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Amperometric l -lysine biosensor based on carboxylated multiwalled carbon nanotubes-SnO2 nanoparticles-graphene composite
Accepted Manuscript Title: Amperometric L-lysine biosensor based on carboxylated multiwalled carbon nanotubes-SnO2 nanoparticles-graphene composite Authors: Ceren Kac¸ar, Pınar Esra Erden, Esma Kılıc¸ PII: DOI: Reference:
S0169-4332(17)31433-2 http://dx.doi.org/doi:10.1016/j.apsusc.2017.05.120 APSUSC 36055
To appear in:
APSUSC
Received date: Revised date: Accepted date:
13-3-2017 3-5-2017 14-5-2017
Please cite this article as: Ceren Kac¸ar, Pınar Esra Erden, Esma Kılıc¸, Amperometric L-lysine biosensor based on carboxylated multiwalled carbon nanotubes-SnO2 nanoparticles-graphene composite, Applied Surface Sciencehttp://dx.doi.org/10.1016/j.apsusc.2017.05.120 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Amperometric L-lysine biosensor based on carboxylated multiwalled carbon nanotubes-SnO2 nanoparticles-graphene composite Ceren Kaçar, Pınar Esra Erden*, Esma Kılıç Ankara University, Faculty of Science, Department of Chemistry, Ankara, TURKEY *Corresponding author e-mail: [email protected]; Tel.: +90(312)2126720/1278; Fax: +90(312)2232395
Graphical abstract
Research Highlights
L-lysine biosensor based on c-MWCNTsSnO2graphenechitosan composite was fabricated
Wide linear range, low detection limit and excellent long term stability were achieved
L-lysine was successfully determined in dietary supplements
1
Abstract
A
novel
matrix,
carboxylated
multiwalled
carbon
nanotubes–tin
oxide
nanoparticlesgraphene‒chitosan (c-MWCNTsSnO2GR‒CS) composite, was prepared for biosensor construction. Lysine oxidase (LOx) enzyme was immobilized covalently on the surface of c-MWCNTsGRSnO2‒CS composite modified glassy carbon electrode (GCE) using N-ethyl-N-(3-dimethyaminopropyl) carbodiimide (EDC) and N-hydroxyl succinimide (NHS). Effects of electrode composition and buffer pH on biosensor response were investigated to optimize the working conditions. The biosensor exhibited wide linear range (9.9×10-7 M1.6×10-4 M), low detection limit (1.5×10-7 M), high sensitivity (55.20 μA mM-1 cm-2) and fast amperometric response (<25 s) at +0.70 V vs. Ag/AgCl. With good repeatability and longterm stability, the c-MWCNTsSnO2GRCS based biosensor offered an alternative for L-lysine biosensing. The practical applicability of the biosensor in two dietary supplements has also been addressed.
Keywords: Amperometry, biosensor, L-lysine, SnO2 nanoparticles, carbon nanotubes, graphene
Introduction
L-lysine is an essential amino acid for animal feed and human nutrition [1]. Accurate determination of L-lysine is of great importance in food industry since its level is an important indicator of the nutritional quality of food [2]. Many analytical methods such as reverse-phase liquid chromatography [3], liquid chromatography with fluorescence detection [4], amino acid analysis [5], high performance liquid chromatography [6], densitometry [7], capillary electrophoresis [8, 9], chemiluminescence [10], flow injection analysis [11] and electrochemical biosensors [1, 12, 13] have been developed for L-lysine determination. Among these methods, development of electrochemical biosensors has been the subject of considerable interest because of their high sensitivity, good selectivity, low cost and direct, rapid and simple protocols [14].
2
Since their discovery by Iijima [15] CNTs have been extensively studied for the development of electrochemical biosensors. These fascinating nanomaterials exhibit extraordinary properties such as functional surface, good conductivity, biocompatibility, high reactivity, fast electron transfer and high surface area [16]. GR, a single layer of sp2-bonded carbon atoms closely packed into a two-dimensional honeycomb arrangement, has also attracted much attention in biosensing applications due to its fast electron transfer ability, good thermal conductivity, large specific surface area and biocompatibility [17, 18]. Metal oxide nanoparticles (MONPs) is an other important group of nanomaterials that have long been used for fabricating high performance electrochemical biosensors being mainly due to their large surface-to-volume ratio, excellent electron conductance, high chemical stability, high surface reaction activity, good mechanical strength, and biocompatibility [19, 20, 21]. Tin oxide (SnO2) is a metal oxide semiconductor with a large band–gap of 3.6 eV at room temperature [22]. SnO2 nanoparticles show excellent photoelectronic properties, high gas sensitivities and relatively higher conductivity than TiO2 and SiO2 [23, 24]. Furthermore, its low cost, chemical stability and favorable electrical properties made it a promising compound in many fields [25]. Tin oxidebased materials have demonstrated great potentials in the applications of solar cells [26], lithium ion batteries [27], humidity sensors [28] and gas-sensing devices [29]. The use of SnO2 nanoparticles for several biosensing applications were also reported [22, 24, 25, 30–32]. Ansari et al. developed an electrochemical cholesterol sensor based on chitosan–SnO2 nanobiocomposite film modified indium–tin oxide glass plate [30]. Jia et al., reported the direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized on sol–gel derived nano-SnO2/gelatin composite film [24]. Liu et al., fabricated a SnO2 nanorod array-based biosensor for the electrochemical detection of hydrogen peroxide [31]. Zhou et al., used SnO2 nanoparticles–carboxylic graphene–nafion composite to develop an acetylcholinesterase biosensor for pesticide detection [32]. Ansari et al., reported an urea sensor based on urease immobilized SnO2 thin films [25]. Mahadeva and Kim developed a conductometric glucose biosensor using glucose oxidase immobilized cellulose‒SnO2 hybrid nanocomposite [22].
The nanocomposites combining different components possess properties of the individual components with a synergistic effect, leading to promising application in biosensors [33]. Various combinations of nanomaterials such as GR-MONPs [34], CNTs-MONPs [35] and CNTs-GR-MONPs composites [36] were reported to show favorable analytical characteristics. Moreover, the nanocomposites of SnO2 nanoparticles and carbon nanomaterials were reported to show better electrochemical performance than the pure SnO2 [37, 38]. 3
In continuation of our earlier work on the preparation and applications of carbon and metal oxide nanoparticle based biosensors [35, 39, 40], we have presented here a sensitive amperometric L-lysine biosensor based on SnO2 nanoparticles, GR, c-MWCNTs composite modified GCE. These nanomaterials have been selected for biosensor fabrication due to their unique properties such as, high conductivity, good catalytic efficiency and large surface area. Chitosan, a natural biopolymer with good film forming ability, biocompatibility, and nontoxicity [41] was used as an efficient matrix to disperse SnO2 nanoparticles, GR and cMWCNTs and to immobilize LOx. To the best of our knowledge, this is the first report of Llysine biosensor based on this composite. The resulting biosensor is expected to combine the advantages of c-MWCNTs, GR, and SnO2 and to show good analytical performance.
Materials and methods
Reagents
L-Lysine--oxidase from Trichoderma viride, SnO2 nanoparticles (<50 nm particle size), K3Fe(CN)6, K4Fe(CN)6.3H2O, L-methionine, chitosan (90% deacetylation, medium molecular weight), acetic acid, sodium acetate, Nafion (5 wt % in lower aliphatic alcohols) and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxy-succinimide (NHS), were obtained from Sigma-Aldrich (St. Louis, MO, USA). c-MWCNTs (O.D. 8 nm and length 1030 m) were purchased from Cheap Tubes Inc. (Brattleboro, USA). Graphene solution (in N,NDimethylformamide and deionized water 2 mg mL-1) was purchased from Dropsens (Llanera, Spain). Boric acid, phosphoric acid, L-arginine, L-asparagine monohydrate, L-glutamic acid, L-glutamine, L-isoleucine, L-leucine, L-phenylalanine, L-proline, L-serine, L-threonine, Lornithine and L-valine were purchased from Merck. The supporting electrolyte used in this study was 0.025 M Britton-Robinson (BR) buffer solution, pH 10.0. All aqueous solutions were prepared with deionized water (ELGA Purelab Option-S).
