Nanobiotechnology Scanning Probe Microscopies Jan Přibyl CEITEC MU Kamenice 5/A35, CZ-62500 Brno pribyl@nanobio.cz Sample preparation for AFM AFM sample preparation Concentration – surface density Substrates for preparation of AFM samples Atomically flat surfaces 1. HOPG Highly Ordered Pyrolytic Graphite •Kish’s graphite, waste in steel production •Hexagonal planar structure •C-C bond142 pm, layer-layer distance335 pm •Conductive, highly hydrophobic •Planar structure •Synthetic form of graphite, high chemical purity •Traditionally – substrate for SEM, STM i AFM (→ conductivity) •Immobilization – spontaneous adsorption (→ hydrophobicity) Large areas visible layers Small areas atomically flat Atomically flat surfaces 1. HOPG Highly Ordered Pyrolytic Graphite •„Cat’s silver“, muscovite acc. to city of Moscow •Chem. structure: K2O·Al2O3·SiO2 •Hydrophilic surface •Easy to be modified by chemical synthesis •Immobilization by chemical bonding as well as ionic interaction •pKa ~ 3, physiological pH  negative surface charge •Mica = silicate, hydrated SiO2 (~ Si-OH) from the chemical point of view Atomically flat surfaces 2. Mica (muscovite) Extremely flat on small and larger areas Atomically flat surfaces 2. Mica (muscovite) •Inert metal •Traditionally in (bio)electrochemistry (i.e. biosensors) - electrodes •Conductive - STM + AFM •Hydrophobic: spontaneous non-selective adsorption of molecules (proteins, DNA, …) •Specific chemical binding of thiols (-SH) – organic molecules + cysteine •Prepared usually by evaporation •Adhesion layer for operation in liquids (Al/Cr/Ti) Sputtered gold layer image by tapping mode AFM Other surfaces 3. Gold •Amorphous noncrystalline structure •Lab glass composition: 75% SiO2 plus Na2O, CaO, borate and minor additives •Si-OH  from chemical point of view •Less hydrophilic comparing to mica •Roughness much higher comparing to mica (production by pressing) •Not suitable for individual molecules imaging with AFM •Typically used together with optical microscopy – cell compartments, whole cells Other surfaces 4. Glass AFM – optical image overlap Whole cells on glass under AFM glass A. Thiooxidans on glass Human sperm Fibroblast on polystyrene (PS) • Most of lab supplies made of plastic (PP, PE, PS) • No functional groups to be used in covalent binding • PS – hydrophobic  spontaneous non-specific adsorption of proteins  usually as underlying support (i.e. for cell attachment) Non-modified polystyrene (PS) Other surfaces 5. Plastic materials mica Compare roughness Immobilization procedures + + + 1. Proteins Surface: mica or HOPG (extremely flat) P pHIEP no charge P + + + + + + + + P - - - - - - - OH OH OH OH SiSi - - - Si Protein: charge is given by IEP + pH Immobilization on mica: pKa (mica) < pH < IEP P + + + + + + + + - - - Si Protein immobilization on HOPG HOPG A. Spontaneous (non-specific) adsorption of protein  hydrophobic surface (best results at zero charge pH = IEP) P PP Lysozyme molecules on HOPG HOPG COO-COO- COO- HOPG Electrochemical oxidation (E ~ 2 V vs. Ag/AgCl) Adsorption of long chain double-sided ions (C16/C18) •Ionic interation •Covalent binding (i.e. via NHS-esters) NH2 NH2 NH2 NH2 NH2 HOOC HOOC HOOC HOOC HOOC HOPG HOPG B. Ionic (specific) binding of molecules  creation of charge/chem. groups on HOPG surface - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - slída Immobilization problem: DNA (sugar-phosphate bone) as well as mica – negative charge under physiological pH → surface introduction of positive charge 2. DNA Surface: mica or HOPG (extremely flat) SiO O O CH3 CH3 CH3 R OH Si R OO OH - CH3 OH N(Et)3 OH OH OH OHOH OH OHOH OH OO O OO O R-propyltrimethoxysilane + cat. Si Si Si Silanization = chemical (covalent) modification of mica surface - Aim: introduction of functional group - Applicable also for: glass, quartz, silicon, titanium, … - Strong basis catalysis - Procedure can monitored by water contact angle measurement silanization hydrophobization A. DNA on mica SiO O O NH2 Si O NH2 SiO O O SH (3-Mercaptopropyl)trimethoxysilane MPTS(3-Aminopropyl)trimethoxysilane APTES 3-(Ethoxydimethylsilyl)propylamine APDMES Examples of alkoxysiloxanes Self-polymerization practical complication SiO O O R2 R1 R1 R1 SiO O O R2 R1 R1 R1 H O H Si OO R2 SiO O O R2 R1 R1 Si O O R2 + -R1OH • Especially with APTES during liquid silanization • Even vapors of water can cause this effect • Fixation for optical microscopy – expected factor • In contrary – in fixation for AFM – very disturbing • Solution: - silanization in vapours under vacuum (i.e. in desiccators) - monoalkoxysilanes – can not polymerize Si O NH2 3-(Ethoxydimethylsilyl)propylamine APDMES Self-polymerization examples NH2 NH2 NH2 NH2 NH2 HOPG HOPG B. DNA on HOPG Adsorption of long chain double-sided ions (C16/C18) Substrates for immobilization: mica / HOPG (smooth surfaces), also gold, glass in selected cases. Example: gold nanoparticles (AuNP) mercapto-silanized mica (SH-mica): OH Si SH OO OH OH OH OH SiO O O SH Si (3-Mercaptopropyl)trimethoxysilane MPTS 3. Nanoparticles SH-mica Si - - - - - - - - - - - - - - - - Au+ + + + + + + + P P P P P P + + + + Au+ + + + + + + + P P P P P P + + + + Gold nanoparticles (AuNP) conjugated with protein molecules: protein = immobilization bridge glass + + ++ + + + + ++ ++ Protein adhesive layer, i.e. pLL (poly-L-lysine  introducing positive charge) pLL Standard coating on glass glass 3. Bacteria, spores A. Standard culturing on polystyrene dishes Adhesive protein layers usually takes place (i.e. pLL, RGD adhesion factors, fibronectin, etc.) 5. Eukaryotic cells Cell culturing equipment BioAFM incl. Petri dish heater for in-vitro imaging of cell cultures • Adhesion of cells out of incubator (37oC, 5% CO2) is mostly problematic • Allows study of cells in long term periods after removal from incubator • Cell wall destruction • Example: EtOH, acetic acid, paraformaldehyde, glutardialdehyde B. Fixation agents AFM spectroscopy Force Distance curves (FD curves) Force Distance curves (FD curves) Minimum value: Area under the curve: Slope: SQ Evaluation of curves containing binding ‘event’ Types of FD curves Containing single binding event No interaction between tip and surface (Young’s modulus can be determined) Useless curve Containing multiple binding events ScanAssyst – automatic AFM ScanAssyst - principle (A) Typical force–distance curves for hard (green) and soft (blue) materials. (B) Adhesion on a hard surface. (C) Molecule–molecule and cell–surface detachment process with three unbinding events. Phys. Chem. Chem. Phys., 2015, 17, 2950-2959 QI mode Hertzian fit QI-imaging examples JPK supporting info Quantitative NanoMechanics (QNM) PeakForce QNM = quantitative nanomechanical information (biological samples without damaging) Based on Peak Force Tapping technology - probe is oscillated (~TappingMode), res. freq 1 - 8 kHz (=sampling rate) depending on the tool). Difference: Tapping Mode – const. amplitude, Peak Force Tapping maximum peak force on the probe (much lower comparing to contact mode – biological samples) attractive forces (capillary, VdW, elstat) negative forces > cantilever’ stiffness indentation Peak force  feed back control withdrawing PeakForce QNM on Bacteria (A) PeakForce QNM (250Hz) Sneddon modulus (B) PeakForce curves (C) Force volume Sneddon modulus image of the same bacteria collected at a ramp rate of 2Hz. (Standard DNP-A probe in water with 300nm modulation amplitude, Scan size 5µm.) BrukerNano supporting info AFM force mapping Examples Material properties mapping by AFM Young’s modulus mapping Young’s modulus of materials http://www-materials.eng.cam.ac.uk/ Methods for YM measurement Reference: Battelle PNNL MST Handbook, U.S. Department of Energy, Pacific Northwest Laboratory Olympus 38DL PLUS Measure the longitudinal and shear wave sound velocity of the test piece using the appropriate transducers and instrument setup. Acta Biomater. 2007 Jul; 3(4): 413–438. Cell Young’s modulus - methods Hertzian fit Measured curves were fitted to following function: where F is force, E is Young modulus, α – face angle, δ – tip-sample separation, ν – Poisson ratio: Tip-sample separation = correction of measured curve (height) for cantilever bending Parabolic tip shape Four sided pyramid Spherical tip Examples of Force-distance curves and Force Maps evaluation Force-distance curves Height Slope Adhesion Height Adhesion Young modulus With Giancarlo Forte, ICRC AFM in biomechanical characterization of cardiomyocytes Tyrod Beta Iso Caffeine 10 s 10 nN AFM CoreFacility CEITEC MU CEITEC AFM CoreFacility JPK NanoWizard3 Bruker FastScan Bio NTMDT NTgra Vita NTMDT Solver Next AFM visualization of biomolecules and bioobjects Bare metal (gold) electrodesBacteria on glass slide SpermWhole cells (cell synapse)Individual biomolecules IgG Nanoparticles (gold nanoparticles BSA modified) DNA  J. Hejátko – YM mapping  P. Bouchal – YM mapping  J. Paleček - DNA  M. Pešl, V. Rotrekl CMCs J. Sládková – CMCs  A. Meli - CMC  M. Kalbáčová – TiO2 NT  H. Kolářová - DNA  I. Crha - sperms Cooperation: Thank you for your attention!