MICRO/NANOSTRUCTURED SUBSTRATES FOR CELL TYPING … · Micro/Nanostructured Substrates for Cell Typing, Isolation and Disease Diagnostics 55 Figure 3. Conceptual figures depicting

In: ISBN: 978-1-62808-020-9 Editors: M. J. A. Shiddiky, E. J. H. Wee, S. Rauf et al. © 2013 Nova Science Publishers, Inc.

Chapter 3

MICRO/NANOSTRUCTURED SUBSTRATES FOR CELL TYPING, ISOLATION AND DISEASE DIAGNOSTICS

Mohammed A. I. Mahmood,1,2,3 Yuan Wan4 and Samir M. Iqbal1,2,3,5,6,

1Nano-Bio Lab, 2Nanotechnology Research and Education Center,

3Department of Electrical Engineering, University of Texas at Arlington, Arlington, TX, US

4Mawson Institute, University of South Australia, Mawson Lakes, Adelaide, SA, Australia

5Department of Bioengineering, 6Joint Graduate Committee of Bioengineering Program,

University of Texas at Arlington and University of Texas Southwestern Medical Center at Dallas,

University of Texas at Arlington, Arlington, TXs, US

ABSTRACT

Circulating tumor cells (CTCs) are the cells shed from primary and/or metastatic tumors. These cells often enter into peripheral blood circulation system and travel along. These are important in understanding biology of metastasis, cancer prognosis, disease monitoring and personalized therapy. A number of approaches have been reported for the direct or indirect isolation of celloss of cell viability after isolation, and possible perturbations in the cell microenvironment during capture make cell typing and isolation quite challenging. A number of approaches have been developed at micro/nanoscale for cell-level diagnostics in last few years that require very little sample preparation while enabling multiplexed analyses, adding improved sensitivity and promising lower cost of analysis in drastically reduced turnaround time. This chapter introduces and reviews many important features of

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Mohammed A. I. Mahmood, Yuan Wan and Samir M. Iqbal 52

micro and nanoscale substrates and devices especially more recent ideas of nano-texturing towards ultra-sensitive and very specific cell detection and isolation.

1. INTRODUCTION Cancer is one of the diseases that has high mortality rate even after decades of research.

Undesired genetic mutations result in abnormal cell growth and tissue re-organization, causing cancer. In 2012, in the United States only, 1.6 million men and women were projected to be diagnosed with cancer [1]. Billions of dollars have been invested in healthcare and research toward development of effective cancer diagnosis and therapy. Patients diagnosed at early cancer stages have high survival rates. However mortality increases exponentially with delays in diagnosis. This underwrites the significance of cancer diagnosis at early stages. Early detection and localization dictate the effectiveness in combating this disease. In addition, cancer relapse even after surgical removal of tumors is very common. Thus continued monitoring of cancer related biomarkers in patients increases the survival rate by manyfold. Cancer biomarkers are statistical in nature and a blurry boundary exists between healthy and tumorous concentrations with no definite threshold. The situation is aggravated by having no single marker for cancers of various types. As a result a multimodal approach is important for effective diagnosis.

Tumors start to spread and invade to otherwise healthy organs/tissues distant from the primary tumor via blood stream, a condition called metastasis. Australian pathologist Thomas Ashworth in 1869 first observed the presence of abnormal cells in blood resembling more to the tumors than the healthy cells [2]. Its importance was immediately perceived as a potential cause of cancer spreading as well as early diagnosis. Since then, several strategies for the detection and isolation of circulating tumor cells (CTCs) from blood stream have been reported. Numerous techniques relying on mechanical/hydrodynamic forces and affinity interactions have been developed. Methods such as dielectrophoresis [3, 4], flow cytometry [5] and magnetic attraction [6-8] have been employed to enumerate and isolate tumor cells from the blood stream. The race is on with many novel and interesting strategies coming up rapidly. This chapter focuses on these micro and nanoscale approaches towards cancer detection.

2. MECHANICAL AND HYDRODYNAMIC SEPARATION Mutated cells have distinguishing physical characteristics than healthy cells in terms of

deformation, increased nuclear size as well as changes in other internal organizations. It was previously reported that cell lines derived from liver, prostate, breast, and cervical human carcinomas had significantly larger cell sizes compared to peripheral blood leukocytes [9].

