*3.3. Cobalt Oxide (Co3O4) and Nickel Oxide (NiO) Nanocomposite*

Cobalt oxide exists in three polymorph forms as cobaltous oxide (CoO), cobaltic oxide (Co2O3), and cobalt oxide (Co3O4). Among them, Co3O<sup>4</sup> has been studied for non-enzymatic glucose and H2O<sup>2</sup> sensing because of its biocompatibility, and pseudo electro catalytic property. A few research articles are available for the dual sensing of NEGH based on Co3O<sup>4</sup> nanomaterial. The synthesis of Co3O<sup>4</sup> NPs

using metal-organic frameworks (MOFs) as a template was investigated by Hou et al. (2012). For this, the Co3O<sup>4</sup> NPs of 20 nm diameter were drop casted on GCE and tested NEGH sensing in alkaline media. The modified electrode also showed efficient practicable performance in human serum for glucose and in disinfectant solution for H2O2. Overall, the Co3O<sup>4</sup> NPs showed a satisfactory performance when compared with traditional results [73]. Furthermore, Karuppiah et al. (2014) adopted the hydrothermal method to fabricate graphene/Co3O4-NP composite for the electrochemical sensing of glucose and H2O2. SEM images revealed a uniform distribution of Co3O<sup>4</sup> nanoparticles on graphene nanoflakes due to the strong interaction of Co-O-C bonds as a result of the highly reactive sp<sup>2</sup> carbon atoms of the graphene flakes and the electron-rich oxygen species of Co3O<sup>4</sup> nanoparticles. The modified electrode exhibited excellent stability, repeatability and reproducibility [74]. The nickel oxide (NiO) nanomaterial also holds great promise as an electrode material for non-enzymatic sensing due to its low toxicity, excellent electro catalytic activity, and stability. Ni forms hydroxide species (NiOOH) in alkaline medium and catalyzes the analytes' oxidation during the sensing process. When the Ni-based materials as nanoparticles or nanocomposites are grown on specific substrates, the synergistic effect of particle–substrate combination enhances the efficiency of electro catalytic sensing [75]. Many different ways are adopted to develop the Ni-based sensors, a few of which are discussed in this section. GoO et al. (2014) proposed a conventional electrodeposition technique for NEGH sensing based on Ni (OH)2/electro reduced graphene oxide (ERGO)-MWCNTs. In this nanocomposite, graphene oxide (GO) nano sheets served as a surfactant to stabilize MWCNTs, whereas MWNTs functioned as connecting bridges between ERGO sheets and GCE to enhance the electron transfer mechanism, and Ni (OH)<sup>2</sup> acts as a suitable electro catalyst for glucose and H2O<sup>2</sup> sensing. This sensor exhibited a very high sensitivity due to the synergistic interaction and further confirmed the practical application in urine and milk samples. The glucose showed 106% recovery with a relative standard deviation of 3.72% in urine and, the H2O<sup>2</sup> sensor retained 104.9% with a standard deviation of 2.39% in milk. This work opens new avenues for NEGH sensors as non-enzymatic biosensors [76].

The current review addressed the unique metal oxides, such as Cu2O, CuO and NiO Co3O4, in NEGH sensing. CuO- and Cu2O-based nanomaterials are the most popular modified electrodes in NEGH sensing due to its efficient catalytic property, stability and runnable working potential to avoid interference during sensing. However, these electrodes have a few limitations, such as poor conductivity and structural instability during operation. To overcome these limitations and to realize the practical applications, researchers have focused on designing unique morphologies and combined them with carbon/polymer materials. The metal oxide nanocomposite in neutral/acid/alkaline media have performed with high sensitivity and selectivity. In a few reports, different morphologies of CuO electrode have demonstrated high sensitivity, especially with low interference phenomena due to its tunable working potential. Therefore, researchers need to focus on novel metal oxide electrode materials, such as MnO, CeO2, TiO<sup>2</sup> and Fe3O4/Fe2O3, for dual sensing of glucose and H2O2.

