**2. Nanorod manufacturing method**

Most of the research to fabricate SERS-based chips focuses on optimizing the surface of substrates through nanomaterials and nanostructures synthesized using sophisticated techniques such as lithographic patterning or high-temperature processes [16, 19–22]. Other research groups deposit Au/Ag nanoparticles on papers [23–25] or coat metal on a Si nanowire structure [26] to make a porous SERS substrate suitable for biological or liquid samples. Such Si nanowires are too dependent on the substrate and are difficult to combine with common cell culture substrates such as glass and petri dishes, due to their amorphous and manufacturing nature. In the case of paper-based SERS substrate, porosity and nanogaps could not be adjusted. On the other hand, if a ZnO nanorod-based platform is introduced, the substrate can be manufactured at a temperature below 100°C. Furthermore, homogeneous nanostructures can be formed without any lithography process on amorphous substrates such as glass and plastic, which are common in bioscience applications [27, 28].

To make the SERS substrates, the Si wafer were scribed with a size of 1 × 1 cm2 for substrate of ZnO nanorods initially. It was cleaned in ethanol and deionized (DI) water for 5 min, respectively. The 30 nm ZnO seed layer was deposited on the surface of as-prepared samples by using RF magnetron sputtering for 5 min under 100 W power to grow the vertically aligned ZnO nanorods utilizing by the hydrothermal synthesis. The ZnO growth solution was prepared by dissolving 10 mM zinc nitrate hexahydrate (Sigma-Aldrich Co.) and 0.9 mL of ammonium hydroxide (Sigma-Aldrich Co.) in 30 mL DI water. A homogeneous aqueous solution was achieved using mildly stirred vortexer for 5 min at room temperature. Then, the as-prepared samples were immersed into the aqueous solution in an oven at 90°C for 50 min.

**101**

**Figure 1.**

*Surface-Enhanced Raman Spectroscopy (SERS) Based on ZnO Nanorods for Biological…*

After ZnO growth, the substrates were cleaned with DI water and dried with nitrogen gas. Finally, the ZnO nanorods (NRs) were coated with Au using a thermal evaporator (Alpha Plus Co., Ltd., Korea). The thickness monitor for 100 and 200 nm deposition was standardized. The morphological and structural properties of the Raman measured samples were observed by using a field-emission scanning electron microscope (FE-SEM) (S-4700, HITACHI, Japan) with 15 kV beam voltage. The procedure of the experiment including the measurement analysis is

To obtain adequate porosity for the solution sample, the ZnO seed layer was modified and deposited such that the preferential growth direction of the zinc oxide nanorods was within about 10° from vertical. A volume of gold having a height of 100 and 200 nm per unit area was deposited on nanorods having length distribution of 300–450 nm or 500–650 nm, respectively. These four specimens were displayed with FE-SEM images of the 45° tilt view as shown in **Figure 2**. The top and bottom of the **Figure 2e** show the substrates with gold deposited (top) and not deposited (bottom) for **Figure 2a**, and ZnO is fully covered even when only 100 nm of gold is deposited. When the gold deposition is increased to 200 nm, the rod thickness is distributed about 10–30 nm thicker than when the gold deposition is not performed. Also, since the nanorod length distribution has a standard deviation of 50 nm and the deposited gold is clustered at the head of the nanorods, the height distribution of the gold clusters undergoes a similar variation. Therefore, when confocal Raman spectroscopy measurements are focused on the gold clusters, the

The Raman enhancement effect of SERS substrate based on ZnO nanorods was confirmed using 1 mM Rhodamine B drop, and the signals were measured after natural drying. Rhodamine B (RhB, >95%) purchased from Sigma-Aldrich was used as a standard for Raman measurements due to its refined condition. Raman measurements (LabRam Aramis, Horiba) were carried out using a 785 nm diode laser in a confocal geometry with a 0.5 NA, x50 objective lens and beam spot diameter ~1.9 μm. The spectrum of each point was measured in the range of 400–2500 cm<sup>−</sup><sup>1</sup>

*Schematic of the experiment involving zinc oxide nanostructure-based SERS substrate fabrication.*

and an integration time of 30 s at room

*DOI: http://dx.doi.org/10.5772/intechopen.84265*

**3. SERS metal growth and enhancement test**

schematically shown as a diagram in **Figure 1**.

with a spectral resolution of 5 cm<sup>−</sup><sup>1</sup>

head size can be a key factor in the Raman enhancement effect.

*Surface-Enhanced Raman Spectroscopy (SERS) Based on ZnO Nanorods for Biological… DOI: http://dx.doi.org/10.5772/intechopen.84265*