A number of separation techniques are based on measuring the effects of mechanical and/or hydrodynamic forces on the cells, usually in a microfluidic environment. These techniques separate target cells based on their physical attributes and do not employ any ligand-receptor affinity. Methods of such separation have evolved from simple porous sieve devices. Power of such techniques lie in their simplicity since cells do not have to undergo


Micro/Nanostructured Substrates for Cell Typing, Isolation and Disease Diagnostics 53

any pre- or post-treatment by Size of Epithelial Tumor cells using filtration

through a "sieve" [10,11]. A filter-based micro-device exploits the cell size differences between CTCs and the smaller normal cells for efficient isolation and on-chip analysis (Figure 1). A speedy CTC identification is claimed by this approach. The captured cells are reported to remain viable for further manipulation such as electrolysis or immunofluoresence.

A multistage microfabricated sieve device was reported to fractionate cancer cells from human blood [12]. The device consisted of successive channels, each having two dimensional arrays of columns with varying dimensions. Huang et al. reported a method of particle separation based on size, through lateral zigzag displacement and by bifurcation of laminar flow around obstacles [13]. Tan et al. created multiple arrays of crescent shaped isolation wells for trapping cancer cells [14]. The gap in the crescent ensured that smaller blood cells and other cells effectively sieved through these and the cancer cells got trapped. Cell enrichment based on lift force difference between larger and smaller size has also been employed for separating cells [15]. This method is somewhat superior to sieve-based separation where filters get clogged. Reservoirs fabricated along the length of microchannels create microvortices and larger cells are subjected to greater lift force while passing by the reservoir. This results in the accumulation of larger cells in the respective reservoirs (Figure 2).

Inertial microfluidics based separation has been reported to separate spiked CTCs [16]. The preferential cell focusing was achieved in high aspect ratio microchannels. Much more has been detailed regarding this in the chapter of Hyun et al. in this book. Pinched flow dynamics was used to keep the low abundance cells at the center of the channel to be separated out. In another microfabricated device, isolation was accomplished by allowing the cell suspension and the buffer solution to flow in parallel in two separate channels connected by a vertical trapping route [17].

Figure 1. (A) shows a sieve based cell isolation approach using a parylene membrane sandwiched between PDMS slabs. (B) shows the uniformly spaced pores and (C) trapped cell in a pore. Reproduced from [10] with permission from the American Association for Cancer Research.



Figure 2. Cell separation with micro-vortex: (a) describes the device dimension as well as the schematic where larger cells are trapped due to lift force whereas smaller cells easily pass through; (b) shows the complete device with multiple parallel channels; (c) demonstrates parallel trapping of fluorescent particles; (d) shows the trapping mechanism and forces acting on the particles of various sizes. Reproduced from [15] with permission from the American Institute of Physics.

Flow rate difference between the channels allowed cells to enter the trapping routes and separation was achieved. A major drawback of the sized based sensors is that the selectivity is relatively low due to absence of size threshold of the target cells. A prior centrifugation step is often practiced as a first level separation.

Measuring temporal changes in individual cell response, when exposed to unfavorable or harmful conditions, is important. This can give insights about disease progression by observing the ability of cells in adapting to these conditions. Overexpression or abundance of certain proteins, metabolite secretions, intracellular enzyme activity or instability during incubation process can give important clues about the causes or states of a disease. Immobilization of single cell is difficult due to morphological variations of cells as well as their viability.

Eyer et al. fabricated parallel microchambers that could be pneumatically sealed and operated independent of each other [19]. The chambers had microhurdles that were used for cell capture. The captured cells were subjected to chemical treatment and the released biomolecules were extracted for further analysis. Many more approaches have evolved to use microfluidic chambers to study behavior of living cells. Figure 3 shows a few sketches of cell-trapping arrays. The sizes of traps are made so that only one cell can be captured [18].

Mechanical or Hydrodynamic methods, although simple, are not always effective, due to numerous types of cells and their overlapping behavioral traits. As a result, reliable selectivity is questionable when only these methods are used. These principles can be, however, conjugated with other techniques to improve selectivity and sensitivity.



Figure 3. Conceptual figures depicting cell traps for large-scale cell analysis. (a-c) Self-explanatory sketches show cells moving along flow and occupying the traps for later analysis; (d) captured HeLa Cells. Reproduced from [18] with permission from the Nature Publishing Group.

3. AFFINITY BASED ASSAYS

In many cases, cells are not solely differentiable based on their physical and mechanical

attributes. This results in poor selectivity in capture and isolation. It is thus imperative to employ other modes for effective detection. Cancer cells usually demonstrate abnormal expression of several proteins and receptors, when compared to healthy cells. Abnormality is statistical in nature in both the number of the proteins and the extent of their mutation from the original forms. Several membrane proteins such as epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), prostate-specific membrane antigen (PSMA), etc. have been used for cancer cell isolation [20]. For instance, overexpression of EGFR has been associated with cancer, especially lung and glioblastoma. EGFRs are widely expressed on cellular surfaces of tumor cells with density ranging from 40,000 to 100,000 per cell. A mutant EGFR, EGFRvIII, has been observed in lung and glio-carcinomas which is responsible for cells' proliferative nature [21].

