Large-Scale Single Particle and Cell Trapping ... - ACS Publications

Article pubs.acs.org/ac

Large-Scale Single Particle and Cell Trapping based on Rotating Electric Field Induced-Charge Electroosmosis Yupan Wu,† Yukun Ren,*,†,‡ Ye Tao,† Likai Hou,† and Hongyuan Jiang*,†,‡ †

School of Mechatronics Engineering, Harbin Institute of Technology, Harbin, Heilongjiang PR China, 150001 State Key Laboratory of Robotics and System, Harbin Institute of Technology, Harbin, Heilongjiang PR China, 150001

‡

S Supporting Information *

ABSTRACT: We propose a simple, inexpensive microfluidic chip for large-scale trapping of single particles and cells based on induced-charge electroosmosis in a rotating electric field (ROT-ICEO). A central floating electrode array, was placed in the center of the gap between four driving electrodes with a quadrature configuration and used to immobilize single particles or cells. Cells were trapped on the electrode array by the interaction between ROT-ICEO flow and buoyancy flow. We experimentally optimized the efficiency of trapping single particles by investigating important parameters like particle or cell density and electric potential. Experimental and numerical results showed good agreement. The operation of the chip was verified by trapping single polystyrene (PS) microspheres with diameters of 5 and 20 μm and single yeast cells. The highest single particle occupancy of 73% was obtained using a floating electrode array with a diameter of 20 μm with an amplitude voltage of 5 V and frequency of 10 kHz for PS microbeads with a 5-μm diameter and density of 800 particles/μL. The ROT-ICEO flow could hold cells against fluid flows with a rate of less than 0.45 μL/min. This novel, simple, robust method to trap single cells has enormous potential in genetic and metabolic engineering.

C

like structures24 have also been developed. Jacqueline et al.19 achieved large-scale single cell trapping using microwell arrays, and optimized their approach for fibroblasts and rat basophilic leukemia cells Shohei et al. developed a single cell microarray system to perform high-throughput analysis of cellular responses. This method also facilitated retrieval of a targeted cell by a micromanipulator.21 Chung and colleagues25 designed a microfluidic platform for high-density single cell capture by disposing cell traps carefully in a serpentine channel. However, microwells may induce shear stress and have an unknown influence on cell-surface interactions. In addition, cells are easily dislodged from microwells during reagent exchange unless the microwells are covered.26 Thus, long-term cell cultivations and assays are limited because of the absence of continuous nutrient supply and spatial restriction of growth. As for contactless trapping, optical and acoustic traps always attract cells to the point of highest energy intensity. Henddrik et al.16 and other researchers18,27 have trapped cells at the minimum electric field through negative dielectrophoresis (nDEP), which lowered the physiological effect on the cells. However, there are several limitations in trapping single cells by nDEP: a local heat source is generated, relatively complex apparatus is required, and nDEP is not suitable for the simultaneous analysis of many cells. In addition, the trapping

ells are the basic units of life. To better understand biochemistry and genetics, bulk analysis of large numbers of cells is usually carried out within the available Petri dishes and dedicated bioreactors,1,2 thus requiring enough original material for analysis. However, in population-level assays, the performance of single cells is masked and we cannot obtain a unique insight into the complex interplay between environmental reactor dynamics and cellular activity. The detection and analysis of a single particle or cell may not only allow further investigation and understanding of single cell behavior, but also be of great importance in the areas of genetic and metabolic engineering.3−5 Therefore, it is desirable to confine or trap single cells to designated locations of a substrate. To analyze the behavior of single cells, fluorescence-activated cell sorting (FACS) is quite a good tool for high-throughput screening and sorting populations.6 However, FACS requires multiple complicated sampling routines in advance. Thus, it is not suitable to investigate the time-dependent processes of a single cell.7 Recently, the rapid development of innovative microfluidic systems has provided previously unachievable insights into bioreactor inhomogeneity and population heterogeneity by facilitating the spatiotemporal analysis of single particles or cells.8−10 Numerous methods have been developed that allow single cell manipulation,4,11,12 which are classified into contactless and contact-based methods. Contactless trapping includes optical,13,14 acoustic, magnetic,15 and electric techniques.16−18 Cell-surface contact-based approaches like microwell structures,19−22 adhesion on reagents,23 and gel© XXXX American Chemical Society

Received: August 30, 2016 Accepted: November 2, 2016 Published: November 2, 2016 A

