DNA Dielectrophoresis Theory and Application A Review

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com Page 1 Electrophoresis
1 DNA Dielectrophoresis: theory and applications a review
2 Martina Viefhues+* and Ralf Eichhorn#

+
3 Experimental Biophysics and Applied Nanoscience, Faculty of Physics, Bielefeld University, 33615
4 Bielefeld, Germany
#
5 Nordita, Royal Institute of Technology and Stockholm University, Roslagstullbacken 23, 10691
6 Stockholm, Sweden
*
7 Viefhues@physik.uni-bielefeld.de
8 Keywords
9 Review dielectrophoresis DNA theory applications
10 Corresponding author:
11 Dr. Martina Viefhues
12 Experimental Biophysics and Applied Nanoscience

13 Faculty of Physics
14 Bielefeld University
15 Universittsstr. 25
16 33615 Bielefeld
17 Germany
18 phone +49-521-106-5388
19 fax +49-521-106-2959
20 viefhues@physik.uni-bielefeld.de
21
22
23 Abstract
24 Dielectrophoresis is the migration of an electrically polarizable particle in an inhomogeneous electric

25 field. This migration can be exploited for several applications with (bio)molecules or cells.
26 Dielectrophoresis is a non-invasive technique; therefore, it is very convenient for (selective)
27 manipulation of (bio)molecules or cells. In this review, we will focus on DNA dielectrophoresis as this
28 technique offers several advantages in trapping and immobilization, separation and purification, and
29 analysis of DNA molecules. We present and discuss the underlying theory of the most important
30 forces that have to be considered for applications with dielectrophoresis. Moreover, a review of
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www.electrophoresis-journal.com Page 2 Electrophoresis
1 DNA dielectrophoresis applications is provided to present the state-of-the-art and to offer the
2 reader a perspective of the advances and current limitations of DNA dielectrophoresis.
6 1 Introduction
7 Manipulation of biomolecules is important in various applications in life sciences like separation,
8 purification, or analysis. Often the molecules of interest are present only in small amounts, or the
9 sample volume is in the range of a few microliters. Micro- and nanofluidic technology address those
10 needs and therefore are important systems in life science applications. Micro- and nanofluidic
11 research has made considerable advances in the field of separation, purification, and analysis of
12 biomolecules as shown in recent reviews [1, 2, 3, 4, 5, 6, 7]. The microfluidic manipulations and
13 separations were used to address a wide variation of different molecular characteristics like size,
14 charge, hydrophobicity, affinity, and polarizability [3, 8, 9]. In this review, we will focus on
15 microfluidic manipulation with dielectrophoresis, which is based on the molecules polarizability.
16 Dielectrophoresis is the migration of a polarizable particle in an inhomogeneous electric field [10, 11,
17 12]. It offers great advantages for the controlled manipulation, separation, and analysis of biological
18 samples as it is a noninvasive, nondestructive, and fast technique. Therefore, it plays an important
19 role in life science applications as reviewed in [8, 13, 14, 15, 16, 17, 18, 19, 11, 20, 21]. Various
20 biomolecules were manipulated with dielectrophoresis; they were ranging from nanometer sized
21 proteins over tens to few hundred nanometer sized DNA molecules or DNA origami to micrometer
22 sized prokaryotic or eukaryotic cells as thoroughly reviewed in [8, 15, 16, 20]. The previous reviews
23 about dielectrophoresis and dielectrophoretic applications were focused to specific topics, for
24 example, a focus on insulator based dielectrophoresis of cells, biomolecules and colloidal particles as
25 in [11, 21], a review of dielectrophoretic separation of cells and biomolecules as in [8, 13],
26 summarizing applications of dielectrophoresis as in [14, 15], a survey of the dielectric and
27 dielectrophoretic properties of DNA from a theoretical point of view as given in [22], or a review
28 about the impact of electrode shape on DNA dielectrophoresis as in [23]. Our review is distinct from
29 the previous reviews as we solely focus on DNA dielectrophoresis, presenting the theory important
30 in dielectrophoretic applications and a review of DNA dielectrophoresis applications presented so far
31 with a discussion of the current challenges and future tasks.
32 Dielectrophoresis is based on the polarizability of the sample molecules and the polarization
33 mechanism of DNA has been studied by several groups [24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35],
34 but still is not fully understood. However, commonly, larger DNA molecules exhibit a stronger
35 dielectrophoretic trapping behavior than shorter DNA molecules.
36 The possible applications of DNA dielectrophoresis are manifold like manipulation, concentration,
37 separation, and analysis of DNA molecules. These are important tools in various fields like medicine,

2
1 biotechnological production, and genetics [15, 16, 22, 36]. While manipulation and concentration of
2 DNA not necessarily have to be selective, selective dielectrophoretic forces are indispensable for
3 separation of DNA molecules, and thus, have to be well-balanced.
4 The main advantage of dielectrophoresis over other separation techniques is that the selectivity is
5 only based on the dielectric phenotype, thus an intrinsic molecular specificity [8, 10, 13, 15, 19, 36,
6 37, 38]. Hence, dielectrophoresis enables separation without any matrices, e.g. affinity matrices, and
7 therefore is a label-free technique, which could be used for purification of biotechnological or
8 medically relevant samples replacing the standard purification methods for pharmaceutical-grade
9 DNA [39]. By massive parallelization of microfluidic devices even large-scale DNA purification could
10 be accomplished with dielectrophoresis.
11 Beyond applications of dielectrophoresis in life sciences, dielectrophoresis can be exploited in

12 production of molecular electronics [40, 41]. Nucleic acid based molecular electronics offer a new
13 approach for a bottom-up production of nanometer sized electronics, and thus overcome the
14 physical limitations in size decrease in semiconductor manufacturing [22]. We will review several
15 applications of dielectrophoresis for controlled manipulation and immobilization of (single) DNA
16 molecules and DNA origami.
17 This paper is structured as follows; we will start with presenting the theory relevant for
18 dielectrophoretic applications, thus the various deterministic and stochastic forces. The theory
19 section is followed by a comparison of the two main branches of dielectrophoresis, namely the
20 standard, microelectrode based, and the insulator based dielectrophoresis (DEP and iDEP,
21 respectively). Afterwards, we will present applications of dielectrophoresis divided into the two
22 branches of microelectrode based DEP and iDEP.
23 2 Theoretical Background
24 Dielectrophoresis is the migration of an electrically polarizable particle in an inhomogeneous electric
25 field [42]. The backbone of DNA is negatively charged. In solution, these charges attract positive
26 counter-ions, which form a so-called electric double layer around the DNA strand. For entropic
27 reasons, this screened DNA strand assumes a random-coil like configuration. A (not too short1) DNA
28 fragment in an electrolyte solution can therefore be imagined as an almost spherical, charge-neutral
29 particle with a negatively charged, coiled backbone, which is surrounded by a cloud of positive
30 counter-ions.
31 When an external electric field is applied, a non-uniform charge distribution ( ) is induced

32 within such an object. The resulting force on this charge distribution can be expressed as the integral
33 ( ) ( ) over the volume occupied by the coiled DNA (including the counter-ion cloud)
34 [43]. If variations of the electric field are small on scales of the particle size, we can expand ( ) in
35 the integrand around the particle center and keep only the lowest non-trivial term. Using charge
1
Significantly larger than the persistence length, since on scales of the persistence length, DNA is
essentially stiff.

3
1 neutrality ( ) and the definition ( ) for the dipole moment, this

2 lowest order term reads ( ). The dipole moment is induced by the electric field
3 and thus proportional to (x) (in lowest order), i.e. ( ). The proportionality constant is
4 the polarizability of the DNA/counter-ion complex. We therefore find for the force
5 the electric field exerts on the polarized probe.
6 This is the dielectrophoretic force. We can see that it depends on the gradient of and therefore
7 requires non-uniform electric fields to be non-zero. Such inhomogeneous fields can be generated
8 either by structured microelectrodes, which are integrated into the microfluidic channel, or by a
9 non-trivial geometrical topography, containing constrictions, posts, ridges etc., in a device made of
10 an insulating material (see section 0). Moreover, the dielectrophoretic force is quadratic in , i.e. a
11 second-order effect.
12 In experimental setups which aim to manipulate particles or DNA by dielectrophoresis, we therefore

13 expect linear and other quadratic effects to play a significant role as well. The most prominent ones
14 are electrophoresis (section 0), electroosmotic flow (section 0) and Joule heating (section 0), and
15 result from a coupling between electrodynamic, hydrodynamic and thermodynamic effects
16 (electrohydrodynamic and electrothermal forces) [44]. Whereas electrophoresis subsumes the direct
17 impact of electric fields on particle migration in electrolytes (to linear order) [45], the latter two
18 processes cover indirect effects in form of viscous drag forces on the particle due to fluid motion
19 which is induced by the electric field. In general, these field-induced flows may be superimposed to
20 hydrostatic sources of fluid motion, i.e. pressure-driven flows (section 0, in particular section 0),
21 which exert further hydrodynamic forces on the DNA.
22 The forces listed so far are controlled externally (by applying electric fields or pressure gradients)
23 and are deterministic in nature. However, on the micrometer or nanometer scales relevant in
24 experiments with DNA, thermal fluctuations are omnipresent. The resulting forces are inherently
25 stochastic and induce diffusive (Brownian) motion [46, 47] of the DNA molecules (section 0).
26 In the following we will discuss the physical mechanisms behind all these various deterministic and
27 stochastic forces in more detail. Our focus will be on the most important effects that have been
28 identified in the literature to have relevant contributions to DNA migration and manipulation in
29 dielectrophoretic experiments. We start by presenting some basic properties of the electric field
30 (section 0), fluid flow (section 0) and temperature distribution (section 0) inside micro- and
31 nanostructures. We will then outline how these fields create various forces on the DNA (section 0),
32 and how their relative contributions define distinct operating regimes for DNA applications
33 (section 0).
34 2.1 Electric field

35 Electric fields inside micro- and nanostructures are generated by imposing electric potentials ( ) at
36 electrodes or electrode structures in the device. Typically, these external potentials are composed of
37 a static and a harmonic component with frequencies between several Hz and a few MHz. The
38 wavelength corresponding to a 100 MHz frequency is of the order of 1 meter and thus much larger
39 than the relevant structures in the device on micrometer scales and below. This separation of scales
40 justifies the use of the quasistatic approximation , which is commonly (and often tacitly)

4
1 employed in electric field calculations in micro- and nanodevices [48]. It permits to calculate the
2 generated electrical field ( ) as from the electrical potential ( ) created inside the
3 device. The latter is obtained from solving the Poisson equation
4 (1)
5 for a specific charge distribution ( ) ( is the vacuum permittivity). Boundary conditions have to
6 be provided for the (insulating) solid walls, which confine the electrolyte solution, and for the micro-
7 electrodes integrated in the device. Insulating boundaries in contact with an electrolyte typically
8 carry a surface charge density . Assuming that the interior of the insulator is free of electric
9 currents and fields, the boundary condition at insulator surfaces reads
10 (2)
11 where is the unit vector normal to the boundary, pointing into the electrolyte. On each electrode
( )( ( ) ( )
12 boundary, is prescribed by the potential ) ( ) applied to that electrode,
( ) ( )
13 ( ) (3)
( ) ( )
14 with denoting the static components and the amplitudes of the harmonic component,
15 which all have frequency (and identical phase2).
16 In principle, eq. (2) has to be complemented by a set of equations describing the dynamics of the
17 ions in the solutions [45]. However, for driving frequencies (much) smaller than the charge
18 relaxation frequency (where and are the electrical conductivity and permittivity of the
19 electrolyte) the charge distribution in the electric double layer can be assumed to be quasi-
20 stationary, so that in eq. (1) is time-independent. For typical experimental conditions, the buffer is
21 an aqueous electrolyte solution with and of the order of S/m to S/m, so that is in
22 the range of several MHz for the lowest conductivities up to GHz for high conductivities of the
23 solution. The driving frequencies of the applied electric fields, on the other hand, are typically in the
24 kHz range and do not exceed 30 Mhz. Therefore, the quasi-stationary approximation for the charge
25 distribution is reasonably well fulfilled.
26 The time-variations of the externally applied potential thus enter only parametrically via the
27 boundary conditions at the electrodes, and accordingly result in an electric field of the
28 form
29 ( ) (4)
( ) ( )
30 where is determined by the static bias and by the amplitudes , and both field
31 components have identical dependencies on the spatial position within the electrolyte solution.
2
We therefore do not consider such driving fields as, e.g., traveling waves [13]

5
1 A further simplification arises for electrolyte solutions with relatively high salt concentrations, where
2 the thickness of the electric double layer at the boundaries is much smaller than any other relevant
3 length scale in the device3. In such cases the electric double layer can be viewed as being part of the
4 boundary, and the electric field is of interest only in the regions outside the double layer where
5 there are no net charge accumulations. With , the Poisson eq. (1) therefore reduces to the
6 Laplace equation . For this simplified description, the boundary conditions have to be
7 specified at the interface between double layer and bulk electrolyte solution. At insulating
8 boundaries the surface charge is now screened by the electric double layer, so that the normal
9 component of the electric field has to vanish [11, 49] ( 0) in eq. (2). At the electrode surfaces,
10 one has to take into account the capacitive properties of the double layer by imposing the current
11 balance , where is the area charge density in the double layer[49].
12 Using a simple linear model for the double layer, its charge density is proportional to the voltage
13 drop across the layer ( ( )). The proportionality factor is the capacitance per
14 unit area of the double layer. The new boundary condition at electrode thus reads
( )
15 ( ( )).
16 In insulator based dielectrophoresis, the electrodes supplying the voltage signal are typically inserted
17 into (fluid) reservoirs outside the structured part of the device, where the DNA is actually
18 manipulated in, and are connected to that part by inlet and outlet channels. In the relevant
19 region the electric field is thus determined by the Laplace equation with the boundary
20 condition at all the device walls, and a prescribed, usually time-harmonic, voltage drop
21 between inlet and outlet boundaries [11].
22
23 2.2 Fluid flow

24 On the length-scales of microfluidic experiments with DNA, the Reynolds number is extremely small,
25 usually and below. Hence, inertia effects in the fluid motion do not play any role and flow
26 patterns are laminar [50]. Viscous forces in the fluid flow are balanced by pressure forces and body
27 forces on the fluid, which are induced by the presence of the electric fields. This force balance is
28 expressed by the Stokes equation for the incompressible fluid flow field [45],
29 ( ) (5)
30 where is the dynamic viscosity of the solution, the pressure, and the electrical force per unit
31 volume. At the interface between device walls and electrolyte solution, no-slip boundary conditions
32 are imposed.
33
34 For an incompressible fluid, the electrical body force on the liquid is [44, 51, 43]
3
For instance, for a mMol solution at room temperature the thickness of the electric double layer is
in the order of 10 nm or below.

