Microfluidic Applications of Magnetic Particles for Biological Analysis andCatalysis

Martin A. M. Gijs,* Frederic Lacharme, and Ulrike Lehmann

Laboratory of Microsystems, Ecole Polytechnique Federale de Lausanne, 1015 Lausanne EPFL, Switzerland

Received May 14, 2009

Contents

1. Introduction 15181.1. Microfluidic Systems 15181.2. Basic Characteristics of Magnetic Beads 15201.3. Scope of This Review 1521

2. Magnetic Beads 15212.1. Synthesis 15212.2. Protection and Stabilization 15222.3. Functionalization 1523

3. Forces on Magnetic Beads 15243.1. Magnetic Force 15243.2. Viscous Drag Force 15253.3. Electrical Interaction with the Microchannel

Wall1525

3.4. Other Forces 15264. Magnetic Bead Manipulation 1526

4.1. Retention and Separation 15274.1.1. Systems with Macroscopic Magnets 15274.1.2. Systems with Microscopic Magnets:

Magnetic Templates1528

4.1.3. Systems Using Microelectromagnets 15294.2. Magnetic Transport 15294.3. Magnetic Beads as Labels for Detection 15314.4. Enhancing the Bead-Flow Interaction and

Mixing1532

4.5. Magnetic Droplets 15345. Magnetic Cell Manipulation 1534

5.1. Cell Types 15345.1.1. Cells with Magnetic Character 15345.1.2. Nonmagnetic Cells Labeled with Magnetic

Beads1535

5.2. Magnetophoretic Mobility of a Cell 15365.3. Cell Separation Methods 15365.4. Magnetic Cell Separation and Purification 15375.5. Cell Biophysics 1542

6. Magnetic Nucleic Acid Assays 15436.1. DNA Capture and Purification 15436.2. DNA Processing 15446.3. DNA Detection 15456.4. Integrated DNA Analysis Systems Starting

from Cells1545

7. Magnetic Immunoassays 15477.1. Magnetic Beads as Substrate 1548

7.1.1. Direct Fluorescent Detection 1548

7.1.2. Enzyme Reaction-Based Detection 15497.2. Magnetic Beads as Label for Detection 1551

7.2.1. Surface Coverage Measurement 15517.2.2. Magnetic Properties Measurement 1552

7.3. Agglutination Tests 15538. Catalytic Applications 1554

8.1. Homogenizing Heterogeneous Catalysis UsingMagnetic Particles

1554

8.2. Transition Metal Catalysts 15548.3. Biocatalysts 15558.4. Magnetic Particle-Based Protein Digestion 1556

9. Conclusions and Outlook 155610. Acknowledgments 155711. References 1557

1. Introduction

1.1. Microfluidic SystemsSince the introduction of the concept of Micro Total

Analysis System or TAS in 1990,1 the interest in and impactof such devices, also called lab-on-a-chip or miniaturizedanalysis systems, has grown explosively. Clinical diagnosis,as practiced in blood screening laboratories, is for economicreasons dominated by high-throughput, highly reliable, androbust modular automated systems. Such systems generatea minimum of consumables per test indeed. However,microfluidics has shown to provide attractive solutions formany problems in chemical and biological analysis, espe-cially for in-field use or point-of-care testing. A detailedoverview of the various fluidic operations in microfluidicsystems, such as sample preparation, sample injection, samplemanipulation, reaction, separation, and detection, publishedin the 1990-2008 period, was presented in a series of reviewarticles by the group of A. Manz.2-4 Three of the mostimportant advantages of using fluidic systems of reduceddimension for analytical applications are (i) the possibilityof using only minute quantities of sample and reagents (downto picoliters), as problems of fluidic connectors with largedead volumes can be avoided for an integrated lab-on-a-chip, (ii) comparatively fast reaction times, when moleculardiffusion lengths are of the order of the microchanneldimension, and (iii) a large surface-to-volume ratio offeringan intrinsic compatibility between the use of microfluidicsystems and surface-based assays. The long-range nature ofviscous flows and the reduced device dimensions imply thatthe influence of boundaries or channel walls is verysignificant for a microfluidic system. A variety of strategieshave been implemented to manipulate fluids by exploiting

* To whom correspondence should be addressed. Tel.: +41 21 693 67 34.Fax: +41 21 693 59 50. E-mail: martin.gijs@epfl.ch.

Chem. Rev. 2010, 110, 151815631518

10.1021/cr9001929 2010 American Chemical SocietyPublished on Web 12/04/2009

boundary effects, among them electrokinetic effects, dropletgeneration, acoustic streaming, and fluid-structure interac-tions, topics that were also reviewed extensively.5-8

Multiple technologies for the realization of fluidic micro-systems have been developed, as shown for example in thebook of Madou9 and the review of Reyes et al.10 The basicmicrofabrication sequence of a fluidic chip usually involvesthe patterning of a microchannel structure, a bondingoperation to seal the open channel, followed by surfacecoating or functionalization steps. Glass and silicon have

been the dominating material for the realization of microf-luidic chips in the early years,11 but have been more andmore replaced by polymers. Still, borosilicate glass formsan interesting option for more demanding microreactorapplications in harsh environments, for example, duringcatalytic reactions at high temperatures. Fused silica is amaterial with low autofluorescence in the ultraviolet region,making it a preferred choice for high-resolution fluorescentdetection methods. However, microfabrication of glass9 canbe expensive due to the requirement of clean room-basedtechnologies such as deep plasma etching or hydrofluoricacid-based wet etching. This explains the increasing interestin the replication of microchannel master structures in anelastomeric material like poly dimethylsiloxane (PDMS),12,13which has become a preferred microfluidic technology forthe lab-on-a-chip research community. Also, high-throughputpolymer microfabrication techniques such as microinjectionmolding and hot embossing14 have emerged for affordableand disposable microfluidic analytical applications.

Parallel to the boom of microfluidic systems, nanomaterialsand nanoparticles have become a hot topic in research. Whenbrought into a microfluidic channel, nano- and microparticlesoffer a relatively large specific surface for chemical binding.Such particles are also called in general beads in literature,independent of what their size is. A polymer colloid ormicrosphere solution has a low viscosity as compared tosolutions having the same amount of solid, giving it specialproperties. Also, such small particles can be advantageouslyused as a mobile substrate in catalysis, for bioassays oreven for in vivo applications; they can be easily recoveredfrom a dispersion, reversibly redispersed, etc. Several reviewson the preparation and use of polymer particles and polymercolloids for medical, biological, and optical applicationsexist.15,16 Moreover, the technologies for the realization ofnano- and microparticle handling systems, with a focus onthe integration of the latter with microfluidic systems, werereviewed.17 With respect to open or empty microchannels,microfluidic structures with packed beds of functionalizedbeads or containing bead suspensions profit from an even

Martin A. M. Gijs received his degree in physics in 1981 from theKatholieke Universiteit Leuven, Belgium, and his Ph.D. degree in physicsat the same university in 1986. He joined the Philips Research Laboratoriesin Eindhoven, The Netherlands, in 1987. Subsequently, he has workedthere on micro- and nanofabrication processes of high critical temperaturesuperconducting Josephson and tunnel junctions, the microfabrication ofmicrostructures in magnetic multilayers showing the giant magnetoresis-tance effect, the design and realization of miniaturized motors for harddisk applications, and the design and realization of planar transformersfor miniaturized power applications. He joined the Ecole PolytechniqueFederale de Lausanne (EPFL) in 1997. He presently is a professor in theInstitute of Microengineering, where he is responsible for the MicrosystemsTechnology Group. His main interests are in developing technologies fornovel magnetic devices, new microfabrication technologies for Microsys-tems fabrication in general, and the development and use of microfluidicsfor biomedical applications in particular. He is on the editorial board ofMicrofluidics and Nanofluidics and the Journal of Micromechanics andMicroengineering. He has published over 160 papers in peer-reviewedjournals and holds over 20 patents.

Frederic Lacharme was born in Bordeaux, Gironde, France in 1978. Hereceived his Masters degree in Physical-Chemistry from the Universityof Bordeaux 1 Sciences and Technologies in 2002. In 2003, he joinedthe laboratory of Professor Martin Gijs at the Ecole Polytechnique Federalede Lausanne, where he received his Ph.D. degree in 2008. His researchwas focused on the development of analytical microfluidic systems usingmagnetic nanoparticles. He is currently working on the development ofmicrochips for analytical and diagnostic applications.

Ulrike Lehmann was born in Rostock, Germany in 1978. She obtainedher Dipl-Ing. degree in electrical engineering, with a specialization onMEMS, in 2003, at the Chemnitz Technical University, Chemnitz, Germany.She subsequently joined the group of Prof. Gijs in the Laboratory forMicrosystems at the Ecole Polytechnique Federale de Lausanne to workon her Ph.D. In 2008, she obtained her Ph.D. for her work on themanipulation of magnetic microparticles in liquid phases for application inbiomedical systems, which included the magnetic manipulation of dropletsas well as the direct manipulation of single magnetic microparticles. UlrikeLehmann is currently working at Microsens S.A., where she is involvedin the development of environmental sensor probes based on electro-chemical and resistive microsensors.

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larger surface-to-volume ratio, an enhanced interaction ofreactive surfaces with fluids passing by, and an improvedrecuperation of reaction products.

1.2. Basic Characteristics of Magnetic BeadsIf a magnetic material is placed in a magnetic field of

strength H, the individual atomic moments in the materialcontribute to its overall response, resulting in the magneticinduction B ) 0(H + M),where 0 is the permeability offree space, and the magnetization M ) m/V is the magneticmoment per unit volume, where m is the magnetic momentof a volume V of the material. The magnetic character of amaterial depends on its atomic structure and temperature.Materials may be conveniently classified in terms of theirvolumetric magnetic susceptibility, , where M ) Hdescribes the magnetization induced in a material by H. InSI units, is dimensionless, B is expressed in tesla (T), andboth M and H are expressed in A/m.

