Lab-on-a-chip Thermoelectric and Solid-phase Immunodetection of Biochemical Analytes and Extracellular Vesicles: Experimental and Computational Analysis

Microfluidics is the technology of controlling and manipulating fluids at the microscale. Microfluidic platforms provide precise fluidic control coupled with low sample volume and an increase in the speed of biochemical reactions. Lab-on-a-chip platforms are used for detection and quantification of biochemical analytes, capture, and characterization of various proteins, sensitive analysis of cytokines, and isolation and detection of extracellular vesicles (EVs). This study focuses on the development of microfluidic and solid-phase capture pin platforms for the detection of cytokines, extracellular vesicles, and cell co-culture. The fabrication processes of the devices, experimental workflows, numerical analysis to identify optimal design parameters, and reproducibility studies have been discussed. Layer-by-layer assembly of polyelectrolytes has been developed to functionalize glass and stainless-steel substrates with biotin for the immobilization of streptavidinconjugated antibodies for selective capture of cytokines or EVs. Microstructure characterization techniques (SEM, EDX, and fluorescence microscopy) have been implemented to assess the efficiency of substrate functionalization. A detailed overview of current methods for purification and analysis of EVs is discussed as well. Additionally, the dissertation demonstrates the feasibility of a calorimetric microfluidic immunosensor with an integrated antimony-bismuth (Sb/Bi) thermopile sensor for the detection of cytokines with picomolar sensitivity. The developed platform can be used for the universal detection of both exothermic or endothermic reactions. A three-dimensional numerical model was developed to define the critical design parameters that enhance the sensitivity of the platform. Mathematical analyses identified the optimal combinations of substrate material and dimensions that will maximize the heat transfer to the sensor. Lab-on-a-chip cell co-culture platform with integrated pneumatic valve was designed, numerically characterized, and fabricated. This device enables the reversible separation of two cell culture chambers and serves as a tool for the effective analysis of cell-to-cell communication. Intercellular communication is mediated by extracellular vesicles. A protocol for the functionalization of stainless-steel probe with exosomespecific CD63 antibody was developed. The efficiency of the layer-by-layer deposition of polyelectrolytes and the effectiveness of biotin and streptavidin covalent boding were characterized using fluorescent and scanning electron microscopy

Advances in Biosensors Technology for Detection and Characterization of Extracellular Vesicles

Exosomes are extracellular vehicles (EVs) that encapsulate genomic and proteomic material from the cell of origin that can be used as biomarkers for non-invasive disease diagnostics in point of care settings. The efficient and accurate detection, quantification, and molecular profiling of exosomes are crucial for the accurate identification of disease biomarkers. Conventional isolation methods, while well-established, provide the co-purification of proteins and other types of EVs. Exosome purification, characterization, and OMICS analysis are performed separately, which increases the complexity, duration, and cost of the process. Due to these constraints, the point-of-care and personalized analysis of exosomes are limited in clinical settings. Lab-on-a-chip biosensing has enabled the integration of isolation and characterization processes in a single platform. The presented review discusses recent advancements in biosensing technology for the separation and detection of exosomes. Fluorescent, colorimetric, electrochemical, magnetic, and surface plasmon resonance technologies have been developed for the quantification of exosomes in biological fluids. Size-exclusion filtration, immunoaffinity, electroactive, and acoustic-fluid-based technologies were successfully applied for the on-chip isolation of exosomes. The advancement of biosensing technology for the detection of exosomes provides better sensitivity and a reduced signal-to-noise ratio. The key challenge for the integration of clinical settings remains the lack of capabilities for on-chip genomic and proteomic analysis