Thin Film Microextraction Enables Rapid Isolation and Recovery of DNA for Downstream Amplification Assays

Nucleic acid analysis has been at the forefront of theCOVID-19 global health crisis where millions of diagnostic testshave been used to determine disease status as well as sequencingtechniques that monitor the evolving genome of SARS-CoV-2. Inthis study, we report the development of a sample preparationmethod that decreases the time required for DNA isolation whilesignificantly increasing the sensitivity of downstream analysis.Functionalized planar supports are modified with a polymeric ionicliquid sorbent coating to form thinfilm microextraction (TFME)devices. The extraction devices are shown to have a high affinity forDNA while also exhibiting high reproducibility and reusability.Using quantitative polymerase chain reaction (qPCR) analysis, the TFME devices are shown to require low equilibration times whileachieving higher preconcentration factors than solid-phase microextraction (SPME) by extracting larger masses of DNA. Rapiddesorption kinetics enable higher DNA recoveries using desorption solutions that are less inhibitory to qPCR and loop-mediatedisothermal amplification (LAMP). To demonstrate the advantageous features of the TFME platform, a customized leuco crystalviolet LAMP assay is used for visual detection of the ORF1ab DNA sequence from SARS-CoV-2 spiked into artificial oralfluidsamples. When coupled to the TFME platform, 100% of LAMP reactions were positive for SARS-CoV-2 compared to 66.7%obtained by SPME for a clinically relevant concentration of 4.80x106DNA copies/mL

Automated summary

Nucleic acid analysis plays a crucial role in the COVID-19 global health crisis. This study develops a sample preparation method that reduces DNA isolation time and increases downstream analysis sensitivity. The functionalized planar supports modified with a polymeric ionic liquid sorbent coating show high DNA affinity, reproducibility, and reusability. The TFME devices achieve higher preconcentration factors and DNA recoveries compared to SPME devices, resulting in improved detection of SARS-CoV-2.