Focusing and collecting of small particles

A comparison of dielectrophoresis and inertial focusing for the up-concentration and detection

of viruses in water

Kevin Li, Caitlyn Moore, and Tan Pei Leng

6/25/2014

Waterborne viral diseases pose a high risk for global public health as 1-10 viruses/L concentrations of dangerous viruses in the water supply can cause severe diseases, including but not limited to, gastroenteritis, meningitis, norovirus, and hepatitis A and E. For detection of these low concentrations of viruses in water samples, it is important to up-concentrate viruses to a more easily detectable level. Dielectrophoresis (DEP) and inertial focusing are intriguing methods for small particle manipulation that have the potential to be applied to virus up-concentration and subsequent virus detection in water. DEP is a technique that achieves separation of particles with a high level of particle specificity by capitalizing on the polarizability of particles, while inertial focusing utilizes differences in particle physical properties and is advantageous due to its high rate of operation. Therefore, certain aspects of each technique may be extremely useful to increase the concentration of particles in aqueous solution, enabling simpler, more reliable detection in water supplies. Moreover, this report details the theory and practice underlying DEP and inertial focusing and evaluates the potential applicability of each technique to the problem of virus concentration and detection.

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Table of Contents

Division of Report ....................................................................................................................... 1

Introduction ................................................................................................................................. 2

Motivation ............................................................................................................................... 2

Aim ......................................................................................................................................... 3

Dielectrophoresis .......................................................................................................................... 3

Theory ..................................................................................................................................... 3

Current Applications and State-of-the-Art .................................................................................... 5

Illustrative Experiment .............................................................................................................. 6

Method ................................................................................................................................ 6

Results and Discussion ........................................................................................................... 8

Inertial Focusing .........................................................................................................................11

Theory ....................................................................................................................................11

Current Applications and State-of-the-Art ...................................................................................13

Illustrative Experiment .............................................................................................................14

Methods ..............................................................................................................................14

Results and Discussion ..........................................................................................................15

Future Investigation and Conclusion ..............................................................................................18

References ..................................................................................................................................20

Division of Report

Section Written By: Abstract Caitlyn Moore Motivation Caitlyn Moore, Kevin Li Aim Caitlyn Moore DEP Theory Kevin Li DEP Current Applications & State-of-the-Art Kevin Li, Tan Pei Leng DEP Illustrative Experiment Kevin Li Inertial Focusing Theory Caitlyn Moore Inertial Focusing Current Applications & State-of-the-Art Caitlyn Moore, Tan Pei Leng Inertial Focusing Illustrative Experiment Caitlyn Moore Comparison of DEP and Inertial Focusing & Conclusion Caitlyn Moore, Tan Pei Leng DEP Experiment (Laboratory) Kevin Li, Caitlyn Moore Inertial Experiment (Laboratory) Kevin Li, Caitlyn Moore, Tan Pei Leng