Magnetic beads have additional advantages beyond thosementioned for nonmagnetic particles: having embeddedmagnetic entities, they can be magnetically manipulated usingpermanent magnets or electromagnets, independent of normalmicrofluidic or biological processes. This additional degreeof freedom is the basis of a still improved exposure of thefunctionalized bead surface to the surrounding liquid, dueto the increased relative motion of the bead with respect tothe fluid. It is important to note that a magnetic field gradientis required to exert a translation force, because a uniformfield solely gives rise to a torque, but no translational action.The magnetic force acting on a point-like magnetic dipoleor magnetic moment m in a magnetic induction B can bewritten as a function of the derivative of the magneticinduction:18-23

As an example, for a constant moment m in the x-direction,leading to m ) mx(()/(x)), a force will be exerted onthe moment, provided there is a magnetic field gradient inthe x-direction. Figure 1a is a schematic diagram of aferromagnetic microparticle, which can either have a homo-geneous magnetic core or be composed of an ensemble ofprimary magnetic nanoparticles that each have a dimensions that is in the nanometer range. The magnetization measure-ment of an ensemble of such microparticles results in amagnetization loop as shown in Figure 1b, which ischaracterized by a remnant magnetization Mrem close to thesaturation magnetization Msat. Such particles will be subjectedto substantial forces when brought into a magnetic fieldgradient, but suffer from magnetic clustering and agglomera-tion effects due to the magnetic dipole interaction. Super-paramagnetic nanoparticles, as schematically shown in Figure1c, have zero magnetization in the absence of a magneticfield, as their magnetic anisotropy energy (proportional totheir magnetic volume V) is typically smaller than the thermalenergy. They therefore essentially behave as nonmagneticparticles in the absence of a magnetic field and are character-ized by a magnetization curve as shown in Figure 1d. Theslope at low fields is characterized by the magnetic suscep-tibility . The susceptibility of a particle is also calledeffective susceptibility and is related to the intrinsic suscep-tibility of the material mat by ) mat/(1 + Ndmat), with Ndthe demagnetization factor (1/3 for a spherical particle).24

In case of superparamagnetic particles in a biologicalmedium, one can describe the moment at small fields by thelinear relation m ) V0 M ) V0H, with M themagnetization of the particle and the difference inmagnetic susceptibility between the magnetic particle andthe surrounding liquid medium. Using the relation B ) 0H,eq 1 for a superparamagnetic particle in the linear suscep-tibility regime becomes

The magnetic moment of a superparamagnetic nanoparticlegenerally is smaller than that of a larger ferromagneticmicroparticle. Hence, the magnetic force on a superpara-magnetic particle will be smaller, which will result in slowermagnetic separation processes. However, the advantage ofsuperparamagnetic particles is the possibility to simplyswitch off the magnetic effects by removing the magneticinduction field.

Besides actuation of magnetic beads, the latter can alsobe used as labels for detection by a magnetic field sensor.The magnetic dipole stray field of a point-like magnetic beadis schematically shown in Figure 2 and is given by

Fm )10

(m)B (1)

Figure 1. (a) Schematic of a ferromagnetic microparticle contain-ing either a single magnetic core or a core composed of an ensembleof magnetic nanoparticles of size s. (b) Schematic magnetizationcurve of ferromagnetic particles with indication of the saturationmagnetization Msat and the remnant magnetization Mrem. (c)Schematic of a superparamagnetic nanoparticle having a singlemagnetic nanoparticle of size s as a core. (d) Schematic magnetiza-tion curve of superparamagnetic particles with indication of themagnetic susceptibility and the saturation magnetization Msat.

Figure 2. Schematic diagram of the dipole field of a magneticparticle. When a superparamagnetic particle is placed in a magneticfield H, the resulting moment m leads to a stray magnetic inductionat each position vector r on the sensor surface, given by eq 3.

Fm )V0

(B)B (2)

B(r) ) 14[-mr3 + 3(mr)rr5 ] (3)

1520 Chemical Reviews, 2010, Vol. 110, No. 3 Gijs et al.

Typically, the sensor output will be proportional to the totalmagnetic induction integrated over the sensor area. Anotherconsequence of eq 3 is that, when a magnetic bead is in themagnetic induction field of other beads, the particles willinteract via the magnetic dipole interaction, and chain-likeor more complicated magnetic supraparticle structures (SPS)can be formed.

1.3. Scope of This ReviewPankhurst et al.,25 Gijs,26 and Pamme27 reviewed the

applications of magnetic beads, with focus on the underlyingphysics25 and microfluidic aspects.26,27 Here, we intend topresent a comprehensive overview of recent research on themanipulation of magnetic beads in microfluidic systems andtheir utilization for biological analysis and chemical catalysis.We start with a discussion on the production of various typesof magnetic beads and their chemical/biological functional-ization. We then discuss the main forces that act on magneticparticles, that is, magnetic forces that provide the primarymeans of actuation, viscous drag forces in the liquid thatoppose the magnetically induced bead motion, and electro-static forces that can be responsible for agglomeration orsticking of the particles to microchannel walls. Thereafterwe discuss the basic manipulation steps of magnetic particles:retention, separation, mixing, and transport. We also discussthe use of beads as magnetic detection labels, or containedas actuators/substrates in discrete droplets for digital mi-crofluidics. We then focus on the applications of magneticbeads: the handling, detection, and separation of biologicalcells, on-chip nucleic acid assays, and immunoassays.Finally, the promising application area of magnetic micro-and nanoparticles for chemical catalysis is discussed. Through-out this Review, we try to compare the use of magnetic beadsin microfluidic systems with alternative solutions and pointout the advantages. We end this Review with an outlooksection on possible future developments.

2. Magnetic BeadsMagnetic bead suspensions or magnetic fluids are stable

dispersions of magnetic beads or encapsulated magneticnanoparticles in an organic or aqueous carrier medium.Requirements of the bead matrix such as biocompatibility,biodegradability, and stability in different media must becombined with a preferable uniform size distribution and acorrect shape. The magnetic material content determines themagnetic behavior of the beads and must be associated withsuitable measures of protection against corrosion. Forexample, the primary magnetic nanoparticles (schematicallyshown as the black dots on the left of Figure 1a and in Figure1c) can be coated with polymers or surfactants, with silicaor carbon, or can be embedded in a chemically inertprotective matrix. Moreover, for use in specific targetapplications, the bead surface needs to be functionalized(hydrophilicity versus hydrophobicity, chemical functionalityat the surface, etc.) to allow covalent bonding or simpleaspecific adsorption of biomolecules (proteins, antibodies,nucleic acids) or cells. Many types of magnetic beads arecommercially available nowadays and are usually tailor-madefor a specific final application. The synthesis, protection, andfunctionalization of magnetic nanoparticles were reviewedby Lu et al.28 and Horak et al.29 Also, the articles ofLandfester and Ramrez,30 Bergemann et al.,31 Gruttner etal.,32,33 and Latham et al.34 present specific short review

sections on the synthesis and chemical modifications ofmagnetic beads. Therefore, we only provide representativeexamples of bead synthesis, protection, and functionalizationin this Review, rather than covering this vast field of literaturein a comprehensive way.

2.1. SynthesisReviews of the synthesis of inorganic nanoparticles in the

liquid phase (not limited to magnetic materials) werepresented by Grieve et al.,35 Trindade et al.,36 Murray et al.,37and Lu et al.,28 while a review of the synthesis of suchparticles from the vapor phase was presented by Swihart.38The synthesis and applications of (nonmagnetic) polymermicoparticles were reviewed by Kawaguchi.15 The synthesisof magnetic beads is also a well-covered subject in patentliterature. Magnetic nanoparticles have been synthesized witha number of different compositions that include iron oxides,such as magnetite (Fe3O4) and maghemite (-Fe2O3),39,40 puremetals,41,42 such as Fe and Co, or alloys,43,44 such as CoPt3and FePt. Among the practiced magnetic nanoparticlesynthesis methods, coprecipitation, thermal decomposition,microemulsion, and hydrothermal technologies are mentionedhere. To date, magnetic nanoparticles prepared from copre-cipitation and thermal decomposition are the most widelystudied. Synthesis in microfluidic chips is an alternativetechnique for very controlled realization of magneticparticles.45-47

Coprecipitation

In early publications, magnetic bead solutions (ferroflu-ids) were produced by grinding magnetite with long-chainhydrocarbons and a grinding agent.48 Later, magnetic fluidswere produced by precipitating an aqueous Fe3+/Fe2+ solutionwith a base, coating these particles with an adsorbed layerof oleic acid, and then dispersing them in a nonaqueousfluid.49 Both types of processes result in very small magneticparticles with a surfactant coating in a nonaqueous liquidcarrier, in which the hydrophobic magnetite particles aredispersed. However, a lot of applications of magnetic beadsrely on water as the continuous phase. A water-basedmagnetic fluid was realized by conversion of iron productsto magnetic iron oxide in an aqueous medium undercontrolled pH conditions.50 The experimental challenge inthe synthesis of Fe3O4 by coprecipitation is in the control ofthe particle size and the achievement of a narrow particlesize distribution. To produce monodisperse iron oxidemagnetic nanoparticles in a coprecipitation reaction, a processwith a short burst of nucleation followed by a slow controlledgrowth is necessary.

