title: Highly sensitive electrochemical detection of genomic DNA based on stem loop probes structured for magnetic collection and measurement via metalised hollow polyelectrolyte shells.
abstract: We report a highly sensitive method for the electrochemical detection of genomic DNA, based on the employment of two sub-micron oligonucleotide labels - one for magnetic collection and the other for voltammetric detection - and their incorporation onto a stem loop DNA probe. The magnetic label consists of a latex particle of mean diameter 441±6 nm, bearing magnetic Fe3O4 particles and approx. 3.5×10(5) anti-DIG antibodies. The voltammetric label is a hollow polyelectrolyte shell containing approx. 1.0×10(11) Au atoms in the form of well dispersed Au nanoparticles. A DIG tag on one arm of the stem loop enables binding to the magnetic label, while a thiol tag on the other arm enables attachment to the Au nanoparticles. Due to steric hindrance from the two relatively large labels, attachment of both moieties is dependant on target-probe hybridisation straightening the loop. Once attached, sensitive DNA measurement is facilitated by magnetic collection of the DNA into a small volume and by the high quantity of Au atoms available for detection. Using differential pulse anodic stripping voltammetry we calibrated a 30 mer sequence common to 71 strains of Escherichia coli across the concentration range from 0.1 aM to 100 pM with a LOD of 1.8 aM. Three strains of E. coli, BL 21, ATCC8739, O157:H7, when spiked into UHT milk and fermented palm juice, could be detected with LODs of approx. 2-4 CFU mL(-1) in an assay time of approx. 140 min.
authors.affiliation: School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand.
authors.forename: Naphat
authors.initials: N
authors.lastname: Khunrattanaporn
authors.affiliation: Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand.
authors.forename: Patsamon
authors.initials: P
authors.lastname: Rijiravanich
authors.affiliation: Biochemical Engineering and Pilot Plant Research and Development Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand. Electronic address: mithran.somasundrum@gmail.com.
authors.forename: Mithran
authors.initials: M
authors.lastname: Somasundrum
authors.affiliation: School of Bioresources and Technology, King Mongkut's University of Technology Thonburi, Bang Khun Thian, Bangkok 10150, Thailand. Electronic address: werasak.sur@kmutt.ac.th.
authors.forename: Werasak
authors.initials: W
authors.lastname: Surareungchai
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics; D055664:Electrochemical Techniques / Q000379:methods* / Q000706:statistics & numerical data; D004573:Electrolytes; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005516:Food Microbiology; D006046:Gold; D057230:Limit of Detection; D008280:Magnetics; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D011108:Polymers
chemical_list: D015342:DNA Probes; D004573:Electrolytes; D011108:Polymers; D006046:Gold; D004247:DNA
keywords: DNA biosensor; E. coli; Latex; Nanoparticle; Voltammetry