Anal Bioanal Chem (2014) 406:6933–6942 DOI 10.1007/s00216-014-7999-x

RESEARCH PAPER

Rapid and simple electrochemical detection of morphine on graphene–palladium-hybrid-modified glassy carbon electrode Nada F. Atta & Hagar K. Hassan & Ahmed Galal

Received: 23 January 2014 / Revised: 2 June 2014 / Accepted: 25 June 2014 / Published online: 11 July 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract A hybrid of reduced graphene oxide–palladium (RGO–Pd) nano- to submicron-scale particles was simultaneously chemically prepared using microwave irradiation. The electrochemical investigation of the resulting hybrid was achieved using cyclic voltammetry and differential pulse voltammetry. RGO–Pd had a higher current response than unmodified RGO toward the oxidation of morphine. Several factors that can affect the electrochemical response were studied, including accumulation time and potential, Pd loading, scan rate, and pH of electrolyte. At the optimum conditions, the concentration of morphine was determined using differential pulse voltammetry in a linear range from 0.34 to 12 μmol L−1 and from 14 to 100 μmol L−1, with detection limits of 12.95 nmol L−1 for the first range. The electrode had high sensitivity toward morphine oxidation in the presence of dopamine (DA) and of the interference compounds ascorbic acid (AA) and uric acid (UA). Electrochemical determination of morphine in a spiked urine sample was performed, and a low detection limit was obtained. Validation conditions including reproducibility, sensitivity, and recovery were evaluated successfully in the determination of morphine in diluted human urine.

Published in the topical collection Graphene in Analytics with guest editors Martin Pumera, Ronen Polsky, and Craig Banks. Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-7999-x) contains supplementary material, which is available to authorized users. N. F. Atta : H. K. Hassan : A. Galal (*) Department of Chemistry, Faculty of Science, Cairo University, Giza 12613, Egypt e-mail: [email protected] Present Address: A. Galal Chemistry Department, Faculty of Science, Kuwait University, P.O. Box 5969, Safat 13060, Kuwait

Keywords Electrochemical sensor . Reduced graphene oxide . Palladium nanoparticles . Morphine

Introduction Morphine (MO), a narcotic pain-relieving drug, is a highly effective and preferred drug for moderate treatment of severe pain [1]. It is frequently used to relieve severe pain, especially for patients who undergo a surgical procedure or carcinomatosis. The most common uses of morphine in biological samples are monitoring therapeutic levels in patients and drug concentrations in human and animal pharmacokinetics studies [2]. MO can also be used to investigate opiate abuse, for epidemiological purposes of drug-abuse control, and for forensic cases as an indicator of heroin use [3]. When taken in overdose or abused, MO is toxic and can disrupt the central nervous system [1, 4–7]. Different methods have been used for the determination of morphine in plasma, urine, and opium samples. These methods include gas chromatography (GC) [8], liquid chromatography (LC) [9], high-performance liquid chromatography (HPLC) [10], ultraviolet (UV) spectroscopy [11], gas chromatography–mass spectroscopy (GC–MS) [12], fluorimetry [13], chemiluminescence [14], surface plasmon resonance (SPR) [15], capillary electrophoresis [16–19], and diffuse reflectance near-infrared spectroscopy [15]. These techniques often have disadvantages, including sample pretreatment requirements, time-consuming nature, and cost of operation. Therefore, rapid and simultaneous determination of MO in pharmaceutical and illicit samples has remained a great challenge in analytical chemistry [20]. Electrochemical techniques, especially voltammetric methods, have been widely used for individual determination of opium alkaloids because they have improved simplicity and selectivity [21–26]. The use of bare, unmodified electrodes for detecting

