Figure 4 show the SEM images of GLAD Ag nanorods fabricated on the (a-c) top and (d-f) bottom area of micropost structure on the UV-imprinted substrate, with varying the deposition angles of (a, d) 81°, (b, e) 85°, and (c, f) 89°. It was noted that the well-developed Ag nanorods were generated on the top of the micropost structure (Figure 4. (a-c)) and less developed Ag nanorods were formed on the bottom of the micropost structure due to the shadowing effect of micropost structure (Figure 4 d-f). Figure 4 (a-c) clearly show that the increasing deposition angle results in the increase of the distance between the nanorods and the decrease of the number of nanorods per unit area. Figure 4 (d-f) show that the increasing deposition angle decreased the formation of Ag nanorods on the bottom area due to the increase of shadowing effects of the microposts.  

2.3 Microarray chip preparation

The AgNMPA MEF substrates were functionalized with amino groups to improve immobilization efficiency of the capture antibodies. Silanes are well-proven effective coupling reagents with hydroxyl (OH)-functionalized surfaces, such as glass, silica, and the oxides of aluminum and silver [29]. 3-aminopropyl triethoxy silane (APTES), are used as coupling agents to covalently bind proteins to inorganic surfaces [30]. Surface functionalization was performed using a vapor-phase deposition method with a 200-ml glass bottle as the reaction chamber. Fifty milliliters of the APTES solution was added to the bottle, and the AgNMPA MEF substrate, fabricated by varying the deposition angle (81°, 85°, and 89°), was set in the bottle. After sealing, the bottle was placed on a hot plate and heat treated at 80 °C for 30 min. After the amine treatment process, the prepared chips were washed with distilled water and dried with nitrogen flux. As the reference substrates, the general GLAD substrate, which was composed of GLAD Ag nanorods on bare glass substrate, and the bear glass slide substrate, were also amine-treated in the same way as in the AgNMPA MEF substrate.
The capture antibody, antigen, and detection antibody, used for the evaluation of the developed AgNMPA MEF substrate, were taken from the MPIF-1 enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, cat. No. DY131). Firstly, MPIF-1 capture antibodies were diluted with the reagent diluent to a final concentration of 320 μg/ml. For spotting, the non-contact spotting system (pipejet nanodispenser, BioFluidix, Germany) was used. The arrayer tool was programmed to deliver approximately 15 nL of a capture antibody solution per spot in the 5 × 5 array, each spot was placed on top of the micropost structures with the alignment. In total, six arrays were spotted on each substrate; the spotted substrates were maintained at 4 °C for 15 h for immobilization.