DOI: 10.1021/acs.analchem.6b03413 Anal. Chem. XXXX, XXX, XXX−XXX

Article

Analytical Chemistry

Figure 1. (a) Schematic of the trapping device. The side channel with the inlet (I2) and outlet (O2) allows growth medium exchange, and the yellow regions depict the indium tin oxide electrodes. (b) Schematic illustration of the trapping principle in the central floating electrode area: randomly distributed particles are focused to the center of the floating electrodes and immobilized by the interaction between ROT-ICEO flow and gravitational force. (c and d) The generation process of ICEO around the central electrode. (e) Photograph of an experimental chip. (f) Microscope image of the central floating electrode (50-μm diameter) array area. Scale bar = 150 μm. (g) A mechanical equilibrium analysis of vertical force components acting on a single particle (or cell) trapped on the floating electrode surface. Fb is the buoyancy force, Fdrag is the drag force derived from the ROT-ICEO flow, and G is the gravitational force.

technique is simple and inexpensive, capable of trapping various cells using ROT-ICEO flow regardless of their shape and size, permits continuous investigation of numerous individual cells in parallel, and allows the retrieval of cells for further analysis. The ROT-ICEO method is optimized numerically and experimentally to achieve single particle or cell trapping in a large microarray by investigating the main parameters that influence the single particle or cell trapping rate; for example, the size of the central floating electrode, the particle or cell density, the applied electrical potential and frequency, and the conductivity of the medium.

force for smaller particles is weak because the DEP trapping force scales with the third power of particle radius.18 Bazant and Squires28,29 proposed induced-charge electroosmosis (ICEO), in which the double-layer charge induced by the applied field is driven by the same field (Figure 1c and d). ICEO has received extensive attention from many researchers28,30−34 because of its notable feature of microflow generation near a polarizable conducting surface in an external electric field. A device using ICEO to trap single particles or cells has not been reported to date. Here, we report a novel microfluidic chip to trap single particles or cells based on rotating-field induced charged electro-osmosis (ROT-ICEO).35 Cells are immobilized by the interaction between ROT-ICEO flow and buoyancy flow, which holds them against continuous fluid flows. This

■

THEORY Traditional electroosmotic flows (known as alternating-current electroosmosis (ACEO)) occur when an applied electric field B


Article


Figure 2. (a) Plot of the ROT-ICEO flow in the x−z plane when frequency f = 1200 Hz and amplitude voltage A = 10 V (unit, m/s). (b) DEP velocity (in m/s) of a PS microbead with a diameter of 20 μm in the x−z plane at f = 100 kHz and A = 10 V on logarithmic scale. (c) Surfaceaveraged velocity of the ROT-ICEO flow on the surface of the central floating electrodes plotted against the applied frequency at A = 30 V for different diameters and particle numbers. (d) DEP and ROT spectra of PS microbeads with diameters of 5 and 20 μm in a low-conductivity buffer with σm = 0.001 S/m. (e) Frequency-dependent surface-averaged velocity of ICEO flow, particle cDEP motion, and twDEP motion for a PS microbead with a diameter of 20 μm at A = 10 V. (f) Frequency-dependent surface-averaged velocity of ICEO flow, particle cDEP motion, and twDEP motion for a PS microbead with a diameter of 5 μm at A = 5 V.

portion of the total double layer voltage ψD = Δϕ/(1 + δ), where CD = ε/λD is the capacitance of the diffuse layer, and CS is the capacitance of the Stern layer. The ratio of the diffuse layer capacitance to that of the Stern layer is δ = CD/CS, while ε is the permittivity (ε = 7.08 × 10 −10 F/m) and λD = Dε /σ = 37.6nm is the Debye screening length, where D = 2 × 10−9 m2/s is the bulk diffusivity. A capacitance-like boundary condition closes the equivalent RC circuit and the normal current from the bulk charges of the diffuse layer

forces the thin ionic clouds that screen charged surfaces into motion. In contrast, ICEO occurs when a diffuse double-layer charge is induced around a polarizable surface by an applied electric field. The same electric field subsequently drives the induced charge into motion.36 Therefore, the fundamental difference between these processes is the origin of the diffuse double-layer charge. In ICEO, the double layers are provided by electrical conductors that may not be energized, whereas the double layers are formed on the electrode surfaces in ACEO.34 The standard model of ICEO encompasses the Poisson− Nernst−Planck equations of ion transport coupled with the Navier−Stokes equations of viscous fluid flow. On the basis of the assumption of linear or weakly nonlinear charging dynamics, this model can be simplified by decoupling the electrokinetic problem into electrochemical relaxation and viscous flow. Electrochemical Relaxation. The electric potential in the fluid bulk can be achieved by solving the Laplace equation ∇·(σ E) = −σ ∇2 ϕ = 0

C0

dψ0 dt

= −σn·̂ ∇ϕ = σ En

(2)

