2025 AIChE Annual Meeting

(343e) Ultrasensitive and Long-Lasting Bioluminescence Immunoassay for Point-of-Care Viral Antigen Detection

Authors

Giwon Cho, Brigham and Women's Hospital
Jaebaek Lee, Brigham and Women's Hospital
Khushi Doshi, Brigham and Women's Hospital, Harvard Medical School
Daniel Kuritzkes, Brigham and Women’s Hospital, Harvard Medical School
Jonathan Li, Brigham and Women’s Hospital, Harvard Medical School
Athe Tsibris, Brigham and Women’s Hospital, Harvard Medical School
Hadi Shafiee, Brigham and Women’s Hospital, Harvard Medical School
Bioluminescence holds notable promise as a modality in diagnostics due to its high signal-to-noise ratio and absence of incident radiation. However, challenges arise from rapid signal decay and reduced enzyme activity when linked to targeting molecules, limiting its reliability in point-of-care diagnostic applications. Here, we introduce LUminescence CAscade-based Sensor (LUCAS), an enzyme cascade system capable of detecting analytes with ultrahigh sensitivity and prolonged bioluminescence. Utilizing a sequential enzymatic reaction, our assay achieves more than a 500-fold increase in bioluminescence signal and maintains an 8-fold improvement in signal persistence compared to conventional bioluminescence assays. Implemented on a portable, fully automated device designed for point-of-care settings, our system facilitates rapid (< 23 min) sample-to-answer analysis of viruses without an external power supply. Its accuracy surpasses 94% in the qualitative classification of 177 viral-infected patient samples and 130 viral-spiked serum samples, various pathogens including the respiratory virus SARS-CoV-2, and blood-borne pathogens such as HIV, HBV, and HCV as clinical models. The decentralized, rapid, sensitive, specific, and cost-effective of LUCAS positions it as a viable diagnostic tool for low-resource environments.

Figure | LUCAS schematic overview in the detection module. a, Target virus capture is achieved using a MgBeads-based immunoassay, where cAb-MgBeads and dAb-GAL capture the viruses. A strong bioluminescence signal is produced via an enzyme cascade reaction mediated by GAL and Fluc. b, Overview of a user-friendly automated diagnostic device and process: i) Collecting a virus sample, mixing it with assay materials, and transferring the mixture to the automated device. ii) Initiating the automated assay by pressing the start button on the smartphone application, with bioluminescence signal detection using a CMOS sensor inside the automated device. iii) Displaying the infection result on the smartphone application. c, Conjugation of GAL to antibodies, enabling GAL to maintain its activity, facilitating the conversion of LUGAL to luciferin, followed by natural Fluc-mediated oxidation, resulting in highly sensitive bioluminescence. d, Comparison with non-LUCAS systems, where Fluc is linked to antibodies, generating bioluminescence in the presence of luciferin. Abbreviations: LUGAL = D-Luciferin-6-O-beta-D-galactopyranoside, cAb-MgBeads = capture antibody conjugated magnetic beads, dAb = detection antibody, GAL = β-galactosidase, Fluc = firefly luciferase.