| Gamma-aminobutyric acid (GABA) is one of two main inhibitory neurotransmitters in the central nervous system that plays an important role in neuronal function and dysfunction. However current techniques used to measure the extracellular concentrations of GABA require GABA to be either labeled, or modified prior to detection, due to its non-fluorescent and non-electroactive nature. In this study we proposed a piezoelectric immunosensor based method for detection of g-aminobutyric acid (GABA). Due to its small molecular weight of GABA molecule, a competitive assay design is appropriate for achieving high sensitivity for small molecules. As the first part of the study, a novel strategy for immobilization of bioactive GABA on gold substrates is proposed. Immobilization of GABA molecules on a rigid surface in an ordered fashion will provide an opportunity to understand some of the fundamental properties related to its structure and function. GABA was immobilized in three consecutive steps, namely gold substrate amination, dextran covalent attachment and GABA immobilization. Surface chemistry was verified at each step using XPS and FTIR. Bioactivity of GABA immobilized on the gold surface was studied using atomic force microscopy (AFM) to reveal antigen-antibody binding. Non-specific protein adsorption on the bioactive surface was analyzed quantitatively using anti-GABA antibody and an enzyme linked non-specific anti-immunoglobulin-G antibody in an ELISA assay. All these data support the presence of a bio-functional immobilized GABA on the gold surface. Next, a piezoelectric immunosensor was developed using surface modified GABA in a competitive assay. The affinity binding of monoclonal anti-GABA antibody on the modified piezoelectric crystals was studied in real time without any additional labels. For the binding interactions, the equilibrium association constant Keq was 14.5 mug·ml -1 for the anti-GABA antibody. Standard curve for measurement of GABA is also determined and the expected detection limit for GABA is approximately 42muM. However compared with the indirect sensing techniques such as optical absorption, and fluorescence, this sensitivity is still very limited. Theoretically, the mass sensitivity depends on the square of resonance frequency which is achieved by using thin resonators. Thus, the last part of our study is focused towards the development of a miniaturized quartz crystal by using standard photolithographic and wet etching technologies. Also finite element modeling by using FEMLAB and theoretical studies are incorporated in our study in optimizing the design of our piezoelectric sensor. |
