DESIGN OF GLUCOSE BIOSENSOR USING IMMOBILIZATION OF GLUCOSE OXIDASE ON EGG SHELL MEMBRANE

Ishwar Chandra1, Ms. Badrunnisa2*

1

Student, M.Tech, Biotechnology, Amity University, Sector-125, Noida, UP ishwar.bt016@gmail.com 2* Lecturer, Department of Biotechnology, BITM, Bellary, Karnataka zakiya_28@yahoo.co.in ABSTRACT

A glucose biosensor using an enzyme glucose oxidase immobilized over an egg shell membrane was fabricated. Glucose oxidase an enzyme from A. Niger was covalently immobilized on egg shell membrane, a waste from poultry industry used as a polymer for immobilization using glutaraldehyde as a cross linker. The enzyme-membrane detachable unit was developed in laboratory. The membrane of the commercially available strip was replaced by egg shell membrane, and it was attached to the working electrode, consists of a platinum cathode at which oxygen is reduced and a silver/silver chloride acting as reference electrode. Detection scheme was based on the depletion of dissolved oxygen content upon exposure to glucose solution. So the reading on the glucometer will be the amount of glucose concentration oxidized over the membrane. Glucose oxidase (GOx) was isolated from A.niger and characteristic studies on Glucose oxidase carried out in lab. Egg shell membrane can reduce the cost for the sensor by a big margin because this membrane is a waste from the poultry industry and it is cheaper than other polymer membrane. Key words: Biosensor, Glucose Oxidase, A.niger, Egg shell membrane

1. INTRODUCTION
Sensor that makes use of biological or living material for its sensing function called biosensor. Biosensor consist of, biological detection element, transducer, and signal processing system, which recognize the substance of interest, converts biorecognition events in to measurable signals and the signals been converted in to a workable form respectively. Biosensor studies created the challenge in the field of microelectronics and optoelectronics to offer powerful new analytical tools with major applications in medicine, environmental diagnostics food and processing industries. Most studied and developed biosensor application is glucose biosensor. Glucose is of special interest as it involve in human metabolic processes [1]. (fig1) indicate the mechanism of sensing. GOx is a dimeric protein (fig 2) with a molecular weight of 160 kDa, containing one tightly bound flavin adenine dinucleotide (FAD) per monomer as cofactor, made up of two identical subunits. The dimeric protein displays an ellipsoidal shape with a high content of secondary structure (28% helix, 18% sheet). The tertiary structure of the enzyme is characterized by two separate and distinctly different -sheet systems. One forms part of the FAD binding domain.

The second is a large six stranded anti parallel -sheet supported by 4 -helices (fig 3). This sheet forms one side of the active site [2]. Present study utilizes a source of A. Neiger one of the major source of glucose oxidase enzyme.

It catalyses the reaction addd some more points on its reaction (GOX)
-D-Glucose + O2+ H2O D-glucono- -lactone + H2O2 2 H2O2 + p-hydroxybenzoic acid + 4-aminoantipyrine (POD) Quinoneimine dye + 4 H2O Aspergillus species are a ubiquitous group of filamentous fungi that are commonly isolated from soil, plant debris, and indoor air environments. Many species of Aspergillus are xerophilic, thermo tolerant and show a remarkable tolerance to freezing [3] .A. Niger is one of the most common species of the genus Aspergillus. It causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a common contaminant of food. It is ubiquitous in soil and is commonly reported from indoor environments, where its black colonies can be confused with those of Stachybotrys (species of which have also been called "black mold"). Some strains of A. Niger have been reported to produce potent mycotoxins called ochratoxins. Colonies on potato dextrose agar at 25C are initially white, quickly becoming black with conidial production. Reverse is pale yellow and growth may produce radial fissures in the agar. In this study A. Niger is taken as the source of glucose oxidase enzyme. This microorganism is capable to produce variety of metabolites and industrially important enzymes.