Using complex amplitudes, the above condition can be written as jwC0

(1)

assuming the electrolyte has a constant conductivity σ. A compact Stern layer is often assumed to act as a capacitor in series with a diffuse-layer capacitor; the total induced double layer (IDL) capacitance is C0 = CSCD/(CS + CD) = CD/(1 + δ). The voltage across the diffuse layer capacitor only occupies a

ϕ ̃ − ϕ0̃ 1+δ

= σn·̂ ∇ϕ ̃

(3)

where ϕ̃ is the potential in the bulk outside the IDL, and ϕ̃ 0 is the potential at the metal surface. The boundary condition at the insulating surface can be given by C


Article


∂ϕ =0 ∂y

(cDEP) force, which signifies that a particle can be either repelled from or attracted to a stronger electric field depending on whether Re[K(w)] is negative or positive, respectively. The second term is the traveling wave DEP (twDEP) force, which only occurs in an electric field with spatial variation of phase and propels the particle toward the regions where the phases of the electric field component are larger or smaller depending on whether Im[K(w)] is positive or negative, respectively. Plots of Re[K(w)] and Im[K(w)] for polystyrene (PS) microbeads with diameters of 5 and 20 μm against frequency are shown in Figure 2d. The time-average electrorotational torque can also be calculated as follows when applying a rotating electric field to the driving electrodes.

(4)

Viscous Flow. The incompressible Navier−Stokes equations of low Reynolds number can be solved as follows: 0 = η∇2 v − ∇p , ∇·v = 0.

(5)

The time-averaged flow, given by the Helmholtz−Smoluchowski boundary condition for the effective slip on a polarizable surface, is −ε ⟨vs⟩ = Re(ζ ̃ E͠ t*) 2η −ε 1 1 ̃ E ̃ − E ̃·n ·n)*) = Re((ϕ͠ 0 − ϕ)( η 1+δ2 (6)

⟨T ⟩ = −4πr 3Im[K (w)]E ̃

where Et is the tangential electric field, ζ is the zeta potential, η is the fluid viscosity, p is the pressure, and the asterisk signifies complex conjugation. The no-slip boundary condition was applied to the other insulating surface and driving electrode surface, that is, The evolution of an IDL is shown in Figure 1c and d. In view of the equipotential nature of the electrode surface, the electric field lines intersected the central electrode surface at right angles as soon as an electric field was applied. Ions in solution were then driven along field lines and gradually accumulated on the electrode surface to form a double layer. There was no net charge in the IDL. The positive and negative ions were deposited close to the field source and field sink, respectively. When the applied electric frequency was low, the IDL reached steady state and all field lines were expelled from the electrode; thus, the conducting surface can be treated as an insulator. In this case, the tangential electric field drove the induced charge into motion and generated microvortices around the electrode surface, resulting in a stagnant region at the center of the floating electrodes, as illustrated in Figure 2a. However, most of the applied voltage was dropped across the bulk if the frequency was high, and the central electrode recovered to a perfect conductor. The characteristic frequency of a transition (1 + δ)D 1 can be estimated as f0 = 2πτ = 2πLλ (L denotes a 0

■

MATERIALS AND METHODS Device Fabrication. The microfluidic chips were fabricated by soft lithography. As shown in Figure 1a and b, the distance between the driving electrodes was 2 mm. Central floating electrode arrays with different electrode diameters (d = 15, 20, 30, 50, and 80 μm) were fabricated. The channel system consisted of a main channel with one inlet (I1) and one outlet (O1) and a side channel with one inlet (I2) and one outlet (O2). Transparent indium tin oxide (ITO) was selected as the electrode material. The ITO electrode pattern was produced through a standard etching process. A polydimethylsiloxane (PDMS) channel with a depth of 500 μm was polymerized using a poly(methyl methacrylate) pattern as a mold. A PDMS channel and glass slide patterned with ITO electrodes were aligned and bonded together (Figure 1e) after being treated with an oxygen plasma cleaner (ZEPTO, Diener, Germany). A microscope image of a central floating electrode array with a diameter of 50 μm is shown in Figure 1f. The manufacturing process of this trapping chip was described in detail in our previous work.30 Sample Preparation. The particles used in our study were PS microbeads (Fluka) with diameters of 5 and 20 μm and the cells were yeast. Yeast cells were obtained by reactivating 20 mg of dry baker’s yeast in deionized (DI) water (5 mL) at 50 °C for 2 h. Yeast suspension (1 mL) was transferred to a centrifuge tube and washed three times with DI water. Yeast cells were resuspended in aqueous solutions with different conductivities (σm) of 0.02 and 0.001 S/m, which were prepared by adding KCl to DI water and measured using a conductivity meter. PS microbeads were added to aqueous solutions with σm = 0.001

