**5. Metal**/**Metal Oxide-Metal Oxide Nanocomposites for NEGHS**

A combination of two metal/metal oxides is another effective approach to improve the electro catalytic activity for NEGH sensing [93]. In recent years, researchers have focused on perovskite oxide (ABO3)-type nanomaterials as an attractive non-noble metal alternative in the electrochemical field, mainly due to the presence of oxygen vacancies in the crystal structure. Some perovskites have a strong electrocatalytic activity to oxygen reduction and oxidation phenomena and are most suitable for NEGH sensing. Liotta et al. (2015) investigated low cost, commercial carbon screen-printed electrodes (CPEs) and modified perovskite nanomaterials for NEGHS. La0.6Sr0.4Fe0.8O3-d (LSF) and La0.6Sr0.4Co0.2Fe0.8O3-d (LSCF) perovskites were synthesized by the citrate method (citric acid/metal ratio=1:5) in the presence of NH4OH at pH 9 to 10. The fabricated modified electrodes showed enhanced oxidation current attributed to the mixed vacancy states of Co and Fe ions, which are accountable for charge transfer in the electro-oxidation of glucose and H2O2. In addition, the LSCF sensor attained good selectivity due to lower anodic potential in human serum samples [94]. Similarly, Zhang et al. (2012) synthesized LaNi0.6Co0.4O<sup>3</sup> (LNC) via a sol-gel method and demonstrated the NEGHS based on LNC/CPEs modified electrode, which avoids the stability problems, complex fabrication process and limited lifetime. LNC NPs showed excellent electrocatalysis to the oxidation of H2O<sup>2</sup> and glucose due to the increased electroactive surface area, intrinsic peroxidase-like activity, and the existence of abundant active sites. The sensor demonstrated good sensitivity and a low level of detection (for H2O2, the concentration range was 10 nM–100 µM with 1.0 nM detection limit; for glucose, the concentration range was 0.05–200 µM with 8.0 nM detection limit). Moreover, this prepared sensor was able to detect glucose in serum and H2O<sup>2</sup> in toothpaste samples [95].

The same research group also prepared novel Co0.4Fe0.6LaO<sup>3</sup> (CFL) NPs via a sol-gel method, and the CFL NPs revealed smooth surfaces with uniform thickness and a particle size of 30 to 70 nm. The enhanced electrocatalytic activity of the composite was attributed to the active sites, which are the transition metal ions with partially occupied d orbitals. The Co0.4Fe0.6LaO<sup>3</sup> catalyst has transition metals in mixed oxidation states as Fe(II)Co(II)/Fe(III)Co(III), and cyclic electron transfer happens while detecting H2O2. During the sensing process, the strong oxidizing agents, Fe (III) and Co (III) electrochemically oxidize CFLs to FeOOH and CoOOH. The OH<sup>−</sup> ions formed along with the species act as the reactive units for glucose sensing. This sensor also offers a fast response, high stability, good reproducibility, and reasonable selectivity. The authors proposed the use of these perovskite structure oxide-based, low-cost, non-enzymatic sensors for public health and environmental applications [96]. In order to increase the performance of perovskite, the rGO has been used for NEGHS application by He et al. (2017). This group synthesized LSC, LSCF, and LNC perovskite via a sol gel process using Ethylenediaminetetraacetic acid (EDTA) citrate as complexing agent. Among them, LSC showed superior electro-oxidation of glucose and H2O2. This work revealed the possible electro chemical mechanism and its pathways of redox activity and formation of Co3+/Co4<sup>+</sup> redox couple via oxygen vacancies and made a route to elucidate the theoretical framework to design new perovskite sensing electrodes. They finally proposed the combination of perovskite with rGO acting as a unique sensing electrode with notable sensitivity, selectivity, stability, and reproducibility through a synergistic effect [97]. Furthermore, the morphology of perovskite was improved by Wang et al. (2013) based on LaNiO<sup>3</sup> nanofibers (LNFs) by electro spinning and subsequent calcination and achieved a high surface area because of the porous structure. This type of effective combination of synthesis method and electrochemical sensing developed reliable NEGH biosensors, with an ultra-low detection limit and wide linear range (33.9 nM and 0.05 to 1000 µM for glucose). This LNFs/CPE performed enhanced catalytic activity and high selectivity and sensitivity in the presence of AA, UA, and DA, in addition to the long-term stability [98]. Xia and coworkers (2018) utilized the same electro spinning process without using a template as an added advantage for NEGH sensing application to avoid contamination problems. This group prepared highly porous CuFe2O<sup>4</sup> nanotubes on nickel foam and achieved a high sensitivity and low detection methods in alkaline solutions. The excellent porosity, flexibility, surface area, inner and outer surfaces and good conductivity of the nanotubes provide many active sites and

transmission of electrons. This leads to the high electrochemical activity of the material at a typical voltage of 0.5 mV in alkaline media [99]. Furthermore, Ensafi et al. (2016) formulated the NEGH sensor based on Ag/SiO<sup>2</sup> nanostructures. These were prepared by decorating the surface of organic functionalized SiO<sup>2</sup> with silver nanoparticles. Modifications of CPE allowed for easier electron transfer when compared to the unmodified electrode as the functionalized SiO2/decorated Ag nanoparticles achieved electrocatalytic effects. Synthesized compounds were used to analyze H2O<sup>2</sup> and glucose levels in commercial UHT dairy products and plasma samples, showing a remarkable selectivity towards H2O<sup>2</sup> and glucose levels, respectively [100]. Zhao et al. (2017) exploited a facile hydrothermal technique for the in situ deposition of CuO/rGO on copper foil. Structural and morphological characterization confirmed that the nanocomposite contained three types of interfaces, namely CuO/rGO, rGO/Cu2O, and Cu2O/Cu. This facilitated redox reactions between GO and the copper foil, resulted in the electrostatic attraction of (+) vely charged copper ions and (–) vely charged rGO. The modified foil achieved an amperometric response of glucose (at 0.45 V vs. SCE) with a low detection limit of 0.10 mM, a linear range of 0.5 to 8.3 mM, an ultrahigh sensitivity of 3401 mA mM−<sup>1</sup> cm−<sup>2</sup> , and a response time of <0.5 s. With regards to H2O<sup>2</sup> sensing at an applied potential of −0.2V, the modified electrode had a low detection limit of 0.05 mM, a linear range of 0.5 to 9.7 mM, a sensitivity of 366.2 mA mM−<sup>1</sup> cm−<sup>2</sup> , and a response time of 0.8s. Moreover, the prepared CuO/rGO/Cu2O/Cu electrode was applied to detect glucose levels in human serum determined to be 4.86 mM, consistent with the laboratory-based value of 5.01 mM [101].

