Assessment of Sub-Micron Particles by Exploiting Charge Differences with Dielectrophoresis

micromachines

Article Assessment of Sub-Micron Particles by Exploiting Charge Differences with Dielectrophoresis

Maria F. Romero-Creel, Eric Goodrich, Danielle V. Polniak and Blanca H. Lapizco-Encinas * ID

Microscale Bioseparations Laboratory and Biomedical Engineering Department, Rochester Institute of Technology, Rochester, NY 14623, USA; [email protected] (M.F.R.-C.); [email protected] (E.G.); [email protected] (D.V.P.) * Correspondence: [email protected]; Tel.: +1-585-475-2773

Received: 7 July 2017; Accepted: 30 July 2017; Published: 2 August 2017

Abstract: The analysis, separation, and enrichment of submicron particles are critical steps in many applications, ranging from bio-sensing to disease diagnostics. Microfluidic electrokinetic techniques, such as dielectrophoresis (DEP) have proved to be excellent platforms for assessment of submicron particles. DEP is the motion of polarizable particles under the presence of a non-uniform electric field. In this work, the polarization and dielectrophoretic behavior of polystyrene particles with diameters ranging for 100 nm to 1 µm were studied employing microchannels for insulator based DEP (iDEP) and low frequency (<1000 Hz) AC and DC electric potentials. In particular, the effects of particle surface charge, in terms of magnitude and type of functionalization, were examined. It was found that the magnitude of particle surface charge has a significant impact on the polarization and dielectrophoretic response of the particles, allowing for successful particle assessment. Traditionally, charge differences are exploited employing electrophoretic techniques and particle separation is achieved by differential migration. The present study demonstrates that differences in the particle’s surface charge can also be exploited by means of iDEP; and that distinct types of nanoparticles can be identified by their polarization and dielectrophoretic behavior. These findings open the possibility for iDEP to be employed as a technique for the analysis of submicron biological particles, where subtle differences in surface charge could allow for rapid particle identification and separation.

Keywords: dielectrophoresis; electrical double layer; electrokinetics; particle polarization

1. Introduction The separation and assessment of submicron particles are essential processes in chemical and biological analysis, with particular importance in the fields of nanotechnology and biotechnology. Microfluidic devices, due to their small size, are an ideal platform for the examination of submicron particles. Electrokinetic (EK) methods have proven to be one of the leading microfluidic techniques for the assessment, separation, and enrichment of nano- to micron-sized particles. Electric field driven techniques offer great flexibility, since a single stimulation force can be used to move both the particles and the suspending medium. Electroosmotic flow (EOF) is commonly used as a liquid and particle pumping mechanism due to the attractive advantage of requiring no mechanical parts, as this phenomenon exploits the electrical double layer (EDL) of the device substrate material [1]. However, EOF requires high voltages, which can lead to undesirable effects such as electrolysis and Joule heating [2–4]. Electrophoresis (EP) is another important EK mechanism that refers to the motion of charged particles relative to the suspending medium, i.e., EP exploits particles’ charge differences to enable particle separations; where distinct particles exhibit differential migration under the influence of an electric field. EP is a well-known phenomenon, used commonly as gel EP for the separation of proteins and DNA in many applications. Other variations of EP, such as capillary EP, isoelectric

Micromachines 2017, 8, 239; doi:10.3390/mi8080239 www.mdpi.com/journal/micromachines Micromachines 2017, 8, 239 2 of 14 focusing, isotachophoresis, electrochromatography, and micellar electrokinetic chromatography, are also commonly used successful techniques for separating nano and micron-sized particles by exploiting electrical charge differences [5,6]. Dielectrophoresis (DEP) is a peculiar electric-field driven technique since it exploits particle polarization effects, not electrical charge, when particles are exposed to a non-uniform electric field [1]. Particles acquire a dipole moment under the effect of an electric field, the poles in this dipole moment are subjected to several forces which results in a net dielectrophoretic force acting on the particle [7]. This method has received significant attention [8–15] due to its flexibility, as DEP can occur in AC and DC electric fields and it can be used to manipulate charged and non-charged particles. There is a plethora of reports available in the literature focused on the dielectrophoretic analysis of macromolecules, viruses, and other submicron particles [16–21]. DEP offers great potential for the separation and enrichment of particles by exploiting slight differences in particle polarization—which is dictated by particle characteristics such as shape, size, and dielectric properties. Depending on the sign of the dipole moment, particles move in distinct directions [7] and can exhibit positive or negative dielectrophoretic behavior. A particle that is more polarizable than the suspending medium will migrate towards the regions of higher electric field gradient under positive DEP; while particles with lower polarizability than the medium would migrate away from these regions due to negative DEP [22]. Particle polarization is a complex phenomenon; many groups have studied the polarization of submicron particles and its relation to particle dielectrophoretic behavior [7,21–30]. Differences in dielectric properties, such as surface charge, can be exploited to achieve effective particle separation. Green and Morgan [25] separated 93 nm diameter particles into two subpopulations by means of DEP with AC potentials at high frequencies (MHz range). In later studies [26,27], this group characterized the dielectrophoretic response of sub-micron particles as a function of electrolyte composition and conductivity, frequency of the applied potentials and particle size. In particular, they analyzed the effects of interfacial polarization mechanism and EDL polarization. The effect of surface functionalization has also been studied [28] demonstrating that a reduction on particle surface conductance leads to changes in the dielectrophoretic behavior of the particles. The DEP response of submicron particles under the influence of high frequency (>1000 Hz) electric potential is well documented in the literature by excellent studies [25–30]. However, the behavior of these particles at low frequencies (<1000 Hz) is still an unexplored area. Insulator-based DEP (iDEP), a dielectrophoretic mode where insulating structures are employed to create non-uniform electric fields [31,32]; usually employs low frequency AC and DC electric potentials to achieve particle manipulation [15,23,33–36]. In iDEP microsystems, particles are exposed to several forces simultaneously (EOF, EP, and DEP), and the resulting particle motion is the net migration caused by the combination of all present forces [37,38]. Particles can be “trapped” between the insulating structures if the applied electric field is high enough to produce DEP forces than can overcome all other present forces [31]; and particles will “stream” along the electric field lines when the generated DEP forces are just comparable to EOF and EP [32]. The interplay between these forces can be fine-tuned to achieve a desired dielectrophoretic process, from particle identification to the enrichment of low-abundant species [39]. The present work is focused on studying how differences in a particle’s electrical charge can be used to achieve dielectrophoretic separation of particles or detect changes in a particle’s surface composition, employing DC and low frequency (<1000 Hz) electric potentials. This low frequency range has not been fully explored in dielectrophoretic microsystems. This work is organized as follows: We first discuss the fundamentals of dielectrophoretic force and particle polarization and the acquisition of a dipole moment. We follow this discussion with mathematical predictions obtained with COMSOL Multiphysics® (Version 4.4, COMSOL Inc., Burlington, MA, USA) of the motion of submicron polystyrene particles under positive and negative DEP. Our experimental results demonstrate that is possible to identify and separate submicron particles with similar characteristics (same size, same Micromachines 2017, 8, 239 3 of 14 shape, and same substrate material) by means of iDEP by exploiting differences in surface charge. These findings open the possibility for iDEP to be employed as a technique for the analysis of submicron biological particles, where subtle differences in surface charge could allow for rapid particle identification and separation.

