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CRITICAL REVIEW View Article OnlineView Journal | View Issue

Department of Biomedical Engineering, The University of Texas at Austin, Austin,

Texas, USA. E-mail: john.zhang@engr.utexas.edu

† The authors contributed equally to this work.

Peng Chen

Peng Chen received his B.S. degreein Biomedical Engineering fromZhejiang University, Hangzhou,China, in 2010. As an under-graduate student, he worked onthe development of LAPS basedbiosensors for taste cell charac-terization. He is currently a Ph.D.student studying in the Depart-ment of Biomedical Engineeringat the University of Texas atAustin. His current researchincludes theoretical modelingand multi-physical optimization

of the microchip based immunomagnetic assay for rare circulatingtumor cell detection.

Yu-Yen Huang

Yu-YedegreefromUnive2003,EnginNatioTainais curthe DEnginof TeHis mon t

immunomagnetic microfluidic chip brare cancer cell study. He has more ththe design and fabrication of micro(MEMS) technology for optical and bio

446 | Lab Chip, 2014, 14, 446–458 This journal is © The R

Cite this: Lab Chip, 2014, 14, 446

Received 30th September 2013,Accepted 12th November 2013

DOI: 10.1039/c3lc51107c

www.rsc.org/loc

Multiscale immunomagnetic enrichment ofcirculating tumor cells: from tubes to microchips

Peng Chen, Yu-Yen Huang,† Kazunori Hoshino and Xiaojing Zhang*

We review the rare cancer cell sorting technologies, with a focus on multiscale immunomagnetic

approaches. Starting from the conventional magnetic activated cell sorting system, we derive the scaling

laws of immunomagnetic assay and justify the recent trend of using downscaled systems for CTC studies.

Furthermore, we introduce recent work on combining the immunomagnetic assay with microfluidic

technology for enhanced separation. We summarize different types of in-channel micro-magnetic

structures that can further increase the local magnetic field without lowering the system throughput.

Related design concepts, principles, and microfabrication techniques are presented and evaluated.

Introduction

Metastasis, which is the major cause of carcinoma-relateddeath in cancer patients, occurs often through blood circula-tion and the lymphatic system. During this process, cancercells spread from a primary tumor site to distant organs,which leads to the development of another solid tumor.In 1889, the English surgeon Stephen Paget introduced the“seed and soil” theory to describe metastasis, in which cancer

cells were considered to be the seed, while the organ micro-environments were considered to be the soil.1–4

Circulating Tumor Cells (CTCs) are cells that have escapedfrom the primary tumor site and now circulate in the periph-eral bloodstream. Viable CTCs cause the carcinoma metastasisthrough a series of steps, namely local invasion, intravasation,circulation, arrest, extravasation, proliferation, and angio-genesis.5 The presence of CTCs is believed to be an importantindicator of carcinoma progression and metastasis. Accuratecounting of CTCs contributes to cancer diagnosis, prognosis,and therapeutic response monitoring.6,7 Due to the natural rare-ness of CTCs in the peripheral blood sample, only 1–100 CTCscan be found from 1 mL of whole blood, which usually con-tains one billion normal blood cells.8,9 Enriching, characterizing,

n Huang received his B.S.in Mechanical Engineering

the National Chung Chengrsity, Chia-yi, Taiwan, inand his M.S. degree in

eering Science from thenal Cheng Kung University,n, Taiwan, in 2005. Herently a Ph.D. student inepartment of Biomedicaleering, The Universityxas at Austin, TX, USA.ain research is focused

he development of theased detection system foran 10 years' experience in-electro-mechanical systemlogical applications.

oyal Society of Chemistry 2014

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and analyzing CTCs in such low concentrations is challenging.Therefore, an effective CTC detection system with high sensi-tivity, high throughput, high purity, and low cost is neededfor advancing biological and clinical cancer studies.

In the past decade, emerging CTC separation technolo-gies have attracted significant attention from the scientificcommunity and biotechnology industry. Various mechanismshave been proposed and successfully demonstrated, as sum-marized in previous reviews.10–12 Based on the separationprinciples, these methods can be categorized as (1) separationbased on physical parameters (sizes and density gradients,13–18

dielectrophoretic separation,19,20 and hydrodynamic separation)21–24

and (2) affinity mediated separation (immunoassay25,26 andimmunomagnetic separation).7,27–30

Separation based on physical parameters distinguishesCTCs from normal blood cells by relying on one or moreintrinsic properties of the CTCs (such as size, density,deformability, and electrical charges) without external labels.The size filtration method works under the assumption thatcarcinoma cells are generally larger than normal bloodcells.31–33 Density gradient centrifugation for the enrichmentof cells is based on the differences in cellular buoyantdensity.34,35 Other techniques based on cell deformabilitycan provide additional information for cell separation.15,16

