ARTICLE IN PRESS

Journal of Magnetism and Magnetic Materials 321 (2009) 1440–1445

Contents lists available at ScienceDirect

Journal of Magnetism and Magnetic Materials

0304-88

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.com/locate/jmmm

Design, fabrication and demonstration of a magnetophoresis chamber with25 output fractions

Chris Carr a, Michelle Espy a,�, Pulak Nath a, Sara L. Martin a, Michael D. Ward b,d, John Martin c

a Applied Modern Physics, Los Alamos National Laboratory, Los Alamos, NM 87545, USAb Acoustic Cytometry Systems Inc., Los Alamos, NM 87544, USAc B-9, Los Alamos National Laboratory, Los Alamos, NM 87545, USAd Life Technologies, Eugene, OR 97403, USA

a r t i c l e i n f o

Available online 20 February 2009

Keywords:

Field-flow fractionation

Magnetophoresis

Magnetic microparticle

Magnetic microsphere

Laser microfabrication

53/$ - see front matter Published by Elsevier

016/j.jmmm.2009.02.064

esponding author. Tel.: +1505 665 6218; fax:

ail address: espy@lanl.gov (M. Espy).

a b s t r a c t

Our goal is to develop an instrument for parallel and multiplexed bioassay using magnetic labels.

Toward this end we are developing a multi-outlet magnetophoresis instrument incorporating a fluidic

flow chamber placed inside a magnetic field gradient. Magnetic microparticles are sorted by their

magnetic moment for eventual use as biological labels based on magnetic signature. In this paper,

we concentrate on developments in our flow chamber fabrication methods that have allowed us to

scale the number of sorting channels from 8 to 25. We present data for instrument performance and

reproducibility of sorting.

Published by Elsevier B.V.

In conventional laser-based flow cytometry the number ofdistinct biological labels is limited by the availability of differentfluorescent dyes. By utilizing variations in the magnetic momentof microparticles, we have the potential to produce orders ofmagnitude more biological labels for multiplexed in-vitro studies.Much research has been carried out on biomolecular labelingand sorting with techniques ranging from electrophoresis [1] toelutriation [2] to flow cytometry based cell scanning/sorting [3]and magnetic sorting based on immunomagnetic labeling [4].Sorting based on magnetophoresis has also been demonstrated inmany forms, see for example [5–10], and it is one approach to thistechnique that is detailed in this paper.

In our previous work [11] we described our magnetophoresisinstrument, including the magnetic microparticles used (bothferromagnetic and superparamagnetic), the magnet quadrupoleand the flow chamber design. We analyzed the forces acting on asingle microparticle in flow and from this we were able to modeltheir trajectory for various transit times. The modeled andexperimental results compared favorably, though the spread inthe experimental data highlighted certain hydrodynamic issuesthat we were forced to address such as reducing the aspect ratiobetween the sample inlet and the chamber depth, and hencestabilizing the sheath flow. Having eliminated many of thehydrodynamic instabilities, we then were able to re-sort magneticmicroparticles with a high degree of reproducibility.

B.V.

+1505 665 4507.

In this paper, we report on the design and a new fabricationprocess for our flow chambers with the number of outputchannels scaled up to a maximum of 25, limited by the featuresize we are able to cut with our existing laser cutter. A key featureof the fabrication process is the simplicity of design and assemblysuch that we essentially are able to consider the chambers asdisposable. As with the 8-channel flow chambers we have sortedand re-sorted populations of magnetic microparticles, thoughhere we only consider superparamagnetic microparticles.

When magnetic microparticles are placed in a magnetic fieldgradient they experience a force that is proportional to theirmagnetic moment. The sum of the forces acting on the micro-sphere leads to the following equations:

Fy ¼ ma@vy

@t¼ �raVg þ rsheathVg þ 6pZaðvsheath þ vyÞ (1)

Fx ¼ ma@vx

@t¼

MVG

m0

� 6pZavx (2)

where vx and vy are the microparticle velocity (m s�1) in the x andy direction, respectively (see Fig. 1a), ma is the microparticle mass(kg), qv/qt is the acceleration (m s�2), ra and rsheath are thedensities of the microparticle and the sheath fluid, respectively(kg m�3), V is the microparticle volume (m3), g is the accelerationdue to gravity (m s�2), Z is the sheath fluid viscosity (kg m�1 s�1),and a is the microsphere radius (m). Note that the sheath velocityis assumed to be constant. M is the volume magnetization of themicrosphere in A m2, m0 is the free space permeability (4p�10�7

