2.1. Immobilization of ascorbate oxidase on membrane Ascorbate

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Immobilization of ascorbate
oxidase on membrane

Ascorbate oxidase act as the biological compound or
biorecognition system where it will be immobilized on the surface of the
membrane. 10?L of ascorbate oxidase was mixed with 20?L of glutaraldehyde
solution 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.


Assembly the based
carbon-electrode (membrane biosensor)

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Bare carbon electrode (CE), CE-nylon,
CE-nylon-ascorbate oxidase is prepared with ascorbate oxidase is being
immobilized on the surface of the nylon membrane corresponding to the
preparation in 3.1.


2.3.      Preparation
of different test:

3.3.1. Different testing solution

There are four different testing solutions being
prepared, which are buffer solution, buffer-ascorbic acid, buffer-polyaniline,
buffer-ascorbic acid-polyaniline. Buffer solution that is being used in this
study is phosphate buffer solution with 0.1 mol L-1 of pH 6 and
ascorbic acid of 50?L of 3×10-3mol L-1 (Pisoschi, et al., 2010).
For the chemical preparation of polyaniline, it is obtained using electrolysis
of 0.2 M of aniline in 1 M of hydrochloric acid aqueous solution at a constant
potential of 0.70 V. (Wang & Mu, 1997).

3.3.2. Different concentration of
ascorbic acid

Different concentration of ascorbic acid is used in
the working solutions at 0.1 M, 0.5 M and 1mM. It is prepared for the
determination of ascorbic acid in buffer-ascorbic acid-polyaniline solution
with different concentration of ascorbic acid. Ascorbate oxidase will be immobilized
on the surface on the nylon membrane and will be deposited on the carbon

3.3.3. Different membrane physical

Different membrane physical structure of 16wt% and
25wt% nylon membrane is deposited on the surface of the carbon electrode. Ascorbate
oxidase will be immobilized on the surface of the nylon membrane and the testing
solution being used is a mixture of buffer solution, ascorbic acid and

3.3.4. Determine the selectivity of
ascorbic acid in noise solution

Ascorbic acid is mixed with citric acid to provide a
noise solution with different ratio of each species. The ratio provided for
this determination is at 50:50, 80:20 and 20:80 of citric acid and ascorbic
acid respectively.


2.4.      Characterization
of membrane and sample

3.4.1. Scanning Electron Microscopy
(SEM) of nylon membrane

nylon 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 membrane

membrane before and after the immobilization of ascorbate oxidase was placed
between two KBr disks for the mid-IR characterization using a FTIR
spectrometer. This to confirm the functional group of nylon membrane to capture
the ascorbate oxidase for it to bind the ascorbic acid in the analyte provided. (Narang, et
al., 2011)

3.4.3. Double-beam UV-Visible
spectrometer (UV-Vis) study of ascorbic acid

absorption spectra are performed to study the conjugation between polyaniline
and ascorbic acid when polyaniline is present in the analyte.


2.5.      Sample

3.5.1. Cyclic voltammetry (CV)

voltammetry measurement is studied on different membrane biosensor involved
with cyclic voltammograms.  It is
recorded with the cycling potential between -0.4 V to +0.8 Vat the scan rate of
100mV s-1 (Zhang, et al., 2017)

3.5.2. Electrochemical Impedance
Spectroscopy (EIS)

determination of ascorbic acid was performed
on 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,

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