Single molecule force distance measurement

Figure 1 Schematic illustration of single-molecule force-distance measurement of a supramolecular host-guest pair exploiting a surface-attached single-polymer chain. The conversion of the recorded cantilever deflection-piezo displacement signal (a) into a force-extension curve of the single-polymer chain (b) is shown. (Redrawn from Ref. 21. Wiley-VCH, 2006.)...

Single Molecule Force Spectroscopy - Force-Distance Measurements Measuring the force as a function of tip-sample distance can yield information on the elasticity of individual supermolecules, on conformational transitions (e.g. of proteins), on the mechanical stability of chemical bonds and secondary structures, as well as of the desorption of the molecules from the solid substrate [82, 92-94]. Moreover, information on the chemical bond formation of the tip cluster with a particular bonding site on the sample surface can be obtained [95]. 

We only mention here that the probe used in AFM is a sharp tip, which is attached to a flexible microbeam (microcantilever). In AFM various forms of interactions between the apex of the tip (with a radius between approximately 10-100 nm) and the sample surface are measured, either as a function of tip location with respect to the surface, or at a fixed (x,y) position as a function of the cantilever deflection or tip-sample distance. In most conventional instruments the cantilever-tip assembly is attached to a piezo controller, which positions the tip in the (x,y) scanned plane and adjusts the vertical position (piezo travel) to accommodate sample height, or to measure tip-sample force curves. The latest generation instruments can also be equipped with active x-y-z distance feedback control loops, which enable one to perform lithography, vertical positioning of the tip (e.g. for single molecule force spectroscopy), etc. 

The capability of AFM to measure forces in the range of tens of picoNewtons has stimulated its application to study intermolecular and intramolecular forces stabilizing the fold of membrane proteins. The protein of interest is pulled out of the biological membrane by the AFM probe, and the force-distance spectrum depicts the force required to unfold the molecule in distinct steps as well as the contour length of the unfolded pol q)eptide. BR is one of the bestcharacterized membrane proteins by single-molecule force spectroscopy (SMFS). - When force spectroscopy measurements were applied to polytopic membrane proteins, the technique was shown to be sufficiently sensitive to detect forces between the transmembrane helices. - ... 

Even when taking into account that van der Waals forces for macroscopic objects fall slower than the fast decay law for single molecules, the direct measurement of van der Waals forces is demanding with respect to precise distance control and... 

Fig. 5 (a) Force induced ring opening of gem-dihalocyclopropane (gDHC). X=F, Cl, and Br. (b) Force-distance curve measured upon extension of gDBC by single molecule AFM at a constant tip withdrawal velocity of 3 pm/s. Adapted with permission from [87], Copyright 2010 American Chemical Society... 

New techniques are being developed that are able to measure the force-distance relationships of single molecules as they are stretched. Such techniques include atomic force microscopy and optical tweezers. Atomic force microscopy utilizes a microscale cantilever that has a probe to scan the specimen surface. Force is measured in piconewtons and the distance in nanometers. An example of some results from this technique is shown in Figure 6.5, where the force required to stretch poly(ethyleneglycol) molecules of different lengths is shown (Oesterhelt, Rief, and Gaub 1999). 

The principle behind SFM is that the lateral or shear force between an oscillating probe tip and the sample increases as the distance decreases. The probe is usually mounted in a support such that several millimeters of the aperture end of the optical fiber extends beyond the clamping point. The probe thus forms a cantilever having one fixed and one free end. It is driven transversely at a so-called tip resonance , which indicates that the resonance is due to the cantilever rather than the support structure of the microscope, with an amphtude 5nm. Shear forces between the probe tip and sample surface damp the oscillation. The amplitude is measured and fed back to the sample height position so as to maintain constant oscillation amplitude and presumably constant tip-sample distance. The amplitude was measured, originally, with optical deflection methods. Recently, a number of electrical measurement schemes have been demonstrated that may prove to have a number of advantages in speed, sensitivity or ease-of-use [12]. In near-field single molecule experiments the bandwidth of the feedback is not an issue as scan rate is limited by... 

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