1 © 2016 IOP Publishing Ltd Printed in the UK

Journal of Physics D: Applied Physics

B Lim et al

Printed in the UK

033002

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© 2016 IOP Publishing Ltd

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J. Phys. D: Appl. Phys.

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Journal of Physics D: Applied Physics

1. Introduction

The precise manipulation of small particles, vesicles, and cells in micro!uidic environments has a myriad of applications in biochemical detection, gene sequencing, chemical synthesis, and in the highly parallel analysis of single cells [1–5]. Such techniques would not only open new and ef"cient ways to investigate single-molecule and cell biophysics, i.e. the study of biomolecular interactions at the level of the individual bio-logical entity, but would also offer fascinating opportunities to directly probe individual molecules and cells at work. These capabilities would offer the possibility of identifying and measuring intermediates and following the time-dependent

pathways of chemical reactions and folding mechanisms that are dif"cult or impossible to synchronize at the ensemble level. Such advances will help unveil the fundamental molec-ular mechanisms underlying biological processes and address key issues in protein, nucleic acid, and cellular kinetics and functions.

Likewise, methods for precise immobilization and manipu-lation of large ensembles of living cells offer new possibilities in biology, medicine, and biotechnology that could ultimately lead to the development of reproducible, automated, and ef"cient systems to be employed in research laboratories as well as in industrial and clinical applications. For example, immobilization of cells on a patterned substrate or a scaffold

Nano/micro-scale magnetophoretic devices for biomedical applications

Byeonghwa!Lim1, Paolo!Vavassori2,3, R!Sooryakumar4 and CheolGi!Kim1

1 Department of Emerging Materials Science, DGIST, Daegu, 42988, Korea2 CIC nanoGUNEConsolider, Tolosa Hiribidea, 76, San Sebastian, 20009, Spain3 IKERBASQUE, The Basque Foundation for Science, 48013 Bilbao, Spain4 Department of Physics, The Ohio State University, Columbus, OH 43210, USA

E-mail: cgkim@dgist.ac.kr (C Kim), p.vavassori@nanogune.eu (P Vavassori) and soory@physics.osu.edu (R Sooryakumar)

Received 13 July 2015, revised 22 September 2016Accepted for publication 14 October 2016Published 13 December 2016

AbstractIn recent years there have been tremendous advances in the versatility of magnetic shuttle technology using nano/micro-scale magnets for digital magnetophoresis. While the technology has been used for a wide variety of single-cell manipulation tasks such as selection, capture, transport, encapsulation, transfection, or lysing of magnetically labeled and unlabeled cells, it has also expanded to include parallel actuation and study of multiple bio-entities. The use of nano/micro-patterned magnetic structures that enable remote control of the applied forces has greatly facilitated integration of the technology with micro!uidics, thereby fostering applications in the biomedical arena. The basic design and fabrication of various scaled magnets for remote manipulation of individual and multiple beads/cells, and their associated energies and forces that underlie the broad functionalities of this approach, are presented. One of the most useful features enabled by such advanced integrated engineering is the capacity to remotely tune the magnetic "eld gradient and energy landscape, permitting such multipurpose shuttles to be implemented within lab-on-chip platforms for a wide range of applications at the intersection of cellular biology and biotechnology.

Keywords: magnetophoresis, magnetic domain walls, cell sorting, micro/nanopatterns

(Some "gures#may appear in colour only in the online journal)

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doi:10.1088/1361-6463/50/3/033002J. Phys. D: Appl. Phys. 50 (2017) 033002 (25pp)

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according to a precise geometry is of major importance for various applications such as cell cultivation and interaction, production and regeneration of tissues, and investigation of cell-surface aggregation and of the dynamics of cellular pro-cesses. Besides applications to biophysical research, such pre-cise manipulation control would open up new possibilities for medical and pharmaceutical applications, e.g. handling small volumes of bio-samples, performing sophisticated sorting of different molecules, or testing the effectiveness of a drug or molecular genetics delivery system.

A vast array of particle-handling tools are currently under development based on hydrodynamic [6–9], optic [10–13], electric [14–16], and magnetic [17–22] trapping forces which can move single particles in arbitrary directions. Methods based on the remote control of magnetic particles via external or local magnetic "elds are receiving great attention because: (i) magnetic "elds are not screened by biological matter and culture media, making the manipulation possible in any chemical environment (at variance with, e.g. dielectropho-retic electric trapping, which requires instead the use of non- conductive media); (ii) magnetic particle manipulation does not involve relevant energy dissipation, which could eventu-ally damage the molecule or cell structure as in the case of optical approaches (e.g. optical tweezers).

Recently, superparamagnetic beads (referred to as magn-etic beads or beads hereafter for simplicity), i.e. micro- and nano-spheres consisting of one or more superparamagnetic cores with a coating matrix of polymer, have enabled highly !exible approaches for controlling the transport and sorting of various biological materials such as cells, DNA, and pro-teins. Magnetic beads do not retain any signi"cant amount of magnetization in the absence of an externally applied magn-etic "eld, and thus do not form aggregates when they are in suspension. They develop a net magnetic moment under exposure to external magnetic "elds and can be thereby manipulated by generating desired magnetic "eld gradients. The polymeric matrix enables easy coating with antibodies or other bio-af"ne ligands for selective binding to target bio-logical entities. These developments have ushered in great progress in magn etically responsive micro- and nanustruc-tures, which can control the movement of magnetic beads on the micrometer and submicrometer scale [18, 19, 23–26]. Colloidal transport on magnetic micro-patterns especially exploits a non-linear dynamic phenomenon to achieve pre-cise position and velocity control of magnetic beads in mas-sively parallel con"gurations. In these instances, the magnetic micro-patterns provide a periodic potential energy landscape which is modulated by an externally applied magnetic "eld that serves to shift the regions of potential energy minima along desired directions and transport magnetic beads along programmable paths.

Several groups have demonstrated different approaches to transporting magnetic beads along tracks composed of hard [18] or soft [20, 22, 27] magnetic materials, and using cur-rent lines [19, 23, 28]. However, the use of current lines for the transport of bead generates heat, which is a hurdle for real biological applications. Separation of different beads based on size or coating has also been demonstrated by manipulating the

non-linear dynamic transition between phase-locked and phase-slipping motion [29]. Separations based on quasi-static adiaba-tic transitions of potential energy landscape textures have also been demonstrated [17]. However, no single technique to our knowledge encompasses the scalability, !exibility, and automa-tion that allow single- and multiple-cell chip devices to perform with the level of integration of computer circuits.

Drawing inspiration from general circuit theory and magn-etic bubble technology, we demonstrate, for example, a class of integrated circuits for executing sequential and parallel timed operations on an ensemble of single particles and cells [30–32]. The circuitry elements are constructed from lithographically de"ned, overlaid patterns of magnetic "lm. The magnetic pat-terns passively control particles similar to electrical conductors, diodes, and capacitors, whose functions are ruled by the magn-etic energy and forces. Current lines producing local magnetic "eld are used to actively switch particles between different tracks similar to gated electrical transistors. When combined into arrays and driven by a rotating magnetic "eld clock, these integrated circuits can implement general multiplexing prop-erties and enable the precise control of arbitrary objects with magnetization contrast relative to its surroundings. Moreover, recent advances in nanolithography allow for the extension of these functionalities to the nanoscale [33–35].

In this article, the applications of nano/micro-scale mag-nets for remotely controlled digital magnetophoresis for the translocation of bare magnetic beads, magnetically labeled molecules, and living cells such as those schematically shown in "gure#1 are reviewed. In order to cover the basic design and fabrication of nano/micro-sized magnets for individual con-trol of beads/cells and their bio-applications, the manuscript is organized into the following sections: In section#2, digital magnetophoretic circuits for many particle and cell manipu-lation are introduced based on magnetophoretic conductor, diode, and trapping capacity concepts. The basic magn etic energy and force pro"les are also analyzed in relation to mag-net size and susceptibility as well as scale of channel. Section#3 discusses remote nanoscale manipulation of individual micro- and nano-sized magnetic beads used as molecules and cells carriers, based on the controlled nucleation, displacement, and annihilation of domain walls in magnetic nanowires. Section#4 presents the integration of patterned magnetic constructs with micro!uidics and engineered platforms to provide useful devices that enable a range of biological applications such as dose-controlled introduction of genetic material—for exam-ple, nucleic acids—into cells (a process called transfection), the sorting and encapsulation of single cells within individual droplets, and the development of biomarker assays, all within lab-on-chip con"gurations. Finally, section#5 summarizes the content of the paper and provides a brief outlook into future directions in this area.

2. Digital magnetophoretic devices

2.1. Experiments

The micro-magnetic patterns were prepared on silicon sub-strates using conventional photolithography and lift-off

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techniques [31]. Negative photoresist patterns of different-sized disks were prepared on the substrate followed by direct current (DC) magnetron sputtering of 100 nm thick Ni82.6Fe17.4 "lm. After lift-off of the "lm, the desired micro-magnet pat-terns became available.

A rotating magnetic "eld was applied to the on-chip by soft magnetic core solenoids arranged along mutually orthog-onal axes (x–y) with respect to the substrate surface [31]. Two

current sources controlled by Lab View software (National Instruments) supply sinusoidal waveforms to each solenoid. The rotating magnetic "eld in the x–y plane was generated by adjusting the current phase to shift by 90°, i.e. H i tcosx ( )! !

!= and H j tcosy ( )! !

!= . A Gaussmeter (Lakeshore 450) was positioned under the sample in order to monitor direction of the rotating "eld [31], where overall instrumental setup with magn etic chip is shown in "gure#2.

Figure 1. (a) Conceptual image for magnetophoretic circuit. (b) and (c) Demonstrated circuitry elements. (d) Conceptual image for individual cell trapping by digital magnetophoretic circuit.

Figure 2. (a) Experimental setup for rotating magnetic "eld for bead manipulation. (b) High-zoom picture of the magnetic coils. (c) Magnetic chip.

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The diameter of the superparamagnetic beads was 2.8 µm (Dynabeads M-280 Streptavidin, Invitrogen cat. no. 142.03), and the beads were functionalized with a covalently coupled streptavidin layer. The bead trajectories were recorded with a video camera (Guppy Pro F-031, Allied Vision) with a frame rate up to 123 fps, which was controlled by the software Fire Capture 2.1.

2.2. Magnetization properties for micro-patterns

As is generally known, demagnetization effects become signif-icant for thin-"lm patterns smaller than a few tenths µm size. Since it is dif"cult to measure the magnetization of a single micro-scale pattern, an array was fabricated for the measure-ment, as illustrated in the SEM image shown in the inset of "gure#3(a). In order to reduce the magnetic dipole interaction

between patterns, the gap separating them is adjusted to be same as their diameter. Figure#3(a) shows the magnetization curves for disk patterns with different radii, where the satur-ation "eld decreases from 200 to 20 Oe with corresponding increase in radius from 2.5 to 25 µm, respectively, as redrawn in "gure#3(b). The inset displays the initial M–H curve of the non-patterned Ni80Fe20 thin "lm from which the saturation magnetization value, 668 emu cc!1, was obtained.

Figure 4(a) is the distribution of induced magnetic "eld around a disk magnet, in which the "nite element method was used for the "eld simulation using Maxwell 3D software (Version 12.2, Ansys). For the applied static "eld, the magn-etic bead is trapped at the local minimum of magnetic energy

for a micro-magnet, ( )! !=! "!µ

E B BV

2v

0, shown in "gure# 4(b).

