Interferometric imaging for pathogenic bacteria identification and antibiotic susceptibility testing

Pathogenic bacterial infections are a serious threat to public health, claiming millions of lives every year. In order to contain the spread of infectious diseases sensitive and timely diagnosis of pathogenic bacteria is of significant importance. The rapid detection of low abundance analytes is still challenging in the most common bacteria detection techniques including, culture and colony counting, Enzyme-linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR). Conventional bacteria detection techniques suffer from limitations such as low sensitivity, cost, long procedural time and requiring complex lab equipment. Thus, there is a critical need for rapid, sensitive and low-cost bacterial detection platform in various applications ranging from water and food safety to medical diagnosis. The quest to overcome these limitations have sparked significant interest in innovative biosensor development, with considerable emphasis on optical techniques. Among optical biosensors, label-free methods are highly desirable over label-based alternatives for eliminating the additional cost and sample processing required for labeling. Also, techniques for whole-cell bacteria detection are preferred to detection of pathogenic molecular components detection due to the requirement for extracting and isolating the desired bacterial components such as nucleic acids or proteins. Overall, label-free whole-cell detection of pathogenic bacteria has a significant advantage of simplicity in sample preparation that translates to time and cost reduction. An additional benefit of detecting whole-cell bacteria without labels, thus in their natural environment, is the ability of monitoring the growth and replication of individual pathogens with a potential application in antimicrobial susceptibility determination. Despite the significant advantages of antibiotics as one of our most powerful tools for fighting infections, their extensive misuse and overuse over the years, have resulted in the emergence of antibiotic resistant bacteria as the global health crisis of our time. The current gold-standard technique for antibiotic susceptibility testing (AST) used in clinics, is culture-based disk diffusion assays. The time-consuming diagnosis method of the common clinical susceptibility testing, which is an inherent limitation of culture-based techniques, have necessitated the need for an alternative AST analysis platform. A clinical diagnosis test that could perform rapid pathogenic bacteria identification and determine its susceptibility to a panel of selected antibiotics, would greatly reduce the hospital stay time for patients with bacterial infection, therefore decreasing mortality and morbidity rate. In addition, it will have a great economic impact on the global healthcare system by advising optimal antibiotic use and maintaining the value of existing drugs. In this dissertation, we describe the design and development of a rapid, sensitive, and multiplexed biosensor platform that can both identify pathogenic bacteria and perform image-based AST on a single reader instrument. The simple and low-cost design of our biosensing platform makes it a perfect candidate as a point-of-care (POC) diagnostic tool in clinical setting. The biosensor presented in this dissertation is based on interferometric enhancement of the visibility of individual biological particles, such as viruses and bacteria, afforded by Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS), previously developed in our group. The integration of SP-IRIS with microfluidic flow cells provides kinetic measurements capability, by enabling in-liquid imaging of the sensor surface in real-time, therefore making it a promising diagnostic platform. Here, we build upon the SP-IRIS platform and utilize it for pathogenic bacteria identification and image-based AST analysis. To validate our biosensor's functionality, we demonstrate E. coli detection and characterization in end-point and real-time measurement modality through particle detection and tracking analysis of the acquired images from sensor surface. In addition, we perform rapid image-based AST analysis for E. coli bacteria against two antibiotics, ampicillin and gentamicin, by monitoring single cell morphological variations and tracking their growth rate under various antibiotic challenges

Highly sensitive and label-free digital detection of whole cell E. coli with interferometric reflectance imaging

Bacterial infectious diseases are a major threat to human health. Timely and sensitive pathogenic bacteria detection is crucial in identifying the bacterial contaminations and preventing the spread of infectious diseases. Due to limitations of conventional bacteria detection techniques there have been concerted research efforts towards development of new biosensors. Biosensors offering label free, whole bacteria detection are highly desirable over those relying on label based or pathogenic molecular components detection. The major advantage is eliminating the additional time and cost required for labeling or extracting the desired bacterial components. Here, we demonstrate rapid, sensitive and label free E. coli detection utilizing interferometric reflectance imaging enhancement allowing for visualizing individual pathogens captured on the surface. Enabled by our ability to count individual bacteria on a large sensor surface, we demonstrate a limit of detection of 2.2 CFU/ml from a buffer solution with no sample preparation. To the best of our knowledge, this high level of sensitivity for whole E. coli detection is unprecedented in label free biosensing. The specificity of our biosensor is validated by comparing the response to target bacteria E. coli and non target bacteria S. aureus, K. pneumonia and P. aeruginosa. The biosensor performance in tap water also proves that its detection capability is unaffected by the sample complexity. Furthermore, our sensor platform provides high optical magnification imaging and thus validation of recorded detection events as the target bacteria based on morphological characterization. Therefore, our sensitive and label free detection method offers new perspectives for direct bacterial detection in real matrices and clinical samples.First author draf

