NanoArrayPAD−X: Nanoprobe Array and 3D-µPAD for the Simultaneous Detection of Respiratory Pathogens and Biomarkers at the Point of Care

Microfluidic paper-based analytical devices (µPADs) are ideal for point-of-care diagnostics due to their low cost, compact size, and ease of use. However, current designs have limited multiplexing capabilities, making it difficult to simultaneously detect pathogens and biomarkers in the same sample. In this work, we introduce NanoArrayPAD-X, a novel µPAD design that combines wax-printed microfluidic networks with an array of nanoprobes for the simultaneous detection of multiple targets. This is achieved by distributing the sample through the microfluidic network containing X detection areas. There, targets are captured through physical interactions and recognized by specific antibody-coated nanoprobes released from the nanoprobe array. This generates X dots whose color depends on the concentration of the targets in the sample. A NanoArrayPAD-5 platform capable of detecting five targets was developed to aid in the diagnosis of ventilator-associated pneumonia (VAP). The sensor array could detect <i>Pseudomonas aeruginosa</i>, <i>Klebsiella pneumoniae</i>, <i>Staphylococcus aureus</i>, <i>Escherichia coli</i>, and the inflammatory biomarker myeloperoxidase (MPO) with a total turnaround time of 25 min, which is faster than waiting for an overnight culture and the results of an ELISA. Notably, our prototype successfully detected the targets in 87 bronchial aspirate (BAS) specimens, thus demonstrating the suitability of the platform for analyzing complex samples with sputum-like qualities. These findings establish NanoArrayPAD-X as a promising tool for the rapid, multiplexed screening of respiratory pathogens and biomarkers, with potential for guiding personalized antimicrobial therapy in suspected cases of nosocomial pneumonia.