Detection of the neurotransmitter dopamine by a glassy carbon electrode modified with self-assembled perovskite LaFeO₃ microspheres made up of nanospheres

Abstract

In this paper we report the detection of the neurotransmitter dopamine by an LaFeO₃ microsphere-modified electrode in which the microspheres are made up of nanospheres. The morphology, structure and composition of the prepared nanostructure were characterized using SEM, TEM, XRD and XPS, and the electrocatalytic properties were investigated using cyclic voltammetry and amperometric studies. The modified electrodes markedly increased the efficiency of the electrocatalytic oxidation of dopamine. The biosensor exhibited high sensitivity at a low detection limit of 59 nM and wide linear range from 2 × 10⁻⁸ to 1.6 × 10⁻⁶ M (R = 0.9983). More importantly, the sensor effectively avoids the interference of ascorbic acid and uric acid. A possible electrocatalytic mechanism has been proposed. The LaFeO₃ microspheres are highly promising for the detection of dopamine because of their high selectivity, fast response and good sensitivity.

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1. Introduction

Electroanalytical methods have been used during the past decade to investigate the role of neurotransmitters in the brain due to their electro-active nature.¹ Dopamine (DA), ascorbic acid (AA) and uric acid (UA) are critically important compounds not only in the field of diagnostic and pathological research, but also for biomedical chemistry and neurochemistry. Dopamine, in particular, is an important catecholamine neurotransmitter in the mammalian central nervous system, and its depletion in neurons results in diseases such as Parkinson's. Dopamine is found in high amounts (50 nmol g⁻¹) in the caudate nucleus (a region of the brain). In the extracellular fluid of the caudate nucleus, however, dopamine occurs in low concentrations for a healthy individual, and in even lower concentrations or is completely depleted for persons affected with Parkinson's disease.^2,3 Selective and sensitive detection of dopamine has been a long-standing goal, and is most advantageously accomplished using electrochemistry. A major problem in dopamine detection, however, is the interference of AA and UA, which are present in biological fluids at much higher concentrations than in dopamine.⁴ Moreover, the electrode surface can be easily fouled by the products of ascorbic acid and uric acid oxidation, which result in poor selectivity and sensitivity in the detection of dopamine, as reported in our previous publication.⁵

Controlling the morphology of a nanomaterial is crucial for modifying its properties. Over the past few years, tremendous effort has been expended to control the size and shape of perovskite materials and a variety of morphologies have been reported for the perovskite LaFeO₃.^6–8 Nanostructures of LaFeO₃ have attracted much attention due to their low band gap energy (∼2.1 eV) as well as good catalytic, optical and magnetic properties, which are useful for applications in visible-light photocatalysis, gas sensing, magnetic data storage, photovoltaic cells, solid oxide fuel cells, and most recently as a biosensor.^9,10 LaFeO₃ nanostructures have been synthesized using a variety of wet chemical techniques such as hydrothermal, sol–gel, co-precipitation, combustion, and sonochemical procedures.^11–13 Reflux condensation, however, is a dominant as well as facile tool for the synthesis of anisotropic nanoscale material. A significant advantage of this method over other wet chemical techniques is the possibility to control the size of the material to achieve different morphologies at low temperature. Moreover, this process is relatively simple and cost effective. Our research group is interested in exploring the underlying connection of soft templates such as citric acid, CTAB and urea with different morphologies using wet chemical processes.¹⁴

In the present work, we report for the first time the facile synthesis of LaFeO₃ microspheres made up of nanospheres via a one-step wet chemical route, as well as the characterization of these microspheres. Furthermore, the fabrication of a modified glassy carbon electrode with LaFeO₃ microspheres and its ability to detect dopamine are also reported.

2. Experimental section

2.1 Preparation of LaFeO₃ microspheres made up of nanospheres

LaFeO₃ microspheres were prepared using analytical-grade lanthanum nitrate hexahydrate (La(NO₃)₃·6H₂O) and potassium ferric cyanide K₃[Fe(CN)₆] as starting materials and polyethylene glycol (PEG, MW 200) as surfactant. In a typical synthesis, starting materials were dissolved in 30 mL double distilled water under magnetic stirring followed by addition of PEG. The molar amount of PEG surfactant added was equal to the total molar amount of metal nitrate. The solution was refluxed with continuous stirring at 90 °C for 12 h in a three-necked refluxing pot. After the reaction mixture was allowed to cool down to room temperature, the green-coloured precipitate obtained was washed repeatedly with ethanol and distilled water to remove unwanted ions, followed by drying at 80 °C and calcination at 800 °C for 2 h to obtain pure LaFeO₃ samples.

