title: Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets: optimization and data analysis.
abstract: A real-time, label free assay was developed for microbial detection, utilizing double-stranded DNA targets and employing the next generation of an impedimetric sensor array platform designed by Sharp Laboratories of America (SLA). Real-time curves of the impedimetric signal response were obtained at fixed frequency and voltage for target binding to oligonucleotide probes attached to the sensor array surface. Kinetic parameters of these curves were analyzed by the integrated data analysis package for signal quantification. Non-specific binding presented a major challenge for assay development, and required assay optimization. For this, differences were maximized between binding curve kinetic parameters for probes binding to complementary targets versus non-target controls. Variables manipulated for assay optimization included target concentration, hybridization temperature, buffer concentration, and the use of surfactants. Our results showed that (i) different target-probe combinations required optimization of specific sets of variables; (ii) for each assay condition, the optimum range was relatively narrow, and had to be determined empirically; and (iii) outside of the optimum range, the assay could not distinguish between specific and non-specific binding. For each target-probe combination evaluated, conditions resulting in good separation between specific and non-specific binding signals were established, generating high confidence in the SLA impedimetric dsDNA assay results.
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Andrei L
authors.initials: AL
authors.lastname: Ghindilis
authors.affiliation: Center for Coastal Margin Observation & Prediction and Division of Environmental and Biomolecular Systems, Oregon Health & Science University, 20000 NW Walker Road, Beaverton, OR 97006, USA.
authors.forename: Maria W
authors.initials: MW
authors.lastname: Smith
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Dean S
authors.initials: DS
authors.lastname: Messing
authors.affiliation: Center for Coastal Margin Observation & Prediction and Division of Environmental and Biomolecular Systems, Oregon Health & Science University, 20000 NW Walker Road, Beaverton, OR 97006, USA.
authors.forename: Vena N
authors.initials: VN
authors.lastname: Haynes
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: George B
authors.initials: GB
authors.lastname: Middleton
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Schwarzkopf
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Carmen E
authors.initials: CE
authors.lastname: Campbell
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Changqing
authors.initials: C
authors.lastname: Zhan
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Bruce
authors.initials: B
authors.lastname: Ulrich
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Frasier
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Schuele
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: David R
authors.initials: DR
authors.lastname: Evans
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Sezan
authors.affiliation: SHARP Laboratories of America, Inc., 5700 NW Pacific Rim Blvd, Camas, WA 98607, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Hartzell
authors.affiliation: Center for Coastal Margin Observation & Prediction and Division of Environmental and Biomolecular Systems, Oregon Health & Science University, 20000 NW Walker Road, Beaverton, OR 97006, USA. Electronic address: simonh@ebs.ogi.edu.
authors.forename: Holly M
authors.initials: HM
authors.lastname: Simon
mesh_terms: D001431:Bacteriological Techniques / Q000295:instrumentation / Q000379:methods / Q000706:statistics & numerical data; D001483:Base Sequence; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D003199:Computer Systems; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D003627:Data Interpretation, Statistical; D017097:Electric Impedance; D017743:Equipment Reuse; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D008828:Microbiological Techniques / Q000295:instrumentation / Q000379:methods* / Q000706:statistics & numerical data; D016133:Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial