*3.1. Electrochemical Impedance Spectroscopy*

EIS is an effective technique for detecting complex formations on the electrode surface by detecting interface phenomena [70,71].

It measures the resistive and capacitive properties of the electrode upon perturbation with a small amplitude AC excitation. The frequency is varied over a wide range to generate the impedance spectrum, and the steady-state electrical impedance of the electrode/electrolyte interface is measured over an appropriate frequency range by applying a small sinusoidal voltage [72]. Impedance can be understood as the ratio of the voltage phasor to the current phasor of the system. When the impedance changes, it indicates that the detection target has been combined with the biometric element fixed on the electrode surface. EIS is usually able to distinguish between two or more electrochemical reactions occurring at the same time, identify diffusion-limiting reactions, mathematically evaluate experimental results using equivalent circuits (EEC), and reliably provide quantitative electrochemical data.

AuNPs are mainly used as electrodes and modification materials in EIS sensors [73–76]. When gold is used as the electrode, the aptamer is usually fixed on the surface of the gold electrode to generate a huge charge transfer resistance (Rct). When the corresponding target substance is added, the aptamer falls off from the surface of the gold electrode and combines with the target to produce an electrical signal transformation. Gu et al. developed an ultra-sensitive As3+ biosensor based on the hybridization chain reaction and RecJf exonuclease catalyzed reaction. In the presence of As3+, the aptamer specifically binds to As3+, resulting in DNA dissociation. The release of the hybrid chain reaction (HCR) product significantly reduced the Rct, and the detection limit was 0.26 nM. Moreover, the sensor has good selectivity. Even though the concentration of potential interfering ions is ten times higher than that of As3+, the change in Rct is negligible and only sensitive to As3+ [77]. Rabai et al. modified the surface of the gold electrode by a diazonium salt (CMA) electrochemical reduction method for the fixation of the aptamer. When Cd2+ was present, the aptamer changed from a random coil structure to a complex. This interaction blocks electron transfer, increasing the surface resistance, which is proportional to the concentration of Cd2+ in the sample [78]. In addition to traditional gold electrodes, the use of inkjet-printed gold electrodes as a reliable method for the detection of trace Hg2+ in water and organic solvents was first proposed in 2019. They applied water droplets of gold ink to a substrate and sintered it under the right conditions to produce printed gold electrodes. The aptamer was fixed to the electrode by the force of disulfide bonds, and then the interface was assembled layer by layer using impedance spectroscopy (PEIS) via RCT under optimal manufacturing conditions. With the increase in layers, RCT increases

gradually. The interfacial resistance increases from an average of 20.6 U to 144.5 U when the aptamer is fixed on the surface. With the addition of Hg2+, the ssDNA aptamer will change its secondary conformation by folding into a hairpin structure, establishing a bridge between the two thymidine residues, and forming a base pair, with a significantly reduced RCT. Figure 6a shows evidence of a directly proportional relationship between the response variable (RCT) and the target concentrations under optimal conditions. The working principle, signal strength, and linearity are also shown in Figure 6. The sensor has good stability and significant repeatability under harsh conditions and can detect Hg2+ at 0.005 ppM in organic solvents. Even when compared with high concentrations of cadmium, lead, and arsenic (50 ppM), it still has good selectivity [79].

**Figure 6.** Inkjet−printed electrochemical apt sensor performance analysis; (**a**) Analytical Faradaic impedance response under optimal fabrication conditions; (**b**) Working electrode diagram; (**c**) The bar chart represents the overall reaction, and the shaded area represents the signal strength after subtracting the background; (**d**) Linear correlation between signal and target concentration; and (**e**) All the results correspond to the mean value from 5 independent replicates and the error bars represent 1 SD from the mean. Reprinted with permission from ref. [79]. Copyright 2019 Elsevier.

AuNPs are commonly used to modify various carbon-based materials such as graphene oxide (GO), CNTs, etc. The high porosity of GO provides a large number of reaction sites for the bonding of AuNPs with aptamers. Wang et al. [80] used gold to modify porous GO (Au@p-rGO) and used it to fix substrate aptamers. Gold-modified GO (AuNPs@GO) was attached to the complementary chain as a signal probe; the current signal was obtained by recording the electrocatalytic conditions of H2O2. With the increase in Pb2+ concentration, the current response showed signal attenuation, and the lower limit of detection was 1.67 pmol/L. Rabai et al. [43] first dispersed CNTs in chitosan (CS) to modify a glassy carbon electrode (GCE). Chitosan improves its solubility and biocompatibility, and AuNPs are then electrically grafted onto the CNT-Cs/GCE. The synergistic effect of the two will make electron transfer easier, have good electrical conductivity, high surface electrical activity, and a detection limit as low as 0.02 ppm. Because the aptamer does not react with other heavy metal ions and the two-dimensional structure does not change, the sensor also has good selectivity. Yadav et al. [81] used silver (Ag)-Gold (Au) alloy nanoparticles (NP)-aptamer on a modified glassy carbon electrode (GCE) to detect Pb2+. Ag-Au bimetallic nanoparticles have electronic polarity which attracts the charged adsorption and promotes

the attachment of adsorption to the loading platform on the surface of GCE. A large number of binding sites can improve the sensitivity and stability of the electrode and reduce its detection limit.
