In situ developed NiCo₂O₄–Ti₃C₂T_x nanohybrid towards non-enzymatic electrochemical detection of glucose and hydrogen peroxide

Abstract

Owing to the adverse consequences of excess glucose (Glu) and hydrogen peroxide (H₂O₂) on humans, it is imperative to develop an electrochemical sensor for detection of these analytes with good selectivity and sensitivity. Herein, a nanohybrid comprising nickel cobaltite nanoparticles (NiCo₂O₄ NPs) embedded on conductive Ti₃C₂T_x nanosheets (NSs) has been prudently designed and employed for the electrochemical detection of Glu and H₂O₂. The developed nanohybrid has been systematically characterized using morphological and spectral techniques, and then immobilized on a glassy carbon electrode (GCE). Under optimized conditions, the developed NiCo₂O₄–Ti₃C₂T_x/GCE based electrochemical sensor has demonstrated an impressive analytical response towards Glu and H₂O₂ with good sensitivity and selectivity. The non-enzymatic sensor has demonstrated a broad linear range from 30 μM to 1.83 mM for Glu, and two linear ranges of 20–100 μM and 100 μM–2.01 mM for H₂O₂. The sensor has exhibited limits of detection (LOD) of 9 μM and 6 μM with sensitivities of 101.2 μA μM⁻¹ cm⁻² and 107.03 μA μM⁻¹ cm⁻², respectively, for Glu and H₂O₂ detection. The impressive analytical performance of the fabricated sensor in terms of linear range, LOD and sensitivity are ascribed to the (i) enhanced conductivity of Ti₃C₂T_x NSs, (ii) mediated electrocatalytic activity of NiCo₂O₄ NPs and (iii) large number of catalytically active sites on the NiCo₂O₄–Ti₃C₂T_x heterostructure. Notably, the NiCo₂O₄–Ti₃C₂T_x/GCE has demonstrated impressive stability and reproducibility, which is mainly due to the in situ uniform growth of NiCo₂O₄ NPs over Ti₃C₂T_x NSs.

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1. Introduction

The design and fabrication of extremely specific and precise diagnostic devices that can be used in analytical assays, and in particular identification of biomolecules, are crucial for the sustainability of human life and the environment. Glucose (Glu) and hydrogen peroxide (H₂O₂) are the most investigated analytes and excessive concentrations of these molecules in bodily fluids can have adverse impacts on human health.^1–3 The Glu level in the human body is regarded as a pathological indicator of diabetes and an increase in blood sugar level causes chronic microvascular problems in diabetes patients.^4,5 Therefore, rapid, reliable and everyday monitoring of Glu in blood is extremely beneficial for the early identification and treatment of diabetes and related ailments.^6–8 Similarly, H₂O₂ is one of the most significant biomarkers of oxidative stress and also serves as a precursor to extremely reactive and possibly hazardous hydroxyl radical formation.⁹ Anomalous H₂O₂ concentrations in both plasma and cells have been associated with a number of illnesses, including cerebrovascular dysfunction and cancer, and can cause gene mutation and DNA damage.^10–12 Hence, researchers are continuously exploring quick and sensitive techniques towards the quantitative determination of H₂O₂ and Glu for clinical diagnosis.

Electrochemical sensors have attracted significant attention among various analytical techniques because of their numerous advantages, including high sensitivity, simplicity, low cost, high throughput, fast operation and the potential for miniaturization.^13–15 The effectiveness of electrochemical sensors depends on the properties of the sensing element, typically an electrode modifier. Initially, enzyme-based materials were utilized as a sensing element for selective and sensitive detection of a targeted analyte.¹⁶ Conversely, there are a few disadvantages associated with enzyme-based sensors, which include the difficulties in modification, instability and tedious immobilization processes.^17,18

Consequently, non-enzymatic sensors, especially those based on transition metal oxides, have attracted much interest and are becoming a replacement for enzymatic sensors.¹⁹ Among the different metal oxides, transition metal spinel cobaltites (MCo₂O₄) exhibit excellent electrocatalytic activity compared to monometallic oxides.²⁰ Recently, various spinel cobaltites have been reported, among which nickel cobaltite (NiCo₂O₄) has been explored to a great extent due to its ease of synthesis, affordability, biocompatibility, strong ionic conductivity, and exceptional electrochemical activity.^21,22 The synergetic effect between the two metals could modulate the electronic structure of NiCo₂O₄ NPs, which in turn would enhance its electrocatalytic properties. The efficacy of NiCo₂O₄ could still be enhanced by converting it to a nano-sized material and uniformly embedding it over appropriate supports. This strategy would offer enough anchoring sites to properly immobilize the NPs and prevent their overgrowth. Different conductive supports such as graphene oxides (GO), MXene, multi-walled carbon nanotubes (MWCNTs) and graphitic carbon nitride (gC₃N₄) could be employed for the growth of NiCo₂O₄ NPs in order to increase the electrocatalytic efficiency and stability.^23–25

