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Gold nanoparticles embedded electropolymerized thin film of pyrimidine derivative on glassy carbon electrode for highly sensitive detection of l -cysteine
Materials Science and Engineering C 78 (2017) 513–519
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Gold nanoparticles embedded electropolymerized thin film of pyrimidine derivative on glassy carbon electrode for highly sensitive detection of L-cysteine Ayyadurai Kannan, Ranganathan Sevvel ⁎ Post Graduate and Research Department of Chemistry, Vivekananda College, Tiruvedakam West, Madurai 625 234, Tamil Nadu, India
a r t i c l e
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Article history: Received 9 November 2016 Received in revised form 16 April 2017 Accepted 18 April 2017 Available online 19 April 2017 Keywords: Poly(4-amino-6-hydroxy-2mercaptopyrimidine) Gold nanoparticles L-cysteine Electrocatalysis
a b s t r a c t This paper demonstrates the fabrication of novel gold nanoparticles incorporated poly (4-amino-6-hydroxy-2mercaptopyrimidine) (Nano-Au/Poly-AHMP) film modified glassy carbon electrode and it is employed for highly sensitive detection of L-cysteine (CYS). The modified electrode was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). SEM images of modified electrode revealed the homogeneous distribution of gold nanoparticles on poly (4-amino-6-hydroxy-2mercaptopyrimidine) thin film modified glassy carbon electrode. The modified electrode was successfully utilized for highly selective and sensitive determination of L-cysteine at physiological pH 7.0. The present electrochemical sensor successfully resolved the voltammetric signals of ascorbic acid (AA) and L-cysteine with peak separation of 0.510 V. To the best of our knowledge, this is the first report of larger peak separation between AA and CYS. Wide linear concentration ranges (2 μM–500 μM), low detection limit (0.020 μM), an excellent reproducibility and stability are achieved for cysteine sensing with this Nano-Au/Poly-AHMP/GCE. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Cysteine (2-amino-3-sulphydrylpropanoic acid) is a sulphur containing amino acid plays a vital role in biological systems [1]. Cysteine (CYS) can be easily oxidized to form disulphide bond which is essential in construction of the secondary protein [2,3]. It acts as an active site in the catalytic activity of certain enzymes such as cysteine proteases and in many other peptides and proteins [4]. It is also called paravitamin, due to its derivatives role similar to vitamins particularly antioxidative vitamins by maintaining oxidant/antioxidant balance and by indirectly regulating the metabolic processes [5]. Hence, CYS has essential role in the human body in protein synthesis, detoxification and metabolism [6]. Many studies reveal that CYS deficiency causes series illness such as slow growth, leucosite loss, hair depigmentation, oedema, skin lesions, liver damage, lethargy and loss of muscle [7,8]. Moreover, it is acting as physiological regulator for various diseases such as heart disease, rheumatoid arthritis and AIDS [9]. Monitoring the concentration of CYS in physiological samples such as blood plasma, saliva and urine is one of the protocols in clinical analysis and the typical concentration range of CYS is 5 μM to 30 μM [10–13]. Variations in the concentrations of CYS in human body have been related to many diseases, such as arteriosclerosis, leukemia and cancer [14, ⁎ Corresponding author. E-mail address: [email protected] (R. Sevvel).
http://dx.doi.org/10.1016/j.msec.2017.04.105 0928-4931/© 2017 Elsevier B.V. All rights reserved.
15]. Wealth of literature available for the determination of CYS in past decades which includes chromatography [16,17], spectrophotometry [18], electrophoresis [19], chemiluminescence [20], spectrofluorimetry [21] and electrochemical [22–24] techniques. Among these, electrochemical method provides simplicity, high sensitivity, rapid detection and cost efficiency [25]. Because of electroactive nature of CYS molecule, various modified electrodes have been developed such as nanomaterial based electrode [26], chemically modified electrode [27], enzyme based biosensors [28]. Even though these modified electrodes showed good electrocatalytic oxidation of CYS, there are still some challenges lingering such as low sensitivity, less electrode stability and high overpotential. Hence, it is necessary to design high sensitive, highly selective and easy methods for the determination of CYS. Most of the time, ascorbic acid (AA) is a main interferent in the determination of CYS and therefore it is essential to determine them either selectively in presence of other or simultaneously. Many different kinds of nanoparticles such as noble metal, metal oxides and semiconductor nanoparticles have been developed for electrochemical sensing and biosensing and they played essential roles in various sensing systems [29–33]. In recent time, active research involving metal nanoparticles incorporated polymer film have drawn attention of researcher tremendously due to their easy and simple method of preparation [34]. Gold nanoparticles (Nano-Au) are having more catalytic tendency than their bulk counterparts which would enable fast electron transfer kinetic and decreases high overpotential. Gold
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nanoparticle modified electrode plays essential role in electroanalysis, catalysis and electrochemical sensing [35]. Electropolymerization is the efficient and promising technique to immobilize the conducting polymer onto GCE in order to prepare the modified electrode. Electrodes modified by electropolymerization are precise control in thickness of the film, wide choice of electrode materials, strong adherence power on surface of the electrode, broad potential window, large surface area which promotes higher turn-over efficiency, ease in preparation, high stability and sensitivity [36,37]. The combination of Nano-Au and electropolymerized thin film produce the synergetic effect which brings the enhanced conductivity, facilitation of electron transfer, amplification of the electrochemical response and improvement of detection limit [35]. By self-assembly process, Nano-Au strongly bound to the polymer functional groups, such as \\CN,\\NH2, or \\SH and gets incorporated into the thin film of modified electrode [38]. 4-Amino-6-hydroxy-2-mercaptopyrimidine (AHMP) is a low molecular compound which has five potential coordination sites: two nitrogen atoms in pyrimidine ring, one \\NH2 group, one \\OH group and one \\SH group. The Poly-AHMP film layer contains numerous functional groups such as\\NH2 and\\SH, which can facilitate the binding of Au-nanoparticles covalently on Poly-AHMP [39,40]. In this paper, we report the preparation of Nano-Au/PolyAHMP composite by using electrochemical method on glassy carbon electrode (GCE) and its usage for the ultrasensitive and selective determination of CYS in the presence of AA. 2. Experimental 2.1. Chemicals 4-Amino-6-hydroxy-2-mercaptopyrimidine, gold chloride (HAuCl4), L-cysteine,
ascorbic acid were purchased from Aldrich and were used as received. All other chemicals were used in this experiment were of analytical grade. Phosphate buffer solutions were prepared with 0.1 M Na2HPO4 and 0.1 M NaH2PO4 and adjusting the pH using 0.1 M H3PO3 and 0.1 M NaOH. Doubly distilled deionized water was used for the preparation of all solutions and washing. 2.2. Instruments All electrochemical experiments were carried out in a CHI 6088D (Austin, TX, USA) electrochemical workstation with a custom-made three-electrode cell setup. A mirror polished BASi glassy carbon electrode (GCE-3 mm diameter) used as the working electrode, platinum wire used as counter electrode. A dry leakless electrode (DRIREF-2) was purchased from World Precision Instruments, USA and used as reference electrode. Glassy carbon discs with a diameter of 1 cm were purchased from HTW, Germany and were used as substrates for SEM (scanning electron microscopy). SEM measurements were performed using FEI Nova™ NanoSEM Scanning Electon Microscope 450 with an accelerating voltage of 10 kV under high vacuum. 2.3. Preparation of Nano-Au/Poly-AHMP modified electrode The GC working electrode was mirror polished with alumina slurry of 0.5 μm and 0.3 μm, respectively and subsequently sonicated in double distilled water for 10 min to get rid of the physically adsorbed alumina particles from the GC surface. The quality of the polished electrode was electrochemically tested using [Fe(CN)6]3−/4− redox couple in 0.1 M KCl. Electropolymerization of AHMP on GC electrode was carried out by 10 successive potential sweeps between − 0.5 V and 2 V at a scan rate 0.05 Vs−1 in 0.1 M H2SO4 solution containing 1 mM AHMP. After electropolymerization, the AHMP polymerized GC electrode (PolyAHMP/GCE) was removed from the solution and washed with ample
amount of 0.1 M H2SO4 to remove the free monomer molecules from the electrode surface [41]. Gold nanoparticle was electrochemically deposited on Poly-AHMP/ GCE by applying −0.2 V for 90 s in 0.5 mM HAuCl4 solution [38]. The obtained modified electrode is denoted as Nano-Au/Poly-AHMP/GCE.
