On Chip Free Flow Magnetophoresis Separation and Detection Of

Journal of Magnetism and Magnetic Materials 307 (2006) 237244

On-chip free-ow magnetophoresis: Separation and detection ofmixtures of magnetic particles in continuous ow

Nicole Pammea,b,, Jan C.T. Eijkela,c, Andreas Manza,d

aDepartment of Chemistry, Imperial College London, South Kensington, London SW7 2AY, UKbDepartment of Chemistry, The University of Hull, Cottingham Road, Hull HU6 7RX, UK

cBIOS/Lab-on-a-Chip Group, University of Twente, P.O. Box 217, 7500 AE Enschede, The NetherlandsdInstitute for Analytical Sciences (ISAS), Bunsen-Kirchhoff Strasse 11, 44139 Dortmund, Germany

Received 17 January 2006

Available online 12 May 2006

Abstract

The complete separation of mixtures of magnetic particles was achieved by on-chip free-ow magnetophoresis. In continuous ow,

magnetic particles were deected from the direction of laminar ow by a perpendicular magnetic eld depending on their magnetic

susceptibility and size and on the ow rate. 2.8 and 4.5 mm superparamagnetic particles with magnetic susceptibilities of 1.1 104 and1.6 104m3 kg1, respectively, could be completely separated from each other reproducibly. The separated particles were detected byvideo observation and also by on-chip laser light scattering. Potential applications of this separation method include sorting of magnetic

micro- and nanoparticles as well as magnetically labelled cells.

r 2006 Elsevier B.V. All rights reserved.

PACS: 75.50.T

Keywords: Magnetic separation; Magnetic microparticles; Microuidics; Magnetophoresis

1. Introduction

Magnetic microparticles are widely used in biomedicalsciences as a solid support surface for immunoassays,DNA sequencing and cell analysis [1,2]. The use ofmagnetic labels greatly facilitates the separation of thedesired biomaterial from the bulk of a reaction mixturesimply by application of an external magnetic eld.Conventionally, such magnetic separations are carriedout in a test tube which is brought in close proximity toa magnet that traps the magnetic particles whilst theremainder of the reaction mixture is pipetted off. Suchconventional separation methods are quite labour intensiveand time consuming. Recently there has therefore been aninterest in continuous ow separation of magnetic particles

for example by means of a quadrupolar magnetic eldacross a tube [3] or a fanned out, inhomegenous magneticeld across a chamber [4,5]. Another approach forcontinuous ow magnetic separation is split ow thin(SPLITT) fractionation [6]. In this technique, two owstreams, one containing magnetic particles the othercontaining a buffer, were introduced into a central channeland were separated into two outlet streams further down-stream. Magnetic particles were dragged away fromtheir stream into the buffer stream by means of an externalmagnetic eld. Miniaturisation of magnetic separationdevices has gained some attention in recent years dueto the anticipated integration of such devices intomicro total analysis systems (mTAS) [710]. In this context,we described on-chip free-ow magnetophoresis [11]as a separation method that is capable of separatingmagnetic particles not only from non-magnetic materialbut also different types of magnetic material fromeach other based on their size and magnetic susceptibility(Fig. 1).

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www.elsevier.com/locate/jmmm

0304-8853/$ - see front matter r 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.jmmm.2006.04.008

Corresponding author. Department of Chemistry, The University ofHull, Cottingham Road, Hull HU6 7RX, UK. Tel.: +44 1482 465027; fax:

+44 1482 466416.

E-mail address: [email protected] (N. Pamme).

One of the challenges associated with on-chip free-owmagnetophoresis is the reproducibility of the particledeection within the separation chamber. Previously, wereported that particles of a given size and susceptibilitywould be spread over as many as four exit channels,making separations of mixtures of particles impossible. Inthis paper, we demonstrate an improved microuidicdesign featuring greater deection reproducibility. Thispermitted the complete separation of different magneticparticles from magnetic particle mixtures. Furthermore, wehave investigated on-chip laser light scattering detection asa detection method for the separated particles.

