Optically Switchable Molecular Machine-Inspired Nanoplasmonic Sensing Platforms for Early Cancer Detection

Abstract

Disease diagnostics enable physicians to diagnose cancer and monitor at-risk disease associated pathology sub-populations enabling implementation of lifesaving treatment at the earliest timepoint to improve patient prognosis. However, limitations in biosensing sensitivity and specificity at the point of disease onset and during the early stages of pathogenic progression have hindered identification of biomarkers capable of early clinical diagnostics. Moreover, it has been well documented in literature that the combination of multiple biomarkers from different bimolecular classes, such nucleic acids, proteins, exosomes and exosomal cargo molecules, increases both sensitivity and specificity while mitigating false responses for early cancer diagnostics when marker concentrations and concentration changes occur at extremely low levels. However, to date, scientists have been limited in this endeavor to combining various laboratory techniques in order to pool assay results of a diverse groups of biomarkers from various bimolecular classes. For example, modern bioanalytical techniques such as drop digital or quantitative reverse transcription polymerase chain reaction (ddPCR, qRT-PCR), next generation sequencing (NGS), mass spectrometry (MS) and electrochemistry have been used to assay nucleic acids, while lateral flow assay (LFA), western blot (WB), SERS and enzyme-linked immunosorbent assay (ELISA) are routinely utilized for detection and quantification of proteins. Furthermore, exosomes and exosomal cargo molecules have been assayed using nanopores, microarrays, immunoassays and fluorescence. However, these techniques are also hindered with high occurrences of false positive responses, are extremely labor intensive, require amplification and/or fluorescent labeling, and have extensive sample processing requirements. To overcome these challenges and improve accuracy, diagnostic technology has sought to develop a single platform with multiplex functionality that is also capable of adaptive detection of multiple types of biomarkers simultaneously using a single instrument. Current literature for multiplexed and multiparametric assay capability has been limited to microRNA and Protein detection with nanopores, plasmonics, PCR or mass spectrometry and detection of exosomes and exosomal cargo molecules achieved using microfluidic devices and fluorescence. Unfortunately, there has yet to be a single platform capable for adaptively assaying microRNA, proteins, exosomes and exosomal cargo molecules simultaneously, under identical device constructs in addition to, a device capable of achieving the unprecedented sensitivity and specificity needed for early cancer diagnostics. In this dissertation, a novel localized surface plasmon resonance (LSPR)-based sensing mechanism is introduced and utilized in the development of a photo-switchable molecular machine-inspired diagnostic platform. LSPR is a highly studied nanoscale phenomenon resulting from the oscillations of free electrons on the surface of metallic noble metal nanostructures when irradiated with light. These oscillations can be collected to produce dipole spectral absorption peaks and result in strong electromagnetic near-field enhancements ideal for developing optoelectronic devices. Consequently, this property is highly dependent, and tunable, based on the size, shape and composition of the nanostructure employed. Sensing mechanisms utilizing this phenomenon are conducted by observing a change in absorbance, bulk refractive index, and local refractive index. In this dissertation, a fourth novel mechanism is identified involving the dipole-dipole coupling interactions between the free electrons on the surface of the nanostructure and a zwitterionic spiropyran/merocyanine-based surface ligand. This innovative mechanism is utilized for the fabrication of an optically switchable molecular machine-inspired nanoplasmonic sensor (OSMINS)-based diagnostic platform capable of highly sensitive and specific adaptable assays for multiparametric analysis of patient biofluids. Additionally, the multiplex functionality on the OSMINS platform is ideal for rapid, and both label and amplification-free sample processing. The work presented in this dissertation is presented in five chapters, including: (1) Introduction. (2) Methods. (3) Dipole-dipole coupling mechanism elucidation and utilization in optoelectronic device fabrication to detect microRNA and protein for bladder cancer diagnosis. In this chapter, a new LSPR-based sensing mechanism was identified and explored through the development of a novel single nanostructure-zwitterionic organic molecule coupled plasmonic ruler (PR). A dipole-dipole coupling mechanism is hypothesized and supported