To meet the global needs of tuberculosis (TB) control, a nanoELIwell device was developed as a multifunctional assay for TB diagnosis and drug susceptibility testing. The device integrates on-chip culturing of mycobacteria, immunoassay, and high-resolution fluorescent imaging. Mycobacterium smegmatis and Mycobacterium kansasii were used as models of Mycobacterium tuberculosis to evaluate device integrity by using antigens, Ag85 and ESAT-6, as biomarkers. As a result, the nanoELIwell device detected antigens released from a single bacterium within 24–48-hour culture. Antimycobacterial drug-treated M. smegmatis showed significant decreased in Ag85 antigen production when treated with ethambutol and no change in antigen production when treated with rifampin, demonstrating drug susceptibility and resistance, respectively. The nanoELIwell assay also distinguished the ESAT-6-secreting M. kansasii from the non-ESAT-6-secreting M. simiae. The combination of microwell technology and ELISA assay holds potential to the development of a rapid, sensitive, and specific diagnostics and susceptibility testing of TB.

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