2. Theory

The principle of free-ow magnetophoresis is shown inFig. 1. The key component of the microuidic chip is ashallow rectangular separation chamber over which alaminar ow is generated in x-direction by a number ofinlet and outlet channels. Perpendicular to the direction ofow, i.e. in y-direction, an inhomogeneous magnetic eld isapplied. The sample inlet is located in the corner farthestaway from the magnet. Any non-magnetic materialentering the separation chamber via the sample inlet willow straight through following the direction of thepumped liquid. Any magnetic material, however, willinteract with the magnetic eld and will be draggedinto the eld and thus deected from the straight ow path.The observed velocity vector uobs (in m s

1) of amagnetic particle in the separation chamber can bedescribed as the sum of the magnetically induced ow onthe particle, umag, and the applied hydrodynamic ow uhyd:

uobs uhydr umag. (1)At excessively high ow rates, the hydrodynamic ow

vector, uhyd, outweighs the magnetically induced owvector, umag, and no deection in y-direction will beobtained. On the other hand, at slow ow rates, the

magnetically induced ow in y-direction dominates overthe applied ow in x-direction and hence, particles wouldbe expected to become immobilised in the separationchamber. Within a certain window, an optimum velocityhas to be found for the particles to be deected and to owsmoothly through the chamber.The magnetically induced ow vector, umag, is the ratio

of the magnetic force, Fmag, exerted on the particle by themagnetic eld over the viscous drag force [12]:

umag Fmag

6pZr DwVpr BB=m0

6pZr, (2)

where Dw is the difference in magnetic susceptibilitybetween the particle and the uid (dimensionless), Vp theparticle volume (m3), B the externally applied ux density(T) and r B its gradient (Tm1), m0 is the permeability ofa vacuum (Hm1), Z the liquid viscosity (kgm1 s1) and rthe particle radius (m).On closer inspection of Eq. (2), it can be seen that, for a

given magnetic eld and a given viscosity, the magneticow vector, umag, is dependent only on the size and themagnetic characteristics of the particle (Eq (3)).

umag / r2wp. (3)

3. Experimental section

3.1. Microchip fabrication

Microchips were fabricated from sodalime glass [11]. Thedesign is shown in Fig. 2. The separation chamber of6mm 6mm was supported by 13 square posts of200 mm 200 mm. 16 buffer inlet channels and one sampleinlet channel were all 100 mm wide and evenly spaced. Thejunction between the inlet channels and the separation

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Fig. 1. The principle of free-ow magnetophoresis. Magnetic particles are

deected from the direction of laminar ow depending on their size and

magnetic susceptibility.

Fig. 2. Design of the microuidic chip, featuring the 6mm 6mmseparation chamber, supported by a number of posts, one sample inlet, 16

buffer inlets as well as 16 outlet channels. The junctions between the

chamber and the channels are tapered.

N. Pamme et al. / Journal of Magnetism and Magnetic Materials 307 (2006) 237244238

chamber was tapered to allow for smooth entering of theuid into the chamber. On the opposite site, 16 outletchannels with 100 mm width were also evenly spaced withtapered junctions. The sample channel had a length of10mm; the buffer inlet system was branched to enable theuse of a single buffer inlet reservoir and to evenly spreadthe liquid over the entire width of the separation chamber.The outlet system was branched in a similar fashion.Although, any separated particles would ultimately be re-combined in the outlet reservoir, this setup requires onlyone pump and was chosen for simplicity and proof ofprinciple.The design as shown in Fig. 2 was patterned onto a wafer

coated with chromium and photoresist. After development,the glass was etched to a depth of 25 mm and thermallybonded to a cover plate. Pipette tips were glued around theinlet holes as reservoirs with epoxy glue and the outlet wasconnected to a syringe pump (PHD 2000, Harvard, Kent,UK) via a short piece of fused silica capillary and Tygontubing. Flow was controlled by applying a withdrawal rateof typically between 200 and 600 mLh1, corresponding tobetween 0.4 and 1.1mm s1 in the separation chamber.Prior to experiments the chip was ushed with 0.5M

NaOH, de-ionised water and running buffer, i.e. a glycinesaline buffer, pH 8.3 containing 1mM glycine and 1.5mMNaCl. Particle suspensions with about 1 105particlesmL1 were introduced into the sample reservoir,running buffer was lled into the buffer reservoir andnegative pressure was applied via the syringe pump.The experiments were lmed with a CCD camera (CX-

999P, Sony, Weybridge, Surrey, UK) connected to aninverted microscope (DMIRB, Leica, Milton Keynes, UK)A photograph of the microchip with reservoirs andconnecting tubing is shown in Fig. 3.

3.2. Reagents, magnets and setup

Uniform, superparamagnetic particles (Dynabeads) con-sisting of an iron oxide core and a polystyrene shellwere obtained from Dynal Biotech (Oslo, Norway).2.8 mm particles with epoxy surface groups (DynabeadsM-270 Epoxy) were delivered as a dry powder of 67 107beadsmg1. Dynabeads M-450 Epoxy with a diameterof 4.5 mm were purchased as a suspension with4 108 particlesmL1. The particles had magnetic sus-ceptibilities of w 1:07 104 and 1.6 104m3 kg1,respectively.

