1. Unità Università degli studi di Milano
Dipartimento di Scienze Molecolari Applicate ai Biosistemi
Via Trentacoste 2 - Milano
Alessandro Lascialfari
Francesco Orsini (tecnico)
Paolo Arosio (post-doc)
Dipartimento di Chimica ed Elettrochimica
Marco Scavini
Serena Cappelli (tecnico)
Altri partecipanti alla ricerca– Dipartimento di Fisica “A. Volta” - Università degli studi di Pavia
Maurizio Corti
Evrim Umut (dottorando)
Andrea Capozzi (laureando, da fine aprile)

2. Experimental apparatuses
DISMAB - Via Trentacoste 2
Atomic force microscopy (AFM)
* Atomic Force Microscopy / Scanning Tunneling Microscopy / Magnetic Force Microscopy - Autoprobe CP Research System - Veeco. Working temperature range 0-60°C. Cell for liquids, measurements in solution.
* AFM Microscope with force spectroscopy facility, Nanoscope IIIA-Multimode, Veeco
Wide-band Nuclear Magnetic Resonance (NMR)
* Fourier transform Stelar Spinmaster spectrometer for NMR (also relaxometry), working in the range 5-70 MHz (with electromagnet , magnetic field Tesla) and flux cryostat (3.8 < T < 350K)
SMARTracer relaxometer by Stelar for NMR-Fast Field Cycling. Frequencies : 10 KHz < f < 10 MHz. Temperature : 150 < T < 300 K.
Muon Spin Rotation  International facilities PSI-Switzerland and ISIS-Didcot(UK)
Dipartimento di Chimica Fisica ed Elettrochimica : slides MARCO SCAVINI
In strict collaboration, Dept. of Physics, Pavia
Wide-band Nuclear Magnetic Resonance (NMR)
* Fourier transform Tecmag and Bruker spectrometers for NMR f-range MHz (SC magnet-electromagnets 0-9 Tesla)
* Cryostats for T-range 1.5<T<1000K, pressure chambers to reach 15 kbar
Magnetometry
* SQUID magnetometer for susceptibility and magnetization measurements in the range 0-7 Tesla and K. 
Magnetic Resonance Imaging (MRI)
Esaote MRI-Imager ARTOSCANfor in-vitro imaging acquisition sequences at low magnetic field 0.2 Tesla
Adiabatic and non-adiabatic calorimeters, Non-conventional Electron Paramagnetic Resonance (EPR)

3. Atomic Force Microscopy technique
Multimode - Nanoscope 3d AFM (Veeco). Working temperature range 0-60 °C. Liquid cell for measurements in solution. PicoForce modulus for force spectroscopy measurements
AutoProbe CP Research AFM (Veeco). Working temperature range 0-60 °C. Liquid cell for measurements in solution.

4. Nuclear Magnetic Resonance (NMR) technique
Smartracer relaxometer
10 kHz<f<10 MHz
(H= Tesla)
WIDE BAND NMR
not high resolution
Stelar Spimaster 5<f<70 MHz
(H= Tesla

5. Nuclear Magnetic Resonance
NMR is a spectroscopic technique :
Resonant (0 =  B0 e B1 << B0)
Microscopic i.e. local probe (it studies the magnetic/electrical interactions between nuclei, atoms and molecules)
At radiofrequency ( MHz)
It’s widely used in solid and liquid state Physics, Chemistry, pharmacological and bio-medical world
From 1975 it is an laternative to TAC; now also better for many applications
It does not use ionizing radiations

6. Nuclear Magnetic Resonance
APPARATUS
A static magnetic field
A rf magnetic field
Electronics
Cryogenics
EXPERIMENTAL PARAMETERS
3 main parameters:
spectrum
nuclear spin-spin relaxation time T2
nuclear spin-lattice relaxation time T1
LOCAL PROBE
Nuclei are local probes  sensitive to local hyperfine interactions
Local spin dynamics (mainly T1 and T2) and spin distribution (mainly spectra) can be studied
In MRI and relaxometry, sensitivity to spin dynamics and molecular “motion”
T1n
Nuclei
(T2n)
Electrons
(T2e)
Phonons
(lattice)
T1e
T1n

7. Nuclear Magnetic Resonance : statics i.e. spectra
NMR spectra : sensitive to local magnetic field
i.e. useful to estimate local field created by e.g. electrons
Examples
High-resolution NMR
(not our group)
Wide-band NMR

8. NMR : spin dynamics i.e. relaxation rates
NMR relaxation rates : sensitive to local electron spin dynamics
through hyperfine nuclei-electron interaction
Spin dynamics vs H at
room temperature
Spin dynamics vs T
at different constant fields

