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Enzyme Immobilization on Glass Surface for the Development of Phosphate Detection Biosensors F

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Enzyme Immobilization on Glass Surface for the Development of Phosphate Detection Biosensors F 1 Enzyme Immobilization on Glass Surface for the Development of Phosphate Detection Biosensors F. Sharmin 1 , S. K. Rakshit 1 , H.P.W. Jayasuriya 2 1 Food Engineering & Bioprocess Technology and 2 Agricultural Engineering, School of Environment, Resources and Development, Asian Institute of Technology, Thailand. [email protected]; [email protected]; [email protected] ABSTRACT Alkaline Phosphatase was immobilized on aminated glass fiber disks by covalent bond in the vacuum process. In this procedure, amide bonds were formed between carboxyl groups on the enzyme and amino groups on the glass surface. 10% Glycidoxypropyle trimethoxysilane was the best coupling reagent which could help form bonds between carboxyl and amino group on the glass fiber disk. A 10% concentration of coupling reagent, pH 9.0 and 2 gram of silica were found to be the best conditions for coupling the enzyme over the glass surface showed the highest enzyme activity. The covalent attached immobilized enzyme not only retained its activity but also could be reused at least 4 times after washing without loss of enzyme activity. Immobilized enzyme showed nearly 16% loss of enzyme activity after the first trial. An average of 1.65 mg of reusable alkaline phosphatase was immobilized per gram of glass fiber. Phosphate elements were measured from water, raw milk and raw shrimp sample by the used of this alkaline phosphatase immobilized disk as a biosensor. Immobilized enzyme can converts substrate to product and then will converts it to a measurable signal. This study demonstrates the possibility of using such a glass disk for the development of biosensors application. Keywords: Aminopropyl trimethoxysilane , Aminopropyle triethoxysilane, Chloroporpyle trimethoxysilane, Glycidoxypropyle trimethoxysilane, Alkaline phosphatase, P- nitrophenyl phoshphate, Immobilization, Biosensor. 1. INTRODUCTION Biosensors based on immobilized enzymes have many applications in areas such as industry, biochemistry and immunology and enzymology , pharmaceutical (Marconi, 1978; Cheetham, 1985), where a wide range of fixation techniques have been developed and are continuously being improved. Of these techniques, covalent coupling on inorganic supports is one of the most commonly used due to its stability in achieving a good enzyme attachment (Weetall, 1993). Enzyme immobilization is carried out by activating a functional group, either on the protein surface or on the solid support, with chemical reagents (Lunblad, 1995). The general formula of covalent attachment with glass, silica, is R-Si-X. R is organofunctional group and X is hydrolysable group. The R group is separated from the silicon atom by a propyl chain and X is alkoxy group,eg : metoxy , ethoxy (Veliky.I.A et al 1994) .The support for enzyme immobilization may be a membrane, a water-insoluble solid, or a polymer matrix. Immobilization on glass surface is cheapest way for the application of biosensor. Alkaline phosphatase has been F. Sharmin, S. Rakshit and H. Jayasuriya. “Enzyme Immobilization on Glass surfaces for the development of Phosphate detection Biosensors”. Agricultural Engineering International: the CIGR Ejournal. Manuscript FP 06 019. Vol. IX. April, 2007. 2 immobilized on a variety of surfaces (Surinenaite et al., 1996; Wiley et al., 2001; Filmon et al., 2002) but, to our knowledge, from the previous report of immobilization on glass (Weetall, 1969), the porosity of the glass beads they used and organic solvents applied led to considerable enzyme inactivation. It was expected that with better flat surface of the glass fibre disk that problem could be overcome and more effective sensor produced. Glass has advantage that is dimensionally stable and easy to clean thoroughly to remove contaminants by sterilization, maximum enzyme loading, economics of its preparation and application and regeneration, easy to handling, low possibility to loss of enzyme activity, and nontoxic. The major objective of this study was to Optimization of the composition of selected coupling reagents for the immobilization of alkaline phosphatase on the glass surface and Application of the immobilized enzyme for analysis of phosphate element in fish, milk and water samples. Kim et al (1996) considered biosensors to be an analytical device composed of biological element in intimate contact with physical transducer, which together relates the concentration of a target analyte to a measurable signal. Usually aim is to produce an electronic signal which is proportional in magnitude or frequency to the concentration of a specific analyte or group of analytes to which the biosensing element binds. Biosensors are composed of a detector and immobilized biocatalyst. Enzyme-based biosensors primarily rely on two operational mechanisms. The first mechanism involves the catalytic transformation of a pollutant (typically from a non-detectable form to a detectable form). The second mechanism involves the detection of pollutants that inhibit or mediate the enzyme's activity (Kim R. Rogers 2006). In this work the alkaline phosphates enzyme was used as a representative enzyme for immobilization on glass surface as its presence is easily selected due to its color reaction. For the same reason it is also tagged or conjugated to antibodies or antigen in Enzyme Linked Immunosorbant Assay (ELISA). 