6
1 . (6)
2 It is induced by the response of free (first term) and bound (second term) electric charges to the
3 external electric field. Note that the contribution due to free charges is linear in the electric field,
4 while the one from bound charges is quadratic in and therefore a higher-order effect.
5 A significant accumulation of free charges in microfluidic devices is built up in the electric double
6 layer at the device walls and around the DNA backbone. According to equations (5) and (6) the
7 electric double layer thus generates a fluid flow at the device walls (electroosmosis) and around the
8 DNA (electrophoresis), once an external electric field is applied. While the electroosmotic flow gives
9 rise to a global flow pattern in the device, the electrophoretic flow perturbs only the immediate
10 neighborhood of the DNA, exerting local stresses, which drive DNA motion (it is discussed in more
11 detail in section 0 below)
12 2.2.1 Electroosmotic flow

13 The electroosmotic flow, being created by the interaction of the electric field with the electric
14 double layer, in general depends on the specific properties of the device, most importantly the
15 properties of the surfaces confining the solution, and the ion concentrations in the electrolyte.
16 Electroosmotic flows can thus be controlled by applying specific surface coatings to the device walls
17 [52]. The most complex flow patterns, including vortices, are generated with inhomogeneous or
18 patterned surfaces.
19 Very much in the same spirit as for the electric field (see section 0), the theory simplifies in case the
20 double layers are small compared to other relevant length scales in the experimental device. Then
21 the density of free charges is zero in the relevant device region (not containing the electric double
22 layer), such that in eq. (5). The driving effect of the quasi-stationary double layer is captured
23 by imposing a slip-velocity boundary condition for at the walls [45],
24 (7)
25 where is the so-called zeta-potential characterizing the (local) properties of the solid-fluid interface
26 containing the electric double layer [45]. If the interface between electrolyte solution and device
27 material is homogeneous throughout the whole device such that is a constant, it can be shown
28 that the electroosmotic flow is proportional to the electric field everywhere in the fluid region [53],
29 (8)
30 This situation is typical for insulator-based DEP devices [11], which do not contain electrodes within
31 the structured region and are usually fabricated from one block of a homogeneous material.
32
33 2.2.2 Electrothermal flows

34 Electric fields drive fluid flows according to the second term in eq. (6), if the permittivity of the
35 electrolyte is space-dependent. Likewise, a contribution from spatially varying conductivities, which
36 deforms the charge distributions, is hidden in the first term in eq. (6) [51]. Given that in typical DNA

7
1 experiments the microfluidic device is uniformly filled with a homogeneous electrolyte solution, we
2 may naively expect that such effects are not relevant. However, like other material properties, the
3 permittivity and conductivity are temperature dependent. This gives rise to so-called electrothermal
4 flows, where spatial temperature variations in the device in combination with electric fields induce
5 fluid motion. It has only recently been acknowledged that such electrothermal flows may play an
6 important role in microfluidic devices due to local Joule heating effects (see section 0) close to
7 electrodes or in narrow device constrictions [54], where the electric fields and field gradients are
8 largest [44, 51, 55].
10 2.2.3 Pressure-driven flows

11 It is obvious from eq. (5) that even without body forces, , fluid flows can be generated by
12 applying hydrostatic pressure differences on the device. The most well-known example of such
13 pressure-induced flow is the parabolic Hagen-Poiseuille profile in a straight channel [45]. Even in
14 general channel designs with non-trivial structures (posts, constrictions etc.), the main
15 characteristics of pressure-driven flows are small flow velocities close to the device boundaries and
16 high velocities in the middle of the fluidic channel.
17
18 2.3 Temperature field

19 The electric current through the device is carried by the ions in the solution (recall that we
20 restrict our considerations to frequencies of the electric field smaller than ). Friction of the
21 ions in the surrounding water therefore creates resistive Joule heating. The local power density
22 generated by the electric current is
23 (9)
24 It increases quadratically in the electric field strength and is proportional to the conductivity of the
25 electrolyte solution. The amount of Joule heating is therefore much more significant for high
26 external fields, but can be controlled by adjusting the conductivity .
27 The increase in temperature associated with the power generation, eq. (9), is spread over the
28 device by a temperature current with a convective component T and a diffusive component
29 , where is the mass density of the electrolyte, its specific heat (at constant pressure),
30 and the thermal conductivity. The temperature balance equation (continuity equation) is therefore
31 [44, 51]
32 ( ) (10)
33 For water with properties , , and ,

34 the diffusive term dominates over the convective term on typical length scales of 1-10 m, and up to
35 convective transport velocities of about 1 mm [44]. The main contribution to temperature
36 transport is thus essentially diffusive with a time-scale given by (where 1-10 m is

8
1 a characteristic length scale in the device). For water, is smaller than s such that a stationary
2 temperature profile is established within the order of milliseconds [44].
3 This inhomogeneous temperature distribution entails gradients in material properties, due to their
4 temperature dependence. Most importantly, permittivity and conductivity of the solution are no
5 longer constant but depend on space with gradients
6 (11)
7 These gradients act as body forces on the surrounding fluid upon application of an electric field (see
8 eq. (6)) and therefore generate an electrothermal fluid flow.
10 2.4 Motion of the DNA

11 The electric field, fluid flow and temperature distribution in the microfluidic device and their
12 interplay (as described in the previous section) induce different kinds of motion for the DNA
13 molecules suspended in the electrolyte solution. In the following we discuss the most prominent
14 driving forces behind DNA migration in DEP devices.
15 2.4.1 Electrophoresis
16 Electrophoresis is induced by the action of an electric field on the charge distribution of the DNA
17 molecule and in the double layer around the DNA backbone. The ions in the double layer are set into
18 motion by the electric field, generating hydrodynamic shear, which contributes to driving DNA
19 motion. The electrophoretic velocity is proportional to the electric field,
20 (12)
21 with the electrophoretic mobility .
22 For electric double layers, which are significantly smaller in size than relevant scales characterizing
23 the DNA molecules (most notably persistence length and radius of gyration), shear is restricted to a
24 thin layer around the DNA backbone and the electrophoretic mobility can be calculated exactly. It is
25 independent of DNA length and conformation, and is given by the famous Helmholtz-Smoluchowski
26 result [56]
27 (13)
28 where is the zeta-potential of the double layer around the DNA backbone. The zeta-potential
29 characterizes the DNAs (electrical) surface properties in the solution; in particular it is proportional
30 to the surface charge density [56]. DNA is uniformly charged, resulting in a surface charge density
31 which is essentially independent of other properties like length or conformation, and thus is the
32 same for different DNA fragments. As a consequence, DNA fragments of different length or
33 conformation all move with the same electrophoretic velocity in free solution. For DNA fractionation
34 by electrophoresis gels are used, which render the electrophoretic velocity length-dependent via
35 steric forces exerted on the DNA by the gel itself [56]. In free solution, selective manipulation of DNA
36 can be achieved by making use of higher-order electric effects such as dielectrophoresis.

9
1 The electric field typically consists of a static DC and an oscillating AC component (see eq. (4) in
2 section 0), the latter having frequencies between several Hz and a few MHz. The AC component thus
3 induces an oscillating back-and forth motion of small amplitude on time-scales such short that they
4 are typically not of interest. It can therefore be averaged out to obtain the relevant electrophoretic
5 motion
6 (14)
7 driven by the DC component of the electric field.
8 Although the electrophoretic DNA motion is force-free in the sense that no net external force is
9 directly applied to the DNA-counterion complex, it can be rewritten as an effective electrophoretic
10 force of the form [57, 58]
11 (15)
12 where is the viscous friction coefficient of the DNA (see also section 0). This force-like
13 representation of electrophoresis is particularly useful for combining electrophoretic effects with
14 other forces acting on the DNA, see section 0.
15
16 2.4.2 Dielectrophoresis
17 DEP is the second-order counterpart to electrophoresis, denoting the contribution to the electric
18 force on a particle in an electrolyte solution, which is quadratic in the electric field . DEP results
19 from the coupling of the electric field to the charge-dipole induced in the DNA and the electric
20 double layer around it by the same field. It can be written as
21 ( ) (16)
22 where is the polarizability of the DNA fragment. Assuming that the DNA is isotropic in good
23 approximation4, and that the frequencies of the external electric field are smaller than the charge
24 relaxation frequency (our standard assumption, valid for almost all the DEP experiments with
25 DNA), the induced dipole will be parallel to the electric field. Moreover, under these
26 conditions we can assume that the polarization process occurs in phase with the external electric
27 field and thus basically lossless5. The polarizability can therefore be modeled as a scalar (isotropic),
28 real-valued quantity. For , the DEP force (16) points towards larger electric fields, such that the
29 particle is attracted to the high field regions. This behavior is called positive DEP. Accordingly,
30 negative DEP denotes the contrary behavior when the DEP force drives the particle towards the low
31 field regions due to . We discuss further properties of the polarizability in more detail in
32 section 0.
4
We expect that this assumption is fulfilled on average when the (very fast) fluctuations in the structure of
the coiled DNA backbone are averaged out.
5
Dissipation due to damping of the charge-oscillations in the double layer (and the DNA molecule) induced by
the applied electric field are negligibly small.

10
1 The electric field in eq. (16) creating the DEP force typically is composed of various components,
2 see section 0. For insulator-based DEP a number of studies using only DC fields are reported in the
3 literature (see, e.g. [21]). Often, however, insulator-based DEP is generated by AC fields, and an
4 additional DC component is used to independently induce electrophoretic motion [11]. In such
5 setups, the DC component is by an order of magnitude smaller than the AC component. In
6 conventional DEP based on micro- or nanoelectrodes, the electric potentials applied to the
7 electrodes typically consist of an oscillating component only; either there is no static potential at all
8 or a static component is superimposed with separate electrodes attached outside the structured
( )
9 region of the device in which the DNA is manipulated. In any case at all the electrodes
10 (see eq. (3)). In all these DEP settings, combining DC and AC fields, the contribution of the DC field is
11 negligibly small as compared to the AC field. Time-averaging over the sinusoidal oscillations of the
12 AC field in eq. (4), we therefore obtain for the DEP force
13 ( )( ) (17)
14 For more general time-periodic driving potentials the time-averaged version of eq. (16) is obtained
15 by replacing the electric field with its rms value [21].
16
17 2.4.3 Hydrodynamic drag

18 The hydrodynamic drag force exerted on a particle when it moves through a fluid is
19 proportional to the deviation of the momentary particle velocity from the flow velocity ( )
20 at the particle position ,
21 ( ) (18)
22 The proportionality constant is the viscous friction coefficient. For a simple model of DNA in
23 solution which assumes that the DNA moves as an almost spherical random-coil like particle (see
24 also beginning of section 0), we can express the friction coefficient with Stokes' formula as
25 (19)
26 where is the hydrodynamic radius of the DNA fragment.
27 Obviously, the nature of the mechanism which creates the fluid flow is irrelevant for the actual
28 drag force exerted on the particle. The flow velocity in eq. (18) may therefore contain any of the
29 contributions described in section 0. In other words, the hydrodynamic drag force covers the effects
30 of electroosmotic, electrothermal and pressure-driven fluid flows.
31
32 2.4.4 Thermal noise

33 At room temperature the thermal energy (with being Boltzmann's constant) is about 4 fN
34 m= 4 pN nm [47]. At micro- and nanometer scales, the associated thermal forces are therefore well
35 comparable in magnitude to other forces acting on the DNA. They become visible as diffusive

11
1 motion, which is characterized quantitatively by the diffusion coefficient . The diffusion coefficient
2 is connected to the friction coefficient by the fluctuation-dissipation theorem6 [47],
3 (20)
4 In order to capture the fluctuating, random nature of the thermal forces , they are commonly
5 modeled as Gaussian white noise processes [46] with zero mean, and time-correlations that
6 reproduce diffusion correctly, i.e.
7 ( ) ( ) (21)
8 Here, the angular brackets denote the average over many realizations of the noise process, and the
9 subscripts and refer to the different spatial components of the force , and denote
10 the Kronecker delta and the Dirac delta-function.
11
12 2.4.5 Force balance

13 At any instance in time, all the various forces acting on the DNA molecule balance each other,
14 (22)
15 In principle, we would have to include inertial forces as well. However, at microscales and below,
16 inertia effects are by orders of magnitude smaller than viscous friction effects and other relevant
17 forces [50], such that in eq. (22) we adopt the so-called overdamped limit by neglecting inertial
18 forces.
19 Using the various expressions for the different forces as derived in the previous sections and solving
20 the resulting equation for the particle velocity , we obtain the equation of motion
21 ( ) ( ) ( ) (23)
22 Since we here used the time-average expressions for the forces, this equation of motion is only valid
23 on time-scales larger than the driving frequency of the external electric field.
24 In the important case that the fluid flow is created electroosmotically from electric double layers
25 much smaller than any other relevant length-scale in the device, the fluid flow is given by eq.(8),
26 where we have to replace by due to the time-average taken in eq. (23). We obtain for the
27 particle dynamics

28 ( ) ( ) (24)
29 with an effective zeta-potential , which combines the zeta-potentials of particle and

30 device walls. This result implies that in a superposition of DC and AC electric fields, the
6
The fluctuation-dissipation relation is usually used for defining the hydrodynamic radius of a polymer as
the radius of a spherical colloid that diffuses at the same rate as the polymer.

