Dendrimer‐Scaffold‐Based Electron‐Beam Patterning of Biomolecules

Microarrays have revolutionized the field of genomics and more recently proteomics and have proven to be an asset in high-throughput screening. As the demand for improved sensitivity and throughput of biomolecular assays increases, considerable research effort has been put into developing microelectronic and nanophotonic biosensors, which are presumably more sensitive than conventional fluorescencebased assays and have a faster response time. The lithographic technology for making the densely packed microelectronic devices in a high-throughput manner is already quite advanced and may be integrated to form biosensors. This is expected to increase the pace of research in early detection of disease biomarkers, discovering cell-signal transduction pathways, and in drug discovery. Denser arrays are also important for reducing reagent volume consumption and to improve sensitivity. Successful biomolecule patterning on sensor chip circuitry requires a number of important steps. First, selective immobilization of the probe and reduction in non-specific binding should be achieved for higher signal-tonoise ratios. Second, reduction in the sensor size reduces the background, enhancing the signal-to-noise ratio, and therefore biomolecule patterns on the same order as the sensors are desirable. Third, the biomolecule pattern should be aligned with the sensor circuitry, which becomes more difficult as sensor size decreases. Finally, a fabrication process should be formulated to ensure that the biomolecules are intact and functional, which is a challenge given the harsh microor nanofabrication processing steps. Here, we have demonstrated an electron-beam (e-beam)-based approach fulfilling the above requirements for patterning biological macromolecules that does not involve the use of resist, hence eliminating the exposure of these biomolecules to harsh resist-stripping processes that are normally employed to remove the resist. A non-biofouling poly(ethylene glycol) self-assembled monolayer (PEG-SAM) was selectively removed by e-beam and patterned with aldehyde-terminated polyamidoamine dendrimer (ald-PAMAM-SAM) in a layer-by-layer (LbL) manner to covalently immobilize the aminated oligonucleotide, which bind only to their complementary sequence targets and can be stripped and reprobed. The Generation-6 (G-6) PAMAM molecule, terminated with 256 primary amine groups and 6.7 nm in diameter, was used to increase the surface density of aldehyde functional groups to increase the oligonucleotide-immobilization efficiency. Current techniques for patterning biomolecules involve the use of polymer-based templates, which can be removed mechanically without the use of organic solvents after biomolecule immobilization. However, serious limitations exist in each case. Poly(dimethylsiloxane) (PDMS)-based soft-lithographic techniques cannot be used to create high-resolution patterns, as aligning the PDMS pattern with sub-micrometer features has been shown to work in a mix-and-match manner with an accuracy of only 2 lm. Although alignment is not an issue for biomolecule patterning based on polymer liftoff, as it is an integrated process, this method involves extra steps of deposition and etching a polymer film, which increases the complexity of the process. Challenges are also encountered as the size of the photolithographic patterns decrease due to the increase in line-edge roughness (LER) and the isotropic nature of oxygen plasma etch. Patterned gold has been used for creating protein patterns using thiolbased linkers, but gold surfaces cannot be tolerated in some biosensors as gold interferes with the optical signal or conductivity of the sensor. This technique also includes extra photolithographic and lift-off processing steps for patterning gold. Protein patterning using fluorescence-tagged proteins C O M M U N IC A IO N S