9. MUSR : spin dynamics through a different local probe
MUSR relaxation rates : sensitive to local electron spin dynamics
through hyperfine muon-electron interaction
Typical muon polarization behaviour
Polarization can be studied vs T and H

10. Magnetic Resonance Imaging
Pavia apparatus
MRI Timeline
1946 MR phenomenon - Bloch & Purcell
1952 Nobel Prize - Bloch & Purcell
NMR developed as analytical tool
1972 Computerized Tomography
1973 Backprojection MRI - Lauterbur
1975 Fourier Imaging - Ernst
1977 Echo-planar imaging - Mansfield
1980 FT MRI demonstrated - Edelstein
1986 Gradient Echo Imaging - NMR Microscope
1987 MR Angiography - Dumoulin
1991 Nobel Prize - Ernst
1992 Functional MRI
1994 Hyperpolarized 129Xe Imaging
2003 Nobel Prize - Lauterbur & Mansfield

11. Research (I) :magnetic nanoparticles in biomedicine
Magnetic Nanoparticles in Theranostics:
Diagnostics : MRI CA, fluorescence
Therapy : Magnetothermia
Biocompatible
shell
Antibody
Fluorescent
molecule
Drug
Molecular
Imaging
Magnetic nucleus 11

12. Research (II) :Molecular nanomagnets
SMM : finite number N of magnetic centers [e.g. Cr(III), Fe(III)], Single molecule behaviour (very weak intermolecular interaction), AF or F interaction  Non magnetic S=0 or low-spin, high-spin ground state
Crystal structure
p-NO2.C6F4CNSSN
Single molecule of Cr7Fe
SCM : magnetic chains with weak intrmolecular interactions and metallic/rare-earth ions alternating to radical groups  slow relaxation of M (NANOWIRES), frustration, peculiar magnetic phases
Slowly relaxing CoPhOMe
SIM : single rare-earth ions very diluted in the lattice. Good systems for study of quantum tunneling of magnetization (electro-nuclear coupled levels)

13. Effects of plasmons/magnetoplasmons
Role in the project
NMR and MUSR
Spectra
Relaxation rates
Electron spin
dynamics
Effects of plasmons/magnetoplasmons
MRI Contrast agents efficiency (r1, r2)
Correlation with magnetic and plasmonic properties

14. Possibly 150/200 mg or more of powders NMR-MRI
Requests for samples
NMR
Possibly 150/200 mg or more of powders
NMR-MRI
Solution with known magnetic ion concentration
Ideal concentration : mg/ml of magn. center
MUSR
500 mg or more of powders

15. Main collaborations and projects
Projects (others : C. Lenardi, F. Orsini)
FIRB “Investigation of protein structure and function by AFM and physiological studies” (ending)
EU FP7 - NANOTHER “Integration of novel NANOparticle based technology for THERapeutics and diagnosis of different types of cancer”
EU FP6 - MAGMANet “Molecular Approach to Nanomagnets and Multifunctional Materials ” (ending)
Fondazione Cariplo “Processi di funzionalizzazione di polimeri per la modifica della biocompatibilità e dell’adesione di proteine ” (ending)
Fondazione Cariplo “Progettazione di nuovi biosensori magnetici per l'applicazione in scienze della salute e ambientali ” (ending)
Currently active main collaborations (others : C. Lenardi, F. Orsini)
LOCAL, NATIONAL, INTERNATIONAL, INDUSTRIES
DISMAB, University of Milano (Italy), Prof. V.F. Sacchi, Dr. P. Perego, Dr. M. Castagna
Dept. Pharmacological Sciences, University of Milano, Prof. R. Paoletti, Prof. E.Tremoli, Dr.U.Guerrini, Dr.G.Sironi
Dept. Chem. And Electrochem., University of Milano, Dr. M. Scavini
S3 CNR-INFM, Modena (Italy) – AFFILIATION – Prof. M. Affronte
Dept. Physics “A. Volta” – University of Pavia (Italy) – F. Borsa, M. Corti, P. Carretta, A. Rigamonti, S. Sanna
Dept. Chemistry – University of Firenze (Italy) – D. Gatteschi, A. Caneschi, C. Sangregorio, R. Sessoli
Dept. Chemistry – University of Cagliari (Italy) – M.F. Casula
Dept. Physics, University of Parma (Italy), Dr. L. Romano’, Prof. G. Amoretti, Prof. P. Santini, Dr. S. Carretta
National Nanotechnology Laboratory, CNR-INFM, Lecce (Italy), Dr. T. Pellegrino
Dept. Physics – University of Firenze, Firenze (Italy) – Prof. A. Rettori
Dept. Physics, University of Milano Bicocca, Milano (Italy), gruppo prof. C. Riccardi
Dept. Chemistry, Manchester University (UK), prof. R. Winpenny
Dept. Physics, University of Zaragoza (Spain), Prof. F. Palacio e Dr. A. Millan
Dept. Chemistry, University of Valencia (Spain), prof. E. Coronado
Department of Physics, Boston College (USA), Prof. M. J. Graf
Inorganic Chemistry Department, University of Bucarest (Romania), prof. M. Andruh
CNRS and University of Montpellier (France), Dr. J. Larionova, Dr. Y. Guari
Centro Ricerche Colorobbia, Vinci (FI) (Italia), Dr. G. Baldi, Dr. D. Bonacchi

