nm. A standard curve of absorbance measurements of Trypan Blue concentrations in PBS was used to quantify the purity. The collection efficiency was calculated by observation of video of the outlet trifurcation, recorded using a high-speed camera (Phantom v711, Vision Research Inc., Wayne, NJ, USA) connected to a Nikon Ti-E inverted microscope. Collection efficiency was defined as 100 × collected particles/total particles observed. The same measurement was performed with HeLa cells in Solution 1. Maintenance of Cell Lines. The HeLa cell line was cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1% pencillin−streptomycin (P/S; Invitrogen Corp., Carlsbad, MA, USA). The MCF7 cell line was cultured in DMEM supplemented with L-glutamine, 4.5 g/L glucose, and sodium pyruvate, with 10% FBS and 1% P/S. HeLa and MCF7 cells were passaged when confluence reached between 70% and 100% with 0.25% porcine trypsin (Thermo Fisher Scientific Inc., Waltham, MA, USA). Jurkat cells were maintained in Roswell Park Memorial Institute (RPMI) Media 1640 with 10% FBS and 1% P/S. Cells were resuspended in fresh media every 3 to 4 days. Measuring Incubation Times. Particle and cell suspensions were prepared as described above. High-speed microscopic videos were captured at the inlet junction, and time taken for cells or beads to transfer across the Trypan Blue stream was measured using either a 10× or 6× magnification at a frame rate, depending on the resolution, ranging from 7532 to 56151 frames per second with an exposure time of 0.29 μs (see Figure 2 and Supporting Information Figure 2). Separation of Reactive Solutions. Polystyrene beads were suspended in Mayer’s hematoxylin (Sigma Aldrich, St. Louis, MO, USA), which contains aluminum ions that form a chelate around the hematoxylin. In the Dip Chip, a coflow of acetic acid was used to separate the hematoxylin solution from the sodium carbonate solution. Importantly, the system was first primed with acetic acid before flowing the other solutions (Figure 3A). These solutions were used for this demonstration

Figure 2. Tunable, rapid incubations. (A) Beads and cells reside in Solution 2 for only a few milliseconds. HeLa cells migrate slower than beads due to their smaller size and increased deformability, resulting in longer exposures. (B) Modifying the flow rate of the incubation solution changes its thickness, increasing the Peclet number and therefore limiting interactions between Solutions 1 and 3 through diffusion. The flow rates of Q1 and Q3 were held constant at 30 and 120 μL/min. (C) Incubation times can be tuned with the flow conditions. Incubation time can be varied from approximately 1 ms to approximately 5 ms. The arrow indicates that under the last condition all particles still resided in the incubation solution within the imaging frame.

due to their applications in automated cellular staining for cytopathology and hematology. Chemical Permeabilization with Triton-X 100. Triton-X 100 (Sigma Aldrich, St. Louis, MO, USA) was prepared at a 2% w/v in PBS. Jurkat cells were transferred through the Triton-X 100 stream into a solution of diluted Trypan Blue (10% v/v in PBS) using the Dip Chip. Trypan Blue is membrane impermeable; therefore, it stains membrane-compromised cells. Collected cells in the Trypan Blue solution were imaged with a color Go-3 CMOS Camera, using QCapture software (QImaging; Figure 3B). Staining of Cytokeratin. MCF7 cells were harvested from culture using standard tissue culture techniques. Cells were fixed with 4% formaldehyde/PBS for 10 min. Using the Dip Chip, cells were transferred through 2% Triton-X 100 into a solution of phycoerythrin−anticytokeratin (Becton Dickinson, Franklin Lakes, NJ, USA) in PBS. Cells were maintained in the anticytokeratin solution for 1 h. For comparison, a similar operation was carried out manually: after fixation, cells were incubated in 0.2% Triton-X 100 for 7 min. They were then 1504

