Catalase biosensor based on the PAni/cMWCNT support for peroxide sensing

Polymeric-based composites can contribute to enhancing the detection, stability, and performance of enzymatic biosensors, due to their high structural stability, conductivity, and biocompatibility. This work presents the fabrication of a nanocomposite of polyaniline (PAni)/gold nanoparticles (AuNP)/carboxylated multiwalled carbon nanotubes (cMWCNT) as functional support for covalently linked catalase (CAT) enzyme. PAni was electropolymerized on a screen-printed carbon electrode (SPCE) and decorated with AuNP to improve charge transfer properties. CAT was bonded through amide formation using the carboxylic groups of cMWCNT, resulting in PAni/AuNP/cMWCNT/CAT biosensor. The structural and electroactive characteristics of the nanocomposite were studied by SEM, FT-IR, and cyclic voltammetry. The optimal performance was achieved after CAT immobilization over PAni/AuNP/cMWCNT/nanocomposite, showing improved analytical features such as a fast amperometric response of 1.28 s, a wide detection range from 0.01 to 6.8 mM, a correlation coefficient (R) of 0.9921, a low detection limit of 2.34 μM, and an average recovery rate of 99.6% when evaluated in milk samples. Additionally, the bioelectrode showed excellent selectivity and retained bioactivity after 30 days of storage. Such remarkable performance proved the synergistic effects of both the high surface area of the cMWCNT and AuNP and the inherent PAni electroactivity, yielding direct electron transfer from CAT.


Introduction
Enzyme-based electrochemical biosensors have been the subject of extensive research due to their promising applications in food safety, clinical diagnosis, healthcare, and environmental monitoring (1). Compared with typical analytical techniques, biosensors offer advantages such as portability, low cost, and facility of use; however, they can suffer from poor stability and limited sensitivity that might affect the analytical performance (2). Commonly, enzymatic biosensors rely on carbon electrodes modified with high-performance nanomaterials like metal nanoparticles, graphene, or conductive polymers that allow enzyme linking and facilitates electron transfer between the enzyme redox center and the electrode. It is expected that the nanostructured support enhances the kinetics and chemical stability of the enzyme (3,4). The design of polymeric-based composites as enzymatic supports contributes to enhancing the detection, stability, and performance of biosensors because of their unique properties at the nanoscale such as high structural stability, conductivity, and biocompatibility (5). In this sense, polyaniline (PAni) has been incorporated in enzymatic biosensors due to its attractive features such as low cost, facile synthesis, conductivity, environmental stability, and biocompatibility, functioning as the transducer, mediator for electron transfer, or immobilization matrix (6). PAni, also, is an effective electroactive platform for enzymatic biosensor design due to its tunable redox properties, which facilitate the charge transfer process from the redox enzyme's catalytic site to the polymeric structure (7). Furthermore, PAni is capable of immobilizing enzymes through different methods such as physical adsorption, covalent linking, or entrapping (8), resulting in an ideal component for developing electrochemical bioelectrodes.
Although the electrochemical properties of PAni are suitable for electrochemical detection, incorporation of electroactive entities into PAni, such as gold nanoparticles and carbon nanotubes, has been shown to allow synergistic effects on improved surface area and conductivity, improving the electrochemical performance of the platform with a faster response (7,9). Thus, to extend the PAni characteristics and tune its performance, composites based on PANi and AuNP have been studied, considering that the high surface to volume ratio of AuNP is highly beneficial for loading biomolecules (10). Besides, AuNP improves the electron transfer rate between the active centers of the immobilized enzyme and the electrode, resulting in enhanced performance (11). In addition, the combination of PAni with carbon-based components such as MWCNT provides advantages to the sensing systems such as chemical stability, enhanced mechanical properties, extensive surface area, and high conductivity (12).
Additionally, MWCNTs can act as an immobilization nano-matrix for covalent coupling through the induction of carboxylic acid groups and the reaction with the free amino groups on the enzyme (13). These modifications facilitate charge transfer and conductivity of the sensing platform (3,14), stabilizing the PAni and favoring retention of its inherent electrochemical activity, which provides more active nucleation sites, contributing, in conjunction, to improve biosensor performance (12).
Heme enzymes, including peroxidase, catalase, cytochrome c, hemoglobin, and myoglobin are involved in several fundamental biological processes. The redox chemistry of the heme iron as an active site and the biochemical diversity has led to the application as biological recognition elements in electrochemical biosensors for H 2 O 2 and phenolic compounds (15).
Catalase, from the bovine liver (CAT, EC 1.11.1.6), is an essential oxidoreductase enzyme (16), with four equal subunits of 60 kDa molecular weight and a heme prosthetic group at its active site with metallic iron Fe(III) (17). CAT is present in almost all aerobically respiring organisms, protecting cells from the toxic effects of hydrogen peroxide (H 2 O 2 ) by decomposing it into oxygen and water without the formation of free radicals (18). According to Lonča and Fraaije (19), catalases are robust enzymes with unusually high catalytic rates and among the highest enzymatic rates. Therefore, CAT is used in diverse industrial applications as an effective H 2 O 2 removal tool.
The fast, reliable, and accurate determination of H 2 O 2 is of great interest given its substantial role in food, pharmaceutical, clinical, industrial, and environmental analyses; additionally, H 2 O 2 is a biomarker of some cells and tumors (20). H 2 O 2 , also, is widely used as an antiseptic agent for preserving milk in the dairy industry. It inhibits microorganism growth and milk spoilage during the storage period. The use of H 2 O 2 must not exceed 0.05% of the milk weight and is approved in the USA by the food and drug administration (FDA) for this function (21). The excess of residual H 2 O 2 is harmful to humans; Hence, determining H 2 O 2 in food and biological systems is very important in health and product pre-treatment (22). Therefore, novel devices and configurations for sensitive H 2 O 2 detection are of significant attention like BiVO 4 /fluorinedoped tin oxide (23) and graphene oxide-poly(amidoamine) dendrimer-Fe 2+ /GCE (24). Also, some authors have designed H 2 O 2 biosensors using CAT as the bioreceptor, linking the CAT with supports like chitosan, single-walled carbon nanotubes, and polyethyleneimine (18). Furthermore, CAT has excellent stability and could be an excellent choice for biosensor design, where shelf stability is important (25).
Because of the interesting properties of CAT as a biocatalyst and the efficient operation of PAni-cMWCNTbased immobilization supports, the development of an electroactive platform capable of both enzyme immobilization, and measure biological interaction between CAT and H 2 O 2 by the electrochemical method is proposed. This work reports the construction of an amperometric bioelectrode departing from a screen-printed carbon electrode modified with electropolymerized PAni, which was subsequently optimized with AuNP and carboxylated MWCNT (cMWCNT). The PAni/AuNP/cMWCNT nanocomposite was applied as functional support for covalent immobilization of CAT used as a bioreceptor and exploited for quantifying H 2 O 2 in PBS and milk samples. The assembled nanocomposite showed high retention of enzyme activity, resulting in improved analytical parameters as accuracy, sensitivity, and low limit of detection (LOD), as well as favorable long-term stability, which evidenced the beneficial synergistic effects of the assembled nanocomposite in the biosensor performance.