16. MAGNETISM: Scientific fields of interest
MAGMANet (Nanother)

17. Superparamagnetic MRI-contrast agents : a scheme
USPIO
SPIO
Small Particle of Iron Oxide
( >20 nm )
UltraSmall Particle of Iron Oxide
( < 15 nm )
Core (Fe3O4+Fe2O3)
Core (Fe3O4+Fe2O3)
Coating (dextran...)
Coating (dextran...)
Problems : low reproducibility, unknown mixing of ferrites, not-controlled microscopic chemico-physical
properties  no real control on the efficacy

18. Magnetic field biosensors
IgG
BSA
AbIgG-c-Biotin
Streptavidin
Fe2O3-c-PMA-c-Biotin
ppAA
They detect
protein-”anti”protein interaction
Plasma Deposited Poly Acrylic Acid (ppAA) [1]
Adsorption of human IgG
Blocking of the unreacted surface groups by BSA
Reaction with biotinated Ab-IgG molecules at different concentrations
Absorption of streptavidin
Absorption of biotinated modified γ-Fe2O3 superparamagnetic nanoparticles [2]
Technique of detection : SQUID. It uses the presence of magnetic nanoparticles
and the labelling of biosensors 18

19. Magnetic field biosensors
Agarose
Fe2O3-c-PMA-c-Biotin
They detect
protein-”anti”protein interaction
Hydrogel of Agarose (1%) directly prepared in the glass tube for NMR measurements
Diffusion of biotinated modified γ-Fe2O3 superparamagnetic nanoparticles with mild shaking
Diffusion of streptavidin <= WORK IN PROGRESS!!!
Technique of detection : NMR.
It uses the presence of magnetic nanoparticles and the labelling of biosensors 19

20. Very recent studies SMM :
Cr7Fe (NMR, ) – heterometallic ring ST=1/2  distribution of magnetic moment,
correlation function
Dimers (NMR, )  basic exchange interactions
Ni10 (NMR, )  resonant phonon trapping
FeCu6, CoCu6 (NMR, ) - ST=1/2 system  single molecule paramagnet
Fe4 (MUSR, ) - high-spin system ST=5  transitions to higher ST states
SCM :
Gd-based chains (NMR)  peculiar phase transitions
(Villain’s conjecture)
Co or Dy-based chains (NMR, MUSR) – slowly relaxing  two mechanisms of relaxation,
size effects
SIM : LiYF4:Ho (MUSR)  spin dynamics and tunneling

21. SUPERCONDUCTORS “Zero” resistivity below a critical T=Tc
Cables, wires
Magnets (MRI !!)
Magnetic Levitation train
“Zero” resistivity below a critical T=Tc
Repulsion of magnetic
field : Meissner effect
“High-Tc“ SC

22. Superconducting “fluctuations”
Susceptibility (already subtracted
of Pauli term)………..
…..and magnetization curves H
T >> TC
T  TC
Mdia
dia
H1/2 SF T
dia
Tc(H)
Tc(0)
[ Theory with exact solution : Prange (1970), but no field quenching of fluctuating pairs ]
First experiments ( Gollub, Tinkham et al.) in metals evidence the breakdown of a description which neglects the effect of the field in suppressing the fluctuating pairs, but only from M(T) curves.