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Introduction

Motivation

Access to potable water is essential to health and development at national, regional, and local levels for both developed and developing countries [1]. Unfortunately, a satisfactory water supply is currently unavailable to the entirety of the global population [1]. In many developing countries, water is not safe or accessible, resulting in adverse health effects as well as personal and social developmental limitations. An overwhelming 88% of diarrhea cases globally are linked to unsafe water, inadequate sanitation or insufficient hygiene, resulting in 1.5 million deaths annually which are generally experienced by young children [2]. For both developed and developing countries, investment in water supply and sanitation has been shown to yield net economic benefit in some regions as the reduction in health care costs due to adverse health effects outweigh the cost of the initial investment [1]. Viruses associated with waterborne disease can cause a wide variety of infections and symptoms involving different routes of transmission, routes and sites of infection, and routes of excretion [1]. Combinations of routes and sites of infection can vary and do not always follow predicted patterns, making viral disease difficult to prevent [1]. In addition, viruses are nanoscale particles. This leads to difficulties in detection and water treatment due partly to their ability to pass through filters [3]. Viral persistence in water supplies is long, resistance to chlorine is moderate, and health significance and relative infectivities are high [1]. Detectable concentrations (viruses/L) of enteric viral pathogens and indicators are 1-10 in lakes and reservoirs, 30-60 in impacted rivers and streams, 0-3 in wilderness rivers and streams, and 0-2 in groundwater [1]. Furthermore, mosquitos and other insects can act as a vector for the transmission of viral waterborne disease, infecting humans through their bite [1]. Prevalent viral waterborne diseases and contaminants include, but are not limited to: adenoviruses, coxsackie virus, Eastern and western equine encephalitis (abbreviated EEE and WEE, respectively), Japanese, La Crosse, and St. Louis encephalitides, enterovirus, hepatitis A and E, meningitis, norovirus, and gastroenteritis [4]. Although symptoms of each specific disease differ, symptoms collectively include fatigue, fever, abdominal pain, pneumonia, nausea, diarrhea, weight loss, jaundice, myalgia, organ damage and failure, and death [5]. Due to the demand for safer global water supplies, methods for detection of viruses in water are becoming increasingly important. Viruses are generally present at very low concentration in large quantities of water, concentrations that may be below the detection limits of current detection methods [6]. The most common detection method is polymerase chain reaction (PCR) [6]. In today’s industry, large water samples (100-1,500 liters) that are suspected to contain viruses are initially concentrated using a filter adsorption and elution method before analysis [6]. At the same time, an increase in the interest in microfluidics has enabled a greater understanding of the behavior of small particles in a fluid. Microfluidic-based techniques for small particle manipulation provide several advantages over more traditional non-microfluidic techniques (i.e. centrifugation, membrane filtration, fluorescence activated cell sorting, and magnetic activated cell sorting, etc), including higher processing rates, lower sample use, enhanced spatial resolution, and increased accessibility as a result of decreased cost [7]. Most significantly, microfluidic-based methods trap particles based on intrinsic physical characteristics such as size, shape, deformability, density, polarizability, and magnetic susceptibility [7]. Further, some of these techniques apply an external force field to the sample to achieve separation including optic, electric, magnetic, acoustic, and hydrodynamic forces; these are thusly referred to as “active” methods [7]. Conversely, some techniques utilize microchannel geometrical effects and nonlinear hydrodynamic forces and are termed as “passive” methods [7].

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Techniques for small particle manipulation have the potential to be applied to the detection of viruses in aqueous solution. Specifically, dielectrophoresis (DEP) and inertial focusing are up and coming techniques that have immense possibilities in the field of small particle manipulation. These methods will be assessed relative to one another to determine their prospective use for the detection of viruses in water.

Aim

The goal of this study is to evaluate DEP and inertial focusing in terms of their relative abilities to up-concentrate and subsequently detect viruses (typically ranging from 30 nm to 80 nm) in water samples. Potential ways DEP and inertial focusing can be adapted to better serve the application will be determined. Through the utilization of DEP and inertial focusing, virus concentrations can be consistently increased in order to more easily detect viruses in water samples. By increasing the ability to determine the safety of drinking water, there will be dramatic increases in global public health.

Dielectrophoresis

Theory

Dielectrophoresis (DEP) is the motion of polarizable particles in an inhomogeneous electric field arising from the differences in dielectric properties between the particles and the suspending fluid [8]. It is an active form of separation in which the movement of the particles is governed by the conductance and the permittivity of both the particle and the medium in which the particle is present [8]. Figure 1 illustrates an inhomogeneous electric field and the resulting electric field lines for two cases: (a) particle being more polarizable than the medium and (b) particle being less polarizable than the medium [8, 9]. When the particle is more polarizable than the medium, the particle will move toward the area where the electric field gradient is the greatest [8]. On the other hand, when a particle is less polarizable than the medium, the particle moves away from the area where the electric field gradient is the greatest [8]. This does not imply that the particle will simply move towards the negative or positive electrode.

Figure 1: The electric field lines are plotted by applying a positive voltage on the left electrode and a negative voltage on the right electrode. (a) Particle is more polarizable than the medium and moves away from the largest electric field gradient . (b) Particle is less polarizable than the medium and moves towards the largest electric field gradient. Adapted from [8].

