NANO LETTERS
Rapid and Dynamic Intracellular Patterning of Cell-Internalized Magnetic Fluorescent Nanoparticles
2009 Vol. 9, No. 8 3053-3059
Peter Tseng,*,† Dino Di Carlo,‡,§,|,⊥ and Jack W. Judy†,‡,§,|,# Department of Electrical Engineering, Department of Bioengineering, Biomedical Engineering Interdepartmental Program, California Nanosystems Institute, UniVersity of California, Los Angeles, Los Angeles, California Received May 13, 2009; Revised Manuscript Received June 18, 2009
ABSTRACT Conjugated magnetic nanoparticles have recently demonstrated potential in activating unique and specific activity within cells. Leveraging microfabrication, we have developed a technique of localizing nanoparticles to specific, subcellular locations by a micropatterned ferromagnetic substrate. Controlled patterns of nanoparticles were assembled and dynamically controlled with submicrometer precision within live cells. We anticipate that the technique will be useful as a compact, simple method of generating localizable, subcellular chemical and mechanical signals, compatible with standard microscopy.
Magnetic particles have gained wide acceptance in biological and medical research as a method of selectively controlling biological environments. Antibody or DNA-conjugated particles, which have been used for highly specific methods of sorting cells, proteins and DNA, have also been used in conjunction with detectors to determine particle conjugation and size.1-3 More recently, researchers have integrated particles with additional characteristics,4 including conjugating particles with quantum dots for ease of detection, and fabricating porous particles (conjugated to enzymes) to be used as highly active reaction templates.5,6 Magnetic beads have been used to remotely generate heat,7,8 control ion channels, mediate signaling, and probe cell mechanics.9,10 Even with these advances, the potential for this technology and its use for the cellular localization of signals remains largely untapped.5,11,12 This is especially important given the high level of localization and compartmentalization important for cellular function. Micro-electromechanical systems (MEMS) technology and its ability to interface with microenvironments is well suited as a means of interaction with magnetically coupled biological matter. While there has been ample investigation into microfabrication as a means of sorting and manipulation of biological material,13-16 its use in generating magnetically * To whom correspondence should be addressed. Phone: 310-206-3995. Fax: 310-794-5956. E-mail:
[email protected]. † Department of Electrical Engineering. ‡ Department of Bioengineering. § Biomedical Engineering Interdepartmental Program. | California Nanosystems Institute. ⊥ E-mail:
[email protected]. # E-mail:
[email protected]. 10.1021/nl901535m CCC: $40.75 Published on Web 07/02/2009
2009 American Chemical Society
mediated, single cell biological activity is scarce.9,10 The capability of generating, in a simple fashion, highly localized chemical and mechanical effects could give biologists a simple method of probing cellular architecture and biochemistry, as well as providing a unique method of studying highly localized cellular signaling. In this report, we detail the use of simple micromachined magnetic substrates as a method of generating and manipulating complex, dynamically controllable, and localized groups of magnetic fluorescent nanoparticles within single living cells. The base elements of our substrate are simple ferromagnetic lines and dots. When combinations of these elements are placed in an incident magnetic field, unique magneticflux-density maxima will be generated as shown in Figure 1a. Within a cell, these maxima attract and coalesce particles, generating designed patterns of ensembles of nanoparticles at high speed. Importantly, the device is engineered for controllable localization (Figure 1a), as the magnetic potential minima can span nearly all x-y points of the substrate depending on orientation of the applied magnet field. Incident fields can be ultimately decomposed into two base modes, normal and tangential fields, combinations of which can be generated by a simple magnet oriented below the substrate. Our substrate design is composed of permutations of ferromagnetic arrays with dots that have a diameter varying from 2 to 8 µm, and a pitch of approximately 0.4-1 times the dot size. As an illustration of the arbitrary-patterning capability of our device, several patterns that spell out letters and words, both with dots and continuous regions of permalloy, were included (Figure 1b).
Figure 1. Ferromagnetic substrates. (a) Diagrams of our magnetic array in its two operation modes, tangential and normal incident field. Combinations of these modes generate unique potential minima spanning the x-y space of the substrate. (b) Schematic of the letters U C L A spelled out with our dot-pattern, followed by SEM images of our pattern (1) after permalloy electroplating and mold strip and (2) upon MFC7 breast cancer cell seeding and fixation. (c) The magnetic potential minima as generated by a strong magnet oriented underneath our substrate.