Characterization
Cyclic voltammetry (CV) and amperometry measurements were measured using an EmStat3+blue potentiostat, whereas electrochemical impedance spectroscopy (EIS) was performed using a Solartron 1260A potentiostat/galvanostat. Fourier-transform infrared spectroscopy (FTIR) was registered using Perkin-Elmer GX-FTIR equipment, and the morphology was analyzed with a JEOL 300-S scanning electron microscope (SEM). FTIR and SEM characterized each stage of electrode modification and enzyme immobilization.

Assembling of PAni/AuNP/cMWCNT
MWCNTs were obtained according to a previous procedure and subsequently carboxylated by acid treatment (26). Briefly, 50 mg of MWCNT was added in a solution of 10 mL of H 2 SO 4 and HNO 3 in a 3:1 volume ratio; the mixture was sonicated for 1 h to obtain a fine dispersion and then refluxed at 90°C for 1 h. The resulting carboxylated multiwalled carbon nanotubes (cMWCNTs) were filtered and washed until neutral pH was achieved and dried at 60°C for 12 h; finally, FTIR was used to verify carboxylation.
A solution of aniline (0.25 M) in H 2 SO 4 (0.25 M) was used for electropolymerization onto the SPCE by CV, applying ten cycles at a potential from −0.5 to 1.0 V at a rate of 50 mV s −1 . The resulting SPCE/PAni was immersed in a mixture of HAuCl 4 (1 mM) in H 2 SO 4 (0.25 M) to perform AuNP electrodeposition. The conditions were three cycles at a potential from −0.5 to 1 V and a rate of 20 mV s −1 , obtaining the SPCE/PAni/AuNP. Then, 4 µL of cMWCNT (1 mg mL −1 ) were dropped cast on SPCE/PAni/AuNP and dried at room temperature. Finally, the modified surface was rinsed with distilled water to remove loose material, obtaining the SPCE/PAni/AuNP/cMWCNT functional support.