H m�1), and G is the magnetic field gradient (constant for aquadrupole magnet in T m�1). For ferromagnetic microparticles M

ARTICLE IN PRESS

Y Y

X X

Flowdirection

F drag (-x)

F gravity (-y)

Field/gradient

gradient

Sample inlet

Sheath2

Sheath1

direction

direction

collection bins

F magnetic (+x)

F drag (+y)+

F buoyant (+y)

Fig. 1. (a) Force diagram for a magnetic microparticle in flow and (b) schematic diagram of the flow chamber.

C. Carr et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1440–1445 1441

is constant, resulting in a linear trajectory. For paramagneticmicroparticles, the value of M is a function of the externalmagnetic field.

This principle can be used to sort magnetic microparticles foruse as biological labels in multiplexed assays (as is our goal) [12].The subsequently magnetically labeled biomolecules could becharacterized in flow by their magnetic signature using a sensorsuch as a giant magnetoresistive sensor (GMR) or superconduct-ing quantum interference device (SQUID) [13] or biomoleculeslabeled with magnetic microparticles can then be sorted inthe same way, with the biomolecule total magnetic momentdepending on the number of bound labels and their individualmagnetization [14]. Typically magnetophoretic sorting of cells hasbeen binary, but our method demonstrates potential for multi-plexed sorting based on magnetic labeling.

Fig. 1a shows the force diagram for a single magneticmicroparticle flowing through our magnetophoresis instrument.Summing the forces on each microparticle allows us to formulateEqs. (1) and (2), and hence model the trajectory [11].

The magnetic separation shown here was based on a quadru-pole magnet. This particular design of magnet was chosen toprovide a linear field gradient for microparticle sorting. TheSmCo5 permanent magnet pieces were 6.25�7�8.5 cm3 whilethe iron pole pieces were 3.75�8.5�8.5 cm3. The length of themagnet is 8.5 cm; considerably shorter than the overall length ofthe sorting chamber. The field gradient was measured using a Hallprobe and was found to be 17 T m�1 within the bore of themagnet. With the flow chamber positioned in the magnet,the sample inlet tube is located just far enough off-center thatthe microparticle trajectory is essentially only parallel to thex-axis (see Fig. 1). We are aware that outside the bore of themagnet the field is non-zero. To reduce the effect of the stray fieldon the magnetic microparticles while they are still in the samplesyringe (i.e. to minimize the possibility of agglomeration), weplace a 1-mm-thick mumetal plate above the magnet to shield thefield. The sorting chamber also protrudes below the magnet, thusthe microparticle trajectory will again be influenced by the strayfield (though to a much lesser extent compared to within the boreof the magnet) as they flow towards the collection bins.

Prior to running experiments to sort magnetic microparticles,we determine the appropriate flow settings by running tests withfood coloring as the sample. This allows us to vary the flowthrough the individual sheath inlets by means of a clamp(note that they are still fed from the same pressure-driven source)

such that a non-magnetic sample in the absence of a magneticfield will appear in a pre-chosen collection bin (or bins). The foodcoloring also allows us to measure the transit time through themagnet. Fig. 2a shows a detail of the collection bin region for the8-channel flow chamber during a pre-test check with foodcoloring. The sample stream (red food color) is focused andchanneled by the dividing walls and goes into bin 2 (with little orno visual evidence of the sample going into bins 1 and 3). Thegreen food color (bins 4–8) is from one of the sheath supply inletsand shows that there is smooth laminar flow with no turbulentmixing in the chamber.

During each experiment we use 10-mm-diameter non-magnetic, latex fluorescent microparticles (Polysciences, Warring-ton, PA) to ensure that any run-to-run variability is kept to withina few percent. These are counted in the same way as the magneticmicroparticles (described below).

