Combining nanobody generation platform, 3D-printed microfluidic chip, and smartphone detection system for monitoring emerging virus-caused diseases.

Rapid in-field detection of emerging viruses is crucial for preventing potential pandemics. This study presents the development of a portable, cost-effective point-of-care testing (POCT) device for detecting emerging pathogens. We took H7N9 avian influenza virus (AIV) as an example, utilizing a combination of novel nanobodies, 3D-printed microfluidic chips, and smartphones to monitor the pathogen. The system employs projection micro-stereolithography (PμSL) 3D printing to fabricate a flower-shaped microfluidic chip, which integrates a micropillar array to enhance the surface area for nanobody immobilization. A pair of nanobodies with high specificity and affinity for H7N9 was generated to develop a sensitive colorimetric immunoassay. The assay results can be visually inspected and further quantified using smartphone imaging, eliminating the need for external lighting devices. The device demonstrated a limit of detection (LOD) of 5.9 × 103 EID50/0.1 mL, comparable to traditional ELISA methods, and was validated on real-world samples. The microfluidic chip's reusability was also established, with up to nine reuse cycles without significant loss of sensitivity. The smartphone-integrated POCT device offers the advantages of portability, simplicity, and rapidity, making it a promising tool for in-field H7N9 virus screening, especially in resource-limited settings. This approach exemplifies the potential of integrating advanced nanotechnology with modern manufacturing techniques and ubiquitous devices to enhance diagnostic accessibility and efficiency.