Synthesis of an ordered nanoporous Fe₂O₃/Au film for application in ascorbic acid detection

Abstract

A simple method based on sputtering and electrochemical deposition onto an anodic aluminum oxide template is presented to fabricate an ordered nanoporous Fe₂O₃/Au film. Investigation results indicated that the prepared porous film consisted of an ordered hexagonal array of nanoholes with a pore diameter of 50 nm and a periodic distance of 120 nm. After dissolving the template, the independent Fe₂O₃/Au film can be transferred onto an ITO substrate to be used as an effective non-enzymatic sensor for detection of ascorbic acid. It exhibited excellent electrocatalytic performance with a high sensitivity of 1281.9 μA mM⁻¹ cm⁻², a wide linear range of 25 μM to 10 mM, and a low detection limit of 1 μM. The satisfactory results obtained indicated that the proposed sensor was promising for the development of a novel strategy for AA detection.

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Introduction

Ascorbic acid (AA), better known as vitamin C, is a most important water soluble compound present in various fruits, vegetables, and soft drinks.^1,2 It is also a medication for scurvy, liver disease, allergic reactions and atherosclerosis, and helps promote healthy cell development, calcium absorption and normal tissue growth.^3,4 Therefore, the development of a simple and rapid method for the determination of AA is essential for diagnostic and food safety applications. Many analytical methods such as titrimetry,⁵ colorimetry,⁶ chemiluminescence,⁷ spectrophotometry⁸ and electrochemical methods⁹ have been developed for the determination of AA. Among them, electrochemical methods are considered to be one of the most suitable approaches because of their ease of monitoring, high sensitivity and simplicity.¹⁰ However, enzymatic sensors are unstable and exhibit poor reproducibility, due to the intrinsic nature of enzymes.^11,12 Therefore, the development of non-enzymatic AA sensors with a low detection limit and wide response range is desired.¹³

Fe₂O₃ is an important oxide material, which is eco-friendly, non-toxic, heat-resistant and corrosion-resistant, and widely used in the field of photocatalyst, solar cells, lithium ion battery, gas sensors and field emission devices.^14–16 In the last few years, researches on the growth and properties of Fe₂O₃ nanomaterials have increased steadily. And many kinds of Fe₂O₃ nanomaterials, such as nanoparticles, nanowires, and nanotubes have been reported.^17–19 Since the discovery of the size-dependent peroxidase-like activity of Fe₃O₄ nanoparticles, iron oxide based materials for the electrochemical sensors have been intensively studied.¹¹ To date, there have been many reports about Fe₂O₃-based glucose, H₂O₂ and dopamine sensors due to its large surface, high stability, and remarkable sensitivity.^20–23 Au nanostructures are also interest for the electrochemical detection of analytics in physiological fluids because of their stability, oxidation resistance, electronic conductivity and biocompatibility.^24–26 Nevertheless, these nanostructured iron oxides or gold were synthesized in powder form and required additional coating procedure for electrode preparation, which makes them easy to fall off partly from the electrode in the test procedure. So developing new morphology of electrode nanomaterial is therefore extremely important.

Ordered nanoporous film, with a large active surface area and an ordered arrangement of pores in nanoscale, has received considerable attention in recent years due to their unique characteristics.^27,28 To the best of our knowledge, there is no report based on the application of nanoporous Fe₂O₃ film for determination of ascorbic acid. In this paper, we reported a simple method based on anodic aluminum oxide (AAO) template to synthesize ordered nanoporous Fe₂O₃/Au film. After dissolving the template, the prepared porous film could keep its integrality and be transferred onto any substrate. If immobilizing the porous Fe₂O₃/Au film onto ITO glass, as an assembly, it can be used as an effective electrochemical sensor for detection of AA. Electrochemical experiments showed that the presented sensor possessed many excellent properties as expected.