12
1 electrophoretic and dielectrophoretic forces can be controlled practically independently of each

2 other by adjusting the amplitudes of the DC and AC components, respectively, a fact that has been
3 exploited, for instance, in DNA separation experiments [59, 60].
4 More generally, the relative strength of the driving forces ( ) and thermal
5 fluctuatons in eq. (23) compared to the dielectrophoretic force ( )( ) define
6 basically three different operating regimes of the DEP device:
7 1.
8 The DNA essentially streams through the device with diffusive motion
9 superimposed, and is deflected or focused by DEP forces.
10 2.
11 The DEP forces are strong enough to significantly slow down the DNA migration
12 through the device. Since the polarizability is expected to depend on the
13 properties of the DNA (such as length or conformation), this regime is sensitive to
14 particle properties and may be particularly useful for DNA separation.
15 3.
16 The DNA is trapped by the dielectrophoretic forces. Escapes from these traps are
17 possible due to thermal fluctuations, but become exponentially rare with increasing
18 ratio
19 between DEP and thermal forces. For very large DEP forces, the
20 DNA is effectively immobilized for times longer than the lifetime of the experiment.
21 Thus, when designing a new application that exploits dielectrophoresis, the DEP force range needs
22 to be adapted to the desired operation regime.
23
24 2.5 Polarizability
25 The polarizability of the DNA molecule in an electrolyte solution describes the tendency to have the
26 charges on the DNA backbone and in the double layer around it displaced by an external electric
27 field , relative to the charge displacements induced in the surrounding solution. The induced
28 electric dipole moment is proportional to the electric field, (see also at the beginning of
29 section 0). Probably due to the lack of a detailed polarization model, the DNA polarizability is
30 commonly treated in a simplifying manner as a scalar, real-valued quantity (see also Section 0). A
31 standard method in the theory of DEP for calculating is the so-called effective moment method
32 [61, 62]7. This method is based on the known analytic expression for the electric field around a
33 point-like dipole with dipole moment . The idea is to compare this expression with the electric
34 field that results from the perturbations of a uniform field around the object of interest (e.g. a DNA
35 molecule) due to the presence of that object. By this procedure one obtains the dipole moment
7
An alternative, more rigorous but also more difficult method is based on the Maxwell stress tensor,
see e.g. [8].

13
1 (of a point-dipole) that would create a perturbation field identical to and indistinguishable from that
2 of the object [62]. This dipole moment is parallel to the undisturbed electric field, and thus has the
3 structure , so that the polarizability can be read off. The challenge, of course, consists in
4 calculating the electric field perturbations around the object of interest, in particular for such
5 complex objects like DNA molecules (or polyelectrolytes) suspended in an electrolyte solution. To
6 the best of our knowledge, this problem has not been solved yet.
8 2.5.1 Clausius-Mossotti factor

9 In general, we may expect that the electric field perturbations due to the presence of a dielectric and
10 conductive object in a dielectric and conductive medium depend on the permittivity and
11 conductivity of the medium, and on corresponding quantities and of the polarizable object.
12 Indeed, for simple rigid particles, like spherical or ellipsoidal colloids, one finds that the polarizability
13 is proportional to the Clausius-Mossotti factor ( ) [10, 13, 63]
14 ( ) (25)
15 given as
16 ( ) (26)
[ ( )]
17 where is a geometrical shape factor which is equal to 1/3 for spherical objects and tends to zero
18 for large aspect ratios between the principal axes of the object. The Clausius-Mossotti factor is
19 complex-valued with the complex permittivities ( ) [10, 13, 64, 63]
20
21 (27)
22 where is the frequency of the electric field (as before).The real part in (26) results from the
23 assumption of a lossless polarization process.
24 Calculating the real part explicitly, we find
( )[ ( )]
( )[ ( )]
25 ( )
(28)
[ ( )] [ ]
26 The sign of ( ) determines the sign of , and hence if the particle shows positive ( , i.e.
27 ( ) ) or negative DEP ( , i.e. ( ) ).
28 In the limit of small frequencies, ( ) simplifies to [13, 17]
29 ( ) ( ) (29)
[ ( )]
30 implying that the polarization process is dominated by free charges with conductivities in eq. (29)
31 being understood as the limiting conductivities. This is the typical situation in DEP
32 experiments, since is usually smaller than the charge relaxation frequency . In contrast,

14
1 high-frequency electric fields induce dipoles due to the relative dielectric properties of particle and
2 medium, as can be seen from the large frequency limit of ( ) [13, 17],
3 ( ) ( ) (30)
[ ( )]
4 where now the permittivities are understood to represent the limiting values.
5 Although the Clausius-Mossotti factor in eq. (26) has been used to describe the dielectric properties
6 of DNA, e.g. in [64, 63], it may be expected that this simple expression for homogeneous ellipsoidal
7 objects does not represent a generally accurate model for polyelectrolytes. Calculating the Clausius-
8 Mossotti factor ( ) of DNA based on realistic polyelectrolyte models, or, especially, the DNA
9 permittivity and conductivity, is obviously very difficult (see also Section 0). For instance, molecule
10 dependent cross-over frequencies from positive to negative polarizability are observed in the 100
11 kHz regime, indicating a complex dependence of the permittivities and conductivities involved in the
12 Clausius-Mossotti factor ( ) on the electric field frequency and the characteristics of the DNA
13 molecule.
14
15 A possible strategy for determining ( ) for more complex objects is to replace the permittivity
16 and conductivity of the simple sphere in eq. (28 (28), as well as the geometrical factor , by
17 effective quantities, which are derived from more accurate particle models taking into account
18 (some of) their non-trivial features. An example of such an approach is the multishell model [17, 13].
19 It is, for instance, used to capture heterogeneous structures in biological cells and similar particles,
20 but cannot be applied to DNA molecules.
21
22 2.5.2 Polarizability of DNA

23 Experimental results on dielectric measurements with DNA show that the polarization of DNA or
24 polyelectrolytes in general, is an extremely complex process [22]. Nevertheless, most of the
25 experiments seem to indicate that the main polarization process is due to the ions around the DNA,
26 even up to 1 GHz frequency of the electric field, in accordance with our rough qualitative arguments
27 from above (see, e.g., [30, 65] and references therein, a notable exception to the counter-ion
28 hypothesis is reported in [66]). In these works, usually two types of counter-ions are distinguished:
29 diffusive counter-ions, which can move freely over the whole extension of the DNA coil, and
30 condensed counter-ions which are restricted to move along the DNA backbone. The relative
31 importance of the two counter-ion types for the polarization process changes with the electric field
32 frequency, where the diffusive counter-ions seem to be dominant in the low frequency-region below
33 a few kHz [30].
34 The theoretical, quantitative description of polarization processes in DNA and polyelectrolytes is

35 very difficult due to the complex nature of the underlying mechanisms. In their 1984 review on
36 Dielectric properties of polyelectrolyte solutions, Mandel and Odijk criticized that the different
37 semi-empirical attempts which have been made to understand the experimental findings lack a
38 rigorous and convincing theoretical basis [67]. On the other hand, the more rigorous approaches
39 which try to explain the polarization processes quantitatively, suffer from the disadvantage to rely

15
1 on models which do not capture the experimental conditions for polyelectrolytes properly. Rather,
2 they were typically developed for rigid spherical objects of sizes much larger than the electric double
3 layer [67]. It seems that this situation has not improved dramatically since then. Recent
4 developments, however, show a trend in the theoretical studies of dielectric particle properties
5 towards more complex particle shapes, such as rod-like polyelectrolytes, (see for instance [29, 31,
6 68, 69]8), which may even be allowed to bend in order to mimic simple model-DNA [70, 71].
7 Moreover, advances in simulation techniques and increased computer power nowadays allow
8 simulating electrokinetic phenomena to such resolution that the mechanisms behind the dielectric
9 response of single polyelectrolyte macromolecules to external electric fields can be studied even
10 down to the molecular level [72, 73]. For instance, in [32, 33, 34, 74] ion distributions around DNA
11 fragments have been computed using different simulation approaches, ranging from Brownian
12 dynamics to all atom techniques.
13 2.6 Concluding remarks

14 For the sake of simplicity, we did not present the equation governing the dynamics of the charge
15 distribution due to the combined action of electric fields, convective transport in the moving fluid
16 and diffusion (see, e.g., Chapter 7 in [45]). This equation complements the fundamental equations
17 for the electric field, the fluid flow and the temperature distribution as discussed in sections 0-0, and
18 is needed for them to become a closed set of dynamical equations. In praxis, this set of coupled
19 partial differential equations has to be solved numerically on a computer in order to accurately
20 quantify the experimental setup at hand. There are powerful commercial software packages or
21 suites available for that purpose, COMSOL [75] being probably the most commonly used one. Such
22 numerical solutions are particularly relevant if the experimental conditions do not allow for
23 simplifying assumptions which (partly) decouple and simplify the governing equations, for instance,
24 when the double layer size is comparable to the dimensions of the electrode or channel structures,
25 or when the driving frequency is too fast to allow for a quasi-stationary description of the charge
26 distributions.
27 The exposition in the theory sections lists the most relevant effects (and their physical basis) which
28 are used to manipulate DNA in DEP experiments.
29 For the sake of completeness we here mention two additional aspects that have not been discussed
30 in the theory sections above. The first one is the deformability of DNA molecules and the effect of
31 electric fields on the shape of the DNA coil. It seems to be commonly believed that DNA molecules
32 get stretched by DEP. However, in [76, 77] Zhou et al. report that double-stranded DNA actually
33 collapses in the presence of AC electric fields at frequencies of a few hundred Hertz, rather than
34 being stretched. They argue [77] that the apparent stretching at high fields is an artifact of the finite
35 frame time in video microscopy.
36
37 The second supplement is a particular electrokinetic effect, which has been observed and
38 characterized in specific electrode designs [49, 78, 79] and which, for instance, has been applied as a
8
In particular, reference [29] provides an excellent overview of the literature on polarization of rod-like
polyelectrolytes until around 1996.

16
1 DNA concentrator [80], namely AC electroosmosis. AC electroosmosis is a flow phenomenon that

2 occurs below the charge relaxation frequency of the electrolyte buffer when the electric field at
3 the electrode has a component parallel to the electrode surface. Then it exerts a tangential force on
4 the charge distribution, which is being accumulated at the electrode surface due to the applied
5 electric potential. The tangential motion of the charges induces a corresponding fluid motion (see
6 eq. (5) and eq. (6)). Under AC conditions the sign of the accumulated charge distribution changes in
7 phase with the sign of the tangential field component so that the direction of the induced fluid flow
8 is stationary in time. For a nice summary of AC-electroosmosis in relation to the other electrokinetic
9 effects, see [81].
10 We finally remark that, much to our surprise, thermophoresis of DNA its migration due to direct
11 effects of temperature gradients does not seem to be considered in connection with Joule heating
12 in DEP experiments. Thermophoresis of DNA has been observed in other contexts [82, 83] and thus
13 may be expected to play a role also for DEP experiments with DNA in cases where Joule heating
14 effects are non-negligible.
15 3 Microelectrodes vs. insulator based DEP

16 As explained in the theory section to gain dielectrophoretic migration an inhomogeneous electric
17 field is indispensable. This inhomogeneous electric field is generated mostly by two different
18 approaches, Figure 1. The first approach exploits micro- or nanoelectrodes that are integrated in the
19 microfluidic channel. [21] The first devices and applications were performed with microelectrode
20 based dielectrophoresis and today most DEP applications rely on that technique. The second
21 approach to generate an inhomogeneous electric field is to use insulating posts or ridges that reduce
22 the channel cross-section area. Since no electrodes are used in the region where the
23 dielectrophoretic migration appears this approach is called electrodeless or insulator based
24 dielectrophoresis (iDEP).
25 Both approaches offer specific advantages and disadvantages, nevertheless both have in common
26 that dielectrophoresis is a versatile tool for manipulation, separation, and analysis of DNA as shown
27 in several reviews [15, 17, 19, 20, 21]. A disadvantage of the microelectrode based DEP is that the
28 production of the devices is time-consuming and expensive [84], due to fabrication of the
29 electrodes. The electrodes in those devices either could be aligned in-plane or out-of-plane. For
30 instance, the in-plane electrodes are both on the same substrate, thus, they can be made in one
31 production step without the need of alignment. This is a fabrication related advantage in comparison
32 to the out-of-plane electrode arrangement, where the two electrodes are opposite to each other,
33 e.g. one at the channel bottom and the second one at the channel ceiling. Therefore, an alignment is
34 needed during production, which is more time-consuming. Nevertheless, from an application point
35 of view the out-of-plane electrodes generate a dielectrophoretic force that reaches more of the
36 channel height and, thus, is better suited for separation processes where all molecules should be
37 affected dielectrophoretically. In contrast, the in-plane electrodes are good for DNA immobilization
38 and, e.g., subsequent conductivity measurements as performed in [64, 85, 86, 63].