Figure 1b shows a schematic diagram and a pair of scanning electron microscopy (SEM) images of our device at various stages. The dots themselves are composed of permalloy (Ni80Fe20, a magnetically soft, high permeability, and moderate saturation ferromagnetic material), electroplated on a seed layer of 40 nm Ti/250 nm Cu/40 nm Ti to a thickness of 3 µm. Upon mold stripping, a thin layer (0.2 µm) of low-stress silicon nitride is plasma deposited as a passivation layer, and a final SU-8 layer planarizes the surface and acts as an adhesion layer for cells. Right before the cell-seeding process, the SU-8 surface is modified in an O2 plasma to encourage cell adhesion to the substrate. The magnetic manipulation force for particles is well known and is proportional to the magnetic field gradient for magnetically saturated particles (our case), Fmag ) VBsat(∇B/ µ0), with volume V, permeability of free space µ0, and saturation magnetization Bsat. Our nanoparticles are superparamagnetic, possess a saturation of close to 0.01 T, and have hydrodynamic diameters of 100 nm (nano-screenMAGDX, Chemicell, Berlin, Germany). Given the small volume and low saturation magnetization of the nanoparticles, which is common for water-soluble magnetic particles, their efficient manipulation requires large magnetic gradients and close proximity to potential minima (both of which are 3054
provided by our design). Essentially, we use ferromagnetic material to “focus” magnetic fields (to the point of magnetically saturating the dots and aligning their magnetization with the surrounding fields) to generate large, highly ordered magnetic field gradients, as shown in Figure 1c. Large shifts in the permanent magnet location generate small shifts in position of the potential minima, and thus the micromachined substrate displays the ability to dynamically modify the position of generated nanoparticle ensembles to highly accurate positions. For future reference, when the position of the magnet is noted as “weak [position],” this refers to the core of the magnet appearing approximately underneath the substrate, while “far [position]” refers to the core of the magnet being offset from underneath the substrate. Because of the shape anisotropy of the ferromagnetic elements, the elements will possess a sheared B-H loop, leading to a small amount of remnance and coercivity in the miniature magnet elements, even despite the soft nature of our permalloy (Supporting Information, Figure 1). However, this effect is relatively minimal and does not affect our technique while it is actively manipulating nanoparticles. The “focusing” effect of the permalloy was numerically simulated with 3D ANSYS FEA software, by modeling the permeability as a hyperbolic tangent function of our permalloy saturation and permeability, and obtaining solutions at various heights above the substrate. The thickness of the SU-8 layer used to planarize our substrate is verified to be approximately 0.5-1 µm above the permalloy dots. Contours of the magnetic flux density for both normal and tangential modes of operation are given in Figure 2a for a plane located 1 µm above magnetic dots placed in a magnetic flux density of approximately 0.6 T. Figure 2b shows the extracted contours (assumed as radially symmetric) and corresponding magnetic field gradients for our normal mode of operation. The peak of the gradient occurs expectedly at the edge of the dot, suggesting that the generated nanoparticle patterns will conform to the underlying ferromagnet definition (this occurs with the tangential mode too). The forces on our magnetic nanoparticles typically vary from close to 0.1 to 1 pN over its range of motion. The response time of a magnetic particle to our base array from any point can be determined directly by examining the equation of motion for the magnetic nanoparticles in our system, m(∂V/∂t) ) Fmag - Fdrag, with Fdrag ) 3πµaV, particle velocity in stagnant fluid is V, hydrodynamic diameter is a, and kinematic viscosity is µ. Because of the negligibly small mass of nanoparticles, the velocity will stabilize at a steady state velocity given by Vss ) Fmag/(3πµa), which is directly proportional to the magnetic field gradient at any position. This equation of motion for the nanoparticles can be solved to obtain a quick estimate of our response time (or time for assembled nanoparticle patterns to become noticeable). We will define this time as the initial formation time constant, τ. Ignoring the z-dimension, we obtain an approximate response time constant of 100 ms in water for a 4 µm diameter dot in 4 µm pitch array. Since the cell cytoplasm has a viscosity that is a complex function of spatial Nano Lett., Vol. 9, No. 8, 2009
Figure 2. Simulation of magnetic nanoparticle response. (a) Magnetic-flux-density contours as simulated in ANSYS at 1 µm above the patterned 4 µm diameter dots arranged in a 4 µm pitch array. Dashed circles indicate the edge of the ferromagnetic dots. (b) Overlayed plots of the magnetic flux density and corresponding radial gradient as we progress away from the dot midpoint for a normal incident field. The inset graph displays the contours of the magnetic flux density as generated by a best-fit polynomial (assumed radially symmetric). The edge is denoted by the dashed line and coincides with the approximate peak of the magnetic field gradient.