A key requirement for magnetic microparticles for use asbiological labels for multiplexed assay is that they have a widerange of magnetic moments to provide a wide range of labels.Permanent magnet material such as SmCo5 is an ideal candidatesince it has a high remnant magnetization, but this can also causeproblems with agglomeration. In general, any ferromagneticmaterial has to be handled much more carefully than eitherparamagnetic or superparamagnetic material and sample con-centrations have to be typically lower. Previously [11], we haveused both commercial ferromagnetic and superparamagneticmicroparticles in sorting experiments to validate performance.These were chosen because they were supplied with a known sizedistribution and had magnetic moments in the range of interest.We have not investigated the variations in magnetic content ofthese commercial beads but they appear to be quite uniformbased on our sorting results. In this paper, we have usedonly 870.23-mm-diameter superparamagnetic particles (BangsLaboratories, Fishers, IN). These particles consist of a functiona-lized polymer impregnated with iron oxide, giving a total densityof 1.2 g cm�3. The microparticles have a calculated magneticmoment of �8�10�13 A m2 (the manufacturer-supplied magneti-zation curve showed a saturation magnetization of 2.54 A m2 kg�1

at �5�104 A m�1).Typical sample volumes for the experiments were 1–2 mL

consisting of 0.5 mL of magnetic microparticles in solution(concentrations of the order of 105–106 microparticles/mL) withthe remainder being fluorescent microparticles in a solution ofde-ionized water and PluronicTM surfactant (MP Biomedicals,

ARTICLE IN PRESS

Fig. 2. (a) Flow characterization for the 8-channel flow chamber using food coloring dyes and (b) experimental setup of the 25-channel flow chamber showing the magnet

quadrupole and the sample/extraction syringes.

C. Carr et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1440–14451442

Solon, OH). The PluronicTM appeared superior to sodium dodecylsulfate (SDS) (the surfactant we used in previous studies) atpreventing agglomeration.

Our experimental sorting method is described elsewhere [11]and described only briefly here. The collection channels on theflow chamber are connected to output collection syringes. Thechamber has a differential sheath design, as shown in Fig. 1b.The larger sheath stream, ‘‘Sheath 1,’’ is provided by a regulatedpressure system. For initial studies, a syringe pump provided‘‘Sheath 2,’’ but this was later changed to a single pressure systemdriving both sheath streams, which greatly improved hydrody-namic stability. The sheath is 0.125% PluronicTM in distilled waterthat is passed through a 0.2-mm filter prior to the chamber.Another single syringe pump provides the sample stream, and amultiple syringe pump withdraws the twenty-five outlet streamsfor the collection bins. We observed that the sheath’s streamspeed in the chamber (and hence microparticle transit timethrough the 8.5 cm of the bore of the magnet and deflection) islargely regulated by the speed of the withdrawal syringe pump.The differential sheath design utilizes standard flow cytometrytechniques that in principle generate uniform fluid flow throughthe chamber [3].

On completion of the sort, the contents of each collection binsyringe were transferred to a test-tube, centrifuged to removeexcess sheath and subsequently counted using a CaliberTM FACsinstrument (Becton Dickinson & Company, Franklin Lakes, NJ). Toreduce any potential loss of microspheres at this step, we are inthe process of modifying the design so the collection bins are feddirectly to the test tubes. The measurement ‘‘gates’’ on the FACswere pre-determined by running a high concentration of micro-particles (both magnetic and fluorescent) through the instrument,and these gates were kept constant throughout the countingprocess. More information can be found in our earlier work [11].

Fig. 1b shows a schematic of the 8-channel flow chamber. Theexternal width of the chamber ranges from 35.0 mm (8-channel)to 38.4 mm (25-channel) to 41.2 mm (14-channel); this dimensionnecessarily changes to permit more collection bins (or wider bins)to be added. The external length of the chamber is kept constantat 173 mm, while the external thickness is approximately 3.8 mmfor all. The internal dimensions of the chamber range from a widthof 18.7 mm (8-channel) to 26.6 mm (14–25 channel), whileall have a depth of 0.8 mm. The chamber dimensions takeinto account various parameters including magnet dimensions,

microparticle diameter, number of discrete populations required,and fabrication processes.