3. Results and discussion 3.1. Surface morphology studies of the modified electrode The morphology of modified electrode was studied by scanning electron microscopy (SEM). Fig. 1A & B shows the typical SEM images of electrodeposited nano-Au particles on Poly-AHMP film. These images reveal that the gold nanoparticles are well distributed on the surface of the Poly-AHMP film with size ranging from 100 nm to 500 nm. Crystallite aggregation of flower-like structured gold nanoparticles caused the increase of porosity and roughness of modified electrode with high electrochemical surface area. Also, the gold nanoparticles look very pretty, and the sharp-edged leaves possess a large specific surface area. Interestingly, the Poly-AHMP film induces the formation flower like structured gold nanoparticle. This was confirmed by electrodeposition of gold on bare GCE (Fig. 1C) which shows homogeneous surface coverage of gold nanoparticles. The SEM images indicated that the gold nanoparticles are effectively immobilized on Poly-AHMP/GC electrode. It is corroborated with the increased oxidation peak currents of AA and CYS.
3.2. Characterization of the modified electrode We firstly investigate the electrochemical behaviour of different modified electrodes using cyclic voltammetry in 1 mM [Fe(CN)6]3−/4− solution containing 0.1 M KCl as redox probe. Fig. 2A describes cyclic voltammetry curves of bare GC (curve a), Poly-AHMP/GC (curve b) and Nano-Au/Poly-AHMP/GC (curve c) electrodes in 1 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1) containing 0.1 M KCl. On bare GC and Poly-AHMP/GC electrodes, a definite redox peaks were obtained without appreciable change in peak potential and peak current. However, the background current of Poly-AHMP/GC electrode (curve b) is slightly higher than that of bare GCE (curve a). But in the case of Nano-Au/PolyAHMP/GCE, (curve c) the peak current was larger than bare GC and Poly-AHMP/GC electrodes, which could be ascribed to the conductivity of gold nanoparticles on Poly-AHMP film and also, increased background current of same electrode implying that the larger electroactive surface area of modified electrode compared to Poly-AHMP/GCE. Electrochemical Impedance Spectroscopy (EIS) is a versatile tool for studying resistivity changes and interface properties of electrode surface during modification process. In the Nyquist plots of EIS contains both semicircle part at high frequency and linear part at low frequency region which correspond to an electron transfer limited process and diffusion process respectively [42]. The diameter of the semicircle in the impedance spectrum is equal to charge transfer resistance, Rct. Fig. 2B shows the results of impedance spectra of bare GCE (curve a), PolyAHMP/GCE (curve b) and Nano-Au/Poly-AHMP/GCE (curve c) in 1 mM [Fe(CN)6]3−/4− solution containing 0.1 M KCl. On the bare GCE, the value of Rct was obtained 6954 Ω (curve a). After fabrication of Poly-AHMP on GCE surface, the value of Rct was obtained 2541 Ω (curve b). The Rct for Nano-Au/Poly-AHMP/GCE (curve c) is estimated to be 830 Ω. The decreased Rct value for Nano-Au/Poly-AHMP/GCE has been indicated that the highly conducting of gold nanoparticles behaves as an electron-transfer channel, which further improved the conductivity of modified electrode [35] and also it can be observed that the gold nanoparticles are homogeneously distributed within the film as conduction centers, which can accelerate the electron transfer between the analyte molecules and surface of the modified electrode.
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Fig. 2. (A) Cyclic voltammograms and (B) Nyquist plots showing Faradaic impedance measurements of bare GCE (a), Poly-AHMP/GCE (b) and Nano-Au/Poly-AHMP/GCE (c) in 1 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) containing 0.1 M KCl, respectively.
3.3. Effect of pH
Fig. 1. A and B. SEM images of Nano-Au/Poly-AHMP modified GCE. (C) SEM image of Au nanoparticles deposited on bare GCE.
The pH of buffer solution significantly influences the oxidation peak current of CYS at Nano-Au/Poly-AHMP/GC electrode. The effect of pH of the solution on peak current and peak potential has been studied by recording the CVs of CYS with concentrations 500 μM in a series of PB solutions of varying pH in the range 4.0–9.0. The plot of the peak currents versus pH for CYS is shown in Fig. 3A. As can be seen, the peak current of CYS increase with the pH from 4.0 to 6.0 and reaches a maximum at pH 6.0, and then the oxidation current remains same at pH 7.0. Further, decreasing of oxidation current was observed when the pH was increased. In aqueous solution, CYS contains various functional groups such as \\COOH, \\SH and \\NH2 with pKa values of 1.92, 8.37 and 10.30, respectively. In aqueous solution, the ionization of CYS depends on pH of the buffer solution. Above pH 5, CYS exists as zwitter ions and/or anionic form that can be oxidized more easily than the cationic form with higher anodic current is observed at the modified electrode [43]. Thus, the physiological value of pH 7.0 was selected as the optimum pH for the determination CYS. Further, it can be seen from Fig. 3B that the oxidation peak potential of CYS shifts less positive side with a gradual increase of pH. The slope of the curve Ep versus pH is − 0.048 V which suggests that the same number of protons and electrons are involved in the electrode reaction. This conclusion is in accordance with the mechanism of electrochemical oxidation of CYS (Scheme 1) [44,45].
