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Research Papers
CamesanoAbuLailBiomacromol2002
bacterial surface (with polymers that range in size from tens to hundreds of nanometers and have adhesion affinities for the AFM tip from 0-1nN)
adhesion between bacteria and surfaces is important for environmental and biomedical applications such as in situ bioremediation, microbially facilitated transport of contaminants and biofilm formation on biomaterials and implants
bacterial adhesion depends on polymers on the surface, but prediction of attachment based on biopolymer properties are not possible
sample prep:
bacterial cells bonded to glass slides (ref. 33)
hydrated
experiment in pure water, 0.01M-0.1M KCl
AFM:
DI Dimension 3100 with Nanoscope III controller
silicon nitride tips (DNPS) from DI
CarrionVazquezProgBiophysMolBio
review
Invention of AFM: Binnig et al., 1986
Imaging most frequent application
Force spectroscopy mode of AFM was developed later (Burnham and Colton, 1989) and applied to receptor-ligand systems (Florin et al., 1994, Science 264, 415-417.)
advantages: few micrograms of protein needed, no size limitation, no crystals needed
review mostly on AFM to study the molecular determinants of the mechanical stability of proteins
comparison of alpha-helical and beta-sheet proteins
(un)folding of mechanical proteins
examples of proteins that are exposed to mechanical tension: titin, extracellular matrix proteins, cytoskeletal proteins
one structural characteristic common to proteins exposed to mechanical tension is that they contain multiple individually folded domains
domain architecture
references to other reviews of AFM
Review
saw-tooth pattern of force peaks is the unmistakable fingerprint of a modular protein
each peak corresponds to the consecutive unfolding of each of the protein domains in a single molecule
can yield unfolding force peaks equal to the number of domains in the protein, but more frequently will yield fewer peaks or no peaks at all
spacing between the peaks reflects the number of amino acids that each unfolding event adds to the total length
molecular dynamics simulations suggest that force=induced unfolding occurs through different pathway than chemical unfolding
GouauxWhiteCurrOpinStructBio2001
GroganBiosensorsBioelectronics2002
AFM and biosensor: development of cantilever-based immunosensors
Definition of biosensor: Biosensors are analytical devices which combine a biologically sensitive element with a physical or chemical transducer to selectively and quantiatively detect the presence of specific compounds in a given external environment (ref. Nicolini et al, 1992)
Applications: diagnostic and therapeutic monitoring in clinical applications (frequent testing with short turnaround times)
Get review for microfabtricated cantilevers to use in bio-chemical sensing: Raiteri and Grattarola (2001) in Sensors and Actuators B.
antibody is the sensing element, microcantilever acts as a mechanical transducer
measure the activity, stability, lifetime and reusability of antibodies to myoglobin covalently immobilized onto microfabricated cantilever surfaces: stable for up to 7 weeks
next stage: use an array of cantilevers - the net differential signal from the array of cantilevers is the sensor signal (get ref. Fritz et al. (2000) Science 288, 316-318.)
major challenges in biosensor design: reproducibility, long term drift of cantilever bending, understanding of the mechanism of binding of analyte to the cantilever surface
use two cantilevers: one as reference, one as sample, can improve the sensitivity
AFM:
silicon nitride (DI)
flowing cell
Sample prep:
Cantilever surface: first, sulfo-LC-SPDP from Pierce react with antibody NH2, plus DTT to reduce SS to SH, then self-assembly on gold surface
amyloid fibrils
sample prep:
C1 sensor chip (BIAcore) in which the carboxymethyl residue was directly coupled to the gold thin layer with no dextran chain
coupling done within the BIAcore flow cell, then take it out, remove the glass support from the plastic support by dissolving the adhesives with ice-cold acetone
AFM:
dynamic force microscope SPI-3800N-SPA400
scanning microcantilever (SI-DF20) (Seiko Instruments)
comparison of X-ray diffraction, electron crystallography and AFM
AFM particularly useful to study the surface properties and dynamics
rhodopsin loop grafted onto br
different salt concentration was necessary than was used with normal br
standard deviation map useful for study of flexible loops
AFM is not able to image segments that are really unstructured
Sample prep:
purple membrane was diluted to 10mg/ml in 300mM KCl pH 7.8, 10mM Tris-HCl prior to adsorption (ref. Mueller and Engel (1997) Biophys. J. 73, 1633-1644.