Apparatus and Measurements
Electrochemical measurements were conducted using Metrohm Autolab electrochemical system (Eco-Chemie, Utrecht, The Netherlands) equipped with BASi C3 cell stand (Bioanalytical Systems, Inc., USA) and NOVA software (Eco-Chemie, Utrecht, The 4
Netherlands). A platinum wire auxilary electrode, an Ag/AgCl (3 M KCl) reference electrode and a glassy carbon working electrode (GCE, 3 mm in diameter) were used for the electrochemical measurements. The amperometric response measurements were carried out at +0.70 V versus Ag/AgCl in BR buffer solution (pH 10.0). Scanning electron microscopy (SEM) images were obtained on a Carl Zeiss, EVO LS model scanning electron microscope (Carl Zeiss SMT Ltd. 511 Coldhams Lane Cambridge UK). The energy dispersive X-ray spectroscopy (EDX) was performed with a Bruker detector. X-ray diffraction (XRD) analysis was performed using Bruker D.8 advance diffractometer with Cu–K1 radiation. Preparation of LOx/c-MWCNTsSnO2GR‒CS/GCE
Scheme 1 illustrates the schematic representation of the procedure for the fabrication of the proposed biosensor. Prior to modification, GCE surface was polished with 0.05 μm Al2O3 slurry and then ultrasonically cleaned in ethanol and deionized water, successively. A chitosan (CS) solution was obtained by dissolving 0.050 g of chitosan in 10.0 mL of acetate buffer solution (pH 5.0) and stirring at room temperature until complete dissolution was achieved. 2 mg of SnO2 nanoparticles and, 8 mg of c-MWCNTs were added into 1 mL of CS solution and the resulting mixture was ultrasonicated for 2 h, in order to obtain a homogenous dispersion. 300 μL of this mixture (c-MWCNTsSnO2‒CS) was mixed with 600 μL of GR solution (2 mg mL1
). c-MWCNTsSnO2‒GRCS suspension was stable for up to two months. This suspension
was sonicated for 10 min before every use. A 9 μL aliquot of c-MWCNTsSnO2GR‒CS composite was drop-casted onto the surface of GCE, and then this electrode was allowed to dry at room temperature. LOx was covalently immobilized onto c-MWCNTsSnO2GR‒CS/GCE using EDC−NHS. For this purpose, 5 μL of 50 mM EDC–200 mM NHS solution (in 0.025 M BR solution) prepared immediately before use was dropped on the c-MWCNTsSnO2GR‒ CS/GCE modified electrode surface to activate the functional groups of c-MWCNTs. 4 µL of LOx (0.10 Units μL−1) was casted onto the electrode surface and left to dry. The resultant enzyme electrode was rinsed with BR buffer solution to remove free enzymes. Finally, 5 μL (0.025%) of Nafion was casted on the enzyme electrode and dried. The L-lysine biosensor was stored at 4 C for further use.
Results and discussion
5
Surface morphology of c-MWCNTsSnO2GR‒CS/GCE
SEM, XRD and EDX analysis were performed to investigate the morphology and structure of the c-MWCNTsSnO2GR‒CS composite. Figure 1 depicts the SEM images of (a) GR‒ CS/GCE,
(b)
SnO2CS/GCE,
(c)
c-MWCNTs‒CS/GCE
and
(d)
c-
MWCNTsSnO2GRCS/GCE. Figure 1ac indicates that graphene, SnO2 nanoparticles or cMWCNTs were well dispersed in CS matrix. Figure 1d shows that c-MWCNTs were the dominant structure of the surface morphology of c-MWCNTsSnO2GR composite. The porous structure of the composite is a promising platform for enzyme immobilization. Elemental composition of the c-MWCNTsSnO2GRCS composite was examined by EDX (Figure 1e). The Sn, N, O and C peaks confirmed the composite contained these elements. Moreover, in Figure 1(f) energy dispersive spectroscopy mapping image of the cMWCNTsSnO2GRCS composite showed the elements of Sn, N, O and C were homogeneously distributed in the resulting composite. Figure 1(g) shows the XRD patterns of c-MWCNTsSnO2GRCS composite. The diffraction peaks observed at about 26.7°, 33.9°, 38.0°, 51.9°, 54.9°, 61.9°, 66.0° and 78.7° are well indexed to the tetragonal structure of SnO2 (Powder Diffraction File PDF 00-041-1445).
Electrochemical studies of c-MWCNTsSnO2GR‒CS composite
Figure 2 illustrates the cyclic voltammograms (CVs) of CS/GCE, GRCS/GCE, SnO2CS/GCE, c-MWCNTsCS/GCE and c-MWCNTsSnO2GRCS/GCE in 0.1 M KCl aqueous solution containing 5.0 mM [Fe(CN)6]3-/4- at 100 mVs−1. Peak separations and peak currents obtained in this study are listed in Table 1. As shown in curve a CS/GCE has a couple of redox peaks corresponding to ferri/ferrocyanide redox probe. Comparing SnO2CS/GCE (curve b) or GRCS/GCE (curve c) with CS/GCE, the redox peak currents increased and peakto-peak separation (Ep) decreased indicating the facilitated electron transfer and higher electroactive surface area of the modified electrodes. After casting c-MWCNTsCS (curve d) onto the GCE surface, a remarkable current increase was observed which can be attributed to the large surface area of c-MWCNTs. The c-MWCNTsSnO2GRCS composite (curve e) 6
modified GCE, showed the highest peak current. This further indicates that cMWCNTsSnO2GRCS composite modified GCE has larger electroactive surface area than the other electrodes and it could provide more conduction pathways for faster kinetics. The average value of the electroactive surface area for CS/GCE, GRCS/GCE, SnO2CS/GCE, c-MWCNTsCS/GCE and c-MWCNTsSnO2GRCS/GCE were found to be 0.048 cm2, 0.145 cm2, 0.127 cm2, 0.185 cm2, and 0.244 cm2, respectively using the Randles–Sevick equation [42]: Ip=2.69×105AD 1/2n3/2v½C where n is the number of electrons involved in the redox reaction, A is the electrode area (cm2), D is the diffusion coefficient of the molecule in solution (cm2 s−1), C is the concentration of the probe molecule in the bulk solution (mol cm−3), and v is the scan rate (V s−1). cMWCNTsSnO2GRCS/GCE exhibited the highest electroactive surface area. The electroactive surface area for this electrode was 1.32 times higher than that of cMWCNTCS/GCE and 5.08 times higher than the one for CS/GCE. These observations also suggest that the c-MWCNTsSnO2GRCS composite provided a large surface area for immobilization of the LOx.
Figure 2(B) depicts the CVs of the c-MWCNTsSnO2GR‒CS/GCE recorded in 0.025 M BR buffer (pH 10.0) in the absence (curve a) and presence of 0.50 M H2O2 (curve b). The cMWCNTsSnO2GR‒CS/GCE exhibits significant electrocatalysis to the oxidation and reduction of H2O2 starting around +0.50 V. The good performance of the fabricated cMWCNTsSnO2GR‒CS/GCE toward the oxidation of H2O2 makes it attractive for L-lysine sensing applications. Electrochemical impedance spectroscopy (EIS) studies of GRCS/GCE, SnO2CS/GCE, cMWCNTsCS/GCE and c-MWCNTsSnO2GRCS/GCE were performed in 5.0 mM [Fe(CN)6]3-/4- with 0.10 M KCl solution in the frequency range, 0.05–105 Hz with 10 mV as the amplitude. In the impedance spectra presented as Nyquist plots, the semicircle part corresponds to the electron transfer limited process and its diameter is equal to the electron transfer 7
resistance, Rct, which can be used to describe the interface properties [43]. Figure 3 presents the the EIS of different modified GCEs. The Rct (95.0 ) for SnO2CS/GCE composite (curve b) is smaller than that of bare GCE (4630 ) (data not shown) indicating that the electron transfer in the SnO2CS/GCE composite film is easier between the solution and electrode. The results for
GRCS/GCE
(43.5
),
c-MWCNTsCS/GCE
(15.0
)
and
c-
MWCNTsSnO2GRCS/GCE (7.5 ) are shown in Figure 3a, c, and d, respectively. The semicircle diameter of the c-MWCNTsCS/GCE and GRCS/GCE decreased due to the facilitated electron transfer by GR or c-MWCNTs [44, 45]. The EIS of the cMWCNTsSnO2GRCS/GCE showed that the semicircle diameter decreased indicating that c-MWCNTsSnO2GRCS composite may provide higher electron conduction pathways than GR‒CS, c-MWCNTs‒CS and SnO2‒CS. Optimization of the electrode composition and buffer pH
In order to optimize the amounts of LOx, c-MWCNTs, SnO2 and GR, different enzyme electrodes were prepared by changing the amount of each component and keeping other parameters constant. Amperometric responses of these enzyme electrodes were recorded in 5.0 mL 0.025 M BR buffer solution (pH 10.0) containing 0.09 mM L-lysine.