Thermal Decomposition

Monodisperse magnetic nanoparticles can be synthesizedthrough the thermal decomposition of organometallic com-pounds in boiling organic solvents that contain stabilizingsurfactants.51 The ratios of the starting reagents, includingthe organometallic compounds, surfactant, and solvent, are,together with the reaction temperature and reaction time, themain parameters for controlling the nanoparticle synthesis.For example, nearly monodisperse iron oxide crystals withsizes adjustable over a wide size range (3-50 nm) have beenprepared.52,53 The reaction system was composed of iron fattyacid salts, fatty acids, hydrocarbon solvents, and activation

Microfluidic Applications of Magnetic Particles Chemical Reviews, 2010, Vol. 110, No. 3 1521

reagents. The thermal-decomposition method can also beused to prepare metallic nanoparticles. A metallic ferromag-net has a larger magnetization as compared to a magneticoxide; therefore, the magnetic force (eq 1) on such particleswill be larger. As an example, the reaction at 150 C of themetal-organic precursor Fe[N(SiMe3)2]2 with H2 in thepresence of a long-chain acid and a long-chain amineproduces monodisperse cubic nanoparticles that have edgesof 7 nm and are incorporated into extended superlattices54(see Figure 3a).

Microemulsion Techniques

A microemulsion is a stable dispersion of two immiscibleliquids in which small-size droplets are stabilized by aninterfacial film of surfactant molecules. In water-in-oilmicroemulsions, the aqueous phase forms droplets (1-50nm diameter) in a continuous hydrocarbon phase. By mixingtwo identical water-in-oil emulsions containing the desiredreactants, the droplets will collide, coalesce, and split andinduce the formation of precipitates. Adding a solvent likeethanol to the microemulsion allows extraction of theprecipitate by filtering or centrifuging the mixture. Also, thepreparation of magnetizable polymer particles from aqueousdispersions is known. The particles can be prepared bydispersing magnetic elements in an organic phase containingan organosoluble initiator and/or monomer(s), mixing thedispersion with an aqueous solution made of water andemulsifier, homogenizing the mixture to give droplets oforganic phase with (sub)micrometer size, and finally poly-merizing the homogenized mixture after the addition ofmonomer(s), if necessary.55 The distribution of the droplets(and hence of the polymer particles) is a function of theproportion of emulsifier present in the aqueous solution, andthe ratio of the organic to the aqueous phase. The sizedistribution of the polymer particles obtained by this processis generally wide. By applying a purification process to theinitial polydisperse crude emulsion, it is possible to obtain a

set of highly monodisperse samples with high magneticcontent (up to 70%).56 The purification method is analogousin principle to a fractionated crystallization process.33,56,57

Hydrothermal Synthesis

Hydrothermal reactions with mixed oxides or hydroxidesof iron and other metals can be carried out in water undersupercritical conditions, that is, at temperatures around orhigher than 200 C under a pressure higher than 14 MPa.Under these conditions, water plays the role of a hydrolyticreactant. The size and morphology of the reaction productsare controlled by the reaction time and temperature. How-ever, hydrothermal reaction procedures are experimentallydemanding.

Synthesis in Microfluidic Chips

Microfluidic devices provide an alternative for the con-trolled generation of monodisperse emulsion droplets bycoflowing two immiscible fluids to induce droplet formation.The size of the monodiperse emulsion droplets can becontrolled by tuning the relative flow rates of the two fluids.In these devices, spherical emulsion droplets are usuallygenerated because the disperse phase seeks to minimize itsinterfacial free energy.45,46 However, also nonsphericalhydrogel microparticles with embedded magnetic nanopar-ticles have been synthesized using ultraviolet-initiated pho-topolymerization.47

2.2. Protection and StabilizationMaintaining the stability of magnetic beads for a long time

without agglomeration or precipitation problems is a pre-requisite for applications. Especially pure metallic particlesare subjected to oxidation or degradation, but also magnetitenanoparticles are not very stable under ambient conditions,and can be easily oxidized to maghemite or dissolved in anacid medium. Particle protection results in magnetic beadshaving a core-shell structure, where the role of the shell isto protect the magnetic core against environmental influence.Also, some particle compounds, in particular cobalt, areconsidered extremely toxic, and thus the leakage of this intothe liquid medium must be avoided. Several coating strategiesexist, ranging from coating the magnetic nanoparticles withorganic shells containing surfactants and polymers, withinorganic compounds like silica, with carbon, to coatingwith precious metals. Besides simply coating individualmagnetic nanoparticles, it is also possible to embed a numberof magnetic nanoparticles or magnetic material in a polymeror silica matrix to form composites. This way, one cansynthesize microbeads that, due to a higher magnetic content,are more easy and rapid to manipulate magnetically than theprimary magnetic nanoparticles. Such types of beads (sizearound 1 m) are therefore often used in microfluidicsystems.

Surfactant and Polymer Coating

To stabilize magnetic nanoparticles after synthesis, elec-trostatic or steric repulsion can be used to keep the nano-particles dispersed in a nonagglomerated colloidal state. Forthe so-called ferrofluids,48 the stability results from a controlof surface charges and the use of specific surfactants; bothwater- and oil-based ferrofluids are available. Surfactants orpolymers can be chemically linked to or physically adsorbed

Figure 3. (a) Three-dimensional superlattice of iron nanocubesas deposited on a carbon-coated copper grid from a toluenedispersion. Bar: 10 nm. (b) TEM photograph of dextran nanopar-ticles; the dextran coating is not visible in the TEM picture, andthe particles have an irregular shape. (c) More regular polystyreneparticles with encapsulated magnetite nanoparticles. (d) SEMphotograph of irregular magnetic glass particles [obtained from theMagNA Pure LC DNA isolation Kit from Roche Diagnostics(Rotkreuz, Switzerland)]. (a) Reprinted with permission from ref54. Copyright 2004 American Association for the Advancementof Science. (b) Reprinted with permission from ref 33. Copyright1999 Elsevier. (c) Reprinted with permission from ref 30. Copyright2003 The Institute of Physics Publishing Ltd.

1522 Chemical Reviews, 2010, Vol. 110, No. 3 Gijs et al.

to the magnetic nanoparticles, creating repulsive forces (dueto steric hindrance) that balance the attractive magnetic,electrostatic, and van der Waals force. Polymers containingfunctional groups, such as carboxylic acids, phosphates, andsulfates, can bind to the surface of iron oxide,58 while surface-modified magnetic nanoparticles with certain biocompatiblepolymers are intensively studied for magnetic-field-directeddrug targeting59 and as contrast agents for magnetic resonanceimaging.60,61 For metallic magnetic nanoparticles, a thinpolymer coating may not be sufficient to prevent oxidationof the highly reactive metal particles, while, more generally,such coating will also be less stable at higher temperatures.

Precious-Metal and Carbon Coating

Several precious-metal coating procedures have beenproposed. Gold seems to be an ideal coating due to its lowreactivity. Coating magnetic particles directly with gold isdifficult due to the dissimilar nature of the two surfaces.However, gold-coated iron nanoparticles with 11 nm coresize and 2.5 nm gold shell thickness have been prepared bya partial replacement reaction in a polar aprotic solvent.62Gold-coated iron nanoparticles were also prepared by areverse microemulsion method.63 Coating with gold isinteresting, because the gold surface can be easily chemicallyfunctionalized with ligands via thiol groups.

Carbon-based coatings can be advantageous over polymersor silica, due to their higher chemical and thermal stability,as well as their biocompatibility. For example, a sonochemi-cal coating procedure was developed that leads to air-stablecobalt nanoparticles.64

Silica Coating

Silica coatings are attractive due to their relatively goodstability under aqueous conditions, their easy surface modi-fication, and control of interparticle interactions via thevariation of shell thickness. The Stober process65 and othersol-gel techniques66,67 are the preferred methods for coatingthe magnetic nanoparticles with silica. The coating thicknesscan be varied by tuning the concentration of ammonium andthe ratio of tetraethoxysilane (TEOS) to water. Silica-coatedmagnetic nanoparticles are hydrophilic and can be easilyfunctionalized with several groups.68 On pure metal nano-particles, direct silica deposition is complicated because ofthe lack of hydroxyl groups on the metal surface. Anotherproblem can be the oxidation of a metal like iron or cobaltin the presence of dissolved oxygen in the reaction medium.Although silica coatings are in general robust, silica isunstable under basic conditions and in addition may containpores through which oxygen can diffuse.

Embedding Nanoparticles in a Protective Matrix

Matrix-dispersed magnetic nanoparticles can be createdin different ways. The nanoparticles can be homogeneouslydispersed in a continuous matrix, they can be dispersed as acoating on other larger particles, or they can form agglomer-ates that are connected through their protective shells. Thepreparation of magnetic polymer microbeads was reviewedin detail by Horak et al.29 For example, polymer-coatedmagnetic beads can be produced by in situ precipitation ofmagnetic materials in the presence of a polymer. In this way,single or multiple magnetic core beads surrounded by ahydrophilic polymer shell have been made, choosing for the

polymer the water-soluble dextran,69 poly-(ethylene imine),70poly-(vinyl alcohol),71 poly-(ethylene glycol),72 etc. The well-known Dynabeads73 are magnetic monodisperse microbeadsdeveloped in a multistep procedure. They contain iron oxidesthat are formed in situ by precipitation in preformed porousand monodisperse polymer microspheres, followed by acoating step.74,75 Such types of polystyrene-coated magneticparticles are known for their excellent size distribution andspherical shape.73,75 However, their hydrophobic surfaceresults in a high amount of aspecific protein and antibodyadsorption on the particle surface, so that it often needs tobe modified chemically. Magnetic silica particles are veryefficient in adsorbing proteins and DNA on their surface,but are hardly available with a small size distribution andan ideal spherical shape.76,77 Magnetic polysaccharide par-ticles are important for many in vivo applications. Theycombine biocompatibility with availability in a size rangebelow 300 nm,33,78 but the particles are irregular in shape,and the soft particle matrix causes them to be sensitive tomechanical stress. Magnetic poly-(lactic acid) particles alsoplay an important role in the in vivo applications:79,80 theyare biodegradable, and their degradation time in the bloodcan be adjusted by their molecular weight and exact chemicalcomposition. However, because of their hydrophobic sur-faces, these particles stick to plastic surfaces found inmicrofluidic systems, resulting in problems with particlehandling and analytical errors. In other work, nonsphericalhydrogel microparticles with embedded magnetic nanopar-ticles have been synthesized in microfluidic chips usingultraviolet-initiated photopolymerisation.47 As an exampleof matrix-dispersed magnetic nanoparticles, Figure 3b is atransmission electron microscopy (TEM) micrograph ofdextran nanoparticles; note that the dextran is not visible inthe TEM picture and that the particles have an irregular(nonspherical) shape. Figure 3c is a TEM photograph of moreregular polystyrene particles with encapsulated magnetitenanoparticles. Figure 3d is a scanning electron microscopy(SEM) micrograph of irregular magnetic glass microparticles.