In an attempt to isolate cells, molecules which can target specific membrane proteins recognizable on the cells of interest should be immobilized onto the devices first, usually in a microfluidic environment. These molecules can then subsequently capture cells. On the contrary, unwanted cells that have low or normal expression of certain membrane protein can pass without any adhesion. The affinity interaction between specific molecules and corresponding cell membrane proteins cannot be altered; therefore, the major challenge is increasing the odds of the contact between immobilized molecules and cells in microfluidic devices. In the last two decades, many designs of microfluidic devices have been reported and more recently, devices with appropriate surface modifications have also been developed for cell isolation. Under optimized flow rate, minimized steric hindrance, controlled shear stress, and suitable topography, cells of interest can be efficiently isolated. Developing better devices is a continuous process. Some of the designs with significant impact are briefly introduced here.

There is increasing recognition of aptamers for their great utility in cancer diagnosis and therapeutics. Aptamers are nucleic acids and have been shown to have affinities and



specificities that are comparable with those of antibodies, but have the advantage of being highly stable at a variety of salt and ionic conditions and can be reversibly denatured. These can be synthesized with labels and functional groups at specific sites, giving much more flexibility in design. Moreover, aptamers are much more hydrophilic than antibodies, thus these may provide surface passivation against nonspecific binding. The development and discovery of aptamers targeting protein rely on Systematic Evolution of Ligands by Experimental Enrichment (SELEX) technique, which is used for isolating biomolecule specific to ligands [22]. Aptamers for EGFR and PSMA have been used for cell isolation. The work is ongoing to produce aptamers for other molecules like EpCAM.

Overexpression of the receptors/antibodies used as biomarker is great for such aptamers but the downregulation poses a challenge as a detection modality with aptamers. There may not be sufficient number of target proteins on cell membranes for aptamers to work as probes. For example, low expression levels of EpCAM on various breast cancer cell lines has been reported [23].

An aptamer that selectively binds to EGFR has been extensively used to isolate and capture human glioblastoma (hGBM) cells (Figure 4). This RNA aptamer has the tendency to fold into 3-dimensional structure which has high affinity to the EGFR, making it suitable for affinity based isolation [24]. Iqbal and coworkers have demonstrated anti-EGFR aptamer capture efficiency (ratio of the number of captured cells to the total number of tumor cells) to be 62% [25]. The specificity of the aptamer was attributed to its ability to bind to the extracellular ligand binding domain III of the receptor which is known to be present in both wild-type and mutant EGFR.

Figure 4. Steps for cancer cell isolation. Capture DNA sequence is first immobilized on the surface of the chip and it is then used to bind the RNA aptamer. RNA molecules fold into 3-D structures that have high affinity to EGFR. Cells with EGFR overexpressed on the cell wall bind strongly to the aptamer on the chip surface. Cells with low EGFR expression (normal cells) are washed away, leaving only cancer cells on the surface.



Figure 5. Device package (upper pane) using microposts made of silicon (lower pane). Whole blood spiked with lung cancer cells flows through the microposts and tumor cells get captured on the pillars. The cells are false colored in red. Reproduced from [27] with permission from the Nature Publishing Group.

Microfluidic devices with functionalized surfaces have been reported to be used for enhanced CTC isolation. These devices have the potential to be used as much better alternatives of present methods. Devices with microposts (Figure 5) and herringbone

-28]. Microfluidic channels functionalized with anti-PSMA aptamers have also been used for isolating prostate cancer cells [29]. The channel dimension was optimized to increase the collision of fluid borne cells with the channel wall. The capture bed was functionalized with anti-PSMA aptamers. Authors reported 90% recovery of the cells. These captured cells were released using trypsin reagent and were counted successfully using a contact cytometer.

EpCAM is the most targeted molecule for cancer cell isolation, which is expressed on almost all carcinomas. Microfluidic channels with anti-EpCAM antibody modified microposts in it were designed to increase capture sites and increased cell capture [27]. Laminar flow inside a microfluidic channel is a major impediment towards achieving maximum cell isolation. Due to minimal mixing in microchannel flows, cells tend to pass by the isolation surfaces without coming into any real contact. In a later report, a herringbone



texture was fabricated to achieve micromixing while using anti-EpCAM antibody for isolation [28]. This design provided an enhanced platform for CTC isolation. The passive mixing of blood cells inside the channel significantly increased the number of collision between target CTCs and the antibody-coated chip surface. Anti-EpCAM antibody modified curved channel also has been used for CTC isolation [30].

