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Towards on-chip controlled single cell drug delivery via magnetic nanoparticles

Sviluppo di una piattaforma "on-chip" che permetta la somministrazione controllata di un farmaco specifico a una cellula bersaglio,attraverso l'impiego di nanoparticelle magnetiche come veicoli per trasportare tali farmaci. Le funzionalità implementate in questa piattaforma sono due: rilevare, attraverso dei sensori, il passaggio delle nanoparticelle in una cella microfluidica e manipolarle con un' alta risoluzione spaziale verso la cellula bersaglio. Due i sensori utilizzati: i primi sono basati sull' effetto di anistropia magnetoresistiva (AMR), mentre i secondi su una misura impedenziale. La tecnica di manipolazione, chiamata "DWT" si fonda sull'accoppiamento tra i "bead" magnetici e le pareti di dominio magnetiche che si formano in condotti di Py a causa della peculiare forma geometrica e la cui posizione puo' essere controllata grazie all'applicazione di un campo magnetico esterno.

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Articoli tecnico scientifici o articoli contenenti case history
Tesi di Laurea, Politecnico di Milano, Anno Accademico 2012-2013

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POLITECNICO DI MILANO Facoltà di Ingegneria dei Industriale e dell'Informazione Corso di Laurea Magistrale in Ingegneria Fisica TOWARDS ON-CHIP CONTROLLED SINGLE CELL DRUG-DELIVERY VIA MAGNETIC NANOPARTICLES Relatore:
Prof. Riccardo BERTACCO Correlatore:
Dott.ssa Daniela PETTI Candidato:
Marco MONTICELLI
matr. 766446 Anno Accademico 2012/2013 Contents Sommario XVI Abstract XIX 1 Introduction and technological background 1 1.1 Bionanotechnology and nanomedicine . . . . . . . . . . . . . . 1 1.2 Drugs delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.2.1 Nanoparticles Transport methods . . . . . . . . . . . . 4 1.2.2 Interaction between nanoparticles and living cells . . . 6 1.3 Lab on a chip . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 1.3.1 Magnetic based LOC's devices . . . . . . . . . . . . . . 11 1.4 Thesis outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 2 Micromagnetics and magnetic nanoparticles 18 2.1 Micromagnetism . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.1.1 Exchange Interaction . . . . . . . . . . . . . . . . . . . 20
2.1.2 Magnetic Anisotropy Energy . . . . . . . . . . . . . . . 21
2.1.3 Magnetostatic and Zeeman Energy . . . . . . . . . . . 24
2.1.4 Magnetic domains . . . . . . . . . . . . . . . . . . . . . 26
2.1.5 Domain walls . . . . . . . . . . . . . . . . . . . . . . . 28
2.1.6 Neel domain walls in thin lm materials . . . . . . . . 29
2.1.7 Pinning and propagation of Domain walls . . . . . . . 31 I 2.2 Magnetic micro and nanoparticles . . . . . . . . . . . . . . . . 33 2.2.1 Interaction forces on magnetic beads . . . . . . . . . . 36
2.2.2 DWs assisted manipulation of magnetic nanoparticles . 38 2.3 Anisotropic Magnetic Resistance . . . . . . . . . . . . . . . . . 39 3 Experimental methods 42 3.1 Micro- and nano-fabrication techniques . . . . . . . . . . . . . 42 3.1.1 Optical and Electron-beam lithography . . . . . . . . . 43
3.1.2 Electron beam evaporation . . . . . . . . . . . . . . . . 46
3.1.3 Capping and microuidic cells . . . . . . . . . . . . . . 48
3.1.4 Process ow . . . . . . . . . . . . . . . . . . . . . . . . 50 3.2 Characterization methods . . . . . . . . . . . . . . . . . . . . 54 3.2.1 Atomic force microscopy(AFM) . . . . . . . . . . . . . 54
3.2.2 Magnetic force microscopy (MFM) . . . . . . . . . . . 57 3.3 Experimental setups . . . . . . . . . . . . . . . . . . . . . . . 59 3.3.1 Setup for biological experiments and beads manipulation 59
3.3.2 Setup for sensors measurements . . . . . . . . . . . . . 63 3.4 Micromagnetic simulations . . . . . . . . . . . . . . . . . . . . 69 4 Manipulation of nanoparticles over magnetic conduits 70 4.1 Single particles manipulation on zig-zag conduits . . . . . . . 71
4.2 Free 2D manipulation of many particles over curved conduits . 74 4.2.1 Working principles . . . . . . . . . . . . . . . . . . . . 74
4.2.2 Micromagnetic simulation . . . . . . . . . . . . . . . . 79
4.2.3 Manipulation of a particles batch on free paths . . . . 85 4.3 Conclusions and perspectives . . . . . . . . . . . . . . . . . . . 88 5 Detection methods for magnetic nanoparticles 89 5.1 AMR sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 5.1.1 Detection experiments . . . . . . . . . . . . . . . . . . 94 II 5.2 Capacitive sensors . . . . . . . . . . . . . . . . . . . . . . . . . 100 5.2.1 Beads Impedance . . . . . . . . . . . . . . . . . . . . . 100
5.2.2 Experiments of single bead detection . . . . . . . . . . 104 5.3 Conclusions and perspectives . . . . . . . . . . . . . . . . . . . 109 6 Controlled administration of nanoparticles to a single cell 111 6.1 Manipulation of MNPs in the cellular medium . . . . . . . . . 112 6.1.1 Micromagnetic simulations . . . . . . . . . . . . . . . . 114
6.1.2 Manipulation experiments . . . . . . . . . . . . . . . . 117 6.2 Passive uptake of magnetic nanoparticles . . . . . . . . . . . . 119
6.3 Manipulation of nanoparticles to a target cell . . . . . . . . . 122
6.4 Conclusions and perspectives . . . . . . . . . . . . . . . . . . . 125 Conclusions and perspectives 126 References 129 III List of Figures 1.1 Scheme of multifunctional nanoparticle for molecular imaging, drug delivery and therapy. Specically functionalized and de-
vised nanoparticles can be realized for individualized diagnosis
and treatments [10]. . . . . . . . . . . . . . . . . . . . . . . . . 5 1.2 Sketch of the main endocytic mechanisms: Phagocytosis, Pinocy- tosis, Endocytosis mediated by receptors [25]. . . . . . . . . . 8 1.3 Sketch of Laboratory on-chip [29]. . . . . . . . . . . . . . . . . 11
1.4 Magnetic separation of labelled biomolecules via magnetic nanopar- ticles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1.5 Manipulation of 2.8 µm magnetic beads on a staircase pat- tern of Permalloy ellipses by in-plane 80 Oe rotating magnetic
eld. The arrows indicate the direction of the eld. After one
complete eld revolution, the beads have moved one step in
the pattern as indicated by the white curve in (f)[33]. . . . . . 13 1.6 Ferromagnetic conduits geometries: zig-zag shaped (a) and curved (b) structures. . . . . . . . . . . . . . . . . . . . . . . . 14 2.1 Uniaxial anisotropic energy density. (left) Anisotropy with easy axis (K1 > 1). (right) Anisotropy with easy plane (K1 <
1). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.2 Domain formation: from left to right the magnetostatic energy is decreased due to domains creation [44]. . . . . . . . . . . . 28 IV 2.3 Two domain wall types, Bloch wall (above) and Neel wall (be- low). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 2.4 Top view of a Permalloy innitely long strip with two opposite domains (red and blue arrows) and a DW which divides the
two regions. Both transverse spin structure and vortex spin
structure DW are shown (a). Phase diagram of a transverse
HH DW in thin magnetic stripe. δ is equal to lex/2 (b) [45]. . 31 2.5 Simulated behavior of a transverse DW at a corner site. The gure shows the magnetization structure of the DW and below
the sketch of the potential well, whose minimum is centered
on the tip of the corner. . . . . . . . . . . . . . . . . . . . . . 32 2.6 Sketch of magnetic bead formed by magnetic nanoparticles in a non-magnetic matrix/shell. . . . . . . . . . . . . . . . . . . 36 2.7 Sketch of the magnetic attractive potential well generated by a HH DW pinned at one corner of the magnetic conduit. Such
potential is able to trap a superparamagnetic antibodies func-
tionalized bead at a distance of 100 nm from the chip surface.
From [49] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 2.8 AMR eect in a ferromagnetic strip in presence of a DW; the resistance is higher when J and M are parallel. Image from
OOMMF. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.1 Microscope images of optical lithography patterning of ex- ternal contacts (a) and EBL patterning of ne contacts (b).
Sketch of the entire lithography process (c) . . . . . . . . . . 44 3.2 Mask aligner Karl SussMA56. . . . . . . . . . . . . . . . . . . 45
3.3 Rings array pattern obtained by EBL. . . . . . . . . . . . . . 50
3.4 Optical image of the AMR device: after the rst (a) and the second (b) step. Picture of the nal device (c). . . . . . . . . . 51 V 3.5 Optical microscope images of the fabrication process for a ca- pacitive sensor. . . . . . . . . . . . . . . . . . . . . . . . . . . 53 3.6 Sketch of the AFM working principle . . . . . . . . . . . . . . 55
3.7 Atomic force microscope VEECO innova . . . . . . . . . . . . 56
3.8 Magnetic force microscopy: lift mode. In the rst step the topography of the surface is recorded, while in the second step,
the magnetic image arising from the stray eld of magnetic
domains in the sample is captured. . . . . . . . . . . . . . . . 57 3.9 MFM image of a zig-zag magnetic conduit: dark and bright spots represent respectively an attractive or repulsive mag-
netic force, acting on the ferromagnetic tip. . . . . . . . . . . . 58 3.10 Experimental setup for biological tests and beads manipula- tion. A indicates the optical microscope (Nikon Eclipse FN-1),
B the entire sample stage, C the stepper motors system and
D the thermostat. . . . . . . . . . . . . . . . . . . . . . . . . . 59 3.11 Image of stepper motors used to control the rotation and the height of a plate containing two couples of permanent magnets
(a). Sketch of the lines of force from the permanent magnets
eld (b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.12 Sketch of the stepper motors working principle. The electro- magnets around the central gear are sequentially powered to
get a single step rotation. . . . . . . . . . . . . . . . . . . . . . 62 3.13 Experimental setup for sensors measurement. A is the four poles electromagnet, B is the Lock-in amplier (HF2LI), C
is a bipolar generator (KepcoTM) and the optical microscope
(Nikon Eclipse FN-1). . . . . . . . . . . . . . . . . . . . . . . 64 3.14 Sample stage employed in electrical measurements. A is the AMR or capacitive device, B the electrical pins connected to
the "tulip" wires. C and D indicate the small electrical boards 64 VI 3.15 Schematic of the equivalent circuit of AMR sensors measure- ments (a) and optical microscope image of the device which
shows the electrical contacts and the magnetic zig-zag shaped
conduit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 3.16 Equivalent circuit for capacitive sensors measurements (a) and schematic transversal section of the device (b). . . . . . . . . 68 4.1 Top: sequence of magnetic force microscopy images and mi- cromagnetic congurations (right) showing the injection and
propagation of a domain wall under the action of external
magnetic elds Hi, Hup, and Hdw directed as sketched in the
gure. The dark and bright re contrast in the image is due
to the inward and outward local stray elds. At the zig zag
corners the stray eld is generated by a domain wall, while in
the case of the injection pad is only due to the magnetization
stray eld. Bottom: sketch of the zig-zag conduit dimensions. 72 4.2 Transport sequence of a 1µm bead over a zig-zag shaped con- duit in an AMR sensor. The DW is nucleated (a) and (b)
and displaced over the nanostructure (c) and (d), dragging
the MNP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 4.3 Square matrix (a) and hexagonal matrix (b) of Permalloy rings. 75
4.4 Outer gures: sketches of the micromagnetic conguration of a circular ring showing the nucleation and displacement along
the perimeter of the two, HH and TT, DWs obtained by ap-
plying a continuous rotating eld HR. The inner images are
the corresponding MFM data.[35] . . . . . . . . . . . . . . . . 76 VII 4.5 Schematic representation of the two rings system, after the ap- plication of the magnetic elds sketched below. Red and blue
arrows show the micromagnetic conguration of the magnetic
structures. Step (a): a magnetic bead, in grey, is trapped by
the HH DW of the left ring. Step (b): the magnetic bead
is moved to the point closest to the next ring. Step (c): by
switching the polarity of the out-of-plane eld and removing
the in-plane eld, the magnetic bead is attracted and coupled
to the TT DW of the second ring. Step(d): the magnetic bead
is moved by rotating again of 180o the in-plane magnetic eld. 77 4.6 OOMMF simulation of the magnetization of a portion of the two rings facing each other from OOMMF. The arrows show
the direction of the magnetization in the xy-plane. Blue and
red pixels indicate positive and negative values of the magne-
tization along y. HH and TT vortex DWs are nucleated by an
external in-plane eld (500 Oe). . . . . . . . . . . . . . . . . 80 4.7 a) z-component of the total eld generated close to the HH DW with respect to the vertical distance z from the magnetic struc-
ture. The eld near HH DW is positive and is enhanced by the
application of the concordant external eld. b) z-component
of the total eld generated close to the TT DW versus z. In
absence of Hz, the vertical component of Htot is negative while
upon the application of the out of plane external eld, Htot
crosses the zero value at a certain distance (around 400 nm). 81 VIII 4.8 Magnetic potential energy wells felt by 1 µm superparamag- netic particle generated by the magnetic conguration illus-
trated in gure 4.6 for dierent values of Hz. When Hz is o,
HH and TT DWs produce two equally deep potential wells.
While, increasing Hz, HH DW becomes a stronger attractive
pole and TT DW a weaker one. When Hz is equal to 60 Oe
the particle is completely decoupled by TT DW. . . . . . . . . 82 4.9 Contour plot of the z-component of the magnetic force calcu- lated at a distance equal to the radius of the bead plus 60 nm.
The left graph illustrates the situation when the out-of-plane
magnetic eld is o while the right graph shows the case in
which the force generated by the HH DW is enhanced by a Hz
eld equal to 60 Oe. . . . . . . . . . . . . . . . . . . . . . . . 83 4.10 Contour plot of the x-component of the magnetic force calcu- lated at a distance equal to the radius of the bead plus 50 nm.
The left graph illustrates the situation when the out-of-plane
magnetic eld is o while the right graph shows the case in
which Hz is equal to 60 Oe. . . . . . . . . . . . . . . . . . . . 84 4.11 Manipulation sequence of a single 1µm bead around a mag- netic ring in an hexagonal matrix by applying a 300 Oe in-
plane eld. The magnetic eld is rotated in an anti-clockwise
direction. The images are recoded from an optical microscope
exploiting a 60x immersion objective. . . . . . . . . . . . . . . 86 4.12 Optical microscope images of a particles batch manipulation. The particles path is highlighted with dierent colour. A 60x
immersion objective and 1µm beads are employed. . . . . . . . 87 5.1 Optical microscope image of an AMR chip: the zig-zags mag- netic structures are illustrated, together with the four electri-
cal contacts for each sensor. . . . . . . . . . . . . . . . . . . . 90 IX 5.2 Magnetization of a zig-zags shaped conduit corner, in absence (a) or presence (b) of a DW. The arrows indicate the direction
of M. Red-white-blue pixels indicate the value of M along the
x-axis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 5.3 Sketch of the AMR sensor. The internal electrodes are em- ployed to measure the voltage drop at one corner of the mag-
netic zig-zag conduit. The external contacts are exploited to
apply the electrical signal. The green arrows represent the
current which ows in the device. . . . . . . . . . . . . . . . . 92 5.4 Sketch of the zig-zag conduit in an AMR sensor. A magnetized bead of moment µ produces a stray eld in the upper corner
of the conduit. . . . . . . . . . . . . . . . . . . . . . . . . . . 93 5.5 Impedantial spectra of the AMR devices, measured by LCR. Curve A represents the resistance between the inner electrodes,
while B the resistance between the outer contacts. . . . . . . . 95 5.6 Sketch of the displacement of a DW to the corner between the inner contacts in AMR sensors (a) and plot of the voltage drop
due to AMR as a function of the external eld. At 179.5±2
Oe (indicated by the red-dashed line) the DW is displaced
(b). The dots represent the experimental values, while the
continuous line is a polynomial t of the data. . . . . . . . . 96 5.7 Sketch of the displacement of a DW beyond the inner contacts in AMR sensors (a) and plot of the voltage drop by AMR
as function of the external eld. At 180±2 Oe, (indicated
by the red-dashed line) the DW is displaced (b). The dots
represent the experimental values, while the continuous line is
a polynomial t of the data. . . . . . . . . . . . . . . . . . . . 97 X 5.8 Optical microscope image of 1 µm bead transition between the inner contacts in an AMR sensor. An immersion objective 60x
has been employed. . . . . . . . . . . . . . . . . . . . . . . . . 98 5.9 Plots of the voltage drop as function of the applied eld when the bead is displaced inside (a) or outside (b) the contacts.
Panel (c) compares two curves which represent the voltage
variation when a DW is displaced outside from the inner con-
tacts in presence (red line) and absence (black line) of a MNP
on the top of it. The ne lines and dots represent the experi-
mental values, while the thicker continuous line is a polynomial
t of the data. . . . . . . . . . . . . . . . . . . . . . . . . . . 99 5.10 Sketch of the electrical contacts in the measurement area of a capacitive device. W=3 mm, G=4 µm, S=4 µm, H=1 mm
and L=12 µm. . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 5.11 Graphic of the resistance variation as function of time when a permanent magnet is used to attract 1 µm particles away
from the electrodes. Green arrows indicate respectively when
the beads are between the electrical contacts (Beads IN), when
they are attracted away by the magnet (Beads OUT) and while
they are sedimenting (Sedimentation). A constant descending
drift (-30 m'/s) in the resistance value is subtracted to the
experimental data. . . . . . . . . . . . . . . . . . . . . . . . . 102 5.12 Optical microscope image of a capacitive sensor. Each chip is provided with three magnetic zig-zags conduits and 24 elec-
trodes. One of such contacts is employed as counter electrode
to set the potential in the solution. The couple of electrodes
labelled by "A" represent the geometry used in the numerical
simulations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 XI 5.13 Image of the 3D geometry employed in FEM simulation. The colours indicate dierent value of the electrical potential. . . . 105 5.14 Plot from FEM simulations which shows the Resistance vari- ation in presence and absence of bead between the electrodes.
In this simulation the double layer capacitance was not con-
sidered. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 5.15 Sketches of the equivalent circuit for a capacitive sensor (a) and the electronic readout (b). CDL is the double layer capac-
itance, RPBS is the liquid resistance and CP is the parassitic
capacitance. In (b) the lock-in amplier (Zurich), the tran-
simpedance amplier (Femto), the Dummy circuit and the
chip mux are illustrated. . . . . . . . . . . . . . . . . . . . . . 107 5.16 Sequence of the transit of a 1µm bead between two electrical contacts in a capacitive sensor. . . . . . . . . . . . . . . . . . . 108 6.1 AFM image (a) and derived 3D view (b) of a sample covered by ZrOX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 6.2 Magnetization of a portion of ring from OOMMF. The arrows show the direction of the magnetization in the xy-plane. Blue
and red pixels indicate positive and negative values of the
magnetization along y. HH vortex DW is nucleated by an
external in-plane eld (500 Oe). . . . . . . . . . . . . . . . . . 114 6.3 Magnetic force on a superparamagnetic bead having a diam- eter of 300 nm and ' equal to 0.39, as a function of the ring
thickness. The distance between the top of the magnetic struc-
ture and the bottom of the bead ranges between 50 nm and
200 nm. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 XII 6.4 Magnetic force on a superparamagnetic bead having a diame- ter of 300 nm and ' equal to 0.39, as a function of the distance
between the top of the Py ring and the bottom side of bead. It
was calculated for dierent values of the ring thickness ranging
between 30-80 nm. . . . . . . . . . . . . . . . . . . . . . . . . 116 6.5 Manipulation sequence of a single 300 nm bead around a mag- netic ring in an hexagonal matrix by applying a 300 Oe in-
plane eld. A sample covered by ZrOX (50 nm) was employed.
The magnetic eld is rotated in an anti-clockwise direction.
The images are recoded from an optical microscope exploiting
a 60x immersion objective. . . . . . . . . . . . . . . . . . . . . 118 6.6 Confocal microscopy image: the green-stained cellular nucleus of an HeLa cell is surrounded by red uorescent (TRITC)
magnetic nanoparticles. It is acquired by a TCS-SP5 Leica
microscope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 6.7 Confocal microscopy image: red-uorescent (TRITC) mag- netic nanoparticles are internalized inside the cells (HeLa).
The green cytoplasm is labelled by a Dextran based marker.
The brightest green circular spot on the left are two cellu-
lar nuclei (some cells have not absorbed the marker). Image
acquisition via a TCS-SP5 Leica microscope. . . . . . . . . . 121 6.8 Manipulation sequence of a 300 nm MNP to a target cell (mammalian-cancer cell). A continuous magnetic eld of 300
Oe is rotated in a clockwise direction to displace the bead. An
immersion 60x objective is used. . . . . . . . . . . . . . . . . . 123 6.9 Frames from a video showing the manipulation of a 300 nm bead dragged to a target cell (epithelial HeLa cell) through
a rotating magnetic eld of 300 Oe. Image acquisition via a
TCS-SP5 Leica microscope. . . . . . . . . . . . . . . . . . . . 125 XIII 6.10 Scheme of the platform made of two reservoirs (RIN and ROUT ) connected by a channel (C). Functionalized MNPs are dis-
pensed in the reservoir RIN (a). From there, beads are ma-
nipulated along the channel (C) and detected by means of an
AMR (or capacitive) device (b). At the end of the channel,
a second reservoir (ROUT where cells are cultured is located.
Beads are manipulated by means of the DWTs to the target
cell to administrate the drug. . . . . . . . . . . . . . . . . . . 127 XIV List of Tables 3.1 Parameters used in the evaporation processes and the relative deposition rate . . . . . . . . . . . . . . . . . . . . . . . . . . 47 3.2 Parameters used in magnetron sputtering processes and the relative deposition rate. . . . . . . . . . . . . . . . . . . . . . . 48 4.1 Sequence of applied eld pulses to nucleate and displace a DW in a zig-zag shaped conduit. . . . . . . . . . . . . . . . . . . . 73 XV Sommario Il lavoro di tesi ha riguardato lo sviluppo di una piattaforma "on-chip" che
permetta la somministrazione controllata di un farmaco specico a una cel-
lula bersaglio, attraverso l'impego di nanoparticelle magnetiche come veicoli
per trasportare tali farmaci.