2.3. FunctionalizationAs was already indicated in the previous section, a

protective shell around a magnetic nanoparticle or a protec-tive matrix not only protects the particle against degradation,but can also be used to functionalize the bead surface withspecific molecules, like those used in catalytic applications,or for use in experiments with proteins, cells, etc. Proteinsmay bind/adsorb to hydrophobic surfaces, such as thosefound in polymer-coated beads, forming a monolayer thatis resistant to washing. It may also be desirable to have astrong covalent binding between the particle surface and theprotein. This is achievable through specific groups at theparticle surface (-COOH-NH2, -CONH2, -OH groups),which via an activating reagent bind to -NH2 or -SH groupson the proteins.81 Also, streptavidin, biotin, histidine, proteinA, protein G, etc., can be grafted onto the bead surface forspecific biorecognition reactions.29 Small antibody-bindingpeptides have also been proposed for surface functionaliza-tion.82 Silica-coated beads can be used as-prepared to recoverand purify the total DNA content of a lysed cell solution.Sample DNA is for example bound to the silica surface afterchemical cell lysis in a guanidine thiocyanate binding buffer.It binds preferentially to the silica surface of the magneticbeads due to the presence of chaotropic salts like guanidineor sodium iodine in the buffer solution.83

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In many catalytic applications, transition metals are usedas catalyst to transform or bond reagents. The good catalyticactivity of these elements is due to their ability to havevarious oxidation states helping electron transfers or, in thecase of metals, to adsorb other substances onto their surfaceand activate them in the process. When used with magneticbeads, different strategies are developed to link the metalatoms to the bead surface. The metal can be either complexedusing ligands, chemically bonded to the surface, or directlyimmobilized on or incorporated into the magnetic beadsurface using a supporting matrix (see section 8.2).

3. Forces on Magnetic Beads

3.1. Magnetic ForceThe magnetic force (eq 1 or 2) is responsible for the unique

possibilities offered by magnetic beads. Particularly interest-ing are small magnetic monodomain nanoparticles, becausethey do not express hysteretic magnetization curves and havezero remnance. Leslie-Pelecky and Rieke84 have reviewedthe relation between the morphology of nanostructuredmaterials and their magnetic properties. Magnetic particlesare single-domain, when they have a dimension that istypically of the order or smaller than the thickness of amagnetic domain wall ) (JS22/Ka)1/2, with J the magneticexchange constant, S the total spin quantum number of eachatom, a the interatomic spacing, and K the magneticanisotropy constant of the magnetic material.19 For iron,assuming that S ) 1, and taking J ) 2.16 10-21 J, a )2.86 10-10 m, and K ) 4.2 104 J/m3, one calculates adomain wall width of 42 nm. The time over which themagnetization of a single-domain particle is stable and willremain in a certain state is of importance for probing themoment and magnetization. The relaxation time of themagnetic moment of a particle can be written as25

with 0 10-9s, Keff the effective anisotropy constant ofthe particle, kB the Boltzmann constant, and kBT the thermalenergy. Equation 4 shows that monodomain magneticparticles become superparamagnetic, that is, their time-averaged magnetization without a magnetic field is zero, overa typically supposed experimental time scale ) 100 s, whentheir magnetic energy Keff (4/3)r3 is lower than about20 times the thermal energy kBT, with r the particle radiusof a supposed spherical particle.84 When the particle magneticmoment does not reverse its magnetic moment over theexperimental time scale, such a particle is in the blockedstate. The temperature that separates the two regimes is theblocking temperature and very sensitively depends on theparticle size. For example, at room temperature, one finds amaximum radius r ) 6 nm for a superparamagnetic sphericalparticle of iron. In the superparamagnetic state, such ananoparticle behaves similar to a paramagnetic spin but witha much larger moment. The induced magnetization of anensemble of such particles is described by the Langevinfunction, which at low fields (mH , kBT) behaves as mH/kBT, and at high fields (mH . kBT), as (1 - (kBT)/(mH)),with m the size of the individual particle moment.19

An additional advantage of using small nanoparticles,besides the possibility to easily switch off the magnetic state

and particle interactions by removing the external magneticinduction, is the minimum disturbance such a particle hason reactions, implying the biomolecules attached to theparticle85 and the large surface-to-volume ratio, which is ofhigh interest for chemical binding. A disadvantage, however,of such a particle may be that the magnetic force is smalldue to the small volume, so that viscous forces dominateand magnetic separations can take a long time (tens ofminutes).

This explains the interest of using larger magnetic particles(typically 0.2-5 m in diameter) in microfluidic applications.These can either have a single magnetic core or have a corecomposed of multiple (non)interacting nanoparticles in anonmagnetic matrix (see Figure 1a). Such microparticlesoften have a multidomain structure and are characterized bya hysteretic magnetization characteristic (see Figure 1b). Forexample, spherical particles with radius r ) 1.5 m, asaturation magnetization 0Msat ) 0.2 T, and a remnantmagnetization 0Mrem ) 1 mT will have the remnantmagnetic moment size mrem ) V0Mrem ) (4/3)r30Mrem )1.4 10-20 T m3. One notes here that the magnetic momentof a magnetic bead not only depends on its volume or size,but also on its magnetic load, that is, the kind and amount(up to 70%) of magnetic material that is present in theparticle. Two of such particles will have the maximummagnetic attraction energy |Umax| ) (mrem2/20)(1/(2r)3) )9.2 10-19 J, much larger than the thermal energy of 4 10-21 J at room temperature, resulting in strong magneticdipolar forces between the particles.19 As a consequence,when exposed to an external magnetic induction, suchmagnetic microparticles coalesce under influence of themagnetic dipole interaction into a SPS consisting of chain-like columnar structures along the field direction. The exactshape of this SPS depends on parameters such as the particleconcentration and applied magnetic field. Even after removalof the external magnetic induction, such particle structurescan stay agglomerated, hindering separation and recoveryof the magnetic beads.

While the magnetic character of a magnetic bead isessential for delivering the magnetic force, it is interestingto note that also diamagnetic objects brought in solutions ofparamagnetic ions can be displaced on the basis of theirrepulsive force from a high magnetic field.86,87 The magneticforce on a magnetic bead can be induced using a magneticfield generated either by permanent magnets or by coils. Apermanent magnet typically is characterized by a magneticinduction Bm ) 0.5-1 T and a field gradient B Bm/w,with w the typical geometrical dimension of the permanentmagnet.88 For a cylindrical permanent magnet with a diameter ) 5 mm, one induces on a spherical particle with radiusr ) 500 nm and ) 1 a magnetic moment m ) 2.6 10-19 T m3, resulting in a magnetic force of about 40 pN.For a current-fed coil, the generated field is much smaller:a flat millimeter-size coil with 10 windings and a current of0.5-1 A generates typically a magnetic induction of 1-10mT, at least 100 times smaller than the permanent magnet.Consequently, the gradient is also a factor 100 lower, so thatthe force of eq 2 is a factor 104 larger, when using apermanent magnet rather than a coil. On the other hand, acoil offers more flexibility in switching the field on and offby simply controlling the current (and heat dissipation!),whereas a permanent magnet requires a mechanical actionto move it away from the microfluidic system containingthe magnetic beads.

) 0 exp(KeffVkBT ) (4)

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3.2. Viscous Drag ForceIn many applications, a magnetically labeled material is

separated from a liquid solution by passing the fluid mixturethrough a region with a magnetic field gradient that canimmobilize the tagged material via magnetic forces. For invivo applications, magnetic particles can be transported bythe blood flow and locally retained with the help of anexternal magnet. In other applications, there is a magnetictranslational driving force, and the liquid solution is static.In the equilibrium state, the magnetic force Fm is opposedto the hydrodynamic drag force Fd acting on the magneticparticle. The hydrodynamic drag force is shown in Figure 4and is a consequence of the velocity difference between themagnetic particle and the liquid v and, for a sphericalparticle with radius r, is given by89

where is the viscosity of the medium surrounding theparticle (for water, ) 8.9 10 -4 N s/m2). fD is the dragcoefficient of the particle and incorporates the influence ofa solid wall in the vicinity of the moving particle and iscalculated as90,91

Here, z is the distance of the particle to the solid wall; in thelimit of small z, fD 3, so that the drag will be a factor 3higher than when no solid wall is in the vicinity. In amicrofluidic system, a particle may be moving at different zfrom the microchannel wall, under influence of the appliedmagnetic forces, so that the viscous force of eq 5 will varyalong the particle trajectory.

Equalizing eqs 2 and 5 permits one to determine themaximum flow rate a particle can withstand when exposedto a magnetic immobilization force, or the maximum particleflow rate that can be generated by a magnetic force in asurrounding static liquid:

with

the magnetophoretic mobility of the particle, a parameterdescribing how magnetically manipulable the particle is. Thequantity (B )B can be a strongly varying function in space,which implies a similar spatial variation for v.