Some of the challenges of the affinity based devices include: (1) Lack of sufficient number of cancer cell biomarkers; (2) Differences in expression levels of target proteins; (3) Effects of operating parameters like flow rate, complex shear forces in channels, changes in binding affinity with ionic concentration, pH, etc; (4) Portability of the system in fields with harsh environments; (5) Variations in the number of biomarker expression within diseased cell population, etc.

Nanostructured scaffolds with nano-scale topography that mimic the natural extracellular matrix (ECM) or basement membrane have been used in tissue engineering. These provide in vitro cell culture environment that promotes cellular organization, cell matrix and cell-cell interactions, cellular proliferation and ECM synthesis [31]. The major advantage of nanostructured scaffolds is that larger biomimetic surface area can facilitate firm cellular attachment, and this feature can also be helpful for isolation and detection of rare cells [32]. As mentioned before, although detection and sorting based on affinity interactions can yield higher efficiency and greater specificity in contrast to mechanical and electrical sorting techniques -EpCAM antibodies in CTC-microchip has been shown to be around 65% [26]. In addition, the false-negative results in CTC isolation and detection are very much possible since all CTCs do not express EpCAM. The combination of affinity interactions and biomimetic nanostructured surface may further improve isolation and detection efficiency; therefore nanostructured surface have been introduced into this field.

Leveraging from development in Microelectromechanical Systems (MEMS), silicon based structures have been used as well. Mircon sized pillars in an array were used to enhance cell adhesive force [34]. Under larger surface-area-to-volume ratio, more adhesive elements were available for binding, and thus van der Waals adhesion was predicted to increase due to geometric features alone [35]. On the other hand, affinity interactions further strengthened bioadhesive forces. Hydrofluoric acid and AgNO3 solution have been employed to produce silicon nanopillars (SiNP) with diameters ranging between 100 200 nm on silicon wafer [36, 37]. The results showed cell capture yield was around 45 65% on SiNP, 10 times more than that achieved on flat silicon. In addition 84 91% cells on SiNP were viable for subsequent analysis. Cell morphology on flat silicon appeared rounded; on the contrary, cellular protrusions were observed on SiNP substrates. However, the length of SiNP had limited effect on cell capture efficiency once it exceeded 6 µm. Wang et al. further integrated SiNPs to microfluidic devices with chaotic micromixers [38]. A serpentine patterned SiNP substrate with antibody coating was overlaid with a chevron-shaped chaotic mixing channel. When cell suspensions were flown through the channel, the micromixers significantly increased cell-SiNP contact frequency by making vortex. It was demonstrated that cell capture efficiency could be up to 95% if flow rate did not exceeed 2 ml/h. It could capture more CTCs compared to CellSearchTM assay. CellSearchTM is a commercially available, FDA approved magnetic system for CTC isolation targeted on EpCAM.

Nanotextured substrates have also received much attention lately towards cancer cell enrichment [39]. It is believed that basement membrane can anchor cancer cells through cell



adhesion molecules; therefore it can improve cell adhesion and growth [40, 41]. In metastasis, before cancer cells enter into peripheral blood, these first have to attach appropriately to basement membrane, proliferate, and then break through the barrier [42]. On the basis of these characteristics, Iqbal and co-workers prepared aptamer functionalized nanotextured PDMS substrates for cancer cell isolation [43]. First, larger surface area improved the total number of immobilized aptamers and increased their density, which favored tumor cell isolation, and more importantly, the nanotextured surface mimicked basement membrane that facilitated cancer cell attachment. The aptamer density on nanotextured PDMS was putatively 20 times more than that on planar surface. Total number of captured cancer cells on nanotextured PDMS was also twice as much as that on glass surface. However, the higher physical absorption decreased isolation specificity, i.e. non-specific cell attachment on nanotextured PDMS also increased.

roughness and increased total surface area [44, 45]. They coated capillary channels with poly-L-lysine (PLL) which carried positively charged ammonium groups, and deposited negatively charged P-selectin grafted silica on PLL via electrostatic interactions. The total amount of P-selectin on silica was increased up to 35% compared to planar surface, which was helpful for cell binding. They found significantly slower rolling velocity of cells on silica coated surfaces. The total number of cells captured was twice as much as on planar control surfaces. Titanium Butoxide and halloysite nanotubes were also investigated with similar results. Wang et al. carefully controlled surface nanotopography over a greater range and studied its influence on cell capture [46].

