1. INTRODUCTION
Recent years, low-cost, simple and automated biomaterials production
methods are widely recognized in enzymatic biosensor
development1,2. In most cases, this requires the
application of sensitive liquid components containing enzymes on the
substrate surface3. For example, such methods include
additive technology that can accurately control the amount of components
applied to a substrate4. At the moment, there are
different additive methods, ranging from microdispensing to
printing5. Inkjet printing is a perspective direction
for material science development as it allows to apply different
reagents from active substances solutions up to complex sol-gel systems
with unique properties in the ink form6. A distinctive
feature of this method is the possibility of a high-precision deposition
of variety materials, the creation of ordered layers and complex
three-dimensional structures of an active substance7.
A relatively new direction in the field of inkjet printing is the
producing of biomaterials and biosensors8,9. The
possibilities of accurate dosing and positioning along with the
autonomous manufacturing largely determine the advantages of a printing
method for the production of high-precision diagnostic
biosensors10. There is a great variety of types of
biomolecules11, bio-inks12 and based
on them biosensors13. The simplest and most widely
recognized biosensing method is still a qualitative enzymatic reaction
based on oxidases due to their high selectivity and
sensitivity14. Oxidases catalyze the
oxidation-reduction reactions15. They specifically
react with the substrate and transfer the hydrogen from substrate to
atmospheric oxygen to form water or hydrogen peroxide. There are many
oxidases meaningful for biochemical analysis and biosensors, such as
Glucose oxidase, Lactate oxidase, Urate oxidase, Cholesterol oxidase,
Glycerol-3-phosphate oxidase, Choline oxidase, Alcohol oxidase and
others14.
In the widely used method, the additional reaction is used to visualize
the substrate oxidation products. In this reaction a chromogenic
substrate (dye) and peroxidase are used for detection the formed
peroxide16. Peroxidase transfers oxygen from the
peroxide to the dye molecule. As a result, the oxidized form of the
chromogen acquires or changes the color. However, many chromogenic dyes
are not stable in the light17, they are readily
oxidized in the air and require additional components to protect against
undesirable oxidation at storage conditions18. This
multicomponent system overloads and complicates the sensor, requires
careful calculation of optimal concentrations and ratios of each
component for the reproducibility of the results, and increases the
costs of creating a biosensor.
In addition to the use of chromogenic dyes, colorimetric biosensors
based on nanoparticles were reported19. These assays
usually use noble metals nanoparticles20 such as gold
or silver. The detection is based on the principle of particles
aggregation or dispersion21. However, most of these
nanoparticles are used to DNA analysis22, only a few
of them being adapted to the enzymatic reaction. For example, the use of
redox ceria nanoparticles as colorimetric probes in bioassay were
reported23. The method is based on changes in the
physicochemical properties of cerium oxide nanoparticles operating as
chromogenic indicator.
Along with ceria nanoparticles, the color qualitative reaction to
titanium with a solution of hydrogen peroxide is stated to
exist24. This method is highly sensitive which allows
the detection of titanium trace amounts, due to the yellow complex Ti
(IV) -H2O2 formation with the
tetravalent titanium and hydrogen peroxide25. In this
work we have used titanium dioxide (titania) for the detection of
hydrogen peroxide. Since hydrogen peroxide is formed in substrate
oxidation reactions, oxidase enzymes are used for glucose and
cholesterol determination.
In this work we for the first time propose an approach to obtain
biosensors based on redox nanostructured material applied by desktop
inkjet printing.
Using ink jet printing and special titania ink we have created a
universal substrate for the oxidase biosensors and successfully tested
it on glucose and cholesterol. The application of inkjet printing
titania substrate as a colorimetric component achieves high
repeatability with improved surface properties for subsequent layer
deposition. The minimum concentration of peroxide that is possible to
detect using this method is 0.4 mM.