To investigate the influence of the c-MWCNTs amount on the enzyme electrode response, different c-MWCNTs amounts as 6, 12, 18, 24, and 30 μg were used for the construction of enzyme electrode. The response current of the enzyme electrode increased with the increase in c-MWCNTs amount until 24 μg c-MWCNTs. Further increase of c-MWCNTs amount did not improve the amperometric response, indicating that the response current of the enzyme electrode reached the highest although excess amount of c-MWCNTs was used. GR amount was increased from 6.0 μg to 15.0 μg for the optimization study. Optimum response current was obtained with 12 μg GR. The effect of SnO2 amount towards the determination of L-lysine was also examined in order to improve the response of the proposed biosensor. SnO2 amount was varied as 6, 12, 18 and 24 μg and amperometric response was measured. The highest response current was achieved with 6 μg SnO2. In conclusion, 24 μg of c-MWCNTs, 12 μg of GR and 6 μg of SnO2 were selected as the optimum values for electrode composition and used for the construction of LOx/c-MWCNTsSnO2GRCS/GCE.
8
The optimization of enzyme amount on the electrode surface is important to achieve the highest sensitivity of the biosensor. Therefore, the effect of enzyme loading on the biosensor response was investigated. Five different LOx/c-MWCNTsSnO2GRCS/GCEs containing 0.2, 0.4, 0.6 and 0.8 U LOx were fabricated and their response to L-lysine was determined. The amperometric response improved by increasing the loading of LOx from 0.2 to 0.4 U (Figure 4A). Higher enzyme loadings, gave a biosensor with a reduced response. Hence, L-lysine biosensor which was prepared with 0.4 U LOx content was used for further experiments.
The pH of the working buffer has an important influence on the performance of enzyme-based biosensors since the activity of the enzymes are pH dependent [46]. Especially, when working with amino acids the interaction between the related enzyme and aminoacid is closely associated with the form of the aminoacid and the charge on the enzyme. Because amino acids may be present in different forms (cationic, anionic or the zwitter ion form) based on their isoelectric points (pI) and the pH [47]. The pH dependency of biosensor was tested by BR buffer solution between pH 5.0 and 12.0 in the presence of 0.09 mM L-lysine (Figure 4B). The biosensor response increased gradually from pH 5.0 to 10 and then dramatically decreased. The highest biosensor response was achieved at pH 10.0. The isoelectric point of LyOx (4.35) indicates that the enzyme is in its negative charged form at pH 10.0. On the other hand, the mol fractions of the various forms of L-lysine were calculated for each pH value using its dissociation constants and the species H3N+(CH2)4CH(NH2)COO- was found to be the most abundant form at pH 10.0. It can be assumed that this form is the one that interacts most easily with negative charged LyOx. Similar increase in the activity of the LyOx in weakly basic media and higher electrode responses at high pH values [48, 49] were also reported in the literature. Analytical performance of the biosensor Fig. 5(A) depicts the current–time curve obtained with LOx/c-MWCNTsSnO2GRCS/GCE for increasing concentrations of L-lysine in 0.025 M BR buffer (pH 10.0) at +0.70 V. The Llysine biosensor has a rapid response to the each addition of L-lysine, which is less than 25 s (95% of the steady-state current). Figure 5(B) inset displays the calibration plot of the biosensor. The linear range of L-lysine was from 9.9×10-7 to 1.6×10-4 M with a correlation coefficient of 0.9963. This linear range is superior to those reported in literature [1, 50‒52]. The detection limit was found to be 1.5×10-7 which is much lower than those reported based on gold-mercaptopropionic acid self-assembled monolayer modified Au electrode
9
[51], Au
nanoparticles-c-MWCNT-polyaniline modified Au electrode [1] and Pt nanoparticlespoly(vinylferrocene) modified Pt electrode [52]. Moreover, the L-lysine biosensor has a high sensitivity of 55.20 μA mM-1 cm-2 (electrode surface area 0.065 cm2). The apparent Michaelis-Menten constant (KMapp), a reflection of the enzymatic affinity, was calculated from the Lineweaver–Burk equation [53]. The KMapp value for the biosensor was estimated to be 0.20 mM. The value is much lower than previously reported 1.70 mM for LOx immobilized on Pt electrode by co-crosslinking [54], 1.58 and 1.63 mM for LOx on a Pt nanoparticles-poly(vinylferrocene) modified Pt electrode and poly(vinylferrocene) modified Pt electrode [52], and in good agreement with the value of 0.33 mM for the covalent immobilization of LOx on gold–platinum nanoparticles modified Au electrode [2]. These results show that the biosensor possesses higher biological affinity to L-lysine.
The repeatability, reproducibility and long-term stability are crucial parameters for the evaluation of the performance of a biosensor. The repeatability of the L-lysine biosensor was studied by determining its calibration sensitivity. The results of five successive calibration plots showed a satisfactory repeatability with a relative standard deviation (R.S.D.) of 5.9%. The biosensor-to-biosensor reproducibility was also investigated from the calibration sensitivities of five different electrodes made independently by the same fabrication procedure. The R.S.D. was 3.8%, confirming the high reproducibility of the preparation method. The long-term stability of the biosensor was tested over a 1-month period. The response of the biosensor was evaluated by performing measurements of 0.09 mM L-lysine in BR buffer solution every 3 days. The biosensor was kept in a BR buffer (pH 10.0) at 4 ◦C when not in use. The biosensor lost about 3%, 5% and 6% of the initial response after 6, 15 and 30 days, respectively, indicating that the biosensor has a very good long-term stability. Good long-term stability can be attributed to the following aspects: (i) The covalent interaction between the c-MWCNTsSnO2GRCS composite and LOx with EDCNHS is strong and little enzyme has leaked out from the electrode surface, (ii) the biocompatible microenviroment provided by the chitosan based cMWCNTsSnO2GR composite is quite efficient for retaining the activity of LOx [32] and (iii) the Nafion membrane also minimized enzyme leakage from the electrodes surface and prolong the stability of the biosensor.
L-lysine-(α)-oxidase catalyzes the oxidation of L-lysine to 2-oxo-6-aminocaproate with the production of H2O2 and NH3. However, the ability of LOx to catalyze the oxidation of various 10
amino acids to varying extents was also reported [55]. Various studies have highlighted that the interfering effect of other amino acids is the basic problem of the selectivity of most L-lysine biosensors based on LOx [13, 50, 56, 57]. Therefore, it is important to investigate the effect of various amino acids on the biosensor response. The selectivity of the LOx/cMWCNTsSnO2GR‒CS/GCE was evaluated amperometrically in the presence of other 13 amino acids. Amperometric responses were obtained in 0.025 M BR buffer solution (pH 10.0) at +0.70 V with sequential additions of 0.02 mM L-lysine with 0.02 mM interferents. The interference effect was evaluated as the percentage of the current response, obtained for determining 0.02 mM L-lysine, which was contributed by the addition of a particular interferent. Table 2 shows the results of the selectivity study. L-valine, L-leucine, L-isoleucine, L-serin, L-threonin, L-asparagine, L-glutamine and L-glutamic acid did not cause any significant interference (<5%) while L-methionine, DL-phenyalanine, L-proline and Lornithine caused 7.3%, 10.2%, 12.9% and 15.6% interference on the response of LOx/cMWCNTsSnO2GR‒CS/GCE respectively. The highest interference effect was observed for L-arginine (31.8%). Significant interference effects of ornithine (from 28% to 90%) [56‒58], arginine (from 14.7% to 32.4%) [50, 57, 58], methionine (18.7%) [50] and phenyalanine (from 8.6% to 15.5%) [50, 58] were also reported in the literature.