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The force exerted by an electric field is governed by equation:

where Γ is a factor depending on the particle geometry, εm is the relative permittivity of the suspending medium, ε0 is the permittivity of free space, and E is the applied electric field [8]. The square of electric field (|E2|) in the governing equation denotes that even when the direction of the field is changed, the force would continue to point in the same direction. This serves as one of the primary advantages of DEP [8]. The geometry factor Γ is given by:

where a is the sphere radius. As previously mentioned, viruses are assumed to be spherical particles. The forces on a spherical particle and its behavior in an electric field is determined by the Claussius-Mossotti factor (Kf factor), given by [8]:

where ε represents the permittivity and the subscripts p and m refer to the particle and the medium respectively [8]. Kf depends on the complex permittivities of the particle and the medium as well as the particle geometry [8]. Complex permittivity, ε*, is given by:

where σ is the conductivity, ε is the relative permittivity, and ω is the angular frequency of the applied electric field [8]. By replacing the complex permittivity into the Claussius-Mossotti factor equation, it can easily be shown that:

The two equations above represent the real, Re(Kf), and the imaginary parts of Kf, Im(Kf). Particles innately have different electrical properties and, consequently, can exhibit different DEP force at the same frequency [8].

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Referring to ForceDEP equation previously mentioned, it is evident that the dielectrophoretic force depends only on the real part of Kf. Therefore, this part of the equation is of more interest. If the real part is positive, the force is oriented towards increasing electric fields, otherwise known as positive DEP [8]. Conversely, if the real part of Kf is negative, then the force on the particle points toward decreasing electric fields [8]. This is referred to as negative DEP. Positive and negative DEP are compared in Figure 1. Importantly, the frequency at which the real part of Kf changes from positive to negative (or negative to positive) is termed turnover frequency [8]. This frequency is the frequency at which the particle will feel no force [8]. Generally, two different types of small particles will have distinct Claussius-Mossotti factors and can therefore be exploited to separate small particles from each other [8]. This dissimilarity in turnover frequencies between particles is illustrated in Figure 2.

Figure 2: The Claussius-Mossotti factor plotted against frequency for two different particles. The two particles show distinct differences in their response to an electric field, enabling the separation of the two particles by using the frequency at which the Claussius-Mossotti factor is opposite in sign. Adapted from [8].

Current Applications and State-of-the-Art

Dielectrophoresis can used to manipulate, transport, separate, and sort different types of particles. Since varying biological particles and cells have different dielectric properties, dielectrophoresis has immense medical applications. Prototypes that use the DEP technology have been developed to separate cancer cells from healthy cells [10]. Also, platelets have been separated from whole blood with a DEP apparatus [11]. Further, DEP has made it possible to characterize and manipulate biological particles such as blood cells, stem cells, neurons, pancreatic beta cells, DNA, chromosomes, proteins, and viruses. Currently, DEP is mainly used for characterizing cells by measuring the change in their electrical properties. To do this, many techniques are available to quantify the dielectrophoretic response, as it now possible to directly measure the DEP force. These techniques rely on obtaining a proportional response of the strength and direction of the force that is scaled to particle of interest. Most models only consider the Claussius-Mossotti factor. Common techniques are:

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1. Collection rate measurements: The simplest and most used technique. Electrodes are submerged in a suspension with a known concentration of particles and the particles that collect at the electrode are counted [12].

2. Crossover measurements: The crossover frequency between positive and negative DEP is measured. This method is used for smaller particles (i.e. viruses), that are difficult to count using the previous technique. [13]

3. Particle velocity measurements: The velocity and direction of the particles are measured in an electric field gradient [14].

4. Measurement of the levitation height: The levitation height of a particle is proportional to the negative DEP force that is applied from opposite sides of a particle. This technique is effective for characterizing single particles and larger particles such as cells [15].

5. Dielectrophoretic Impedance Measurement (DEPIM): Particles that collect at the electrodes edges and create a circuit with another electrode can have an influence on the impedance of the electrodes. This change can be monitored to quantify DEP. Generally used with larger particles [16].

Illustrative Experiment

Method

The experiment was designed to observe the effects of DEP on varying concentrations of green fluorescent polystyrene (PS) beads. No flow was involved. Small gold electrodes were etched on a silicon wafer prior to the start of the experiment. Two specific designs were used: (1) sharp tip electrodes and (2) interdigitated electrodes, shown in Figures 3 and 4, respectively. Each electrode was positioned on an individual chip, which are segments of the larger wafer onto which the electrodes were etched.

Figure 3: Sharp tip electrode uses a simple design where two electrodes are separated by a small distance. A larger view of the tip of the electrode is shown in blue on the right. Image courtesy of Mark Holm Olsen (DTU).

