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Measuring Bond Rupture Forces
with the Atomic Force Microscope


Figure 1. Concept of the atomic force microscope (AFM), an instrument which allows researchers to obtain molecular-scale images of surfaces and information about the specific forces between and within molecules on the surface. (Left) The sample (gray surface) containing the molecules of interest moves underneath the yellow plank ("the cantilever") by means of the piezoelectric tube scanner (silver cylinder) to which it is attached. A laser and position-sensitive detector measure the deflection of the cantilever which has an underlying tip that scratches or taps the moving surface. The cantilever's deflections provide information which can be converted into molecular-scale images of the surface and knowledge on the forces between molecules or within them. (Right) Close-up of the cantilever, which has a length of just 100 micrometers (millionths of a meter).The piezoelectric tube scanner has a diameter of 24 mm (Figure 1, D.R. Baselt, G.U Lee, K.M. Hansen, L.A. Chrisey and R.J. Colton, to appear in Proc. IEEE 85, 1997).

First described in 1986 (G. Binnig, C.F. Quate and Ch. Gerber, Phys. Rev. Lett., 56, 930, 1986), the atomic force microscope can image surfaces both in air and under liquids at a resolution of nanometers, or billionths of a meter. In its contact mode, the AFM lightly touches a tip at the end of a 50 - 300 micrometer long leaf spring (the cantilever) to the sample. As the tip is scanned over the sample, a detector measures the vertical deflection of the cantilever, yielding the precise height of the sample at local points. The deflections of the cantilever are monitored by a laser beam reflected off the cantilever and into a position-sensitive detector (Figure 1). Using microfabricated cantilevers, force sensitivities and position accuracy as small as 10-15 Newtons/Hz1/2 and 0.01 nm, respectively, can be measured. If the tip and sample are coated with two types of molecules, an AFM can measure force of attraction or repulsion between them, potentially at the level of a single hydrogen bond.

Living organisms are composed of cells that are in turn built from macromolecules such as DNA and proteins. These macromolecules make up the scaffolding that holds the cells together and the motors responsible for motion. Each of these molecules is folded into a intricate three-dimensional structure that allows it to perform its specific function. The slightest defect in a macromolecule can result in failure of an organism, for example, genetic diseases such as MS result from defects in a single macromolecule.

Macromolecular structure and function are controlled by the forces between individual units that make up macromolecules and their environment. Our knowledge of these forces has been gathered from indirect x-ray crystallography and nuclear magnetic resonance (NMR) measurements. The ability to directly measure these forces with AFM, and other techniques, is allowing us to understand the detailed mechanisms responsible for living systems. For example, the forces responsible for biological adhesion can now be studied. Bioadhesion controls diverse functions such as cell migration in cancer and the adhesion of barnacles to ship surfaces.


Figure 2. Interaction force between two complementary strands of DNA, measured by AFM. "Relative surface displacement" is the distance between the tip and sample relative to the position at which 1000 piconewtons (pN) of force is reached. Measurements are recorded both as the tip and sample are brought together (thin trace) and as they are separated (thick trace) (Figure 2, D.R. Baselt, et al., to appear in Proc. IEEE 85, 1997).

To date, authors at the Naval Research Laboratory and elsewhere have used AFM to measure interaction forces between single pairs of DNA nucleotides, complementary DNA strands, streptavidin-biotin, adhesion proteoglycans, and antibodies and their antigens. For example, Lee et al. have measured the force required to tear two complementary strands of DNA apart. In one such experiment (Figure 2) (G. U Lee, L. A. Chrisey and R. J. Colton, Science 266, 771, 1994), 20-base-pair long strands of polycytosine (i.e., single stranded DNA) were covalently attached to the tip and sample. Free strands of polyinosine averaging 160 base pairs long were introduced. When the tip and sample were brought together, these strands would sometimes bind to both the polycytosine on the tip and that on the sample, bridging the tip and sample. The tip and sample were then pulled apart. The cantilever does not sense any force until the slack in the DNA is taken up, at which point tension on the DNA begins to pull the cantilever down (starting at 100 nm of separation). The form of the force-distance curve describes the mechanical properties (intramolecular forces) of a single-strand of DNA as it is stretched from a random coil into a linear molecule. When the force is large enough (-600 pN in Figure 2), the intermolecular DNA-DNA bonds at either the tip or sample break, and the force on the cantilever returns to zero. The magnitude of the rupture force has been correlated with number of bases involved in Watson-Crick base-pairing.

Figures and text courtesy of Gil Lee, Naval Research Laboratory.

More information about this work and applications can be found here. .

This research was described by Gil Lee of the Naval Research Laboratory at the 1997 APS March Meeting in Kansas City.