2. Theory

2.1. Dielectrophoretic Force A dielectric particle immersed in dielectric medium will polarize under the presence of an electric ﬁeld. The effective dipole moment for a spherical particle is given by:

→ → 3 m = 4πεmrp fCM E (1) where εm is the medium permittivity, rp is the particle radius, and fCM is the Clausius-Mossotti factor (fCM). The dielectrophoretic force exerted on a spherical particle is derived from the dipole moment and is a function of particle size and its relative polarizability compared to that of the suspending medium: → 3 2 F DEP = 2πεmrpRe( fCM)∇E (2) where ∇E2 refers to the gradient of the electric field squared. Particle polarizability is expressed through the fCM which is estimated employing the complex permittivities of the particle and the suspending medium [40]: ∗ ∗ ! ε p − εm fCM = ∗ ∗ (3) ε p + 2εm ε∗ = ε − (jσ/ω) (4) where σ and ε are real conductivity and permittivity√ values, respectively, ω is the angular frequency of the applied electric potential, and j = −1. For spherical particles the magnitude of the f CM ranges from −0.5 to 1.0 [40]. The frequency of the applied electric potential is a critical parameter that determines the type of dielectrophoretic response of a particle from positive to negative DEP (Equations (3) and (4)). The cross-over frequency—defined as the frequency at which the DEP force and f CM are both zero—has been extensively used to characterize the dielectrophoretic behavior of submicron particles under AC electric potentials at frequencies above 105 Hz [26,28–30]. Figure1a illustrates the behavior of the real part of the f CM for a solid homogeneous spherical particle with properties similar to those of latex at electric field frequencies between 103 and 109 Hz. The image depicts how the particle behavior shifts from positive to negative DEP with a cross-over frequency between 106 and 107 Hz [26]. It has been reported that the magnitude of particle’s surface charge has significant effects on the cross-over frequency. Green and Morgan [25] separated nanoparticles by means of DEP based on surface charge differences by employing an array of electrodes and high frequency (~20 MHz) electric potentials. In their experiments, particles with a high surface charge had a positive dielectrophoretic response and particles with a low surface charge exhibited negative DEP. Similar results were reported in a later study [28] that was carried out with nanoparticles and high frequency (5 MHz) electric fields, where highly charged unmodified carboxylated particles experienced positive DEP and lowly charged antibody-coated particles displayed negative DEP. The aforementioned studies [25,28] clearly illustrate the effect of surface charge on particle dielectrophoretic behavior under conditions of high frequency electric fields. However, the dielectrophoretic behavior of particles at lower frequencies (<1000 Hz) is still an unexplored subject. The present study focuses on the dielectrophoretic behavior of particles employing DC and low frequency (<1000 Hz) electric Micromachines 2017, 8, 239 4 of 14

frequency (<1000 Hz) electric potentials. Under these conditions the fCM can be simplified in terms of

Micromachinesthe real 2017conductivities, 8, 239 of the particle and the suspending medium [41]: 4 of 14 σ −σ p m (5) f = CM σ + 2σ potentials. Under these conditions the fCM can be simpliﬁedp inm terms of the real conductivities of the particle and the suspending medium [41]: The total particle conductivity (σp) of polystyrene or latex particles can be described by the sum

of the bulk conductivity (σb) and the surface conductanceσp − σm (KS) [42]: fCM = (5) σp + 2σm σ = σ + KS p b 2 (6) The total particle conductivity (σp) of polystyrene or latexrp particles can be described by the sum of the bulk conductivity (σb) and the surface conductance (KS)[42]: Typical values for KS are in the order of 1 nS [30]; although this value seems low, small changes in KS can generate significant changes in KdielecS trophoretic behavior of nanoparticles. σp = σb + 2 (6) Hughes et al. [30] demonstrated that particle surfacerp conductivity is the sum of two surface conductance components. The first component accounts for charge movement in the Stern layer and Typical values for K are in the order of 1 nS [30]; although this value seems low, small changes in the second componentS considers charge movement in the diffuse layer of the EDL. Therefore, K can generate significant changes in dielectrophoretic behavior of nanoparticles. Hughes et al. [30] S particle total conductivity can be estimated as: demonstrated that particle surface conductivity is the sum of two surface conductance components. The first component accounts for charge movementK in the SternK layer and the second component σ = σ + Stern + Diff considers charge movement in the diffusep layerb of the2 EDL. Therefore,2 particle total conductivity can(7) rp rp be estimated as: KStern KDi f f where KStern is the Stern layer conductanceσ = σ + 2 and K+Diff2 is the diffuse layer conductance; a detailed(7) p b r r derivation of these two conductance terms canp be foundp elsewhere [30]. In the case of high whereconductivityKStern is the suspending Stern layer media— conductancewhich anddoesK Diffnotis apply the diffuse to this layer study—interesting conductance; a detailedanomalous derivationbehavior of thesehas been two reported conductance at low terms frequency can be foundcondit elsewhereions where [30 particles]. In the caseexhibit of highpositive conductivity DEP [29]. suspendingFor media—whichestimating particle does total not applyconductivity to this study—interesting(σp) the value of σb anomalous can be considered behavior negligible has been for reportedlatex atand low polystyrene frequency [43]. conditions Ermolina where and particles Morgan exhibit determined positive the DEP values [29]. of KStern and KDiff for latex spheresFor estimating with diameters particle of total 44, 216, conductivity and 996 nm. (σp )Their the valuefindings of σillustratedb can be considered that for smaller negligible particles for the latexmagnitude and polystyrene of KStern [is43 only]. Ermolina slightly andlower Morgan than that determined of larger particles. the values However, of KStern andthe magnitudeKDiff for latex of KDiff spheresfor smaller with diameters particles ofis 44,usually 216, andlarger 996 than nm. that Their of findingslarger particles illustrated (this that effect for smallerincreases particles with medium the magnitudeconductivity) of KStern [43].is only These slightly are two lower opposite than that effects of larger that particles.coupled However,together with the magnitude the effect of KparticleDiff for smallerradius (Equation particles is (7)) usually result larger in smaller than that particles of larger having particles a greater (this effect total increases conductivity with mediumthan larger conductivity)particles. [43]. These are two opposite effects that coupled together with the effect of particle radius (Equation (7)) result in smaller particles having a greater total conductivity than larger particles.

Figure 1. Cont.