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Figure 3. Automated processes are enabled by the Dip Chip. (A) High Peclet number barrier between two reactive solutions. (i) Increasing the width of the middle solution (Solution 2) prevents interaction between Solutions 1 and 3, which react to form an insoluble salt (see Supporting Information Figure 3). (ii) A lower Peclet number allows diffusion across Solution 2 forming salt precipitates at the interface and preventing particle transfer (indicated by black arrows). Scale bar = 250 μm. (B) Rapid on-chip chemical permeabilization is achieved by using Triton-X 100 as the incubation solution. (i) High-speed image stack of a single cell transferring across Triton-X 100 (white solution) into Trypan Blue, dye excluded by viable cell membranes (dark solution). Scale bar = 50 μm. (ii) Jurkat cells in-suspension were successfully permeabilized. (C) Automated cellular staining of fixed MCF7 cells. (i) On-chip permeabilization of cells using Triton-X 100 and on-chip introduction of fluorescent anticytokeratin antibodies and comparison with manual permeabilization and addition of fluorescent markers. Look up tables (LUTs) were autoscaled to enhance contrast. Scale bar = 10 μm. (ii) Cytokeratin fluorescent intensity was calculated for two operations of the Dip Chip and two manual runs. A student’s t test was performed between the two Dip Chip runs and the two manual runs. The resulting fluorescence levels were more similar for the automated process than the manual process. Automated staining promises increased uniformity between runs as opposed to manual operations, which might be valuable for quantitative measurements.

of molecules to cells, the same device was used and continuously operated while changing the Solution 2 (saponin) flow rate and collecting new fractions. The device was allowed to run for a minute before collecting cells after any change in flow rate. This time is sufficient to fully replace the volume in the outlet tubing of approximately 8 in. in length (total volume in tubing = 41.19 μL). To measure the time scale of pore closing, MCF7 cells were transferred across a stream of saponin into culture media and then delivered to a well plate. Ten minutes after plating, ethidium homodimer-1 and calcein AM were added to the same final concentrations as above.

resuspended in anticytokeratin solution and also incubated for an hour. During this hour incubation, both manually and Dip Chip processed cells were stained with DAPI (4′,6-diamidino2-phenylindole, Invitrogen, Corp., Carlsbad, CA, USA) at a final concentration of 1 μg/mL for 5 min. Stained cells were imaged using the DAPI and TRITC fluorescent channels of the Nikon TI-E inverted microscope (Nikon Japan) and a Coolsnap HQ2 camera (Photometrics, Tucson, AZ, USA). The highest fluorescent intensity in the TRITC channel (cytokeratin) in each cell was quantified using ImageJ (U.S. National Institutes of Health, Bethesda, Maryland, USA). A Student’s t test for difference in means was performed to characterize similarity between staining runs (Figure 3C). Delivery of Molecules to Viable Cells. Saponin (Sigma Aldrich, St. Louis, MO, USA) was prepared 1% w/v in PBS as the porating chemical. Cells in PBS (Solution 1) were transferred through the saponin stream (Solution 2) into a stream of ethidium homodimer-1 (Solution 3, 1.7 μM) and collected (Figure 4). After collection, calcein AM was added to the cells to a final concentration of 1.0 μM. Cells were imaged after 15 min of staining. Look up tables (LUTs) and exposure times were adjusted to enhance image contrast for merged images describing coincidence of Ethd-1 and calcein AM to indicate viable delivery to cytosol. For all images used in quantitative comparisons, exposure time, gain, and LUTs were identical. In quantifying the ability to deliver varying amounts

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RESULTS AND DISCUSSION Design and Operating Principles of the Dip Chip. The Dip Chip is designed on the basis of principles of rapid inertial solution exchange (RInSE), which involves transfer of cells and particles across laminar streams by wall effect inertial lift forces. In order to achieve the desired lift forces for rapid transfer of cells and particles between layers of a coflow, we used rectangular channels with three coflows occupying different regions of the wide dimension of a low aspect ratio transfer channel in a high Reynolds and high Peclet number system (Figure 1A,B). This device has three inlets to deliver a cell or particle suspension (Solution 1), a solution in which to rapidly incubate these particles or serve as a reaction barrier (Solution 2), and a final receiving solution (Solution 3) (Figure 1A). The 1505