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Figure 4: Interdigitated electrode uses a multiple electrodes that are interweaved into each other in a comb-like structure. A larger view of the comb-like structure is shown in blue on the right. Image courtesy of Mark Holm Olsen (DTU). 2.65% stock solution of 50 nm green fluorescent PS beads was serially diluted by factors of 10 to 1000 with de-ionized (DI) water. The lowest concentration was initially utilized, subsequently increasing with each consecutive solution being tested, in order to demonstrate the effects of DEP on increasing concentrations of particles. The contact pads of the chip were covered in a copper adhesive in order to increase the surface area where the metallic clamps are able to attach. Silver conductive paste was then applied at the junction of the copper plate and contact pad to improve electrical contact between the two components. It is extremely important that the silver adhesive is not applied to the chip in a way that it makes contact with the electrode of opposite charge in order to avoid an electric shortage. A multimeter was used to confirm a current was applied to the electrodes. A 1 mm thick sheet of polydimethylsiloxane (PDMS) was cut into the shape of a window and placed on top of the silicon wafer. This created a small chamber in which the solution was able to be loaded and isolated above the electric fields. Following this, a glass cover-slip was then placed on top of the PDMS window, effectively trapping the solution inside the chamber. Then, two clamps were connected to a function generator where the frequency was adjusted from 10 kHz up to 2500 kHz. The voltage was set at the highest amplitude of 20 volts in order to observe the greatest effect of DEP. The experimental set-up is shown in Figure 5.

Figure 5: Experimental set-up including the silicon wafer, the copper pads, silver conducting paste, PDMS window, and the glass cover slip. The copper pads were connected to a function generator with which the frequency was adjusted.

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Results and Discussion

The theoretical turnover frequency was calculated for both concentrations of PS beads and an example Virus (HSV-1). The graphical representation of these calculations is shown in Figure 6.

Figure 6: The Claussius-Mosotti Factor for PS beads and a HSV-1 virus plotted over a range of frequencies [12].

Based on theoretical calculations, PS beads are predicted to have a turnover frequency around 1.2 x 105 Hz or 120 kHz. Physically, thermal effects and Brownian motion overcoming DEP forces should be observed at this frequency. As the frequency increases past the turnover frequency, forces coming from the electrodes facing the opposite direction should be observed. Similarly, for the example virus, HSV-1, the theoretical turnover frequency should be approximately 7 x 107 Hz or 70000 kHz and as the frequency increases, the force will increase in the opposite direction.

Sharp Tip Electrode The frequency applied by the function generator was varied between 10 kHz up to 2500 kHz. Due to the high voltage applied, thermal effects caused particles to move in a cyclical pattern around the electrode, especially as experiment duration progressed. A collection of particles at one electrode was observed, whereas the other did not show any collection. This result implies that the electrode setup was incorrectly completed and there was no current at the opposite electrode. Unfortunately, images were not able to be acquired of this particular experiment. Figures 7 and 8 are images from a previously performed experiment.

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Figure 7: Sharp tip electrode with an applied voltage at a frequency below 1000 kHz. As expected, a collection of particles at the electrodes was observed. Courtesy of Mark Holm Olsen (DTU).

Figure 8: The sharp tip electrode under a UV lamp and therefore causing the PS beads to fluoresce. The leftmost picture shows the frequency set at lower the turnover frequency and particles collect between the 2 electrodes. The middle picture shows a frequency very close to the turnover frequency, there is no longer a DEP force large enough to over Brownian motion. The rightmost picture shows a frequency above the turnover frequency, the DEP force changes sign and pushes the particles away center of the electrodes. Courtesy of Mark Holm Olsen (DTU).

Interdigitated Electrodes Again, the frequency was varied between 10 kHz upwards to 2500 kHz and thermal effects of the electrodes were observed over the experiment duration. A collection of particles at the electrodes approximately 1 mm from each side of the electrode was observed. Particles did not collect at the edges of the electrodes as expected. As the frequency decreased to approximately 10 kHz - 40 kHz, a sudden movement of particles away from the electrodes was observed. This may have been a result of the thermal

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effects overcoming the DEP forces rather than observing a turnover frequency. Figures 9 and 10 are images from a previous experiment performed by Mark Holm Olsen at DTU.

Figure 9: Interdigitated Electrodes with an applied voltage at a frequency lower than the turnover frequency. There is a collection of PS particles at the end of the electrodes because DEP forces overcome any opposing forces. Courtesy of Mark Holm Olsen (DTU).

