Aliyu Muhammada,
Reza Hajian*b,
Nor Azah Yusof*ac,
Nafiseh Shamsb,
Jaafar Abdullahac,
Pei Meng Woid and
Hamid Garmestanib
aDepartment of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia. E-mail: azahy@upm.edu.my
bSchool of Materials Science and Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA. E-mail: reza.hajian@mse.gatech.edu
cInstitute of Advanced Technology, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysi
dDepartment of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
First published on 11th January 2018
Antibiotic residues in milk are of great concern for health regulatory agencies, milk consumers, and dairy farmers due to their destructive effects, ranging from allergic reactions, antibiotic resistance and the ability to interfere with the production of fermented products (i.e. cheese and yogurt). Therefore, a reliable, fast, and simple method needs to be developed to monitor antibiotic residues in milk samples before distribution to consumers. In this study, the first sensitive electrochemical sensor is presented for the determination of thiamphenicol (TAP), a broad-spectrum antibiotic in bovine milk. In the fabrication process, a screen printed electrode (SPE) was modified with gold nanoparticles (AuNPs) and carbon nanotubes (CNTs) using ethylenediamine (en) as a cross linker. Cyclic voltammetry studies showed an adsorptive control process for the electro-oxidation of TAP at −0.1 V on the modified electrode of SPE/CNT/en/AuNPs. Differential pulse voltammetry (DPV) was applied for the quantitative determination of TAP under optimized conditions (0.1 M citrate buffer, pH 6.0, accumulation potential −0.7 V, and accumulation time 150 s). A DPV study for TAP shows a wide linear calibration range of 0.1–30 μM with the detection limit of 0.003 μM. Furthermore, the developed sensor displays high sensitivity, reproducibility, repeatability, and good stability for the detection of TAP. The proposed sensor was successfully applied for the determination of spiked TAP in bovine milk with satisfactory results.
Several methods, including capillary electrophoresis,2 liquid chromatography coupled with mass spectrometery,4 and high performance liquid chromatography, have been reported for the determination of TAP residues in various samples.4 These techniques have some advantages including high accuracy, precession, and robustness, but they also have some limitations for routine analysis in terms of cost, analysis time, sample size, and tedious sample pretreatment.5 Alternatively, electrochemical methods are characterized by simplicity, fastness, cost-effectiveness, and portability for quantitative analysis.6
The extraordinary electrochemical features of carbon nanotubes (CNTs) in terms of excellent electron transfer and large surface area7 make them suitable for use in modification of electrochemical sensors.8,9 Commonly, gold nanoparticles (AuNPs) are being used as a kind of nanomaterial for fabrication of nanocomposites and enhancement of sensitivity in electrochemical sensors because of excellent catalytic and conductivity properties.10
A nanocomposite of AuNP/CNTs is of particular interest owing to its easy fabrication process and wide potential application, moreover, it combines the excellent physicochemical properties of AuNPs and CNTs,11 which have been reported in several studies for electrochemical analysis.12–14 In general, AuNPs are decorated on the surface of CNTs by either a direct or indirect deposition process. In the direct deposition process, AuNPs are directly coated on the surface of CNTs by reduction of chloroauric acid. In the indirect deposition process, a covalent linkage is formed between AuNPs and CNTs in nanocomposite production.15 Efforts have been made to produce a AuNP/CNT nanocomposite via different techniques.13,16,17 A nanocomposite of AuNP/MWCNT was reported to facilitate electron exchange reactions with free-diffusing redox species.18 However, synthesis of this nanohybrid has been limited in some cases by the aggregation of AuNPs, which blocked the electro-active surface area of the CNTs, thereby reducing the catalytic effect of the resulting material.
In this study, we fabricated a nanocomposite of AuNPs and CNTs with ethylenediamine (en) as a linker between carboxyl groups of CNT and AuCl4− for more distribution of gold ions prior to the fabrication process. The fabricated nanocomposite was used for modification of a screen printed electrode based on an electroless deposition process for voltammetric determination of thiamphenicol. To the best of our knowledge, this is the first report on the electrochemical determination of thiamphenicol in bovine milk samples using a nanocomposite (CNT/en/AuNP)-based electrochemical sensor.
Multi-walled carbon nanotubes (D × L 7–10 nm × 0.5–10 μm), ethylenediamine, and gold(III) chloride hydrate (99.5%) were obtained from Aldrich (USA). Thiamphenicol (98%) was purchased from Tokyo chemical industry (TCI, Japan). All other chemicals were of analytical grade and used as received.
Milk samples were supplied from two different farms in Malaysia (Putra Mart UPM as farm A, and Johor as farm B) and kept in a fridge before analysis.