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Figure 4. Rapid incubations enable molecular delivery to viable cells. (A) Illustration of molecular delivery assay. Viable cells have an intact membrane that prevents ethidium homodimer 1 (Ethd-1) from entering and becoming fluorescent red upon binding to nucleic acids. Calcein AM is membrane permeable and cleaved by esterases to become fluorescently excitable calcein (emitting green) and membrane impermeable. A viably porated cell will have available esterases to cleave calcein AM as well as a porated plasma membrane, allowing Ethd-1 to enter. (B) Optimization of molecular delivery to Jurkat cells: cells were run through the device with varying incubation times in saponin (as modified by Q2). A low flow rate of saponin prevents any poration (no cells positive for both calcein and Ethd-1) whereas a high flow rate (long incubation) leads to cell death (no calcein localized to cells). An intermediate incubation time results in molecular delivery and viability. White arrows indicate cells staining with both dyes (colocalized). The Ethd-1 positive cells in the first image are expected to have been carried over from culture; since Jurkat cells are cultured insuspension, adherence of viable cells to the culture vessel cannot be used to remove dead cells in advance of Dip Chip processing. Scale bar = 200 μm. (C) 40× magnification of Jurkat cells stained positive for both Ethd-1 and calcein. Scale bar = 10 μm. (D) Fluorescent molecules can also be delivered to other cell types. Scale bar = 100 μm. (E) Quantification of molecular delivery and viability. (F) Correlation of poration efficiency with diameter. Larger cells traverse the saponin stream faster, decreasing the amount of porating time. (G) Optimization of molecular delivery to M398 cells: tuning the flow rate, we identified an optimal saponin flow rate of 20 μL/min resulting in 75% poration efficiency. Images are overlays of calcein and Ethd-1. Scale bar = 100 μm. Look up tables were autoscaled to improve contrast.

channels containing the first and second solutions bifurcate after passing through size exclusion filters, parallelizing the system in order to increase throughput. The first meeting of solutions occurs between Solutions 2 and 3 where a stable coflow is established (Figure 1A,D, dark red box). Next, Solution 1, the particle suspension, is joined to these flows (Figure 1A,D, green box). Prior to this junction, the channel containing the suspension is equipped with a series of asymmetric curves, which are designed to rapidly focus cells and particles using a combination of inertial lift forces and secondary Dean flow.31 In the main transfer channel, the Reynolds number (Re = ρUDH/μ) is 42.3, where ρ is fluid density of 1000 kg/m3, U (U = Qin/A) is the mean fluid velocity of 0.92 m/s, DH is the hydraulic diameter calculated as 2WH/(W + H) for a channel width of 100 μm and a channel height of 30 μm, and μ is dynamic viscosity of 0.001 Pa·s. After 1 cm, the channel splits with two outer branches meeting again

to form a waste outlet and the central channel leading to a separate outlet that collects the particles in the final solution (Figure 1D, pink box). The input code depicted in Figure 1C describes the volumetric flow rate in the Dip Chip with the relative width of each section approximately representing the relative flow rate. An important aspect of this technology is the simple modulation and tunability afforded by changing the solutions and flow rates to achieve separate outcomes. The main channel, where the inertial transfer and exposure of cells to chemicals occurs, is designed to be a low aspect ratio (W > H) rectangular channel (Figure 1B) in order to direct cells and particles to the channel centerline. As explored previously,4 particles migrate to two inertial equilibrium positions at the center of the long faces of such a channel. In our design, this results in particle migration to the center of the channel as depicted in Figure 1B when viewing the channel through the wide face. This behavior can be explained through 1506

