Khoi Le
BME PhD Proposal Presentation

Date: 2025-08-12
Time: 9-11 AM
Location / Meeting Link: EBB 5029/ Zoom Link: https://gatech.zoom.us/j/94530583756?pwd=8ItqOOLFsKgtpRzzvLO6a3rh2QRqp4.1

Committee Members:
Leslie Chan, PhD (Advisor); M.G. Finn, PhD; Wilbur Lam, MD, PhD; Nael McCarty, PhD; Peng Qiu, PhD


Title: Volatile-barcoded Probes for Multiplexed Protease Sensing in Respiratory Diseases

Abstract:
Dysregulated protease activities occur early on in disease pathology and thus provide a unique opportunity for sensitive detection of early-stage disease. Additionally, activity measurements for multiple proteases in ex vivo clinical samples or in in vivo diseased tissues can come together into activity-based biomarker signatures, providing enhanced disease specificity over single proteases. Therefore, tools that enable multiplexed sensing of protease activities (i.e. simultaneous measurement of multiple protease activities from a single sample) have promising diagnostic utility for early disease detection. While optical probes are most frequently used to measure protease activities, their shared common reporter molecules and lack of distinct alternative reporters due to spectral crosstalk limit their multiplexing capabilities. Moreover, they are difficult to translate to humans due to light scattering in tissues. Here, we propose the development of a new class of volatile-barcoded probe arrays for multiplexed sensing of protease activities in clinical samples and in vivo. In particular, we focus on applications in lung disease detection. In the lungs, proteases such as elastases, cathepsins, and granzymes, drive inflammation, matrix remodeling, and immune response, respectively, and are relevant in chronic lung allograft dysfunction (CLAD) (i.e., lung transplant rejection) and lung infections. To capture these activities, probe arrays will be developed, with probes for each protease designed to release volatile organic compounds (VOCs) of distinct mass upon cleavage. The benefits of VOC barcodes is three-fold: (1) VOCs of distinct mass are easily resolved at high resolution via mass spectrometry for high-plex detection (2) volatilized VOCs can be rapidly measured in the headspace of clinical samples and (3) VOCs are cleared from the body in breath and enable protease activity measurements via breath analysis after intrapulmonary probe delivery. In this work, I will develop probe arrays by first establishing modular linker chemistries to barcode peptide substrates with VOCs (Aim 1) and demonstrate their use for lung disease classification ex vivo via analysis of bronchoalveolar lavage fluid (BALF) (Aim 2) and in vivo after intrapulmonary delivery (Aim 3). We predict that reactions of probe arrays with BALF will produce “VOC signatures” detectable by mass spectrometry, which can be used with machine learning (ML) to train diagnostic classifiers. Alternatively, we predict probe arrays delivered in vivo in the lung will produce synthetic breath biomarker signatures for lung disease.