3.2.1. Preparation of M-270 beads

Three milligrams of the supplied dry particles (ca.2 108 beads) were washed by dispersing the particles in1mL PBS buffer, vortexing the suspension, magneticallycollecting the particles and taking off the supernatant. Thisprocess was repeated twice. After the last supernatant hadbeen removed, the particles were re-suspended in 1ml of a10 concentrated assay buffer (pH 8.3), containing100mM glycine and 150mM NaCl. The mixture wasincubated for 24 h whilst shaking constantly to maintainthe particles in suspension. During this incubation time,glycine molecules were covalently bound to epoxy func-tional groups on the particles surface. Finally, thesuspension was diluted in 1 concentrated assay bufferto 1 106 beadsmL1 and stored at 4 1C.

3.2.2. Preparation of M-450 beads

Ten microlitres of the Dynabead stock suspension wasmixed with 990 mL of a 10 concentrated glycine salinebuffer (pH 8.3) and left to incubate for 24 h whilst shakingconstantly. The particle surface was thus coated withglycine molecules and the reactive epoxy surface groupswere inactivated. Subsequently, the mixture was dilutedwith glyince saline buffer (1 concentrated) to yield asuspension with 1 106 beadsmL1 and this was storedat 4 1C.Prior to experiments the particle suspensions were 10

diluted in a 0.1 concentrated glycine saline buffer,containing 1mM glycine and 1.5mM NaCl.The magnetic eld perpendicular to the direction of

ow was generated by an assembly for four cylindricalNdFeB magnets (Magnetsales, UK) which were placedon the chip beside the separation chamber. The magnetshad diameters of 6, 5, 4 and 2mm and lengths of 4, 3, 5and 2mm, respectively. This assembly together with asteel back-plate was placed on top of the microchip asshown in Fig. 3. The magnetic ux generated by thisassembly was simulated in two dimensions (x and y)with FEMM-freeware as plotted in Fig. 4. The uxgradient over the separation chamber can clearly be seenin the simulation. The ux density was almost 700mTat the surface of the magnet and about 70mT at thesample inlet.

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Fig. 3. Photograph of the setup showing the microuidic glass chip with

buffer and sample inlet reservoirs as well as the connection to the syringe

pump for application of negative pressure. To generate a magnetic eld

gradient over the separation chamber, assemblies of small NdFeB magnets

were used.

N. Pamme et al. / Journal of Magnetism and Magnetic Materials 307 (2006) 237244 239

3.3. Detection of particles

Detection of the separated particles was undertaken bytwo means: (i) by counting according to visual observationunder the microscope and from video footage and (ii) byusing laser light scattering detection.

3.3.1. Counting by visual observation

Videos were taken of the particles owing through thechamber. The optical eld of view even for the lens with thesmallest magnication (2.5 ) was only about a quarter ofthe 6mm 6mm separation chamber. Hence, only asection of the chamber could be observed and lmed at atime. For counting of the separated particles, it wassufcient to observe a section with several of the outletchannels in the eld of view.

3.3.2. Counting by laser light scattering detection

Particle counting was also attempted by using the laserlight scattering detection system as described earlier [13].Briey, a laser beam was focussed into the microchannel.Two optical bres were positioned above the microchannelat angles of 251 and 351 with respect to the laser beamabove the microchannel. The bres were connected to aPMT and computer. Beads owing through the laser beamcaused laser light scattering, which was collected by theoptical bres. For free-ow magnetophoresis experiments,the laser beam was adjusted to a width of about 100 mmand positioned just upstream of the desired outlet channelto minimise background scattering from the channelwalls. This method allowed for counting in one channelat a time.

4. Results

4.1. Stream lines in the flow chamber

To visualise the ow stream of the sample within themicrochamber in the absence of any magnetic eld, thesample reservoir and the buffer reservoir were lled withblack ink and water, respectively. The uidic design with

tapered channel junctions was compared with a previousdesign featuring 901 junctions between the channels and theseparation chamber [11]. In the previous design (Fig. 5a),some of the liquid from the sample stream was observed toleave via exit 2. For the current design (Fig. 5b), the inkstream was conned to the side of the separation chamberand all ink left the chamber via exit number 1 directlyopposite the sample inlet. A portion of buffer solution isalso dragged into exit number 1. This can be attributed tothe different spacing of channels on each side of theseparation chamber. On the inlet side there are 17 channels,on the outlet side, there are only 16 channels. From theseobservations it can be concluded, that any particles owinginto an outlet channel other than exit 1 would have beeninuenced by the magnetic forces perpendicular to thelaminar ow.

