Fabrication of Gold-Graphitic Carbon Nitride Composite Electrode for the Detection of Pimobendan

1. Introduction

Heart failure is an increasing public health problem, mainly in the elderly, and is one of the important sources of death and hospitalization in elderly patients. The cardiotonic drug pimobendan (C₁₉H₁₈N₄O₂) in Figure 1(a) is a calcium sensitizer and selective inhibitor of phosphodiesterase III, studies have shown that as a canine cardiotonic therapy can increase lifespan [1]. Pimobendan is a drug licensed for veterinary use indicated for the treatment of heart failure in dogs and it is also used in Japan in human patients [1]. Oral pimobendan is used for treating congestive heart failure (CHF) in dogs and cats [2]. Pimobendan is current in patients with chronic heart failure but is less effective in patients with old myocardial infarction (OMI) than in patients with dilated cardiomyopathy (DCM) or other heart diseases [3,4].

Currently used for clinical analysis of pimobendan is by high performance liquid chromatography (HPLC) [5] and Liquid Chromatography Tandem Mass Spectrometry (LC/MS) [6]. It is time-consuming (more than a few minutes), expensive equipment, and the coexistence of many complex sample preparations that interfere with the actual sample, that is not suitable for tests that require a large number of samples in a short analysis time.

To our knowledge, the fast detections of pimobendan by electrochemical method have not been studied. Therefore, it is hoped to develop a cardiotonic drug measurement with high sensitivity, high selectivity and high long-term stability by cyclic voltammetry and differential pulse voltammetry in electrochemical systems. This study is expected to gain a deeper understanding of the mechanisms of adsorption and surface redox reactions during cardiotonic drug sensing. Electrochemical detection technique has been considered as an outstanding candidate for the determination of pimobendan n owing to its advantages such as excellent sensitivity, high selectivity, fast response, simple operation and cost-effective.

Graphite carbonitride (g-C₃N₄) is a planar two-dimensional sheet structure similar to graphene, which is a typical polymer semiconductor. Due to its special structure and excellent properties, g-C₃N₄ has become a popular material for research of some electrochemical reactions [7,8,9]. Since g-C₃N₄ has attracted consideration in various fields due to its excellent optical properties, high thermal stability in basic/acidic media, earth abundance and environmentally friendly nature [10]. However low conductivity of g-C₃N₄ limits its application in electrochemical electrode, to overcome this deficiency to dope the metal is a way to improve its conductivity [11].

Among nanomaterials, gold nanoparticles is functional material that has received increased attention [12]. Gold has been used in promotion of some oxidation reactions and electrochemical reaction system [12]. Until now, the Au-based nanomaterials have been observed as dynamic nanostructured materials and expected to be important element for electrode device [12]. Therefore, the main objective of the present work was to establish a simplified cyclic voltammetry method for determination of pimobendan using electrochemical sensors based on Au/g-C₃N₄/glassy carbon electrode. The morphology, crystal phase, chemical component and microstructure of the as-prepared electrochemical sensors were characterized using various techniques like XRD (X-ray diffraction) and TEM (transmission electron microscopy). Moreover, the electrochemical behavior of the as-synthesized electrochemical sensors was investigated by cyclic voltammetry.

2. Experimental section

2.1. Chemicals and reagent

Melamine (99%), Methyl alcohol (99%) and tetrachloroauric acid trihydrate (99%) were purchased from Alfa Aesar Co., Ltd. Chitosan (>75%), Sodium phosphate dibasic (99%), Potassium phosphate monobasic (99%), Potassium chloride (99%) and Sodium chloride (99%) were obtained from SIGMA ALDRICH Company. The reagents used in the experiment were analytically pure without further purification. The laboratory-grade distilled and deionized water (Millpore, Milli-Q Water Purification System) was used throughout the experiment.

