Ishwar Paper

8/13/2019 Ishwar Paper

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DESIGN OF GLUCOSE BIOSENSOR USING IMMOBILIZATION

OF GLUCOSE OXIDASE ON EGG SHELL MEMBRANE

Ishwar Chandra1, Ms. Badrunnisa2*

1Student, M.Tech, Biotechnology, Amity University, Sector-125, Noida, [email protected]

2*Lecturer, Department of Biotechnology, BITM, Bellary, [email protected]

ABSTRACT

A glucose biosensor using an enzyme glucose oxidase immobilized over an egg shellmembrane was fabricated. Glucose oxidase an enzyme from A. Niger was covalentlyimmobilized on egg shell membrane, a waste from poultry industry used as a polymer forimmobilization using glutaraldehyde as a cross linker. The enzyme-membrane detachable unitwas 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 cathodeat which oxygen is reduced and a silver/silver chloride acting as reference electrode. Detectionscheme was based on the depletion of dissolved oxygen content upon exposure to glucosesolution. So the reading on the glucometer will be the amount of glucose concentration oxidizedover the membrane. Glucose oxidase (GOx) was isolated from A.nigerand characteristic studieson Glucose oxidase carried out in lab. Egg shell membrane can reduce the cost for the sensor bya big margin because this membrane is a waste from the poultry industry and it is cheaper thanother polymer membrane.

Key words: Biosensor, Glucose Oxidase,A.niger, Egg shell membrane

1.INTRODUCTIONSensor 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 measurablesignals and the signals been converted in to a workable form respectively. Biosensor studiescreated the challenge in the field of microelectronics and optoelectronics to offer powerful newanalytical tools with major applications in medicine, environmental diagnostics food andprocessing 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 themechanism of sensing.

GOx is a dimeric protein (fig 2) with a molecular weight of 160 kDa, containing one tightlybound flavin adenine dinucleotide (FAD) per monomer as cofactor, made up of two identicalsubunits. The dimeric protein displays an ellipsoidal shape with a high content of secondarystructure (28% helix, 18% sheet). The tertiary structure of the enzyme is characterized by twoseparate and distinctly different -sheet systems. One forms part of the FAD binding domain.
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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 themajor 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 fromsoil, 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 mostcommon species of the genus Aspergillus. It causes a disease called black mold on certain fruitsand vegetables such as grapes, onions, and peanuts, and is a common contaminant of food. It isubiquitous in soil and is commonly reported from indoor environments, where its black coloniescan be confused with those of Stachybotrys (species of which have also been called "blackmold"). Some strains of A. Niger have been reported to produce potent mycotoxins calledochratoxins. Colonies on potato dextrose agar at 25C are initially white, quickly becomingblack with conidial production. Reverse is pale yellow and growth may produce radial fissures inthe agar. In this study A. Niger is taken as the source of glucose oxidase enzyme. Thismicroorganism 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 werecollected from 7-9 days spoiled coconut (fig4). The microorganism was isolated by enrichmentculture method on PDA media as growth limiting and sole carbon and energy source andincubated for 24-48 hrs at 370C (fig 5). Another three serial sub-cultures were made in the PDAmedium over a period of 15 days. The growth of the fungus was confirmed by the appearance ofturbidity in the culture medium and also by the development of fungal colonies after inoculationof 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 ofA.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 byhanging drop method (Seeley and Van Demark, 1972). Flagellar staining was performed by themethod of Leifson (Benson, 1990).Further, several physiological and biochemical tests were carried out to observe thephysiological characteristics of the organism.2.3.1. Gram staining:


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The stock culture was used to streak the fresh nutrient agar plate. After streaking on to thenutrient agar plate the plate was kept in an incubator at 30 oC for 24 hours. From this plate, fewfungal 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 ofsodium citrate by the organism produces alkalinity in the culture medium, which turns themedium from dark green to blue.Catalase activity:The catalase production was performed by incubating, fresh bacterial cell pellet with 3% ofhydrogen peroxide solution. The positive test was confirmed by the production of theeffervescence from the medium.Acid gas production:

The production of acid and gas from glucose due to fermentation was observed by incubation ofthe organism for a period of one week in the nutrient medium containing 10g of specificcarbohydrate in one liter distilled water. The pH of the medium was adjusted to 7.0 and the pHindicator (Bromocresol purple) was added in to the medium. The acid production can beobserved by the change in the color of the medium from purple to yellow due to the decrease ofpH.MRVP test:The mixed acid fermentation and butandiol fermentation (MR-VP) test was performed in MR-VP medium composed of 5g of glucose with peptone and di potassium phosphate. The tubeswere 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 abilityof 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. Initiallythe pH was 7.0, after incubation of two days the pH of the media increases. This indicates thedecarboxylation 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 wastested by incubating the organism in the medium, which becomes red at pH 8.0, which is anindicative for the production of the ammonia. The results of various physico-chemical tests arepresented in table .

Isolation of GOx from A.niger

The freshly grown A.niger was centrifuged at 10,000rpm. The supernatant part is taken as thesource of crude GOx used for the further study. The activity of glucose oxidase was carried outusing glucose as a substrate. Glucose oxidase catalyses the oxidation of -D-glucose to D-glucono--lactone with the concurrent release of hydrogen peroxides. In the presence ofperoxidase (POD) this hydrogen peroxide H2O2 enters into a second reaction involving p-


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hydroxybenzoic acid and 4- aminoantipyrine with the quantitative formation of a quinoneiminedye complex which is measured at 510 nm.

