Atomic Force Microscopy – More Than Microscopy

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报告题目： Atomic force microscopy more than microscopy

( 原子力显微镜 – 不仅仅是显微镜 )

报告人：陈新镛教授 University of Nottingham

School of Pharmacy

Atomic Force Microscopy– More Than Microscopy

Xinyong ChenLaboratory of Biophysics and Surface Analysis

School of PharmacyThe University of Nottingham

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ConstantForce.swf TappingMode.swf

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Outline

• Force measurement– Force/distance (f/d)– Two-dimensional Force measurement

• AC mode “force” measurement– Amplitude/distance (a/d)– Phase/distance (p/d)– Tapping mode imaging and a-p/d

• Other force-related afm techniques– Force-modulation imaging– Lateral-force (friction) imaging– Lateral resonance (AC mode lateral force)

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Force measurement

ForceCurves.swf

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Force/distance curves

Maximum adhesion

force

Contact region

ab

c

d

Scanner Displacement (nm)

Point of maximum

load

Deflect

ion/F

orc

e

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Experimental f/d curves

Approach Retract

School of PharmacySingle particle interaction between salbutamol, lactose and PTFE

Increase in

strength of

interaction with

salbutamol in air

0

10000

20000

30000

40000

50000

60000

Tip 1 Tip 2 Tip 3

Avera

ge A

dhesi

on F

orc

e/n

N

Lactose

Salbutamol

PTFE

International Journal of Pharmaceutics 238 (2002) 17-27

PTFE

Salbutamol

250 5000Distance (nm)

Forc

e

(nN

) LactoseAFM probe

Salbutamol

Measurement of

particle-particle

interaction

Lactose1µm

School of PharmacyAdhesion between a solid sphere and a solid plane (Hertz Model)

• JKR (Johnson, Kendall, Roberts – 1964-1971) model:

• DMI (Derjagin, Muller, Toropov – 1975) model:

• Maugis (1992):

Radhesion ofwork :Δγ ,

2

3RF

RF 2

F3

1

2

2

0

06.2

K

R

Stiff materials (DMT)

Compliant materials (JKR)

With complex and implicit parameter equations

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Specific interaction

Anti - Anti - Ferritin Ferritin AntibodyAntibody

FerritinFerritin

AFM Cantilever and ProbeAFM Cantilever and Probe

SubstrateSubstrate

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Antigen-antibody interaction

Biochemistry, 36, 7457 – 7463 (1997)

-0.6

-0.2

0.2

0.6

1

1.4

-25 25 75 125 175

Distance moved by the Cantilever (nm)

For

ce (

nN)

ApproachRetract

-0.4

0

0.4

0.8

1.2

1.6

-25 25 75 125 175


For

ce (

nN)

ApproachRetract

School of PharmacyMultiple link in tip/surface modification

Force versus probe-sample separation curves for a BBSA coated probe and (a) a streptavidin coated silicon surface, (b) a streptavidin functionalised surface after incubation in a solution of the bis-biotinylated peptide, and (c) the streptavidin/bis-biotinylated peptide/streptavidin multilayer.

The Analyst, 2000, 125(2), 245 - 250

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Force curve of chain-like molecules

School of PharmacyForce measurement of RNA molecules

Examples of the types of force curves observed between RNA functionalized AFM surfaces and tips; each curve has been corrected to show force against tip-sample displacement. In each example the approach trace is shown in gray and the retract trace in black. (A) No interaction has occurred between the tip and substrate. (B) A nonspecific interaction between the two surfaces, with no observable change in the gradient of the retraction slope. (C) A specific interaction between the two RNA functionalized surfaces, showing a stretch of ~10 nm before the rupture event.

Biophysical Journal 86:3811-3821 (2004)

School of PharmacyRepresentative force-extension traces for DNA molecules of three different lengths

Biochem. Soc. Trans. (2003) 31, (1052–1057)

School of PharmacyAntigen-antibody interaction

Anti - Anti - Ferritin Ferritin AntibodyAntibody

FerritinFerritin

AFM Cantilever and ProbeAFM Cantilever and Probe

SubstrateSubstrate

- Single antigen-antibody bond?

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0

50

100

150

200

250

0 0.25 0.5 0.75

Force (nN)

Antigen-antibody interaction- Single antigen-antibody bond?