dimension, it would yield a value slightly higher than that for water. While our time constant for water was predicted for our 100 nm diameter particles, the manipulation speed for particles half this size (50 nm, which is closing in on the pore size for the nuclear membrane), would yield speeds only four times slower (400 ms), which is still fairly rapid. The magnetic fluorescent nanoparticles (excitation max, 476 nm, blue; emission max, 490 nm, high frequency green) used in our experiments are composed of a magnetic core covered first with a lipophilic dye, and subsequently with a polymer matrix around this dye. Presumably due to the processing of the particles, each individual particle possesses a random fluorescent yield. As seen in Figure 3d, a bottomoriented magnet applied to the letters U C L A (with nanoparticles diluted in water above it) would immediately Nano Lett., Vol. 9, No. 8, 2009
produce green fluorescing patterns directly above the dots on the substrate. However, what should be a fairly uniform cluster of particles generating a relatively smooth signal, produces instead a clearly punctuated signal at the permalloy dots (i.e., certain portions will fluoresce significantly stronger than others). This will also be reflected in our intracellular experiments, as ensembles of our particles will produce varying green fluorescing signals. We discovered, however, that the absorption of the particles in the blue-UV excitation range of the particles gives an excellent indication of nanoparticle presence and density, as these results correspond directly with our expected results. In addition, the nanoparticles were the only object within the cell that absorbed significantly in this region. As such, in our experiments, both the emission and excitation wavelengths of the particles are used to indicate nanoparticle presence and manipulation. The patterning capabilities of our substrate were verified in water under a fluorescent microscope with a setup as shown in Figure 3a, with a 1-T rare-earth magnet providing the incident magnetic flux. Substrates were inverted over a diluted solution of the nanoparticles within a Petri dish and excited under UV and blue light. As in Figure 3d, periodic potential wells were tuned to various x-y positions above the micropatterned substrate depending on magnetic orientation. In addition to basic patterning capability, we tested for manipulation reversibility (i.e., if the system returns to its original state upon field removal), and as expected from the soft magnetic nature of the permalloy elements and magnetic nanoparticles, the system would convert from dispersed, to captured upon magnet arrival, and immediately disperse once the magnet was removed. In water, dependent on the remnant fluidic motion, near full dispersal occurred within 7 min. Thus, in general the substrate displays the ability to reversibly generate a combination of chemical, magnetically mediated mechanical, and fluorescent signals to engineered locations, dependent on the orientation and application of the magnet. Manipulation of nanoparticles internal to live cells was also demonstrated within MFC7 breast cancer cells. The cells were grown in Dulbecco’s Modified Eagle Media (DMEM) and incubated with the nanoparticles for 24-48 h. Beyond 48 h, interactions between the endosomes of the particles would often lead to irreversible aggregation of the nanoparticles.17 After incubation, the cells were then washed multiple times in phosphate buffered saline (PBS), trypsinized, spun down, rewashed and sheared, and resuspended in media. For some experiments, live cells were simultaneously stained with a red lipid stain (diL) for improved membrane visualization. Because of the dextran coating and small size of the particles, the continuous washing resuspends and removes virtually all noninternalized particles from the system. The cells were then placed on the magnetic substrate and left overnight for cell adhesion (Figure 1c). The next day the media was washed and substrate inverted over a PBS-wetted Petri dish, so that it could be viewed with a conventional inverted fluorescent microscope, again as shown in the schematic diagram given in Figure 3a. Figure 3b displays a single cell with magnetic particles patterned by a magnet oriented to the far north and weak 3055
Figure 3. Generation of intracellular nanoparticle patterns. (a) Schematic diagram of our experimental setup. A 1-T rare-earth magnet is mounted onto a glass slide, and applied above our inverted substrate in a Petri dish for viewing with an inverted fluorescent microscope. (b) Blue filter and blue/red filter merged images of a single cell stained with red diL containing magnetically patterned nanoparticles. The nanoparticle absorbance illustrates the modification of patterns at the cell membrane left edge, suggesting intracellular localization. (c) General results indicating the capability of generating various x-y patterns within cells with varying incident magnetic fields. Various visible components are noted on the image. Diffuse cell autofluorescence and individual nanoparticle fluorescence can be seen in the green-fluorescence image, while permalloy elements and nanoparticle absorption contrasting against UV and blue reflected from the substrate can be seen in the UV-blue image. (d) U C L A patterned by nanoparticles in water with a directly normal magnetic field.
