A facile microfluidic chip design for DNA detection using dengue serotypes as a proof-of-concept case study

Abstract

Dengue fever is caused by any of the four serotypes of dengue viruses, DENV-1, DENV-2, DENV-3 and DENV-4 spread by mosquito bites and is important to distinguish between them due to lack of cross-protective neutralizing antibodies for each serotype. Secondary infections also put individuals at higher risk for severe dengue illness than those who have not been previously infected. Current preferred assays include reverse transcription-PCR (RT-PCR) and ELISA. To enable on-field diagnosis of dengue serotypes, the detection process would need to be simplified or at least semi-automated. A downstream detection module was conceptualized and fabricated to detect the amplified DNA from the provided PCR mix (product) of previously developed modular microfluidic chips involving sample loading, cell lysis, RNA extraction and RT-PCR. Further, to ensure accuracy, each serotype assay necessitates a positive control, negative control and test sample, which constitutes 3 separate channels for the diagnosis of just 1 serotype. In this study, a 6-channel bi-assay microfluidic chip was designed with pre-loaded diluent and cyanine dye, sample chamber for loading, sequential fluidic sample mixing, and integrated membranes for simultaneous (6-channel) fluidic manipulation from a single actuation source. Positive samples will turn the dye from blue to violet while the negative controls will remain blue. The integrated membranes provided color contrast and facilitated the manipulation of the samples to the same line of sight for simultaneous analysis, paving the way for automated color analysis via smartphone.