Le funzionalita' implementate in questa piattaforma sono essenzialmente due:
rilevare, attraverso dei sensori, il passaggio delle nanoparticelle in una cella
microuidica e manipolarle con un' alta risoluzione spaziale verso la cellula
bersaglio. Due diversi sensori sono stati utilizzati per questo scopo: i primi
sono basati sull' eetto di anistropia magnetoresistiva (AMR), mentre i sec-
ondi su una misura impedenziale. La tecnica di manipolazione, chiamata
"DWT" (domain walls tweezers) si fonda sull'accoppiamento tra i "bead"
magnetici e le pareti di dominio magnetiche che si formano in condotti di
Py a causa della peculiare forma geometrica e la cui posizione puo' essere
controllata grazie all'applicazione di un campo magnetico esterno. Questo
metodo di manipolazione ha attratto un interesse sempre crescente in am-
bito medico e biologico poiche' le particelle magnetiche vengono, oggigiorno,
comunemente impiegate come veicoli (per sostanze biologiche o farmaci) e/o
marcatori specici.
II lavoro sperimentale svolto puo' essere diviso in 3 parti principali: ' Implementazione della tecnologia "DWT", ottenendo una manipolazione controllata e simultanea di un gruppo di nanoparticelle magnetiche su XVI un chip nanostrutturato dotato di nanocondotti magnetici. La proget-
tazione e la fabbricazione di tali campioni viene inoltre presentata in
questo lavoro. ' Fabbricazione, sviluppo e test dei sensori "AMR" e capacitivi, impiegati per rilevare le nanoparticelle magnetiche. ' Somministrazione controllata di nanoparticelle magnetiche ad una cel- lula bersaglio, facendo uso della tecnologia "DWT". Le nanoparticelle
vengono manipolate su chip dotati di "DWT" e ottimizzati per tale
scopo. Il lavoro sperimentale e' stato realizzato sotto la supervisione del Professore
Riccardo Bertacco, responsabile del gruppo NaBiS presso il centro LNESS-
Dipartimento di Fisica del Politecnico di Milano, Polo Regionale di Como.
Una parte degli esperimenti biologici sono stati svolti al "Istituto Farma-
cologico di Oncologia molecolare" IFOM, a Milano. Hanno collaborato a
questo progetto: per la parte che riguarda la fabbricazione dei chip di sen-
sori il gruppo di "litograa elettronica e grafene" del centro LNESS, per la
fabbricazione dei DWTs Andrea Cattoni di LPN (Parigi) e per le misure per
la detection dei beads il gruppo di "ingegneria elettronica" del Politecnico di
Milano.
Questo elaborato di tesi e' organizzato in sei diversi capitoli. Il capitolo 1
tratta del background tecnologico su cui si fonda il lavoro ed in particolare
aronta le tematiche della "drug-delivery" e del "Lab-on-chip". Il capitolo
2 descrive i principi sici su cui si basa la tecnologia DWT e in quale modo
viene utilizzata per manipolare le nanoparticelle magnetiche. Il capitolo 3 si
occupa delle tecniche e dei metodi sperimentali utilizzati in questo lavoro. Il
capitolo 4 descrive la manipolazione di nanoparticelle magnetiche. Nel capi-
tolo 5 vengono descritti i risultati ottenuti sui sensori AMR e capacitivi. Nel
capitolo 6 viene illustrata la somministrazione di nanoparticelle magnetiche a XVII una cellula bersaglio ed, inne, vengono presentate le conclusioni del lavoro. XVIII Abstract This thesis work deals with the development toward an on-chip platform for
achieving a controlled drug-delivery to a target cell via magnetic nanoparti-
cles exploited as drug carriers.
With this platform two main issue are addressed: sensing the magnetic
nanoparticles transit in a microuidic cell and nely manipulating them at
the nanoscale to a target cell, in order to deliver a specic drug. The mag-
netic beads are detected by means of two dierent sensors based respectively
on the Anisotropic Magnetoresisrance eect (AMR) and on an impedantial
measurement. The handling method is founded on the coupling of magnetic
beads with externally controlled magnetic domain walls and it is called do-
main walls tweezers (DWT).
This recent manipulation technique has attracted a growing interest in biol-
ogy and medicine as functionalized magnetic particles are commonly used as
molecular and cellular carriers or markers.
The experimental work can be divided in three main parts: ' Implementation of the DWTs technology to achieve a synchronized ma- nipulation of a magnetic nanoparticles batch over magnetically pat-
terned magnetic chip. The design and the fabrication of the employed
devices is also described in this work. ' Fabrication, development and test of AMR and capacitive sensors for the detection and transport of magnetic beads. XIX ' Controlled administration of magnetic nanoparticles to a target cell by means of the DWTs technology. The particles are handled over pat-
terned magnetic devices, properly projected and fabricated. The work has been realized under the supervision of Professor Riccardo
Bertacco, responsible for the NaBiS group at the LNESS Center-Dipartimento
di Fisica of the Politecnico di Milano, Polo Regionale di Como. A part of the
biological experiments have been performed at the "Istituto Farmacologico
di Oncologia molecolare" IFOM, in Milan. The fabrication of the sensors has
been executed in collaboration with the "e-beam litography and graphene"
group of LNESS, while sensor testing and detection measurements have been
carried out with the "Electronic engineering" group of the Politecnico di Mi-
lano. The fabrication of DWTs chip has been performed in collaboration
with Andrea Cattoni of LPN (Paris).
The thesis is organized in 6 sections. In chapter 1, an oveview of the tech-
nological background concerning drug-delivery and lab-on-chip devices is il-
lustrated. Chapter 2 discusses the physics principles of the DWT technology
and the principles exploited to handle magnetic nanoparticles. Chapter 3
explains the experimental methods which have been employed. In chapter 4,
the manipulation of magnetic nanoparticles is described. Chapter 5 explains
the results related to the AMR and capacitive sensors. In chapter 6 the con-
trolled administration of nanoparticles to the target cell is illustrated and,
nally, the conclusions are presented. XX Chapter 1 Introduction and technological
background This thesis work deals with the development of an on-chip magnetic-based
platform for achieving a controlled drug-delivery to a single cell by means of
magnetic nanoparticles exploited as drug-carries.
The purpose of this chapter is to illustrate the motivations behind the minia-
turization of medical and biological addressed devices and the use of mag-
netism in this context. Moreover, the current state of the art concerning
drugs-delivery via nanoparticles is illustrated. Finally, the organization of
this thesis and a brief summary of all the chapters is presented. 1.1 Bionanotechnology and nanomedicine Bionanotechnology is a branch of nanoscience which deals with biological ap-
plications of nanotechnologies and with the study of biological entities in the
nanometric scale (i.e.1nm-1µm) [1]. It ranges from the biological application
of nanomaterials, to the development of nanostructured devices for biological
tests (e.g. biosensors), and even to possible future applications of molecular
nanotechnologies. The side of bionanotechnology concerning medical appli- 1 cations is called nanomedicine. It has two main goals: understanding how
the biological entities inside living cells are organized and operate at the
nanoscale, and employing this information to re-engineer these structures
and to develop new technologies applications such as diagnostics, diseases
treatment and/or damaged tissues repairing.[2].
From an economic point of view, nanomedicine represents now a large indus-
try with at least 3.8 billion of dollars invested in research and development
funding every year. In this context this work involves two main branches of
nanomedicine: ' the rst one concerns the use of nanomaterials for drugs encapsulation and delivery. ' the second one is related to the application of nanotechnology to minia- turized laboratories (Lab-on-chip) performing complex biological oper-
ations on minute biological samples quantities. The following sections will describe the state of the art in these two central
topics and will explain in which way they are exploited in the present work. 1.2 Drugs delivery One of the main problems in contemporary medical science is the invasive-
ness of many diseases treatment. In particular, the assimilation of a specic
drug might potentially produce a large amount of side eects mainly due to
the absorption of such drug from the entire organism instead of being local-
ized in the specic area requiring the treatment. Nanomedical approaches
to drug delivery focus on developing nanoscale particles and molecules to
improve drugs eectiveness while reducing their side eects. Optimization
of drug delivery aims to spatially and temporally control the drug release
in the body. This can be potentially achieved by modifying and targeting 2 molecules to specic cells [3],[4] by means of nanoengineered materials. One
of the main goal of this research branch is the non-invasive cancer diseases
treatment. Because of quantum size eects and large surface to volume ra-
tio, nanoparticles and nanomaterials have unique properties compared with
their respective bulk materials. These properties, such as the high chemical
and physical reactivity, can be employed to bind specic molecules to the
particles surface so that they could interact with target cells. In addition
to that, cells usually take up nanoparticles because of their small size with
no specic distinction exploiting various bio-chemical mechanisms described
in section 1.2.2. However, the toxicity of these materials and the long term
eects on the body still remain one of the main issues.
Triggered response is one way for ecient molecules delivery. It can be based
on lipid or polymer nanoparticles [5], designed to improve the pharmacologi-
cal and therapeutic properties of drugs. Moreover, thermo and pH-responsive
nanoparticles which react to an environment modication in temperature
and pH, by releasing drugs molecules to cells, have been developed in the
last years [6] [7].
Nevertheless potential nano-drugs behaviors are dicult to foresee in a very
complex and, in some cases, not well-understood environment such as the
human body. In this framework new in-vitro tools, mimicking the physio-
logical conditions of the human environment are fundamental for testing the
eects of new drug release nanoplatforms and/or new drugs. Many eorts
have been done to develop nanocarriers able to mask the drug molecules and
selectively release the drug content to the disease site [8]. Although drug
encapsulation has been used to reduce the toxicity of many drugs, the low
eectiveness in their accumulation in the area of interest still represents the
main problem. The dilution and dispersion of the carriers in the bloodstream
dramatically reduces their accumulation in the diseased area. Besides passive
accumulation mechanisms, active retention mechanisms that rely on the spe- 3 cic targeting of unhealthy tissues via recognition units at the nanocarrier's
surface have been exploited so far [9]. However, there is a strong interest
in developing alternative ways to remotely enhance drug accumulation and
delivery.
In this framework, magnetic nanoparticles have been widely used. It is due
to the possibility to conne them in a certain area, by means of an external
magnetic eld. 1.2.1 Nanoparticles Transport methods The integration between medicine, biology and nanotechnologies is often re-
alized by the use of functionalized micro- and nanoparticles. New diagnostic
and therapeutic testing procedures, both in-vivo and in-vitro, were intro-
duced by the controlled synthesis and manipulation of such objects, which
can be used as carrier for biological systems such as molecules, cells or anti-
gens. Fig.1.1 shows the concept of a functionalized particle, illustrating in a
single comprehensive picture the dierent goals for which it can be employed.
Many materials are used for various purposes. For example, quantum dots
(QDs) core or gold nanoparticle can be used for nanoimaging, while dielectric
or magnetic materials can be employed for manipulation. Polymeric coat-
ing is common for surface modication protocols in order to covalently bind
biological entities to the particle and thus convey them. By exploiting the
intrinsic properties of particles, the handling can be obtained through the
application of mechanical, uidic, optical, electrokinetic and magnetic forces
and practically allows for the control and transport of the smaller and inert
biological entity. Examples of mechanical manipulators are micro-tweezers
for cells [11] or tips of atomic force microscopes, used to position particles
[12]. However it is dicult to scale down the size of mechanical manipulators
in order to increase the parallel yield of the device.
Moreover in biology non-invasive methods, where there is no contact between 4 Figure 1.1: Scheme of multifunctional nanoparticle for molecular imaging,
drug delivery and therapy. Specically functionalized and devised nanopar-
ticles can be realized for individualized diagnosis and treatments [10]. the probe and the sample, are preferred. The use of hydrodynamic forces is
an example of a non-contact method for cell manipulation. These forces have
been exploited successfully in trapping [13] and selecting [14] individual cells.
However, these devices require complex pumping and control systems and the
high amounts of uid in the microuidic channel can induce shear stresses
to cells, changing their behavior. Cell manipulation can be accomplished via
optical methods, either by radiation pressure [15] or by the force exerted by
the gradient of a highly focused laser beam eld [17],[16]. Optical tweezers
are devices that allow to manipulate particle sizes ranging from tens of mi-
crometers to tens of nanometers. However, the photo-damage to biological
entities and the limited area of action impedes the application of these device
to highly parallel handling and transport of particles for long paths. Dielec-
trophoresis (DEP) depends on the force gradient of an electric eld rather
than an optical one [18]. DEP electrode devices are capable of handling in
parallel, but have a limited exibility; for this reason, it is dicult to isolate 5 a single particle of interest. Another limitation is induced by the fact that
conductive solutions cannot be always used in biology.
The ability of magnets to act on objects at a nite distance makes them valu-
able medical tools since they represent a non-invasive technique. Magnetism
is exploited through the use of magnetic particles to label biological species.
The use of phenomena related to magnetism brings several advantages with
respect to other techniques. First, magnetic particles are poorly aected by
the environment in which are oating and their properties do not depend on
the biological molecules which are bound to. Furthermore they are stable
over time, because magnetism is usually not aected by reagent chemistry
or subject to photo-bleaching (a problem which instead characterizes uo-
rescent labels). There is also no signicant magnetic background present in
biological samples and magnetic elds are not screened by aqueous reagents
or biomaterials. As an example, once a magnetic functionalized particle is
bound to the cellular membrane, it is possible to act indirectly on it in order
to measure its mechanical properties or activating ionic channels.
Magnetic particles have a wide range of applications in modern medicine.
They can be used to deliver an anticancer drug to a previously targeted tu-
mor region of the body. They also can be engineered to resonantly respond
to a time-varying magnetic eld in order to transfer energy to the biologi-
cal target through hyperthermia [19]. In this way the local excess of heat
generated induces the death of the tumor cells. 1.2.2 Interaction between nanoparticles and living cells In the previous chapter dierent methods to manipulate nanoparticles are
illustrated. The next step is understanding the mechanisms of interaction
between nanoparticles and cells. In particular, the internalization processes
of such nanoparticles by living entities are fundamental to deliver drugs.
Because of the cell membrane has in almost the totality of cases a negative 6 Zeta-potential, nanoparticle surface can be functionalized with particular
chemical groups which produce a positive Zeta-potential that favors the elec-
trical interaction between particle and membrane [20]. The Zeta-potential is
the electrical potential in a solution, due to the formation of a double-layer
(DL) interface between a charged surface and ions which are electrically at-
tracted. In other words, Zeta-potential is the potential dierence between
the dispersion medium and the stationary layer of uid attached to the dis-
persed particle or cell. Entities with an opposite Zeta-potential are electri-
cally attracted to each other. For this reason, nanoparticles decorated with
carbossylic acid (COOH'') are widely used in biological applications.
Moreover a large amount of various ligands are exploited to target specc
cells. For example, folic acid is employed to activate folate receptors (in can-
cer cells) which promote the uptake of particles functionalized with such acid
molecule [21].
In general, there are two main factors for which various uptake mechanisms
occur: the nanoparticles size and the stimulations of specic cell receptors in
a mechanical or chemical way. Even if the way in which a cell internalizes an
external body is still not well known, it is possible to distinguish four major
endocytic processes all based on the formation of intracellular vescicles (see
Fig.1.2) [22]: ' Phagocytosis ('cell eating') It is associated with the formation of big vescicles used to uptake large entities such as bacteria or particles larger
than 500nm. ' Pinocytosis ('cell drinking') It is used to internalize uids surrounding the cell through the simoultaneously formation of small vescicles due
to the membrane invagination. ' Endocytosis mediated by clathrin It is the most important endo- cytic mechanism involving a receptor (clathrin). It is used to internal- 7 ize particles that range between 50 and 300 nm, togheter with a large
amount of biological nutrients. ' Endocytosis mediated by caveolae It is employed to internalize particles up to 100 nm and some virus. It is characterized by caveloine-
1 receptors which cover the surface of small invaginations of cellular
membrane. Figure 1.2: Sketch of the main endocytic mechanisms: Phagocytosis, Pinocy-
tosis, Endocytosis mediated by receptors [25]. In literature is possible to nd many others receptors-mediated endocytosis
that can be employed to internalize specic nanoparticles properly function-
alized. Other uptake processes don't involve the formation of vescicles but
the cellular membrane is 'penetrated' by small particles. This mechanism is
called 'passive uptake' to distinguish it from the rst one [23].
Apart from the dierent uptake mechanisms, another important factor is the
uptake rate (i.e. the percentage of particles successfully internalized over
the total number of particles in solution). In-vitro experiments show as it
strongly depends on the nanoparticles size, concentration and surface coating
[24]. It is dicult to nd in literature an uptake rate larger than 20% and 8 it is mainly due to the dispersion of nanoparticles in a liquid environment.
The aim of the thesis is to develop a system able to increase such rate by
mechanically keep a single nanoparticle in proximity to a target cell exploit-
ing a conned magnetic force. This can be achieved thanks to an on-chip
technolgy that will be introduced in the next paragraphs. 1.3 Lab on a chip In the last two decades, the eld of the methodologies applied to the biologi-
cal and chemical laboratories has been revolutionized by the miniaturization
processes. In particular, the way in which the research and the analysis are
carried out was completely upset by the nanoscience progress.
This trend towards miniaturization began in the second half of twentieth
century applied mainly in the eld of electronic components and integrated
electronics. The driving force for miniaturization is the aim of increasing
processing power while reducing the economic cost and environmental im-
pact. Microfabrication technologies were soon applied to other devices, such
as pressure sensors and accelerometers, allowing the development of complex
microelectromechanical systems (MEMS) and, at the end of the 20th cen-
tury, devices for chemical and biological analysis: the so called Lab on-Chips
(LOCs). Their major feature is the integration of one or several laboratory
functions on a single chip of few square centimeters in size. LOCs deal with
the handling of extremely small uid volumes down to less than pico liters.
The controlled motion of the uids, the DNA amplication, drug screen-
ing and many others analytical techniques have been miniaturized in LOC
platforms. Many of today's applications and possibilities of microuidic lab
on-chip systems have been reviewed by Weigl et al. [26] and more recently
by Dittrich et al. [27]. The advantages brought to biology and diagnostics
by the miniaturization are mainly related to the following points: 9 ' Velocity It has been demonstrated that the size reduction brings ad- vantage when transfers of mass and/or heat are involved. Smaller vol-
umes imply faster heating and cooling cycles, shortening the completion
time of a large number of cycles. For example, in the DNA amplica-
tion by PCR [30], the operational time has been reduced from several
hours to few minutes. ' Parallelism Like in the microelectronic industry, the miniaturization can bring to a massive parallelism. The need of increasing parallelism
in the discovery of the genomic information has been the major driving
force. Lithographic techniques allowed the synthesis of more than 500k
samples of DNA on a single chip [28]. ' Low consumption of reagents Another benet of the miniaturiza- tion is the costs reduction in the pharmaceutical test on the potential
eectiveness of certain drugs. This compounds are very expensive and
reducing their volume by order of magnitude implies a signicant sav-
ing. ' Functional integration Although the previous point are important, the most interesting opportunity due to the miniaturization is the func-
tional integration. It allows to carry out complex analytical protocols
involving several steps or tasks in a faster and cheaper way on the same
platform. Several commercial products already exist in the market like the Agilent
LabChip 2100 BioAnalyzer, which uses a lab-on-a-chip approach to perform
capillary electrophoresis and a uorescent dye that binds to RNA to deter-
mine both RNA concentration and integrity. The chip format together with
the computer for the analysis of the results, dramatically reduces sample
consumption but also the time needed for the test. Many application and 10 Figure 1.3: Sketch of Laboratory on-chip [29]. dierent devices can be envisaged in the area of lab-on-chip platforms: Micro
total analysis systems (µTAS) are miniaturized and highly integrated chem-
ical analysis systems initially designed in the late 80's [31]. Micro reactors
are used for chemical synthesis or energy production. They provide methods
to synthesize, on request, unstable, valuable or even dangerous materials.
Finally, exploiting physical and chemical properties that are unique for mi-
crouidic systems (e.g. laminar ow and the high surface to volume ratio)
complex biological experiments, are indeed possible. 1.3.1 Magnetic based LOC's devices The integration of microuidic lab-on-chip devices and magnetic nanoparti-
cles permit a large amount of biological applications that range from sorting
target biomolecules in a solution, detecting the presence of a specic molecule 11 or to manipulate biological entities or cells.
The main commercial use of magnetic manipulators concern the separation
of biological species in solution (see Fig.1.4). In this application, magnetic Figure 1.4: Magnetic separation of labelled biomolecules via magnetic
nanoparticles. nanoparticles are decorated with particular probes which bind a specic
biomolecule. Exploiting a permanent magnet, an external eld is applied to
attract nanoparticle so that the concentration of such molecules is increased
in a certain region of uid [32]. However this system doesn't allow a single
particle control together with an high parallelism. In order to increase the
spatial resolution, a much more conned eld has to be applied to trap a tar-
get particle. This can be achieved patterning the chip surface with magnetic
elements. The joint action of an external magnetic eld and the stray eld
due to the magnetization of such elements, permits to trap the nanoparticles
in a conned region. A variation in the direction of the external magnetic
eld can be employed to manipulate the particles in solution over magnetic
structures (Fig.1.5)[33],[34]. The NanoBiotechnology and Spintronics (NaBiS) group of L-NESS Center in collaboration with the Spanish research centre nanoGUNE, has devel-
oped a system for trapping and moving magnetic nanoparticles by means 12 Figure 1.5: Manipulation of 2.8 µm magnetic beads on a staircase pattern
of Permalloy ellipses by in-plane 80 Oe rotating magnetic eld. The arrows
indicate the direction of the eld. After one complete eld revolution, the
beads have moved one step in the pattern as indicated by the white curve in
(f)[33]. of magnetic domain walls (DWs) conned in magnetic nanostructures [35].
