Recently, Surface-enhanced Raman scattering (SERS) has attracted great attention because of its high sensitivity, label-free detection, and low detection limit. Additionally, it provides a specific fingerprint spectrum of multiplex samples with narrow band width. Because of these advantages, a number of research groups have investigated the SERS based biomolecular detection using the conventional DNA-DNA hybridizations and protein-protein interactions. Here, we demonstrated new approaches to detecting 1) Single Nucleotide Polymorphisms (SNPs) based Ligase Detection Reaction (LDR) and 2) peptides of vasopressin based aptamer-protein interactions techniques. SNPs have recently become key indicators of genetic disease, allelic variation associated with cancer progression, and pharmcogenomics. There are several methodologies such as primer extension, oligonucleotide ligation, invasive cleavage or hybridization for SNP detection. Among them, LDR is more accurate than hybridization approaches, permits parallel analysis of several loci directly on genomic DNA, and requires very low amounts of detection oligonucleotides per genotyping reaction. Therefore, we present a novel SERS-LDR approach by combining LDR reaction with SERS where a Raman spectrum was diagnostic for positive ligation and SNP discrimination. Figure 1 shows a new SERS-LDR schematic representation. In this technique, the upstream primer contains a SERS active dye and the discriminating 3' base, while the downstream primer contains an amine to which a silver nanoparticle is attached. When the two primers are ligated together the dye is brought into close proximity to the nanoparticle and its Raman signature is detectable. Different Raman active fluorophores are placed on the discriminating primers based on their genotype, which produce a signature Raman profile as shown in Fig. 1. To obtain the faster and more sensitive SERS signals, we implement the SERS-LDR method in the optofluidic device that enables us to enhance SERS detection by concentrating the multiplex products from the bulk phase into a confined volume. For the on-chip assays, the SERS-LDR products are concentrated into the 10 ìm wells by applying electric potential. Once concentrated, the well can be interrogated optically through the upper PDMS. To characterize the multiplex SNPs detection based SERS-LDR method, we examined the spectra collected on-chip at various ratios of wild type (TAMRA) to mutant type (fluorescein) templates (5:1, 3:1, 1:1, 1:3 and 1:5). The intensity of the peaks corresponds well with the ratios of the SNP templates. As expected, a near linear curve is generated. Using SERS-LDR products coupled with the electrokinetic device, we detected SERS signals for SNPs with a limit of detection on the order of 20 pM. As another approach, we demonstrate the SERS detection of vasopressin (VP) based aptamer interaction technique. Vasopressin is a peptide hormone, which can serve to rapidly increase arterial pressure thereby stabilizing the patient. As such, VP can be considered both a biomarker of a critical injury state and a therapeutic agent. In this study, to rapidly and specifically monitor VP, we develop the aptamer based SERS detection approach in a SERS-active microfluidic device. Briefly, aptamers are artificial nucleic acid ligands that can bind to a target molecule such as amino acids, drugs, and proteins with high specificity and affinity in a similar manner of antibodies. However, aptamers have significant advantages in that they are synthetically derived, making production predictable unlike antibodies. Figure 2a shows the schematic of the aptamer-VP recognition binding reaction. Briefly, to immobilize the aptamer, the thiolated aptamers were added onto the SERS-active silver substrate in PBS buffer solution. And then, the FITC-labelled VP in PBS buffer solution was introduced into the sample inlet port of the SERS device. To characterize the reaction specificity, the fluorescence intensity of each spot was analyzed with the aptamer-VP reaction procedures (Fig. 2b). From the fluorescent image results, it can be clearly seen that the aptamer has its specific recognition function toward VP. To improve the reproducibility, we examined the SERS spectra of VP based aptamers in a microfluidic device pattered 200 nm metal nano-tube array. For the fabrication of SERS-active chip, Au/Ag/Au (5 nm/190 nm/5 nm) was deposited onto the walls of the AAO membrane pores at an incident angle 45. After evaporation, dissolving the AAO in 3 M NaOH solution removes the template to obtain an array of nanotubes connected by a thin backing of the same metal, followed by changing the dissolving solution to the 3-mercaptopropyl-trimethoxysilane (MPTMS) solution. The MPTMS layer served as an organic adhesion layer aiding with the bonding between nano-tube array and PDMS substrate. For the bonding of the nano-tube array patterned PDMS and the PDMS channels patterned by soft-lithography, the surfaces of both layers were activated in oxygen plasma. To verify the selective binding assay of aptamer, we conducted SERS detection experiments according to the reaction procedures based apatmer (Fig. 2a). Figure 2c shows the SERS spectra collected from on-chip nano-tube arrays region for (1) background control of nano-tube array substrate, (2) aptamers immobilized onto the nano-tube array, (3) FITC-labeled bovine serum albumin (BSA) (negative control), and (4) FITC-labeld VP (positive control). As can be seen in Fig. 2c(3), our results show that small peak around 1600 cm-1 was observed from the negative control sample, but there are almost no detectable SERS signals beyond 1600 cm-1. For the positive control sample, the SERS spectra of VP are generated from the SERS active molecules located in the region of the nano-tube structures. Figure 2c(4) shows the correct spectroscopic fingerprints corresponding to FITC-labeled dye suggesting positive detection. To verify the ability for quantitative analysis, we conducted a series of experiments at different VP concentrations. The intensity of the peaks at 1823 cm-1 corresponds well with the increase of VP concentrations. Using the aptamer as VP recognition agent, we successfully detected SERS signals and demonstrated the ability to quantify the solution concentration based on the linearity of SERS intensity.&'