17
1 In contrast to microelectrode based DEP, iDEP devices offer the fundamental advantage that those
2 mainly could be fabricated monolithically and with mass fabrication techniques like injection
3 molding or hot embossing. Hence, the devices can be produced cost-efficiently [11, 21, 87]. No
4 electrodes are in the vicinity of the dielectrophoretic manipulation consequently no surface fouling
5 or electrochemical side effects, impacting the efficiency or the long-term stability of the DEP
6 manipulation, occur [11, 15, 21].
7 Comparing the electric field of microelectrode based DEP and iDEP the first has the drawback that
8 the DEP force decreases quadratic with distance from the electrode. This results in an
9 inhomogeneous electric field over the channel height for microelectrode based DEP, Figure 1a. Thus,
10 the DEP force strongly depends on the distance to the electrode and the sample has to be in vicinity
11 of the electrodes to get affected by the dielectrophoretic force. Whereas for iDEP the electric field
12 inhomogeneity is generated over the whole channel height, Figure 1b. Therefore, the samples do not
13 necessarily have to flow with small distance from the channel surface but can be distributed over the
14 whole channel height to be affected by the DEP force [11].
15 The AC electric field frequency can be divided into two regimes. The first with frequencies up to kHz,
16 usually for iDEP, with typical electric field strengths of . In the second regime frequencies
17 are hundreds kHz or above, usually for microelectrode based DEP, where the AC electric field
18 strength is one order of magnitude higher. Bubble formation especially becomes a problem in the
19 vicinity of the electrodes for frequencies below 100 kHz, this is more critical for sharp electrode
20 geometries as for the microelectrode based dielectrophoresis [22, 88]. Moreover, electrochemical
21 side effects and surface fouling are critical [11].
22 One advantage of microelectrode based DEP is that alternating current frequencies up to several
23 MHz could be applied, which offers studies over wide frequency ranges as shown in [89, 64, 90].
24 Thus, the impact of the frequency on the polarizability and the direction of the dielectrophoretic
25 force, i.e. the cross-over frequency, can be studied with micro- or nanoelectrodes. Since integrated
26 microelectrodes typically are placed only a few micrometer apart very high electric fields are
27 achieved for low voltages applied to the electrodes. Whereas, a disadvantage of iDEP is that
28 significantly higher electric potentials need to be applied to gain equivalent dielectrophoretic
29 potentials. This limits the frequency range due to the limited slew-rates of high-voltage equipment
30 [91].
31 4 Applications
32 In this section applications of dielectrophoresis are reviewed. The applications are distinguished in
33 microelectrode based dielectrophoresis and electrodeless dielectrophoresis to gain clarity of the
34 achievements that have been obtained in the respective fields.
35 In the theory section the basics of dielectrophoresis, i.e. the dielectrophoretic migration and the
36 trapping forces were discussed. When starting with dielectrophoresis one has to take into account
37 that three regimes can be distinguished by the occurring forces and their balances, see section 0 for
38 more detail. In the following sections we are trying to distinguish the reviewed applications into the
39 regimes, trapping and immobilization, as well as separation. Continuous flow concentration

18
1 according to our definition was not found for DNA dielectrophoresis. However, several groups
2 presented DNA concentration at fixed locations, e.g. [92, 93, 94].
3 Two different performances can be distinguished for separation techniques. The first performance is
4 the sequential or batch performance, where a sample plug is injected into the separation region and
5 selective forces lead to species dependent migration velocities, thus, leading to a separation in time,
6 i.e. the separated species pass a detection point at different times, Figure 2. The next plug can be
7 injected into the separation region after all species passed the detection point. The second
8 separation performance is the continuous separation. The samples are injected continuously in the
9 separation region, where a selective force acts perpendicular to the direction of flow [95]. After the
10 samples have passed the separation region they flow in parallel, thus they are spatially separated.
11 For instance, if the channel is split downstream of the separation region the samples could be
12 collected and harvested in separate outlets. The continuous separation is versatile for scaling-up
13 separation processes, for example to purify gene vaccines or collect rare circulating cells [38, 39].
14 The dielectrophoretic force can be adapted either via the applied voltages and frequencies or by the
15 device geometry, i.e. the electrode shape and conformation or the shape and dimensions of
16 insulating posts. While the applied electric fields can be adapted during an experiment, and thus, the
17 dielectrophoretic force, the device geometry cannot be adapted, besides for a few exceptions like oil
18 insulating posts [96, 97]. Thus, it is important to consider the final application, e.g., continuous
19 separation or immobilization of DNA and the range of necessary dielectrophoretic forces that have
20 to be generated, as those might exclude certain device concepts or require certain device
21 dimensions.
22
23 4.1 Microelectrode based DEP

24 In the next sections we review applications that are based on microelectrode DEP of DNA. The used
25 samples, the parameters of the applied voltages and the buffer are summarized in table 1.
26
27 4.1.1 Trapping / Immobilization

28 In section 0, we described that the force balance of the occurring forces is of high importance for
29 any application. The dielectrophoretic force has to overcome all other forces leading to a motion of
30 the DNA molecule to achieve a sufficient trapping or immobilization.
31 The first demonstration of dielectrophoretic trapping of DNA was presented by Washizu et al. in
32 1990 [98]. They performed dielectrophoresis of DNA at microelectrodes with applied AC electric field
33 strengths of and frequencies around 1 MHz. Washizu et al. observed a stretching and
34 shrinking of an individual molecule as the field was switched on and off and they determined the
35 contour length of the DNA molecules by measuring the stretched length of the molecule [98].
36 Permanent immobilization of dielectrophoretically trapped DNA was presented in [99, 100] by
37 Washizu and co-workers in 1995 and 2000. They exploited the DNA trapping to further analyze the
38 DNA with scanning tunneling microscopy [99]. Dielectrophoretic DNA trapping and immobilization
39 was exploited for space-resolved dissection (molecular surgery) of DNA [100]. The

19
1 dielectrophoretically trapped DNA was anchored on a solid surface, followed by a subsequent

2 enzymatic cutting. The cutting enzymes were modified on microspheres that were manipulated with
3 optical tweezers and brought in contact with the immobilized DNA [100].
4 In 1998, Asbury and van den Engh presented the experimental realization of dielectrophoretic
5 trapping of DNA by fluorescent video microscopy [101]. They structured gold microelectrodes onto
6 quartz chips. After a drop of the sample was placed on the microelectrode array a coverslip was
7 placed on top and the chamber was sealed with agarose. Reversible DNA trapping and release was
8 observed for switching AC voltages on and off [101]. This concept for DNA labeling has been used
9 since then for most applications of DNA DEP.
10 Asbury et al. used a microfluidic flow chamber with integrated microelectrodes to perform size-
11 dependent dielectrophoretic trapping of DNA [102], though they did not actually separate the DNA
12 this proved the general applicability of dielectrophoresis for DNA separation. Asbury et al. observed
13 an increase of the DNA concentration in solution by at least 60-fold [102].
14 Zheng et al. demonstrated the applicability of DNA dielectrophoresis for molecular circuit fabrication
15 [63]. They immobilized DNA molecules between quadrupole electrodes with gaps ranging from only
16 3 m up to large gaps of 100 m. They measured the electric conductivity of DNA molecules trapped
17 in 5 m gaps, allowing the DNA ( -phage, 48.5 kbp, about 17 m long) to bridge the gap, thus, the
18 two electrodes. The lowest conductivity that was measured was 25 nS. Zheng et al. assumed that
19 was the conductivity of a single DNA molecule [63].
20 Bakewell and Morgen characterized the frequency dependent collection of DNA onto interdigitated
21 microelectrodes for high-frequency dielectrophoresis [24]. They quantitatively measured reaction
22 time profiles and characterized the data by two parameters: the initial dielectrophoretic collection
23 rate, and the initial to steady-state collection transition. From the experimental findings they
24 determined that factors like electrohydrodynamic forces also have to be taken into account for
25 sufficient modeling of DEP [24].
26 In 2005, Tuukkanen et al. used fingertip gold nanoelectrodes, with a very small gap size of 100 nm,
27 to trap and immobilize thiol-modified DNA by DEP [40, 85]. After drying the sample AFM images
28 have been taken from the trapped DNA at the nanoelectrodes. Additionally, they measured the full
29 I-V characteristics to measure the conductivity of single DNA molecules [40, 85]. In their work [40]
30 they varied the humidity conditions to study the impact on the electric conductivity [85].
31 Germishuizen et al. also used thiol-modified DNA for immobilization of dielectrophoretically trapped
32 DNA to gold electrodes [103]. Another type of electrodes was used by Tuukkanen et al. with carbon
33 nanotubes [104]. Due to the small diameter of the nanotubes the electric field gradient was very
34 strong, thus even relatively low trapping voltages were sufficient. Tuukkanen et al. compared the
35 efficiency of dielectrophoretic trapping of small DNA (1065 bp) with carbon nanotubes and
36 lithographically fabricated metallic electrodes. The trapping performance of well-confined
37 nanotubes was found to have a better performance compared to lithographically fabricated 100 nm
38 wide fingertip type nanoelectrodes [104]. However, in 2007, Tuukkanen et al. used dielectrophoresis
39 at fingertip metal electrodes to study the trapping efficiency of DNA of varying length [105]. The
40 focus of their study was to determine parameters that impact the trapping performance and

20
1 efficiency. Three parameters were investigated with respect to their impact on the dielectrophoretic
2 trapping efficiency of DNA molecules. First, the impact of the gap size between the electrodes was
3 evaluated. Tuukkanen et al. deduced that the shape of the electrodes had more effect on the
4 trapping efficiency than small changes in the gap size. This also is in accordance with their previous
5 work [104]. Second, they investigated the impact of the DNA length on the trapping efficiency. An
6 increase of the necessary electric field strength for shorter molecules was observed, which is in
7 accordance to the predictions of Ajdari and Prost [106]. Tuukkanen et al. discussed that the decrease
8 of the trapping efficiency was due to a higher Brownian motion and a smaller polarization of the
9 molecule. Third, Tuukkanen et al. determined that the higher the frequencies were the higher the
10 voltages had to be to enable trapping. They explained this with less time to polarize for higher
11 frequencies [105].
12 In 2007, Kumemura et al. exploited two opposing aluminum electrodes to trap and isolate a single -
13 DNA molecule by dielectrophoresis [41]. They discussed that by avoiding electrodes with sharp
14 corners and high conductive buffers, using DI water instead, the Joule heating and electrothermal
15 flow could be reduced. Kumemura et al. presented a fast trapping of DNA 1 to 2 seconds after the
16 molecule was electrophoretically moved towards the trapping electrodes, where an AC voltage was
17 applied. Once the DNA was trapped it permanently anchored to the electrodes, thus forming a
18 bridge [41]. With that experimental realization the electric properties of a single DNA molecule could
19 be determined in contrast to [63] where several DNA molecules were trapped at the same time. In
20 their later work [107], Kumemura et al. successfully captured a single DNA molecule between the
21 tips of silicon nanotweezers by DEP. This approach provided the capability to manipulate a single
22 molecule, e.g. to move DNA molecules to a specific position in a microbioelectronic device. These
23 achievements pave the way for fabrication of a new class of molecular biosensor based on silicon
24 microtechnology [107].
25 An electrokinetic funnel was used to trap and concentrated DNA [108]. The strategy utilized
26 nonlinear electroosmotic flow together with dielectrophoretic trapping at an asymmetric
27 quadrupole electrode. The focusing transformed into a robust funnel, see Figure 3, into which DNA
28 molecules could be trapped forming a compact cone with the aid of short-range dipole-induced self-
29 attraction and dielectrophoresis. The results revealed that DNA molecules were concentrated within
30 a few seconds by several orders [108].
31 Cheng et al. presented dielectrophoresis based microfluidic devices that were capable of enhancing
32 the sensitivity and selectivity of DNA hybridization utilizing an AC electric field and hydrodynamic
33 shear in a continuous flow [109, 110]. They employed a combination of microelectrode based and
34 insulator based dielectrophoresis to rapidly trap ssDNA molecules in a flowing solution to a cusp-
35 shaped nanobead assembly in the vicinity of microelectrodes. The DNA was flushed through the
36 bead assembly and was affected by insulator based dielectrophoresis to concentrate close to the
37 beads surface, and hybridized with target DNA on the beads surface. Thus, the positive DNA
38 dielectrophoresis enhanced the sensitivity [110, 109].
39 In 2013 Martinez-Duarte et al. experimentally and theoretically investigated dielectrophoretic

40 trapping of DNA at carbon electrodes [37]. They studied the frequency dependent dielectrophoretic
41 response of DNA under various flow conditions. Negative DEP was observed at frequencies above 75

21
1 kHz and positive DEP in the range below 75 kHz and down to 5 kHz. Martinez-Duarte et al. further
2 implemented a theoretical model to capture the experimental findings in sufficient detail. Their
3 theoretical considerations, based on reported scaling laws for linear and supercoiled DNA, revealed
4 a flow rate and size depending concentration or trapping of the DNA [37]. Based on these findings an
5 array of carbon electrodes might be applicable for size dependent separation of DNA molecules.
6 Recently, Frusawa and Yoshii presented a DNA dielectrophoresis based fabrication of anisotropic
7 micro-cloths [89]. They used denatured DNA, thus, ssDNA, to stabilize a (carbon nanotube) CNT-
8 suspension followed by dielectrophoretic trapping of the CNTs at tungsten electrode needles.
9 Concentration of CNTs led to growth of an anisotropic CNT-DNA hybrid rectangular shaped film with
10 length scale of 100 m [89].
11 DNA origamis are promising building blocks in micro- and nanobioelectronic circuits. The single
12 building blocks have to be manipulated in a well-defined manner. Dielectrophoretic manipulation
13 could be used for reversible manipulation of the origami structures. Two microelectrode based DEP
14 approaches to trap DNA origami structures have been presented. The first was presented by Kuzyk
15 et al. in 2008. They exploited DEP to immobilize flat DNA origami structures at well-defined positions
16 [111]. AC voltages were applied to gold fingertip nanoelectrodes to trap the origami structures.
17 Immobilization was enabled with thiol-modifications of the DNA for further characterization of the
18 DNA origami structures. After the immobilization the chip was dried and AFM images were taken
19 from the electrodes with the immobilized DNA origami [111, 112]. The second DEP approach to
20 manipulate DNA origami structures was presented in 2015 by Shen et al. [86]. In contrast to the flat
21 origami used by Kuzyk et al., Shen et al. trapped multilayer DNA origami structures. Again the DNA
22 was thiol-modified for immobilization of the origami to the metal nanoelectrodes used for the
23 dielectrophoretic trapping. After the immobilization, AFM imaging was performed to visualize the
24 electrodes and DNA origami structures [86]. Both approaches impressively demonstrate the
25 applicability of dielectrophoresis for manipulation DNA origami structures. These approaches are
26 very promising in the field of molecular electronics and in structural DNA nanotechnology.
27
28 4.1.2 Separation of DNA