Figure 10: The leftmost electrode show a frequency lower than the turnover frequency and particles collect at the end of the electrodes. The center picture shows a frequency near the turnover frequency, you can see signs of brownian motion and lack of DEP forces. The rightmost picture shows a frequency above the turnover frequency, the particles are being pushed away. However, not all of the particles are pushed away due to other forces, greater than DEP force, keeping it in place. Courtesy of Mark Holm Olsen (DTU).

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When this experiment was performed, test conditions were not ideal. The experiment was limited by the testing apparatus as there was no wafer with properly connected sharp tip electrodes. In addition, the chamber holding the sample over the electrode was lesioned, which resulted in leaking of the test solution.

Referring back to the DEP force equation, the geometry factor plays a small part in relation to the other terms in this equation, therefore, the small size of the particle should not significantly the force, especially if the electric field is an order of magnitude greater. The Kf factor plays a major part in determining the magnitude and the direction of the force but can be changed accordingly to your particle’s electrical properties in order to achieve the greatest DEP force applied to overcome any other forces. As there is enough force to contain 10 μm bead, it can be concluded with confidence that viruses can be captured using DEP. DEP can be used as a method of separation for concentrating viruses from large sample volumes on a high throughput method. Of course, a high velocity to achieve high throughput cannot be viable as the shear forces would over DEP forces. Therefore, high throughput would be achieved by having multiple DEP electrode devices covering a large surface area and a slow velocity of the medium in order to prevent the shear forces from overcoming the DEP forces.

Inertial Focusing

Theory

Inertial microfluidic systems capitalize on the invariable effects of fluid inertia for applications in enhanced mixing, particle separation, and particle focusing. Traditionally, microfluidics has been associated with negligible inertia, implying that fluid flow in microfluidic channels is assumed to occur at a low Reynolds number [17]. Reynolds number (Re = ρUH/μ) describes the ratio between inertial and viscous forces in a flow, where ρ is density in kg/m3, U is mean flow velocity in m/s, H is the characteristic dimension of the channel in m, and μ is viscosity in Pa-s [18]. However, in the case of a microchannel at Re between 1 and 100, using an estimation in which inertia is neglected by equating the inertial components on the left side of the Navier-Stokes equation:

to zero produces incorrect results because although Stokes flow implies laminar flow, laminar flow does not necessarily imply Stokes flow [17]. Furthermore, flows in microchannels are laminar and predictable because turbulence is generally observed for Reynolds numbers over 2300 [17]. The two primary hydrodynamic ways particles are focused in microfluidics are through inertial migration of particles or secondary flow in curved channels. Inertial migration utilizes inertial lift forces to focus particles. Particles in a flow experience forces parallel (drag forces) and perpendicular (lift forces) to their surfaces due to shear and normal stresses acting over their surfaces [17]. By utilizing aforementioned intrinsic inertial lift forces, precise particle manipulation is possible. Channel dimensions (aspect ratio), particle diameter, and flow rate can be altered in order to control the magnitude and direction of lift forces, as lift scales as:

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where Fl is lift force where ρ is density, U is the average flow velocity, a is particle diameter, and H is channel dimension [17]. Lift force is also dependent on the cross-sectional position of a particle in the channel; an equilibrium position observed and predicted at approximately 0.6 times the channel radius of cylindrical channels that is otherwise known as the Segre-Silberberg annulus shown in Figure 11A [17]. Additionally, in square channels, particles are focused at an equilibrium position at each face of the channel as shown in Figure 11B [17]. Similarly, in rectangular channels, equilibrium positions are focused to two longer faces of the channel [17].

Figure 11: Inertial focusing in the cross-sectional view of the outlet of a (A) cylindrical channel and a (B) rectangular channel. Adapted from [17]. These equilibrium positions dominate because of a balance between two different components of inertial lift: “wall-effect” lift that acts away from the walls toward the channel centerline and “shear-gradient” lift that acts from where flow rate is highest toward the walls, shown in Figure 12 [17].

Figure 12: Inertial lift forces that contribute to inertial migration of particles in fluid flow. Wall-effect lift forces act from the walls toward the center while shear-gradient lift forces work from near the channel centerline toward the walls. Adapted from [17]. It has been shown that lift scales as [17]:

near the channel centerline and,

near the channel wall These relations reveal a strong dependence of inertial lift forces on particle size and channel dimensions. For instance, as particle sizes increase near-wall lift increases; as channel height increases near-wall lift decreases. Secondary flow occurs in fluid flow through a curved channel because of a mismatch in velocity of the fluid in the center and fluid at the near-wall regions of a channel in the downstream direction [17].