There are three inlets at the top of the chamber: one for thesample and two for the sheath supply. The sample inlet tubeis a 362mm OD, 248mm ID polyimide-coated glass capillary(Polymicro Technologies, Phoenix, AZ) though for added mechan-ical durability, it is glued inside a piece of 750mm OD, 500mm IDPEEKTM tubing (Upchurch Scientific, Oak Harbor, WA). The sampleis supplied from a 3 mL plastic syringe using a single syringe drive(Harvard Apparatus, Holliston, MA). Each sheath supply isconnected via SilasticTM tubing (Dow Corning, Midland, MI) tothe same pressure-driven supply. One of the problems associatedwith the chamber design is the occurrence of small air bubblescollecting at the sheath inlets that affect the hydrodynamic flow.To alleviate this, each sheath inlet has a ‘‘bleed off’’ valveconnected to it (with both the inlet itself and the valve madefrom PEEKTM tubing). At the end of the collection channels, a pieceof PEEKTM tubing glued into the chamber is connected to a pieceof SilasticTM tubing for sample collection.

At the other end of the flow chamber, below the collection binsare the extraction syringes. In our experience, having the outlettubes merely drain into collection vials led to problems withreproducibility. The syringe drives (KD Scientific, Holliston, MA)are specifically designed for multiple syringes and have beenmodified to take up to 20 syringes per drive. At present we runthem with at most 13 in any one drive. If we are successful atmodifying the set up to draw directly into test tubes, thisextraction protocol may be simplified. Fig. 2b shows the experi-mental set up.

Since the critical flow chamber dimensions are sub-mm, usinga standard milling machine is not a reliable option for chamberconstruction. In our lab we have access to a CO2 laser cutter(M360, Universal Laser Systems, Scottsdale, AZ) that allow us tofabricate chambers with a minimum feature dimension of�100mm. Fig. 3a and b shows a detail of the design for the inletand outlet regions of the middle layer of the flow chamber.

The chambers were fabricated using double-sided tapes(ACcares 8570, Adhesive Research Inc., Glenrock, PA) and a threesets of acrylic sheets (top layer, middle layer, and bottom layer).The tape is first applied on both sides of a 0.5-mm-thick acrylicsheet. One side of the double-sided tape has a protective filmwhich is not removed during this process. The laser cutter is thenutilized to cut the acrylic sheet and the double-sided tapes

ARTICLE IN PRESS

C. Carr et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1440–1445 1443

according to the channel layout. The power and the writingspeed of the laser cutter is optimized such that the acrylic sheet/double-sided tape assembly is cut all the way with out excessiveburning. A second sheet of acrylic (1.5 mm thick, Optixs acrylicsheet, Paskolite Inc, Columbus, OH) is then utilized to obtain thetop and bottom plate for the fabrication of the enclosed chamber.The same CO2 laser cutter is utilized to cut out acrylic pieces fromthe bulk sheet. The top plate is cut such that it has through holesto form inlets and outlets connections. For a 25-channel outputdesign, special optimization of the laser cutter power and speedwas required so that 25 through holes can be fit into the width ofthe channel. The acrylic section with the double-sided tapes isthen used as the middle layer. The enclosed chamber is formed byremoving the protective films and sandwiching this layer with thetop and bottom acrylic. The top layer is aligned with the bottommiddle layer such that the inlet and outlet holes are aligned withthe channel layout. A special clamping mechanism is utilized toapply uniform pressure throughout the stack for improvedbonding by the double-sided tapes. Chambers fabricated thisway with all layers having the same width were found to bemoderately robust and able to flow at chamber pressures up to

sample inlet

Fig. 3. Schematic of the flow chamber design (a) inlet of the middle layer and (b)

outlet of the middle layer.

Start with 0.5 mm thick acrylic sheet. This serves as the middle layer of the channel and dictates the thickness of the channel

Apply double sided tape on both sides of the acrylic sheet

Use CO2 laser based laser cutter to cut patterns on the acrylic sheet according to the design

Remove cut-out acrylic and remove protective film of the double sided tapes

Plontach(dflow

z

Fig. 4. Schematic cross-section showing dif

�2 psi. However, we observed this was not adequate for thereliable performance at the highest of our flow rates. In order toobtain higher bonding strength, the top and bottom plates werecut slightly bigger (�1–2 mm on all four sides). This designallowed us to obtain additional space between the bottom and thetop plates throughout the perimeter of the channel. This spacewas then filled up with epoxy cement (Loctite Marine Epoxy,Hankel Consumer Adhesives Inc., Avon, OH) and cured for about2–4 h. The epoxy allowed additional strength to the bonding andthe chamber was able to withstand flow at chamber pressures upto �15 psi. A step by step schematic diagram showing the crosssection of the channel at different fabrication steps is presentedin Fig. 4.