Transverse Neel DW (see chapter 2) generates a considerable stray eld that
attract a single nanoparticle on the top of the structures. Besides DWs dis-
placement along ferromagnetic conduits applying a proper sequence of mag-
netic elds, is employed to achieve an high spatial control (down to 100nm)
of the nanoparticles which follow the DWs movement. Two dierent nanos-
tructures geometries are used to this goal: zig-zag and curved structures as
illustrated in gure 1.6. This topic will be deeply discussed in the next chap-
ters, considering both the theoretical aspects (Chapter 2), the development
and the applications of this technology (Chapter 3,4,5 and 6).
Another fundamental application of LOC devices is to detect and record
the presence of biomolecules in solution. A large amount of high resolution
and miniaturize sensors for this purpose can be found in literature [37]. In
particular, sensors which combine electrical and magnetic properties such 13 Figure 1.6: Ferromagnetic conduits geometries: zig-zag shaped (a) and
curved (b) structures. as giant-magnetic resistance (GMR), tunneling-magnetic resistance (TMR),
anisotropic magnetic resistance (AMR) and hall-eect are widely employed
in nanomedicine [38],[39],[41],[40]. In this work, in order to exploit magnetic
nanoparticles properties, not only as biomolecules carriers, but also as labels,
two dierent MNPs detection paradigms have been developed: ' Anisotropic-magnetic resistance based sensors This system is able to electrically detect the crossing of a DW between two micro
electrical contacts by an electrical signal variation through a magne-
toresistive eect. The presence of a MNP on the top of a DW is de-
tected, because of it induces a variation of the depinning eld (the
minimum eld required to displace the DW) that can be recorded. ' Electrical sensors based on impedance measurements A particle driven between two micro-electrical contacts in a buer solution pro-
duces a voltage drop due to the high impedance of such nanoparticle
that can be detected by a capacitance measurement. A full analysis of the working principle, fabrication and application of such
sensors is illustrated in chapters 2,3,5. 14 1.4 Thesis outlook The aim of this thesis is to develop an innovative on-chip platform for drugs-
delivery through magnetic nanoparticles. This technology permits the con-
trolled administration of a single particle to a target cell by means of mag-
netic tweezers based on magnetic DWs, briey introduced in section 1.2.2.
Superparamagnetic nanoparticles are injected in a microuidic cell and their
ow in a channel is magnetically activated via the manipulation of DWs in
magnetic conduits. The passage along the channel is then detected by a sen-
sor (AMR or impedance-variation based). Then, particles are trapped and
manipulated to an area of chip where living cells are cultured, studying the
interaction between a single particle and cell. The nal goal is to observe a
particle uptake in order to complete the vehiculation of the drug inside cell
membrane.
Despite the complexity of the presented work, involving dierent area of
knowledge and competences, some relevant progress have been done during
this thesis. In particular the activation and detection of the passage of the
bead over a magnetic conduit has been demonstrated. Furthermore prelimi-
nary experiments of cellular uptake have been carried out. The experimental
activity has been performed mainly in the L-NESS laboratory under the su-
pervision of Prof. Riccardo Bertacco. The sensors development has been
performed in collaboration with the 'Electronic engineering' group of 'Po-
litecnico di Milano' leaded by Prof. Marco Sampietro. The biological part of
this work has been carried out in collaboration with 'IFOM' (Istituto Farma-
cologico di Oncologia Molecolare) and 'Istituto farmacologico Mario Negri'. Here an overview of each chapter is presented: ' Chapter 1: Introduction and scientic background This chapter gives a brief description of the scientic background and a summary of 15 this thesis work. ' Chapter 2: Micromagnetics and magnetic nanoparticles In this chapter the theoretical aspects such as the physics and the working
principles of magnetic nanostructures are illustrated. Besides, a de-
scription of magnetic nanoparticles behavior and the interaction of such
particles with DWs is provided, together with an explanation of the
AMR eect. ' Chapter 3: Experimental methods This chapter shows the exper- imental techniques used in this work. The fabrication of devices, the
simulations performed to optimize the working conditions, the charac-
terization techniques, the dierent setups used for measurements and
tests are presented here. ' Chapter 4: Movimentation of nanoparticles over magnetic conduits The manipulation of magnetic nanoparticles over thin lm
nanostructure is presented here. The rst part is a description of a
single-particle manipulation on zig-zag shaped and curved structures.
In the second part, the simultaneous motion of a nanoparticle batch
over curved conduits is illustrated. ' Chapter 5: Detection methods of magnetic nanoparticles In this chapter the detection and counting of MNPs exploiting two dier-
ent sensors, based on AMR and electrical impedance, is described. ' Chapter 6: Controlled administration of nanoparticles to a single cell This chapter deals with the use of magnetic manipulation
system described in Chapter 4 to bring a single magnetic nanoparticles
in proximity of a living cell, studying the interaction between particles
and cells as fundamental aspect of drug-delivery. 16 ' Conclusions and Perspectives This section summarizes the conclu- sions of this thesis and outlines the future perspectives. 17 Chapter 2 Micromagnetics and magnetic
nanoparticles The behavior and the properties of a magnetic material are described by the
relation between the magnetization vector and the magnetic eld M(H). The
calculation of this parameter is a complex matter because there are several
physics phenomena that compete to determine what is the magnetic cong-
uration for a certain material. This chapter has the goal to describe these
dierent contributions through simple equations and parameters which are
basic to explain the behavior of a magnetic body. The knowledge of these
parameters allows to foresee the behavior of nanostructured magnetic mate-
rials and thus to properly design the devices employed in this thesis work.
The rst part of the chapter deals with the existence of magnetic domains
with a focus on magnetic domain walls (DW) conned in nanostructures. In
the second part, instead, a second ingredient of the employed manipulation
methods will be discussed: the superparamagnetic beads and their interac-
tion with the DWs stray eld. Finally, the anisotropic magnetic resistance
eect exploits in AMR sensors will be described. 18 2.1 Micromagnetism The main mechanisms leading the formation of the dierent magnetic con-
gurations are exchange interaction, magnetic anisotropy, magnetostatic self
energy (due to dipole-dipole interaction) and Zeeman Energy.
In a thermodynamic approach, the most stable conguration of M arises
from the minimization of a Free Energy functional which include all the con-
tributions listed before. Domains formation helps to minimize the energy
in most cases. The main issue of this approach is that each energy term
(in particular exchange and anisotropy) depends on the atomic structures
of materials, consequently the energy minimization has to be calculated in
an innite dimensional space including innite spatial coordinates. This ap-
proach belongs to the theoretical framework of micromagnetism. It is based
on the idea that a magnetic body can be divided into small volume ele-
ments ''V in which the value of Magnetization vector M can be considered
uniform. These partitions of the volume are small compared to the char-
acteristic length of variation of the magnetization, but they are big enough
so that thermodynamics and statistics rules can be applied. Free Energy in
micromagnetic theory is expressed in the continuum approximation, where
atomic structure is averaged away and M(r) is a smoothly varying function.
Morover, another assumption of micromagnetism is that the relaxation time
for which a single volume element ''V reaches the thermal equilibrium is
much shorter than the relaxation time for the whole system.
The micromagnetism history starts in 1935 with a paper of Landau and Lifs-
chitz on the structure of the wall between two antiparallel domains. William
Fuller Brown gave this theory the name micromagnetism in 1963 to distin-
guish it from Domain theory which considers domains but neglects walls.
In the following sections there will be a description of all the physics mech-
anisms that lead magnetization in a magnetic material. 19 2.1.1 Exchange Interaction The exchange energy, which is the base of ferromagnetism, is a quantum
mechanic eect concerning the spin-spin interaction. It favors the parallel
(ferromagnetic) or antiparallel (antiferromagnetic) orientation of spins along
interatomic distances. It can be expressed by the Heisenberg Hamiltonian: Hexchange = '' N '' i=j=1 JijSi · Sj (2.1) where Si is the spin angular moment related to an ion located in position i
in a certain lattice and the exchange parameter Jij gives information on the
strengthness of the interaction between spins i and j. The exchange inter-
action decreases quickly with the interatomic distance, so that the exchange
energy can be calculated considering only the rst neighboring atoms.
Considering spin operators as classical vectors, it can be written: Eexchange = ''JS2 N '' i=j=1 cos'ij (2.2) where 'ij is the angle between two classical spins vectors and J is considered
uniform for all the coupled spins in a lattice (taking into account only the
rst neighbors). The angle between neighbors spins is usually very small,
consequently, the cosine function can be written as a Taylor sum and the
equation 2.2 becomes: Eexchange = 1 2 J S 2 N '' i=j=1 ' 2
ij (2.3) The zero point energy of exchange energy is removed neglecting the constant
term of cosine expansion. Equation 2.3 can be usually expressed in another
form adopting the unitary magnetic moment m = M/Ms. For small angles
|'ij| = |mi '' mj| = |(rij · '')m| where rij is the vectorial distance between
ions in position i and j. In this way the 2.3 becomes: Eexchange = ''JS2 N '' i,j '' rij [(rij · '')m]2 (2.4) 20 Substituting the sum with an integral on small volume portions, equation
2.4 can be written as follow: Eexchange = ' V A[( ''mx)2 + (''my)2 + (''mz)2]dV (2.5) It is the proper form of free energy term due to exchange. A [J/m] is the
exchange constant dened by: A = 2J S2c a (2.6) where a is the lattice parameter and c is an integer depending on the crys-
talline structure of a body. For example, it is 1 for a cubic lattice, 2 for a
BCC lattice and 4 for a FCC structure. From the exchange energy contribu-
tion we can derive the most important length scale in micromagnetism: the
exchange length lex. lex = '' A µ0M 2s (2.7) It represents the distance over which the magnetization can twist by consider-
ing only exchange and magnetostatic interaction, while neglecting magnetic
anisotropies. It gives also the boundary between "small" objects with uni-
form magnetization (single domain state) and "large" objects which tends
to develop a complex and non-uniform magnetization (multi-domain). Fur-
thermore, the size of the cells ''V in which the structure is divided should
be smaller than lex in order to consider magnetization constant inside each
cell. 2.1.2 Magnetic Anisotropy Energy Magnetic anistropy is the tendency for magnetization in a ferromagnet or
antiferromagnet to lie along preferred directions, called easy axis. If the
magnetization of a certain body is oriented along another direction an en-
ergy penality has to be considered in the total free energy (which is exactly 21 the work to align M with a direction dierent from an easy one). As dis-
cussed before, considering an uniform value of magnetization (M = Msm) in
each small volume portion, it is possible to write an expression of anisotropy
energy density eAN(m) as a series of trigonometric functions: eAN = K0 + K1sin 2θ + K 2sin 4θ (2.8) where θ is the angle between M and the anisotropy axis. K0, K1, K2 have the
unit of J·m''3 Values for K1 may range from less than 1 kJm''3 to 30 MJm''3.
It depends on temperature and must tend to zero at the Curie temperature
TC.
This expression is valid only for an uniaxial anisotropy. There are dierent
expressions of anisotropy energy in dierent symmetries (depending on the
atomic lattice geometry) which can be found in literature. Figure 2.1: Uniaxial anisotropic energy density. (left) Anisotropy with easy
axis (K1 > 1). (right) Anisotropy with easy plane (K1 < 1). K1 can be bigger or smaller than zero. In the rst case there will be a preferred (easy) axis for magnetization, in the second one a preferred (easy)
plane (see Fig. 2.1).
For small values of θ (small angle between magnetization and easy direction), 22 equation (2.8) becomes: eAN (m) = 2K1sin 2θ '' µ0M · HAN (2.9) where HAN is the anisotropy eld dened as follow: HAN = 2K1 µ0MS (2.10) It is not a real eld but simply a parameter which measures the strengthness
of the anisotropic eect.
In order to write an equation for the entire body an integration on the total
volume has to be performed: EAN = ' V eAN (m)dV (2.11) In a three dimensional picture, easy directions are related to minima of
anisotropy energy density function instead maxima and saddle points are
associated with hard and medium axis.
So far, magnetic anisotropy has been treated without considering what is the
origin of the phenomenon. There are two main sources of anisotropy related
to sample shape and crystalline structure. Magnetocrystalline Anisotropy Magnetocrystalline anisotropy is an intrinsic property due to the fact that
the magnetization process is dierent when the eld is applied along dif-
ferent crystallographic directions because the anisotropy reects the crystal
symmetry. Its origin is in the crystal eld interaction and in the spin-orbit
coupling. There are two dierent mechanisms which drive magnetocrystalline
anisotropy: single ion contributions and two ions contributions
Single ion anisotropy is essentially due to the electrostatic interaction
of the orbitals containing the electrons responsible of magnetism with the
potential create at the atomic site by the rest of the crystal. The crys-
tal eld interaction tends to stabilize a particular orbital, and by spin-orbit 23 interaction, the magnetic moment is aligned in a particular crystallographic
direction. In a ferromagnetic crystal the contributions of all ions are summed
producing a macroscopic eect.
Two ions anisotropy reects the anisotropy of the dipole-dipole interac-
tion. Comparing the broadside and head-to-tail congurations of two dipoles,
each with moment m, the head-to-tail conguration is lower in energy (mag-
nets tend to align head-to-tail). However, the dipole sum has to be extended
over the entire lattice, and it vanishes for certain lattices (including all the
cubic lattices). In noncubic lattices, the dipole interaction is an appreciable
source of ferromagnetic anisotropy. Shape Anisotropy This phenomenon is strictly related to the geometry of the body. The origin
of the shape anisotropy is the magnetostatic energy, in particular it arises
from the demagnetizing eld at which the self-energy of the body is associ-
ated. In order to minimize the magnetostatic energy, magnetization pointing
perpendicular to the surface is not favored in thin lms because an high
energetic cost should be paid. In the nanoworld this contribution is often
fundamental to drive the magnetic congurations. 2.1.3 Magnetostatic and Zeeman Energy Magnetostatic Energy is dened as the mechanical work required to bring
all the magnetic moments forming the body from the innity to their nal
positions, so as to form the macroscopic material. It is essentially due to
dipole-dipole interactions. Compared to exchange, magnetostatic interac-
tions are long range ones.
Considering a magnetic body in a certain region of space, it obeys to Maxwell 24 equations so that in absence of currents: '' ' H = 0 (2.12) H is the eld induced by the magnetization of the body. From 2.12 is possible to write: H = ''''U (2.13) where U is magnetostatic potential. Introducing the fundamental relation
B = µ0(H + γBM) (where γB is the Brown constant, equal to 1 for the "international system of units" -S.I.-) and remembering that B is a diver-
genceless eld ('' · B = 0), it is possible to obtain the following expression: ''2U = γBM (2.14) The last equation is dierent to zero only inside the magnetic material (M ̸=
0 ). From the previous expressions is possible to obtain the following boarding conditions: Uint = Uext (2.15) and ''Uint ''n '' ''Uest ''n = γBM · n (2.16) in which n is the direction perpendicular to the surface. The solution of 2.14
is unique with the adequate conditions and it is possible to demonstrate the
following expression for the magnetostatic energy: U = Emagstat = '' 1 2 ' ' M · Hdd' (2.17) where the integration is over the whole magnetic material. Hd is the demag-
netizing eld induced by magnetization and dened as follow: Hd = ''NM (2.18) N is the shape dependent demagnetizing tensor. Equation 2.17 represents the
so called "self-energy" because is the energy associated to a magnetic body 25 in absence of an applied eld and in a static condition. When an external
magnetic eld is applied, the magnetic moment will try to reduce its energy
by aligning itself parallel to it. The energy that describes the interaction of
a magnetic moment with an external eld Ha is called Zeeman energy and
it is given by the following relation: FZeeman = ' V Ha · MdV (2.19) It is a long range interaction. 2.1.4 Magnetic domains In 1906 Weiss rst identied the presence in magnetic materials of small re-
gions, called domains, where the Magnetization is almost uniform and the
orientation of M varies from one domain to another [42]. Domains are sepa-
rated by domain walls. However Weiss didn't properly explain why domains
exist. In 1935 Landau and Lifshitz showed that domains formation is the
consequence of minimization of the total free energy functional (ETOT ) of a
magnetic system [43], which gives rise to the dierent magnetization con-
gurations. ETOT can be written by adding up the energy contributions
arising from the exchange coupling, magnetocrystalline anisotropy energy,
demagnetization and the external eld as: ET OT = ' V {A[(''mx)2 + (''my)2 + (''mz)2]+ +eAN '' 1 2 µ0MSm · Hd '' µ0MSm · Ha}dV (2.20) where m is the magnetic moment, Hd and Ha are respectively the demag-
netizing eld and the external magnetic eld and M is expressed as MSm.
As mentioned before, the local energy minima correspond to the metastable
states of the system. Therefore, the existence of domains is the result of the
combination of all the energy terms that appear in this functional.
The exchange energy favors the parallel allignment of m throughout the 26 whole volume. The direction of m is not important for such contribution,
but each conguration in which m spatially varies costs in terms of energy.
The magnetocrystalline anisotropy promotes local alignment of m with easy
axis or planes within the material. This means that an energy cost has to be
paid if m is not directed in that way. Moreover, the magnetostatic energy
favors any congurations in which magnetization follows close paths inside
the object in order to minimize the stray eld outside. This fact can be
in competition with requirements for lowering the exchange energy and the
anisotropic energy contribution. A parameter to evaluate the relative im-
portance of the magnetostatic interaction respect to the anisotropic term is: kM''A = 2K1 µ0MS (2.21) Generally, the magnetostatic contribution leads in isotropic materials where
kM''A' 1. Instead, the anisotropic energy is the dominant term in crystalline
material where kM''A' 1.
The break up of magnetization in localized regions which provides for ux
closure at the ends of the specimen, is mainly due to the need of minimizing
the magnetostatic energy. On the other hand, a large number of domains are
associated to a large number of domain walls (region between two neighboring
domains) that implies an exchange energy cost.
As an example, gure 2.2 shows three dierent congurations for the domain
structure in a ferromagnetic sample. The single domain structure (a) has no
domain walls, but has a large dipolar energy because the magnetic moments
are directed perpendicular to two edges of the object. The dipolar energy can
be reduced by the formation of dierent antiparallel domains (b). The so-
called closure domain structure (c) eliminates the dipolar energy, since there
are no "magnetic charges" on the edges, but introduces a larger number of
domain walls. 27 Figure 2.2: Domain formation: from left to right the magnetostatic energy
is decreased due to domains creation [44]. 2.1.5 Domain walls In order to create domains, a certain work has to be performed against ex-
change interaction which would favor spin alignment. To limit this energetic
penalty domain walls (DWs) exist. DW is a region between two magnetic
domains where the magnetization changes gradually from one direction to
another. This is true since the exchange energy, as explained in section
2.1.1, is a short range term. Considering a piece of material with two do-
mains, where the orientation of magnetization is in opposite directions (see
Fig. 2.2(b), there is not a substantial variation of anisotropic energy if the
two antiparallel domains are aligned along the uniaxial anisotropy easy axis
compared to the single domain case (Fig. 2.2(a)).
Anisotropy tends to form thin walls (where only few spins are not aligned to
the easy axis), while the exchange interaction promotes thicker walls. The
more gradual is the angle variation between spins, the lower is the exchange
energy cost.
Depending on the spin rotation across the wall, two main types of domain 28 walls can be distinguished: Bloch and Neel wall. In a Bloch wall, the spins
rotate in a plane perpendicular to the domain wall plane. In a Neel wall the
spins rotate in the domain wall plane. Both types are illustrated in gure
2.3. The former one is promoted in bulk samples. The Neel wall is instead
favored when thin lms are considered, since it removes the magnetostatic
energy cost due to spins pointing out of the lm plane. The contribution
of magnetic poles on the two surfaces is eliminated, but the conguration of
the elementary magnetic moments inside the wall creates an additional stray
eld. Figure 2.3: Two domain wall types, Bloch wall (above) and Neel wall (below). 2.1.6 Neel domain walls in thin lm materials In thin lms, the magnetic conguration is not only determined by the in-
trinsic material properties but also the morphology and geometrical shape
plays a fundamental role. This is particularly true for soft magnetic mate-
rial in which the magnetocrystalline anisotropic energy is negligible and the 29 equilibrium magnetic state is related essentially to the geometric shape. This
permits to properly x the magnetic conguration and the magnetization re-
versal by choosing the morphology and the applied external eld.
It is due to the fact that, in soft magnetic nanostructures, the magnetostatic
energy promotes the alignment of magnetization along the edges of the struc-
tures, in order to minimize the stray eld.
In elongated elements, such as stripes or conduits, the magnetization is
aligned to the long axis and this mono-domain state is very often the low-
est energy magnetic state. In multi-domain congurations, instead, domain
walls (DWs) are created, forming mobile interfaces that separate regions with
opposite magnetization. Two spin structures can be found, classied as Neel
wall variants: the transverse DW and the vortex DW, shown in gure 2.4(a).
A DW can be either head-to-head (HH) or tail-to-tail (TT), if the magneti-
zation is pointing towards or away from the wall. As an example, in gure
2.4(a) both the conguration are HH. In the transverse DW, spins rotate in
the plane of the structure, from one domain to the adjacent one. The vortex
wall shows a completely dierent conguration. The spins rotates clockwise
or counterclockwise around the vortex core where the magnetization points
out of plane. The energy of the two congurations varies with the dimensions
of the stripes and with the material used. The phase diagram between the
transverse and vortex state was calculated by McMichael et al. [45]
They considered only the magnetostatic and exchange energy terms for a pat-
terned stripe of Permalloy of thickness t and width w coming to the equation: wt = const · δ2 (2.22) The results, expressed as function of the dimensionless variables t/δ and
w/δ , where δ = lex/2, are shown in gure 2.4(b).The graph shows that for relative thin and narrow geometries, the transverse wall is preferred, whereas
the vortex wall is favored in thicker and wider stripes. For example, in a 30 Figure 2.4: Top view of a Permalloy innitely long strip with two opposite
domains (red and blue arrows) and a DW which divides the two regions.
Both transverse spin structure and vortex spin structure DW are shown (a).