2.2 Characterization and property measurements

The structure and purity of the prepared nanostructures were determined using an XRD – Bruker D8 Advance X-ray diffractometer with Cu-Kα radiation (λ = 0.15418 nm). The thermal analysis was determined using thermogravimetric/differential thermal analysis (TG/DTA) (carried out on an SDT Q 600 V20) from RT to 1000 °C under N₂ atmosphere. The X-ray photoelectron spectroscopy (XPS) measurement was performed using an ESCA + Omicron UK XPS system containing a Mg Kα source with a photon energy of 1486.6 eV. Scanning electron microscopy (using a JEOL JSM-6380LV microscope) and transmission electron microscopy (using a JEM-2100F microscope), performed with an acceleration voltage of 200 kV by placing the powder on a copper grid, were utilized to observe the morphology and size of the prepared samples. N₂ adsorption–desorption was determined by Brunauer–Emmett–Teller (BET) measurements using Micromeritics ASAP 2020 nitrogen adsorption apparatus and the pore size distribution was determined by the Barrett–Joyner–Halenda (BJH) method.

2.3 Electrochemical measurement and fabrication of a modified glassy carbon electrode

Electrochemical measurements were performed (using an EG & G Instrument model 6310 work station) in a conventional two-compartment three-electrode cell with a mirror-polished 0.07 cm⁻² glassy carbon (GC) as the working electrode, Pt wire as the counter electrode and 3 M KCl Ag/AgCl as the reference electrode. All the measurements were carried out in phosphate buffer solution (pH = 7.2) under N₂ atmosphere at RT. The GC electrode was polished to a mirror-like surface with 0.05 μM alumina powder and sonicated in double distilled water for 10 min. 100 mg of LaFeO₃ nanospheres was dispersed in 10 mL of anhydrous alcohol and ultrasonicated for 30 min. 10 μL of this alcohol-dispersed LaFeO₃ was dropped onto the GC surface and air dried at ambient temperature.

3. Results and discussion

3.1 Morphological analysis of LaFeO₃ microspheres

According to the SEM images (Fig. 1(a)), the synthesized LaFeO₃ samples exhibit microsphere-like morphology with size between 0.5 and 1.5 μm. No other morphology was observed, indicating high yield of microspheres. The higher magnification image in Fig. 1(b) clearly reveals that individual LaFeO₃ microspheres are made up of numerous nanospheres with a uniform size of ∼60 nm (Fig. 1(c)). This value is in close agreement with the crystalline size determined from XRD discussed in a later section. The TEM image (Fig. 1(d)) of a particular area of an LaFeO₃ microsphere shows several anisotropically directed nanospheres self-assembled to form microstructures due to local supersaturation.¹⁵ Furthermore, these microspheres are stable, and do not break into scattered individual nanospheres even at high temperatures for long periods of time, which confirms that these nanosphere units are tightly connected with each other to form the entire LaFeO₃ microsphere.

Fig. 1 SEM image (a) low magnification, (b and c) high magnification (inset – schematic illustration of microspheres), (d) TEM image, (e) HRTEM and (f) SAED pattern of a microsphere made up of LaFeO₃ nanospheres.

A high-resolution transmission electron microscopy (HRTEM) image of an LaFeO₃ nanosphere is shown in Fig. 1(e). The orderly and clear lattice fringes parallel to each other show that the microsphere building blocks are well crystallized, and the interplanar distance between adjacent lattice planes is 0.27 nm, corresponding to the d-spacing of the (121) LaFeO₃ plane. In the corresponding selected-area electron diffraction (SAED) pattern (Fig. 1(f)), the major diffraction spots corresponding to (121), (220) and (202) indicate the high crystalline nature. No diffraction spots were attributed to a secondary phase or impurity. The observed results obtained by comparing the TEM and HRTEM images are thus in good agreement with the results of SEM and XRD.

3.2 Formation of LaFeO₃ microspheres

When the surfactant PEG was added to the precursor solution containing (La(NO₃)₃·6H₂O) and K₃[Fe(CN)₆], the metal ions could be easily absorbed on the surface of the non-ionic PEG surfactant because of strong interaction between activated oxygen in the PEG molecular chains and the metal ions. Because of the long-chain structure and flexibility of PEG, the La3d and Fe2p–PEG complex forms a network structure of polymers, and due to hydrogen-bonding effect the complex forms spherical aggregates in water, which act as nucleation centers for the formation of LaFeO₃ nanospheres.¹⁶ These nanospheres nucleate and mineralize on the surface of LaFeO₃ aggregates, forming the microspheres.

