Browsing by Author "Langlais, Sarah R."

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    Design of Anisotropically Shaped Plasmonic Nanocrystals from Ultrasmall Sn-Decorated In2O3 Nanoclusters Used as Seed Materials
    (American Chemical Society, 2022-12-07) Davis, Gregory A., Jr.; Prusty, Gyanaranjan; Hati, Sumon; Lee, Jacob T.; Langlais, Sarah R.; Zhan , Xun; Sardar, Rajesh; Chemistry and Chemical Biology, School of Science
    Ultrasmall inorganic nanoclusters (<2.0 nm in diameter) bridge the gap between individual molecules and large nanocrystals (NCs) and provide the critical foundation to design and prepare new solid-state nanomaterials with previously unknown properties and functions. Herein, for the first time, we report the monodispersed colloidal synthesis and successful isolation of metastable, rhombohedral-phase, <2.0 nm indium oxide (In2O3) nanoclusters. Ultrasmall nanocluster formation is controlled by a kinetically driven growth process, as evaluated through the variation of metal-to-passivating ligand concentrations. Although <2.0 nm-diameter In2O3 nanoclusters are synthesized in the presence of tin (Sn) precursors, they do not display typical localized surface plasmon resonance (LSPR) properties, which are commonly observed in Sn-doped In2O3 (Sn:In2O3) NCs. Our Raman and X-ray photoelectron spectroscopy and high-resolution transmission electron microscopy (HRTEM) analyses support the existence of Sn-decorated In2O3 nanoclusters, where Sn complexes reside on the surface of the nanocluster as Z-type ligands, as opposed to the formation of Sn:In2O3 nanoclusters, which behave as wide band gap (∼5.5 eV) nanomaterials. The experimentally determined band gap is in good agreement with the theoretical effective mass calculations. The newly synthesized Sn-decorated, 1.7 nm-diameter In2O3 nanoclusters are further used as reactive monomers for the seeded growth synthesis of bcc-phase, plasmonic Sn:In2O3 NCs via ex situ injection of In precursors without the addition of any Sn precursors. The LSPR peak of Sn:In2O3 NCs, which appear to form nanoflower assemblies, is tunable in the 1800–4000 nm region and possibly even the deep-IR region. In addition to altering the size and assembly of the spherical Sn:In2O3 NCs by introducing different amounts of indium acetylacetonate, injection of indium chloride precursors in the reaction mixture results in the formation of rod-shaped NCs. Surprisingly, Sn-decorated, <1.5 nm-diameter In2O3 nanoclusters do not grow into large plasmonic Sn:In2O3 NCs. Taken together, the results presented here contribute to the fundamental understanding of the surface free energy of ultrasmall metal oxide nanoclusters and further advance the knowledge on the phase transformation and growth of plasmonic NCs.
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    Optically Switchable Molecular Machine-Inspired Nanoplasmonic Sensing Platforms for Early Cancer Detection
    (2025-05) Langlais, Sarah R.; Sardar, Rajesh; Naumann, Christoph; Deng, Yongming; Goodpaster, John
    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.
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    β Cell microRNAs Function as Molecular Hubs of Type 1 Diabetes Pathogenesis and as Biomarkers of Diabetes Risk
    (bioRxiv, 2023-06-15) Syed, Farooq; Krishnan, Preethi; Chang, Garrick; Langlais, Sarah R.; Hati, Sumon; Yamada, Kentaro; Lam, Anh K.; Talware, Sayali; Liu, Xiaowen; Sardar, Rajesh; Liu, Jing; Mirmira, Raghavendra G.; Evans-Molina, Carmella; Pediatrics, School of Medicine
    MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in modulating gene expression and are enriched in cell-derived extracellular vesicles (EVs). We investigated whether miRNAs from human islets and islet-derived EVs could provide insight into β cell stress pathways activated during type 1 diabetes (T1D) evolution, therefore serving as potential disease biomarkers. We treated human islets from 10 cadaveric donors with IL-1β and IFN-γ to model T1D ex vivo. MicroRNAs were isolated from islets and islet-derived EVs, and small RNA sequencing was performed. We found 20 and 14 differentially expressed (DE) miRNAs in cytokine- versus control-treated islets and EVs, respectively. Interestingly, the miRNAs found in EVs were mostly different from those found in islets. Only two miRNAs, miR-155-5p and miR-146a-5p, were upregulated in both islets and EVs, suggesting selective sorting of miRNAs into EVs. We used machine learning algorithms to rank DE EV-associated miRNAs, and developed custom label-free Localized Surface Plasmon Resonance-based biosensors to measure top ranked EVs in human plasma. Results from this analysis revealed that miR-155, miR-146, miR-30c, and miR-802 were upregulated and miR-124-3p was downregulated in plasma-derived EVs from children with recent-onset T1D. In addition, miR-146 and miR-30c were upregulated in plasma-derived EVs of autoantibody positive (AAb+) children compared to matched non-diabetic controls, while miR-124 was downregulated in both T1D and AAb+ groups. Furthermore, single-molecule fluorescence in situ hybridization confirmed increased expression of the most highly upregulated islet miRNA, miR-155, in pancreatic sections from organ donors with AAb+ and T1D.