Ferrocene-Grafted Carbon Nanotubes for Sensitive Non-Enzymatic Electrochemical Detection of Hydrogen Peroxide

Abstract

Sensitive detection of hydrogen peroxide (H₂O₂) residue in aseptic packaging at point of use is critical to food safety. We present a sensitive non-enzymatic, amperometric H₂O₂ sensor based on ferrocene-functionalized multi-walled carbon nanotubes (MWCNT-FeC) and facile screen-printed carbon electrodes (SPCEs). The sensor utilizes the covalently grafted ferrocene as an effective redox mediator and the MWCNT networks to provide a large active surface area for efficient electrocatalytic reactions. The electrocatalytic MWCNT-FeC modified electrodes feature a high-efficiency electron transfer and a high electrocatalytic activity towards H₂O₂ reduction at a low potential of −0.15 V vs. Ag/AgCl. The decreased operating potential improves the selectivity by inherently eliminating the cross-reactivity with other electroactive interferents, such as dopamine, glucose, and ascorbic acid. The sensor exhibits a wide linear detection range from 1 μM to 1 mM with a detection limit of 0.49 μM (S/N=3). The covalently functionalized electrodes offered highly reproducible and reliable detection, providing a robust property for continuous, real-time H₂O₂ monitoring. Furthermore, the proposed sensor was successfully employed to determine H₂O₂ levels in spiked packaged milk and apple juice with satisfactory recoveries (94.33–97.62%). The MWCNT-FeC modified SPCEs offered a facile, cost-effective method for highly sensitive and selective point-of-use detection of H₂O₂.

1. Introduction

Over the past few decades, the determination of hydrogen peroxide (H₂O₂) residue has been critical in the food industry and environmental analysis [1–3]. Hydrogen peroxide is commonly used as a preservative in milk and applied for sterilization in aseptic food packaging due to its inherent bactericidal and sporicidal properties [4]. However, repeated exposure to H₂O₂ residue in food or beverage may increase the risk of many health problems, including skin irritation and gastrointestinal tract, cyanosis, and cardiac arrest [5]. According to the U.S. Food and Drug Administration (FDA) regulation (21 CFR 178.1005), the H₂O₂ residue level in the aseptically-packaged products must not exceed 0.5 ppm (~15 μM) [1, 6]. Thus, real-time monitoring of H₂O₂ levels in food production or detecting H₂O₂ residue at point of use is critical to food safety in the food and beverage industry. Conventional H₂O₂ sensing relies on spectroscopy [7, 8] and enzyme-based fluorescence or chemiluminescence assays [9, 10]. Although these techniques provide sensitive and selective detections, they generally rely on expensive lab-based equipment, require sample pre-treatments, and suffer from low stability due to enzymes’ intrinsic nature, making them unsuitable for on-site routine analysis of H₂O₂ [11]. On the contrary, electrochemical sensors offer rapid, simple, cost-effective approaches for sensitive and selective detection [12–15]. Furthermore, they also allow direct real-time analysis without sample pre-treatment procedures [11, 16, 17]. Among them, enzyme-based electrochemical biosensors provide high sensitivity and low detection limits. However, the complicated enzyme immobilization and stabilization processes, the environment-dependent enzyme activity, and the high cost hinder their general applications [18]. Therefore, it is highly desirable to develop an enzyme-free H₂O₂ sensor that can overcome these issues by incorporating electrocatalytic nanomaterials.