#### **4. Metal-Metal Nanocomposites for NEGH Sensing**

Bimetallic nanoparticles (BNPs) have been extensively investigated in various applications because of their unique properties, and they are more efficient catalysts than most mono metal NPs. The presence of synergistic interactions between two metals within a bimetallic system can potentially improve NEGH sensing performance and reduce surface poisoning, interfering effects of electrodes and bimetallic structures, including alloys such as Pt-Co, Au-Ag and Pd-Ni. BNP-based sensors have shown better sensing parameters due to enhanced electron transfer and surface area dependent tunable electro catalytic activity. In recent years, BNP-based sensor reports have increased, reflecting a change in the trend of engineered nanomaterials. Bimetallic systems have been combined with graphene/CNT in NEGH sensing, leading to significant advances in this area.

### *4.1. Platinum Bimetallic Nanocomposite*

Bimetallic platinum-gold nanoparticles are one of the preferable alloys in catalytic and bio-sensing studies. The three-dimensional nano spongy architecture for the PtAu alloy was developed with a size of 5 nm by Wang et al. (2014) and utilised for electrochemical sensing. The nanoporous metals possessed an interconnected network backbone and hollow channels, large internal surface and high electrocatalytic activity. PtAu NP catalysts were obtained at much higher current densities than commercial Pt/C and Pt NPs due to the synergetic catalytic effect of Pt and Au. The homogeneity of PtAu NPs facilitated mass transport and electrical conductivity, leading to enhanced chloride ion resistance, showing high sensitivity, a good detection limit and a wider linear range to H2O2. However, with glucose, the values were slightly less (0.5 µM detection limit and 0.2–5.4 mM linear range), but comparable stability, durability and selectivity [77]. Though the bi-continuous nano scaled skeletons and interconnected hollow channels within the particle promoted the electrochemical sensing responses, an additional nafion coating, when applied, decreased the selectivity by allowing the interferents to react.

In order to decrease the cost of the electrode and explore the catalytic activity of Co, Au was replaced with Co and fabricated Nano porous PtCo NPs with a size distribution of 3 nm by dealloying PtCoAl in a mildly alkaline solution. The simple dealloying process pre-defines the nanoparticles' bimetallic composition without losing the target metal, compared with the traditional chemical synthesis through which the reduction in individual metal salts occurs. The current density for H2O<sup>2</sup> oxidation by the PtCo nanoparticle (0.90–1.2 V) was 10 times higher than that of the Pt/C catalyst due to the catalysis effect on the H2O<sup>2</sup> electro oxidation. Higher scan rates also increased the oxidation current, attributed to the diffusion-controlled process happening on the PtCO alloy. The PtCo alloy showed a high sensitivity response, and wide linear range due to its synergetic electro catalytic activity on electrochemical reactions. Besides, the PtCo alloy also exhibited good anti-interference towards AA, UA, and DA. The authors also reported effective detection of ethanol in addition to glucose and H2O<sup>2</sup> and claimed advantages such as easy preparation, improved precious metal utilization, and large-scale preparation [78]. Furthermore, in the NEGH sensing of Pt BNP, Mei et al. (2016) designed a novel PtNi/MWCNTs nanocomposite using a chemical reduction method. Ni NPs in the PtNi alloy had a dramatic synergetic effect on the electrochemical activity. In contrast, the CNTs enhanced the electro catalytic activity of the alloy and prevented alloy precipitation or aggregation, resulting in the acceleration of electron transfer and enhanced sensitivity. Extraordinarily, the Pt/Ni/MWNCTs-based sensor exhibited superior electro catalytic activity in neutral solutions towards H2O<sup>2</sup> and glucose at a positive working potential of 0.45 V and +0.1 V [79]. To further boost the catalytic and electronic properties of Pt BNP, Mei et al. (2016) have developed a core-shell nanoparticle's morphology using Pt as a shell and Fe as a core part with carbon (Fe@Pt/C) for the sensing of glucose in human serum samples and H2O<sup>2</sup> in lake water and antibacterial lotion (3%). The Fe@Pt/C core-shell nanoparticles were prepared by spontaneous replacement reactions using Vulcan XC-72 carbon as supportive material. The superb electrical conductivity and great electro catalytic activity of Fe@Pt/C make them sensitive and rapid electrochemical sensing platforms for the reduction of H2O<sup>2</sup> and oxidation of glucose. In fact, the lower electronegativity of Fe compared to Pt changes the electronic properties of Pt and its d-band density is lowered in energy in the Fe@Pt-skin structure. This induces changes in chemisorption energies and increases the number of analyte adsorption active sites. These surface structural and electronic effects (in other words strain and ligand effect) are responsible for the electrochemical sensing mechanisms in response to various analytes. The H2O<sup>2</sup> sensor retained 92% of the current response in 30 days, indicating long-term stability and reproducibility with a relative standard deviation of 1.2%. Practical applicability was also studied by testing the presence of H2O<sup>2</sup> in lake water and antibacterial lotion and glucose in human blood, and in all cases, very similar values were obtained with recorded data. In addition, this sensor also exhibited good reproducibility, long-term stability, and selectivity in the presence of interference compounds [80].