Recently, (2D) single-layer MXene has been established as a promising material in electrochemical sensing. Titanium carbide MXene (Ti₃C₂T_x) is one of the heavily investigated 2D MXenes by virtue of its distinct characteristics, including high conductivity, large surface area, remarkable electrochemical stability and abundant functional groups (relative to other synthesized MXenes).^26–29 Interestingly, the terminal groups, such as –O, –OH, –F, and –Cl, on the surface of Ti₃C₂T_x NSs could serve as growth sites for the dispersion and stabilisation of metal oxide NPs.^30,31 MXenes and NiCo₂O₄ materials have been individually utilized for various applications in the past, whereas limited research has been devoted towards their combined functionalities. This motivated us to develop a nanostructure comprising NiCo₂O₄ NPs and Ti₃C₂T_x NSs to trigger the catalytically active sites and further to employ it for the electrochemical detection of biomolecules. To the best of our knowledge, the combination of a NiCo₂O₄ NPs decorated Ti₃C₂T_x MXene based nanohybrid has not been probed for the detection of both Glu and H₂O₂. With the aforementioned background, a robust non-enzymatic sensor has been constructed in this work, utilizing a NiCo₂O₄ NPs decorated Ti₃C₂T_x NSs modified GCE for Glu and H₂O₂ sensing. In the sensor setup, MXene provides an active surface towards the uniform growth of NiCo₂O₄ NPs and the in situ growth prevents the agglomeration of NPs. The prepared nanohybrid was thoroughly characterized using different morphological and spectral investigations. The developed sensor exhibited outstanding selectivity and sensitivity towards Glu oxidation and H₂O₂ reduction. Additionally, the fabricated sensor was employed to detect the targeted analyte in food, serum and urine samples, where it displayed impressive recoveries.

2. Experimental

All chemicals and instrumentation details are given in the ESI.†

2.1. Synthesis of Ti₃C₂T_x NSs

Ti₃C₂T_x NSs were synthesized by selective etching of the Al layer from readily available bulk MAX phase Ti₃AlC₂ MAX, using an in situ HF etching method³² (Fig. 1). Briefly, 2 g of LiF was added to 40 mL of HCl (9 M) with rapid stirring, and then 2 g of Ti₃AlC₂ powder was slowly added to the above solution to avoid overheating. Subsequently, Al etching was carried out over 24 h with continuous stirring of the aforementioned solution at 35 °C. The obtained suspension was subjected to washing several times using deionized water and centrifuged until the pH of the solution reached 6. The obtained sediment was mixed with deionized water and sonicated for 1 h with continuous Ar bubbling. Ti₃C₂T_x NSs were obtained after the solution was freeze-dried for 24 h at −50 °C.

Fig. 1 Schematic representation for the preparation of the NiCo₂O₄–Ti₃C₂T_x nanohybrid.

2.2. Synthesis of the NiCo₂O₄–Ti₃C₂T_x nanohybrid

To synthesize the proposed nanohybrid,³³ 10 mg of Ti₃C₂T_x NSs were ultrasonically dispersed in deionized water (10 mL) for 1 h. Furthermore, 20 mmol and 40 mmol of cobalt(II) chloride hexahydrate and nickel(II) chloride hexahydrate were mixed in 25 mL of deionized water and poured into the above mentioned Ti₃C₂T_x NS solution with stirring (30 min) to obtain a homogenously dispersed solution. This homogeneous solution was transferred into a 50 mL stainless steel autoclave for hydrothermal treatment and kept at 160 °C for 6 h. After the reaction was completed, the resulting product was repeatedly washed with deionized water and ethanol before being dried overnight in an oven at 80 °C. Finally, the synthesised material was calcined at 400 °C for about 3 h with a heating rate of 3 °C per minute to finally obtain the NiCo₂O₄–Ti₃C₂T_x nanohybrid material (Fig. 1). The same synthesis method was followed to prepare NiCo₂O₄ without adding Ti₃C₂T_x.

2.3. Fabrication of the NiCo₂O₄–Ti₃C₂T_x nanohybrid modified electrode

The GCE surface was successively polished before every electrode modification using 1, 0.3, and 0.05 μm sized alumina powder on a leather polishing pad. The surface residues were then removed by ultrasonication in Milli-Q water and ethanol (1 : 1). The pre-cleaned GCE surface was then drop-casted with 10 μL of NiCo₂O₄–Ti₃C₂T_x nanohybrid (2 mg mL⁻¹), and was air dried to obtain NiCo₂O₄–Ti₃C₂T_x/GCE. Similarly, NiCo₂O₄ NPs and Ti₃C₂T_x NSs modified GCEs (NiCo₂O₄/GCE and Ti₃C₂T_x/GCE) were also prepared for comparison.

