# Heavy Metal Ion Detection Platforms Based on a Glutathione Probe: A Mini Review
## 2 Recognition of HMIs by GSH  
Glutathione (GSH) is a ubiquitous tripeptide biomolecule (\(\gamma\)-Glu-Cys-Gly) widely found in animals and plants with a concentration from 0.1 mM to 10 mM [31]. GSH is known as an antidot to prevent cells from toxicosis of heavy metal ions by forming complexes as a key step in biological detoxification processes [30]. The chemical structure of GSH is shown in Figure 1. If oxidized, GSH will form disulfide bonds between themselves, and become what is known as glutathione disulphide (GSSG). GSSG lacks the ability to bind with electrodes or nanoparticles. In practice, GSH in its reduced form is used to bind with HMIs. Reduced GSH is a linear tripeptide of L-glutamine, L-cysteine, and glycine, so reduced GSH is also labeled as L-GSH. GSH contains one amino (-NH2), one sulfhydryl (-SH), and two carboxyl (-COOH) groups, all with the ability to bind with HMIs. Therefore, GSH is a versatile ligand with several binding modes. GSH has a sulfhydryl group on the cysteinyl portion. The sulfhydryl group is known to coordinate with a wide range of metal ions. For surface-based or nanoparticle-based HMI sensors, GSH is usually immobilized on gold nanoparticles or electrodes. Numerous experiments indicate that there is a strong binding force between gold and the (-SH) group. It is expected that, during probe immobilization, Au-S bonds are formed to link GSH molecules onto the gold surface [32; 33; 34; 35]. As a result, only one amino (-NH2) and two carboxyls (-COOH) are still available to bind with metal ions. A number of published papers have demonstrated that via the two free (-COOH) groups, strong binding with Pb\({}^{2+}\) is preferred over that with other HMIs [36; 37; 35]. It was found that the solution pH has a strong effect on the affinity of the GSH-Pb\({}^{2+}\) complex. At around pH 7, (-COOH) is deprotonated to (-COOH). Known as an oxyphilic ion, Pb\({}^{2+}\) can combine with four acetate molecules or eight oxygen atoms [33]. Within a pH range of 4-8, binding between Pb\({}^{2+}\) and (-COOH) will predominate over other reactions. Under the same pH condition, (-NH2) is protonated to (-NH3\({}^{+}\)), which is hypothesized to prevent GSH from combining with other ions due to its charges [33; 37]. Therefore, good affinity and selectivity can be achieved for GSH-Pb\({}^{2+}\) binding. There exist a number of reports on using GSH to specifically detect Pb\({}^{2+}\) from a mixture of multiple HMIs. Nevertheless, detection of metal ions other than Pb\({}^{2+}\) can also be achieved using GSH-based sensors. The GSH support material and the solution pH value play an important role in the HMI-GSH complexation pattern. Research has shown that GSH's conformational structure is strongly influenced by the solution pH [38]. Various functional groups in GSH can participate in complexation under different working conditions, leading to a certain selectivity. For example, GSH-capped quantum dots (QDs) are often used to detect HMIs other than Pb\({}^{2+}\). In [39], GSH immobilized on sulfur- and nitrogen-co-doped carbon QDs presented free (-SH) groups for binding, which showed strong affinity to Hg\({}^{2+}\) as opposed to other metal ions (Ag\({}^{+}\), Al\({}^{3+}\), Ba\({}^{2+}\), Cu\({}^{2+}\), Pb\({}^{2+}\), etc.) and realized specific detection of Hg\({}^{2+}\). Simultaneous detection of Cr\({}^{2+}\) and Pb\({}^{2+}\) was reported using GSH-coated magnetic nanoparticles (NPs) [40], as GSH molecules were immobilized onto Fe\({}_{3}\)O\({}_{4}\) NPs through a hydrogen bond. In this detection scheme, NH\({}^{4+}\), Na\({}^{+}\), Mg\({}^{2+}\), Hg\({}^{2+}\) and other MIs caused no obvious responses, except for Cd\({}^{2+}\) and Pb\({}^{2+}\). This was attributed in part to the negative charges on the functional groups of GSH [40], since the testing was done at a pH of 4.5. Simultaneous detection of multiple HMIs is thus possible by adjusting the solution pH and the GSH immobilization strategy.