Phase diagram of a transverse HH DW in thin magnetic stripe. δ is equal to
lex/2 (b) [45]. Permalloy conduit 20 nm thick the transverse wall is favored for width under
200 nm. 2.1.7 Pinning and propagation of Domain walls In this section, the quasi-particle approach to describe the DW behavior will
be presented. As discussed in the previous sections, the particular spin con-
guration of the DW is the result of the energy minimization process. The
terms, involved in the process, do not only depend on the material but also
on its shape and on the external magnetic elds. Shape variations change
the potential landscape that a DW feels. By using dierent geometries we
are able to engineer well-dened attractive potentials which acts as stable
positions for DWs. Moreover, such potential landscape can be modied by
applying external magnetic eld, so that equilibrium stable positions depend
both on geometry and on the applied eld. Intuitively, DWs should achieve
an energy minimum in positions where the wall is small, since, in rst approx- 31 imation, the DW energy scales with its dimension . Thus the DW nucleation
is energetically favored in constriction and in narrowed conduits. A con-
striction pins a DW, generating an attractive potential well. Every well is
characterized by a characteristic length and depth. The depth is related to
the magnetic eld intensity needed to push the DW out of the constriction
and move it to a new stable position, or annihilate it. An example of rele-
vant pinning sites for DWs are corners. Figure 2.5 shows the transverse DW
conguration in a corner and the potential well generated by this geometry. Figure 2.5: Simulated behavior of a transverse DW at a corner site. The
gure shows the magnetization structure of the DW and below the sketch of
the potential well, whose minimum is centered on the tip of the corner. The magnetization and DW dynamics are described by the Gilbert equa- tion: ''M ''t = γM ' Heff '' αγM ' ''M ''t (2.23) where γ is the electron gyromagnetic ratio, α is the experimental damping
coecient and Heff is the eective eld. It is a ctitious magnetic eld that
takes into account all the energetic contributions: the external and mag- 32 netostatic eld, but also the exchange interaction and the anisotropy. The
eective eld exerts a torque to the magnetization vector. The rst term de-
scribes a pure gyroscopic eect. If M is not at the equilibrium, it will start to
precede continuously around the eld without reaching the equilibrium. The
second term, experimentally introduced, takes into account the dissipative
eects. The thermodynamic forces lead M to the equilibrium. Similarly to
the viscous forces, the damping term is proportional to the temporal varia-
tion of the quantity of interest, in this case the magnetization.
When an external magnetic eld is applied to the system, the DW feels a
change in the potential landscape. If the eld intensity is high enough to
overcome the local pinning site, the DW will propagate towards the new
equilibrium position. The propagation process of a transversal DWs along a
conduit has been studied by Walker and Schryer [46]. They found that the
DW moves with a velocity v: v = γ'' α · H (2.24) where '' is the DW length. The velocity is thus proportional to the applied
eld. The relationship is valid until a critical eld, called Walker eld HW ,
is reached. Above this eld the DW magnetic conguration is transformed
and it reduces the wall velocity. 2.2 Magnetic micro and nanoparticles In this section, the magnetic particles and their interaction with magnetic
elds in a liquid environment will be described.
Many properties of magnetic materials can be explained by their volume
magnetic susceptibility ', which describes the magnetic response, i.e. its
magnetization, to an applied eld: M = 'H. In presence of a magnetic 33 material the total ux density B results to be: B = µ0(M + H) = µ0(1 + ')H = µ0µrH (2.25) where µ0 is the permeability constant in vacuum and µr the relative perme-
ability of the material. Depending on the interaction of the solid material
with an external magnetic eld, various types of magnetic behaviour can be
distinguished, such as diamagnetism (µr <1), paramagnetism (µr >1), fer-
romagnetism (µr '1).
When the size of a magnetic object is reduced below a certain dimension, as
in the case of nanoparticles, a dierent magnetic eect can arise. It is called
superparamagnetism, corresponding to the situation in which each particle
behaves as a macrospin (where spins are ferromagnetically aligned) uctuat-
ing in an external eld as in the case of magnetic moments within a param-
agnet.
Considering a magnetic spherical particle of volume V with an uniaxial
anisotropy and two possible magnetization states: parallel or antiparallel
to the easy axis. They are separated by an energy barrier proportional to
the anisotropy constant K1 of the material and to the volume V. If the activa-
tion energy (K1V) for ipping the magnetization from parallel to antiparallel
is small compared to the thermal energy kBT, where kB is the Boltzmann
constant, the magnetization is continuously inverted by thermal uctuations.
The average time between two thermally activated transitions is given by: ' = '0exp( k1V kBT ) (2.26) where '0 ranges 10''9 10''11 s for isolated particles and T is the absolute tem-
perature. By reducing the particle size, the energy barrier K1V decreases
and, inversely, the ipping rate increases. Superparamagnetism is a size ef-
fect that depends both on the temperature T and on the observation time t.
A magnetic particle appears blocked, i.e. ferromagnetic, if the observation 34 time t is much smaller than '. Above the blocking temperature, dened
as the temperature at which ' is equal to the measuring time, the particles
behave as a superparamagnet. At room temperature the typical diameter
of a superparamagnetic particle is in the range from 5 to 128 nm [47].The
superparamagnetic state provides to nanoparticles high saturation magneti-
zation MS, comparable to those of ferromagnets, and no remanence MR as a
paramagnet. Those properties make them suitable for dierent biological ap-
plications. The magnetic behavior of such superparamagnetic particles can
be described through the Langevin function (L(x) = cot(x) '' 1/x) so that: M(H) = mL( µ0mH kBT ) (2.27) where m is the magnetic moment of the particle.
A typical nanoparticle is formed by a superparamagnetic core surrounded by
a nonmagnetic coating. The last one is necessary to form selective bonds with
biomaterials of interest. Standard particles have a total diameter between
5 and 50 nm. Iron oxides such as magnetite (Fe3O4) or maghemite (Fe2O3)
are employed for the core because they are biocompatible and potentially
non-toxic for biological entities.
In order to maintain the properties of superparamagnetic particles, but achiev-
ing a greater volume and magnetic moment, larger magnetic bead (0.1 to 5
µ m in diameter) have been introduced. They are obtained by embedding several superparamagnetic nanoparticles, not magnetically interacting, in a
non-magnetic matrix. A sketch of a magnetic bead made by superparamag-
netic particles core in a polymeric shell is illustrated in gure 2.6. In this
thesis work, three main dierent types of commercial superparamagnetic
beads are employed. Dynabead My-One (Invitrogen) are widely used for the
experiments described in chapter 4 and 5. They have a diameter of 1 µm
and they are functionalized with COOH. In chapter 6 nanomag-CLD-redF
(Nanomag) particles are employed. They have a diameter of 300 nm and
are functionalized with COOH. The latter are also covered by a TRITC red 35 Figure 2.6: Sketch of magnetic bead formed by magnetic nanoparticles in a
non-magnetic matrix/shell. uorescent marker to get images in uorescence. In chapter 5, micromer-
M-Streptadivin (Nanomag) particles are also used to test capacitive sensors.
They have a diameter of 2 µm and are fuctionalized with the Streptavidin
protein. COOH and Streptavidin functionalizations are highly hydrophillic
and, consequently, they are suitable for experiments in wet environments.
Moreover, they oer a low non-specic binding, excellent dispersion abilities
and easy handling in a wide variety of buers. 2.2.1 Interaction forces on magnetic beads To understand how the magnetic eld can be used to transport and manip-
ulate magnetic beads it is important to know which forces act on a particle
suspended in a uid. In all the experiments magnetic bead are subjected to
gravity forces, to the viscous force and magnetic force.
The buoyancy force Fb opposes to the gravity force Fg giving a net contri-
bution expressed by: Fg '' Fb = ''V (ρbead '' ρfluid)g (2.28) 36 where ρbead and ρfluid are respectively the density of bead and liquid. V is
the volume of the particle and g the gravitational acceleration. For Dynabead
My-One particles which have a density of about 4 g*cm''3, this contribution
is around 80 fN in water.
The magnetic interaction between a bead and a certain magnetic eld can
be described by the magnetic potential energy density ub = Mb · B, where
Mb is the bead magnetization.
The force per unit of volume that is related to the magnetic interaction is
fM = ''''ub. it is possible valuate the total force acting on the entire particle by integrating fm over the bead volume: Fm = ''µ0 ' V (Mb · '')HdV (2.29) It is valid if '' ' B = 0 as in the magnetostatic case, and exploiting the
equivalence of the equation 2.25. In this thesis work, this integral has been
calculated through numerical methods via Matlab (see chapter 4 and 6).
A particle moving with velocity v in a uid of velocity u is subjected to the
hydrodynamic drag force. This force Fdrag obeys to the Stokes law: Fdrag = ''6'ηRb(v '' u) (2.30) where η is the uid viscosity and Rb the bead radius. It opposes the magnetic
interactions in the magnetic handling process.
Finally, Brownian motion leads to superimposed velocities in random direc-
tions which become more important as the size of the particles decreases.
The diusion constant D and the related diusion length ldif for a spherical
particle in a uid at temperature T are inversely proportional to the bead
radius and are respectively equal to [48]: ldif = '' Dt (2.31) with D = KB ξ = kBT 6'ηRb (2.32) 37 where ξ is the friction coecient. For example, after 1s of diusion in water
at room temperature, a 1 µm diameter particle travels an average distance
of 700 nm. For a magnetic particle with a diameter of 100 nm the diusion
length on the same time scale is almost three times larger.
In the next section, the description of the physical mechanism of the DW
assisted manipulation of magnetic particles will be given. 2.2.2 DWs assisted manipulation of magnetic nanopar- ticles As discussed in section 2.1.8 the DWs can be nucleated, pinned and prop-
agated along magnetic conduits. Moreover, Neel walls produce a highly in-
homogeneous stray eld, up to few kOe which can be employed to trap a
MNP in suspension. This stray eld is spatially localized at the nano scale Figure 2.7: Sketch of the magnetic attractive potential well generated by a
HH DW pinned at one corner of the magnetic conduit. Such potential is able
to trap a superparamagnetic antibodies functionalized bead at a distance of
100 nm from the chip surface. From [49] on the plane of the conduit thanks to the very conned geometry of the DW. 38 The DW, nucleated by the application of an external magnetic eld, acts as
movable attracting pole (see Fig.2.7). The gradient, together with the inten-
sity of the stray eld produces an attractive force on a superparamagnetic
particle which is moving in the proximity of the DW location. The DW eld
induces a magnetic moment in the particle that then is attracted towards the
minimum of the potential. In this way, when the DW is depinned from one
pinning site to another, applying an external eld, the particle also moves,
following the modication of the attractive landscape potential. This tech-
nology allows a maximum transport speed of 15µm/s which is aected by the
viscosity of the medium where particles are diluted. This technology, called
"domain wall tweezers" (DWT), has been invented by the NaBis group of
LNESS in collaboration with nanoGUNE research center [60]. In this the-
sis work, two dierent geometries for the magnetic conduits are employed:
zig-zag shaped and curved conduits; they are illustrated in chapter 4. 2.3 Anisotropic Magnetic Resistance Magnetoresistance refers to the change in electrical resistance of magnetic
materials with the application of a magnetic eld and was rst discovered
by William Thomson (Lord Kelvin) in 1851. Changes in resistance of up
to 5% were observed and this discovery was later referred to the Ordinary
Magnetoresistance (OMR). More recently anisotropic (AMR), giant (GMR),
colossal (CMR), and tunnelling magnetoresistance (TMR) eects have been
discovered with larger changes in resistance reported. In this thesis the AMR
will be employed in magnetic based sensors in order to count the particle tran-
sit in lab on chip for drug delivery.
Anisotropic Magnetoresistance is the property of a material in which the
electrical resistance depends on the angle between the direction of electri-
cal current and orientation of magnetization. The AMR eect is based on 39 the anisotropic scattering of conduction electrons of the band with uncom-
pensated spins (e.g., 3d electrons for the rst series of transition metals Fe,
Co and Ni). Theoretical analysis of AMR are given in terms of the elec-
tron density-of-states diagram at the Fermi level. The diculty is that the
anisotropic part of the resistance depends on the exact three-dimensional
shape of the Fermi surface (3d-envelope of the Fermi level), which is not
precisely known except for very few magnetic materials. If we indicate the
resistivity in the direction parallel and perpendicular to the direction of M
as ρ' and ρ', in most of the magnetic materials is observed that ρ'>ρ'. In
this case, the AMR is dened as: RAMR = ρ' '' ρ' ρ (2.33) where ρ is dened as ρ = ρ' + 2ρ' 3 (2.34) For example, let us consider a ferromagnetic strip in presence or absence of
a DW as illustrated in gure 2.8. Figure 2.8: AMR eect in a ferromagnetic strip in presence of a DW; the
resistance is higher when J and M are parallel. Image from OOMMF. If a current density J ows in the strip and a DW is present in the conduit segment considered, the magnetization in the DW points perpendicularly to 40 the current ow. This means that the resistance is lowered respect to the
case when there is no DW and J and M are parallel. Sensors based on the
AMR eect will be described in Chapter 5. 41 Chapter 3 Experimental methods In this chapter an overview of all the experimental methods used in this thesis
work is presented, starting from the fabrication of samples and devices, to the
device characterization techniques but also the experimental setups exploited
for biological assays.
Three main dierent microuidic-devices have been developed, produced and
tested in this work: nanopatterned samples for particles manipulation, AMR
sensors and capacitive sensors for bead detection. 3.1 Micro- and nano-fabrication techniques The fabrication of microuidic-devices requires the patterning of magnetic
nanostructures over a SiO2 (1000nm)/Si substrate. The material mainly used
for the magnetic micro and nanostructures is Permalloy (Ni80Fe20, Py). The
choice of this material is particularly suitable for the fabrication of the mag-
netic conduits, since Py has many favorable properties such as a negligible
magnetocrystalline anisotropy, high saturation magnetization, low coerciv-
ity. Due to these characteristics, the magnetization is mainly driven by the
shape of the nanometric conduits (See section 2.1.6) and the magnetic stray
eld gradient generated by DWs is large enough to trap magnetic particles 42 in suspensions over the magnetic structures. Moreover, Py thin lms display
a high AMR.
The micro and nanostructures employed in this work are fabricated by means
of electron beam lithography (EBL) and optical lithography and a lift o
procedure. The deposition of Py and Au contacts of the AMR devices are
deposited by electron beam evaporation. Besides, nanopatterned chips are
capped with an uniform thin lm of dierent materials (such as SiO2 or
Al2O3), grown by magnetron sputtering, to prevent damages from the wet
environment. Finally, a Poly-DiMethylSiloxane (PDMS) cell is placed on the
summit of the chips to contain the uid in which MNPs are dispersed.
In the next sections, the dierent techniques above mentioned are in depth
described. 3.1.1 Optical and Electron-beam lithography Once the geometries of the devices have been designed, the patterns are
transferred on the top of the substrate by means of electron beam or optical
lithography. The EBL has been used to write patterns with submicrometer
resolution (such as the magnetic structures and ne electrical contacts), in-
stead optical lithography is employed to transfer structures tipically larger
than 1 µm (such as the external electrical contacts -see Fig. 3.1(a)-). The
EBL fabrication of the magnetic rings array presented in chapter 4 was car-
ried out by the LPN-CNRS (Laboratoire de Photonique et de Nanostructures,
A. Cattoni) of Paris. The zig-zag shaped magnetic nanostructures (see Chap-
ter 4 and 5) and the "ne" electrical contacts were made by the EBL group
in the L-NESS laboratory (see Fig. 3.1(b)). The optical lithography process
to pattern the external electrical contacts is performed by me at the L-NESS
clean room.
In principle the lithography process always consists of the following steps: a)
wafer cleaning from contaminants b) prime coating for improving the resist 43 adhesion to the substrate, c) resist spin coating, d) resist soft bake (only for
optical lithography), e) exposure of specic part of the resist to UV light or
electron beam, f) development: the soluble zones of the exposed resist are
dissolved by using a developer (solvent) while leaving the others undisturbed.
The entire lithographic process is illustrated in Fig. 3.1(c). Figure 3.1: Microscope images of optical lithography patterning of external
contacts (a) and EBL patterning of ne contacts (b). Sketch of the entire
lithography process (c) Two types of photoresist (PR) can be used: "positive resist" which is removed in the exposed area or "negative resist" whch is removed in non-
exposed area. The patterned resist can be employed as an open mask for
metallization (as it is always the case in this thesis) or as a protective mask
for etching. After that, the resist can be completely removed leaving on the 44 substrate the metallic nanostructures or the non-etched parts. This process
is called Lift-o and the resist is dissolved in an organic solvent like acetone,
AZ 100 remover (Sigma Aldrich, USA) or n-ethyl-pirrolidone (NEP) for e-
beam resist, removing the resist and consequently all the material parts not
directly in contact with the substrate. This procedure is performed heating
the remover at 60oC on an hot plate and leaving the sample immersed for
a time varying between 1h, for the AZ 100 remover or acetone, and 2h for
NEP.
For the optical lithography a mask aligner is exploited to align the mask
with sample and in this case it is Karl Suss MA56 (gure 3.2), which can
work both in full-contact and in proximity mode; it can support wafers of
4" and masks of 5". The lamp used for expositions mainly emits light at a
wavelength of 365 nm, with a power density of about 13 mW/cm2. Figure 3.2: Mask aligner Karl SussMA56. For the fabrication of the external contacts in AMR and capacitive sensors (see Fig. 3.1(a)), the image reversal AZ5214E photoresist (Microchemicals,
USA) was used. This positive resist can be inverted through an extra backing 45 after exposition and a "ood exposure" before developing. In order to obtain
an undercut prole suitable for the lift-o technique, the inverted mode has
been exploited. All the parameters of the process have been optimized in
order to be able to precisely dene features with dimension of 2 µm or smaller: ' Substrate cleaning with acetone and isopropanol, drying with Nitrogen. ' Prime spinning and subsequent bake on hot plate at 1200 C for 120 s ' Spinning of AZ5214E at 5000 rpm ' Soft bake at 1100C for 50 s ' Exposure with positive mask at broadband light at a dose of 29.9 mJ/cm3 ' Reversal bake (the most critical step): 1170C for 100 s. ' Flood exposure at a dose bigger than 455 mJ/cm3 ' Development: 25 s in AZ726 MIF developer (Microchemicals, USA) 3.1.2 Electron beam evaporation Once the resist is developed a thin lm of magnetic material or a bilayer of
Ti (as adhesive layer) and Au is deposited by electron beam evaporation.
This process consists in an electron beam heating a crucible containing the
evaporation material above the melting temperature in a vacuum chamber.
Evaporated atoms can move from the crucible (made of refractory material
in order to prevent evaporation if the electron beam is misaligned) to the
substrate along an almost linear path, since atoms do not undergo collisions
in vacuum. In fact, the mean free path (the distance covered by atoms be-
tween two collisions), at a pressure of less than 10''5 mTorr, is greater than
the typical distances of a deposition chamber. 46 The deposition process has many limitations imposed by the resist: it must,
for example, occur at temperatures not exceeding 200oC because of the ther-
mal stability of the resist. The deposition rate, once the target-substrate dis-
tance and the material are xed, just depends on the electron beam power.
The parameters used during the evaporation are listed in table 3.1. Material Base pressure Deposition distance Rate E-beam power Permalloy 2*10''6 mTorr 10cm 0.1nm/s 9% of the full scale Gold 2*10''6 mTorr 10cm 0.1-0.2nm/s 6% of the full scale Titanium 2*10''6 mTorr 10cm 0.1nm/s 7% of the full scale Table 3.1: Parameters used in the evaporation processes and the relative
deposition rate In the L-NESS lab, the machine employed is a Leybold "Heraeus L560" evaporator.
The deposition rate was monitored with an integrated thickness monitor
(a quartz microbalance) which has been calibrated by direct measuring the
thickness of the deposited lms.
In order to facilitate the resist removal, the deposition technique should be
strongly directional with low capacity of step coverage. In this view the
evaporation technique is better for lift-o than the sputtering deposition
(described in the next section)which is more isotropic. The process described
in the last two sections (3.1.1 and 3.1.2) can be repeated to grow complex
structures in which many lithographic steps have to be performed or dierent
materials have to be deposited on the same surface. For example, AMR
and capacitive sensors require two EBL steps (for the deposition of magnetic
structure and the ne electrical contacts) and one optical step for the external
contacts. 47 3.1.3 Capping and microuidic cells Once the devices are properly patterned with the nanostructures, a "cap-
ping" thin lm is deposited on the top of the entire chip to prevent it from
damages due to the wet environment, to favor the adhesion of the PDMS
cells and to planarize the surface. Two dierent materials have been tested
as capping layer: SiO2 and Al2O3. All of these are deposited by Magnetron
Sputtering.
Magnetron Sputtering is a physical deposition method where ions from an
argon (Ar) plasma are focused through a magnetic eld to transfer momen-
tum to the atoms of a target, which are then deposited onto the substrate in
a vacuum chamber. A detailed description of the sputtering process can be
found in [50]. The machine employed for this purpose is the AJA Orion 8
system aviable at the L-NESS.
The system is provided with 10 confocal magnetron sputtering sources which
can be also used for co-deposition. In order to deposit the insulating cover
layers a radio-frequency (RF) source is used, in order to avoid the formation
of a charged layer on the top of the target which would diminish the eec-
tiveness of the sputtering process. The deposition parameters are illustrated
in table 3.2. Material Base pressure [mTorr] Rate [A/min] Power density [W/cm2] Al2O3 3 4.84 15.79 SiO2 2 12.46 9.87 Table 3.2: Parameters used in magnetron sputtering processes and the rela-
tive deposition rate. After the protective layer deposition, a PDMS microuidic cell is placed 48 on the summit of the chips to contain the liquid. The PDMS (Poly-DiMethylSiloxane)
is a exible and transparent polymer which is bound to the surface of the
samples after being properly preparated. PDMS is prepared using the Syl-
gard 184 Silicone Elastomer Kit, according to the following protocol: ' The elastomer is mixed with its curing agent. The elastomer and curing agent are carefully mixed in the 10:1 ratio for few minutes; ' The mixture is placed in a vacuum chamber until all the bubbles, due to the mixing, disappear; ' The PDMS is then slowly poured to a smooth and clean surface (for example, a silica wafer was exploited for this purpose). Bubbles should
be avoided because they would otherwise create defects in the solidied
sample; ' The PDMS sample is put in the oven for about 3 hours at 65oC ' Once the PDMS is cured and cooled to room temperature, it can be cut as the shape of the chip substrate and peeled out. The PDMS gasket is then bonded to the capping layer on the chip, with the
use of an oxigen plasma in a Plasma Asher Machine. In such equipment, the
RF plasma at low energy and sucient pressure is used to assist and control
surface chemical reactions, modifying the hydrophobicity of both PDMS and
capping layer surface to hydrophilic ones. The chip and the PDMS are placed
in the vacuum chamber of the plasma asher and exposed to oxygen plasma at
1 mbar of pressure and 150 W of power for 2 minutes. After this treatment,
the PDMS is placed directly in contact with the capped sample within 1
minute, since the surface modication is temporary. Additionally, the entire
structure could be put on a hotplate at 75oC for 15 minutes in order to
increase the bond force. 49 3.1.4 Process ow In the previous sections an overview of the fabrication techniques exploited
in this work, was performed. In the next sections the process ow to fabricate
samples for beads manipuation, AMR sensors and impedantial based sensors
will be described. Devices for beads manipulation The results described in chapter 4 and 6 are obtained by means of magneti-
cally patterned chips that permit to manipulate superpara magnetic nanopar-
ticles as described in chapter 2.
