Abstract
Bioassays relying on magnetic read-out using probe-tagged magnetic nanobeads are potential platforms for low-cost biodiagnostic devices for pathogen detection. For optimal assay performance it is crucial to apply an easy, efficient and robust bead-probe conjugation protocol. In this paper, sensitive (1.5 pM) singleplex detection of bacterial DNA sequences is demonstrated in a portable AC susceptometer by a magnetic nanobead-based bioassay principle; the volume-amplified magnetic nanobead detection assay (VAM-NDA). Two bead sizes, 100 and 250 nm, are investigated along with a highly efficient, rapid, robust, and stable conjugation chemistry relying on the avidin–biotin interaction for bead-probe attachment. Avidin-biotin conjugation gives easy control of the number of detection probes per bead; thus allowing for systematic investigation of the impact of varying the detection probe surface coverage upon bead immobilization in rolling circle amplified DNA-coils. The existence of an optimal surface coverage is discussed. Biplex VAM-NDA detection is for the first time demonstrated in the susceptometer: Semi-quantitative results are obtained and it is concluded that the concentration of DNA-coils in the incubation volume is of crucial importance for target quantification. The present findings bring the development of commercial biodiagnostic devices relying on the VAM–NDA further towards implementation in point-of-care and outpatient settings.
Highlights
Novel biodiagnostic principles for rapid, sensitive and cost-efficient detection of various types of biomolecules such as proteins, enzymes, and DNA, in particular DNA oligomers having sequences specific for pathogenic bacteria and viruses, are increasingly needed in today’s society [1,2,3,4,5,6,7]
We provide a first-time proof-of-concept of biplex detection of Vibrio cholerae (VC) and Escherichia coli (EC) targets in the portable AC susceptometer
Padlock probes, and detection oligonucleotides used in the singleplex (VC) and biplex (VC, EC) experiments can be found in Supporting information, Table S1
Summary
Novel biodiagnostic principles for rapid, sensitive and cost-efficient detection of various types of biomolecules such as proteins, enzymes, and DNA, in particular DNA oligomers having sequences specific for pathogenic bacteria and viruses, are increasingly needed in today’s society [1,2,3,4,5,6,7]. Magnetic beads functionalized with a second type of probe having affinity for the target bind to the sensor surface and the stray field from the beads is detected by the sensor Another category, sometimes referred to as lab-on-a-bead, instead is based on a strategy relying on changes in the frequency-dependent (dynamic) response of the magnetic beads upon changes in the bead hydrodynamic size caused by a probe-target binding reaction occurring on the surface of the beads. Sometimes referred to as lab-on-a-bead, instead is based on a strategy relying on changes in the frequency-dependent (dynamic) response of the magnetic beads upon changes in the bead hydrodynamic size caused by a probe-target binding reaction occurring on the surface of the beads To this belongs the Brownian relaxation biosensor principle theoretically outlined in 2001 by Connolly and St Pierre [19] and later on experimentally demonstrated in a protein bioassay [20]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
