Liquid photopolymerizable compositions as immobilized matrix of biosensors.
Series of liquid photopolymerizable compositions (LPhPC) based on oligouretanemetacrylate (OUM-1000T and OUM-2000T) and oligocarbonatemetacrylate (OCM-2), monomethacrylic ether of ethylene glycol and vinylpyrrolidone (VP) were tested. It was shown that the LPhPC, which contained VP (as basic hydrophylic matrix), OCM-2 (cross-linking agent) and OUM-2000T (to increase adsorption of polymer) was the most optimal. The blend contained 3 g/100 ml of enzyme. ISFET based biosensors for analysis of glucose and urea had the following characteristics: linear response in the range of concentrations 0.1-10 mmol/l, 0.05-20 mmol/l, angle of slope of concentration curve--30 mV/pC, 38 mV/pC, and response time of approximately 10-15, 5-10 min, correspondingly. The value of Km for immobilized urease and beta-glucose oxidase (GOD) achieved 0.85 and 3.1 mmol/l, respectively. It was established that under immobilization conditions at 20 degrees C the residual activity of GOD was about 35% from the initial level, the residual activity of horseradish peroxidase (HRP) and urease was 42% and 20%, respectively. In case of an immobilization of GOD at -50 degrees C its residual activity reached almost 50% from the initial level. It was investigated how different sources of UV radiation and different substances (including specific and non-specific substrates) influenced stability of the enzymes in the LPhPC and in the prepared membrane at storage. Dynamics of changes of enzyme activity at the process of photo immobilization was characterized, and requirements for enzyme maximal storage were selected. The proposed LPhPC may be prepared in advance since enzymes do not lose their activity during 2 months. Therefore, two processes, i.e. manufacturing of a transducer and preparation of a biological membrane on its surface, can be combined in one. In order to achieve this, approaches of modern electronics, such as for example photolithography, can be used. The developed LPhPC is homogenous, non-active to biological substances, permeable for the analyzed sample, can be prepared using a simple immobilization procedure, and has a defined hydrophobic-hydrophilic balance and sufficient level of adhesion to transducer surfaces. These all cover the requirements to modern biosensors.
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A.V. Palladin's Institute of Biochemistry of the National Academy of Sciences of Ukraine, 9 Leontovicha Street, Kyiv, Ukraine. nikstarodub@yahoo.com
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D015374:Biosensing Techniques / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004795:Enzyme Stability / Q000528:radiation effects; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005947:Glucose / Q000032:analysis; D005949:Glucose Oxidase / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D010777:Photochemistry; D011108:Polymers / Q000737:chemistry*; D013696:Temperature; D014508:Urea / Q000032:analysis; D014510:Urease / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D011108:Polymers; D014508:Urea; D005949:Glucose Oxidase; D006735:Horseradish Peroxidase; D014510:Urease; D005947:Glucose