Label-free biosensor of phagocytosis for diagnosing bacterial infections

Phagocytic cells recognize and phagocytose invading microbes for destruction. However, bacterial pathogens can remain hidden at low levels from conventional detection or replicate intracellularly after being phagocytosed by immune cells. Current phagocytosis-detection approaches involve flow cytometry or microscopic search for rare bacteria-internalized phagocytes among large populations of uninfected cells, which poses significant challenges in research and clinical settings. Hence it is imperative to develop a rapid, non-disruptive, and label-free phagocytosis detection approach. Using deformability assays and microscopic imaging, we have demonstrated for the first time that the presence of intracellular bacteria in phagocytic blood cells led to aberrant physical properties. Specifically, human monocytes with internalized bacteria of various species were stiffer and larger compared with uninfected monocytes. Taking advantage of these physical differences, a novel microfluidics-based biosensor platform was developed to passively sort, concentrate and quantify rare m onocytes with i nternalized p athogens (MIP) from uninfected monocyte populations for phagocytosis detection. The clinical utility of the MIP platform was demonstrated by enriching and detecting bacteria-internalized monocytes from spiked human blood samples within 1.5 h. Patient-derived clinical isolates were used to validate the utility of the MIP platform further. This proof-of-concept presents a phagocytosis detection platform that could be used to rapidly diagnose microbial infections, especially in bloodstream infections (BSIs), thereby improving the clinical outcomes for point-of-care management. • Development of the first label-free biosensor to detect phagocytosis, based on physical differences in ininfected cells. • Circumvent search for rare infected cells. Identification of internalized bacteria-positive samples could be completed within 1.5 h. • Broad applications to infection caused by different bacteria (gram-positive and gram-negative, extracellular and intracellular bacteria). • Demonstration of clinical relevance with P. aeruginosa -based pneumonia isolates from cystic fibrosis patients. • Application for clinically relevant infected blood samples at low sample volume (~1 ml).