Although filter based approaches are not limited by celltypes, these techniques cannot differentiate between cancercells and normal blood cells that are physically similar.When passing through filtration systems, CTCs may poten-tially become damaged or lost due to the increased shearstress. Additionally, low pore density and pore fusion of filterstructures are other disadvantages that fundamentally limitthe system throughput.8,36,37

A promising alternative to the filtration systems is to sepa-rate cells in a streamline by manipulating the hydrodynamicforces acting on cells. Cells with different sizes or shapes

Dr. Kazunori Hoshino

Dr. Kazunori Hoshino obtainedhis Ph.D. from the University ofTokyo in 2000. He worked forthe University of Tokyo from2003 to 2006 as a lecturer inthe Department of Mechano-Informatics. In 2006, he joinedthe University of Texas atAustin, where he currentlyworks as a Senior ResearchAssociate in the Departmentof Biomedical Engineering.His research interests include(1) nano/micro-electro-mechanical

systems based detection and analysis of cancer cells, and(2) nano/microscale mechanical sensing and optical imaging.He has more than 100 peer reviewed publications and is theinventor of 6 US patents and 12 Japanese patents.

This journal is © The Royal Society of Chemistry 2014

behave differently in the streamline and can be sortedaccordingly.21 The continuous flow deterministic array is oneof the earliest methods used to separate blood componentsby hydrodynamic size.23 In asymmetric pinched flow frac-tionation, cells with different sizes and shapes move alongdifferent streamlines and enter different collection outlets.38

When micro-scale vortices are applied to separate CTCsusing size as the biomarker, target cells with larger diametersmigrate to and are enriched in the expansion–contractiontrapping reservoirs.22,39 Spiral microchannels focus largerCTCs against the inner wall of a curvilinear chamber and usethe inherent Dean vortex flow to collect the CTCs in the inneroutlet.40,41 A stream based separation operates with highflow rates and does not suffer from an increased shear stressbecause no physical filtration is present. However, to keepthe carrier liquid consistent in properties such as viscosity,the sample usually needs to be diluted appreciably beforebeing screened, which limits the separation efficiency.

Electrical properties of cells can also be used to performseparation. When an external alternating current electric fieldis applied, cells are polarized by the field and affected by theapplied dielectrophoretic (DEP) force. The magnitude of theapplied DEP force is dependent on the electric field, thefluid medium, dielectric, and density of the cells.42 CTCs andnormal blood are increased to different heights and aretransported with different velocities in a parabolic flowprofile. Blood cells move faster near the center, whereasCTCs move slower near the edge, and become separatedthereby.19,43 DEP systems are appealing because a largeamount of blood sample can be treated in a flow-throughdevice without labeling, and target cells can be collected forfurther analysis. However, to increase the separation resolu-tion, a large chamber size is preferred, and the DEP systemrequires an external power supply, both of which lower theportability of the system.

Dr. John X. J. Zhang

Dr. Xiaojing (John) Zhang is anAssociate Professor at theUniversity of Texas of Austin inthe Department of BiomedicalEngineering. He received hisPh.D. from Stanford Universityand was a Research Scientistat Massachusetts Institute ofTechnology (MIT). Zhang's researchfocuses on exploring bio-inspirednanomaterials, scale-dependentbiophysics, and nanofabricationtechnology, towards developingnew diagnostic devices and

methods on quantitative biological measurements critical tounderstand development and diseases. Dr. Zhang is a recipientof the Wallace H. Coulter Foundation Early Career Award inBiomedical Engineering, the British Council Early Career RXPAward, NSF CAREER Award, and DARPA Young Faculty Award.

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Other than the physical parameters, CTCs can also be dif-ferentiated from normal blood cells based on the surfacemarkers. In affinity mediated immunoassays, affinity ligandsprovide direct capture force and usually display better selec-tivity than physical separation due to the nature of antibody–antigen interactions. Affinity immunoassay is first demonstratedby coating the three-dimensional micro-post structures withanti-EpCAM antibodies.25 Afterwards, a number of deviceswith carefully engineered three-dimensional structures areintroduced to help increase the separation efficiency by takingadvantage of the enhanced surface to volume ratio.26,44 How-ever, the affinity mediated immunoassay is limited by therelatively slow transport of target cells to the capture surface.Besides, selecting the appropriate shear stress that facilitatesreactions between CTCs and surface molecules, dissociatesthe non-specific bonds and removes non-target cells isdifficult. To address these challenges, a fluid-permeablenanoporous membrane has been recently integrated to theimmunoassay to promote surface reactions and reduce non-target fouling.45,46

Meanwhile, affinity ligands can be used solely as labels inmechanisms such as fluorescence activated cell sorting(FACS) and magnetic activated cell sorting (MACS). FACS hasbeen widely used in past decades, and the method sorts aheterogeneous mixture of cells into multiple containersbased on fluorescent signals.47 Recent developments in nano-technology have enabled miniaturized FACS systems basedon microfluidics and pulsed lasers.48,49 However, FACS isusually designed to sort cells with large sub-populations andlacks the sensitivity to separate cells as rare as CTCs.