Subsequently, the trapped bead synchronously moves around the perimeter of the disk pattern along with the rotating "eld,

Figure 3. (a) Magnetization curves for different NiFe pattern size (NiFe disk diameter). (b) Saturation "eld dependence on the pattern size (inset: initial magnetization curve for thin "lm).

Figure 4. (a) Magnetic !ux lines, (b) energy of disk pattern, and (c) radial force to attract the beads to pattern. (d) Tangential force to transport the beads with susceptibility, v! of 0.7 (SI).

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where a phase of the bead is lagged from the "eld direction [32]. Here, the phase lag angle is de"ned as the difference between the orientation of the bead and the rotating external "eld direction. The magnetic forces to describe the dynamics of the moving bead are considered next.

With the magnetic susceptibility of the bead v! = 0.7, the components of the magnetic forces acting on the beads were obtained by numerical differentiation of the induced "eld components using the following relationship in cylindrical coordinate as depicted in inset of "gure#4(c) [32]:

( )! ! !!µ

= ! " = + +""

# # #F

VB B F e F e F e

2v r

rz

z0

mag mag mag (1)

Here, V is the volume of the magnetic bead (m3), v! is the volume susceptibility of the magnetic bead (the magnetization of the surrounding medium is neglected), 0µ is the permeabil-ity of vacuum (4! " 10!7 N A!2), and Fr

mag, Fmag! , Fz

mag are the radial, tangential, and vertical components of the magnetic force, respectively. The magnetic forces are the functions of r and !. Here, the Fr

mag, Fmag! , are related to the attraction of

force to the pattern edge and the tangential driving force for the circular bead moving, respectively.

As for the transportation of a few-µm-sized bead, a force of ~10 pN is required to overcome the frictional and viscous forces. Figure#4(c) shows the radial force which depends on the distance d between pattern and bead, as in inset of "g-ure#4(c). The simulated values of initial force at d = 0 were used and then plotted against 1/r3 [32]. For constant volume susceptibility, it is noted that a larger-size bead has advantages on the moving velocity because the force is proportional to carrier volume as in equation#(1). After the bead arrives at the pattern edge, it begins to circulate around the pattern perim-eter with the help of the tangential force. Figure#4(d) shows the maxim tangential forces for different bead sizes and pat-tern radii, where the maximum force is revealed at the pattern

radii equal to twice the bead radii. The maximum value for the force related to ratio of bead size and pattern size indi-cates that the optimum pattern size is twice that of the moving object.

2.3. Circuitry elements for digital magnetophoresis

Figure 5 shows the trajectory of beads for separated, full-disk, and half-disk patterns as a function of the time-varying "eld angle. There is a phase lag behind the "eld direction and also from the minima of energy. In fact, the bead follows the mini-mum energy trace, in which the phase lag angle is governed by the balancing of tangential and drag forces. Phase-locked motion, i.e. constant phase lag angle, results from the balance between the magnetic force and the viscous and frictional drag [33]. As the driving frequency is increased, the higher bead velocity causes it to experience a stronger viscous !uid drag, which increases the bead phase. Over a critical frequency, the bead enters the ‘phase-slipping regime,’ which is described as such because the phase difference between the external "eld direction and the bead position can no longer be constant, and the bead is unable to synchronously follow the "eld [31].

When the pattern gap is over ~20% of the diameter of the moving bead in phase-locked motion, it just circulates around a pattern, as shown in "gure# 5(a). If the gap is small, then a bead positioned on the upper track of the full-disk pattern moves in the positive x-direction, as revealed for the clock-wise rotational "eld as in "gure#5(b). However, a bead posi-tioned on the lower track of the pattern moves in the negative x-direction. With the full-disk patterns, the beads con"ned to the upper portion of the track would move in the opposite

Figure 5. Moving of beads under clockwise "eld rotation depending on the geometrical pattern shapes: (a) isolated disk, (b) connected full disk, and (c) connected half disks. Figure 6. The potential energy landscape for a 2.8 µm diameter

magnetic bead, as it moves around the half-disk track in a clockwise rotating "eld, is presented in (a)–(d). Blue and red colors designate the energy minima and maxima, respectively. Corresponding experimental images for the magnetic bead trajectory are shown as the dotted white line in (e)–(h).

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direction of those on the lower portion, thereby yielding no net current !ow. This indicates that it is not possible to control the moving direction of beads because of dif"culty in "nd-ing the bead location—for example, either on upper or lower tracks of the patterns. However, the bead for half-disk patterns in "gure#5(c) always moves in the positive x-direction.

The more detailed analysis on bead trajectory for a half-disk pattern track as a function of the time-varying "eld angle is presented in "gure#6, corresponding to a clockwise rotating "eld. Simulation of the potential energy distribution ("gures 6(a)–(d)) compared with the experimental trajectory ("gures 6(e)–(h)) demonstrates that the bead position is cor-related to the traces of potential energy minima. The beads move along the upper section#of the track because it has the deepest energy minima. The linear segment connecting adja-cent magnets provides an energy barrier that con"nes the local energy minima to one side of the magnetic track. For driv-ing frequencies lower than the critical frequency, the bead moves exactly two array periods for each complete cycle of "eld rotation, demonstrating the linear relationship between frequency and magnetic bead current. Clockwise "eld rotation leads to particle motion along the positive x-direction whereas counterclockwise rotation produces motion along the negative x-direction. The asymmetry in the track design is essential for a net bead current using controlled "eld rotation as summa-rized in table#1.

Recti"cation, as in the function of a diode, is accom-plished by introducing additional asymmetry in the

conductor paths, such as by joining two magnetic tracks in a T-shaped junction ("gure 7). In the forward biased mode ("gures 7(a)–(h)), the bead initially moves from left to right on the horizontal track in the positive x-direction. When the bead reaches the T-junction, the two potential energy minima on either side of the junction merge together, which permits the particle to jump over the linear vertical seg-ment. After surmounting this barrier, the particle continues its horizontal path along the positive x-direction in harmony with the rotating "eld.

An example of the reversed bias recti"cation mode can be observed in the counterclockwise driving "eld ("gures 7(i)–(p)), in which the bead moves along the negative x-direction of the

Table 1. Moving direction of superparamagnetic beads as function of rotation "eld direction for full- and half-disk patterns.

Pattern shape Location

Field rotation

Moving direction

Field rotation

Moving direction

Upper + !Lower + !Upper + + ! !Lower + ! ! +Upper + + ! !Lower + + ! !

Note: Movement: (+, ! ) represents positive and negative x-directions. Field rotation: (+, ! ) represents the clockwise and counterclockwise directions. Bead location: (+, ! ) represents upper and lower position of the track.

Figure 7. The potential energy landscape in a clockwise rotating "eld in the forward-biased diode mode is shown when the external "eld has angular orientation of 180°, 105°, 45°, and 0°, as presented in (a)–(d). The red arrows denote the instantaneous external "eld direction. The blue energy minima correspond to the particle locations in the experimental images of (e)–(h). The potential energy landscape for the reverse conditions for "eld angles of 75°, 135°, and 180° is presented in (i)–(l) along with the associated experimental images in (m)–(p).

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Figure 8. Capacitor behaviors of closed patterns for bead trapping in (a) clockwise direction and (b) bead stored in closed pattern irrespective of "eld rotation direction.

Figure 9. (a) Selection of immune B- and T-cells using MACS. (b) Schematic representation of loading magnetic 50 nm nanoparticles onto lymphocytes used in antigen–antibody interaction. (c) SEM image of B-cells. (d) SEM image of nanoparticle-bonded cells. (e) Fluorescence images of labeled B- (orange) and T-cells (green)5.

5 A schematic representation of isolation of lymphocytes from mice through negative magnetic-activated cell sorting (MACS) is shown in "gure#9(a). Spleens removed from mice were converted into single-cell suspensions by squeezing through a cell strainer. Isolated splenocytes were washed twice with BSA-PBS (0.5% BSA and 0.2 mM EDTA in PBS), incubated with biotin-conjugated antibodies at 4 °C for 10 min, then incubated with the microbead-conjugated anti-bodies at 4 °C for 15 min. After washing twice, the labeled cells were applied to a LS separation column and Midi-MACS magnet. The column was rinsed with 9 ml of BSA-PBS to !ush out unbound cells. Then, isolated lymphocytes were incubated with 1 µM calcein AM green (T lymphocytes) and red/orange (B lymphocytes) (Invitrogen, Grand Island, NY, USA) at 37 °C for 20 min. After washing twice, calcein-labeled lymphocytes were harvested and used for the next experiments.

Figure 9(b) is a schematic representation of the loading of magnetic nanoparticles onto lymphocytes used in antigen–antibody interaction. Here, 0.8 ml aliquots of T- and B-lymphocyte cell suspensions (1 " 106 cells ml!1) were centrifuged in an Eppendorf tube and the supernatant was discarded. The T- and B-lymphocytes were re-suspended in PBS buffer (0.8 ml and pH 7.4). The washed cells were mixed with 0.2 ml of nanoparticle conjugated with mono-clonal rat anti-mouse CD90.2 (Thy1.2; T lymphocytes) and CD45R (B220; B lymphocytes) antibodies (MiltenyiBiotec Inc.), respectively, and incubated with rotation for 15 min at room temperature. The resulting magnetic nanoparticles on lymphocytes were isolated from unbounded lymphocytes with an external magnetic "eld. The isolated lymphocytes loaded with magnetic nanoparticles were diluted with 1 ml of PBS buffer (pH 7.4) and used in the present experiments. The T- and B-cells were labeled with calcein dyes, green and red/orange respectively.

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horizontal track. When the bead reaches the T-junction, the bead crosses over to the linear vertical segment. Instead, the bead fol-lows the deeper energy minimum, which moves in the positive y-direction and enforces the asymmetry of the conduction mech-anism. The overall effect is that beads can conduct along the posi-tive x-direction in a clockwise rotating "eld, but cannot conduct along the negative x-direction in a counterclockwise rotating "eld.

Recti"cation and conduction pathways can be geometrically arranged in closed loops to store beads and cells in well-de"ned spatial regions individually, operating in a manner similar to an electrical capacitor. Figure#8(a) shows an array of magnetic conduction tracks and square apartments, which are con"g-ured such way that the T-junction entryway is in the forward bias mode and allows beads or cells to enter the apartment. In a clockwise driving "eld, single bead is moved into the apart-ments as in "gure#8(b), where they subsequently remain trapped in a closed spatial orbit. Upon reversing the "eld rotation, the beads remain trapped in their apartments due to the reverse bias of the diode junction. The isolation and conjugation of cells with magnetic nanoparticles are shown in "gure 9.

2.4. Performance for cell trapping

Figure 10(a) shows the separation procedure of two living cells labeled with 50 nm superparamagnetic nanoparticles using the serial application of clockwise and counterclockwise rotating "elds. Under the clockwise rotation, two cells ("rst and sec-ond) apart from two patterns synchronously move in the posi-tive x-direction. After the "rst bead enters into the apartment after passing the diode junction, the "eld rotation is changed to counterclockwise rotation. Then the "rst bead is moved to the positive y-direction and trapped onto an apartment, but the second cell moves in the negative x-direction as shown in "g-ure#10(b). Thus, it is possible to separate the trapped "rst cell from subsequent cells, and thus one cell or several cells in an apartment allows for long-term analysis of the cells and visu-alization of biological processes.