Micromachines 2017, 8, 239 5 of 14 Micromachines 2017, 8, 239 5 of 14

Figure 1.Figure(a) Real 1. (a) partReal part of the of the Clausius-Mossotti Clausius-Mossotti factorfactor as as a afunction function of the of theelectric electric field frequency field frequency for a for a solid spherical particle. For this plot, relative permittivities of 2.55 and 78.4 for the particle and the solid spherical particle. For this plot, relative permittivities of 2.55 and 78.4 for the particle and the suspended medium, respectively, as well as conductivities of 10−2 and 10−3 S/m for the particle and −2 −3 suspendedmedium, medium, respectively, respectively, were considered. as well as(b) conductivities Schematic representation of 10 and of the 10 polarizationS/m for of the the particleEDL and medium,for respectively, a negatively werecharged considered. particle immersed (b) Schematic in an representationelectrolyte solution, of the with polarization the formation of theion EDL for a negativelyconcentration charged gradients. particle immersed(c) Schematic in representation an electrolyte of solution, the EDL withpolarization the formation caused by ion particle’s concentration gradients.electrophoretic (c) Schematic motion, representation the arrow coming of the EDLfrom polarizationthe particle represents caused by the particle’s direction electrophoretic of the motion,electrophoretic the arrow coming motion. fromIn both the illustrations particle the represents electric field the direction direction is from of theleft to electrophoretic right. Reprinted motion. from [44], with the permission of AIP Publishing. In both illustrations the electric field direction is from left to right. Reprinted from [44], with the permission2.2. Particle of Polarization AIP Publishing. and Dipole Moment Particle polarization—the core of dielectrophoretic force—is the result of the interaction 2.2. Particlebetween Polarization the non-uniform and Dipole electric Moment field and the induced dipole moment on the particle. The dipole Particlemoment polarization—the depends on the frequency core of of dielectrophoretic the electric field, particle force—is surface the charge result and of the ratio interaction of dielectric between permittivities of the particle and suspending medium. Differences in the dipole moment can be the non-uniform electric field and the induced dipole moment on the particle. The dipole moment exploited to separate or identify distinct types of particles [7]. depends onIn thethis frequencystudy, the polarization of the electric and dielectrop field, particlehoretic response surface of charge several andtypes ratio of charged of dielectric permittivitiespolystyrene of theparticles particle were and assessed. suspending When particle medium.s are immersed Differences in an in electrolyte the dipole solution, moment they can be exploiteddevelop to separate an EDL. or The identify ions contained distinct in typesthe EDL of expe particlesrience [migration7]. under the action of an electric Infield. this This study, generates the polarization an EOF on the andparticle dielectrophoretic surface and produces response an ionic ofcurrent several along types the surface of charged polystyreneof the particlesparticle—resulting were assessed. in the polarization When particles of the EDL—which are immersed in turn inalters an the electrolyte magnitude solution, of the they dipole moment. Figure 1b depicts a schematic representation of the EDL polarization for a develop an EDL. The ions contained in the EDL experience migration under the action of an electric negatively charged particle [44], where there is migration and convection of ions along the electric field. Thisfield generates direction ( an is EOF from on left the to right), particle and surface there is an and exchange produces of ions an between ionic current the diffuse along layer the and surface of the particle—resultingthe electrolyte. Negative in the polarization co-ions (blue of color) the EDL—whichmigrate upstream in turn while alters positive the magnitudecounter-ions of (red the dipole moment.color) Figure migrate1b depicts downstream, a schematic producing representation a depletion of ofions the upstream EDL polarization of the particle for (salt a negatively sink) and an charged excess of ions downstream of the particle (salt source). This produces a phenomenon called “ion→ particle [44], where there is migration and convection of ions along the electric field direction (E is from concentration polarization”, which is a gradient in ion concentration established along the length left to right),scale of and the thereparticle is [7,44,45]. an exchange Furthermore, of ions the between charged the particle diffuse will layer also exhibit and the an electrolyte.electrophoretic Negative co-ionsmotion (blue color)under the migrate influence upstream of the electric while field; positive for a negatively counter-ions charged (red particle color) the migrate EP migration downstream, is producingtowards a depletion the upstream of ions direction. upstream The electrophoretic of the particle motion (salt enhances sink) and the polarization an excess of of ionsthe EDL, downstream as of the particledepicted (saltin Figure source). 1c, where This producesan arrow coming a phenomenon from the calledparticle “ionis used concentration to illustrate particle polarization”, which iselectrophoretic a gradient invelocity ion concentration [7,44,45]. established along the length scale of the particle [7,44,45]. The thickness of the EDL—relative to particle size—plays a major role in particle polarization. Furthermore, the charged particle will also exhibit an electrophoretic motion under the influence of Several important theories have been developed to describe the interplay between the EDL the electricthickness, field; the for ion a negatively exchange between charged the particle EDL, theand EPthe migrationbulk electrolyte is towards and the the dipole upstream moment direction. The electrophoretic[7,44,45]. For our motion current enhances operating the conditions polarization of low of thefrequency EDL, as(<1000 depicted Hz) electric in Figure fields,1c, the where an arrow comingDunkhin-Shilov from the (DS) particle theory isis usedrelevant. to illustrateThis theory, particle which is electrophoretic restricted to thin velocity EDLs, assumes [7,44,45 that]. Thethe thickness EDL is at of quasi the EDL—relativeequilibrium with to the particle suspending size—plays electrolyte, a major since rolethe inions—due particle to polarization. low frequency condition—have enough time to reach local equilibrium. That is, the DS theory Several important theories have been developed to describe the interplay between the EDL thickness, successfully predicts low frequency dispersion [22,44]. the ion exchange between the EDL, and the bulk electrolyte and the dipole moment [7,44,45]. For our current operating conditions of low frequency (<1000 Hz) electric fields, the Dunkhin-Shilov (DS) theory is relevant. This theory, which is restricted to thin EDLs, assumes that the EDL is at quasi equilibrium with the suspending electrolyte, since the ions—due to low frequency condition—have enough time to reach local equilibrium. That is, the DS theory successfully predicts low frequency dispersion [22,44]. Micromachines 2017, 8, 239 6 of 14