east. The image is notable because the magnetic nanoparticle patterns adjacent to the cell membrane appear modified, indicating constraint internal to the membrane and intracellular patterning. Confocal microscopy also indicated internalization of nanoparticles, both with a larger signal in the particle emission band (490 nm) and the significant presence of lipids within the cell (stained by diL and which suggests internalization within endosomes). Figure 3c displays our patterning results for various permalloy elements and at different magnet orientations. The various images from this figure indicate three disparate signals that are clearly viewable: (1) blue-excited and green-emitting fluorescent particles, (2) diffuse green autofluorescence of the cells, and (3) reflected short wavelengths from the substrate under the blue filter that reveals the array of micromachined ferromagnetic elements, and whose absence indicates the blue absorption of both the fluorescent dye and magnetic core of the nanoparticles. In addition to fluorescence microscopy, we performed SEM imaging of focused ion beam (FIB) etched cells with patterned magnetic nanoparticles. Cells were seeded onto our substrate as in previous experiments, and patterns were allowed to stabilize over an extended period of time in incubation (approximately 45 min in order to form a highly stable group of particles to survive the cell fixation). Cells 3056
were then fixed while the magnet was held in place, dried with the supercritical dryer, and imaged under the fluorescent microscope. Shown on the left in Figure 4a is a single cell (magnet applied to the weak south and weak east) observed under UV excitation, displaying enhanced cell autofluorescence induced by cell fixation, the permalloy posts, and the patterned, absorbing nanoparticles. Unfortunately, the fixation process induces significant additional background in the UV image; despite this, several nanoparticle ensembles are noticeably patterned according to our expected results. The substrate was sputtered with a thin layer of AuPd, and imaged and etched with a SEM/FIB (NOVA 600, FEI, Hillsboro, Oregon). Figure 1a highlights four generated ensembles and their correspondences under both top-down SEM and fluorescence microscopy of a single cell. FIB etching was initiated at the southern portion of the cell and progressed north, while sequential images of the cell cross-section were simultaneously captured (Supporting Information Video 1). Two of these cuts and the corresponding top-down image are shown in Figure 4b and display the morphology of the nanoparticle ensemble. The ensemble forms a type of hemisphere, as one would expect from the magnetic field simulations. A close-up of an ensemble is displayed at the bottom of the Figure 4, and the respective portions of our Nano Lett., Vol. 9, No. 8, 2009
Figure 4. Combined SEM imaging and focused ion beam (FIB) etching of stimulated cells. (a) Top-down images of the same fixed cell under UV exposure-blue emission and SEM imaging. Four clear ensembles are noted on each image. (b) Top-down and coinciding cross-sectional view of generated magnetic nanoparticle ensemble at two lines of cuts. The ensemble tends to resemble a hemisphere, as would be suggested from the magnetic potential landscape generated by the underlying ferromagnetic elements. At the bottom is a close-up of a single created ensemble. The nanoparticles are densely packed due to the long exposure time of the particles to the magnetic field and generate a noticeable bump in the cell.
technique, including the permalloy, SU-8, cell, and nanoparticle ensembles are highlighted. Preliminary statistics for the patterns generated by our device indicate accurate focusing of nanoparticle clusters. Figure 5a gives four separate scatter plots of the ensemble centroids corresponding to differing magnetic orientations, and include the mean and standard deviation of position. Two of these plots are scatter plots from groups of cells with internal patterned nanoparticles excited as in Figure 3c. We also introduce two more cases: cells above patterns with 4 µm diameter dots and arranged in a square array with a 4 µm pitch size, and a control situation above unpatterned portions of the substrate with no permalloy. As one would Nano Lett., Vol. 9, No. 8, 2009
Figure 5. Manipulated nanoparticle pattern statistics. (a) Scatter plots of the localization of the centroids of our patterned particle ensembles. Four separate cases are given: (1) cells above 4 µm diameter dots arranged in a 4 µm pitch array, (2 and 3) cells from Figure 3c with completed results from additional cells excited by the same field, and (4) cells above unpatterned portions of the substrate without permalloy. Included is the standard deviation, which indicates that the centroids typically occupy around a 600 nm to 1 µm radius around the mean position, whereas above the unpatterned substrate, the particles appear approximately randomly around the cell for the time scales of our experiment. In addition, the orientation of the magnet is notated by the letters “F, W” and “N, S, E, W” to represent the orientation of the magnet with respect to the permalloy dots. The image above the unpatterned substrate was contrast enhanced to better illustrate the cell and its encompassing diffuse particles. (b) Combined scatter and box plots of the fill factor of our particles (defined as the pattern area divided by the pattern unit cell area). Groups of particles are typically localized to around 15-25% of the area, indicating our ability to gather particles to areas significantly smaller than the size of the substrate.