2. MATERIALS AND METHODS Glass fiber filter disk (2.4 cm dia.) purchased from Whatman company (cat no is 1820024). Silicon powder, four coupling agents Aminopopyl trimethoxy silane (APTMS), Aminopropyl triethoxy silane (APTES), chloropropyl trimethoxy silane (CPTMS) and Glycidoxypropyl trimethoxy silane (GOPS), Enzyme : Alkaline phosphatase, Substrate : P nitrophenyl phosphate and fish sample (shrimp),milk and water where all used in the cause of the experiment. 2.1 Optimization of enzyme immobilization on glass surface by using selected coupling reagents on the glass surface 2.1.1 Activation of silica filter disks The glass fiber disks were refluxed in 12N HCl for 3 hours. With the help of a Buchner funnel, Glass fiber filter disks washed with excess distilled water and dried in oven at 80°C for about 40 minutes. 2.1.2 Amination process The activated glass filter disks were animated as described by Russel et al. (2005) the glass fibre disks were fitted over a pressure equalizer funnel. Then different amount of silicon powder (2gm, 4gm, and 6gm) and 0.05 ml triethylamine was added over the disks. Then 0.1 M NaCl, toluene and 2 ml of coupling reagents (APTMS, APTES, CPTMS, and F. Sharmin, S. Rakshit and H. Jayasuriya. “Enzyme Immobilization on Glass surfaces for the development of Phosphate detection Biosensors”. Agricultural Engineering International: the CIGR Ejournal. Manuscript FP 06 019. Vol. IX. April, 2007. 3 GOPS) were mixed by using magnetic stirrer. This solution was added on the disk as a washing solution of silica by syringe in the pressure equalizer flask. Three different concentrations 10 %( v/v), 20 %( v/v) and 30 %( v/v) solutions of APTMS, APTES, CPTMS, GOPS in toluene were used for the second time refluxed of glass disk after amination for 18 hours. After that toluene, acetone and then distilled water was used respectively to rinsed the disks. 2.1.3 Immobilization of alkaline phosphatase A 10 mg/ml aqueous stock of bovine intestinal mucosa alkaline phosphatase was prepared and adjusted to different pH 7.0, 8.0 and 9.0 by using 1N NaOH. In 200 μl of these stock solutions glass fibre disks was soaked for approximately 10 second. These moist activated filter disks were lyophilized. After that the dried ALPase-disks were sealed under vacuum ( ≈ 50 m Torr) and incubated at 80°C for 96 hours as described by Russel et al. (2005). Immobilized disks were rinsed in a Buchner funnel with excess 0.1M NaCl and distilled water and then lyophilized and then stored at 4°C. 2.1.4 Immobilized alkaline phosphatase (ALPase) activity assay Standard method with p-nitrophenylphosphate (pNPP) as a substrate was used to determine ALPase activity. A stock solution of 350 μM pNPP in 25 mM glycine was prepared and adjusted to pH 9.6 with 1 N NaOH. The immobilized glass fiber filter disks were put into the test tube. At time zero, 20 ml of pNPP solution was added to the test tube with constant stirring. Every two minutes, a 90 μl aliquot was removed from the mix and placed in a well of a microtiter plate containing 10 μl of a stop solution composed of 0.1M NaOH and 0.1M EDTA. The appearance of the color indicated enzyme reaction. The enzyme activity of the sample was determined from the absorbance at 405nm of the ensuing solution measured using a spectrophotometer. The activity of immobilized ALPase was calculated using the formula: A (V /V ) Enzyme Activity = 405 reaction aliquot β Where, A405 is the absorbance at 405 nm Valiquot The volume of the aliquot read (0.1ml) Vreaction The volume of the reaction (20ml) and β is the extinction coefficient for the para-nitrophenol (pNP (18.5 at 405nm)) EU is the enzyme activity in micromoles of pNPP. 2.1.5 Quantification of enzyme in soluble form 20mL of the 350 μM pNPP substrate and free soluble ALPase were used to generate a standard activity curve. Initially the substrate was stirred vigorously and 0.5, 1, 5, 20, 100 μg of soluble ALPase from stock solution were added and incubated for 30 minutes and then the change in absorbance at 405nm measured. 5 different concentrations of soluble ALPase were examined, each in duplicate, and the mean values plotted on a standard curve. This standard curve was used to estimate the amount of immobilized ALPase activity. Between each trial the filter disk containing the immobilized ALPase were rinsed F. Sharmin, S. Rakshit and H. Jayasuriya. “Enzyme Immobilization on Glass surfaces for the development of Phosphate detection Biosensors”. Agricultural Engineering International: the CIGR Ejournal. Manuscript FP 06 019. Vol. IX. April, 2007. 1 three times with excess (approximately 3× 10 mL) 1M NaCl and distilled water by placing the disk in a Buchner funnel attached to a water aspirator.
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