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these compounds also has several limitations, including low sensitivity and reproducibility, slow electron-transfer kinetics, low stability over a wide range of solution compositions, high overpotentials at which the electron-transfer processes occur, or high overlapping of peak potentials of the compound under analysis and other species that may be present in solution [23, 26, 27]. Different modified electrodes have been developed for morphine detection. For example, Jin and co-workers prepared a cobalt-hexacyanoferrate-modified carbon-paste electrode combined with HPLC, and successfully detected morphine in vivo [21]. Ho et al. devised a Prussian-bluemodified indium-tin-oxide (ITO) electrode [28] and molecularly imprinted electrodes for morphine determination [29–31]. Multiwalled carbon nanotubes (MWCNTs) [25] and a MWCNT-modified preheated glassy carbon electrode have also been used for morphine detection [32]. Goldnanotube arrays attached on to the surface of a glassy carbon electrode [33], palladized-aluminium electrode [34], orderedmesoporous-carbon-modified glassy carbon electrode [27], and graphene-modified glassy carbon electrode [20] have recently been used for morphine detection. Graphene, an innovative carbon material, was discovered by Andre Geim’s research group at the University of Manchester in 2004, through the so-called “scotch-tape” technique [35]. It is a two-dimensional array of carbon atoms which looks like a honeycomb [36–38]. Graphene is one of the most interesting materials now available because of its unique properties, including large theoretical surface area of approximately 2620 m2 g−1 [37–39], chemical stability and near-impermeability to gases, ability to withstand large current densities, high thermal [36] and chemical conductivity [40, 41], outstanding mechanical properties [41, 42], large amount of edge planes and/or defects [43], and low production cost. Deposition of metallic nanoparticles on to the graphene sheet (GS) surface to obtain nanoparticle–GS hybrid materials with exciting properties [43] can be performed by two approaches: solution-phase approaches using reducing agents [44–48], or the electrochemical method [49]. The large theoretical surface area of graphene facilitates the deposition of nanoparticles with low aggregation, and graphene should therefore be investigated as a support material to improve electrocatalytic activity of catalyst particles [38]. In this work, a stable and sensitive electrochemical sensor is prepared from a hybrid of chemically converted graphene and Pd nanoparticles (RGO–Pd) via in-situ reduction using a microwave method. This prepared hybrid is well characterized using x-ray diffraction (XRD), atomic force microscopy (AFM), and field-emission electron microscopy (FE-SEM). The electrochemical behavior of MO in the presence of interfering compounds at the optimized modified electrode is investigated using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). Then the suitability of this

N.F. Atta et al.

composite as a sensor for the determination of MO in real samples of urine is established.

Experimental Chemicals and materials All chemicals were used as received without further purification. Graphite powder, sulfuric acid, hydrazine hydrate, palladium (II) chloride, dopamine (DA), uric acid (UA), and ascorbic acid (AA) were supplied by Aldrich Chem. Co. (Milwaukee, WI. USA). Morphine (MO) sulfate was supplied by the National Organization for Drug Control and Research of Egypt. Preparation of reduced graphene oxide–palladium hybrid Graphene oxide (GO) was prepared by oxidation of highpurity graphite powder following the modified method of Hummer and Offeman [50]. Graphene was prepared by reduction of GO using a microwave method in the presence of hydrazine hydrate (HH) as reducing agent, as in [51], and the RGO–Pd hybrid was prepared by the same method, starting with 20 wt % Pd salt and 80 wt % GO. Briefly: 0.08 g GO and 0.02 g PdCl2 in 20 mL deionized water was sonicated until a homogeneous solution was obtained (approximately 2 h). 150 μL HH was added to the homogeneous solution of GO, which was then subjected to microwave irradiation using a conventional microwave oven (MC-9283 JLR, 900 W) operated at full power (900 W) for total reaction time 200 s (20 s on and 30 s off, repeated four times). After microwave irradiation the yellow color of the solution was converted into black, indicating the complete reduction of GO into RGO. The graphene flakes modified with Pd nanoparticles were separated by use of a Mark IVauto bench centrifuge operated at 5000 rpm for 15 min, and were dried overnight. Surface characterization The x-ray diffraction pattern (XRD) was recorded by use of Panlytical X’Pert using Cu-Kα radiation (λ=1.540Ǻ), FESEM by use of JEOL JSM-6360LA and Philips XL30, and AFM by use of Shimadzu Wet-SPM (scanning probe microscope). All samples were prepared in the same way as for electrochemical measurements, and using GC electrode as a support. Electrochemical cells and equipment The working electrode was a GC electrode (diameter: 3 mm), the reference electrode was Ag–AgCl (4 mol L−1 KCl), and a