A recent report investigated a one-step anodization process to construct a self-supporting Co3O4/nanoporous gold (NPG) composite. This electrode effectively worked in alkaline solutions with an ultra-sensitivity of 4470.4 mA mM−<sup>1</sup> cm−<sup>2</sup> , a low detection limit of 0.085 mM, and a linear range of 2 µM to 2.11 mM for the detection of glucose. With the same electrode, H2O<sup>2</sup> showed a sensitivity of 1338.7 mA mM−<sup>1</sup> cm−<sup>2</sup> with a linear range of 20 to 19.1 mM, and both the sensing results are comparable to the hospital laboratory results [102]. Direct growth of nanostructures on Cu foil via a hydrothermal method was the subject of research for generating tubular hierarchical structures. The morphological studies of synthesized Ni (OH)2/rGO/Cu2O revealed that Cu substrate modified to Cu2O with uniform cubic structure and tubular hierarchical structures of Ni (OH)<sup>2</sup> are grown on the surface of reduced graphene oxide as shown in Figure 1f. The outstanding electro catalytic activity of this material was ascribed to the synergistic interactions of rGO, Ni (OH)2, and Cu2O. Ni (OH)<sup>2</sup> structure had promoted the effective diffusion of glucose molecules, while the wrinkled graphene functioned as an excellent electric conductor. The sensor showed remarkable reproducibility and superior stability for long-term applications [40]. Long et al. (2018) reported CuO/NiO hallow nanocomposite via the solvothermal process and subsequent thermal treatment. This work developed core-shell, yolk-shell, or hollow structure of CuO/NiO by adjusting the amount of NiCl<sup>2</sup> during synthesis. A porous hallow structure showed outstanding electrochemical properties due to the synergetic interaction of CuO and NiO, porous hallow, and large void spaces. The electrode exhibited a high sensitivity towards glucose and for H2O2. Furthermore, it was applied in human serum to estimate practical feasibility [103]. Wang et al. (2018) produced a novel combination of nano hybrids through a two-step process for NEGH sensing. A particle size of 7 nm ZnO/CoO nanoparticles was decorated over a graphene sheet to achieve high conductivity and abundant active sites. This modified ZnO/CoO/rGO/GCE showed a remarkable selectivity in the presence of AA, UA and KCl, due to the low working potential of the electrode [104]. Lu et al. (2019) fabricated a highly conductive and large surface area electrode for NEGH sensing based on 3D nitrogen-doped graphene hydrogel (NHGH) decorated with NiCo2O<sup>4</sup> nanoflowers using the hydrothermal method. The novel NHGH/NiCo2O<sup>4</sup> nanocomposites performed an excellent electro catalytic activity to detect glucose and H2O<sup>2</sup> due to the presence of abundant active sites. The redox reactions of Co and Ni species in alkaline solution explains the same as mentioned in previously reported work. The sensitivity and high selectivity parameters have been used to detect glucose in blood. Taken together, the results suggest that the hybrid nanocomposite is a promising non-enzymatic electrochemical sensor [105].

From the above discussions, it is clear that researchers have focused on synthesizing bifunctional catalysts using metal (oxide)/metal oxide nanocomposites for NEGH sensing. The morphology, dimensions, surface area, grain and pore size have primarily effected electrochemical NEGH sensing. The peculiar nanostructures such as porous nanotubes, and nano fibers enhance inner and outer surface area, high porosity, excellent flexibility, facilitate the sluggish kinetic process (oxidation of inactive glucose), improve mass and electron transfer between electrode and electrolyte, and maximize the number of active sites. These properties have potentially improved the linear ranges, sensitivity and selectivity. The use of screen-printed electrodes have motivated researchers to elaborate NEGH sensing research in real-time applications and overcome the limitations of portability and instability. A few research groups focused on perovskite-type ferrites for sensing both glucose and H2O<sup>2</sup> because of their fascinating physical and chemical properties such as dual catalytic property (catalysis and peroxidase activity), superior electro catalytic activity, low cost, biocompatibility, rapidness and sensitivity. By considering rGO incorporation, it has solved the aforementioned problems of agglomeration, stability and poor conductivity. The mixed metal oxide exhibited higher conductivity as the activation energy required to transfer electrons from cations is relatively low, which further enhanced the sensing parameters. A significantly smaller number of publications are reported for metal/metal oxide combinations due to wide band gap and homogeneity issues. Based on the literatures, a limited number of transition mixed metal oxide was used for NEGH sensing even though these are low cost and exhibit good electrochemical behavior. Therefore, nanostructures with a high surface area and enhanced charge transfer electrode would be desirable for NEGH sensing in the future. The NEGH sensing parameter with the same electrode-based metal (oxide)/metal oxide nanocomposite are compared in Table 4.



#### *Biosensors* **2020**, *10*, 151