Antibody modified silica beads from 100 to 1150 nm in diameter were deposited on hydrophobically treated glass slide surfaces. Cell suspension flowed through deposited silica beads surface at various flow rates. The results showed that cell capture profiles were complicated. One one hand, nanoscale surface did increase cell capture efficiency, but on the other hand wall shear stress was more pronounced.

The cell capture efficiency does not show linear relationship with increased nanoscale surface area. It could be due to mechanical deformation of cells at the cellular and subcellular levels. Zhang et al. coated silicon wafer with TiO2 nanofibers (100 to 300 nm in size) by electrospinning. The basic idea was the same, utilizing nanostructured surface for improved cell isolation efficiency [47]. They studied electrospining time and incubation time, and found that after 60 min of electrospinning and incubation, maximum cell capture yields could be achieved. It indicated that local topographic interactions were correlated to characteristics of horizontally packed nanofibers. Kim et al. coated wafer surface with polystyrene as etching mask, and dry etched with CF4 plasma to obtain nanopillars [48]. Their results also showed nanostructured surfaces could improve cell attachment and therefore increased cell isolation efficiency.

These research directions prove that nanostructured surfaces can increase cell isolation efficiency; meanwhile these are also affected by flow rate and wall shear stress. However, jury is still out on the best nanotopography roughness. Cell isolation is a complicated process. It involves many factors like cell toughness, density and quantity of adhesive elements, immunoaffinity interactions, substrate material, surface topography, flow rate, channel dimensions, various flow conditions, and so on. More work is needed to explore best conditions for all the various factors.



Figure 7. Isopotential lines around a circular electrode in a DEP device. The field gradient is maximum near the pole and hence cells will be subjected to greater force and deflection, determined by the given equations.

As the equation suggests, the force applied on the cell depends on the gradient of the applied electric field [59]. Maximum gradient is available in the vicinity of the electrode (Figure 7), thus a cell would feel maximum DEP force in close proximity of the electrode. A triangular pole electrode fabricated by electrodeposition of metal inside a microfluidic channel has been shown to create a high gradient electric field [59, 60].

Electrodes have also been fabricated inside the walls to form 3-D structures [61]. Such a device has been used to successfully separate malignant breast tumor cells from a two cell population. Nuttawut et al. fabricated a 3-D electrode in combination with hydrodynamic focusing to maximize the effect of the dielectric gradient [62]. Gascoyne et al. have optimized electrode design towards such separation [3]. They fabricated a DEP electrode array for cell separation employing a force balancing between DEP and hydrodynamic forces inside microfluidic channel.

DEP effectiveness has been shown to increase by labeling cells with particles of differing polarizability [63]. Hu et al stained rare bacteria bacteria was separated from a mixture of non-target bacteria that did not express this marker. The enrichment was more than 200-fold in a single run of sorting a mixture of 10,000 cells/s through a single-channel.

DEP offers certain advantages over conventional methods. In case of magnetic and fluorescence based separations, cells have to be tagged. The tags are then removed later in order to restore natural functions of cells. On the other hand, DEP uses intrinsic cell properties to isolate them. As a result, cells are not expected to become abnormal once these are separated. The efficiency of DEP depends on individuality of the cells. However, although as high as 90% recovery of tumor cells has been reported, it must be kept in mind that when cell density becomes high the dipole approximation is disturbed [3]. In addition, the effects emanating from interactions with other cells and boundary surfaces, and the effect of electrical double-layer polarizations, must be considered. As a result, DEP separation efficiency falls quite badly with increasing concentration. Moreover, since the gradient falls drastically as one move away from the electrode, the performance is seriously impaired if the



channel is too wide. There are also some undesired effects due to exposure of metal electrodes to the sample. Some reported side effects include heat generation and bubble formation, chemical interactions of metal with the fluid and hence degradation of electrodes. Fouling and contamination inside the channel that leads to unpredictable behavior is also a common issue.

An approach to alleviate the drawbacks of metal-electrode DEP is insulator-based DEP (iDEP) [4, 65, 66]. The electric field from a DC source is varied inside a microchannel by strategic positioning of insulators giving rise to non-uniform fields (Figure 8) [64, 67, 68]. This technique uses metal macroelectrodes, at very high potential, positioned at both end of an array of insulating microstructures. The non-uniform electric field is created in the vicinity of the insulators, creating the DEP forces.