Real Sample Analysis
To illustrate the feasibility of the L-lysine biosensor in real sample analysis, it was employed to determine L-lysine in two different dietary supplements. L-lysine tablets which contains 730 mg L-lysine per tablet were used as the Sample 1. Five tablets were weighed and powdered finely in a mortar. The powder equivalent to one tablet was weighed and transferred to a 100.0 mL calibrated flask. 80 mL of deionized water was added to this flask and sonicated for 1 hour. The content of flask was centrifuged at 4000 rpm for 5 min and the clear supernatant liquor was quantitatively diluted to 100.0 mL with deionized water. The final solution was diluted 1:10 with BR buffer (pH 10.0). Aliquots from stock L-lysine tablet solution were transferred to electrochemical cell containing 5.0 mL of BR buffer solution and direct calibration method was applied to determine the L-lysine content. L-lysine in Sample 1 determined by LOx/c11
MWCNTsSnO2GR‒CS/GCE, was 731.18.5 mg tablet-1. The content of L-lysine measured by LOx/c-MWCNTsSnO2GR‒CS/GCE correspond to the average of 5 replicate assays and the mean recovery was found as 100.11.2%.
Sample 2 was a multi amino acid capsule which contains 75 mg of each amino acids namely Llysine.HCl, L-leucine, L-histidine, L-isoleucine, L-methionine, L-phenyalaline, L-threonine and L-valine. Each capsule corresponds to 60.0 mg of L-lysine. Since L-methionine and Lphenyalaline present in this capsule showed an intererence effect of 7.3% and 10.2%, on biosensor response respectively, standard addition method was used to determine the L-lysine content in Sample 2 in order to minimize the matrix effects. Five capsules were weighed and mixed finely in a mortar. The amount equivalent to one capsule was taken, dissolved in deionized water by sonication for 1 hour and centrifuged at 4000 rpm for 5 min. The clear supernatant liquor was quantitatively diluted to 100.0 mL with deionized water. The final solution was diluted 1:10 with BR buffer (pH 10.0). Aliquots from stock L-lysine capsule solution were transferred to electrochemical cell containing 5.0 mL of BR buffer solution. Then, additions of standard L-lysine solution were made to the electrochemical cell and a multiple addition calibration curve was obtained. L-lysine concentration in capsule solution was calculated from this calibration curve and found to be 62.0±0.6 mg capsule-1 (N=5) with a mean recovery of 103.4±1.1%. The results given above illustrate the ability to employ LOx/c-MWCNTsSnO2GRCS/GCE for L-lysine determination in dietary supplements without any interference effect of various amino acids.
Conclusion The results demostrated that the composite of c-MWCNTsSnO2GR‒CS is an efficient platform for L-lysine biosensor fabrication providing an excellent enviroment for LOx immobilization. The presence of the c-MWCNTs, SnO2 nanoparticles and GR promoted the electron transfer so as to improve the sensitivity of the L-lysine biosensor. The high sensitivity, low detection limit, wide working range, good reproducibility and excellent long-term stability make this biosensor a suitable device for L-lysine determination. The biosensor has satisfied performance for L-lysine determination in dietary supplements with very good recoveries, 12
despite of the potential amino acid interferences. The biosensor developed did therefore provide a promising method for monitoring L-lysine in different food samples. Moreover, the proposed composite can be used as a new electrochemical platform for designing a variety of biosensors.
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17
Scheme 1. The fabrication procedure for the LOx/c-MWCNTsSnO2GRCS/GCE.
(a)
(b)
(c)
(d)
18
(e)
(f)
1400 (g)
1200 (110)
200
(321)
400
(310) (301)
600
(211) (220)
(101)
800
(200)
Intensity
1000
0 0
20
40 60 2θ (degree)
80
100
Figure 1. SEM images of (a) GR‒CS/GCE, (b) SnO2‒CS/GCE, (c) c-MWCNTs‒CS/GCE, (d) c-MWCNTsSnO2GRCS/GCE (10 μm; EHT = 20.00 kV; Mag = ×1.00 KX). (e) EDX spectra, (f) elementel mapping for the elements Sn, N, O, C and (g) XRD patterns of cMWCNTs‒SnO2‒GR‒CS composite.
19
300
400 (A)
(B) b
250
100
Current, µA
Current, µA
200
0 a ↓ e
-100 -200
100 a
-50
-300 -400
-200 -1
-0.5
0 0.5 Potential, V
1
1.5
-1
-0.5
0 0.5 Potential, V
1
1.5
Figure 2. (A) CVs of (a) CS/GCE, (b) SnO2CS/GCE, (c) GRCS/GCE, (d) cMWCNTsCS/GCE and (e) c-MWCNTsSnO2GRCS/GCE in 0.10 M KCl aqueous solution containing 5.0 mM [Fe(CN)6]3-/4- at 100 mV s−1. (B) CVs of the c-MWCNTsSnO2GR/GCE electrode in 0.025 M BR buffer solution (pH 10.0) in the (a) absence and (b) presence of 0.50 mM H2O2.
150
(a) (b) (c) (d)
-Z'' (Ω)
120 90
ᴏ □ Δ җ
60 30 0 50
150 Z' (Ω)
250
Figure 3. The Nyquist curves of (a) GRCS/GCE, (b) SnO2CS/GCE, (c) cMWCNTsCS/GCE and (d) c-MWCNTsSnO2GRCS/GCE in 0.10 M KCl aqueous solution containing 5.0 mM [Fe(CN)6]3-/4-.
20
0.25
0.8 (A)
(B) 0.6
ΔI, µA
ΔI, µA
0.20 0.4
0.15 0.2 0.10
0 0
0.2
0.4 0.6 Enzyme, U
0.8
1
4
6
8 pH
10
12
Figure 4. The effect of (A) enzyme loading and (B) buffer pH on the response of L-lysine biosensor (Error bars show standard deviation of three measurements).
2.0
1.8 705 µM
(A)
(B)
410 µM
1.5
160 µM
1.0
55 µM
0.5
0.8
ΔI, µA
I, µA
1.2
y = 3.60x + 0.01 R² = 0.9963
0.6 0.4
0.6
0.2
14 µM
0 0
0.0
0.1
0.2
0.0 0
500 1000 1500 2000 2500 Time, s
0.0
0.5 1.0 1.5 Concentration, mM
2.0
Figure 5. (A) Typical current-time response of the LOx/c-MWCNTsSnO2GRCS/GCE to successive addition of L-lysine into a stirred solution of 0.025 M BR buffer solution at (pH 10.0) +0.70 V (B) The effect of the L-lysine concentration on biosensor response. Inset: calibration curve of the biosensor (Error bars show standard deviation of three measurements).