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compared to analytical solutions.4,33,35 We can define the transfer time across the incubation stream, τ, as τ = w/Up, where w is the incubation stream width. w is related to the ratio of flow rates (w/W = Qratio = Q2/Qtotal). Accordingly, we can control the transfer time by modulating several parameters as τ scales proportionally to Qratio, 1/a5, and 1/U2. Note that over a small range of Reynolds numbers fc remains relatively constant. The migration time scales with both the square of the mean flow velocity and with the relative flow rate of the middle solution. However, since the middle solution velocity contributes only slightly to the combined downstream velocity of the coflowing streams, changing this flow rate leads to a larger effect on migration time based on an increase in the stream thickness compared to the effect of the migration velocity. Experimental measurements of the incubation time for both cells and particles with a constant flow in Solution 2 at Q2 = 15 μL/min qualitatively agree with theoretical predictions (Figure 2A). Definitions of incubation start time and end time are provided in Figure 1C. As predicted, the incubation time is size dependent because the magnitude of the inertial lift force scales strongly with particle size. Larger beads reside in the middle solution for a shorter time than smaller beads (Supporting Information Figure 2). As expected, beads also reside in the middle stream for a shorter time than cells (due to deformability differences).34 Nonetheless, incubation times on the order of a few milliseconds are achieved for both types of particles. Next, we demonstrate the range of incubation times that can be achieved by varying the ratio of the three flow rates over a wide range while operating at the same Reynolds number. Increasing the thickness of the incubation stream (w) by increasing Q2/Qtotal (Figure 2B) is expected to result in a longer incubation time. Increasing Solution 2 thickness (with Q2) is observed to increase the incubation time more significantly from 1.4 to >5 ms especially for lower relative Q1 flow rates (Figure 2C). With a lower Q1, presumably the particle can enter the incubation stream early and wall effect lift force dominates only after the new steady flow profile has been established. Prior to a blunted profile being established, the shear gradient lift force holding the particle in its initial entry position and acting toward the wall will contribute to lower the net force toward the channel center. Additionally, as described above, the initial migration due to inertial lift will occur along the zdirection. Therefore, if the incubation stream is contacted prior to the z-direction motion (when Q1 is small) being completed, the incubation time will be longer (as first the particles will travel in the z-direction and then begin migration across the incubation stream). Therefore, incubation time appears to be quite strongly influenced by the initial contact position between the particle and incubation solution. Accordingly, in the case where Q1 dominates, the particles encounter the middle solution further downstream. This enables the rapid transit of particles across Solution 2 for the reasons mentioned above. Increasing Solution 2 thickness while Q1 is high enables further expansion of the incubation time, but not an exceedingly high variation is achieved under these conditions (1.1 to 1.4 ms). Across six conditions, we were able to achieve an incubation time ranging from 1.16 ms to greater than 5 ms (Figure 2C). For the condition with greater than 5 ms exposure, our measurements were limited by the field of view of the camera, as all beads were still present in the middle solution at the edge of our imaging frame.

an understanding of how the channel geometry defines the shape of the velocity field and how this velocity field defines the lift forces acting on the particles. Two dominant lift forces act on neutrally buoyant particles: the shear gradient lift, which is directed down shear rate gradients, and the wall effect lift, which is directed away from the channel wall. The shear gradient lift force is proportional to the product of shear gradient and shear rate whereas the wall effect is proportional to the square of the shear rate. The blunted parabolic velocity profile, characteristic of low aspect ratio rectangular microfluidic channels and critical for inertial transfer across laminar streamlines, develops approximately 100 μm downstream of the joining of microfluidic flows for the designed geometry (Supporting Information Figure 1). As the particles enter the transfer channel and, shortly thereafter, following the development of the blunted velocity profile, they experience a weak shear gradient lift force and a dominant wall effect lift force. The wall effect lift forces from opposing walls balance in the ydirection at the center of the wide face of the channel. There, however, remain large shear gradients in the z-axis, which results in the formation of two stable equilibrium positions along the z-axis (Figure 1B). Therefore, the strongest lift forces act in the z-direction causing first out-of-plane motion and then lateral migration toward the channel center (Figure 1C, inset). This has previously been confirmed numerically and experimentally.4 Accordingly, within the main channel, cells and particles migrate to the center of the wide face of the microchannel (Figure 1C, purple box). Characterization of the Dip Chip Efficiency and Speed. The collection efficiency (particles collected/total particles observed) for both polystyrene beads (diameter, a = 19 μm) and HeLa cells (a = 15 μm) were observed at the outlet junction under optimal flow conditions. Bead collection efficiency was 100%, and cell collection efficiency was 81.4%. This can be explained on the basis of decreased size for a subpopulation of cells compared to the polystyrene beads as inertial lift forces scale with particle diameter.32,33 Additionally, cells are more deformable than the hard polystyrene particles, which results in focusing closer to channel centerlines in the zdimension, away from the largest y-direction lift forces near the walls.34 Final solution purity from the collect channel was found to be 98.5%. This was measured by flowing a dye as Solution 2 (Trypan Blue) and measuring contamination of Solution 3 by the dye in the collect outlet via absorbance measurements. Cells and particles travel rapidly across Solution 2 enabling short incubation times. An understanding of the variables that affect the lateral migration velocity is critical to tuning incubation times. For example, one cannot expect a linear increase in incubation time due to a linear increase in the flow rate of the middle solution as the particle migration velocity, UP, is dependent on the mean flow velocity, U, in a complex manner, and incubation time is related to (i) the particle migration velocity (Up), (ii) the solution layer thickness, and (iii) the initial point of contact of the particle with the incubation solution. The particle migration velocity, assuming Stokes drag, is defined by Up = [ρU2a5/3πμH4]fc(Rec, yc), where fc(Rec, yc) is a lift coefficient that is dependent on channel Reynolds number (Rec), the velocity field shape, and particle position in the channel cross-section (yc).29 Our previous work demonstrated that particle migration in low aspect ratio rectangular channels was dominated by the wall effect lift force, yielding the modified scaling with a higher power of a/H 1507

