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

ABSTRACT

Digital nucleic acid amplification testing (dNAAT) and analysis techniques, such as digital polymerase chain reaction (PCR), have become useful clinical diagnostic tools. However, nucleic acid (NA) 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 for dNAAT. Therefore, we demonstrate a simple workflow which automates magnetic bead-based extraction of NAs with a one-step transfer to dNAAT. Specifically, we leverage droplet magnetofluidics (DM) to automate the movement of magnetic beads between small volumes of reagents commonly employed for NA 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 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 coinfection. 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 and then perform dHRM to identify the two bacterial species.