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Fig. 4. CV for the oxidation of 0.5 mM of AA and 1 mM of CYS at bare GCE (a), Poly-AHMP/ GCE (b) and Nano-Au/Poly-AHMP/GCE (c) in 0.1 M PB solution (pH 7.0). Scan rate: 0.05 Vs−1.
Fig. 3. Variation of anodic peak current (A) and peak potential (B) with different pH of 0.1 M PBS at Nano-Au/Poly-AHMP/GCE for CYS. Scan rate: 0.05 Vs−1.
3.4. Simultaneous detection of cysteine and ascorbic acid AA is one of the main interferents in the determination of CYS. Therefore, determination of CYS in the presence of AA is very important. The cyclic voltammetric behaviours of CYS and AA at different electrodes were investigated in 0.1 M PBS of pH 7.0. As can be seen from Fig. 4, the bare GC electrode showed a broad oxidation wave around 0.4–0.6 V which is attributed to the overlapped oxidation signals of AA and CYS (curve a). However, the Poly-AHMP film separates and resolves the oxidation peak currents of AA and CYS with potential difference of
Scheme 1. Electrochemical oxidation of CYS.
0.450 V (curve b). Poly-AHMP/GCE shows oxidation peaks for AA and CYS, at 0.160 V and 0.610 V, respectively. More interestingly, NanoAu/Poly-AHMP/GC electrode enhances the oxidation peak currents of both AA and CYS in dramatic manner with a peak separation of 0.510 V (curve c). Nano-Au/Poly-AHMP/GC modified electrode shows the well-defined and distinguished oxidation peaks were observed for AA and CYS at 0.150 V and 0.660 V, respectively with three fold excess of higher oxidation peak currents. To the best of our knowledge, this is the first report with large peak separation (0.510 V) between AA and CYS at physiological pH of 7.0 [46,47]. The utilization of the Nano-Au/Poly-AHMP modified GC electrode for the simultaneous determination of AA and CYS was demonstrated by simultaneously changing the concentration of AA and CYS, and recording DPV measurements. The DP voltammetric results exhibited well-defined and distinguished two anodic peaks at potentials of 0.040 V and 0.510 V corresponding to the oxidation of AA and CYS (Fig. 5 curve a), respectively. This indicates that simultaneous determination of these compounds is feasible at Nano-Au/Poly-AHMP modified GC electrode in 0.1 M PBS (pH 7.0) as shown in Fig. 5. No shift in the oxidation peak potentials of AA and CYS were observed on the further addition of respective analyte to 0.1 M PBS (Fig. 5 curve b–p). The peak current of AA increased linearly with correlation coefficients of 0.999 and 0.998 for lowest and highest concentration ranges respectively, when increasing the concentration AA from 2 μM to 500 μM. Similarly, the oxidation peak current of CYS increased linearly with correlation coefficients of 0.996 and 0.989 for lowest and highest concentration ranges respectively, when increasing the concentration CYS from 2 μM to 500 μM. Generally our body fluids contain higher concentration of AA which coexists with CYS. Therefore, it is very essential to detect CYS in the presence of large excess of AA. Fig. 6 shows DPVs for the oxidation of 20 μM CYS in the presence of 0.5 mM AA. A distinguished oxidation peak was observed for CYS in the presence of 250 fold excess of AA (Fig. 6 curve a). The observed results suggest that the Nano-Au/PolyAHMP modified GC electrode is very sensitive towards the CYS determination even in the presence of high concentration of AA. With the incremental addition of 20 μM CYS to 0.5 mM AA, the oxidation peak current of CYS was linearly increased with correlation coefficient 0.994. No shift of anodic peak potentials of AA and CYS were found. The detection limit of CYS is found to be 0.020 μM (S/N = 3). The performance of the fabricated CYS sensor is very much comparable to the literature values (Table 1) [48–55]. It is evident from Table 1, the newly designed Nano-Au/Poly-AHMP modified electrode exhibited relatively low detection limit, high sensitivity and wide linear range.
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Fig. 5. (A) Differential pulse voltammogramms for the simultaneous determination of a) 2, b) 10, c) 20, d) 30, e) 40, f) 50, g) 60, h) 70, i) 80, j) 90, k) 100, l) 150, m) 200, n) 300, o) 400, p) 500 μM of AA and CYS at Nano-Au/Poly-AHMP/GC electrode in 0.1 M PB solution (pH 7.0). (B) Plot of AA oxidation current vs. concentration and (C) Plot of CYS oxidation current vs. concentration. Amplitude = 0.05 V, Pulse Width = 0.2 s, Sample Width = 0.02 s and Pulse Period = 0.5 s.
3.5. Interference study The selectivity of the Nano-Au/Poly-AHMP/GCE for sensitive determination of CYS was analysed and several compounds such as biologically important amino acids and some metal ions were checked as potential interfering substance. The electrochemical response of 20 μM CYS in presence of epinephrine, norepinephrine, glycine, alanine, serine, leucine, tyrosine, lysine, tryptophan, methionine, dopamine, uric acid, glucose, K+, Ca2+, Zn2+ and Mg2+ is tabulated in Table 2. This table indicates that the coexisting substances do not interfere in the oxidation
Table 1 Comparison of some modified electrodes in the determination of CYS.
Fig. 6. Differential pulse voltammogramms for the oxidation of CYS at Nano-Au/PolyAHMP/GC electrode in different concentrations a) 20, b) 40, c) 60, d) 80, e) 100, f) 120, g) 140, h) 160, i) 180, j) 200 μM in the presence of 0.5 mM of AA in 0.1 M PBS. (Inset: Plot of CYS oxidation current vs. concentration). Amplitude = 0.05 V, Pulse Width = 0.2 s, Sample Width = 0.02 s and Pulse Period = 0.5 s.
