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.