(I-326) Integration of a paper analytical device for isothermal nucleic acid amplification and automated detection of V. cholerae

Introduction:: Waterborne pathogens, such as Vibrio cholerae (V. cholerae), manifest as a severe gastrointestinal infection affecting 7.15 million people per year¹. Detection and treatment can be difficult because the incubation time can range from five hours to five days and concentrations in the environmental water sources can be as low as 10 cells/mL². Developing a water monitoring platform capable of detecting V. cholerae and other waterborne pathogens in the environment would enable monitoring and prevention for resource-limited communities where these pathogens are endemic.

Currently, methods for detecting V. cholerae in the environment require laborious protocols, laboratory expertise and equipment such as polymerase chain reaction (PCR)². Nonetheless, advances in molecular biology, such loop mediated isothermal amplification (LAMP), can reduce the required infrastructure needed to detect diseases. We have previously demonstrated a limit of detection of in pond water using a lateral flow assay (LFA) readout ³. To enable in-field detection, however, a platform is required to perform LAMP and enable automatic detection directly from pond water. We have developed a microfluidic paper analytical device (μPAD) for filtration, capture, amplification, and detection of V. cholerae (Fig. 1). Here we show the capability of filtrating the bacterium onto the reaction pad achieving high capture for amplification and downstream detection on LFA.













Materials and Methods::

Materials and Methods: The mPAD is built by carefully assembling the cellulose and glass fiber membranes into a pressure sensitive adhesive sheet and enclosing it. Fluid control is achieved in the device by using thermally actuated wax valves⁴Toxigenic V. cholerae strain N16961 was cultured overnight on an orbital shaker at 37°C and diluted into pond water. We first evaluated material inhibition of the LAMP assay, prior to selecting membranes to include. Samples are then filtrated through the device, amplified on a hot block, and detected on an LFA. Amplified samples are also validated using gel electrophoresis. Test band intensity of the LFA readouts is asssesed using a customized MATLAB script and statistically evaluated using using a one-way ANOVA with Dunnett’s post hoc.




Results, Conclusions, and Discussions:: We demonstrated a limit of detection of 100 cells/reaction in pond water. Pond water was used as an alternative to molecular biology water as it better represents a natural environment for V. cholerae. We tested various membranes for their capture efficiency and found that 0.22m PES yielded high capture while maintaining stability and not inhibiting amplification. We have demonstrated detection of off-chip amplicons added to the integrated device and then detected on the LFA



Next steps in the integration of the platform will further improve the wax valve geometry to increase repeatability and enable the full filtration, capture and amplification and detection of the sample on the device.






Acknowledgements (Optional): :

References (Optional): : ¹DeFlorio-Barker. Emerging Infectious Diseases 2021, ²Alam. PLoS ONE 2015, ³Phillips, E. A., Moehling, T. J., et al. (2018). Analytical Chemistry, ⁴Phillips (2016). Lab on a Chip