**1. Introduction**

Biological particles on the nanometer and submicrometer scale, such as proteins, lipids, nucleic acids, exosomes, and metabolic content, have attracted much attention as biomarkers for diagnosing diseases from biologically generated fluids such as blood, urine, and lymph. These biomarkers are now understood to be fundamental to healthy intercellular communication and can be produced in diseased cells. Label free Raman spectroscopy is useful for verification of biological samples ranging from nanoscale to millimeter size, such as tissue [1, 2], cells [3–5], bacteria [6, 7], exosomes [8, 9], and proteins [10, 11]. After incident laser emission with a single wavelength, Raman spectroscopy can identify biomarkers with the spectral peak position as a fingerprint because the molecular vibrations of the sample are

represented by spectra due to inelastic scattering. Electromagnetic enhancement can be achieved on rough surface of metal such as a gold or silver nanoparticle that causes amplification of the light by local surface plasmon resonance (LSPR) effects [12, 13].

A "hot spot" is formed on the surface of the SERS particles, and the Raman signal is dramatically increased at the nano-sized gap. Surface-enhanced Raman spectroscopy (SERS) is an approach for cell analysis and identification that applies a wide range of chemical spectroscopy to nanometer-sized biomarkers. Recent studies on monomolecular scales have been made possible through surface-enhanced Raman techniques [14–18]. According to finite element method (FEM) analysis, when the colloid is separated by 2 nm between a diameter of 30 nm colloids, a "hot spot" is formed which gives a surface-enhanced effect of about 108 degrees [13]. In biomedical applications, biomarkers suitable for these nanogaps are very rare, and due to the size and shape of biomolecules, research on nanogap and signal enhancement of the SERS structure is needed to optimize the LSPR effect.

In this chapter, we fabricated SERS substrate based on ZnO nanorods and improved the SERS effect by forming selective growth clustering of gold nanoparticles, which could be formed in specific condition of ZnO nanorod-based SERS substrate. To control the porosity and gold nanostructure, the length and density of the ZnO nanostructures and the thickness of the deposited gold were modified morphologically. The SERS enhancement mechanism was described based on finite element analysis. Cell viability was also evaluated to determine the presence or absence of toxicity for cancer cell applications. In other bio-applications, we demonstrate early diagnostic possibilities with Raman signals and statistical analyses from nano-sized biomarkers of intractable inflammatory diseases that cause patient pain.