4.2. Separation of magnetic particles

Particle suspension was lled into the sample reservoir,buffer into the buffer reservoir and a magnetic eld wasapplied as outlined in the experimental section. Adsorptionof the beads to the microchip walls as well as non-specicadsorption of beads to each other had to be minimised. Forthis, the bead surface had been rigorously coated withglycine. The pH of the buffer (pH 8.3) was signicantlyhigher than the pI of glycine (pI 6.1) such that theparticle surfaces as well as the channel walls werenegatively charged and electrostatic repulsion was domi-nant. Furthermore, the buffer featured a low ionic strengththus increasing electrostatic repulsion between beads andbetween beads and wall. This approach proved valuable, asagglomeration of particles within the sample reservoir did

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Fig. 4. Two-dimensional simulation of the magnetic eld generated by the

assembly of four small NdFeB magnets over the separation chamber with

freeware FEMM (http://femm.foster-miller.net).

Fig. 5. Flow visualisation with black ink in the sample reservoir: (a) for a

uid cell design with 901 junctions between the channels and theseparation chamber some of the sample stream was observed to leave

via exit number 2 and (b) for the design with tapered 451 junctions, alluid from the sample stream was found to leave via exit number 1.


not occur and also sticking of the particles to the channelwalls was not prevalent.The deection behaviour of 2.8 and 4.5 mm particles was

observed for different ow rates as summarised in Table 1.At slow ow rates of 200 and 300 mLh1, the 2.8 mmparticles were found to be deected towards exits 2 and 3.However, the 4.5 mm particles did not ow smoothlythrough the chamber, as the magnetically induced owvector umag (see Eq. (1)) on these particles was too strong incomparison to the applied ow vector uhyd. Morespecically, the magnetic ow vector has a component inz-direction, i.e. pulling the particles upwards. At these lowpumping rates, the drag of the applied ow was not strongenough to pull the particles along through the chamber.For 400 and 500 mLh1, both types of particles weredeected, the smaller particles mainly into exit 2, the largerparticles mainly into exit 3 or 4. Hence, separation of amixture of particles could be achieved at these ow rates.At the faster ow rate of 600 mLh1, the 2.8 mm Dynabeadswere hardly deected anymore and some of them even leftvia outlet number 1. In this case, the applied ow vectoruhyd was dominant over the magnetically induced owvector umag and thus almost no deection was observed.Hence, this ow rate was not suitable for the separation ofthe particles from each other and from non-magneticmaterials at the same time.Statistical data was obtained from video footage by

counting the number of particles per exit. This wasundertaken for the 2.8 and 4.5 mm particles individuallyat ow rates of 400 and 500 mLh1 over a period of 10mineach. The results are given in Fig. 6a for 400 mLh1 andFig. 6b for 500 mLh1. At 400 mLh1 the two particlepopulations could be differentiated, the smaller particleswere deected towards exits 2 and 3, the larger particlesdeected towards exits 35. However, there was someoverlap at exit 3. For 500 mLh1, a complete differentiationwas achieved. The 2.8 mm particles went almost exclusivelyinto exit 2, while the 4.5 mm particles were deectedtowards exits 3 and 4. The particle throughput at the owrate of 400 mLh1 was approximately 20 particlesmin1,for 500 mLh1 the throughput was approximately 25particlesmin1.Mixtures of 2.8 mm particles and 4.5 mm particles were

separated repeatedly at ow rates of 400 and 500 mLh1.

Photographs from a video sequence are shown in Fig. 7afor 400 mLh1 and in Fig. 7b for 500 mLh1.

4.3. Magnetic force on particles

As described in the theory section, the magnetic force onthe particles can be estimated according to Eq. (2), bymeasuring the magnetically induced velocity, umag. At400 mLh1, the particles took about 12 s to ow throughthe chamber. The 2.8 mm particles were deected towardsoutlet number 2, corresponding to a deection in y-direction of between 0.40 and 0.55mm. The 4.5 mmparticles were deected towards outlet number 4 and 5,corresponding to approximately 1.201.45mm. In the caseof 500 mLh1, the particles took about 9 s to pass throughthe chamber. The smaller particles were deected towardsoutlet number 2, corresponding to 0.350.45mm, the largerparticles towards outlet 3 and 4, corresponding to0.91.1mm. The calculated values for the magneticallyinduced velocity, umag, and the magnetic force, Fmag arelisted in Table 2.