3. Results and discussion

3.1. Characterization of the NiCo₂O₄–Ti₃C₂T_x nanohybrid

The PXRD patterns of Ti₃AlC₂, Ti₃C₂T_x NSs, NiCo₂O₄ NPs and the NiCo₂O₄–Ti₃C₂T_x nanohybrid were obtained to confirm the formation of the NiCo₂O₄–Ti₃C₂T_x nanohybrid, and are demonstrated in Fig. 2. After etching out Al from Ti₃AlC₂ (MAX phase), the peak at 9.2° corresponding to the (002) plane in Ti₃AlC₂ was broadened and shifted towards a lower 2θ angle at 6.74° in Ti₃C₂T_x NSs. Furthermore, the distinctive peak for Al vanished (2θ = 39°), signifying the successful etching of Al from the MAX phase to form Ti₃C₂T_x NSs (Fig. S1, ESI†). These findings strongly indicate the formation of Ti₃C₂T_x NSs.³⁴ The PXRD pattern of NiCo₂O₄ NPs (green) exhibited hkl planes of (311), (400), (511) and (440) of NiCo₂O₄ (JCPDS:00-002-1074).³⁵ The prominent peak of Ti₃C₂T_x NSs related to the (002) plane is constrained in the NiCo₂O₄–Ti₃C₂T_x nanohybrid (blue), which could be due to the minimal amount of MXene used for nanohybrid synthesis. The peaks corresponding to NiCo₂O₄ appeared in the PXRD patterns of the NiCo₂O₄–Ti₃C₂T_x nanohybrid, which clearly indicates the successful formation of the nanohybrid.

Fig. 2 PXRD patterns of NiCo₂O₄ NPs (green), Ti₃C₂T_x NSs (red) and the NiCo₂O₄–Ti₃C₂T_x nanohybrid (blue).

Additionally, HRTEM with SAED and FESEM investigation with elemental mapping were performed to probe the morphology of Ti₃AlC₂, Ti₃C₂T_x NSs, NiCo₂O₄ and the NiCo₂O₄–Ti₃C₂T_x nanohybrid. The FESEM image of Ti₃AlC₂ (Fig. S2a, ESI†) exhibits a packed layered structure, while Ti₃C₂T_x MXene demonstrates a single nanosheet like morphology (Fig. S2b, ESI†). The FESEM image displayed in Fig. S2c (ESI†) indicates the formation of NiCo₂O₄ NPs. Furthermore, the FESEM image of the NiCo₂O₄–Ti₃C₂T_x nanohybrid³⁶ (Fig. S2d, ESI†) clearly indicates the in situ formation of NiCo₂O₄ NPs on Ti₃C₂T_x NSs. Elemental mapping (Fig. S3, ESI†) reveals the uniform distribution of NiCo₂O₄ NPs embedded over Ti₃C₂T_x NSs, with all the elements present in nanohybrid. The TEM images of the in situ grown NiCo₂O₄ NPs over Ti₃C₂T_x NSs are shown in Fig. 3a and b. Fig. 3a demonstrates the uniform distribution of NiCo₂O₄ NPs over the Ti₃C₂T_x NSs and the average size of the NiCo₂O₄ NPs is found to be around 12 nm. The TEM image of the prepared NiCo₂O₄–Ti₃C₂T_x nanohybrid in Fig. 3b indicates the presence of both the NiCo₂O₄ NPs and thin Ti₃C₂T_x MXene NSs. In Fig. 3c, the HRTEM image of NiCo₂O₄–Ti₃C₂T_x nanosheets clearly exhibits d-spacings of 0.43 and 0.26 nm, corresponding to (002) and (311) planes of Ti₃C₂T_x MXene and spinel NiCo₂O₄ NPs, respectively. The SAED pattern (Fig. 3d) reveals well-defined rings that correspond to the crystal planes of the NiCo₂O₄ NPs, which are well indexed to (311), (220), and (440).

Fig. 3 (a) and (b) TEM images of the NiCo₂O₄–Ti₃C₂T_x nanohybrid. (c) and (d) HRTEM image and SAED patterns of the NiCo₂O₄–Ti₃C₂T_x nanohybrid.

