2.1. Immobilization of ascorbate oxidase on membrane Ascorbate

2.1.     Immobilization of ascorbateoxidase on membraneAscorbate oxidase act as the biological compound orbiorecognition system where it will be immobilized on the surface of themembrane. 10?L of ascorbate oxidase was mixed with 20?L of glutaraldehydesolution 0.5% (v/v).

It was covalently immobilized onto nylon membrane. (Tomita, et al., 2005)and is stored overnight at 4°C, washed with phosphate buffer solution. 2.2.

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     Assembly the basedcarbon-electrode (membrane biosensor)Bare carbon electrode (CE), CE-nylon,CE-nylon-ascorbate oxidase is prepared with ascorbate oxidase is beingimmobilized on the surface of the nylon membrane corresponding to thepreparation in 3.1. 2.3.      Preparationof different test:3.3.1. Different testing solutionThere are four different testing solutions beingprepared, which are buffer solution, buffer-ascorbic acid, buffer-polyaniline,buffer-ascorbic acid-polyaniline.

Buffer solution that is being used in thisstudy is phosphate buffer solution with 0.1 mol L-1 of pH 6 andascorbic acid of 50?L of 3×10-3mol L-1 (Pisoschi, et al., 2010).For the chemical preparation of polyaniline, it is obtained using electrolysisof 0.

2 M of aniline in 1 M of hydrochloric acid aqueous solution at a constantpotential of 0.70 V. (Wang & Mu, 1997).3.3.2. Different concentration ofascorbic acidDifferent concentration of ascorbic acid is used inthe working solutions at 0.1 M, 0.

5 M and 1mM. It is prepared for thedetermination of ascorbic acid in buffer-ascorbic acid-polyaniline solutionwith different concentration of ascorbic acid. Ascorbate oxidase will be immobilizedon the surface on the nylon membrane and will be deposited on the carbonelectrode.3.3.3. Different membrane physicalstructureDifferent membrane physical structure of 16wt% and25wt% nylon membrane is deposited on the surface of the carbon electrode. Ascorbateoxidase will be immobilized on the surface of the nylon membrane and the testingsolution being used is a mixture of buffer solution, ascorbic acid andpolyaniline.

3.3.4. Determine the selectivity ofascorbic acid in noise solutionAscorbic acid is mixed with citric acid to provide anoise solution with different ratio of each species.

The ratio provided forthis determination is at 50:50, 80:20 and 20:80 of citric acid and ascorbicacid respectively. 2.4.

      Characterizationof membrane and sample3.4.1.

Scanning Electron Microscopy(SEM) of nylon membraneThenylon membrane with and without the immobilization of biomolecule compound,which is ascorbate oxidase, was subjected to Scanning Electron Microscopy (SEM)to study the physical structure of the membranes. (Narang, et al., 2011)3.

4.2. Fourier Transform Infrared(FTIR) study of nylon membraneNylonmembrane before and after the immobilization of ascorbate oxidase was placedbetween two KBr disks for the mid-IR characterization using a FTIRspectrometer. This to confirm the functional group of nylon membrane to capturethe ascorbate oxidase for it to bind the ascorbic acid in the analyte provided. (Narang, et al., 2011)3.4.3.

Double-beam UV-Visiblespectrometer (UV-Vis) study of ascorbic acidUV-Visibleabsorption spectra are performed to study the conjugation between polyanilineand ascorbic acid when polyaniline is present in the analyte. 2.5.      Sampleanalysis3.

5.1. Cyclic voltammetry (CV)Cyclicvoltammetry measurement is studied on different membrane biosensor involvedwith cyclic voltammograms.  It isrecorded with the cycling potential between -0.

4 V to +0.8 Vat the scan rate of100mV s-1 (Zhang, et al., 2017)3.5.2. Electrochemical ImpedanceSpectroscopy (EIS)Electrochemicaldetermination of ascorbic acid was performedon different membrane biosensor by sweeping the potential between -0.45 V to+0.65 V at a scan rate 100mVs-1, under OFN (oxygen-free nitrogen)atmosphere and at 25°C (Parsa & Salout, 2014).

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