Nanotools for Materials Nanotools for Materials ScienceScience
Animations by Marc DuseillerAnimations by Marc Duseiller
Nicholas D. SpencerNicholas D. Spencer
Dept. Of Materials, ETH-ZürichDept. Of Materials, ETH-Zürich
•STM and AFM
•AFM as a chemical probe: Oxides
•AFM as a chemical probe: Polymers
•Designer molecules for Biosensors
Outline
•STM and AFM
•AFM as a chemical probe: Oxides
•AFM as a chemical probe: Polymers
•Designer molecules for Biosensors
Outline
s
y-piezo
z-piezo
x-piezo
tunnel
current
Tipz-displacement
(image)
sample
surface
Inhomogeneity
Scanning Tunneling Scanning Tunneling MicroscopyMicroscopy
€
j ∝Φ
s⋅V ⋅exp − k ⋅ Φ ⋅s( )
Binnig and Rohrer:
Nobel Prize for Physics, 1986
Evac1
Evac2
EF1
EF2
Φ1
Φ2
V
( )D E
( )D E
M1
M2
s
Scanning Tunneling Scanning Tunneling MicroscopyMicroscopy
STM: Constant-Height ModeSTM: Constant-Height Mode
STM: Constant-Current STM: Constant-Current ModeMode
A series of time-lapse STM topographic images at room temperatureshowing a 40nm x 40nm area of Au(111). The time per frame is 8 min,and each took about 5 min to scan. The steps shown are one atomicunit in height. The second frame shows craters left after tip-samplecontact, which are 2 and 3 atoms deep. During a 2h period the smallcraters have filled completely with diffusing atoms, while the largecraters continue to fill.R.C. Jaklevic and L. Elie Phys. Rev. Lett. 60 (1988) 120
STM Constant current 50nm x 50nm image of a Cu(111) surface held at4K. Three monatomic steps and numerous point defects are visible.Spatial oscillations (electronic standing waves) with a periodicity of~1.5nm are evident.M.F. Crommie, C.P. Lutz, and D.M. Eigler Nature 363 (1993) 524
Plasmid DNA (pUC18) on mica imaged by STM at high resolution. Theinset is a cutout of a zoomed-in image taken immediately after theoverview. R. Guckenberger, M. Heim, G. Cevc, H.F. Knapp, W.Wiegräbe, A. Hillebrand Science 266 (1994) 1538
Spatial image of the eigenstates of a quantum corral. 48-atom Fe ringconstructed on a Cu(111) surface. Average diameter of ring is 14.3nm. The ring encloses a defect-free region of the surface.M.F. Crommie, C.P. Lutz, and D.M. Eigler Science 262 (1993) 218
Atomic Force Atomic Force MicroscopyMicroscopy
AFM: Photodiode detectionAFM: Photodiode detection
AFM: Attractive and AFM: Attractive and Repulsive Force CurvesRepulsive Force Curves
AFM: TappingModeAFM: TappingModeTMTM
The compound eye of a housefly (Musca domestica), seen by TappingMode AFM. The detail image reveals channel-like
features on the surface. 60µm scan courtesy P. Gorostiza, I. Diez, F. Sanz, Universitat de Barcelona, Spain.
AFM: Phase-Contrast ModeAFM: Phase-Contrast Mode
TappingMode AFM Phase image of a PMMA-b-polybutylacrylate-b-PMMA symmetric triblock copolymer partly covering a mica substrate. The PMMA component forms cylindrial microdomains (located 40 nm apart) that
appear as cones on the phase image. 1.5µm scan courtesy P. LeClere and R. Lazzaroni, Universite de
Mons-Hainaut, Belgium.
AFM: Lateral Mode (LFM)AFM: Lateral Mode (LFM)
AFM as NanoindenterAFM as Nanoindenter
"The world's smallest turbine." Densely packed assembly of proton driven rotors of the chloroplast ATP synthase imaged in
buffer solution. Rotors are incorporated in both orientations with respect to the membrane plane. At a lateral resolution
better than 1nm, the fourteen subunits of the wide connector end (diameter ~7.6nm) can be seen. 70nm scan courtesy of H.
Seelert, A. Poetsch, N. Dencher, A. Engel, H. Stahlbert and D.J. Müller, Max-Planck-Institute of Molecular Cell Biology and
Genetics, Dresden, Germany.