21
Table 1. Peak separations and peak currents for different electrodes in 5 mM [Fe(CN)6]4-/3- with 0.1 M KCl at 100 mVs-1. Electrodes
ipa, μA
ipc, µA
ΔEp, V
CS/GCE GRCS/GCE SnO2CS/GCE c-MWCNTsCS/GCE c-MWCNTsSnO2GRCS/GCE
54.0 158.0 139.7 202.4 268.0
-55.0 -201.4 -167.5 -262.6 -317.1
0.37 0.15 0.21 0.17 0.14
Table 2. Selectivity of LOx/c-MWCNTsSnO2GRCS/GCE Amino acid Intereference %* Valine 1.1 Leucine 1.1 Isoleucine 4.6 Phenyalanine 10.2 Methionine 7.3 Proline 12.9 Serine 1.6 * Values are the average of three determinations
22
Amino acid Threonin Asparagine Glutamine Glutamic acid Arginine Ornithine
Intereference %* -2.9 -2.9 1.2 1.0 31.8 15.6
S0169-4332(17)31433-2 http://dx.doi.org/doi:10.1016/j.apsusc.2017.05.120 APSUSC 36055
To appear in:
APSUSC
Received date: Revised date: Accepted date:
13-3-2017 3-5-2017 14-5-2017
Please cite this article as: Ceren Kac¸ar, Pınar Esra Erden, Esma Kılıc¸, Amperometric L-lysine biosensor based on carboxylated multiwalled carbon nanotubes-SnO2 nanoparticles-graphene composite, Applied Surface Sciencehttp://dx.doi.org/10.1016/j.apsusc.2017.05.120 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Amperometric L-lysine biosensor based on carboxylated multiwalled carbon nanotubes-SnO2 nanoparticles-graphene composite Ceren Kaçar, Pınar Esra Erden*, Esma Kılıç Ankara University, Faculty of Science, Department of Chemistry, Ankara, TURKEY *Corresponding author e-mail: [email protected]; Tel.: +90(312)2126720/1278; Fax: +90(312)2232395
Graphical abstract
Research Highlights
L-lysine biosensor based on c-MWCNTsSnO2graphenechitosan composite was fabricated
Wide linear range, low detection limit and excellent long term stability were achieved
L-lysine was successfully determined in dietary supplements
1
Abstract
A
novel
matrix,
carboxylated
multiwalled
carbon
nanotubes–tin
oxide
nanoparticlesgraphene‒chitosan (c-MWCNTsSnO2GR‒CS) composite, was prepared for biosensor construction. Lysine oxidase (LOx) enzyme was immobilized covalently on the surface of c-MWCNTsGRSnO2‒CS composite modified glassy carbon electrode (GCE) using N-ethyl-N-(3-dimethyaminopropyl) carbodiimide (EDC) and N-hydroxyl succinimide (NHS). Effects of electrode composition and buffer pH on biosensor response were investigated to optimize the working conditions. The biosensor exhibited wide linear range (9.9×10-7 M1.6×10-4 M), low detection limit (1.5×10-7 M), high sensitivity (55.20 μA mM-1 cm-2) and fast amperometric response (<25 s) at +0.70 V vs. Ag/AgCl. With good repeatability and longterm stability, the c-MWCNTsSnO2GRCS based biosensor offered an alternative for L-lysine biosensing. The practical applicability of the biosensor in two dietary supplements has also been addressed.
Keywords: Amperometry, biosensor, L-lysine, SnO2 nanoparticles, carbon nanotubes, graphene
Introduction
L-lysine is an essential amino acid for animal feed and human nutrition [1]. Accurate determination of L-lysine is of great importance in food industry since its level is an important indicator of the nutritional quality of food [2]. Many analytical methods such as reverse-phase liquid chromatography [3], liquid chromatography with fluorescence detection [4], amino acid analysis [5], high performance liquid chromatography [6], densitometry [7], capillary electrophoresis [8, 9], chemiluminescence [10], flow injection analysis [11] and electrochemical biosensors [1, 12, 13] have been developed for L-lysine determination. Among these methods, development of electrochemical biosensors has been the subject of considerable interest because of their high sensitivity, good selectivity, low cost and direct, rapid and simple protocols [14].
2
Since their discovery by Iijima [15] CNTs have been extensively studied for the development of electrochemical biosensors. These fascinating nanomaterials exhibit extraordinary properties such as functional surface, good conductivity, biocompatibility, high reactivity, fast electron transfer and high surface area [16]. GR, a single layer of sp2-bonded carbon atoms closely packed into a two-dimensional honeycomb arrangement, has also attracted much attention in biosensing applications due to its fast electron transfer ability, good thermal conductivity, large specific surface area and biocompatibility [17, 18]. Metal oxide nanoparticles (MONPs) is an other important group of nanomaterials that have long been used for fabricating high performance electrochemical biosensors being mainly due to their large surface-to-volume ratio, excellent electron conductance, high chemical stability, high surface reaction activity, good mechanical strength, and biocompatibility [19, 20, 21]. Tin oxide (SnO2) is a metal oxide semiconductor with a large band–gap of 3.6 eV at room temperature [22]. SnO2 nanoparticles show excellent photoelectronic properties, high gas sensitivities and relatively higher conductivity than TiO2 and SiO2 [23, 24]. Furthermore, its low cost, chemical stability and favorable electrical properties made it a promising compound in many fields [25]. Tin oxidebased materials have demonstrated great potentials in the applications of solar cells [26], lithium ion batteries [27], humidity sensors [28] and gas-sensing devices [29]. The use of SnO2 nanoparticles for several biosensing applications were also reported [22, 24, 25, 30–32]. Ansari et al. developed an electrochemical cholesterol sensor based on chitosan–SnO2 nanobiocomposite film modified indium–tin oxide glass plate [30]. Jia et al., reported the direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized on sol–gel derived nano-SnO2/gelatin composite film [24]. Liu et al., fabricated a SnO2 nanorod array-based biosensor for the electrochemical detection of hydrogen peroxide [31]. Zhou et al., used SnO2 nanoparticles–carboxylic graphene–nafion composite to develop an acetylcholinesterase biosensor for pesticide detection [32]. Ansari et al., reported an urea sensor based on urease immobilized SnO2 thin films [25]. Mahadeva and Kim developed a conductometric glucose biosensor using glucose oxidase immobilized cellulose‒SnO2 hybrid nanocomposite [22].
The nanocomposites combining different components possess properties of the individual components with a synergistic effect, leading to promising application in biosensors [33]. Various combinations of nanomaterials such as GR-MONPs [34], CNTs-MONPs [35] and CNTs-GR-MONPs composites [36] were reported to show favorable analytical characteristics. Moreover, the nanocomposites of SnO2 nanoparticles and carbon nanomaterials were reported to show better electrochemical performance than the pure SnO2 [37, 38]. 3
In continuation of our earlier work on the preparation and applications of carbon and metal oxide nanoparticle based biosensors [35, 39, 40], we have presented here a sensitive amperometric L-lysine biosensor based on SnO2 nanoparticles, GR, c-MWCNTs composite modified GCE. These nanomaterials have been selected for biosensor fabrication due to their unique properties such as, high conductivity, good catalytic efficiency and large surface area. Chitosan, a natural biopolymer with good film forming ability, biocompatibility, and nontoxicity [41] was used as an efficient matrix to disperse SnO2 nanoparticles, GR and cMWCNTs and to immobilize LOx. To the best of our knowledge, this is the first report of Llysine biosensor based on this composite. The resulting biosensor is expected to combine the advantages of c-MWCNTs, GR, and SnO2 and to show good analytical performance.
Materials and methods
Reagents
L-Lysine--oxidase from Trichoderma viride, SnO2 nanoparticles (<50 nm particle size), K3Fe(CN)6, K4Fe(CN)6.3H2O, L-methionine, chitosan (90% deacetylation, medium molecular weight), acetic acid, sodium acetate, Nafion (5 wt % in lower aliphatic alcohols) and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxy-succinimide (NHS), were obtained from Sigma-Aldrich (St. Louis, MO, USA). c-MWCNTs (O.D. 8 nm and length 1030 m) were purchased from Cheap Tubes Inc. (Brattleboro, USA). Graphene solution (in N,NDimethylformamide and deionized water 2 mg mL-1) was purchased from Dropsens (Llanera, Spain). Boric acid, phosphoric acid, L-arginine, L-asparagine monohydrate, L-glutamic acid, L-glutamine, L-isoleucine, L-leucine, L-phenylalanine, L-proline, L-serine, L-threonine, Lornithine and L-valine were purchased from Merck. The supporting electrolyte used in this study was 0.025 M Britton-Robinson (BR) buffer solution, pH 10.0. All aqueous solutions were prepared with deionized water (ELGA Purelab Option-S).
