Size-controllable quartz nanostructure for signal enhancement of DNA chip
Introduction
Recently, numerous studies on fabrication of nanostructures and their applications to biotechnology have been reported (Kaji et al., 2003, Ogawa et al., 2007, Oillic et al., 2007b, Rosi and Mirkin, 2005, Anandan et al., 2006). A nanopattern of well-defined height and spacing generally offers several advantages for improving the sensing ability of a biosensor with several reasons. The first is increased surface area with high aspect ratio for the immobilization of more sensing probes. Second, appropriate spacing between the immobilized probes on the nanostructure enhances the accessibility of target materials (Oillic et al., 2007b). Finally, in an optical sensing system, a patterned surface can reduce the quenching effect of fluorescent signal materials by controlling immobilization and spacing. The fabrication of silicon-based high-aspect-ratio nanostructures is performed either by nanolithography followed by deep RIE (Reactive Ion Etching) or nanomolding (Choi et al., 2009, Fu et al., 2009). However, such methods still remain costly and problematic in the point that those methods still require expensive masks or master molds, and the cost for fabricating a mask or a master mold increases exponentially as the required resolution gets smaller and smaller in nanometer scale. Therefore, production of a nanopattern with well-defined height and spacing via a simple low-cost method is a crucial requirement for the successful construction of a highly sensitive biosensor system.
We previously developed an effective method for fabricating high-aspect-ratio pillar-like nanostructures on a quartz surface (Lee et al., 2010). Our method finely controls the spacing and height of the resulting nanopattern in nanometer-scale resolution over several centimeters by simple two-step reactive ion etching (RIE) with O2 and CF4 plasma without any expensive mask, additional equipment or complicated technology. The spacing was controlled by the O2 RIE time, and the height and shape of features in the nanopattern were mainly controlled by CF4 RIE time.
DNA chips, also called as DNA microarrays, have been developed to analyze the concentration of specific DNA of which the sequences are related to genetic disease, pathogenic microorganism, or gene expression (Bittel et al., 2005, Cho et al., 2006, Ito et al., 2007, Wen et al., 2004). This technology using immobilized DNA oligonucleotides allows highly parallel analysis by hybridization process, after which the DNA chip is analyzed by various methods such as surface plasmon resonance, electrochemical signaling or fluorescence level (Ahmed et al., 2007, Bin Lim et al., 2008, Lao et al., 2009, Wakai et al., 2004). Although DNA chips have great potential as a high-throughput detection method, their sensitivity on planar substrates is not particularly high due to the limitation of mixing efficiency and probe immobilization capacity (Oillic et al., 2007a). Therefore, pillar-like nanostructures have been synthesized on solid substrate for the detection of biomolecules (Kuwabara et al., 2008, Murthy et al., 2008, Park et al., 2009). In this study, we controlled the height and spacing of pillar-like nanostructures and examined their impact on DNA detection sensitivity. By adjusting the O2 and CF4 plasma treatment time of the two-step RIE procedure, the morphology of the structure was successfully modified. After immobilization of probe DNA and hybridization with fluorescently-labeled target DNA, the effect of morphology changes on signal intensity and detection limit were examined.
Section snippets
Nanostructure fabrication
Various quartz nanostructures were fabricated according to previously reported procedures (Lee et al., 2010). Fig. 1(a)–(d) shows brief schemes of the nanostructure fabrication procedures. (a) A 0.5 mm thick quartz wafer (Buysemi, Seoul, Korea) was spin-coated with PMMA A8 (Microchem, USA) resist. The thickness of the PMMA layer was estimated to be about 500 nm. (b) The resist-coated quartz substrates were exposed to O2 RIE for 1 min, 2 min or 3 min. During this process, dot-like nanostructures of
Preparation of nanopillar array
Fig. 2 shows the SEM images of the obtained nanostructure substrates with various heights and pillar densities (spacing). The heights of the nanopillars were 118 (±10), 182 (±24), and 269 (±26) nm for exposure times of 2, 5, and 10 min to CF4 RIE, respectively (Fig. 2(a)).The average distances between the centers of the pillars were 107 (±13), 127 (±14), and 161 (±19) nm for exposure times of 2, 5, and 10 min to O2 plasma, respectively. The diameter of the pillar was 61 (±5) nm for all substrates.
Morphology effect on the signal of DNA chip
Conclusions
In summary, a size-controllable nanopillar structure using only O2 and CF4 RIE was constructed. This structure had high surface density compared to that of planar quartz substrate. A DNA chip using the nanopillar structure had increased fluorescence intensity and specificity due to a high surface to volume ratio. The morphology of the nanostructure with various heights and spacing affected the sensitivity and specificity of the DNA chip experiment. Especially, the height and spacing of the
Acknowledgements
This study was supported in part by the Seoul R&BD Program (10543 and 10920), by the Korea Foundation for International Cooperation of Science and Technology (KICOS, No. M20601000002-09E0100-00200), by the National Research Foundation of Korea through the Pioneer Research Center Program (No. 2010-0002190) and by a grant of the Korea Health Care Technology R&D Project, Ministry of Health, Welfare and Family Affair, Republic of Korea (A084204).
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These authors contributed equally to this work.