Development and application of glucose biosensor by using enzyme polymer conjugates

Enzymes are widely used in medicine, many fields of biotechnology and industry. In these fields, enzymes being stable against high temperature, pH and other media conditions are desired. For this reason, genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. Enzymes can be modified chemically by using water-soluble polymers or some chemicals. Modified enzymes, produced by this way, whose optimum pH range and optimum temperature is changed and also stable against chemicals found in media, are advantageous for various applications. Conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. The commercial enzyme, Glucose oxidase (β-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) is flavoprotein structured enzyme catalyzing a reaction between β-D-glucose and hydrogen peroxide and generating D-glucono-δ-lactone as a product. In this reaction molecular oxygen is used as an electron acceptor. Each GOX subunit contains one mole of flavin adenine dinucleotide (FAD) as cofactor which is not covalently bonded and loosely found in the structure. In this study, Glucose Oxidase (GOD) - polyethylene glycol (PEG) covalent conjugates were synthesized whose resistance was increased against high temperature. Firstly polyethylene glycol aldehyde derivative was synthesized and enzyme-polymer conjugates with different molar ratios (nGOD/nPEG: 1/1, 1/5, 1/10, 1/20) were synthesized using enzyme (GOD). The obtained conjugates were examined with GPC system and FTIR spectroscopy. Activities of synthesized conjugates and free enzyme at different temperatures and pH values are determined and results are compared. The conjugates showed lower activity compared to purified enzyme in all temperatures and pHs. However, the conjugates against increasing temperatures showed greater activity values when it is compared to free enzyme. The conjugate displayed quite well thermal stability against high temperatures. Storage stability of GOD-PEG conjugate was also studied and showed better stability than free enzyme. In this thesis study, the development of a glucose sensitive biosensor has been purposed by using micro sized potentiometric pH sensitive composite membrane sensor first time produced in Bioengineering's laboratories by the Prof.Dr. İbrahim Işıldak. The enzyme is be used to immobilize for composite pH sensor membrane surface or inside while biosensor is being prepared. The glucose-sensitive biosensor is not include inner reference electrode and inner reference solution. The biosensor is thought to be more suitable to make stable readings for a long time and to be used for continuous measurement systems. Potentiometric performance of biosensor will be examined with a computer-contolled measurement system designated.