20 min adsorption
washing gently with imaging buffer: 10mM Tris-HCl, pH 7.8, 100-150mM KCl
light-powered molecular machines are probably essential constituents of future nanoscale devices
new development: optically lengthen and contract individual polymers by switching the azo groups between trans/cis configs.
polymer contracts against an external force acting along the polymer backbone, thus delivering mechanical work
Optomechanical energy conversion in a single-molecule device
Bacteriorhodopsin
after photobleaching in presence of hydroxylamine: loss of crystalline properties, but br is still a trimer
note on comparison to rhodopsin
Sample prep:
before use, the fluid cell was cleaned with conventional dish cleaner
rinsed with ultra pure water and ethanol
muscovite mica was punched to 6 mm diameter and glued onto 12 mm Teflon disc with water-insoluble epoxy glue
Mica New York Corp., New York, USA:
1412-T W. Broadway
New York
, NY 10018
Tel: 212-354-2664
Fax: 212-354-2860
teflon disc glued onto a slightly smaller steel disc to allow magnetic fixing of the sample on to the piezo scanner
Review
resolution of AFM ~0.5nm
imaging of a statistically significant number of single proteins by AFM allows their structural variability to be assessed
determine the principal modes of protein motion
observe directly conformational changes of single proteins and of their assemblies
conformation changes can be induced in a controlled manner to identify flexible protein structures
Review of br results
Bacteriorhodopsin:
purple membranes at 50 mg/ml suspended in 150 mM KCl, 10mM Tris, pH 9.2
Sample preparation:
purple membrane suspension 50mg/ml was absorbed to freshly cleaved mica for 30 min
wash to remove not absorbed material
AFM:
Nanoscope III (Digital Instruments, CA)
15mm or 130mm piezo scanner
oxide sharpened silicon nitride tips mounted on a 100mm cantilever (Olympus Ltd. Tokyo, Japan)
SEMPER image processing system (Saxton et al., 1979)
Bacteriorhodopsin:
purple membranes isolated from Halobacterium salinarum ET1001 according to Oesterhelt and Stoeckenius, 1974
orthorhombic 2D crystals prepared as described by Michel et al, 1980
Sample preparation:
adsorbed onto freshly cleaved mica in buffer 300mM KCl, 10mM Tris-HCl, pH 7.8 according to Mueller, Amrein and Engel (1997) Adsorption of biological molecules to a solid support for scanning probe microscopy. J. Struct. Biol. 119, 172-188.
Experimental Conditions:
100mM KCl, 10mM Tris-HCl, pH 7.8
AFM:
Nanoscope III (DI) with E-scanner (12mm) and oxide sharpened Si3N4 tips on a cantilever with a spring constant of 0.1N/m (Olympus)
Bacteriorhodopsin:
- purple membranes of H. salinarum strain S1 isolated as described in Oesterhelt and Stoeckenius, Methods Enzymol. 31, 667 (1974).