### *4.2. Palladium Bimetallic Nanocomposite*

Three-dimensional and bicontinuous nanospongy PdCr alloy of ligament size, ∼5 nm was reported for the significant effect of Cr in improving the stability and exhibiting synergetic catalytic effect on electro catalytic reactions for NEGH sensing. The as-synthesized PdCr alloy exhibited a wide linear range (0.1 to 1.9 mM) with low detection limit (3.1 µM) towards H2O<sup>2</sup> sensing with no loss in electro catalytic activity after long-term storage for two weeks. The sensor also showed high sensing properties for glucose with wide linear ranges (1–38 mM) [81]. With the same synthesis method, the same research group also developed nano porous PdFe and studied the effect of Fe on the sensing performance of both glucose and H2O2. Dealloying PdFeAl here also produced similar nanospongy architecture with 5 nm ligament size. When Fe is combined with Pd, the electrochemical properties modify due to the smaller electronegativity of the Fe as opposed to the Pd and enhances the d-band electron density in Pd for the generation of OHads species on a PdFe nanocomposite surface. The desorption of OHads or reduction of Pd/Fe generates the active metallic surface for the electro-oxidation of glucose. The added advantage of this sensor is the high resistance to interference species such as Cl<sup>−</sup> ions, AA, UA, and DA [82]. Using the same dealloying method, Dianyun et al. (2015) generated nanoporous PdNi alloy composite for NEGH sensing. The nanoporous network with hallow interconnections made a bicontinuous skeleton nature for the nanocomposites. The electrochemical parameters revealed a high catalytic activity of as-synthesized PdNi alloy rather than Pd NPs and Pd/C catalyst. This work provided a simple and green route to construct efficient electrodes for glucose and H2O<sup>2</sup> non-enzymatic sensing [83]. Furthermore, researchers combined unique 2D molybdenum disulfide (MoS2) nano sheets and the high electro catalytic activity of Au-Pd BNP using a facile thermal co-reduction method to achieve a wide linear range, low detection limit, and good stability. A low working potential of −0.3 V for the reduction of H2O<sup>2</sup> in a neutral solution and the −0.1 V for glucose in alkaline medium was reported using Au-Pd/MoS<sup>2</sup> nanocomposites. Both glass electrode and MoS<sup>2</sup> nanosheets did not show any oxidation peak in the presence of glucose, indicating their non-electrocatalytic activity. Strong peak currents of glucose electrooxidation was observed for Au-Pd/MoS<sup>2</sup> electrode. Glucose on exposure to the electrode surface, is adsorbed on to it due to the dehydrogenation of the anomeric C1 atom. Such adsorbed moieties occupy the Pd active sites and inhibit further electrooxidation of glucose. During a positive potential scan, the Pd-OH species developed in the presence of aqueous NaOH catalyze the adsorbed intermediate oxidation, which makes the Pd active sites free for the direct electrooxidation of glucose. Further positive potential scan decreases the peak current as Pd oxide is formed to inhibit the electrooxidation. The reduction of this Pd oxide occurs during the negative potential scan and almost simultaneously, the surface Pd active sites become available for the electrooxidation process. All these significant reactions are attributed to the synergetic interaction between the MoS<sup>2</sup> and Au-Pd bimetallic combination [84]. A highly electroactive material was fabricated for the first time without a pretreatment approach based on in situ Pd-Co alloy supported over carbon nanotubes (Pd-CoCNTs) via a one-pot pyrolysis process as shown in Figure 4a. The nanostructure prevented agglomeration due to in situ formation and has much more stability than the previously reported Pd nanocomposite, and the morphology of the TEM image is depicted in Figure 4b. The low concentration of Pd and small size (diameters of 2–4 nm) on Pd-CoCNTs reduced the competition among active sites and resulted in good selectivity, good stability and sensitivity. While the lower concentration and detection limit for glucose were 10 µM and 1 µM, respectively, the sensor exhibited a 0.3 µM detection limit for H2O<sup>2</sup> [85].

