Highly Sensitive SERS Immunosensor via Extrinsic Nano-Stress Sensing
- Author(s): Apontti, Leonard James
- Advisor(s): Yan, Ruoxue
- et al.
Biosensors have begun to attract and find widespread use in the medical field due to their potential for ultrasensitive detection, and as a reliable, high-throughput, and nondestructive diagnostic immunoassay, specifically aimed towards the early screening of cancer. Specifically, there is need to screen the early stages of tumor growth, as 90% of all cancer related deaths are due to metastasis. Matrix metalloproteinase-14 (MMP-14) is an important target for cancer research due to its ability to induce tissue remodeling, extracellular matrix degradation, tumor invasion and angiogenesis. Surface Enhanced Raman Spectroscopy (SERS) has emerged as a popular method of detection due to the high optical enhancement offered by the surface plasmon resonances (SPRs) of metallic nanostructures while preserving the structural specificity, rapid screening, and high flexibility of Raman spectroscopy. The direct SERS analysis of biomolecules often suffer from poor selectivity and sensitivity due to their weak Raman activities, lack of characteristic functional groups/polarizable moieties, and poor surface selection rules with the nanoparticle surface. This study hopes to take advantage of a novel indirect approach termed “nano-stress” sensing which provides an excellent alternative to conventional direct sensing. In short, a Raman reporter with a highly resolved, well characterized spectrum is chemically functionalized with a biorecognition element (e.g. antibodies) and undergoes a change in peak intensity or position in response to a molecular recognition event (e.g. antibody-antigen binding). Chapter 2 describes the synthesis of silver octahedral nanoparticles using a modified polyol process and their assembly into a large-scale, close-packed, two-dimensional, thin film SERS platform, using a homemade Langmuir-Blodgett (LB) trough. SEM images reveal many well-ordered, close-packed polycrystalline domains scattered throughout. Chapter 3 presents the fabrication of the SERS immunosensor using a self-assembled monolayer (SAM) of 4-MBA, its chemical modification to bind to Fab3A2, and the detection of cdMMP-14 through extrinsic nano-stress sensing. Optimization experiments using TRIS Buffer indicate that a 0.1M/0.1M EDC/NHS solution at pH 6.0 with stirring gives the highest 4-MBA conjugation (~50%). About 38.58% Fab3A2 conjugation was observed along with the successful sensing of the biorecognition event between Fab3A2 and cdMMP-14 at various concentrations. A maximum frequency shift of 0.95±0.37cm-1 was observed for 5x10-7M cdMMP-14. A linear semilog relationship (R2=97.79%) was also observed giving the equation y = 0.31136x + 2.9109 for the concentration range 5x10-7M to 5x10-10M. Control experiments shows slight to relatively no binding with cdMMP-9 as well as minimal non-specific protein adsorption therefore confirming the specificity of the immunosensor.