2.4 Fluorescence signal measurements
After immobilization, the AgNMPA MEF substrate, fabricated by varying the deposition angles (81°, 85°, 89°), was placed into the hybridization cassette (Arrayit, cat. no. AHC1×16) and blocked with the reagent diluent, 1% (v/v) BSA diluted in 0.1 M phosphate buffered saline(PBS), pH 7.2–7.4 (R&D Systems, Catalog # DY995), with 200 μL in each well for 1 hour at room temperature and subsequently washed thrice with 200 μL of washing buffer containing 0.05% Tween in PBS, pH 7.2–7.4 (R&D Systems, Catalog # WA126). MPIF-1 standard solution (antigen) with concentrations of 500 pg/ml were prepared in reagent diluent buffer and incubated in each well with 100 μL for 2 hours at room temperature for antibody-antigen reaction, and subsequently washed thrice with washing buffer. 100 μL of biotinylated anti-MPIF-1 detection antibody (100 ng/ml) was added to each well and incubated for 2 hours at room temperature. After three washes, Cy-3-conjugated streptavidin (100 μL per well) was added at a concentration of 0.002 mg/ml, and incubated for 20 min at room temperature, followed by another three washes with washing buffer and drying with filtered nitrogen. Fluorescence measurements were performed at room temperature by employing a microarray scanner (GenePix 4000B, Molecular Devices, USA) at an excitation wavelength of 532 nm. Figure 5 shows the schematics of AgNMPA MEF microarray chip for fluorescence measurement. We spotted the antibody every 2 micropost to check the difference between spotting area on micropost and non-spotting area on micropost. Ideally there are no Ag nanorods on bottom of micropost, less developed Ag nanorods were formed on the bottom of micropost due to the limited shadowing effects of micropost depending on the deposition angle as shown in Fig. 4(d-f).