properties for glucose with wide linear ranges (1–38 mM) [81]. With the same synthesis method, the same research group also developed nano porous PdFe and studied the effect of Fe on the sensing performance of both glucose and H2O2. Dealloying PdFeAl here also produced similar nanospongy architecture with 5 nm ligament size. When Fe is combined with Pd, the electrochemical properties modify due to the smaller electronegativity of the Fe as opposed to the Pd and enhances the d-band electron density in Pd for the generation of OHads species on a PdFe nanocomposite surface. The desorption of OHads or reduction of Pd/Fe generates the active metallic surface for the electrooxidation of glucose. The added advantage of this sensor is the high resistance to interference species such as Cl- ions, AA, UA, and DA [82]. Using the same dealloying method, Dianyun et al. (2015) generated nanoporous PdNi alloy composite for NEGH sensing. The nanoporous network with hallow interconnections made a bicontinuous skeleton nature for the nanocomposites. The electrochemical parameters revealed a high catalytic activity of as-synthesized PdNi alloy rather than Pd NPs and Pd/C catalyst. This work provided a simple and green route to construct efficient electrodes for glucose and H2O2 non-enzymatic sensing [83]. Furthermore, researchers combined unique 2D molybdenum disulfide (MoS2) nano sheets and the high electro catalytic activity of Au-Pd BNP using a facile thermal co-reduction method to achieve a wide linear range, low detection limit, and good stability. A low working potential of −0.3 V for the reduction of H2O2 in a neutral solution and the −0.1 V for glucose in alkaline medium was reported using Au-Pd/MoS2 nanocomposites. Both glass electrode and MoS2 nanosheets did not show any oxidation peak in the presence of glucose, indicating their non-electrocatalytic activity. Strong peak currents of glucose electrooxidation was observed for Au-Pd/MoS2 electrode. Glucose on exposure to the electrode surface, is adsorbed on to it due to the dehydrogenation of the anomeric C1 atom. Such adsorbed moieties occupy the Pd active sites and inhibit further electrooxidation of glucose. During a positive potential scan, the Pd-OH species developed in the presence of aqueous NaOH catalyze the adsorbed intermediate oxidation, which makes the Pd active sites free for the direct electrooxidation of glucose. Further positive potential scan decreases the peak current as Pd oxide is formed to inhibit the electrooxidation. The reduction of this Pd oxide occurs during the negative potential scan and almost simultaneously, the surface Pd active sites become available for the electrooxidation process. All these significant reactions are attributed to the synergetic interaction between the MoS2 and Au-Pd bimetallic combination [84]. A highly electroactive material was fabricated for the first time without a pretreatment approach based on in situ Pd-Co alloy supported over carbon nanotubes (Pd-CoCNTs) via a one-pot pyrolysis process as shown in Figure 4a. The nanostructure prevented agglomeration due to in situ formation and has much more stability than the previously reported Pd nanocomposite, and the morphology of the TEM image is depicted in Figure 4b. The low concentration of Pd and small size (diameters of 2–4 nm) on Pd-CoCNTs reduced the competition among active sites and resulted in good selectivity, good stability and sensitivity. While the lower concentration and

