Zepto w6

The fabricated
nanoneedles were oxidized by O₂ plasma for 10 min in the
Low-pressure Plasma System (ZEPTO-W6, Diener electronic GmbH &
Co). The oxidized surfaces of nanoneedle were functionalized with
amine group by incubating with 2% APTES in ethanol for 3 h. The nanoneedles
were then washed three times with ethanol and dried for 1 h at 100
°C. Next, NHS-Biotin (100 μg/mL) was coated to the nanoneedles
for 1 h. After washing with PBS five times, the biotinylated nanoneedles
were reacted with poly streptavidin (50 μg/mL) for 18 h and
washed with DI water five times. Finally, the biotinylated locked
activator probes (5 μM) were added to the polystreptavidin/biotin/nanoneedles
array. After washing with PBS three times, ATP aptamer-1 and aptamer-2
(10 μM) in a hybridization buffer solution(5X SSC, 750 mM NaCl
and 75 mM trisodium citrate) were added to the locked activator functionalized
nanoneedles and incubated for 6 h, followed by washing twice with
the rinsing buffer solution (2X SSC and 0.05 wt % Tween-20).

The ceramic mold was first coated with a thin layer of oil (WD40-silicone) to reduce adhesion. Next, the first polymer was prepared and degassed in a standard vacuum chamber (Tarson, 402020) for ~10 min to remove bubbles. The first polymer was then poured onto the lubricated mold and placed into the vacuum chamber to facilitate additional bubble removal. The first layer was allowed to cure for 24 h at room temperature on a leveled surface to ensure flatness. The polymerized first layer was then carefully separated from the ceramic mold, and the edges removed to achieve the desired size of the first layer. Then a brief oxygen plasma treatment (duration ~1 min, power ~7, Diener Electronic, Zepto W6) was done to the flat side of the first layer and to a high optical quality glass (Siegert Wafer, fused silica substrate, 35 × 35 mm² with a thickness of 500 μm), then the two components were attached and placed in an oven at 70 °C for 30 min for bonding.