The ability to trap pairs of cells in individual apartments, including a homogeneous pair of B-lymphocyte cells ("gure 10(c)) or a heterogeneous pair of B- and T-lymphocyte cells, can serve as a platform for studying single cell–cell commu-nication. In addition to the basic circuit elements, integrated circuits allow for the cell trapping in the multiplexing arrays in "gure# 10(d), where the cells are trapped in 4 " 4 apart-ments using the serial combination of "eld rotations in clock-wise and counterclockwise directions in each row and column junctions. This procedure demonstrates a milestone towards achieving highly scalable digital circuitry for performing mas-sively parallel operations on individual cells. This affords a breakthrough in single-cell analysis for their heterogeneous drug resistance function and cellular contents.

In summary, section# 2 discussed how, stimulated by general circuit theory and magnetic data storage technologies, a new class of "eld-driven integrated magnetic circuits were developed to execute sequential and parallel as well as timed operations. These miniature circuits, easily integrated with micro!uidic platforms, were shown to operate on an ensemble

of superparamagnetic particles or magnetically labeled cells with speci"c response analogous to those of electrical conductors, diodes, and capacitors that are the cornerstone of today’s microelectronic revolution. These experiments have laid an exciting foundation towards achieving highly scalable digital circuitry for performing massively parallel operations on individual cells for single cell analysis.

3. Individual particle manipulation

3.1. Constrained domain walls in magnetic nanoconduits

In the following section, we will present and discuss a recently demonstrated approach [34–36] to con"ne and control the position and movement of an individual bio-functionalized magnetic nanoparticle in a liquid by remotely controlling the motion of constrained magnetic domain walls (CDWs) in patterned nano-wires. This is possible since CDWs can magnetostatically couple to !uid-borne magnetic nanopar-ticles [37–41]. The approach shares some similarities with other thin-"lm-based devices reported in the literature that have been developed to trap and transport large populations of magnetic microparticles via strong localized "elds and gradients found at conventional domain walls in garnet "lms [42–44]. However, such thin-"lm-based approaches do not allow for the single-entity manipulation. Successful attempts in this direction were recently reported using discrete pat-terned magn etic thin-"lm micro-structures with "eld-control-lable magnetization [45], tailored to form transport lines that enable the programmable motion of single micrometer-sized magnetic particles by the application of time-variable external magnetic "elds [20, 46]. However, they require that a magn-etic "eld be constantly applied so as not to lose the coupling with the magnetic particle.

The methodology presented here allows us to overcome these limitations and would open up new possibilities towards single-molecule biomedical applications such as handling small volumes or performing sophisticated sorting of differ-ent targets attached to different particles on a chip device. Particularly relevant in view of medical applications is the easy integration of such bio-manipulation systems on a single chip, with sensors able to detect the presence of magnetic nanopar-ticles for ‘lab-on-chip’ diagnostic applications. The present approach is based on recent developments of electron-beam (e-beam) nanolithography techniques that make it possible to fabricate magnetic nano- or micro-conduit structures (wires and rings) with well-de"ned geometry on a chip surface.

A schematic of the e-beam lithography process is shown in "gure#11, together with some examples of the micro- and nanustructured conduits that will be described later. The mat-erial used for the conduits is Permalloy (Fe20Ni80, Py). Even narrower magnetic conduits, in the single-nm range, can be nowadays fabricated using other advanced tooling such as focused electron-beam induced deposition [47]. In very nar-row ferromagnetic nano-wires a CDW is a mobile interface which separates regions of oppositely aligned magnetization, which is generally parallel to the wire axis due to shape aniso-tropy [48], as schematically shown in "gure#12.

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Each magnetic domain has a head (positive or north pole) and a tail (negative or south pole). The resulting CDW is there-fore either head-to-head (HH) or tail-to-tail (TT), as shown in "gure#12. Successive CDWs along the nanowire alternate between HH and TT con"gurations. Submicrometer planar

strips (planar nanowires) made from a soft magnetic mat-erial such as Py have been shown to form excellent conduits (hereafter referred to as nanoconduits or nano-conveyors) for CDWs that can be nucleated at selected positions in a con-trollable way [48]. In addition, it has been shown that, under

Figure 10. Separation of two living cells. (a) Synchronous movement in the positive x-direction of two cells for clockwise rotating "eld, indicating "rst cell entrance into apartment after passing diode junction. (b) Movement of trapped cell in positive y-direction, but movement in negative x-direction of second cell for counterclockwise rotation. (c) Trapped cells in an apartment of B- and T-cells (left: bright image; right: !uorescence image). (d) Trapped B-cells in 4 " 4 apartments (bright and !uorescence images). In !uorescence image, T- and B-cells are green and red/orange, respectively.

Figure 11. Schematic of the sequence of steps in the e-beam nanolithography process (top) utilized to nanofabricate the ferromagnetic nanostructured conduits discussed in this manuscript. An electron-sensitive "lm called resist (‘Resist’) is shortly spin-coated on top of a substrate, typically of Si. A focused beam of electrons is then used to draw custom-shaped nanostructures (‘E-beam Write’). The sample is then immersed in a solvent, enabling selective removal of the e-beam exposed regions of the resist (‘Develop’). A thin layer of the desired material is then deposited on top of the sample (‘Deposition’). Eventually, the sample is immersed in another solvent, typically acetone, to remove all remaining resist, leaving on the substrate only the custom-shaped nanostructures of the desired material (‘Lift-off’). The bottom panel shows relevant examples of fabricated magnetic (the material is Fe20Ni80) conduit nanostructures that are discussed in this manuscript.

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the action of an externally applied magnetic "eld, CDWs can be propagated through complex 2D and 3D networks of such nanoconduits that contain bifurcation and intersection points [49–51]. Due to geometrical con"nement, the size of a CDW is controlled by the lateral dimension and thickness of the nanoconduit (W and t in "gure#12). For instance, the lateral width of a CDW, L in "gure#12, is of the order of the strip width W and therefore can be easily compressed far below the

micron scale by reducing W. Figure#12 shows the internal spin structure of such CDWs in the case of thin (thickness t below ~40 nm for Py) and narrow (conduit width W below ~200 nm for Py) conduits. Indeed, according to the CDW ‘phase dia-gram’ [52], for 25 nm < t < 40 nm Néel-type, i.e. CDWs in which the spins rotate in-plane are systematically observed in !at Py wires with W ! 200 nm (also called transverse CDWs). For thicker conduits, or if the constituent material possesses

Figure 12. Spin structure of constrained domain walls in thin and narrow ferromagnetic nanoconduits. The two cases of head-to-head (HH CDW, top) and tail-to-tail (TT CDW, bottom) constrained domain walls are shown. Labels W and t mark the width and thickness, respectively, of the ferromagnetic nanoconduit, while L denotes the width of the constrained domain wall.

Figure 13. (a) Constrained domain wall pinned at a corner of a Py nanoconduit (width 100 nm and thickness 25 nm) with a superparamagnetic nanoparticle of radius R placed at a distance D from the conduit surface. (b) Vector plot of the force exerted on a superparamagnetic particle in an arbitrary position in a plane at distance D from the conduit surface. (c) Force on superparamagnetic particles of radius 25 and 50 nm at various distances D from the top surface of the conduit (D = 200, 100, and 50 nm).

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an out-of-plane magnetic anisotropy, the rotation of the spins inside the CDW can occur out of plane (Bloch-type structure). For wider conduits, the internal spin con"guration gradually turns into a vortex structure as the width W, and thus the CDW size L, increase (above ~500 nm for Py and for t = 25 nm) [38, 53]. The "eld-induced displacement of a CDW in a nano-conduit can be "nely controlled in various ways, namely by creating arti"cial pinning points for the CDW such as notches or corners [54–57], using localized sources of magnetic "eld provided by nearby nanomagnets [58], and using curved nanoconduits, in which case any position is a stable one for a CDW [59]. It is also possible to induce a controlled and reproducible CDW displacement in such nanostructures by injection of polarized current pulses rather than by using an external magnetic "eld. The current-induced CDW motion is due to a spin-torque effect, where the electrons transfer angu-lar momentum to the CDW when passing through it, pushing it in the direction of the electron !ow [60, 61].

3.2. Coupling energy between a CDW and a superparamagn etic nanoparticle

The important element of the application discussed here is the highly inhomogeneous magnetic stray "eld, of up to several hundreds of mT, generated by a CDW. This stray "eld is spa-tially localized on the nanometer scale due to the CDW’s very con"ned geometric structure. More precisely, the "eld generated by a HH CDW is directed outwards from the CDW, with a posi-tive out-of-plane component. Conversely, a TT CDW creates a stray "eld directed inwards to the CDW and with a negative out-of-plane component. The coupling energy E, between the inho-mogeneous stray "eld H generated by a CDW and the magnetic moment µ of a nanoparticle, de"nes an attractive potential well. As discussed in section#1, equation#(1) shows that the coupling force between a magnetic particle and the CDW is proportional to B B( )! "! ! . The dependence of the coupling force on the gra-dient of B2 indicates that both HH and TT CDWs can equally attract and trap a superparamagnetic !uid-borne nanoparticle moving in their vicinity. The examples shown in "gure#13 for a CDW trapped at a corner of a nanoconduit (w = 100 nm and t = 25 nm) indicate that the attractive force can vary of two orders of magnitude depending on both the distance of the par-ticle from the conduit and the size of the particle, namely M(rc). The former is usually controlled by depositing a layer of SiO2 that serves also as a protective layer for in-liquid operations. For the cases displayed in "gure#13, the trapping force varies from a few hundreds of fN to a few tens of pN. The force intensity plots have been calculated using the equation#above by comput-ing, with the object-oriented micromagnetic framework [62], the magnetic "eld H created in the surrounding space by the HH CDW and considering a typical value of µ of commercial superparamagnetic nanoparticles (material Fe2O3).

Interestingly, the coupling between a CDW and a super-paramagnetic nanoparticle can be used to sense the presence of individual nanoparticles. The sensing concept is illustrated in "gure#14, in which two magnetic random access memory devices, a simple corner and a square ring with a slit, both

made of Py and with Au contacts have been used for demon-stration purposes [63, 64]. The Au contacts are used to meas-ure the magnetoresistance of selected portions of the structure. The magnetoresistance in these structures is dominated by the anisotropic magnetoresistance and a maximum resistance is observed when the spins are parallel or antiparallel to the injected current !ow. This corresponds to a state when there is no CDW present between the two sensing leads and the magnetization follows the direction of the perimeter of the structure. If a CDW is present between the measuring leads, some of the magnetization of the CDW points perpendicularly to the current !ow and hence the resistance is lowered. As a result, magnetoresistance could be used to determine the loca-tion of a CDW. If a CDW is generated in a corner between two sensing leads, the measurement of magnetoresistance can be used to determine the value of the external "eld Hext that needs to be applied to depin the CDW from the corner. When a magnetic nanoparticle is placed over a CDW previously posi-tioned at the sensing corner of the structure, and a magnetic "eld Hext is applied to displace the CDW, a magnetic dipole moment µ is generated in the superparamagnetic particle, as shown in "gure#14. The stray "eld generated by µ opposes the applied "eld Hext below the bead, causing an increase of the value of the "eld Hext required to displace the CDW.