However, the effects of particle electrophoretic motion on the dipole moment are not fully considered by the DS theory, since it assumes that particles are stationary, which can only be justified when λD ≥ 0.1 (λD is Debye length normalized by the particle radius) [7,22,44]. For the current experimental conditions the values of λD for 100 nm and 200 nm diameter particles are 0.267 and 0.133, respectively, considering the 0.02 mM NaCl suspending medium employed. Therefore, for these particles, the electrophoretic contribution to the dipole moment has to be considered, since λD ≥ 0.1. In contrast, for the larger particles utilized in this study (500 nm and 1 µm in diameter), electrophoretic effects are not as significant since λD ≤ 0.1. This is in agreement with previous results by our group, where particles with diameter ≤200 nm exhibited positive dielectrophoretic behavior and particles with diameter ≥500 nm showed negative DEP [23]. However, in that previous report [23], all particles had a high surface charge. In the present study, some of the particles analyzed possess lower surface charge. The magnitude of the particle’s surface charge alters the particle zeta potential (ζp) and the particle dipole moment. Zhao [44] analyzed the effect of ζp on the dipole moment coefficient; for these predictions a normalized zeta potential (ζ) was employed, which is defined as ζp normalized with the thermal voltage (~25 mV). Zhao reported that at low values of ζ the dipole moment coefficient shifts from negative to positive, almost in a linear fashion, and as ζ increases, it reaches a maxima at ζ ~ 4 (ζp ~ 100 mV). These estimations were valid for λD = 0.1 and λD = 0.3, which apply to our system [44]. Considering these findings, a particle with a lower surface charge would have a lower value for ζp, which in turn would lead to a lower and perhaps negative value for the particle dipole moment coefficient, possibly producing a negative dielectrophoretic force on the particle. On the contrary, particles with higher surface charge would have a higher value of ζp, resulting in a positive particle dipole moment coefficient, leading to a positive dielectrophoretic force on the particle. For a detailed discussion and derivation of the particle dipole moment, the reader is encouraged to review the report by Zhao [44]. In summary, the smaller particles in our study (100 nm and 200 nm in diameter) can exhibit positive or negative DEP depending on the magnitude of their surface charge. If the particle bears a sufficiently high surface charge (high ζp), then electrophoretic motion can further enhance the particle dipole moment producing positive DEP effects on the particle. For a lowly charged particle (low ζp), the particle’s electrophoretic motion is not sufficient to enhance the particle dipole moment, leading to a negative dipole moment and a negative dielectrophoretic particle motion [44]. Under our experimental conditions, larger particles (500 nm and 1 µm diameter) will always exhibit negative dielectrophoretic behavior due to their size.

2.3. Particle Behavior in iDEP In iDEP systems, particles are exposed to linear EK (superposition of EP and EOF) and DEP forces. Many iDEP systems usually consist of a microchannel with an embedded array of insulating structures, such as the one illustrated in Figure2a. Our group has studied particle polarization in these types of devices employing a mathematical model built with COMSOL Multiphysics® and extensive experimentation [23]. Since particles are affected by linear EK and DEP, particle velocity, which accounts for both forces, is an ideal parameter for predicting the regions where particles will gather under the influence of an electric field in an iDEP system. Considering a microchannel with cylindrical insulating structures, positive DEP would lead to particle clustering at the center of the constriction between the posts, where the particle velocity is the highest—since particles are attracted to these regions, as depicted in Figure2b. In contrast, negative DEP would produce a region of particle depletion at the constriction between the posts, since the particles are being repelled from the regions with the higher particle velocity, as illustrated in Figure2c. Details about the COMSOL model are published elsewhere [46]. These particular simulations are for 100 nm particle under the effect of 3000 V applied across the length of the microchannel illustrated in Figure1a. For the positive dielectrophoretic behavior image, a combined zeta potential ζc (defined as ζc = ζp + ζw) of −15 mV was assumed, considering a particle with high negative charge, where ζw represents the wall zeta Micromachines 2017, 8, 239 7 of 14

Micromachines 2017, 8, 239 7 of 14 potential. For the negative dielectrophoretic behavior image, it was assumed that the particle has a lowparticle charge has depicted a low charge by a ζ cdepicted= −75 mV. by Thea ζc = use −75 of mV. the The zeta use potentials of the zeta ensured potentials that aensured ﬂat velocity that a proﬁleflat wasvelocity considered profile for was EOF considered in the COMSOL for EOF in simulations. the COMSOL simulations.

FigureFigure 2. 2.(a ()a Schematic) Schematic representation representation ofof thethe iDEPiDEP micr microchannelochannel used used for for experimentation, experimentation, the the image image depicts the channel, the array of cylindrical insulating posts, the inlet and outlet reservoirs, as well as depicts the channel, the array of cylindrical insulating posts, the inlet and outlet reservoirs, as well the location of the electrodes. (b) Modeling of particle velocity with COMSOL Multiphysics® to as the location of the electrodes. (b) Modeling of particle velocity with COMSOL Multiphysics® to illustrate positive DEP where particles cluster at the center of the constriction between the cylindrical illustrate positive DEP where particles cluster at the center of the constriction between the cylindrical posts. (c) Modeling of negative DEP where particles are repelled from the constriction region, posts. (c) Modeling of negative DEP where particles are repelled from the constriction region, creating creating a region depleted of particles at the constriction between the cylindrical posts. a region depleted of particles at the constriction between the cylindrical posts. 3. Materials and Methods 3. Materials and Methods 3.1. Microfluidic Devices 3.1. Microfluidic Devices Experiments were conducted with microchannels made from Polydimethylsiloxane (PDMS, DowExperiments Corning, Midland, were conducted MI, USA) withusing microchannelsstandard soft lithography made from techniques Polydimethylsiloxane [47]. A mold for (PDMS, the Dowmicrochannels Corning, Midland, was created MI, USA)using usinga silicon standard wafer soft (Silicon lithography Inc., Boise, techniques ID, USA) [47 and]. A moldSU-8 3050 for the microchannelsphotoresist (MicroChem, was created usingNewton, a silicon MA, USA). wafer PDMS (Silicon wa Inc.,s then Boise, casted ID, onto USA) the and SU-8 SU-8 negative 3050 photoresist replica (MicroChem,and once cured, Newton, sealed MA, onto USA). a 10-cm PDMS PDMS was coated then glass casted wafer onto using the a SU-8 plasma negative corona replica wand (Electro and once cured,Technic sealed Products, onto a Chicago, 10-cm PDMS IL, USA) coated to activate glass wafer both PDMS using asurfaces. plasma The corona microchannels wand (Electro employed Technic Products,consisted Chicago, of 10.16 IL,mm USA) long tochannels, activate 1 bothmm wide, PDMS 40 surfaces. μm deep Theand microchannelscontained one inlet employed and one consisted outlet ofliquid 10.16 mmreservoirs. long channels, The cylindrical 1 mm posts wide, employed 40 µm deep were and 200 contained μm in diameter one inlet and and arranged one outlet 220 liquidμm reservoirs.center to Thecenter. cylindrical The spacing posts between employed the wereposts 200and µthem inchannel diameter wall and was arranged 10 μm. The 220 arrayµm center of tocylindrical center. The insulating spacing betweenstructures the consisted posts and of 72 the posts channel located wall in the was middle 10 µm. of Thethe channel, array of arranged cylindrical insulatingas 18 rows structures of four posts consisted each (Figure of 72 posts 2a). The located first incolumn the middle of posts of featured the channel, a “dove arranged tail” geometry as 18 rows ofwhich four posts was eachadded (Figure to prevent2a). The clogging first column in the ofchan postsnel featuredby having a “doveparticles tail” splattered geometry on which the first was addedcolumn to prevent of posts. clogging in the channel by having particles splattered on the first column of posts.

