Принципы нанометрологии (1027506), страница 44
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This relationship is valid up to an approximately 30 nN repulsive force [44]. Frictionalforces vary on an atomic scale, and with temperature, scan velocity, relativehumidity, and tip and sample materials.7.3.7.1 Tip functionalisationInter- and intra-molecular forces affect a variety of phenomena, includingmembrane structure, molecular recognition and protein folding/unfolding.AFM is a powerful tool for probing these interactions because it can resolveforces that are several orders of magnitude smaller than the weakest chemicalbond, and it has appropriate spatial resolution.
In recent years, researchers havetaken advantage of these attributes to create chemical force microscopy [45].AFM probes (i.e. cantilevers or tips) are functionalised with chemical functionalgroups, biomolecules or living, fully functional cells to make them sensitive tospecific interactions at the molecular to cellular level (see Table 7.3).There are many ways to functionalize an AFM tip or cantilever. Allfunctionalization methods are constrained by one overriding principle – thebonds between the tip/cantilever and the functionalizing substance (i.e.
theforces holding the substance of interest to the tip/cantilever) must be muchstronger than those between the functionalizing substance and the sample(i.e. the forces that are actually measured by the AFM). Otherwise, thefunctionalizing substance would be ripped from the tip/cantilever duringforce measurements.Table 7.3Various substances that have been linked to AFM tips or cantileversSubstance linked to tip/cantileverLinkage chemistryProteinNucleic acidPolysaccharideGlass or latex beadLiving microbial cellDead microbial cellEukaryotic cellOrganic monolayerNanotubeadsorption, imide, glycol tether, antibody-antigenthioladsorptionepoxysilane, poly-lysinegluteraldehydeepoxy, adsorptionself-assembling monolayer, silaneepoxy195196C H A P T ER 7 : Scanning probe and particle beam microscopySingle, colloidal-size beads, a few micrometres in diameter, can beroutinely attached to a cantilever using an epoxy resin [46].
Such beads maybe simple latex or silica spheres, or more complex designer beads imprintedwith biomolecular recognition sites. Care must be taken to select an epoxythat is inert in the aqueous solution and that will not melt under the laser ofthe optical lever detection system [47].Simple carboxylic, methyl, hydroxyl or amine functional groups can beformed by self-assembling monolayers on gold-coated tips [45] or by creatinga silane monolayer directly on the tip.
Organosilane modification of a tip isslightly more robust because it avoids the use of gold, which forms a relatively weak bond with the underlying silicon or silicon nitride surface of thetip in the case of self-assembling monolayers. Carbon nanotubes (CNTs) thatterminate in select functional groups can also be attached to cantilever tips[48]. The high aspect ratio and mechanical strength of CNTs creates functionalized cantilevers with unprecedented strength and resolution capabilities. Direct growth of CNTs onto cantilevers by methods such as chemicalvapour deposition [49] will probably make this method more accessible toa large number of researchers.Biomolecules such as polymers, proteins and nucleic acids have beenlinked to AFM tips or deposited directly on the cantilever [50].
One of thesimplest attachment techniques is by non-specific adsorption betweena protein, for example, and silicon nitride. The adsorbed protein can thenserve as a receptor for another protein or ligand. Virtually any biomoleculecan be linked to a cantilever either directly or by means of a bridging molecule. Thiol groups on proteins or nucleic acids are also useful becausea covalent bond can be formed between sulfulhydrol groups on the biomolecule and gold coatings on a tip. Such attachment protocols have been veryuseful: however, there are some disadvantages.
The linkage procedure maydisrupt the native conformation or function of the biomolecule, for example,if the attachment procedure disrupts a catalytic site. It is well known thata protein attached to a solid substrate (a cantilever or tip) may exhibita significantly different conformation, function and/or activity relative to itsnative state within a membrane or dissolved in solution.