Catalase immobilization
CAT was covalently immobilized onto SPCE/PAni/AuNP/ cMWCNT, using the EDC/NHS chemistry. First, the COOH groups of the cMWCNT were activated by casting 4 µL of a solution of EDC (20 mM) and NHS (20 mM) in PBS (0.01 M) over the electrode surface at room temperature. Finally, 5 µL of CAT (5 mg mL −1 ) were deposed on the modified electrode and allowed to dry at room temperature. The electrode was washed with PBS to remove unbound enzymes, obtaining the SPCE/PAni/AuNP/cMWCNT/CAT biosensor. Figure 1 illustrates the fabrication stages, which were characterized by FTIR, SEM, CV, and EIS.

Electrochemical measurements
The quantitative detection of H 2 O 2 was performed using PBS (0.01 M) as the supporting electrolyte (pH 7.4) at 25°C. Before measurements, the PBS was deoxygenated by bubbling nitrogen for 10 min to protect the solution from oxygen. CV was evaluated with concentrations from 0.1 to 4 mM, using a potential from −1 to 1 V and a scan rate of 20 mV s − 1 . The amperometric response of the modified electrode toward H 2 O 2 was determined by applying a constant potential of −0.4 V in 25 mL of PBS at 0.01 M. During the measurements, the solution was kept under continuous magnetic stirring. After reaching a steady-state, a given amount of H 2 O 2 solution was added to the PBS electrolyte and the change in current was recorded. Measurements were conducted by triplicate under the same conditions. Finally, the proposed biosensor was used to determine H 2 O 2 in spiked samples of whole milk purchased at a local supermarket. The milk samples were used as they came from the packing. Known concentrations of H 2 O 2 were spiked using the whole milk as a matrix. The determination of H 2 O 2 in the milk samples was carried out by amperometry.   shows CAT immobilization on SPCE/PAni/AuNP/cMWCNT, with the enzyme forming a thin layer over the cMWCNT as seen in the inset. The change in morphology revealed a successful enzyme immobilization onto the support, in agreement with previous observations for CAT binding (28).

Functional group characterization
The reaction of the strong acids with the unstable carbon atoms of the MWCNT generated COOH groups (29). The spectrum of the cMWCNT ( Figure A1 in Appendix) shows absorption bands at 3,298 and 1,727 cm −1 , which correspond, respectively, to the stretching vibrations of the O-H and C]O bonds of the carboxyl group. The band at 1,627 cm −1 corresponds to the skeletal vibrations of the C]C bond of cMWCNT (30). Thus, the presence of these bands confirmed carbon nanotube's carboxylation, which has been reported for equivalent treatments (29). Figure 3a shows the spectrum of PAni/AuNP/cMWCNT showed the bands at 3,298, 1,727, and 1,627 cm −1 , as described for the cMWCNT. Besides, a pair of bands at 1,542 and 1,463 cm −1 , attributed, respectively, to the stretching vibrations of the C]C bond of the quinoid ring and the benzenoid ring, confirmed the PAni electropolymerization (31). The band at 1,370 cm −1 was related to the C-N stretching vibration in the neighborhood of a quinoid ring (32), suggesting that the π-bonded surface of the carbon nanotubes might interact actively with the conjugated structure of the PAni, primarily through the quinoid rings (33). Figure 3b illustrates the spectrum of PAni/AuNP/ cMWCNT/CAT. As seen, peaks are present at 1,640 and 1,572 cm −1 , these bands correspond on amide I and amide II bands, respectively (34). These peaks are located close to the amides bands of catalase (see Figure A2). According to Zuccarello et al. (15), the alterations of the relative intensities, positions, and shapes of the amide I and amide II bands of the enzyme indicate structural changes upon immobilization; Thus, these bands might be attributed to the formation of an amide bond between the NH 2 groups of CAT and the COOH groups of cMWCNT. In addition, the band at 1,727 cm −1 practically disappeared, and the band at 3,298 cm −1 showed a considerable intensity decrease, which was also associated with the interaction between COOH and NH 2 groups, confirming the covalent link of CAT and cMWCNT.

