Nucleic acid (NA) analysis has been at the forefront of responding to the SARS-CoV-2 pandemic. Prior to analysis, NAs are acquired from infected individuals through a wide variety of biological samples. Samples are often shipped to laboratories with ample resources, equipment, and technicians for subsequent testing. Functionalized magnetic beads or silica-based spin columns are employed to purify large quantities of NAs from biological samples. However, these extraction kits are consumables, require many steps, and have high cost-per-use. Methods that facilitate point-of-care (POC) testing increase availability of NA diagnostic assays and provide nearly instant results. Typically, POC testing centers have less available equipment and may require the use of nasopharyngeal swabs. However, swabs co-extract matrix components and cannot preconcentrate NAs, providing less sensitive analysis. New extraction devices that preconcentrate NAs, reduce the number of required steps, and are compatible with NA amplification techniques are needed. Additionally, devices with high rates of NA extraction and desorption enable more sample throughput for laboratories.<br/>Herein, novel thin film microextraction (TFME) devices were prepared featuring polymeric ionic liquid (PIL) sorbents for the extraction of DNA fragments. The TFME devices containing PILs are selective for DNA, robust, and highly reproducible. The planar geometry of TFME was compared to a previously developed PIL-SPME platform. Sorption-time profiles indicate that TFME devices provided higher extraction rates and isolated an order of magnitude more total mass of DNA than SPME devices with the same sorbent coating. The enhanced desorption kinetics offered by TFME enabled higher recoveries of DNA using desorption solutions that are less inhibitory to downstream analysis. A colorimetric LAMP assay was used for downstream detection of a ORF1ab DNA fragment, originating from SARS-CoV-2, in artificial oral fluid. By isolating higher DNA concentrations, a 100% positivity rate was achieved with TFME devices compared to only 66.7% with SPME.

Rapid DNA isolation and analysis by thin film microextraction

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ACS Fall 2022

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