It is known that protein attachment to surfaces depends sensitively upon the local structure and environment of the binding sites at the nanometer scale. Using nanografting and reversal nanografting, both atomic force microscopy (AFM) - based lithography techniques, protein binding sites with well-defined local environments are designed and engineered with nanometer precision. Three proteins, goat-anti-biotin Immunoglobulin G (IgG), lysozyme and rabbit-Immunoglobulin G, are immobilized onto these engineered surfaces. Strong dependence on the dimension and spatial distribution of protein binding sites are revealed in antibody recognition, covalent attachment via primary amine residues and surface bound aldehyde-groups. This investigation indicates that AFM based nanolithography enables the production of protein nanostructures and more importantly, protein-surface interactions at a molecular level can be regulated by changing the binding domains and their local environment at nanometer scale.

Nanopatterns of thiolated single-stranded DNA (ssDNA) are produced by using an atomic force microscopy (AFM)-based lithography technique known as nanografting. Under high shear force, AFM tips displace resist molecules within a self-assembled monolayer, while ssDNA molecules adsorb chemically onto the exposed gold area through the sulfur headgroup. Nanostructures of ssDNA are characterized directly and in situ by using the same tip. Lines as narrow as 10 nm have been produced. The ssDNA molecules stand up on the gold surfaces and adapt a stretched conformation. In situ and real-time imaging studies have revealed that DNA molecules within the nanostructures are accessible by enzyme molecules.Arrays of DNA patterns are important in gene mapping, drug discovery, DNA sequencing and disease diagnosis. 1 Various approaches have been taken to create DNA arrays on surfaces. One approach is to use light-directed oligonucleotide synthesis to attach DNA nucleotides at mask-defined areas and build subsequent DNA strands by coupling. 2 Another method is to attach presynthesized DNA strands onto designated sites of a solid support. 3,4 A broad range of solid supports have been used, such as gold, conductive polymer, SAMs, and carbon paste. 5-15 Typically, the size of the DNA patterns is tens to hundreds of micrometers. 16,17 Further miniaturization is essential for the development of ultrasmall biosensors and biochips. The performance of chips or sensors can be enhanced after miniaturization because of the higher density of receptor elements, higher detection sensitivity, and smaller amounts of reaction reagents. New generations of nanochips also offer the hope of faster analysis time, less waste of costly reagents, and massive parallelization. 18 Scanning probe microscopy (SPM) techniques are well known for their ability to visualize surfaces of materials with the highest spatial resolution. 19-21 Taking advantage of the sharpness of the tips and strong and local interactions between the tip and surface molecules, SPM has also been used to produce nanostructures on surfaces. 22-30 "Dip-pen" nanolithography (DPN) has been used to pattern nanostructures of DNA on a gold surface. The size of the DNA pattern depends on the substrate, humidity of the environment, and fabrication speed, thus making it difficult to reach high spatial precision. The tip coating process is relatively difficult, and a different tip needs to be used to characterize the produced pattern. 31 Recently, a meniscus force nanografting method was used to pattern DNA on surfaces, and the patterns have been coupled with complementary oligonucleotides tagged by gold particles. 32 The spatial precision and selectivity is not sufficiently high and the method used to characterize the DNA patterns involves tagging. We have developed three AFM-based lithography techniques for creating nanopatterns of self-assembled monolayers (SAMs) and biosensors: nanoshaving, nanografting, and nanopen reader and writer (NPRW). 25-29 Using these methods, nanostructures of thiols as s...

This article reveals the enabling aspects of nanografting (an atomic force microscopy-based lithography technique) in surface physical chemistry. First, we characterize self-assembled monolayers and multilayers using nanografting to place unknown molecules into a matrix with known structure or vice versa. The availability of an internal standard in situ allows the unknown structures to be imaged and quantified. The same approaches are applied to reveal the orientation and packing of biomolecules (ligands, DNA, and proteins) upon immobilization on surfaces. Second, nanografting enables systematic investigations of size-dependent mechanics at the nanometer scale by producing a series of designed nanostructures and measuring their Young's modulus in situ. Third, one can investigate systematically the influence of ligand local structure on biorecognition and protein immobilization by precisely engineering ligand nanostructures. Finally, we also demonstrate the regulation of the surface reaction mechanism, kinetics, and products via nanografting.

Condensation of DNA by multivalent cations can provide useful insights into the physical factors governing the folding and packaging of DNA in vivo. In this work, local ordered structures of spermidine-DNA complexes prepared from different DNA concentrations have been examined by using atomic force microscopy (AFM) and polarizing microscopy (PM). Two types (I and II) of DNA condensates, significantly different in sizes, were observed. It was found that for extremely dilute solutions (DNA concentrations around 1 ng/microl or below), the DNA molecules would collapse into toroidal structures with a volume equivalent to a single lambda-DNA (type I). In relatively dilute solutions (DNA concentrations between 1 and 10 ng/microll), a significantly larger structure of multimolecular toroids (circular and elliptical, type II) were formed, which were constructed by many fine particles. Measurements show that the average diameter of these fine particles was similar to the outer diameter of the monomolecular toroids observed in extremely dilute solutions, and the thickness of the multimolecular toroids had a distribution of multi-layers with height increments of 11 nm, indicating that the multimolecular toroidal structures have lamellar characteristics. Moreover, by enriching the DNA-spermidine complexes in very diluted solution, branch-like structures constructed by subunits were observed by using AFM. The analysis of the pellets in polarizing microscopy reveals a liquid-crystal-like pattern. These observations suggest that DNA-spermidine condensation could have multiple stages, which are very sensitive to the DNA and spermidine concentrations.