A digital microfluidic platform coupled with colorimetric loop-mediated isothermal amplification for on-site visual diagnosis of multiple diseases

Abstract

Traditional diagnosis of infectious diseases relies on advanced analyzers in central hospitals, which is not sufficient for rapid control of epidemics, especially in resource-limited areas, raising the importance of the development of diagnostic systems for point-of-care testing (POCT). Here, we developed a simple and cost-effective digital microfluidic (DMF) platform in combination with colorimetric loop-mediated isothermal amplification (LAMP) for simple on-site diagnosis of diseases by the naked eye. The DMF chip contained four parallel units for the simultaneous detection of multiple genes and samples at a time. After amplification, the results were visualized by endpoint detection with concentrated dry neutral red on the chip. The whole process could be completed within 45 min and the on-chip LAMP reaction was shortened to 20 min. The analytical performance of this platform was evaluated by the detection of Enterocytozoon hepatopenaei, infectious hypodermal and hematopoietic necrosis virus, and white spot syndrome virus genes of shrimp. The DMF-LAMP assay showed a detection limit of 10¹ copies per μl for each target, exhibiting comparable sensitivity to the conventional LAMP assay but was more efficient. The sensitivity was also competitive with that of microfluidic-based LAMP assays using other POCT devices such as centrifugal discs regarding the detection of the same targets. Moreover, the proposed device possessed a simple chip structure and high flexibility to integrate multiplex analysis, which was beneficial for further widespread use in POCT. The practicability of the DMF-LAMP assay was verified by the testing of field shrimp. The result of the DMF-LAMP assay indicated a good agreement with the qPCR method, with Cohen's kappa values ranging from 0.91 to 1.00 depending on different targets. For the first time, an image processing method was established based on RGB analysis under different lighting conditions, and a universally adaptable positive threshold was determined regardless of lighting conditions. Paired with a smartphone, the objective analytical method was facile to implement in the field. Moreover, the DMF-LAMP system is easy to extend for a broad range of bioassays, with the advantages of low-cost, rapid detection, ease of use, good sensitivity, and easy readout.