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Table 1

The deection of 2.8 and 4.5mm Dynabeads observed at different owrates

Flow rate (mLh1) Outlet channel 2.8 mmbeads

Outlet channel 4.5 mmbeads

200 3 Intermittent ow

300 23 Intermittent ow

400 23 35

500 2 34

600 12 3

Fig. 6. The separation of mixtures of 2.8 and 4.5mm Dynabeads at owrates of (a) 400mLh1 and (b) 500mLh1.


The ow velocity and force values in the table are givenas a range. They were calculated for the lowest and highestdeection typically observed for a given particle populationat a given ow rate. The magnetically induced force on theparticles, Fmag, is in the order of pN. Force values for agiven particle type are similar at both tested ow rates. Thisshould be expected, as the magnetic force as such isgiven by the magnetic eld, which is constant andindependent of a change in ow rate. The force on the2.8 mm particles is considerably lower than the force on the4.5 mm particles.The magnetic force as shown in Eq. (2) is proportional to

the particles magnetic susceptibility, w, and volume. Incase of the 2.8 mm diameter particle, wr3 equals2.9 1022m6 kg1, for the 4.5 mm diameter particles, thisvalue is 1.8 1021m6 kg1. The ratio of wr3 for the 4.5 mmparticles to the 2.8 mm particles is 4:1. This would be thetheoretically expected ratio of the magnetic forces onthe two particles. The experimentally observed ratio of theforces is closer to 6:1. A more thorough investigationwould have to be carried out in order to draw conclusions.One contributing factor would be, that the exact size of themagnetite core within the Dynabead is not known. Onlythe outer diameter of the particle, including the polymershell is given by the manufacturer.

4.4. Laser light scattering detection

So far, particle counting had been undertaken byobservation from video footage. This was feasible for therelatively slow ow rates employed in these experiments,i.e.o1mm s1. However, the particles were rather difcultto see, as they are fairly small in comparison to the channelwidth. Alternative particle counting methods includecounting based on uorescence and scattering signals[1416] or counting based on the Coulter principle[1719]. In our laboratory, a setup was readily availablefor particle counting based on laser light scatteringdetection [13]. This method, however, was optimised forcounting of particles at much faster ow rates (1m s1) andfor particles that were hydrodynamically focussed into themiddle of a microchannel.In the free-ow magnetophoresis chip, no hydrodynamic

focussing of the particle stream is possible. To avoidbackground scattering from the channel walls, the laserbeam was positioned just upstream the outlet channeljunction and the laser beam diameter was adjusted toabout 100 mm, i.e. the width of the outlet channel.The scattering signal intensity of the particles wasexpected to be widespread, depending whether a particlewould ow through the centre of the laser beam oroff the centre. A typical example of a scattering traceis shown in Fig. 8 obtained from deected 4.5 mmmagnetic particles at outlet channel number 4. Datawas acquired over a period of 5min. In Fig. 8, asection of 35 s is presented for clarity. As expected,the intensities of the signals varied considerably.Standard deviations of around 100% were observed,however, the number of particles passing through thebeam could clearly be detected. Only those particlescreeping along the channel wall or passing through theperiphery of the laser beam could not be distinguishedfrom the background noise.

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Fig. 7. Photograph sequence of the separation of a mixture of 2.8 and 4.5 mm magnetic particles at (a) 400mLh1 and (b) 500mLh1.

Table 2

Estimated force on magnetic particles based on the observed deection

Particle

diameter (mm)Flow rate

(mLh1)umag (mms

1) Fmag (pN)

2.8 400 0.030.05 0.81.3

2.8 500 0.040.05 1.11.3

4.5 400 0.100.12 4.35.1

4.5 500 0.100.12 4.35.1


Laser light scattering signals were measured for amixture of 2.8 mm Dynabeads and 4.5 mm Dynabeads, thatwere magnetophoretically separated at a ow rate of500 mLh1. The laser was positioned in front of exits 1, 2,3, 4 and 5 and data was collected for 5min at each exit. Thenumber of detected signals per exit is plotted in Fig. 9.It can be seen that most signals were detected at outlet

channel number 2 and 4, i.e. where the 2.8 and 4.5 mmDynabeads would be expected to exit (see Fig. 6b). Somesignals were also detected at outlets 3 and 5 and at outletnumber 1. On visual inspection, however, it becameapparent, that salt crystals had formed within the samplereservoir and these were pulled through the separationchamber alongside the magnetic particles. The salt crystals

from the sample inlet owed straight through the chamberinto exit number 1. While this result demonstrates howfree-ow magnetophoresis can separate magnetic particlesand non-magnetic crystals from each other, it also high-lights that laser light scattering detection is not a specicdetection method but rather universal. For more speciccounting of magnetic particles, new methods are currentlybeing developed, for example, miniaturised Hall sensors[2022] or miniaturised giant magnetoresistive sensors[2325].