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Demonstration of Separation of Reactive Streams in the Dip Chip. The Dip Chip is a convection-dominated system that has the ability to separate reactive streams with an intermediate solution. By increasing the thickness of the middle stream by increasing Q2, diffusion between Solutions 1 and 3 is limited. This is described by the Peclet Number (Pe = LU/D) where L is the characteristic distance for diffusion, U is the velocity, and D is the diffusivity of dissolved solutes assumed to be 10−9 m2/s. Changing the flow rate most significantly affects the characteristic diffusion distance for interactions between Solutions 1 and 3 preventing unwanted solution interactions (Figure 2B). To test this, we separated two solutions that form an insoluble salt. Aluminum in a Mayer’s hematoxylin stain reacts with carbonate to form aluminum carbonate, which quickly precipitates out of solution36 (Supporting Information Figure 3). These chemicals are often used in conjunction in cytopathology for colorimetric staining of cells.37 Polystyrene beads were added to hematoxylin solution containing aluminum and introduced to the Dip Chip as Solution 1, while acetic acid was introduced as Solution 2 and sodium carbonate as Solution 3. Acetic acid was chosen since it did not contain any salt that would react with the aluminum ions. The choice of the separating solution can be tailored for specific applications. When the acetic acid stream is wide with Peclet number ≈13 000, there is no precipitation. This allows successful inertial transfer of the suspended polystyrene particles across to the sodium carbonate solution (Figure 3Ai). However, when the acetic acid stream thickness is decreased (Peclet number ≈5000), precipitates form at the interface of the fluid streams (Figure 3Aii). This precipitate (indicated by black arrows) prevents the transfer of the particles across the solutions. Such a system will have applications in automation for biological assays by eliminating wash steps and in chemical synthesis on bead surfaces. Demonstration of Chemical Permeabilization of Cells. Permeabilization of cells is used in a wide range of molecular and cellular biology applications such as staining intracellular components with antibodies, introduction of specific chemical stimuli, and exposing the intracellular milieu. In typical permeabilization protocols, cells are incubated with either an organic solvent or a detergent for several minutes.36 For example, Triton-X 100 may be used at a concentration of 0.2% w/v and incubated with cells for several minutes. We postulated that a more concentrated Triton-X 100 solution would be able to achieve permeabilization faster in flow with the few millisecond exposure enabled by the Dip Chip and in a uniform manner. Jurkat leukemia cells suspended in media were injected as Solution 1, while Triton-X 100 (2%) was injected as Solution 2, and Trypan Blue was injected as Solution 3. Trypan Blue is a membrane impermeable dye that is used to determine if a cell has a compromised cell membrane. Jurkat cells were inertially transferred through the Triton-X 100 solution into the Trypan Blue and were collected (Figure 3Bi). All cells permeabilized with the Dip Chip were positive for Trypan Blue (Figure 3Bii). This result demonstrates a 100 000-fold decrease in incubation time needed for permeabilization with only a 10-fold concentration increase in Triton-X 100. Automation of Cellular Staining. Chemical permeabilization of cellular membranes is an essential step for delivery of large or charged fluorescent dyes. The process of labeling cells manually is time-consuming and suffers from user-to-user variations. We applied the Dip Chip to the intracellular labeling of cytokeratin in MCF7 breast cancer cells. Cytokeratin is often