Various approaches have been reported to construct a non-enzymatic sensor based on oxidation or reduction of H₂O₂ [19–21]. The oxidation of H₂O₂ often suffers from high over-potential, which simultaneously produces oxidative responses from any non-target species in the biofluids, including ascorbate, uric acid, dopamine, etc., leading to poor sensing specificity [4]. The sensors based on Prussian Blue for catalytic reduction of H₂O₂ have been demonstrated to exhibit a low detection limit and good selectivity. However, Prussian Blue-contained electrodes were found to exhibit low operational stability and risk of cyanide leakage, limiting their practical application [19, 22]. On the other hand, ferrocene is an alternative catalyst that supports a low over-potentials for the redox reaction of H₂O₂. Ferrocene becomes one of the most exploited catalysts for non-enzymatic sensors because it supports fast electron-transfer rates, good stability of two redox states, and, more importantly, excellent electrocatalytic activity towards both the reduction and oxidation of H₂O₂ [23]. Efforts have been made for immobilizing ferrocene onto the sensor surface. Physical adsorption of ferrocene/ferrocenium (FeC/FeC⁺) redox couples on electrodes were applied to facilitate H₂O₂ detection [21] but yielded limited stability because they are likely to detach from the electrode surface [24]. Polymers with covalently attached FeC redox mediators have been reported to overcome the stability issue with proper electrochemical properties [1, 25]. However, the polymer backbone tends to swell during sensing, increasing the distance between FeC mediators and hindering the electron transfer [26–28]. Moreover, these sensors require tedious and complicated fabrication processes [29]. Therefore, developing a facile and robust sensor that covalently immobilizes redox reagents is crucial to enhance sensing performance and reliability. Conductive nanomaterials covalently bound with redox mediators, such as ferrocene-graphene nanosheets [21], can be promising in accelerating electron transfer processes. Compared to graphene, multi-walled carbon nanotubes have been proven superior in supporting a larger surface area and strong mechanical property [30]. Electrode material aside, most H₂O₂ sensors were demonstrated on stand-alone glassy carbon electrodes [31–33], which can hardly be miniaturized for point-of-use applications. In comparison, screen-printed carbon electrodes (SPCEs) can be scaled and integrated to form a low-cost, disposable coplanar three-electrode system suitable for mass production [34].

In this work, we demonstrate a facile, sensitive, non-enzymatic H₂O₂ sensor based on ferrocene-grafted multi-walled carbon nanotube (MWCNT-FeC) nanocomposites. The nanocomposite offers several advantages, including strong catalytic properties towards H₂O₂ reduction, outstanding electron transfer property, and large electroactive surface area. The unique features enable the MWCNT-FeC modified SPCE to detect H₂O₂ with high sensitivity, wide dynamic range, and high stability, compared with the H₂O₂ sensors reported to date. More importantly, the sensor operates at a low reduction potential of −0.15 V for H₂O₂ sensing that inherently avoids the cross-reactivity with common electroactive interferences, such as glucose, ascorbic acid, and dopamine, leading to a highly selective H₂O₂ detection. The practical application was validated by determining the H₂O₂ levels in spiked aseptic milk and juice. The results suggest that the proposed MWCNT-FeC modified SPCEs are promising for practical H₂O₂ detection.

2. Materials and methods

2.1. Reagents and Apparatus

Ferrocene carboxaldehyde (FeC-CHO), ferrocyanide/ferricyanide ([Fe(CN)₆]^3/4−), glucose, and L-ascorbic acid were purchased from Sigma-Aldrich. Hydrogen peroxide (H₂O₂, 30% solution) was purchased from Macron Fine Chemicals. Chitosan (CS, 85% deacetylated), sodium cyanoborohydride (NaCNBH₃) was purchased from Alfa Aesar. 3-Hydroxytyramine hydrochloride (dopamine, DA) was purchased from Acros Organics. Phosphate-buffered saline (PBS, 10X, pH 7.4) was purchased from Fisher Scientific. Plasma-created amine-functionalized multi-walled carbon nanotubes (MWCNT-NH₂) were procured from Cheap Tubes, Inc. (USA). Carbon ink (CI-2042) and silver/silver chloride ink (CI-4002) were purchased from Engineered Materials Systems, Inc. All other chemicals were of analytical grade and used without further purification. All the solutions were prepared using double distilled water.

2.2. Synthesis of MWCNTs-FeC nanocomposite

Ferrocene grafted multi-walled carbon nanotubes (MWCNTs-FeC) were synthesized using a procedure modified from a reported method for conjugation of FeC on polymer [35]. The process started with dispersing 30 mg MWCNT-NH₂ in 2.5 mL 0.1 M acetic acid solution. FeC-CHO (35 mg) was dissolved in 5 mL methanol solution and added to the MWCNT-NH₂ dispersion. After being fully sonicated for 10 min, the reaction mixture was stirred for 4 hours at 35 °C, followed by adding NaCNBH₃ (50 mg) to the mixture and continuing the reaction for another 24 hours. Subsequently, the products were extracted by centrifugation and rinsed thoroughly with double distilled water and methanol three times. Finally, the products were dried under vacuum and stored as a powder for further use.