TappingModeTM AFM image of CD surface
Contact mode AFM of cholera toxin oligomers bound to lipid bilayer under buffer. Note that a clear pentameric
structure is resolved for many of the cholera toxin oligomers, while others appear hexameric or
unstructured. 80 nm scan size. Courtesy of Shao lab, University of Virginia.
Normal and sickled human red blood cells. Sample preparation consisted of a standard smear on a glass slide. The rigid contour of the sickled cell
(center) contrasts with the normal red blood cells; note the sickled cell has indented the softer red blood cell. Three spicules (top left of cell),
approximately 0.5 to 1.0µm long, project out from the sickled cell, denoting rearrangement of intracellular hemoglobin molecules. Sample
courtesy of Sansum Medical Clinic, Santa Barbara, CA.
•STM and AFM
•AFM as a chemical probe: Oxides
•AFM as a chemical probe: Polymers
•Designer molecules for Biosensors
Outline
Spatial Resolution Range of Imaging Surface Methods Spatial Resolution Range of Imaging Surface Methods
for Insulating Samplesfor Insulating Samples
1Å1Å 1nm1nm 10nm10nm 100nm100nm 1µm1µm 10µm10µm 100µm100µm
SpatialSpatial ResolutionResolution
Chemical
Morphological
iXPSiXPS
SAMSAM
ToF-SIMSToF-SIMS
SEMSEM
STMSTM
AFMAFM
InformationGap
Experimental SetupExperimental Setup
Si3N4 tip
+++++++++++1 mM NaCl solutionSampleliquid cellFnFlatFlat
Effect of pH on Force CurvesEffect of pH on Force Curves
SiSi33NN44 tip, Si/SiO tip, Si/SiO22 Sample Sample
pH 4 pH 8.5
Tip-Sample Separation [nm]
a)
0 20 40 0 20 40-5
0
5
10N
orm
al Fo
rce [
nN
]
A. Marti, G. Hähner, and N.D. Spencer, A. Marti, G. Hähner, and N.D. Spencer, LangmuirLangmuir 1111 (1995) 4632-5 (1995) 4632-5
Lateral Force for SiLateral Force for Si33NN44/SiO/SiO22 and Si and Si33NN44/Al/Al22OO33
3 4 5 6 7 8 9 10 11pH
Late
ral Fo
rce [
arb
. un
its]
tip Si3N4Al 2O3
SiO2
G. Hähner, A. Marti, and N.D. Spencer, G. Hähner, A. Marti, and N.D. Spencer, Tribology Letters Tribology Letters 33 (1997) 359-65 (1997) 359-65
Chemical Imaging with pH-Dependent AFM/LFM—1Chemical Imaging with pH-Dependent AFM/LFM—1
G. Hähner, A. Marti, and N.D. Spencer, G. Hähner, A. Marti, and N.D. Spencer, Tribology Letters Tribology Letters 33 (1997) 359-65 (1997) 359-65
Friction pH 8.2 5 μm Friction 3.8pH 5 μm > pH pKs < pH pKs S COOH S CH3 -Si wafer Au - 3CH terminated -COOH terminated Height 5 μm 0 30 nm 0 5 nm
Chemical Imaging with pH-Dependent AFM/LFMChemical Imaging with pH-Dependent AFM/LFMSelf-assembled MonolayersSelf-assembled Monolayers
No height difference observableNo height difference observableµCP-generatedµCP-generatedthiol patternthiol pattern
Chemical (frictional) contrast in the lateral force image can be changed Chemical (frictional) contrast in the lateral force image can be changed by modifying pH, due to a pKa value of 4-5 of the -COOH groups.by modifying pH, due to a pKa value of 4-5 of the -COOH groups.