D

geometric length scale characterizing the bulk resistor and is equal to 0.115 d here), which is much smaller than the charge σ D relaxation frequency of the electrolyte f = 2πε = 2πλ 2 . For an D

AC field of frequency w, the steady-state flow decays above the 1 .29,37 It is clear that the flow velocity RC frequency as 2 1 + (wτ0)

halved at f 0 = 1609 Hz, as shown in Figure 2c. Dielectrophoretic Force. The time-average dielectrophoretic (DEP) force can be written in terms of the complex expressions38,39 ⟨FD⟩ = πr 3Re[K (w)]∇(E ̃·E*̃ ) − 2πr 3Im[K (w)] ̃ (∇ × Re(E)̃ × Im(E))

(9)

Note that the torque depends on Im[K(w)], and positive or negative torque implies that the particle can rotate in the same direction as the field rotation or in the opposite direction, respectively. To predict trapping conditions, numerical simulations were carried out based on experimental device geometries and buffer conditions using COMSOL Multiphysics 5.0 software. The Laplace eq 1 subject to eq 3 and 4 was solved using the inherent electric current module. The Navier−Stokes eq 5 subject to eq 6 and 7 was solved using the inherent creeping flow module. The two modules were solved in sequence because of their weak coupling. The effective slip velocity on the polarizable surface (eq 6) was inserted as a boundary condition into the mechanical problem after the electric potential was obtained.

(7)

v=0

2

(8)

where K(w) = (εp* − εm *)/(εp* − 2εm *) is the complex frequencydependent Clausius−Mossotti factor, εp* and εm* are the complex permittivities of the particle and medium, respectively, Re(Ẽ ) and Im(Ẽ ) denote the real and imaginary parts of the electric field phasor, respectively, and r is the particle radius. The first term on the right-hand side is the conventional DEP D


Article


Fb, and the drag force arising from the ROT-ICEO flow Fdrag, as shown in Figure 1g. When f was increased to between 2.5 and 8 kHz, it took a long time (more than 10 min) to trap a single particle and the trapping efficiency was poor. This is because the surfaceaveraged velocities of the nDEP force and ROT-ICEO flow were almost equal. When a high f (greater than 10 kHz) far beyond f 0 was applied to the driving electrodes, the effect of ROT-ICEO flow weakened dramatically because of the relaxation process in the electric double layer. The particle behavior will be dominated by the nDEP force instead of ROTICEO flow at high f (Figure 2e). The particles located near the floating electrode rim are transported by the nDEP force toward the center of the floating electrode, where the electric field has a local minimum (Figure 2b). Meanwhile, the nDEP force will repel the particles initially located in the area between the neighboring floating electrodes away from the floating electrodes, resulting in a poor trapping efficiency (data not shown). For trapping experiments using PS microbeads with a diameter of 20 μm, PS microbead suspensions with concentrations (ρ) of 100−500 particles/μL were prepared. Floating electrodes with different diameters (d = 30, 45, and 80 μm) and a distance between two adjacent electrodes of 2d were also studied. Figure S2 shows the results of typical experiments under different conditions. We did not consider the cases in which two or more particles were always trapped in one central floating electrode, which occurred when d was larger than 80 μm and the concentration of PS microbeads exceeded 500 particles/μL, or when most of the floating electrodes tended to be empty, which was observed when d and ρ were less than 20 μm and 100 particles/μL, respectively (data not shown). Statistic characterization of the trapping efficiency was assessed by the ratio of the number of particles located in the center floating electrodes to the number of center floating electrodes through three independent experiments. Figure 3

S/m to make a colloidal suspension for the experiments. To obtain the required cell or particle density, the yeast and microbead solutions were diluted with KCl solution of the same conductivity. Experimental Setup. Prior to each experiment, the microchannel was coated with a solution of 2% bovine serum albumin (BSA) in phosphate-buffered saline for 1 h to remove any air bubbles and prevent cell adhesion. The BSA solution was then removed by thoroughly washing the microchannel with KCl solution. Cell trapping experiments were then conducted by flowing the prepared single particle or cell suspension into the chip. Constant medium flow conditions were achieved using syringe pumps (PHD Ultra, Harvard, USA) attached to the side channel. The ITO driving electrodes were energized with a traveling wave signal using a voltage generator (TGA12104, TTi, UK). The left, top, right, and bottom driving electrodes were energized with V1(t) = A cos(wt), V2(t) = A cos(wt + 90°), V3(t) = A cos(wt + 180°), and V4(t) = A cos(wt + 270°), respectively. The trapping experiments were observed using a microscope (CKX41, Olympus, Japan) with a CCD camera (DP27, Olympus, Japan).