Electrochemical characterization
The voltammogram of PAni, Figure 4a, showed the typical oxidation peaks due to the transition from leucoemeraldine to emeraldine and from emeraldine to pernigraniline (37), and only one cathodic peak was observed. This behavior may be attributed to the neutral pH and the saline phosphate buffer used as electrolyte support. According to Mažeikien et al. (38), the redox activity depends not only on the pH but also on the composition of the electrolyte solution. They found that the redox properties of sulfonated polyaniline were reduced in sodium salt solutions due to the exchange of hydrated sodium cations between the electrolyte solution and the polymer film during the electrochemical process, producing a less effective transition between the oxidation states of PAni. However, the electropolymerized PAni used in this work showed the ability to undergo an electrochemical redox process even in neutral pH, which is suitable for this application.
After the AuNP electrodeposition, the current intensity of the anodic peaks increased, suggesting the increase of the active surface of PAni and the formation of a more effective surface area as a charge transfer mediator (39). For PAni/AuNP/cMWCNT, the high surface area of cMWCNT and their tubular structure generated a more energetically favorable pathway for the charge transfer, improving the inherent PAni electroactivity (3). Finally, the biosensor showed a small decrease in the electroactive area, indicating a slower electron transfer at the electrode surface by the immobilized enzyme. This confirmed the enzyme binding over the PAni/AuNP/cMWCNT functional support while retaining the electroactivity of the system. Similar behavior displayed the insulating property of the protein layer (40). Figure 4b illustrates the charge transfer at the electrode interface determined by EIS. The semicircle diameter at the high-frequency region represents the resistance charge transfer (R ct ), caused by electrochemical reactions in contact with the interface between the electrode and the electrolyte, and the linear part of the low-frequency region corresponds to the diffusion process (41). Figure 4b shows the Nyquist plots of the electrodes at each modification stage. As seen, the R ct of PAni was more extensive than that of PAni/AuNP and PAni/AuNP/cMWCNT, suggesting that the electrode modified by AuNP and cMWCNT decreased the R ct and promoted the electron transfer between the electrolyte and the modified electrode. These results agreed with CV; consequently, the cMWCNT not only played the role of the functional nanomatrix for the enzyme immobilization but also improved the electroactivity and charge transfer capacity in the nanocomposite. The R ct of the biosensor increased in comparison with the previous stage due to the non-conductive nature of CAT. Another study attributed a similar behavior because the electrode surface suffers from blockage by the poorly enzyme conductivity (42). PAni/AuNP/cMWCNT/CAT exhibited a highly defined cathodic peak at −0.52 V and an anodic peak of lower intensity not well defined at −0.26 V, suggesting that the intrinsic electroactivity of PAni overlapped it. These redox peaks were attributed to the heme group Fe III/IV from the CAT enzyme (43). To corroborate the direct electron transfer from CAT to PAni/AuNP/cMWCNT, CAT was immobilized on the nanocomposite without PAni to verify the overlap of the anodic peak. The AuNP/cMWCNT/CAT revealed a cathodic peak, at −0.4 V and a well-defined anodic peak at −0.22 V (Figure 5a, inset), corroborating the overlapping. The cathodic peak intensity was considerably more prominent than the anodic peak in both voltammograms, attributed to the conformational changes of CAT due to the immobilization process (44). This behavior was comparable with recent observations with catalase, where the presence of the redox peaks of the heme group from catalase was taken as evidence of the direct electron transfer (45).

Direct electron transference of catalase
Thus, the nanocomposite properties, such as high surface area, improved electroactivity, conductivity, and functional sites for covalent linking, lead to good electron transfer communication between the center of CAT with the modified PAni/AuNP/cMWCNT electrode.