The XPS survey spectrum (Fig. 4a) of the as synthesized nanohybrid confirmed the existence of Ti, C, Ni, Co and O. In Fig. 4b the Ni 2p spectrum of NiCo₂O₄–Ti₃C₂T_x MXene exhibits two binding energy states at 871.45 eV and 853.44 eV and satellite peaks at 878.82 eV and 859.86 eV are associated with Ni 2p3/2 and Ni 2p1/2, respectively.³⁷ The high resolution spectrum of Co 2p (Fig. 4c) exhibits two peaks that are in agreement with Co 2p3/2 and Co 2p1/2. The binding energy values obtained for NiCo₂O₄–Ti₃C₂T_x NSs at 795.45 eV and 780.06 eV are attributed to Co²⁺ and those observed at 793.54 eV and 778.17 eV are ascribed to Co³⁺. Furthermore, the peaks observed at 798.99 eV and 784.81 eV correspond to satellite peaks.³⁸Fig. 4d shows the core level spectrum of Ti in the NiCo₂O₄–Ti₃C₂T_x nanohybrid. The presence of titanium carbide and titanium oxide in the nanohybrid could be ascertained from the peaks observed in the Ti 2p state, wherein the peaks at 457.65 and 463.34 eV correspond to Ti–C, and those at 459.41 and 464.51 eV correspond to Ti–O. Ti displays different valence states due to the surface terminal groups like –OH and –F introduced during the etching process. In the C 1s spectrum (Fig. 4e), the binding energies at 280.17 eV and 283.5 eV are assigned to C–Ti and C–C species, and the peaks appearing at 284.82 eV and 286.97 eV are due to C–O and O–C=O bonds.^39,40 Similarly, the O 1s spectrum was fitted over three subpeaks and presented in Fig. 4f. The peak observed at 528 eV correlated well with M–O (Ni–O, Ti–O and Co–O). Furthermore, the concordant peak for –OH on MXene and observed water molecules appeared at the binding energy values of 529.3 eV and 531.1 eV, respectively.³³ The results observed from XPS revealed the presence of Ni and Co in the nanohybrid with +2 and +3 oxidation states, respectively. All these morphological and spectral investigations show that NiCo₂O₄ NPs have been uniformly distributed on the Ti₃C₂T_x NSs and the in situ growth of NiCo₂O₄ NPs over Ti₃C₂T_x NSs would suitably modulate the electronic structure of the nanohybrid. This in turn would facilitate the electrochemical characteristics of the NiCo₂O₄–Ti₃C₂T_x nanohybrid modified electrode and its electrocatalytic activity towards Glu and H₂O₂ determination.

Fig. 4 (a) Survey spectrum of the NiCo₂O₄–Ti₃C₂T_x nanohybrid. Core level spectra of (b) Ni 2p, (c) Co 2p, (d) Ti 2p, (e) C 1s, and (f) O 1s in the NiCo₂O₄–Ti₃C₂T_x nanohybrid.

3.2. Electrochemical behaviour of the NiCo₂O₄–Ti₃C₂T_x nanohybrid modified electrode

To assess the electrochemical characteristics of the fabricated NiCo₂O₄–Ti₃C₂T_x/GCE, cyclic voltammetry (CV) was carried out in 0.1 M NaOH electrolyte at a scan rate of 20 mV s⁻¹ within the potential range of +0.2 V to +0.7 V as illustrated in Fig. 5a. The NiCo₂O₄–Ti₃C₂T_x/GCE exhibited distinct redox peaks, with an anodic peak potential (E_pa) observed at +0.56 V and a cathodic peak potential (E_pc) at +0.38 V with E° of +0.47 V. The modified electrode prepared in the absence of MXene, i.e., NiCo₂O₄/GCE, exhibited redox peaks with E_pa and E_pc of 0.58 V and 0.42 V with E° of −0.5 V. The obtained well-defined redox peaks are due to the faradaic response of Ni³⁺/Ni²⁺ and Co³⁺/Co²⁺ present in the NiCo₂O₄ NPs.⁴¹ Notably, the NiCo₂O₄–Ti₃C₂T_x nanohybrid modified electrode exhibits lower redox potentials with higher anodic and cathodic peak currents than the original NiCo₂O₄ redox spinel oxide NPs. The enhanced electrochemical performance in the case of NiCo₂O₄–Ti₃C₂T_x/GCE could be due to the 2D MXene NSs that afford higher surface area and also a platform for the uniformly grown NiCo₂O₄ NPs (without any aggregation). This heterostructure results in the generation of abundant catalytic sites and is expected to trigger the electrocatalytic activity of the modified electrode. Conversely, the bare and MXene modified GCE exhibited no discernible redox behaviour in the investigated potential window.

Fig. 5 (a) CVs of the NiCo₂O₄/GCE and NiCo₂O₄–Ti₃C₂T_x/GCE in the presence and absence of 150 μM Glu. Inset: CVs of the unmodified GCE and Ti₃C₂T_x/GCE in the presence and absence of 150 μM Glu. (b) CVs of the NiCo₂O₄–Ti₃C₂T_x/GCE with increasing addition of Glu at 20 mV s⁻¹. (c) Amperometric response of NiCo₂O₄/GCE and NiCo₂O₄–Ti₃C₂T_x/GCE for the successive spikes of Glu in 0.1 M NaOH. (d) Corresponding calibration plots.