■

RESULTS AND DISCUSSION Single Microsphere Trapping. The ROT-ICEO flow field patterns formed above the floating electrodes with a diameter of 50 μm are shown in Figure 2a. Particles are transported to the floating electrode center by the flow vortices. The stagnant area at the center of the floating electrodes is suitable to trap a single particle. Moreover, the variation of the surface-averaged velocity with frequency is identical for the experiments with different numbers (n = 1, 9, and 25) of central floating electrodes of identical diameter (d = 50 μm), and the flow velocity is halved at the same double-layer relaxation frequency (f 0 = 1609 Hz), as shown in Figure 2c. Thus, the double-layer polarizations over central floating electrodes are independent of each other, making it possible to arrange large-scale electrode arrays in the center area to trap multiple single cells simultaneously. For the systems with the same number of central floating electrodes (n = 9) of different diameters (d = 15, 20, 30, 50, and 80 μm), the characteristic f 0 decreases with increasing electrode diameter. When the applied frequency is much lower than f 0, the ROTICEO effect will gradually become stronger. At a frequency f of 500 Hz and amplitude voltage A of 10 V, the fast ROT-ICEO flow will rapidly transport the particles toward floating electrode surface, where they are trapped erratically at the center of the flow vortex (Figure 2e) above the rim of each floating electrode, as shown in Figure S1. The trapped particles revolve slowly and synchronously around the floating electrodes with the applied rotating field at the electrode edges, because the particles experience a twDEP force along the direction of field travel originating from the negative Im[K(w)] (Figure 2d). The trapping experiments were then conducted at intermediate frequencies (in the vicinity of the characteristic frequency f 0 = 1609 Hz). When the driving electrodes were energized by a traveling wave signal of f = 1.5 kHz and A = 10 V, the ROT-ICEO flow still plays a dominant role over the nDEP force because of the negative Re[K(w)] (Figure 2d and e). The particles can be stably trapped at the center of floating electrodes because of the balanced interplay of the vertical force components, including gravitational force G, buoyancy force

Figure 3. Microbead occupancy statistics for different microelectrode diameters and microbead densities after applying an electric signal. Microbead diameter = 20 μm.

shows microbead occupancy statistics for different microelectrode diameters and microbead densities after applying an electric signal with a peak-to-peak voltage of 20 V and f = 1.5 kHz for 5 min. Figure 3 reveals that the maximal trapping efficiency of a single particle (75%) was obtained using an optimized particle density of 250 particles/μL and central electrode diameter of 50 μm. Cell−cell interactions and intercellular behavior have attracted considerable attention from researchers, and the E


Article

Analytical Chemistry influence of cell− cell contact on cell differentiation can be illustrated by controlling the cell−cell contact at a single level.40 One other thing to note here is that a large number of pairs of single particles can also be trapped accurately by adjusting the relevant parameters (d and ρ), as illustrated in Figure 3. The central floating electrode array with a diameter of 50 μm yielded an occupancy of particle pairs of almost 40% at a particle density of 350 particles/μL. Trapping experiments using single PS microbeads with a diameter of 5 μm were also performed. As shown in Figure 2f, when f was far lower than f 0, we were unable to stably trap a single particle at the center of the floating electrodes because of the fast ROT-ICEO flow. When f was raised to around f 0, single particle trapping was achieved because of the double-layer relaxation process. When f was increased to much higher than f 0, the nDEP force dominated over the drag force arising from ROT-ICEO flow (Figure 2d and f). In this case, the singleparticle trapping performance was poor because the nDEP force repelled the particles initially situated above the floating electrodes from the center of the electrodes. Therefore, the following trapping experiments of single PS microbeads with a diameter of 5 μm were carried out at f = 10 kHz and A = 5 V. The particle suspensions had concentrations of 500−2500 particles/μL and the floating electrodes possessed different diameters (d = 15, 20, and 30 μm). Figure 4 shows the results

Figure 5. Microbead occupancy statistics for different microelectrode diameters and microbead densities after applying an electric signal. Microbead diameter = 5 μm.

