A magneto-DNA nanoparticle system for rapid detection and phenotyping of bacteria Hyun Jung Chung 1 , Cesar M. Castro 1,3† , Hyungsoon Im 1† , Hakho Lee 1 * and Ralph Weissleder 1,2,3 * So far, although various diagnostic approaches for pathogen detection have been proposed, most are too expensive, lengthy or limited in specificity for clinical use. Nanoparticle systems with unique material properties, however, circumvent these problems and offer improved accuracy over current methods. Here, we present novel magneto-DNA probes capable of rapid and specific profiling of pathogens directly in clinical samples. A nanoparticle hybridization assay, involving ubiquitous and specific probes that target bacterial 16S rRNAs, was designed to detect amplified target DNAs using a miniaturized NMR device. Ultimately, the magneto-DNA platform will allow both universal and specific detection of various clinically relevant bacterial species, with sensitivity down to single bacteria. Furthermore, the assay is robust and rapid, simultaneously diagnosing a panel of 13 bacterial species in clinical specimens within 2 h. The generic platform described could be used to rapidly identify and phenotype pathogens for a variety of applications. T he rapid and sensitive detection of pathogenic bacteria is crucial for improving patient care with appropriate antibiotic treatment, preventing the spread of disease, and identifying the source of infection in hospital, home or field settings 1–3 . So far, a variety of diagnostic approaches have been proposed, each varying in sensitivity, specificity, cost and efficacy 4–7 . Strategies based on polymerase chain reaction (PCR) and sequencing have shown particular promise as highly sensitive tools for microbiologi- cal identification 8–11 . However, quantitative real-time PCR (qPCR)- based systems are often too expensive for resource-limited environ- ments 12 , and current sequencing techniques still lack practical applicability to patient care 5 . Bacterial culture and biochemical staining remain the clinical gold standard, despite their long procedural times (up to several days) and limitations in identifying certain species. There is therefore a need for generic, accurate and point-of-care platforms that allow both pathogen detection and phenotyping. Such systems could have far-reaching benefits in other sectors, including food industries, shipping and export businesses, defence and agriculture. Here, we report a new diagnostic platform for the rapid detection and phenotyping of common clinical pathogens. The assay makes use of magnetic nanoparticles (MNPs) and oligonucleotide probes to specifically detect target nucleic acids from the pathogen. In particular, we hypothesized that ribosomal RNA (rRNA) sequence information from microorganisms could be used in a robust magneto-DNA assay. Because this magnetic detection strategy allows near background-free sensing, the assay steps are greatly sim- plified and detection is much faster. For bacterial detection, we selected 16S rRNA (a component of the 30S small subunit of bac- terial ribosomes 13 ) as the target marker, because a single bacterium contains many 16S rRNA strands (1 × 10 3 to 1 × 10 5 strands) 14 . Furthermore, the strands have a high degree of sequence consensus across species (important for general bacterial detection) as well as species-specific variable regions (important for species typing) 15,16 . For bacterial phenotyping (for example, identifying drug resistance), targeting of specific mRNA sequences was carried out in parallel with species detection. In this study, rather than sequencing the whole RNA strand, we established a series of primers and probes for amplification and detection of specific regions of interest within common bacterial types. For signal readout we used a miniaturized micro-NMR (mNMR) system, which requires only small volumes of sample for detection (≏2 ml) and is also capable of supporting rapid, high-throughput operations in point-of-care settings 17–19 . Design and validation of the assay The magneto-DNA assay is based on a sandwich hybridization technique wherein two oligonucleotide probes bind to each end of the target nucleic acid (Fig. 1a). Total RNA is extracted from a speci- men, and target regions within the 16S rRNA are amplified by asymmetric reverse transcription-PCR (RT-PCR) to produce large numbers of single-strand DNA with only sense (or antisense) sequences. The resultant DNAs are then captured by polymeric microspheres conjugated with probe oligonucleotides (the bead- capture probe). Subsequently, the overhanging edges of the target DNA are hybridized with MNP-detection probe conjugates (the MNP-detection probe). These magnetically labelled beads shorten the transverse relaxation rate (R 2 ) of a sample, which is detected by a miniaturized mNMR device. This detection method is both robust and highly sensitive, not only because there are multiple 16S rRNA strands per bacterium (as opposed to a single strand of genomic DNA), but also because there are three steps of signal amplification: (i) PCR amplification of the target nucleic acids; (ii) bead capture and enrichment of target nucleic acids; and (iii) mag- netic amplification (because a single MNP can affect billions of sur- rounding water molecules 17 ). Probes specific to each bacterial target were designed through comparative analyses of 16S rRNA gene sequences from different types of bacterial species (see Supplementary Table S1 for details). By aligning multiple sequences from several different genera, we identified both conserved and variable regions; both types of region were subsequently selected as targets. Primers were designed 1 Center for Systems Biology, Massachusetts General Hospital, 185 Cambridge Street, CPZN 5206, Boston, Massachusetts 02114, USA, 2 Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA, 3 Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA; † These authors contributed equally to this work. *e-mail: rweissleder@mgh.harvard.edu; hlee@mgh.harvard.edu ARTICLES PUBLISHED ONLINE: 5 MAY 2013 | DOI: 10.1038/NNANO.2013.70 NATURE NANOTECHNOLOGY | VOL 8 | MAY 2013 | www.nature.com/naturenanotechnology 369 © 2013 Macmillan Publishers Limited. All rights reserved.