Figure 14. Magnetic random access nanodevice, namely a Py square nanoring with Au contacts, utilized for the detection of individual magnetic nanoparticles via magnetoresistance measurements. Contacts labeled 1 and 2 are used to inject a current (1–10 µA) in the nanoring, while the other contacts are used to measure the resistance of selected portions of the nanoring. The measurement reported in the bottom panel shows the resistance variation between contacts labeled 3 and 4 as a function of an applied "eld Hext applied as sketched in the top-right panel in order to displace a CDW initially nucleated in the nanoring corner !anked by the contacts. The resistance between contacts 3 and 4 increases abruptly once the CDW is removed by the selected corner. The magnetostatic interaction between a CDW and a magnetic nanoparticle results in a higher value of Hext that is required to remove the CDW from its initial position in the corner. Reproduced with permission from [63]. Copyright 2008, AIP Publishing LLC.

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Another potentially interesting approach for sensing indi-vidual nanoparticles, is based on the coupling between a superparamagnetic nanoparticle and the stray "eld generated by the core of a vortex magnetic con"guration occurring in cylindrical disks of Py. The coupling is expected to affect the amplitude and frequency of the gyrotropic motion of the vor-tex, which can be precisely detected through magnetoresist-ance measurements [65].

3.3. Nanodevices based on nanoconduits for the remote manipulation of !uid-borne superparamagnetic nanoparticles

A setup similar to that shown in "gure# 2 was used for the experiments devoted to nanoparticles and cell manipulation, in which a micro!uidic cell is positioned at the center of four PC-controlled quadrupolar electromagnets; an additional ver-tical coil placed below the micro!uidic cell is used for experi-ments requiring an out-of-plane magnetic "eld. Pulsed or rotating weak magnetic "elds are applied to move the beads under the objective of an optical microscope. The micro!uidic system used for experiments was made by laser ablation of polymethylmethacrylate [66].

The central idea of the proposed strategy for nanoparti-cle manipulation relies on the highly precise controllable of the motion of CDWs that can be achieved in ferromagnetic nanostripes (magnetic nanoconduits) of even complex paths and on the robust coupling between a DW and a superpara-magnetic nanoparticle in suspension over the conduits [63, 64]. In this way the injection, displacement, and annihilation of a single CDW in a ferromagnetic nanoconduit results in the cap-ture, displacement, and release of a !uid-borne nanoparticle. The method has been successfully applied to the manipula-tion of both biomolecules [37] attached to a magnetic carrier

and of cells [66] decorated with magnetic particles. The "rst device utilized for demonstrating the concept is shown in the scanning electron microscopy (SEM) image in "gure# 15(a). It consists of a zigzag-shaped Py nanostripe structure (width 250 nm and thickness 25 nm), with a tapered end to transport the CDW towards the bifurcation and then annihilate it, and a CDW injector made of an initial pad having a width of 500 nm to allow for the creation and injection of a single CDW.

This device implements a sort of a controllable magnetic CDW step motor that can be used to displace magnetic beads over large distances. The magnetic force microscopy (MFM) images in "gure# 15(a) show the sequence of applied "eld pulses, H0 and H1, required to initialize the device (nucleate and position a CDW marked by a dashed white circle in the initial corner of the device). The CDW is then displaced along the wire by a second sequence of "eld pulses (Hup–Hdw), which makes the CDW jump from one corner to the next (cor-ners are the only stable position for a CDW) in a sequence of steps. Eventually, the CDW annihilates when it reaches the tapered end of the conduit. The direction of the CDW dis-placement can be reversed at any time by reversing the direc-tion and sequence, i.e. their order, of Hup and Hdw. The idea at the base of the utilization of such a device for nanoparticle manipulation is sketched in "gure#15(b). A zigzag nanowire similar to that in "gure#15(a), but covered with a 70 nm SiO2 protecting layer, has been used for the displacement of bare magnetic nanoparticles. The SiO2 layer, apart from allow-ing the utilization of the device in a wet environment, can be easily ‘neutralized’ in order to avoid non-magnetic binding between particles and the chip surface due to hydrophobic and/or electrostatic interactions [37]. The device, placed inside the micro!uidic cell under the objective of a microscope, is prepared with a CDW in it "rst corner and then a solution containing the nanoparticles is injected. When a nanoparticle

Figure 15. (a) MFM image sequence showing the "eld-induced DW displacement in a zigzag conduit. (b) Sketch of the principle of the digital displacement of magnetic particles along the zigzag conduit. (c) Optical microscopy images of the "eld-induced displacement of an individual magnetic particle in suspension (diameter of the particle 1 µm). Reproduced with permission from [39]. Copyright 2010, AIP Publishing LLC.

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couples with the CDW, the "eld pulse displacement sequence Hup–Hdw is applied. Figure#15(c) shows a sequence of opti-cal microscopy images of the transport of a single magnetic particle along the entire conduit. Aside from transport of bare magnetic nanoparticles in suspension [39], the device has been used to transport protein-coated magnetic nanoparti-cles [37] as well as individual cells decorated with magnetic nanoparticles [66]. Note that micron-sized nanoparticles have been used only because the investigation of their motion with a high-speed camera coupled to an optical microscope is easier, but the method can be applied to ‘true’ nanoparticles with diameter below 100 nm (see [64] for the demonstration of capture of 80 nm nanoparticles in suspension by a CDW). It is worth noting here that the time scale of CDW motion in Py conduits is very short, of the order of one ns over a length of a few microns [52], while in our experiment the displacement of the magnetic nanoparticles is much slower (a few hundreds of milliseconds). In this sense, the nanoparticles do not strictly move with the CDW but rather follow it, with a drift motion superposed to Brownian motion towards the potential energy minimum generated at the new position occupied by the CDW after the application of Hup/Hdw. It is also worth pointing out that the corners of the zigzag are a stable position for the DW so that a nanoparticle, molecule, or cell can be held in a selected position inde"nitely.

A continuous control of the nanoparticle displacement at the nanoscale, synchronous to that of the coupled CDW, can be achieved using curved nanoconduit structures, as illus-trated in the next example for an 8 µm diameter circular Py ring (conduit width 300 nm) displayed in "gure#16.

Two CDWs, one HH and the other TT, are initially gener-ated in the ring by applying a saturating magnetic "eld H0. Once created, the CDWs can be moved around the circumfer-ence by the application of a smaller "eld Hr. By rotating the "eld, both the CDWs are displaced with an angular speed equal to that of the rotating "eld, thus achieving a synchronous and fully controllable CDW motion, as shown in the MFM images in "gure#16(a). The required magnitude for Hr is determined by the ring radius and the local CDW pinning sites due to edge irregularities and material inhomogeneities. The utilization of this device for nanoparticle remote and synchronous manipu-lation is sketched in "gure#16(b), while "gure#16(c) shows a sequence of optical microscopy images of the "eld-induced displacement of the two particles in suspension. Also, in this case the device was capped with a 70 nm SiO2 protecting layer and a similar setup as that shown in "gure# 2 was used for the experiment. The device was used to displace nanoparticles together with the attached biological cargo (proteins [37] or even cells [66]).

Based on the manipulation capabilities achievable via the precise engineering of a magnetic ring size and shape, the next step was to devise a reliable and controlled way to exchange nanoparticles among adjacent rings arranged on a matrix, as sketched in "gure#17. The underlying idea is to overcome one limitation of the presented devices, namely that the manipulation capabilities of this class of devices were limited to prede"ned patterned paths, albeit more than one

path could be selected [40]. By using an array of circular rings and enabling the hopping of nanoparticles among adjacent rings, the local 2D manipulation on the individual ring is readily extended to the total surface of the device covered by the array. This allows for the capture, manipulation, and release of individual or multiple !uid-borne magnetic nanoparticles without any prior "xed pathway [66]. As shown in "gures# 17(a) and (b), a magnetic nanoparticle initially coupled to a CDW on a ring can be transferred to an adjacent ring by simply stopping the rotation when opposite types of CDWs are aligned along either the x or y direction and applying momentary perpendicular magnetic "eld Hz with the right polarity. In order to induce a hopping from a HH CDW to an adjacent TT CDW, Hz ought to be directed along !z, while a Hz directed along +z induces a hopping of a particles from a TT CDW to an adjacent HH CDW. The effect of Hz on the double potential landscape of two adjacent CDWs is shown in

Figure 16. (a) Schematic (top panel) and magnetic force microscopy images (bottom panel) of the displacement of two CDWs in a circular ring by applying a rotating "eld Hr of 300 Oe. The ring is made of Py and fabricated by e-beam lithography on a Si substrate. The ring diameter is 15 µm, its width is 200 nm, and the thickness is 30 nm. (b) Sketch of the principle of the continuous displacement of magnetic nano-beads. (c) Optical microscopy images of the "eld-induced displacement of beads in suspension (the ring diameter in this case is 10 µm and the diameter of beads is 1 µm). Panel (a) is reproduced from [37]. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission. Panel (c) is reproduced with permission from [39]. Copyright 2010, AIP Publishing LLC.

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"gure#17(a). As it can be appreciated, Hz lifts one of the two potential wells and deepens the other one, making the trapped nanoparticles ‘slide’ from one ring to the adjacent one.

Figure 17(c) presents selected frames of a video recorded using an optical microscope equipped with a CCD camera, showing the programmable manipulation of an individual magnetic nanoparticle from point A to point B marked in the "gure#[67]. In order to facilitate the utilization of the devices, a graphical interface is implemented that allows a user to draw the desired path on the real-time image provided by the CCD camera using the mouse of the computer connected to the camera. A LabVIEW code was developed to gener-ate automatically a path code, viz., the sequence of "elds (rotating in plane and perpendicular to the plane) required to displace the selected bead along the desired and prede"ned path. In the devices described here, the diameter of the rings (10 µm) is much bigger than the size of the particle (500 nm) and therefore not any position on the chip surface is acces-sible. However, both the ring diameters and particle sizes discussed here have been chosen for demonstration purposes only, in order to make it possible to follow the manipulation

using an optical microscope. One property of magnetic rings that makes them very appealing is their scalability without loss of performance: rings of submicron diameter, down to 100 nm, can be nowadays fabricated and they are found to behave exactly the same as the much bigger ones utilized here. Thereby, manipulation devices with rings of radius compa-rable to the typical size of the magnetic nanoparticles to be manipulated can be readily fabricated.

Figure 18 shows other potential applications of the con-cepts presented above. Figure# 18(a) shows a portion of an array of zigzag conduits used to trap and monitor cell divi-sions. In this experiment, an in-plane magnetic "eld was initially applied to create CDWs in all corners (HH and TT in sequence in each conduit). A suspension of magn etically labeled yeast cells was introduced in the !uidic system until a few cells were trapped. The same growing media as used for the cell culture was introduced with at a !ow rate of 6 µl min!1 for 16 h while continuously monitoring part of the device through an optical microscope [66]. The sequence of images in "gure#18(a) demonstrates that neither the labeling with magnetic beads nor the CDW trapping are affecting the

Figure 17. (a) Principle of 2D manipulation. (b) Hopping of magnetic beads between adjacent DWs. (c) Frames of a video recorded using an optical microscope (bottom panel) showing the programmable manipulation of a single !uid-borne magnetic particle (diameter 500 nm, black dot in the images marked with a red dashed circle) following a prede"ned and arbitrary path (dashed red line) over the array of a Py ring matrix (ring diameter 10 µm), dragged by the "eld-induced CDW displacements.

