A LEGO®-inspired pipette-free approach for a fully-integrated molecular diagnostic kit isothermally operated at near body temperature for the detection of antibacterial resistance

Abstract

Herein, we introduce a LEGO®-inspired molecular diagnostic microdevice fully integrated with DNA extraction, loop-mediated isothermal amplification (LAMP), and colorimetric detection functionalities for rapid detection of antibacterial resistance in a pipette-free manner. The microdevice system is composed of a ready-to-use microdevice containing all necessary reagents and stamps that offer sample-to-answer diagnosis in a pipette-free manner. In particular, antimicrobial resistance was analyzed through LAMP at a significantly reduced temperature of approximately 40 °C, combined with DNA extraction and detection, which were performed at room temperature (RT). Semi-solid colorimetric detection of LAMP amplicons was introduced using polyethylenimine (PEI)-coated paper and AuNP solutions to enable stable storage and color discrimination between negative and positive samples. To use the introduced microdevice system, a commonly encountered antibiotic-resistant bacterium – Enterococcus faecium – was successfully identified in 93 min, and a limit of detection as low as 10¹ CFU mL⁻¹ was obtained by naked-eye colorimetric detection of LAMP amplicons. The introduced device offers point-of-care applications that accurately and rapidly detect antimicrobial-resistant bacteria. The simple operation and colorimetric detection strategy can help implement point-of-care testing (POCT) in resource-limited areas.