29 In this section DNA separation applications are reviewed. Especially for sufficient separation the
30 occurring forces of driving forces and dielectrophoresis have to be well balanced, see also section 0.
31 In 1991, Ajdari and Prost firstly proposed the theoretical possibility of dielectrophoretic separation
32 of molecules [106]. Due to their theoretical model they proposed that the dielectrophoretic trapping
33 time would depend exponentially on the polarizability of the molecules, which again depends on the
34 molecules length. This basic concept of dielectrophoretic separation of DNA of different length has
35 been and is nowadays exploited for numerous separation applications. Moreover, it has been
36 extended to distinguish DNA molecules that differ only in the amount of proteins bound to the DNA.
37 The first experimental realization of a size selective dielectrophoretic separation was presented in
38 2005 by Lao and Hsing [113]. They presented size-dependent manipulation and separation of DNA
39 molecules exploiting dielectrophoresis. Their results revealed the retention time of dielectrophoretic
40 traps increased with the length of DNA molecules (pUC18 and -DNA), which was consistent with

22
1 the theory from Ajdari and Prost. With their experiments, Lao et al. observed a dielectrophoretic
2 separation of DNA for the first time, though they did not achieve a base-line separated resolution.
3 Thereby, they demonstrated the general applicability of dielectrophoresis for matrix-free DNA
4 separation [113].
5 Recently, Song et al. presented a new type of dielectrophoresis device [114]. They have fabricated
6 micropipette tip devices containing a 2% agarose gel plug. Electrodes were positioned inside the
7 micropipette and outside to generate a dielectrophoretic potential. Large genomic DNA (>50 kbp)
8 was separated from 10 m microbeads in high conductivity 1x PBS buffer, with transfer of the
9 collected DNA to another solution [114].
10 Cell-free circulating (CFC) DNA is nowadays considered an important biomarker for early detection of
11 cancer, residual disease, monitoring chemotherapy and other aspects of cancer management [38,
12 115, 116, 117, 94]. The isolation of CFC-DNA from plasma as a liquid biopsy may begin replacing
13 more invasive tissue biopsies as a means to detect and analyze cancer mutations. Sonnenberg et al.
14 developed a dielectrophoresis based microfluidic device to collect the CFC-DNA from whole blood
15 with at the same time detection of fluorescently labeled DNA. The microfluidic detection device
16 consisted of an array of circular microelectrodes. When AC electric fields were applied the CFC-DNA
17 was dielectrophoretically trapped at the electrode edges, see Figure 4 [115, 116, 117, 94]. All other
18 components of the whole blood were removed by a washing step, thus the CFC-DNA was completely
19 separated. PCR was performed after collection of the DNA to gain the DNA sequences. The PCR
20 results of the DEP collected DNA were comparable to results using conventional sample preparation,
21 thus the dielectrophoresis did not affect the DNA integrity. Moreover, the sequencing results were
22 accurate for all patient samples [115, 116, 117, 94]. Thus, the dielectrophoretic approach provides a
23 rapid isolation and collection of CFC-DNA. This, DEP technique also is applicable to a wide variety of
24 biomarkers important in health care [38, 115, 116, 117, 94].
25
26 4.1.3 Molecular detection

27 Dielectrophoresis based detection of molecular binding was firstly shown by Kawabata and Washizu
28 in 2001. For detection of molecular binding of sample DNA they modified single stranded target DNA
29 (ssDNA) to microbeads, mixed those with sample DNA and flushed the mixture through an
30 interdigitated electrode array [118]. The applied electric field settings were chosen such that the
31 unbound DNA exhibited positive dielectrophoresis, while the target DNA bead-complex exhibited
32 negative dielectrophoresis, enabling direct observation of complex formation by the position of the
33 dielectrophoretically trapped DNA [118].
34 Another application of DEP for the detection of molecular binding was presented by Gagnon et al.
35 [119, 120]. They prepared microbeads and sample ssDNA similar to [118] and flushed the mixture in
36 to a quadrupole electrode array. By determining the dielectrophoretic response over an AC electric
37 field frequency range of 50 kHz to 5 MHz they found specific crossover frequencies. Gagnon et al.
38 employed DEP to detect both, hybridization of DNA and the conformation of the hybridized DNA.

23
1 The DEP molecular binding applications reviewed here, are pointing out that dielectrophoresis can
2 be exploited to detect specific nucleic acid sequences due to hybridization to target DNA. Thus, this
3 proves that DNA dielectrophoresis can be used for numerous applications in genetics and medicine.
4 Besides hybridization detection, Rmon-Azcon et al. designed a DEP based competitive

5 immunodevice to detect pesticides [121]. They modified microparticles with ssDNA and pesticide
6 specific antigens. Additionally, they modified electrode arrays with complementary ssDNA. Then the
7 beads were mixed with fluorescent labeled antibodies and the sample, containing pesticides, and
8 flushed through the electrode array. The DNA modified beads were trapped by positive
9 dielectrophoresis and immobilized specifically to complementary DNA. The fluorescence signals
10 decreased with increasing atrazine concentration because of the competitive effect of the
11 immunoreaction between particle-bound antigens and antigens in solution see Figure 5. Rmon-
12 Azcon et al. were able to detect multiple pesticides simultaneously with this DEP based
13 immunoassay.
14 All applications presented in this section utilized microbeads with immobilized ssDNA to determine
15 molecular binding [85, 121, 118]. Thus, the detection was neither on a single molecule base nor
16 label-free. However, it is expected by the authors that label-free single molecule binding detection is
17 possible with microelectrode based DEP.
18
19 4.2 Applications of insulator based DEP

20 In the following sections, applications of insulator based dielectrophoresis are presented. They are
21 subdivided into trapping and concentration of DNA, separation of different DNA samples and
22 detection of molecular binding events. The used samples, the parameters of the applied voltages
23 and the buffer are summarized in table 2.
24
25 4.2.1 iDEP Trapping and concentration of DNA

26 The first realization of dielectrophoretic trapping of DNA molecules by iDEP was shown by Chou et
27 al. in 2002 [25]. They structured a microfluidic channel with insulating posts of trapezoidal shape,
28 Figure 6. They demonstrated frequency and electric field strength dependent trapping of DNA
29 samples with lengths of 368 bp, 1137 bp, 4361 bp, and 39936 bp. These findings were in good
30 agreement to the size dependent polarizability found in dielectrophoretic applications with
31 microelectrodes. With their work, they demonstrated the general applicability of iDEP for DNA
32 manipulation and the potential DNA concentration.
33 Ros et al. presented trapping of large genome DNA (T2-DNA with 164 kbp) in an array of rectangular
34 posts similar to Chou et al. [122]. They showed that the DNA trapping process was reversible by
35 switching AC voltages on and off for trapping and release of DNA molecules. With this work Ros et al.
36 demonstrated the applicability of iDEP for manipulation of large genome DNA, which is hardly
37 addressable with capillary electrophoresis.

24
1 In 2008, Salieb-Beugelaar et al. presented the dielectrophoretically affected migration of DNA in 20

2 nm high nanoslits [123]. They observed an intermitted migration through the nanoslits when the
3 applied electric field was increased to values above 30 kV/m. The intermitted migration was
4 assumed to be dielectrophoretic trapping due to the surface roughness inside the nanoslits. They
5 observed a size dependent behavior, which was in good agreement to the size dependent
6 polarizability of DNA molecules.
7 Swami et al. exploited iDEP trapping of ssDNA molecules to enhance hybridization kinetics [124].
8 Their device consisted of constrictions between sharp triangular posts. They integrated electrodes
9 into the microfluidic device 250 m away from the constriction, different from other iDEP devices
10 where the electrodes were about 1 cm away from the dielectrophoretic active area. They exploited
11 the dielectrophoretic trapping to pre-concentrate DNA tenfold before performing DNA hybridization.
12 Therefore, the hybridization velocity of target and sample ssDNA was significantly enhanced [124].
13 Gallo-Villanueva et al. presented an iDEP device consisting of insulating cylindrical posts to generate
14 dielectrophoretic trapping potentials [84]. In contrast to most of the dielectrophoretic applications,
15 where AC electric fields were used, they exploited a DC electric field. The DNA molecules were
16 immobilized due to negative dielectrophoretic trapping for applied electric fields between 500 and
17 1500 V/cm. Gallo-Villanueva determined concentration factors varying from 8 to 24 times after
18 concentration time-periods of 20 to 40 seconds [84]. Moreover, they investigated if their
19 dielectrophoretic treatment impacted the DNA, i.e. if DNA molecules were denatured during the
20 experiments. A gel electrophoresis after the dielectrophoretic treatment of linear DNA proved that
21 the DNA was not destroyed [84].
22 In 2010, Regtmeier et al. investigated the impact of the spatial conformation and the ionic strength
23 of the buffer on the polarizability and the trapping behavior of DNA [60]. Their device consisted of an
24 array of rectangular insulating posts. They determined the DNA polarizability by the
25 dielectrophoretic trapping lifetimes. Their results depicted that the ionic strength of the buffer
26 impacts the polarizability of DNA molecules, and, therefore the trapping of DNA. Regtmeier et al.
27 found a scaling property of of the polarizability with the ionic strength of the buffer
28 solution. Thus, the polarizability of DNA molecules decreased for increasing ionic strength of the
29 buffer, resulting in a poor trapping for high conductive buffers
30 Gan et al. demonstrated the first trapping of DNA origami by iDEP [125, 126]. They used a device
31 that consists of an array of insulating cylindrical posts. Evaluation of the experimental data and
32 simulations of the dielectrophoretic and electrophoretic migration behavior of the DNA origami
33 structures revealed that the migration of the origami structure in a dielectrophoretic trap was
34 related to electrophoretic migration [126]. Their results allow careful design and optimization of
35 applications of DNA origami such as nanoassembly [126]. In 2015, Gan et al. determined the
36 polarizability of DNA origami structures, which strongly depended on the structure of the origami
37 [125]. Hence, a controlled assembly of specific DNA origami out of a mixture of differently shaped
38 origami should be possible.
39 In 2015, Li et al. investigated DNA trapping behavior in nanoslits for different medium conductivities
40 [92]. They observed that the electrokinetic effects of electrophoresis, electroosmotic flow, and

25
1 dielectrophoresis were affected by the buffer conductivity, similar to Regtmeier et al. A comparison
2 of the experimental trapping results with numerical simulations revealed that those were in good
3 agreement. Thus, the forces by electrophoresis, electroosmotic flow, and dielectrophoresis were the
4 main forces and lead to a sufficient numerical simulation. Moreover, Li et al. determined that the
5 type of dielectrophoresis, i.e. negative or positive dielectrophoresis, depends on the buffer
6 conductivity [92], which is in good agreement to the theory (see section 0).
7 The majority of the devices for trapping DNA with iDEP consist of posts or constrictions in
8 microfluidic devices [25, 126, 125, 92, 60, 123, 124]. Another way for generating an inhomogeneous
9 electric field is by using a nanopipette. Ying et al. used a nanopipette for trapping short DNA-
10 fragments [127]. They used tapered nanopipettes with an inner diameter of 100 nm and a strong
11 voltage drop occurring within a few micrometers at the very tip. With that device a pulsatile delivery
12 of DNA molecules was realized, such that DNA features could be written on surfaces with high
13 precision [127].
14
15 4.2.2 iDEP Separation of DNA

16 In 2006, Li et al. utilized a nanostructured surface to distinguish between open circle and supercoiled
17 DNA [128]. They exploited surface electrophoresis where dielectrophoresis led to conformation
18 specific trapping forces and therefore to different migration velocities. The device consisted of a
19 separation chamber into which a silicon plate, consisting Au nanopattern, was placed, Figure 7. A
20 droplet of DNA solution was loaded on the surface and let dry in air, followed by resolution in buffer
21 and application of an electric field for 60 minutes to gain a baseline separated resolution [128]. The
22 device differs from all other devices reported in this review as it was the only open chamber device.
23 Thus, it lacks of a well-defined micro- or nanochannel and is not applicable for DNA purification.
24 Regtmeier et al. presented a dielectrophoretic separation of DNA in a microfluidic channel for the
25 first time in 2007 [59]. They structured a microfluidic device with insulating rectangular posts and
26 injected a plug of a mixture containing two different sized DNA. The different species exhibited
27 specific migration velocities through the array due to the size dependent dielectrophoretic trapping
28 times. Therefore, the injected plug separated into two distinct bands, representing the two species,
29 respectively, if the applied voltages were balanced carefully with respect to the thermal noise [59].
30 In 2010, Regtmeier et al. successfully demonstrated the separation of DNA with different spatial
31 conformation [60]. They led a mixture of linear and supercoiled DNA through an array of posts and
32 achieved almost baseline-separated resolution of the two conformations within 210 s. This was
33 significantly faster than the well-established gel electrophoresis with typical separation times of >30
34 minutes [60].
35 Parikesit et al. presented a microfluidic device where DNA samples flew around a sharp corner,
36 experiencing a dielectrophoretic force [26]. They stated that a continuous separation would be
37 achieved at the sharp corner due to different dielectrophoretic manipulation of the samples. The
38 device consisted of a 100 m wide supply channel that widened into an arc shaped chamber.
39 Although the trajectory of DNA, of a fixed starting position, was proven to be size dependent, the
40 width of the supply channel was critical for the separation resolution as the dielectrophoretic force