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cell viability. This system methodology is a valid and fast tool to monitor the cell division over time and even to create a template for tissue regeneration. Figure#18(b) illustrates how the combination of zigzag and curve conduits can be used to create a single particle multiplexer. The proposed device com-bines all the concepts described above, namely the ‘digital’ motion of zigzag conduits, the synchronous motion of curved conduits, and the hopping of nanoparticles induced by vertical "eld pulses [40].

A further step towards integration of optimized magnetic nanoconduit structures into a real ‘lab-on-a-chip’ design, including a micro!uidics system, is illustrated in "gure#19. In detail, we have developed a technique to transfer and embed high-quality functional patterned magnetic nanostructures into !exible and stretchable polymeric and biocompatible substrates [68]. The transfer process is schematized in "g-ures#19(a) and (b). We demonstrated the high "delity of the transfer–embed process in terms of preservation of shape, size, surface roughness, and separation of the nanostructures, at a scale down to the limit of resolution of state-of-the-art e-beam lithography. As shown in "gure# 19(c), we demon-strated the transfer of functional nanostructures directly inside polymer PDMS microchannels. An all-polymeric, bio-com-patible magnetic particle ‘Y’-shaped separator with magnetic nanostructures embedded at the !oor and ceiling sides of a closed channel has been realized by bonding together two such channels. By embedding Py circular rings, a continuous, in-!ow sorting of a suspension of nanoparticles was induced by creating pairs of HH and TT CDWs in each ring and mak-ing them rotate fast enough to prevent their magnetostatic coupling with the !uid-borne particles. In this way, the !ux of particles is diverted in the selected channel by selecting the

sense of rotation of the CDWs, remotely driven by a rotating external magnetic "eld, as shown in "gure#19(d).

Before concluding, we just mention a few very recent advances that demonstrate the feverish activity in this research "eld. One is the development of an on-chip plat-form suitable for the simultaneous manipulation using CDW propagation and integrated magnetoresistance detection of a single magnetic particle in transit [69]. Another example is the development of 3D magnetic nanodevices for remote actuation that can be used in a liquid environment for bio-logical investigations [70]. Another interesting development is the remotely controllable transport of magnetic nanopar-ticles above a topographically !at exchange-bias thin-"lm system, magnetically patterned into parallel stripe domains [71]. As a conclusive remark, the advances of the CDW-based methodology reviewed here could open up new pos-sibilities towards single-molecule and single-cell biomedical applications thanks to its ability to handle small volumes or perform sophisticated sorting of different targets attached to different particles on a chip device. Particularly relevant in view of medical applications is the easy integration of such bio-manipulation systems on a single chip with sensors able to detect the presence of magnetic nanoparticles for ‘lab-on-chip’ diagnostic applications.

4. Cell manipulation, encapsulation, electroporation, and biomarker assays

4.1. High throughput gene transfection in living cells

High throughput transfection, i.e. the delivery of genetic material such as nucleic acids into mammalian cells without

Figure 18. Other CDW conduit devices and applications. (a) Zigzag Py conduits in which CDWs are nucleated at each corner, thereby creating a template for cell reproduction and tissue regeneration. Reproduced from [66], with permission from the Royal Society of Chemistry. (b) Magnetic bifurcation realized, combining multiple zigzag Py conduits for multiplexing of beads. Reproduced with permission from [40]. Copyright 2012, AIP Publishing LLC. (Full videos available as supplementary material of [40, 66]).

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compromising the integrity or viability of the cells, is criti-cal for investigating the effects of gene delivery for funda-mental studies and clinical applications. Techniques based on micro-injection [72], gene gun [73, 74], laser irradiation [75, 76] and sonoporation [77] are some of the current trans-fection approaches. Bulk electroporation [78, 79] is another approach, where millions of cells are simultaneously exposed to a high voltage to momentarily open the cell membrane to introduce the transfectant. However, a serious drawback to this latter approach is that a large fraction of the cells are damaged during the process due to the non-uniform and haz-ardous electric "elds that adversely affect individual cells. Thus, four critical aspects—targeting individual cells, con-trolling dosage, achieving high selectivity, and throughput as well as retaining cell viability—are not guaranteed with bulk electroporation.

Transfection based on a micro-magnetic patterned platform with an array of microscopic pores overcomes these draw-backs by enabling remotely activated protocols to arrange individual cells in a planar array directly above the openings to allow delivery of regulated dosages through the tiny pores that spatially focus the electric "elds [80]. The dosage each cell receives is determined and controlled by the duration of a single electrical pulse and the number of consecutive pulses delivered. Moreover, in this con"guration, low voltages (<10 V) are suf"-cient for cell poration due to con"nement of the electric "eld to a small microscopic area of the cell membrane de"ned by each pore which, in turn, ensures cell viability. The higher through-put of this 3D micro electroporation (3D MEP) device is an attractive feature over nanochannel-based electroporation [81] and serves as a single platform for multi-cell loading, alignment with micropores, gene delivery, and subsequent transport for

Figure 19. (a)–(c) Direct transfer of e-beam lithographed magnetic conduits to polymer substrates. The illustration (a) shows a sketch of the substrate (Si/Ti/Au/SiO2) on which the magnetic structures made of Py are patterned. In (b), the desired polymer is poured on top of the substrate, cured, and "nally peeled off from the substrate by immersion in water. The SiO2 and the Au layer separate, and the magnetic structures end up being transferred and embedded into the polymeric receiving free-standing membrane. Illustration (c) shows the same process, in which a Si mold is used to transfer Py structures in polymeric membrane shapes to create a micro!uidic channel. (d) Example of a ‘Y’-shaped micro!uidic channel with embedded ferromagnetic nanorings for particle !ow control via an external rotating magnetic "eld. Panels (a)–(c) are reproduced from [68]. Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission.

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analysis. Central to the recent progress in transfection of cells have been the developments in !uorescence techniques associ-ated with selected molecules to speci"c sequences of nucleic acids. For instance, molecular beacons are hairpin-shaped mol-ecules that possess an internally quenched !uorophore whose emission is restored when they bind to a target nucleic acid sequence—thereby offering a convenient method for identify-ing speci"c sequences of nucleic acids. Similarly, a family of blue !uorescent dyes has been used to stain and identify DNA, a process often called Hoechst staining.

Figure 20 illustrates the underlying setup and chip fabri-cated using cleanroom techniques. To ef"ciently place an indi-vidual cell at each of the micropores, an array of 50 nm thick Permalloy (Py, NiFe) disks are fabricated on a silicon wafer to function as an effective multiplexed conveyor system to maneuver each of the magnetically labeled cells to the pora-tion sites directly above the micropore. The cells were linked via CD45 antigen with 1 µm diameter Dextran-coated super-paramagnetic beads to enable remote control of the cells by weak external magnetic "elds. Once transfected, the cells are subsequently transported through magnetic protocols across the platform. Four orthogonal electromagnets and surrounding solenoid generate the external "eld Hxy + Hz to create attrac-tive traps (magnetic potential energy wells) or repulsive cent-ers at the periphery of the disks whose strengths are tuned with Hz. The typical energy landscape near a Py disk is illustrated in "gure#4, showing the energy minima that lead to capture of magnetically labeled cells [82]. The use of Py disks for trans-port is in contrast to utilizing Py rings as described in "gure#16, for example. Parallel transfection and manipulation [80] ena-bles easy scale-up of the device (currently capable of 40 000 cells cm!2) to address millions of cells by merely increasing the number of micropores and associated magnetic disks. Typical voltage characteristics utilized for transfection were ~5 V, 10 ms pulse duration with 5 pulses delivered in about a minute [80]. Additionally, the weak magnetic "elds (<150 Oe) do not generate heat or adversely damage the cells, concerns that may arise with some other manipulation techniques [5].

As illustrated in "gure#21 for two cell types and transfection reagents, the versatility of the magnetic alignment approach with its potential for pre-clinical studies and gene therapy has been demonstrated [80]. The delivery of the GATA2 molecu-lar beacon (MB) for detection of GATA2 mRNA expression is signi"cant for the study of heterogeneities of hematopoietic stem cells (HSCs) [83] since it is highly expressed in HSCs and its disorder has been implicated in the onset of leuke-mia [84]. In the experiment, a GATA2 molecular beacon was delivered into both K562 (a common myeloid, pertaining to bone marrow, progenitor with high GATA2 expression, "g-ure#21(f)) and Jurkat (a T-cell mature leukemic cell line with low level of GATA2 mRNA) cells (not shown) in order to detect the regulation of the GATA2 gene [85]. The transfected GATA2-MB speci"cally hybridizes to GATA2 mRNA in the cytosol (!uid in which organelles of the cell reside) and subse-quently !uoresces by unzipping its hairpin structure, thereby distinguishing cells with high GATA2 levels from those with low levels. The 3D MEP chip, in combination with molecular beacon-based probes, thus offers a !uorescence biosensing system for detecting explicit intracellular markers within indi-vidual living cells, with the potential to provide insight into the expression of genes and their role in diseases initiated by speci"c cells.

To test the highly selective gene-delivery capabilities of this system, ODN-FAM (oligonucleotide with carboxy!uo-rescein !uorescent dye) was delivered into an array of KG1a cells ("gure 21). Ef"cient alignment of the cells with the array of micropores is con"rmed with phase-contrast imaging and Hoechst staining ("gure 21(g)). The cells were transfected with low voltage (4 V), and the green cellular !uorescence reported from FAM (Fluorescein, a !uorescent label) was visualized after pulse generation, indicating the intracellular delivery as well as the relatively high throughput (40 000 cells cm!2 min!1) func-tionality of the chip for gene delivery ("gure 21(b)). Quantitative results in "gure#21(h) con"rm viability of 92% for K562 cells and 96% for KG1a cells, validating that magnetic labeling and the low-voltage pulses do not damage the cell membranes.

Figure 20. Schematic top view of experimental setup showing four electromagnets (to create in-plane x, y "eld) and solenoid (out-of-plane z "eld). (a) Magnetic setup. (b) 3D micro-electroporation device located at center of magnets. A gold substrate functions as bottom electrode beneath a PDMS container that holds the transfection reagents. The location of the magnetic disks on the upper surface is indicated. (c) The transfection wafer has arrays of magnetic (Permalloy) disks patterned on its upper surface for holding magnetically labeled cells while the micropore arrays enable the transfection reagents to be delivered to cells.

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These results illustrate a promising highly selective gene transfection device based on magnetic alignment of labeled cells and electroporation with the added advantages of retaining high cell viability and uniform transfection at the single-cell level. By transporting the transfected cells through magnetic protocols across the Py disks [82], a uni-"ed platform can be fully integrated into experiments aimed at not only delivering and tracking intracellular molecular probes for gene detection but also to subsequently evaluate the processes associated with gene therapy outcomes and regenerative progressions in large populations of individual cells.