An investigation on the effect of scan rate was conducted to examine the nature of the electrochemical process that occurs at the fabricated NiCo₂O₄–Ti₃C₂T_x/GCE (Fig. S5a, ESI†). As the scan rate increased, the peak currents for both oxidation and reduction gradually increased. As shown in Fig. S5b (ESI†), a linear relationship was obtained for the plot of the square root of scan rate vs. peak current, which reveals that the redox process is diffusion-controlled. Additionally, we further planned to examine the number of catalytically active sites by calculating the electrochemical active surface area (ECSA). The ECSA was calculated using the Randles–Sevcik equation (eqn (1)) by investigating the effect of the scan rate of the modified electrode in 5 mM K₃Fe(CN)₆.⁴¹

i_p = 2.69 × 10⁵ × A × C × n^3/2 × D^1/2 × ν^1/2 (1)

where i_p represents the anodic peak current, A is the ECSA, C refers to the concentration of K₃Fe(CN)₆, D is the diffusion coefficient of K₃Fe(CN)₆, n refers to the number of electrons involved in the redox reaction and ν is the scan rate. The ECSA values were calculated for Ti₃C₂T_x/GCE, NiCo₂O₄/GCE and NiCo₂O₄–Ti₃C₂T_x/GCE, and found to be 0.094, 0.162 and 0.462 cm². The remarkably higher ECSA value obtained with NiCo₂O₄–Ti₃C₂T_x/GCE demonstrates that a larger number of catalytically active sites are being generated upon the formation of the NiCo₂O₄–Ti₃C₂T_x heterostructure.

The influence of pH on the electrochemical behaviour of NiCo₂O₄–Ti₃C₂T_x/GCE was tested in the pH range from 3 to 13 to optimize the working conditions (Fig. S4, ESI†). As observed from the figure, no redox peaks could be obtained in the pH range from 3 to 9, whereas a small oxidation peak was observed at pH 11. However, well-defined redox peaks corresponding to Ni³⁺/Ni²⁺ and Co³⁺/Co²⁺ could be observed when the pH was further increased to 13 (eqn (2)). No change in the redox response was observed when the pH was increased beyond 13 and hence pH 13 was chosen as the optimum pH for all the electrochemical measurements.

3.3. NiCo₂O₄–Ti₃C₂T_x modified electrode towards glucose detection

3.3.1. Investigation of the electrocatalytic performance. As NiCo₂O₄–Ti₃C₂T_x/GCE has demonstrated distinct electrochemical characteristics, we planned to explore its electrocatalytic activity towards the detection of Glu. The electrocatalytic activity was systematically explored towards Glu detection by recording the CV response of bare-GCE (inset), MXene-GCE (inset), NiCo₂O₄/GCE and NiCo₂O₄–Ti₃C₂T_x/GCE in the presence and absence of Glu as depicted in Fig. 5a. The bare-GCE and Ti₃C₂T_x/GCE did not exhibit any catalytic response, whereas the NiCo₂O₄/GCE showed a slight but negligible response towards Glu oxidation. Conversely, the NiCo₂O₄–Ti₃C₂T_x/GCE displayed a noticeable increase in the anodic current around 0.55 V upon the addition of 150 μM Glu. Interestingly, upon injections of Glu in 50 μM increments, a linear enhancement in the anodic peak current was observed, demonstrating the electrocatalytic oxidation of Glu on the surface of NiCo₂O₄–Ti₃C₂T_x/GCE (Fig. 5b). The enhanced electrocatalytic performance of NiCo₂O₄–Ti₃C₂T_x/GCE could arise from the intrinsic properties of redox-active NiCo₂O₄ spinel metal oxides and high number of active sites and excellent conductivity of the 2D MXene sheets. At an applied potential of 0.5 V, the Ni²⁺ and Co²⁺ ions in the NiCo₂O₄ will be electrochemically oxidized to Ni³⁺ and Co³⁺ (eqn (2)). Upon addition of glucose, these metal ions in the oxidized form in turn will oxidize the glucose to gluconolactone and will be reduced to the Ni²⁺ and Co²⁺ states (eqn (3)). Both these reactions (eqn (2) and (3)) will continue in a cycle as long as glucose is available at the electrode surface, which results in an enhanced oxidation current, and this indicates the mediated electrocatalytic oxidation of glucose.⁴²

NiCo₂O₄ + H₂O + OH⁻ → NiOOH + 2CoOOH + e⁻ (2)

Ni³⁺ + Co³⁺ + Glucose → Ni²⁺ + Co²⁺ + Gluconolactone (3)

