Multiplexed Breath Biopsy of Lung Disease by Controlled Chemical Signal Activation

Breath testing is a fast, non-invasive diagnostic method that can link specific volatile organic compounds (VOCs) in exhaled breath to medical conditions. Complementary to blood and urine assays in clinical disease diagnosis, breath is an informative analyte that can provide real-time information of changes in the body’s metabolome affected by disease activity. However, few breath tests are currently used in the clinic to monitor disease due to bottlenecks in biomarker identification and differentiation of endogenous VOCs in complex breath signatures. One approach to overcome this challenge is the in vivo administration of orthogonal exogenous agents with controlled properties that can be expelled from breath upon being metabolized by disease-specific molecular processes. Exogenous agents have been used in clinical breath tests for gastrointestinal and liver diseases and are more recently developed for respiratory disease. As the direct source of breath, the lungs are particularly suited for enzymatic activity-based breath tests. The multiplexed capabilities of designing reliable in vivo probes that release detectable and distinguishable exogenous VOCs are still in the early stages. Here, we report engineered breath biomarkers by protease-sensing nanoparticles for multiplexed detection of dysregulated activities of pulmonary proteases in response to disease and produce volatile reporters that can be detected in the exhaled breath. We show that the nature of proteolytic hydrolysis generating peptide fragments can be engineered to activate and release various types of volatile reporters. Using viral infection and <i>Alk</i>-mutant lung cancer mouse models, we show that multiplexed volatile-releasing activity-based nanosensors (vABNs) can detect and dynamically monitor diseases and treatment response.