Abstract
Metabolites are the reactants, intermediates, and products of metabolism. It is estimated that metabolism of a biological cell comprises 7900 metabolites. Metabolites are important in many biological functions and involve in the normal cell growth, development, reproduction, energy generation and transfer. Hence, development of a selective and sensitive analytical method for the detection of metabolites can provide important insights for many cellular processes. For the metabolites detection using electrochemical method, one of the fundamental limitations is the requirement of redox enzymes to generate electrochemical signals. However, there are only limited numbers of redox enzymes in nature which are applicable for metabolites detection by using electrochemical method. Many metabolites and drug molecules which do not have the corresponding redox enzymes are important in medical diagnosis and pharmaceutical industry. For example, sulfa drugs which are important for the treatment of many diseases and killing bacteria, have no corresponding redox enzymes to catalyze redox reaction to deliver the electrochemical signals. The objective of this thesis is to develop an electrochemical sensor which does not require a redox enzyme to generate electrochemical signal to overcome the fundamental limitation of electrochemical method for the selective detection of metabolites and drug molecules. The electrochemical sensor is constructed by using a recombinant protein, SNAP-hCAII and a dialkyldisulfide linker (Lip-BG) which consists of a disulfide moiety for the self-assembly on gold electrode and a benzylguanine (BG) for SNAP-tag protein labeling. The addition of electrochemical probe Ferrocene-Linker-Sulfonamide (Fc-Linker-SFA) will result in the binding of SFA to the hCAII protein in which the ferrocene will be in the close proximity to the gold electrode to enhance the electrochemical current. Upon addition of sulfonamide drug, the drug will displace the Fc-Linker-SFA from the hCAII binding and as a result, the electrochemical signals will decrease.