Apparatus and Measurements
Electrochemical measurements were conducted using Metrohm Autolab electrochemical system (Eco-Chemie, Utrecht, The Netherlands) equipped with BASi C3 cell stand (Bioanalytical Systems, Inc., USA) and NOVA software (Eco-Chemie, Utrecht, The 4
Netherlands). A platinum wire auxilary electrode, an Ag/AgCl (3 M KCl) reference electrode and a glassy carbon working electrode (GCE, 3 mm in diameter) were used for the electrochemical measurements. The amperometric response measurements were carried out at +0.70 V versus Ag/AgCl in BR buffer solution (pH 10.0). Scanning electron microscopy (SEM) images were obtained on a Carl Zeiss, EVO LS model scanning electron microscope (Carl Zeiss SMT Ltd. 511 Coldhams Lane Cambridge UK). The energy dispersive X-ray spectroscopy (EDX) was performed with a Bruker detector. X-ray diffraction (XRD) analysis was performed using Bruker D.8 advance diffractometer with Cu–K1 radiation. Preparation of LOx/c-MWCNTsSnO2GR‒CS/GCE
Scheme 1 illustrates the schematic representation of the procedure for the fabrication of the proposed biosensor. Prior to modification, GCE surface was polished with 0.05 μm Al2O3 slurry and then ultrasonically cleaned in ethanol and deionized water, successively. A chitosan (CS) solution was obtained by dissolving 0.050 g of chitosan in 10.0 mL of acetate buffer solution (pH 5.0) and stirring at room temperature until complete dissolution was achieved. 2 mg of SnO2 nanoparticles and, 8 mg of c-MWCNTs were added into 1 mL of CS solution and the resulting mixture was ultrasonicated for 2 h, in order to obtain a homogenous dispersion. 300 μL of this mixture (c-MWCNTsSnO2‒CS) was mixed with 600 μL of GR solution (2 mg mL1
). c-MWCNTsSnO2‒GRCS suspension was stable for up to two months. This suspension
was sonicated for 10 min before every use. A 9 μL aliquot of c-MWCNTsSnO2GR‒CS composite was drop-casted onto the surface of GCE, and then this electrode was allowed to dry at room temperature. LOx was covalently immobilized onto c-MWCNTsSnO2GR‒CS/GCE using EDC−NHS. For this purpose, 5 μL of 50 mM EDC–200 mM NHS solution (in 0.025 M BR solution) prepared immediately before use was dropped on the c-MWCNTsSnO2GR‒ CS/GCE modified electrode surface to activate the functional groups of c-MWCNTs. 4 µL of LOx (0.10 Units μL−1) was casted onto the electrode surface and left to dry. The resultant enzyme electrode was rinsed with BR buffer solution to remove free enzymes. Finally, 5 μL (0.025%) of Nafion was casted on the enzyme electrode and dried. The L-lysine biosensor was stored at 4 C for further use.
Results and discussion
5
Surface morphology of c-MWCNTsSnO2GR‒CS/GCE
SEM, XRD and EDX analysis were performed to investigate the morphology and structure of the c-MWCNTsSnO2GR‒CS composite. Figure 1 depicts the SEM images of (a) GR‒ CS/GCE,
(b)
SnO2CS/GCE,
(c)
c-MWCNTs‒CS/GCE
and
(d)
c-
MWCNTsSnO2GRCS/GCE. Figure 1ac indicates that graphene, SnO2 nanoparticles or cMWCNTs were well dispersed in CS matrix. Figure 1d shows that c-MWCNTs were the dominant structure of the surface morphology of c-MWCNTsSnO2GR composite. The porous structure of the composite is a promising platform for enzyme immobilization. Elemental composition of the c-MWCNTsSnO2GRCS composite was examined by EDX (Figure 1e). The Sn, N, O and C peaks confirmed the composite contained these elements. Moreover, in Figure 1(f) energy dispersive spectroscopy mapping image of the cMWCNTsSnO2GRCS composite showed the elements of Sn, N, O and C were homogeneously distributed in the resulting composite. Figure 1(g) shows the XRD patterns of c-MWCNTsSnO2GRCS composite. The diffraction peaks observed at about 26.7°, 33.9°, 38.0°, 51.9°, 54.9°, 61.9°, 66.0° and 78.7° are well indexed to the tetragonal structure of SnO2 (Powder Diffraction File PDF 00-041-1445).
Electrochemical studies of c-MWCNTsSnO2GR‒CS composite
Figure 2 illustrates the cyclic voltammograms (CVs) of CS/GCE, GRCS/GCE, SnO2CS/GCE, c-MWCNTsCS/GCE and c-MWCNTsSnO2GRCS/GCE in 0.1 M KCl aqueous solution containing 5.0 mM [Fe(CN)6]3-/4- at 100 mVs−1. Peak separations and peak currents obtained in this study are listed in Table 1. As shown in curve a CS/GCE has a couple of redox peaks corresponding to ferri/ferrocyanide redox probe. Comparing SnO2CS/GCE (curve b) or GRCS/GCE (curve c) with CS/GCE, the redox peak currents increased and peakto-peak separation (Ep) decreased indicating the facilitated electron transfer and higher electroactive surface area of the modified electrodes. After casting c-MWCNTsCS (curve d) onto the GCE surface, a remarkable current increase was observed which can be attributed to the large surface area of c-MWCNTs. The c-MWCNTsSnO2GRCS composite (curve e) 6
modified GCE, showed the highest peak current. This further indicates that cMWCNTsSnO2GRCS composite modified GCE has larger electroactive surface area than the other electrodes and it could provide more conduction pathways for faster kinetics. The average value of the electroactive surface area for CS/GCE, GRCS/GCE, SnO2CS/GCE, c-MWCNTsCS/GCE and c-MWCNTsSnO2GRCS/GCE were found to be 0.048 cm2, 0.145 cm2, 0.127 cm2, 0.185 cm2, and 0.244 cm2, respectively using the Randles–Sevick equation [42]: Ip=2.69×105AD 1/2n3/2v½C where n is the number of electrons involved in the redox reaction, A is the electrode area (cm2), D is the diffusion coefficient of the molecule in solution (cm2 s−1), C is the concentration of the probe molecule in the bulk solution (mol cm−3), and v is the scan rate (V s−1). cMWCNTsSnO2GRCS/GCE exhibited the highest electroactive surface area. The electroactive surface area for this electrode was 1.32 times higher than that of cMWCNTCS/GCE and 5.08 times higher than the one for CS/GCE. These observations also suggest that the c-MWCNTsSnO2GRCS composite provided a large surface area for immobilization of the LOx.
Figure 2(B) depicts the CVs of the c-MWCNTsSnO2GR‒CS/GCE recorded in 0.025 M BR buffer (pH 10.0) in the absence (curve a) and presence of 0.50 M H2O2 (curve b). The cMWCNTsSnO2GR‒CS/GCE exhibits significant electrocatalysis to the oxidation and reduction of H2O2 starting around +0.50 V. The good performance of the fabricated cMWCNTsSnO2GR‒CS/GCE toward the oxidation of H2O2 makes it attractive for L-lysine sensing applications. Electrochemical impedance spectroscopy (EIS) studies of GRCS/GCE, SnO2CS/GCE, cMWCNTsCS/GCE and c-MWCNTsSnO2GRCS/GCE were performed in 5.0 mM [Fe(CN)6]3-/4- with 0.10 M KCl solution in the frequency range, 0.05–105 Hz with 10 mV as the amplitude. In the impedance spectra presented as Nyquist plots, the semicircle part corresponds to the electron transfer limited process and its diameter is equal to the electron transfer 7
resistance, Rct, which can be used to describe the interface properties [43]. Figure 3 presents the the EIS of different modified GCEs. The Rct (95.0 ) for SnO2CS/GCE composite (curve b) is smaller than that of bare GCE (4630 ) (data not shown) indicating that the electron transfer in the SnO2CS/GCE composite film is easier between the solution and electrode. The results for
GRCS/GCE
(43.5
),
c-MWCNTsCS/GCE
(15.0
)
and
c-
MWCNTsSnO2GRCS/GCE (7.5 ) are shown in Figure 3a, c, and d, respectively. The semicircle diameter of the c-MWCNTsCS/GCE and GRCS/GCE decreased due to the facilitated electron transfer by GR or c-MWCNTs [44, 45]. The EIS of the cMWCNTsSnO2GRCS/GCE showed that the semicircle diameter decreased indicating that c-MWCNTsSnO2GRCS composite may provide higher electron conduction pathways than GR‒CS, c-MWCNTs‒CS and SnO2‒CS. Optimization of the electrode composition and buffer pH
In order to optimize the amounts of LOx, c-MWCNTs, SnO2 and GR, different enzyme electrodes were prepared by changing the amount of each component and keeping other parameters constant. Amperometric responses of these enzyme electrodes were recorded in 5.0 mL 0.025 M BR buffer solution (pH 10.0) containing 0.09 mM L-lysine.
