We have used self-assembled purines and pyrimidines on planar gold surfaces and on gold-coated atomic force microscope (AFM) tips to directly probe intermolecular hydrogen bonds. Electron spectroscopy for chemical analysis (ESCA) and thermal programmed desorption (TPD) measurements of the molecular layers suggested monolayer coverage and a desorption energy of about 25 kcal/mol.Experiments were performed under water, with all four DNA bases immobilized on AFM tips and flat surfaces. Directional hydrogen-bonding interaction between the tip molecules and the surface molecules could be measured only when opposite base-pair coatings were used. The directional interactions were inhibited by excess nucleotide base in solution. Nondirectional van der Waals forces were present in all other cases. Forces as low as two interacting base pairs have been measured. With coated AFM tips, surface chemistry-sensitive recognition atomic force microscopy can be performed.Direct measurements of intermolecular forces are of great interest because such forces dominate the behavior of all materials and biological systems. The atomic force microscope (AFM) (1) has been primarily employed to examine the topography of surfaces with atomic or molecular resolution (2-7). It has also been used to measure molecular friction (8, 9) and elasticity (10). In addition, it has been applied to measure colloidal forces between a sphere and a planar silica surface (11,12). Further, the ability to directly measure forces between molecules suggests the possibility to probe for the chemical interaction between tips and surface molecules. Consequently, investigators measured the forces between metal tips and metal surfaces (10, 13) and the forces between tungsten tips and molecular films on surfaces (14,15). IHoh et al (15, 16) investigated the pH dependence of forces between a silicon nitride tip and a glass substrate in aqueous solutions. However, in the previous studies, the surface chemistry of sample and probe was not well defined and was poorly characterized. The interpretation of the measured forces is further complicated because the glass surface properties under such conditions may be closer to those of a silica gel than to those of the original glass (17). Molecular recognition forces have been measured by using the specific interactions between the ligand-receptor pair streptavidin-biotin (18, 19) as well as between complementary DNA strands (20). Here we use DNA base pairing to demonstrate that the AFM can directly measure directional hydrogen bonding in well-defined, ordered systems. Although hydrogen bonding is generally nondirectional (21), the shape of the nucleotide bases renders the hydrogen-bonding interaction remarkably orientation specific (22). We present evidence that we can measure this specific interaction between bases that have obvious biological significance.EXPERIMENTAL PROCEDURES AFM Tips. Mica (Structure Probe) and AFM silicon nitride (Digital Instruments, Santa Barbara, CA) pyramidal tips were gold coated by a dc a...