used to stain for circulating tumor cells (CTCs) after capture and is used for validation of capture. Measurement of cytokeratin intensity might be better incorporated into an automated isolation procedure with a system for automated staining, but such inline analysis is limited by the need for manual procedures. We fixed MCF7 cells in formaldehyde and injected the cells as Solution 1, while simultaneously injecting Triton-X 100 as Solution 2 and fluorescent anticytokeratin antibodies as Solution 3. Cells and anticytokeratin in PBS were collected and incubated for an hour off-chip. In parallel, cells were prepared manually with a 7 min permeabilization in Triton-X 100 and an hour incubation in anticytokeratin. The automated workflow reduced the permeabilization time and also eliminates the need for two wash and centrifugation steps (resulting in approximately 20 min in operation time). DAPI was also added to the cells off-chip for better visualization of cells (Figure 3Ci). Despite the manual operation being performed using a precise protocol, there was greater variability in samples stained manually versus with the Dip Chip (Figure 3Cii). Two samples of staining for both manual and the Dip Chip were analyzed for fluorescent intensity with the same exposure time and gains for each set. The two samples operated with the Dip Chip were more similar to each other as the p-value for rejecting the null hypothesis that the samples were from the same population as measured by a Student’s t test was closer to 1 (p = 0.71) in comparison to the p value statistic for the two samples that were prepared manually (p = 0.20). This may be attributed to the manual procedure requiring several more steps, such as centrifugations and washes. Increased uniformity in such processes will make the analysis from antibody labeled cells more uniform. Additionally, while this demonstration was applied to a less quantitative system (fluorescence microscopy), such a technique might serve to increase uniformity upstream of more quantitative tools such as flow cytometers. Molecular Delivery to Viable Cells. The success with chemical permeabilization with Triton-X 100 led us to postulate that the extremely short incubation times might not kill the cell, enabling delivery of molecules to viable cells. We tested this using calcein AM and ethidium homodimer-1 (Ethd1). These are commonly used as part of a live-dead assay as Ethd-1 fluoresces upon binding to nucleic acids but is membrane impermeable so will not enter normal live cells, while calcein AM requires live cell activity as intracellular esterases cleave it to calcein, which is fluorescent. A successfully porated, viable cell would therefore contain Ethd-1 delivered on-chip and also contain calcein converted from calcein AM following pore closure (Figure 4A). Triton-X 100, however, is a harsh detergent that is nonselective in its extraction of lipids and proteins. We found that chemical treatment with Triton-X 100 resulted in nonviable cells (Supporting Information Figure 4A). As a control, we used phosphate buffered saline (PBS) as the incubation solution, and as expected, there was no cell permeabilization (Supporting Information Figure 4B). Using the milder saponin as the porating agent yielded improved cell viability. Saponin selectively removes cholesterol molecules from the cell membrane, enabling a gentler poration method.36 We found that tuning the saponin flow rate was also critical in achieving viable poration. At Q2 = 10 μL/min, the incubation time was too short resulting in no successful molecular delivery and Q2 = 30 μL/min resulted in a very large degree of cell death. When the saponin flow rate was controlled at Q2 = 15 μL/min, successful poration of Jurkat cells was 1508