26
1 was strongly dependent on the distance from the corner; already 20 m away from the channel wall
2 the migration was almost size-independent. Thus, this device would be insufficient for a continuous-
3 flow separation or purification of DNA samples, but an enhanced design of the supply channel or a
4 focusing of the DNA towards the channel wall could lead to an easy realization of a continuous-flow
5 separation device.
6 Li et al. and Regtmeier et al. demonstrated the general applicability of DNA separation with iDEP.
7 Nevertheless, their approaches lack a continuous performance that allows further downstream
8 applications or harvesting of the separated samples. In 2013, Viefhues et al. presented for the first
9 time a sufficient dielectrophoresis based continuous-flow separation of DNA molecules [129]. Their
10 microfluidic device consisted of a channel-spanning ridge reducing the flow through height, thus
11 generating a shallow slit of 670 nm height, Figure 8. The continuousflow separation approach was
12 according to Hawkins et al. [51], who separated microspheres of different diameters. Depending on
13 the electrophoretic, electroosmotic, and dielectrophoretic forces the sample followed one of two
14 possible pathways. The first possibility was to pass the slit unaffected. The second possibility was
15 that the sample was deflected along the ridge and traveled further down the channel after reaching
16 the opposite channel wall laterally separated from the first pathway (see Figure 8). Especially for
17 the second pathway the interplay of the electrokinetic forces was crucial. In [95] the main effects
18 that appear at such slit have been simulated and compared with the experimental results of
19 continuous-flow nano bead separation, both, simulation and experiment were in very good
20 agreement. This continuous-flow approach also allows analysis of the purity of minicircle production
21 [130].
22 Later in 2013, Viefhues et al. presented an enhanced microfluidic device for continuous-flow
23 separation of three different DNA species [39]. The enhanced device consisted of two insulating
24 ridges, providing a two-step separation. First, the smallest species were separated from the sample
25 mixture and, second, the middle sized species were separated from the mixture due to weaker DEP
26 forces, see Figure 9. Viefhues et al. achieved a baseline separated resolution with about 100%
27 efficiency for both steps [39]. Due to branching of the channel each species was collected in a
28 separate reservoir. A possible application of this separation is in purification of gene vaccines or
29 analysis of their purity during the production. Although the continuous separation performance
30 offers applications in production, the volume throughput so far was very low. Thus, strategies for
31 (massive) parallelization have to be developed.
32 Recently, Jones et al. presented iDEP based continuous-flow separation of linear DNA molecules at a
33 2D axisymmetric channel constriction [131]. Due to the geometry localized electric field
34 inhomogeneities were generated at the corners. The microfluidic channel split into different
35 branches downstream of the constriction. Thus, depending on the direction of the dielectrophoretic
36 force, i.e. positive or negative dielectrophoresis, the DNA was collected in different reservoirs. Jones
37 et al. reported sorting efficiencies of 10.2 kbp and 48.5 kbp DNA of 32% (+/- 9%) and 43% (+/- 18%),
38 respectively, at 2000 V and 100 Hz [131].
39 The monitoring of the separation techniques presented so far was performed with fluorescence
40 microscopy. The DNA species were labeled with one type of fluorophore, mainly YOYO-1, except for
41 [131]. Thus, a distinction of the different species was not possible. Future tasks will be to either use

27
1 monitoring techniques that distinguish between the species or to investigate the separation
2 products to determine the process efficiency and selectivity. Furthermore, if applications of the
3 dielectrophoresis based separations are purification, for example in biotechnological or medical
4 production processes, a label-free monitoring is indispensable. A promising alternative monitoring
5 method is described in section 0.
6 4.2.3 Detection of molecular binding

7 The general applicability of dielectrophoresis to detect molecular binding exploiting microelectrodes
8 has been described in section 0. In 2012, Viefhues et al. demonstrated for the first time a
9 dielectrophoresis based application for single-molecule, label-free detection of molecular binding
10 [132]. Their device consisted of an array of insulating ridges that reduced the channel height from 6
11 m down to 180 nm generating a very high electric field gradient. Viefhues et al. injected a plug
12 containing the sample and a reference DNA into the array of ridges and observed the
13 dielectrophoresis dependent migration velocities. A shift between the reference DNA and the
14 sample DNA was observed within 30 seconds for complex-formation of the DNA with specific
15 proteins, like RNA-polymerase or ActinomycinD [132]. They named the dielectrophoresis based
16 molecular binding detection dielectrophoretic mobility shift assay (DEMSA) in accordance to the
17 electrophoretic mobility shift assay (EMSA).
18 In 2013, Viefhues et al. demonstrated a continuous-flow detection of molecular binding by iDEP

19 [129]. Their device consisted of an insulating ridge spanning the microfluidic channel laterally. The
20 sample DNA, i.e. DNA that was incubated with proteins, was continuously led towards the ridge
21 together with a reference DNA. The DNA-complex was deflected at the ridge, whilst the reference
22 DNA was not, thus molecular binding was detected in continuous-flow operation [129, 133]. The
23 advantage of that approach is that complex formation could be observed over time when started
24 directly after mixing the samples.
25 5 Alternative detection of DNA trapping and separation

26 Dielectrophoresis has been described as being label-free, nevertheless, for most applications with
27 DNA fluorescent labeling has been used to monitor the dielectrophoretic response. Especially for
28 applications of DEP in purification or production for biotechnology or medicine the fluorescent
29 labeling is a huge drawback as DNA that has been labeled fluorescently is forbidden for medical
30 applications and does not fulfill the requirements for biotechnological applications. Furthermore,
31 the fluorescent monitoring always needs large and expensive optical components for the detection.
32 Thus, alternative detection methods are necessary.
33 For dielectrophoretic trapping of DNA with a subsequent immobilization AFM or TEM imaging is an
34 alternative imaging method. Kuzyk et al. and Shen et al. immobilized DNA, which was
35 dielectrophoretically, trapped at gold electrodes, and used AFM and TEM imaging to visualize the
36 trapped DNA [111, 112, 86]. This imaging could be used for control of positioning of DNA or DNA
37 origami structures, which are used as nanobioelectronic elements. However, this imaging approach
38 is not suitable for monitoring for most of the applications described above, especially for separation
39 or concentration of DNA samples.

28
1 Another monitoring method exploits impedance or capacity measurements, which both are strongly
2 dependent on the existence of charged objects in the measurement area [134, 135, 136, 137, 138].
3 Thus, concentrations of biomolecules can be detected by these methods.
4 Basuray et al. exploited impedance measurements to detect matching and miss-matching DNA
5 hybridization [134]. They demonstrated an open-flow impedance-sensing platform for DNA
6 hybridization on carbon nanotube (CNT) surfaces by DEP. Basuray et al. were able to detect
7 hybridization events in less than 20 min with picomolar target DNA concentrations in a label-free
8 microfluidic detection platform [134].
9 Henning et al. measured the concentration of DNA by monitoring the capacitance between
10 interdigitated electrodes [137, 136]. Via the capacitance measurement they determined the
11 dielectrophoretic response of DNA without the need for any chemical modification of the analyte,
12 making fluorescent labeling obsolete. DNA concentrations from below 0.1 g/mL were detected and
13 the capacitance response correlated significantly with the applied electric field frequency (3 kHz to 3
14 MHz) as well as with the length of the DNA molecule (100 bp to 48.5 kbp) [136]. Being purely
15 electronic the monitoring method can be easily integrated into lab-on-a-chip systems. Neither
16 optical nor mechanical access to the sample is needed in the course of the measurement [136, 137].
17 Furthermore, the method allows for an uncomplicated, automatic acquisition of the
18 dielectrophoretic properties of DNA.
19 In 2013, Li et al. utilized impedimetric changes to detect DNA concentrations during microelectrode
20 based DEP applications [138]. They used physiological solutions with high electric conductivity (154
21 mM Na+) for pursuing DNA-based physical applications. An array of interdigitated electrodes was
22 utilized for the dielectrophoretic manipulation of DNA. The impedimetric measurements were
23 performed with a high precision impedance analyzer. Parallel to the impedimetric measurement Li
24 et al. used fluorescent microscopy to observe the dielectrophoretic response. A comparison of the
25 fluorescent images and the impedance measurements revealed a correlation between a change in
26 the impedance and dielectrophoretic trapping [138].
27 6 Summary
28 The aim of this review was to give an overview about the basic theory of DNA dielectrophoresis as
29 well as important considerations, like Joule heating and fluid dynamics, when starting with DNA
30 dielectrophoresis. Furthermore, we wanted to provide the reader an overview of the experimental
31 realizations of DNA dielectrophoresis, their applications and future challenges.
32 A comparison of the experimental trapping results of the reviewed papers with their numerical
33 simulations revealed that those were in good agreement. Thus, the forces induced by
34 electrophoresis, dielectrophoresis, electroosmotic flow, and pressure driven flow were the main
35 forces and lead to sufficient numerical simulation. The respective forces were described and
36 discussed in the theory section.
37 Various applications of DNA dielectrophoresis have been presented for microelectrode based
38 dielectrophoresis as well as for insulator-based dielectrophoresis. Those applications were for DNA

29
1 trapping, concentration, and molecular binding detection with huge impact on future medicine and
2 diagnostics. Furthermore, DNA dielectrophoresis provides new separation and purification methods
3 in pharmaceutical or biotechnological applications. Thus, DNA dielectrophoresis was proven a very
4 versatile and successful approach for manifold DNA manipulation and investigation methods.
5 7 Conflict of interest statement

6 The authors declare no conflict of interest.
8 8 References
9 [1] Kler, P. A., Sydes, D., Huhn, C., Anal. Bioanal.Chem. 2015, 407, 119138.
10 [2] Shariatgorji, M., Astorga-Wells, J., Ilag, L. L., Anal. Bioanal.Chem. 2011, 399, 191195.
11 [3] Tetala, K. K. R., Vijayalakshmi, M. A., Anal. Chim. Acta 2016, 906, 721.
12 [4] Rahong, S., Yasui, T., Kaji, N., Baba, Y., Lab. Chip 2016, 16, 11261138.
13 [5] Acquah, C., Moy, C. K. S., Danquah, M. K., Ongkudon, C. M., J. Chromatogr. B Analyt.
14 Technol. Biomed. Life. Sci. 2016, 1015-1016, 121134.
15 [6] Slouka, Z., Senapati, S., Chang, H.-C., Annu Rev Anal Chem (Palo Alto Calif) 2014, 7, 317335.
16 [7] de Kort, B. J., de Jong, G. J., Somsen, G. W., Anal. Chim. Acta 2013, 766, 1333.
17 [8] Jubery, T. Z., Srivastava, S. K., Dutta, P., Electrophoresis 2014, 35, 691713.
18 [9] Tegenfeldt, J. O., Prinz, C., Cao, H., Huang, R. L., Austin, R. H., Chou, S. Y., Cox, E. C., Sturm,
19 J. C., Anal. Bioanal.Chem. 2004, 378, 16781692.
20 [10] Pethig, R., Dielectrophoresis: Theory, methodology, and biological applications, John Wiley &
21 Sons, Inc, Hoboken, NJ, 2017.
22 [11] Regtmeier, J., Eichhorn, R., Viefhues, M., Bogunovic, L., Anselmetti, D., Electrophoresis 2011,
23 32, 22532273.
24 [12] Zhang, C., Khoshmanesh, K., Mitchell, A., Kalantar-Zadeh, K., Anal. Bioanal.Chem. 2010, 396,
25 401420.
26 [13] Dash, S., Mohanty, S., Electrophoresis 2014, 35, 26562672.
27 [14] Kuzyk, A., Electrophoresis 2011, 32, 23072313.
28 [15] Lapizco-Encinas, B. H., Rito-Palomares, M., Electrophoresis 2007, 28, 45214538.

30
1 [16] Meighan, M. M., Staton, S. J. R., Hayes, M. A., Electrophoresis 2009, 30, 852865.
2 [17] Pethig, R., Biomicrofluidics 2010, 4.
3 [18] Pethig, R., Adv Drug Deliv Rev 2013, 65, 15891599.
4 [19] Qian, C., Huang, H., Chen, L., Li, X., Ge, Z., Chen, T., Yang, Z., Sun, L., Int. J. Mol. Sci. 2014, 15,
5 1828118309.
6 [20] Sia, S. K., Whitesides, G. M., Electrophoresis 2003, 24, 35633576.
7 [21] Srivastava, S. K., Gencoglu, A., Minerick, A. R., Anal. Bioanal.Chem. 2011, 399, 301321.
8 [22] Hlzel, R., IET Nanobiotechnol. 2009, 3, 2845.
9 [23] Ghonge, S., Banerjee, S., Defence Science Journal 2016, 66, 307315.
10 [24] Bakewell, D. J., Morgan, H., IEEE Trans. Nanobioscience 2006, 5, 139146.
11 [25] Chou, C.-F., Tegenfeldt, J. O., Bakajin, O., Chan, S. S., Cox, E. C., Darnton, N., Duke, T., Austin,
12 R. H., Biophys. J. 2002, 83, 21702179.
13 [26] Parikesit, G. O. F., Markesteijn, A. P., Piciu, O. M., Bossche, A., Westerweel, J., Young, I. T.,
14 Garini, Y., Biomicrofluidics 2008, 2, 24103.
15 [27] Cuervo, A., Dans, P. D., Carrascosa, J. L., Orozco, M., Gomila, G., Fumagalli, L., PNAS 2014,
16 111, E3624E3630.
17 [28] Rau, D. C., Charney, E., Biophys. Chem. 1983, 17, 3550.
18 [29] Mohanty, U., Zhao, Y., Biopolymers 1996, 38, 377388.
19 [30] Saif, B., Mohr, R. K., Montrose, C. J., Litovitz, T. A., Biopolymers 1991, 31, 11711180.
20 [31] Forns, J. Colloid Interface Sci. 2000, 222, 97102.
21 [32] Cerutti, D. S., Wong, C. F., McCammon, J. A., Biopolymers 2003, 70, 391402.
22 [33] Grandison, S., Penfold, R., Vanden-Broeck, J.-M., Phys. Chem. Chem. Phys. 2005, 7, 3486
23 3495.
24 [34] Zavadlav, J., Podgornik, R., Praprotnik, M., J. Chem. Theory Comput. 2015, 11, 50355044.
25 [35] Ambia-Garrido, J., Vainrub, A., Pettitt, B. M., Comput. Phys. Commun. 2010, 181, 20012007.
26 [36] Dorfman, K. D., King, S. B., Olson, D. W., Thomas, J. D. P., Tree, D. R., Chem. Rev. 2013, 113,
27 25842667.
28 [37] Martinez-Duarte, R., Camacho-Alanis, F., Renaud, P., Ros, A., Electrophoresis 2013, 34, 1113
29 1122.

