Strategies for patterning biomolecules with dip-pen nanolithography

Dip-pen nanolithography (DPN) is an atomic force microscopy (AFM)-based lithography technique, which has the ability to fabricate patterns with a feature size down to approximately 15 nm using both top-down and bottom-up approaches. DPN utilizes the water meniscus formed between an AFM tip and a substrate to transfer ink molecules onto surfaces. A major application of this technique is the fabrication of micro- and nano-arrays of patterned biomolecules. To achieve this goal, a variety of chemical approaches has been used. This review concisely describes the development of DPN in the past decade and presents the related chemical strategies that have been reported to fabricate biomolecular patterns with DPN at micrometer and nanometer scale, classified into direct- and indirect DPN methodologies, discussing tip-functionalization strategies as well.     

Polymer pen lithography (PPL)-induced site-specific click chemistry for the formation of functional glycan arrays

Polymer pen lithography (PPL) can be combined with the Cu(I) -catalyzed azide-alkyne click reaction to create molecular arrays with control over orientation and sub-1 μm feature sizes over cm(2) areas. The process has been applied to the deposition of carbohydrates to form functional glycochips.     

Methods for fabricating microarrays of motile bacteria

Motile bacterial cell microarrays were fabricated by attaching Escherichia coli K-12 cells onto predesigned 16-mercaptohexadecanoic acid patterned microarrays, which were covalently functionalized with E. coli antibodies or poly-L-lysine. By utilizing 11-mercaptoundecyl-penta(ethylene glycol) or 11-mercapto-1-undecanol as passivating molecules, nonspecific binding of E. coli was significantly reduced. Microcontact printing and dip-pen nanolithography were used to prepare microarrays for bacterial adhesion, which was studied by optical fluorescence and atomic force microscopy. These data indicate that single motile E. coli can be attached to predesigned line or dot features and binding can occur via the cell body or the flagella of bacteria. Adherent bacteria are viable (remain alive and motile after adhesion to patterned surface features) for more than four hours. Individual motile bacterial cells can be placed onto predesigned surface features that are at least 1.3 microm in diameter or larger. The importance of controlling the adhesion of single bacterial cell to a surface is discussed with regard to biomotor design.     

Biochips for Cell Biology by Combined Dip‐Pen Nanolithography and DNA‐Directed Protein Immobilization

A general methodology for patterning of multiple protein ligands with lateral dimensions below those of single cells is described. It employs dip pen nanolithography (DPN) patterning of DNA oligonucleotides which are then used as capture strands for DNA‐directed immobilization (DDI) of oligonucleotide‐tagged proteins. This study reports the development and optimization of PEG‐based liquid ink, used as carrier for the immobilization of alkylamino‐labeled DNA oligomers on chemically activated glass surfaces. The resulting DNA arrays have typical spot sizes of 4–5 μm with a pitch of 12 μm micrometer. It is demonstrated that the arrays can be further functionalized with covalent DNA‐streptavidin (DNA‐STV) conjugates bearing ligands recognized by cells. To this end, biotinylated epidermal growth factor (EGF) is coupled to the DNA‐STV conjugates, the resulting constructs are hybridized with the DNA arrays and the resulting surfaces used for the culturing of MCF‐7 (human breast adenocarcinoma) cells. Owing to the lateral diffusion of transmembrane proteins in the cell's plasma membrane, specific recruitment and concentration of EGF receptor can be induced specifically at the sites where the ligands are bound on the solid substrate. This is a clear demonstration that this method is suitable for precise functional manipulations of subcellular areas within living cells.     

Development of Dip‐Pen Nanolithography (DPN) and Its Derivatives

Dip‐pen nanolithography (DPN) is a unique nanofabrication tool that can directly write a variety of molecular patterns on a surface with high resolution and excellent registration. Over the past 20 years, DPN has experienced a tremendous evolution in terms of applicable inks, a remarkable improvement in fabrication throughput, and the development of various derivative technologies. Among these developments, polymer pen lithography (PPL) is the most prominent one that provides a large‐scale, high‐throughput, low‐cost tool for nanofabrication, which significantly extends DPN and beyond. These developments not only expand the scope of the wide field of scanning probe lithography, but also enable DPN and PPL as general approaches for the fabrication or study of nanostructures and nanomaterials. In this review, a focused summary and historical perspective of the technological development of DPN and its derivatives, with a focus on PPL, in one timeline, are provided and future opportunities for technological exploration in this field are proposed.