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.