A. MartiA. MartiG.HähnerG.HähnerETH-LSSTETH-LSST
•STM and AFM
•AFM as a chemical probe: Oxides
•AFM as a chemical probe: Polymers
•Designer molecules for Biosensors
Outline
AFM of Polyesterurethane Block CopolymerAFM of Polyesterurethane Block CopolymerSpin-Coated from a 1% NMP Soln. onto a Si-WaferSpin-Coated from a 1% NMP Soln. onto a Si-Wafer
K. Feldman - ETH-LSSTK. Feldman - ETH-LSST
Polymers used in this StudyPolymers used in this StudyPSPS
PolystyrenePolystyrene
Avg. MW=250,000Avg. MW=250,000
TTgg = 100°C = 100°CPolyacrylonitrilePolyacrylonitrile
Avg. MW=200,000Avg. MW=200,000
PANPAN
TTgg = 87°C = 87°C
Poly(acrylic acid)Poly(acrylic acid)
Avg. MW=150,000Avg. MW=150,000
PAAPAA
TTgg = 102°C = 102°C
Poly(methyl methacrylate)Poly(methyl methacrylate)
Avg. MW=150,000Avg. MW=150,000
PMMAPMMA
TTgg = 102°C = 102°C
Poly(vinylidene fluoride)Poly(vinylidene fluoride)
Avg. MW=534,000Avg. MW=534,000
PVDFPVDF
TTgg = 38°C = 38°C
FEPFEP
Avg. MW=50,000Avg. MW=50,000
FEPFEP
Isotactic PolypropyleneIsotactic Polypropylene
TTgg = 22°C = 22°C
Avg. MW=250,000Avg. MW=250,000
iPPiPP
Increasing Increasing H-bonding H-bonding InteractionInteraction
Non-PolarNon-Polar PolarPolar
Comparison of Force Comparison of Force Curves, Pull-On, and Pull-Curves, Pull-On, and Pull-
Off Forces Off Forces Between a SiOx Probe Between a SiOx Probe and a PMMA Surface in and a PMMA Surface in
Different MediaDifferent Media
Israelachvili’s Approximation to Lifshitz’ Theory Israelachvili’s Approximation to Lifshitz’ Theory for Calculation of the Non-Retarded Hamaker Constantfor Calculation of the Non-Retarded Hamaker Constant
ATotal = Aν=0 + Aν> 0 ≈3
4kT
ε 1 − ε3( )ε 1 + ε3( )
ε 2 −ε 3( )ε 2 + ε3( )
+3 hνe
8 2
n12
− n32( ) n2
2− n3
2( )
n12
+ n 32( )
1 / 2n22
+ n32( )
1 / 2n12
+ n32( )
1 / 2+ n 2
2+ n3
2( )1/ 2
[ ]
HamakerHamakerConstantConstant
dipole-dipole anddipole-dipole anddipole-induced-dipole dipole-induced-dipole
componentscomponents(from dielectric constants)(from dielectric constants)
dispersion (London) componentdispersion (London) component(from refractive indices)(from refractive indices)
Work of Adhesion, W, calculated fromWork of Adhesion, W, calculated fromW≈AW≈ATotalTotal / (12D / (12Doo
22), ), where Dwhere Doo=0.165nm=0.165nm
is the commonly used value for the cut-off separationis the commonly used value for the cut-off separation
== ++
2.98±0.16 nN
2.07±0.15 nN
0.62±0.20 nN
0.18±0.08nN
PS
iPP
PVDF
FEP
Non-Polar Polymer Adhesion is Due to London Interaction OnlyNon-Polar Polymer Adhesion is Due to London Interaction Only(all measurements under perfluorodecalin)(all measurements under perfluorodecalin)
(Lifshitz Theory)(Lifshitz Theory)
Yields tip radius of 50 nm,assuming JKR Theory holds
(confirmed by FESEM)
K. Feldman, T. Tervoort, P. Smith, N.D. Spencer, K. Feldman, T. Tervoort, P. Smith, N.D. Spencer, LangmuirLangmuir 1414(1998)372(1998)372
Towards a Force Spectroscopy of PolymersTowards a Force Spectroscopy of Polymers
K. Feldman, T. Tervoort, P. Smith, N.D. Spencer, K. Feldman, T. Tervoort, P. Smith, N.D. Spencer, LangmuirLangmuir 1414(1998)372(1998)372
Silica TipSilica Tip Gold TipGold Tip
HEIGHTFRICTIONHEIGHTFRICTION
Chemical Imaging of PS:PMMA Blend (1:10)Chemical Imaging of PS:PMMA Blend (1:10)(Spin-coated from toluene, 2 wt.% total) (Spin-coated from toluene, 2 wt.% total)
Plasma-CleanedPlasma-CleanedSiSi33NN44 Tip Tip
Plasma-CleanedPlasma-CleanedAu TipAu Tip
K. Feldman, T. Tervoort, P. Smith, N.D. Spencer, K. Feldman, T. Tervoort, P. Smith, N.D. Spencer, LangmuirLangmuir 1414(1998)372(1998)372
•STM and AFM
•AFM as a chemical probe: Oxides
•AFM as a chemical probe: Polymers
•Designer molecules for Biosensors
Outline
Biosensors, ProteomicsBiosensors, Proteomics
Challenge: Challenge:
How to immobilize proteins How to immobilize proteins in an active, well-defined state?in an active, well-defined state?