2.3. Preparation of screen-printed carbon electrodes

The flexible SPCEs were fabricated on polyethylene terephthalate (PET) substrates using a homemade stainless-steel screen-printing mask. The three-step manufacturing process started with transferring carbon ink through a mask onto the PET substrate to provide electrical contacts. The electrode connections, 3 mm diameter working electrode, and auxiliary electrode were subsequently baked at 110 °C for 15 min. The reference electrode was printed with silver/silver chloride ink, then cured at 110 °C for 10 min [36]. Finally, polyimide (Kapton) tape was used to delimit the sensor area, leaving contact pads and connections on the other side of the sensor.

2.4. Preparation of MWCNTs-FeC/CS composite-modified electrode

The 1 mg synthesized MWCNT-FeC was dispersed in a 1 mL, 0.5 mg mL⁻¹ chitosan solution prepared in 0.2 M acetic acid, followed by a 20 min probe sonication. The resultant 4 μL MWCNT-FeC/CS homogeneous solution was drop-casted onto the working electrode of SPCE, followed by 30 min baking at 50 ℃. CS improved the uniformity of the MWCNT-FeC in the drop-casting process. The modified electrodes were stored at 4 ℃ for future use. The fabrication process of the MWCNT-FeC/CS composite-modified SPCE is presented in Fig. S1. Control samples (MWCNT-NH₂/SPCE) prepared by drop-casting untreated MWCNT-NH₂/CS composite on SPCE were created to investigate the effect of FeC modification on the electrocatalytic property of the modified electrodes.

2.5. Material and device characterizations

Scanning electron microscopy (SEM) images and energy-dispersive X-ray spectroscopy (EDX) were acquired on a QUANTA 600F scanning electron microscope (FEI, USA). Electrochemical measurements, including cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry (CA), were performed with a three-electrode system using potentiostat (Gamry Reference 600+) electrochemical system driven by Gamry Framework™ Applications Version 6.3. CV measurements were conducted with a scan rate of 100 mV s⁻¹ between −0.3 V and 0.5 V in 1X PBS solution at pH 7.0. The CV results indicate that the sensor performed most efficiently at an optimum voltage of −0.15 V vs. Ag/AgCl, selected as the applied potential for amperometric measurements. The sensor’s amperometric response to H₂O₂ was recorded at a working potential of −0.15 V (vs. Ag/AgCl) with various H₂O₂ concentrations in a 1X PBS solution (scheme shown in Fig. 1). The sensing data was taken at 30 seconds when the current reached a steady state.

Figure 1.

Printable H₂O₂ sensor enabled by ferrocene grafted multi-walled carbon nanotubes (MWCNT-FeC) and its H₂O₂ sensing mechanism.

2.6. H₂O₂ detection in commercial products

Milk and apple juice purchased from a local grocery store were firstly spiked with H₂O₂ to reach concentrations above, equal, and below 15 μM. After thoroughly mixing for 5 min, the H₂O₂ spiked samples were diluted with 10X PBS in a 9:1 ratio. After 5 min shaking, the mixture was centrifuged at 7000 rpm for 15 min, and the top supernatant was collected for H₂O₂ detection under the optimized conditions.

3. Result and discussion

3.1. Morphology Characterization of MWCNTs-FeC modified SPCE

The SEM image of a bare SPCE surface in Fig. 2(a) shows the porous surface morphology of carbon black. The coating of MWCNT-FeC composite on SPCE in Fig. 2(b) resulted in excellent coverage of woven mesh-like carbon nanotube networks over the electrode surface, creating a three-dimensional structure that increased the electroactive area for electrochemical reaction. The EDX analysis of the bare SPCE in Fig. 2(c) displays the presence of 83.67 at. % carbon and 16.29 at. % oxygen. On the other hand, the MWCNT-FeC modified SPCE surface contained additional peaks in the EDX spectrum corresponding to irons as shown in Fig. 2(d), yielding 92.81 at. % carbon, 6.97 at. % oxygen and 0.23 at. % iron. The presence of iron confirms the existence of ferrocene on the modified electrode. Also, the increased carbon percentage on the MWCNT-FeC modified electrode resulted from the presence of high-density carbon nanotubes.

Figure 2.

SEM images of (a) SPCE and (b) MWCNT-FeC modified SPCE. Scale bars indicate 1 μm. EDX spectra of (c) SPCE and (d) MWCNT-FeC modified SPCE.