2. MATERIAL AND METHODS

Source of micro organism was 7-9 days spoiled coconut (fig-4). Colonies of microorganism were collected from 7-9 days spoiled coconut (fig4). The microorganism was isolated by enrichment culture method on PDA media as growth limiting and sole carbon and energy source and incubated for 24-48 hrs at 370C (fig 5). Another three serial sub-cultures were made in the PDA medium over a period of 15 days. The growth of the fungus was confirmed by the appearance of turbidity in the culture medium and also by the development of fungal colonies after inoculation of loop-full of culture medium by quadrant streak method on PDA-agar plates (fig6). Biochemical test were performed in order to identify the strain. Thus the characterization of A.niger was done. Following are some of the test carried out in order to characterize A.niger. Freshly prepared broth culture was used for the biochemical studies. Biochemical characterization of A. Niger The colorless colonies were observed on the PDA medium. The motility test was performed by hanging drop method (Seeley and Van Demark, 1972). Flagellar staining was performed by the method of Leifson (Benson, 1990). Further, several physiological and biochemical tests were carried out to observe the physiological characteristics of the organism. 2.3.1. Gram staining:

The stock culture was used to streak the fresh nutrient agar plate. After streaking on to the nutrient agar plate the plate was kept in an incubator at 30 oC for 24 hours. From this plate, few fungal colonies were picked up for the Gram staining test. 2.3.2. Starch hydrolysis: Starch hydrolysis was examined by incubating plates of the Yeast Extract, agar containing 1% soluble starch for 2 days and flooding with Lugols iodine solution. Citrate utilization: The citrate utilization was observed by streaking the microorganism on Simmons citrate agar, which contains indicator, bromothymol blue. After incubation for 24 hours, the utilization of sodium citrate by the organism produces alkalinity in the culture medium, which turns the medium from dark green to blue. Catalase activity: The catalase production was performed by incubating, fresh bacterial cell pellet with 3% of hydrogen peroxide solution. The positive test was confirmed by the production of the effervescence from the medium. Acid gas production: The production of acid and gas from glucose due to fermentation was observed by incubation of the organism for a period of one week in the nutrient medium containing 10g of specific carbohydrate in one liter distilled water. The pH of the medium was adjusted to 7.0 and the pH indicator (Bromocresol purple) was added in to the medium. The acid production can be observed by the change in the color of the medium from purple to yellow due to the decrease of pH. MRVP test: The mixed acid fermentation and butandiol fermentation (MR-VP) test was performed in MRVP medium composed of 5g of glucose with peptone and di potassium phosphate. The tubes were incubated over a period of 2-4 days and the tests were observed by adding color reagents. Indole production: Indole test was carried out by incubating the organism with tryptone broth (1%) to test the ability of the organism to split the tryptophan in to indole by the addition of Kovacs reagent, p dimethyle amino-benzaldehyde in amyl alcohol (Benson, 1990). Decarboxylation test: Decarboxylation test was carried by out incubating the bacterium in the Tryptone broth. Initially the pH was 7.0, after incubation of two days the pH of the media increases. This indicates the decarboxylation of the amino acid L-tyrosine and arginine. Urea hydrolysis: The ability of this organism to produce enzyme urease, which hydrolysis urea in to ammonia was tested by incubating the organism in the medium, which becomes red at pH 8.0, which is an indicative for the production of the ammonia. The results of various physico-chemical tests are presented in table . Isolation of GOx from A.niger The freshly grown A.niger was centrifuged at 10,000rpm. The supernatant part is taken as the source of crude GOx used for the further study. The activity of glucose oxidase was carried out using glucose as a substrate. Glucose oxidase catalyses the oxidation of -D-glucose to Dglucono--lactone with the concurrent release of hydrogen peroxides. In the presence of peroxidase (POD) this hydrogen peroxide H2O2 enters into a second reaction involving p-