Experimental section

Chemicals and reagents

Nafion solution, ascorbic acid, uric acid, and dopamine were obtained from Sigma-Aldrich Chemical Company. All other chemicals were of analytical grade and purchased from Chongqing Chuandong Chemical Company. Deionized water was used throughout. Electrochemical measurements were carried out on CHI 660E electrochemical workstation (CH Instruments, China) with a three-electrode system, a modified electrode as working electrode, a Ag/AgCl electrode as the reference electrode and a platinum electrode as the counter electrode. All experiments were performed at ambient temperature (20 ± 1 °C).

The crystalline structure of the synthesized samples were identified by using X-ray diffraction (XRD, Shimadzu 7000) with Cu Kα radiation (k = 1.5406 Å) operated at 30 kV. Morphologies and elemental composition were characterized by scanning electron microscopy (SEM, JSM-7100F) equipped with an energy dispersive X-ray spectroscope (EDS), and transmission electron microscopy (TEM, FEI Tecnai G20).

Synthesis of Fe₂O₃/Au film

The AAO templates were homemade by an anodization process as described in previous reports.^29,30 In this work, the anodization was carried out at 50 V for 6 h in 0.3 mol L⁻¹ oxalic acid electrolyte. As a result, the distance between two adjacent channels in the templates would be about 120 nm and the diameter of channels would be about 70 nm. Then, the scheme of the preparation for Fe₂O₃ nanoporous film was illustrated in Scheme 1. According to a series of experiments and demonstrations, the optimized synthesis condition is obtained. Specifically, a thin Au layer of about 30 nm in thickness was sputtered onto the top side of the template by an advanced sputter coater to serve as conductive electrode and substrate. Then porous Fe film was firstly electrodeposited onto the surface of Au layer using a two-electrode system with a carbon plate working as counter electrode. The direct current electrodeposition was carried out in an aqueous solution containing 160 g L⁻¹ FeSO₄, 60 g L⁻¹ (NH₄)₂SO₄, 3.5 g L⁻¹ ascorbic acid, 2 mL L⁻¹ glycerol and 14.3 g L⁻¹ orthoboric acid. The electrodeposition process lasted for 10 min at a constant current of 0.8 mA cm⁻². If Au layer or Fe film is too thick, the final product would lose its porous structure. The samples were then thermal annealed at 150 °C for 3 h in air to convert Fe into Fe₂O₃. It should be pointed out that the higher annealing temperatures would change the structural forms of ferric oxides and reduce the electrocatalytic performance of the final product. More work is underway to better understand the formation mechanism of these ferric oxides and their relations to electrochemical properties.

Scheme 1 Schematic illustration of the fabrication of nanoporous Fe₂O₃/Au film and the architecture of the AA sensor.

Fabrication of the electrode

The ordered nanoporous Fe₂O₃/Au film was separated from the template through floating on the surface of 2 mol L⁻¹ NaOH aqueous solution for few minutes to dissolve the AAO and then form a soft, free and continuous film. After losing the support of AAO template, the Fe₂O₃ film combined with Au layer could be transferred through floating-transfer method.³¹ More importantly, once the transferred film completely dried, there would form a strong binding force (van der Waals' force) between the film and the substrate – ITO glass, which can promise the feasibility of practical application of the film. Then, a quantity of epoxy resin was encapsulated on the ITO glass to prevent the shedding of porous film and avoid the contact between ITO and the test solution. As a result, the effective area of Fe₂O₃/Au film was about 2.4 mm². For comparison, Fe₂O₃/Au smooth film was deposited directly on a planar ITO glass and then encapsulated by epoxy resin to keep a similar effective area.