3.3.2. Amperometric detection of glucose. The impressive catalytic response obtained with NiCo₂O₄–Ti₃C₂T_x/GCE motivated us to substantiate the catalytic activity towards Glu oxidation under dynamic conditions. Thus, the amperometric response (Fig. 5c) was measured for NiCo₂O₄/GCE and NiCo₂O₄–Ti₃C₂T_x/GCE in 0.1 M NaOH by applying an optimized potential of +0.5 V and dynamic conditions (350 revolutions per minute). The amperometric (i–t) curve is presented in Fig. 5c, wherein the NiCo₂O₄–Ti₃C₂T_x/GCE responded swiftly to every spike of Glu addition over the linear range of 30 μM to 1.83 mM. The estimated LOD and sensitivity of the fabricated NiCo₂O₄–Ti₃C₂T_x/GCE based non-enzymatic sensor were calculated to be 9 μM and 101.2 mA μM⁻¹ cm⁻², respectively. It could also be observed from Fig. 5d that the NiCo₂O₄–Ti₃C₂T_x/GCE demonstrated remarkably improved current response and catalytic activity compared to those of NiCo₂O₄/GCE. The obtained electrochemical performance of NiCo₂O₄–Ti₃C₂T_x/GCE in terms of low LOD, high sensitivity and wide detection ranges is comparable or better than that of the recently reported non-enzymatic sensors for Glu detection (Table S3, ESI†). This significant catalytic activity and enhanced current response attained with NiCo₂O₄–Ti₃C₂T_x/GCE is attributed to the large surface area afforded by 2D-Ti₃C₂T_x NSs, inherent properties of uniformly distributed NiCo₂O₄ NPs on MXenes and the large number of catalytic sites on the NiCo₂O₄–Ti₃C₂T_x heterostructure.

3.3.3. Stability and reproducibility. The effectiveness of the produced sensor heavily depends on its operational stability, long term durability and reproducibility. The NiCo₂O₄–Ti₃C₂T_x/GCE based sensor was subjected to 50 continuous cycles at 20 mV s⁻¹ with the potential ranging from +0.2 V to +0.7 V. The results demonstrated that the sensor was extremely stable with no discernible changes to the anodic and cathodic peak potentials as well as peak currents. The long-term performance of the designed sensor was tested over a period of 30 days with and without 0.05 mM Glu. The sensor maintained 96% of the response in the absence of Glu and 93% of the response while 0.05 mM Glu was present, as represented in the inset of Fig. 6c. These results indicate that the constructed sensor NiCo₂O₄–Ti₃C₂T_x/GCE has excellent operational consistency and long-term durability for the detection of Glu. Additionally, five identically modified sensors were prepared and used to evaluate the repeatability of the synthetic procedure of NiCo₂O₄–Ti₃C₂T_x/GCE. A reproducible current response was achieved at the sensors, in the presence and absence of Glu with relative standard deviations (RSD) of 1.86 and 1.56, respectively, as illustrated in Fig. 6d. The results establish firmly that the constructed NiCo₂O₄–Ti₃C₂T_x/GCE sensors also display good electrode-to-electrode repeatability.

Fig. 6 (a) Interference effect towards Glu detection (20 μM) in the presence of various interfering analytes (0.2 mM) in 0.1 M NaOH. (b) Corresponding bar diagram. (c) Stability investigation for 50 continuous cycles. Inset: Long-term stability of the fabricated sensor for 30 days in the presence and absence of 50 μM Glu. (d) Bar diagram corresponding to the current response in the presence and absence of Glu (50 μM) for five identically prepared sensors.

3.3.4. Interference and real sample analysis. To examine the selectivity of the developed sensor for the real time electrochemical detection of Glu, the effect due to interference has been examined by employing amperometry in 0.1 M NaOH at +0.5 V. Fig. 6a illustrates the amperometric detection response of NiCo₂O₄–Ti₃C₂T_x/GCE for the detection of 20 μM Glu in the presence of 10 times higher concentrations of other coexisting biomolecules, including ascorbic acid (AA), dopamine (DA), uric acid (UA), cholesterol (Chol), fructose (Fru), adenine (Ade), H₂O₂, resorcinol (RS), hydroquinone (HQ), hydrazine (HZ), and cysteine (CyS). The bar graph of the corresponding catalytic current in Fig. 6b reveals that the fabricated non-enzymatic sensor did not show significant interference and exhibited high selectivity towards Glu. The applicability of the developed sensor for real time application has been examined by monitoring Glu in serum and urine samples (human). In a typical procedure, a known amount of Glu was spiked into a real sample, and the recovery was obtained from amperometry. The non-enzymatic detection of Glu using the NiCo₂O₄–Ti₃C₂T_x nanohybrid decorated GCE resulted in a recovery range of 97.6% to 100.8%, as shown in Table S1 (ESI†). These findings showed the suitability of the constructed system for Glu detection in a biological medium owing to the selectivity and sensitivity acquired from the rationale design of the non-enzymatic sensor by decorating NiCo₂O₄ over highly conducting MXene.