3.2. Electrochemical characterization of MWCNTs-FeC/SPCE

CV measurements were carried out to further verify the successful conjugation of ferrocene on the carbon nanotubes. Fig. 3(a) shows the CVs of the bare SPCE, MWCNT-NH₂ modified SPCE, and MWCNT-FeC modified SPCE characterized in 1X PBS at a sweep rate of 100 mV s⁻¹ without redox reagents in the solution. Both bare SPCE and MWCNT-NH₂ modified SPCE did not present any indication of redox activity. However, the MWCNT-NH₂ modified SPCE exhibited an increased baseline current due to the enlarged electroactive surface area contributed by the dense MWCNT networks. A pair of stable and well-defined redox peaks were observed on the MWCNT-FeC modified SPCE with the anodic and cathodic peak potentials at +0.117 V and +0.079 V vs. Ag/AgCl, respectively. The redox peaks were the electrochemical signatures of the immobilized FeC/FeC⁺ redox couple on MWCNTs, implying the successful conjugation of ferrocene to the MWCNTs [21, 37]. The broadening of the redox peaks was probably due to the interactions of π-π stacking between the FeC groups [38]. The modified electrode yielded a +98 mV formal potential calculated from the average of the cathodic and anodic peak potentials and 42 mV potential separation between reduction and oxidation peaks. The formal potential and peak potential separation of this work were lower than the counterparts reported previously [39–41] due to the improved electron transfer rate and a larger electroactive surface area [42]. Fig. 3(b) shows the CVs of the MWCNT-FeC modified SPCE under various potential scan rates. The oxidation and reduction peak currents increase linearly with the scan rate in the range from 10 to 600 mV s⁻¹, as shown in Fig. 3(c). The peak current I_p as a function of scan rate v agrees with Laviron’s theory, I_p = n²F²vAΓ/4RT, with A, F, R, and T being the surface area, Faraday constant, the universal gas constant, and temperature in Kelvin, respectively [43]. The n is the number of electrons involved in the catalytic reaction, and Γ is the electroactive coverage of the catalyst on the electrode. The linear relationship between the peak current I_p and scan rate v implies that the surface-confined redox reagents were confined on the electrode surface due to the covalently immobilized ferrocene on the MWCNT modified electrode. Compared to the previously reported CNT-FeC nanocomposites modified electrodes [44–46], the proposed sensor provided a higher electroactive coverage of ferrocene (Γ = 2.12 × 10⁻⁹ mol cm⁻²) and a more prominent redox peak. The results can be attributed to the high-density amine functional groups created on the MWCNT surface, offering abundant conjugation sites for ferrocene grafting [47, 48]. The short functional linkers reduced the distance between the FeC redox center and the conductive MWCNTs, thus promoting electron transfer efficiency [49]. The interfacial properties of the electrodes were analyzed using electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)₆]^3/4− redox reagents in the solution. As shown in Fig. 3(d), the charge transfer resistance (R_ct) of the bare SPCE decreased from 2.3 kΩ to 146 Ω after being modified with MWCNT-NH₂ due to the introduction of a high-density conductive MWCNTs network. The R_ct further reduced to 97 Ω after FeC grafting. The reduced R_ct across the electrode-electrolyte interface could result from the covalent conjugation of FeC on MWCNTs that brought the high-density FeC/FeC⁺ redox centers very close to the MWCNTs, further improving the charge transfer channel to the electron [41].

Figure 3.

(a) CVs of (1) SPCE, (2) MWCNT-NH₂ modified SPCE, and (3) MWCNT-FeC modified SPCE measured in 1X PBS at 100 mV s⁻¹ scan rate. (b) CVs of MWCNT-FeC modified SPCE measured in 1X PBS at various scan rates (from inner to outer curves: 10, 50, 100, 200, 300, 400, 500, 600 mV s⁻¹). (c) Anodic and cathodic peak currents vs. scan rate. (d) EIS of (1) SPCE, (2) MWCNT-NH₂ modified SPCE, and (3) MWCNT-FeC modified SPCE measured in 0.1 M KCl containing 1.0 mM [Fe(CN)₆]^3−/4− (1:1). Inset: Randles equivalent circuit model for the extraction of charge transfer resistance R_ct.