Results and discussion

Characterization of porous film

The overview SEM image at the surface of the product is illustrated in Fig. 1a. It shows that the as-prepared porous film is arranged in an ordered pores array retaining the geometrical ordering of the AAO template. The magnified image of Fig. 1b exhibits that the covered porous film is mountain-shaped, with ups and downs on the surface. The period distance of the adjacent pores is about 120 nm in agreement with that of the template. Every pore with an average diameter of about 50 nm surrounds by some humps just like a plum-blossom. It also should be noted that the porous film consists of fine grains, leading to a highly rough and loose surface topography, which increase its surface area greatly. More observations (inset of Fig. 1a) reveal that the vertical height of this rough film is about 100 nm.

Fig. 1 (a) Low-magnification SEM image of the nanoporous Fe₂O₃/Au film, (b) the high-magnification SEM image.

In order to make it easier for XRD characterization and analysis, AAO template was etched by 2 mol L⁻¹ NaOH solution. Then, the independent porous film was transferred onto (111) oriented single-crystal silicon wafer through the above floating-transfer method. XRD pattern of the porous film is shown in Fig. 2a. An obvious Au diffraction peak can be observed corresponding to the cubic Au phase (JCPD no. 65-8601), which should come from Au layer sputtered onto the template. The other main peaks found in Fig. 2a are all closely matched with α-Fe₂O₃ reflections (JCPDS no. 84-0311). It reveals that the synthesized Fe₂O₃ has rhombohedral structure. The XRD peaks of Fe₂O₃ film show broadening, indicating the ultrafine nature of the film. On the basis of Scherrer's equation, the average Fe₂O₃ crystalline size was estimated and the calculated particle size was about 12 nm. Two weak peaks existed in the XRD pattern correspond to NaOH coming from the residue in the etching process. The EDS analysis indicates that the product was made up of Au, Fe and O (Fig. 2b). Among them, the average atomic ratio of Fe : O is about 38.4 : 58.3 and consistent with stoichiometric Fe₂O₃ within experimental error. The carbon signature is from the carbon conductive tape coating on the SEM supports. We also conduct the EDS in the mode of area mapping to detect the spatial distribution of specific element. Fig. 3 indicates that the Au, Fe and O element are all uniformly distributed within the nanoporous film.

Fig. 2 XRD pattern (a) and EDS data (b) of the nanoporous Fe₂O₃/Au film.

Fig. 3 (a) A SEM image of the nanoporous Fe₂O₃/Au film, (b), (c) and (d) the elemental chemical maps of Au, Fe, and O respectively.

A typical TEM image of Fe₂O₃/Au porous film is depicted in Fig. 4a. It indicates that the sample shows the uniform pore size and long-distance ordered arrangement of hexagonal structure, which is typically ordered nanoporous structure. It can be found from the high magnification TEM image of Fig. 4b that Fe₂O₃/Au porous film is composed of many polycrystalline grains. These grains are randomly oriented with a dimension of ten to twenty nanometers. The corresponding SAED (selected area electron diffraction) pattern (Fig. 4c) indicates that Fe₂O₃ porous film appears to be polycrystalline. The diffraction rings in the pattern corresponding to the rhombohedral structure α-Fe₂O₃ are seen in agreement with the XRD result.

Fig. 4 (a) TEM image of the nanoporous Fe₂O₃/Au film, (b) high-magnification TEM image, (c) corresponding SAED pattern.

Properties of the sensor

After immobilizing the nanoporous Fe₂O₃/Au film onto ITO glass, as an assembly, it can be used as a non-enzymatic sensor for detection of AA. Fig. 5a shows the cyclic voltammograms (CVs) curves of Fe₂O₃ film in the absence and presence of AA in 10 mL 0.2 M NaOH solution at a scan rate of 100 mV s⁻¹. When AA is added (curve b and c), an increase of the oxidation peak is observed, compared to the system without AA. This effect shows that Fe₂O₃/Au film electrode has a strong electrocatalytic activity towards the oxidation of AA. The anodic and cathodic peak potentials positioned at 0.50 and 0.06 V can be assigned to the Fe(II)/Fe(III) redox couple.²⁰ The possible catalytic mechanism of Fe₂O₃ surface layer to AA oxidation can be explained by the following scheme:

