In Chemical Force Microscopy (CFM), the AFM tip is modified with specific chemical functional groups . This enables the tip to generate contrast dependent on the chemical properties of the sample from the friction signal in contact mode or the phase lag signal in tapping mode simultaneously with topography . Functionalized tips have also been employed in force spec-troscopy. In this mode of operation the tip is brought into contact with a surface, then retracted. The forces applied to the tip during retraction are due to the interactions of tip and sample molecules. Force spectroscopy has been used to measure a variety of interactions including the intermolecular adhesion between fundamental chemical groups [90,92,93,94,96], the unfolding of protein molecules , antigen-antibody interactions , and DNA stretching and unbinding .
Despite the progress made in chemically sensitive imaging and force spec-troscopy using silicon and silicon nitride tips, these probes have important limitations. First, the tips have a large radius of curvature making it difficult to control the number of active tip molecules and limiting the lateral resolution. Second, the orientation and often the spatial location of the attached molecules cannot be controlled, leading to uncertainty in the reaction coordinate for force spectroscopy, and increased non-specific interactions. Carbon nanotube tips can overcome these limitations. They have small radii of curvature for higher resolution and can be specifically modified only at their very ends, creating fewer active molecular sites localized in a relatively controlled orientation. Modified SWNT tips could lead to subnanometer resolution in chemical contrast and binding site recognition.
Nanotube tips etched in air are expected to have carboxyl groups at their ends based on bulk studies of oxidized nanotubes , although conventional analytical techniques have insufficient sensitivity to observe this for isolated tubes. Chemical species present at the ends of nanotube tips can be studied with great sensitivity by measuring the adhesion of a nanotube tip on chemically well-defined self assembled monolayers (SAMs). Wong et al. [75,78] demonstrated the presence of carboxyl groups at the open ends of manually assembled MWNT and SWNT tips by measuring force titrations as shown in Fig. 23. In the force titration, the adhesion force between a nanotube tip and a SAM surface terminating in hydroxyl groups is recorded as a function of solution pH, thus effectively titrating ionizable groups on the tip [95,96]. Significantly, force titrations recorded between pH 2 and 9 with MWNT and SWNT tips were shown to exhibit well-defined drops in the adhesion force at ca. pH 4.5 that are characteristic of the deprotonation of a carboxylic acid.
Wong et al. [75,78] also modified assembled SWNT and MWNT bundle tips' organic and biological functionality by coupling organic amines to form amide bonds as outlined in Fig. 23a. Nanotube tips modified with benzy-lamine, which exposes nonionizable, hydrophobic functional groups at the b 'i-n b 'i-n
tip end, yielded the expected pH-independent interaction force on hydroxyl-terminated monolayers (Fig. 23). Moreover, force titrations with ethylene diamine-modified tips exhibit no adhesion at low pH and finite adhesion above pH 7 (Fig. 23b), consistent with our expectations for exposed basic amine functionality that is protonated and charged at low pH and neutral at high pH.
Covalent reactions localized at nanotube tip ends represent a powerful strategy for modifying the functionality of the probe. However, the linking atoms that connect the tip and active group introduce conformational flexibility that may reduce the ultimate resolution. In an effort to develop a chemically sensitive probe without conformation flexibility, Wong et al.  explored the modification of the tips during the electrical etching process in the presence of O2, H2, or N2. Significantly, force titrations carried out on tips modified in O2, N2 and H2 exhibited behavior consistent with the incorporation of acidic, basic and hydrophobic functionality, respectively, at the tip ends.
Wong et al. [75,78] used functionalized nanotube probes obtain chemically sensitive images of patterned monolayer and bilayer samples [75,78]. Tapping mode images recorded with -COOH and benzyl terminated tips exhibit greater phase lag on the -COOH and -CH3 sample regions, respectively. The "chemical resolution" of functionalized manually assembled MWNT and SWNT tips was tested on partial lipid bilayers . Significantly, these studies showed that an assembled SWNT tip could detect variations in chemical functionality with resolution down to 3 nm, which is the same as the best structural resolution obtained with this type of tip. This resolution should improve with CVD SWNT tips, and recent studies bear this idea out .
Lastly, modified nanotube probes have been used to study ligand-receptor binding/unbinding with control of orientation, and to map the position of ligand-receptor binding sites in proteins and on cell surfaces with nanometer or better resolution. To illustrate this point, Wong et al.  examined the biotin-streptavidin interaction, which is a model ligand-receptor system that has been widely studied . Biotin-modified nanotube tips were used to probe the receptor binding site on immobilized streptavidin as shown in Fig. 24a . Force spectroscopy measurements show well-defined binding force quanta of ca. 200 pN per biotin-steptavidin pair (Fig. 24b). A key feature of these results compared to previous work [102,103], which relied on nonspecific attachment of biotin to lower resolution tips, is the demonstration that a single active ligand can be localized at the end of a nanotube tip using well-defined covalent chemistry. With the current availability of individual SWNT tip via surface CVD growth, it is now possible to consider the direct mapping of ligand binding sites for a wide range of proteins.
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