LabyrinTH based approachES for samplE preparation, cleanUp, Screening

Abstract

Over the course of the Theseus project, extensive efforts were dedicated to the development and optimization of a microfluidic system for the detection of soybean protein as a model analyte. Technological challenges prompted the replacement of gluten with soybean protein and led to the refinement of the assay workflow. Key decisions included the adoption of a sandwich immunoassay format utilizing two antibodies, monoclonal and polyclonal, coupled with fluorescent microbeads and a lateral flow membrane. Throughout the project's duration, significant progress was made in various aspects. Antibodies were characterized and purified, while a sample preparation protocol involving denaturing extraction and desalting clean-up was established and optimized. Microfluidic operations were designed and integrated, with thermoformed prototypes successfully produced and tested before transitioning to injection-moulding. An injection-moulding tool was developed, with simulations aiding in optimizing the injection process. Challenges were encountered in the coupling of purified antibodies to fluorescent microbeads, leading to the exploration of alternative strategies such as controlled thiol group addition and N-glycan remodelling. While the former faced aggregation issues, the latter showed promise in mitigating aggregation while maintaining functionality. Additionally, an engineered affinity protein (EAP) demonstrated effectiveness in minimizing aggregation, although concerns regarding false positives necessitated further investigation. Testing of functionalized beads in dipstick experiments revealed aggregation challenges, particularly with non-selective coupling strategies, whereas coupling via N-glycan groups displayed potential. Optimization efforts led to the selection of concentrations for the final assay, which demonstrated the capability to detect soy protein levels as low as 0.01 ppm in buffer. Simultaneously, the microfluidic layout transitioned to injection-moulded cartridges, incorporating modifications to enhance functionality. Testing affirmed consistent fluidic performance, albeit with challenges in spin column integration and readout adaptation. Efforts to integrate optical beam expansion into the system's detector enhanced functionality, with successful production and refinement of the injection moulded lab disk. Final testing of the cartridge with pre-stored functionalized fluorescent beads showcased its potential in simplifying sample preparation and detecting allergens in complex food matrices. With a reduced incubation time compared to dipsticks, the system demonstrated sensitivities of 1 ppm and 4 ppm for soy protein in various samples, highlighting its utility in food safety applications. In conclusion, the Theseus project's outcomes represent a significant leap forward in microfluidic-based allergen detection, laying a solid foundation for the advancement of portable assays with broader applications in food safety monitoring and beyond. Datei-Upload durch TIB