ABSTRACT
Homogenized mushroom (Agaricus bisporus) tissue based biosensors by using plant tissue materials is a relatively new development in the biosensor technology. A simple in dip stick kind of visual ethanol biosensor that based on immobalised alcohol oxidase (AOX) homogenized from mushroom tissue onto polyaniline (PANI) film for ethanol (> 5% concentration ) detection in toiletries products. The colour going to change from green to blue due to response of biosensor to the ethanol and the change of colour can be seen by naked eyes. Regarding the enzymatic reaction of ethanol, acetaldehyde and hydrogen peroxide will produced, then PANI film is latter going to be oxidised. The method used to immobalise AOX onro the PANI film is by adsorption. Regarding immobilisation process, AOX solution need to deposit on the PANI film and then left it at room temperature within 30 minutes until it dry. Scan and analyse the changes film’s colour to obtain the biosensor’s response characteristics toward the ethanol. The biosensor respond. Therefore, this simple visual biosensor is suitable for all-range-aged community to determine the safeness of certain toiletries products from the ethanol.
Keywords : Biosensor; Alcohol Oxidase Ethanol; Mushroom Tissue; Polyaniline; Toiletries
6.0 EXPERIMENTAL
6.1 Chemicals
10-40 units/mg protein of Alcohol Oxidase or simply called (AOX) (A2404, EC1.1.3.13) which extract from mushroom (A. Bisporus). These mushroom can be bought at fresh market as culture vegetables. Before use make sure store it at 4°C. In order to immobalization use 225 bloom of gelatin from calf skin and 25.0% glutaraldehyde. Ascorbic acid, 2-propanol, D-glucose, n-butanol and many more chemicals needed can be purchased from Sigma, St. Louis, USA. Aniline with AR-grade, gallic acid (G7384) and l-cysteine (W326305) can be purchased from Sigma – Aldrich (Saint Louis, MO, USA). Ethanol which contain >99.5%, methanol, orthophosphoric acid (85%) and sodium hydroxide (pellets) can be delivered by Merck (Nottingham, UK). All needed chemicals are from commercial source which in analytical grade. Millipore Direct-QTM 5 purification system provide the Milli-Q water. Prepare ethanol’s stock solutions in 0.1 M phosphate buffer at suitable pH daily and store it in refrigerator at 4 °C. For pH studies, 0.1 M of the phosphate buffer solutions with pH values between 4 and 8 were can be used and to measure the pH value, use commercial glass electrode and pH-meter (model 9318, Hanna Instruments, Woonsocket, RL, USA) and calibrate it at the pH values of 4.00, 7.00 and 9.00.
6.2 Homogenization of Mushroom Tissue
Homogenize 200 mg mushroom tissue in the 400ml phosphate buffer. Mix this 300 ml homogenate and 10 mg gelatin. Incubate it for 5 minutes at 38°C to make sure the gelatin is dissolved.
6.3 Preparation of Polyaniline (PANI) Film
In order to prevent bumping in the aniline, purify the aniline with vigorous and rapid stirring by distilled under vacuum. Prepare PANI dispersion as nanofibre and Huang and Karner (2006) already mention the method to should to be used to build it. Mix 3.2 mmol or 0.3 g of purified aniline with 10 mL of 1.0M Hydrochloric Acid (HCL). Mix 0.8 mmol or 0.18 g of Ammonium peroxydisulfate into another 10 mL aliquot acid solutions. Add aniline-acid to oxidant and these two solution will mix rapidly within 30 seconds and then allow it to react in undisturbed overnight condition. On the next day, wash the polyaniline by using water and centrifuged. Supernatant liquor with pH 3.3 and strong green colour will form and it is indicate as the PANI particles which can be observed after several times of washing. Any remaining particles with size larger than 1μm must be removed before casting by passing the dispersion through a 55-mm glass fiber filter (Whatman GFA, Kent, UK) which is attach to vacuum source. Cast directly PANI dispersion on a substrate of polystyrene and then, left the thin film of PANI that on the sheet of polystyrene in the dark to dry after cut it into individual in 10mm2 size. Next, store that ready film at 4°C. The thickness must be 0.7μm and use SEM images to determine it. To make sure the thickness of the film is always in the same magnitude order, it must be determined routinely. Then, choose 0.7 μm thickness of PANI film to use for the further experiment result in good of PANI film fabrication reproducibility.
6.4 Enzyme Immobilization
Immerse the PANI film in 0.1 M phosphate buffer which have pH 7.0 to make sure the condition of PANI film is at natural condition which means at pH 7.0. After that, deposit appropriate concentration about 10 μL of AOX solution which is from homogenate of mushroom tissues and phosphate buffer on the PANI film and left it to dry within 30 minutes. For the further use, store this PANI film with immobilised AOX at 4 °C.
6.5 Biosensor Construction
Construct a dip stick test visual biosensor of PANI film with immobilized AOX as Figure 1, connect AOX/PANI film with a handle which made by cellulose paper or can use transparent plastic tape. To way to use this this dip-stick format visual biosensor by just dipping this kind of biosensor into the toiletries sample solution for a several seconds (±5 s), then the change of colour can be seen by baked eye if the concentration of ethanol is >5% since that only amount allowed to be in toiletries products and use image analysis for the quantitative measurement change of colour.

(a)(b)
Figure 1: (a) dip stick format of biosensor
(b) dip biosensor into toiletries sample solution (Kuswandi et.al,2014)
6.6 Colour Change Recording
See the change of colour by naked eye during alcohol detection since this biosensor is kind of visual mode. Use scanner for example Canon, Cano Scan, Japan and Tokyo for quantification of colour measurements. The presence of ethanol exceed 5% of concentration in toiletries sample solutions will change the colour of the biosensor from green to blue. The detection can be done by dipped the biosensor which in the form of dip stick test in the sample solutions in 5 seconds. Use ImageJ program which can be used as online applet, free download application or can be used in any computer together with Java 5 (Dougherty, 2009 & Rueden et.al, 2007) in order to assess the colour after it has been scanned. The purpose s to determine the mean RGB colour value.
7.0 EXPECTED RESULT
The expected result from this research is the colour of dip stick AOX/PANI film biosensor will change from green to blue if there presence of >5% of ethanol in toiletries products after the biosensor is dipped into the toiletries sample solutions for 5 seconds. Since this is the visual mode biosensor, the change of colour can easily seen by naked eyes. On the hand, use scanner (Canon, Cano Scan, Japan, Tokyo) for quantitative colour measurement and then use ImageJ program to assess the colour change of biosensor and to determine the mean RGB colour value. (Collins, 2007)