Analysis of the distribution of adhesive forces (n = 140) obtained with three functionalized AFM probes. Each data point represents an individual force measurement performed at any position on the sample surface. The force distribution data is plotted, using data bin sizes from 15 to 35 pN, as a stacked area graph (smallest bin size plotted lowermost). In this way, common features of the distributions are reinforced by the stacking process. A quantization in the forces, with a period of 49 ± 10 pN, is suggested by this analysis.

Biochemistry, 36, 7457 – 7463 (1997)

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“Spacers” – a way to measure single molecular interaction

PEG attached Antibody

Mono-functional Spacer PEG molecules

Silicon Substrate

Covalently attached hCG

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Force measurements between PEG-antibody coated probes and hCG coated surfaces

-2

0

2

4

6

8

0 100 200 300

Distance Moved by the Cantilever (nm)

ApproachRetractF

orce

(n

N)

-1

-0.5

0

0.5

1

1.5

2

2.5

0 50 100 150

For

ce (

nN)

ApproachRetract


ApproachRetract

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 50 100 150 200


For

ce (

nN)

-1

-0.5

0

0.5

1

1.5

2

2.5

0 50 100 150


ApproachRetract

For

ce (

nN)

(a) (b)

(c) (d)

Low probability in detectingsingle molecular pair interaction!

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Antibody-antigen interaction using polymeric linkers

Polymeric linker e.g. agarose, PEG

Antigen

Biotinylatedantibody

Streptavidin

PLA-PEG-Biotin

AFM probe

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Exploring surface hardness

Z

F

Z: Indentation on soft surfaces

Deflection onhard surfaces(no indentation)

0

5

10

15

20

25

0.30 1.06 1.82 2.58 3.34 4.09 4.85 5.61 6.37 M ore

You ng 's M o dul u s (G pa )

Fre

qu

en

cy

0

2

4

6

8

1 0

1 2

1 4

0.32 0.56 0.80 1.04 1.2 8 1.5 2 1.7 6 2.0 0 2.24 M or e

You n g's Mo du lus (Gpa )

Fre

qu

en

cy

Hertz Model:

2

1

2

3

2

4

)1(3

RZ

FE

crystalline budesonide Lactose

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Searching for specific interaction sites: Two dimensional force measurement

Functionalized probe

Array of Potential Interaction Sites

Specific Molecular Interaction

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0

20

40

60

80

100

120

0 10 20

Force-Volume (FV) mode

0

20

40

60

80

100

120

0 10 20

0

20

40

60

80

100

120

0 10 20

0

20

40

60

80

100

120

0 10 20

0

20

40

60

80

100

120

0 10 20

0

20

40

60

80

100

120

0 10 20

0

20

40

60

80

100

120

0 10 20

School of PharmacyFV measurement of salbutamol particles on lactose-coated surfaces

AFM probe

Salbutamol

Scan probe whilst

recording adhesion events

Lactose1µm

A

B

A

B

A BForce distance curves extracted from the marked locations on the force volume image. The Salbutamol particle has greater adhesion to the lactose carrier at position B.

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FV measurement on BSA partially coated surfaces with a BSA-coated tip

Si

PS

BSA

Buffer

500 nm

Topography Adhesion

Langmuir 13, 4106 (1997)

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FV measurement on BSA partially coated surfaces with a BSA-coated tip:Time evolution

Langmuir 13, 4106 (1997)

Dynamic observation of BSA adsorption onto apolystyrene surface. Retract f-d curves were measured on apolystyrene spin-cast surface with a BSA-coated probe afterthe surface was exposed to a BSA solution (0.01 mg/mL, pH 7,100 mM phosphate buffer) for 0 min (I), 4 min (II), 5 min (III),7 min (IV), 10 min (V), and 60 min (VI) without movement ofthe sampling location.

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Pulsed-Force AFM

Topography

AdhesionDrive

Maximum force(Topography)

Adhesion

Stiffness

Sample: a silicon surface AFM tip: silicon, 4 N/m PF frequency: 905Hz

Tip oscillation

School of PharmacyPF-AFM images of PLMA/PmMl6 blend in water

PLMA: poly (lauryl methacrylate)PmMl6: 2-methacryloyloxyethyl phosphorylcholine-co-lauryl methacrylate (1:6)

1 m

Height Adhesion Stiffness

School of PharmacyPF-AFM images of a polystyrene surface partially coated with albumin

Height Adhesion5 m

0102030

0 4 8 12

Hei

gh

t (n

m)