Electrodes
Method pH Linear ranges (μM)
Detection limits (μM)
References
GO/CNTs/AuNPs@MnO2/GCE Ag-PDA/ITO Au-SH-SiO2@Cu-MOF/GCE NPG/GCE Pt/VACNTs/GCE PB-AuNPs-Pd/CPE Zinc Bismuthate NRs/GCE DMBQ/ZnO/NPs/CPE Nano-Au/Poly-AHMP/GCE
DPV LSV DPV Amp Amp CA CV SWV DPV
0.340 0.023 0.008 0.050 0.050 0.180 0.074 0.050 0.020
[48] [49] [50] [51] [52] [53] [54] [55] This work
7.0 5.0 5.0 7.0 6.5 4.0 7.0 5.0 7.0
0.01–7 0.05–300 0.02–300 1.0–400 1.0–500 0.3–400 0.01–200 0.09–340 2.0–500
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Table 2 The influences of some interferents on the peak current of 20 μM CYS oxidation at NanoAu/Poly-AHMP/GCE. Interferents
Concentration (μM)
Signal change CYS (%)
Epinephrine Norepinehrine Glycine Alanine Serine Leucine Tyrosine Lysine Tryptophan Methionine Dopamine Uric acid Glucose K+ Ca2+ Zn2+ Mg2+
50 50 50 50 50 50 50 50 50 50 50 50 50 500 500 500 500
5.7 6.1 2.2 3.5 2.0 3.0 1.5 4.5 6.2 4.4 18.0 22.1 0.8 1.3 2.1 1.5 1.4
samples were in the range of 96.50–100.25%. It is clearly observed that a good recovery was obtained suggesting the practical applicability of this electrochemical sensor. 4. Conclusion In conclusion, a novel electrochemical sensor has been fabricated by electrodeposition of Nano-Au into Poly-AHMP thin film modified glassy carbon electrode and utilized for highly sensitive, selective and stable determination of CYS. Nano-Au/Poly-AHMP/GC modified electrode was characterized by SEM, CV and EIS. This modified electrode exhibited excellent electrocatalytic activity towards CYS at physiological pH 7.0. The present electrochemical sensor successfully resolved the voltammetric signals of AA and CYS with peak separation of 0.510 V. To the best of our knowledge, this is the first report of larger peak separation between AA and CYS. The lowest detection limit of 0.020 μM (S/N = 3) was achieved for cysteine sensing at Nano-Au/Poly-AHMP/ GCE. Acknowledgements
current and peak potential of CYS significantly at modified electrode whereas modest interference of dopamine and uric acid is observed [40]. 3.6. Reproducibility, repeatability and stability of the Nano-Au/Poly-AHMP/ GCE Reproducibility, repeatability and long term stability are very essential for any electrochemical sensor. The repeatability of Nano-Au/PolyAHMP/GC electrode was examined by 10 repetitive measurements for 20 μM of CYS in 0.1 M PB solution by DPV method. The result indicated that the oxidative current of CYS remains the same with a relative standard deviation of 4.8%. In addition, the reproducibility of three modified electrodes has been evaluated under optimized condition at same concentration of CYS which gives current response with 7% of RSD. The long term stability of the modified electrode was monitored by taking DPV method at 10 days intervals for 30 days. The current response was decreased 2.8% in first 10 days, 4.5% in second 10 days and 5.1% for third 10 days from initial current response, which indicating that the NanoAu/Poly-AHMP/GC electrode exhibited long term stability. These results suggested that the modified electrode has no significant memory effect in the determination of Cysteine. However, to eliminate the memory effect of the proposed sensor, the modified electrode was renewed by potential scanning from −0.2 to 0.7 V in 0.1 M PBS (pH 7.0) for 5 cycles. 3.7. Real sample analysis To assess the practical utility of proposed sensor to the determination of CYS in real samples, urine and human blood serum were selected for analysis. Urine samples were collected from healthy volunteers and the urine samples were centrifuged. The obtained supernatants were diluted 100 times with 0.1 M phosphate buffer solution. The human blood serum samples were collected from clinical laboratory and the collected samples were diluted 10 times with 0.1 M PBS (pH 7.0). The recoveries of CYS were determined by standard addition method. The results are summarized in Table 3, which suggests that the recoveries of the spiked
Table 3 Real sample analysis. Sample
Added (μM)
Found (μM)
Recovery (%)
R.S.D (n = 3) %
Urine 1 Urine 2 Human serum 1 Human serum 2
20 40 20 40
19.9 40.1 19.3 40.0
99.50 100.25 96.50 100
1.89 1.14 3.61 2.45
Financial support from the University Grants Commission – South Eastern Regional Office (UGC – SERO), Hyderabad, for the award of Minor Research Project (No. F MRP-5855/15) (SERO-UGC) is gratefully acknowledged by AK. The authors would like to thank The Management and The Principal, Vivekananda College, Tiruvedakam West for their support and encouragement. The Authors would also thank Dr. P. Veluchamy, Senior Consultant Engineer, First Solar, USA, for his donation of CHI electrochemical workstation to Vivekananda College. References [1] Y.H. Bai, J.J. Hu, H.Y. Chen, Biosens. Bioelectron. 24 (2009) 2985. [2] D.L. Nelson, M.M. Cox, Lehninger Principles of Biochemistry, fourth ed. W. H. Freeman, New York, 2005. [3] R. Lill, U. Muhlenhoff, Trends Biochem. Sci. 30 (2005) 133–141. [4] Y.T. Lai, A. Ganguly, L.C. Chen, K.H. Chen, Biosens. Bioelectron. 