Atomic Force Microscopy

@

LGM/CIMaINa - UniMI

Alessandro Podestà

Centro Interdisciplinare MAteriali e Interfacce NAnostrutturati

& Dipartimento di Fisica,

Molecular Beam and Nanocrystalline Materials Laboratory

Università degli Studi di Milano

COST Action TD1002 AFM4NanoMed&Bio, Working Groups Meeting, Lamorlaye, France 10-13 May 2011

Alessandro Podestà

Born in 1973, Italy

http://www.researcherid.com/rid/E-6568-2010

Present position

Assistant professor (since 2006), Università degli Studi di Milano, Italy.

Research interests

AP is the scientific coordinator of the Atomic Force Microscopy group of the Molecular Beam

and Nanocrystalline Materials Laboratory (LGM) and of the Interdisciplinary Centre for

Nanostructured Materials and Interfaces (CIMAINA) of the University of Milano.

Research topics:

-Development and implementation of atomic force microscopy techniques and methods;

-Characterization of nanoscale physical and chemical properties of interfaces and systems

by atomic force microscopy techniques.

AFM @ LGM/CIMaINa - UniMIMolecular Beam and Nanocrystalline Materials Laboratory (LGM)

Interdisciplinary Centre for Nanostructured Materials and Interfaces (CIMAINA)

AFM since 2001

-2 AFMs (Multimode N4 & Bioscope Catalyst N5, Bruker)

- Multimode AFM is strongly interfaced for adv. charact.

-Sample preparation facilities

- Sputter-coater & evaporator

- Chemistry lab.

-Other complementary techniques

- SEM, XPS, UV-VIS & FTIR spectroscopy

- Stilus profilometer

-Nanostructured (biocompatile) materials synthesis

Synthesis of nanostructured materials by cluster-assembling

Supersonic Cluster Beam Deposition

Bottom-up

Nanostructured material

Clusters

• Nanometer-sized building blocks

• Assembled materials show

interesting features from the very

nanoscale

• Building block properties determine

film properties

Supersonic Cluster Beam Deposition

(Ulta-)high vacuum

Pulsed Microplasma Cluster Source

J. Phys. D 32, L105 (1999)

Surface morphology of nanostructured TiOx deposited by SCBD

• Carrier gas controls grain size and

deposition rate (i.e. thickness)

• Thickness controls morphological

parameters

Carrier gas: He

Carrier gas: Ar

smaller clusters

larger clusters

AFM@LGM/CIMaINa: Research topics

- Development and implementation of atomic force microscopy techniques and

methods

- Statistical analysis of surface morphology of thin films and nanostructure;- Metrology of nanometer-sized objects (proteins, nanoparticles);- Nanotribological characterization;- Nanomechanical testing of thin films;- Scanning electrical impedance microscopy;- Calibration of atomic force microscopy probes.

- Characterization of physical and chemical properties of interfaces and systems at the nanometre scale using atomic force microscopy techniques.- Structural/configurational properties of biomolecules and their interaction with

nanostructured surfaces- Configurational and elastic properties of DNA;- Aggregation of proteins and enzymes;- Force spectroscopy of single-molecule interactions with biocompatible

surfaces.– Study of nanostructured interfaces and inorganic systems

– Evolution of surface morphology, wettability, electrical properties of thin nanostructured oxide films;

– Interfacial properties of supported thin films of ionic liquids.

Bio-related activities

and tentative WGs attribution

• Study of the interaction of biological entities (DNA, DNA-protein complexes, and cells) with biocompatible (nanostructured) surfaces– High-resolution imaging for the characterization of configurational

and structural properties of biomolecules, biocomplexes, biofilms and nanoparticles (WG1,WG4?);

– Force-spectroscopy and force-imaging (force-distance and Force-Volume analyses) for the direct characterization of protein-surface interactions and of relevant surface properties (elasticity, adhesion, charge density, PZC…) (WG2 ?, WG3?).

• Development of AFM-based techniques– Nanoscale electrical characterization of nanostructured interfaces

(WG3 ? See Gomila’s note);

– Nanomechanical characterization of thin films and nano- micro-systems (WG3).

