Design, fabrication and characterization of multisensing microfluidic paper-based analytical devices

[eng] Biosensors have become highly relevant tools in various fields, providing a decentralized and cost-effective alternative to conventional methods for analyte detection. Their applications extend across diverse areas such as the food industry, environmental monitoring, and, most notably, healthcare. Among the emerging strategies for biosensor development, the use of paper as a substrate has attracted increasing attention due to its intrinsic advantages: low cost, ease of storage, structural versatility, and favorable chemical properties. In particular, its hydrophilic nature, derived from the abundance of hydroxyl groups on the surface, together with its high capacity for biomolecule retention, makes paper an ideal material for the fabrication of analytical platforms. Nevertheless, despite its versatility, it is often necessary to modify paper properties to optimize its performance in specific analytical devices. In this context, the incorporation of polymers and wax represents an efficient and affordable strategy, given the wide variety of compounds available. Such modifications enable, on the one hand, enhanced selective retention of analytes or reagents through the functionalization of active surface groups, and on the other hand, the creation of barriers or valves capable of directing liquid flow. This latter application has fostered the development of so-called microfluidic paper-based analytical devices (µPADs), which exploit hydrophilic channels and the intrinsic capillarity of paper to broaden analytical capabilities in rapid diagnostic contexts. This thesis explores different strategies for creating multisensing microfluidic PADs for applications in the healthcare field. First, a wearable device was developed, capable of performing time-sequenced detection of biomarkers in sweat during physical exercise. Subsequently, two analytical platforms were designed for the multiplexed detection of pathogenic bacteria and biomarkers in respiratory samples. All the proposed platforms rely on colorimetric signal transduction, either through acid–base reactions or through biorecognition processes based on antibody-functionalized nanoparticles. The first approach introduces a chrono-sampling method that enables the fabrication of portable devices entirely constructed from filter paper. By employing dried polymers as time-gated valves, fluid flow can be directed in a controlled manner towards different transducers, thereby allowing successive measurements that reveal variations in analyte concentration. This concept was validated with a multisensor capable of performing nine consecutive pH measurements, and further adapted to design an enzymatic urea biosensor, demonstrating its ability to monitor sweat pH during exercise with accuracy comparable to laboratory-based methods. The second approach describes the design of three-dimensional origami biosensors (OriPlex), which exploit the foldability of filter paper to integrate multiplexed analytical systems into a single device. The detection principle relies on the controlled release of antibody-functionalized nanoparticles into a hydrophilic central channel delineated by wax, where pathogens are recognized through specific interactions. The device proved effective in the simultaneous detection of Pseudomonas aeruginosa and Klebsiella pneumoniae in spiked real bronchial aspirate samples, achieving limits of detection of 3.4·10³ and 1.4·10² CFU·mL⁻¹, respectively, with a total turnaround time of 25 minutes. These results demonstrate the usefulness of Oriplex for the analysis of complex biological matrices and highlight its potential as a rapid screening tool for respiratory infections. Finally, the development of NanoArrayPAD−X is presented, a µPAD that combines wax-printed microfluidic networks with nanoprobe arrays for the simultaneous detection of multiple pathogens and biomarkers in a single sample. The NanoArrayPAD−5 prototype enabled the identification of five clinically relevant targets in the diagnosis of ventilator-associated pneumonia: Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, Escherichia coli, and the inflammatory enzyme myeloperoxidase (MPO). The platform achieved a total response time of 25 minutes and was validated using 87 clinical bronchial aspirate samples, demonstrating its suitability for the diagnosis of respiratory infections in clinical settings. Taken together, the results presented herein pave the way for the design of versatile, multiplexed, and portable paper-based colorimetric sensors capable of monitoring biomarkers and detecting clinically relevant pathogens in real samples. These platforms represent a significant advancement towards the development of accessible tools for point-of-care diagnostics and the monitoring of personalized antimicrobial therapies.