3.2.3.2. Particles Particles and and Suspending Suspending Media Media FluorescentFluorescent polystyrene polystyrene microspheresmicrospheres of sizes ranging ranging from from 0.1 0.1 to to 1.0 1.0 μmµm obtained obtained from from two two different providers—Invitrogen (Eugene, OR, USA) and Magsphere (Pasadena, CA, USA)—were different providers—Invitrogen (Eugene, OR, USA) and Magsphere (Pasadena, CA, USA)—were employed. The microspheres ranged in surface charge magnitude, fluorescence color, type of surface employed. The microspheres ranged in surface charge magnitude, fluorescence color, type of surface functionalization and concentrations employed. Specific details of the microparticles used are listed functionalization and concentrations employed. Specific details of the microparticles used are listed in in Table 1. Table1. The suspending medium used in experimentation consisted of diluted salt solutions of K2HPO4 or NaCl.The The suspending pH and conductivity medium used were in experimentationadjusted by the addition consisted of a of 0.1 diluted N KOH salt solution solutions to a ofpH K 2~HPO 7.5 4 orand NaCl. a conductivity The pH and in conductivitythe range of 35–100 were μ adjustedS/cm. Tween by the 20 surfactant addition ofwas a also 0.1 Nadded KOH to solution the solution to a at pH ~7.5a concentration and a conductivity of 0.05% in vol/vol the range in order of 35–100 to minimiµS/cm.ze aggregation Tween 20 of surfactant particles. wasSpecific also information added to the solutionabout the at abuffer concentration solution used of 0.05% in each vol/vol experiment in order is toincluded minimize in the aggregation figure legends of particles. in the results Specific informationsection. In aboutorder theto ensure buffer stable solution electroosmotic used in each flow experiment and avoid is adhesion included of in particles the figure to legends the surface in the of the channel, devices were soaked in buffer two hours prior to experimentation.

Micromachines 2017, 8, 239 8 of 14 results section. In order to ensure stable electroosmotic ﬂow and avoid adhesion of particles to the surface of the channel, devices were soaked in buffer two hours prior to experimentation.

Table 1. Fluorescent polystyrene microspheres information including diameter (µm), fluorescence (Ex/Em), provider, charge (meq/g), type of surface functionalization and particle concentration employed.

Diameter Fluorescence Charge Concentration Provider Surface Functionalization (µm) (Ex/Em) (meq/g) (Particles/mL) 0.1 Green (480/501) Magsphere Not Available Amine 9.09 × 1011 0.1 Green (480/501) Magsphere 0.0680 Carboxyl 9.09 × 1011 0.1 Red (580/605) Invitrogen 0.3850 Carboxyl 9.09 × 1011 0.2 Green (505/515) Invitrogen 0.5810 Carboxyl 5.68 × 1010 0.2 Red (538/584) Magsphere Not Available Amine 5.68 × 1010 0.5 Green (480/501) Magsphere Not Available Amine 1.46 × 109 0.5 Red (580/605) Invitrogen 0.3160 Carboxyl 1.46 × 109 1.0 Green (505/515) Invitrogen 0.0227 Amine 2.18 × 108 1.0 Green (505/515) Invitrogen 0.1826 Carboxyl 2.18 × 108 1.0 Green (505/515) Invitrogen 0.0170 Sulfate 2.18 × 108

3.3. Equipment and Software All experiments were imaged using a Leica DMi8 inverted microscope (Leica Microsystems Wetzlar, Wetzlar, Germany) and a Leica DFC7000 T video camera. The microscope was equipped with two fluorescence filter sets: a GFP filter set and a Texas Red (TXR) filter set. Two distinct voltage supplies were employed to apply AC or DC electric potentials. A waveform generator (Agilent 33500 Series, Santa Clara, CA, USA) and amplifier (Model: PZD700A2, Trek Inc., Lockport, NY, USA) were used to apply AC electric potentials up to 3600 V peak-to-peak (Vpp). DC potentials up to 6000 V and low frequency (<1000 Hz) AC potentials were applied with a voltage sequencer (Model HVS6000D; LabSmith, Livermore, CA, USA).

3.4. Experimental Procedure Experiments were conducted using a clean microchannel ﬁlled with the selected suspending medium. The channels were reversibly sealed to a vacuum chuck manifold (LabSmith, Livermore, CA, USA) employing a vacuum pump (Model 400–3910; Barnant Company, Barrington, IL, USA). Platinum wire electrodes were placed at the reservoirs and an electric potential was applied to generate a dielectrophoretic response in the particles. For all experiments, a sample of 10 µL of the particle suspension was employed (Table1). Particle response was observed and recorded for every trial using the inverted microscope and video camera. The microchannels were re-conditioned in between uses by soaking them in 0.1 N KOH for 2 h and then soaked in DI water for 1 h in order to ensure a negative surface charge on the PDMS surface and a stable electroosmotic ﬂow. To avoid any undesired effects (e.g., electrolysis, Joule heating, etc.) due to the high applied voltages, all experiments were limited to 30–60 s. During this time window no negative effects were observed.

4. Results and Discussion

4.1. Effect of Particle Size on Polarization The effect of size in dielectrophoretic trapping has been explored by our group before [23]. It was shown that under DC and low frequency AC potentials sub-micron particles could exhibit positive or negative dielectrophoretic behavior. Highly charged polystyrene particles with diameters <200 nm exhibited positive DEP, while particles with diameters >500 nm exhibited negative DEP [23], a particle size threshold for the shift from positive to negative DEP was observed to be between 200 nm and 500 nm diameter Micromachines 2017, 8, 239 9 of 14

In order to assess the effect of the magnitude of the particle’s surface charge and type of surface functionalization on dielectrophoretic behavior, experiments with particles over the size threshold of 500 nm in diameter were conducted. Due to their size, these particles are expected to depict negative DEP. Particles with amine, carboxyl, and sulfate surface functionalizations, with diameters Micromachines 2017, 8, 239 9 of 14 of 500 nm and 1 µm, and surface charges ranging from 0.01 to 0.18 meq/g (Table1) were exposed to low frequencyDEP. Particles AC potentials. with amine, Ascarboxyl, illustrated and sulfate in Figure surface3, allfunctionalizations, five distinct particle with diameters types employedof 500 in this setnm of experimentsand 1 μm, and exhibited surface charges negative ranging dielectrophoretic from 0.01 to 0.18 motion.meq/g (Table A clear 1) were depletion exposed ofto particleslow is frequency AC potentials. As illustrated in Figure 3, all five distinct particle types employed in this observed in the constriction between the insulating posts; where the highest electric field gradient is set of experiments exhibited negative dielectrophoretic motion. A clear depletion of particles is present.observed Figure3 ina,b the show constriction the behavior between for the 500-nminsulating particles, posts; where and the Figure highest3c–e electric display field thegradient behavior is for 1 µm particles.present. Figure It is important 3a,b show the to notebehavior that for a 500-nm 0.4 mM particles, K2HPO and4 buffer Figure with3c–e display a higher the ionicbehavior strength for was used for1 theμm experimentsparticles. It is important conducted to note with that 1 µam 0.4 particles, mM K2HPO to4 strengthenbuffer with a thehigher negative ionic strength dielectrophoretic was behavior;used in for particular the experiments the behavior conducted shown with 1 inμm Figure particles,3e to is strengthen a very well-defined the negative dielectrophoretic depiction of negative DEP. Frombehavior; these in findings, particular itthe is behavior demonstrated shown in that Figure due 3e tois a their very largewell-defined size these depiction particles of negative will exhibit DEP. From these findings, it is demonstrated that due to their large size these particles will exhibit negative dielectrophoretic motion regardless of particle functionalization (amine, carboxyl, or sulfate) negative dielectrophoretic motion regardless of particle functionalization (amine, carboxyl, or or magnitudesulfate) ofor surfacemagnitude charge. of surface These charge. results These are results in agreement are in agreement with awith previous a previous publication publication from our group [17from]. Theour group next section [17]. The reports next section the experiments reports the experiments conducted conducted with submicron with submicron particles particles (diameter < 500 nm),(diameter where two< 500 distinct nm), where types two of distinct dielectrophoretic types of dielectrophoretic behaviors behaviors were observed. were observed.