The fabrication process is the following: ' Patterning of rings array by electron beam lithography on a Si/SiO2 substrate (see Fig. 3.3). The rings have a diameter of 5 or 10 µm and
width of 300 nm. Figure 3.3: Rings array pattern obtained by EBL. ' Deposition of a thin lm (30-60 nm) of Permalloy by electron beam evaporation 50 ' Lift-o procedure to remove the resist (2 h in NEP at 60oC) ' Sputtering of a capping layer (50-70 nm) by magnetron sputter. Dif- ferent materials were tested to this purpose (such as Al2O3 or SiO2) AMR sensors The devices used to detect the beads transit, through a magneto-electrical
measurement based on AMR eect, are fabricated as follows: ' Patterning of ne electrodes by EBL on a Si/SiO2 substrate (see Fig. 3.4(a)). ' Deposition of Ti as adheisve layer (3 nm) and Au (20 nm) by electron beam evaporation. Figure 3.4: Optical image of the AMR device: after the rst (a) and the
second (b) step. Picture of the nal device (c). 51 ' Lift-o procedure (2 h in NEP at 60oC). ' Patterning of zig-zag shaped structures by EBL (see Fig. 3.4(b)). Zig- zag segments are long 2 µm and the conduit width is 200 nm. ' Deposition of Permalloy (25-30 nm) by e-beam evaporation. ' Lift-o procedure (2 h in NEP at 60oC). ' Patterning of external electrical contacts by optical lithography. ' Deposition of Ti (5 nm) and Au (65 nm) by e-beam evaporation. ' Lift-o (1 h in AZ remover at 60oC). ' Sputtering of a capping layer (40-50 nm) of SiO2. ' Positioning of a PDMS gasket on the top of the chip (see Fig. 3.4(c)). Finally, it is worth to notice that the thickness of magnetic conduits is 25-
35nm, lower compared to rings structure, in order to guarantee the formation
of completely transverse Neel walls, enhanching the AMR eect (see section
2.3 ). Capacitive sensors The sensors based on impedential detection of beads have been manufactured
in the following way: ' Patterning of zig-zags shaped conduits by EBL (see Fig. 3.5(a)). ' Deposition of Ni80Fe20 (30-40 nm) by electron beam evaporation. ' Lift-o procedure (2 h in NEP at 60oC). ' Sputtering of a thin lm of SiO2 (30 nm) to cap zig-zags. 52 Figure 3.5: Optical microscope images of the fabrication process for a capac-
itive sensor. ' Patterning of ne electrical contacts by EBL (see Fig. 3.5(b)). Various geometries of coupled electrodes have been tested. ' Deposition of Ti (5 nm) as adhesive layer and gold (65 nm) by e-beam evaporation. ' Lift-o procedure (2 h in NEP at 60oC). ' Patterning of external electrical contacts by optical lithography. ' Deposition of Ti (5 nm) and Au (80 nm) by e-beam evaporation. ' Lift-o (1 h in AZ remover at 60oC). ' Patterning of the protective layer: micrometric strips are patterned by optical lithography in order to protect the chip by the uids, while 53 maintaining exposed the sensing area (contacts near the zig zag). (see
gure 3.5(c)). ' Sputtering of SiO2 or Al2O3 (50 nm) as capping layer. ' Lift-o procedure (1 h in AZ remover at 60oC). ' Positioning of a PDMS gasket on the top of the chip (see Fig.3.5(d)). As alternative, the capping layer was replaced by the resist used for the last
litographic step. It is employed because, due to its thickness (2µm), it guar-
antes a good insulation of contacts from the liquid. An extra backing (at
120oC on a hot plate) was performed in order to increase the hardness of the
resist. 3.2 Characterization methods The main technique employed to characterize the samples morphology is
atomic force microscopy. In order to study and draw the magnetic con-
guration of such samples a variant of this method called magnetic force
microscopy has been used. 3.2.1 Atomic force microscopy(AFM) AFM is a scanning probe microscopy technique which permits to provide
topographical maps of the sample surface with sub-nanometric resolution.
A sharp tip (few angstrom in width) mounted on a cantilever is positioned
within a few nanometers above the surface of the sample, then the probe is
moved laterally (in the plane of the sample) by a piezoelectric manipulator
which can provide sub-nanometric motion increments. The change in height
of the surface causes a variation in the interatomic forces between tip and 54 sample. As a consequence, deections are produced in the cantilever. These
deections are measured using a laser beam which reects on the cantilever
and is collected by a photodetector. Figure 3.6 shows the schematic of an
AFM working principle. Figure 3.6: Sketch of the AFM working principle The interatomic forces between tip and sample is described by the em- pirical Lennard-Jones potential: V (r)LJ = A r12 '' B r6 (3.1) where the r''6 term describes attractive interaction due to the Van der Waals
force and the r''12 component represent the Pauli repulsion due to the over-
lap of interatomic orbitals. Three possible operational modes for the AFM,
depending on the the tip sample distance, exist: contact mode, non-contact
mode and tapping mode. In contact mode, the tip scans the sample in close
contact with the surface and thus experiences strong repulsive forces. Due
to the large variation of the force with distance, it is possible to achieve ex-
tremely high resolutions. However, the dragging motion of the probe tip,
combined with adhesive forces between the tip and the surface, can cause
substantial damage to both sample and probe and create wrong images. In
non-contact mode the tip is kept from 5nm to 15nm above the surface and is 55 forced to oscillate at a frequency slightly above its resonance frequency with
a typical oscillation amplitude of a few nanometers (<10 nm). The increased
tip-sample distance lead to a reduced damaging of the sample but, since
the force on the tip is far less intense than in contact mode, the resolution is
much lower. The last operational mode for the characterization of the surface
topology, is the tapping mode [52]. In this mode, the cantilever is driven to
oscillate up and down near its resonance frequency with an oscillation ampli-
tude which typically ranges from 100 nm to 200 nm and a frequence between
50 and 70 KHz. A tapping AFM image is therefore produced by imaging the
force of the intermittent contacts of the tip with the sample surface. Due to
the forces acting on the cantilever when the tip comes close to the surface,
the amplitude of the oscillation is reduced. A feedback cycle then restores
the nominal amplitude by adjusting the tip height through the piezoelectric
vertical motion so that it provides point-by-point the information about the
surface topography. The machine used in this work is a VEECO innova
system illustrated in gure 3.7. Figure 3.7: Atomic force microscope VEECO innova 56 3.2.2 Magnetic force microscopy (MFM) The magnetic force microscope [51] is a variant of the AFM in which the
magnetic force or the force gradient is employed to visualize the magnetic
conguration of a sample with a high resolution. The tips are covered by
a ferromagnetic material and the basic idea is measuring the cantilever de-
ection due to the interaction between the tip and the stray eld generated
by the sample. The competition between Van der Waals forces and mag-
netic forces has to be considered: for short distances (tipically less than 10
nm) the Van der Waal forces lead, instead for larger distances the magnetic
forces (the magnetic force gradient) are the dominant term in the tip-sample
interactions. Two dierent working behaviour occur as a function of the
distance between the tip and the sample: the so called far eld regime and
the near eld regime. In the rst case the signal, related to the force gradi-
ent, is dominated by the surface magnetic properties of the sample, while in
the second case by the changes in the sample topography. As in the AFM
case the tapping mode is used. In order to separate the magnetic image
from the topography, the signal detection by the MFM is realized through a
two-step procedure, also called lift mode (see Fig.3.8)[54]. During the rst Figure 3.8: Magnetic force microscopy: lift mode. In the rst step the topog-
raphy of the surface is recorded, while in the second step, the magnetic image
arising from the stray eld of magnetic domains in the sample is captured. step, outwards, the cantilever oscillates close to the surface, recording the 57 topography. In the second step, on the way back, the cantilever is lifted up
at a selected height in the range of 10-200 nm and the magnetic image is
registered at constant height (without feedback) on the basis of the topog-
raphy saved outwards. The distance is kept at sucient high value in order
to make the Van der Walls force contribution negligible; thus the cantilever
is sensitive to the magnetic forces only. The magnetic image gives us direct
information about the presence of surfaces domain walls and in general on
the magnetization of the thin lm sample. Note that the magnetic force can
be attractive or repulsive for a tip with permanent magnetic moment. The
MFM image contrast tells us the nature of the interaction. Considering, for
example, in gure 3.9, the tip repulsion leads to brighter spots while attrac-
tive forces means darker spots. Figure 3.9: MFM image of a zig-zag magnetic conduit: dark and bright spots
represent respectively an attractive or repulsive magnetic force, acting on the
ferromagnetic tip. The tip height while registering the magnetic signal was set from 80 nm to 200 nm, depending on the fabrication quality of the structures tested.
Fabrication defects like impurities bumps or high peaks at the structure edge
require to set high tip-surface distance in order to avoid detrimental contacts
with magnetic material during the backward scan. For the image acquisition
MAGT magnetic tips (App Nano, USA) have been used. They are covered
by a thin lm of 50±5 nm of CrCo with a magnetic moment (109-1011 Am2)
and a minimum coercivity of 500 Oe. 58 3.3 Experimental setups In the following sections a description of the experimental setups employed in
this work is illustrated. In particular, the next paragraph shows the system
used for manipulation of beads and for biological tests, instead in section
3.3.2 the setups for magneto-electrical measurement on AMR and capacitive
sensors are presented. 3.3.1 Setup for biological experiments and beads ma- nipulation The experimental results shown in chapter 4 and 6 have been mainly obtained
by means of the setup illustrated in Figure 3.10. Figure 3.10: Experimental setup for biological tests and beads manipulation.
A indicates the optical microscope (Nikon Eclipse FN-1), B the entire sample
stage, C the stepper motors system and D the thermostat. In such system an alluminium (Al) sample stage is mounted on a metallic arm which is connected to a micromanipulator, allowing a ne control of the 59 in-plane sample position. Besides the sample stage is able to contain 800 µl
of liquid so that experiments can be performed in a wet environment (as it
always the case).
For biological tests in which the temperature control is fundamental to guar-
antee the cells viability, a thermo-couple is placed close to the sample in order
to measure the temperature in the liquid. A PID thermostat, connected to
the thermo-couple, is employed to control and set the temperature by means
of a thin conductive heater made of silicon rubber and properly mounted
just under the alluminum stage. A good thermal contact is ensured by a
heat-sink paste.
The nano-objects and the cells have been monitored by the optical micro-
scope "Nikon Eclipse FN-1" equipped with 60x water immersion objective
(Nikon NIR-APO, numerical aperture 1.0) so that a water environment or, at
least, a drop of water as focusing lens is required between the objective and
the sample. A second 4x objective was used to center the area of interest.
The light was provided to the microscope by an arc-discharge mercury lamp
(NIKON intensilight C-HGFI). The microscope is equipped by an electronic
ne controller of the objectives displacements along the direction perpen-
dicular to the plane. Two additional lters have been installed in the light
pathway of the microscope: FITC (Fluorescine IsoThio-Cyanate) and TRITC
(Tetramethyl Rhodamine Iso-Thiocyanate) uorescence lter blocks. FITC
lter provides an excitation light bandwidth of 465-495 nm (blue spectral
region) and an absorption bandpass from 515 nm to 555 nm (green spec-
tral region), TRITC lter block have an excitation bandpass of 525-540 nm
(green spectral region) and an absorb in the red spectral region from 605
nm to 655 nm. Fluorescence has been employed in biological experiments to
distinguish magnetic nanoparticles, functionalized by a TRITC marker and
cells "coloured" with a FITC one. The data acquisition is performed by a
fast electron-multiplying charge-coupled device (EM-CCD) camera (ANDOR 60 Luca-S, Belfast UK) which has a resolution 658 x 496 pixel, max 50 fps. It is
Peltier cooled down to -20oC in order to reduce the thermal electronic noise.
The external in plane magnetic eld which is needed for beads manipulation
through the DWs formation and displacement is applied exploiting two cou-
ple of permanent cilindric magnets made of NdFeB and properly placed over
an Al plate as illustrated in gure 3.11. The maximum stray eld produced Figure 3.11: Image of stepper motors used to control the rotation and the
height of a plate containing two couples of permanent magnets (a). Sketch
of the lines of force from the permanent magnets eld (b) in-plane by the magnets (located at 2 cm of distance each other) is 400 Oe
at 5 mm over the plate, in the center of it. In order to achieve a variation
of the magnetic eld direction, the plate is connected to a stepper motor
which allows a controlled rotation. A similar stepper motor is employed to
nely regulate the height of the plate by means of a screw connected to the
motor shaft. A stepper motor is a DC powered component that divide a full
rotation in an equal number of steps. The working principle is the following
(see Fig.3.12)[53]: a multiple "toothed" electromagnet is arranged around a
central iron gear. The electromagnets are sequentially energized by an exter-
nal control circuit. In order to turn the motor shaft, the rst electromagnet
is powered so that the gear's teeth are magnetically attracted to the electro- 61 Figure 3.12: Sketch of the stepper motors working principle. The electro-
magnets around the central gear are sequentially powered to get a single step
rotation. magnet's teeth. When the gear's teeth are aligned to the rst electromagnet,
they are slightly oset from the second electromagnet. In this way when the
second electromagnet is turned on and the rst is turned o, the gear rotates
slightly to align with the second one, and from there the process is repeated.
Each one of those pieces of rotations is called "step", with an integer number
of steps making a full rotation. In that way, the motor can be turned by a
precise angle. In this work SM-42BYG011-25 stepper motors are used. A
precise control of the plate rotation down to 1,8o (200 steps for the entire
revolution) is achieved.
The stepper motors are powered through proper drivers and they are con-
trolled by means of an Arduino-uno microcontroller. Arduino can be easily
interfaced with a labview software to achieve an ecient and remote control
of the whole system.
In order to apply an out of plane magnetic eld, a small hollow cilinder
shaped coil was designed and placed around the microscope objective. This 62 electromagnet exercises a maximum eld of 100 Oe, 1 cm far from the bottom
side (where the sample is located). The current is supplied by a KepcoTM
bipolar power generator and controlled by a Labview software via GPIB-USB
connection. A part of the experimental results described in chapter 6 have been per- formed at the IFOM center using the same setup described in this chapter
except for the optical microscope that has been substituted with TCS-SP5,
LEICA, a confocal microscope. This machinery permits a higher spatial res-
olution and sharpness of uorescent images, by using a point illumination
based on a laser and a spatial pinhole to eliminate out-of-focus light in spec-
imens that are thicker than the focal plane.
A full descrption of confocal microscope working principle can be found in[55]. 3.3.2 Setup for sensors measurements The setup employed to test the AMR and capacitive sensors is illustrated in
gure 3.13. A four coils electromagnet is used, replacing the stepper motors
system, because a ne control of the eld absolute value has to be performed.
This electromagnet was projected to apply a maximum eld of 700 Oe when
the distance between poles is 4 cm. It is powered by two KepcoTM bipolar
generators.
The sample stage is shown in gure 3.14. A small electrical board transfers
the electrical signal from coaxial cablets to smaller electrical wires which end
with "tulip" shaped contacts. They are connected to electrical pins of a sec-
ond small board. The chip glued on the top of it is electrical connected via
wire bonding to the boards pads. The bonding procedure deals with the con-
nection of two electrical pads through a gold wire having a diameter of some
micrometers, whose extremities are sealed by thermal heating to the contacts.
This procedure was performed by the "electronic engineering" group in Milan. 63 Figure 3.13: Experimental setup for sensors measurement. A is the four poles
electromagnet, B is the Lock-in amplier (HF2LI), C is a bipolar generator
(KepcoTM) and the optical microscope (Nikon Eclipse FN-1). Figure 3.14: Sample stage employed in electrical measurements. A is the
AMR or capacitive device, B the electrical pins connected to the "tulip"
wires. C and D indicate the small electrical boards The entire sample stage is held up by four plexiglass legs and it is mounted
on a micromanipulator to control the position of the device in plane. 64 The microscope used to monitor the sensors is the same described in the
previous section and it is employed in bright eld mode (BF). AMR sensors measurements AMR sensors, whose fabrication process is described in section 3.1.4, are
used to detect and count the MNPs which are trapped and manipulated over
a zig-zag shaped magnetic nanoconduit. A magneto-electrical measurement
which is based on the AMR eect described in section 2.3 is performed. The
working principle of such sensors is the following: when a magnetic DW,
which is in this case a Neel trasverse wall with the magnetization pointing
perpendicular to the conduit direction, is located between two electrical con-
tacts. A voltage drop along that piece of conduit is measured due to the
AMR eect. The zig-zag structure permits to control the displacement of a
single DW from one corner to another by means of a sequence of magnetic
elds applied in the direction of the straight segments (see chapter 4). In this
way the crossing of a DW between two electrical contacts can be electrically
detected. If a MNP is trapped over the DW, a variation of the depinning eld
(the minimum eld required to displace a DW from one corner to another)
occurs. In this way, synchronizing the electrical signal acquisition and the
applied eld, it is possible count the beads passing over the conduit.
The electrical measurements are performed through a four point probes tech-
nique in which a couple of contacts are used to apply a certain signal, and the
other two contacts are employed to detect the electrical response. In order
to reduce the electrical noise, a modulation technique and a lock-in amplier
(HF2LI Zurich instrument) was used. A schematic view of the equivalent
circuit is illustrated in gure 3.15. The external contact A is supplied by the HF2LI instrument with an AC signal, instead D is connected to ground. The applied voltage amplitude is 65 Figure 3.15: Schematic of the equivalent circuit of AMR sensors measure-
ments (a) and optical microscope image of the device which shows the elec-
trical contacts and the magnetic zig-zag shaped conduit. 50 mV-200 mV (the current that ows in the zig-zags is 1-4 µA) and the
frequence is set between 10-100 kHz. The voltage drop between B and C is
measured by means of the HF2LI machinery.
R1 , R2 and R3 are mainly related to the zig-zag shaped nanostructure resis- tivity, because the gold electrodes conductivity is very high. RAMR (which is
the resistance encountered by the current owing along the conduit) varies
if the magnetization is parallel or perperndicular to the nanostructure direc-
tion, as it is the case when a DW is located between B and C. The output
signal is acquired and demodulated by the lock-in amplier, in order to re-
move the periodic, low frequency noise.
The external magnetic eld is controlled remotely by a Labview software.
A proper sequence of magnetic elds is applied to nucleate and displace a
single DW in the zig-zag structure to the corner between electrodes B and C
(the full description of this procedure can be found in chapter 4). Moreover 66 magnetic eld sweeps along a xed direction can be performed. In that way
it is possible to synchronize the voltage acquisition with the applied eld
value so that the depinning eld can be electrically taken over. The AMR
experiments are executed in a H2O environment where beads are diluited
with a concentration of 1µg/ml. The variation of depinning eld in presence
or absence of bead over the DWs is exploited to detect and count the number
of MNPs which transit on the top of the zig-zag (see chapter 5). Impedantial sensors measurements Capacitive sensors exploits an impedantial measurement to record the transit
of MNPs diluted in a buer saline solution (PBS). The fabrication of such
sensors is described in section 3.1.4. The idea behind these detectors is the
following: beads are trapped and manipulated applying external magnetic
elds along the same magnetic zig-zag shaped nanoconduit used for the AMR
sensors. When a MNP crosses a couple of electrodes, the low value of bead
conductivity, compared to PBS, produces a voltage drop that can be mea-
sured.
Dierently from AMR measurements, only two electrodes are involved ( two
point probes technique). The same HF2LI instrument is used to apply the
electrical signal and to detect the electrical response. The applied voltage
amplitude ranges between 100 mV and 500 mV and the frequence is set at
2 MHz. A schematic view of the equivalent circuit is illustrated in gure
3.16(a).
In that picture CDL is the "double layer capacitance" which is formed at
the interface between the electrical contacts and the saline solution. It is
due to the ions in PBS that are attracted by the electrical eld generated
at the contacts extremities. This produce a charge accumulation that has
to be taken into account. RL is related to the conductivity of the liquid. It
depends on the ions concentration and valence, together with the distance 67 between the electrodes immersed in PBS, which was set to 2µm.
The chip is aected by some parasitic capacitances due mainly to the in-
terfaces between dierent material layers on the devices (see Fig.3.16(b)). Figure 3.16: Equivalent circuit for capacitive sensors measurements (a) and
schematic transversal section of the device (b). At the working frequence, the eect of parasitisms has to be negligible, in order not to disrupt the electrical response. However an active compen-
sation circuit can be used in order to eliminate all the capacitive eects not
related to beads detection. A Dammy circuit was employed to this purpose
(see chapter 5).