Electrocatalytic behavior of CAT-modified electrode toward H 2 O 2
The catalytic activity of the nanocomposite for each step of modification toward H 2 O 2 was evidenced by CV. Figure A3  shows the voltammograms of each electrode modification in the presence of 2 mM of H 2 O 2 . The modifications without CAT presented a broad reduction peak around −0.75 V, which is attributed to reducing O 2 generated by the applied potential in the modified electrode at the H 2 O 2 solution (46). While PAnI/AuNP/cMWCNT/CAT exhibited higher current intensity due to a cathodic peak at −0.4 V, such response is a probe of the enhanced catalytic activity from the anchored enzyme. Thus, incorporating the CAT in the nanocomposite led to an improved response towards H 2 O 2 with less potential needed for the electron transfer process. Figure 5b displays that the cathodic peak at −0.49 V manifested a proportional increase in current as the H 2 O 2 concentration raised. The CAT mechanism to H 2 O 2 was proposed by Alfonso-Prieto et al. (47). According to this mechanism, CAT catalyzes the decomposition of H 2 O 2 into water and oxygen (2H (2) The active species responsible for oxidation and/or oxygenation reactions is a high-valent iron intermediate, oxoferryl porphyrin cation radical (Por ·+ -Fe IV ]O, Cpd I) obtained by the reaction with H 2 O 2 . Once Cpd I is formed (Eq. 1), it immediately reacts with a second molecule of H 2 O 2, reducing Cpd I to regenerate CAT (Por-Fe III ) with water and oxygen as by-products (Eq. 2). The reaction involves a two-electron redox process (48); therefore, the linked CAT over the support provided a fast direct electron transfer reaction between the heme group of CAT and the modified electrode surface and, consequently, an electrocatalytic activity toward H 2 O 2 .
It is worth saying that all components of the composite play a very important role in the electrochemical platform. The electrochemical characterization demonstrated that both cMWCNTs and AuNP helped to increase the active surface of PAni, improving the inherent PAni electroactivity. But also the incorporation of PAni on the nanocomposite is essential since it helps to maintain the direct electron transfer from the CAT ( Figure A4). The nanocomposite without PAni presented a definition loss of the cathodic peak of CAT as the concentration of H 2 O 2 increased. Whereas, with the presence of PAni, the cathodic peak of CAT remained defined. This behavior could be attributed to the biocompatible properties of PAni, which contribute to an optimal environment for enzyme performance. Hence, besides working as a transducer surface, the PAni could anchor some enzymes by physical adsorption or covalent linkage (6). Thus, a synergistic effect was formed in conjunction with the cMWCNTs and AuNP protecting the native structure of CAT and maintaining the accessibility of the catalytic sites and, therefore, facilitated the direct transfer of the CAT center to the electrode surface.

Amperometric determination of H 2 O 2
According to the response observed in CV, the biosensor was evaluated for the detection of H 2 O 2 by amperometry at −0.4 V. Figure A5 shows the amperometric response toward H 2 O 2 at every stage of the nanocomposite assembly. After the modification of the SPCE with PAni and PAni/AuNP, a small current was recorded at the selected potential. However, PAni/AuNP/cMWCNT nanocomposite showed a clear improvement due to the electrocatalytic activity of cMWCNT toward H 2 O 2 . This step improved the electron transfer rate enabling the H 2 O 2 reduction through electron transfer, which is the principle of non-enzymatic sensors (49). Indeed, some nonenzymatic amperometric sensors for H 2 O 2 detection have been developed (50)(51)(52). Although these kinds of sensors show good electrochemical response and have the advantage of avoiding enzymatic susceptibility, they usually need a higher potential for H 2 O 2 reduction.
With the immobilization of CAT, it was evident that the biosensor exhibited the best amperometric response for H 2 O 2 detection. Furthermore, the incorporation of CAT into the nanocomposite led to an improved response toward H 2 O 2 with less working potential needed for the electron transfer process. The electrode modified with PAni/AuNP/cMWCNT exhibits an enhanced catalytic activity toward H 2 O 2 with the CAT loading, corroborating the results obtained by CV. The direct electron transfer between the heme group of CAT and the electrode surface significantly improved the device sensitivity because of the effective covalent linkage of CAT and the synergistic effects of the PAni/AuNP/cMWCNT support. Hence, CAT immobilization was critical in the biosensor assembly to enhance the amperometric response.
The biosensor capacity was evaluated by amperometry with successive additions of H 2 O 2 at −0.4 V under optimized conditions. Figure 6a and b illustrates the i versus t curve and the calculated calibration curve (n = 3), respectively. The current as a function of H 2 O 2 concentration showed a linear response of i to the increasing level of H 2 O 2 , with R 2 = 0.9921. The biosensor exhibited a linear range from 0.01 to 6.8 mM, a sensitivity of 58.8 µA mM −1 cm −2 , and a LOD of 2.34 µM (LOD = 3.3 * σ/S, where σ and S are the standard deviation and sensitivity, respectively). Notably, the device showed a fast amperometric response after only 1.28 s. Table 1 reports a comparison of electrocatalytic values of some electrochemical (bio)sensors for H 2 O 2 . The comparative data showed that the biosensor properties are comparable or even better concerning previously reported catalase biosensors, especially considering LOD. For instance, the biosensor based on CAT/Fe@G-MWCNTs showed a similar linear range, but higher LOD (45). In another report, CAT immobilized onto an inorganic hybrid microflower HMFs (Cu 3 (PO 4 ) 2 )-modified GCE exhibited a wide linear concentration range but higher LOD (53). Similarly, the CAT/PNR Ethaline /Fe 2 O 3 NP/GCE biosensor showed a higher LOD and smaller linear range (54). It is worth saying that the notable performance of the PAni/AuNP/cMWCNT/CAT biosensor expressed the synergistic effects of both the high surface area of the cMWCNT and AuNP and the PAni inherent electroactivity.

