Development of immunosensors and immunoassays using metallic nanostructures




Tuckmantel Bido, Ariadne

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Nanoplasmonic biosensors exploit the unique properties that arise from the interaction of light with free electrons of metallic nanostructures. They offer several advantages over the currently most employed methods of detection. These biosensors can be used for screening of infectious diseases, early diagnosis, management of chronic conditions, food quality and safety, among others. In this dissertation, the focus is on the development of surface-enhanced spectroscopy (SERS) and localized surface plasmon resonance (LSPR)- based immunosensors. A SERS-based immunoassay was developed for detection of IgG from human serum. A method for the sensor construction using poly(dimethylsiloxane) (PDMS) masks in 3D-printed platform, and extrinsic Raman labels (ERLs) were developed. SERS-based immunoassays are well-established in the research community. However, SERS-based sensors are questioned regarding their reproducibility and robustness. This is due to the large fluctuations in intensities in SERS measurements caused by inhomogeneous hotspots distributions in the sensing areas. The substantial local optical field variation within nanometers can affect reliability and reproducibility of these sensors. This effect particularly affects assays at low to ultralow concentrations (pM and lower) since there are less probed species per area in those conditions. Additionally, the common assumption that the analytical SERS intensities at all concentrations follow a Gaussian distribution can be detrimental to the reliable implementation of SERS-based assays. In this work, extensive SERS measurements were taken from the constructed sensors and the SERS intensities were shown to be log-normal distributed, particularly at low concentrations. Assessment of the number of measurements (sample size) per sensor at a particular concentration taken at different positions on the sensor surface were discussed. This study is a step forward towards the determination of best practices for a more reliable and robust employment of quantitative SERS-based immunoassays. A digital SERS protocol was also developed for the determination of SARS-CoV-2 (2019-nCoV) spike S1 + S2 ECD-His recombinant protein in saliva. In general, SERS-based immunoassays rely on a linear relationship between the average Raman intensity of a molecular reporter embedded in a SERS probe with the concentration of the analyte. As the concentration of the analyte decreases, the probability of a statistically significant number of SERS probes to be illuminated by the laser excitation also decreases. Additionally, there is large variation of intensities inherent to SERS and the inhomogeneous distribution of hotspots in the sensor being probed. These factors contribute to a loss in linearity of the assay at low concentrations of analyte and/or extrinsic Raman labels (ERLs). Here, an immunoassay was developed for determination of concentration of SARS-CoV-2 in saliva, and the conventional data treatment was compared to a digital protocol. The digital protocol generated a calibration curve with good linearity whereas the conventional approach did not. This approach is simple and can be employed in heterogeneous SERS immunoassays to improve both the limit-of-detection (LOD) and the dynamic working range. Finally, a low-cost localized surface plasmon resonance (LSPR)-based sensor was developed for SARS-CoV-2 screening in saliva. The sensor was built on plastic well plates for high throughput, but they can also be constructed in individual (discardable) plastic strips. The results were accessed using a plate reader, a ubiquitous equipment in laboratories and research centers. The sensor was challenged with 16 patient samples, being half COVID positive and half COVID negative. The sensor was developed in two modalities: 1) viral detection in saliva; and 2) antibody against COVID in saliva. Both sensors successfully classified all COVID positive patients (among hospitalized and non-hospitalized), and 7/8 COVID negative patients. This sensor is of low cost and of easy construction and is an alternative for SARS-CoV-2 screening in underserved communities. This sensor can be adapted to be used with other screening tests, by changing the element of recognition for other viral particles or antigens.



Biosensors, Immunosensors, Immunoassays, Plasmonics, Nanoparticles, Extrinsic Raman Lables, SERS, Raman, SERRS, Metallic Nanoestructures, LSPR, Digital sensing, SARS-CoV-2, Viral Detection, Screening tests, SERS hotspots