Ag–Ag2S Hybrid Nanoprisms: Structural versus Plasmonic Evolution

Recently, Ag–Ag2S hybrid nanostructures have attracted a great deal of attention due to their enhanced chemical and thermal stability, in addition to their morphology- and composition-dependent tunable local surface plasmon resonances. Although Ag–Ag2S nanostructures can be synthesized via sulfidation of as-prepared anisotropic Ag nanoparticles, this process is poorly understood, often leading to materials with anomalous compositions, sizes, and shapes and, consequently, optical properties. In this work, we use theory and experiment to investigate the structural and plasmonic evolution of Ag–Ag2S nanoprisms during the sulfidation of Ag precursors. The previously observed red-shifted extinction of the Ag–Ag2S hybrid nanoprism as sulfidation occurs contradicts theoretical predictions, indicating that the reaction does not just occur at the prism tips as previously speculated. Our experiments show that sulfidation can induce either blue or red shifts in the extinction of the dipole plasmon mode, depending on reaction conditions. By elucidating the correlation with the final structure and morphology of the synthesized Ag–Ag2S nanoprisms, we find that, depending on the reaction conditions, sulfidation occurs on the prism tips and/or the (111) surfaces, leading to a core(Ag)–anisotropic shell(Ag2S) prism nanostructure. Additionally, we demonstrate that the direction of the shift in the dipole plasmon is a function of the relative amounts of Ag2S at the prism tips and Ag2S shell thickness around the prism.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted versio

Ag–Ag₂S Hybrid Nanoprisms: Structural <i>versus</i> Plasmonic Evolution

Recently, Ag–Ag₂S hybrid nanostructures have attracted a great deal of attention due to their enhanced chemical and thermal stability, in addition to their morphology- and composition-dependent tunable local surface plasmon resonances. Although Ag–Ag₂S nanostructures can be synthesized <i>via</i> sulfidation of as-prepared anisotropic Ag nanoparticles, this process is poorly understood, often leading to materials with anomalous compositions, sizes, and shapes and, consequently, optical properties. In this work, we use theory and experiment to investigate the structural and plasmonic evolution of Ag–Ag₂S nanoprisms during the sulfidation of Ag precursors. The previously observed red-shifted extinction of the Ag–Ag₂S hybrid nanoprism as sulfidation occurs contradicts theoretical predictions, indicating that the reaction does not just occur at the prism tips as previously speculated. Our experiments show that sulfidation can induce either blue or red shifts in the extinction of the dipole plasmon mode, depending on reaction conditions. By elucidating the correlation with the final structure and morphology of the synthesized Ag–Ag₂S nanoprisms, we find that, depending on the reaction conditions, sulfidation occurs on the prism tips and/or the (111) surfaces, leading to a core­(Ag)–anisotropic shell­(Ag₂S) prism nanostructure. Additionally, we demonstrate that the direction of the shift in the dipole plasmon is a function of the relative amounts of Ag₂S at the prism tips and Ag₂S shell thickness around the prism

Ag–Ag₂S Hybrid Nanoprisms: Structural <i>versus</i> Plasmonic Evolution

Recently, Ag–Ag₂S hybrid nanostructures have attracted a great deal of attention due to their enhanced chemical and thermal stability, in addition to their morphology- and composition-dependent tunable local surface plasmon resonances. Although Ag–Ag₂S nanostructures can be synthesized <i>via</i> sulfidation of as-prepared anisotropic Ag nanoparticles, this process is poorly understood, often leading to materials with anomalous compositions, sizes, and shapes and, consequently, optical properties. In this work, we use theory and experiment to investigate the structural and plasmonic evolution of Ag–Ag₂S nanoprisms during the sulfidation of Ag precursors. The previously observed red-shifted extinction of the Ag–Ag₂S hybrid nanoprism as sulfidation occurs contradicts theoretical predictions, indicating that the reaction does not just occur at the prism tips as previously speculated. Our experiments show that sulfidation can induce either blue or red shifts in the extinction of the dipole plasmon mode, depending on reaction conditions. By elucidating the correlation with the final structure and morphology of the synthesized Ag–Ag₂S nanoprisms, we find that, depending on the reaction conditions, sulfidation occurs on the prism tips and/or the (111) surfaces, leading to a core­(Ag)–anisotropic shell­(Ag₂S) prism nanostructure. Additionally, we demonstrate that the direction of the shift in the dipole plasmon is a function of the relative amounts of Ag₂S at the prism tips and Ag₂S shell thickness around the prism