31
1 [38] Hyun, K.-A., Jung, H.-I., Electrophoresis 2013, 34, 10281041.
2 [39] Viefhues, M., Wegener, S., Rischmller, A., Schleef, M., Anselmetti, D., Lab. Chip 2013, 13,
3 31113118.
4 [40] Tuukkanen, S., Kuzyk, A., Toppari, J. J., Hytnen, V. P., Ihalainen, T., Trm, P., Appl. Phys.
5 Lett. 2005, 87, 183102.
6 [41] Kumemura, M., Collard, D., Yamahata, C., Sakaki, N., Hashiguchi, G., Fujita, H.,
7 ChemPhysChem 2007, 8, 18751880.
8 [42] Pohl, H., Dielectrophoresis: The Behavior of Neutral Matter in Nonuniform Electric Fields,
9 Cambridge University Press, Cambridge, 1978.
10 [43] Stratton, J. A., Electromagnetic Theory, New York, McGraw Hill, 1941.
11 [44] Ramos, A., Morgan, H., Green, N. G., Castellanos, A., J. Phys. D: Appl. Phys. 1998, 31, 2338
12 2353.
13 [45] Bruus, H., Theoretical Microfluidics, Oxford University Press, Oxford, 2007.
14 [46] Mazo, R. M., Brownian Motion: Fluctuations, Dynamics and Applications, Oxford University
15 Press, Oxford, 2002.
16 [47] Frey, E., Kroy, K., Ann Phys (Leipzig) 2005, 14, 20.
17 [48] Haus, H. A., Melcher, J. R., Electromagnetic Fields and Energy, Prentice-Hall, Englewood
18 Cliffs, 1989.
19 [49] Green, N. G., Ramos, A., Gonzalez, A., Morgan, H., A., Castellanos, Phys Rev E 2002, 66,
20 026305.
21 [50] Purcell, E. M., Am. J. Phys 1977, 45, 3.
22 [51] Hawkins, B. G., Smith, A. E., Syed, Y. A., Kirby, B. J., Anal. Chem. 2007, 79, 72917300.
23 [52] Viefhues, M., Manchanda, S., Chao, T.-C., Anselmetti, D., Regtmeier, J., Ros, A., Anal.
24 Bioanal.Chem. 2011, 401, 21132122.
25 [53] Cummings, E., Griffiths, S., Nilson, R., Paul, P., AnalChem 2000, 72, 25262532.
26 [54] Sridharan, S., Zhu, J., Hu, G., Xuan, X., Electrophoresis 2011, 32, 2274?2281.
27 [55] Xuan, X., Electrophoresis 2008, 29, 3343.
28 [56] Viovy, J.-L., Rev. Mod. Phys. 2000, 72, 813872.
29 [57] Eichhorn, R., in preparation.
30 [58] Long, D., Viovy, J.-L., Ajdari, A., Phys. Rev. Lett. 1996, 76, 38583861.

32
1 [59] Regtmeier, J., Duong, T. T., Eichhorn, R., Anselmetti, D., Ros, A., Anal. Chem. 2007, 79, 3925
2 3932.
3 [60] Regtmeier, J., Eichhorn, R., Bogunovic, L., Ros, A., Anselmetti, D., Anal. Chem. 2010, 82,
4 71417149.
5 [61] Jones, T. B., Electromechanics of Particles, Cambridge University Press, Cambridge, 1995.
6 [62] Jones, T. B., IEEE Engineering in Medicine and Biology 2003, 33.
7 [63] Zheng, L., Brody, J. P., Burke, P. J., Biosens. Bioelectron. 2004, 20, 606619.
8 [64] Mohamad, A. S., Jeynes, J. C. G., Hughes, M. P., IEEE Trans. Nanobioscience 2014, 13, 5154.
9 [65] Tomic, S., Babic, S. D., Vuletic, T., D., S. K., Ivankovic, Griparic, L., Podgornik, R., Phys Rev E
10 2007, 75, 021905.
11 [66] Porschke, D., Biophys. Chem. 1985, 22, 237247.
12 [67] Mandel, M., Odijk, T., Ann Rev Phys Chem 1984, 35, 75108.
13 [68] Manning, G. S., Eur. Phys. J. E 2011, 34, 39.
14 [69] Dhont, J. K. G., Kang, K., Eur. Phys. J. E 2011, 34, 40.
15 [70] Bertolotto, J. A., Corral, G. M., Farias de La Torre, E. M., Roston, G. B., J Phys Condens Matter
16 2010, 22, 494101.
17 [71] Porschke, D., Antosiewicz, J. M., J Chem Phys B 2005, 109, 10341038.
18 [72] Zhou, J., Schmid, F., Soft Matter 2015, 11, 67286739.
19 [73] Rotenberg, B., Pagonabarraga, I., Mol. Phys. 2013, 111, 827842.
20 [74] Savelyev, A., MacKerell, A. D., Jr, J. Phys. Chem. B 2014, 118, 67426757.
21 [75] Comsol, http://www.comsol.com.
22 [76] Zhou, C., Reisner, W. W., Staunton, R. J., Ashan, A., Austin, R. H., Riehn, R., Phys. Rev. Lett.
23 2011, 106, 248103.
24 [77] Zhou, C., Riehn, R., Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2015, 92, 012714.
25 [78] Green, N. G., Ramos, A., Gonzalez, A., Morgan, H., A., Castellanos, Phys Rev E 2000, 61,
26 40114018.
27 [79] Gonzalez, A., Ramos, A., Green, N. G., A., Castellanos, Morgan, H., Phys Rev E 2000, 61,
28 40194028.
29 [80] Bown, M. R., Meinhart, C. D., Microfluid. Nanofluid. 2006, 2, 513?523.

33
1 [81] Kakac, S., Kosoy, B., Li, D., Pramuanjaroenkij, A. (Eds.), Microfluidics Based Microsystems
2 Fundamentals and Applications, Springer, New York, 2010.
3 [82] Duhr, S., Braun, D., PNAS 2006, 103, 1967819682.
4 [83] Reichl, M., Herzog, M., Gtz, A., Braun, D., Phys. Rev. Lett. 2014, 112, 198101.
5 [84] Gallo-Villanueva, R. C., Rodrguez-Lpez, C. E., Daz-de-la Garza, R. I., Reyes-Betanzo, C.,
6 Lapizco-Encinas, B. H., Electrophoresis 2009, 30, 41954205.
7 [85] Tuukkanen, S., Toppari, J., Hytonen, V., Kuzyk, A., Kulomaa, M., Torma, P., Int. J.
8 Nanotechnol. 2005, 2, 280291.
9 [86] Shen, B., Linko, V., Dietz, H., Toppari, J. J., Electrophoresis 2015, 36, 255262.
10 [87] Simmons, B. A., McGraw, G. J., Davalos, R. V., Fiechtner, G. J., Fintschenko, Y., Cummings,
11 E. B., MRS Bull. 2006, 31, 120124.
12 [88] Sung, K. E., Burns, M. A., Anal. Chem. 2006, 78, 29392947.
13 [89] Frusawa, H., Yoshii, G., Nanoscale Res. Lett. 2015, 10, 107.
14 [90] Loucaides, N., Ramos, A., Georghiou, G., J. Electrostat. 2011, 69, 111 118.
15 [91] Hawkins, B. G., Kirby, B. J., Electrophoresis 2010, 31, 36223633.
16 [92] Li, S., Ye, Z., Hui, Y. S., Gao, Y., Jiang, Y., Wen, W., Biomicrofluidics 2015, 9, 054115.
17 [93] McCanna, J. P., Sonnenberg, A., Heller, M. J., J. Biophotonics 2014, 7, 863873.
18 [94] Sonnenberg, A., Marciniak, J. Y., Skowronski, E. A., Manouchehri, S., Rassenti, L., Ghia, E. M.,
19 Widhopf, G. F., 2nd, Kipps, T. J., Heller, M. J., Electrophoresis 2014, 35, 18281836.
20 [95] Viefhues, M., Eichhorn, R., Fredrich, E., Regtmeier, J., Anselmetti, D., Lab. Chip 2012, 12,
21 485494.
22 [96] Barbulovic-Nad, I., Xuan, X., Lee, J. S. H., Li, D., Lab. Chip 2006, 6, 274279.
23 [97] Thwar, P. K., Linderman, J. J., Burns, M. A., Electrophoresis 2007, 28, 45724581.
24 [98] Washizu, M., Kurosawa, O., IEEE Trans. Ind. Appl. 1990, 26, 11651172.
25 [99] Washizu, M., Kurosawa, O., Arai, I., Suzuki, S., Shimamoto, N., IEEE Trans. Ind. Appl. 1995, 31,
26 447456.
27 [100] Yamamoto, T., Kurosawa, O., Kabata, H., Shimamoto, N., Washizu, M., IEEE Trans. Ind. Appl.
28 2000, 36, 10101017.
29 [101] Asbury, C. L., van den Engh, G., Biophys. J. 1998, 74, 10241030.
30 [102] Asbury, C. L., Diercks, A. H., van den Engh, G., Electrophoresis 2002, 23, 26582666.

34
1 [103] Germishuizen, W. A., Tosch, P., Middelberg, A. P. J., Wlti, C., Davies, A. G., Wirtz, R., Pepper,
2 M., J. Appl. Phys. 2005, 97, 014702.
3 [104] Tuukkanen, S., Toppari, J. J., Kuzyk, A., Hirviniemi, L., Hytnen, V. P., Ihalainen, T., Trm, P.,
4 Nano Lett. 2006, 6, 13391343.
5 [105] Tuukkanen, S., Kuzyk, A., Toppari, J. J., Hkkinen, H., PHytnen, V., Niskanen, E., Rinki, M.,
6 Trm, P., Nanotechnology 2007, 18, 295204.
7 [106] Ajdari, A., Prost, J., PNAS 1991, 88, 44684471.
8 [107] Kumemura, M., Collard, D., Sakaki, N., Yamahata, C., Hosogi, M., Hashiguchi, G., Fujita, H., J.
9 Micromech. Microeng. 2011, 21, 054020.
10 [108] Du, J.-R., Juang, Y.-J., Wu, J.-T., Wei, H.-H., Biomicrofluidics 2008, 2, 44103.
11 [109] Cheng, I.-F., Senapati, S., Cheng, X., Basuray, S., Chang, H.-C., Chang, H.-C., Lab. Chip 2010,
12 10, 828831.
13 [110] Cheng, I.-F., Han, H.-W., Chang, H.-C., Biosens. Bioelectron. 2012, 33, 3643.
14 [111] Kuzyk, A., Yurke, B., Toppari, J. J., Linko, V., Trm, P., Small 2008, 4, 447450.
15 [112] Kuzyk, A., Toppari, J. J., Trm, P., Methods Mol. Biol. 2011, 749, 223234.
16 [113] Ieng Kin Lao, A., Hsing, I.-M., Lab. Chip 2005, 5, 687690.
17 [114] Song, Y., Sonnenberg, A., Heaney, Y., Heller, M. J., Electrophoresis 2015, 36, 11071114.
18 [115] Sonnenberg, A., Marciniak, J. Y., Krishnan, R., Heller, M. J., Electrophoresis 2012, 33, 2482
19 2490.
20 [116] Sonnenberg, A., Marciniak, J. Y., McCanna, J., Krishnan, R., Rassenti, L., Kipps, T. J., Heller,
21 M. J., Electrophoresis 2013, 34, 10761084.
22 [117] Sonnenberg, A., Marciniak, J. Y., Rassenti, L., Ghia, E. M., Skowronski, E. A., Manouchehri, S.,
23 McCanna, J., Widhopf, G. F., 2nd, Kipps, T. J., Heller, M. J., Clin. Chem. 2014, 60, 500509.
24 [118] Kawabata, T., Washizu, M., IEEE Transactions on Industry Applications: Dielectrophoretic
25 detection of molecular bindings, volume 37, 2001.
26 [119] Gagnon, Z., Senapati, S., Gordon, J., Chang, H.-C., Electrophoresis 2008, 29, 48084812.
27 [120] Gagnon, Z., Senapati, S., Chang, H.-C., Electrophoresis 2010, 31, 666671.
28 [121] Ramn-Azcn, J., Yasukawa, T., Mizutani, F., Anal. Chem. 2011, 83, 10531060.
29 [122] Ros, A., Hellmich, W., Regtmeier, J., Duong, T. T., Anselmetti, D., Electrophoresis 2006, 27,
30 26512658.

