All the SERS spectra were collected using × 50, NA = 0.5, long working distance INK-128 objective and the laser spot size is about 2 μm. SERS spectra were recorded with an accumulation time of 10 s. After the SAM of benzene thiol was formed on the substrate surface, a single scan was performed. To get an accurate approximation of the enhancement factors, we measured the neat Raman spectrum of benzene thiol. For the measurement of the neat Raman spectrum of benzene thiol, the power of the 785-nm laser was 1.031 mW, the accumulation time was 10 s, the spot size was 20 μm, and the depth of focus was 18 μm. Figure 2a

shows the Raman spectra of the benzene thiol SAM on the P-AAO-Au (black), W-AAO1-Au (green), and W-AAO2-Au (red) with all having been normalized to account for the accumulation time and laser power. To characterize the SERS performance of our substrates, commercial Klarite® substrates were used as reference samples which consists of gold-coated textured

silicon (regular arrays of inverted pyramids of 1.5-μm wide and 0.7-μm deep) mounted on a glass microscope slide. Figure 2b shows the normalized Raman spectra of the benzene thiol SAM on the W-AAO2-Au (red), on the Klarite® substrate (blue), and neat thiophenol (black). Figure 2 Comparison of substrates and neat benzene thiol, Raman spectra, and spatial mapping. (a) Comparison of the SERS of substrates P-AAO-Au, W-AAO1-Au, and W-AAO2-Au. (b) Comparison of the SERS of substrates W-AAO2-Au (red), Klarite® (blue), and neat Raman spectra (black) of benzene thiol collected at 785-nm incident. (c) Zoomed-in region of the spectra showing Selleck OSI 906 the three primary modes located near 1,000 cm-1, with the 998 cm-1 used for calculation of the SERS enhancement factor. The number of molecules of benzene thiol that each measurement is probing is denoted in the figure. (d) Spatial mapping of the SERS intensity at 998 cm-1

of SERS substrate W-AAO2-Au over an area larger than 20 μm × 20 μm. The background is the optical reflection image of substrate W-AAO2-Au photographed through a microscope with a × 50 objective. The calculation of EF The average EFs were calculated from the following equation Protein tyrosine phosphatase [8, 42]: where I SERS and I Raman are the normalized Raman intensity of SERS spectra and neat Raman spectrum of benzene thiol, respectively. N SERS and N Raman represent the numbers of molecules contributing to SERS signals and neat Raman signals of benzene thiol, respectively. I SERS and I Raman can be measured directly from the Raman spectra. N Raman is defined as follows [42]: where ρ = 1.073 g mL-1 and MW = 110.18 g mol-1 are the density and molecular weight of benzene thiol, respectively, and V is the collection volume of the liquid sample monitor. N A is Avogadro’s number. N SERS is defined as follows [42]: where ρ surf is the surface coverage of benzene thiol which has been reported as approximately 0.544 nmol cm-2[8, 42], and S surf is the surface area irradiated by exciting laser.