Parallel dilution microfluidic device for enabling logarithmic concentration generation in molecular diagnostics

In this study, we present a genetic diagnostic device with a four-stepwise logarithmic dilution capability for rapid and reliable detection of target nucleic acids in a single operation using the colorimetric loop-mediated isothermal amplification (LAMP) method. An innovative feature is the confluent point with differing microchannel heights ensuring the synchronized inflow of liquids while preventing backflow, even under large volumetric flow rate variations (10-10 000-fold). This enabled the independent generation of each dilution factor under constant pressure. Furthermore, an integrated asymmetric micromixer effectively mixed two liquids under laminar flow conditions, enabling simultaneous dispensing of the mixed solution at uniform concentrations into five microchambers for each dilution factor. Additionally, a permanent stop valve in the outlet of each microchamber prevented leakages, minimizing the waste of valuable samples and reagents. We demonstrate that diluted samples were accurately prepared at the intended logarithmic dilution factors in a single operation using purified cannabis seed DNA, achieving detection sensitivity similar to that of conventional turbidity-based LAMP assays. Moreover, we used crudely extracted cannabis resin DNA, which contains several gene amplification inhibitors, successfully detecting the target nucleic acids in a single test. Overall, this versatile device eliminates extensive manual sample preparation and has potential for on-site genetic testing in applications such as infectious disease detection, food safety, and illegal drug testing.