Immobilization of motile bacterial cells via dip-pen nanolithography.

A strategy to bind bacterial cells to surfaces in a directed fashion via dip-pen nanolithography (DPN) is presented. Cellular attachment to pre-designed DPN generated microarrays was found to be dependent on the shape and size of the surface feature. While this observation is likely due in part to a dense, well formed mercaptohexadecanoic acid (MHA) monolayer generated via DPN, it may also simply be due to the physical shape of the surface structure. Motile Pseudomonas aeruginosa bacterial cells were observed to bind to DPN generated mercaptohexadecanoic acid/poly-L-lysine (MHA/PLL) line patterns, 'blocks' made up of eight lines with 100 nm spacings, with approximately 80% occupancy. Cellular binding to these 'block' surface structures occurs via an electrostatic interaction between negatively charged groups on the bacterial cell surface and positively charged poly-L-lysine (PLL) assemblies. These data indicate that these DPN generated 'block' surface structures provide a promising footprint for the attachment of motile bacterial cells that may find utility in cell based biosensors or single cell studies.

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.

Self-leveling two-dimensional probe arrays for Dip Pen Nanolithography.

Scanning probe lithography (SPL) has witnessed a dramatic transformation with the advent of two-dimensional (2D) probe arrays. Although early work with single probes was justifiably assessed as being too slow to practically apply in a nanomanufacturing context, we have recently demonstrated throughputs up to 3x10(7) microm(2)/h--in some cases exceeding e-beam lithography--using centimeter square arrays of 55,000 tips tailored for Dip Pen Nanolithography (DPN). Parallelizing DPN has been critical because there exists a need for a lithographic process that is not only high throughput, but also high resolution (DPN has shown line widths down to 14 nm) with massive multiplexing capabilities. Although previous methods required non-trivial user manipulation to bring the 2D array level to the substrate, we now demonstrate a self-leveling fixture for NanoInk's 2D nano PrintArray. When mounted on NanoInk's NLP 2000, the 55,000 tip array can achieve a planarity of <0.1 degrees with respect to the substrate in a matter of seconds, with no user manipulation required. Additional fine-leveling routines (<2 min of user interaction) can improve this planarity to <0.002 degrees with respect to the substrate-a Z-difference of less than 600 nm across 1 cm(2) of surface area. We herein show highly homogeneous etch-resist nanostructure results patterned from a self-leveled array of DPN pens, with feature size standard deviation of <6% across a centimeter square sample. We illustrate the mechanisms and methods of the self-leveling fixture, and detail the advantages thereof. Finally, we emphasize that this methodology brings us closer to the goal of true nanomanufacturing by automating the leveling process, reducing setup time by at least a factor of 10, enhancing the ease of the overall printing process, and ultimately ensuring a more level device with subsequently homogeneous nanostructures.

Attachment of motile bacterial cells to prealigned holed microarrays.

Construction of biomotors is an exciting area of scientific research that holds great promise for the development of new technologies with broad potential applications in areas such as the energy industry and medicine. Herein, we demonstrate the fabrication of prealigned microarrays of motile Escherichia coli bacterial cells on SiOx substrates. To prepare these arrays, holed surfaces with a gold layer on the bottom of the holes were utilized. The attachment of bacteria to the holes was achieved via nonspecific interactions using poly-l-lysine hydrobromide (PLL). Our data suggest that a single motile bacterial cell can be selectively attached to an individual hole on a surface and bacterial cell binding can be controlled by altering the pH, with the greatest occupancy occurring at pH 7.8. Cells attached to hole arrays remained motile for at least 4 h. These data indicate that holed surface structures provide a promising footprint for the attachment of motile bacterial cells to form high-density site-specific functional bacterial microarrays.

Electrochemical attachment of motile bacterial cells to gold.

Selective attachment of Escherichia coli K-12 bacterial cells to charged gold surfaces was demonstrated. Electrostatic binding of E. coli K-12 bacterial cells to positively charged surfaces was observed starting at +750mV. The binding of E. coli K-12 cells to positively charged gold surfaces is proposed to occur due to long-range electrostatic interactions between the negatively charged O-chain of lipopolysaccharide (LPS) molecules protruding the bacterial cell body and the electrode surface. Removing LPS alters the cellular surface charge and results in cellular attachment to negatively charged surfaces. Thus, applying an electrical potential allows for the direct, real time detection of live, dead or damaged bacterial cells. The attachment of E. coli K-12 bacterial cells to surfaces with an applied potential substantiates the hypothesis that an electrostatic interaction is responsible for the binding of bacterial cells to positively charged molecular assemblies on surfaces used for building bacterial microarrays.