Scheme 1 The chemical process for the synthesis of CNT/en/AuNP nanocomposite under reflux conditions. |
The proposed method for the fabrication of the electrochemical sensor was based on drop casting the synthesized nanocomposite on the surface of a working electrode (screen-printed electrode). For this purpose, 0.01 g of the synthesized nanocomposite (CNT/en/AuNP) was mixed with dimethylformamide (DMF, 10 mL) and dispersed in an ultrasonic bath for 30 min. Then, 5 μL of the suspension was drop casted carefully on the working electrode SPE and dried in air to evaporate the solvent.
Fig. 1 FESEM images of (a) CNTs and (b) CNT/en/AuNP and the respective EDS spectrum for the nanocomposite (c). |
The composition of the modified electrode was studied using EDS. The result (Fig. 1c) shows the EDS result of CNTs after their decoration with AuNPs (CNT/en/AuNP) and their respective peaks for carbon (C), oxygen (O), nitrogen (N), and gold (Au) due to the covalent bonding of the amine group to the carboxylic group on the surface of CNTs and formation of AuNPs. The morphology of the synthesized nanocomposite was further evaluated by transmission electron microscopy. The result (Fig. S1-a†) shows the presence of carbon nanotubes on the surface of SPE, whereas Fig. S1-b† displays a high density and an even distribution of AuNPs on the surface of CNTs.
Electrochemical impedance spectroscopy (EIS) investigations of the electron transfer between an electrode surface and an electrolyte commonly contain a semicircular part (high frequency) and linear part (low frequency). The low frequency part of the plot shows the conductivity and electron transfer limited process. The semicircle diameter is equivalent to the charge transfer resistance (Rct), whereas the linear part at a higher frequency relates to the diffusion process of the electron transfer.21,22 Fig. 2B shows the EIS results for the bare SPE (a), SPE/CNTs (b), and SPE/CNT/en/AuNPs using a solution containing a redox probe with 1.0 mM K3Fe(CN)6 and 0.1 M KCl. The results obtained from Rct show that the modification of the electrode with CNTs (b) causes a significant decrease in Rct in comparison with the case of bare SPE (a), from 127 kΩ for the bare to 5.45 kΩ for the SPE/CNTs; this indicates that pretreated MWCNTs has high conductivity and electron transfer. The Rct of SPE/CNT/en/AuNPs (c) was obviously decreased to 0.75 kΩ as an evidence of the combination of AuNPs with carbon nanotubes.
Fig. 3 CVs of TAP (10 μM) on (a) SPE, (b) SPE/CNTs, and (c) SPE/CNT/en/AuNPs in 0.1 M citrate buffer (pH 6.0). |
The influence of pH on the oxidation peak of TAP was studied in 0.1 M citrate buffer in the range from 3 to 10 (Fig. 4a). It was observed that by increasing the pH from 3.0 to 6.0, the oxidation peak height increased, and then, the peak current declined. The results indicate that at pH > 6, the TAP molecule has a negative charge due to the de-protonation of amide group, and the repulsion forces with electrode surface decreases the mass transfer of TAP to the surface of electrode. It seems that by increasing the pH from 3 to 6, the adsorption process becomes predominant.10,11 As a result, pH 6.0 was chosen as the optimum pH for the electrochemical oxidation of TAP. The plot of Ep against pH (Fig. 4b) has a slope of 0.0590, close to the standard value of 0.060 V, revealing that the same number of H+ and e− are participating in the oxidation of TAP.11
The kind of buffer on the anodic peak current of TAP was also studied at pH 6.0. The best result was obtained in citrate buffer (Fig. 4c); this showed that the TAP oxidation was more favored in a citrate buffer solution. Therefore, citrate buffer (0.1 M, pH 6.0) was chosen as the supporting electrolyte for practical analysis of TAP.
The relationship between the peak current of TAP and the amount of CNT/en/AuNPs on the SPE was also investigated. As a result, it was observed that by increasing the drop casting volume of nanocomposite suspension in the range of 2–10 μL, the oxidation signal of TAP increased due to the enhancement in surface area and complete covering of SPE. Inversely, the peak height decreased at >6 μL due to the thickness of the nanocomposite and decrease in conductivity (Fig. 4d).
In a further study, the influence of accumulation parameters (accumulation potential and accumulation time) was investigated on the sensitivity of fabricated electrochemical sensors to wards detection of TAP because of the effect of these parameters on the sensitivity of the electrochemical sensor during analysis.23 Accumulation time (tacc) was an effective parameter for the measured response of the proposed sensor. The effect of tacc on the voltammetric detection of the analyte at the electrode was investigated between 30 and 300 s, as shown in Fig. 5a. The result shows that the anodic peak current enhanced after increasing the accumulation time of TAP on the surface of SPE/CNT/en/AuNPs in the range from 30 to 150 s. The peak current leveled off at tacc > 150 s due to the saturation of TAP on the electrode surface. Therefore, tacc of 150 s was selected as the optimum preconcentration time for TAP analysis.
