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School of Pharmacy 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.

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Presentation on theme: "School of Pharmacy 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."— Presentation transcript:

1 School of Pharmacy 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

2 School of Pharmacy Atomic Force Microscopy – More Than Microscopy Xinyong Chen Laboratory of Biophysics and Surface Analysis School of Pharmacy The University of Nottingham

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5 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)

6 School of Pharmacy Force measurement

7 School of Pharmacy Force/distance curves Maximum adhesion force Contact region a b c d Scanner Displacement (nm) Point of maximum load Deflection/Force

8 School of Pharmacy Experimental f/d curves ApproachRetract

9 School of Pharmacy Single particle interaction between salbutamol, lactose and PTFE Increase in strength of interaction with salbutamol in air Tip 1 Tip 2 Tip 3 Average Adhesion Force/nN Lactose Salbutamol PTFE International Journal of Pharmaceutics 238 (2002) PTFE Salbutamol Distance (nm) Force (nN) Lactose AFM probe Salbutamol Measurement of particle- particle interaction Lactose 1µm

10 School of Pharmacy Adhesion between a solid sphere and a solid plane (Hertz Model) JKR (Johnson, Kendall, Roberts – ) model: DMI (Derjagin, Muller, Toropov – 1975) model: Maugis (1992) :  R F Stiff materials (DMT)  Compliant materials (JKR)  With complex and implicit parameter equations

11 School of Pharmacy Specific interaction Anti - Ferritin Antibody Ferritin AFM Cantilever and Probe Substrate

12 School of Pharmacy Antigen-antibody interaction Biochemistry, 36, 7457 – 7463 (1997) Distance moved by the Cantilever (nm) Force (nN) Approach Retract Distance moved by the Cantilever (nm) Force (nN) Approach Retract

13 School of Pharmacy Multiple 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),

14 School of Pharmacy Force curve of chain-like molecules

15 School of Pharmacy Force 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: (2004 )

16 School of Pharmacy Representative force-extension traces for DNA molecules of three different lengths Biochem. Soc. Trans. (2003) 31, (1052–1057)

17 School of Pharmacy Antigen-antibody interaction Anti - Ferritin Antibody Ferritin AFM Cantilever and Probe Substrate - Single antigen-antibody bond?

18 School of Pharmacy 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)

19 School of Pharmacy “Spacers” – a way to measure single molecular interaction PEG attached Antibody Mono-functional Spacer PEG molecules Silicon Substrate Covalently attached  hCG

20 School of Pharmacy Force measurements between PEG-antibody coated probes and hCG coated surfaces Distance Moved by the Cantilever (nm) Approach Retract Force (nN) Force (nN) Approach Retract Distance Moved by the Cantilever (nm) Approach Retract Distance Moved by the Cantilever (nm) Force (nN) Distance moved by the Cantilever (nm) Approach Retract Force (nN) (a)(b) (c) (d) Low probability in detecting single molecular pair interaction!

21 School of Pharmacy Antibody-antigen interaction using polymeric linkers Polymeric linker e.g. agarose, PEG Antigen Biotinylated antibody Streptavidin PLA-PEG- Biotin AFM probe

22 School of Pharmacy Multi-cantilever sensor Different DNA-drug complexes have different ‘fingerprints’ From Cantisens® Research from Concentris

23 School of Pharmacy Exploring surface hardness Z F  Z: Indentation on soft surfaces Deflection on hard surfaces (no indentation) Hertz Model: crystalline budesonide Lactose

24 School of Pharmacy Forces in the “snap-in” region

25 School of Pharmacy Searching for specific interaction sites: Two dimensional force measurement Functionalized probe Array of Potential Interaction Sites Specific Molecular Interaction

26 School of Pharmacy Force-Volume (FV) mode

27 School of Pharmacy FV measurement of salbutamol particles on lactose-coated surfaces AFM probe Salbutamol Scan probe whilst recording adhesion events Lactose 1µm A B A B Force 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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29 FV measurement on BSA partially coated surfaces with a BSA-coated tip Si PS BSA Buffer 500 nm TopographyAdhesion Langmuir 13, 4106 (1997)

30 School of Pharmacy FV measurement on BSA partially coated surfaces with a BSA-coated tip: Time evolution Langmuir 13, 4106 (1997) Dynamic observation of BSA adsorption onto a polystyrene surface. Retract f-d curves were measured on a polystyrene spin-cast surface with a BSA-coated probe after the 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 of the sampling location.

31 School of Pharmacy Pulsed-Force AFM Topography Adhesion Drive Maximum force (Topography) Adhesion Stiffness Sample: a silicon surface AFM tip: silicon, 4 N/m PF frequency: 905Hz Tip oscillation

32 School of Pharmacy PF-AFM images of PLMA/PmMl6 blend in water PLMA: poly (lauryl methacrylate) PmMl 6 : 2-methacryloyloxyethyl phosphorylcholine-co-lauryl methacrylate (1:6) 1  m HeightAdhesionStiffness

33 School of Pharmacy PF-AFM images of a polystyrene surface partially coated with albumin HeightAdhesion 5  m Albumin Polystyrene Buffer

34 School of Pharmacy Si PS PF-AFM images of a polystyrene surface partially coated with fibrinogen Fibrinogen HeightAdhesion 1  m Polystyrene Fibrinogen pH7 Phosphate Buffer

35 School of Pharmacy 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)

36 School of Pharmacy Force measurement in AC mode

37 School of Pharmacy Amplitude-phase/distance curves on a mica surface

38 School of Pharmacy PS gel A-p/d curves measured on different material surfaces mica

39 School of Pharmacy Phase imaging

40 School of Pharmacy Tapping mode images of gelatine on a mica surface HeightPhase 250 nm Ultramicroscopy 75, 171 (1998) Dark phase

41 School of Pharmacy Tapping mode images of gelatine on a polystyrene surface HeightPhase 500 nm Light phase Ultramicroscopy 75, 171 (1998)

42 School of Pharmacy Phase imaging and a-p/d curves Ultramicroscopy 75, 171 (1998)

43 School of Pharmacy Physical meaning of phase contrast = + J.P. Cleveland et al., Appl. Phys. Lett. 72 (1998) Energy dissipation model (J. Cleveland et al., 1998) = – Free of tip/sample interactions:  = 90

44 School of Pharmacy TM-AFM imaging of an EDMPC partially- coated mica surface HeightPhase Height Phase

45 School of Pharmacy Physical meaning of phase contrast Bistable status of tapping tips Height Phase

46 School of Pharmacy 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)

47 School of Pharmacy Force-modulation imaging

48 School of Pharmacy FM-AFM images of a two-phase block copolymer 900nm scans HeightForce Modulation Veeco Instruments

49 School of Pharmacy Lateral-Force AFM

50 School of Pharmacy Lateral-Force AFM HeightLateral Force

51 School of Pharmacy Lateral resonance (LR) AFM HOPG surface imaged with interleaved TappingMode height (left) and TRmode phase (right). 3µm scan. (Supplied by Veeco.)

52 School of Pharmacy 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 王宽诚教育基金会


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