HRFS : GENERAL EXPERIMENT DESCRIPTIONHRFS : VERIFICATION OF SURFACE FUNCTIONALIZATION : IMAGING OF PLANAR SUBSTRATESHRFS : QUANTIFIED VERIFICATION OF SURFACE FUNCTIONALIZATION 2HRFS : VERIFICATION OF SURFACE FUNCTIONALIZATION 3 : LIPID BILAYERSREQUIREMENTS FOR SINGLE MOLECULE FORCE SPECTROSCOPYHIGH RESOLUTION FORCE SPECTROSCOPY : RAW DATA CONVERSIONHIGH RESOLUTION FORCE SPECTROSCOPY EXPERIMENT (HRFS): RAW DATA CONVERSION TO FORCE-DISTANCEHIGH RESOLUTION FORCE SPECTROSCOPY : DATA INTERPRETATIONHIGH RESOLUTION FORCE SPECTROSCOPY : CALCULATION OF RUPTURE DISTANCEINTRA- AND INTERMOLECULAR POTENTIALSIN VITRO VERSUS IN VIVO3.052 Nanomechanics of Materials and Biomaterials Tuesday 04/03/07 Prof. C. Ortiz, MIT-DMSEILECTURE 13: MIDTERM #1 SOLUTIONS REVIEWOutline :HIGH RESOLUTION FORCE SPECTROSCOPY.................................................................................2-10 General Experiment Description...........................................................................................2 Verification of Surface Functionalization:Imaging of Planar Substrates...............................3 Quantified Verification of Surface Functionalization 2..........................................................4 Verification of Surface Functionalization 3 : Lipid Bilayers..................................................5 Requirements for Single Molecule Force Spectroscopy.......................................................6 High Resolution Force Spectroscopy : Raw Data Conversion....................7-8 Data Interpretation : Adhesion..............................................................................................9 Calculation of Rupture Forces............................................................................................10INTRA- AND INTERMOLECULAR POTENTIALS....................................................................................11IN VITRO VERSUS IN VIVO....................................................................................................................12Objectives: To review the solutions to Midterm #1 in detail with your corrected exams to see where questions were answered correctly and incorrectly.Readings: Dupres, et al. Nature Methods 2, 7, 2005, 515, Midterm Solutions (posted on stellar). Multimedia : MP3 of this Lecture Posted13.052 Nanomechanics of Materials and Biomaterials Tuesday 04/03/07 Prof. C. Ortiz, MIT-DMSEHRFS : GENERAL EXPERIMENT DESCRIPTIONFigure 1a is a schematic of a HRFS experiment to measure the interactionbetween a protein found on the surface of M. tuberculosis bacterial cellcalled HBHA (heparin-binding haemoglutinin adhesion) and heparin. Theexperiments are performed in aqueous solution.→ the latter statement indicates that capillary forces are minimal, hence one canmeasure accurately intersurface forces (e.g. van der Waals, bioadhesion, ionic,etc.)→ Functionalized probe tip and surface (i.e. attaching chemical groups/moleculesto a surface)→ since the authors draw one molecule on the probe tip, can assume they aresuggesting this is a single molecule experiment→EG3 linker is 1) to increase mobility molecule on tip to allow easier binding withheparin, 2) to allow HBHA-Heparin binding when the tip is far away from surface toavoid tip-surface nonspecific interactions→ 4 monolayers on substrate :1) biotinylated-BSA which is physisorbed to Au (remember we talked about whathappens to proteins when adsorbed to surfaces (biocompatibility), they candenature due to hydrophobic interactions)2) Streptavidin binds to biotin with ligand-recepter lock and key mechanism3) Biotinylated-Heparin binds to streptavidin4) Heparin- negatively charged sulfate groups, since HBHA + charged, assumeionic interactions dominant→ Each layer can potentially contribute to the net intersurface interaction23.052 Nanomechanics of Materials and Biomaterials Tuesday 04/03/07 Prof. C. Ortiz, MIT-DMSEHRFS : VERIFICATION OF SURFACE FUNCTIONALIZATION : IMAGING OF PLANAR SUBSTRATES1. The authors state that they have verified their surface chemistry by first carrying it out on planar surfaces (Figure 1b = tip functionalization scheme, Figure 1c = substrate functionalization scheme) and then AFM imaging these functionalized surfaces at very high force (within the internal square regions shown in Figure 1b,c), and then zooming out and imaging at very low force.33.052 Nanomechanics of Materials and Biomaterials Tuesday 04/03/07 Prof. C. Ortiz, MIT-DMSEHRFS : QUANTIFIED VERIFICATION OF SURFACE FUNCTIONALIZATION 21. Explain what the data is in Figures 1b,c (both images andplots) and where it comes from in the instrument.-The images in Figure 1b, c are AFM height images as seen from the 2D section profile plots (1D line scans along the white line in each image) underneath each image which are labeled "height" on the vertical axis. -AFM height images come from the movement of z-piezo (z=direction perpendicular to the sample x/y plane) controlled bythe feedback loop.What the observed features are. -When the tip scans at high force within the smaller squareregion in the center, of the image, the imaging force is highenough to scratch off and remove all of the attached moleculesto the surface revealing the atomically flat underlying goldsubstrate (notice there is no recordable roughness in the 2Dsection profile). In the larger low force scan, one can see thefunctionalized region with molecules attached around the "bare"internal square. How these images quantitatively verify the success of the surface chemistry.The 1D section profile gives us the height of the functionalizedmolecular layer as the tip scans from the functionalized to bareregion. This height should correlate well with the known sizes ofthe molecules being attached to the surface. This is how thesurface chemistry is verified quantitatively. 43.052 Nanomechanics of Materials and Biomaterials Tuesday 04/03/07 Prof. C. Ortiz, MIT-DMSEHRFS : VERIFICATION OF SURFACE FUNCTIONALIZATION 3 : LIPID BILAYERSDPPCDOPCHiggens, et al. Biophys. J. 2006 91, 2532. 53.052 Nanomechanics of Materials and Biomaterials Tuesday 04/03/07 Prof. C. Ortiz, MIT-DMSEREQUIREMENTS FOR SINGLE MOLECULE FORCE SPECTROSCOPY2.