Rapid and simple electrochemical detection of morphine

Pt wire was used as the auxiliary electrode. The GC electrode was polished using an alumina (2 μm)–water slurry until no scratches were observed. The electrochemical characterizations were performed using a BAS-100B electrochemical analyzer (Bioanalytical systems, BAS, West Lafayette, USA). All experiments were performed at 25±0.2 °C. For preparation of modified GC electrodes, 1 mL DMF was added to 1.5 mg RGO or RGO–Pd and was sonicated until a homogeneous suspension was obtained. The GC electrode was polished well, rinsed with distilled water, casted with 10 μL RGO or RGO–Pd suspension, and left to dry at 70 °C. The electrode was then ready for electrochemical tests. The GC-Pd electrode was prepared by electrodeposition of Pd nanoparticles on the GC electrode using repeated cyclic voltammetry from −0.25 V to +0.65 V for 25 cycles at a scan rate of 50 mV s−1, using a 2.5 mmol L−1 solution of PdCl2 dissolved in 0.1 mol L−1 HClO4. The cyclic voltammograms of MO over different modified electrodes were recorded in 2.3×10−4 mol L−1 MO dissolved in 0.1 mol L−1 PBS of pH 7.4. They were recorded from + 200 mV to +700 mVat a scan rate of 50 mV s−1, after applying an accumulation potential of −300 mV for 2 min and after stirring had been stopped for 20 s. DPV of GC-Pd, GC-RGO, and GC-RGO–Pd electrodes were recorded in the same concentration of MO (DPV conditions: pulse amplitude=50 mV, scan rate=20 mV s−1, sample width=17 ms, pulse width= 50 ms, pulse period=200 ms, and quiet time=2 s.). Analysis of urine Use of the proposed method for real-sample analysis was also investigated by direct analysis of MO in human-urine samples. Standard MO provided by the National Organization for Drug Control and Research of Egypt was dissolved in urine to make a stock solution with a concentration of 5 × 10−4 mol L−1. Successive additions of 5×10−4 mol L−1 MO in urine were added to 10 mL 0.1 mol L−1 PBS of pH 7.4.

Results and discussion Surface and spectral characterization The XRD of RGO has no peak around 8.7 Å, which would be associated with GO, nor around 26 Å, which would be associated with graphite; this indicates the successful preparation of GO and its complete reduction into RGO (Fig. 1). However, the XRD pattern of RGO–Pd has three peaks associated with Pd nanoparticles. The FE-SEM images of both RGO and RGO–Pd are shown in Electronic Supplementary Material Figs. S1a and b, respectively. The RGO sheets are rippled and crumpled on a scale of several nm to a few μm, and it is

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Fig. 1 XRD of RGO and RGO–Pd hybrid

proved that Pd nanoparticles are well distributed on the RGO; the corresponding EDX analysis reveals that Pd is present in the hybrid (RGO–Pd) in the same ratio as in the initial synthesis step, i.e. approximately 20 wt %. Atomic-force microscopy was also used to investigate the structure of RGO and the RGO–Pd hybrid; typical 3D images are shown in Fig. 2a, b. The particle size varies from nano scale to submicron scale with an average particle size of 99.8 nm, as indicated by AFM particle-size analysis (data not shown). The average roughness factors of RGO–Pd and of RGO are comparable at 1.60 and 1.67, respectively. The presence of Pd increases the separation between graphene layers, as revealed by the fact that the thickness of RGO–Pd is nearly twice that of RGO. Both platinum [52] and glassy carbon electrodes [23, 53] are frequently used in the electrocatalytic oxidation of MO. After discovering graphene in 2004, the researchers started to investigate the electrochemical activity of graphene toward some neurotransmitters and other biological compounds. Recently the electrochemical activity of graphene sheets toward MO electro-oxidation was investigated [20]. In this study the effect on MO oxidation of dispersing palladium nanoparticles on the graphene mat was investigated. Figure 3 shows the cyclic voltammogram and DPV for GC, GC-RGO, GC-Pd, and GC-RGO–Pd. As clearly seen, the use of a bare GC electrode gives broad oxidation peak around +560 mV, with a low current of oxidation. Modification of GC with RGO resulted in a sharper and well-defined oxidation peak with higher current and a negative shift in peak potential. Using GC-RGO–Pd revealed that the oxidation-current response increases to nearly double that obtained with GCRGO and approximately five times that obtained with bare a GC electrode. To investigate the effect of RGO, Pd was electrochemically deposited on the surface of a GC electrode and the signal obtained was compared with that from electrodes modified

