Development of Reproducible Analytical Platform for Highly Sensitive SERS-based Assay

Abstract

Spectroscopy, which observes and interprets phenomena between light and matter, has been used in many diagnostic and sensing applications nowadays. Among them, Raman scattering is useful to biochemical and biomedical analysis. Raman scattering itself, however, has a very small optical cross-section which is problematic for the reproducibility of signal acquisition. This problem can be solved through surface-enhanced Raman scattering (SERS) which uses the properties of the metal surface. Raman scattering improved by SERS has larger optical cross-section than fluorescence. SERS is a phenomenon in which the scattering of a material located on the surface is amplified through the inherent optical properties of the metal surface. The mechanism of this phenomenon is explained by electromagnetic field enhancement and chemical enhancement. In particular, there is a singularity in which SERS is greatly enhanced, which is called a hot spot. This singularity appears between the junctions, gaps, and crevices of metal nanoparticles or substrates. The quantitative value correlated with the enhancement of SERS is expressed by calculating and comparing enhancement factor (EF). Since the majority of EFs in SERS is heavily influenced by hotspots, sensitivity is determined by how hotspots are formed. Metal nanoparticles are used to obtain the SERS. Metal nanoparticles form localized surface plasmons (LSP) on the surface, a feature associated with SERS. Hot spots can be easily derived at junctions or gaps caused by clustering of nanoparticles. However, uncontrollable aggregation of nanoparticles is the anxiety factor for analysis and sensing such as quantitative detection. Hot spots caused by the aggregation of particles allow for highly sensitive detection, but unintentionally this leads to a detection error. For example, unintended strong SERS responses affect the correlation to the analyte concentration and the result is a loss of reliability of detection. In other words, this problem causes an issue in the reproducibility of SERS-based sensing.

Unintentional aggregation of particles has a tendency from the macro perspective. In order to reflect this tendency in the detection result, a method of obtaining an averaged pattern from the SERS response of the entire analysis solution is required. This measurement can be achieved by improving the ensemble-averaged effect. Microfluidic devices can implement micro or nanometer-sized fluids. Using this device, it is possible to induce a uniform reaction continuously in the flowing state and to improve the ensemble-averaged effect. It is also called Lab-on-a-Chip (LOC) or micro total analysis system (μTAS) because it enables laboratory-scale experiments and comprehensive analysis on the chip itself. Microfluidic devices types are divided into a single-phase microfluidic and a microdroplet using two different phase fluids according to fluid phase combination. The problem with this technique is that highly sensitive detection techniques are required to measure small scale reactions in the channel. Therefore, SERS is the most suitable technique to overcome this problem.

SERS-based imaging analysis, is difficult to exploit the ensemble-averaged effect, which is a feature of the platform presented above. Measuring the nanoparticles in their essentially fixed state determines the reproducibility of the assay based on the state of the nanoparticles themselves. In this case, both strong SERS response and stability of individual single particles must be considered. Cellular imaging is the most widely used field in SERS-based imaging analysis. The solvent environment in which the diversification of biological elements in cellular imaging occurs has a great impact on nanoparticle stability. For example, changes in salt concentrations, pHs and temperatures can cause serious problems in particle stability and consistency of SERS response. Therefore, SERS nanotags for biological applications require stability against external factors. In addition, there is a need for nanotags with a uniform SERS response.

Chapter 2 introduced a microfluidic device for detecting two different DNA markers mixed at different concentrations through nanoparticle-based SERS detection. The continuous flow of the channel was measured under conditions in which the SERS signal was generated by uncontrolled hot spots from the aggregation of silver nanoparticles. At this time, since the ensemble-averaged effect was maximized, the reproducible analysis could be performed. Based on this feature, highly sensitive detection of two different DNA markers could be performed using silver nanoparticles. This is a successful result showing the excellent synergy between nanoparticle-based highly sensitive detection and programmed microfluidic channels.

Chapter 3 introduced microdroplet devices which minimized the repetitive process required by conventional immunoassays. The immunoassay has performed on the channel using nanoparticles and magnetic beads to which the corresponding antibodies were conjugated, respectively. The immunocomplexes were separated through the magnetic field inside the channel without any washing steps. This programmed device implemented assay method does not require a washing process by separating the droplets containing immunocomplexes and the droplets of supernatant. This SERS-based droplet device for immunoassay has no memory effect and automatically performs a complex immunoassay on one device. This platform is expected to be a potential new immunoassay method.

Chapter 4 introduced novel nanotags which can be used stably in various biological environments with strong SERS responses. Unlike conventional spherical gold nanoparticles, this new nanotags were synthesized using hollow gold nanoparticles with unique optical properties due to the pinhole structure generated during particle synthesis. The hollow nanoparticles have a consistent SERS response capability in a single particle state by pinholes. The silver shell was formed on the surface of the hollow nanoparticles to form nanogaps between the shell and core, and to obtain an improved of SERS response. This, unlike gold nanoparticles, shows strong improvement along with unique properties of the hollow nanoparticles which were obtained by pinholes. However, as the surface was changed to silver, problems of stability loss and cytotoxicity appeared. To solve this problem, the surface was treated with poly(ethene glycol) (PEG) and successfully improved stability and biocompatibility. This stable nanotag was used for imaging analysis to distinguish the phenotypes of two different breast cancer cells.

The platform techniques introduced in this thesis could improve the reproducibility of nanoparticle-based SERS detection. The convergence platform of these techniques has the greatest potential for SERS-based sensing systems. In addition, it is expected to provide a new platform as a highly sensitive detection technique in the field of point-of-care testing (POCT), such as on-site diagnostics.