Figure 3.FigureNegative 3. Negative dielectrophoretic dielectrophoretic response response ofof 500500 nm nm and and 1 μ 1mµ mparticles particles under under AC potentials—all AC potentials—all images show particle depletion at the constrictions between the cylindrical posts. All of these images show particle depletion at the constrictions between the cylindrical posts. All of these particles particles are from Invitrogen. (a) 500 nm green aminated particles at 2800 Vpp and 70 Hz. (b) 500 nm are fromred Invitrogen. carboxylated (particlesa) 500 nmat 2800 green Vpp aminatedand 70 Hz. ( particlesc) 1 μm green at 2800carboxylated Vpp and particles 70 Hz. 2800 (Vppb) 500 and nm red carboxylated1000 Hz. particles (d) 1 μm atgreen 2800 aminated Vpp andparticles 70 Hz. at 2800 (c) Vpp 1 µ mand green 1000 Hz. carboxylated (e) 1 μm green particles sulfated particles 2800 Vpp and 1000 Hz.at (2000d) 1 Vppµm and green 1000 aminated Hz. The suspending particles atmediums 2800 Vpp employed and 1000 were Hz.0.02 (mMe) 1 NaClµm greenwith pH sulfated = 7.5 and particles at 2000 Vppconductivity and 1000 = 35 Hz. μS/cm The for suspending the 500 nm particles mediums (images employed a–b); and were 0.4 mM 0.02 K mM2HPO NaCl4 with withpH =7.5 pH and = 7.5 and conductivity = 100 μS/cm for the 1 μm particles (images c–e). conductivity = 35 µS/cm for the 500 nm particles (images a–b); and 0.4 mM K2HPO4 with pH =7.5 and conductivity = 100 µS/cm for the 1 µm particles (images c–e). 4.2. Effect of Particle Charge Magnitude for Smaller Particles (dp < 500 nm)

4.2. Effect ofThe Particle effect Charge of surface Magnitude charge on forthe Smallerdielectrophore Particlestic motion (dp < 500of particles nm) with diameters <500 nm was studied experimentally as a function of surface charge magnitude and type of functionalization The(amine effect and of surface carboxyl). charge Microspheres on the dielectrophoretic obtained from two motion different of particles providers, with Invitrogen diameters and <500 nm was studiedMagsphere experimentally (Table 1) were as aemployed, function where of surface Invitr chargeogen particles magnitude were observed and type to of have functionalization higher surface charge magnitudes than Magsphere particles by an order of magnitude, as shown in the (amine and carboxyl). Microspheres obtained from two different providers, Invitrogen and Magsphere Supplementary Information (Table S1). Submicron particles, 100 nm and 200 nm diameter in size, (Table1) were employed, where Invitrogen particles were observed to have higher surface charge magnitudes than Magsphere particles by an order of magnitude, as shown in the Supplementary Micromachines 2017, 8, 239 10 of 14

Information (Table S1). Submicron particles, 100 nm and 200 nm diameter in size, from both providers were observed to exhibit distinct types of dielectrophoretic behavior under low frequency AC signals.

It was assumedMicromachines that 2017 particles, 8, 239 provided by Magsphere had low surface charge, while particles10 of provided14 by Invitrogen carried higher surface charges, as defined by data provided by the manufacturer (Table1). The resultsfrom fromboth providers these experiments were observed are to includedexhibit dist ininct Figure types of4. dielectrophoretic It was observed behavior that particlesunder low with a frequency AC signals. It was assumed that particles provided by Magsphere had low surface charge, low surface charge magnitude (Figure4a,d) exhibit negative dielectrophoretic behavior—as depicted while particles provided by Invitrogen carried higher surface charges, as defined by data provided by particleby the depletion manufacturer at the (Table constriction 1). The results between from thethese posts. experiments This is are in included agreement in Figure with 4. the It was discussion presentedobserved in Section that particles2.2, where with it wasa low explained surface charge that lowmagnitude charge (Figure leads to4a,d) a lowexhibitζp fornegative the particle, which indielectrophoretic turn decreases behavior—as the electrophoretic depicted by contribution particle depletion to the at the dipole constriction moment. between Although the posts. the exact charge magnitudeThis is in agreement is unknown, with the these discussion aminated presente particlesd in Section are expected2.2, where it to was have explained lower surfacethat low charge charge leads to a low ζp for the particle, which in turn decreases the electrophoretic contribution to magnitude than carboxylated particles. Microsphere manufacturers have very robust processes and the dipole moment. Although the exact charge magnitude is unknown, these aminated particles are experienceexpected producing to have highly lower charged surface carboxylatedcharge magnitude particles, than whilecarboxylated usually particles. lowercharge Microsphere densities are achievedmanufacturers for other functional have very groupsrobust processes such as and amine experience and sulfate. producing Meanwhile, highly charged carboxylated carboxylated particles which possessparticles, a while higher usually surface lower charge charge magnitude densities are (Figure achieved4b,c,e) for presentedother functional positive groups dielectrophoretic such as particleamine capture, and depicted sulfate. Meanwhile, by the formation carboxylated of a clusterparticles of which particles possess at thea higher center surface of the charge constrictions betweenmagnitude two posts. (Figure These 4b,c,e) findings presented are in agreementpositive dielec withtrophoretic the discussion particle includedcapture, depicted in Section by 2.2the. Highly formation of a cluster of particles at the center of the constrictions between two posts. These findings charged particles possess a higher ζ , which leads to enhancement of the particle dipole moment due are in agreement with the discussionp included in Section 2.2. Highly charged particles possess a to particlehigher electrophoretic ζp, which leads motion. to enhancement These resultsof the particle illustrate dipole that moment the magnitude due to particle of theelectrophoretic surface charge is the mainmotion. parameter These dictating results illustrate the dielectrophoretic that the magnitud responsee of the ofsurface the particles charge is in the this main size parameter range, while the type of thedictating surface the functionalizationdielectrophoretic response does not of seemthe part toicles have in a this significant size range, effect. while That the is,type particles of the below the sizesurface threshold functionalization of 500 nm could does not exhibit seem positiveto have a orsignificant negative effect. dielectrophoretic That is, particles behaviorbelow the dependingsize on the magnitudethreshold of of500 their nm could surface exhibit charge, positive while or nega largertive particles dielectrophoretic (Figure 3be)havior would depending exhibit only on the negative magnitude of their surface charge, while larger particles (Figure 3) would exhibit only negative dielectrophoreticdielectrophoretic behavior behavior dictated dictated by by their their size,size, rather than than the the magnitude magnitude of the of surface the surface charge charge presentpresent on the on particle. the particle.

