*3.5. Discussions on DNA-Based Ag+ Sensors*

As surveyed from the literature, DNA-based Ag+ sensors can be generally classified into three types: (i) mismatch-containing DNA functionalized with nanomaterials [34–37], (ii) mismatch-containing DNA only [26,29,30,32], and (iii) DNAzyme [22] (Table 2). The ensemble of mismatch-containing DNA and nanomaterials is an effective strategy to improve the detection limit by taking advantage of amplified local DNA concentration and interaction surfaces. Recently, Pal et al. have reported an electrochemical Ag+ sensor based on DNA hairpin-functionalized nanoflakes with a detection limit of 0.8 pM [38]. Comparing with the detection limits of other sensors using only mismatch-containing DNA (i-motifs and hairpins), detection limit of the *M-DNA* sensor was the lowest. In addition, the *M-DNA* sensor exhibited a response time of less than 2 s, which is kinetically much faster than those using i-motifs and hairpins (Table 2). However, the *M-DNA* sensor requires a controlled acidic pH to work, and this limitation may be further improved by chemical modification, such as cytosine methylation, to enhance the thermodynamic stability of the CCTG MDB. Overall, the *M-DNA* sensor uses an ultrashort oligonucleotide to achieve a high sensitivity and fast response for Ag+ detection.

**Table 2.** Literature survey on DNA-based sensors for Ag+ detection.



**Table 2.** *Cont.*

<sup>a</sup> The kinetic data was derived from time-dependent fluorescence spectra. <sup>b</sup> There was no kinetic data available.

<sup>c</sup> The kinetic data was derived from time-dependent electrochemical change.

#### **4. Conclusions**

In sum, we have designed a smart DNA sensor for Ag+ detection using a new form of non-B DNA, i.e., a minidumbbell, apart from the previously used hairpins and i-motifs. Owing to its small size, it shows fast response, high sensitivity, high selectivity, and good anti-interference capability for Ag<sup>+</sup> sensing. The performance of this *M-DNA* sensor may be further improved by chemical modification to further enhance the thermodynamic stability of the CCTG MDB. A successful demonstration of this *M-DNA* sensor provides new insights into Ag+ detection, and paves the way for designing DNA-based tools to sense other metal ions and molecules.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/bios13030358/s1, Figure S1: CD changes of *M-DNA* with SGI in 10 mM NaPi at pH 6 before and after adding Ag+; Figure S2: Fluorescence changes of variousconcentration *M-DNA* in 10 mM NaPi at pH 6, with different SGI:*M-DNA* ratios before and after adding Ag+; Figure S3: Normalized fluorescence intensity at 520 nm of the *M-DNA* and *C-DNA* upon titrating various concentrations of Ag+.

**Author Contributions:** J.Z.: methodology, investigation, formal analysis, data curation, and writing—original draft; Y.L.: methodology, investigation, formal analysis, and writing—original draft; Z.Y.: investigation, formal analysis, and writing—original draft; Y.W.: conceptualization, methodology, formal analysis, and writing—review and editing; P.G.: conceptualization, methodology, formal analysis, writing—review and editing, supervision, and project administration. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (22004038), the Natural Science Foundation of Guangdong Province, China (2021A1515010102), the Guangdong Basic and Applied Basic Research Foundation (2021A1515111174), the Science and Technology Project of Guangzhou (202201010471), the China Postdoctoral Science Foundation (2022M722112), and a start-up fund from the Institute of Basic Medicine and Cancer (IBMC) of the Chinese Academy of Sciences (2022QD13).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in supplementary material.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


**Disclaimer/Publisher's Note:** The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