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We demonstrate its utility in automating the process of staining cells with fluorescent antibodies with increased uniformity and decreased preparation time versus manual processes as well as molecular delivery to viable cells. This strategy will also be useful for controlling kinetic solid phase reactions and probing molecular binding affinities and also as a method for rapid measurements of protein folding. The system operates simply without the need for external forces or complex fabrication, which will facilitate manufacturing and enable broad adoption.

achieved (Figure 4B, white arrows indicate viably porated cells). A high magnification image of several stained Jurkat cells shows that the Ethd-1 molecules were successfully delivered to Jurkat cells, which still retain esterase activity to cleave calcein AM. The localization of the stains also confirms successful poration as the Ethd-1 seems to have localized tightly in the nucleus and the calcein staining is present in the cytosol. We note that calcein staining in some cases looked to be localized to subcellular vesicles, which may be an effect of the quick poration time leading to pore formation in the external membrane, but intact internal membranes leading to greater accumulation of calcein in such compartments. We were also able to deliver Ethd-1 molecules to MCF7 epithelial breast cancer cells and M398 cells, a melanoma cell line (Figure 4D, white arrows indicate porated cells). We further tested the pore closing time scale to be on the order of several minutes. MCF7 cells were transferred across a stream of saponin into cell culture media on the Dip Chip. Ten minutes after collection, calcein AM and Ethd-1 were added. None of the cells were positive for Ethd-1 showing that there were no longer any pores, suggesting transient poration. Additionally, there was no background fluorescence of calcein AM suggesting that there was not a significant amount of esterase leakage (Supporting Information Figure 5). Quantification of efficiency of molecular delivery across three cell lines showed that there was a high efficiency of viable poration (>40%) (Figure 4E). Efficiency is defined as % of viable cells with delivery of Ethd-1 to the nucleus. Additionally, a trend was observed between efficiency of molecular delivery and cell size (Figure 4F). Jurkats (a = 15 μm) had the highest efficiency of 55.4%. MCF7 cells are slightly larger (a = 18 μm) and had an efficiency of 52.2%. The largest of the cell types were the M398 cells (a = 22 μm) which had the lowest efficiency of 43.6%. These results were for the same flow rates but different saponin incubation times. As larger cells feel a greater inertial lift force, they transfer across the saponin solution faster, decreasing their incubation time. As described earlier, application specific parameters can be determined by simple modulation of flow rates. Additionally, using different poration agents is feasible as needed for the application. We further determined that we could control the quantity of molecules delivered by simply tuning exposure time. By increasing the flow rate of the saponin stream, we could increase the length of poration and thus the amount of molecules that were delivered. Ethd-1 molecules were delivered over a range of saponin flow rates (Q2, 15−30 μL/min), and we observed increases in intracellular fluorescent intensity due to increased amount of Ethd-1 molecules delivered to the cell (Supporting Information Figure 6). Finally, we found we could optimize the viable poration efficiency of M398 cells by tuning flow rates. We found that, by increasing the flow rate of the saponin solution to Q2 = 20 μL/ min, we could significantly increase poration efficiency to 75%. Increasing Q2 further, however, lead to an increase in cell death and again reduced efficiency (Figure 4G).

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (310) 983-3235. Fax: (310) 794-5956. Present Addresses ⊥

J.S.D.: Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA. ¶ D.R.G.: CytoVale Inc., South San Francisco, California 94080, USA. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Dr. Henry T.K. Tse and Dr. Westbrook M. Weaver for valuable feedback and insight. This work was partially supported by a Defense Advanced Research Projects Agency Young Faculty Award #N66001-11-1-4125 (D.D.C.), a David and Lucile Packard Fellowship for Scientists and Engineers (D.D.C.), a Goldwater Scholarship (J.S.D.), and a grant from the Howard Hughes Medical Institute to UCLA through the Precollege and Undergraduate Science Education Program (J.S.D.).

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REFERENCES

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CONCLUSIONS In this work, we developed a continuous flow microfluidic platform for tunable rapid incubation (a few milliseconds) of cells and beads into and then out of a solution. We applied the platform to several critical applications involving rapid exposures of cells and particles to reactants and solution exchange through diffusion barriers between reactive solutions. 1509

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