Sample preparation using multiple microbial pattern recognition proteins and magnetic bead ratcheting

Sample preparation (i.e., isolation and purification of pathogens from samples) remains one of the bottlenecks limiting deployment of diagnostic tests. Two recent examples highlighting these limitations include the difficulties in widespread nucleic acid testing during COVID-19 and in identifying drug-resistant infections. Unmet needs include systems that work with different bodily fluids and samples, multiple types of suspected pathogen, and function in a rapid and semi-automated fashion. Advances such as these could accelerate the deployment of novel diagnostic tests by eliminating upstream sample preparation bottlenecks. Herein, we tested the feasibility of combining multiple pathogen-binding paramagnetic beads with magnetic ratcheting-based enrichment to directly isolate microbes from samples. We demonstrate effective use of three different paramagnetic bead-conjugated proteins (mannose binding lection [MBL], C-reactive protein [CRP], and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin [DC-SIGN]) to capture 18 different bacteria species and model virus particles/peptides. We used magnetic ratcheting to isolate bead-bound microbes from milliliters (mLs) of sample at concentrations of 4 - 4000 microbes per mL. The flow-through system was operated up to 1 mL per minute and enabled pathogen isolation from 10 mL samples in <30 min. Demonstration of post-ratcheting PCR-based microbe analysis was also performed, showing that the technology may have applicability across different infectious agents, sample types/volumes, and analytical assays. Based on these results, further studies are warranted to test clinical samples, compare results to current gold-standard diagnostic methods, and test the sample preparation technologies across additional pathogen types.