Abstract

Low-cost, portable, and easy-to-use analytical devices are essential for applications ranging from healthcare and environmental monitoring to biotechnology and forensics. Microfluidic paper-based analytical devices (µPADs) and tablet-based assays represent promising platforms for developing such detection tools, particularly for resource-limited settings. However, challenges and gaps remain. Improvements in µPAD fabrication are needed to enhance accessibility, while enhanced precision and sensitivity are required for colorimetric and electrochemical sensing. For tablet-based assays, further progress is required to expand applicability to viscous media and to establish integration of bioreagents into auto-mixing tablets.
This thesis originated during pandemic-related laboratory shutdowns with a comprehensive review of COVID-19 diagnostic methods, published in Clinical Microbiology Reviews. The review surveys molecular, antigen, and serological assays, providing insights into pandemic preparedness and identifying gaps in diagnostic technologies. Building on these findings, the main body of the thesis comprises four chapters, each corresponding to a manuscript focused on µPADs and tablet-based assays.
First, a critical review examines the history, fabrication, and detection strategies of µPADs and tablet-based assays, presenting the first dedicated literature review of tablet-based assays. Next, a simplified µPAD fabrication method combining Parafilm® heating and CO2 laser cutting enabled high-resolution devices. Novel multi-inlet and segmented detection zones reduced the color gradient by more than half, improving precision in colorimetric glucose detection in urine. Subsequently, a do-it-yourself screen-printing approach for electrochemical electrode fabrication was developed using portable equipment and laser-cut vinyl masks. A simple water treatment enhanced signal intensity by over 75-fold, further amplified by sandwiching electrodes between two paper layers, and the devices were validated for glucose detection in cell culture media. Finally, rapid auto-mixing tablets were developed, with effective operation in viscous media up to 1700 mPa·s. A proof-of-concept application was demonstrated for nitrite detection in saliva. Additionally, a simple mist-sprayer method for reagent encapsulation in trehalose enabled incorporation of bioreagents into tablets without specialized equipment. Using this approach, the first tablet-based biosensor with rapid auto-mixing was validated for glucose detection in human urine.
Overall, this work simplifies fabrication, enhances analytical performance, and broadens applicability, further advancing the translation of µPADs and tablet-based assays toward diverse real-world uses.