Therefore, caremust be taken to design control experiments that test the specificity ofa particular biomolecule as it occurs in its natural state.7.3.8 Tip sample distance measurementTo obtain the distance or separation part of the force–distance curve, a pointof contact (i.e. zero separation) must be defined and the recorded piezoelectricscanner position (i.e. displacement) must be corrected by the measuredAtomic force microscopydeflection of the cantilever.
Simply adding or subtracting the deflection of thecantilever to the movement of the piezoelectric scanner determines thedisplacement. For example, if the sample attached to the piezoelectricscanner moves 10 nm towards the cantilever, and the cantilever is repelled 2nm due to repulsive forces, then the actual cantilever–sample separationchanges by only 8 nm. The origin of the distance axis, the point of contact, ischosen as the beginning of the region of constant compliance, i.e. the pointon the force curve where cantilever deflection becomes a linear function ofpiezoelectric scanner displacement (see Figure 7.6). Just as it was difficult toconvert photodiode voltage to displacement units for soft, deformablematerials, it is not always easy to select the point of contact because there isno independent means of determining cantilever–sample separation.
Fordeformable samples, the cantilever indents into the sample such that theregion of constant compliance may be non-linear and the beginning pointcannot be easily defined. Recent research has developed an AFM with independent measurement of the piezoelectric scanner and the cantileverdisplacements [51].7.3.9 Challenges and artefacts in AFM force measurementsThere are a number of artefacts that have been identified in force curves.Many of these artefacts are a result of interference by the laser, viscosityeffects of the solution or elastic properties of soft samples. When the sampleand the cantilever are relatively remote from each other, such that there is nointeraction, the force curve data should be a horizontal line (i.e.
the region ofnon-contact; see Figure 7.6). However, the laser has a finite spot size thatmay be larger than the size of the cantilever such that the laser beam reflectsoff the sample as well as the cantilever. This is particularly troublesome forreflective substrates, often resulting in optical interference, which manifestsitself as a sinusoidal oscillation or as a slight slope in the non-contact regionof the force curve [52]. This affects the way in which one defines attractive orrepulsive forces. A simple solution is to realign the laser on the cantileversuch that the beam does not impinge upon the underlying sample.
Alternatively, the oscillation artefact may be removed from the force curve withknowledge of the wavelength of the laser. This optical problem has beenlargely solved in commercial AFMs by using superluminescent diodes, whichpossess high optical power and low coherence length.A further artefact is the hysteretic behaviour between the approach andretraction curves in the non-contact area. The approach and retractioncurves often do not overlap in high-viscosity media due to fluid dynamiceffects [53]. Decreasing the rate at which the piezoelectric scanner translates197198C H A P T ER 7 : Scanning probe and particle beam microscopythe samples towards and away from the cantilever can help to minimizehysteresis by decreasing the drag caused by the fluid.Another frequently observed artefact in the force curve is caused by theapproach and retraction curves not overlapping in the region of contact butrather being offset laterally.
Such artefacts make it difficult to define the pointof contact, which is necessary to obtain separation values between the sampleand the tip. Such hysteresis artefacts are due to frictional effects as the tip(which is mounted in the AFM at an angle of typically 10 to 15 relative to thesample) slides on the sample surface. This hysteresis is dependent upon thescan rate and reaches a minimum below which friction is dominated by stickslip effects and above which friction is dominated by shear forces.
This artefactmay be corrected by mounting the sample perpendicular to the cantilever,thereby eliminating lateral movement of the cantilever on the sample.Viscoelastic properties of soft samples also make it difficult to determinethe point of contact and to measure accurately the forces of adhesion. Whenthe cantilever makes contact with a soft sample, the cantilever may indent thesample such that the region of contact is non-linear. It is then difficult todetermine the point at which contact begins.
The rate at which the sampleapproaches or retracts from the tip also affects the adhesive force measured onsoft samples. This is because the tip and sample are weakly joined over a largecontact area that does not decouple fast enough as the tip is withdrawn at veryhigh scan rates. Thus, the membrane deforms upward as the tip withdraws,causing an increased force of adhesion. Contact between a soft sample and thetip also affects the measured adhesion force in other ways.
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