Electrochemical techniques for characterization of stem-loop probe and linear probe-based DNA sensors Rebecca Y. Lai ⇑ , Bryce Walker, Kent Stormberg, Anita J. Zaitouna, Weiwei Yang Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE 68588-0304, USA article info Article history: Available online 8 August 2013 Keywords: Stem-loop probe E-DNA sensor Linear probe E-DNA sensor Alternating current voltammetry Cyclic voltammetry Differential pulse voltammetry Methylene blue abstract Here we present a summary of the sensor performance of the stem-loop probe (SLP) and linear probe (LP) electrochemical DNA sensors when interrogated using alternating current voltammetry (ACV), cyclic vol- tammetry (CV), and differential pulse voltammetry (DPV). Specifically, we identified one critical param- eter for each voltammetric technique that can be adjusted for optimal sensor performance. Overall, the SLP sensor displayed good sensor performance (i.e., 60 + % signal attenuation in the presence of the tar- get) over a wider range of experimental conditions when compared to the LP sensor. When used with ACV, the optimal frequency range was found to be between 5 and 5000 Hz, larger than the 5–100 Hz range observed with the LP sensor. A similar trend was observed for the two sensors in CV; the LP sensor was operational only at scan rates between 30 and 100 V/s, whereas the SLP sensor performed well at scan rates between 1 and 1000 V/s. Unlike ACV and CV, DPV has demonstrated to be a more versatile sen- sor interrogation technique for this class of sensors. Despite the minor differences in total signal attenu- ation upon hybridization to the target DNA, both SLP and LP sensors performed optimally under most pulse widths used in this study. More importantly, when used with longer pulse widths, both sensors showed ‘‘signal-on’’ behavior, which is generally more desirable for sensor applications. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction One of the major driving forces for the development of DNA and RNA sensing methods is the growing demands for point-of-care medical diagnostics [1–5]. Nucleic acid sensors have also found applications in the agriculture and food industries, as well as in na- tional defense [6,7]. However, despite the research efforts, very few of these sensors have been successfully commercialized, presum- ably due to the sensors’ inability to simultaneously fulfill a set of stringent criteria that includes high sensitivity, specificity, selectiv- ity, user-friendliness, and cost-effectiveness. While many of the sensing approaches feature impressive sensitivity, a DNA sensing technology that is highly sensitive and selective, does not use exogenous reagents, and is operationally convenient has yet to be realized. Among the recently developed sensing platforms, the electrochemical DNA (E-DNA) sensor first reported by Fan et al. in 2003 has garnered substantial attention, owing to its unique attributes [8]. The stem-loop probe (SLP) E-DNA sensor is the elec- trochemical equivalent of the fluorescent molecular beacon assay [9–13]. The signaling of this sensor originates from the binding-in- duced change in the conformation of the stem-loop probe and the efficiency with which the attached redox label transfers electrons to the electrode. In the absence of the target DNA, the stem-loop structure holds the redox label, methylene blue (MB), in close proximity to the electrode, enabling efficient electron transfer. Upon hybridization to the perfect match target DNA, the probe-tar- get duplex positions the redox label away from the electrode, impeding electron transfer, resulting in a detectable reduction in the MB redox current [9–12]. An alternate version of the original E-DNA sensor that utilizes a redox-labeled linear DNA probe was reported by Ricci et al. in 2007 [13]. The signaling of this linear probe (LP) sensor is based on the binding-induced changes in the dynamics of the redox-labeled probe, which is dampened due to the limited flexibility of the probe-target duplex, leading to a sig- nificant reduction in the redox current [14–16]. Independent of the sensor architecture, both sensors have been demonstrated to be sensitive and specific, and more important, they are selective enough to be interrogated directly in a wide range of complex bio- logical matrices [10,12]. Although most reported sensors focused on the detection of DNA, this class of sensors is well-suited for detection of RNA. In addition, the folding- or dynamics-based sens- ing motive can be used in the design of electrochemical aptasen- sors for protein and small molecule detection [17–19]. This article summarizes the sensor performance of both SLP and LP E-DNA sensors when interrogated using three different electro- chemical techniques [20]. Here we systematically characterized both sensors (Scheme 1) using alternating current voltammetry 1046-2023/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ymeth.2013.07.041 ⇑ Corresponding author. Fax: +1 (402) 472 9402. E-mail address: rlai2@unl.edu (R.Y. Lai). Methods 64 (2013) 267–275 Contents lists available at ScienceDirect Methods journal homepage: www.elsevier.com/locate/ymeth