The magnetic nanoparticles manipulation is achieved using the same electro-
magnet described for AMR measurements, applying a rotating continuous
eld of 250 Oe. In this way an alternate sequence of head-to-head and tail-to-
tail DWs in the zig-zag corners are simultaneously displaced along the corners
in the conduit, driving the magnetic nanoparticles captured on the top. The
experimental results concerning capacitive sensors are illustrated in chapter 5. 68 3.4 Micromagnetic simulations Since the functionalities of magnetic DW conduits are strongly related to the
geometry of the nanostrips, micromagnetic simulations were performed in
order to study the magnetization equilibrium states and the magnetic force
generated. Public OOMMF (Object Oriented Micro Magnetic Framework)
platform has been used with the following parameters for Permalloy: satu-
ration magnetization Ms =800*103 Am''1, exchange stiness constant A =
1.3*10''11 Jm''1, damping coecient = 0.5, no magneto-crystalline anisotropy
has been considered. The damping coecient is high, compared to the ex-
perimental one (0.01); this leads to a faster computation of the equilibrium
state, that is what we are interested in, even though a lower accuracy in
describing the transient behavior must be taken into account. A unit cell
of 20 nm side and 10 nm high is used for the simulation of zig-zag shaped
and curved conduits. This is greater than the exchange length of 5.2 nm but
still the smallest ones compatible with the computational time. In chapters
4 and 6 the results of simulations are illustrated and discussed. 69 Chapter 4 Manipulation of nanoparticles
over magnetic conduits This chapter presents the results related to the implementation of an on chip
magnetic based technology to trap and manipulate micro and nanoparticles
over a surface in a microuidic device. This technology, called domain walls
tweezers (DWTs), can be potentially used for a large amount of interesting
applications. It is a central part of this thesis work because it can be em-
ployed to control the position of magnetic beads in a 2D space with a high
spatial resolution, down to 100 nm.
In this work, it is employed to bring MNPs in close proximity with a tar-
get cell, studying the interaction with the cellular membrane (see chapter 6)
which is a fundamental aspect of the drug-delivery process. Curved geome-
tries are used to this purpose.
Moreover, AMR and capacitive sensors exploits zig-zag shaped magnetic con-
duits to capture and transport magnetic nanoparticles while detecting their
passage through a given point.
DWTs have been developed by the NaBiS group at L-NESS, in collaboration
with Nanogune Center in San Sebastian [60] [59]. In the rst part of the chap- 70 ter, a description of the optimization of a single particle manipulation over
zig-zag conduits is performed. Instead, in the second part, curved structures
are employed to achieve an innovative, simultaneous and controlled motion
of a nanoparticles batch over a single chip.
The physics behind DWTs technology is fully discussed in chapter 2. 4.1 Single particles manipulation on zig-zag con- duits In order to magnetically control the transit of a bead over AMR or capacitive
sensors, zig-zag shaped nanostructures are used. DWTs approach relies on
the precise control of the motion of DWs that can be achieved in ferromag-
netic stripes (magnetic conduits) and on the robust coupling between a DW
and a magnetic particle in suspension over the conduits [58]. The injection
and displacement of a single DW in a zigzag shaped stripe results in the
capture and dragging of a particle. In order to create a DW in a magnetic
conduit an injector pad is needed. The injector pad is the initial part and
the element capable to inject the DW in the conduit. Starting from a mono-
domain conguration, the injector pad, wider than the conduit, reverses its
magnetization, upon the application of a small eld, while leaving unchanged
the conduit magnetization, thus creating a DW (see Fig.4.1). A sequence of
magnetic elds propagates the DW from one corner to the following one,
since every corner represents a pinning site for the DW. The magnetic par-
ticle, in suspension above the conduit, is dragged by the motion of the DW
along the conduit. This reduces the average velocity of transportation which
is limited by the diusion of the magnetic bead from one pinning site to the
following one.
In this thesis work, the zig-zag shaped conduits in AMR and capacitive sen-
sors are made of Py, they have a width of 200 nm, a thickness of 25-35 nm 71 and the zig-zag segments are 2 µm long. The injector is 600 nm large and
long 4 µm. In gure 4.1 a MFM image shows the single H-H DW (dark) Figure 4.1: Top: sequence of magnetic force microscopy images and micro-
magnetic congurations (right) showing the injection and propagation of a
domain wall under the action of external magnetic elds Hi, Hup, and Hdw
directed as sketched in the gure. The dark and bright re contrast in the
image is due to the inward and outward local stray elds. At the zig zag
corners the stray eld is generated by a domain wall, while in the case of the
injection pad is only due to the magnetization stray eld. Bottom: sketch of
the zig-zag conduit dimensions. nucleation and displacement applying a sequence of magnetic eld pulses, 1
second long, in a specic direction. The applied eld changes the magnetic
conguration because the equilibrium condition is dierent due to the adding
of an extra energy term (Zeeman interaction) to the total energy functional
(see chapter 2). The sequence of eld pulses applied to nucleate and displace
a single DW is listed in table 4.1. 72 Field name Field value [Oe] Angle [o] H0 1000 180o Hi 150±10 15o Hup 180±10 45o Hdw 180±10 -45o Table 4.1: Sequence of applied eld pulses to nucleate and displace a DW in
a zig-zag shaped conduit. Experiments are executed in a wet environment: water (AMR sensors) or PBS (capacitive). A 0,5% (V/V) of SDS (Sodium Dodecyl Sulfate) which is
a surfactancts based solution (soap) was added to reduce the friction and the
non-magnetic interactions between beads and surface. MyOneTM dynabeads
with a diameter of 1 µm have been diluted to reach a MNPs concentration
of 1µg/ml. The chip surface was previously treated with an Oxigen plasma
to make it hydrophillic (3.1.3). Figure 4.2: Transport sequence of a 1µm bead over a zig-zag shaped conduit
in an AMR sensor. The DW is nucleated (a) and (b) and displaced over the
nanostructure (c) and (d), dragging the MNP. 73 Figure 4.2 shows the displacement sequence of a single bead recorded by
means of the optical microscope, applying the sequence of eld pulses de-
scribed in table 4.1. The electromagnet used to apply the magnetic eld is
described in section 3.3.2 together with the whole experimental setup. 4.2 Free 2D manipulation of many particles over curved conduits The major limitation of zig-zag shaped nanostructures is that the particle is
forced to follow the predetermined path of the conduit, even if more than
one pathway is available [61]. In this paragraph a method for transporting
magnetic particles without any a priori determined path will be presented
which is based on array of nanometer sized rings of Py.
Such system allows to implement an innovative and simultaneous controlled
motion of many particles thus achieving, an high parallelism in beads trans-
port. In this way, some processes like the drug delivery to cell cultures could
be not only well controlled, but also ecient.
In the following, the operation principles of this new manipulation technique
will be illustrated and discussed. 4.2.1 Working principles The basic element of the device is a soft-magnetic ring made of Py. The
diameter of the designed and tested rings is variable between 5 and 10 µm,
the width is 300 nm and the thickness ranges between 30 nm and 60 nm.
The pattern design consists of a matrix array of such rings. The design
parameters are the diameter of the ring, its width and the minimum dis-
tance between neighboring rings. The rings are "packed" in two dierent
ways: square matrices in which every rings has four neighbors and hexagonal 74 matrices in which there are 6 neighbours. The entire fabrication process is
described in section 3.1.4. The total patterned area was 600 µm x 600 µm on
a chip of 150 mm2. The gure 4.3 shows the optical images of two portions of Figure 4.3: Square matrix (a) and hexagonal matrix (b) of Permalloy rings. square and hexagonal arrays with rings of 5 µm (left side) and 10 µm (right
side) diameter.
The distance between the center of two neighbor rings, is 6 µm for the left
side array and 12 µm for honeycomb array of magnetic rings. The minimum
distance between the four neighbours rings has been always chosen smaller
than 2 µm with a lower limit of 1 µm. This parameter is fundamental for
a correct and reliable manipulation of the beads. The elemental step of this
manipulation method relies on the motion of beads over the magnetic rings
with a geometry that allows the nucleation of two Neel magnetic domain
walls.
By applying an in-plane external magnetic eld (Hsat), two DWs, one HH
and the other one TT, are generated. The DWs lie along the direction in
which the eld has been applied. As shown in gure 4.4 by the MFM images
and by the sketch of the micromagnetic conguration, once created, the two
DWs can be moved around the circumference by a smaller in-plane magnetic
eld HR. By rotating the eld, both the DWs rotate by the same angle, thus
achieving smooth and fully controllable DW motion. 75 Figure 4.4: Outer gures: sketches of the micromagnetic conguration of a
circular ring showing the nucleation and displacement along the perimeter of
the two, HH and TT, DWs obtained by applying a continuous rotating eld
HR. The inner images are the corresponding MFM data.[35] However up to now, the manipulation is strictly conned on a single ring.
In order to achieve a free 2D motion, an out-of-plane external magnetic
eld, called Hz, must be applied, in order to modify the magnetic poten-
tial landscape felt by the magnetic beads in suspension above the DWs. In
the absence of an out-of-plane magnetic eld, a superparamagnetic bead is
equally attracted by the stray eld emanated from the HH DW and the TT
DW because it does not possess any spontaneous magnetic moment. This is
at variance with what happens for the MFM tip, being coated with a hard
magnetic material, which has a xed magnetic dipole moment and thus is
able to distinguish between the two DW type (white and black contrast). In
fact, HH DW generates a magnetic stray eld with a positive out-of-plane
component, i.e. pointing outward; while the TT DW creates a stray eld
with a negative out-of-plane component, i.e. pointing inward. The applica-
tion of an external magnetic eld Hz, which can be considered homogeneous
over the volume of the bead-DW interaction, increases the magnetic poten-
tial well created by the DW with the stray eld parallel to the z-eld. On
the contrary, the potential well of a DW with stray eld antiparallel to the
external one is weakened. Furthermore, by properly choosing the intensity 76 of Hz, the total eld (HTT + Hz) can cancel out the potential energy well felt
by the magnetic bead, at the typical interaction distance. In this condition,
even though the ring always possesses two DWs of opposite polarity, only
one of them is able to trap a superparamagnetic particle in suspension.
In the following a simple system of two aligned magnetic rings separated by
the same distance of the matrix is considered. This system represents the ba-
sic unit model to describe the free 2-dimensional manipulation method. The
rst step (see Fig. 4.5 (a)) is the application of an in-plane magnetic eld
Hxy in the -x direction and of a positive Hz eld. The intensity of Hz is 60
Oe while, Hxy has a value of 300 Oe. This combination brings the magnetic Figure 4.5: Schematic representation of the two rings system, after the appli-
cation of the magnetic elds sketched below. Red and blue arrows show the
micromagnetic conguration of the magnetic structures. Step (a): a mag-
netic bead, in grey, is trapped by the HH DW of the left ring. Step (b):
the magnetic bead is moved to the point closest to the next ring. Step (c):
by switching the polarity of the out-of-plane eld and removing the in-plane
eld, the magnetic bead is attracted and coupled to the TT DW of the sec-
ond ring. Step(d): the magnetic bead is moved by rotating again of 180o the
in-plane magnetic eld. 77 rings in a state with two DWs but allows for the trapping of a bead only by
the HH DW. Let's suppose now that the left ring of g. 4.5 (a) captures a
magnetic bead on its HH DW, as show in gure. By continuously rotating
Hxy by 180o the magnetic bead can be nely displaced to the opposite point
of the ring, facing the neighboring structure. The situation, named step (b),
in which the bead coupled to the HH DW is close to the TT DW of the other
ring, is illustrated in gure 4.5 (b). In this condition the in-plane magnetic
eld Hxy is decreased to zero and the polarity of Hz is reversed, from posi-
tive to negative. This modies the attractive potential landscape felt by the
bead, that jumps from the HH DW to the TT DW of the next ring (gure
4.5 (c)).
Indeed, in this conguration the magnetic potential well of the HH DW is
now negligible while the TT DW becomes strongly attractive and drags the
magnetic bead on the right. This is possible because the new attractive well
is spatially close enough to the bead for capturing it. After that, the in-plane
eld Hxy is restored and can be continuously rotated by other 180o in order
to displace the bead to the diametrically opposite site of the second ring.
The situation is illustrated in gure 4.5 (d).
Upon application of this sequence of elds, the bead undergoes a displace-
ment of approximately two ring diameters. The key point of the method is
the switching of the polarity of Hz which allows for the jump of the bead
from a structure to the closest one, so that the bead motion is no longer
limited to a rotation along a single circular ring but, in principle, to the
whole rings-patterned space. The example illustrated before shows the way
for moving along a linear array of rings. However, in a squared matrix of
rings any of the four directions (±x,±y) in the 2-dimensional plane can be
addressed by rotating the magnetic eld Hxy by 180o and 90o respectively. In
an hexagonal matrix, instead, 6 dierent directions are allowed by rotating
the in-plane eld of a multiple of 30o. 78 4.2.2 Micromagnetic simulation In the previous section, the free 2-dimensional manipulation method has
been illustrated and discussed qualitatively. However, in order to under-
stand deeply the physical mechanisms at the basis of the presented method,
a more quantitative analysis has been carried out.
The most critical step in the manipulation process is the decoupling between
the bead and the underlying DW, and the subsequent coupling with the DW
of the next ring.
By means of OOMMF simulations, the micromagnetic conguration of two
adjacent magnetic rings was studied, upon saturation with a strong in-plane
magnetic eld (500 Oe). The two simulated rings are made of Py, have a
diameter of 5 µm and a width of 300 nm. The paramters used in the simula-
tions are listed in section 3.4. The minimum distance between the two rings
is 1 µm along the x axis. The saturating magnetic eld creates a couple of
DWs in both rings, in order to obtain two DWs facing each other. Upon the
application of the eld the magnetic spin structure of the DWs assumes a
"vortex" conguration due to the balance between the shape anisotropy of
the structures and the magnetostatic energy contribution (see g.4.6).
In particular, in the area of interest where the two rings are closer, we obtain
a vortex-like DW with a HH feature on the right ring and a vortex-like DW
with a TT feature on the left ring, as it is illustrated in gure 4.6. The image
displays the magnetization inside the two adjacent rings upon the applica-
tion of a strong saturating magnetic eld in the x direction in the case of
no out-of-plane magnetic eld. The tendency to close the eld lines of the
magnetization on the upper and lower part of the two arches of the rings is
an artifact due to the nite area of the simulation which did not consider the
entire ring. 79 Figure 4.6: OOMMF simulation of the magnetization of a portion of the
two rings facing each other from OOMMF. The arrows show the direction of
the magnetization in the xy-plane. Blue and red pixels indicate positive and
negative values of the magnetization along y. HH and TT vortex DWs are
nucleated by an external in-plane eld (500 Oe). The simulations show that, applying an out-of plane eld Hz of 60 Oe, the
nanostructures produce a stray eld Hdw only slightly dierent (within 5 Oe)
from the case without Hz. Nevertheless the total magnetic eld Htot = (Hdw+
Hz) is directly aected by Hz and it can even change sign when Hz is applied.
This is illustrated in gure 4.7 where the maxima in absolute value of the
z-component of the total eld are plotted with respect to the z position. The
total eld near the HH vortex DW is positive and increased by Hz. The total
eld near the TT vortex DW is instead negative and the application of Hz
reverses its polarity at a distance of 400 nm. 80 Figure 4.7: a) z-component of the total eld generated close to the HH DW
with respect to the vertical distance z from the magnetic structure. The eld
near HH DW is positive and is enhanced by the application of the concordant
external eld. b) z-component of the total eld generated close to the TT
DW versus z. In absence of Hz, the vertical component of Htot is negative
while upon the application of the out of plane external eld, Htot crosses the
zero value at a certain distance (around 400 nm). This leads to a modication of the magnetic potential energy and ultimately
of the force exerted on a magnetic bead by the DWs in that spatial region.
From the total eld Htot, the magnetic interaction energy is calculated via
MatLab for dierent values of Hz. The magnetic potential energy has been
calculated through the following equation: Eb = ''µ0 ' V mb · HtotdV (4.1) where mb is the induced magnetic moment of the superparamagnetic particle
of volume V. The magnetic bead, acting as a probe of the magnetic inter-
action, has a diameter of 1µm and a magnetic susceptibility ' of 0.39. The
magnetic potential energy has been valuated in a plane at constant height
hz equal to the radius of the bead plus 50 nm, which represents the typical 81 experimental distance of a particle from the magnetic conduits because a
capping layer of 40-50 nm is deposited on the top of the structures. Figure 4.8: Magnetic potential energy wells felt by 1 µm superparamagnetic
particle generated by the magnetic conguration illustrated in gure 4.6 for
dierent values of Hz. When Hz is o, HH and TT DWs produce two equally
deep potential wells. While, increasing Hz, HH DW becomes a stronger
attractive pole and TT DW a weaker one. When Hz is equal to 60 Oe the
particle is completely decoupled by TT DW. Figure 4.8 shows the proles of potential energy landscapes varying Hz. In the absence of Hz the two magnetic potential wells related to HH and TT
DWs are almost equal in shape and intensity. At a distance of 50 nm from
the surface (which is the thickness of the capping layer), the minimum of the
wells is around 6.0*10''18 J. When the out-of-plane eld is switched on, the 82 equilibrium between the DWs is broken and the potential well generated by
the HH DW is greatly deepened (1.1*10''17 J). On the contrary, the potential
well created by the TT DW is weakened by the external eld down to a value
close to 0. Applying a Hz of 60 Oe (the optimal value) the coupling of a
magnetic bead to the TT DW is unfavorable with respect to the coupling to
the HH DW.
A valuation of the force exerted on the magnetic bead is simulated through
Matlab according to the following equation: F = ''µ0' ' V Htot · ''HtotdV (4.2) where the magnetization of the superparamagnetic bead is written as 'Htot. Figure 4.9: Contour plot of the z-component of the magnetic force calculated
at a distance equal to the radius of the bead plus 60 nm. The left graph
illustrates the situation when the out-of-plane magnetic eld is o while the
right graph shows the case in which the force generated by the HH DW is
enhanced by a Hz eld equal to 60 Oe. Figure 4.9 shows the force value along z, comparing the situation with Hz equal to 0 Oe and 60 Oe. Without an out-of-plane eld, the force suered 83 by the MNP is almost the same for HH and TT DW, that is 43 pN. Instead,
if Hz is 60 Oe, the HH DW produce a higher attracting force of 89 pN while
the TT DW a lower force equal to 5 pN.
Finally, gure 4.10 illustrates the x-component of the magnetic force on the
bead, which is the dragging force making the bead to "jump" from one ring
to the other. For Hz set to 0, HH and TT DWs are attracting poles for the
MNPs. Instead, if Hz is equal to 60 Oe the bead is decoupled from the TT
DW and it is strongly attracted by HH DW. Figure 4.10: Contour plot of the x-component of the magnetic force calcu-
lated at a distance equal to the radius of the bead plus 50 nm. The left graph
illustrates the situation when the out-of-plane magnetic eld is o while the
right graph shows the case in which Hz is equal to 60 Oe. 84 4.2.3 Manipulation of a particles batch on free paths Magnetic array of rings made of Py have been employed to achieve a simulta-
neous and synchronized manipulation of a magnetic beads batch in solution
over the patterned substrate. For all the experiments of free manipulation,
superparamagnetic beads with 1 µm diameter (myOneTM, Invitrogen) and
COOH surface functionalization have been used. The arrays were fabricated
on Si/SiO2 chips (15 mm X 15 mm) and covered by 30-50 nm of SiO2. More-
over an oxigen plasma has been used in a plasma asher machine to make
the surface hydrophillic (see 3.1.4). The experimental setup is described in
section 3.3.1. The stepper motors system is used to apply the in-plane mag-
netic eld of 300 Oe (enough to nucleate and displace DW over the single
ring). The out-of-plane magnetic eld is generated by a hollow cylindrical
coil placed around the objective of the the microscope and connected to a
generator, controlled via a labview software. It can provide a magnetic eld
up to 100 Oe.
The experiments have been executed in a wet environment of D.I. water and
SDS (Sodium Dodecyl Sulfate) at 0.5% (V/V) employed to reduce the friction
between particles and surface. Beads are diluited to reach a concentration of
1-5 µg/ml.
The manipulation of MNPs has been monitored by the optical microscope in
the Bright Field (BF) mode.
Once beads are sedimented on the surface, they are trapped by DWs and they
can be displaced over the single ring by rotating the in-plane eld (generated
by the permanent magnets) through the stepper motors system (see 3.3.1).
Figure 4.11 shows the motion of a captured bead by applying a rotating eld
of 300 Oe. 85 Figure 4.11: Manipulation sequence of a single 1µm bead around a magnetic
ring in an hexagonal matrix by applying a 300 Oe in-plane eld. The mag-
netic eld is rotated in an anti-clockwise direction. The images are recoded
from an optical microscope exploiting a 60x immersion objective. In order to achieve the decoupling of MNPs from HH or TT DWs, a negative or positive value of Hz has been applyed. Hz is switched from
a positive value of 60 Oe to a negative one of -60 Oe (or vice versa) to
allow a "jump" of the particles from one ring to the adjacent one, as deeply
discussed in the previous sections. Note that there is an intrinsic uncertainty
on the value of Hz (±30 Oe) due to the out-of-plane component generated
by permanent magnets which are used to apply the in-plane eld. They are
located just under the sample stage (see 3.3.1) so that they create a small
out-of-plane eld (maximum 30 Oe) which has to be compensated by the coil
eld in order to achieve the 60 Oe value.
However, the combination of the in-plane and the out-of-plane elds permits
to manipulate the particles all over the 2-dimensional space. If more than one
particle is captured by the DWs in dierent rings, it is possible to synchronize
the motion of such particles along parallel paths. This happens because the
potential energy landscape is periodic and it is the same for each ring. A
modication of such landscape by an external eld will simultaneously aect
all the beads. 86 Figure 4.12: Optical microscope images of a particles batch manipulation.
The particles path is highlighted with dierent colour. A 60x immersion
objective and 1µm beads are employed. In gure 4.12 a sequence of the synchronized manipulation of 3 beads is illustrated. Again, the in-plane eld is employed to displace the DWs in the
curved conduit, dragging the particles where the adjacent rings are closer.
Hz is switched from a negative (-60±30 Oe) to a positive value (60±30 Oe)
or vice versa, so that particles are decoupled by TT DWs and attracted by
HH DWs (or vice versa). From that point the process is repeated. 87 4.3 Conclusions and perspectives In this chapter a method to achieve a synchronized and controlled manipu-
lation of many particles over curved conduits in a 2-dimensional space has
been developed and demonstrated.
