Evaluation of Optical Sensor Platforms for Multiplexed Detection of Proteins

This work investigated optical sensor platforms for protein multiplexing, the ability to analyze multiple analytes simultaneously. Multiplexing is becoming increasingly important for clinical needs because disease and therapeutic response often involve the interplay between a variety of complex biological networks involving multiple, rather than single, proteins. Moreover, one biomarker may be indicative of more than one disease, similar diseases can manifest with similar physical symptoms, and monitoring a disease requires the ability to detect subtle differences over time. Multiplexing is generally achieved through one of two routes, either through spatial separation on a surface (different wells or spots) or with the use of unique identifiers/labels (such as spectral separation - different colored dyes, or unique beads - size or color). We looked into combining both spatial separation and unique labels to further expand the multiplexing capabilities of surfaces. Our original research resulted in one of the few demonstrations of reactive semiconductor nanocrystal immunoassays for multiplexed analysis within a single well on a microtiter plate. Innovative planar surface fluorescent immunoassays were developed for both spatial and spectral multiplexing using Quantum Dots and prospective incorporation into a Point-of-Care (POC) device involving an evanescent wave scanner. These assays used standard microscope slides combined with flow cells and were designed to markedly reduce the amount of sample and reagents needed as compared to standard 96-well plate assays. The platform was optimized for detecting Chicken IgG and Staphylococcal Enterotoxin B (SEB); SEB is commonly used in the literature to characterize the performance of biosensor platforms. The planar surface fluorescent immunoassays were applied to a real-world public health need to detect renal injury. Two emerging novel biomarkers, Kidney Injury Marker-1 (KIM-1) and Neutrophil Gelatinase Associated Lipocalin (NGAL), were investigated for their potential to detect injury earlier and with more specificity than current methods using serum creatinine (SCr). Detecting these medically-relevant markers using planar surface fluorescence immunoassays could potentially allow for more rapid diagnosis of acute kidney injury (AKI), among other uses