Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples

Anja Mezger, Jeppe Fock, Paula Soares Martins Antunes, Frederik Westergaard Østerberg, Anja Boisen, Mats Nilsson, Mikkel Fougt Hansen, Annika Ahlford, Marco Donolato

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    We demonstrate a nanoparticle-based assay for the detection of bacteria causing urinary tract infections in patient samples with a total assay time of 4 h. This time is significantly shorter than the current gold standard, plate culture, which can take several days depending on the pathogen. The assay is based on padlock probe recognition followed by two cycles of rolling circle amplification (RCA) to form DNA coils corresponding to the target bacterial DNA. The readout of the RCA products is based on optomagnetic measurements of the specific agglutination of DNA-bound magnetic nanoparticles (MNPs) using low-cost optoelectronic components from Blu-ray drives. We implement a detection approach, which relies on the monomerization of the RCA products, the use of the monomers to link and agglutinate two populations of MNPs functionalized with universal nontarget specific detection probes and on the introduction of a magnetic incubation scheme. This enables multiplex detection of Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa at clinically relevant concentrations, demonstrating a factor of 30 improvement in sensitivity compared to previous MNP-based detection schemes. Thanks to the universal probes, the same set of functionalized MNPs can be used to read out products from a multitude of RCA targets, making the approach truly scalable for parallel detection of multiple bacteria in a future integrated point of care molecular diagnostics system.
    Original languageEnglish
    JournalA C S Nano
    Volume9
    Issue number7
    Pages (from-to)7374-7382
    Number of pages9
    ISSN1936-0851
    DOIs
    Publication statusPublished - 2015

    Keywords

    • magnetic nanoparticles
    • padlock probes
    • rolling circle amplification
    • molecular diagnostics

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