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Subsequently, fluid near the wall centerline has increased inertia relative to fluid near the channel walls and, therefore, tends to flow outward around a curve in the channel, creating a radial pressure gradient across the channel [17]. As the channel is enclosed, the relatively stagnant fluid near the walls is recirculated inward as a result of the aforementioned centrifugal pressure gradient [17]. This recirculation effect creates two symmetric vortices [17]. This phenomenon, shown in Figure 13, is referred to as Dean flow. Dean vortices are often utilized to focus and separate particles with varying size or density [17].

Figure 13: Representation of Dean flow, in which faster moving fluid near the channel centerline has higher inertia than the fluid along the walls. The fluid at the channel center has a tendency to move outward around a bend in the channel, and to conserve mass, more stagnant fluid near the walls recirculates inward, creating two counter-rotating vortices perpendicular to the primary flow direction. Adapted from [17].

Current Applications and State-of-the-Art

As the appreciation for inertial lift and ordering effects in microfluidic flows occurred recently, there are few commercial applications of inertial migration in microfluidic systems. Applications for particle and cell separation and filtration are logical because inertial lift forces scale with a power of particle size and the ability to operate at higher flow rates is a significant advantage for most separation and filtration applications [17]. Importantly, it is possible to achieve separation in straight microchannels due to differences in migration times for particles of different sizes as well as separation based on differences in equilibrium positions for particles of different sizes [17]. Capitalizing on this principle in conjunction with geometry-induced secondary flow, Papautsky and colleagues developed a spiral lab-on-a-chip device for size-dependent focusing of particles at distinct equilibrium positions across the microchannel cross-section from a multi-particle mixture, collecting individual particle streams with an appropriately designed outlet system [19]. This particular device was capable of separating neuroblastoma and glioma cells with 80% efficiency and above 90% relative viability at a rate of 1 million cells/min [19]. Utilizing both straight microchannel focusing and geometry-induced secondary flows introduced previously, Di Carlo and colleagues designed a novel inertial focusing platform that creates a single-stream microparticle train in a single-focal plane [20]. The design consists of a low-aspect ratio straight channel containing a series of constrictions in height arranged orthogonally, making use of inertial

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focusing and geometry-induced secondary flows, similar to that shown in Figure 8 [21]. With throughput as high as 36,000 particles/second, focusing efficiency upwards of 99% is achieved for a variety of different sized particles and cells [20]. Besides this Di Carlo and collegues separated pathogenic bacteria and blood cells by size using a linear channel that can be parallized radically and potentially stacked in 3D. This device is arrayed in 40 parallel devices with a single inlet and two “ring” outlets that filters mL volumes of blood continuously at 200 L/min per channel, removing contaminating bacteria without labeling [22].

Illustrative Experiment

Methods

An inertial focusing experiment was conducted in order to observe the focusing phenomenon experienced by particles in a straight microchannel. A straight 80 μm x 30 μm x 5 cm PDMS microchannel was provided, shown in Figure 14. A 5 cm channel allows the microparticles sufficient distance to travel downstream and focus into streamlines. The PDMS microchannel used was fabricated using photolithography, a technique in which a photoresist solution is applied to the surface of the PDMS, covered by a mask that covers desired areas of the construct, and exposed to UV radiation. Following UV exposure, the areas covered by the mask remain (positive photoresist) or the areas not covered by the mask remain (negative photoresist). This technique allows for intricate patterns to be fabricated.

Figure 14: 80 μm x 30 μm x 5 cm PDMS channels utilized for the inertial focusing experiments. Inlet channels are located inside the green rectangle. Outlet channels are located inside the orange rectangle. Three separate microchannels are present on the PDMS chip. At both ends of each main microchannel, a small channel is connected perpendicularly (out-of-plane in Figure 14) through which a relatively rigid polyether ether ketone (PEEK) tube is inserted. At the inlet, this tube is connected to a syringe pump. At the outlet, the tube is inserted into a vial in order to collect the used fluid. Due to the creation of the small perpendicular inlet channel, a series of mechanical filters enable the filtration of PDMS residue from the fluid flow, ensuring that only the desired particles will be observed in the flow. The rigidity of the tube and the microchannels are important for decreasing the compliance of the overall experimental system, subsequently minimizing interference of the desired microfluidic effects. Following the insertion of the tubes into the inlet and outlet channels, the PDMS device was affixed to the microscope stage with tape in order to deter unwanted movement. 2.65% solution of 10 μm diameter particles was serially diluted by a factor of 10 and 1000. 5 ml of the dilutions were loaded into 10 ml HSW Soft-Ject Syringes. A needle was inserted into syringe and then into the end of the rigid tube protruding from the PDMS microchannel inlet. The microparticle-filled

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syringe was then loaded into the syringe pump. The tube protruding from the microchannel outlet was placed into a collection tube. The experimental setup is shown in Figure 15.