2.2. Electrochemical sensors preparation

A facile method for the fabrication of electrochemical sensors and the construction process is as behind. Take melamine for gradient calcining to obtain g-C₃N₄ of light yellow powder. Then take g-C₃N₄, add appropriate amount of tetrachloroauric acid (HAuCl₄) solution and methanol (Methyl alcohol), heat and stir until powdery. Then the powers were dried, and carried out gradient calcination to obtain a light gray powder of various percentages of Au/g-C₃N₄ products. To shake and mix Au/g-C₃N₄ and chitosan solution evenly, and then was coating on the surface of the glassy carbon electrode. After drying to form a film, the preparation of the working electrode was completed.

2.3. Characterization apparatus

The crystal structures of the preparation samples were characterized by X-ray diffraction (XRD) using a Bruker D8 focus diffractometer operating at 35 kV and 35 mA with a scan step of 2º/min between 10º and 80º (2θ), using Cu Kα radiation (Cu Kα1, 1.5404Å). The microstructure and morphology of the samples were performed using JEM-2010 (JEOL, Japan, 200 kV) transmission electron microscope and scanning electron microscope FESEM/ Energy-dispersive X-ray spectroscopy (EDS) (JEOL JSM-7500F, JSM-6500F, Japan, 15 kV). Fourier transform infrared (FTIR) spectroscopy was by using an Agilent Cary 630 ATR-FTIR spectrophotometer (USA).

2.4. Electrode preparation and electrochemical measurements

Prior to use, the surface of the bare electrode (glasses carbon electrode, GCE) was successively polished with aluminum oxide powder on a polishing cloth to a mirror finish and rinsed with water. As shown in Figure 1(b), the electrochemical sensing system includes three electrodes and an electrochemical workstation. Pt and Ag/AgCl were used as the counter and reference electrodes, respectively. G-C₃N₄ and Au/g-C₃N₄ electrodes were used as the working electrode. All electrochemical measurements were performed out on a ZIVE SP1 compact type electrochemical workstation (Won A Tech/ZIVE LAB). The scanning range was from -1.0 to 1.0 V at a rate from 10 to 200 mVs^-1 by using cyclic voltammetry (CV) method. The detection limit (DL) is measured as DL = (3×SD)/m, where SD is the standard deviation of the blank sample signal and the m is revealed the slope of the response curve versus pimobendan concentration (range from 0.0 to 55 μM).

3. Results and Discussion

3.1. Characterized the sensing materials

To investigate the structure of g-C₃N₄ and Au/g-C₃N₄, the XRD patterns of g-C₃N₄ and Au/g-C₃N₄ with different Au contents (1%, 3% and 5%) were studied. As can be seen in Figure 2, g-C₃N₄ XRD has clear peaks (100) and (002) at 2θ = 13.1° and 27.3°, respectively. These represent the accumulation of conjugated π-π bonds in the aromatics of graphite carbonitrides [7]. Furthermore in Figure 2, the characteristic peaks at 37.7°, 44.0°, 64.3°, and 77.4° correspond to the crystalline planes of Au(111), Au(200), Au(220) and Au(311), respectively [8]. From this, it can be seen that the characteristic peak of Au (111) increases with the increase of the proportion of Au doping, so it is known that Au has been successfully doped onto the g-C₃N₄ flakes. By using Scherer’s formula, the calculated average crystalline sizes of Au (111) of 3% Au/C₃N₄ was found to be approximately 18.6 nm.

Figure 3(a) presents a g-C₃N₄ TEM with a magnification and scale bar 1000 nm, and (b) shows a g-C₃N₄ TEM with a magnification and the scale bar 500 nm. It can be seen from the figure that g-C₃N₄ presents a multilayer sheet structure. Figure 4(a) is a g-C₃N₄ TEM with a magnification and scale bar 500 nm, and (b) is a g-C₃N₄ TEM with a magnification and scale bar 10 nm. We can see from Figure 4(b) where Au is doped on the surface of flake g-C₃N₄. The lattice line is used to calculate the lattice spacing of Au and compared with the literature. It is found that the Au (111) crystal plane has a lattice spacing of d = 0.225 nm [9]. It can be seen that the spherical Au is successfully doped on the flake g-C₃N₄, the average crystalline size of Au (111) is calculated as 19.2 nm. It reveals similar result of XRD spectra in Figure 2.