**Figure 4.** (**a**) Schematic diagram for preparation of Pd-CoCNT; (**b**) HRTEM images of Pd-CoCNT [85]. **Figure 4.** (**a**) Schematic diagram for preparation of Pd-CoCNT; (**b**) HRTEM images of Pd-CoCNT [85].

#### *4.3. Copper Bimetallic Nanocomposite 4.3. Copper Bimetallic Nanocomposite*

Noh et al. (2012) fabricated a hierarchical Cu–Co alloy dendrite by electrochemical synthesis. The Co2<sup>+</sup> ions that formed on the alloy dendrite contributed to glucose oxidation, and Co3<sup>+</sup> was the main species involved in the reduction of H2O<sup>2</sup> with Cu2<sup>+</sup> ions contributing to the electrocatalytic process. The major oxidation product/number of an electron that participated in the conversion of glucose was identified to 97% of formate (12-electron oxidation product) and the remaining 3% with other minor products through coulometry and High pressure liquid chromatography–mass spectrometry (HPLC-MS) analysis. The electrochemical properties analyzed at different pH conditions and temperatures achieved a dynamic detection limit, and long-term stability. Compared to the single metal dendrite, the Co bimetallic dendrite enhanced the catalytic property by 10 times [86]. Silver nanodendrites on Cu rods were synthesized by a facile displacement reaction with the absence of any surfactants. In this work, the dendritic Ag structures offered a large surface area for good conductivity of Cu-Ag BNP and reported an ultra-low detection limit for glucose and H2O<sup>2</sup> sensing. The advantage of this work lies in its good reproducibility as the electrode can be regenerated under hydrodynamic conditions without any extra treatment method. Five successive cycles of sensing experiments demonstrated a relative standard deviation of 3.59% to 4.22%. In addition, good selectivity and long-term stability over 30 days were also achieved by the dendrite sensor [87]. Mei et al. (2016) prepared three-dimensional nanoporous copper (Al75Cu25) and carbon black by dealloying Al-Cu ribbons to make a clean surface highly conductive material. In addition to the low cost and simple preparation, the dealloying process possesses control over structural uniformity of the synthesized materials. Other advantages of the prepared sensor include environmental protection by dealloying and good selectivity through synergistic interaction between the nanoporous copper and carbon black. The sensor operated at a working voltage of 0.6 and 0.75 V (vs. saturated calomel electrode (SCE)) for glucose and H2O2, respectively. The numerous conducting channels present in carbon black help to transfer electrons and the Cu-carbon black structure allows for electronic transfer between their active sites. The working electrode has a wide linear analytical range, good selectivity, stability, and sensitivity in the positive potential window. The detection limits for glucose and H2O<sup>2</sup> were 2.6 µM and 1.2 µM, respectively. Real-time analysis was also performed in some commercial beverages for glucose and different contact lens solution for H2O<sup>2</sup> and achieved good correlation with existing values [88]. The different combination of bimetallic AuCu, PtCu, and Fe, Ni-CNTs were successfully tested for NEGHS and achieved high sensing parameters [89–91].

