2. Experimental
2.1 Fabrication of Ag nanorods on micropost array (AgNMPA) metal-enhanced fluorescence (MEF) substrates
Figure 2 shows a schematic diagram of the fabrication process for the proposed AgNMPA MEF substrate. A silicon master, containing a micropost array with 300-m diameter, 50-m height, 600-m pitch, and 75 × 25 mm2-pattern area (size of a glass slide), was fabricated by the photolithography and reactive ion etching process (Figure 2(a)); a self-assembled monolayer was applied on it, as an anti-adhesion layer, to prevent the adhesion of following PDMS replication process by dipping the silicon master in a solution of 2% dimethydichlorosilane dissolved in octamethylcyclooctasilane (Repel-Silane ES, GE Healthcare Co., Ltd., Chicago, IL, USA). To fabricate a polymer micropost array on a glass slide substrate, a UV-imprinting process with PDMS mold was carried out. First, a replicated PDMS mold with microhole structures was obtained from the silicon master. The PDMS mold was obtained by the curing of a mixture of PDMS elastomer (Sylgard 184 A, Dow Corning Korea Ltd., Seoul, Korea) and curing agent (Sylgard 184 B, Dow Corning Korea Ltd., Seoul, Korea) at a weight ratio of 10:1. The mixture of PDMS was poured onto the Si master, which was then cured at room temperature (~ 20 °C) for 24 hours to minimize shrinkage (Figure 2(b)). After curing, the PDMS mold was released from the Si master. The UV-imprinting process was conducted on a glass slide substrate (25 × 75 mm2) using a UV-curable urethane acrylate photopolymer (UP088, SK Chemicals Co., Ltd., Seongnam, Korea) and the PDMS mold (Figure 2(c)). The UV-curable photopolymer was dropped on the glass slide substrate and the PDMS mold was carefully covered on it. The imprinting process was conducted with a UV exposing dose of 2000 mJ and compression pressure of 50 kPa. After the imprinting process the PDMS mold was peed off from the UV-imprinted micropost substrate. For microarray bio-chip applications, where several washings are required, adhesion between the imprinted micropost layer and GLAD Ag nanorods is an important factor. To improve the adhesion between Ag nanorods and the substrate, sequential layers of 10-nm nickel (Ni) and 50-nm Ag were deposited on the substrate using the electron-beam (E-beam) evaporator before the GLAD process (Figure 2(d)). Finally, the Ag nanorod structures were fabricated on a UV-imprinted micropost substrate via the GLAD technique, using an E-beam evaporator (Modified SEE-7, Ultech, Co., Ltd., Daegu, Korea) equipped with a sample holder capable of deposition angle and rotation speed control[23]. The substrates were mounted on a substrate holder in an E-beam evaporator during the deposition process; high vacuum conditions of approximately 5 × 10−6 Torr were achieved, and the growth rate during the deposition was monitored using a quartz crystal microbalance positioned at an incidence angle normal to the vapor source. The distance between the evaporation source and the rotating substrate was 680 mm. Ag pellets were used as the main source of the fabrication of the nanorods on the top of micropost array. During the GLAD process, the vertical Ag nanorods were formed due to the substrate rotation and the Ag nanorods were deposited only on the top of the micropost array due to the shadowing effects of pre-existing micropost strucutres (Figure 2(e)). Finally an Ag NMPA MEF substrate was obtained (Figure 2(f)). In each experimental conditions, the Ag nanorods were deposited on two imprinted substrates and a glass slide substrate. One of the GLAD deposited imprinted micropost substrates was used for antibody-antigen reaction experiment whereas the other was used for scanning electron microscopy (SEM, FESEM-6330F, JEOL, Ltd., Tokyo, Japan) measurements. To examine the effects of surface morphology on fluorescence enhancement and background noise reduction, various Ag nanorod structures were fabricated at the fixed deposition rates of 5 Å/s, substrate rotation speed of 5 rpm, deposition thickness of 500 nm, and varying deposition angles of 81°, 85°, and 89°. Finally, the reference GLAD substrate, fabricated by the same process as for the optimized AgNMPA MEF substrate, was comparatively examined to assess the performance of the optimized AgNMPA MEF substrate.