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Fig. 2 AFM of (a) RGO and (b) RGO–Pd hybrid

with RGO and Pd. The result reveals that the current signal obtained by use of RGO–Pd is double that for GC-Pd electrodes. The increase in oxidation current obtained with RGO– Pd is attributed to a synergistic effect of both RGO and Pd nanoparticles. Another important factor is that the electroactive area greatly increases, as can be seen by comparing the surface area and surface roughness of RGO and of RGO–Pd. On the basis of the surface roughness estimated from the AFM measurements, the increase in current signal cannot be solely attributed to the increase in surface area; a second reason for the increase is that Pd particles take part in the oxidation process, and this brings about the inherent catalytic effect of Pd surfaces. The oxidation peak of MO at the RGO–Pd electrode is attributed to the oxidation reaction of the phenolic group (−OH) at the 3-position, which involves one electron transfer and is responsible for the major peak appearing around 400 mV [22, 28, 31]. The oxidation of the phenolic group

leads to the formation of pseudomorphine (PM-OH) as the main product. The structure of pseudomorphine includes two phenolic groups that make its further oxidation possible by means of the mechanism described by Eqs. 1–5 [25, 54]. The second oxidation peak for morphine occurred at a more positive potential value and is associated with oxidation of the tertiary amine group [55] by means of the proposed mechanism. For morphine, only the first oxidation peak was investigated. So, the peak at 0.42 V is ascribed to the oxidation of the phenolic groups in morphine: M−OHðmorphineÞ↔M−O− þ Hþ

ð1Þ

M−O− ↔M−O˙ þ e−

ð2Þ

2M−O˙↔PM−OH

ð3Þ

PM−OH↔PM−O− þ Hþ

ð4Þ

PMO− ↔PM−O˙ þ e−

ð5Þ

Effect of accumulation time and potential Fig. 3 Cyclic voltammetry of GC (black), GC-Pd (red), GC-RGO (green), and GC-RGO–Pd (yellow) in 2.3×10−4 mol L−1 MO from + 200 to +700 mV at scan rate 50 mV s−1

MO has a pKa value of 9.4 [56], and in 0.1 mol L−1 PBS of pH 7.4 MO acquires a positive charge. Accumulation of MO at the surface of the electrode can be

Rapid and simple electrochemical detection of morphine

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achieved by applying a negative potential. This potential was varied from −200 mV to −500 mV for different accumulation times from 30 s to 5 min, and the effect on the electrochemical response of RGO–Pd was investigated (figure not shown). The results reveal that, as the accumulation potential shifts to more negative values up to −300 mV, the current response increases. After −300 mV the current response starts to stabilize, with no further increase. As the accumulation time increases from 30 s to 2 min the current response increases; after 2 min it becomes nearly constant. Therefore the optimized accumulation potential and time were −300 mV for 2 min. Effect of Pd loading RGO–Pd was prepared with different Pd loading of 15, 20, and 30 wt %, and the electrochemical response toward MO electro-oxidation at each wt % was tested by use of CV in 2.3×10−4 mol L−1 MO after applying an accumulation potential of −300 mV for 2 min (figure not shown). The result revealed that as Pd loading increases the oxidation peak current response increases linearly. When using higher loading (30 wt % Pd) the surface becomes unstable and the current response decreases from the 2nd cycle onwards. Therefore 20 % Pd was chosen as the best loading for electrochemical oxidation of MO at the GC-RGO–Pd electrode surface under the applied experimental conditions. Effect of scan rate A wide scan-rate range (from 2 to 500 mV s−1) was selected to determine whether morphine oxidation is a diffusion-controlled process. The results revealed an increase of peak current of MO electro-oxidation as scan rate increases, as shown in Electronic Supplementary Material Fig. S2. A plot of the anodic peak-current values versus the square root of the scan rate (inset of S2) results in a straight-line relationship which proves that the electro-oxidation of MO at GC-CCG–Pd is a diffusion-controlled process, with the linear regression equation:
I pa ðAÞ ¼ 1:99  10−4 ν 1=2 −5:57  10−6 R2 ¼ 0:9927 ð6Þ