expect, the standard deviation of the position of nanoparticle centroids above the unpatterned substrate is large because for the time scales of our experiments, they are not significantly manipulated (for longer manipulation times they form long chains oriented randomly around the cells). These data suggest that the particles are scattered approximately randomly about the cell, as compared to being localized 3057
significantly when patterned by our substrate and field. Figure 5b introduces a new, useful value: the pattern fill factor (the size of the ensembles divided by the area of the unit cell of the particular corresponding array). This value is useful as a gauge of how localized our nanoparticle ensembles are in the x-y plane. Unfortunately, due to the random nature of cell internalization, cells internalize varying densities of magnetic nanoparticles. As such, the variance of these statistics derived from the biology is large, as shown in the combined scatter and box plots of our data. However, we can gather that the groups of particles typically appear to occupy around 15 to 25% of the unit-cell area, indicating an ability to gather particles into space significantly smaller than the size of our substrate, even for larger densities of particles. This would allow us greater control over the effective imprint of the nanoparticle ensembles within cells. In addition to basic patterning, we were able to actively manipulate ensembles of nanoparticles with micrometer-scale resolution. Displayed in Figure 6a is a single cell with four noticeable groups of nanoparticles. The leftmost group conforming to the membrane edge is an irreversible nanoparticle aggregate caused by extended incubation time (as noted in an earlier paragraph) and undergoes very little morphology change during the time frame of our manipulation. Interestingly, the three other groups undergo both morphological and positional changes during magnet reorientation, including the rightmost group (flanking the cell membrane). Images were taken immediately upon magnet reorientation and subsequent refocusing, indicating the speed of the transformation is below the 10 s required to acquire an image (exposure time + refocusing). Upon image capture, the magnet is immediately reoriented, yielding six images spaced approximately 10 s apart each. Between images 1-2 and 4-5, we show the difference between that image and the subsequent image, and the approximate translational shift to better illustrate changes occurring at each step. The initial image displays the cell at the first state with magnet located weak south, and the magnet was progressively manipulated in order to display both manipulation and merging and separation of the groups of nanoparticles. Of additional note is the rightmost group, because it flanks the cell membrane, its centroid appears significantly modified as compared to the other two ensembles of particles, again indicating the intracellular nature of the ensembles. This is particularly noticeable in images 4-5, while the left two groups manipulated as expected, the rightmost saw no translation toward its expected location at all. Notice also in the red filter image (Figure 6a), as also is suggested by our confocal data, the nanoparticles colocalize to large signal in the green excitation/red filter images, which indicate that the nanoparticles enter the cell cytoplasm through an endosomal pathway (the diL stain targets lipids).18,19 This may also indicate the potential to dynamically modify the locations of endosomes internal to single cells. In summary, we have demonstrated the first engineered, simultaneous precision control of multiple groups of magnetic fluorescent nanoparticles within single cells. Uniquely aligned patterns within single cells were generated through 3058
Figure 6. Dynamic manipulation of intracellular nanoparticles. (a) UV excitation-blue filter; green excitation-red filter and merged images of a single cell with patterned nanoparticles to be manipulated. Four groups are strongly visible: a group flanking the left edge of the membrane, a group flanking the right edge of the membrane, and two internal, completed groups. (b) Consecutive images of the particles taken at 10 s intervals upon shifting the permanent magnet. The left group is an irreversible nanoparticle aggregate17 and sees little morphological changes during the time scales of the experiment (these result from extended incubation time). The rightmost group, from images 1 to 2 and 4 to 5, sees limited movement as restricted by the cell edge, and its centroid is incapable of reaching its expected potential minima in a manner as suggested by our statistics. The differences between images 1-2 and 4-5 are shown between those particular images and clearly illustrate the shift in position of the nanoparticles during these manipulations (for the rightmost group in 4-5 it appears to have no shift). In addition, during these manipulations, various merge and separations were accomplished, demonstrating additional versatility in the technique. Nano Lett., Vol. 9, No. 8, 2009
a lithographically patterned micromagnetic substrate composed of fine ferromagnetic tips and lines and in general can span much of the x-y space of the array through a combination of a normal and tangential incident magnetic fields. The methodology displays potential to dynamically control intracellular environments and signaling, locally and with submicrometer accuracy and movement, while simultaneously performing rapid operations on nanoparticles ensembles. Finally, the array and its overhead generates no heat to the biological sample and is compact enough to be workable with nearly any microscopy setup. Such tools could be used to generate highly localized magnetic and biochemical signals, allowing studies on localizable probing and actuation in single cells, and expanding on existing microfabricated tools that probe single cell dynamics. Our next step is to study cell behavior with injected or electroporated conjugated nanoparticles. Acknowledgment. The authors acknowledge Eric Tsang and Hyowon Lee for materials and protocols aide, Andrew Fung for assistance with confocal microscopy, Noah Bodzin for assistance with the FIB, and Ira Goldberg for helpful discussions on magnetic particle manipulation. Supporting Information Available: Supporting figure of an M-H loop of our electroplated permalloy and supporting movie of sequential cross-sectional images generated by our FIB etching. This material is available free of charge via the Internet at http://pubs.acs.org.
