Effect of Signaling Probe Conformation on Sensor Performance of a Displacement-Based Electrochemical DNA Sensor

Abstract

Here we report the effect of the signaling probe conformation on sensor performance of a "signal-on" folding-based electrochemical DNA sensor. The sensor is comprised of a methylene blue (MB)-modified signaling probe and an unlabeled capture probe that partially hybridize to each other at the distal end. In presence of the full-complement target which binds to the unlabeled capture probe, the labeled signaling probe is released. Two different signaling probes were used in this study, in which one is capable of assuming a stem-loop conformation (SLP-MB), whereas the other probe adopts a flexible linear conformation (LP-MB). In the presence of the full complement target DNA, both sensors showed a large increase in MB current when interrogated using alternating current (ac) voltammetry, verifying the release of the signaling probe. Overall, the SLP-MB sensor showed higher % signal enhancement; the LP-MB sensor, however, showed distinctly faster binding kinetics when interrogated under the same experimental conditions. The SLP-MB sensor displayed a wider usable ac frequency range when compared to the LP-MB sensor. Despite these differences, the detection limit and dynamic range were found to be similar among the two sensors. In addition to 6-mercapto-1-hexanol, longer chain hydroxyl-terminated alkanethiols were used to construct these sensors. Our results showed that sensors fabricated with longer chain diluents, independent of the sensor architecture, were not only functional, the signaling capability was significantly enhanced.

A versatile immobilization-free photoelectrochemical biosensor for ultrasensitive detection of cancer biomarker based on enzyme-free cascaded quadratic amplification strategy

“…For instance, to obtain good assay performance for target analyte, the orientation and density of the surface-bounded probes on the modified electrode should be exactly controlled through careful optimizations (Farjami et al, 2012;Lubin et al, 2009;Ricci et al, 2007). In addition, the immobilization processes usually restricted the probe's configurational freedom and even change its geometry due to the steric hindrance effect of the modified electrode surface (Ricci et al, 2007;Yu and Lai, 2013), which results in the much lower binding efficiency and rate between the targets and bio-recognition probes on modified electrode surface than that in solution. Furthermore, the probeimmobilization strategy should vary with the size and configuration of probes, thus no uniform strategy is available for the immobilization of different probes, which may actually limit the routine use of PEC biosensors.…”

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“…Conformational changes of the probe may be varied in a facile manner by changing the DNA probe sequence and spacing regions, as signal-off/signal-on methods can take the form of stem-loop formations [32] or deformations [35, 36], respectively. There are a number of factors that may influence the performance of this class of sensors, including the probe conformation/orientation [37, 38], diluent length [38], probe and target flexibility [39], and redox tether length and flexibility [40]. Ion chelating effects of DNA heteroduplex pairs have also been shown to affect the probe rigidity, and this aspect has recently been applied toward Hg(II) mediated glutathione sensing [41], Ag(I) detection [42], and Au(III) detection [43].…”

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“…This trend is often seen with the linear probe electrochemical DNA (E-DNA) sensor and can be explained using a previously developed theory. [37][38][39][40][41][42][43] In brief, as the applied frequency approaches a critical value (i.e., "threshold" frequency) above which electron transfer can no longer keep pace with the rapidly oscillating potential, the peak current diminishes relative to the background current. 29,42,43 The AC frequency-dependent profiles suggest that the peptide probes assume a somewhat linear and unstructured conformation.…”