5. Discussion

In this paper the separation of magnetic particles fromeach other as well as from non-magnetic material wasdemonstrated. Differentiation between particles is based onthe particles magnetic susceptibility and its radius. Singleparticles of different magnetic characteristics were sepa-rated. A rst investigation into detection of separatedparticles was also undertaken.A number of further improvements could be incorpo-

rated into the system in order to achieve (i) less distributionin particle deection for a given particle population and(ii) a higher throughput of particles. The distribution indeection is caused by (a) a wide spread in startingpositions when the particles enter the separation chamberthrough the 100 mm wide inlet channel and (b) byvariations in the size and magnetic susceptibility of a givenparticle population. The uidic design could thus befurther improved by signicantly reducing the width ofthe sample inlet stream from its current 100 mm in order toachieve identical start positions for each particle whenentering the separation chamber. The distribution inparticle deection however also depends on the homo-geneity of the magnetic particles themselves. Some authorshave reported that the magnetic properties, at least of2.8 mm Dynabeads show signicant variations [23,26]. Thethroughput of particles in these experiments, i.e. a few tensof particles per minute, was somewhat low. Increasing thethroughput by increasing the particle concentration be-yond 105 particlesmL1 is not easily feasible, since non-specic particle agglomerates would then be readilyformed. The throughput could be improved by increasingthe ow rate. This is only possible, if at the same time themagnetic force on the particles can be increased, i.e. bydesigning a magnetic eld with larger eld gradients. Themagnetic design used in these experiments was fairlyprimitive with eld gradients of 100200mTmm1. Thereare many examples of more sophisticated magnetic designsin combination with microuidic applications [10]. Some ofthese feature tapered magnet tips in order to achieve higherux gradients [27,28], others use pairs of magnets with theirpoles facing each other in order to achieve gradients ofseveral thousand mT per mm [29] Assuming an improve-ment of the magnetic force on the particles by a factor 10,the ow rate could and thus the throughput could beincreased by factor 10.

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Fig. 8. Traces of scattering signals obtained for the 4.5mm diameterDynabeads at outlet number 4. The traces were detected by two optical

bres positioned at 251 and 351 with respect to the incident laser beam.

Fig. 9. Signals detected by both optical bres for a mixture of 2.8 and

4.5 mm particles at the respective outlets.


The described form of magnetic separation has a varietyof possible applications. Different types of magneticparticles could, for example, be coated with differentantibodies and incubated with a sample. The targetedantigens would attach themselves to the appropriate beadsand after magnetophoretic separation could be separatedfrom each other for downstream applications. Similarly,the particles could be targeted for cells, DNA sequences,RNA, organelles and so forth. Another potential applica-tion is the separation of magnetic cells, not only cellslabelled with magnetic particles but also naturally magneticcells, such as red blood cells, containing iron, andmagnetotactic bacteria, containing small magnetic crystals[30]. Free-ow magnetophoresis offers a valuable additionto the currently employed immuno-magnetic separationtechniques.

Acknowledgements

Nicole Pamme would like to thank Helke Reinhardt forassistance with chip fabrication, Alexander Iles for com-ments on the manuscript and Asahi Kasei Corp., Fuji,Japan, for funding.

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ARTICLE IN PRESSN. Pamme et al. / Journal of Magnetism and Magnetic Materials 307 (2006) 237244244

On-chip free-flow magnetophoresis: Separation and detection of mixtures of magnetic particles in continuous flowIntroductionTheoryExperimental sectionMicrochip fabricationReagents, magnets and setupPreparation of M-270 beadsPreparation of M-450 beads

Detection of particlesCounting by visual observationCounting by laser light scattering detection

ResultsStream lines in the flow chamberSeparation of magnetic particlesMagnetic force on particlesLaser light scattering detection

DiscussionAcknowledgementsReferences

On Chip Free Flow Magnetophoresis Separation and Detection Of

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