In a MACS system, magnetic force is applied to separateCTCs labeled with magnetic tags.50 Magnetic tags, such asmagnetic microbeads or nanoparticles, are usually conju-gated specifically to cancer cells through antibody–antigenbinding without affecting normal blood cells. Depending onthe desired field intensity, either permanent magnets29 orelectromagnets51 are used to provide the magnetic field.Compared with other CTC enrichment methodologies, animmunomagnetic assay has several advantages that make itespecially suitable for rare CTC separation.

(a) Selectivity: similar to affinity mediated immunoassays,immunomagnetic assays have good sensitivity that arises fromantibody–antigen binding. Currently, many immunoassaysare based on the over-expression of an epithelial cell adhe-sion molecule (EpCAM), a common biomarker found onepithelium-derived cancer cells. However, EpCAM may bedown-regulated during metastasis. Increased separationsensitivity can be achieved by expanding the type of antigensthat can distinguish CTCs and by using magnetic tagslabeled with multiple antibodies. As a recent example,we have successfully demonstrated the capture of A-431(skin cancer cell) using EpCAM/EGFR, SK-BR-3 (breast cancer cell)using EpCAM/HER2, and BT-20 (breast cancer cell) usingEpCAM/MUC1.52

(b) Specificity: using magnetic force as the retaining force,an immunomagnetic assay increases the contrast between

448 | Lab Chip, 2014, 14, 446–458

target and non-target cells in terms of the surface attach-ment. High shear stress can be applied in the flushing stepsto remove the non-target cells.

(c) Throughput: unlike an affinity mediated immunoassaywhere direct contact between CTCs and surface molecules isessential for successful capture, an immunomagnetic assaycan attract cells over a broader spatial domain. Larger separationchamber space and higher flow rates (up to tens of ml h−1)29

can be adopted without sacrificing the separation performance.(d) Tunability: compared to the fixed filtration structure or

the surface molecule immobilization, the magnetic field canbe easily and accurately modulated, especially when an electro-magnet is used as the magnetic field source. The field intensityand distribution can be adjusted based on the cell types andthe magnetic tag properties.

(e) Integration: a magnetic field can be introduced withoutdirect contact with cells within the immunomagnetic assay,and the platform can be integrated seamlessly with other sepa-ration methods. It has been demonstrated that integratingthe immunomagnetic assay with deterministic flow and iner-tial focusing in series can notably increase the separationefficiency by removing red blood cells (RBCs) and plateletsin advance.53

Macroscopic magnetic activatedseparation system

Various types of immunomagnetic assays for cell separationhave been invented over the past decades. In conventionalmagnetic-activated cell isolation systems, samples are storedin conical tubes and the screening/flushing steps are usuallydone manually. Fig. 1(a) shows the principle of a MACSsystem. Cells labeled with supermagnetic microbeads areattracted to the tube wall and unlabeled cells are eluted.Once the external magnetic field is removed, the labeled cellscan be released.27 Bulky permanent magnets provide themagnetic field and are arranged either as dipole or quadru-pole separators. Dipole separators drive cells across thestreamlines with a constant magnetostatic energy gradient,while quadrupole separators deflect and capture cells in theradical direction.54 Commercial magnetic-activated cell sorting(MACS) systems have been developed (such as Manual MACSby Miltenyi Biotec), in which a high magnetic field gradientis generated in a column tube. Separating rare cell sampleswith a conventional MACS system is usually a time-consumingprocess with low throughput because the system is manuallyoperated and the magnetic field intensity is limited.

Recently, a new magnetic cell sorting system designcalled the MagSweeper technology was reported. In thesystem, magnetic rods covered with plastic sheaths are sweptthrough the well to magnetically attract microbead-labeledtarget cells.28,55 Fig. 1(b) shows the operation principlesof the MagSweeper system. The diluted blood samples arepre-labeled with magnetic particles, and the samples areloaded into the capture wells. Sheath-covered magnetic rodsare swept a few millimeters above the bottom of the wells.

This journal is © The Royal Society of Chemistry 2014

Fig. 1 Major macroscopic cell separation technologies using magnetic force. (a) MACS system. Cells labeled with superparamagnetic beads (black dots)are attracted to the tube wall by the external magnetic field, while unlabeled cells (white dots) are eluted. The attracted cells can be released once themagnets are removed. (b) Operational schematic of the MagSweeper system used to isolate rare CTCs. Magnetic rods covered with plastic sheaths areswept through the well to magnetically attract microbead-labeled target cells. (c) Work flow of the FDA-proved CTC detection system – CellSearch.Reproduced from ref. 27, 55 and 8 with permissions from Wiley and Elsevier.

