Plasma-assisted nanoscale protein patterning on Si substrates via colloidal lithography.

Selective immobilization of proteins in well-defined patterns on substrates has recently attracted considerable attention as an enabling technology for applications ranging from biosensors and BioMEMS to tissue engineering. In this work, a method is reported for low-cost, large scale and high throughput, selective immobilization of proteins on nanopatterned Si, based on colloidal lithography and plasma processing to define the areas (<300 nm) where proteins are selectively immobilized. A close-packed monolayer of PS microparticles is deposited on oxidized Si and, either after microparticle size reduction or alternatively after metal deposition through the PS close-packed monolayer, is used as etching mask to define SiO2 nanoislands (on Si). C4F8 plasma was used to selectively etch and modify the SiO2 nanoislands while depositing a fluorocarbon layer on the Si surface. The plasma-treated surfaces were chemically characterized in terms of functional group identification through XPS analysis and reaction with specific molecules. Highly selective protein immobilization mainly through physical adsorption on SiO2 nanoislands and not on surrounding Si was observed after C4F8 plasma-induced chemical modification of the substrate. The thickness of the immobilized protein monolayer was estimated by means of AFM image analysis. The method reported herein constitutes a cost-efficient route toward rapid, large surface, and high-density patterning of biomolecules on solid supports that can be easily applied in BioMEMS or microanalytical systems.

Creating highly dense and uniform protein and DNA microarrays through photolithography and plasma modification of glass substrates.

We demonstrate a method to create high density protein microarrays with excellent spot uniformity using photolithography and plasma processing on low cost commercially available microscope glass slides. Protein deposition and fluorescence signal evaluation on these substrates are performed by standard arrayers and scanners. To this end, spots of commercial photoresists (AZ5214, SU8 and Ormocomp(®)) were defined through lithography on glass substrates followed by short SF(6) plasma treatment and selective protein adsorption on these spots with respect to glass (spot to background fluorescence signal ratios 30:1 to 40:1) was demonstrated using model protein binding assays. Among the photoresists tested, Ormocomp was selected since it provided the highest protein binding capacity. No ageing of Ormocomp/glass substrates in terms of protein binding capacity was observed for at least two months. Besides to protein microarrays, DNA microarrays were also developed by spotting streptavidin-biotinylated oligonucleotide conjugates corresponding to wild- and mutant-type sequences of four deleterious BRCA1 gene mutations. For all of the examined mutations, higher specific hybridization signals (1.5-4 times) and improved discrimination ratios between wild- and mutant-type sequences as well as higher spot uniformity and repeatability were demonstrated on Ormocomp/glass substrates with intra- and inter-spot CVs of 8.0% and 4.5%, respectively, compared to commercial polystyrene (intra- and inter-spot CVs 36% and 18%) and epoxy-coated glass (intra- and inter-spot CVs 26% and 20%) slides. Thus, the proposed substrates can be readily applied to protein and DNA microarrays fabrication and, moreover, the described method for selective protein adsorption can be advantageously implemented in various analytical microdevices for multi-analyte detection.