4.2. Cell sorting and encapsulation and single-cell electroporation using nanowires

Analysis of cellular content such as proteins and nucleic acid molecules typically requires a priori cell lysis, the breaking down of the outer membrane of cell by rupture. Given that, even within an isogenic cell population, stochastic gene expressions exist among cells [86–88], analyzing an ensemble of cells at an individual level with high spatiotemporal resolutions will lead to a better understanding of such cell-to-cell variations [89, 90]. The three key processes required for performing single-cell analyses and study of its cellular

Figure 21. Cell transfection achieved with 3D MEP chip in KG1a and K562 cells with ODN and GATA2 molecular beacons, respectively. The phase contrast images ((a) and (d)) show the location of the through-pores on the chip. Hoechst staining ((b) and (e)) reveals the cell nuclei and thus the cell location. The green !uorescence from (c) the ODN or (f) GAT2 molecular beacon is illustrated. (g) Trapping ef"ciency of aligning KG1a and K562 using the 3D MEP chip in comparison to random seeding (cell number = 300). (h) Quantitative analysis of using negative !uorescence from propidium iodide (PI), showing high levels (>90%) of cell viability. Reproduced from [80], with permission from Wiley.

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components are (i) sorting of cells into subpopulations, (ii) compartmentalization of the cells of interest with dedicated reagents into individually isolated environments, and (iii) disruption of the membrane leading to lysis and release of biological comp onents from the cell.

4.2.1. Cell sorting and encapsulation in droplets. Differ-ent cell-sorting techniques such as !ow cytometry [88, 91] and hydrodynamic [6–9], electric "eld [14–16], and optic trap [10–13] methods have been developed. Magnetic "eld-based sorting (e.g. via marker-speci"c magnetic bead labeling approaches), utilizes external magnets [91–96], ferromagnetic channels [97], strips, or patterns [29, 30, 46, 66, 98–102] to manipulate cells to desired locations.

Compartmentalization of the sorted cells of interest into individually isolated environments, such as within individ-ual droplets, is a crucial step towards single-cell analysis. Moreover, it is advantageous to integrate this step directly with the sorting protocols in the same setup to maintain the native environment from which the cells were derived. Since the number of droplets generated is practically unlimited, scale-up thus becomes easy. Figure#22 illustrates such an inte-grated platform where the magnetic sorting and droplet encap-sulation occur on the same chip [101].

Using the magnetic setup shown in "gure#20(a), the labeled cells were transported across the Py disk array by rotation of the in-plane "eld H1, i.e. Hext = (H1 · cos", H1 · sin", Hz), " = 0°–180°, followed by reversing the out-of-plane Hz [82]. These steps resulted in the transport of the cell around the disk periphery (e.g. from –x end to +x end) during the "eld-rotation phase followed by its transfer to the adjacent disk (e.g. from +x end of one disk to –x end of next) when Hz was reversed. Such transport is analogous to the bead trajectories

described in section#2.3 for isolated and connected disks as illustrated in "gure#5. With the ratio of the |Hz| to H1 set "xed, the two central parameters for magnetic manipulation of the labeled cells are the magnitude of |H1| and the transport rate f (the number of disks traversed by the cell per unit time).

Separation of a heterogeneous mixture of labeled breast cancer cell line (BT-474) and unlabeled red blood cells under optimal !ow rate enabled separation of the BT-474 with greater than 75% separation ef"ciency and 100% purity (i.e. no unlabeled cells were encapsulated) [101]. Cell viability assay subsequent to encapsulation was also demonstrated, permitting analysis on a single-cell basis rather than averag-ing properties over bulk populations.

4.2.2. Cell lysis. In order to lyse the isolated cell(s), they must then be positioned above the electroporation element. A variety of methods have been used to controllably position cells, including patterning [103], !uidic traps and wells [104], optical traps [105], and dielectrophoresis [106]. A label-free and trapless technique is where magnetic beads are simply rolled to push untethered or non-speci"cally bound cells into a desired position [82]. With optimization of the platform surface, applied "eld strength, and frequency, magnetic beads can reliably push the cells into position directly above the transistors or nanowires for subsequent lysis as shown in "gure# 23 [107]. The cell transport and positioning was thus accomplished without the need for labeling the cells or special surfaces or patterns by utilizing the programmability of magnetic "eld routines for magnetic manipulation of 7.9 µm diameter magnetic microspheres.

In electroporation, an applied electric "eld applied at the site of the positioned cell creates an electric potential differ-ence across the cell membrane. Once this transmembrane

Figure 22. (a) Schematic of layout of micro!uidic channels used to input and sort targeted cells as well as create encapsulation droplets. The magnetic disk array underlies the sorting. (b) Image showing layout of Py disks. Q1–Q4 are !uid !ow rates within different channels, T1, T2, T3 identify the three junctions where labeled cells are sorted, moved towards the droplet, and encapsulated. (c)–(e) Snapshots show encapsulation of a cell (indicated by the box) mixed with the reagent from !ow Q3. Reference [101], reproduced by permission of The Royal Society of Chemistry.

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potential exceeds a threshold of ~0.2–1.0 V, pores form in the membrane and, depending on their size and number, the pores either reseal as in the transfection approach discussed in sec-tion# 4.1 or remain open, resulting in lysis with the cellular contents being released and the cell dying.

In the case of cell lysis above a Si nanowire [107], the posi-tioned cells were allowed to adhere to the device surface for ~30 min prior to application of the lysing voltages at 10 MHz and up to 900 mVpp between the shorted source–drain and the back gate of the transistor. A live–dead assay monitored the cell membrane integrity where propidium iodide (PI) dye, a membrane impermeable dye, was used. Upon breakdown of the cell membrane, PI enters the cell and intercalates with the cellular DNA. An increase in PI !uorescence was used to determine cell lysis.

Several methods for single-cell electroporation have been developed [108], utilizing platforms such as microfabricated chips [109] and vertical nanopillars [110]. The advantages of these approaches are they do not require (1) transparent substrates often needed for optical tweezers or (2) high on-chip voltages and strict control of media conductivity as in the case of dielectrophoresis-based manipulation. Moreover, the micro-magnetic cell positioning technique is, as noted, easily integrated with "eld effect transistor platforms to offer enhanced use and portability with the added opportunity for "eld effect sensing of the released cellular components [107].

4.3. Magnetic nano-conveyor platform for hybrid nanostructures and biomarker assays

While controlling multiple individual nanostructures is central for bottom-up assembly strategies, two signi"cant challenges arise in realizing their ordered transport: (1) large external "elds may be required to target and manipulate each nanoparticle, and (2) dif"culties of visually tracking

Figure 23. (a)–(c) Schematic showing magnetic beads pushing unlabeled cell into position above a Si nanowire. (d)–(f) Corresponding images of an unlabeled MCF-7 cell being pushed atop a nanowire for lysis. Reference [107], reproduced by permission of The Royal Society of Chemistry.

Figure 24. Schematic of the nano-conveyor technology to transport multiple individual nano-containers simultaneously with external control and real-time tracking. Top row: micelle nano-containers, linker molecule (e.g. Avidin or DNA), quantum dot (QD), and superparamagnetic iron oxide nanoparticle (SPION). Row 2: micelle with encapsulated QD and SPION transported on Py disks. Row 3: a composite micelle–protein–micelle construct moved along and between FeCo wires. Row 4: actual images of composite structure showing detection !uorescent tracking and transport of biomolecule along and across zigzag wire tracks.

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the motion of sub-200-nm structures. These concerns are addressed by the ‘nano-conveyer’ platform technology [111], which is based on two key components: polymeric micelle nano-containers (~35 nm) encapsulating both quantum dots (QDs) for !uorescent observation and superparamagnetic iron oxide nanoparticles (SPIONs) to permit controlled manipula-tion using patterned magnetic tracks [46, 82]. This technology thus permits simultaneous observation and control of nano-structure movement and can open new avenues in nanofabrica-tion, nano!uidics, biomechanics, drug delivery, and magnetic actuation. As illustrated in "gure#24, by using two micelles to separately hold the QD and magnetic nanoparticle along with speci"c capture probes designed to bind to a target biomol-ecule, an assay to detect and isolate molecular biomarkers also becomes feasible [112].

Figure 24 illustrates the nano-conveyor arrays to transport multiple individual nano-containers plus any nano-cargo they may carry with external control and real-time tracking. The conveyors are composed of microfabricated Py magnetic disk [82] or FeCo zigzag wire [46] patterns coupled with electro-magnets. The nano-containers are ~35 nm polymeric micelles that are formed through interfacial instability [113]. The num-bers of QDs and SPIONs in each micelle are controlled by the molecular structure of the polymer and quantities of polymer, QDs, and SPIONs used. Coupled with strong QD !uorescence and resistance to photobleaching, the combination of SPIONs

and QDs provides for investigator controlled nano-container manipulation with long-term optical tracking capability. Note that, as evident from the last row in "gure#24, the magnetic construct is not restricted to moving along the wire conduits but can also be maneuvered from one zigzag wire to a neigh-boring wire. This feature stems from the magnetic properties of Fe0.5Co0.5, where the magnetization of the wires remains substantially unchanged during transport for the external "elds (<150 Oe) used in the experiment. The use of Fe0.5Co0.5 is in contrast to the Py wires described in section#3. One important difference, for example, is that in the former (Py), the domain walls are mobile (as shown in "gure#15) while in the Fe0.5Co0.5 case the domain walls are stationary and the energy landscape is modi"ed by the "elds that are applied to move the magnetic entities. In such cases, through programmed external "eld sequences, the trapping sites (i.e. energy minima), along with the trapped micelles, can be transported across the platform to desired locations away from a given vertex—for example, to a neighboring wire vertex.

The magnetization pro"le for a 380 nm wide 40 nm thick zigzag Fe0.5Co0.5 wire was derived from the object-oriented micro-magnetic framework program and shown in "gure#25. The force F on a nano-container with 10 encapsulated Fe3O4 nanoparticles in the absence of an external magnetic "eld was

calculated using F N V Hz

12 0

2! ( )µ != !!

, where N = 10 is the

Figure 25. Calculation of (a) magnetic "eld and (b) "eld gradient from the vertex of a 380 nm wide, 40 nm thick Fe0.5Co0.5 wire. (c) Forces from this vertex on a typical nano-container as a function of height directly above the vertex. (d) Results of OOMMF simulation showing the head-to-head and tail-to-tail domain structure of zigzag wire with arm length of 2 µm. Color indicates the divergence of the magnetization (red, negative divergence; blue, positive divergence). Areas of large negative and positive divergence localized at the wire vertices correspond with sources and sinks of stray "eld respectively, which may be used to trap particles. Inset: detail of domain structure at central vertex of zigzag wire.

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number of micelle-encapsulated Fe3O4 nanoparticles, µ0 is the permeability of free space, # = 1.3 is the magnetic sus-ceptibility of the nanoparticles, V is the volume of each encap-sulated nanoparticle, and H is the calculated magnetic "eld arising from the wire. The magnetic "elds and "eld gradients from the Fe0.5Co0.5 wires, as a function of height directly above the vertex, are shown in "gure#25.

Several noteworthy points are drawn from "gure#25 for the given wire dimensions and for heights within 100 nm directly above the vertex:

(1) Fields generated by the Fe0.5Co0.5 wires (~800 Oe) can be modulated by external "elds from an electromagnet.

(2) Field gradients exceeding 105 T m!1 are created, a desir-able feature for manipulation of magnetic nanoparticles.

(3) Magnetic forces on a few (~10) Fe3O4 nanoparticles embedded in a 50 nm micelle or linked to a biomolecule are ~0.1 pN—which is adequate to achieve magnetic "eld guided transport of the particles.

These sandwich-type micelle constructs, together with the conveyor tracks, have enabled (last row of "gure#24) detec-tion and separation of protein (e.g. Avadin) and DNA [112]. An important advance in such biomarker detection has been the capability of multiplex detection on the same chip, for instance by utilizing QDs that have different emission wave-lengths. In these cases, the QDs are encapsulated in micelles having different capture probes on their surface that bind to the target molecule and form a !uorescent-magnetic nano-composite only in the presence of the targeted analyte.