To investigate the influence of the c-MWCNTs amount on the enzyme electrode response, different c-MWCNTs amounts as 6, 12, 18, 24, and 30 μg were used for the construction of enzyme electrode. The response current of the enzyme electrode increased with the increase in c-MWCNTs amount until 24 μg c-MWCNTs. Further increase of c-MWCNTs amount did not improve the amperometric response, indicating that the response current of the enzyme electrode reached the highest although excess amount of c-MWCNTs was used. GR amount was increased from 6.0 μg to 15.0 μg for the optimization study. Optimum response current was obtained with 12 μg GR. The effect of SnO2 amount towards the determination of L-lysine was also examined in order to improve the response of the proposed biosensor. SnO2 amount was varied as 6, 12, 18 and 24 μg and amperometric response was measured. The highest response current was achieved with 6 μg SnO2. In conclusion, 24 μg of c-MWCNTs, 12 μg of GR and 6 μg of SnO2 were selected as the optimum values for electrode composition and used for the construction of LOx/c-MWCNTsSnO2GRCS/GCE.
8
The optimization of enzyme amount on the electrode surface is important to achieve the highest sensitivity of the biosensor. Therefore, the effect of enzyme loading on the biosensor response was investigated. Five different LOx/c-MWCNTsSnO2GRCS/GCEs containing 0.2, 0.4, 0.6 and 0.8 U LOx were fabricated and their response to L-lysine was determined. The amperometric response improved by increasing the loading of LOx from 0.2 to 0.4 U (Figure 4A). Higher enzyme loadings, gave a biosensor with a reduced response. Hence, L-lysine biosensor which was prepared with 0.4 U LOx content was used for further experiments.
The pH of the working buffer has an important influence on the performance of enzyme-based biosensors since the activity of the enzymes are pH dependent [46]. Especially, when working with amino acids the interaction between the related enzyme and aminoacid is closely associated with the form of the aminoacid and the charge on the enzyme. Because amino acids may be present in different forms (cationic, anionic or the zwitter ion form) based on their isoelectric points (pI) and the pH [47]. The pH dependency of biosensor was tested by BR buffer solution between pH 5.0 and 12.0 in the presence of 0.09 mM L-lysine (Figure 4B). The biosensor response increased gradually from pH 5.0 to 10 and then dramatically decreased. The highest biosensor response was achieved at pH 10.0. The isoelectric point of LyOx (4.35) indicates that the enzyme is in its negative charged form at pH 10.0. On the other hand, the mol fractions of the various forms of L-lysine were calculated for each pH value using its dissociation constants and the species H3N+(CH2)4CH(NH2)COO- was found to be the most abundant form at pH 10.0. It can be assumed that this form is the one that interacts most easily with negative charged LyOx. Similar increase in the activity of the LyOx in weakly basic media and higher electrode responses at high pH values [48, 49] were also reported in the literature. Analytical performance of the biosensor Fig. 5(A) depicts the current–time curve obtained with LOx/c-MWCNTsSnO2GRCS/GCE for increasing concentrations of L-lysine in 0.025 M BR buffer (pH 10.0) at +0.70 V. The Llysine biosensor has a rapid response to the each addition of L-lysine, which is less than 25 s (95% of the steady-state current). Figure 5(B) inset displays the calibration plot of the biosensor. The linear range of L-lysine was from 9.9×10-7 to 1.6×10-4 M with a correlation coefficient of 0.9963. This linear range is superior to those reported in literature [1, 50‒52]. The detection limit was found to be 1.5×10-7 which is much lower than those reported based on gold-mercaptopropionic acid self-assembled monolayer modified Au electrode
9
[51], Au
nanoparticles-c-MWCNT-polyaniline modified Au electrode [1] and Pt nanoparticlespoly(vinylferrocene) modified Pt electrode [52]. Moreover, the L-lysine biosensor has a high sensitivity of 55.20 μA mM-1 cm-2 (electrode surface area 0.065 cm2). The apparent Michaelis-Menten constant (KMapp), a reflection of the enzymatic affinity, was calculated from the Lineweaver–Burk equation [53]. The KMapp value for the biosensor was estimated to be 0.20 mM. The value is much lower than previously reported 1.70 mM for LOx immobilized on Pt electrode by co-crosslinking [54], 1.58 and 1.63 mM for LOx on a Pt nanoparticles-poly(vinylferrocene) modified Pt electrode and poly(vinylferrocene) modified Pt electrode [52], and in good agreement with the value of 0.33 mM for the covalent immobilization of LOx on gold–platinum nanoparticles modified Au electrode [2]. These results show that the biosensor possesses higher biological affinity to L-lysine.
The repeatability, reproducibility and long-term stability are crucial parameters for the evaluation of the performance of a biosensor. The repeatability of the L-lysine biosensor was studied by determining its calibration sensitivity. The results of five successive calibration plots showed a satisfactory repeatability with a relative standard deviation (R.S.D.) of 5.9%. The biosensor-to-biosensor reproducibility was also investigated from the calibration sensitivities of five different electrodes made independently by the same fabrication procedure. The R.S.D. was 3.8%, confirming the high reproducibility of the preparation method. The long-term stability of the biosensor was tested over a 1-month period. The response of the biosensor was evaluated by performing measurements of 0.09 mM L-lysine in BR buffer solution every 3 days. The biosensor was kept in a BR buffer (pH 10.0) at 4 ◦C when not in use. The biosensor lost about 3%, 5% and 6% of the initial response after 6, 15 and 30 days, respectively, indicating that the biosensor has a very good long-term stability. Good long-term stability can be attributed to the following aspects: (i) The covalent interaction between the c-MWCNTsSnO2GRCS composite and LOx with EDCNHS is strong and little enzyme has leaked out from the electrode surface, (ii) the biocompatible microenviroment provided by the chitosan based cMWCNTsSnO2GR composite is quite efficient for retaining the activity of LOx [32] and (iii) the Nafion membrane also minimized enzyme leakage from the electrodes surface and prolong the stability of the biosensor.
L-lysine-(α)-oxidase catalyzes the oxidation of L-lysine to 2-oxo-6-aminocaproate with the production of H2O2 and NH3. However, the ability of LOx to catalyze the oxidation of various 10
amino acids to varying extents was also reported [55]. Various studies have highlighted that the interfering effect of other amino acids is the basic problem of the selectivity of most L-lysine biosensors based on LOx [13, 50, 56, 57]. Therefore, it is important to investigate the effect of various amino acids on the biosensor response. The selectivity of the LOx/cMWCNTsSnO2GR‒CS/GCE was evaluated amperometrically in the presence of other 13 amino acids. Amperometric responses were obtained in 0.025 M BR buffer solution (pH 10.0) at +0.70 V with sequential additions of 0.02 mM L-lysine with 0.02 mM interferents. The interference effect was evaluated as the percentage of the current response, obtained for determining 0.02 mM L-lysine, which was contributed by the addition of a particular interferent. Table 2 shows the results of the selectivity study. L-valine, L-leucine, L-isoleucine, L-serin, L-threonin, L-asparagine, L-glutamine and L-glutamic acid did not cause any significant interference (<5%) while L-methionine, DL-phenyalanine, L-proline and Lornithine caused 7.3%, 10.2%, 12.9% and 15.6% interference on the response of LOx/cMWCNTsSnO2GR‒CS/GCE respectively. The highest interference effect was observed for L-arginine (31.8%). Significant interference effects of ornithine (from 28% to 90%) [56‒58], arginine (from 14.7% to 32.4%) [50, 57, 58], methionine (18.7%) [50] and phenyalanine (from 8.6% to 15.5%) [50, 58] were also reported in the literature.
