Small Molecule Sensing by Local pH Modulation
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Our laboratory has previously invented and characterized a technique for studying ligand-receptor interactions on supported lipid bilayers (SLBs) without fluorescently labeled analytes. This technique utilizes a pH-sensitive dye labeled phospholipid, ortho-rhodamine B conjugated POPE (1-hexadecanoyl-2-(9Z-octa-decenoyl)-sn-glycero-3-phosphoethanolamine), as a membrane imbedded reporter molecule to monitor ligand-receptor interactions on SLBs by measuring the fluorescence intensity change. Also, this technique has been demonstrated to operate as either a “turn-on” or a “turn-off” sensor depending on the analytes to be detected. Based on these preliminary studies, it is necessary to give further development and application for this new technique. Firstly, as a potential application of this sensing technique, we utilized this sensing platform to detect the interactions between tetracaine, a positively charged small molecule used as a local anesthetic drug, and planar supported lipid bilayers (SLBs). Studies with membranes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) yielded an equilibrium dissociation constant value of Kd = 180±47 µm for this small molecule-membrane interaction. And the influences of cholesterol, phosphatidylethanolamine, and three negatively charged lipids on the interaction between tetracaine and the bilayers have been studied. Secondly, in order to fully develop the power of this local pH modulation technique, it has been extended to a homogeneous platform from an SLB based heterogeneous platform. Specifically, a pH sensitive dye, 5-(and-6)-carboxyfluorescein, was conjugated to free lysine residues on the surfaces of designated capture proteins. The fluorescein intensity was found to change upon target molecule binding. The assay was used to follow the binding of immunoglobulin G (IgG) to streptavidin, thiamin monophosphate (ThMP) to thiamin binding protein A (TbpA), and Ca^(2+) to calmodulin (CaM). Finally, as an instrumental development for this sensing platform, an inexpensive fluorescence detector has been built as a cheap substitute for the epi-fluorescence microscope. This device is composed of a compact camera, a schott glass color filter, and a green (532 nm) laser pointer. This device has been tested to be able to obtain fluorescence images with decent signal to noise ratio. And this device has been demonstrated in measuring tetracaine-POPC SLB interaction using local pH modulation method.
Huang, Da (2013). Small Molecule Sensing by Local pH Modulation. Doctoral dissertation, Texas A & M University. Available electronically from