#### *4.4. Other Bimetallic Nanocomposite*

Metal nitrides are reported for their applicability in designing glucose and H2O<sup>2</sup> sensors due to their superior electrical conductivity, exceptional redox properties and mechanical strength. Zhou et al. (2017) fabricated a Fe3N-Co2N nanowire array on carbon cloth, which is an attractive bifunctional catalyst for NEGH sensing because of a large surface area and easy accessibility. This transition metal

nitride had metalloid characteristics with superior electrical conductivity and had not yet been reported for NEGH sensing. The fabricated Fe3N-Co2N/carbon cloth sensor obtained a respective response time of 8 and 2 s for glucose and H2O2. The prepared sensor exhibited a high selectivity, specificity, and reproducibility [92]. Furthermore, Deepalakshmi et al. (2018) prepared core-shell nanostructures based on nitrogen-doped graphene encapsulated nickel-cobalt nitride (NixCo3-xN/NG) via a simple, scalable, and cost-effective pyrolysis technique. This work suggested that Ni was the best choice in combination with Co as opposed to Fe transition metals for sensing applications. It successfully controlled the molar ratio of Ni/Co to achieve ahigh electrocatalytic activity, and nitrogen-doped graphene provided a high conductive nature and long-term cycling stability of a working electrode, as shown in Figure 1e. Due to the synergistic effect of the NiCo2N core and the NG shell, highly sensitive and selective properties were obtained for the electrodes. The practical feasibility of the prepared electrode was tested in human serum, and proved to be efficient for the determination of glucose and H2O<sup>2</sup> [39].

Palladium (Pd) nanocomposites have attracted researchers' interest due to their high electro catalytic activity, lower price, and abundant yield when compared with Au, Ag and Pt materials. Palladium bimetallic nanocomposite improved the electro catalytic performance by modifying the structure, correction in ligand and strain effect. However, the surface of Pd metal can easily be poisoned by chloride and intermediate species and remains unstable during electrochemical reactions leading to a decrease of sensing parameters. Many researchers synthesized Pd alloy in the form of interconnected porous nanostructure using advanced fabrication techniques. The porous channels prepared by the dealloying method are preferred for rich surface chemistry, unique catalytic activity, easy mass and electron transport, unlimited electron conductivity, and synergetic effect. The nano porous Pd-based alloys prepared by the dealloying method, which gained a valid fabrication route to construct highly effective electrochemical sensors and had advantages such as easy handling, no particles aggregation, clean metal surface, and eco-friendliness compared to other reported synthesis methods. Platinum nanoparticles are widely applied in the analysis of non-enzymatic glucose and H2O2, but there are several limitations like slow kinetics, low sensitivity, and poor selectivity. In a few reports modified platinum with Au, Co, Fe, and Ni and altered surface catalytic activity have been mentioned. Furthermore, these alloys are decorated with carbon/MWCNTs to enhance stability and sensitivity. In the same way, palladium incorporated with transition metals such as Fe, Cr, Ni, Co and noble metal Au has been reported. By considering the effective combination such as CNTs, MOS<sup>2</sup> etc., specific preparation methods have been applied to overcome the limitations of Pd BNP for sensing both glucose and H2O2. The bifunctional properties of Cu bimetallic nanocomposites also contributed to the enhancement of NEGH sensing application. The obtained morphological changes offered the best electro catalyst for NEGH sensing. Modifying the morphology of copper-based nanomaterials into nanowires, nanoplates, nanospheres, and nanofibers altered the potential window to avoid etching and interference of electrodes in alkaline solutions and showed significant performance in achieving high electrocatalytic activity and selectivity. Among the various nano morphologies, metallic dendrites structures are attracted in NEGHS due to their high surface area and a high degree of connectivity with the main stem and many side branches. Researchers have further focused on enhancing the surface area of Cu-based bimetallic nanostructures for sensing both glucose and H2O2. Finally, bimetallic nitrides (BMN) have attracted attention due to their exceptional redox property, superior conductivity, interstitial alloy behavior and exceptional mechanical strength. Among BMN, Co nitrides showed superior electrical conductivity, high chemical stability and extraordinary corrosion resistance. The exchange of nitrogen with oxygen in BMN prefers large electron donating ability for higher electrical conductivity. The BMN in electrochemical reactions suffers from poor stability due to easy oxidation. To overcome these issues, researchers have combined BMN with supporting materials such as graphene, activated carbon and CNTS. Table 3 shows bimetallic nanocomposites in NEGH sensing with their electrochemical performances.



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


**Table 3.** *Cont.*

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