For T=298 K (temperature at which the experiments were conducted), the following equality holds true:
ð8Þ I p ¼ 2:687  105 n3=2 ν 1=2 D1=2 AC o In Eqs. (7) and (8): Ip is the peak current (A), n is the number of electrons exchanged in oxidation (n=1), ν is the scan rate (V s−1), F is Faraday’s constant (96,485 C mol−1), Co is the analyte concentration (2.3 × 10−7 mol cm−3), A = 0.1136 cm2 is the electrode area taking into account the surface roughness calculated from AFM results, R is the universal gas constant (8.314 J mol−1 K−1), and T is the absolute temperature (K) [25]. At relatively slow voltage scans the diffusion of the analyte to the electrode surface is slow. As the scan rate increases the diffusion of analyte to the electrode surface greatly increases. D, calculated from Eqs. (6) and (8), is 8.03×10−4 cm2 s−1. The diffusion coefficient at the bare GC electrode surface is 1.31× 10−4 cm2 s−1. Increased charge transfer is expected at the RGO–Pd electrode surface compared with the bare electrode when comparing the two values relatively. Effect of pH The pH of the supporting electrolyte (0.1 mol L−1 PBS) is an important condition in determining the performance of electrochemical sensors [27]. Altering the pH of the supporting electrolyte altered both the peak potential and the peak current of MO, indicating that oxidation of MO at the GC-RGO–Pd electrode is a pH-dependent reaction (Electronic Supplementary Material Fig. S3a). The anodic peak potential shifted negatively with increased solution pH [21, 25]. A linear relationship was observed between the anodic peak potential and the solution pH in the pH range 2.8 to 9.0 (Electronic Supplementary Material Fig. S3b). The slope is −0.057 V pH−1 units, which is close to the theoretical value of −0.059 V pH−1 units. This indicates that the overall process is proton dependent, with an equal number of protons and electrons involved in morphine oxidation [2, 54]. Because MO oxidation is a one-electron process the number of protons involved was predicted to also be one, indicating a 1e−/1H+ process [28, 31]. The reduction of the peak current in alkaline medium might be caused by the deprotonation of MO, where the pKa value of MO is 9.4 [58]. Calibration curve

The diffusion coefficient (D) of MO (in cm2 s−1) was calculated from the Randles–Sevcik equation [57] and for the oxidation process (O): I p ¼ 0:4463 F3 =RT


1=2

n3=2 ν 1=2 D1=2 AC o

ð7Þ

Figure 4a shows the DPV of MO at the GC-RGO–Pd electrode for a variety of MO concentrations from 0.34–100 μmol L−1 in 0.1 mol L−1 PBS of pH 7.4, where the current response increases as MO concentration increases. The corresponding calibration curve is shown in Fig. 4b; the anodic peak current has a linear relationship with MO concentration from 0.34 to 12 μmol L−1

6938 Fig. 4 (a) DPV of MO at GCRGO–Pd electrode at a variety of MO concentrations from 0.34– 100 μmol L−1 in 0.1 mol L−1 PBS (pH 7.4), and (b) Relationship between the anodic peak current (μA) and concentration of MO (μmol L−1) in ranges 0.34 to 12 μmol L−1 and 14 to 70 μmol L−1 (inset)

N.F. Atta et al.

a

b

and 14 to 70 μmol L−1, with regression equations ipa (μmol L−1) =0.466c (μmol L−1)+0.6716 (R2 =0.9970) and ipa (μA)= 0.135c (μmol L−1) +5.06 (R2 =0.9890), respectively. The detection limit was 12.95 nmol L−1 for the first concentration range. The limit of detection (LOD) was calculated from the oxidation peak currents of the two linear ranges using the equation:

LOD ¼ 3s=m

ð9Þ

where s is the standard deviation of the oxidation peak current (three runs) and m is the slope of the related calibration curve (μA μmol−1 L).

