*3.2. Differential Pulse Voltammetry*

The working principle of DPV is the constant voltage pulse amplitude overlayed on the step potential; immediately before each potential change measuring the electric current. The implementation response is the pulse strength at the beginning of the two currents between the resulting peak response; the difference between the two currents minimizes the amount of capacitance current, generating a higher signal-to-noise ratio of voltammograms [82]. DPV is a universal technique that can be used for both quantitative chemical analysis and the study of the mechanism, kinetics, and thermodynamics of chemical reactions. DPV is very sensitive and can routinely detect analytes at a part per billion level with a resolution higher than that achieved by cyclic voltammetry (CV), therefore, DPV is superior to CV when higher selectivity is required.

Reduced graphene (rGO) is commonly used in DPV sensors. It can be coupled with other nanomaterials as signal amplifiers or directly used as electrodes, which can significantly change the electrochemical properties. Luo et al. [83] used Fe3O4/rGO nanocomposite as a signal amplifier and many directional platinum nanotube arrays (PtNAs) crystallized in situ on flexible electrodes as sensing interfaces; a Hg2+ sensor was prepared. The schematic illustration of the assembly process and the detection strategy is shown in Figure 7. Due to its large surface area, it facilitates electrochemical performance and fixation of captured DNA (cDNA) and reporter DNA (rDNA). In the presence of Hg2+, part of the junction DNA binds closely to cDNAs through a thymine nucleotide pair (T-Hg2+-T). The Fe3O4/rGO nanoprobes attached to rDNAs were then fixed to the electrode by matching the remaining linker DNA with the rDNAs. Under optimal conditions, the Hg2+ aptamer sensor showed synergistic amplification performance with a linear range from 0.1 nM to 100 nM and a detection lower limit of 30 pM. When the heavy metal ions were not mercury ions, the aptamers could not undergo conformational changes through thymine nucleotides so this sensor had good selectivity. In addition, the E-apt sensor also showed reliable performance in the detection of real lake water samples.

**Figure 7.** Schematic illustration of the assembly process and the detection strategy; (**A**) Preparation of nano-probe; (**B**) Modification of flexible electrode. Reprinted with permission from ref. [83]. Copyright 2018 Elsevier.

When used as an electrode, aptamers can be fixed on rGO electrodes (ERGO) by π-π interaction. The ERGO electrode modified by aptamers can improve the value of Rct. Lee et al. [84] fixed the aptamer probe (Apt) labeled with methylene blue (MB) and part of its complementary DNA (cDNA) on the ERGO electrode to form the aptamer double-stranded structure, which blocked the effective electron transfer of MB to the electrode. After adding Cd2+, the aptamer unlocked the link and released the cDNA. This can quantitatively promote the electron transfer efficiency of MB, leading to the enhancement of electrochemical signals. Su et al. [85] also used this strategy to detect ultratrace Pb2+, as the existence of Pb2+ could make Apt fold into a G-quadruplex structure. The formation of the G-quadruplex leads to the separation of Apt from ERGO/GCE, which changes the REDOX current of MB labels with a detection limit of 0.51 fM. The sensor was tested in the presence of various metal ions (Cd2+, Co2+, Ag+, Cu2+, Mg2+, Ni2+, Zn2+, and Fe2+), but only Pb2+ resulted in a significant change in voltammetry response and had good repeatability. In addition to MB, toluidine blue (TB) molecules are also commonly used for electron migration in sensors, and the peak current of TB interacting with double-stranded DNA (dsDNA) is higher than that of single-stranded DNA (ssDNA). Ding et al. [86] used the composites of Au nanoparticles and a Polypyrene (Au@Py) modified screen printing electrode to amplify the current signal, fix the complementary chain on the electrode, and combined it with the aptamer to form a double chain structure. When Pb2+ was combined with an aptamer, the double chain structure was destroyed, and the peak current decreased continuously. Ma et al. [87] developed a sensor for Hg2+ ultra-sensitive determination, also using TB to characterize electron migration, using mesoporous silica nanocontainers (MSNs) as containers. MSNs have a rich porous structure that can trap TB molecules using AuNPs to link specific ssDNA. Hg2+ induces ssDNA to form a hairpin structure and the stored tuberculous molecules are released from MSNs. The electron transfer signal of TB was stably detected by micro DPV, which was correlated with the concentration of Hg2+, with a low detection limit of 2.9 pM. Jin et al. [88] developed an electrochemical adaptive sensor for Pb2+ detection using porous carbon (PCs) loaded platinum nanoparticles (PtNPs) to catalyze the hydroquinone-H2O2 system in the form of simulated enzymes. PtNPs@PCs were fixed on the electrode surface by the specific binding of streptavidin and biotin and catalyzed the oxidation of hydroquinone in the presence of Pb2+ and H2O2. The resulting electrochemical signal was dependent on the concentration of Pb2+.