3.3. Electrical Interaction with the MicrochannelWall

Both electrostatic and electrodynamic (van der Waals)forces act on a particle in solution. The van der Waals forceoriginates from attractive electromagnetic interactions be-tween permanent electrical dipoles and/or induced dipoles.The van der Waals force between a particle and a substrateor the wall of a fluidic microchannel is expressed in theHamaker approach as91,92

with A123 the Hamaker constant of particle of material 1 ona substrate of material 2 in a medium of material 3, and ezthe unit vector in the vertical direction; ret is a characteristiclength scale of the interaction, often assumed to be 100 nm.For example, one finds for an acrylate-coated particle closeto a SiO2 substrate in water a value of A123 ) 3.4 10-21J.91,93

When immersed in an ion-containing aqueous solution,the substrate and particle acquire a surface charge throughthe adsorption of ions present in the solution and/or thepresence of charged surface groups. This surface charge isneutralized by mobile ions of opposite charge also presentin the solution, thus forming the well-known double layer.When the double layers of two surfaces overlap, an elec-trostatic interaction occurs, resulting in either a repulsive oran attractive force. This electrical force Fel is given, for aconstant surface charge density on substrate and particle, by94

with the permittivity of the medium (7 10-10 F/m forwater), and s and p the zeta-potential of the substrate andparticle, respectively.95 The electrical force is acting over aspatial range given by the Debye-Huckel double layerthickness -1:

with NA Avogardos number, and Ic the ionic strength inmol/L of the solution. -1 is typically in the 10 nm range.Wirix-Speetjens et al.91 showed how, by changing the pHand ionic strength of the solution, it is possible to obtainrepulsive electrical force conditions between the particle andthe substrate surface, considering combined van der Waalsand electrostatic forces. This is important, if one wants toavoid the issue of unwanted particle sticking to the micro-fluidic channel. Higher pH values (6-10) and low ionicstrength (1-10 mM) are shown to favor repulsive electrical

Figure 4. Simplified schematic diagram of the main directions ofthe most important forces acting on a magnetic particle with radiusr and positioned at a distance z from a surface. The particle is ina fluid and is subjected to a magnetic force Fm, a viscous dragforce Fd, a gravitational force Fg, and an electrical force Feloriginating from the surface.

Fd ) 6rvfD (5)

fD ) [1 - 916( rr + z) + 18( rr + z)3 - 45256( rr + z)4 -1

16( rr + z)5]-1 (6)

v ) 2r2(B)B90fD

1

0fD

(B)B (7)

2r29 )

V6r (8)

FVdW ) -A123r

6z2 [ 11 + 14(z/ret)]ez (9)

Fel )2r

1 - exp(-2z)[2sp exp(-z) +(s2 + p2) exp(-2z)]ez (10)

-1 ) kBT2000NAe2Ic (11)

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forces for the acrylate-coated particle/SiO2 substrate system.91Alternatively, at the cost of more complex technology, it ispossible to coat the microchannel wall with proteins orpolymers to avoid sticking of beads or molecules.96

3.4. Other ForcesIn addition to the magnetic, viscous, and electrical forces

on magnetic beads, several other forces exist. As indicatedin Figure 4, a gravitational force acts on a magnetic beadand is given by

with g the gravitation constant, and mb ) V(F - Ffl) thebuoyant mass that represents the effective mass of themagnetic bead by taking into account the average densityof the bead F and the surrounding fluid Ffl. For example, 1.0m diameter MyOne Dynabeads73 have a volume of 0.52m3, 26% iron content, and a density of 1.8 g/cm3, resultingin a gravitational force of 0.004 pN, which is much smallerthan the typical magnetic force generated by a permanentmagnet, but of the order of the force generated by a simplecoil. When only gravitation is taken into account, thesemagnetic beads will sink to the bottom of the microchannel,hindered by a viscous force (eq 5) in the z-direction, resultingin a sinking speed of 0.5 m/s. As the ratio of gravitationalto viscous forces is proportional to r2, smaller particles willsink much slower.

When the size of the magnetic bead is in the submicrome-ter range, stochastic Brownian forces become relatively moreimportant, as the magnetic (and gravitational) forces, beingproportional to r3, are fastly decreasing. In a long-timedynamics, the friction coefficient 6r of eq 5 is related tothe diffusion coefficient D of the particle by the well-knownStokes-Einstein relation:97

For example, a MyOne Dynabead has a diffusion coefficientin water of 4.8 10-13 m2/s at room temperature, resultingin the time-dependent diffusion length (Dt); for example,after 1 s of diffusion, the particle will have moved an averagedistance of 0.7 m. Taking a magnetic nanoparticle withradius of 100 nm instead, the diffusion coefficient becomes2.4 10-12 m2/s, resulting in a more than 2 times increaseddiffusion. Analysis by optical microscopy of the Brownianmotion of magnetic nanoparticles in a magnetic field gradientwas used to characterize the basic magnetic parameters ofthe particles.98,99 A rotating magnetic field was used to applya controlled torque on superparamagnetic beads, leading toa tunable bead rotation frequency in fluid, as observed viaoptical microscopy.100 It is suggested that control of torqueand measurement of the rotation will enable future torsion-based protein assays as well as nucleic acid assays on a singlebead.

A combination of magnetic and dielectrophoretic forcesto discriminate specific and nonspecific molecular bindingsfor on-chip magnetic bioassays was reported.101 Conjugatedto the analytes, magnetic particles are used as the agents fordielectrophoretic force generation. Because of a weakerbinding strength, the nonspecifically bound particles areremoved, while specific bindings remained intact. Thistechnique can not only be used to improve the specificity of

on-chip bioassays, but also be developed as a tool of forcespectroscopy for the study of biomolecular binding physics.

Additional forces act on magnetic beads, such as hydro-dynamic, magnetic, and electrostatic interaction forces orig-inating from other beads,102 especially when bead concen-trations are high and the particles come close to each other.Magnetic interactions between beads lead to the formationof bead clusters or SPS, which have their own dynamics, aswell as magnetic and viscous forces.98,103,104 Finally, todescribe the motion of a magnetic bead, one should solveNewtons law involving all forces present, which becomesextremely complex and leads to cumbersome numericalprocedures. Therefore, a practical approach is to consideronly the most important forces (for example, the magneticand viscous forces for magnetic microparticles), whendesigning magnetic bead manipulation systems.

4. Magnetic Bead ManipulationThe spatial and temporal combination of the forces

introduced in the previous section allows designing proce-dures or manipulation protocols, which are at the basis ofthe applications of magnetic beads in bioanalysis or catalysis.Figure 5 shows diagrams representing basic manipulationsof magnetic beads in microfluidic systems. In separation(Figure 5a), magnetic beads are retained from a flow byfocusing a magnetic field over the channel using, electro-magnets, coils, or permanent magnets. Also, systems withminiaturized soft magnetic microstructures exist, which canbe coupled to a macroscopic electromagnet or placed in themagnetic induction field of a macroscopic permanent magnet.Magnetic transport (Figure 5b) is more difficult, as it requiresstronger and long-range magnetic forces to move themagnetic beads within the liquid, without the need of amicrofluidic flow. The use of magnetic beads as labels fordetection is shown in Figure 5c: here, a magnetic bead isbound to the surface of the microchannel, and a magneticfield sensor monitors its stray field, when the particle isplaced in an external magnetic induction. A particularlyinteresting property of magnetic beads is that they can bemagnetically suspended in a microfluidic channel usingmagnetic forces, without the requirement of having asupporting substrate. Such trapping of beads or SPS can beadvantageous, if one wants to have a high exposure of thebeads to a liquid flow. Also, when locally alternating

Figure 5. Schematics of basic manipulations of magnetic beadsin a microfluidic channel. (a) Separation of magnetic beads from aflow by actuation of electromagnets or positioning of magnets. (b)Transport of magnetic beads using long-range magnetic forcesprovided by an electromagnet or magnet. (c) Detection of the strayfield of a bead by a magnetic field sensor, after specific binding ofthe labeled bead on the sensor surface. (d) Mixing of two laminarstreams by dynamic agitation of a supraparticle structure using alocally applied alternating magnetic field between two soft magnetictips.

Fg ) -mbgez (12)

D )kBT

6r (13)

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magnetic fields are applied, for example, via soft magneticfield focusing structures, a dynamic agitation of the magneticbeads is possible that can be used to mix the essentiallylaminar flow patterns within a microfluidic channel (Figure5d).

4.1. Retention and Separation4.1.1. Systems with Macroscopic Magnets

Separation of magnetic bead-labeled biomolecules or cellsfrom a liquid solution is a well-documented and a widelypracticed application today. Many types of magnetic particleshave been developed for use in separation processes,including purification processes and immuno-assays.105-109Magnetic separator design can be as simple as the applicationand removal of a permanent magnet to the wall of a testtube to cause particle aggregation, followed by removal ofthe supernatant. However, it is preferable to increase theseparator efficiency by producing regions with a highmagnetic field gradient to capture the magnetic beads as theyfloat or flow by in the carrier medium. Magnetic cell sorting(MACS) is a commonly used magnetic cell sorting techno-logy,78,110 in which cells bound to superparamagnetic particlesare retained in a high-gradient magnetic field, generated byplacing a porous magnetic column in the field of externalmagnets. After flowing the sample solution through thecolumn, the latter is removed from the separator, and theretained particles can be analyzed using flow cytometry ormicroscopy. For such application, it is necessary to looselypack a flow column with a stationary magnetizable matrixof thin wires or beads.111,112 Such approach has the drawbackthat close monitoring of the separation process is difficultdue to the nature of the filter geometry. Continuous flowseparation in microfluidic devices was reviewed by Pamme(not restricted to magnetic beads).113 Considering magneticbeads, separation in a microfluidic capillary without magneticstationary phase has been demonstrated.114 In this separationtechnique, also known under the name magnetic field-flow

fractionation,115 a magnetic force acts on the magnetic beads,forcing them toward the capillary wall. The magneticretention force is opposed by diffusion back into the flowstream, so that a steady-state particle distribution and anensemble of beads of specific height is formed at the capillarysurface. Hydrodynamic forces act perpendicular to theapplied field and laterally displace the magnetic plugs alongthe capillary wall. Less-magnetic species will be less closelypacked, extend more to the center of the capillar, and willtherefore experience larger hydrodynamic forces. Hence, less-magnetic species will move faster and elute first from thecapillary.