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Loosely bound contaminating cells are removed, while thesheathed rods are washed. An external magnetic field isapplied to facilitate the release of labeled cells and excessivemagnetic particles. Using multiple rods, the system canprocess different samples at the same time. However, duringthe screening process, sheaths considerably reduce themagnetic force on the magnetic rods and may lower thecapture efficiency.

CellSearch™ is a commercial system used to detect CTCsand is based on the MACS method. CellSearch™ is amongthe few systems that have been approved by the U.S. Foodand Drug Administration (FDA) for clinical diagnostics ofbreast, colorectal, and lung cancers. The system consists ofa CellSave™ preservative tube (Immunicon, HuntingdonValley, PA) for sample collection, preservation, and transporta-tion. A CellSearch™ profile kit (Veridex) containing ferrofluidnanoparticles and a capture enhancement reagent is used forthe screening test. Fig. 1(c) illustrates the working process ofthe CellSearch™ system.8 CTCs labeled with ferrofluids conju-gated with EpCAM are isolated from unlabeled blood cells. Asemi-automated microscope is utilized for sample scanning

This journal is © The Royal Society of Chemistry 2014

and data acquisition (CellSpotter Analyzer™, Veridex).56,57

There have been a number of studies carried out usingCellSearch™ to study the correlation between the CTC countlevel and the survival rates of cancer patients.58–64 The limita-tions of the CellSearch™ system include the fixed targetassay designed only for EpCAM surface marker expressionwhich may lead to low capture efficiency in many clinicalcases. Besides, instead of collecting captured cells, the systemre-suspends the captured cells in the original screening solu-tion after fluorescence imaging. It is difficult for the suspendedcells to be retrieved on slides for further analyses.

Conventional magnetic activated cell sorting systemsplay significant roles in cancer biology and clinical studies,as revealed by the high level of interest and research activi-ties in CTCs. The field has been expanding remarkably fastin the past few years, and the enrichment techniques areimproving.65,66 At present, the conventional MACS systemcannot provide the performance needed for sophisticatedmolecular studies. In addition, the bulky size and lowportability restrict their applications for point-of-care medicalsystems. New technology is needed to provide more control of

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the magnetic and hydrodynamic forces acting on the targetcells to improve separation.

Scaling law of immunomagneticseparation

The physical principles of immunomagnetic assays evolveover the length-scale. To elucidate key design parameters,we discuss the scaling laws of the immunomagnetic assay.Considering a virtual immunomagnetic capture region asillustrated in Fig. 2, the motion of target cells in a flow envi-ronment, under an external magnetic field, is affected by themagnetic force (Fmag), hydrodynamic drag force (Fdrag), gravi-tational force (G), and buoyancy force (Fbuo). According toour previous estimation based on the properties of thenanoparticles and the labeling efficiency, the magnetic force(on the order of 10−10 N) is nearly 100 times larger than thegravitational force (on the order of 10−12 N) on the CTCs.67

Therefore, to simplify the calculation, we only consider Fmag

and Fdrag.More specifically, Fdrag is closely related to the flow field

inside the capture region, which is determined by the flowrate (FR) and the dimensions of the capture region, includinglength (L), width (W) and height (H), whereas Fmag is depen-dent on the magnetic field generated by the magnetic fluxsource (B0), the thickness of the substrate (S), the magneticsusceptibility of the magnetic tags (Δχp), and the labeling effi-ciency which is represented as the number of tags on eachcell (N). Biological properties of the screening samples andother parameters may also influence the system, but they arebeyond the scope of the discussions here.

If the dimensions of the permanent magnets are muchgreater than the capture region, the magnetic field within the

Fig. 2 Physical model and scaling laws of the immunomagnetic cellseparation system. Cell motion is affected by the magnetic force,gravitational force, buoyancy force and hydrodynamic force. However,the magnetic force is approximately 2 orders of magnitude larger thanthe gravitational force, hence dominating the movements of the cells.Reducing the height (H) or increasing the cross-section area (W × L) ofthe capture region can help improve the immunomagnetic separationefficiency, as shown in eqn (10).

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capture region falls off inversely with the square of thedistance to the magnet (y) and can be represented as:

B By

02 0S y

y H (1)

The average time (τ) a cell travels inside the capture regioncan be estimated using the flow rate and the dimensions ofthe region:

W H LFR

(2)

The magnetic force acting on a nanoparticle (Fp) is depen-dent on the magnetic dipole m of the particles and themagnetic field B, given by the equation:68

Fp = (m · ∇)B (3)

The total magnetic moment of a nanoparticle can beexpressed using:

m BVp p

0(4)

Here, Vp is the volume of a single nanoparticle, μ0 = 4π ×10−7 T m A−1 is the magnetic permeability of vacuum. Weassume ∇ × B = 0, and the magnetic force on a magneticnanoparticle can be simplified to:

FV

pp p

2 0

2B (5)

Eventually, the magnetic force applied to a nanoparticle-labeled cell (Fc) is simply the summation of the forces fromall the nanoparticles.