3.4. NiCo₂O₄–Ti₃C₂T_x modified electrode towards detection of H₂O₂

3.4.1. Investigation of the electrocatalytic performance. The ability of the NiCo₂O₄–Ti₃C₂T_x nanohybrid modified electrode to efficiently detect Glu allowed us to envisage the development of a non-enzymatic sensor for the electrochemical detection of H₂O₂. Thus, the electrochemical behaviours of the constructed electrode NiCo₂O₄–Ti₃C₂T_x/GCE towards H₂O₂ sensing were investigated by recording CV responses in the absence and presence of the analyte in the potential range from 0 V to −0.6 V at a sweep rate of 20 mV s⁻¹ in N₂ saturated 0.1 M NaOH. No redox peaks were observed for any of the electrodes in the applied potential range in the absence of H₂O₂. The electrocatalytic activities of the fabricated sensors including bare/GCE, Ti₃C₂T_x/GCE, NiCo₂O₄/GCE, and NiCo₂O₄–Ti₃C₂T_x/GCE were studied in the presence of 50 μM H₂O₂. No change in the voltammetric behaviour could be observed for bare/GCE, Ti₃C₂T_x/GCE, or NiCo₂O₄/GCE in the absence and presence of the analyte, indicating that these electrodes are catalytically inactive. Interestingly, the hybrid nanomaterial NiCo₂O₄–Ti₃C₂T_x decorated electrode displayed an increase in the cathodic current around −0.25 V in the presence of 50 μM H₂O₂ and the catalytic current was found to vary linearly with the increase in the concentration of added H₂O₂. These results clearly demonstrate that the electrocatalytic activity towards the reduction of H₂O₂ occurs only at the NiCo₂O₄–Ti₃C₂T_x/GCE owing to the presence of a catalytically active heterostructure. At the applied potential (−0.25 V), the metal ions would already exist in the reduced form. Upon addition of H₂O₂, these metal ions will reduce H₂O₂ to H₂O (eqn (4)), and they are oxidized to Ni³⁺ and Co³⁺, which again will be electrochemically reduced at a potential of −0.25 V to Ni²⁺ and Co²⁺ (eqn (5)). This results in an enhanced reduction current of ∼−0.25 V, corresponding to the NiCo₂O₄ mediated electrocatalytic reduction of H₂O₂.⁴³

Ni²⁺ + Co²⁺ + H₂O₂ + 2H⁺ → Ni³⁺ + Co³⁺ + 2H₂O (4)

Ni³⁺ + 2Co³⁺ + 3e⁻ → Ni²⁺ + 2Co²⁺ + 2H₂O (5)

3.4.2. Amperometric detection of H₂O₂. The electrochemical performance of the nanohybrid loaded GCE was investigated using amperometry under dynamic conditions. An applied potential of −0.25 V was used as the CVs of NiCo₂O₄–Ti₃C₂T_x/GCE demonstrated high cathodic peak current at this potential. Fig. 7c exhibits typical amperometric (i–t) curves of NiCo₂O₄/GCE and NiCo₂O₄–Ti₃C₂T_x/GCE upon sequential addition of H₂O₂ in N₂ saturated 0.1 M NaOH electrolyte solution. The current response obtained for both the modified electrodes gradually decreased for each addition of H₂O₂. A comparatively poor response with no linear variation in current was obtained for NiCo₂O₄/GCE. Notably, the NiCo₂O₄–Ti₃C₂T_x/GCE exhibited a sharp response for every addition of H₂O₂. Linearity was observed in two concentration ranges of 20 to 100 μM and 100 μM to 2.01 mM with sensitivities of 57.2 μA μM⁻¹ cm⁻² and 107.03 μA μM⁻¹ cm⁻², respectively, and the sensor achieved a LOD of 6 μM. The outcomes are comparable with or even superior to those of the recently published non-enzymatic sensors towards H₂O₂ reduction (Table S3, ESI†). The steady-state current was rapidly achieved within 3s during the electrocatalytic reduction of H₂O₂ at NiCo₂O₄–Ti₃C₂T_x/GCE. The analytical characteristics obtained in the absence of any enzyme make it obvious that the proposed sensor functions as a robust H₂O₂ sensing platform.

Fig. 7 (a) CVs of the NiCo₂O₄/GCE and NiCo₂O₄–Ti₃C₂T_x/GCE in the presence and absence of 50 μM H₂O₂. Inset: CVs of the unmodified GCE and Ti₃C₂T_x/GCE in the presence and absence of 50 μM H₂O₂. (b) CVs of the NiCo₂O₄–Ti₃C₂T_x/GCE with increasing additions of H₂O₂ at 20 mV s⁻¹. (c) Amperometric response of the NiCo₂O₄/GCE and NiCo₂O₄–Ti₃C₂T_x/GCE for successive spikes of H₂O₂ in N₂ saturated 0.1 M NaOH. (d) Corresponding calibration plots.

3.4.3. Stability and reproducibility. The stability of the NiCo₂O₄–Ti₃C₂T_x/GCE non-enzymatic sensor was assessed by measuring its CV response at a scan rate of 20 mV s⁻¹ for 50 continuous potential cycles in NaOH (0.1 M; N₂ saturated), which is presented in Fig. 8c. The modified sensor shows good stability for 50 cycles with no noticeable change in potential and current response. In addition, the non-enzymatic sensor was tested over 30 days towards the detection of 0.05 mM H₂O₂. As seen in the inset of Fig. 8c, the biosensor demonstrated an acceptable long-term stability, retaining its activity up to 96.2% and 95.7% for 30 days, when recorded without and with H₂O₂. Additionally, the replicability of the sensor was studied by fabricating 5 different NiCo₂O₄–Ti₃C₂T_x/GCE sensors, and their electrocatalytic efficacy was investigated towards 0.02 mM H₂O₂. A RSD of 2.8% was detected across the individual electrodes, establishing the good electrode-to-electrode electrocatalytic performance replicability (Fig. 8d). High stability and good reproducibility of the developed non-enzymatic sensor is achieved due to the judicious uniform growth of NiCo₂O₄ on the large surface of the MXene NSs.

Fig. 8 (a) Interference effect toward H₂O₂ detection (20 μM) in the presence of various interfering analytes (0.2 mM) in N₂ saturated 0.1 M NaOH. (b) Corresponding bar diagram. (c) Stability investigation for 50 continuous cycles. Inset: Long-term stability of the fabricated sensor for 30 days in the presence and absence of 50 μM H₂O₂. (d) Bar diagram corresponding to the current response in the presence and absence of H₂O₂ (50 μM) for five identically prepared sensors.

3.4.4. Interference and real sample analysis. The selectivity of the modified non-enzymatic sensor was evaluated by adding 0.2 mM of interfering species, including Leu, L-tryp, Chol, Val, KNO₂, Cys, Glu, AA, DA and UA along with 20 μM H₂O₂. The investigation was carried out amperometrically at an applied potential of −0.25 V. Fig. 8a illustrates that even in the presence of ten times greater concentration of these interfering species, the current response did not change significantly and only the addition of H₂O₂ produced a notable current response. The observed outcomes indicate that the NiCo₂O₄–Ti₃C₂T_x/GCE has an acceptable selectivity for determination of H₂O₂. As H₂O₂ is commonly used as a preservative in food products, in this work, the applicability of NiCo₂O₄–Ti₃C₂T_x/GCE has been assessed by detecting H₂O₂ in milk and apple juice samples. By employing a conventional addition method, known amounts of H₂O₂ were injected to apple juice and milk samples and amperometry was used to determine their recovery at an operating potential of −0.25 V. The results (Table S2, ESI†) demonstrated recoveries ranging from 97.8 to 105.6%, indicating that the fabricated H₂O₂ sensor has the ability to detect the analyte in complex real samples.

4. Conclusion

The NiCo₂O₄–Ti₃C₂T_x nanohybrid was rationally designed and utilized towards the fabrication of a facile electrochemical sensor for the detection of glucose and H₂O₂. The NiCo₂O₄–Ti₃C₂T_x/GCE sensor demonstrated good electrocatalytic activity along with good selectivity, remarkable stability and reproducibility. The inherent catalytic properties of NiCo₂O₄ NPs, augmented conductivity of Ti₃C₂T_x NSs and increased number of catalytically active sites due to the NiCo₂O₄–Ti₃C₂T_x heterostructure collectively result in promising analytical performance of the sensor. Furthermore, the practical applicability of the developed sensor for H₂O₂ and Glu was tested using food (milk and apple juice) and biological samples (serum and urine), respectively, and it exhibited satisfactory recovery results. As a result, the fabricated sensor could be potentially utilized for medical diagnosis and environmental monitoring. With the increase in the development of a wide variety of 2D materials, many of novel high-performing nanohybrids and heterostructures could be designed not only for sensor design, but also for other task specific applications.

Author contributions

Devarasu Mohanapriya: conceptualization, methodology, investigation, validation, writing – original draft. Kathavarayan Thenmozhi: conceptualization, resources, supervision, project administration, writing – review & editing.

Data availability

The data supporting this article have been included as part of the ESI.† The original files will be made available on request.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors acknowledge the Central Research Facilities (CRF), Vellore Institute of Technology (VIT) for providing instrumentation facilities.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Materials, instrumentation, XRD patterns of Ti₃AlC₂ and Ti₃C₂T_x NS, FESEM image of Ti₃AlC₂, Ti₃C₂T_x NSs, NiCo₂O₄ NPs and NiCo₂O₄–Ti₃C₂T_x nanohybrid, FESEM image and elemental analysis of the NiCo₂O₄–Ti₃C₂T_x nanohybrid, Effect of pH, effect of scan rate and corresponding calibration plot, Real time detection of Glu and H₂O₂, comparison table of NiCo₂O₄–Ti₃C₂T_x/GCE sensor with previously reported Glu and H₂O₂ sensors. See DOI: https://doi.org/10.1039/d4tb02265c

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