Investigation and validation of the use of surface enhanced Raman spectroscopy for minimally invasive, affordable detection of Alzheimer's disease biomarkers
Creators: name: Scher, Kaleigh Marie Ryan
Publisher: name: No Publisher Supplied
Year: 2023

description: There is an unmet need for affordable, minimally invasive detection methods ofAlzheimer’s disease (AD) biomarkers. Amyloid-β oligomers (AβOs) are highly correlated to disease onset and progression. However, their low concentration in blood has hindered their quantification in clinical settings. Surface enhanced Raman spectroscopy (SERS) based sensing platforms are an ideal choice for diagnostics, due to their ultra-high sensitivity and multiplexed detection capability. It was hypothesized that a SERS-based sensing platform could offer high enough sensitivity to enable detection of AβOs at the concentration at which they are expected to exist in blood. To ensure the development of a diagnostic tool with highest sensitivity and lowest deviation, different factors that impact the SERS signal were investigated. SERS in a tensorial technique, so the XYZ spatial orientation of the molecules impacts the resulting spectrum. Nanostars have demonstrated ultra-high sensitivity when used in SERS tags, so two different nanostar morphologies with different surface chemistries were studied. Three analytes, crystal violet, 4- aminothiophenol, and thiophenol, were selected as Raman reporters. It was found that analyte orientation changes can result in SERS signal intensity changes, depending on the surface chemistry of the nanoparticle, the analyte-nanoparticle mode of interaction, and preferred bond angle between analyte and nanoparticle surface, which could be mistaken as due to analyte concentration changes, impacting the reliability of the sensor. Through this study, one nanostar morphology, the 6-branched nanostar, demonstrated higher enhancement factor and lower coefficient of variation, and was therefore selected to synthesize the SERS tag for AβO capture and detection. The lower deviation in signal afforded by these stars is attributed to the uniform spike length, width, and number of spikes per star. Nanoparticle morphology is a key factor in the resulting enhancement factor and deviation of a SERS tag. Conventional methods of nanoparticle visualization consist of electron microscopy, which are 2D imaging techniques. When a 2D image is taken of a 3D object, valuable morphological information is lost. A 3D reconstruction technique would be highly advantageous in extracting nanoparticle features in all dimensions. 3D TEM is a method that enables 3D visualization of nanoparticles, but there are a number of limitations to this method. Few particles can be imaged at a time, and a small total volume can be analyzed by this method. This method does not expose internal morphology, due to the fact that the particle is imaged from different angles, rather than taking cross sectional images. FIB-SEM can be used to acquire images for 3D reconstruction, but to the best of our knowledge, reconstructions of particles smaller than 500 nm has not been achieved. In addition, there are many types of particles, such as porous particles or silica particles, that are difficult to reconstruct by the classic thresholding method due to voxel misidentification. FIB-SEM is applied here to enable reconstruction of particles down to 167 nm in diameter, core-shell structures, hollow, and porous silica particles. This is enabled by the high resolution of acquired images with a voxel size of 5 nm x 5 nm x 5 nm. The final chapter reviews the design and validation of an indirect SERS-based sensor for ultra-sensitive detection of AβOs. Unique capture of the oligomeric form of Aβ is enabled through the implementation of an aptamer that has demonstrated sensitivity and selectivity to AβOs, but not to monomeric or fibrillar Aβ. The sensor is then applied to a low concentration of AβOs in F-12 cell culture media, used to prepare AβOs from the monomeric state, and artificial cerebrospinal fluid (aCSF). Sensitivity at pg/mL concentrations was demonstrated in both systems. Next, the concentration of AβOs was varied in F-12. A good correlation was established between the concentration of AβOs and SERS peak intensity at 1440 cm-1, corresponding to the δ(CH) + ν(CC) vibrational modes. The R2 value is 0.9776 when plotted on a log(x) scale, where x is the oligomer concentration. An inverse correlation, rather than a direct correlation, is established between peak intensity and AβO concentration under these conditions. This is attributed to continuous aggregation of Aβ, which is most rapid at elevated concentrations, resulting in a final concentration that is different from the initial concentration. A high deviation is observed at the intermediate AβO concentrations, which was hypothesized to be attributed to the aggregation during the AβO incubation period with the sensor surface. Therefore, Aβ aggregation behavior at incubation conditions (one hour at room temperature) was investigated with three different solutions, aCSF, F-12, and phosphate buffered saline (PBS), using dynamic light scattering (DLS). It was found that AβOs aggregate at a much faster rate in F-12 than aCSF and PBS, and that aggregation is most rapid at higher initial AβO concentrations. Overall, the work presented here highlights experimental factors that can impact the reliability of a SERS sensor, expands the 3D reconstruction capabilities using FIB-SEM to allow for reconstructions of particles smaller than 200 nm in diameter, and establishes SERS as a useful tool in detecting low concentrations of AβOs.