Microfabricated iDEP devices have been shown to remove and concentrate bacterial cells, spores and viruses [4]. The applied electric field was 2,000 V/cm DC. The device trapped particles selectively when dielectrophoretic force overcame electrokinesis. Trapped particles were then released by removing the electric field. A sawtooth geometry was designed to isolate cells from whole blood [69]. The dimension of the geometry was varied at steps so that DEP forces increased monotonically. Cells passing through channel were subjected to pumping action, electrokinesis, electroosmosis and DEP, not necessarily in the same direction but at the same time. If the resultant force was zero the cell was trapped.

The iDEP approach offers low-cost fabrication since no deposition of metal microelectrodes is required [4], and the insulator structures are inexpensive to manufacture. Moreover, the sample does not necessarily come in contact with the electrodes, thus the fouling or chemical instability is absent, unlike for conventional metal electrode DEP. However, the drawback of using iDEP includes requirement of a very high voltage source to produce an effective DEP force, making it hazardous to use.

Figure 8. iDEP using insulated electrodes and field focusing due to the strategically positioned posts. The shades around the posts indicate the intensity of the electric field. Reprinted with permission. Reproduced from [64] with permission from the Elsevier.

Carbon based electrodes have been proposed to eliminate the drawbacks of DEP/iDEP, because of higher chemical stability and ability to use lower potential. A 3-D carbon electrode design (instead of planar electrode) has been shown to increase efficacy [70]. Another approach to improve performance is using contactless DEP (cDEP) which employs the advantages of DEP while mitigating the drawbacks [71]. In this case, a highly conductive



charged fluid channel is fabricated in order to create the electric field, instead of a solid metal electrode [72].

The channel geometry is manipulated to create a non-uniform field necessary for DEP force. This technique has been claimed to minimize joule heating, bubble formation, electrochemical effects as well as touted to be inexpensive to fabricate. cDEP has been reported to successfully trap and selectively isolate viable leukemia cells from non-viable cells. Michael et al. have developed a model to also evaluate the effects of geometry in cDep devices [73].

5. MAGNETIC ISOLATION Magnetic cell isolation techniques offer simplicity in terms of fabrication. At slow flow

rate, charged ions present in the solution would not be affected by the applied magnetic field which is mostly static, hence providing better selectivity. Again when compared to vigorous force applied in centrifuging or even filtering, magnetic sorting is milder and thus prevents any damage to cells during processing [7]. However, tagging the cells with magnetic labels and later un-tagging introduce added complexity. Also, complete release of magnetic tags is sometimes difficult. This raises a question of cell viability for further processing.

Some cells are inherently paramagnetic because of their iron content [74]. As a result, these can demonstrate sufficient response to magnetic field without any external tag. For example, red blood cells contain high concentration of paramagnetic hemoglobin. There are some bacteria which show similar behavior. However, CTCs lack any such inherent magnetism. To separate CTCs from blood flow, it is important to selectively tag the tumor cells with magnetic labels while keeping the healthy cells unaffected. This creates a magnetic contrast between the healthy and tumor cells. Conventional magnetic isolation can then be applied for isolation and sorting.

Response of a magnetic material subjected to a magnetic field depends on the atomic structure and temperature. Such materials susceptibility ( ) as . This equation gives the magnetization level induced in a material by a magnetic field of intensity H.

Magnetic particles used in cell isolation are usually confined within coatings (polymer or oxide). The coating can be functionalized to suitably link with the targetted moiety. Paramagnetic or super-paramagnetic materials are used for cell tagging. It is highly desired that tagging materials have the least amount of hysteresis or magnetic memory (Figure 9). This prevents any aggregation of the target cells along the process and hence a homogenous suspension is possible. Super-paramagnetic materials show magnetic property only in the presence of applied field. Without an applied magnetic flux, these behave as regular particles and hence do not aggregate. Particles sized below superparamagnetic limit fail to show magnetic property at room temperature as thermal energy exceeds the energy required for maintaining magnetic anisotropy. So the net magnetization is zero. This switchable magnetic property is important for successful attachment of the particles to the cells without interactions between themselves.

The size of the magnetic particles has to be carefully selected as well. The diameters of the magnetic particles usually are in the range of 1-5 micron. Smaller particles or molecular

M H



labels are easy to attach, as these do not need agitation and can get attached quickly to the cells by Brownian motion.