hydroxybenzoic acid and 4- aminoantipyrine with the quantitative formation of a quinoneimine dye complex which is measured at 510 nm. Isolation of Peroxidase from Radish Radish was taken as the Peroxidase source by crushing about 10gm in 50 ml phosphate buffer of pH 6.9 centrifuged at 10.000 rpm for 30min. supernatant was used as the source of Peroxidase for further studies. Glucose oxidase Assay table 03: Reaction mixture containing 0.5 ml of glucose as substrate was taken and made up to two ml adding phosphate buffer of pH 6.9 and 0.5 ml of oxidase enzyme incubate for five min then add one ml of p-hydroxy benzoic acid and Peroxidase enzyme incubate for two min then reaction mixture is inhibited by adding 2ml of 4-aminoantipyrine the .color intensity was measured at 510 nm. Activity and specific activity was calculated using standard graph of H 2O2. Activity can be defined as the amount of mg of Glucose oxidizes per ml of an enzyme Standard curve for H2O2 (table 2) (figure 9): Reaction mixture containing various concentrations of H2O2 ranging from 0.2 to 2.0 ml made up to one ml with phosphate buffer to which one ml of p-hydroxy benzoic acid and Peroxidase enzyme was added reaction mixture allowed to stand for two min. the reaction was inhibited by adding 2ml of 4-aminoantipyrine the .color intensity was measured at 510 nm. Obtained optical density Vs concentration of H2O2 was plotted and used as the standard H2O2 graph for the further study. Estimation of protein content of enzyme preparation. The protein content of enzyme preparation was estimated according to the method described by Lowry et al., (1951). A 1-ml solution of diluted enzyme was added with equal volume of trichloroacetic acid (TCA 10 %, w/v) and incubated at room temperature for 20 min. The precipitate so formed is removed by centrifugation at 10,000 rpm for 15 min at 4oC the precipitate was redissolved in small volume of phosphate buffer pH 7.0 (1-2 ml) and used for the estimation of protein content. Standard curve of protein (BSA): The standard curve of protein was constructed using the method of Lowry et.al (1951). In a series of test tubes 0-0.5 ml of standard solution of BSA (20 mg/100ml) was pipetted out and the volume was adjusted to 1 ml by adding required amount of distilled water. Then 5 ml of alkaline copper reagent was added to each tube and the tubes were incubated for 20 min at 37oC in dark. A 0.5 ml of Folin Ceocalteaus reagent (1N) was added to all the tubes and incubated in dark for further 20 min. The optical density of blue color developed was measured at 660 nm. Graph of absorbance at 660 nm versus concentration of BSA was plotted (fig 2.2), and was used to estimate the concentration of protein in the enzyme solution. Effect of pH and temperature on Gox activity. The effect of physical parameters such as pH and temperature on the protease was carried out as follows.

The effect of pH on the activity of the partially purified enzyme was analyzed as follows. A 10 fold diluted enzyme in buffer was assayed in 20mM of buffers of varying pH values; potassium phosphate buffer at pH 6.0, 7.0; Tris-HCl buffer of pH 7.0, 8.0, 9.0 and Glycine NaOH buffer of pH 9.0, 10.0, 11.0, and 12.0. The effect of temperature on partially purified enzyme diluted up to 10 folds (using Tris-HCl buffer of pH 9.0),was performed by assaying the activity at different temperatures of 30oC, 37oC, 40oC, 45oC, 50oC, 60oC, and 70oC. 2.4 IMMOBILIZATION OF GLUCOSE OXIDASE OVER EGG SHELL MEMBRANE

1. Weigh about 1ml glucose oxidase solution and 60 mg bovine serum albumin into a test tube.
Dissolve the mixture carefully in 1 ml of 20 mM phosphate buffer pH 7.0, by careful stirring (do not cause excessive foaming as this causes denaturation of the enzyme which reduces its activity).

2. how u prepare egg shell membrane Egg shell membrane was placed on an
exceptionally clean, flat and horizontal glass plate. 0.1 ml of 25% w/v glutaraldehyde was added to the enzyme solution. The solution was mixed and Carefully transferred onto the egg shell membrane forming a single.large damp area.

3. After allowing polymerising until it is set (about one hour), the membranes was flooded with
distilled water and carefully pealed away from the glass. If the membranes are still fragile at this stage then a further polymerization period is indicated.

4. The commercial available strip was taken and the enzyme layer was replaced by the prepared
membrane enzyme layer.[5] (figure 10).