Figure 4.FigureDielectrophoretic 4. Dielectrophoretic response response ofof 100 nm nm and and 200 200nm particles nm particles under AC under potentials. AC potentials.Images a and Imagesd a and d correspondcorrespond to lowly charged charged particles particles and and illustra illustratete negative negative dielectrophoretic dielectrophoretic response response with a with a depletion of particles at the constriction between the posts. Images b, c, and e correspond to highly depletion of particles at the constriction between the posts. Images b, c, and e correspond to highly charged particles and illustrate positive dielectrophoretic response, where particles form a cluster at chargedthe particles constriction and illustratebetween positivethe posts. dielectrophoretic (a) Negative DEP—100 response, nm wheregreen aminated particles particles form a clusterfrom at the constrictionMagsphere between at 2800 the Vpp posts. and (a 1000) Negative Hz. (b) Positive DEP—100 DEP—100 nm green nm green aminated carboxylated particles particles from from Magsphere at 2800 VppMagsphere and 1000at 2800 Hz. Vpp (b )and Positive 1000 Hz. DEP—100 (c) Positive nm DEP—100 green carboxylated nm red carboxylated particles particles from from Magsphere at 2800Invitrogen Vpp and 1000at 2800 Hz. Vpp (c )and Positive 1000 Hz. DEP—100 (d) Negative nm redDEP— carboxylated200 nm red aminated particles particles from Invitrogen from at 2800 VppMagsphere and 1000 at Hz. 3600 (d Vpp) Negative and 200 DEP—200Hz. (e) Positive nm red DEP—200 aminated nm particlesgreen carboxylated from Magsphere particles atfrom 3600 Vpp and 200Invitrogen Hz. (e) Positiveat 3000 Vpp DEP—200 and 200 Hz. nm All greenexperiments carboxylated were carried particles out with from0.02 mM Invitrogen NaCl with at pH 3000 = Vpp 7.5 and conductivity = 35 μS/cm as suspending medium. and 200 Hz. All experiments were carried out with 0.02 mM NaCl with pH = 7.5 and conductivity = 35 µ S/cm as suspending medium. Micromachines 2017, 8, 239 11 of 14

4.3. Application: Dielectrophoretic Differentiation of Sub-Micron Particles by Exploiting Charge Differences MicromachinesThe previous 2017, experiments8, 239 demonstrated that particles with different surface charge will11 of 14 exhibit distinct4.3. Application: dielectrophoretic Dielectrophoretic behaviors. Differentiation To further of Sub-Micron assess the Particl potentiales by Exploiting of exploiting Charge charge Differences difference by means of iDEP, a set of DC-iDEP experiments were conducted using a mixture of two types of particlesThe of theprevious same experiments size, same demonstrated shape, and samethat partic substrateles with material, different surface but different charge will surface exhibit charge distinct dielectrophoretic behaviors. To further assess the potential of exploiting charge difference by magnitude (Table1). The mixture contained 100 nm-red-carboxylated particles by Invitrogen and means of iDEP, a set of DC-iDEP experiments were conducted using a mixture of two types of 100 nm-green-aminatedparticles of the same size, particles same shape, by Magsphere. and same substrate The red-carboxyl material, but particles different insurface the mixturecharge are consideredmagnitude to have (Table a higher1). The surfacemixture charge contained than 100 the nm-red-carboxylated green-amine particles, particles as depicted by Invitrogen by the and results in Figure1004 a,cnm-green-aminated under AC electric particles fields. by Magsphere. The red-carboxyl particles in the mixture are consideredFigure5 illustrates to have a thehigher behavior surface of charge the mixture than the of green-amine two types ofparticles, 100 nm as particles depicted exposedby the results to 1300 V DC andin Figure observed 4a,c under under AC two electric different fields. fluorescence filter sets (GFP filter and TXR filter). The use of two differentFigure filters 5 illustrates allowed the forbehavior visualization of the mixture of each of two individual types of 100 type nm of particles particle exposed within to the 1300 mixture. FigureV DC5a showsand observed the mixture under two under different the TXR fluorescence filter to focusfilter sets only (GFP on filter the red and particles, TXR filter). while The use Figure of 5b wastwo taken different with the filters GFP allowed filter toforfocus visualization only on of the each green individual particles. type Furtherof particle analysis within the of thesemixture. particle Figure 5a shows the mixture under the TXR filter to focus only on the red particles, while Figure 5b images suggests that the red-carboxyl particles—which bear a higher surface charge—exhibit positive was taken with the GFP filter to focus only on the green particles. Further analysis of these particle DEP;images as these suggests particles thatare the fully red-carboxyl trapped partic at theles—which applied potential—thesebear a higher surface are no charge—exhibit particles escaping the dielectrophoreticpositive DEP; as these trap. particles This illustrates are fully thattrapped DEP at isthe able applied to overcome potential—these EK under are theseno particles conditions, meaningescaping that the electrophoretic dielectrophoretic motion trap. (whichThis illustrate is opposites that DEP to EOF) is able is sufficientto overcome to decreaseEK under thethese overall magnitudeconditions, of EKmeaning towards that theelectrophoretic outlet. In motion contrast, (which Figure is opposite5b shows to EOF) the behavior is sufficient of to lowly decrease charged particles,the overall where magnitude DEP is not of EK able towards to overcome the outlet. EK In completely contrast, Figure (EP does5b shows not sufficientlythe behavior decreaseof lowly EK), andcharged particles particles, are escaping where the DEP dielectrophoretic is not able to overcome traps—as EK evidenced completely by the(EP greendoes not fluorescent sufficiently particle streamdecrease that comes EK), and out ofparticles each particleare escaping cluster. the It dielectrophoretic is important to notetraps—as that theevidenced clusters by of the green green particles featurefluorescent a “round” particle back, stream thismeans that comes that out these of particleseach particle are cluster. more affected It is important by EK motionto note that than the the red clusters of green particles feature a “round” back, this means that these particles are more affected ones. As previously reported by our group [46], clusters of particles that exhibit negative DEP have by EK motion than the red ones. As previously reported by our group [46], clusters of particles that a “back”exhibit (upstream negative DEP direction) have a that“back” is defined(upstream by direction) EK, which that results is defined in a by round EK, which shape. results The clusters in a of greenround particles shape. also The feature clusters a of flatter green “front” particles than also the feature red particles,a flatter “front” which than further the red supports particles, that which the green particlesfurther are supports exhibiting that negative the green DEP particles [46]. Figureare exhibi5c presentsting negative the stacking DEP [46]. of Figure the images 5c presents in Figure the 5a,b, wherestacking the leakage of the images of green in Figure particles 5a,b, is where evident. the leakage of green particles is evident.

