A lab-on-a-chip system integrating DNA purification and loop-mediated isothermal amplification for the quantification of the toxic diatom Pseudo-nitzschia multistriata

Abstract

Microfluidic technology can expedite nucleic acid testing by converting the functions of bulky laboratory instruments and protracted bench methodologies into easy-to-use and inexpensive miniaturised systems without compromising speed and reliability. We developed a lab-on-a-chip (LOC) platform that integrates a dimethyl adipimidate (DMA)-based functionalised silica DNA isolation and pre-concentration method with a rapid and real-time loop-mediated isothermal amplification (LAMP) for detecting domoic acid-producing phytoplankton, Pseudo-nitzschia. An optimised design of a lab on a chip extraction module achieved a maximum DNA capture capacity of 61.73 ± 0.98 ng μL⁻¹. The DMA-based method reduced reagent costs per sample by 97% compared to a commercial nucleic acid isolation kit. A subsequent on-chip LAMP process was capable of sensitively quantifying cytochrome P450 homologous to the dabD gene, coding for a component of the domoic acid toxin production pathway, with a limit-of-detection of 10 cells per mL. LAMP-based detection of the target gene was achieved using dry-preserved reagents with a shelf-life of five months without refrigeration. There was no significant difference in assay performance between the preserved LAMP and freshly prepared LAMP mixtures. The total analysis time at the LOD of 10 cells per mL, from sample to result, was achieved within one hour. Our results demonstrate the long-term stability of assay reagents, rapid turnaround, and cost-effectiveness, offering a simple and economical approach to environmental monitoring and environmental bio-hazard diagnostics.