TY - JOUR
T1 - Development of real-time assays for impedance-based detection of microbial double-stranded DNA targets
T2 - Optimization and data analysis
AU - Ghindilis, Andrei L.
AU - Smith, Maria W.
AU - Messing, Dean S.
AU - Haynes, Vena N.
AU - Middleton, George B.
AU - Schwarzkopf, Kevin R.
AU - Campbell, Carmen E.
AU - Zhan, Changqing
AU - Ulrich, Bruce
AU - Frasier, Michael J.
AU - Schuele, Paul J.
AU - Evans, David R.
AU - Sezan, Ibrahim
AU - Hartzell, John W.
AU - Simon, Holly M.
N1 - Funding Information:
Funding for M.W.S., V.N.H. and H.M.S. was supplied by National Science Foundation grant OCE-0424602 .
PY - 2012/5/15
Y1 - 2012/5/15
N2 - 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.
AB - 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.
KW - Double-stranded DNA detection
KW - Impedance sensor array
KW - Real-time label-free detection
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U2 - 10.1016/j.bios.2012.02.021
DO - 10.1016/j.bios.2012.02.021
M3 - Article
C2 - 22405908
AN - SCOPUS:84862818892
SN - 0956-5663
VL - 35
SP - 87
EP - 93
JO - Biosensors and Bioelectronics
JF - Biosensors and Bioelectronics
IS - 1
ER -