Assay developments for detection of biological analytes

• Main Author: Tan, Jiajun
• Other Authors: Sierin Lim
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Engineering::Bioengineering
• Online Access: https://hdl.handle.net/10356/143757
• Institution: Nanyang Technological University

Various ligands and analytes interact with biological systems in many ways. In certain cases, they may cause harm to the body. The detection of these ligands and analytes are therefore crucial. Their different physical and chemical properties require unique strategies and methods for the detection of these substances. This thesis demonstrates three different methods for different classes of substances such as ions and molecules of sizes below 100 Daltons to proteins in the range of 100 kilo-Daltons. Iron ions are important as intermediaries in bodily chemical reactions. Furthermore, they also play a crucial role in haemoglobin for oxygen transport in blood. Distinction between the two oxidation states is crucial as the homeostasis of iron ions is crucial for bodily functions. Here, a tannic acid shell, gold core nanocomposite was developed to detect Iron (III) ions. The nanocomposites are synthesized by a one-step one-pot synthesis method wherein the reduction of gold salt and oxidation of tannic acid to poly tannic acid shell takes place simultaneously. The chelation of Iron (III) (but not Iron (II)) to tannic acid results in the aggregation of the nanocomposites driving a distinct colour change from red to blue that is visible with the naked eye at a limit of detection of 5 M. The chelation is further exploited to detect H2O2 as H2O2 oxidizes Iron (II) ions to Iron (III), driving the chelation. The naked eye visible colour change for H2O2 detection is 0.4 M. While tannic acid-gold core nanocomposites show good utility for iron ion detections, other chemicals such as odorants require different approaches. Odorants are hydrophobic and do not chelate with tannic acid. In nature, these chemicals require the odorant binding protein for detection where they enter the binding pockets of odorant binding protein. To detect these odorants outside their natural environment, pig odorant binding protein (OBP) was produced recombinantly using an E. coli expression system. We hypothesized that the deletion of the alpha helix tail domain the in pig OBP would improve binding of odorants to the pig OBP and increase the pig OBP’s sensitivity to the odorants. This is because the tail is positioned at the entrance of the binding pocket and appears to act as a gate hindering odorants from readily entering the binding pocket. DNA cloning was done to delete the alpha helix tail. We found that the absence of the tail domain in fact lowers the binding affinity and increases the dissociation constant by between 32% and 434%. This data suggests that the alpha helix tail is crucial in providing stability for the ligand attachment to the binding pocket of the protein. In other words, removing the tail lowers the entropy of the system. Detection of more complex molecules, such as protein biomarkers will require proteins other than odorant binding proteins as these molecules are too large to enter the binding pocket of the OBPs. Their binding to other proteins that act as receptors also depend on their geometry charges and hydrophilicity. Here we worked to develop a cell-based assay for detecting such molecules. Cell-based assays are more advantageous then conventional system such as SPR and ELISA as it eliminates signals from unspecific binding. The cell-based assay is operated by presenting a CAR on the surface of the cell. The scFv antibody domain of the CAR serves as the receptor site. When a molecular biomarker binds to the CAR, it should cause a cross-linking and phosphorylation of the CAR followed by an intracellular signalling that can be coupled to various outputs (e.g. calcium influx, gene transcription). Here, our designed CAR is intended to bind the ligand epidermal growth factor receptor (EGFR-CAR). We show using confocal images that the CAR is presented on the surface of HEK293FT cells. Additionally, EGFR also binds to the expressed CAR. However, the expected calcium influx and phosphorylation upon ligand binding were not apparent suggesting that the absence of intracellular cell signalling taking place. The data suggest challenges for connecting the extracellular binding event of the EGFR-CAR to the intracellular signalling in the intended cell that need to be overcome for future development of cell-based assay.