Nitrite Biosensing Using Cytochrome C Nitrite Reductase: Towards a Disposable Strip Electrode - Biomedical Engineering Systems and Technologies - page 25

conductive ink), one can see that the thermal treatment does not have a strong influ-
ence on I max (Fig. 4). On the other hand, the sensitivity of the sensor, as given by the
slope of the linear range of the plot, decreased about 55%. This indicates that partial
protein denaturation has occurred.

3.2 Electrode Optimization

Enzyme/Carbon Ink Ratio. Different proportions of enzyme and carbon ink sus-
pended in acetone were early tested in order to choose the best composition. The one
using the highest amount of protein (1:2 ratio, corresponding to 3.3

g ccNiR) dis-
played the best results (not shown) without relevant loss of activity, and was selected
for further studies.
Organic Solvent and Curing Temperature. Normally, inks for screen printing con-
tain organic solvents that are later evaporated by heating. If other ingredients like
ccNiR need to be included, it is highly recommended to lower the viscosity of the
paint in order to facilitate the mixing process. For this reason, prior to enzyme incor-
poration, the carbon ink used in this work was diluted with two different organic sol-
vents - MEK or acetone. It is worth noting that acetone is less commonly used for

μ

0 2000 4000 6000 8000

0,0E+00

-2,0E-05

-4,0E-05

-6,0E-05

-8,0E-05

-1,0E-04

-1,2E-04

Fig. 5. Electrocatalytic response to nitrite of ccNiR (3.3 μg) modified PG electrodes, pre-mixed
with the carbon conductive ink, prepared and treated in the following ways: (  ) diluted in
MEK and cured at 40°C; (  ) diluted in MEK and cured at 60°C; (  ) diluted in acetone and
cured at 60°C. (  ) diluted in acetone and cured at 40°C.

[Nitrite] μ M

inks dilution than MEK, although it has a lower boiling point that could permit the
use of lower curing temperatures. Actually, the response to nitrite was much higher
when this solvent was used instead of MEK (Fig. 5) whereas the linear range was
wider. Therefore, acetone has proved to be less harmful to the protein.
In order to evaporate residual organic solvents, most CPSPEs have to be dried
thermally. Although a temperature of 60 C is normally selected for the curing process

of those used in the present work, due to the presence of the biocatalyst, we have also
tested the lowest permitted heating temperature, i.e., 40

°

C. Interestingly, the differ-
ences on nitrite reducing activity were generally small, except when MEK was used
for ink dilution, which generated much lower catalytic currents. Most likely, this

°