μm), as illustrated in Figure S3. As the distance between electrodes increased, it became difficult to collect particles effectively because they were located beyond the range of ROTICEO flow. Conversely, the mutual effect and influence of the ROT-ICEO flow between two neighboring central electrodes impaired the trapping performance when the distance between two adjacent electrodes was too small. Single Cell Trapping. To verify the usability of this device, we carried out trapping experiments using yeast cells, which is a preferred model system for eukaryotic cells.41 Given that the diameter of a yeast cell is similar to that of a PS microbead (∼5 μm), cell trapping was carried out based on the optimal results for the 5-μm PS microbeads (d = 20 μm and ρ = 800 particles/ μL). Yeast cells were suspended in KCl aqueous solutions with σ m = 0.02 and 0.001 S/m. Cells flowing within the microchannel were attracted to the floating electrode by the long-range ROT-ICEO convective microvortices, then stable cell trapping was achieved around the center of the floating electrodes based on the balance of downward Fb and G, and upward Fdrag. A typical trapping experiment was first conducted by stably trapping yeast cells in a low-conductivity medium (σm = 0.001 S/m). As shown in Figure 6d, a single cell was successfully isolated on the central floating electrode surfaces when A = 3.5 V and f = 20 kHz. Cells began to be attracted to the edges of central floating electrodes and rotate when the cDEP and twDEP forces dominated over ROT-ICEO flow as f increased. At A = 3.5 V and f = 100 kHz, the cells were directed by the twDEP force to move around the edges of the central floating electrodes in the opposite direction to the field rotation. Meanwhile, the electrorotational torque also made the cell revolve on its axis counter to the field rotation direction (Figure 6e) because of the positive Im[K(w)] (Figure 6a and b). When a higher f of 3 MHz was applied to the driving electrodes, the cells were propelled to move around the edges of the central floating electrodes and rotated in the same direction as the field rotation because of the negative Im[K(w)] (Figure 6f). The rotational speed of cells located at the center of floating electrodes reached 6−7 rpm (at A = 3.5 V and f = 100 kHz) according to the values determined for ten cells. Trapping experiments were also conducted in KCl aqueous solutions with σm = 0.02 S/m. Equation 6 indicates that the ROT-ICEO flow will weaken as σm increases. Therefore, cell trapping was achieved when A = 10 V and f = 100 kHz, as presented in Figure 6g. The cell solution was then exchanged by pumping cell culture medium in another inlet (I2). The cells were retained at their initial positions upon adding fresh culture medium and the byproducts from cell metabolism were

Figure 4. Nine representative images of PS microbeads with a diameter of 5 μm trapped in central electrodes with different diameters (d = 15−30 μm) and densities of microbeads (ρ = 500−2500 particles/μL). Scale bar = 20 μm.

for typical experiments under different conditions. The maximum single-particle occupancy of 73% was obtained using a floating electrode array with a diameter of 20 μm and PS microbead concentration of 800 particles/μL (Figure 5). The optimal distance between two adjacent electrodes was verified by conducting trapping experiments using PS microbeads with a 5-μm diameter, d = 20 μm, and different distances between adjacent electrodes (1.5d = 30 μm, 2d = 40 μm, and 2.5d = 50 μm). Good trapping performance was obtained when the distance between two adjacent electrodes was set as 2d (40 F


Article


the membrane can also be ignored considering that the yeast cells are mainly trapped at the center of the floating electrode surface where the field intensity is extremely small around f 0. Therefore, the cell viability during trapping can be guaranteed. This trapping device is suitable for not only stably trapping single cells, but also cultivating cells in a constant medium flow. Trapping experiments demonstrated good agreement with simulation results. The trapping device can be used for multiplexing experiments by integration with upstream microfluidic parts.25 Using this device, we can observe the performance of cells under different conditions. The realization of electrical connectivity to each trap is a major challenge in designing large-area arrays for single cell trapping.18,42 Our trapping device is simpler because the electrical signals just need to be applied to the driving electrodes instead of the central floating electrode arrays as a result of the ROT-ICEO mechanism. Importantly, this trapping method based on ROTICEO can also be used to trap and handle nonspherical particles or cells, which are more challenging than spherical objects.43

■

CONCLUSIONS We developed a novel simple approach to trap single particles or cells based on ROT-ICEO. The performance of our chip was demonstrated by trapping PS microspheres with diameters of 5 and 20 μm and yeast cells. Cells were immobilized on the floating electrode surface through the interaction of ROTICEO and buoyancy flows without requiring bulky or expensive controls. More than 100 particles with a diameter of 5 μm (20 μm) could be trapped on the floating electrode array with a diameter of 20 μm (50 μm) using A = 5 V (10 V) and f = 10 kHz (1.5 kHz), yielding an optimal single particle occupancy of 73% (75%) when the concentration of PS microbeads was 800 particles/μL (250 particles/μL). Given that the diameter of a yeast cell is similar to that of a PS microbead, yeast cell trapping was carried out using the chip in KCl aqueous solutions with different conductivities (0.02 and 0.001 S/m). Our results indicated that the ROT-ICEO flow could hold the cells against a fluid flow with a rate of less than 0.45 μL/min. The developed method should be suitable to trap various cells via the interaction of ROT-ICEO and buoyancy flows regardless of their shape and size, allowing both continuous investigation of numerous individual cells in parallel and cell retrieval for further analysis. Thus, we envision this device will be used for diverse applications.