23. SOME RECENT PUBLICATIONS
A.Figuerola, A. Fiore, R. Di Corato, A. Falqui, C. Giannini, E. Micotti,A.Lascialfari, M. Corti, R. Cingolani, T. Pellegrino,
P. D. Cozzoli and L.Manna. J. Am. Chem. Soc. 130, 1477 (2008)
M. Corti, A. Lascialfari, M. Marinone, A. Masotti, E. Micotti, F. Orsini, G. Ortaggi, G. Poletti, C. Innocenti,
C. Sangregorio, J. Magn. Magn. Mater. 320 , e316 (2008)
M. Corti, A. Lascialfari, E. Micotti, A. Castellano, M. Donativi, A. Quarta, P.D. Cozzoli, L. Manna, T. Pellegrino,
C. Sangregorio, J. Magn. Magn. Mater. 320, e320 (2008)
A Boni, M Marinone, C Innocenti, C Sangregorio, M Corti, A Lascialfari, M Mariani, F Orsini,
G Poletti and M F Casula, J. Phys. D: Appl. Phys. 41, (2008)
Y. Guari, J. Larionova, M. Corti, A. Lascialfari, M. Marinone, G. Poletti, K. Molvinger and C. Guérin,
Dalton Trans. 28, 3658 (2008), DOI: /b808221a 2)
A.Masotti, A. Pitta, G. Ortaggi, M. Corti, C. Innocenti, A. Lascialfari, M. Marinone, P. Marzola, A. Daducci, A. Sbarbati,
E. Micotti, F. Orsini, G. Poletti, C. Sangregorio, Magn Reson Mater Phy 22, 77 (2009)
P. Sánchez, E. Valero, N.Gálvez, J. M. Domínguez-Vera, M. Marinone, G. Poletti, M. Corti and
A. Lascialfari, Dalton Transactions, (2009)
L. Lartigue, K. Oumzil, Y. Guari, J. Larionova, C. GueLrin, J.-L. Montero, V. Barragan-Montero, C. Sangregorio, A. Caneschi,
C. Innocenti, T. Kalaivani, P. Arosio and A. Lascialfari, Organic Letters 11, 2992 (2009) SC
E. Bernardi, A. Lascialfari, A. Rigamonti, and L. Romanó, Phys. Rev. B 77, (2008)
S. Cagliero, A. Agostino, M. Mizanur Rahman Khan, M. Truccato, F. Orsini, M. Marinone,
G. Poletti, A. Lascialfari, Appl Phys A 95, 479 (2009)
A. Lascialfari, A. Rigamonti, E. Bernardi, M. Corti, A. Gauzzi, and J. C. Villegier, Phys. Rev. B 80, (2009)
MNM
F. Cinti, A. Rettori, M. G. Pini, M. Mariani, E. Micotti, A. Lascialfari, N. Papinutto, A. Amato, A. Caneschi,
D. Gatteschi, and M. Affronte, Phys. Rev. Lett. 100, (2008)
F. Borsa, Y. Furukawa, A. Lascialfari, Inorg. Chim. Acta 361, 3777 (2008)
M.Belesi, E.Micotti, M. Mariani, F.Borsa, A. Lascialfari, S. Carretta, P. Santini, G. Amoretti,
E. J. L. Mcinnes, I. S. Tidmarsh, J. Hawkett, Phys. Rev. Lett. 102,  (2009)

24. Some general considerations about Magnetism in bio-medicine
Techniques
Magnetic Resonance Imaging (clinic and research) and NMR
Magnetic nanoparticles joint to drug delivery/targeting, markers
Magnetic hyperthermia
Magnetic driven transport of particles
Magnetic biosensors’ detectors (NMR, SQUID,....)
Magnetoencelography or neuromagnetism (SQUID detection of cerebral activity), Magnetocardiography (by means of SQUIDs)
Materials (nanosized)
Paramagnetic systems
Ferrites (e.g. hard disk in computers)
Superparamagnetic systems
Molecular nano-particles

25. Aims of the work on MRI contrast agents
To obtain MRI contrast agents based on superparamagnetic cores coated in different ways
Samples with controllable size, shape, kind of magnetic ion of the core – narrow distributions of sizes – and coating/functionalization
MOTIVATION
a) reproducilibity of physical properties/performances ;
b) optimization of the chemico-physical characteristics ;
c) feedback to synthetic process to optimize the performances / study role of different parameters
d) Systematic study of nuclear relaxivity (the efficiency) as a function of the core dimensions, shape, coating, bulk anisotropy, kind of magnetic ion
e) Model for the dependence of the nuclear relaxivities of novel contrast agents on anisotropy and molecular reorientation
f) Multifunctionality (molecular targeting, hyperthermia, fluorescence....)
g) Low toxicity of Fe-oxides

26. Highly-sensitive magnetic field biosensors (MFB)
The scheme of MFB , molecular result of steps I + II :
I) biomolecular probe attached to a (nanostructured and functionalized) surface
II) complementary biomolecule labelled with a magnetic nanoparticle
Examples : antibody-antigene, protein-protein, DNA-DNA, enzyme reactions, etc.
APPLICATIONS
Labelling to “in vivo” molecular interactions (imaging)
Reagents in miniaturized microfluidic systems
Affinity ligands for rapid and high-throughput magnetic readouts of arrays
probes for Magnetic Force Microscopy
Scientific/technological related problems, to be optimized :
Nanostructuring (regular) of surface
Functionalization of surface
Magnetic labelling of biomolecule (choice of magnetic NP) (surface characteristics of NP allow covalent and stoichiomteris attachment of oligonucleotides, nucleic acids, small molecules, peptides, receptor ligands, proteins, antibodies,.....)
Choice of system of magnetic detection (Hall s., NMR/MRI, SQUID,....)
Development for applications