**1. Introduction**

Silver ion (Ag+) has been widely used as an antiseptic in cosmetics, building materials, and medical products owing to its antibacterial properties [1–4]. However, overuse of Ag+ inevitably leads to environmental pollution. Human exposure to Ag+ pollution mainly comes from the release of airborne silver nanoparticles and natural water contaminated by industrial sources [5,6]. The tolerable concentration of Ag+ in drinking water is ~927 nM as recommended by the World Health Organization [7]. Excessive Ag+ ingestion can cause certain serious health consequences, such as respiratory system injury, organ failure, and even cancer [6,8–11]. Various methods have been developed for detecting low concentrations of Ag+ in environmental samples and drinking water sources. At present, Ag<sup>+</sup> detection is mainly carried out by conventional analytical methods such as inductively coupled plasma mass spectrometry [12], optical emission spectrometry [13], atomic absorption spectrometry [14,15], and laser ablation microwave plasma torch optical emission spectrometry [16]. These conventional methods are sensitive and selective, but they rely on expensive instruments and intensive labor.

In recent years, nucleic acid molecules have gained prominence in the fields of sensing and material science because of their programmability and predictability by forming complementary base pairs [17]. DNA molecules have been used to design sensors for detecting metal ions such as Ag+, UO2 2+, Cu2+, Ca2+, Mg2+, Hg2+, and Pb2+ [18–26]. In general, there are mainly two DNA-based strategies for Ag+ detection. The first strategy utilizes an Ag+ dependent DNAzyme that can irreversibly cleave an RNA or DNA substrate in the presence

**Citation:** Zhang, J.; Liu, Y.; Yan, Z.; Wang, Y.; Guo, P. A Novel Minidumbbell DNA-Based Sensor for Silver Ion Detection. *Biosensors* **2023**, *13*, 358. https://doi.org/10.3390/ bios13030358

Received: 10 January 2023 Revised: 2 March 2023 Accepted: 3 March 2023 Published: 8 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of Ag+ [22]. The second strategy is based on the well-established knowledge that Ag+ binds to cytosine (C) at the N3 site to coordinate and stabilize a C·C mismatch [27,28]. Ag+ can induce the formation of DNA i-motif or hairpin structures that contain C·C mismatch(es), thus giving reporting signals upon DNA conformational change [26,29–33]. Moreover, the duplex or hairpin-forming strands can also be assembled onto nanomaterials for signal amplification [34–38]. The second strategy can achieve a low detection limit, but the reported ones generally used relatively long oligonucleotides, which might make the Ag+-induced DNA conformational change slow. For instance, a DNA sensor based on a 20-nucleotide (nt) hairpin required an incubation time of at least 10 min for Ag+ detection. Therefore, a DNA sensor using a short oligonucleotide is expected to have advantages of fast response, easy operation, and probably anti-interference capability in a complex environment, which allow for the further development of on-site environmental detection devices [33,39].

Minidumbbell (MDB) is a type of non-B DNA structure formed by 8–10-nt sequences [40,41]. The MDB structure was initially found to form in CCTG tetranucleotide repeats, which are associated with the neurodegenerative disease of myotonic dystrophy type 2 [40,41]. The CCTG MDB is simply composed of two repeats, i.e., 5'-CCTG CCTG-3', and each repeat folds into a type II tetraloop. The C1-G4 and C5-G8 adopt Watson-Crick loop-closing base pairs; C2 and C6 fold into the minor groove, whereas T3 and T7 stack on the C1-G4 and C5-G8, respectively (Figure 1) [40]. One of the most interesting features of this MDB is that the two minor groove residues formed a unique reverse wobble C2·C6 mispair containing one/two hydrogen bond(s) or Na+-mediated electrostatic interactions at neural pH [42], or a C2+·C6 mispair containing three hydrogen bonds with C2 being protonated at acidic pH (Figure 1) [43]. Upon lowering the pH from 7 to 5, the melting temperature (*Tm*) of the CCTG MDB was increased from ~20 ◦C to <sup>46</sup> ◦C [43]. Apart from pH, we wondered if Ag+ could coordinate the C2·C6 mispair to stabilize the MDB and then induce a DNA conformational change for Ag+ sensing. Here we report a novel and minimal DNA sensor, based on the CCTG MDB, for Ag+ detection with high sensitivity and fast kinetics.

**Figure 1.** The averaged solution nuclear magnetic resonance (NMR) structure of the CCTG MDB at pH 7 (PDB ID: 5GWL) and pH 5 (PDB ID: 7D0Z). C2 and C6 formed predominantly a one-hydrogenbond mispair at pH 7, whereas they formed a stable three-hydrogen-bond mispair at pH 5 with C2 being protonated.