3.3. Hydrogen peroxide detection

The H₂O₂ electrocatalytic activity of the developed sensor was evaluated using cyclic voltammetry and chronoamperometry. The CVs in Fig. 4 show that the MWCNT-FeC modified SPCE significantly increased both reduction and oxidation of H₂O₂ compared with the MWCNT-NH₂ modified SPCE. The feature was not observed in most of the reported modified electrodes, which only promotes either oxidation [50, 51] or reduction [52, 53] of H₂O₂. After introducing 1 mM H₂O₂, the current of the MWCNT-FeC modified electrode increased by 8.3 μA at −0.15 V and 2.8 μA at 0.6 V, whereas the current of the non-functionalized MWCNT-NH₂ modified electrode increased by only 1 μA at −0.15 V and 0.8 μA at 0.6 V. The elevated current in MWCNT-FeC electrodes suggests ferrocene’s strong electrocatalytic activity towards H₂O₂ on the functionalized MWCNT surfaces for both the oxidation of H₂O₂ to O₂ and the reduction to H₂O. Overall, the improved electrocatalytic activity can be attributed to the large electron transfer through the modified carbon nanotube, the shorter distance between the mediators and electrode, and FeC’s strong electrocatalytic activity towards H₂O₂ reduction.

Figure 4.

CVs of (a) MWCNT-NH₂ modified SPCE and (b) MWCNT-FeC modified SPCE in response to various H₂O₂ concentrations: (1) 0 mM, (2) 1 mM, and (3) 10 mM in 1X PBS at 100 mV s⁻¹ scan rate.

Apart from detection sensitivity, the operating potential for the amperometric measurements must be properly chosen to eliminate interferences from the common electroactive species and achieve highly selective H₂O₂ detection. The CVs of the MWCNT-FeC modified SPCE in the absence and presence of 0.5 mM H₂O₂ and other interferents, including glucose, ascorbic acid, and dopamine, are shown in Fig. 5(a–d), respectively. Fig. 5(e) and 5(f) summarize the CV currents acquired at −0.15 V and 0.6 V electrode potentials vs. Ag/AgCl. At 0.6 V electrode potential, ascorbic acid and dopamine were easily oxidized, yielding the current changes larger than that contributed by the oxidation of H₂O₂ at the same potential, which agrees with our previous observation [54]. In contrast, at −0.15 V, the electrode actively reduced H₂O₂ while showed negligible responses to glucose and ascorbic acid, leading to a substantial current change compared with those produced by the three interferents, as shown in Fig. 5(e). The MWCNT-FeC modified electrode efficiently reduced H₂O₂ to H₂O at a potential as low as −0.15 vs. Ag/AgCl and allowed specific detection of H₂O₂ without the simultaneous oxidization or reduction of the interferents, significantly improving the detection selectivity. Applying a more negative electrode potential did not considerably increase the current response. Therefore, this electrode potential was then chosen to examine the amperometric response against H₂O₂ reduction. The MWCNT-FeC modified SPCE enables a highly selective H₂O₂ detection with a much lower electrode potential than the previous works [55, 56]. The low operating potential for H₂O₂ detection also prevented the interference of O₂ reduction in solution. As presented in Fig. S2, detection of H₂O₂ in the analytes purged with air and nitrogen gas for 10 min yielded similar responses. Because FeC did not present strong electrocatalytic activity towards oxygen reduction and the detection was performed at a small reduction potential, we did not observe the impact of O₂ reduction on H₂O₂ detection.

Figure 5.

CVs of MWCNT-FeC modified SPCE in the absence and the presence of 0.5 mM (a) H₂O₂, (b) glucose, (c) ascorbic acid, and (d) dopamine in 1X PBS at 100 mV s⁻¹ scan rate. CV current change in response to 0.5 mM analytes obtained at −0.15 V (e) and 0.6 V (f) vs. Ag/AgCl as indicated by the circles in the CV curves (a-d).