Ag–Ag₂S Hybrid Nanoprisms: Structural <i>versus</i> Plasmonic Evolution

Recently, Ag–Ag₂S hybrid nanostructures have attracted a great deal of attention due to their enhanced chemical and thermal stability, in addition to their morphology- and composition-dependent tunable local surface plasmon resonances. Although Ag–Ag₂S nanostructures can be synthesized <i>via</i> sulfidation of as-prepared anisotropic Ag nanoparticles, this process is poorly understood, often leading to materials with anomalous compositions, sizes, and shapes and, consequently, optical properties. In this work, we use theory and experiment to investigate the structural and plasmonic evolution of Ag–Ag₂S nanoprisms during the sulfidation of Ag precursors. The previously observed red-shifted extinction of the Ag–Ag₂S hybrid nanoprism as sulfidation occurs contradicts theoretical predictions, indicating that the reaction does not just occur at the prism tips as previously speculated. Our experiments show that sulfidation can induce either blue or red shifts in the extinction of the dipole plasmon mode, depending on reaction conditions. By elucidating the correlation with the final structure and morphology of the synthesized Ag–Ag₂S nanoprisms, we find that, depending on the reaction conditions, sulfidation occurs on the prism tips and/or the (111) surfaces, leading to a core­(Ag)–anisotropic shell­(Ag₂S) prism nanostructure. Additionally, we demonstrate that the direction of the shift in the dipole plasmon is a function of the relative amounts of Ag₂S at the prism tips and Ag₂S shell thickness around the prism

Ag–Ag₂S Hybrid Nanoprisms: Structural <i>versus</i> Plasmonic Evolution

Recently, Ag–Ag₂S hybrid nanostructures have attracted a great deal of attention due to their enhanced chemical and thermal stability, in addition to their morphology- and composition-dependent tunable local surface plasmon resonances. Although Ag–Ag₂S nanostructures can be synthesized <i>via</i> sulfidation of as-prepared anisotropic Ag nanoparticles, this process is poorly understood, often leading to materials with anomalous compositions, sizes, and shapes and, consequently, optical properties. In this work, we use theory and experiment to investigate the structural and plasmonic evolution of Ag–Ag₂S nanoprisms during the sulfidation of Ag precursors. The previously observed red-shifted extinction of the Ag–Ag₂S hybrid nanoprism as sulfidation occurs contradicts theoretical predictions, indicating that the reaction does not just occur at the prism tips as previously speculated. Our experiments show that sulfidation can induce either blue or red shifts in the extinction of the dipole plasmon mode, depending on reaction conditions. By elucidating the correlation with the final structure and morphology of the synthesized Ag–Ag₂S nanoprisms, we find that, depending on the reaction conditions, sulfidation occurs on the prism tips and/or the (111) surfaces, leading to a core­(Ag)–anisotropic shell­(Ag₂S) prism nanostructure. Additionally, we demonstrate that the direction of the shift in the dipole plasmon is a function of the relative amounts of Ag₂S at the prism tips and Ag₂S shell thickness around the prism

Ag–Ag₂S Hybrid Nanoprisms: Structural <i>versus</i> Plasmonic Evolution

Recently, Ag–Ag₂S hybrid nanostructures have attracted a great deal of attention due to their enhanced chemical and thermal stability, in addition to their morphology- and composition-dependent tunable local surface plasmon resonances. Although Ag–Ag₂S nanostructures can be synthesized <i>via</i> sulfidation of as-prepared anisotropic Ag nanoparticles, this process is poorly understood, often leading to materials with anomalous compositions, sizes, and shapes and, consequently, optical properties. In this work, we use theory and experiment to investigate the structural and plasmonic evolution of Ag–Ag₂S nanoprisms during the sulfidation of Ag precursors. The previously observed red-shifted extinction of the Ag–Ag₂S hybrid nanoprism as sulfidation occurs contradicts theoretical predictions, indicating that the reaction does not just occur at the prism tips as previously speculated. Our experiments show that sulfidation can induce either blue or red shifts in the extinction of the dipole plasmon mode, depending on reaction conditions. By elucidating the correlation with the final structure and morphology of the synthesized Ag–Ag₂S nanoprisms, we find that, depending on the reaction conditions, sulfidation occurs on the prism tips and/or the (111) surfaces, leading to a core­(Ag)–anisotropic shell­(Ag₂S) prism nanostructure. Additionally, we demonstrate that the direction of the shift in the dipole plasmon is a function of the relative amounts of Ag₂S at the prism tips and Ag₂S shell thickness around the prism