through theoretical calculations on dipole polarizability using an inorganic-organic heterodimer model and experimentally by determining work function and interfacial dipole values. A PR is first fabricated utilizing different Au nanostructures (triangular nanoprisms (TNPs), bipyramids (BiPs) and rods (NR)) and then when TNPs and BiPs are found to generate a superior LSPR response, further optimization of the spiropyran (SP) surface concentration via SP-spacer self-assembled monolayer (SAM) ratios is investigated. Given the synergistic relationship between LSPR-based optoelectronic device fabrication and light activated molecular machines, the new dipole-dipole coupling mechanism and PR construct is employed to fabricate an adaptable photo-switching (APS) nanoplasmonic biosensor. The singleplex-based APS biosensor is employed to detect microRNA and protein in human plasma and urine, respectively, for bladder cancer diagnosis. This regenerative and reusable APS biosensor is shown to achieve a femtomolar limit of detection (LOD) assaying 10-healthy control (HC) and 10-metastatic bladder cancer patients attaining p values ranging from 0.0002-0.0001. (4) Fabrication and optimization of an optically-switchable molecular machine-inspired nanoplasmonic sensor (OSMINS)-based diagnostic platform is achieved and utilized in performing microRNA and protein singleplexing assays for early diagnosis of pancreatic cancer (PDAC) from at-risk disease associated pathologies. In this chapter, alkylthiol linker length is optimized for SP bound to TNPs to achieve an ultrasensitive attomolar concentration LOD for detecting circulating microRNA and protein. Two-dimensional conditioned cellular media studies and orthotopically implanted PDAC cell NOD scid gamma (NSG) mouse model study is conducted to assess OSMINS diagnostic potential for early PDAC diagnosis. The OSMINS platform is then deployed to assay oncogenic microRNA and protein in 11-PDAC, 20-chronic pancreatitis (CP), 6-intraductal papillary mucinous neoplasm (IPMN), and 20-HC patients achieving p values of 0.0001 (PDAC vs. HC, IPMN vs. HC), 0.0332 (CP vs. HC) and 0.1234 (PDAC vs. CP, IPMN vs. CP). Biostatistical analysis is used to pool biomarker results, meaning microRNA + protein, to improve CP vs. HC, PDAC vs. CP and IPMN vs. CP comparison p values to 0.0001. Cross-validation of the OSMINS platform is also presented using ddPCR and electrochemiluminescence (ECL) for microRNA and protein assays, respectively, showing excellent correlation. (5) Fabrication of a multiplexed and multiparametric OSMINS-based platform with receptor structure engineered molecular machine-enabled fully customizable assays of circulating microRNA, protein, exosomes and exosomal cargo molecules for early pancreatic cancer detection and prediction of Neoadjuvant chemotherapy (NAC) treatment response. Based on the results, a predictive model is developed for early cancer detection and patient monitoring. In this work, the previously presented OSMINS technology from chapter 4 is expanded and deployed to fabricate a 96 multi-well, high-throughput device for simultaneous assays of multiple biomarkers from various biomolecular classes in a single instrument run, allowing for direct comparison of results for the first time. OSMINS development from a singleplex solid-state biosensor into a multiplexed and multiparametric diagnostic platform is reported and assessed via three-dimensional conditioned cellular media study and a PDAC specific Patient-Derived Xenograft (PDX-21) mouse model study. The multiplexed and multiparametric OSMINS platform is then used to analyze 20-PDAC, 14-low grade IPMN, 6-high grade/invasive IPMN and 20-HC patient plasma samples for direct assay of microRNA, protein and exosomes as well as isolation of exosomes and assay of exosomal lysate for protein and microRNA cargo molecules. This work achieved p values ranging from 0.0001 to 0.1234, which is discussed in detail with regard to type of assay and marker biomolecular classes designation. Validation of multiplexed and multiparametric OSMINS platform is conducted via ddPCR, ELISA, and nanoparticle tracking analysis for microRNAs, proteins and exosomes, respectively. Finally, multiplexed and multiparametric OSMINS-based platform is utilized for 15-PDAC patients before and during NAC treatments to evaluate microRNA and protein biomarkers for their effectiveness in predicting NAC treatment response. Taken together, our multifaceted detection approach utilizing a novel multiplexed and multiparametric OSMINS-based sensing platform represents a paradigm shift in accessing the full diagnostic potential of current and future identified circulating biomarkers and their biomolecular cargo for early cancer diagnosis, monitoring of at-risk associated pathogenic conditions, and as predictive markers for patient treatment response.