Abstract
Electrochemiluminescence (ECL) is a powerful transduction technique that has rapidly gained importance as a powerful analytical technique. Since ECL is a surface-confined process, a comprehensive understanding of the generation of ECL signal at a nanometric distance from the electrode could lead to several highly promising applications. In this work, we explored the mechanism underlying ECL signal generation on the nanoscale using luminophore-reporter-modified DNA-based nanoswitches (i.e., molecular beacon) with different stem stabilities. ECL is generated according to the “oxidative-reduction” strategy using tri-n-propylamine (TPrA) as a coreactant and Ru(bpy)32+ as a luminophore. Our findings suggest that by tuning the stem stability of DNA nanoswitches we can activate different ECL mechanisms (direct and remote) and, under specific conditions, a “digital-like” association curve, i.e., with an extremely steep transition after the addition of increasing concentrations of DNA target, a large signal variation, and low preliminary analytical performance (LOD 22 nM for 1GC DNA-nanoswtich and 16 nM for 5GC DNA-nanoswitch). In particular, we were able to achieve higher signal gain (i.e., 10 times) with respect to the standard “signal-off” electrochemical readout. We demonstrated the copresence of two different ECL generation mechanisms on the nanoscale that open the way for the design of customized DNA devices for highly efficient dual-signal-output ratiometric-like ECL systems.
Highlights
Electrochemiluminescence (ECL) is a powerful analytical technique widely studied and applied from both the academic and industrial points of view.[1−4] ECL is a luminescent phenomenon triggered by electrochemical stimulus, and thanks to the combination of electrochemical and spectroscopic methods, it shows excellent signal-to-noise ratios in complex matrices such as cell lysates, urines, and blood
The stepwise procedure to build the ECL platform is reported in detail in Figure 1: we have employed a pair of DNA nanoswitches composed of a common loop sequence (i.e., 15 nucleotides) flanked by two short self-complementary portions with a different content of guanosine (G) and cytosine (C) to obtain DNA nanoswitches with different stabilities (1GC and 5GC base pairing, respectively, Figure 1A)
Our working hypothesis is that the conformational changes of the DNA nanoswitch can provide information on the mechanism of the ECL signal generation on the nanoscale
Summary
Electrochemiluminescence (ECL) is a powerful analytical technique widely studied and applied from both the academic and industrial points of view.[1−4] ECL is a luminescent phenomenon triggered by electrochemical stimulus, and thanks to the combination of electrochemical and spectroscopic methods, it shows excellent signal-to-noise ratios in complex matrices such as cell lysates, urines, and blood. We designed a couple of DNA nanoswitches (i.e., molecular beacons) that share a common recognition loop but differ in the GC base pair content of their double-stranded stem (1GC and 5GC DNA nanoswitch), resulting in a different stem stability (i.e., different free energies of their nonbinding state) and target−probe relative affinity.[11,16] for less stable DNA nanoswitches (i.e., 1 GC base pair in the stem, Ks > 0.1), a significant fraction of these probe is in the extended, binding-competent state even in the absence of a target This generally produces a small signal gain of the biosensing platform upon target binding.[16] In contrast, an overly stabilized stem (i.e., 5GC DNA-nanoswitch) reduces the observed binding affinity because it must overcome a higher free energy barrier. These findings could be useful hints for the design of customized DNA structures to construct highly sensitive ECL sensor platforms
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