Figure 9. Hysteresis diagram for magnetic material showing non-existence of hysteresis for smaller particles, essential for magnetic cell isolation. FM is for ferromagnetic particles and SPM stands for superparamagnetic particles. Reproduced from [75] with permission from IOP Publishing Ltd.

Larger particles aggregate and hence it is important to agitate them constantly so that articles also tend to form cages around the cells and only few

particles get bound to the surface. There is also a possibility for the larger particles to pull out the targeting moiety from the cell surface once subjected to magnetic force. However, larger particles have larger stray magnetic field and these are easy to manipulate. These are also relatively inexpensive. Nanoscale magnetic particles need very strong magnetic field or field gradient to manipulate.

In usual magnetic experiments, Magnetic force (Fmag) acting on a paramagnetic particle, known as Lorentz force, depends on the magnetic dipole created by the applied field, given by

Here B is the magnetic field strength applied on a particle with magnetic moment of m.

This force has to be balanced with the following forces in order to manipulate the cells. (i) Hydrodynamic Force stems from the drag force exerted by the medium molecules on

the particles. Due to low Reynold's number in microfluidic channels, fluid flow is inherently laminar. This facilitates transport and manipulation of cells inside the channel. In a pressure driven system, the velocity in the channel is parabolic. Flow in

long a microfluidic channel also affects this force. Since viscosity depends on temperature this force changes significantly with the variation in the ambient temperature as well.

(ii) Derjaguin Landau Verwey Overbeek (DLVO) Force refers to the sum of van der Waals force and the electrostatic forces between particles and the device surface. A good design aims at making DLVO force repulsive to avoid aggregation of the particles and adhesion between particles and the surface.

( . )magF m B



(iii) Brownian motion is caused by the random movement of the molecules. It is governed by the Stokes-Einstein equation which says that the displacement amplitude of a sphere is inversely proportional to the diameter. Magnetic particles are in constant collision with the medium particles resulting in random movement of the particles themselves. Brownian motion thus depends not only on the distance between the particles themselves but also between particles and the surface. Because of the difference in densities between the medium and the particle, gravity also plays a role while manipulating the magnetic particles. For example, a low speed fluid flow through a microfluidic channel can cause sedimentation due to gravity.

Liu et al. gave a comparative analysis of forces acting on the magnetic particles based on

their size (Figure 10) [76]. They demonstrated that as particle size becomes smaller, force from magnetic field becomes smaller and it become difficult to control the particles with the magnetic field only. As a result, smaller particles require larger magnetic field and higher gradients for successful manipulation.

Figure 10. A comparative study of balancing forces in a magnetic particle experiment. Reproduced from [76] with permission from the American Institute of Physics.

Physical and chemical structure is also another important parameter needed to be taken into account. Due to micro-roughness differences on the surfaces of the solids, the hydrophobicity of the surfaces can vary. The air can also be trapped on the surfaces which may in turn increase the hydrophobicity. This phenomenon is important to decrease cell adhesion to the surfaces and hence to increase the efficiency.



A simple method to separate out targeted cells from a cell mixture is to selectively tag the cells with magnetic particles. In the presence of the magnetic field, the tagged cells move towards the magnetic field and after removal of the non-specific cells, the field is removed and the target cells are separated out. This simple principle has been modified into many advanced techniques.

Figure 11. Magnetic Cell separation. In (a) a permanent magnet is used to attract and separate the magnetically tagged cells from the group. In (b) a controllable steel magnetic wool is used to immobilize cells on to the surface. When supernatant and non-target cells are washed out, the field is removed to recover target cells. Reproduced from [75] with permission from IOP Publishing Ltd.

A similar approach was reported back in 1990 by Miltenyi et al (Figure 11) [77]. A high gradient magnetic field was created in a channel by using a permanent magnet and steel wool matrix was inserted. Due to magnetization of the matrix, the magnetically activated cells were immobilized on the steel wool and these cells were eluted out by removing the permament magnet. A continuous cell sorter has been shown made with a quadrupole magnetic field [78]. The magnetic field acting on the cells in this arrangement pushed the cells towards the channel wall hence enriching the targeted cells at the periphery of the channel which were continuously separated out. FDA approved CellSearchTM technology employs magnetic method to selectively tag cells overexpressing EpCAM protein using magnetic nanoparticles coated with antibody [2]. Sensitivity has been reported to be on the order of 1 CTC per 7.5 ml of whole blood and this technology has been shown to be reproducible, sensitive and has been validated.

