Multiplexed detection at the point-of-care remains challenging when sample volumes are limited, and robust multiplexing methods within a single reaction cavity are lacking. Electrochemical detection of loop-mediated amplification (LAMP) reactions is a promising approach to make sensitive, miniaturized molecular diagnostics available at the point-of-care. Emerging probe-based LAMP methodologies have adopted universal designs that increase analytical specificity and significantly simplify the development process. However, these approaches are limited to the detection of only one target per working electrode. Here we introduce duplex multiplexing on a single electrode using generic surface probes and methylene blue and anthraquinone redox labels to overcome this limitation, thereby expanding the electrochemical mediator displacement (eMD) LAMP toolbox. The uidA E. coli housekeeping gene and the CTX-M1 antibiotic resistance gene found in ESBL E. coli are both successfully detected in singleplex down to 10 CFUs per reaction and show semi-quantitative amplification signals. Our novel electrochemical real-time LAMP assays show equal sensitivity and similar time-to-positive as their optical counterparts while improving the signal-to-noise ratio for the used primer sets. The combined duplex LAMP detects both targets within 40 min, and its clinical matrix compatibility was preliminarily validated using five clinical wound swab samples. The duplex LAMP specifically detects the E. coli–positive sample in presence of eleven other wound-associated bacterial species. Our label-based real-time multiplexing approach doubles the detection capabilities per working electrode and presents the first real-time probe-based electrochemical signal-on duplex LAMP detection, paving the way for miniaturized sample-to-answer point-of-care devices with an increased degree of multiplexing.
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