Fc = N × Fp (6)

When the magnetic force drives cells along the laminarflow inside the capture region, we assume a quasi-staticmotion, equating Stokes' drag force (Fdrag = 6πηRΔν) to themagnetic force. Therefore, the instant relative velocity of thecells can be calculated as

v R

c c2

0

29

B (7)

where Rc is the radius of the cell, η is the viscosity of themedium, and Δχc is the effective magnetic susceptibility ofthe cell, represented as

cp

cp N

R

R

3

3(8)

Here, Rp is the radius of the nanoparticle. Therefore, theaverage velocity of cells inside the channel can be calculatedby averaging across the height of the channel

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vHR x x

Have

c c d 19

2

0

2

0

B (9)

To determine the field strength required for a successfulcapture, we equate the vertical distance moved by the cells tothe height of the channel (τ · νave = H), yielding

B0 04 4

9 1

R

HW L S H Sc

2c

FR (10)

Enhancing the magnetic force by increasing the magnetic

field intensity can improve the cell capture rate. For otherparts of the immunomagnetic system, we make the assess-ment based on the required minimum magnetic field. Thelower this value, the easier the cells are captured. There areseveral ways to reduce this value, such as decreasing thescreening flow rate (FR) or using stronger magnetic tags andincreasing the labeling efficiency. However, from the per-spective of scale, the immunomagnetic separation systemcan be optimized by decreasing the height (H) of the captureregion and increasing the cross-section area (W × L) ofthe device. The result indicates that using miniaturizedimmunomagnetic devices may be critical to increase CTCdetection efficiency.

Microchip based immunomagneticassay

With the recent advancement of nanotechnologies andmicrofabrication techniques, researchers are able to makeminiaturized tools to observe, measure and manipulateextremely small objects. For rare cell detections usingmicrofluidics, such microsystems provide precise controlof the flow behavior, transportation, and biological inter-actions in the microchannel environment. Integration ofmicrochip technology with immunomagnetic assay has beenwell pursued for separation of rare cells.

Similar to the conventional MACS system, a microchipbased immunomagnetic assay uses magnetic beads/particlesthat are conjugated with cancer specific antibodies to labeltarget cells and uses an external magnetic field for capturing.Depending on the direction of the magnetic field andthe final status of the target cells, the microchip basedimmunomagnetic assays work either in a retaining mode,where CTCs are captured and fixed on the substrate,29 or ina deflection mode, where CTCs are magnetically driven todifferent streamlines to be collected at different outlets ascell suspension.51,69 There are also hybrid microchannelsbeing reported that consist of multiple functionalities orseparating mechanisms integrated on the same chip.53,70

Early work on combining microfluidic and immunomagneticassays to study cells in the retaining mode began with analyticalmagnetapheresis, which was proposed to compare magneticproperties of iron-rich protein (ferritin)-labeled human lympho-cyte and magnetite-doped dynabeads.68 Fig. 3a shows the

This journal is © The Royal Society of Chemistry 2014

setup of the analytical magnetapheresis. The samples arestored in a syringe before they are pumped, at a controlledflow rate, into a carrier medium-filled microchannel, whichis placed over permanent magnets. Magnetized cells anddynabeads are magnetically attracted to the interpolar gap ofthe magnets. Cell samples fixed and stained on the slidescan be used for microscopic analysis. Magnetic susceptibilityof the ferritin-labeled lymphocyte and the necessary number offerritin molecules per lymphocyte for the magnetic separationare measured and calculated using the developed analyticalmagnetapheresis.

Recent work on a microchip-based immunomagneticseparation system in retaining mode has been successfullydemonstrated with CTCs (Fig. 3b).30 The automated systemyields high capture efficiency and high throughput withsamples from breast, prostate, and lung cancer patients. CTCsare labeled with ferrofluids, which are conjugated with anti-EpCAM. During the screening process, nanoparticle-labeledCTCs suspended in whole blood sample are pumped into theinverted microchip and are captured on the channel substrate.Other cells such as red blood cells (RBCs) and white blood cells(WBCs) escaping the magnetic entrapment are collected in thewaste syringe. The CTCs are permanently fixed on the channelsubstrate for immunofluorescence staining and microscopicobservation/identification. Clusters of CTCs, which might beclinically important, are found in patient samples using thissystem. The system takes advantage of the cell sedimentationand a motion-control program to keep the system working inthe optimum inverted orientation.