Figure 15: Experimental setup. High-speed camera mounted above microscope allows for the observation of microparticles in fluid flow through designated segments of the microfluidic channel on the computer screen. Following the completion of the experimental setup, the microscope and high-speed camera attachment were turned on. By opening the Andor Solis software, the view from the microscope can be seen on the computer screen. Then, using the 5X objective, the microscope was focused onto the appropriate microchannel in the PDMS device. Ideally, the 20X objective would have been utilized in order to more gain higher resolution. Due to limitations of the camera and subsequent data collection constraints, the 5X objective was used to decrease the resolution and increase data acquisition. When desired focus was achieved, the syringe pump was activated and began pumping microparticle-filled fluid through the inlet tube and into the PDMS microchannel. The flow rates of the syringe pump were variable in this experiment, and were set to 10 μl/min, 100 μl/min, 200 μl/min, 300 μl/min, and 1 ml/min (Re of 4.85, 48.51. 97.02. 145.53, and 485.10, respectively. Images of microparticles flowing through the channel were collected 4.5 cm from the inlet at 1240 frames per second (fps) for each flow rate.

Results and Discussion

The frames that were acquired by the high-speed camera were analyzed as a stack of individual images using ImageJ. The standard deviation of each stack was then evaluated, meaning that the standard deviation of pixel intensity across the individual images was summated and displayed in a single image.

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Moreover, these images depict the most traveled path of the particles through the microchannel at 4.5 cm downstream from the inlet. The results from this particular analysis are shown in Figure 16.

A B C D E Figure 16: Standard deviation of image stacks performed using Image J for 10 μl/min (A), 100 μl/min (B), 200 μl/min (C), 300 μl/min (D), and 1 ml/min (E) flow rates at 4.5 cm downstream in 80 μm x 30 μm x 5 cm microchannel. In these black and white images, the lighter the location in the image, the higher the frequency with which the microparticle is present at that location in the microchannel. From the standard deviation of the stacks, it is evident that as flow rate increased, focusing of the particle into a path at the center of the observed face of the channel increased. At the 10 μl/min flow rate, no particle focusing was observed as the flow rate was not sufficient to induce inertial lift forces significant enough to sharply focus the particles. Conversely, at higher flow rates such as 300 μl/min and 1 ml/min, particles were definitively focused at the center of the observed face of the channel as the inertial lift forces dominate the flow behavior through the channel. Although these results demonstrated the effects of increased flow rate on the degree of microparticle focusing, the results were greatly limited by the high-speed camera used for data collection. In order to collect a stack of images of the desired length, the resolution of the images had to be decreased. This resulted in the observation of lines flowing through the microchannel rather than distinct, circular particles. Therefore, the use of a more sophisticated high-speed camera would enable the collection of more high quality images to be used for analysis. Additional analysis was conducted using ImageJ in order to determine the frequency of microparticle Y-positions in the flow. The 8-bit image stack was adjusted to the desired black and white contrast level and threshold. Next, the walls were cropped out of the stack in order to give a more accurate particle count, as the walls can be often mistaken as particles by the program. Following this, ImageJ was set to measure centroids and the “Analyze Particles” action was performed. This action allows for particle counting as well as particle X- and Y-position determination in each individual image of the stack measured in pixels. These Y-positions in pixels were then exported to MATLAB where the mode of the data was determined. By knowing the dimensions of the image in pixels and assuming that the height of the image was 80 μm, the mode in pixels was converted to μm through a simple proportion. The histogram, shown in Figure 17, revealed that the particles were most often located at a Y-position of 41.01 μm. This supports aforementioned theory and previous experiments as 41.01 μm is approximately at the centerline of the long face (80 μm) of a rectangular channel (80 μm x 30 μm x 5 cm).