Continuous flow magnetic particle and cell separationsystems have been intensively studied by groups at The OhioState University and The Cleveland Clinic Foundation.116These systems have a macroscopic dimension from themagnet point of view, but, from a liquid transport point ofview, they may be called microfluidic, as their functioningis based on the presence of laminar flow patterns. Figure 6ais a schematic diagram of a so-called quadrupole magnetconfiguration, where four magnets are arranged to induce amaximum magnetic field gradient toward the outer side of aliquid carrying tube, inserted in the free space between themagnets.117 Williams et al. described how such a quadrupolemagnet can be combined with an annular fluidic circuit.118The separation takes place within a laminar flow of the carrierfluid along a thin annular channel. A magnetic field gradientis imposed across the thin dimension of the channel,perpendicular to the direction of the flow. The samplemixture is arranged to enter the system close to one of thechannel walls, and, as the sample is carried along the channelby the flow of the fluid, those components that interact morestrongly with the field gradient are carried transverse acrossthe channel thickness. A division of the flow at the channeloutlet using a stream splitter completes the separation intotwo fractions. The radial particle separation velocity isinduced by the field gradient and can be calculated using eq7. The technique was given the name split-flow thin

Figure 6. (a) Schematic diagram of a magnetic quadrupole separator. The magnetic force increases linearly in the radial direction. Amagnetic particle suspension is injected from the top in the inner annular flow channel, while a buffer solution flows within the outerannular flow channel. As the magnetic particle suspension flows through the separator, the magnetic particles are deflected in the radialdirection. If sufficiently deflected, the particles are caught in the lower outer annular flow channel. Undeflected or weakly deflected particlesfollow the inlet streamlines and are collected in the inner annular waste flow channel. (b) Schematic diagram of a magnetic dipole separator.A magnetic force directed to the right is created, and a magnetic particle suspension injected in the bottom port migrates to the right.Different types of magnetic particles will be deflected into different outlet ports. (c) Distribution of the magnetically induced velocity andmagnetophoretic mobility of four different types of magnetic beads in a magnetic dipole separator. (c) Reprinted with permission from ref124. Copyright 2001 Springer.

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(SPLITT) fractionation, a derivative of field flow fraction-ation, as separation is obtained within a mobile phase withoutthe use of a stationary phase.119,120 This separation isessentially a binary one, in the sense that the input samplestream is split into a first exit stream of highly magnetic anda second stream of less or nonmagnetic particles. Besidesannular liquid flow channels, SPLITT fractionation deviceswith rectangular geometry121,122 and a magnetic field-flowfractionation device with helical microfluidic channel placedin a quadrupole magnetic field123 have been proposed.

Permanent magnets arranged in a dipole configuration havebeen used for separation of magnetic beads as well. Figure6b is a schematic diagram of a magnetic dipole separator.Magnetic beads migrate perpendicular to the direction of theflow at a rate proportional to their magnetic content. Thispermits separation of the injected sample into multiple outletstreams on the basis of the magnetic properties of each beadspecies. By combining a separator design with a constantmagnetic energy gradient, an optical monitoring of the beads(or bead-labeled cells), and a theoretical model for interpreta-tion, it is possible to trace back the linear bead trajectoriesto basic magnetic properties of the magnetic beads. Thistechnique was coined cell tracking velocimetry.124-126Figure 6c is a histogram of the distribution of four differenttypes of magnetic beads with different magnetically inducedvelocities, as measured by cell tracking velocimetry, togetherwith the derived magnetophoretic mobilities. In this case,the magnetic energy gradient of the dipole separator wasdesigned to be (1/0)(B )B ) 198 T A/mm2 in the regionof interest (see eq 7). In other work, the combination ofmagnetic dipole separation with microfluidics has beenproposed,127 experimentally demonstrated on-chip128 andanalytically modeled.129 Other permanent magnet configura-tions have been proposed for separation as well.130-133

4.1.2. Systems with Microscopic Magnets: MagneticTemplates

While the separation systems discussed so far have amacroscopic dimension from the magnet point of view, aninteresting option for generating a local and strong magneticfield gradient is to bring micropatterned soft magneticmaterial into the field generated by a larger permanentmagnet (as done in a MACS column134). Because of the highmagnetic permeability of the magnetic material, the magneticflux from the permanent magnet is focused into the magneticmicrostructure and will create strong gradients at certainmagnetic interfaces, at which magnetic beads can beseparated from a passing solution. Magnetic inductiongradients of 0.1-1 T/mm have typically been realized inmicrofluidic channels using soft magnetic structures com-bined with permanent magnets.135,136 In principle, usingarrangements of bulk permanent magnets, 10 T/mm gradientsare possible.137 A first reported magnetic separation structurewas based on electrodeposited nickel posts of 7 m heightand 15 m diameter. Once magnetized by an externalneodymium-iron-boron permanent magnet, these nickelpost generated the strong magnetic field gradient thatefficiently trapped 4.5 m superparamagnetic beads sus-pended in water flowing past the posts.138 This experimentwas later confirmed by other authors.139 Using a similar idea,magnetic separation was demonstrated by evaporating a thinlayer of cobalt on a preformed microstructure realized viasoft lithography or classical lithography.140,141 In one case,liquid PDMS polymer mixed with magnetic particles was

allowed to polymerize in the presence of a magnetic field, aprocess during which the magnetic particles can be trappedin the polymerized PDMS microchannel wall. Hereafter,these embedded magnetic beads act as anchors that cantrap magnetic beads that are flowing in the microchannel.Also, the packing of Ni particles within a channel for thegeneration of high magnetic field gradients in a nearbymicrofluidic channel was reported.142 Several papers existon the analytical and numerical modeling of the magnetictrapping by a micropatterned magnetic structure.140,143-146 Amagnetic separator consisting of micropatterned permalloyelements adjacent to a microfluidic channel was presentedby Smistrup et al.147 When an external magnetic field isapplied transverse to the channel, the elements are magne-tized and generate magnetic field gradients within thechannel, such that the magnetic beads are attracted to andcaptured at the channel side walls. Two types of magneticbeads could be selectively placed at either side of the channel,with negligible cross-talk, by use of hydrodynamic focusing.This system also enabled the simultaneous exposure of aDNA sample to two types of DNA probes.148 A variant ofthis separation system was presented, in which a staggeredherringbone microfluidic mixer was integrated in the micro-channel.149 This proved to increase the capture-and-releaseefficiency by up to a factor of 2. Simulations of beadtrajectories, capture efficiencies, and capture distributionswere also presented. Following a similar idea, a system withstainless steel wires placed close to a capillary in themagnetic field of permanent magnets showed magnetic fluxfocusing at the wire ends and was able to separate magneticbeads from a solution flowing through the capillary.150

An original idea for continuous magnetic separation(without accumulation of magnetic beads at certainpositions in a microchannel) was presented by Berger etal.136 It is based on the magnetic force generated by an arrayof magnetized stripes that are positioned at an angle withrespect to a hydrodynamic flow direction. These stripes createa series of magnetic field gradients that trap the magneticbeads and alter their flow direction. Figure 7a is a schematicdiagram of the microfluidic chip with indication of theoblique magnetic stripes or wires. The sum of the hydrody-namic force and the magnetic force acting on a magneticbead creates a resultant force vector, which moves the

Figure 7. (a) Schematic diagram of the microfluidic chip withmagnetic wires at an angle with respect to the hydrodynamic flowdirection. (b) Because of the vector sum of hydrodynamic andmagnetic force, a magnetic bead or magnetically labeled cell willmove along the direction of the magnetic stripe, while nonmagneticobjects will move along the hydrodynamic force direction. Reprintedwith permission from ref 136. Copyright 2001 Wiley-VCH,Weinheim.

