Figure 4. Colorimetric assay of titania coating: a) light
absorbance spectra of titania layer under different peroxide
concentrations; b) color intensity dependence of
H2O2 concentration; c) repeatability of
titania layer color response
The experiment with the optical response of inkjet printable titania
layer showed not only good repeatability but also high sensitivity of
the system to hydrogen peroxide. As shown in Figure 4, we are able to
observe an intense response with peroxide concentrations below 1 wt. %
and down to a value of 0.0013 wt. %. According to the data, we
identified the relationship between the color intensity and the
concentration of peroxide (Figure 4b), and this dependence was linear at
small values of the concentration that indicated the high sensitivity of
this method. In addition, it is worth noting that the color reaction of
titania layer with peroxide proceeded almost immediately, within the
first minute the color reached its maximum saturation and remained
stable for several hours.
Testing hydrogen peroxide convinced us in our substrate applicability
and reproducibility. The next step was to put bio-ink containing oxidase
enzymes on the surface of titania substrate. For the flexibility of
producing approach and monitoring the amount of applied substance,
enzyme layer was also deposited using ink-jet printing method. For this
purpose, we used bio-inks based on oxidases with optimal rheological
properties (see “Production of bio-ink”).
We have tested two oxidases: glucose oxidase and cholesterol oxidase. It
is known that glucose oxidase (GOD) catalyses the oxidation of glucose
to gluconic acid, as shown in formula (1), and cholesterol oxidase in
the presence of oxygen oxidizes cholesterol to 4-cholestenone (2).
Both of these processes take place with the formation of hydrogen
peroxide, which in our case reacted with tetravalent titanium contained
in the sensitive layer of the biosensor to form a yellow product.