26 (2010) 1688–1691. [5] W. Droge, Philos. Trans. R. Soc. B 360 (2005) 2355–2372. [6] C. Ray, S. Dutta, S. Sarkar, R. Sahoo, A. Roy, T. Pal, J. Mater. Chem. B 2 (2014) 6097–6105. [7] W. Wang, O. Rusin, X. Hu, K.K. Kim, J.O. Escobedo, S.O. Fakayode, K.A. Fletcher, M. Lowry, C.M. Schowalter, C.M. Lawrence, F.R. Fronczek, I.M. Warner, R.M. Strongin, J. Am. Chem. Soc. 127 (2005) 15949–15958. [8] L. Shang, C.J. Qin, T. Wang, M. Wang, L.X. Wang, S.J. Dong, J. Phys. Chem. C 111 (2007) 13414–13417. [9] V.V. Kumar, S.P. Anthony, RSC Adv. 4 (2014) 18467–18472. [10] Y.M. Go, D.P. Jones, Free Radic. Biol. Med. 50 (2011) 495–509. [11] G. Hignett, S. Threlfell, A.J. Wain, N.S. Lawrence, S.J. Wilkins, J. Davis, R.G. Compton, M.F. Cardosi, Analyst 126 (2001) 353–357. [12] T. Fiskerstrand, H. Refsum, G. Kvalheim, P.M. Ueland, Clin. Chem. 39 (1993) 263–271. [13] A. Pastore, R. Massoud, C. Motti, L. Russo, G. Fucci, C. Cortese, G. Federici, Clin. Chim. Acta 44 (1998) 825–832. [14] D.P. Jones, J.L. Carlson, J.V.C. Mody, J. Cai, M.J. Lynn, J.P. Sternberg, Free Radic. Biol. Med. 28 (2000) 625–635. [15] H. Refsum, F. Wesenberg, P.M. Ueland, Cancer Res. 51 (1991) 828–835. [16] G. Chwatko, E. Bald, Talanta 52 (2000) 509–515. [17] T.D. Nolin, M.E. McMenamin, J. Chromatogr. B 852 (2007) 554–561. [18] J. Chrastil, Analyst 115 (1990) 1383–1384. [19] W. Jin, Y. Wang, J. Chromatogr. A 769 (1997) 307–314. [20] Y. Wang, J. Lu, H. Chang, J. Li, Anal. Chem. 81 (2009) 9710–9715. [21] X. Chen, H. Liu, H. Hu, Y. Wang, X. Zhou, J. Hu, Talanta 138 (2015) 144–148. [22] M.C.C. Areias, K. Shimizu, R.G. Compton, Analyst 141 (2016) 5563–5570. [23] R.R. Moore, C.E. Banks, R.G. Compton, Analyst 129 (2004) 755–758. [24] Z. Liu, H. Zhang, S. Hou, H. Ma, Michrochim. Acta 177 (2012) 427–433. [25] S. Yang, G. Li, Y. Wang, G. Wang, L. Qu, Michrochim. Acta 183 (2016) 1351–1357. [26] M. Murugavelu, B. Karthikeyan, Superlattice. Microst. 75 (2014) 916–926. [27] W.Y. Su, S.H. Cheng, Electrochem. Commun. 10 (2008) 899–902. [28] M. Santhiago, I.C. Vieira, Sensors Actuators B Chem. 128 (2007) 279–285. [29] M.A. Khalizadeh, Z. Arab, Curr. Anal. Chem. 13 (2017) 81–86. [30] M.A. Khalizadeh, M. Borzoo, J. Food Drug Anal. 24 (2016) 796–803. [31] B. Nikahd, M.A. Khalizadeh, J. Mol. Liq. 215 (2016) 253–257. [32] H. Karimi-Maleh, M. Hatami, R. Moradi, M.A. Khalilzadeh, S. Amiri, H. Sadeghifar, Michrochim. Acta 183 (2016) 2957–2964. [33] M. Najafi, M.A. Khalilzadeh, H. Karimi-Maleh, Food Chem. 158 (2014) 125–131. [34] S. Thiagarajan, R.-F. Yang, S.–M. Chen, Bioelectrochemistry 75 (2009) 163–169.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Gold nanoparticles embedded electropolymerized thin film of pyrimidine derivative on glassy carbon electrode for highly sensitive detection of L-cysteine Ayyadurai Kannan, Ranganathan Sevvel ⁎ Post Graduate and Research Department of Chemistry, Vivekananda College, Tiruvedakam West, Madurai 625 234, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 9 November 2016 Received in revised form 16 April 2017 Accepted 18 April 2017 Available online 19 April 2017 Keywords: Poly(4-amino-6-hydroxy-2mercaptopyrimidine) Gold nanoparticles L-cysteine Electrocatalysis
a b s t r a c t This paper demonstrates the fabrication of novel gold nanoparticles incorporated poly (4-amino-6-hydroxy-2mercaptopyrimidine) (Nano-Au/Poly-AHMP) film modified glassy carbon electrode and it is employed for highly sensitive detection of L-cysteine (CYS). The modified electrode was characterized by scanning electron microscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). SEM images of modified electrode revealed the homogeneous distribution of gold nanoparticles on poly (4-amino-6-hydroxy-2mercaptopyrimidine) thin film modified glassy carbon electrode. The modified electrode was successfully utilized for highly selective and sensitive determination of L-cysteine at physiological pH 7.0. The present electrochemical sensor successfully resolved the voltammetric signals of ascorbic acid (AA) and L-cysteine with peak separation of 0.510 V. To the best of our knowledge, this is the first report of larger peak separation between AA and CYS. Wide linear concentration ranges (2 μM–500 μM), low detection limit (0.020 μM), an excellent reproducibility and stability are achieved for cysteine sensing with this Nano-Au/Poly-AHMP/GCE. © 2017 Elsevier B.V. All rights reserved.
1. Introduction Cysteine (2-amino-3-sulphydrylpropanoic acid) is a sulphur containing amino acid plays a vital role in biological systems [1]. Cysteine (CYS) can be easily oxidized to form disulphide bond which is essential in construction of the secondary protein [2,3]. It acts as an active site in the catalytic activity of certain enzymes such as cysteine proteases and in many other peptides and proteins [4]. It is also called paravitamin, due to its derivatives role similar to vitamins particularly antioxidative vitamins by maintaining oxidant/antioxidant balance and by indirectly regulating the metabolic processes [5]. Hence, CYS has essential role in the human body in protein synthesis, detoxification and metabolism [6]. Many studies reveal that CYS deficiency causes series illness such as slow growth, leucosite loss, hair depigmentation, oedema, skin lesions, liver damage, lethargy and loss of muscle [7,8]. Moreover, it is acting as physiological regulator for various diseases such as heart disease, rheumatoid arthritis and AIDS [9]. Monitoring the concentration of CYS in physiological samples such as blood plasma, saliva and urine is one of the protocols in clinical analysis and the typical concentration range of CYS is 5 μM to 30 μM [10–13]. Variations in the concentrations of CYS in human body have been related to many diseases, such as arteriosclerosis, leukemia and cancer [14, ⁎ Corresponding author. E-mail address: [email protected] (R. Sevvel).
http://dx.doi.org/10.1016/j.msec.2017.04.105 0928-4931/© 2017 Elsevier B.V. All rights reserved.