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Figure 17: Histogram of Y-position of microparticles in a 80 μm x 30 μm x 5 cm microchannel. The most frequent Y-position is 41.01 μm, approximately along the channel centerline of the largest face (80 μm), as predicted by theory and previous experiments. It is important to be cognizant of the fact that the experiment was conducted using a 10 μm diameter particle. Hence, the results of this experiment can only be extended to microscale particles. As previously stated, viruses have nanoscale diameters. Therefore, these results cannot be applied to viruses, as the behavior of nanoparticles may differ from those of microparticles. In order to observe the effects of nanoparticle inertial focusing, experiments must be performed using nanoparticles. If the results obtained for both scales were equivalent, it could be concluded that this technique holds theoretical potential for virus up-concentration and detection. If the results differ, which is predicted from theoretical and empirical investigations, this technique would not be applicable. In terms of virus up-concentration and detection, the high-throughput nature of inertial focusing is extremely advantageous. From these results, it is clear that microparticles can be focused into distinct streams. This behavior can be applied to a larger microfluidic system in which these streams are collected and focused further, increasing the concentration of particles per volume. Conversely, as mentioned above, based on both theory and results, inertial focusing alone would not be an effective method of virus up-concentration due to the behavior of nanoparticles in a high rate of fluid flow because lift forces acting on a particle on the nanoscale are sufficiently small that they can be considered negligible. Therefore, the forces would not act in the desired manner to focus the particles into a streamline, and moreover, not be up-concentrated in aqueous solution.

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Future Investigation and Conclusion A comparison of DEP and inertial focusing is shown in Table 1. The advantages and limitations of each technique are assessed relative to the application of viral detection in aqueous solution. From this, the most useful aspects are extracted for the techniques’ potential applicability. Table 1. DEP and Inertial Focusing Comparison

DEP Inertial Focusing

Particle size for

effectiveness

Microscale Nanoscale

Microscale

Advantages and

Characteristics

Applicable to

Virus Up-

Concentration

and Detection

1. Specificity of particle separation as technique is dependent upon intrinsic polarizability of particles and the solution (viruses can be isolated for up-concentration/detection) 2. Shown to work on nanoparticles

1. High rate of operation due to high flow rates through microchannels 2. Passive technique (no external force necessary; able to focus particles based on physical properties) 3. No complex fabrication (lithography is low cost and simple process)

Limitations and

Constraints

1. Low-throughput relative to inertial focusing 2. Active technique (external electric force must be applied for technique to be effective)

1. Operation with small volumes is limitation of application to a large-scale system (possible to assemble channels in parallel, but number of devices needed would be extremely large depending on volume through system) 2. Focusing behavior of nanoparticles in fluid flow not comparable to behavior of microparticles due to differences in action of lift forces based on particle diameter, possibly affecting effectiveness of technique for virus manipulation application

The detection of virus in water has high relevance in water utilities, as techniques for small particle manipulation, specifically dielectrophoresis and inertial focusing, can decimate the risk of contracting water borne diseases. Based on theoretical and empirical investigation, DEP and inertial focusing are both impractical for the up-concentration and subsequent detection of viruses in water samples. Although DEP has the ability to isolate a desired particle based on its intrinsic polarizability which would be extremely advantageous for the viral application discussed, the technique does not operate at a high enough rate or scale that it would be able to be applied to large sample volumes. In addition to this, an external electric force must be applied in order for DEP phenomena to occur which further constrains its implementation.

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On the other hand, inertial focusing is beneficial in that it is able to separate particles based on innate physical properties without the application of an external force. However, the inertial focusing of nanoparticles is not comparable to that of microparticles due to the dependence of lift forces in the microchannel on particle diameter. Though these techniques are both limited in their ability to solve the problem of virus up-concentration alone, DEP and inertial focusing may be more effective when implemented in a combinatorial manner. As both techniques have many advantages and aspects that could be applied to the physical manipulation of viruses, the development of a system in which these aspects can be utilized could create an entirely new area of research. For example, in order to overcome the negligible lift forces experienced by nanoparticles in a microchannel in inertial focusing and the low rate of operation of DEP, insertion of a system of electrodes and application of an electric force of a specific frequency could potentially be implemented in a microchannel. Further research in this area is certain to yield a solution to the detection of viruses and, consequently, mitigate the incidence of viral waterborne diseases across the globe.

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