During the preconcentration step on the oxidation of TAP, it was shown that the accumulation potential (Eacc) was an effective parameter to quantify the response of the sensor. The effects of Eacc on the voltammetric determination of TAP at the AuNP/en-MWCNTs electrode were evaluated in the potential range from 0.2 to −0.9 V. CV was performed between −0.5 and 0.5 V at a scan rate of 50 mV s−1, measurements were conducted, and results were obtained. The result (Fig. 5b) shows that by applying the Eacc from 0.2 to −0.7 V, the peak current of TAP increases sharply due to the fact that TAP molecules adsorb better on a more negatively charged surface. Moreover, the reduced state of TAP accumulated on the electrode surface, and its oxidation peak current is bigger. However, at Eacc < −0.7 V, the peak current declined due to the instability of the electrode surface during hydrogen evolution. Therefore, Eacc of −0.7 V was chosen as the optimum value for the sensor to reach higher sensitivity during electro-oxidation of TAP.
Furthermore, there was a good linear relationship between logIpa and logν with a corresponding equation that is expressed as logIpa (μA) = 0.941logν (V s−1) + 0.935; (R2 = 0.991). The slope of 0.941 is close to the expected theoretical value of 1.0 that is typical of an adsorption control process.24
In eqn (1), Ipa is anodic peak current (μA), n is electron transfers number, F is a constant value (96450 C mol−1), Q (C) is the charge of electrode during TAP oxidation, and ν (V s−1) is the scan rate of cyclic voltammogram. The value of n has been calculated from the slope of Ipa vs. ν as 1.86 ≈ 2.0; this means that two electrons are attributed to the oxidation of TAP. Hence, it can be concluded that the oxidation reaction of TAP at SPE/CNT/en/AuNPs involves two protons and two electrons (Scheme 2). Moreover, Fig. 6A shows that the oxidation peak potential (Ep,a) shifted to anodic values with the increasing scan rate. As a theoretical concept in irreversible electrochemical reactions, the plot of Ep versus logν, where ν is scan rate, yields a straight line with the slope of for the anodic currents.25,26 As a result, the value of α (electron transfer coefficient) was estimated to be 0.31, showing asymmetry of the transition state energy of TAP during oxidation on the surface of the modified electrode.
(1) |
Scheme 2 The proposed mechanism for determination of TAP based on the adsorptive stripping voltammetry on the surface SPE/CNTs/en/AuNPs. |
In a further study, differential pulse voltammetry (DPV) was employed to plot the calibration curve of the fabricated sensor against TAP concentration under the optimum conditions. The results (Fig. 7a) showed that the oxidation peak potential shifted around 10 mV to negative direction due to the faster of electron transfer with the surface of the electrode in the presence of adsorbed TAP. As shown in Fig. 7b, there are two linear calibration ranges between anodic peak current and TAP concentration (0.1–10 μM and 10–30 μM) with the equations of Ipa (μA) = 0.9888C + 1.2563 (R = 0.9948) and Ipa (μA) = 0.216C + 7.36 (r = 0.9972), respectively (Fig. 7b). This observation is due to the saturation of some activation sites on the surface of modified electrode at concentrations more than 10 μM.27 The limit of detection (LOD) and limit of quantification (LOQ) were 0.003 μM and 0.01 μM, respectively, based on the following equations:
LOD = 3Sm−1 and LOQ = 10Sm−1; (S, standard deviation of blank solution; m, slope of calibration curve). |
The stability of the fabricated sensor was evaluated on a daily basis for a period of twelve days. The results showed that the electrode retained 90% of its initial signal for the determination of 10 μM TAP within seven days, and then, the response decreased by 15% up to 12 days. These studies indicate that the fabricated sensor is repeatable and reproducible with satisfactory stability and can be applied as a reliable sensor for the analysis of TAP content in milk samples.
Compounds | Tolerancea (mol/mol) | Current change (%) |
---|---|---|
a The maximum mol ratio of each species that cause ≤5% change in the determination of TAP. | ||
Amoxicillin | 50 | +4.8 |
Penicillin G | 50 | +4.6 |
Ampicillin | 50 | +4.7 |
Sulfadiazine | 100 | +2.8 |
Florfenicol | 15 | +8.2 |
(Amoxicillin, penicillin G, ampicillin, florfenicol, sulfadiazine) | 40:45:50:12:85 | +5.0 |
Lactose | 100 | +3.4 |
Casein protein | 100 | +3.6 |
K+ | 200 | +4.5 |
Ca2+ | 200 | −4.0 |
Mg(II) | 200 | −4.6 |
Zn(II) | 200 | −4.8 |
Mn(II) | 200 | −4.7 |
Fe(II) | 200 | −4.0 |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7ra07544h |
This journal is © The Royal Society of Chemistry 2018 |