title: Scalable DNA-Based Magnetic Nanoparticle Agglutination Assay for Bacterial Detection in Patient Samples.
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.
authors.affiliation: †Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, 11418 Stockholm, Sweden.
authors.forename: Anja
authors.initials: A
authors.lastname: Mezger
authors.affiliation: ‡DTU Nanotech, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Jeppe
authors.initials: J
authors.lastname: Fock
authors.affiliation: ‡DTU Nanotech, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Paula
authors.initials: P
authors.lastname: Antunes
authors.affiliation: ‡DTU Nanotech, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Frederik W
authors.initials: FW
authors.lastname: Østerberg
authors.affiliation: ‡DTU Nanotech, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Anja
authors.initials: A
authors.lastname: Boisen
authors.affiliation: †Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, 11418 Stockholm, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Nilsson
authors.affiliation: ‡DTU Nanotech, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Mikkel F
authors.initials: MF
authors.lastname: Hansen
authors.affiliation: †Science for Life Laboratory, Department of Biochemistry and Biophysics, Stockholm University, 11418 Stockholm, Sweden.
authors.forename: Annika
authors.initials: A
authors.lastname: Ahlford
authors.affiliation: ‡DTU Nanotech, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
authors.forename: Marco
authors.initials: M
authors.lastname: Donolato
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D055620:Optical Phenomena; D011513:Proteus mirabilis / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000235:genetics; D016482:Urinalysis / Q000379:methods*
chemical_list: D004269:DNA, Bacterial; D058185:Magnetite Nanoparticles
keywords: magnetic nanoparticles; molecular diagnostics; padlock probes; rolling circle amplification