In summary, some examples of utilizing investigator-con-trolled local magnetic "elds to achieve (1) selective and rela-tively high throughput gene transfection without cell damage, (2) cell encapsulation within individual droplets, (3) nanow-ire-based cell lysis, and (4) a nano-conveyor assay for detect-ing and isolating molecular biomarkers were discussed in this section. The broader bene"ts of these approaches include use of small sample volumes, minimal loss of biological speci-mens, portability, and remote control of magnetic forces that are not screened in solution and are independent of many experimental conditions such as pH, temperature, or solvent compositions. Such features thus allow more freedom and !exibility in the experimental design and operation of emerg-ing manipulation, detection, and analysis platforms.

5. Summary and outlook

Emerging advances in nano- and biotechnology will require the capability to capture, transport, assemble, and spatially localize targeted multifunctional nanoparticles. This review addresses some recent progress in magnetism-based manipulation, separation, and detection methods and highlights a range of engineering and biological applications. The utilization of superparamagnetic particles as the force-transmitting handle has been particularly promising where their non-hysteric magnetization loops and absence of remanence or coercivity at room temperature render predictable forces

and do not promote particle clustering in the absence of an external "eld. While the magnetic "elds and their gradients emanating from the miniature surface pro"les and conduits discussed here play a central role in steering these particles across a surface, the weak external magnetic "elds necessary for manipulation do not interfere with chemical or biological interactions. These characteristics thereby lend themselves as attractive features for integration into micro!uidic platforms for particle and cell sorters, as well as a range of next-generation biomedical devices. Their prospect for scale-up to centimeter-sized platforms offers the potential for large-scale multiplex operations that would also offer attractive approaches for rapid sequential or parallel analysis operations that do not rely on ensemble averaging.

Magnetic nano- and microparticles also offer exciting opportunities as an intracellular probe of crucial physical parameters of living cells such as, for example, the viscoe-lasticity of the cytoplasm (the substance between the cell membrane and the nucleus) and as surrogate vehicles that are traf"cked along microtubule networks (tubular polymers forming a network throughout the cytoplasm, which is part of the cytoskeleton that maintains the structure of a cell and governs intracellular transport of substances crucial for the cellular activity) [114–116]. Since magnetic particles that enter the cell by phagocytosis (the process by which a cell engulfs a solid particle) are wrapped by a bilayer membrane, they essentially exhibit the same outer composition as the cell membrane. These particles are thus expected to mimic the behavior of intracellular bodies. For instance, they could move along micro-tubular tracks—a process mediated by kinesin and dynein motors (proteins that moves along microtubules transporting a cargo between the nucleus to the membrane and both directions). The response of the endocytosed magnetic particles (particles engulfed by a cell through phagocytosis) to external magnetic "elds also renders them as effective micro-rheology probes to quantitatively study local viscoelastic properties that underlie many structural and dynamic prop-erties of the cell. On the other hand, for magnetic particles injected (and not engulfed) into the cell, they may not actively interact with the surrounding cell machinery and yet func-tion as passive probes. Their spontaneous, thermally driven, motions have been used to deduce the mechanical properties of their cellular surroundings. Such studies with intracellu-lar magnetic nanoparticles are continuing to emerge as useful avenues for investigating numerous cellular processes such as cell division, adhesion to substrates, and locomotion. When coupled with the micro- and nano-magnetic patterned plat-forms as those presented in this review, new understanding of cellular activities will likely be forthcoming.

With the ability to utilize digital magnetophoresis to remotely manipulate single as well as large numbers of cells, it is reasonable to expect that one of the next major advance-ments in this area could be the targeting and detection of sub-cellular entities. Such optimism arises from the successes in the transport and isolation of single cells, creation of templates to track cell reproduction and tissue regeneration, sorting of cells and transfection of genetic material across the cellular membrane, and cell rupturing to release cellular content that

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are all carried out through magnetic shuttle technologies in lab-on-chip con"gurations coupled to micro!uidics. Such futuristic biomedical advancements targeting the subcellular level are thus certainly within reach.

Regarding real-life medical applications, the capability of the novel methodologies reviewed in this paper to manipulate even a large population of magnetically tagged bio-entities would open up new and unforeseen possibilities as to handling small volumes or performing sophisticated sorting of differ-ent targets attached to different particles on a chip device. Particularly relevant in view of medical applications is the easy integration of such biomanipulation systems on a sin-gle chip, with sensors able to detect the presence of magnetic nanoparticles for ‘lab-on-chip’ diagnostic applications.

An example of another biomedical opportunity is the criti-cal need for an inexpensive, accessible, point-of-care (POC) assay that quanti"es diseases such as HIV. Many emerging POC devices are being developed and based for implementa-tion as an effective diagnostic tool. There are, of course, many challenges to utilizing the approaches such as those discussed in this article for integration into a low-cost POC device. These hurdles include separating and accessing the pathogens or viruses from blood samples and approaches to gather the related genetic information, their binding to magnetic parti-cles, and subsequent detection within micro!uidic environ-ments. While each of these steps will require contributions from scientists and medical professionals across different dis-ciplines, the integration of the different functionalities into a single unit would be also challenging. However, if realized, the impact on future medical diagnostics could be enormous.

Acknowledgment

This work was supported by the BioNano Health Guard Research Center, funded by the Ministry of Science, ICT and Future Planning (MSIP) of Korea (Grant Number H-GUARD_2013M3A6B2078959). C K acknowledges Dr S R Torati for his editing of the manuscript. Work at The Ohio State University was supported by the US National Science Foundation (EEC-0914790) and the Army Research Of"ce (W911NF-10-1-0353 and W011NF-14-1-0289). PV acknowl-edges support from the Basque Government under Project No. PI_2015_1_19 as well as the Spanish Ministry of Economy and Competitiveness under Project No. FIS2015-64519_R (MINECO/FEDER).

References

[1] Prakash#M and Gershenfeld#N 2007 Science 315#832 [2] Unger#M#A, Chou#H#P, Thorsen#T, Scherer#A and Quake#S#R

2000 Science 288#113 [3] Love#K#R, Bagh#S, Choi#J and Love#J#C 2013 Trends

Biotechnol. 31#280 [4] Yin#H#B and Marshall#D 2012 Curr. Opin. Biotechnol.

23#110 [5] Lindstrom#S and Andersson-Svahn#H 2010 Lab Chip

10#3363

[6] Skelley#A#M, Kirak#O, Suh#H#Y, Jaenisch#R and Voldman#J 2009 Nat. Methods 6#147

[7] Di Carlo#D, Irimia#D, Tompkins#R#G and Toner#M 2007 Proc. Natl Acad. Sci. USA 104#18892

[8] Kuntaegowdanahalli#S#S, Bhagat#A#A#S, Kumar#G and Papautsky#I 2009 Lab Chip 9#2973

[9] Herzenberg#L#A, Parks#D, Sahaf#B, Perez#O, Roederer#M and Herzenberg#L#A 2002 Clin. Chem. 48#1819

[10] Ashkin#A 1997 Proc. Natl Acad. Sci. USA 94#4853 [11] Block#S#M, Golstein#L#S#B and Schnapp#B#J 1990 Nature

348#348 [12] Roichman#Y, Wong#V and Grier#D#G 2007 Phys. Rev. E

75#011407 [13] Chiou#P#Y, Ohta#A#T and Wu#M#C 2005 Nature 436#370 [14] Kortmann#H, Chasanis#P, Blank#L#M, Franzke#J, Kenig#E#Y

and Schmid#A 2009 Lab Chip 9#576 [15] Gascoyne#P#R#C and Vykoukal#J 2002 Electrophoresis

23#1973 [16] Pethig#R 2010 Biomicro!uidics 4#022811 [17] Gao#L, Tahir#M#A, Virgin#L#N and Yellen#B#B 2011 Lab Chip

11#4214 [18] Gao#L, Gottron#N III, Virgin#L and Yellen#B#B 2010 Lab Chip

10#2108 [19] Kose#A#R, Fischer#B, Mao#L and Koser#H 2009 Proc. Natl

Acad. Sci. USA 106#21478 [20] Gunnarsson#K, Roy#P#E, Felton#S, Pihl#J, Svedlindh#P,

Berner#S, Lidbaum#H and Oscarsson#S 2005 Adv. Mater. 17#1730

[21] Venu#R, Lim#B, Hu#X#H, Jeong#I, Ramulu#T#S and Kim#C#G 2013 Micro!uid. Nano!uid. 14#277

[22] Anandakumar#S, Rani#V#S, Oh#S, Sinha#B#L, Takahashi#M and Kim#C 2010 Biosens. Bioelectron. 26#1755

[23] Lee#H, Purdon#A#M, Chu#V and Westervelt#R#M 2004 Nano Lett. 4#995

[24] Tierno#P, Sagues#F, Johansen#T#H and Fischer#T#M 2009 Phy. Chem. Chem. Phys. 11#9615

[25] Rapoport#E and Beach#G#S#D 2012 Appl. Phys. Lett. 100#082401

[26] Gijs#M#A#M 2004 Micro!uid. Nano!uid. 1#22 [27] Chen#A, Vieira#G, Henighan#T, Howdyshell#M, North#J#A,

Hauser#A#J, Yang#F#Y, Poirier#M#G, Jayaprakash#C and Sooryakumar#R 2011 Phys. Rev. Lett. 107#087206

[28] Lee#H, Liu#Y, Ham#D and Westervelt#R#M 2007 Lab Chip 7#331

[29] Yellen#B#Bet"al2007 Lab Chip 7#1681 [30] Lim#B, Reddy#V, Hu#X, Kim#K, Jadhav#M, Abedini-Nassab#R,

Noh#Y, Lim#Y#T, Yellen#B#B and Kim#C 2014 Nat. Commun. 5#3846

[31] Hu#X, Abedini-Nassab#R, Lim#B, Yang#Y, Howdyshell#M, Sooryakumar#R, Yellen#B#B and Kim#C#G 2015 J Appl. Phys. 118#203904

[32] Hu#X, Lim#B, Jeong#I, Sandhu#A and Kim#C#G 2013 IEEE Trans. Magn. 49#408

[33] Hu#X, Goudu#S#R, Torati#S#R, Lim#B, Kim#K and Kim#C#G 2016 Lab Chip 16#3485

[34] Bertacco#R and Vavassori#P 2012 European Patent Application EP2274633B1

[35] Bertacco#R, Cantoni#M, Donolato#M, Gobbi#M, Brivio#S, Vavassori#P and Petti#D 2015 European Patent Application EP2396118B1

[36] Vieira#G, Henighan#T, Chen#A, Hauser#A, Yang#F, Chalmers#J and Sooryakumar#R 2009 Phys. Rev. Lett. 103#128101

[37] Donolato#M et"al 2010 Adv. Mater. 22#2706 [38] Laufenberg#M et"al 2006 Appl. Phys. Lett. 88#052507 [39] Vavassori#P, Metlushko#V, Ilic#B, Gobbi#M, Donolato#M,

