Single-molecule protein arrays enabled by scanning probe block copolymer lithography

Abstract

The ability to control the placement of individual protein molecules on surfaces could enable advances in a wide range of areas, from the development of nanoscale biomolecular devices to fundamental studies in cell biology. Such control, however, remains a challenge in nanobiotechnology due to the limitations of current lithographic techniques. Herein we report an approach that combines scanning probe block copolymer lithography with site-selective immobilization strategies to create arrays of proteins down to the single-molecule level with arbitrary pattern control. Scanning probe block copolymer lithography was used to synthesize individual sub-10-nm single crystal gold nanoparticles that can act as scaffolds for the adsorption of functionalized alkylthiol monolayers, which facilitate the immobilization of specific proteins. The number of protein molecules that adsorb onto the nanoparticles is dependent upon particle size; when the particle size approaches the dimensions of a protein molecule, each particle can support a single protein. This was demonstrated with both gold nanoparticle and quantum dot labeling coupled with transmission electron microscopy imaging experiments. The immobilized proteins remain bioactive, as evidenced by enzymatic assays and antigen-antibody binding experiments. Importantly, this approach to generate single-biomolecule arrays is, in principle, applicable to many parallelized cantilever and cantilever-free scanning probe molecular printing methods.dip-pen nanolithography | scanning probe lithography | single molecule array | gold nanoparticles | protein nanoarray P rotein immobilization on solid substrates with nanoscale control has been utilized in a variety of applications, including chip-based bioassays (1), proteomics (2, 3), drug discovery (3), and cellular biology studies (4). In cellular biology research, the ability to fabricate protein nanostructures on surfaces has enabled the study of many basic cellular functions including growth, signaling, and differentiation (4-7). For combinatorial molecular biology, the miniaturization of protein nanoarrays allows for smaller and higher density chips and the need for smaller sample volumes; in certain cases, this can translate into diagnostic systems with higher sensitivity and the ability to track disease and biological processes more efficiently (2, 3). The ability to site-specifically isolate single biomolecules can also facilitate molecular level studies of such structures (8, 9). Therefore, being able to nanofabricate biomolecular features at a resolution of 10 nm or less is of significant interest because this length scale approaches the dimensions of single protein molecules and offers an opportunity to address many previously unexplored biological phenomena.The use of dip-pen nanolithography (DPN) (4, 10) for the generation of arrays of biomolecules either by a direct deposition of proteins (11-13) or indirect methods (14, 15), through DPN writing of patterns followed by capture of proteins onto the patterns, has been widel...

Strategies for enhancing the sensitivity of plasmonic nanosensors

“…Plasmonic nanoparticles are ideal building blocks for the fabrication of nanoarrays because individual nanoparticles can serve as independent sensing units. Approaches for fabricating biomolecular nanoarrays, such as DPN [269][270][271][272][273][274][275][276][277][278][279] in combination with recently developed tools to track down individual nanoparticles of plasmonic nanoarrays and the method to normalize the signal discrimination could enable the generation of applicable plasmonic nanoarrays for the high-throughput detection of different types of biomolecules. L. Guo et al Anisotropic nanostructures exhibit higher refractive index sensitivity than spherical nanoparticles.…”

Delineating the pathways for the site-directed synthesis of individual nanoparticles on surfaces

“…The technique uses concepts from the block copolymer community (16) and the positional control offered by dip-pen nanolithography (DPN) (17) to deliver attoliter volumes of a metalcoordinated block copolymer onto a surface, which then can be used to synthesize individual nanoparticles (18,19). Importantly, SPBCL allows one to directly synthesize arbitrary patterns of single nanoparticles over large areas on a surface, which has been useful for immobilizing single proteins (20) and studying nanoparticle coalescence in situ (21). Thus far, the technique has been extensively used to synthesize Au nanoparticles, and there is one example of cadmium sulfide nanoparticle synthesis (18,19); however, when other metals and oxides are targeted, either they typically do not form or the technique results in multiple nanoparticles per feature.…”

Nano-bioelectronics via dip-pen nanolithography

“…The technology provides a means to fabricate novel structures. We have highlighted capabilities such as the arbitrary patterning of individual 5 nm nanoparticles, 116 or individual proteins, 135 and the patterning of multiple different proteins within subcellular areas 142 which are not achievable by other means. These tailored nanoenvironments, and others, will continue to justify DPN and PPL as enabling tools; using these techniques researchers can design new experiments and answer fundamental questions in bioelectronics and fundamental cell biology.…”