Reagent filled hydrogel reservoirs for the detection of bacterial pathogens in foil based microfluidic

Bacteria are responsible for diseases around the world. The treatment of bacterial infections is crucial for the survival and wellbeing of humanity but comes along with the development of resistances due to extensive antibiotics usage. One approach to counteract this problem is the specific detection of the pathogens to inhibit unnecessary antibiotic prescription.
The project “HydroChip2” (FFG funded project, No.: 883914) contributes to this issue, by developing a roll-to-roll fabricated foil based rapid test system for the detection of ribosomal RNA to identify five periodontal germs. This system aims to achieve lower complexity in sample preparation and number of handling steps. Sample preparation should be facilitated by using bacterial lysis at room temperature with ionic liquids, removing the need for additional equipment such as external heaters for conventional lysis. Sample handling steps are reduced by integrating reagent filled hydrogel reservoirs that can release different assay reactants by diffusion for detection of ribosomal RNA into a fluidic channel. In this thesis, the realization of Streptavidin-Horseradish Peroxidase (STA-HRP) hydrogel reservoirs for the electrochemical detection of bacterial pathogens was targeted. Acrylate-based hydrogel precursor inks were pipetted manually onto PET films and empty hydrogel reservoirs were created by the following subsequent steps: i) UV-crosslinking (effects studied at wavelengths of 254nm or 365nm), ii) washing with ultrapure water to remove monomers and iii) drying to create an empty hydrogel network (process studied under increased temperature or in vacuum chamber). As a result, it was found that the generated hydrogels processed under different conditions led to quite similar swelling ratios (~8), which enables flexible production in a cost-effective roll-to-roll process which employs an automated dispensing system to integrate biofunctionality in the microfluidic channels. In this thesis, a BioDOT automated dispenser was used to optimize the dispensing of hydrogel inks in cavities on fluidic test foils, which are arranged on a sheet with 8x10 chambers. The empty hydrogel network structures were characterized according to the homogeneity using optical methods and software supported image analysis. Subsequently, the filling of dried hydrogel reservoirs with HRP and STA-HRP solution was studied, with filling either by manual pipetting for preliminary tests (e.g. concentration rows, biomolecule mobility, activity, storability), or with the automated dispensing system for e.g. the final detection of RNA. For storage, the enzyme containing hydrogel reservoirs, were dried and stored vacuum-packed at 4-8°C in the fridge.
As result of optical and electrochemical studies it was found, that the HRP enzyme can move out of the reservoir and is active for up to three weeks. By using STA-HRP solutions it was found that the formulation of the solution plays a critical role, and high concentrated solutions are necessary for the microfluidic based assay. Finally, the filled hydrogel reservoirs can be used to demonstrate the detection of rRNA extracted from Escherichia coli as a parameter for the total germ load.