The deflection channel is also a popular design to collectrare CTCs. A microchip system is developed based oncontinuous-flow ferrofluid hydrodynamics to sort a mixtureof particles and live cells simultaneously.71 Fig. 3c showsthe sorting device with a stack of permanent magnetsplaced close to a microfluidic channel on the side face tosort different sizes of cells based on ferrohydrodynamics.The deflection system manipulates cells within the ferrofluidsuspension under the external magnetic fields in the latterpart of the channel. Escherichia coli (strain MG1655),Saccharomyces cerevisiae (Baker's yeast), and two differentsizes of fluorescent polystyrene microparticles are testedfor the sorting experiments. The device shows high through-put (107 cells h−1) and high capture efficiency (~100%). How-ever, the device needs to be further optimized in terms ofseparation resolution for target cells with smaller differencesfor clinical applications.

Viable CTCs can be retrieved using an immunomagneticassay for cell culture. Integration of microstructures with amicrofluidic magnetic separation device is developed toisolate CTCs from mammary cancer-bearing mice suspendedin whole blood sample, followed by the culturing of isolatedCTCs.70 Fig. 3d shows the device composed of a tilt inletchannel at a certain angle in a main microfluidic channelwith two rows of dead-end side chambers to store attractedCTCs and protect them from being damaged by shear stress.A permanent magnet is placed directly beneath the lower

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Fig. 3 Microchip based immunomagnetic assay for cell isolation purposes. (a) Setup of the analytical magnetapheresis system, which is used tostudy human lymphocyte. Cells are trapped at the inter-polar gap of the magnets. (b) Microchip based immunomagnetic CTC screening device inretaining mode. Permanent magnets are placed beneath the microchannel to capture and retain the cells on the substrate. (c) Microchip basedimmunomagnetic CTC separation system in deflecting mode. Permanent magnets are placed to the side-wall of the microfluidic channel to sortdifferent cell lines into different streamlines and are collected at the end of the channel. (d) Immunomagnetic microchip integrated with a cellstorage chamber to retrieve and culture CTCs. (e) Hybrid immunomagnetic microchip for CTC detection, including hydrodynamic cell sorting,inertial focusing, and magnetophoresis separation. RBCs, platelets and other blood components are removed in advance. Reproduced fromref. 68, 30, 71, 70, and 53 with permissions from ACS, Springer, RSC and AAAS.

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row of side chambers to attract and collect magnetic bead-binded CTCs. The device displays good isolation efficiency(87%) using spiked mouse metastatic M6C breast cancercells. Collected CTCs along with RBCs are cultured for molecu-lar analysis (RT-PCR) after the RBC lysis. The developed isola-tion platform can be scaled up to process large sample

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volumes, multiple types of CTCs, and other molecules forclinical applications.

Immunomagnetic assays can be assembled with otherseparating mechanisms to achieve better performance. Amicrofluidic chip (CTC-iChip) containing three separationstages, namely debulking, inertial focusing, and immunomagnetic

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separation, is fabricated for CTC detection.53 The system(Fig. 3e) incorporates three microfluidic functions to replacebulk RBC lysis and centrifugation, hydrodynamic sheath flowin flow cytometry, and magnetic-activated cell sorting. Hydro-dynamic size-based filtration is performed in the first stageusing an array of micropillar structures, in which RBCs, plate-lets, plasma proteins, free magnetic beads, and other bloodcomponents are discarded through the top outlet. Theremaining CTCs and WBCs are then flowed to the secondstage for inertial focusing before going to the third stagewhere immunomagnetic separation is performed. The devel-oped CTC-iChip is capable of immunomagnetically sortingepithelial and non-epithelial cancer cells in both negativeand positive modes. The captured cells are then used forRNA-based single-cell molecular analysis. The microfluidicdevice exhibits high capture efficiency for different humancancer cell lines expressing different levels of EpCAM.

From conventional MACS to microchip based immuno-magnetic assay, the downscaled systems provide betterconfinement of the flow field and magnetic field. Using aminiaturized microchip with a short vertical height anda large cross-sectional area helps the magnetic capture.However, the miniaturization is limited by our need toavoid cell clogging and a desire to maintain laminar flowinside the channel. In addition, the channel has to be madelarge enough to keep a substantial system throughput.

Approaches based on micromagnets

To enhance the separation efficiency while maintainingthe throughput, researchers have come up with differentapproaches, including the local magnetic field control byintegrating small-scale magnets inside the microchannel.This is to precisely modulate the magnetic field gradient andfacilitate cell distribution after capture. The micro- andnanoscale magnets (to simplify the notation, the term“micromagnets” is used later in the paper to represent allthe micro/nanoscale magnetic structures) are engineered togenerate a periodic, strong localized magnetic field. Thearray of magnetic elements inside the immunomagneticchannel enhances the interactions between the cells andmagnetic field.29,30,67 Integration of these micro-flux sourcesopens up a new way to optimize CTC separation withoutsacrificing the system throughput.