Figure 6. (a) DEP and ROT spectra of yeast cells in aqueous solutions with different conductivities (σm = 0.02 and 0.001 S/m). (b) Frequency-dependent surface-averaged velocity of ICEO flow, particle cDEP motion, and twDEP motion for yeast cells (σm = 0.001 S/m and A = 3.5 V). (c) Frequency-dependent surface-averaged velocity of ICEO flow, particle cDEP motion, and twDEP motion for yeast cells (σm = 0.02 S/m and A = 10 V). (d−f) The trapping performance of yeast cells suspended in aqueous solutions with σm = 0.001 S/m at A = 3.5 V and f = 20 kHz, 100 kHz, and 3 MHz, respectively. (g) and (h) The trapping performance of yeast cells suspended in aqueous solutions with σm = 0.02 S/m at A = 10 V, flow rate 45 μL/min and f = 80 kHz and 1 MHz, respectively. Scale bar: 20 μm.

■

ASSOCIATED CONTENT

S Supporting Information *

removed at the same time. The experimental results indicated that at a flow rate of less than 0.45 μL/min (20 μm/s, which is medium velocity), the ROT-ICEO flow could hold the cells against a fluid flow with an applied signal of A = 10 V and f = 100 kHz. Similarly, the cDEP and twDEP forces prevailed over the ROT-ICEO flow as f increased (Figure 6c). For instance, at A = 10 V and f = 10 MHz, the cells are attracted to the edges of central electrodes by the positive DEP (pDEP) force and directed by the twDEP force to move around the edges of the central floating electrodes in the opposite direction to the field rotation (Figure 6h). Meanwhile, the electrorotational torque also makes the cells rotate counter to the field rotation direction because of the positive Im[K(w)] (Figure 6a) (see Movie 1). Note that because the single cell trapping process is conducted in low-conductivity medium, it is rational to neglect Joule heating. In addition, the alternating voltage imposed upon

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03413. Movements of the microbeads above the central floating electrodes, pictures of PS microbeads in central electrodes for different diameters of central electrodes, influence of the distances between two adjacent electrodes on the trapping performance (PDF) Trapping single yeast cells AVI)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86 451 86418028. Fax: +86 451 86402658. G


Article

Analytical Chemistry *E-mail: [email protected].

(29) Squires, T. M.; Bazant, M. Z. J. Fluid Mech. 1999, 509, 217−252. (30) Ren, Y.; Liu, W.; Jia, Y.; Tao, Y.; Shao, J.; Ding, Y.; Jiang, H. Lab Chip 2015, 15, 2181−2191. (31) Ren, Y.; Liu, J.; Liu, W.; Lang, Q.; Tao, Y.; Hu, Q.; Hou, L.; Jiang, H. Lab Chip 2016, 16, 2803−2812. (32) Nam, S.; Cho, I.; Heo, J.; Lim, G.; Bazant, M. Z.; Moon, D. J.; Sung, G. Y.; Kim, S. J. Phys. Rev. Lett. 2015, 114, 114501. (33) Yossifon, G.; Frankel, I.; Miloh, T. Phys. Fluids 2007, 19, 068105. (34) Harnett, C. K.; Templeton, J.; Dunphy-Guzman, K. A.; Senousy, Y. M.; Kanouff, M. P. Lab Chip 2008, 8, 565−572. (35) Liu, W.; Shao, J.; Ren, Y.; Liu, J.; Tao, Y.; Jiang, H.; Ding, Y. Biomicrofluidics 2016, 10, 034105. (36) Squires, T. M. Lab Chip 2009, 9, 2477−2483. (37) Ramos, A. Electrokinetics and Electrohydrodynamics in Microsystems; Springer Science & Business Media, 2011; Vol. 530. (38) Jones, T. B. IEEE Eng. Med. Biol. Mag 2003, 22, 33−42. (39) Green, N. G.; Ramos, A.; Morgan, H. J. Electrost. 2002, 56, 235− 254. (40) Tang, J.; Peng, R.; Ding, J. Biomaterials 2010, 31, 2470−2476. (41) Falconnet, D.; Niemisto, A.; Taylor, R. J.; Ricicova, M.; Galitski, T.; Shmulevich, I.; Hansen, C. L. Lab Chip 2011, 11, 466−473. (42) Rosenthal, A.; Voldman, J. Biophys. J. 2005, 88, 2193−2205. (43) Chung, K.; Kim, Y.; Kanodia, J. S.; Gong, E.; Shvartsman, S. Y.; Lu, H. Nat. Methods 2011, 8, 171−176.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work is supported by the National Natural Science Foundation of China (No. 51305106, 11672095 and 11372093), the Fundamental Research Funds for the Central Universities (No. HIT. NSRIF. 2014058 and HIT. IBRSEM. 201319), Self-Planned Task (No. 201510B and SKLRS201606C) of State Key Laboratory of Robotics and System (HIT) and the Programme of Introducing Talents of Discipline to Universities (No. B07018).

