DNA-Based Nanoswitches: Insights into Electrochemiluminescence Signal Enhancement

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.

High-performance liquid chromatography (HPLC)-purified DNP sequences were purchased from IBA GmBH (Göttingen, Germany). The hairpin variants were modified with a thiol-C6 group at its 5′ end and three-carbon linked to an amine group at its 3′ end. For the electrochemical detection the same hairpin sequences had a methylene blue (MB) attached by a six-carbon linker to an amine at their 3′ end. In the above-reported sequences the underlined bases represent the stem portion. In bold are the GC bases of the loop.

Sensor Fabrication on Screen-Printed Gold
Electrodes. The gold screen-printed electrodes (Dropsense®) have a gold working electrode (4 mm diameter) and counter electrode and silver reference electrode. Electrodes were clean electrochemically through a series of cyclic voltammetry first in NaOH 0.5M scanning from 1.35 to 0.35V (500 scans, scan rate 2Vs -1 ), incubating for 1h in H 2 SO 4 0.5M and then scanning in the same solution from 0 to 1.25V (scan rate 0.1Vs -1 ) until the gold peak was stable (approximately 20 scans). Then, after S3 carefully rinsing with deionized (DI) water, we dried them without touching the gold surface.
We then deposited a 10 µL drop of the 3 µM DNA probe onto the dry electrodes and incubated for 2 h at room temperature. To prevent the evaporation of the solution, we placed the electrodes inside a Petri dish with a wet piece of paper to maintain humidity. After a final rinse with DI water (to remove the loosely adsorbed DNA), we incubated the electrodes with 2 mM 6-mercaptohexanol dissolved in Phosphate Buffer (PB, pH 7) for 1 hour at room temperature.
Subsequently N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC, 98%) was added to the solution in proportion 2:1 with respect to initial RuCO-OSU amount and 100µL of this solution were dropcasted on the electrode surface. The sensor was incubated over night at 37°C following by a five time cleaning process in Tris buffer, pH 7. The electrode was then incubated in 2 mM 6-mercaptohexanol dissolved in PB for 1 hour at room temperature and rinse carefully in PB before the hybridization with the complementary target probe.
Electrochemical and ECL Measurements. ECL and electrochemical measurements were carried out with an AUTOLAB electrochemical station (Ecochemie, Mod. PGSTAT 30).
All electrochemical measurements were performed at room temperature using an EmStatMUX potentiostat multiplexer (Palmsens Instruments, Netherland). Experimental data were collected using square wave voltammetry from 0.0 to -0.5V in increments of 0.001V vs.
Ag/AgCl, with an amplitude of 10 mV and a frequency of 50 Hz. Peak currents were fit using the manual fit mode in the PSTrace software (of Palmsens Instrument).
For ECL measurements we exploited a home-made transparent plexiglass filled with 100 mM tri-n-propylamine (TPrA) solution in phosphate buffer (PB, pH 7) as oxidative coreactant. The sensor was connected to a specific Boxed Connector (Dropsense®) and ECL S4 signal was generated in cyclic voltammetry applying a potential from 0 to 1.6V and measured with a photomultiplier tube Acton PMT PD471 placed at a constant distance in front of the cell and inside a dark box. A voltage of 750 V was supplied to the PMT. The light/current/voltage curves were recorded by collecting the pre-amplified PMT output signal (by an ultralow-noise Acton research model 181) with the second input channel of the ADC module of the AUTOLAB instrument.
ECL -DNA sensor performance. The ECL platform with either 1GC or 5GC DNA-nanoswitch functionalized with RuCO-OSU was incubated with different concentration of DNA target (0-3000 nM) complementary to their loop sequence. The same procedure was done for 1GC and 5GC probe functionalized with MB.
ECL signals obtained using 5GC DNA-switch as transducer were calculated by means of a ratiometric ratio using the following formula [1] : Where I II ecl and I I ecl are the ECL peak intensity gained through mechanism II and I respectively; I II 0ecl and I I 0ecl are the initial ECL intensity for each mechanism.
ECL and EC signals were analyzed and compare by calculating the Signal Gain %, through the following formula: Where I ecl(T) and I ec(T) are the ECL and EC peak intensity at a specific target concentration (0-3000nm) and I ecl(0) and I ec(0) are the ECL and EC intensity without target (close conformation).

ECL -DNA EIS characterization. EIS was performed on bare gold electrode showing a low
charge electron transfer resistance that switch to an increased resistance of the double layer after the formation of the Self Assembled Monolayer (SAM) of DNA-nanoswitch. After ECL emission at 1.6 V the EIS profile went back to a low charge electron transfer resistance profile due to the formation of the oxide layer on the gold electrode which disassemble the DNAnanoswitch SAM.