Figure 5. Dielectrophoretic response of a mixture of two types of 100 nm particles under a DC electric Figure 5. Dielectrophoretic response of a mixture of two types of 100 nm particles under a DC electric potential of 1300 V. The particle mixture is composed of 100 nm red carboxylated particles (high potentialcharge) of 1300from V.Invitrogen The particle and mixture100 nm isgreen composed aminated of 100particles nm red from carboxylated Magsphere particles(low charge). (high (a charge)) fromImage Invitrogen showing and only 100 the nm red green polystyrene aminated particles particles (obtained from Magsphere with the TXR (low filter) charge). exhibiting (a) Image positive showing onlyDEP. the red(b) Image polystyrene showing particles only the (obtainedgreen aminated with partic the TXRles (obtained ﬁlter) exhibiting with the GFP positive filter) DEP.exhibiting (b) Image showingnegative only DEP. the (c green) Image aminated depicting particlesboth particle (obtained types, obtained with the by stacking GFP ﬁlter) the two exhibiting previous negative images. DEP. (c) ImageThis experiment depicting bothwas carried particle out types, with obtained0.02 mM byNaCl stacking with pH the = two7.5 and previous conductivity images. = 35 This μS/cm experiment as was carriedsuspending out medium. with 0.02 mM NaCl with pH = 7.5 and conductivity = 35 µS/cm as suspending medium.

This mixture of two distinct types of 100 nm particles (low charge vs. high charge) was also analyzedThis mixture under ofAC two potentials; distinct these types results of 100 are nm included particles in Figure (low charge6. It can vs. be observed high charge) from wasthe also analyzedfigure under that the AC red potentials; particles (positive these results DEP) areare includedtrapped at in the Figure center6 .of It the can constriction be observed between from the two ﬁgure that the red particles (positive DEP) are trapped at the center of the constriction between two posts.

MicromachinesMicromachines 20172017,, 8,, 239 239 1212 of of 14 14 posts. Conversely, the green particles (negative DEP) exhibit a weaker dielectrophoretic response andConversely, are not clustered the green at particles the center (negative of the DEP)constriction; exhibit ainstead weaker they dielectrophoretic are gathered responseto the left and of the are red not particlesclustered at at one the of center the constrictions. of the constriction; Furthermore, instead they no appreciable are gathered dielectrophoretic to the left of the redresponse particles from at onethe greenof the particles constrictions. is found Furthermore, at the other no constrictions appreciable dielectrophoreticbetween the posts. response Since negative from the DEP green is particlesparticle repulsion—notis found at the otherparticle constrictions trapping—it between is more the challenging posts. Since to negative observe, DEP as highlighted is particle repulsion—notby Green and Morganparticle trapping—it[25] in their is morework challengingwith similar to observe,subpopu aslations highlighted of particles by Green of andthe Morgansame size [25 ](93 in theirnm diameter).work with similarOur 100 subpopulations nm particles of particlesare small, of thewhich same makes size (93 it nm difficult diameter). to Ourgenerate 100 nm sufficient particles dielectrophoreticare small, which makesforce itto difﬁcult produce to discernible generate sufﬁcient particle dielectrophoretic movement and forcecreate to produceregions of discernible particle depletionparticle movement in our current and createexperimental regions ofset-up. particle depletion in our current experimental set-up.

FigureFigure 6. Dielectrophoretic responseresponse ofof a a mixture mixture of of two two types types of of 100 100 nm nm particles particles under under an appliedan applied AC ACelectric electric potential potential of 3200 of Vpp3200 at Vpp 1000 at Hz. 1000 The Hz. particle The mixtureparticle ismixture composed is ofcomposed 100 nm redof carboxylated100 nm red carboxylatedparticles (high particles charge—positive (high charge—positive DEP) from Invitrogen DEP) from and Invitrogen 100 nm green and 100 aminated nm green particles aminated from particlesMagsphere from (low Magsphere charge—negative (low charge—negative DEP). The red DEP) particles. The exhibit red particles a clear exhibit positive a clear DEP behaviorpositive DEP and behaviorare clustered and atare the clustered center of at each the constriction, center of each while constriction, the green particles while the depict green a weaker particles response depict ofa weakernegative response DEP (slight of negative repulsion DEP from (slight the constriction repulsion from region). the Thisconstriction experiment region). was This carried experiment out with was0.02 mMcarried NaCl out with with pH 0.02 = 7.5 mM and NaCl conductivity with pH = = 35 7.5µS/cm and asconductivity suspending = medium.35 μS/cm as suspending medium. The results from this experiment with a particle mixture demonstrate how submicron particles The results from this experiment with a particle mixture demonstrate how submicron particles that are the same size, same shape, and made from the same substrate material can have different that are the same size, same shape, and made from the same substrate material can have different dielectrophoretic behaviors as a result of distinct surface charge magnitudes. These findings reveal dielectrophoretic behaviors as a result of distinct surface charge magnitudes. These findings reveal the potential in exploiting differences in surface charge magnitude to separate very similar submicron the potential in exploiting differences in surface charge magnitude to separate very similar particles based on their polarization behavior and dielectrophoretic response by employing simple submicron particles based on their polarization behavior and dielectrophoretic response by iDEP microdevices under DC and low frequency (<1000 Hz) AC potentials. employing simple iDEP microdevices under DC and low frequency (<1000 Hz) AC potentials. Supplementary Materials: The following are available online at www.mdpi.com/2072-666X/8/8/239/s1, SupplementaryTable S1: Fluorescent Materials: polystyrene The following microspheres are available information onlineincluding at www.mdpi.com/2072-666X/8/8/239/s1, diameter (µm), provider, charge Table (meq/g) S1: Fluorescentand type of polystyrene surface functionalization. microspheres information including diameter (μm), provider, charge (meq/g) and type of surfaceAcknowledgments: functionalization.The authors would like to acknowledge the financial support provided by the National Acknowledgments:Science Foundation (AwardThe authors CBET-1336160). would like to acknowledge the financial support provided by the National ScienceAuthor Foundation Contributions: (AwardM.F.R.-C., CBET-1336160). E.G., D.V.P., and B.H.L.-E. conceived and designed the experiments as well analyzed the data; M.F.R.-C., E.G., and D.V.P. performed the experiments; M.F.R.-C., D.V.P., and B.H.L.-E. wrote Authorthe paper. Contributions: M.F.R.-C., E.G., D.V.P., and B.H.L.-E. conceived and designed the experiments as well analyzed the data; M.F.R.-C., E.G., and D.V.P. performed the experiments; M.F.R.-C., D.V.P., and B.H.L.-E. Conflicts of Interest: The authors declare no conflict of interest. wrote the paper. ConflictsReferences of Interest: The authors declare no conflict of interest.

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