15]. Wealth of literature available for the determination of CYS in past decades which includes chromatography [16,17], spectrophotometry [18], electrophoresis [19], chemiluminescence [20], spectrofluorimetry [21] and electrochemical [22–24] techniques. Among these, electrochemical method provides simplicity, high sensitivity, rapid detection and cost efficiency [25]. Because of electroactive nature of CYS molecule, various modified electrodes have been developed such as nanomaterial based electrode [26], chemically modified electrode [27], enzyme based biosensors [28]. Even though these modified electrodes showed good electrocatalytic oxidation of CYS, there are still some challenges lingering such as low sensitivity, less electrode stability and high overpotential. Hence, it is necessary to design high sensitive, highly selective and easy methods for the determination of CYS. Most of the time, ascorbic acid (AA) is a main interferent in the determination of CYS and therefore it is essential to determine them either selectively in presence of other or simultaneously. Many different kinds of nanoparticles such as noble metal, metal oxides and semiconductor nanoparticles have been developed for electrochemical sensing and biosensing and they played essential roles in various sensing systems [29–33]. In recent time, active research involving metal nanoparticles incorporated polymer film have drawn attention of researcher tremendously due to their easy and simple method of preparation [34]. Gold nanoparticles (Nano-Au) are having more catalytic tendency than their bulk counterparts which would enable fast electron transfer kinetic and decreases high overpotential. Gold
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nanoparticle modified electrode plays essential role in electroanalysis, catalysis and electrochemical sensing [35]. Electropolymerization is the efficient and promising technique to immobilize the conducting polymer onto GCE in order to prepare the modified electrode. Electrodes modified by electropolymerization are precise control in thickness of the film, wide choice of electrode materials, strong adherence power on surface of the electrode, broad potential window, large surface area which promotes higher turn-over efficiency, ease in preparation, high stability and sensitivity [36,37]. The combination of Nano-Au and electropolymerized thin film produce the synergetic effect which brings the enhanced conductivity, facilitation of electron transfer, amplification of the electrochemical response and improvement of detection limit [35]. By self-assembly process, Nano-Au strongly bound to the polymer functional groups, such as \\CN,\\NH2, or \\SH and gets incorporated into the thin film of modified electrode [38]. 4-Amino-6-hydroxy-2-mercaptopyrimidine (AHMP) is a low molecular compound which has five potential coordination sites: two nitrogen atoms in pyrimidine ring, one \\NH2 group, one \\OH group and one \\SH group. The Poly-AHMP film layer contains numerous functional groups such as\\NH2 and\\SH, which can facilitate the binding of Au-nanoparticles covalently on Poly-AHMP [39,40]. In this paper, we report the preparation of Nano-Au/PolyAHMP composite by using electrochemical method on glassy carbon electrode (GCE) and its usage for the ultrasensitive and selective determination of CYS in the presence of AA. 2. Experimental 2.1. Chemicals 4-Amino-6-hydroxy-2-mercaptopyrimidine, gold chloride (HAuCl4), L-cysteine,
ascorbic acid were purchased from Aldrich and were used as received. All other chemicals were used in this experiment were of analytical grade. Phosphate buffer solutions were prepared with 0.1 M Na2HPO4 and 0.1 M NaH2PO4 and adjusting the pH using 0.1 M H3PO3 and 0.1 M NaOH. Doubly distilled deionized water was used for the preparation of all solutions and washing. 2.2. Instruments All electrochemical experiments were carried out in a CHI 6088D (Austin, TX, USA) electrochemical workstation with a custom-made three-electrode cell setup. A mirror polished BASi glassy carbon electrode (GCE-3 mm diameter) used as the working electrode, platinum wire used as counter electrode. A dry leakless electrode (DRIREF-2) was purchased from World Precision Instruments, USA and used as reference electrode. Glassy carbon discs with a diameter of 1 cm were purchased from HTW, Germany and were used as substrates for SEM (scanning electron microscopy). SEM measurements were performed using FEI Nova™ NanoSEM Scanning Electon Microscope 450 with an accelerating voltage of 10 kV under high vacuum. 2.3. Preparation of Nano-Au/Poly-AHMP modified electrode The GC working electrode was mirror polished with alumina slurry of 0.5 μm and 0.3 μm, respectively and subsequently sonicated in double distilled water for 10 min to get rid of the physically adsorbed alumina particles from the GC surface. The quality of the polished electrode was electrochemically tested using [Fe(CN)6]3−/4− redox couple in 0.1 M KCl. Electropolymerization of AHMP on GC electrode was carried out by 10 successive potential sweeps between − 0.5 V and 2 V at a scan rate 0.05 Vs−1 in 0.1 M H2SO4 solution containing 1 mM AHMP. After electropolymerization, the AHMP polymerized GC electrode (PolyAHMP/GCE) was removed from the solution and washed with ample
amount of 0.1 M H2SO4 to remove the free monomer molecules from the electrode surface [41]. Gold nanoparticle was electrochemically deposited on Poly-AHMP/ GCE by applying −0.2 V for 90 s in 0.5 mM HAuCl4 solution [38]. The obtained modified electrode is denoted as Nano-Au/Poly-AHMP/GCE.
3. Results and discussion 3.1. Surface morphology studies of the modified electrode The morphology of modified electrode was studied by scanning electron microscopy (SEM). Fig. 1A & B shows the typical SEM images of electrodeposited nano-Au particles on Poly-AHMP film. These images reveal that the gold nanoparticles are well distributed on the surface of the Poly-AHMP film with size ranging from 100 nm to 500 nm. Crystallite aggregation of flower-like structured gold nanoparticles caused the increase of porosity and roughness of modified electrode with high electrochemical surface area. Also, the gold nanoparticles look very pretty, and the sharp-edged leaves possess a large specific surface area. Interestingly, the Poly-AHMP film induces the formation flower like structured gold nanoparticle. This was confirmed by electrodeposition of gold on bare GCE (Fig. 1C) which shows homogeneous surface coverage of gold nanoparticles. The SEM images indicated that the gold nanoparticles are effectively immobilized on Poly-AHMP/GC electrode. It is corroborated with the increased oxidation peak currents of AA and CYS.
3.2. Characterization of the modified electrode We firstly investigate the electrochemical behaviour of different modified electrodes using cyclic voltammetry in 1 mM [Fe(CN)6]3−/4− solution containing 0.1 M KCl as redox probe. Fig. 2A describes cyclic voltammetry curves of bare GC (curve a), Poly-AHMP/GC (curve b) and Nano-Au/Poly-AHMP/GC (curve c) electrodes in 1 mM K4[Fe(CN)6]/K3[Fe(CN)6] (1:1) containing 0.1 M KCl. On bare GC and Poly-AHMP/GC electrodes, a definite redox peaks were obtained without appreciable change in peak potential and peak current. However, the background current of Poly-AHMP/GC electrode (curve b) is slightly higher than that of bare GCE (curve a). But in the case of Nano-Au/PolyAHMP/GCE, (curve c) the peak current was larger than bare GC and Poly-AHMP/GC electrodes, which could be ascribed to the conductivity of gold nanoparticles on Poly-AHMP film and also, increased background current of same electrode implying that the larger electroactive surface area of modified electrode compared to Poly-AHMP/GCE. Electrochemical Impedance Spectroscopy (EIS) is a versatile tool for studying resistivity changes and interface properties of electrode surface during modification process. In the Nyquist plots of EIS contains both semicircle part at high frequency and linear part at low frequency region which correspond to an electron transfer limited process and diffusion process respectively [42]. The diameter of the semicircle in the impedance spectrum is equal to charge transfer resistance, Rct. Fig. 2B shows the results of impedance spectra of bare GCE (curve a), PolyAHMP/GCE (curve b) and Nano-Au/Poly-AHMP/GCE (curve c) in 1 mM [Fe(CN)6]3−/4− solution containing 0.1 M KCl. On the bare GCE, the value of Rct was obtained 6954 Ω (curve a). After fabrication of Poly-AHMP on GCE surface, the value of Rct was obtained 2541 Ω (curve b). The Rct for Nano-Au/Poly-AHMP/GCE (curve c) is estimated to be 830 Ω. The decreased Rct value for Nano-Au/Poly-AHMP/GCE has been indicated that the highly conducting of gold nanoparticles behaves as an electron-transfer channel, which further improved the conductivity of modified electrode [35] and also it can be observed that the gold nanoparticles are homogeneously distributed within the film as conduction centers, which can accelerate the electron transfer between the analyte molecules and surface of the modified electrode.