The future perspective could be the complete automation of such method:
drawing a random path on a PC, a sequence of elds can be automatically
applied to drive the particles along the desired path. Moreover, this technol-
ogy should be tested and optimized for dierent biological applications such
as drug delivery. 88 Chapter 5 Detection methods for magnetic
nanoparticles In this chapter a description of the results related to the detection of MNPs
by means of AMR and capacitive sensors is presented. The recording of
beads transit is a fundamental topic of this thesis work because it allows to
count the number of particles which ow in a microuidic channel and whose
motion is activated by DWs propagating in magnetic conduits within such
channel. In this way, for instance, a controlled amount of particles (loaded
with specic drugs) could be administered to target cells (see chapter 6),
according to the general scheme presented in the introduction of this thesis.
In the rst part of the chapter the performances of AMR sensors placed along
a magnetic conduit to detect the transit of a single particle will be discussed.
Instead, in the second part, capacitive sensors will be presented. A detailed
description of the fabrication process for each class of devices can be found
in chapter 3, together with an illustration of the measurements setups. 89 5.1 AMR sensors AMR sensors are employed to detect the transit of MNPs, over a magnetic
conduit exploiting an impedance variation on the portion of the conduit
where the particle is passing due to the Anisotropic Magnetoresistance eect
(see section 2.3)
The sensors are zig-zags shaped nanoconduits made of Py, with 200 nm of
width, 25 nm of thickness and segment length of 2 µm. The injector pad
is 600 nm large and 4 µm long. Additional Au electrodes, 500 nm wide,
are also patterned under the magnetic conduits to perform the electrical
measurements. Fig.5.1 shows a microscope image of the device. Figure 5.1: Optical microscope image of an AMR chip: the zig-zags magnetic
structures are illustrated, together with the four electrical contacts for each
sensor. The fabrication has been made by EBL and optical lithography; after the evaporation of Py and Au, the lift-o procedure was employed to dissolve
the resist. The chip was nally capped by 40-50 nm of Al2O3 or SiO2 to
prevent nanostructures from damages due to the liquid and it is equipped 90 with a PDMS gasket to contain the uid. The entire fabrication process can
be found in section 3.1.4.
The device presents 6 sensors, each one provided with 4 electrodes that are
used to apply and record signals from the zig-zag conduit (see Fig.5.1). Two
inner electrodes are used to measure the electrical signal, while the outer
contacts are employed to apply the external potential.
Zig-zag conduits are used to nucleate and displace a single DW thanks to
the application of external eld pulses, as discussed in paragraph 4.1. The
nanoparticles attracted by the stray eld arising from the DW are dragged
over the zig-zag structures (see Fig. 4.2).
The AMR eect allows to electrically detect the crossing of the DWs between
two electrical contacts. In fact, the resistance of a magnetic and conductive
material (such as Py) is aected by the relative orientation between the mag-
netization vector M and the owing current density J. In particular, almost
the totality of materials present a resistivity for M parallel to J (ρ') bigger
than the resistance for M perpendicular to J (ρ'). In this way, if the mag-
netization vector M is directed perpendicular to the owing current density
J, a lower value of the voltage drop across a magnetic structure is measured
compared to the case in which the magnetization points in the same direction
of the current.
In zig-zag shaped nanostructures of Py, the magnetization vector is sponta-
neously oriented in the same direction of the conduit, in order to minimize
the total energy of the system (shape anisotropy is the leading contribution
-see section 2.1.6-). On the contrary, the DWs nucleated in such structures,
are transverse Neel walls with M locally perpendicular to the conduit as
illustrated in Fig.5.2. 91 Figure 5.2: Magnetization of a zig-zags shaped conduit corner, in absence
(a) or presence (b) of a DW. The arrows indicate the direction of M. Red-
white-blue pixels indicate the value of M along the x-axis. In this way, if a DW is nucleated and positioned on top of the corner located between the inner electrical contacts of our device, the resistance of
the corner decreases and a lower voltage drop is measured as depicted in
gure 5.3. This procedure permits to electrically detect the transit of a DW. Figure 5.3: Sketch of the AMR sensor. The internal electrodes are employed
to measure the voltage drop at one corner of the magnetic zig-zag conduit.
The external contacts are exploited to apply the electrical signal. The green
arrows represent the current which ows in the device. 92 The detection of the transit of a MNP bound to said DW is more complex,
essentially because the variation of the DW micromagnetic conguration due
to the magnetic nanoparticle is not enough to produce a measurable variation
of the impedance across the corner. In this case a simple voltage measurement
is not sucient. However, when a particle is trapped over a DW, a dierent
value of the depinning eld is measured. The depinning eld Hdep is the
lowest magnetic eld that is required to displace a DW from one pinning site
to an adjacent one. In a zig-zag shaped structure, it is the minimum eld to
move a DW from one corner to another.
When a bead is placed over a DW at one corner in the zig-zag nanostructure
and the magnetic eld Hdep is applied to displace the DW along one conduit
segment, an additional magnetic dipole moment eld µ is generated in the
superparamagnetic bead. The stray eld generated by µ opposes the applied
eld below the bead, causing an increase in the value of the eld Hdep required
to displace the DW as illustrated in gure 5.4. This discrepancy in the value
of Hdep can be used to distinguish the presence of a MNP over a DW. Figure 5.4: Sketch of the zig-zag conduit in an AMR sensor. A magnetized
bead of moment µ produces a stray eld in the upper corner of the conduit. 93 In this way, a synchronized measurement of the voltage drop and the Hdep
value is employed to count the number of beads which transit between the
electrodes. 5.1.1 Detection experiments The experiments have been carried out exploiting the setup described in sec-
tion 3.3.2. A four probes technique is employed. The inner contacts (see
g.5.1) are used to record the voltage drop across the corner, while the outer
contacts are supplied by an AC voltage, with an amplitude of 50-200 mV and
a frequency which ranges between 10-100 kHz. The output signal from the
inner contacts was demodulated by a lock-in amplier, in order to increase
the signal to noise ratio.
The magnetic eld is applied by means of a four poles electromagnet which
generates an in-plane magnetic eld that can be nely controlled in direction
and absolute value (up to 700 Oe). The experiments have been executed in
a wet environment of D.I. water and SDS (Sodium Dodecyl Sulfate) at 0.5%
(V/V) to reduce the friction between particles and surface. Superparamag-
netic beads with 1 µm diameter (myOneTM, Invitrogen) and COOH surface
have been used. They are diluted to reach a concentration of 1 µg/ml.
The frequency of the AC signal was set to a relative high value because it
permits to better reduce the electric noise (thanks to the lock-in). Moreover,
the impedantial spectra of the device shows a smooth and constant value of
R for a frequency lower than 200 kHz, as shown in Fig 5.5. 94 Figure 5.5: Impedantial spectra of the AMR devices, measured by LCR.
Curve A represents the resistance between the inner electrodes, while B the
resistance between the outer contacts. The impedantial spectra were measured exploiting an LCR Agilent E4980A. The current which ows in the device ranges between 1-4 µA. The applied
voltage was set not to exceed 4 µA which is the upper limit of the current,
in order to avoid the disruption of contacts by Joule eect. The equivalent
circuit can be found in Fig.3.15
The rst part of the experiment was carried out in absence of MNPs. A
single DW is nucleated and displaced by applying a sequence of eld pulses
as discussed in section 4.1. Once the DW is located at the corner preceding
the inner electrical contacts (see Fig.5.6(a)), we made a sweep of magnetic
eld (0-300 Oe), applied in the direction of the segment leading to the corner
surrounded by the electrodes. When the value of HdepIN =179.5±2 Oe is
reached, the DW is depinned and jump to the corner sandwiched by inner
electrodes so that a lower voltage drop is measured due to AMR. 95 Figure 5.6: Sketch of the displacement of a DW to the corner between the
inner contacts in AMR sensors (a) and plot of the voltage drop due to AMR
as a function of the external eld. At 179.5±2 Oe (indicated by the red-
dashed line) the DW is displaced (b). The dots represent the experimental
values, while the continuous line is a polynomial t of the data. The anisotropic magnetic resistance has been quantied for the employed
device, as the maximum percentage variation of the resistance when passing
from parallel to perpendicular orientation of M and J. The results show a
voltage variation (VAMR) of 1.1±0.2 µV over a baseline of 175±10 µV before
the DW jump. It corresponds to a percentage variation of the resistance of
around 0.6 %. 96 In gure 5.7 another eld perpendicular to the previous one is applied to
displace the DW to the corner beyond the electrodes. During this sweep an
increase in the voltage drop is measured when the depinning eld HdepOUT
reaches a value of 180±2 Oe. Figure 5.7: Sketch of the displacement of a DW beyond the inner contacts
in AMR sensors (a) and plot of the voltage drop by AMR as function of
the external eld. At 180±2 Oe, (indicated by the red-dashed line) the
DW is displaced (b). The dots represent the experimental values, while the
continuous line is a polynomial t of the data. Even if the direction of HdepIN and HdepOUT is dierent, the absolute value is almost the same, as it can be expected by the symmetry of the conduit.
The same experiments have been executed adding the MNPs (1 µg/ml) in 97 solution and trapping one bead over a DW. Particles are manipulated over
the zig-zag shaped conduit as it is illustrated in g.5.8. The presence of Figure 5.8: Optical microscope image of 1 µm bead transition between the
inner contacts in an AMR sensor. An immersion objective 60x has been
employed. beads over DWs change the depinning eld value (as discussed in the pre-
vious paragraph). Figure 5.9 shows the variation in the voltage drop as
function of the applied eld when a bead is displaced to the corner between
the contacts (a) or outside from it (b). In this case, HdepIN =193.5±2 Oe and
HdepOUT =195±2 Oe. Again, a sweep of magnetic eld (0-300 Oe) is applied
to move the DW. A noticeable increase of HdepIN and HdepOUT is measured
in presence of beads. The variation of the depinning eld is ''HdepIN =14±3
Oe and ''HdepOUT =15±3 Oe. This is illustrated in gure 5.9(c). In this way,
if Hdep in presence and absence of beads is known, it is possible to precisely
record and detect the number of beads crossing between the electrical con-
tacts. 98 Figure 5.9: Plots of the voltage drop as function of the applied eld when the
bead is displaced inside (a) or outside (b) the contacts. Panel (c) compares
two curves which represent the voltage variation when a DW is displaced
outside from the inner contacts in presence (red line) and absence (black line)
of a MNP on the top of it. The ne lines and dots represent the experimental
values, while the thicker continuous line is a polynomial t of the data. It can be observed from the graphics how the measurements are aected by a periodic electrical noise which doesn't signicantly alter the experimen-
tal results. The SNR (signal to noise ratio) is around 3, calculated as follows:
SN R = VAMR/VNOISE where VAMR (1.1±0.2 µV) is the voltage variation passing from parallel to perpendicular orientation of M and J and VNOISE 99 is the amplitude of electrical noise signal (0.35±0.1 µV). The electrical noise
is a periodic signal mainly due to a spurious eect of the lock-in that can't
be eliminated with the current setup. The "Electronic engineering group" is
actually developing a dedicated lock-in able to reduce such noise gure. 5.2 Capacitive sensors Capacitive sensors exploit an impedantial measurement to detect the transit
of a bead between two electrical contacts. The idea behind these detectors is
the following: the conductivity of magnetic beads is much smaller than PBS
(Phospate Buer Saline) conductivity ('PBS =1.5 S/m) and consequently
when a bead, diluted in PBS, transits between two electrodes, a voltage
drop is measured. The controlled motion of MNPs between the electrodes is
achieved by means of the well known zig-zag shaped conduits which allow to
trap and displace nanoparticles applying a sequence of magnetic elds.
In the next paragraph a preliminary experiment will be described; it is ex-
ecuted to valuate the impedantial response of beads. Finally, in the last
section of this chapter, the preliminary results related to capacitive sensors
will be presented. 5.2.1 Beads Impedance A rst experiment has been carried out to estimate the impedantial response
of the magnetic nanoparticles. In fact, capacitive sensors are founded on the
idea that beads behave as insulating body or, at least, they present a much
lower conductivity compared to PBS. The goal of this paragraph is to conrm
this assumption. 1µm MNPs (myOneTM, Invitrogen) and 2µm (micromer-M-
Streptadivin, Micromod) have been tested. The impedantial measurement
is performed through a simple microuidic device which exploits bigger elec-
trodes compared to the capacitive sensors described in the next section and 100 employed for the nal experiment. Once MNP, diluted in PBS, are sed-
imented between such electrodes an external permanent magnet is used to
attract them far from the contacts and, a variation of impedance is measured
through an LCR meter. A quantication of the resistance variation permits
to calculate the impedantial eect of a single bead if the concentration of the
particles is known. This chip is made by a glass substrate patterned with Au Figure 5.10: Sketch of the electrical contacts in the measurement area of a
capacitive device. W=3 mm, G=4 µm, S=4 µm, H=1 mm and L=12 µm. electrical contacts. Their extremities are 3 mm wide (W), 300 nm thick and
located at a distance of 4µm (G), as illustrated in Figure 5.10. The sensitive
volume can be calculated as follow: V = W '' H '' (2S + G) (5.1) where S is the sensitive contacts length (4µm) and H (1 mm) is the sensitive
height for the measurement (evaluated through FEM simulations). The total
sensitive volume is equal to 3,6*10''11 m3. The beads are diluted in a PBS
solution with 0.5% of SDS to reach a concentration of 0.5 mg/ml for 1µm
beads and 4 mg/ml for MNPs of 2 µm. A drop of solution is placed on 101 the top of the device and after 5 minutes, the beads sedimented between
the electrodes are attracted away from the contacts thanks to a permanent
magnet. The measurement frequency was set at 1 MHz in order to be in
a spectral region not aected by the double layer capacitance. Figure 5.11
shows the measured resistance. When the beads are placed between the Figure 5.11: Graphic of the resistance variation as function of time when a
permanent magnet is used to attract 1 µm particles away from the electrodes.
Green arrows indicate respectively when the beads are between the electrical
contacts (Beads IN), when they are attracted away by the magnet (Beads
OUT) and while they are sedimenting (Sedimentation). A constant descend-
ing drift (-30 m'/s) in the resistance value is subtracted to the experimental
data. electrodes the resistance is high. Instead, when the magnetic eld is applied,
attracting away the beads, the resistance drops. Once the magnetic eld
is removed, the beads sediment and the resistance is restored to the initial
value, following an exponential trend. A constant descending drift is observed 102 in the measurements which is related to the evaporation of the liquid which
increases the ions concentration.
The conductance variation ''GTOT , when beads are attracted away, has been
quantied by the experimental data. The average value is 1.67*10''4 S for 1
µ m beads and 3.48*10''4 S for 2 µm MNPs. The number of particles in the sensible volume is calculated by the following
equation: N = V '' C 4
3 'r 3 '' ρ (5.2) where C is the concentration of beads, V the sensible volume, r the radius of
MNP and ρ ('4 g/cm3) the density of beads. In this way, N1µm is 8.57*10''4
and the same number is found for 2 µm MNPs because a concentration
8 times higher is balanced by a volume of a single particle 8 times larger
respect with the 1µm MNPs. Finally, the value of the conductance variation
associated to a single bead has been calculated. In fact ''Gbead = ''GTOT /N
and it is equal to 19.4 nS for 1µm bead and 40.6 nS for 2 µm particle. A
bigger dierence between the values of ''Gbead for 1 and 2 µm beads would
be expected because the volume of the second is 8 times higher and the
variation of G should be of the same order. However, the low mobility of
2 µm nanoparticles in the electric eld, together with the creation of big
particles clusters could aect the result.
The value of ''Gbead has been compared with the result of FEM simulations.
In these simulations, beads are considered perfect insulators. The simulated
variation of G for 1µm bead is equal to 25 nS in good agreement with the
value calculated from the experiments. Instead the simulated ''Gbead for 2µm
particles is 170 nS around 4 times bigger than the experimental value. 103 5.2.2 Experiments of single bead detection The fabrication of capacitive sensors is described in section 3.1.4. The chip
is patterned with the magnetic zig-zags nanostructures of Py and with a set
of coupled electrical contacts employed for the measurements. Dierent elec-
trodes geometries have been tested. Each chip is provided with 3 magnetic
zig-zag conduits and 24 electrodes as illustrated in gure 5.12. One of such
contacts is employed as counter electrode to set the potential of the solution. Figure 5.12: Optical microscope image of a capacitive sensor. Each chip is
provided with three magnetic zig-zags conduits and 24 electrodes. One of
such contacts is employed as counter electrode to set the potential in the
solution. The couple of electrodes labelled by "A" represent the geometry
used in the numerical simulations. The magnetic nanostructures and the extremities of the electrical contacts are exposed to the liquid in order to permit the impedantial measurements. 104 This is achieved by means of 3 openings obtained by optical lithography and
Lift-o process. Two dierent capping layers have been employed: SiO2 (40-
50 nm) or the resist exploited for optical lithography (about 2 µm). The last
one is treated with an extra backing process to be hardened and it has the
advantage to be thicker compared to Silica, potentially favoring the insula-
tion of the contacts from the liquid (more details in 3.1.4).
FEM (nite element method) simulations on a 3-dimensional model of the
device have been performed with COMSOL software to evaluate the theoret-
ical electrical response of the sensor in presence and absence of bead. Figure 5.13: Image of the 3D geometry employed in FEM simulation. The
colours indicate dierent value of the electrical potential. In these simulations, electrical contacts, zig-zags shaped nanostructures and the PBS environment have been considered. An image of the 3D model
is shown in gure 5.13. The simulated variation of the resistance in presence
and absence of 1 µm bead between the contacts is 32 k' as illustrated in
gure 5.14 . 105 Figure 5.14: Plot from FEM simulations which shows the Resistance varia-
tion in presence and absence of bead between the electrodes. In this simula-
tion the double layer capacitance was not considered. The equivalent circuit for the capacitive sensor is illustrated in gure 5.15(a). CDL is the double layer capacitance (9.8 pF from simulations) cre-
ated at the interface between electrodes and the PBS solution; it depends on
the area exposed to the liquid together with the amount of ions in solution.
RL is the resistance of the liquid. The latter depends on the ions concen-
tration and valence in PBS together with the distance between electrodes.
For standard values of PBS solution and considering the electrodes couple
labelled by A in gure 5.12, the simulated value is 163 k' without beads.
The working frequency was calculated to be in a spectral region not aected
by the CDL in order to valuate only the variation of RL. It is achieved for
frequencies higher than 105 KHz. CP represents the eect of parassitic ca-
pacitances and it is related to the interfaces between the dierent materials
in the chip. The eect of CP has to be negligible at the working frequency in 106 order not to hide the detection signal. The value of CP extimated by means
of an LCR measurement is 120 pF. Figure 5.15: Sketches of the equivalent circuit for a capacitive sensor (a) and
the electronic readout (b). CDL is the double layer capacitance, RPBS is
the liquid resistance and CP is the parassitic capacitance. In (b) the lock-in
amplier (Zurich), the transimpedance amplier (Femto), the Dummy circuit
and the chip mux are illustrated. The experiments have been performed in a wet environment of PBS with 0.5% SDS (Sodium Dodecyl Sulfate) to reduce the friction between particles
and surface. The external magnetic eld is applied by means a four poles
electromagnet (the same employed for AMR measurements). The capacitive
sensor is mounted on the sample stage described in section 3.3.2. A two
probes technique is exploited for the electrical measurement and the device
is powered via a lock-in amplier with an AC signal having an amplitude
that ranges between 50 mV and 100 mV and a frequence set at 2 MHz. The
output current from the chip is read by means of a trans-impedance ampli-
er through a virtual ground. The signal is then transferred to the lock-in
which demodulate it and, thanks to a really narrow pass-band lter, is able
to reduce the electrical noise. However, the eect of the phase noise due to 107 uctuations in the input signal phase, coupled with CP , creates a high noise
in the device output which can mask the signal of interest.
In order to reduce the eect of CP , an active compensation circuit has been
introduced. It is a Dummy circuit which has the goal to reproduce the trans-
fer function of the device in absence of bead. The signal generated by lock-in
is splitted between the the chip and the compensation circuit. The output
signals from the device and Dummy circuit are retransferred to the lock-in
that subtractes them and cleans the signal of interest from all the eects
related to parassitisms. The schematic view of the whole electronic setup is
illustrated in gure 5.15(b).
The MNPs are monitored by means of an optical microscope trough a 60x
immersion objective. The particles (myOneTM, Invitrogen) have been suc-
cessfully displaced between the electrical contacts applying a continuous mag-
netic eld of 250 Oe which is rotated in a clockwise or anti-clockwise direc-
tion. In this way an alternate sequence of HH and TT DWs nucleated at the
zigs-zags corners is simultaneously displaced along the structure dragging the
particles on the top of DWs. Figure 5.16 illustrates the crossing of a bead
between two electrical contacts in a capacitive sensor. However, the experiments don't show an appreciable variation of the elec- Figure 5.16: Sequence of the transit of a 1µm bead between two electrical
contacts in a capacitive sensor. 108 trical signal when the bead is located between the electrodes. This can be
related to several reasons that are actually under investigation. Firstly, the
capping layer employed on the chip doesn't allow a perfect insulation of the
device. If the liquid penetrates and goes in contact with the electrodes, the
electrical measurements is expected to be be strongly altered by a big paras-
sitic double layer capacitance. A second reason can be the position of the
bead along the z-axis. If the bead uctuated over the magnetic conduit above
100 nm, the electrodes (which are 65 nm thick) would not be able to e-
ciently detect the impedential variation due to the MNP because the electric
eld lines would not intercept it. Moreover, the electrical noise associated
to this measurement is still not completely negligible and further work has
to be done to understand its origin and to delete it. Finally, a high signal
drift during the measurements is observed. It is probably due to variations in
temperature or ions concentration and it could negatively aect the electrical
detection. 5.3 Conclusions and perspectives In this chapter two dierent sensors to detect beads have been developed and
illustrated. The AMR sensors permit to achieve a precise recording of MNPs
that transit over magnetic zig-zags conduits. Instead, capacitive sensors have
to be further developed and improved.
The results display how the magnetic detection is much less sensitive to per-
turbations than capacitive measurements. The main reason is that most of
the media (such as PBS or biological medium) are not magnetic (except for
a negligible diamagnetism or paramagnetism), while they present an electric
behavior which can disrupt the impedantial measurement.
