Browsing by Author "Masterson, Adrianna Nichole"

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    Fabrication of Lspr-Based Multiplexed and High-Throughput Biosensor Platforms for Cancer and Sars-Cov-2 Diagnosis
    (2022-05) Masterson, Adrianna Nichole; Sardar, Rajesh; Long, Eric; Manicke, Nicholas; Naumann, Christoph
    Designing and developing a diagnostic technology that is capable of highly sensitive and specific, multiplexed, high-throughput, and quantitative biomarker assays for disease diagnosis and progression is of the upmost importance in modern medicine and patient care. Current diagnostic assays capable of multiplexed and high-throughput analysis include mass spectrometry, electrochemistry, polymerase chain reaction (PCR), and fluorescence-based techniques, however, these techniques suffer from a lack in sensitivity, false responses, or extensive sample processing that are detrimental to clinical diagnostics. To overcome these sensitivity challenges, the field of nanoplasmonics has become utilized when developing diagnostic assays. Plasmonic-based diagnostic tests utilize the unique optical, chemical, and physical property of nanoparticles to increase the sensitivity of the assay. In this dissertation, novel diagnostic platforms that utilize nanoparticles and their localized surface plasmon resonance (LSPR) property will be introduced. LSPR is an optical property in noble metallic nanoparticles that is referred to as the collective oscillation of free electrons upon light irradiation. It is highly dependent on the shape, size, and dielectric constant (refractive index) of the surrounding medium of the nanoparticle and LSPR sensing is based on a change in these properties. In this dissertation, the LSPR property is utilized to fabricate nanoplasmonic-based diagnostic platforms that are capable of multiplexed and high-throughput biomarker assays, with high sensitivity and specificity. The work presented in this dissertation is presented as six chapters, (1) Introduction. (2) Methods, (3) Fabrication of a LSPR-based multiplexed and high-throughput biosensor platform and its application in performing microRNA assays for the diagnosis of bladder cancer. In this chapter, the advancement of single-plex solid state LSPR-based biosensors into a multiplexed and high-throughput diagnostic biosensor platform is reported for the first time. The diagnostic biosensor platform is first fabricated utilizing different gold nanoparticles (spherical nanoparticles, nanorods, and triangular nanoprisms), and then with the gold triangular nanoprisms as the nanoparticle of choice, microRNA assays were performed. The developed biosensor platform is capable of assaying five different types of microRNAs simultaneously at an attomolar limit of detection. Additionally, five microRNA were assayed in 20-bladder cancer patient plasma samples. (4) Development/optimization of the biosensor platform presented in Chapter 3 for the detection of COVID-19 biomarkers. In this chapter, the biosensor platform utilized in Chapter 3 was designed to assay 10 different COVID-19 specific biomarkers from three classes (six viral nucleic acid gene sequences, two spike protein subunits, and two antibodies) with limit of detections in the attomolar range and with high specificity. The high-throughput capability of the biosensor platform was advanced, with the platform performing analysis of a single biomarker in 92 patient samples simultaneously. Additionally, the biomarker platform was utilized to assay all 10 biomarkers in a total of 80 COVID-19 patient samples. (5) Further optimization of the biosensor platform for the development of a highly specific antibody detection test for COVID-19. During the COVID-19 pandemic, knowledge was gained on the specificity of antibodies produced against COVID-19. In this chapter, that knowledge was applied towards the optimization of the biosensor platform presented in Chapter 4 in order to assay SARS-CoV-2 neutralizing antibody IgG. The optimization of the biosensor platform included the size of the gold triangular nanoprisms and the receptor molecule of choice. The biosensor platform assayed this highly specific COVID-19 IgG antibody with a limit of detection as low as 30.0 attomolar with high specificity and no cross reactivity. Additionally, as a proof of concept, the biosensor platform was utilized in a high-throughput format to assay SARS-CoV-2 IgG in a large cohort of 121 COVID-19 patient samples simultaneously. (6) Advancement of the biosensor platform from a 96-well plate to a 384-well plate and its application in assaying microRNA for early diagnosis of pancreatic cancer. In this chapter, the high-throughput capabilities of the biosensor platform presented in Chapters 3-5 was expanded by increasing the sensor amount in one platform from 92 to 359. The 384-well plate biosensor platform was designed, optimized, and utilized to perform microRNA assays for early-stage pancreatic cancer diagnosis. The optimization of the biosensor platform included the manipulation of LSPR-based parameters and the -ssDNA receptor molecule in order to obtain low limit of detections (high sensitivity). Additionally, the biosensor platform assayed two microRNA in a large cohort (n=110) of pancreatic cancer and chronic pancreatitis patient samples.