Biomedical Engineering Reference
In-Depth Information
22.3
Experimental Design
22.3.1
Reagents and Materials
22.3.1.1 For Hanta Virus
22.3.1.1.1 Experimental Procedure
A “sandwich” scheme immunoprinciple of detection is employed [13,15]. The protocol for
the experimental procedure using HRP-labeled enzyme marker is described previously
[11]. The amperometric measurements were performed at a fixed electrode potential of 127
mV vs. Ag/AgCl to detect iodine produced by NaI and H 2 O 2 in the presence of HRP. The
modified procedure using AP-labeled conjugate involves the following stages.
phosphate buffer
(pH 7.8) containing 0.15 M NaCl and 0.1% Tween 20 (PBST) through the immu-
noelectrode for 1 min.
Prewashing stage : Flow of a washing solution 0.02 M Na
First stage of incubation : Flow of PBST-containing human blood plasma to be
tested for Hanta virus IgG antibodies (target analyte) through the immunocol-
umn for 3 min, thereby allowing the target analyte to be bound to the surface of
the immunoelectrode.
First washing procedure : Flow of the washing solution (PBST) for 3 min.
Second stage of incubation : Flow of PBST containing AP-labeled antihuman IgG
(immunoconjugate AP-labeled, concentration: 0.5
g/mL) against the target
analyte through the immunocolumn for 8.5 min, allowing immunoconjugate to
form an immunosandwich with the antigen
antibody complex on the surface of
the immunoelectrode.
Second washing stage : Flow of a washing solution 0.05 M carbonate buffer (pH
9.6) containing 0.15 M NaCl through the immunoelectrode for 5 min. This
washing allows the removal of conjugate molecules attached elsewhere except
to the target analyte. An electrode working potential of
100 mV is applied to
the immunoelectrode before starting the second wash causing polarization of
the electrode.
Amperometric measurement stage : Flow of 0.05 M carbonate buffer (pH 9.6) con-
taining 0.15 M NaCl and 1 mM naphthyl phosphate (substrate of AP label), or 0.1
M acetate buffer solution (pH 4.5) containing 0.15 M NaCl, 1 mM KI, and H 2 O 2
(substrate for HRP label), through the immunocolumn for 3 min.
The output is an amperometric signal determined by the extent of naphthol or iodine
formed as a result of the enzymatic oxidation of naphthyl phosphate or reduction of iodine.
The amperometric output was obtained as a steady state current due to electrooxida-
tion/reduction of naphthol or iodine. The background current signal was measured using
blood sample without antibodies to Hanta virus. The flow rate of the reagents through the
sensor during all stages is 125 µL/min. The procedure for coating the Ag/AgCl electrode
with Nafion layer contained two stages. (1) The Ag/AgCl electrode is prepared by anodiz-
ing silver (99.9%) electrode at
5.0 V for 5 min in a 0.15 M NaCl solution. (2) Then the
Ag/AgCl electrode is coated with several layers of Nafion film by dipping in a series of
Nafion solutions. The electrode is dipped in 0.5% Nafion solution, removed, and air-dried
for 30 min. This step is repeated once with a 3% Nafion solution and then four times with
5% Nafion solution. Nafion solutions of 0.5 and 3% are prepared by dilution with 1:1 2-
propanol and water. The final Nafion coating was cured in a drier at 40°C overnight.
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