Real Sample Analysis
To illustrate the feasibility of the L-lysine biosensor in real sample analysis, it was employed to determine L-lysine in two different dietary supplements. L-lysine tablets which contains 730 mg L-lysine per tablet were used as the Sample 1. Five tablets were weighed and powdered finely in a mortar. The powder equivalent to one tablet was weighed and transferred to a 100.0 mL calibrated flask. 80 mL of deionized water was added to this flask and sonicated for 1 hour. The content of flask was centrifuged at 4000 rpm for 5 min and the clear supernatant liquor was quantitatively diluted to 100.0 mL with deionized water. The final solution was diluted 1:10 with BR buffer (pH 10.0). Aliquots from stock L-lysine tablet solution were transferred to electrochemical cell containing 5.0 mL of BR buffer solution and direct calibration method was applied to determine the L-lysine content. L-lysine in Sample 1 determined by LOx/c11
MWCNTsSnO2GR‒CS/GCE, was 731.18.5 mg tablet-1. The content of L-lysine measured by LOx/c-MWCNTsSnO2GR‒CS/GCE correspond to the average of 5 replicate assays and the mean recovery was found as 100.11.2%.
Sample 2 was a multi amino acid capsule which contains 75 mg of each amino acids namely Llysine.HCl, L-leucine, L-histidine, L-isoleucine, L-methionine, L-phenyalaline, L-threonine and L-valine. Each capsule corresponds to 60.0 mg of L-lysine. Since L-methionine and Lphenyalaline present in this capsule showed an intererence effect of 7.3% and 10.2%, on biosensor response respectively, standard addition method was used to determine the L-lysine content in Sample 2 in order to minimize the matrix effects. Five capsules were weighed and mixed finely in a mortar. The amount equivalent to one capsule was taken, dissolved in deionized water by sonication for 1 hour and centrifuged at 4000 rpm for 5 min. The clear supernatant liquor was quantitatively diluted to 100.0 mL with deionized water. The final solution was diluted 1:10 with BR buffer (pH 10.0). Aliquots from stock L-lysine capsule solution were transferred to electrochemical cell containing 5.0 mL of BR buffer solution. Then, additions of standard L-lysine solution were made to the electrochemical cell and a multiple addition calibration curve was obtained. L-lysine concentration in capsule solution was calculated from this calibration curve and found to be 62.0±0.6 mg capsule-1 (N=5) with a mean recovery of 103.4±1.1%. The results given above illustrate the ability to employ LOx/c-MWCNTsSnO2GRCS/GCE for L-lysine determination in dietary supplements without any interference effect of various amino acids.
Conclusion The results demostrated that the composite of c-MWCNTsSnO2GR‒CS is an efficient platform for L-lysine biosensor fabrication providing an excellent enviroment for LOx immobilization. The presence of the c-MWCNTs, SnO2 nanoparticles and GR promoted the electron transfer so as to improve the sensitivity of the L-lysine biosensor. The high sensitivity, low detection limit, wide working range, good reproducibility and excellent long-term stability make this biosensor a suitable device for L-lysine determination. The biosensor has satisfied performance for L-lysine determination in dietary supplements with very good recoveries, 12
despite of the potential amino acid interferences. The biosensor developed did therefore provide a promising method for monitoring L-lysine in different food samples. Moreover, the proposed composite can be used as a new electrochemical platform for designing a variety of biosensors.
References
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Scheme 1. The fabrication procedure for the LOx/c-MWCNTsSnO2GRCS/GCE.
(a)
(b)
(c)
(d)
18
(e)
(f)
1400 (g)
1200 (110)
200
(321)
400
(310) (301)
600
(211) (220)
(101)
800
(200)
Intensity
1000
0 0
20
40 60 2θ (degree)
80
100
Figure 1. SEM images of (a) GR‒CS/GCE, (b) SnO2‒CS/GCE, (c) c-MWCNTs‒CS/GCE, (d) c-MWCNTsSnO2GRCS/GCE (10 μm; EHT = 20.00 kV; Mag = ×1.00 KX). (e) EDX spectra, (f) elementel mapping for the elements Sn, N, O, C and (g) XRD patterns of cMWCNTs‒SnO2‒GR‒CS composite.
19
300
400 (A)
(B) b
250
100
Current, µA
Current, µA
200
0 a ↓ e
-100 -200
100 a
-50
-300 -400
-200 -1
-0.5
0 0.5 Potential, V
1
1.5
-1
-0.5
0 0.5 Potential, V
1
1.5
Figure 2. (A) CVs of (a) CS/GCE, (b) SnO2CS/GCE, (c) GRCS/GCE, (d) cMWCNTsCS/GCE and (e) c-MWCNTsSnO2GRCS/GCE in 0.10 M KCl aqueous solution containing 5.0 mM [Fe(CN)6]3-/4- at 100 mV s−1. (B) CVs of the c-MWCNTsSnO2GR/GCE electrode in 0.025 M BR buffer solution (pH 10.0) in the (a) absence and (b) presence of 0.50 mM H2O2.
150
(a) (b) (c) (d)
-Z'' (Ω)
120 90
ᴏ □ Δ җ
60 30 0 50
150 Z' (Ω)
250
Figure 3. The Nyquist curves of (a) GRCS/GCE, (b) SnO2CS/GCE, (c) cMWCNTsCS/GCE and (d) c-MWCNTsSnO2GRCS/GCE in 0.10 M KCl aqueous solution containing 5.0 mM [Fe(CN)6]3-/4-.
20
0.25
0.8 (A)
(B) 0.6
ΔI, µA
ΔI, µA
0.20 0.4
0.15 0.2 0.10
0 0
0.2
0.4 0.6 Enzyme, U
0.8
1
4
6
8 pH
10
12
Figure 4. The effect of (A) enzyme loading and (B) buffer pH on the response of L-lysine biosensor (Error bars show standard deviation of three measurements).
2.0
1.8 705 µM
(A)
(B)
410 µM
1.5
160 µM
1.0
55 µM
0.5
0.8
ΔI, µA
I, µA
1.2
y = 3.60x + 0.01 R² = 0.9963
0.6 0.4
0.6
0.2
14 µM
0 0
0.0
0.1
0.2
0.0 0
500 1000 1500 2000 2500 Time, s
0.0
0.5 1.0 1.5 Concentration, mM
2.0
Figure 5. (A) Typical current-time response of the LOx/c-MWCNTsSnO2GRCS/GCE to successive addition of L-lysine into a stirred solution of 0.025 M BR buffer solution at (pH 10.0) +0.70 V (B) The effect of the L-lysine concentration on biosensor response. Inset: calibration curve of the biosensor (Error bars show standard deviation of three measurements).
21
Table 1. Peak separations and peak currents for different electrodes in 5 mM [Fe(CN)6]4-/3- with 0.1 M KCl at 100 mVs-1. Electrodes
ipa, μA
ipc, µA
ΔEp, V
CS/GCE GRCS/GCE SnO2CS/GCE c-MWCNTsCS/GCE c-MWCNTsSnO2GRCS/GCE
54.0 158.0 139.7 202.4 268.0
-55.0 -201.4 -167.5 -262.6 -317.1
0.37 0.15 0.21 0.17 0.14
Table 2. Selectivity of LOx/c-MWCNTsSnO2GRCS/GCE Amino acid Intereference %* Valine 1.1 Leucine 1.1 Isoleucine 4.6 Phenyalanine 10.2 Methionine 7.3 Proline 12.9 Serine 1.6 * Values are the average of three determinations
22
Amino acid Threonin Asparagine Glutamine Glutamic acid Arginine Ornithine
Intereference %* -2.9 -2.9 1.2 1.0 31.8 15.6