Fig. 6(a) shows the amperometric recordings of H₂O₂ detection at various concentrations. The H₂O₂ analyte solution was loaded for detection at time zero with the sensor biased at −0.15 V. The resulting calibration curve shown in Fig. 6(b) summarizes the current levels acquired in Fig. 6(a) at 30 s. The sensor exhibited a linear response to H₂O₂ (R² = 0.997) in a broad concentration range from 1 μM to 1 mM. The amperometric current change ΔI (μA) in response to H₂O₂ concentration c (mM) follows the relation ΔI = 9.26 c + 0.38. A high current density of 136.4 μA mM⁻¹ cm⁻² and a low detection limit of 0.49 μM H₂O₂ were obtained in the linear range at a signal-to-noise ratio of 3, low enough to detect H₂O₂ in practical applications. We observed a noticeable increase in current when H₂O₂ concentration increases from zero to 1 μM in the inset of Fig. 6(a), resulting in a current offset in the calibration curve after a straight-line fitting as shown in Fig. 6(b). The offset could be related to the reactive reduction of H₂O₂ on the MWCNT-FeC modified electrode. We observed that the presence of H₂O₂ in buffer solutions can produce a detectable net current even at zero electrode bias. The zero-bias current slightly increased with H₂O₂ concentration. The result implies that part of the sensing current measured at −0.15 V was contributed by the intrinsic reduction of H₂O₂ at zero voltage bias. At low concentrations, the zero-bias reduction current accounted for a relatively large share of the total sensing current. Further study is required to clarify the effect.

Figure 6.

(a) Amperometric response of MWCNT-FeC modified SPCE to various H₂O₂ concentrations in 1X PBS (pH 7.0). (b) Calibration curve for H₂O₂ detection. (c) Calibration curves for glucose, ascorbic acid (AA), and dopamine (DA). The sensor electrode was biased at −0.15 V vs. Ag/AgCl.

Compared to the ferrocene-based H₂O₂ sensors reported previously [24], the MWCNT-FeC modified electrode showed a superior H₂O₂ detection sensitivity, lower detection limit, and improved selectivity due to the operation at low reduction potential. Fig. 6(c) shows that glucose and ascorbic acid displayed imperceptible amperometric current changes. Dopamine yielded a current level of less than a quarter of the sensing response of H₂O₂ of the same concentrations. It is worth noting that the dopamine concentrations in the actual samples are much lower than the values demonstrated here for selectivity validation.

The MWCNT-FeC modified SPCE also exhibited highly reproducible and repeatable H₂O₂ detection. As shown in Fig. 7(a), repeated measurements on the same device show a negligible change in the sensing results. Fig. 7(b) shows that the device-to-device variation collected from five sensors was measured to be only 3.7%, indicating the reproducibility of the H₂O₂ sensors. The sensor interrogation-regeneration plots in Fig. 7(c) show repeatable H₂O₂ sensing results during four cycles of alternating detection and refreshing processes in 1X PBS buffer, implying that the sensor can be reused after a simple regeneration process by rinsing it with 1X PBS buffer. Overall, the sensors were found to be reasonably stable over time. Fig. 7(d) shows a ~ 8% drop in sensing signal for the devices stored for ten days at 4°C. The shelf life is expected to be further improved with proper humidity control. The reproducible, stable, and reliable detection results signify that the proposed sensor is a promising candidate for practical measurements.

Figure 7.

(a) Repeatability tests of the sensor. (b) Device-to-device variation tests. (c) Interrogation-regeneration plot for the sensor during alternating H₂O₂ sensing (S) and PBS rinsing (R) processes. (d) Stability of the sensor over 10 days. The sensors were tested in 0.5 mM H₂O₂ in 1X PBS.

Real-time, continuous H₂O₂ sensing was further evaluated using a sensor integrated with a PDMS microfluidic channel, as shown in Fig. 8(a). The printed sensor with the working electrode biased at −0.15 V vs. Ag/AgCl continuously monitored the H₂O₂ levels in an analyte inflow. The analyte was delivered to the sensor area with a constant flow rate using a syringe pump. Interferents, including ascorbic acid, glucose, and dopamine, were added at different time points to evaluate the detection selectivity. Fig. 8(b) shows the amperometric recordings of successive additions of H₂O₂ and the interferents. The results suggest that even the concentrations of the interferents were four times higher than H₂O₂, the interferents’ current responses are much lower than those contributed by H₂O₂. It implies the MWCNT-FeC modified SPCE sensor has an excellent selectivity towards H₂O₂.

Figure 8.

(a) Image of a real-time sensing setup; (b) Chronoamperometry of the sensor responding to H₂O₂ and interferents. The working electrode is biased at −0.15 V vs. Ag/AgCl.