Magnetic flow cytometer works in a similar fashion as fluorescence activated cell sorting (FACS) technique [8]. Magnetically tagged cells are passed through microfluidic channel in between a spin-valve magnetic sensor. A spin-valve device consists of two conducting

-magnetic conducting layer [79]. When an external of the magnetic layers towards alignment, spin-



dependent electron scattering is reduced at the interfaces within the device. This decreases the electrical resistance of the interfaces. As cells pass through the sensor, these create a definite pulse indicating their presence. Increasing the speed and accuracy is the main challenge here.

Field flow fractionation (FFF) technique (Figure 12) involves interaction between hydrodynamic and magnetic forces [80]. FFF is usually done in a channel where flow profile is parabolic and laminar in nature.

Figure 12. Field Flow Fractionation. The separation is due to the combined action of both the flow in the channel and the applied electric field. Reproduced from [81] with permission from Elsevier.

Figure 13. Cell separation using magnetic beads. The self-explanatory sequence shows functionalized magnetic beads after injection in the channel. These are arranged along the magnet islands. After target antigens are injected, these get attached to the beads. Deactivation of magnetic field releases the target along with the magnetic beads. Reproduced from [83] with permission from Elsevier.

In FFF, a magnetic field perpendicular to the flow is applied. Magnetically active cells are drawn out towards the field and due to concentration gradient these tend to flow in opposite directions. At equilibrium, there will be a layer enriched with cells at certain distance from the center and because of the velocity profile these will have distinct flow velocity. This method gives a scope to determine and discriminate cells based on a whole range of properties, size distribution of the particle, etc.



Travelling magnetic field cell sorting has also been reported. Here, strips are alternately magnetized and this gradually separates the cells away from the flow [82]. Due to strong gradient, this gives better performance. The switching frequency is another controlling parameter in this method.

A magnetic beads based separation process has been reported by Choi et al [83]. Antibody coated magnetic beads were immobilized inside a channel by creating local electromagnets (Figure 13). Once fluid passed through the channel, antibody-specific cells got attached to the beads. The beads were then released for further processing. The chamber could be reused for multiple detection cycles. Saliba et al. created self-assembled hexagonally organized posts using magnetic beads [84]. These beads were functionalized for specific targets. Due to enhanced surface area, yield was reported to be much higher than previous approaches.

Magnetic field has been reported to be arranged to create varying forces on the magnetically activated cells [85]. Based on their magnetization, cells were levitated at different levels and then trapped. However, once the traps were occupied more cells could not be captured hence severely limiting the usefulness of the approach. Magnetic nanowires, because of their size and geometry as well as enhanced optical, mechanical and magnetic properties, are seen to gain growing importance in diagnostic and therapeutic applications. These are compatible with cells and living organisms and hence cell proliferation and gene expressions are not affected. Because of their anisotropic shape, these allow large forces and torques to be applied. One drawback of such metal nanowires is the hysteresis, which is mainly due to size. Their stray magnetization causes aggregation even after the driving magnetic field has been removed.

Nickel nanowires have been functionalized with antibody against mouse endothelial cells or fibroblast cells through self-assembled monolayer [86, 87]. The performance comparison of the Ni nanowire as a separation medium with magnetic beads has shown better results for nanowires. Ni nanowires has shown less cytotoxity with minimal use of sample as well as better cell viability.

CONCLUSION Large resources have been invested in cancer research but cancer related mortality has

remained somewhat constant for decades. The economical and social burden of cancer on patients and families is tremendous and hence it has become increasingly important to develop reliable, rapid and preferably non-invasive point-of-care (POC) systems for cancer diagnosis. The isolation of cancer cells and false-free identification has potential to fulfill the needs but also face some challenges.

Multifaceted expression of cancer renders it difficult to use one method for all types of cancers. An effective do-it-all cancer screening method is yet to be found. Having no perceivable symptoms before cancer becomes fairly malignant, the statistical nature of cancer progression and the absence of a definite threshold between cancerous and healthy cells in terms of their physical properties as well as their expression of cancerous biomolecules, all have made it difficult to develop such effective system.



A combined method is deemed necessary that can be efficient and provides quick decision from the collective data. Interdisciplinary systems employing engineering tricks at various levels are being used in the battle against cancer. However, being assisted by other technologies, biological methods need to be at the forefront of cancer detection at early stages. With that in mind, increasing insights in micro and nano-scale materials and their properties as well as their manipulation techniques have opened the door of development of highly reliable and much more effective arsenal of devices.

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MICRO/NANOSTRUCTURED SUBSTRATES FOR CELL TYPING … · Micro/Nanostructured Substrates for Cell Typing, Isolation and Disease Diagnostics 55 Figure 3. Conceptual figures depicting