Protein
Analyte/Antigen
Linker species
Active site
Denaturing Interactions
Poly-l-lysine (PLL)-g-polyethylene glycol (PEG)
PLL backbonePLL backbone• MW: 20,000 to 350,000MW: 20,000 to 350,000• Positively charged at pH<10 Positively charged at pH<10
(R= –NH (R= –NH33++))
• Approximate length of backbone: Approximate length of backbone: 90 to 1000 nm90 to 1000 nm
PEG side chainPEG side chain• MW: 2000 to 5000MW: 2000 to 5000• Adsorbs water and has properties Adsorbs water and has properties
similar to watersimilar to water• Protein resistantProtein resistant• Approximate length of side Approximate length of side
chain:20chain:20 nm nm
J. Hubbell, D. Elbert, Chem Biol 5: (3) 177-183 (1998)
j
CH
CH2
CH2
CH2
CH2
C NH
O
CH C NH
O
CH2
CH2
CH2
CH2
NH2
CH C NH
O
CH2
CH2
CH2
CH2
CH C OH
O
CH2
CH2
CH2
CH2
H2
N
NH2
CH3
O
CH2
CH2
O
CH2
CH2
CO
NH
m≈100
NH2
k
PLL PLL backbonebackbone
PEG side PEG side chainchain
PEG PEG side side chainchainss
PLL PLL back-back-bonebone
Oxide Oxide surfacesurface
Attachment of the Comb-like Co-Polymer Attachment of the Comb-like Co-Polymer to a Negatively Charged Surfaceto a Negatively Charged Surface
HydrophilicHydrophilic
UnchargedUncharged
Flexible chainsFlexible chains
High water contentHigh water content
Steric repulsionSteric repulsion
BiocompatibleBiocompatible
Positive chargePositive charge
High coverageHigh coverage
Kinetic inertnessKinetic inertness
pH dependencepH dependence
MMONITORING ONITORING BBIOMOLECULE IOMOLECULE AADSORPTIONDSORPTIONMMONITORING ONITORING BBIOMOLECULE IOMOLECULE AADSORPTIONDSORPTION
Waveguide
Bulk
Protein layer
• refractive index and thickness of adlayer can be calculated refractive index and thickness of adlayer can be calculated
• highly sensitive (~1 ng/cmhighly sensitive (~1 ng/cm22))
• 3-second time resolution3-second time resolution
• refractive index and thickness of adlayer can be calculated refractive index and thickness of adlayer can be calculated
• highly sensitive (~1 ng/cmhighly sensitive (~1 ng/cm22))
• 3-second time resolution3-second time resolution
00 100100 200200
Time [min]Time [min]
1.60761.6076
1.57221.5722
1.57261.5726
1.57301.5730
1.57341.5734
1.57381.5738
1.57421.5742
1.60721.6072
1.60681.6068
1.60641.6064
Eff
ec
tive
re
fra
cti
ve i
nd
ex T
EE
ffe
cti
ve r
efr
ac
tive
in
dex
TE
Effe
ctive
refra
ctive
ind
ex T
ME
ffec
tive re
frac
tive in
dex
TM
Antigen
Antibody
Buffer
Buffer
Optical grating couplerOptical grating coupler
SiOSiO22/TiO/TiO22 waveguide waveguide
Full human serum Full human serum
HEPES bufferHEPES buffer
Without (bare oxide)Without (bare oxide) and with adlayer of and with adlayer of PLL-PEG PLL-PEG
Surface Modification with PLL-PEG – Effect on Serum AdsorptionSurface Modification with PLL-PEG – Effect on Serum Adsorption
G.L.Kenausis,J.Vörös,D.L.Elbert,N.Huang,R.Hofer,L.Ruiz,M.Textor J.A.Hubbell, and N.D.Spencer, J.Phys.Chem.B 104 (2000)3298-3309