RESULTS AND DISCUSSIONS

1. 2. A.niger was successfully cultured and charecterised. Glucose oxidase was isolated from the A.niger. 3. Activity of glucose oxidase was calculated and it was 1.2110 -4/min and Specific activity =2.57410 -4 /min. 4. Glucose oxidase was successfully immobilized over an egg shell using glutaraldehyde as a cross linker. The sensor chip was successfully prepared and working was demonstrated in the lab. 5. Advantages and Applications of the chip A. The membrane used in this chip is cheaper as compared to other chip, because membrane is a waste product of the poultry industry and it is readily available row product.It requires minimum processing as compared to other commercially available costly membrane. It is clinical indicator of diabetes. B. This membrane is more effective in case of the diabetic checking strips. It is used only for one time and does not cost more even it is used for single time. C. Same type of the set up of the chip can be used for the construction of the biobattery. Bio-battery is a device which generates electricity from the biological molecule.

D. This biosensor is also used for the quality testing is the food processing industry.It is also applicable in the bioprocess industry for the online measurement of the glucose concentration in the culture vessels and the fermentation process. E. It is biodegradable and easy to decompose without causing any harm to environment.

REFERENCES
1. Afrin Sultana, Student ECE 730-13 NANOELECTRONICS, COURSE PROJECT, APRIL, 2004. 2. J. Raba, H.A. Mottola, Glucose oxidase as an analytical reagent. Critical Rev. Anal. Chem,1995, 25, 1-42. And Google search 3. 2005 fungal genomics project, Concordia university 4. Megazyme international Ireland ltd, Bray Business park,Bray,co,Wicklon,Ireland.. 5. Created and Produced by QA International, 2007 Other reverences A. Riklin, E. Katz, I. Willner, A. Stoker, A.F. Bckmann Reconstitution of flavoenzyme- derived apoproteins with ferrocene-modified FAD cofactor yields electroactive enzymes. Ademark, P.; Larsson, M.; Tjerneld, F. and Stalbrand, H. (2001), Multiple a- galactosidases from Aspergillus niger: purification, characterization and substrate specificities. Enzyme Microb. Technol. by Adya, S. and Elbein, A. D. (1977), Glycoprotein enzymes secreted Aspergillus niger: purification and properties of a-galactosidase. J. Bacteriol.

Bergmeyer, H. U. and Bernt, E. (1974), Determination of glucose with oxidase and peroxidase. In: Bergmeyer, H. U. (Eds.). Methods of Enzymatic Analysis. pp.1205-1215 Blum, H., Beier, H. and Gross, H. (1987), Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis, Bradford, M. M. (1976), A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle dye biding. Anal. Biochem., Bulpin, P. V.; Gidley, M. J. and Jeffcoat, R. (1990), Development of a biotechnological process for the modification of galactomannan polymers with plant -galactosidase. Carbohydr. Polym., DANIEL R. THEVENOT Pure Appl. Chem., Vol. 71, No. 12, pp. 23332348, 1999.

 Dey, P. M. and Pridham, J. B. (1972), Biochemistry of galactosidases. Adv. Enzymol. I. Willner, A. Riklin, B. Shoham, D. Rivenzon, E. Katz Development of novel biosensor enzyme-electrodes: glucose oxidase multilayer arrays immobilized onto selfassembled monolayers on electrodes.. Institute of Advanced Chemistry, School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, China. London south bank university, faculty of science engineering and built environment updated by Martin Chaplint. R. Hirsh, E. Katz, I. Willner Magneto-switchable bioelectrocatalysis. J. Am. Chem. Soc. 2000, 122, 12053-12054. R. Wilson, A.P.F. Turner, Glucose oxidase - an ideal enzyme. Biosens. Bioelectron. 1992, . S. Marx-Tibbon, E. Katz, I. Willner Chiral recognition in mediated electrontransfer in redox proteins. Stevens, R. B., editor. 1981. Mycology Guidebook. University of Washington Press, Seattle. Thesis submitted to university of Pune for the degree of doctor of philosophy in chemistry by SUMANT B. PHADTARE