35
1 [123] Salieb-Beugelaar, G. B., Teapal, J., Nieuwkasteele, J. v., Wijnperl, D., Tegenfeldt, J. O.,
2 Lisdat, F., van den Berg, A., Eijkel, J. C. T., Nano Lett. 2008, 8, 17851790, pMID: 18393468.
3 [124] Swami, N., Chou, C.-F., Ramamurthy, V., Chaurey, V., Lab. Chip 2009, 9, 32123220.
4 [125] Gan, L., Camacho-Alanis, F., Ros, A., Anal. Chem. 2015, 87, 1205912064.
5 [126] Gan, L., Chao, T.-C., Camacho-Alanis, F., Ros, A., Anal. Chem. 2013, 85, 1142711434.
6 [127] Ying, L., White, S. S., Bruckbauer, A., Meadows, L., Korchev, Y. E., Klenerman, D., Biophys. J.
7 2004, 86, 10181027.
8 [128] Li, B., Fang, X., Luo, H., Seo, Y.-S., Petersen, E., Ji, Y., Rafailovich, M., Sokolov, J., Gersappe, D.,
9 Chu, B., Anal. Chem. 2006, 78, 47434751.
10 [129] Viefhues, M., Regtmeier, J., Anselmetti, D., Analyst 2013, 138, 186196.
11 [130] Rischmller, A., Viefhues, M., Dieding, M., Schmeer, M., Baier, R., Anselmetti, D., Schleef, M.,
12 Minicircle and Miniplasmid DNA vectors. The future of non-viral and viral gene transfer, Wiley-VCH,
13 Weinheim, 2013.
14 [131] Jones, P. V., Salmon, G. L., Ros, A., Analytical Chemistry 2017, 0, null, pMID: 27936618.
15 [132] Viefhues, M., Regtmeier, J., Anselmetti, D., J. Micromech. Microeng. 2012, 22, 115024.
16 [133] Viefhues, M., Regtmeier, J., Anselmetti, D., Methods Mol. Biol. 2015, 1274, 99110.
17 [134] Basuray, S., Senapati, S., Aijian, A., Mahon, A. R., Chang, H.-C., ACS Nano 2009, 3, 1823
18 1830.
19 [135] Basuray, S., Chang, H.-C., Biomicrofluidics 2010, 4, 13205.
20 [136] Henning, A., Bier, F. F., Hlzel, R., Biomicrofluidics 2010, 4.
21 [137] Henning, A., Henkel, J., Bier, F. F., Hlzel, R., PMC Biophysics 2008, 1, 112.
22 [138] Li, S., Yuan, Q., Morshed, B. I., Ke, C., Wu, J., Jiang, H., Biosens. Bioelectron. 2013, 41, 649
23 655.
24
25
26
27
28 Figure legends:
29 Figure 1: Illustration of the concepts of electrodeless (A) and microelectrode-based (B) dielectrophoresis. The white lines
30 indicate the electric field lines and the color code represents (increasing from blue to yellow). Adapted from Ref.
31 [11] with permission from WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, copyright 2011.

36
4 Figure 2: Separation modes. a) Sequential separation mode, i.e. a sample plug is led into the separation region and split
5 in the different species. Thus, the species are separated in time. b) Continuous-flow separation mode, i.e. the sample is
6 continuously led in the separation region and the species are spatially separated.
10 Figure 3: (a) Long-range electrokinetic funnel under an AC field of 20 Vpp (peak-to-peak voltage) at 1 kHz. The DNA
11 concentration is 1g/ml. The inset is the blow-up view of a 1 mm-long concentrated DNA thread. The sequential images
12 on the west electrode in (a) are taken at (b) 0.0, (c) 0.8, and (d) 3.3 s after turning on the field. Reprinted from [108],
13 with the permission of AIP Publishing.

37
6 Figure 4: DEP isolation and fluorescent detection of CFC-DNA in blood samples from CLL Patients, for detailed
7 information we refer to [116]. About 20 L of whole blood was applied to the DEP microarray device. An AC field was
8 then applied at 10 kHz and 20 Vpp to the nine microelectrodes in columns 2, 3, and 4 (yellow dotted area) for 15
9 minutes. No voltage was applied to the three microelectrodes in column 1 (left side), which serve as a negative control.
10 The DEP microarray was washed three times after which epifluorescent microscope imaging detection was carried out.
11 Figures (A), (B), and (C) show the results for blood samples from three normal (non-CLL) individuals, figures (D), (E), (F),
12 (G), and (H) show the results for five different CLL patient blood samples, and (I) shows the result for a normal
13 disrupted buffy coat blood sample. Reproduced from Ref. [116] with permission from WILEY-VCH Verlag GmbH & Co.
14 KGaA, Weinheim, copyright 2013.
15

38
4 Figure 5: Schematic representation of the competitive immunodevice operation system. Reprinted with permission from
5 [121]. Copyright 2011 American Chemical Society.

39
4 Figure 6: (AD) Optical micrographs of DEP trapping of 368-bp dsDNA with driving voltage of 1 kV (corresponding to 5 V
5 p-p across each unit cell) and applied frequencies of; 200, 400, 800, and 1000 Hz. The frame size is 80 x 80 m. The
6 images shown here were each averaged over three consecutive frames, starting with the first one taken 1 min after the
7 AC electric field parameters were changed, and at 1-min intervals for each of the following images to allow equilibrium
8 densities to be achieved. Reprinted with permission from [25]. Copyright 2002 The Biophysical Society. Published by
9 Elsevier Inc.
10

40
3 Figure 7: Principle of surface electrophoresis as used in [128]. A silicon wafer is structured with Au gold nano pattern,
4 indicated by the semicircles. A droplet of sample solution is dried on the structured wafer and resolved in a buffer
5 medium while an electric field is applied to the sample chamber.

41
3 Figure 8: Scheme of continuous-flow separation as used in [129]. The DNA mixture is led towards the insulating ridge
4 where selective dielectrophoretic forces led to two distinct pathways.
10 Figure 9: Separation of the parental plasmid, miniplasmid and minicircle DNA. False-color collage of the fluorescence
11 microscopy images. A mixture of the parental plasmid, miniplasmid and minicircle DNA is injected towards the ridges
12 from a side channel (each yellow spot depicts one single DNA molecule). At the first ridge, the minicircle DNA is

42
1 separated out of the mixture and led into a separate channel. The parental plasmid and miniplasmid DNA are deflected
2 and migrate towards the second ridge, where only the parental plasmid DNA is deflected. Thus, all three species are
3 collected in separate channels. Adapted from Ref. [39] with permission from The Royal Society of Chemistry, copyright
4 2013.
proces voltag frequenc conductivit

ref. s samples time mode e Vpp y y [mS/cm] buffer
~ 30 10-250
[37]
conc. 48.5 kbp min 16 kHz 0.187
48.5 kbp
[93] DNA from
conc. blood 20 min 10 kHz 1.1 - 6.6 whole blood
blood/plasm
[94]
conc. CFC-DNA 3 min 11 10 kHz a
[136 100 bp to 48 1 kHz - 3
] conc. kbp DNA 4.2 MHz DI water
[137 2961 bp
] conc. DNA 11.3 1 MHz
[138 2.7 and 48.5 20 kHz - 5
] conc. kbp DNA 10 MHz
[141 48.5 kbp
] conc. DNA 1 min 20 5 MHz DI water
DNA
[118
hybridizatio
]
detec. n 2.5 MHz 0.2
DNA
[119
hybridizatio 50 kHz - 5
]
detec. n 10 MHz
pesticides
[121 via DNA
] modified sequentia
detec. beads ~ 3 min l 8 2 MHz
[134 DNA < 20 400 kHz -
] detec. hybridizatio min 1 MHz

43
n
[113 2.7 and 48.5 ~ 50 sequentia 10 Hz to
] sep. kbp DNA min l 1.7 10 kHz DI water
> 50 kbp
[114
DNA from 10 sequentia
]
sep. m beads l 160 10 kHz 1x PBS
CFC DNA
[115
from whole sequentia
]
sep. blood 15 min l 20 10 kHz 7.4 whole blood
CFC-DNA
[116
from whole sequentia blood/plasm
]
sep. blood 15 min l 20 10 kHz 1.1 - 6.6 a
[117 sequentia
] sep. CFC-DNA 3 min l 11 10 kHz whole blood
100 kHz -
[24]
trap. 12 kbp DNA 6-7s 4.5 5 MHz
[41] trap. 48.5 kbp 1-2s 900 kHz DI water
100 kHz-
[63]
trap. 48.5 kbp ~ 30 s 8 30 MHz DI water
0.6 to
[86]
trap. DNA origami 1-5 min 1.5 8-13 MHz 0.275
500 kHz
[88] 48.5 kbp to 2.3
trap. DNA 2 to 25 MHz 0.002
carbon NT 1 kHz - 20
[89]
trap. with DNA ~ 900 s MHz
48.5 kbp 60 to 40 kHz - 2
[98]
trap. DNA 150 MHz 0.002 DI water
17, 1.5, 30,
[99] and 48.5 kbp
trap. DNA 1 MHz <0.002
[101 48.5 kbp 1 Hz - 1
] trap. DNA 1 min 200 kHz 0.63
164, 48.5,
[102
35.9, and 4.4 10 Hz - 2
]
trap. kbp DNA 30 s 200 kHz 12.5 mM Tris
15, 25, 35,
[103
and 48.5 kbp 100 kHz -
]
trap. DNA 20 1 MHz 1 DI water
145 and ~
[104
1065 bp minute 0.2 to 0.2 - 10
]
trap. DNA s 8 MHz 0.02
[105 27 to 8461 0.2 - 10
] trap. bp DNA 0.2 MHz 0.02
[107 48.5 kbp
] trap. DNA 50 ms 10 1 MHz 0.11
[108 169 kbp
] trap. DNA ~3 s 5 to 20 1 kHz 0.15 1 mM Tris
[109 800 kHz -
] trap. 1 kbp ssDNA 30 s 1.2 MHz 0.5 - 1.55
[110 500 bp 900 kHz -
] trap. ssDNA 30 -40 s 20 10 MHz
[111
] trap. DNA origami 1 12.5 MHz <1
[140 trap. 48.5 kbp 15 100 kHz -

44
] DNA 1 MHz
1 Table 1: Overview of microelectrode based DEP applications, electric field settings, and buffer conditions.
2
3
voltage conductivity
ref. process samples time mode Vpp frequency [mS/cm] buffer
20 to
[84]
conc. 5.3 kbp 40 s DC 0.104
1
[92]
conc. 2 kbp DNA min 5 DC > 10
1500 to
[139]
conc. 48.5 kbp DNA 3000 DC 0.001
DNA-protein and
[129] DNA-drug ~1 600 to 300 to 650 1 mM
detec. complexes min continuous 650 Hz PBS
DNA-protein
complexes and
[132]
DNA-drug ~1 325 to 350 - 550 1 mM
detec. complexes min sequential 475 Hz PBS
DNA-protein/ ~2 1 mM
[133]
detec. drug complexes min continuous 200 350 Hz PBS
48.5 and 165.5 1 Hz - 1 DI
[26]
sep. kbp DNA continuous 15.3 MHz water
ccc DNA (2257,
[39] 4509, and 6766 ~2 1 mM
sep. bp) min continuous 250 350 Hz PBS
48.5 and 164 kbp
[59] linear DNA, 7 and 150 to 10 mM
sep. 12 kbp ccc DNA 240 s sequential 240 60 Hz PBS
12.2 kbp ccc and 270 to 10 mM
[60]
sep. linear DNA 210 s sequential 420 60 Hz PBS
5386 bp ccc and 60 0.3 M
[128]
sep. linear DNA min sequential DC TBE
2.7 and 6 kbp
linear DNA, 2247
[129]
and 6766 bp ccc ~1 200 to 1 mM
sep. DNA, min continuous 650 350 Hz PBS
ccc DNA (2257, ~1 DI
[130]
sep. and 6766 bp) min continuous 200 350 Hz water
1.0, 10.2, 19.5, 200 to 50 Hz to 5 mM
[131]
sep. 48.5 kbp continuous 2400 20 kHz PBS
368, 1137, 4361,
[25] and 39936 bp ~1 200 - 1000 0.5 X
trap. DNA min 1000 Hz TBE
10 mM
[122]
trap. 164 kbp 144 60 Hz PBS
48.5 and 2.8 kbp
[123]
trap. DNA DC
5 10 to 100 - 1000 50 mM
[124]
trap. DNA hybridization min 200 Hz 1000 NaCl
[126] trap. DNA origami 500 to 60 Hz - 1.5 1 40 mM

45
2100 kHz Tris

10 mM
[127]
trap. 40-mer, 1 kbp <1s 0.5 to 4 0.5 Hz Tris
600 to 200 - 300 5 mM
[125]
trap. DNA origami 1800 Hz 1 PBS
1 Table 2: Overview of iDEP applications, electric field settings, and buffer conditions.

46

DNA Dielectrophoresis Theory and Application A Review

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www.electrophoresis-journal.

com Page 1 Electrophoresis

1 DNA Dielectrophoresis: theory and applications a review

2 Martina Viefhues+* and Ralf Eichhorn#

9 Review dielectrophoresis DNA theory applications

11 Dr. Martina Viefhues

12 Experimental Biophysics and Applied Nanoscience

24 Dielectrophoresis is the migration of an electrically polarizable particle in an inhomogeneous electric

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11 Beyond applications of dielectrophoresis in life sciences, dielectrophoresis can be exploited in

31 When an external electric field is applied, a non-uniform charge distribution ( ) is induced

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1 neutrality ( ) and the definition ( ) for the dipole moment, this

12 In experimental setups which aim to manipulate particles or DNA by dielectrophoresis, we therefore

34 2.1 Electric field

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23 2.2 Fluid flow

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12 2.2.1 Electroosmotic flow

33 2.2.2 Electrothermal flows

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10 2.2.3 Pressure-driven flows

18 2.3 Temperature field

33 For water with properties , , and ,

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10 2.4 Motion of the DNA

21 with the electrophoretic mobility .

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7 driven by the DC component of the electric field.

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17 2.4.3 Hydrodynamic drag

26 where is the hydrodynamic radius of the DNA fragment.

32 2.4.4 Thermal noise

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12 2.4.5 Force balance

29 with an effective zeta-potential , which combines the zeta-potentials of particle and

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1 electrophoretic and dielectrophoretic forces can be controlled practically independently of each

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8 2.5.1 Clausius-Mossotti factor

24 Calculating the real part explicitly, we find

28 In the limit of small frequencies, ( ) simplifies to [13, 17]

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22 2.5.2 Polarizability of DNA

34 The theoretical, quantitative description of polarization processes in DNA and polyelectrolytes is

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13 2.6 Concluding remarks

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1 DNA concentrator [80], namely AC electroosmosis. AC electroosmosis is a flow phenomenon that

15 3 Microelectrodes vs. insulator based DEP

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23 4.1 Microelectrode based DEP

27 4.1.1 Trapping / Immobilization

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1 dielectrophoretically trapped DNA was anchored on a solid surface, followed by a subsequent

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39 In 2013 Martinez-Duarte et al. experimentally and theoretically investigated dielectrophoretic

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28 4.1.2 Separation of DNA

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