3.3 Structural, thermal, composition and surface area analysis of LaFeO₃ microspheres

The crystal phase of LaFeO₃ microspheres was investigated by XRD. As shown in Fig. 2(a), it is clearly evident that all the diffraction peaks are consistent with the standard data for bulk LaFeO₃ crystals (JCPDS 37-1493) which has an orthorhombic perovskite structure with lattice constants a = 5.658, b = 7.855 and c = 5.689 Å. No characteristic peaks arising from reactants, impurities, La₂O₃/Fe₂O₃ or other phases were detected. The strong and narrow diffraction peaks observed indicate high crystallinity of the LaFeO₃ samples. The average crystallite size was found to be 65 nm using Scherrer's formula.¹⁷

Fig. 2 (a) XRD pattern, (b) TG/DTA curves, (c) XPS survey spectrum (inset – high resolution spectra of Fe and O) and (d) N₂ adsorption–desorption isotherm of microsphere composed of LaFeO₃ nanospheres.

Fig. 2(b) shows the TG/DTA data obtained for LaFeO₃ microspheres conducted at a heating rate of 20 °C min⁻¹. The 1.9% weight loss accompanying the TGA process observed between RT and 380 °C is attributed to the evaporation of absorbed water, also evident from the exothermic peak at 370 °C in the DTA curve. A weight loss of about 2% occurs from 380 to 510 °C and the corresponding DTA peak appears at 500 °C which can be assigned to the decomposition of nitrates and other organic impurities.¹⁸ A weight loss of 1.8% between 510 and 760 °C is due to the complete decomposition of oxycarbonates and the corresponding DTA peak at 590 °C may be attributed to the gradual crystallization of LaFeO₃. At higher temperatures, no obvious weight loss is observed indicating there is no additional phase or structural change in LaFeO₃. Therefore, in order to obtain the LaFeO₃ samples with high purity, 800 °C was chosen as the calcination temperature.

The elemental makeup and the oxidation state of LaFeO₃ microspheres were studied using an XPS survey spectrum (Fig. 2(c)). No peaks other than La(3d), Fe(2p), O(1s) and C(1s) were observed, which indicates that the synthesized LaFeO₃ microspheres are of high purity. All the peaks were calibrated using C1s (284.6 eV) as the reference. La peaks were observed at 845.8 eV which corresponds to spin–orbit splitting of 3d_5/2 and 3d_3/2 of La³⁺ ions in the oxide form. The peaks at 719.8 eV correspond to 2p_3/2 and Fe 2p_1/2, which is consistent with Fe³⁺ ions in the oxide form.¹⁹ The binding energy at 525.8 eV of the O(1s) XPS signal is due to the contribution of La–O and Fe–O in the LaFeO₃ crystal lattice. From the relative intensities of the XPS spectra, the atomic ratio was calculated as 1 : 1 : 3 between La, Fe and O.

A N₂ adsorption–desorption isotherm (Fig. 2(d)) of LaFeO₃ microspheres exhibit a type-IV isotherm with a hysteretic loop in the range 0.6–1.0 P/P_o, indicating the presence of mesoporosity. The BJH pore diameter distribution (inset in Fig. 2(d)) shows a pronounced peak, confirming a high degree of uniformity of the pores. The specific BET surface area is 95.80 m² g⁻¹ and the total pore volume is 0.105 cm³ g⁻¹. The generated mesoporosity in the material is due to the inter-nanosphere space. The large surface area and pore volume indicate that the LaFeO₃ microspheres would possess a fascinating ability to adsorb analytes for biosensing.

3.4 Electrocatalytic properties of an electrode modified with LaFeO₃ microspheres

Recently, research in the development of perovskite oxide nanostructures has emphasized its application in biosensing. The present study confirms that the LaFeO₃ microsphere-modified GC electrode can sense dopamine (DA), ascorbic acid (AA) and uric acid (UA). The electrocatalytic mechanism of the electrode modified with LaFeO₃ microspheres (Fig. 3(a)) for dopamine biosensing involves the electrochemical oxidation of Fe(III), producing an Fe(IV) complex on the surface of electrode followed by the electron transfer of dopamine and consequent regeneration of Fe(III) in the complex. The oxidation of dopamine to dopaminequinone by liberating two hydrogens can be catalyzed by the Fe(IV)/(III) redox couple in alkaline medium, which is also confirmed from the oxidation and reduction peaks in Fig. 3(b). The modified electrode exhibited high electrocatalytic activity towards dopamine oxidation, which improves the reversibility and enhances the electron transfer kinetics. The incorporation of the Fe(IV) complex in the LaFeO₃ microspheres helped to improve the dopamine electrocatalytic activity.

Fig. 3 (a) Schematic illustration of the electrocatalytic mechanism for dopamine oxidation at the LaFeO₃ microsphere-modified GCE. (b) Cyclic voltammetry (CV) plot recorded for a low concentration of DA and high concentrations of AA and UA (inset – current versus concentration for UA and AA) and (c) for different concentrations of DA (1 μM to 10 μM). (d) Amperometric i–t curve for the determination of DA concentration by the LaFeO₃ microsphere-modified electrode (inset – current versus concentration for DA).

