Biomedical Engineering Reference
In-Depth Information
the sensitivity and selectivity needed for the analysis of physiological samples [4-7]. For
example, urine test strips are routinely used for screening nitrites in patients with infec-
tion, but results are just qualitative as they are obtained by visual comparison to a color
chart. Plasma analysis is much less frequent, owing to limitations of the analytical me-
thods, including blood sampling and processing [5,8]. As a consequence, there is a
growing demand for improved analytical tools, increasingly sensitive, reliable and,
preferentially, easy-to-use and inexpensive.
An alternative approach relies on the construction of biosensing devices using
stable enzymes with high catalytic activity and specificity for nitrite. Due to its high
selectivity, turnover and stability, the multihemic cytochrome
c
nitrite reductase
(ccNiR) from the sulphate reducing bacterium
Desulfovibrio desulfuricans
ATCC
27774, which performs the six electron reduction of nitrite to ammonia (eq. 1) [9], has
proven to be a promising candidate for the development of an electrochemical nitrite
biosensor [10-15].
NO
2
-
+ 8 H
+
+ 6 e
-
NH
4
+
+ 2 H
2
O
(1)
→
Miniaturization is critical for both health care and physiological studies. The screen-
printing technology has been widely used for the large-scale fabrication of disposable
biosensors. Besides the portable dimensions, screen-printed electrodes (SPEs) are
low-cost and versatile in terms of formats and materials [16].
In this study, the working electrodes were modified with a layer of a carbon based
conductive ink previously diluted in propanone or methyl ethyl ketone (MEK) and
mixed in different proportions with ccNiR. The enzyme activity after immobilization
in this harsh environment (solvents exposure and heat dry) was evaluated by cyclic
voltammetry and has proved to be highly satisfactory. The electrode preparation was
further optimized and applied on thick-film strip electrodes which were fabricated
beforehand by printing a similar conductive carbon paste on plastic supports.
2
Materials and Methods
2.1
Reagents
Acetone (propanone; 99%; b.p. 56
°
C) and propanone (methylethylketone, MEK;
99%, b.p. 79
C) were from Pronalab. The remaining chemicals were analytical grade
and were used without further purification. Solutions were prepared with deionized
(DI) water (18 M
°
cm) from a Millipore MilliQ purification system.
The graphite conductive ink was obtained from Acheson. Alumina slurries (0.05
and 1.0 µm) were from Buehler.
ccNiR was purified from
Desulfovibrio desulfuricans
ATCC 27774 cells grown in
nitrate, as previously described by Almeida and co-workers [9].
Ω
2.2
Electrochemical Measurements
For the optimization studies, a conventional three-electrode electrochemical cell
was used, with an Ag/AgCl reference electrode, a Pt counter electrode (both from
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