The MWCNT-FeC modified SPCE sensor presented in this paper exhibited a superior sensing performance and promising applications for continuous, real-time sensing. Table 1 compares this work with several representative electrochemical H₂O₂ sensors reported to date. The comparison indicates that the proposed MWCNT-FeC modified SPCE sensor featured superior sensing performance with low detection limit, wide linear dynamic range, high selectivity, low operating potential, and reproducibility.

Table 1.

Comparison of representative electrochemical H₂O₂ sensors with this work

Electrode	Working voltage (V)	Dynamic range (μM)	LoD (μM)	Interferents	Real samples	CH2O2 in real samples (μM)	Ref.
RGO/CNTs-Pt/GCE	−0.2	0.3–4000	0.31	DA, Cys, Fru, GSH, GLC, AA	Milk	32–1218	[57]
[Cu(bmtc)2(H2O)]/SPCE	−0.6	10–524	0.57	Urea, AA, GLC, UA	Milk	10–30	[58]
LSG/AgNPs	−0.56	100–10000	7.9	AA, GLC, KCl, NaCl	Milk	200–500	[59]
BiVO4/TiO2	5	5–200	5	UA, AA, B12, GLY, ASN	N/A	N/A	[60]
GNPs-PB/BG/GCE	−0.05	9.6–143	3.2	AA, UA, GLC, GLY, APAP	N/A	N/A	[61]
MWCNTs-FeC/SPCEs	−0.15	1–1000	0.49	DA, AA, GLC	Milk, apple juice	10–50	This work

GCE: glassy carbon electrode; Fru: fructose; Cys: cysteine; GSH: glutathione; [Cu(bmtc)2(H2O)]: copper (II) 1-(3-bromobenzyl)-5-methyl-1H-1,2,3-triazole-4-carboxylate; UA: uric acid; LSG: laser scribed graphene electrode; AgNPs: silver nanoparticles; NaCl: sodium chloride; GLY: glycine; PB: prussian blue; BG: bucky gel; GNPs: gold nanoparticles; ASN: asparagine; APAP: acetaminophen.

3.4. Determination of H₂O₂ in the real samples

The feasibility of sensing H₂O₂ in real samples using MWCNT-FeC modified SPCE was evaluated through a recovery study using commercial milk and apple juice samples. The samples were spiked with three known H₂O₂ concentrations, including 15 μM, the maximum H₂O₂ level allowed by U.S. FDA for aseptic packaging. As shown in Table 2, the recovery rates and RSD values were in the range of 94.33–97.62% and 1.93–4.46%, respectively, suggesting that the MWCNT-FeC modified SPCE sensor can be readily used to analyze H₂O₂ residues in commercially packaged beverages.

Table 2.

Determination of H₂O₂ in commercially packaged milk and apple juice samples using MWCNT-FeC/SPCE.

Samples	Spiked (μM)	After dilution (μM)	Found (μM)	RSD (%)	Recovery (%)
Milk #1	10	9.0	8.52	4.20	94.66
Milk #2	15	13.5	12.95	2.36	95.93
Milk #3	50	45.0	42.87	1.93	95.27
Juice #1	10	9.0	8.49	3.84	94.33
Juice #2	15	13.5	12.99	2.78	96.22
Juice #3	50	45	43.93	4.46	97.62

Relative standard deviation (RSD) of 3 measurements.

4. Conclusion

In summary, we have successfully demonstrated a sensitive printable H₂O₂ sensor based on ferrocene-grafted carbon nanotubes for enzyme-free amperometric H₂O₂ sensors prepared by a facile and cost-effective method. Owing to the high-density FeC redox centers on MWCNT, efficient electron transfer between FeC and MWCNT, and large electrical conductance of the MWCNT network, the sensor exhibits excellent electrocatalytic activity towards H₂O₂ reduction with a low detection limit of 0.49 μM and a wide linear range from 1 μM to 1 mM covering the interested H₂O₂ level in aseptically-packaged products. Moreover, the sensor can operate at a much lower electrode potential of −0.15 V vs. Ag/AgCl, further improving detection selectivity. Furthermore, the high repeatability of the sensor allowed for real-time, continuous monitoring of H₂O₂ concentration in analyte flows. Successful determination of H₂O₂ in spiked beverages also displayed satisfactory recoveries. The H₂O₂ sensor based on MWCNT-FeC modified SPCE offers an excellent balance between simplicity, cost, and performance, which appears as a good candidate for routine sensing of H₂O₂ at the point of use.