Fig. 3(b)(1) shows that modified GCE has no redox peaks for DA, AA and UA, and the oxidation peak potentials are close to each other. The cathodic peak at −60 mV and anodic peak at 140 mV appear for 2 μM DA (Fig. 3(b)(2)), leading to a peak potential separation (ΔE) of about 200 mV for modified GCE. Oxidation of dopamine to dopaminequinone results in the oxidation peak and the reverse reaction leads to the appearance of the reduction peak.

In the cases of 50 μM AA and 100 μM UA, broader oxidation peaks at 188 mV (AA, Fig. 3(b)(3)) and 235 mV (UA, Fig. 3(b)(4)) were observed. The low separation between the oxidation peaks at 48, 47 and 95 mV observed for DA–AA, UA–AA and UA–DA are considered to be insufficient for simultaneous detection of these species. Note that the concentrations of AA and UA are found to be respectively 25 and 50 times higher than the concentration of DA. Finally, the negative surfaces of LaFeO₃ microspheres attract the DA cation and simultaneously repel AA and UA anions, which is clearly evident from the redox peak at −50 mV. Therefore, the LaFeO₃ microspheres exhibit strong electrocatalytic activity in response to dopamine.

The dopamine peak current was found to increase with dopamine concentration in the range 1.5 × 10⁻⁷ to 5.6 × 10⁻⁶ M as shown by the linear plot with a correlation coefficient of 0.9979 in Fig. 3(d) (inset), and the sensitivity analysis was studied using amperometry.²⁰ The current response for the addition of each 100 nM is presented in Fig. 3(d). The steady state current response was attained within 5 s with a sample interval of 180 s. The dependence of response current on the concentration of DA was linear with a correlation coefficient of 0.9983 as shown in the inset of Fig. 3(c). In the present report, the low detection limit of 59 nM at an S/N = 3 for dopamine concentrations in the range 2 × 10⁻⁸ to 1.6 × 10⁻⁶ M was obtained for the LaFeO₃-modified electrode, confirming high selectivity and good sensitivity towards DA. The fabricated LaFeO₃-modified electrode has been compared with other reported modified electrodes to examine its superiority and the results are shown in Table 1.

Table 1 Comparison of LaFeO₃ dopamine sensor with other reported sensors

Electrode	pH	Linear concentration	Detection limit (M)	Ref.
MCPE	4.0	2.6 × 10^−4–1.2 × 10^−3	2.5 × 10^−5	21
Ru-red/NaY/CPE	4.8	1.2 × 10^−4–1.0 × 10^−2	8.5 × 10^−5	22
Ionic liquid carbon	6.8	2.6 × 10^−6–1.5 × 10^−3	—	23
GNP–MEA–NIHCF	7.0	8.2 × 10^−7–2.5 × 10^−3	53 × 10^−8	24
WO3·H2O–GCE	7.2	1.0 × 10^−7–1.0 × 10^−6	12 × 10^−8	25
PEDOT–SWNT	7.0	2.0 × 10^−5–1.0 × 10^−1	10 × 10^−8	26
LaFeO3	7.2	2 × 10^−8–1.6 × 10^−6	59 × 10^−9	This work

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The reproducibility of the LaFeO₃ microsphere-modified electrode was evaluated from a concentration of 0–10 μM in the linear range of 3.6 × 10⁻⁷ to 4.3 × 10⁻⁶ M by CV measurements. The relative standard deviation of the LaFeO₃ biosensor at 1 μM response for 5 successive measurements was 2.7%, indicating good reproducibility. The stability of the biosensor was studied by comparing the CV peak current at an interval of 4 h. The decrease in the cathodic peak current was less than 3.4%, indicating good stability. Moreover, the biosensor was able to retain 97.6% of the initial response after one week, suggesting good long-term stability.

4. Conclusion

In summary, we report for the first time the use of reflux condensation to form LaFeO₃ microspheres consisting of nanospheres. A GCE modified with these microspheres showed excellent electrocatalytic properties with high selectivity and good sensitivity for DA detection with a wide linear range and without interference of AA and UA despite their concentrations being orders of magnitude higher than that of DA. Therefore, the results highlight the promising use of LaFeO₃ microspheres in the construction of new dopamine biosensors.

Acknowledgements

One of the author S.T gratefully acknowledges Jawaharlal Nehru Memorial Fund for Doctoral studies (Ref: SU-A/270/2011-2012/388 dated 09-12-2010) and also Brazilian research financing institutions: CAPES, FAPESP/CEPID 2013/19049-0, INCTMN/CNPq and FAPESP for financial aid.

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