In the next year we are planning to scale up the number ofsorting bins further, with the goal of achieving 50 outlet channels.This will require the use of a laser cutter capable creating ofsmaller features in the flow chamber (our present device islimited to features �100mm in width). An excimer laser-basedmicrofabrication tool available at the Los Alamos Center forIntegrated Nanotechnologies will be used. Such a device has theability to obtain features as small as 5mm. This laser cutter couldbe used to obtain channels containing up to 100 outlets within thespace of our magnet bore.

The chamber fabrication process we have described is flexibleand allows easily for changes in chamber dimensions and numberof outlet bins. The most time consuming step of our chambermanufacturing process is gluing the inlet and outlet PEEKTM

tubing. Several chambers can be cut and fabricated at one timesuch that the devices can be essentially considered disposable.

To determine the flow chamber performance we sortedthe 8-mm-diameter superparamagnetic microspheres describedabove. Because of the size and magnetic monodisperse propertiesof the microspheres, we expect these microspheres to (under idealconditions) all sort to a single bin, with the deflection proportionalto the moment (which does not change) and the transit time inthe magnet, which we can vary. We then collected populationsfrom selected outlet bins, and re-sorted that material under thesame conditions. The reproducibility of sorting (i.e. microspheres

ace top and bottom cover plates (acrylic) the acrylic sheet with the double sided

pe and apply pressure to form enclosed annels. The top plate has through holes efined by the laser cutter) to establish fluid w into the channel and therefore is aligned

ith the layout of the middle layer

Apply epoxy around the bonded edges

x

ferent steps of the fabrication process.

ARTICLE IN PRESS

C. Carr et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1440–14451444

from bin ‘‘A’’ returning to bin ‘‘A’’) can thus be characterized. Fig. 5shows two sets of data for sorting (a) and re-sorting (b) in ourchambers.

The 8-channel chamber data (left) were sorted at a 0.25 mL/min-channel flow rate corresponding to a transit time through themagnet of �40 s. The fluorescent (non-magnetic) microsphereswhich were flowed as controls (data not shown) arrived incollection bin #2, while the magnetic microspheres are observedto be collected primarily in bin #5. As one would expect, ourexperiments indicate increasing deflection with transit time ormagnetic moment of the particles. In preliminary 8-channelchamber designs we also observed an increasing spread in thedistribution of microparticles [11] as the transit time increased.The origin of this effect was ruled out as being from microparticlediffusion and variations in magnetic content. We ascertained thatthe dominant source of this spread was hydrodynamic in originand alterations to the sheath supply method and also to thedimensions of the sample inlet tube made the distribution muchsharper [11]. Some of these hydrodynamic effects had to do withthe microparticles experiencing different flow velocities dueto a spread in the sample stream, which was partially solvedby changing the aspect ratio of the inlet tube and chamberthickness [11]. As shown in Fig. 2 with food coloring, anddetermined by our non-magnetic microspheres, it appears thatthe sample stream is narrow (i.e. within one bin), however thesetests do not rule out a broadening of the sample stream in themagnetic microspheres which could be caused by agglomerationor other interactions.

The data at right in Fig. 5 are from a 25-outlet chamber at a flowrate of 0.2 mL/min-channel corresponding to a transit time throughthe magnet of�16 s. Sorting experiments in the 25-channel chamberindicate the same features of increasing deflection with transit time.