Osteoblast attachment to Osteoblast attachment to untreated SiOuntreated SiO22/TiO/TiO22 surface surface
Osteoblast attachment to SiOOsteoblast attachment to SiO22/TiO/TiO22
surface treated with PLL-surface treated with PLL-gg-PEG-PEG
Cell Attachment on PLL-PEG-coated Optical WaveguidesCell Attachment on PLL-PEG-coated Optical Waveguides
J. Vörös, G. Kenausis - ETH-LSSTJ. Vörös, G. Kenausis - ETH-LSST
OOURUR A APPROACHPPROACH TOTO B BIOAFFINITYIOAFFINITY S SENSINGENSINGOOURUR A APPROACHPPROACH TOTO B BIOAFFINITYIOAFFINITY S SENSINGENSING
Controlled Controlled orientation and orientation and concentrationconcentration
Controlled Controlled orientation and orientation and concentrationconcentration
RECEPTORRECEPTORIMMOBILIZATIONIMMOBILIZATION
RECEPTORRECEPTORIMMOBILIZATIONIMMOBILIZATION
Biosensor surfaceBiosensor surface
Optimum Optimum sensitivity,sensitivity,
low non-specific low non-specific adsorptionadsorption
Optimum Optimum sensitivity,sensitivity,
low non-specific low non-specific adsorptionadsorption
ANALYTE ANALYTE DETECTIONDETECTIONANALYTE ANALYTE
DETECTIONDETECTION
Biosensor surfaceBiosensor surface
INITIAL SENSOR INITIAL SENSOR SURFACESURFACE
INITIAL SENSOR INITIAL SENSOR SURFACESURFACE
Passive:Passive:resistant to non-resistant to non-specific bindingspecific binding
Passive:Passive:resistant to non-resistant to non-specific bindingspecific binding
Biosensor surfaceBiosensor surface
SSTARTING TARTING SSENSOR ENSOR SSURFACEURFACESSTARTING TARTING SSENSOR ENSOR SSURFACEURFACE
Biosensor surface
TODAYTODAYTODAYTODAY
Bioactive:Bioactive:nonspecific nonspecific bindingbinding
Bioactive:Bioactive:nonspecific nonspecific bindingbinding
Biosensor surface
OUR OUR APPROACHAPPROACH
OUR OUR APPROACHAPPROACH
Passive:Passive:resistant to resistant to nonspecific nonspecific bindingbinding
Passive:Passive:resistant to resistant to nonspecific nonspecific bindingbinding
IIMMOBILIZATIONMMOBILIZATION OF OF RRECEPTOR ECEPTOR MMOLECULESOLECULESIIMMOBILIZATIONMMOBILIZATION OF OF RRECEPTOR ECEPTOR MMOLECULESOLECULES
Controlled Controlled orientation and orientation and concentrationconcentration
Controlled Controlled orientation and orientation and concentrationconcentration
Biosensor surface
The “Glue”: BIOTIN-STREPTAVIDINThe “Glue”: BIOTIN-STREPTAVIDIN
Control of Non-specific Adsorption and Specific InteractionsControl of Non-specific Adsorption and Specific Interactions
PL
L-P
EG
oxideoxide
= Specific binding functionality
PLL(20)-g[3.5]-PEG(2)/PEGbiotin(3.4)
NH
HN
NH
HN
NH
HN
NH
HN
NH
OHO
O
O
O
O
O
O
O
O
O
NH
NH2
NH2
NH2
NH
NH2
NH2
NH2
NH
O
OCH3
OO
O
O
O
O
OCH3
O
14
nm
NH2
H2N
n
HN
O
S
NHHN
O
MODEL SYSTEM: BIOTIN - STREPTAVIDINMODEL SYSTEM: BIOTIN - STREPTAVIDIN
Specific binding of Specific binding of streptavidin to the streptavidin to the
biotinylated PLL-biotinylated PLL-gg-PEG-PEG
Specific binding of Specific binding of streptavidin to the streptavidin to the