Determination of MO in the presence of DA, UA, and AA The increase of plasma catecholamines that occurs during surgery can be reduced by administration of MO. This is because MO specifically blocks nociceptive stimulation during surgery. The mechanism of action of MO may have its etiology in the concurrent modulation of more than one neurotransmitter. In invertebrates, DA is the main molecule used in neural systems [21]. In this work, we report the simultaneous determination of MO and DA at a GC-RGO–Pd electrode. In the presence of DA two well-defined oxidation peaks were obtained at +184 mVand +428 mV, corresponding to the oxidation of DA and MO, respectively. This proves that it is possible to discriminate MO from DA with good separation in

Rapid and simple electrochemical detection of morphine

peak potential (ΔEp =+244 mV) and with relatively high oxidation-current values. This is illustrated in Fig. 5, which represents the DPV of the simultaneous determination of DA and MO, each at concentrations of 0.5 mmol L−1 in PBS of pH 7.4. The corresponding CV appears in inset. Experimental evidence suggests that AA may modulate central dopaminergic transmission. AA is not synthesized in the brain. However, it is found in high concentrations throughout the mammalian brain, where it diffuses to the blood–brain barrier site. AA is a very active component of the neuronal antioxidant pool because it is rapidly oxidized by reactive oxygen species (ROS), and it is the main scavenger of ROS generated from catecholamine oxidation in vivo. Large doses of AA have been reported to suppress withdrawal symptoms of opiate addicts and to prevent the development of tolerance of and physical dependence on MO. Moreover, MO increases UA levels and AA oxidation. Therefore, the electrochemical behavior of MO, UA, and AA in a mixture solution is an extremely important topic of investigation [2]. For a successful procedure for the detection of MO in practical clinical applications, good selectivity and high sensitivity are the two most important requirements [2]. To assess the selectivity of the GC-RGO–Pd electrode, DPV was used for the characterization of a solution containing a mixture of 100 μmol L−1 AA, 1.0 μmol L−1 UA, and 1.0 μmol L−1 MO. As shown in Fig. 6a, there were three well-defined oxidation peaks related to AA, UA, and MO at +92, +300, and +456 mV, respectively. The large separation of the peak potentials enables simultaneous determination of MO, UA, and AA in the mixture. Increasing the concentration of MO (1–15 μmol L−1) in the presence of constant concentrations of AA (100 μmol L−1) and UA (6.8 μmol L−1) was also studied, and the result reveals that when the concentration of MO increases the current oxidation peak of MO increases linearly (Fig. 6b). Investigation of the sensitivity and selectivity of the GC-RGO–Pd

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Fig. 6 (a) DPVof GC-RGO–Pd in 1 μmol L−1 MO+100 μmol L−1 AA+ 1 μmol L−1 UA in 0.1 mol L−1 PBS (pH 7.4), and (b) DPVof GC-RGO– Pd in 1–15 μmol L−1 MO+100 μmol L−1 AA+6.8 μmol L−1 UA in 0.1 mol L−1 PBS (pH 7.4)

electrode was performed by simultaneously changing the concentrations of MO (1–30 μmol L −1 ), AA (100– 300 μmol L−1), and UA (6.8–35.8 μmol L−1) in 0.1 mol L−1 PBS of pH 7.4. The results revealed that when the concentration of MO increases the oxidation peak current increases linearly, and when the concentration of UA increases the current increases asymptotically. The subsequent increase in the concentration of AA results in a constant current response because of the broadness of the response (Electronic Supplementary material Fig. S4). All of these results indicate that the GC-RGO–Pd electrode has high sensitivity and selectivity towards MO oxidation even in the presence of DA, AA, and UA. Analysis of MO in spiked urine samples

Fig. 5 DPV representing the simultaneous determination of 0.5 mmol L−1 DA and 0.5 mmol L−1 MO in 0.1 mol L−1 PBS (pH 7.4); the corresponding CV is shown in the inset

MO was also determined in the presence of urine as a real matrix. In this set of experiments, morphine was mixed into urine to make a stock solution of 5 × 10 −4 mol L −1

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Table 1 Recovery data from determination of MO in urine samples using GC-RGO–Pd. These data were collected from repeating three concentrations three times each Urine sample

Spike (μmol L−1)

Found (μmol L−1)

Recovery (%)