DNA elasticity investigated

at the single-molecule scale

• Charge separation << Bjerrum length β• Strong electrostatic repulsion � chain rigidity

• Electrostatics contribution to DNA elasticity

ΘΘ Θ Θ Θ

Θ

Θ

ΘΘ Θ Θ

Θ0.17 nm

E(β)=kBT

βH20 = 0.71 nm

- +

DNA in H2O @ 300K

In collaboration with D.Dunlap, L.Finzi, G.Manning

BJ 89(4), 2558 (2005)

Elasticity of polymers in 2D

Contour length L

⟨cosθ(∆l)⟩ ~ exp(-∆l/2P)P is the Persistent Length

−−=

−

−P

L

WLCDe

L

PPLR 2

2

2 12

14...)(2

2 +∆=∆P

ll

Dθ

Sample preparation

Positively-charged mica DNA Sample

dried dried

Polyamine coating

+ + ++

Spotting DNA

Quantitative AFM study of DNA conformational properties

• High-resolution imaging in dry N2 environment

• Track molecules and obtain DNA backbone

• Calculate average contour length, angles, end-to-end distances

DNA on poly-L-ornithine -coated Mica

DNA retain the physiological B-form on poly-L-ornithine-coated mica, even when dried for AFM imaging.

Hydrated B-DNA

[PO] = 100 µg/ml

[PO] = 0.018 µg/ml

Positive charge softens DNA

P = 11 nm

P = 32 nm

Characterization of DNA rigidity

−−=

−

−P

L

WLCDe

L

PPLR 2

2

2 12

14

P=56 nm

P=42 nm

P=37 nm

P=32 nm

P=27 nm

P=11 nm

Poly-L-ornithine 49 kDa

5-fold reduction of P by increasing [PO]

Buckled DNA – counterion correlation(GS Manning, J. Biomol. Struct. Dyn. 7:41–61, 1989)

β Bjerrum lengthb separation between phosphatesξ=β/bk-1 Debye-Huckel screening length

x – fraction of neutralized phosphate charges on DNA backbone

null

DNA elasticity - Conclusions

• Direct phosphate charge neutralization modulates DNA rigidity on

positively charged surfaces

• Polyamine-coated surfaces keep DNA hydrated even in dry

environment, while reducing its persistent length up to a factor of 5

• Fast and reliable protocol for controlling DNA rigidity on surfaces

� Application in biomedical devices

ADHESIVE-FREE COLLOIDAL PROBES FOR NANOSCALE FORCE

MEASUREMENTS: PRODUCTION AND CHARACTERIZATIONM. Indrieri et al., Rev. Sci. Instrum. 82, 023708-10 (2011)

Production of Colloidal Probes: the recipe

Strong adhesion

Weak adhesion

Characterization of colloidal probes

Colloidal Probes - Conclusions

- Quick and simple production of epoxy-free colloidal probes for force spectroscopy and nanomechanical measurements based on capillary adhesion and sintering;

- Quantitative statistical characterization of the probe radius and geometry based on inverse imaging of the probe;

- AFM is the only instrument used for both production and characterization of the colloidal probes.

Related publications

POSITIVELY CHARGED SURFACES INCREASE THE FLEXIBILITY OF DNA, A. Podestà et al., Biophysical Journal

89(4), 2558 (2005).

EARLY EVENTS IN INSULIN FIBRILLIZATION STUDIED BY TIME-LAPSE ATOMIC FORCE MICROSCOPY, A.

Podestà et al., Biophysical Journal 90, 589 (2006).

ATP-DEPENDENT LOOPING OF DNA BY ISWI, G. Lia et al., Journal of Biophotonics 4, 280 (2008).

ADSORPTION AND STABILITY OF STREPTAVIDIN ON CLUSTER-ASSEMBLED NANOSTRUCTURED TIOX

FILMS, L. Giorgetti et al., Langmuir, 24, 11637 (2008).

ADHESIVE-FREE COLLOIDAL PROBES FOR NANOSCALE FORCE MEASUREMENTS: PRODUCTION AND

CHARACTERIZATION, M. Indrieri et al., Rev. Sci. Instrum. 82, 023708-10 (2011), doi:10.1063/1.3553499.

PROBING NANOSCALE INTERACTIONS ON BIOCOMPATIBLE CLUSTER-ASSEMBLED TITANIUM OXIDE

SURFACES BY AFM, V. Vyas et al., J. Nanosci. Nanotechol., in press.

THE EFFECT OF SURFACE NANOMETRE-SCALE MORPHOLOGY ON PROTEIN ADSORPTION, P.E. Scopelliti et

al., PlosOne 5, e11862 (2010).

NANOSCALE ELECTRICAL PROPERTIES OF CLUSTER-ASSEMBLED PALLADIUM OXIDE THIN FILMS, V. Cassina

et al., Phys. Rev. B 79, 115422 (2009).