The microstructure and distribution of elements on samples can be characterized by SEM and EDS instruments. Figure 5a showed a g-C₃N₄ SEM image with a scale bar of 50 μm, and 5b is the g-C₃N₄ SEM image of a scale bar of 20 μm. The surface of g-C₃N₄ exhibited an aggregation from unregular like smaller particles structure in Figure 5a and b. To study the element distribution and composition of as-prepared samples, energy-dispersive X-ray spectroscopy (EDS) was employed. It is revealed a g-C₃N₄ EDS and element mapping images in Figure 6. Figure 6 presents the carbon and nitrogen elements are coexisted in the g-C₃N₄, and the two elements are homogeneously distributed on the surface of the g-C₃N₄. Figure 7a is a 3% Au/g-C₃N₄ SEM image with a scale bar of 50 μm, and 7b is a 3% Au/g-C₃N₄ SEM image with scale bar 20 μm. The 3% Au/g-C₃N₄ showed an aggregation structure from smaller particles in Figure 7a and 7b. Figure 8 shows 3% Au/g-C₃N₄ EDS and mapping images. Figure 8 reveals the carbon, nitrogen and gold elements are coexisted on the 3% Au/g-C₃N₄, and the three elements are homogeneously distributed on the surface of the 3% Au/g-C₃N₄.

Figure 9 revealed the FTIR spectra of g-C₃N₄, 1% Au/g-C₃N₄, 3% Au/g-C₃N₄ and 5% Au/g-C₃N₄. Where the broadened peaks between 3000 and 3500 cm⁻¹ (highest at 3092 cm⁻¹) were correlated to the stretching vibration [13] of remaining free N-H in the bridging C-NH-C units and O-H initiated from physically adsorbed water species on g-C₃N₄ surface, respectively. As shown, the main characteristic group peaks observed in the region from 900 to 1700 cm−1 were usually assigned to stretching vibration signals of aromatic heptazine-derived repeating units, including the typical sp² C=N stretching modes at 1627 cm^-1 and out-of-plane bending vibrations of the sp³ C-N bonds at 1536, 1393, 1311 and 1229 cm^-1[14]. The sharp absorption peak located at about 800 cm⁻¹ was attributed to the characteristic ring mode of tri-s-triazine cycles [15], and the absorption band at 881 cm⁻¹ was assigned as the deformation mode of N-H in amino groups [13,14,15], respectively. In Figure 9, no obvious peak was observed by addition of gold on g-C₃N₄.

3.2. Sensing properties

3.2.1. Scan rate effect

To conclude whether the redox process is controlled by a diffusion or adsorption, this study considered the relationship between oxidation current and scan rate. If it appears a linear relationship of peak current with sweep rate, then it usually means an adsorbed pimobendan molecule is controlled [16]. At pH =7.0 the CV method was used of scan rates ranging from 10 to 200 mVs^-1 on oxidation current intensity at a pimobendan concentration at 55 μM. In Figure 10A, the oxidative current peaks were located at around -0.40 V and 0.40 V and reduction current was found near 0.05 V of 3 %Au/g–C₃N₄ increased with the various scan rates. To determine the relationship between pimobendan current and scan rate, plots of the scan rate versus current intensity in Figure 10B. The scan rate was presented a linear relationship with the anodic peak current (Ipa) at 0.4 V and cathodic peak current (Ipc) at 0.05 V, and it showed the R² were obtained as 0.9965 and 0.9968, respectively. In Figure 10C the square root of scan rate exhibited a linear relationship with the anodic peak current (Ipa) and cathodic peak current (Ipc), it presented R² = 0.9823 and 0.9822, respectively. Above results showed the redox reaction of pimobendan on the modified electrode was an adsorption-controlled process, indicating that pimobendan was directly adsorption on the electrode surface [16]. We choose the condition of scan rate at 50 mVs^-1 for it is easily for the peaks separations in Figure 10A.