Micromagnetic field generation

There are two ways to induce the localized micro-magneticfield: using micro-electromagnets72,73 or static micromagnets.74–76

For chip-scale implementations, the micro-electromagnetsoften involve the fabrication of 3-D micro-coils on the planarsubstrate inside the microfluidic channel77 and the usage ofan additional current source to actuate the coil and generatemagnetic field. Using electromagnets, the intensity and distri-bution of the magnetic field can be easily tailored by control-ling the actuation current. However, the external power source

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hinders the portability of the entire system. Strength of themicro-electromagnet is limited due to the relatively weakmagnetic field and heating issues.

In practice, the static magnetic approach is more widelyadopted due to its simplicity in implementation. In recentstudies, soft magnetic materials are directly integrated ontothe substrate of the microchannel using conventional semi-conductor fabrication techniques such as photolithographyand deposition.74–76 Upon application of an external magneticfield, these static micromagnets can be easily magnetized andcan generate a localized magnetic field that is 100 timesstronger than those magnetic fields produced by electro-magnets. Using strong permanent magnets (like the NdFeBmagnet) as the magnetization field source, the static magneticapproach can be made compact. Static micromagnets exhibitgreat potential for serving as a portable diagnostic system andwill be the main scope of our discussions below.

Micromagnet fabrications

Recent micromagnet fabrication technologies presented in theliteratures include 1) semiconductor fabrication techniques,which usually consist of photo-patterning using a photoresistto define the locations of the micromagnets. The micromagnetelements are integrated through techniques like sputtering,thermal deposition or electroplating, depending on therequired size, thickness and resolution.72–76,78–80 2) Shrinkinduced micromagnets, in which Ni is deposited ontoshape memory polymer films. Upon heating, the polymerfilm shrinks in lateral dimension, causing the Ni film tobuckle and wrinkle and producing the micromagnets.81

3) Thermomagnetically patterned micromagnets, which useheat irradiation through a mask to selectively switch themagnetization direction of a thin film magnet and form anarray of oppositely magnetized micromagnets.82,83 4) Ferro-magnetic material encapsulation, in which PDMS is used toencapsulate ferroferric oxide powder into the master templatemade with SU-8. A micromagnet array is formed upon PDMSdemoulding from the master template.84

Given the scales of these fabricated micromagnets, theycan be easily integrated within microfluidic channels tofulfill target sorting, focusing, and isolating functions. Thetargets are usually magnetic microbeads, nanoparticlesor cells labeled with magnetic tags.74,78,83,85 To choosethe proper technique for immunomagnetic cell isolationpurposes, we should consider various factors such as mate-rial costs, fabrication equipment availability, reproducibility,and large scale medical/clinical applications.

Micromagnet-based designs and applications

For chip-scale immunomagnetic cell separation, micro-magnet structures are particularly important in the sense thatthey affect the magnitude and distribution of the local magneticfield, which directly determines the surface retaining force.For example, for small targets with weak magnetic response,large micromagnet elements are needed to provide sufficient

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attracting and retaining force. Therefore, it is essential toadapt the structures for specific applications, depending onthe size, magnetic properties of the target, dimensions of themicrochannels, and flow rates.

As illustrated in Fig. 4, the demonstrated structuresinclude (1) micro-strips: the Ni strip is sputtered onto thesubstrate to alter the directions of cells with differentmagnetic labeling. Leukocytes are successfully separated andcollected at the end of the microchannel.75,78,85,86 (2) Micro-pillars: ferromagnetic post structures are integrated insidethe microchannel and can be used to capture targets movingpast them in the flow. The system is demonstrated by sepa-rating magnetic micro-beads from non-magnetic beads.84,87–90

(3) Micro-grooves: the groove structure is formed using theshrinking mechanism and aligned randomly on the substrate.The system can work either in positive extraction mode, wheretargets are directly captured inside the channel, or in negativecollection mode, where non-targets are retained. DNA canbe collected from a small amount of solution for profilingpurposes.81 (4) Micro-chessboard: fabricated using heatirradiation, the resolution and alignment can be well con-trolled in an alternating pattern. Similarly, the system hasonly been demonstrated to be capable of separating mixed

Fig. 4 Micromagnet technologies used for cell separation. (a) Micromagnenickel inside a PDMS microchannel. (c) Groove micromagnet made usingusing thermal patterning techniques. (e) The patterned thin film made usifrom blood. Reproduced from ref. 85, 97, 81, 82, and 92 with permission fro

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solutions of magnetic and non-magnetic microparticles.82,83,91

(5) Patterned thin-film: compared with previous structures,a patterned thin film has been specially designed for andsuccessfully applied for CTC separation. The thin filmmicromagnet, with a thickness of 200 nm, can generate suffi-cient magnetic force to fix the CTCs in a certain range. Thepatterned array not only helped increase the capture rate,but also distributed the cells to make full usage of the spaceinside the channel.74,92