■

REFERENCES

(1) Vasdekis, A. E.; Stephanopoulos, G. Metab. Eng. 2015, 27, 115− 135. (2) Lecault, V.; White, A. K.; Singhal, A.; Hansen, C. L. Curr. Opin. Chem. Biol. 2012, 16, 381−390. (3) Kim, H. S.; Devarenne, T. P.; Han, A. Lab Chip 2015, 15, 2467− 2475. (4) Weibel, D. B.; Diluzio, W. R.; Whitesides, G. M. Nat. Rev. Microbiol. 2007, 5, 209−218. (5) Cai, L.; Friedman, N.; Xie, X. S. Nature 2006, 440, 358−362. (6) Brehm-Stecher, B. F.; Johnson, E. A. Microbiol Mol. Biol. Rev. 2004, 68, 538−559 table of contents. (7) Grunberger, A.; Paczia, N.; Probst, C.; Schendzielorz, G.; Eggeling, L.; Noack, S.; Wiechert, W.; Kohlheyer, D. Lab Chip 2012, 12, 2060−2068. (8) Grunberger, A.; Wiechert, W.; Kohlheyer, D. Curr. Opin. Biotechnol. 2014, 29, 15−23. (9) Zhan, M.; Chingozha, L.; Lu, H. Anal. Chem. 2013, 85, 8882− 8894. (10) Wu, Y.; Ren, Y.; Jiang, H. Electrophoresis 2016, DOI: 10.1002/ elps.201600106. (11) Lindstrom, S.; Andersson-Svahn, H. Lab Chip 2010, 10, 3363− 3372. (12) Nilsson, J.; Evander, M.; Hammarstrom, B.; Laurell, T. Anal. Chim. Acta 2009, 649, 141−157. (13) Redding, B.; Pan, Y. L. Opt. Lett. 2015, 40, 2798−2801. (14) Kotnala, A.; Gordon, R. Nano Lett. 2014, 14, 853−856. (15) Han, K. H.; Frazier, A. B. Lab Chip 2006, 6, 265−273. (16) Kortmann, H.; Chasanis, P.; Blank, L. M.; Franzke, J.; Kenig, E. Y.; Schmid, A. Lab Chip 2009, 9, 576−585. (17) Bhattacharya, S.; Chao, T. C.; Ariyasinghe, N.; Ruiz, Y.; Lake, D.; Ros, R.; Ros, A. Anal. Bioanal. Chem. 2014, 406, 1855−1865. (18) Thomas, R. S.; Morgan, H.; Green, N. G. Lab Chip 2009, 9, 1534−1540. (19) Rettig, J. R.; Folch, A. Anal. Chem. 2005, 77, 5628−5634. (20) Ochsner, M.; Dusseiller, M. R.; Grandin, H. M.; Luna-Morris, S.; Textor, M.; Vogel, V.; Smith, M. L. Lab Chip 2007, 7, 1074−1077. (21) Yamamura, S.; Kishi, H.; Tokimitsu, Y.; Kondo, S.; Honda, R.; Rao, S. R.; Omori, M.; Tamiya, E.; Muraguchi, A. Anal. Chem. 2005, 77, 8050−8056. (22) Huang, L.; Chen, Y.; Chen, Y.; Wu, H. Anal. Chem. 2015, 87, 12169−12176. (23) Anderson, D. G.; Levenberg, S.; Langer, R. Nat. Biotechnol. 2004, 22, 863−866. (24) Kim, M. S.; Yeon, J. H.; Park, J. K. Biomed. Microdevices 2007, 9, 25−34. (25) Chung, K.; Rivet, C. A.; Kemp, M. L.; Lu, H. Anal. Chem. 2011, 83, 7044−7052. (26) Wang, Y.; Shah, P.; Phillips, C.; Sims, C. E.; Allbritton, N. L. Anal. Bioanal. Chem. 2012, 402, 1065−1072. (27) Voldman, J.; Gray, M. L.; Toner, M.; Schmidt, M. A. Anal. Chem. 2002, 74, 3984−3990. (28) Bazant, M. Z.; Squires, T. M. Phys. Rev. Lett. 2004, 92, 066101. H


Large-Scale Single Particle and Cell Trapping ... - ACS Publications

Recommend Documents