Development of fluorescence and radiolabel-free detection methods with enhanced sensitivity
In this thesis study, we present 1) sensitivity-amplified label-free biodetection and 2) label-free detection of airborne viruses with quartz crystal microbalance (QCM). Today, most sensitive and commonly used biosensors involve the use of fluorescent or radio labels. In an attempt to alleviate the additional cost and time required by these assays, numerous label-free detection techniques have been developed. However, most of these sensors have relatively poor sensitivity. This thesis study focuses on enhancing the sensitivity of platforms that do not require the use of fluorescent or radio labels. In this regard, chapter 2 and 3 comprise amplification schemes that include the use of nanoparticles and aptazymes (allosteric nucleic acids with enzymatic activity) and their application to a model label-free detection platform such as Quartz Crystal Microbalance (QCM). In chapter 2, we present the development of a quartz crystal microbalance biosensor for detection of folate binding protein (FBP). Using a simple folate-BSA conjugate adsorbed onto a Au-coated quartz sensor, a detection limit of 30 nM was achieved. A sandwich assay using an anti-FBP antibody and protein-A-coated gold nanosphere lowered detection limit to 50 pM (∼3 orders-of-magnitude improvement). In chapter 3, we demonstrate the use of aptazymes to enable mass-enhanced detection of small analytes (HIV-1 Rev peptide (MW ∼ 2.4 kDa) and theophylline (MW = 180 Da)) using a QCM sensor. In chapter 4, we present microbead-based rolling circle amplification (RCA) as a signal enhancement method to achieve a sensitive self-assembled optical diffraction biosensor. The bead-based diffractometry has the sensitivity of 10 pM for detecting platelet-derived growth factor B-chain (a disease marker indicative of various cancers). Recently, exposure assessment of nanoscale airborne organisms has become an important issue due to their potential hazards ranging from mild irritation to diseases. Hence, it is highly desirable to measure concentration of the airborne organism in a sensitive and rapid manner. In chapter 5, as a 'spinoff' from our previous QCM studies, we present real-time detection of airborne Vaccinia viruses using QCM. This study demonstrates the capture rate varies linearly with the concentration of virus suspensions (8.5 x 10 8 to 8.5 x 1010 particles/ml) at flow rates of 2.0 l/min and 1.1 l/min, indicating the general potential of mass sensitive detection of nanoscale biological entities in air. This dissertation first introduces the problems from a research point of view (chapter 1), then presents an overview of the experiments, and the results in detail (chapter 2-5). Finally it presents a summary and our plans for future work (chapter 6).
Savran, Purdue University.
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