biotinylated PLL-biotinylated PLL-gg-PEG-PEG
A protein-resistant surface A protein-resistant surface with streptavidin binding with streptavidin binding
sitessites
A protein-resistant surface A protein-resistant surface with streptavidin binding with streptavidin binding
sitessites
biotibiotinnbiotibiotinnPLL-PLL-PEGPEGPLL-PLL-PEGPEG
streptavidistreptavidinnstreptavidistreptavidinn
Biosensor surfaceBiosensor surface Biosensor surfaceBiosensor surface
Specific binding of streptavidin on a Specific binding of streptavidin on a PLL-g-PEG-biotin surfacePLL-g-PEG-biotin surface
Specific binding of streptavidin on a Specific binding of streptavidin on a PLL-g-PEG-biotin surfacePLL-g-PEG-biotin surface
Mass
of
Pro
tein
s [n
g/c
mM
ass
of
Pro
tein
s [n
g/c
m22]]
Mass
of
Pro
tein
s [n
g/c
mM
ass
of
Pro
tein
s [n
g/c
m22]]
BUFFERBUFFERBUFFERBUFFERSERUMSERUMSERUMSERUM
BUFFERBUFFERBUFFERBUFFER
BUFFERBUFFERBUFFERBUFFER
STREPTAVIDINSTREPTAVIDINSTREPTAVIDINSTREPTAVIDIN
PLL-PLL-gg-PEG-PEG50% biotin50% biotinPLL-PLL-gg-PEG-PEG50% biotin50% biotin
400400400400
300300300300
200200200200
100100100100
0000
Time [min]Time [min]Time [min]Time [min]
100100100100 1801801801800000 120120120120 140140140140 16016016016080808080606060604040404020202020
J. Vörös - ETH-LSSTJ. Vörös - ETH-LSST
AAPPROACHINGPPROACHING AA R REALEAL D DETECTIONETECTION S SYSTEMYSTEMAAPPROACHINGPPROACHING AA R REALEAL D DETECTIONETECTION S SYSTEMYSTEM
11stst step step11stst step step
Specific binding of Specific binding of streptavidin to biotinylated streptavidin to biotinylated
PLL-PLL-gg-PEG-PEG
Specific binding of Specific binding of streptavidin to biotinylated streptavidin to biotinylated
PLL-PLL-gg-PEG-PEG
Biosensor surfaceBiosensor surface
22ndnd step step22ndnd step step
Using streptavidin to Using streptavidin to immobilize biotinylated immobilize biotinylated
receptor moleculesreceptor molecules
Using streptavidin to Using streptavidin to immobilize biotinylated immobilize biotinylated
receptor moleculesreceptor molecules
Biosensor surfaceBiosensor surface
Ad
sorb
ed
Mass [
ng
/cm
Ad
sorb
ed
Mass [
ng
/cm
22]]
Ad
sorb
ed
Mass [
ng
/cm
Ad
sorb
ed
Mass [
ng
/cm
22]]
500500500500
400400400400
300300300300
200200200200
100100100100
0000100100100100 200200200200 300300300300 4004004004000000
Time [min]Time [min]Time [min]Time [min]
BUFFERBUFFERBUFFERBUFFER
PLL-PLL-gg-PEG-biotin-PEG-biotinPLL-PLL-gg-PEG-biotin-PEG-biotin
StreptavidinStreptavidinStreptavidinStreptavidin
biotin-aIgGbiotin-aIgGbiotin-aIgGbiotin-aIgG
IgGIgGIgGIgG
TTYPICALYPICAL E EXPERIMENTXPERIMENT FORFOR B BIOAFFINITYIOAFFINITY A ASSAYSSAYTTYPICALYPICAL E EXPERIMENTXPERIMENT FORFOR B BIOAFFINITYIOAFFINITY A ASSAYSSAY
J. Vörös, N. Huang - ETH-LSSTJ. Vörös, N. Huang - ETH-LSST
Bioaffinity Assay ApproachBioaffinity Assay Approach
Biosensors
STM/AFM
AFM and Oxides
HEIGHTFRICTIONHEIGHTFRICTION
AFM and Polymers
Oberstdorf, September, 2001