To combine the micromagnet with the immunomagnet CTCdetection assay, the fundamental requirement is to enhancelocal magnetic force to facilitate surface cell retaining. Addi-tionally, an appropriate micromagnet system should (1) mini-mize possible physical damage to the target cells inside themicrochannel; (2) reduce the aggregation of cells and freenanoparticles; (3) provide retrievable cells after capture forsubsequent cellular imaging and downstream molecularstudies; and (4) have robust fabrication processes so we canscale up for mass production. We summarize the fabricationprinciples, materials, micromagnet dimensions, capture rangeand applications in Table 1. Here, the capture range is themaximal distance away from the magnetic element where thetargets can still be attracted in the immunomagnetic assay.

tic strips used to guide and sort cells. (b) Micro-pillar structure made ofthermal shrinking techniques. (d) Chessboard micromagnets fabricatedng thermal deposition. It has been used to successfully separate CTCsm AIP and IEEE.

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Table 1 Comparison of different micromagnet structures

Micromagnetstructure Fabrication technology Materials Dimensions

∇B2

(T2 m−1)Capturerange (μm)

Demonstratedapplications

Strips85 Etching and sputtercoating

Ni 10 μm wide, 2 μm thick, 35 μmperiod distance

~50 10 Separation of leukocyteand red blood cells

Pillars87 Soft lithography,electrodeposition

Ni 7 μm height, 15 μm diameter ~500a 50 Microbeads filtration

Chessboard83 Deposition, pulse laserirradiation

NdFeB film Feature size 50–100 μm, reversaldepth 1.1 μm ± .12 μm

~5 40–70 Microparticles separation

Grooves81 Deposition, heating andshrinking

Ni, polyolefin(PO) film

20 μm height ~30 60–70 Microbeads separationand DNA extraction

Micromagnetarray92

Photolithography andthermal deposition

Ni 200 nm thickness, 20 μm × 20 μmlateral dimension

~45 20–30 Detection of CTCs

a Dimensions are found from the respective paper cited.

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Different designs use different targets in their demonstrationexperiments. To eliminate the impact of these variations ofthe targets and only compare the capabilities of generatingthe retaining force of different micromagnet structuresobjectively, we estimate the magnetic gradient ∇B2, whichcan be considered as a direct reflection of the magnetic forcein eqn (5).

In a conventional MACS system, like CellSearch™, fluo-rescent signal is the only available outcome. However, byusing a microchip-based immunomagnetic detection assayand by integrating in-channel magnetic structures, CTCs canbe captured and retrieved, allowing more in-depth analysis.Meanwhile, with the development of single cell profiling tech-niques, CTCs have made significant contributions to oncologyand clinical studies. Potential downstream molecular studiesfor CTC can be performed using a set of advanced toolsincluding polymerase chain reaction (PCR), reverse transcription–polymerase chain reaction (RT-PCR), quantitative reversetranscription–polymerase chain reaction (qRT-PCR), digitalPCR, and fluorescence in situ hybridization (FISH) on selectedcells.25,26,53,93,94 The correlation between primary tumors andcorresponding CTCs95 or heterogeneity of different cancer celllines55,96 may have clinical implications for discoveries ofnew drugs and targeted therapeutic strategies in patients.Moreover, breakthroughs regarding the gene expression ofCTCs97 and epithelial–mesenchymal transition (EMT)65 couldhelp provide new insight into metastasis.

Conclusion

Magnetic cell separation plays a significant role in biologyand medicine, where cells can be sorted under the combinedforce of biological bindings and magnetic fields. It is particu-larly valuable for rare cell separation such as for CirculatingTumor Cells or CTCs, which poses a unique challenge interms of the required high sensitivity and specificity forprecise clinical interpretations, without tremendous con-sumption of samples and assay. In this paper, we review therare cancer cell sorting technologies based on magnetic-activated and immunomagnetic assays over multiple scales.

This journal is © The Royal Society of Chemistry 2014

We begin the discussions on the macroscopic cell detectionsystems such as magnetic-activated cell sorting systems,followed by the scaling laws on miniaturization towardsmicrofluidic chip based immunomagnetic assay for cell sepa-ration. Furthermore, we introduce recent work that integratesmicro-magnets in separation experiments. Related designconcepts, principles, and microfabrication techniques arepresented and evaluated. Effective enrichment of raretumor cells across glass slides, combined with downstreammolecular analyses, may provide a minimally invasive toolto monitor cancer diagnosis and prognosis.

Acknowledgements

We thank our collaborators Professor Konstantin V. Sokolov atthe University of Texas at Austin, Professor Eugene P. Frenkel,Professor Jonathan W. Uhr, Nancy Lane, and Dr. MichaelHuebschman at the University of Texas Southwestern MedicalCenter for the discussions related to nanoparticles developmentand clinical applications. We are grateful for the financialsupport from the National Institutes of Health (NIH) NationalCancer Institute (NCI) Cancer Diagnosis Program under grant1R01CA139070.

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