The NaBiS group had previously demonstrated how nanometric Py corners
and micrometric rings can be employed to trap and detect magnetic nanopar- 109 ticles through an AMR measurement [62],[41]. However, the AMR sensors
fabricated and developed in this thesis work are completely innovative de-
vices. They exploit the same magnetic conduit both for manipluation and
detection, adding a new important functionality to DWTs: the integrated
feedback on the manipulation, because particles can be detected while mov-
ing. In this way, we are going towards a close-loop system that clearly oers,
a large amount of advantages.
Besides, the measurements have been carried out on 1 µm beads, but the
SNR ratio suggests us that smaller particles can be employed. By xing the
problem of the "oscillating noise" on the base-line, a particle with 250 nm
diameter or lower could be detected, considering that the calculated depin-
ning eld variation is 14-15 Oe for 1 µm bead. At the current state of the
art, there is not a compact and integrated device capable to such a detection
of a bead crossing thorough a constriction.
For example, Magnetic Tunnel Junction sensors (MTJs) [63] could be used
to detect beads with the same (or even larger) sensitivity but their complex-
ity is much higher compared to AMR sensors and they could not be easily
integrated with a system for beads manipulation.
The future perspective for AMR measurements and setup are related with
the simplication of the electronic readout system by projecting an integrated
lock-in which will be also able to remove the electrical noise due to a spurious
eect of the lock-in currently in use. Moreover, the sensors can be equipped
by a more complex microuidic cell to better control the ux of uid over
the chip. 110 Chapter 6 Controlled administration of
nanoparticles to a single cell This nal chapter describes one of the most important results of this thesis
work which concerns the controlled administration of magnetic nanoparticles
to a target cell. The DWTs technology is employed to nely manipulate
MNPs on a chip where living cells are cultured. In this way, a single particle
can be displaced in close proximity to the cell membrane. This tool permits to
study the interaction between a single bead with a single cell and to evaluate
in which conditions the up-take of particles occur, which is a fundamental
topic in the drug-delivery process (see chapter 1).
In the rst part of the chapter the optimization of MNPs manipulation in
the cellular medium, together with the preparation of the chip to achieve
a good cellular adhesion and viability is described. In the second part, the
"passive" up-take of MNPs is demonstrated. Finally, the results related to
the controlled administration of nanoparticles to a target cell are described. 111 6.1 Manipulation of MNPs in the cellular medium Magnetic martrix of Py rings can be employed to trap and drag magnetic
nanoparticles all over a 2D space as discussed in chapter 4. Similar devices
are used to perform the experiments in presence of biological entities. The
rst test has been made to study the eciency of beads manipulation in the
cellular medium.
The magnetic nanoparticles used for all the experiments (nanomag-CLD-
redF, Micromod) have a diameter of 300 nm, they are functionalized with
carbossylic acid (COOH'') and they are red-uorescent, exploiting a TRITC
marker. The functionalization with COOH'', that creates a positive Zeta-
potential, is chosen to favor the electrostatic interaction with the cellular
membrane which presents a negative Zeta-Potential as illustrated in chapter 1.
The Py nanostructures are patterned over a Si/SiO2 wafer by EBL (see sec-
tion 3.1.4). Magnetic Rings are 300 nm wide with a diameter of 5-10µm and
they are packaged in a square or hexagonal matrix (see Fig. 4.3). Three
dierent capping layers have been tested in order to achieve two goals: pre-
venting the chip from damages due to the liquid and favoring the cellular
adhesion on the surface. The rst one is a 50 nm SiO2 coating, properly
treated with an oxigen of plasma in a plasma asher machine to make the
surface hydrophillic (see section 3.1.3). The second one is a double layer of
Al2O3 (25 nm) and SiO2 (25 nm) also treated with an Oxigen of plasma. The
last one is a composed layer made of 10 nm of Al2O3 and 60 nm of nanostruc-
tured Zirconia ZrOX (deposited through supersonic cluster beam deposition
technique [64] at "Fondazione Filarete", in Milan). Nanostructured ZrOX
is a porous material which favor the cellular adhesion on the surface. The
roughness of nanostructured Zirconia layer was evaluated by means of an
AFM scan and it ranges between 10-20 nm (RMS) in 1 µm2, for dierent
samples (see Fig. 6.1). 112 Figure 6.1: AFM image (a) and derived 3D view (b) of a sample covered by
ZrOX. The manipulation experiments have been performed in the cell culture medium DMEM. It is a basal medium consisting of Amino Acids, Glucose,
pH indicator, Salts and Vitamins. DMEM was enriched by antibiotics to
increase the cellular viability and a serum that provides the nutrients for the
cells.
The employed MNPs have a diameter (300 nm) which is smaller compared
to the beads used in the rest of the work. The attracting magnetic force due
to the stray eld generated by DWs depends on the volume of the super-
paramagnetic body (see eq 2.29), therefore 300 nm particles suered a force
around 27 times lower than 1µm beads. Moreover, DMEM presents a higher
viscosity than PBS or water and this negatively aects the manipulation.
In order to overcome these problems, a set of micromagnetic simulations
have been performed to optimize the thickness of magnetic rings so that the
trapping force is maximized. The next section describes the results of such
simulations. 113 6.1.1 Micromagnetic simulations The force generated by a single magnetic DW in a Py Ring on a 300 nm super-
paramagnetic bead has been simulated as function of the conduit thickness
and the distance between MNP and nanostructures. By means of OOMMF
simulations, the stray eld generated by a DW in a Py ring with a diameter
of 5 µm and width of 300 nm was calculated. Firstly, upon the application of
a saturating in-plane eld of 500 Oe, the magnetic conguration of a single
ring has been found. Figure 6.2: Magnetization of a portion of ring from OOMMF. The arrows
show the direction of the magnetization in the xy-plane. Blue and red pixels
indicate positive and negative values of the magnetization along y. HH vortex
DW is nucleated by an external in-plane eld (500 Oe). A vortex conguration of magnetization in the DW is observed for all the dierent values of thickness simulated. It is due to the balance between the
shape anisotropy of the structures and the magnetostatic energy contribution. 114 The gure 6.2 displays the magnetization inside the ring upon the application
of a strong saturating magnetic eld in the x direction. The tendency to close
the eld lines of the magnetization on the upper and lower part of the arch
of the ring is an artifact due to the nite area of the simulation which did not
consider the entire ring in order to save computation time. Starting from the
magnetic conguration, the stray eld generated by the DW was simulated by
OOMMF. From that, the attracting force is calculated via Matlab, according
to the following equation: F = ''µ0' ' V ''(Hdw · Hdw)dV (6.1) where Hdw is the stray eld generated by the DW and the integration is over
the superparamagnetic bead volume. The 300 nm MNPs considered in this
simulation have a magnetic susceptibility ' of 0.39.
The force along the z-axis was calculated for dierent values of ring thickness
(30-80 nm) and considering a distance from the top of the ring and the bot-
tom of the bead which ranges between 50 nm and 600 nm. The lower limit
was xed at 50 nm because it is the minimum distance if a capping layer of
50 nm is placed on the top of magnetic structures.
The results illustrated in gure 6.3 show that a maximum value of the force
along z is obtained for a thickness of 60 nm, independently on the distance
between bead and surface. For example, considering a distance of 50 nm,
the force for a 60 nm thick structure (45.4 pN) is more than three times
higher compared with the force exerted by a 30 nm thick ring (12.8 pN). For
a thickness larger than 60 nm the force suered by particles decreases. It is
probably due to the rotation of magnetization also in a direction perpendic-
ular to the xy-plane when the DW is nucleated in thicker structures. In this
situation, the DW is not more a "pure" Neel wall (with the magnetization
rotating only in the xy plane) and a lower stray eld is generated. 115 Figure 6.3: Magnetic force on a superparamagnetic bead having a diameter
of 300 nm and ' equal to 0.39, as a function of the ring thickness. The
distance between the top of the magnetic structure and the bottom of the
bead ranges between 50 nm and 200 nm. Figure 6.4 shows the force (z-component) trend when the distance be- tween bead and surface ranges between 50 and 600 nm. Figure 6.4: Magnetic force on a superparamagnetic bead having a diameter
of 300 nm and ' equal to 0.39, as a function of the distance between the top
of the Py ring and the bottom side of bead. It was calculated for dierent
values of the ring thickness ranging between 30-80 nm. 116 An exponential decay is observed independently by the ring thickness. To summarize, with these simulations two conclusions can be drawn: rstly
the force suered by 300 nm particles is maximized when the thickness of
magnetic rings is 60 nm. Secondly, an ecient manipulation is possible only
if beads are in close proximity to the surface, because the force exponentially
decrease with the distance. 6.1.2 Manipulation experiments In this paragraph, the manipulation experiments of 300 nm nanoparticles in
DMEM medium will be described. The setup employed is presented in section
3.3.1. The temperature, set at 37 oC, is controlled by means of a thermostat
connected to a thermocouple placed in contact with the medium. A small
heating plate, mounted just under the sample stage, is employed to heat
the sample and the medium. The stepper motors system is used to apply
the rotating magnetic eld in-plane. 300 nm beads (nanomag-CLD-redF,
Micromod) are diluted in DMEM to reach a concentration of 0.5 µg/ml. An
optical microscope with a 60x immersion objective has been used to monitor
the beads displacement.
The idea behind this experiment is to understand the eect of the cellular
medium on the manipulation over a period of 6 hours (which is the typical
duration of a biological experiment) in which the sample remains in contact
with the DMEM.
Three dierent devices have been tested, each one patterned with a matrix
of 30 nm thick rings, with a diameter of 10 µm. Even if the force is not
maximized with a nanostructures thickness of 30 nm, it has been initially
preferred to have a atter surface. The rst chip is covered by Al2O3 (10
nm) and nanostructured ZrOX (60 nm). The second one is capped by SiO2
(50 nm) and the third device is coated by a composed layer of Al2O3 (25 nm)
and SiO2 (25 nm). 117 In the rst 3.5 hours, despite the viscosity of the medium, it is possible to
achieve such a kind of ne manipulation, for all the devices, even with a
corrugated surface like nanostructured ZrOX lms. The rotating magnetic
eld generated by the permanent magnets controlled through the stepper
motors, allows to nely displace the MNPs on a single ring as illustrated in
the panels of gure 6.5 which are frames of a video taken under the optical
microscope. Figure 6.5: Manipulation sequence of a single 300 nm bead around a magnetic
ring in an hexagonal matrix by applying a 300 Oe in-plane eld. A sample
covered by ZrOX (50 nm) was employed. The magnetic eld is rotated in an
anti-clockwise direction. The images are recoded from an optical microscope
exploiting a 60x immersion objective. Regarding the eect of time and medium over the manipualation, for the rst and the second device (terminated respectively with 60 nm of ZrOX
and 50 nm of SiO2), between 3.5 and 5 hours the manipulation becomes less
ecient; some particles appear to be only sightly bounded to DWs and they
begin to uctuate over the magnetic conduits. After 5 hours, it is not more
possible to attract and displace the particles over the nanostructures.
Instead, over the chip coated with Al2O3 (25 nm) and SiO2 (25 nm), an
ecient manipulation of beads is possible for the rst 5 hours. After 5.5 h
the manipulation becomes ineective also for this device. The reasons for
the manipulation becoming less ecient after a certain time are essentially
related to an ineective insulation of the magnetic nanostructures from the 118 liquid. In fact, the employed capping layers do not prevent the magnetic rings
from going in contact with DMEM. This produces the oxidation of Py which
signicantly alter the magnetic properties of the conduits whose thickness is
only 30 nm.
The results show that a comparable impermeability is observed for the rst
chip (coated with 10 nm of Al2O3 and 60 nm of ZrOX) and the second device
(capped by 50 nm of SiO2), despite the dierent material layers employed
to coat them. Moreover, it can be observed that Al2O3 guarantees a better
insulation from the liquid compared to SiO2 but it is still not enough to
prevent nanostructures from damages due to the liquid for a long time (6 h).
In order to completely overcome the problem, we are actually developing a
process to deposit Silicon Nitride (SiN) as capping layer which oers better
insulating properties and impermeability to saline solutions. 6.2 Passive uptake of magnetic nanoparticles Before performing "active" uptake experiments, one as to demonstrate that
the employed magnetic nanoparticles can be internalized by cells. Tests of
cellular uptake have been performed at the IFOM center where the confocal
microscope TCS-SP5 Leica was used; its high resolution allows to visual-
ize the relative position of particle and cell also in z-direction, so that the
internalization of beads can be properly observed. Epithelial human cells
from the HeLa line are employed to this purpouse. The cellular nucleus is
stained with the green-uorescent marker H2B-GFP which is a basic nu-
clear protein responsible for the nucleosome structure of chromatin. Cells
have been incubated at 37oC in an atmosphere of 5% CO2, with magnetic
and red-uorescent nanoparticles (nanomag-CLD-redF, Micromod) diluted
to 10µg/ml.
After 4 hours, few ml of cells have been washed in PBS to remove the particles 119 dispersed in the medium; then, the cells have been placed on a microscope
slide and investigated under the confocal microscope. Figure 6.6: Confocal microscopy image: the green-stained cellular nucleus of
an HeLa cell is surrounded by red uorescent (TRITC) magnetic nanoparti-
cles. It is acquired by a TCS-SP5 Leica microscope. Figure 6.6 shows the green-uorescent cell nucleus which is crowned by red-uorescent magnetic nanoparticles. However, in this experimental con-
dition it is not possible to unambiguously demonstrate the complete particle
internalization; since it is not possible to properly see the cellular membrane,
it is dicult to evaluate if the particles are inside the cytoplasm or they are
bounded outside the cytoplasmic membrane.
In order to understand where nanoparticles are exactly located, cytoplasm
has been labelled with a Dextran-based green uorescent marker. Dextran is
a polysaccharide made of many glucose molecules, which is quickly absorbed
by cells. The cellular solution was enriched with this marker and, after 30
minutes, it has been washed in PBS and placed on a microscope slide. In 120 this way, the entire cell appears green and red magnetic nanoparticles are
observed inside the cytoplasm as illustrated in gure 6.7. Figure 6.7: Confocal microscopy image: red-uorescent (TRITC) magnetic
nanoparticles are internalized inside the cells (HeLa). The green cytoplasm
is labelled by a Dextran based marker. The brightest green circular spot on
the left are two cellular nuclei (some cells have not absorbed the marker).
Image acquisition via a TCS-SP5 Leica microscope. In this way, the uptake of 300 nm particles functionalized with COOH has been demonstrated. 121 6.3 Manipulation of nanoparticles to a target cell One of the goals of this thesis work is to manipulate nanoparticles to admin-
istrate a drug to a target cell. In this paragraph, it will be demonstrated
how magnetic nanoparticles can be nely moved in close proximity to a spe-
cic cell where they interact with the cellular membrane. Two dierent cell
types have been employed for these experiments: rat's mammalian cancer
cells supplied by "Istituto Farmacologico Mario Negri" and epithelial human
cells from HeLa line, provided by the "IFOM center". The samples used
are the same described in section 6.1: Py rings (300 nm wide, 30 nm thick
and with a diameter of 10µm) arranged in a hexagonal or square matrix and
capped with SiO2 (50 nm). The external in-plane eld exploited to manip-
ulate beads is applied by means of the stepper motors system, described in
section 3.3.1 together with the entire setup used for the biological experi-
ments. Two main tests will be described in this paragraph. In the rst one,
800 µl of mammalian cells diluted in the cellular medium MEM are cultured
for 2 hours on the top of the chip which is placed in the sample stage where
the temperature is set at 37oC by means of a PID thermostat connected to a
thermo-couple and to a heating plate mounted just under the sample stage
(more details in section 3.3.2). In order to compensate the evaporation of the
medium, 100 µl of DMEM are added every half an hour. After 2h, magnetic
nanoparticles with a diameter of 300 nm (nanomag-CLD-redF, Micromod)
and functionalized by COOH are added to the cell environment, after being
properly diluted to reach a concentration of 1 µg/ml.
The system is monitored by means of an optical microscope with a 60x im-
mersion objective. The beads, trapped on the DWs generated by an in-plane
eld of 300 Oe, are successfully manipulated on a single ring by rotating the
magnetic eld (see section 4.2); a single bead can be driven in contact with 122 the membrane of a target cell, as illustrated in gure 6.8. Figure 6.8: Manipulation sequence of a 300 nm MNP to a target cell
(mammalian-cancer cell). A continuous magnetic eld of 300 Oe is rotated
in a clockwise direction to displace the bead. An immersion 60x objective is
used. Once the bead is in contact with the cell, the magnetic eld can be even removed because, also in absence of the attracting force generated by the
DW, nanoparticles are attracted to cells by electrostatic interactions (due to
the opposite values of the Zeta-potential). A detailed study of the eect of
the magnetic force applied to the bead on the uptake has not been fully ac-
complished during this thesis work. Generally speaking, the magnetic force
exercised by the DW could unfavor the internalization mechanisms since it
attracts the beads on the chip surface outside the cell.
In some cases, it has been possible also to manipulate the particles under
the cell membrane, in contact with the surface. This means that the cellular
adhesion on the chip is not optimal. Besides, exploiting a normal optical
microscope, it is dicult to evaluate the height of the particle relative to
the cell and the chip surface; consequently the internalization process is not
easily detected.
In these experimental conditions, without a CO2 incubator and in DMEM
medium, after some hours (4-5 h) from the cells culturing, the viability of
cells decrease and they begin to show signs of apoptosis. This can be due 123 to two main factors: rstly the cells employed are not suitable to live in the
CO2 decent environment like that imposed by the experimental setup (the
CO2 amount inside the body of a living mammal is around 5%) and secondly
the SiO2 surface preparation on the chip could not guarantee an adequate
sterilization.
In order to solve these problems, a second experiment has been executed at
the IFOM center. In this test, epithelial cells from HeLa line are used. They
can be cultured on various surfaces maintaining a high viability. Moreover, a
confocal microscope (TCS-SP5 Leica) which oers a high spatial resolution
along z-direction, has been used. The same setup described in the previous
experiment is employed for manipulation, except for the microscope. In or-
der to obtain a good cellular adhesion on the chip, the cells are cultured for
5 h. During this time, the chip is incubated at 37oC in an atmosphere with
5% of CO2. Then, it is placed on the sample stage where beads are added to
reach a concentration of 1µg/ml in a DMEM medium.
In this experimental condition the cell viability is optimal also after 4-5
hours, and we indeed observed an excellent cellular adhesion on the surface.
However, the long incubation compromises the manipulation eciency of
the nanoparticles, due to the aforementioned problem of magnetic structure
degradation due to medium penetration in the capping layer. Nevertheless,
in the rst hour after the incubation it was still possible to work. So, it has
been possible to trap and manipulated MNPs on a single Py ring applying a
rotating magnetic eld of 300 Oe. Therefore, beads are dragged in contact
with the cellular membrane as illustrated in gure 6.9. 124 Figure 6.9: Frames from a video showing the manipulation of a 300 nm bead
dragged to a target cell (epithelial HeLa cell) through a rotating magnetic
eld of 300 Oe. Image acquisition via a TCS-SP5 Leica microscope. Once the MNP is placed in contact with the cell, the particle is attracted to the cellular membrane by the electrostatic interaction due to the opposite
value of the Zeta-potential and the magnetic eld can be removed. 6.4 Conclusions and perspectives In this chapter, it is described how the DWTs technology can be exploited
to achieve a controlled administration of MNPs to a target cell, manipulat-
ing the beads in close proximity to the cellular membrane. The next step,
currently under development, is to demonstrate the internalization of MNPs
loaded with specic drug and to investigate the nal details of uptake using
our magnetic tweezers. This technology can be potentially used for a large
amount of biological and medical applications as described in chapter 1. 125 Conclusions and Perspectives In this thesis work, it has been developed the building blocks of a microuidic
on-chip platform for controlled drug-delivery to a single cell through mag-
netic nanoparticles. Three main results have been obtained. The rst one
concerns the implementation of the DWTs domain wall tweezers technology
to achieve a synchronized manipulation of a magnetic nanoparticles batch in
a 2-dimensional space over a matrix of magnetic rings.
The second one is the development of on-chip sensors for the detection of
beads transit over magnetic conduits in lab on chip for controlled drug de-
livery. Two dierent sensors have been employed: the AMR and capacitive
detectors.Concerning the rst ones, the eective and successful detection of
beads in manipulation experiments has been demonstrated, while the capac-
itive sensors need to be further developed, since only the preliminary results
have been presented.
Finally, the controlled administration of magnetic nanoparticles to a target
cell has been demonstrated. The particles are eciently manipulated in the
cellular medium and they are moved in contact with cell membrane.
The future perspectives are related, rst of all, with the ne optimization of
devices and measurement setup already developed in this thesis work. For
the DWTs technology, a complete automation could permit to draw a ran-
dom path on a PC which controls and actuates a sequence of magnetic eld
to displace the magnetic nanoparticles along the chosen direction.
For AMR sensors, the perspectives are related with the integration of the 126 electronic readout system by projecting an integrated lock-in with better
performances in terms of noise rejection respect the instruments currently
in use. Moreover, the sensors can be equipped by a more sophisticated mi-
crouidic cell to better control the uxes over the chip.
Both the sensors and the chip with the magnetic nanostructured used to
achieve the controlled administration of magnetic nanoparticles to a target
cell need further works in order to optimize their impermeability to the bio-
logical medium, also after a long exposure time to it. Moreover, it has to be
unequivocally demonstrated how nanoparticles carrying drugs, controlled by
means of the DWTs technology, can be eciently internalized by cells.
The nal perspective is the integration of the sensors and the devices for
2D manipulation in a single on-chip platform. Such system would be able
to count and transport magnetic nanoparticles in a microuidic channel in
order to displace a controlled amount of functionalized particle, to the target
cells (through the 2D manipulation devices). The schematic principle of such
platform is illustrated in gure 6.10. Figure 6.10: Scheme of the platform made of two reservoirs (RIN and ROUT )
connected by a channel (C). Functionalized MNPs are dispensed in the reser-
voir RIN (a). From there, beads are manipulated along the channel (C) and
detected by means of an AMR (or capacitive) device (b). At the end of the
channel, a second reservoir (ROUT where cells are cultured is located. Beads
are manipulated by means of the DWTs to the target cell to administrate
the drug. 127 This platform could be employed for a large number of biological and medical
assays, such as testing the eectiveness of a certain drug on a target cell or
studying the bio-chemical mechanisms that occcur when a cell interact with
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