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Fig. 2. (A) Cyclic voltammograms and (B) Nyquist plots showing Faradaic impedance measurements of bare GCE (a), Poly-AHMP/GCE (b) and Nano-Au/Poly-AHMP/GCE (c) in 1 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) containing 0.1 M KCl, respectively.
3.3. Effect of pH
Fig. 1. A and B. SEM images of Nano-Au/Poly-AHMP modified GCE. (C) SEM image of Au nanoparticles deposited on bare GCE.
The pH of buffer solution significantly influences the oxidation peak current of CYS at Nano-Au/Poly-AHMP/GC electrode. The effect of pH of the solution on peak current and peak potential has been studied by recording the CVs of CYS with concentrations 500 μM in a series of PB solutions of varying pH in the range 4.0–9.0. The plot of the peak currents versus pH for CYS is shown in Fig. 3A. As can be seen, the peak current of CYS increase with the pH from 4.0 to 6.0 and reaches a maximum at pH 6.0, and then the oxidation current remains same at pH 7.0. Further, decreasing of oxidation current was observed when the pH was increased. In aqueous solution, CYS contains various functional groups such as \\COOH, \\SH and \\NH2 with pKa values of 1.92, 8.37 and 10.30, respectively. In aqueous solution, the ionization of CYS depends on pH of the buffer solution. Above pH 5, CYS exists as zwitter ions and/or anionic form that can be oxidized more easily than the cationic form with higher anodic current is observed at the modified electrode [43]. Thus, the physiological value of pH 7.0 was selected as the optimum pH for the determination CYS. Further, it can be seen from Fig. 3B that the oxidation peak potential of CYS shifts less positive side with a gradual increase of pH. The slope of the curve Ep versus pH is − 0.048 V which suggests that the same number of protons and electrons are involved in the electrode reaction. This conclusion is in accordance with the mechanism of electrochemical oxidation of CYS (Scheme 1) [44,45].
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Fig. 4. CV for the oxidation of 0.5 mM of AA and 1 mM of CYS at bare GCE (a), Poly-AHMP/ GCE (b) and Nano-Au/Poly-AHMP/GCE (c) in 0.1 M PB solution (pH 7.0). Scan rate: 0.05 Vs−1.
Fig. 3. Variation of anodic peak current (A) and peak potential (B) with different pH of 0.1 M PBS at Nano-Au/Poly-AHMP/GCE for CYS. Scan rate: 0.05 Vs−1.
3.4. Simultaneous detection of cysteine and ascorbic acid AA is one of the main interferents in the determination of CYS. Therefore, determination of CYS in the presence of AA is very important. The cyclic voltammetric behaviours of CYS and AA at different electrodes were investigated in 0.1 M PBS of pH 7.0. As can be seen from Fig. 4, the bare GC electrode showed a broad oxidation wave around 0.4–0.6 V which is attributed to the overlapped oxidation signals of AA and CYS (curve a). However, the Poly-AHMP film separates and resolves the oxidation peak currents of AA and CYS with potential difference of
Scheme 1. Electrochemical oxidation of CYS.
0.450 V (curve b). Poly-AHMP/GCE shows oxidation peaks for AA and CYS, at 0.160 V and 0.610 V, respectively. More interestingly, NanoAu/Poly-AHMP/GC electrode enhances the oxidation peak currents of both AA and CYS in dramatic manner with a peak separation of 0.510 V (curve c). Nano-Au/Poly-AHMP/GC modified electrode shows the well-defined and distinguished oxidation peaks were observed for AA and CYS at 0.150 V and 0.660 V, respectively with three fold excess of higher oxidation peak currents. To the best of our knowledge, this is the first report with large peak separation (0.510 V) between AA and CYS at physiological pH of 7.0 [46,47]. The utilization of the Nano-Au/Poly-AHMP modified GC electrode for the simultaneous determination of AA and CYS was demonstrated by simultaneously changing the concentration of AA and CYS, and recording DPV measurements. The DP voltammetric results exhibited well-defined and distinguished two anodic peaks at potentials of 0.040 V and 0.510 V corresponding to the oxidation of AA and CYS (Fig. 5 curve a), respectively. This indicates that simultaneous determination of these compounds is feasible at Nano-Au/Poly-AHMP modified GC electrode in 0.1 M PBS (pH 7.0) as shown in Fig. 5. No shift in the oxidation peak potentials of AA and CYS were observed on the further addition of respective analyte to 0.1 M PBS (Fig. 5 curve b–p). The peak current of AA increased linearly with correlation coefficients of 0.999 and 0.998 for lowest and highest concentration ranges respectively, when increasing the concentration AA from 2 μM to 500 μM. Similarly, the oxidation peak current of CYS increased linearly with correlation coefficients of 0.996 and 0.989 for lowest and highest concentration ranges respectively, when increasing the concentration CYS from 2 μM to 500 μM. Generally our body fluids contain higher concentration of AA which coexists with CYS. Therefore, it is very essential to detect CYS in the presence of large excess of AA. Fig. 6 shows DPVs for the oxidation of 20 μM CYS in the presence of 0.5 mM AA. A distinguished oxidation peak was observed for CYS in the presence of 250 fold excess of AA (Fig. 6 curve a). The observed results suggest that the Nano-Au/PolyAHMP modified GC electrode is very sensitive towards the CYS determination even in the presence of high concentration of AA. With the incremental addition of 20 μM CYS to 0.5 mM AA, the oxidation peak current of CYS was linearly increased with correlation coefficient 0.994. No shift of anodic peak potentials of AA and CYS were found. The detection limit of CYS is found to be 0.020 μM (S/N = 3). The performance of the fabricated CYS sensor is very much comparable to the literature values (Table 1) [48–55]. It is evident from Table 1, the newly designed Nano-Au/Poly-AHMP modified electrode exhibited relatively low detection limit, high sensitivity and wide linear range.