- stock solution of protein (5 mg/ml) in ultrapure water at 4oC
AFM:
Nanoscope III (Digital Instruments, Santa Barbara, CA) equipped with a J-scanner (80mm) and oxide-sharpened Si3N4 tips on a cantilever (Olympus, Tokyo)
spring constant of cantilever as estimated by analysis of the thermal noise spectra 0.1N/m
Experimental Conditions:
300mM KCl, 10mM Tris-HCl, pH 7.8
RT
Experiment:
position the tip, apply force to absorb protein on stylus and pull out
Reference in Forbes and Lorimer commentary: use AFM to study folding and MISFOLDING: Oberhauser et al. (1999) Nature Struct Biol. 6, 1025
antigen-antibody immune complex
Sample preparation:
antibodies in PBS at a few mg/ml
deposit on freshly cleaved mica surface for 15 min at RT
rinse with deionized water to remove salts and weakly adsorbed antibodies
dry under Nitrogen
for binding of antigen: surface was exposed to buffer with antigens in same way as for antibodies
References for different substrates for flat solid surface immobilization: silicon oxide, gold, mica, polystyrene etc. , native and modified mica are the most commonly used
Experimental conditions
air, RT
AFM
Nanoscope IIIa (DI)
in non contact (tapping) mode and multi channel data acquisition
silicon lever
keratin fibers
Experiment:
imaging AND indentation
AFM imaging:
multimode AFM with J-scanner and Nanoscope IIIa controller (DI)
imaging in tapping mode in air with 125mm etched silicon microcantilevers (TESP)
AFM indentation:
diamond tip cantilever (DI)
tip was a single crystal of natural diamond bonded with epoxy to the stainless steel cantilever
Sample prep:
slices cut were mounted on 100 mesh Formvar-coated PELCO copper locator grids where each mesh point is uniquely identified
spectrin
unfolding
Monte Carlo simulations
picoNewton
Sample prep:
spectrin from Sigma
adsorb onto a freshly evaporated gold surface or a freshly cleaved mica surface from a 50 ml drop of the solution for 10min
rinse with PBS
measure in PBS
AFM
homebuilt
silicon nitride cantilevers (Park Scientific, Stanford) were used as force sensors
picoNewton instrumentation
dextran
MD calculations
Sample prep:
dextrans linked to gold surface through epoxy-alkanethiols, activated with one carboxymethylgroup per glucose, reacted with streptavidin so that they can be bound to Sensor chip SA5, Pharmacia Biosensor AB, Uppsala, Sweden
AFM cantilever with biotin bound to the AFM tip was used to pull the dextran through the biotin-streptavidin bond
all measurements in PBS
cantilevers were silanized in deimethyl-dichlorosilane (Sigma) vapor and rinsed with acetone
commercially available carboxymethylated dextran films on gold (Sensor Chip CM5, Pharmacia), Pharmacia Biosensor AB, Upppsala, Sweden) were used as samples
AFM:
homebuilt AFM
cantilevers (DI)
biosensor application of AFM: detect change in surface characteristics of E. coli before and after binding of antibodies
biosensor can detect with limit 6000 cells/ml
previous detection methods:
1. quartz crystal microbalance as the electronic transfer means for binding event
2. surface plasmon resonance
3. electrochemical impedance
Challenge in immunosensor development: signal amplification of the binding event
AFM:
Nanoscope IIIa (DI) in tapping mode
Sample prep:
ITO-coated glass (CG-80IN-1515) was purchased from Delta Technologies (Stillwater, MN) was cut into 5x0.8cm slices
clean with acetone, ethanol, water
10 min 1M HCl
wash with water
1:1:5 (v/v) H2O2/NH4OH/H2O for 1 h
rinse with water
before the silanization, the chips were dried under a stream of nitrogen
etc., also see previous ref. 19
after silanization the surface had many exposed active epoxy groups that react readily with protein amino groups
anti-E coli antibodies in drop 5 ml of 1mg/ml antibody onto substrate chip (8mmx8mm) 24 h at 4oC
middle part of the ITO chips was coated with 5-min Epoxy to avoid contact with the solution
antibody chips were rinsed with water and incubated with 50 ml of heat-killed E. coli (10000-70000 cells/ml) for 1 h at 37oC)
ZlatanovaProgBiophysMolBio2000
excellent review!!
what forces govern specific molecular interactions? they result from multiple weak, non-covalent bonds
how to probe such weak interactions?
1. surface force apparatus
2. pipette suction
3. magnetic beads
4. flow chamber apparatus
5. optical traps and tweezers
© 2002 Judith Klein-Seetharaman, University of Pittsburgh. This website was created December 12, 2002 and updated January 21, 2003. For questions or comments please contact Judith Klein-Seetharaman at jks33@pitt.edu.