1528 Chemical Reviews, 2010, Vol. 110, No. 3 Gijs et al.

magnetic bead or cell labeled with beads out of the mainsample stream,151 as illustrated in Figure 7b. A similarseparation principle was later presented by others.152-154

4.1.3. Systems Using Microelectromagnets

An important difference between the use of a permanentmagnet and an electromagnet is the much lower magneticinduction generated by the latter. A permanent magnet easilygenerates a magnetic induction of 0.5-1 T, while themagnetic induction of a simple planar coil is typically inthe mT range. Following the discussion in section 3.1, oneunderstands that a microelectromagnet will produce a muchsmaller magnetic force (in the fN range for a simple spiralcoil and the chosen parameters for the magnetic beads), sothat fluidic flow in the microchannels needs to be stronglylimited or that magnetic beads need to pass at an extremelyclose distance to the planar coil. On the other hand, coilsoffer flexibility, as the magnetic field can be simply switchedoff by setting the coil current to zero. The group of C. H.Ahn has pioneered the use of microelectromagnets for theseparation of magnetic beads.155-157

Figure 8a presents a schematic diagram of a microelec-tromagnet consisting of a planar coil and a soft magneticyoke structure. Copper spiral coils were electroplated intophotoresist molds on a Si substrate, and, when actuated bycurrents in the order of 0.3 A, could effectively retain andseparate micrometer-size superparamagnetic beads from aflow. The integrated fluidic microchannel was realized byanodically bonding a microstructured Pyrex wafer to the Sisubstrate. For enhancing the generated magnetic field (byabout a factor of 2 with respect to a simple coil, as half ofthe space with r ) 1 around the coil is replaced by a softmagnetic material with high magnetic permeability), thespiral coil is combined with an electroplated permalloymagnetic yoke microstructure (Figure 8b), leading to effec-tive magnetic bead separation from a flow (Figure 8c). Suchsoft magnetic microstructures have also been used incombination with micropatterned current-carrying wires formagnetic field enhancement.158 However, the spiral designresults in stronger magnetic fields and forces as compared

to other current-carrying geometries.159 Similar work existson the separation of magnetic beads by microelectromagnetsmade of copper spiral coils and various wire microstructuresin combination with nickel soft magnetic yokes.160-163Magnetic bead capture by current-carrying wires also hasbeen studied theoretically, and optimum separation condi-tions, as influenced by parameters like microchannel dimen-sion, magnetic field magnitude, and flow speed, weredetermined.164,165 Besides separation via electromagnetsrealized directly underneath a microfluidic channel, alsoexternal microelectromagnets or magnetic tweezers havebeen proposed to trap magnetic beads in a liquid solution.The tweezers were made by winding 25 m thick copperwire around a 50 m diameter soft magnetic needle and canbe scanned to effectively displace the magnetic beads in afluidic reservoir.166

Coils offer flexibility, but only relatively small magneticforces can be generated. Combining a coil with a softmagnetic yoke structure can, at maximum, increase the forceby a factor of 2 with respect to the simple coil. Also, ohmicheating of a coil can pose problems and require active coolingof the microfluidic system.167 An interesting option togenerate larger forces is to combine a current-carrying coilor wire for local magnetic field gradient generation with auniform external magnetic field for imposing a largemagnetic moment to the superparamagnetic beads (see eq1).168-170 Such setup allowed for the application of relativelystrong pN forces (100 enhancement) for magnetic beadsin the micrometer range and on a spatial scale correspondingto the microfluidic channel width.

4.2. Magnetic TransportMagnetic separation is different from magnetic transport

in the sense that, in separation, the beads are retained(separated) by action of a magnetic field, but transportedusing a liquid flow. In magnetic transport, magnetic forceseffectively transport the particle, which is a bigger challenge:it requires magnetic fields and magnetic forces that act on alonger range than necessary for separation, where magnetic

Figure 8. (a) Schematic diagram of a microelectromagnet used for separation of magnetic beads. (b) Micrograph of the fabricatedelectromagnet (coil width ) 50 m). (c) Separation of 1 m diameter magnetic beads out of a flow by actuating the second coil with acurrent of 0.3 A. Reprinted with permission from ref 156. Copyright 2001 Elsevier.

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beads approach very closely the magnetic actuation regionby the fluid motion. Transport is a difficult task, as themagnetic susceptibility of the magnetic beads is ratherweak (typically e1), due to small magnetic core volumesand demagnetization effects of the particles (see eq 2). Thisexplains why mostly the large field of permanent magnetshas been used for the separation and positioning of magneticbeads.78

In an approach toward miniaturization and automation ofanalytical applications, a system has been proposed in whichliquid movement is substituted by magnetically inducedmovement of magnetic particles.171 Fluidic channels wererealized on a plastic cartridge of centimeter size, andmagnetic transport was induced by mechanically movingexternal permanent magnets. In another approach, magneticparticles have been transported over millimeter distances ina microfluidic channel using an array of electromagnetsactuated in a four-phase scheme.172 Each electromagnetconsisted of a 0.3 mm diameter magnetic needle core witha wire-wound coil of 300 turns. For coil currents of the orderof 0.5 A, forces of 0.1 pN were possible. Also, alginatemicrodroplets with encapsulated magnetic particles have beentransported using this method.173

Besides these still macroscopic fluidic transport systems,also miniaturized solutions have been proposed for beadtransport, thereby taking advantage of batch microfabricationtechnologies. Typically, the size of the (micropatterned)magnets determines the spatial range, where appreciablemagnetic forces act on the microbeads. Serpentine gold wiresmicropatterned on silicon substrates have been combinedwith microfluidic structures realized in PDMS to transport4.5 m polystyrene-coated magnetic beads.170 By engineeringthe magnetic field generated by different current-carryingwires, a microsystem was realized that could generate localmagnetic field maxima able to trap the magnetic beads (seeFigure 9). When the field maxima change location, the beadsfollowed those maxima. The device allowed precise position-ing and transport over a distance of 100 m in a singleactuation event, which is partly due to the presence of apermanent magnet placed in proximity of the microfluidicchip. The main role of this magnet was to enhance themagnetic force by inducing a magnetic moment in themagnetic beads. A microelectromagnet wire matrix, basedon two layers of mutually orthogonal arrays of linear wires,has demonstrated magnetic transport of 1-2 m sizemagnetic particles over 20 m distances in a single actuationevent.174 This is a typical working range for the magneticforce, generated by a current-carrying conductor, when noexternal permanent magnet is used to induce a magneticmoment in the beads. A transporting device consisting of aset of two tapered conductors shifted linearly over half aperiod was demonstrated by Wirix-Speetjens et al.91,175 Thisdevice was fabricated using standard semiconductor technol-ogy and photolithography techniques. Current conductors(TiW 10 nm/Au 150 nm/TiW 10 nm) were evaporated andpatterned using a lift-off process on an oxidized Si wafer. A250 nm thick SiO2 layer was then sputtered onto the waferfor passivation. By sending a dc current through one of thecurrent conductors, a magnetic field is generated thatmagnetizes the particles. Once the particles are magnetized,they moved toward the edge of the conductor, where thefield is highest. Subsequently, they were attracted to thenearest narrow cross section, driven by the field gradient thatis created by tapering the conductor. This way, one-

dimensional stepwise transport was realized by applying lowfrequency nonoverlapping clock pulses to both conductorsalternatively. The average speed of a magnetic particle wasdefined as the distance over which the particle is transportedparallel to the conductor edge divided by the minimal timeneeded to reach the next minimal cross-section and is of theorder of several 10 m/s. In another approach, a planar coilarray-based magnetic transport system has been proposed,in which an individual coil is capable of displacing beadsover millimeter distances in a liquid-containing capillary.176A drastic increase of the magnetic energy and magneticforces acting on the beads was obtained by placing thecomplete coil array in a uniform static magnetic field thatimposes a permanent magnetic moment onto the beads. Thevery small magnetic field (gradient) of a simple planar coilproved then to be sufficient to displace 1 m diameter beadsover a distance of the order of the coil size. The coils wererealized using simple Printed Circuit Board (PCB) technology(100 m Cu winding width, 35 m winding height, 200 mwinding pitch) and had a small number of windings (N )4-10). Arranging adjacent coils with spatial overlap overtwo layers of the PCB circuit and actuating them in a specificthree-phase scheme assured the long-range displacement ofthe beads. Moreover, it was found that these polarized beadsformed cylindrical columns with a length of the order of themicrofluidic channel size, due to magnetic dipole interactions.This column formation helped to generate the strongmagnetic force. On a smaller length scale, arrays of 50-100m-size microcoils were made on silicon substrates byelectroplating soft magnetic NiCoP pillars at the center ofelectroplated Cu coils.163,177 These microelectromagnetsgenerated sufficient magnetic force to transport 1 mdiameter beads from coil to coil.

Figure 9. Sequence of schematics and corresponding microscopicimages, showing the stepwise transport of magnetic field maximaand magnetic beads, by arranging the relative positions and currentsi of two serpentine wires. Reprinted with permission from ref 170.Copyright 2001 American Institute of Physics.

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Also, a transport system was proposed, which employeda translating periodic potential energy landscape to movemagnetic beads horizontally over a substrate. The potentialenergy landscape was created by superimposing an externalrotating magnetic field on top of the local magnetic fielddistribution of a periodic arrangement of Co micromagnets.At low driving frequencies of the external field rotation (afew Hz), the magnetic beads become locked into the potentialenergy landscape and move at the same velocity as thetraveling magnetic field wave. Therefore, this technique wascoined traveling wave magnetophoresis.178 Also, complexpatterns of soft magnetic microstructures placed in a rotatingmagnetic field were able to trap, transport, and releasemagnetic particles. The translatable local magnetic fieldmaxima were created here by variations in local radii ofcurvature at structural edges of the microstructures.179

4.3. Magnetic Beads as Labels for DetectionA common approach to detecting biological molecules is

to attach a label that produces an externally observable signal.The label may be a radio-isotope, enzyme, fluorescentmolecule, or charged molecule, but also magnetic beads canbe used as labels for biosensing. Magnetic labeling, detection,and the combination of a sensor chip with microfluidicsystem integration aspects were reviewed by severalauthors.180-182 Magnetic labels have several advantages overother labels. The magnetic properties of the beads are stableover time, especially because the magnetism is not affectedby reagent chemistry or subject to photobleaching (a problemwith fluorescent labeling). There is also no significantmagnetic background present in a biological sample, andmagnetic fields are not screened by aqueous reagents orbiomaterials. In addition, magnetism can be used to remotelymanipulate the magnetic particles. Finally, a number of verysensitive magnetic field detection devices have been devel-oped during recent years, like giant magnetoresistance(GMR)183 and spin-valve184,185 magnetic sensors that enablethe measurement of extremely weak magnetic fields, suchas the magnetic stray field generated by the magnetizationof a single magnetic bead. A basic GMR or spin-valve deviceconsists of a pair of magnetic films separated by a nonmag-netic conducting layer.186 When an external magnetic fieldrotates the magnetizations of the magnetic layers towardalignment, spin-dependent electron scattering is reduced atthe interfaces within the device, thus decreasing its electricalresistance. GMR sensors can be microscopic in size andsensitive to the presence of micrometer and smaller sizemagnetic particles, when in close proximity; even singlemicrometer-size particles immobilized down to a fewhundred nanometer above the sensor could be detected.182Also, magnetic particles passing by in a flow, rather thanbeing immobilized above the sensor area, have been de-tected.187 Besides GMR sensors, measurements of singlemagnetic beads have been demonstrated using miniaturizedHall sensors188,189 and planar Hall effect sensors based onpermalloy thin films.190 A possible inconvenience of themagnetic sensor-based detection systems for commercialapplications, however, is that the cost of such a system canbe an issue, due to sensor fabrication (often on a siliconsubstrate) and integration of the sensor in a microfluidicpackage.