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Fig. 5. (A) Differential pulse voltammogramms for the simultaneous determination of a) 2, b) 10, c) 20, d) 30, e) 40, f) 50, g) 60, h) 70, i) 80, j) 90, k) 100, l) 150, m) 200, n) 300, o) 400, p) 500 μM of AA and CYS at Nano-Au/Poly-AHMP/GC electrode in 0.1 M PB solution (pH 7.0). (B) Plot of AA oxidation current vs. concentration and (C) Plot of CYS oxidation current vs. concentration. Amplitude = 0.05 V, Pulse Width = 0.2 s, Sample Width = 0.02 s and Pulse Period = 0.5 s.
3.5. Interference study The selectivity of the Nano-Au/Poly-AHMP/GCE for sensitive determination of CYS was analysed and several compounds such as biologically important amino acids and some metal ions were checked as potential interfering substance. The electrochemical response of 20 μM CYS in presence of epinephrine, norepinephrine, glycine, alanine, serine, leucine, tyrosine, lysine, tryptophan, methionine, dopamine, uric acid, glucose, K+, Ca2+, Zn2+ and Mg2+ is tabulated in Table 2. This table indicates that the coexisting substances do not interfere in the oxidation
Table 1 Comparison of some modified electrodes in the determination of CYS.
Fig. 6. Differential pulse voltammogramms for the oxidation of CYS at Nano-Au/PolyAHMP/GC electrode in different concentrations a) 20, b) 40, c) 60, d) 80, e) 100, f) 120, g) 140, h) 160, i) 180, j) 200 μM in the presence of 0.5 mM of AA in 0.1 M PBS. (Inset: Plot of CYS oxidation current vs. concentration). Amplitude = 0.05 V, Pulse Width = 0.2 s, Sample Width = 0.02 s and Pulse Period = 0.5 s.
Electrodes
Method pH Linear ranges (μM)
Detection limits (μM)
References
GO/CNTs/AuNPs@MnO2/GCE Ag-PDA/ITO Au-SH-SiO2@Cu-MOF/GCE NPG/GCE Pt/VACNTs/GCE PB-AuNPs-Pd/CPE Zinc Bismuthate NRs/GCE DMBQ/ZnO/NPs/CPE Nano-Au/Poly-AHMP/GCE
DPV LSV DPV Amp Amp CA CV SWV DPV
0.340 0.023 0.008 0.050 0.050 0.180 0.074 0.050 0.020
[48] [49] [50] [51] [52] [53] [54] [55] This work
7.0 5.0 5.0 7.0 6.5 4.0 7.0 5.0 7.0
0.01–7 0.05–300 0.02–300 1.0–400 1.0–500 0.3–400 0.01–200 0.09–340 2.0–500
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Table 2 The influences of some interferents on the peak current of 20 μM CYS oxidation at NanoAu/Poly-AHMP/GCE. Interferents
Concentration (μM)
Signal change CYS (%)
Epinephrine Norepinehrine Glycine Alanine Serine Leucine Tyrosine Lysine Tryptophan Methionine Dopamine Uric acid Glucose K+ Ca2+ Zn2+ Mg2+
50 50 50 50 50 50 50 50 50 50 50 50 50 500 500 500 500
5.7 6.1 2.2 3.5 2.0 3.0 1.5 4.5 6.2 4.4 18.0 22.1 0.8 1.3 2.1 1.5 1.4
samples were in the range of 96.50–100.25%. It is clearly observed that a good recovery was obtained suggesting the practical applicability of this electrochemical sensor. 4. Conclusion In conclusion, a novel electrochemical sensor has been fabricated by electrodeposition of Nano-Au into Poly-AHMP thin film modified glassy carbon electrode and utilized for highly sensitive, selective and stable determination of CYS. Nano-Au/Poly-AHMP/GC modified electrode was characterized by SEM, CV and EIS. This modified electrode exhibited excellent electrocatalytic activity towards CYS at physiological pH 7.0. The present electrochemical sensor successfully resolved the voltammetric signals of AA and CYS with peak separation of 0.510 V. To the best of our knowledge, this is the first report of larger peak separation between AA and CYS. The lowest detection limit of 0.020 μM (S/N = 3) was achieved for cysteine sensing at Nano-Au/Poly-AHMP/ GCE. Acknowledgements
current and peak potential of CYS significantly at modified electrode whereas modest interference of dopamine and uric acid is observed [40]. 3.6. Reproducibility, repeatability and stability of the Nano-Au/Poly-AHMP/ GCE Reproducibility, repeatability and long term stability are very essential for any electrochemical sensor. The repeatability of Nano-Au/PolyAHMP/GC electrode was examined by 10 repetitive measurements for 20 μM of CYS in 0.1 M PB solution by DPV method. The result indicated that the oxidative current of CYS remains the same with a relative standard deviation of 4.8%. In addition, the reproducibility of three modified electrodes has been evaluated under optimized condition at same concentration of CYS which gives current response with 7% of RSD. The long term stability of the modified electrode was monitored by taking DPV method at 10 days intervals for 30 days. The current response was decreased 2.8% in first 10 days, 4.5% in second 10 days and 5.1% for third 10 days from initial current response, which indicating that the NanoAu/Poly-AHMP/GC electrode exhibited long term stability. These results suggested that the modified electrode has no significant memory effect in the determination of Cysteine. However, to eliminate the memory effect of the proposed sensor, the modified electrode was renewed by potential scanning from −0.2 to 0.7 V in 0.1 M PBS (pH 7.0) for 5 cycles. 3.7. Real sample analysis To assess the practical utility of proposed sensor to the determination of CYS in real samples, urine and human blood serum were selected for analysis. Urine samples were collected from healthy volunteers and the urine samples were centrifuged. The obtained supernatants were diluted 100 times with 0.1 M phosphate buffer solution. The human blood serum samples were collected from clinical laboratory and the collected samples were diluted 10 times with 0.1 M PBS (pH 7.0). The recoveries of CYS were determined by standard addition method. The results are summarized in Table 3, which suggests that the recoveries of the spiked
Table 3 Real sample analysis. Sample
Added (μM)
Found (μM)
Recovery (%)
R.S.D (n = 3) %
Urine 1 Urine 2 Human serum 1 Human serum 2
20 40 20 40
19.9 40.1 19.3 40.0
99.50 100.25 96.50 100
1.89 1.14 3.61 2.45
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