Nano Lett., Vol. 9, No. 8, 2009
References (1) Guanxiong, L.; Joshi, V.; White, R. L.; Wang, S. X.; Kemp, J. T.; Webb, C.; Davis, R. W.; Shouheng, S. J. Appl. Phys. 2003, 93, 7557. (2) Centi, S.; Laschi, S.; Franek, M.; Mascini, M. Anal. Chim. Acta 2005, 538, 205. (3) Stromgberg, M.; Goransson, J.; Nilsson, M.; Svedlindh, P. Nano Lett. 2008, 8, 816. (4) Lu, Y.; Liu, G. L.; Kim, J.; Mejia, Y. X.; Lee, L. P. Nano Lett. 2005, 5, 119. (5) Gao, J.; Zhang, W.; Huang, P.; Zhang, B.; Zhang, X.; Xu, B. J. Am. Chem. Soc. 2008, 130, 3710. (6) Nomura, A.; Shin, S.; Medhi, O. O.; Kauffman, J. M. Anal. Chem. 2004, 76, 5498. (7) Babincova, M.; Leszczynska, D.; Sourivong, P.; Cicmanec, P.; Babinec, P. J. Magn. Magn. Mater. 2001, 225, 109. (8) Hilger, I.; Hergt, R.; Kaiser, W. A. IEEE Proc. Nanobiotechnol. 2005, 152, 33. (9) Dobson, J. Nat. Nanotechnol. 2008, 3, 139. (10) Mannix, R.; Kumar, S.; Cassiola, F.; Montoya-Zavala, M.; Feinstein, E.; Prentiss, M.; Ingber, D. E. Nat. Nanotechnol. 2007, 3, 36. (11) de Vries, A.; Krenn, B.; Driel, R.; Kanger, J. Biophys. J. 2005, 88, 2137. (12) Park, J. H.; von Maltzahn, G.; Zhang, L.; Derfus, A. M.; Simberg, D.; Harris, T. J.; Ruoslahti, E.; Bhatia, S. N.; Sailor, M. J. Small 2009, 5, 694. (13) Ahn, C.; Allen, M.; Trimmer, W.; Jun, Y.-N.; Erramilli, S. J. MEMS 1996, 5, 151. (14) Do, J.; Choi, J.-W.; Ahn, C. IEEE Trans. Magn. 2004, 40, 3009. (15) Zborowski, M.; Sun, L.; Lee, R.; Williams, P.; Chalmers, J. J. Magn. Magn. Mater. 1999, 194, 224. (16) Lee, H.; Purdon, A. M.; Westervelt, R. M. Appl. Phys. Lett. 2004, 85, 1063. (17) Kodali, V. K.; Roos, W.; Spatz, J. P.; Curtis, J. E. Soft Matter 2007, 3, 337. (18) Bertorelle, F.; Wilhelm, C.; Roger, J.; Gazeau, F.; Menager, C.; Cabuil, V. Langmuir 2006, 22, 5385. (19) Miyauchi, K.; Kim, Y.; Latinovic, O.; Morozov, V.; Melikyan, G. B. Cell 2009, 137, 433.
NL901535M
3059