Isolation of Peroxidase from RadishRadish 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 Peroxidasefor further studies.

Glucose oxidase Assay table 03:Reaction mixture containing 0.5 ml of glucose as substrate was taken and made up to two mladding phosphate buffer of pH 6.9 and 0.5 ml of oxidase enzyme incubate for five min then addone ml of p-hydroxy benzoic acid and Peroxidase enzyme incubate for two min then reactionmixture is inhibited by adding 2ml of 4-aminoantipyrine the .color intensity wasmeasured at 510 nm. Activity and specific activity was calculated using standard graph of H2O2.Activity can be defined as the amount of mg of Glucose oxidizes per ml of an enzyme

Standardcurve for H2O2(table 2) (figure 9):Reaction mixture containing various concentrations of H2O2 ranging from 0.2 to 2.0 ml made upto one ml with phosphate buffer to which one ml of p-hydroxy benzoic acid and Peroxidaseenzyme was added reaction mixture allowed to stand for two min. the reaction was inhibited byadding 2ml of 4-aminoantipyrine the .color intensity was measured at 510 nm. Obtainedoptical density Vs concentration of H2O2was plotted and used as the standard H2O2 graph for thefurther study.

Estimation of protein content of enzyme preparation.

The protein content of enzyme preparation was estimated according to the method described byLowry 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. Theprecipitate so formed is removed by centrifugation at 10,000 rpm for 15 min at 4oC theprecipitate was redissolved in small volume of phosphate bufferpH 7.0 (1-2 ml) and used for theestimation of protein content.

Standard curve of protein (BSA):

The standard curve of protein was constructed using the method ofLowry et.al(1951). In a seriesof test tubes 0-0.5 ml of standard solution of BSA (20 mg/100ml) was pipetted out and thevolume was adjusted to 1 ml by adding required amount of distilled water. Then 5 ml of alkalinecopper reagent was added to each tube and the tubes were incubated for 20 min at 37 oC 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 ofabsorbance at 660 nm versus concentration of BSA was plotted (fig 2.2), and was used toestimate 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 asfollows.


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The effect ofpH on the activity of the partially purified enzyme was analyzed as follows. A 10fold diluted enzyme in buffer was assayed in 20mM of buffers of varyingpH values; potassiumphosphate buffer at pH 6.0, 7.0; Tris-HCl buffer of pH 7.0, 8.0, 9.0 and Glycine NaOH buffer ofpH 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 (donot cause excessive foaming as this causes denaturation of the enzyme which reduces itsactivity).

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

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

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

RESULTS AND DISCUSSIONS

1. A.niger was successfully cultured and charecterised.2. Glucose oxidase was isolated from the A.niger.

3. Activity of glucose oxidase was calculated and it was 1.2110 -4/min andSpecific activity =2.57410 -4/min.

4. Glucose oxidase was successfully immobilized over an egg shell usingglutaraldehyde as a cross linker. The sensor chip was successfully prepared and workingwas 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, becausemembrane is a waste product of the poultry industry and it is readily available row product.Itrequires 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 usedonly 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 bio-battery. Bio-battery is a device which generates electricity from the biological molecule.


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D. This biosensor is also used for the quality testing is the food processing industry.Itis also applicable in the bioprocess industry for the online measurement of the glucoseconcentration in the culture vessels and the fermentation process.

E. It is biodegradable and easy to decompose without causing any harm toenvironment.

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 search3. 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, 2007Other reverences A. Riklin, E. Katz, I. Willner, A. Stoker, A.F. Bckmann Reconstitution offlavoenzyme- derived apoproteins with ferrocene-modified FAD cofactor yields electroactiveenzymes.

Ademark, P.; Larsson, M.; Tjerneld, F. and Stalbrand, H. (2001),Multiple a- galactosidases from Aspergillus niger: purification, characterization

and substrate specificities.Enzyme Microb. Technol. Adya, S. and Elbein, A. D. (1977), Glycoprotein enzymes secretedby Aspergillus niger: purification and properties of a-galactosidase.J. Bacteriol.

Bergmeyer, H. U. and Bernt, E. (1974), Determination of glucosewith oxidase and peroxidase. In: Bergmeyer, H. U. (Eds.).Methods of EnzymaticAnalysis.pp.1205-1215

Blum, H., Beier, H. and Gross, H. (1987), Improved silver staining ofplant proteins, RNA and DNA in polyacrylamide gels.Electrophoresis,

Bradford, M. M. (1976), A rapid and sensitive method forquantification of microgram quantities of protein utilizing the principle dye biding.Anal. Biochem.,

Bulpin, P. V.; Gidley, M. J. and Jeffcoat, R. (1990), Development ofa biotechnological process for the modification of galactomannan polymers withplant -galactosidase. Carbohydr. Polym.,

DANIEL R. THEVENOT Pure Appl. Chem., Vol. 71, No. 12, pp.23332348, 1999.


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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 novelbiosensor enzyme-electrodes: glucose oxidase multilayer arrays immobilized onto self-assembled monolayers on electrodes..

Institute of Advanced Chemistry, School of Chemistry and ChemicalEngineering, Shanxi University, Taiyuan 030006, China Department of Chemistry, HongKong Baptist University, Kowloon Tong, Hong Kong SAR, China.

London south bank university, faculty of science engineering and builtenvironment 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 electron-transfer in redox proteins. Stevens, R. B., editor. 1981. Mycology Guidebook. University of WashingtonPress, Seattle. Thesis submitted to university of Pune for the degree of doctor of philosophyin chemistry by SUMANT B. PHADTARE

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