0.0

0.3

0.6

0 4 8 12

Ah

esio

n (

V)

Albumin

Albumin

Polystyrene Buffer

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Si

PS

PF-AFM images of a polystyrene surface partially coated with fibrinogen

Fibrinogen

Height Adhesion1 m

0

2

4

0.0 1.0 2.0

Heigh

t (nm

)

0.00.51.01.5

0.0 1.0 2.0

Ahes

ion (V

)

Polystyrene

Fibrinogen

pH7 Phosphate Buffer

-30

-25

-20

-15

-10

-5

0

-100 0 100 200 300 400 500

-30

-20

-10

0

10

20

-100 100 300 500 700 900

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Outline




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Force measurement in AC mode

PhaseCurves.swf

AmplitudeCurves.swf

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30

35

40

45

50

-30 -20 -10 0 10

Relative scanner displacement (nm)

15

40

65

90

PS

PS30

35

40

45

50

-30 -20 -10 0 10


15

40

65

90

gel

gel

A-p/d curves measured on different material surfaces

30

35

40

45

50

-30 -20 -10 0 10


15

40

65

90

mica

mica

3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0

Re la tive s ca nne r dis pla ce me nt (nm)

1 5

4 0

6 5

9 0

PS

PS3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

PS

PS

PS

PS3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

gel

gel

3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

gel

gel

gel

gel

3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

mica

mica

3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

mica

mica

mica

mica

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Phase imaging

Phase Imaging

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Tapping mode images of gelatine on a mica surface

Height Phase

250 nm

Ultramicroscopy 75, 171 (1998)

Dark phase

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Tapping mode images of gelatine on a polystyrene surface

Height Phase

500 nm

Light phase


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3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

PS

PS3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

PS

PS

PS

PS3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

gel

gel

3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

gel

gel

gel

gel

3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

mica

mica

3 0

3 5

4 0

4 5

5 0

-3 0 -2 0 -1 0 0 1 0


1 5

4 0

6 5

9 0

mica

mica

mica

mica

Phase imaging and a-p/d curves

Height Phase

Height Phase


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Physical meaning of phase contrast

= +

J.P. Cleveland et al., Appl. Phys. Lett. 72 (1998) 2613.

Energy dissipation model (J. Cleveland et al., 1998)

= –

Free of tip/sample interactions: = 90

School of PharmacyTM-AFM imaging of an EDMPC partially-coated mica surface

Height Phase

-5

0

5

10

15

20

25

13 15 17 19

RMS Amplitude (nm)

Dis

sip

atio

n P

ow

er

(pW

)

0

20

40

60

80

100

120

Pha

se L

ag

(D

eg

ree

s)

Mica, DissipationLiposome, DissipationMica, PhaseLiposome, Phase

Height Phase

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Physical meaning of phase contrast

Bistable status of tapping tips

0

5

10

15

20

25

0 3 6 9 12 15 18

Relative Z Scanner Displacement (nm)

RM

S A

mp

litud

e (

nm)

0

20

40

60

80

100

120P

hase

La

g (

de

gre

es)

Amplitude

Phase

ADRL

Setpoint

0

150

300

450

600

Co

unt

Mica

050

100150200250

0 4 8 12 16 20ADRL (nm)

Liposome

b

0

50

100

150

200

Liposome/Mica

c

a

Height Phase

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Outline




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Force-modulation imaging

ForceModulation.swf

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FM-AFM images of a two-phase block copolymer

900nm scansHeight Force Modulation

Veeco Instrumentswww.veeco.com

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Lateral-Force AFM

LateralForce.swf

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Lateral-Force AFM

Height Lateral Force

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Lateral resonance (LR) AFM

HOPG surface imaged with interleaved TappingMode height (left) and TRmode phase (right). 3µm scan. (Supplied by Veeco.)

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Acknowledgements

Funding: BBSRC, EPSRC, RSPGB, GSK, Pfizer Global R&D, Nektar,

Molecular Profiles.

• Dr Ardeshir Danesh,• Dr Giles Sanders, Dr

Jianxin Zhang, Dr Wenke Zhang

• Prof Martyn Davies, Prof Saul Tendler, Prof Clive Roberts, Dr Phil Williams, Dr Steph Allen

• NT-MDT http://www.ntmdt.ru/

王宽诚教育基金会

Atomic Force Microscopy – More Than Microscopy

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