R.S.D. (%)a

1 2 3

5.0 20.0 100.0

5.11 20.22 101.79

102.2 101.13 101.79

3.99 5.07 2.26

a

%RSD = standard error of three measurements divided by the estimated value multiplied by 100

concentration. Standard additions of 5×10−4 mol L−1 morphine in urine to 10 mL PBS pH 7.4 were performed, and the corresponding DPV was recorded. The results reveal that the oxidation peak current increases linearly as the MO concentration increases (see Electronic Supplementary Material Fig. S5) within two ranges of 2–9 μmol L−1 and 10– 200 μmol L−1, with correlation coefficients 0.9906 and 0.9918 and linear regression equations Ipa (μA)=0.223c (μmol L−1)−0.075 and Ipa (μA)=0.026c (μmol L−1)+2.55, respectively. The detection limit was 39.9 nmol L−1 for the first concentration range. Validation method in urine The proposed electrochemical sensor was validated by performing recovery tests for MO in spiked urine samples [27]. Three different concentrations on the calibration curve were chosen to be repeated to evaluate the accuracy and precision of the proposed method; results are shown in Table 1. The recovery of the spiked samples ranged from 101.13 % to 102.2 % and the results were acceptable, indicating that this

procedure is free from interferences caused by the urine matrix. From all of the above results we can conclude that MO can be selectively and sensitively determined in urine samples at the GC-RGO–Pd electrode. Stability and storage of the GC-RGO–Pd electrode Long-term stability of the GC-RGO–Pd electrode over one week was studied using two different storage methods: storing the electrode in the open air (the electrode is covered only using its cup), and storing it in 0.1 mol L−1 PBS of pH 7.4 in the refrigerator. During this week the electrode was tested in MO in 0.1 mol L−1 PBS of pH 7.4 on three days. The results revealed that the electrode kept in 0.1 mol L−1 PBS in the refrigerator produced 78.63 % of the original current after one week, whereas the electrode kept at room temperature produced 97.4 % of the original current. On the basis of these results the modified electrode has excellent stability, and the best storage method is simple storage in the open air at room temperature. A comparison between our proposed method and other methods reported in literature is given in Table 2. In

Table 2 Comparison between our proposed method and other methods reported in literature Detection method (Electrode)

LOD (nmol L−1)a

Sample

Recovery (%)

LDR (μmol L−1) LOD (nmol L−1)b (Calibration)

Sensitivity (μA μmol−1 L)

Ref.

Voltammetry (Pt-PEDOT)

67

Urine

94–100.1

0.28

[2]

HPLC-EC (cobalt hexacyanoferrate) Amperometry (molecularly imprinted polymer) Voltammetry (mesoporous carbon-GC) Voltammetry (vinylferroceneMWCNTs-GC) Voltammetry (graphene nanosheets-GC) Voltammetry (Au-ferrocene-CPE)

–

Not applicable

–

0.3–8 and 10–60 (50 and 68) 1–500 (50×104)

–

[21]

–

Not applicable

–

100–2000 (20×10 )

0.041

[31]

50

Urine

96.4

0.1–20 (10)

1.74

[59]

–

–

–

–

[60]

–

–

–

0.2–250 and 5.0–600 (90) 0–65 (400)

0.28

[20]

Voltammetry (GC-RGO–Pd)

4

4.3

Urine

99.9–103

1.0–1800 (3.5)

0.023

[61]

40

Urine

101.13–102.2

0.34–12 and 14–100 (12.9)

0.43

This work

a

Limit of detection from samples’ analyses as indicated

b

Limit of detection from calibration curves using standard solutions

Rapid and simple electrochemical detection of morphine

comparison with some other voltammetric methods of MO determination, our method has several advantages. The construction of our electrode is simple and cheap, and it has high stability. It can be reused without pretreatment, and can even be used in real samples.

Conclusion Graphene–palladium hybrid was chemically prepared by simultaneous reduction of GO and palladium salt using microwave irradiation in presence of hydrazine hydrate. The prepared RGO–Pd hybrid had a good electrochemical response toward the electrochemical oxidation of MO in PBS pH 7.4, where RGO–Pd provides an oxidation peak current twice that of chemically converted graphene alone. Several factors which affect the electrochemical response of the electrode during MO determination were studied, including palladium loading, accumulation time and potential, scan rate, pH of electrolyte, and MO concentration. The main advantages of the proposed method are that it is simple, cheap, and fast compared with other determination methods. Furthermore, a low detection limit and a wide range of concentrations make it suitable for standard analytical uses. It also has the advantages of short time and low cost of analysis, and no pretreatment needed before measurement. This sensor also had high resistance to interferences; it was used to determine MO in human urine and satisfactory results were obtained, with a low detection limit of approximately 39.9 nmol L−1 and good recovery data of 101.13–102.2 %. Acknowledgment The authors would like to acknowledge the financial support from Cairo University through the Vice President Office for Research Funds.

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