Cantoni#M and Bertacco#R 2010 J. Appl. Phys. 107#09B301

J. Phys. D: Appl. Phys. 50 (2017) 033002

Topical Review

24

[40] Torti#A, Mondiali#V, Cattoni#A, Donolato#M, Albisetti#E, Haghiri-Gosnet#A#M, Vavassori#P and Bertacco#R 2012 Appl. Phys. Lett. 101#142405

[41] Ehresmann#A, Lengemann#D, Weis#T, Albrecht#A, Langfahl-Klabes#J, Göllner#F and Engel#D 2011 Adv. Mater. 23#5568

[42] Helseth#L#E, Fischer#T#M and Johansen#T#H 2003 Phys. Rev. Lett. 91#208302

[43] Tierno#P, Reddy#S#V, Roper#M#G, Johansen#T#H and Fisher#T#M 2008 J. Phys. Chem. B 112#3833

[44] Holzinger#D, Lengemann#D, Göllner#F, Engel#D and Ehresmann#A 2012 Appl. Phys. Lett. 100#153504

[45] Johnson#J, Metlushko#V, Grimsditch#M, Vavassori#P, Ilic#B, Neuzil#P and Kumar#R 2000 Appl. Phys. Lett. 77#4410

[46] Erb#R#M, Sebba#D#S, Lazarides#A#A and Yellen#B#B 2008 J. Appl. Phys. 103#063916

[47] Nikulina#E, Idigoras#O, Vavassori#P, Chuvilin#A and Berger#A 2012 Appl. Phys. Lett. 100#142401

[48] Klaüi#M 2008 J. Phys.: Condens. Matter 20#313001 [49] Zhu#F, Chern#G, Tchernyshyov#O, Zhu#X, Zhu#J and Chien#C

2006 Phys. Rev. Lett. 96#027205 [50] Allwood#D#A, Xiong#G, Faulkner#C#C, Atkinson#D, Petit#D

and Cowburn#R#P 2005 Science 309#1688 [51] Atkinson#D, Allwood#D#A, Xiong#G, Cooke#M#D and

Cowburn#R#P 2003 Nat. Mater. 2#85 [52] Beach#G#S#D, Nistor#C, Knutson#C, Tsoi#M and Erskine#J#L

2005 Nat. Mater. 4#741 [53] Donolato#M, Lo"nk#F, Hankemeier#S, Porro#J#M, Oepen#H#P

and Vavassori#P 2012 J. Appl. Phys. 111#07B336 [54] Faulkner#C#C, Cooke#M#D, Allwood#D#A, Petit#D,

Atkinson#D and Cowburn#R#P 2004 J. Appl. Phys. 95#6717 [55] Klaüi#M, Vaz#C#A#F, Bland#J#A#C, Wernsdorfer#W, Faini#G

and Cambril#E 2003 J. Appl. Phys. 93#7885 [56] Vavassori#P, Grimsditch#M, Novosad#V, Metlushko#V, Ilic#B,

Neuzil#P and Kumar#R 2003 Phys. Rev. B 67#134429 [57] Libál#A, Grimsditch#M, Metlushko#V, Vavassori#P and

Jankó#B 2005 J. Appl. Phys. 98#083904 [58] Vavassori#P, Bisero#D, Bonanni#V, Busato#A, Grimsditch#M,

Lebecki#K, Metlushko#V and Ilic#B 2008 Phys. Rev. B 78#174403

[59] Vaz#C#A#F et"al 2007 J. Phys.: Condens. Matter 19#255207 [60] Klaüi#M, Vaz#C#A#F, Bland#J#A#C, Wernsdorfer#W, Faini#G,

Cambril#E, Heyderman#L#J, Nolting#F and Rüdiger#U 2005 Phys. Rev. Lett. 94#106601

[61] Vavassori#P, Metlushko#V and Ilic#B 2007 Appl. Phys. Lett. 91#093114

[62] Donahue#M#J and Porter#D#G 1999 OOMMF User’s Guide, Version 1.0 Interagency Report NISTIR 6376, National Institute of Standards and Technology, Gaithersburg, MD

[63] Vavassori#P, Metlushko#V, Ilic#B, Gobbi#M, Donolato#M, Cantoni#M and Bertacco#R 2008 Appl. Phys. Lett. 93#203502

[64] Donolato#M, Gobbi#M, Vavassori#P, Cantoni#M, Leone#M, Metlushko#V, Ilic#B, Zhang#M, Wang#S#X and Bertacco#R 2009 Nanotechnology 20#385501

[65] Vavassori#P, Grimsditch#M, Metlushko#V, Zaluzek#N and Ilic#B 2005 Appl. Phys. Lett. 86#072507

[66] Donolato#M, Torti#A, Kostesha#N, Deryabina#M, Sogne#E, Vavassori#P, Hansen#M#F and Bertacco#R 2011 Lab Chip 11#2976

[67] Sarella#A, Torti#A, Donolato#M, Pancaldi#M and Vavassori#P 2014 Adv. Mater. 26#2384

[68] Donolato#M, Porro#J#M, Tollan#C and Vavassori#P 2013 Adv. Mater. 25#623

[69] Monticelli#M et"al 2016 Small 12#921 [70] Vavassori#P, Pancaldi#M, Perez-Roldan#M#J, Chuvilin#A and

Berger#A 2016 Small 12#1013

[71] Holzinger#D, Koch#I and Ehresmann#A 2015 ACS Nano 9#7323

[72] Diacumakos#E#G 1973 Methods in Cell Biology (New York: Academic)

[73] Klein#T#M, Wolf#E#D, Wu#R and Sanford#J#C 1987 Nature 327#70

[74] Yang#N#S, Burkholder#J, Roberts#B, Martinell#B and McCabe#D 1990 Proc. Natl Acad. Sci. USA 87#95681

[75] Shirahata#Y, Ohkohchi#N, Itagak#H, Satomi#S and Investig#J 2001 J. Investigative Med. 49#184

[76] Tirlapur#U#K and Konig#K 2002 Nature 418#290 [77] Kim#H#J, Greenleaf#J#F, Kinnick#R#R, Bronk#J#T and

Bolander#M#E 1996 Hum. Gene Ther. 7#1339 [78] Hoover#F and Magne#K#J 2000 Anal. Biochem. 285#175 [79] Gehl#J 2003 Acta. Physiol. Scand. 177#437 [80] Chang#L, Howdyshell#M, Liao#W-C, Gallego-Perez#D,

Yang#Z, Lu#W, Byrd#J#C, Muthusamy#N, Lee#L#J and Sooryakumar#R 2015 Small 11#1818

[81] Boukany#P#E et"al 2011 Nat. Nanotechnol. 6#747 [82] Henighan#T, Chen#A, Vieira#G, Hauser#A#J, Yang#F#Y,

Chalmers#J#J and Sooryakumar#R 2010 Biophys. J. 98#412 [83] Nagai#T et"al 1994 Blood 84#1074 [84] Ikonomi#P, Rivera#C#E, Riordan#M, Washington#G,

Schechter#A#N and Noguchi#C#T 2000 Exp. Hematol. 28#1423

[85] Klein#E, Ben-Bassat#H, Neumann#H, Ralph#P, Zeuthen#J, Polliack#A and Vánky#F 1976 Int. J. Cancer 18#421

[86] Elowithz#M#B, Levine#A#J, Siggia#E#D and Swain#P#S 2002 Science 297#1183

[87] Avery#S#V 2006 Nat. Rev. Microbiol. 4#577 [88] Dalerba#P et"al 2011 Nat. Biotechnol. 29#1120 [89] Fritzsch#F#S#O, Dusny#C, Frick#O and Schmid#A 2012 Annu.

Rev. Chem. Biomol. Eng. 3#129 [90] Ohnuma#K, Yomo#T, Asashima#M and Kaneko#K 2006 BMC

Cell Biol. 7#25 [91] Pamme#N and Wilhelm#C 2006 Lab Chip 6#974 [92] Zhou#Y, Wang#Y and Lin#Q 2010 J. MEMS 19#743 [93] Robert#D, Pamme#N, Conjeaud#H, Gazeau#F, Iles#A and

Wilhelm#C 2011 Lab Chip 11#1902 [94] Schneider#T, Karl#S, Moore#L#R, Chalmers#J#J, Williams#P#S

and Zborowski#M 2010 Analyst 135#62 [95] Zborowski#M and Chalmers#J#J 2011 Anal. Chem. 83#8050 [96] Jin#X, Abbot#S, Zhang#X, Kang#L, Voskinarian-Berse#V,

Zhao#R, Kameneva#M#V, Moore#L#R, Chalmers#J#J and Zborowski#M 2012 PLoS One 7#e39491

[97] Jung#Y, Choi#Y, Han#K-H and Frazier#A#B 2010 Biomed. Microdevices 12#637

[98] Adams#J#D, Kim#U and Soh#H#T 2008 Proc. Natl Acad. Sci. USA 105#18165

[99] Adams#J#D, Thévoz#P, Bruus#H and Soh#H#T 2009 Appl. Phys. Lett. 95#254103

[100] Lee#H, Jung#J, Han#S-I and Han#K-H 2010 Lab Chip 10#2764

[101] Chen#A, Byvank#T, Chang#W-J, Bharde#A, Vieira#G, Miller#B, Chalmers#J#J, Bashir#R and Sooryakumar#R 2013 Lab Chip 13#1172

[102] Rapport#E, Montana#D and Beach#G#S#D 2012 Lab Chip 12#4433

[103] Bajaj#P, Reddy#B, Millet#L, Wei#C, Zorlutuna#P, Bao#G and Bashir#R 2011 Integr. Biol. 3#897

[104] Lindstrom#S, Mori#K, Ohashi#T and Andersson-Svahn#H 2009 Electrophoresis 30#4166

[105] Ramser#K and Hanstorp#D 2010 J. Biophoton. 3#187 [106] Bhattacharya#S, Salamat#S, Morisette#D, Banada#P, Akin#D,

Liu#Y#S, Bhunia#A#K, Ladisch#M and Bashir#R 2008 Lab Chip 8#1130

[107] Jokilaakso#N et"al 2013 Lab Chip 13#336

J. Phys. D: Appl. Phys. 50 (2017) 033002

Topical Review

25

[108] Fox#M, Esveld#D, Valero#A, Luttge#R, Mastwijk#H, Bartels#P, van den Berg#A and Boom#R 2006 Anal. Bioanal. Chem. 385#474

[109] Khine#M, Ionescu-Zanetti#C, Blatz#A, Wang#L#P and Lee#L#P 2007 Lab Chip 7#457

[110] Xie#C, Lin#Z, Hanson#L, Cui#Y and Cui#B 2012 Nat. Nanotechnol. 7#185

[111] Ruan#G, Vieira#G, Henighan#T, Chen#A, Thakur#D, Sooryakumar#R and Winter#J#O 2010 Nano Lett. 10#2220

[112] Mahajan#K#D, Vieira#G, Ruan#G, Miller#B, Lustberg#M, Chalmers#J#J, Sooryakumar#R and Winter#J#O 2012 Chem. Eng. Prog. 108#41–6

[113] Zhu#J#T and Hayward#R#C 2008 J. Am. Chem. Soc. 130#7496 [114] Wilhelm#C 2008 Phy. Rev. Lett. 101#28101 [115] Robert#D, Nguyen#T#H, Gallet#F and Wilhelm#C 2010 PLoS

One 5#e10046 [116] Bausch#A#R, Moller#W and Sachmann#E 1999 Biophys. J.

76#573

J. Phys. D: Appl. Phys. 50 (2017) 033002