2Fe(III) + C₆H₈O₆ + 2OH⁻ → 2Fe(II) + C₆H₆O₆ + 2H₂O (1)

2Fe(II) → 2Fe(III) + 2e⁻ (2)

Fig. 5 (a) CV curves of the nanoporous Fe₂O₃/Au film in the absence (curve a) and presence (curve b and c) of AA in 0.2 M NaOH solution. (b) Typical CVs of the modified electrode in 0.2 M NaOH solution containing 1.0 mM AA at different scan rates (from 20 to 240 mV s⁻¹). Inset: plots of peak currents vs. ν^1/2.

The reaction (1) provides more Fe(II) cations in oxidation cycle, resulting in a significant increase in the peak current. Thus, the proposed electrode has an electrocatalytical effect on AA determination. Additional studies on the kinetics of this reaction are in development. The effect of potential scan rate was also characterized in 0.2 M NaOH solution containing 1 mM AA and the results are shown in Fig. 5b. It can be seen that the reduction and oxidation peak currents are both proportional to the square root of scan rate in the range of 20–240 mV s⁻¹. This indicates that a diffusion-controlled process occurred at the modified electrode.²⁹

The amperometry, which is one of the most widely used techniques for evaluating sensors, was also used to study the properties of Fe₂O₃/Au nanoporous film electrode. Fig. 6a and b display the amperometric current responses for the modified electrode to successive additions of AA in a stirring NaOH solution at operating potential of 0.6 V. As the AA was injected, the current of electrode rapidly increased and achieved stable value in less than 10 s, displaying a sensitive response to the change of AA concentration. A wide linear response range of the electrode to AA concentration was from 25 μM to 10 mM and the linear regression equation was I (μA) = 1.927 + 30.765C (mM), with a correlation coefficient of 0.9992. The limit of detection was estimated to be 1 μM at a signal/noise ratio of 3. The sensitivity can be further determined to be as high as 1281.9 μA mM⁻¹ cm⁻². Table 1 demonstrates the comparison in the performances of the previously reported AA sensors fabricated based on the use of micro/nanomaterials to modify the ITO electrodes. It reveals that our sensor shows excellent performance in terms of wide linear range and low detection limit compared with rGO–CNT/ITO,³² PtAu hybrid/ITO³⁸ and etc.

Fig. 6 (a) Typical steady-state current–time response for the nanoporous Fe₂O₃/Au film electrode to successive additions of different concentrations of AA in a stirring 0.2 M NaOH solution at an applied potential of 0.6 V vs. Ag/AgCl, (b) the current–time responses for the nanoporous Fe₂O₃/Au film, the smooth Fe₂O₃/Au film, and the nanoporous Au film, to successive additions of 1 mM AA, inset: the linear relationship of the nanoporous Fe₂O₃/Au film between the catalytic current and the concentration.

Table 1 Comparison of the present Fe₂O₃/Au/ITO electrode with other ascorbic acid sensors

Type of electrode	Linear range (μM)	Detection limit (μM)	Reference
rGO–CNT/ITO	10–200	5.31	32
Silica mesochannels/ITO	49–2651	11	33
P2W12V2/Au–Pd/ITO	1.2–1610	0.67	34
Tm2O3 nanoparticles/ITO	200–8000	420	35
Cys/Au–Pt NPs/ITO	2–400	1	36
βCD–nanoAu/Fc–ITO	53–3000	4.1	37
PtAu hybrid/ITO	103–1650	103	38
Fe2O3/Au/ITO	25–10 000	1	This work

