| | | | iomicrodevices including biochips, BioMEMS, micro-total analysis system (μ-TAS), and lab-on-a-chip (LOC) are of great interest today, and much research is focused on smart technologies to fabricate such devices. A critical and often limiting step for the fabrication of | biomicrodevices is the immobilization and patterning of biomolecules and their perfect alignment to microstructures on the device. Some state-of-the-art biomolecule patterning techniques offer very high resolution but cannot give precise alignment between biomolecules and the underlying microstructures. Some techniques can potentially solve the alignment problem but they are "slow" and not suitable for mass fabrication. More importantly, the biomolecule patterning step is always separated from the microfabrication of structural elements, and is therefore not integrated.
Recently, our group developed a novel approach termed "Focused Ion Beam (FIB) Biolithography" for micro/nanopatterning of DNA and protein and its integration into microfabrication. FIB-Biolithography is a maskless approach which allows fast fabrication of various desired biomolecule patterns with high resolution and perfect alignment of biomolecule patterns with other microstructures. It is a top down approach, based on Ga+ ion FIB direct writing/milling of patterns. The desired DNA or protein is first covalently immobilized onto a substrate material (e.g. silicon/SiO2) and a gold film is deposited onto the immobilized biomolecules. The gold film acts as a protection layer for the FIB milling process where the pattern is created by FIB milling of biomolecules inclusive of the protective gold film at defined areas, and leaves behind non-milled areas of gold protected biomolecules, forming the desired pattern. After FIB milling the protective gold film is removed by treatment with a cyanide solution and a functional pattern of DNA or protein is created.
Figure 1 shows fluorescent micrographs and their corresponding fluorescence intensity plots of "stripe like" patterns of oligonucleotide, NeutrAvidin and anti-Mouse IgG fabricated by FIB-Biolithography after bioaffinity assays were performed. The biomolecules show high biofunctionality retention, biofunctional specificity, and uniformity after FIBBiolithography. So far, a feature size of better than 500 nm was demonstrated for FIB-Biolithography.
During the DNA and protein patterning, integrated microfabrication of structures was achieved simultaneously by FIB-Biolithography. Figure 2 shows SEM and AFM images of DNA (oligonucleotide) patterns and microchannels fabricated by FIB biolithography. | | | As a result of the integrated biomolecule patterning and microfabrication, an inherently perfect alignment of the DNA patterns and the channels was achieved. 
The FIB-Biolithography approach is fast, flexible and controllable, and can be applied for integrated biomolecule patterning and microfabrication in prefabricated structures or devices where precise local manipulation is needed. Importantly, this integrated approach may be combined with other existing FIB microfabrication techniques to fabricate microstructures of electrodes, sensors or conductive heaters etc, and potentially allows the fabrication of complex integrated biomicrodevices. 
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