Biomedical Engineering Reference
In-Depth Information
of the transducer. Critical parameters such as the size of the immunosorbent, amount of
immobilized antigen molecules (Hanta virus RNP) and their surface orientation, the effect
of pH, and the stability of the immunosensing elements have been investigated and opti-
mized previously [9,12
14].
22.2.2
Detection of Parainfluenza and Influenza A Viruses
The immunosensor design concept, previously described [9,13], has been modified and
optimized using the immunoassay prototype system for detection of parainfluenza virus
(PIV) and IAV.
However, the immunocolumn, which is the disposable biosensing element, is prepared
differently than the Hanta virus. This column consists of a plastic tube with a 0.22
m pore
size filter membrane at the bottom. The immunosorbent is deposited on the filter mem-
brane (by vacuum) resulting in dispersed graphite immunosorbent forming the measur-
ing (working) immunoelectrode. Another difference is the immunocolumns prepared by
adding 100
250
L of 20 mg/mL immunosorbent to the microcentrifuge filters.
22.2.3
Immunoassay Scheme and Amperometric Detection
A previously described technique, the “sandwich” scheme of immunoassay, was
employed [13,15]. It is illustrated in Figure 22.1.
22.2.4
Optimization of the Assay Parameters
The parameters such as the flow rate of the reagents, working potential, pH of conjugate,
and substrate solutions and their concentrations, and incubation times are investigated and
optimized earlier [9,10,12,14]. Using these same conditions promising results were obtained
during the application of the device in detecting antibodies against Hanta virus in human
plasma samples. However, several parameters have been changed due to the hemolysis of
blood in the case of mice blood samples including the labeled conjugate to prove the feasi-
bility of the device under operating-field conditions. The parameters affecting both
immunological reaction stage and amperometric transduction stage, type of conjugate,
namely working potential, pH of substrate solutions, and substrate concentrations, were
investigated. Optimization of the electrochemical detection was based on the following
criteria: maximum enzymatic activity of the horse-radish peroxidase (HRP) enzyme, maxi-
mum substrate concentrations, and maximum spontaneous oxidation of substrates.
22.2.5
Optimization of the Amperometric Measurement Stage
22.2.5.1 The Cyclic Voltammetry of Horse-Radish Peroxidase and Alkaline Phosphatase
Enzymatic Products
The cyclic voltammetry apparatus consisted of a BAS CV-1 Voltammograph (Bioanalytical
Systems, Inc.), a BK Precision model 2832 digital multimeter (Dynascan Corporation), a
Huston Instrument model 100XY recorder, and an electrochemical cell. The electrochemi-
cal cell with 10 mL of electrolyte volume included a graphite disc working electrode (5 mm
diameter), a carbon counter electrode, and an Ag/AgCl reference electrode. For cyclic
voltammetry experiments of HRP and alkaline phosphatase (AP) products the 0.1 M
acetic-acid buffer solution pH 4.5 with 0.15 M NaCl (AcBS) and 0.05 M bicarbonate buffer
solution pH 9.5 with 0.15 M NaCl (BcBS) was used accordingly. The scan rate potential