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These perfect characteristics of our modified electrode should be related to the specific structure of nanoporous Fe₂O₃/Au film. Firstly, Fe₂O₃ is an ideal sensor material due to its proper redox potentials. Fe₂O₃ is also demonstrated to show both reversible reduction and reversible oxidation of Fe(III). There are many sensitive sensors reported by using Fe₂O₃ materials to modify the electrodes, including AA sensor.^12,20–23 In contrast, it can be found in Fig. 6b that the nanoporous Au film without deposition of Fe₂O₃ in our experiment shows very low activity for AA oxidation. Secondly, the specific surface of nanoporous film produced here is a key factor. As shown in Fig. 6b, Fe₂O₃/Au smooth film deposited directly on ITO glass only exhibited a weak response to the addition of AA. According to this result, we speculate the nanoporous structure can provide a larger contact area between sensing materials and sensed species, thus leading to an excellent sensing performance. We have estimated the electroactive surface area and find that the nanoporous film was several times larger than the area of the equivalent smooth electrode by using ferricyanide as a redox probe. Thirdly, Au layer between Fe₂O₃ film and ITO can provide high electron communication features to enhance the electron transfer between the active sites of Fe₂O₃ and the underlying electrode. So the simple fabrication of nanoporous Fe₂O₃/Au film and its good electrocatalytic ability make it an excellent material for AA detection in alkaline medium.

The effect of potential interference on the nanoporous Fe₂O₃/Au film was examined. As shown in Fig. 7, when 0.5 mM AA was added into 10 mL 0.2 M NaOH solution, the current significantly increased with great response sensitivity. Comparison to AA, when 0.5 mM H₂O₂, glucose, uric acid (UA) and L-cysteine were added, no interference from these substances was observed. The normal physiological level of AA is general much higher (100–1000 times) than that of dopamine (DA).³⁹ Thus, the concentration of DA added was 0.05 mM and the result showed that such concentration of DA could not produce an obvious amperometric response. Thus, the selective determination of AA in the presence of DA is feasible at the developed electrode. The long-time stability of the AA sensor is an important parameter for the evaluation of its performance. Because the sensor fabricated in this study was enzyme-less, the long-term stability of the sensor was evaluated by measuring its sensitivity to AA with the sensor stored in dry conditions. After three weeks of storage, the current response of sensor retained about 94% of its original response, showing long-term stability. In order to study the applicability of the nanoporous Fe₂O₃/Au film, the content of AA in vitamin C tablets was analyzed. The sample of vitamin C tablets was purchased at a local drug-store and each tablet contained 100 mg of AA according to the label. In the analysis, the standard addition method was applied, by which a known amount of AA in water was added into the test solution. The recoveries for the determination of AA were in the range of 98.2–103.9% for three samples (Table 2), showing the potential application of the nanoporous film for the determination of AA.

Fig. 7 Amperometric curve of addition of 0.5 mM AA, 0.5 mM H₂O₂, glucose, uric acid, L-cysteine and 0.05 mM dopamine in 10 mL 0.2 M NaOH solution.

Table 2 Determination of AA in vitamin C tablets (n = 3)

Sample	Detected (μM)	Added (μM)	Found (μM)	Recovery (%)
1	205.8	400	611.0	101.3
2	401.7	400	817.3	103.9
3	585.3	400	978.1	98.2

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Conclusions

In this work, ordered nanoporous Fe₂O₃/Au film was synthesized by a simple method based on AAO template. After dissolving the template, the nanoporous Fe₂O₃/Au film can be transferred onto an ITO substrate to be used as an effective non-enzymatic sensor for detection of ascorbic acid. It was found that the resulted sensor exhibited a high electrocatalytic activity for the AA detection in alkaline media. A high sensitivity, a wide linear range, and a low detection limit were also obtained. We expect that the nanoporous Fe₂O₃/Au film will provide a promising platform for electrocatalysis and sensing of more biomolecules.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (11204246), the Natural Science Foundation Project of CQ CSTC (cstc2014jcyjA50027, cstc2016jcyjA0125) and the Fundamental Research Funds for the Central Universities (XDJK2014B022, XDJK2016C063).

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