80

60

40

20

01 2 3 4 5 6 7 8

% m

icro

parti

cles Sort

80

100

60

40

20

01 2 3 4 5 6 7 8

% m

icro

parti

cles

Re-Sort

collection bin

collection bin

% m

icro

parti

cles

% m

icro

parti

cles

Fig. 5. Data from the initial sort and re-sort (a) 8

In this chamber the fluorescent beads (not shown) primarily land inbin #7. The 25-channel data shown in Fig. 5 indicate that we are stillsomewhat limited by hydrodynamic spread in the sample stream.For the 8-channel chamber bin #5 was re-sorted and here 89% of themicroparticles that originally ended up in bin 5 return to the samebin when re-sorted. For an extraction rate of 0.4 mL/min, the initialpeak is in bin 3% and 84% of those microparticles return to bin #3(data not shown). More data on the performance of the 8-channelchamber is presented in [11]. For the 25-channel chamber weobserved both in the sort and re-sort data that 85% of themicrospheres re-sort over three bins. The 25 chamber data shownwas obtained in a flow chamber fabricated prior to shifting themethod of adding epoxy around the bonded channels (see right sideof Fig. 4), which were only able to withstand about 2 psi pressure.We have qualitatively observed that those previous chamber designsoften were not as hydrodynamically stable as the new chamberdesign appears to be (likely due to sheath seeping into the sidewalls) while flowing. Tests with the new chambers are underwayand we are optimistic that with the appropriate chamber design wewill be able to continue to narrow our collection capabilities to asingle bin for monodisperse microspheres.

The ultimate aim of the work described here is to producereproducible populations of microspheres sorted by their magneticmoment for use in multiplexed bioassay. Rather than binary(magnetic or not) methods, we have demonstrated that it is possibleto build chambers capable of reliable sorting of a continuum ofmagnetic moments. Such applications could be multiplexed assaysuch as we attain [13], collection of immunomagnetically labeledcells [14], or precise control of microspheres for manipulation inflow [15], or others. We have demonstrated that with relativelysimple fabrication methods and flow chamber design, magneto-phoresis with up to 25 unique populations is possible.

80

100

60

40

20

05 10 15 20 25

Sort

collection bin

80

100

60

40

20

05 10 15 20 25

Re-Sort

collection bin

-channel and (b) 25-channel flow chambers.

ARTICLE IN PRESS

C. Carr et al. / Journal of Magnetism and Magnetic Materials 321 (2009) 1440–1445 1445

Acknowledgments

This work was funded by the US NIH under Grant RR019626-01. The authors also acknowledge the support of the NationalFlow Cytometry Resource and Los Alamos National Laboratoryunder NIH Grant RR-01315. The authors wish to thank ChristinaHanson, Henrik Sandin, Yonathan Araya, and John Gomez for theirassistance.

Reference

[1] W. Schutt, H. Klinkmann (Eds.), Cell Electrophoresis, Walter de Gruyter, Berlin,1985.

[2] J. Bauer, J. Chromatogr. B 722 (1999) 55.[3] H. M Shapiro, Practical Flow Cytometry, Wiley-Liss, New York, 1995.[4] A. Thiel, A. Scheffold, A. Radbruch, Immunotechnology 4 (1998) 89.

[5] J.J. Chalmers, M.J. Zborowski, L.P. Sun, L. Moore, Biotechnol. Prog. 14 (1998)141.

[6] L.R. Moore, M. Zborowski, L. Sun, J.J. Chalmers, J. Biochem. Biophys. Meth. 37(1998) 11.

[7] L.P. Sun, M. Zborowski, L.R. Moore, J.J. Chalmers, Cytometry 33 (1998) 469.[8] A.H. Latham, R.S. Freitas, P. Schiffer, M.E. Williams, Anal. Chem. 77 (2005)

5055.[9] F. Carpino, L.R. Moore, M. Zborowski, et al., J. Magn. Magn. Mater. 293 (2005)

546.[10] N. Pamme, A. Manz, Anal. Chem. 76 (2004) 7250.[11] M.A. Espy, et al., Cytometry Part A 69A (2006) 1132.[12] H. Sandin, C. Carr, A.N. Matlachov, et al., Int. Congr. Ser. 1300 (2007) 271.[13] C. Carr, A.N. Matlachov, H. Sandin, et al., IEEE Trans. Appl. Supercond. 17 (1)

(2007) 808.[14] T. Schneider, L.R. Moore, Y. Jing, et al., J. Biochem. Biophys. Methods 68

(2006) 1.[15] N. Pamme, et al., Magnetic microparticles as maneuverable solid supports for

multi-step (bio)reactions in continuous flow, presented at the MagneticCarriers Conference, May 22–24, 2008, Vancouver, BC.