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Facile Coupling of Droplet Magnetofluidic-Enabled Automated Sample Preparation to Digital Nucleic Acid Amplification Testing and Analysis.

Digital nucleic acid amplification testing (dNAAT) and analysis techniques, such as digital PCR, have become useful clinical diagnostic tools. However, nucleic acid sample preparation preceding dNAAT is generally laborious and performed manually, thus creating the need for a simple sample preparation technique and a facile coupling strategy to dNAAT. Therefore, we demonstrate a simple workflow which automates magnetic bead-based extraction of nucleic acids (NAs) with a 1-step transfer to a dNAAT. Specifically, we leverage droplet magnetofluidic (DM) to automate the movement of magnetic beads between small volumes of reagents commonly employed for nucleic acid extraction and purification. Importantly, the buffer typically used to elute the NAs off the magnetic beads is replaced by a carefully selected PCR solution enabling direct transfer from sample preparation to a dNAAT. Moreover, we demonstrate the potential for multiplexing using a digital high-resolution melt (dHRM) after the digital PCR (dPCR). The utility of this workflow is demonstrated with duplexed detection of bacteria in a sample imitating a co-infection. We first purify the bacterial DNA into a PCR solution using our DM-based sample preparation. We then transfer the purified bacterial DNA to our microfluidic nanoarray to amplify 16S rRNA using dPCR, then perform dHRM to identify the two bacterial species.