title: Single-molecule enzymology based on the principle of the Millikan oil drop experiment.
abstract: The ability to monitor the progress of single-molecule enzyme reactions is often limited by the need to use fluorogenic substrates. A method based on the principle of the Millikan oil drop experiment was developed to monitor the change in charge of substrates bound to a nanoparticle and offers a means of detecting single-enzyme reactions without fluorescence detection. As a proof of principle of the ability to monitor reactions that result in a change in substrate charge, polymerization on a single DNA template was detected. A custom oligonucleotide was synthesized that allowed for the attachment of single DNA templates to gold nanoparticles with a single polymer tether. The nanoparticles were then tethered to the surface of a microfluidic channel where the positions of the nanoparticles, subjected to an oscillating electric field, were monitored using dark field microscopy. With short averaging times, the signal-to-noise level was low enough to discriminate changes in charge of less than 1.2%. Polymerization of a long DNA template demonstrated the ability to use the system to monitor single-molecule enzymatic activity. Finally, nanoparticle surfaces were modified with thiolated moieties to reduce and/or shield the number of unproductive charges and allow for improved sensitivity.
authors.affiliation: Caerus Molecular Diagnostics, Mountain View, CA 94043, USA.
authors.forename: Danielle L
authors.initials: DL
authors.lastname: Leiske
authors.affiliation: Promega BioSystems, Sunnyvale, CA 94085, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Chow
authors.affiliation: Caerus Molecular Diagnostics, Mountain View, CA 94043, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Dettloff
authors.affiliation: Caerus Molecular Diagnostics, Mountain View, CA 94043, USA. Electronic address: javier_farinas@caerusmdx.com.
authors.forename: Javier
authors.initials: J
authors.lastname: Farinas
mesh_terms: D004247:DNA / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry; D004798:Enzymes / Q000378:metabolism*; D006046:Gold / Q000737:chemistry; D053768:Metal Nanoparticles / Q000737:chemistry; D046210:Microfluidic Analytical Techniques / Q000379:methods; D009821:Oils / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D059629:Signal-To-Noise Ratio; D013499:Surface Properties
chemical_list: D004220:Disulfides; D004798:Enzymes; D009821:Oils; D009841:Oligonucleotides; D006046:Gold; D004247:DNA; D004259:DNA-Directed DNA Polymerase
keywords: DNA polymerase; Microfluidic; Nanoparticle; Single enzyme