3.2.2. Various kind materials of sensing properties

Figure 11A presents CV plots which indicating the current signals of the GCE, g-C₃N₄ electrode and 3%Au/g–C₃N₄ electrode at a scan rate of 50 mV/s and pimobendan concentration of 55 μM at pH=7.0. No significant CV current signal was observed on the GCE, a reduction peak was observed at 0.0 V on g–C₃N₄ electrode in Figure 11A. The 3%Au/g-C₃N₄ composite had the highest oxidation peak current at approximately 0.4 V and reduction peak at 0.05 V.

In Figure 11B, the oxidation peaks were placed at around -0.40 V and 0.40 V which were identified as the oxidative products from pimobendan in Figure 11B (a) and (b) [17]. And reduction peak was found at 0.10 V which was identified as the pimobendan from the oxidative products [17].

3.2.3. CV current versus concentration

Figure 12A revealed the CV currents versus pimobendan concentrations (ranging from 0.0 to 55 μM). It showed the 3 %Au/g-C₃N₄ at higher concentrations (Figure 12A) the reduction peak current (cathodic peak current) exhibited a higher current value. From low concentration of pimobendan (0.0–0.84 μM) indicating a linear relationship between the cathodic peak current and the pimobendan concentrations in Fig. 12B. It was obtained the R² as 0.9642, and the detection limit was calculated to 0.28 μM.

3.2.4. Interference effect

From the literature, the interferences of 55 μM dopamine (DA), 55 μM epinephrine (EP) and 55 μM uric acid (UA) are naturally coexist in physiological samples. Accordingly, the interference tests by plotting the CV reduction current versus some interferences materials of DA, EP and UA was displayed in Figure 13. The CV current of pimobendan (4.7 µA) was higher than those of DA (0.38 µA), EP (0.51 µA) and UA (0.49 µA) at the same concentration in Figure 13. It presented the sensing material of pimobendan detection was high selectivity and little interferences with DA, EP and UA.

3.3. Sensing mechanism

The 3 %Au/g-C₃N₄ composite had the highest oxidation peak current at approximately 0.4 V and reduction peak at 0.05 V in Figure 11A. The redox reaction mechanisms of the pimobendan sensing under the amide oxidation and amide oxide reduction reactions on the Au/g-C₃N₄ electrode are illustrated in Figure 14 [17,18,19,20,21]. The C₃N₄ showed a flake-like morphology of TEM images in Figure 3 and Figure 4. Graphitic carbon nitride (g-C₃N₄) is a two-dimensional organic semiconductor with a high specific surface area, additionally g-C₃N₄ owns high electrical conductivity for its sole delocalized conjugated structure [22,23,24,25,26]. Au is applied in promotion of some oxidation-reduction reactions and electrochemical reaction system [12,16,21]. The binding of pimobendan molecules on nano Au increased the electron transfer, yielding the modification in response current. The presence of Au, therefore, enhanced the detection of oxidation and reduction current sensitivity [26,27,28,29,30]. The high sensing circuit of the electrode was feasibly (Figure 11) because the Au nanoparticles on the surfaces of C₃N₄ efficiently collected the pimobendan oxidation–induced electrons during the electrocatalytic reaction and transferred them to the C₃N₄ composite and then to the surface of the GCE [18]. Two possible products of pimobendan (amide) oxidation on the Au catalyst are presented in Figure 14 [17,19,20].

4. Conclusions

The composite 3% Au/g-C₃N₄ sensing electrode was fabricated for detection pimobendan. The Sensing materials characterization by XRD and TEM. The cyclic voltammetry was applied to detect the concentration and redox properties of pimobendan. It can be seen that when the voltage value is 0.05 V, and a reduction peak was presented and the pimobendan concentration from 0 to 55 μM has a relationship with the reductive currents. At low concentrations of pimobendan concentration from 0.0 to 0.8 μM, the linearity R² = 0.9642 with a detection limit of 0.28 μM. Little interferences of DA, EP and UA were detected with pimobendan. A possible pimobendan sensing mechanism on 3% Au/g-C₃N₄ was proposed.