Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets

Optimization and data analysis

Andrei L. Ghindilis, Maria W. Smith, Dean S. Messing, Vena N. Haynes, George B. Middleton, Kevin R. Schwarzkopf, Carmen E. Campbell, Changqing Zhan, Bruce Ulrich, Michael J. Frasier, Paul J. Schuele, David R. Evans, Ibrahim Sezan, John W. Hartzell, Holly Simon

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)87-93
Number of pages7
JournalBiosensors and Bioelectronics
Volume35
Issue number1
DOIs
StatePublished - May 15 2012

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Electric Impedance
Assays
DNA
Oligonucleotide Probes
Surface-Active Agents
Buffers
Sensor arrays
Temperature
Kinetic parameters
Oligonucleotides
Labels
Surface active agents
Electric potential

Keywords

  • Double-stranded DNA detection
  • Impedance sensor array
  • Real-time label-free detection

ASJC Scopus subject areas

  • Biophysics
  • Biomedical Engineering
  • Biotechnology
  • Electrochemistry

Cite this

Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets : Optimization and data analysis. / Ghindilis, Andrei L.; Smith, Maria W.; Messing, Dean S.; Haynes, Vena N.; Middleton, George B.; Schwarzkopf, Kevin R.; Campbell, Carmen E.; Zhan, Changqing; Ulrich, Bruce; Frasier, Michael J.; Schuele, Paul J.; Evans, David R.; Sezan, Ibrahim; Hartzell, John W.; Simon, Holly.

In: Biosensors and Bioelectronics, Vol. 35, No. 1, 15.05.2012, p. 87-93.

Research output: Contribution to journalArticle

Ghindilis, AL, Smith, MW, Messing, DS, Haynes, VN, Middleton, GB, Schwarzkopf, KR, Campbell, CE, Zhan, C, Ulrich, B, Frasier, MJ, Schuele, PJ, Evans, DR, Sezan, I, Hartzell, JW & Simon, H 2012, 'Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets: Optimization and data analysis', Biosensors and Bioelectronics, vol. 35, no. 1, pp. 87-93. https://doi.org/10.1016/j.bios.2012.02.021
Ghindilis, Andrei L. ; Smith, Maria W. ; Messing, Dean S. ; Haynes, Vena N. ; Middleton, George B. ; Schwarzkopf, Kevin R. ; Campbell, Carmen E. ; Zhan, Changqing ; Ulrich, Bruce ; Frasier, Michael J. ; Schuele, Paul J. ; Evans, David R. ; Sezan, Ibrahim ; Hartzell, John W. ; Simon, Holly. / Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets : Optimization and data analysis. In: Biosensors and Bioelectronics. 2012 ; Vol. 35, No. 1. pp. 87-93.
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