Kinetic study
When the concentration of H 2 O 2 increases, a response plateau appears ( Figure A6(a)), which is characteristic of the typical Michaelis-Menten kinetic behavior (60).
To study the enzyme-substrate kinetics, the apparent Michaelis-Menten constant (k m ) was calculated from the linear dependence observed in Figure A6

Sensor stability and specificity
Common interfering substances, such as glucose, ascorbic acid, KCl, and NaCl, were tested for confirming sensor selectivity. Figure 7a shows no significant responses for the evaluated species but a repeatable current response after H 2 O 2 injection, confirming high biosensor selectivity toward the analyte. The biosensor was kept at 4°C and assessed for 30 days to evaluate stability (Figure 7b). After  storage, the biosensor retained almost 85% of the initial current. Such favorable long-term stability expressed the stable enzyme bonding with the cMWCNT at the electrode surface and the favorable biocompatible environment provided by the assembled nanocomposite.

Determination of H 2 O 2 in real samples
H 2 O 2 is widely used as an antiseptic agent for preserving milk in the dairy industry. For practical purposes, the standard addition method was used to determine H 2 O 2 concentrations in commercial milk (whole fat milk) at PAni/AuNP/cMWCNT/CAT by amperometric method. The standard addition method (regularly denoted as "spiking" the sample) is commonly used to determine the analyte concentration in complex samples containing other components that could interfere with the analyte signal, causing inaccuracy determination. The idea is to spike the sample and monitor the amperometric response change, assuming the response is only due to a change in analyte concentration (61). The analysis was carried out by spiking a known concentration of H 2 O 2 in the milk samples without prior treatment and recording their recoveries. Before analysis, a blank was verified without the addition of H 2 O 2 in the milk sample, which presented no signal. For every sample, three individual measurements were performed. Table 2 shows the excellent recovery rates obtained, in the range of 92-104.8%. Besides, the H 2 O 2 concentrations used, 0.2, 0.5, and 0.7 mM correspond to 0.00068%, 0.0017%, and 0.0023%, respectively; The use of H 2 O 2 is approved in the USA by the FDA for treating milk, and it cannot exceed 0.05% of the milk weight (21). Therefore, in terms of detecting peroxide in milk for quality control, concentrations smaller than the minimum required were achieved.
It should be noted that in this experiment, undiluted milk samples were used; the milk samples were used as they came from the packaging. This presents an advantage since the peroxide was detected in milk without a previous dilution step. Other similar biosensors have been studied to detect peroxide in milk samples; however, they diluted the milk in PBS to perform the detection (62)(63)(64). These results evidenced the biosensor suitability for the analysis of H 2 O 2 in real samples, confirming its practical application for food quality control.

Conclusion
Electrochemical characterization demonstrated that both cMWCNT and AuNP facilitated the increase in the active surface of PAni, and PAni helped maintain direct electron transfer from CAT. The assembly of a PAni/AuNP/cMWCNT platform manifested a synergistic effect as functional support for catalase immobilization allowed retaining the catalytic activity and facilitated the direct electron transfer between the CAT and the modified electrode. The biosensor exhibited notable electrocatalytic activity toward H 2 O 2 with high sensitivity, low LOD, wide linear range, excellent recovery with real milk samples, and favorable long-term stability. Such performance evidenced the catalytic activity of CAT due to the effective covalent linkage on the support. Therefore, the electrode, modified by the PAni/AuNP/cMWCNT nanocomposite, is functional as support for covalent immobilization of enzymes, with high performance, sensitivity, and selectivity.

Conflict of interest:
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Figure A1: FTIR spectra of (a) MWCNT and (b) cMWCNT.