A research group at the Naval Research Laboratory,Washington, DC,191-194 followed by others,195,196 has devel-oped a microsystem for the capture and detection of

micrometer-sized, paramagnetic beads on a chip containingan array of GMR sensors, the so-called bead array counter(BARC). Bound beads are detected by the GMR sensors byapplying a uniform magnetic field perpendicular to thesubstrate, and therefore imposing a magnetic moment to thesuperparamagnetic beads. These induced moments generatean in-plane magnetic field component that is measured bythe GMR sensor. Applying the uniform field normal to theplane of the GMR sensor rather than in-plane has theadvantage that, due to demagnetization effects, a much largermagnetizing field can be applied to the beads withoutsaturating the sensor.197 Figure 10 shows optical micrographsthat represent a sensor chip with 64 individually addressableGMR sensors and two reference sensors. Each sensor is aserpentine resistor trace that is 1.6 m wide and with a 4.0m pitch (see Figure 10c), with a total length of 8 mm withina 200 m diameter circular zone. The zone was matched toan arraying system for probe deposition (250 m spots) toselectively capture beads from a flow. The sensor chips arecovered with a silicon nitride passivation layer of about 1m thick to protect the circuitry from the corrosive andconductive media and biochemical reagents. This nitride layerwas etched down to a final thickness of 250 nm over eachsensor zone leading to a larger signal due to the smallerimmobilized bead-sensor distance. Using Dynal M-280 2.8m diameter beads, the threshold for detection was ap-proximately 10 beads per 200 m diameter sensing area. TheGMR sensor sensitivity increases with decreasing surfacearea of the sensor; however, the chemical sensitivity, ornumber of analyte molecules that can bind to the surface,increases with increasing surface area. Theoretical modelingshowed that a GMR sensor can detect a single superpara-magnetic bead of any size,197-199 as long as all systemdimensions (bead size, sensor size, distance between beadand sensor) scale down proportionally. When the sensor sizeapproaches the size of the bead, it should be possible to detectbeads with a radius down to 100 nm or smaller.

A group at Philips Research has proposed a compactbiosensor platform with GMR sensors suited for the detection

Figure 10. Optical micrographs of a third-generation bead arraycounter (BARC) sensor chip. (a) Photograph of the 68 pin-outchip, including a central sensing area with 64 sensors and tworeference sensors, and a number of test structures. (b) Closer viewof the central sensing area. (c) Close-up of one serpentine GMRsensor trace encompassing a 200 m diameter sensing zone.Reprinted with permission from ref 193. Copyright 2000 Elsevier.

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of superparamagnetic nanoparticle labels.200,201 The platformconsists of a disposable microfluidic cartridge, a GMR chip,and an electronic reader. Wires integrated in the silicon ofthe sensor chip are used to generate a well-defined magneticfield on the sensor surface, thus removing the need formechanical alignment with an external apparatus. To opti-mize the signal-to-noise ratio, the magnetic labels are excitedat high frequency (1 MHz). These wires also apply forcesonto the beads to attract the latter toward the sensor surface.A signal modulation scheme is applied to obtain optimaldetection accuracy. Experimental results indicated that threebeads of 300 nm can be detected on a sensor surface of 1500m2 for a measurement time of 1 s. In an effort of costoptimization, the GMR sensors were deposited on a passivesilicon substrate, with the electronic circuitry placed on anexternal chip.202 Despite this, the use of comparativelyexpensive silicon as substrate and the microfluidic packagingof the chip203 might raise cost issues for a (single-use)diagnostic test, unless this particular approach would proveto outperform others, by, for example, a much highersensitivity.

A lot of bead-sensing experiments today have been donewith micrometer-size beads composed of magnetic -Fe2O3and Fe3O4 nanoparticles dispersed in a polystyrene matrix.To maximize the sensor response, the magnetic beads shouldhave a magnetization as high as possible and yet remainnonremnant to avoid clustering when suspended in solution.With the goal of achieving larger signals from the magneticlabels, one has developed soft ferromagnetic beads with100% magnetic content and much higher saturation magne-tization.192 One micrometer diameter NiFe beads showed asusceptibility of 3, the maximum obtainable value for auniformly magnetized sphere. Because of this property,smaller solid ferromagnetic beads could effectively be usedas biomagnetic labels, which would also increase the dynamicrange of biosensor assays by allowing more labels per unitarea.

4.4. Enhancing the Bead-Flow Interaction andMixing

Most of the magnetic bead-related phenomena discussedso far were based on the interaction of individual beads withthe magnetic field. An interesting property of magnetic beadsis that they can form linear-like chain structures or morecomplex SPS, when placed in a magnetic field due to themagnetic dipole interaction between beads. When themagnetic induction can be very locally applied, it is pos-sible to have a small-size magnetic SPS or bead plugspanning the cross-section of a microchannel, which will becharacterized by a strong perfusion by a liquid flow. This isof importance for both biomolecule capture and catalysisapplications. Hayes et al.204 used a varying magnetic fieldgenerated by a mechanically moved permanent magnet tocreate a relatively large magnetic SPS plug composed of 1-2m diameter superparamagnetic beads (see Figure 11a). Theplug could be rotated through all axes in a microfluidicchannel, without loss of structural form, allowing dynamicmicrometer-scale movement without direct mechanical,electrical, or photonic interactions.204 A number of potentialapplications of this phenomenon were described, such asbinding biomolecules on the magnetic particles for immuno-assays,207 studying subcellular biomechanics, and microflu-idic mixing in pico- and femtoliter volumes.

The shape of a SPS depends on different parameters, likethe size of the magnetic moment of the beads and themagnetic dipolar interaction between different beads. Theseproperties are linked to the amplitude and frequency of theapplied magnetic field, the shape and magnetic content ofthe beads, the concentration of the magnetic particles inthe fluid, the temperature, etc. Despite the complexity of theaggregation process of a magnetic fluid into a SPS, thephysical effects of a magnetic field on such a structure arenow very well understood.208,209 When exposed to a strong,continuous magnetic field, the magnetic fluid will rapidlyform a cross-linked network. The continuous-field structureis determined by the kinetics of bead aggregation, and theparticles are prohibited from rearranging to minimize energy,as long as the field persists. In contrast, it was found thatthe application of a pulsed field (square wave alternatingbetween field-on and field-off states) to a magnetic fluid didproduce an energetically determined suspension structure.210,211By allowing particle diffusion during the field-off state, apulsed field enables minimization of energy through struc-tural rearrangements, and the SPS consists of one-dimen-sional periodic patterns composed of high-concentrationregions of magnetic particles (columns), aligned in the fielddirection and sharply separated from low-concentrationregions.208,212,213 Qualitatively, the dependence of the structurecomplexity on the tuning parameters can be understood fromthe time it takes for the particles to aggregate, which dependson the ratio of the magnetic interaction energy betweenparticles relative to thermal energy. Applying a biaxialrotating magnetic field, produced by two orthogonal pairsof Helmholtz coils in quadrature, induces a rotation of dipolar

Figure 11. (a) Superparamagnetic particles in a cylindricalmicrochannel (20 m diameter), as observed by optical microscopy:(i) High-volume fraction 1-2 m diameter particles without appliedexternal magnetic field. (ii) Induced column-like structures that formimmediately upon application of an external magnetic field in theplane of the page. (iii) Upon rotation of the applied magnetic fieldperpendicular to the page, the top of the column-like structures isvisible. (b) Video microscopy images of (i) a dense SPS kept overthe microchannel cross-section in an alternating magnetic field bytwo soft magnetic tips, at a field oscillation frequency f ) 5 Hz, aliquid flow velocity V0 ) 0 cm/s, and a magnetic field amplitudeB0 ) 100 mT, (ii) the expanded SPS of (i) for f ) 5 Hz, V0 ) 0cm/s, and B0 ) 5 mT, (iii) a snapshot image of the oscillating SPSat the most downstream position in the channel for f ) 5 Hz, V0 )0.4 cm/s, and B0 ) 25 mT, and (iv) a snapshot image of theoscillating SPS at the most downstream position in the channel forf ) 5 Hz, V0 ) 0.4 cm/s, and B0 ) 20 mT. (c) Optical image of amicrochannel showing the retention of 18 self-assembled nanopar-ticle magnetic chains that are perfectly positioned in the geometricaltraps of the microchannel, while applying a flow from the left tothe right. (a) Reprinted with permission from ref 204. Copyright2001 American Chemical Society. (b) Reprinted with permissionfrom ref 205. Copyright 2004 American Institute of Physics. (c)Reprinted with permission from ref 206. Copyright 2008 AmericanChemical Society.

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chains of superparamagnetic particles and subjects theaggregates to magnetic forces causing them to rotate withinthe suspending fluid. It has been shown, with both scatterningdichroism214 and video-microscopy experiments,215 thatmagnetic fluids have the capacity of reducing the size of thestructures they are composed of, to decrease their viscousdrag while rotating synchronously with the field.

For an application where low-concentration biomoleculesneed to be recovered from a flow by the SPS, it is useful tohav