SWCNTs composite film modified electrode for sensitive determination of picroside II

Author’s Accepted Manuscript A novel voltammetric sensor based on poly(lCitrulline)/SWCNTs composite film modified electrode for sensitive determination of picroside II Wenjing Wang, Lu Wang, Lina Zou, Gaiping Li, Baoxian Ye www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(15)30579-8 http://dx.doi.org/10.1016/j.talanta.2015.12.055 TAL16224

To appear in: Talanta Received date: 14 October 2015 Revised date: 11 December 2015 Accepted date: 19 December 2015 Cite this article as: Wenjing Wang, Lu Wang, Lina Zou, Gaiping Li and Baoxian Ye, A novel voltammetric sensor based on poly(l-Citrulline)/SWCNTs composite film modified electrode for sensitive determination of picroside II, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.12.055 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 galley proof before it is published in its final citable 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.

A novel voltammetric sensor based on poly(L-Citrulline)/SWCNTs composite film modified electrode for sensitive determination of picroside II

Wenjing Wanga, Lu Wanga,b, Lina Zoua, Gaiping Lia, Baoxian Ye*a

a College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, P R China b Department of Environmental Engineering and Chemistry, Luoyang Institute of Science and Technology, Luoyang 471023, PR China

* Corresponding author. Tel.: +86 0371 67781757; Fax: +86 0371 67763654. E-mail address: [email protected]

Abstract A novel voltammetric sensor was constructed by simple dripping single-walled carbon nanotubes (SWCNTs) on to the glass carbon electrode (GCE) firstly and electro-polymerizing L-Citrulline film subsequently. The resulting poly(L-Citrulline)/SWCNTs/GCE showed a significant voltammetric response to picroside II due to the synergistic effect of SWCNTs and poly(L-Citrulline) film. The first electroanalytical method of picroside II was proposed with detection linear range from 8.0×10-8 to 5.0×10-6 mol L-1 and a detection limit of 3×10-8 mol L-1.The high sensitivity, selectivity and long-term stability made the sensor suitable for the determination of picroside II. Moreover, based on the systematically investigation and some kinetics parameters calculated in the experimentation, the reaction mechanism of picroside II at the poly(L-Citrulline)/SWCNTs modified GCE was obtained reliably. Lastly, the proposed sensor 1

was used for the determination of picroside II in real sample with satisfactory results. This work promoted the potential applications of amino acid materials and SWCNTs in electro-chemical sensors.

Keywords: L-Citrulline; picroside II; synergistic effect; SWCNTs; Voltammetric sensor

2

1. Introduction

Picroside II (Scheme 1), is one of the most effective components extracted from picrorhizae (family: Scrophulariaceae [1]) which used as traditional medical systems in India, China, Tibet, Nepal and Sri Lanka for various immune-related diseases [2]. Previous studies have shown that picroside II has a wide range of pharmacological effects, including antioxidant [2], anti-inflammation [3], neuroprotective [4]. It can also improve accelerated atherosclerosis [1], inhibit hepatocyte apoptosis [5], protect myocardial ischemia reperfusion injury [6], protect cardiomyocyte [7], and decrease oxidative stress [8]. Analytical methods for picroside II have been developed using liquid chromatography-electrospray ionization ion-trap mass spectrometry (LC-ESI-IT-MS) [9], high-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) [10], LC-MS [11], LC-PDA [12], liquid chromatographic separation with tandem mass spectrometric detection (LC-Tandem-MS) [13], HPLC [14, 15], high-performance

liquid

chromatography

with

evaporative

light

scattering

detection

(HPLC-ELSD) [16], and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) [17]. However, there has no report about the determination of picroside II by electrochemical techniques. As we know, electrochemical method has many advantages comparing with above mentioned techniques, such as high sensitivity, simple equipment, cheapness and easy to realize automation. The mechanism of the electrochemical oxidation of pricroside II was investigated, providing important information about its pharmacological actions. Therefore, it is valuable to develop an electroanalytical method for picroside II. Here is for Scheme 1 3

Amino acids are the most fundamental material of the organism. They have many unique properties for containing amino and carboxyl functional groups. Amino acid modified electrodes have caused the extensive research interest for their strong electrochemical response, good stability and simple preparation [18-22]. L-Citrulline (Scheme 2), a kind of α-amino acid isolated from watermelon juice, is beneficial to the cardiovascular system [23, 24]. At present, the reports of L-citrulline are mainly focused on its pharmacological aspects [25, 26]. Nevertheless, literature about L-citrulline modified electrodes has not been reported so far. Here is for Scheme 2 Because of the larger specific surface area and good electrical conductivity, carbon nanotubes (CNTs) have caused great interest in the field of electrochemistry since their first report in 1991 [27]. CNTs modified electrodes have been applied to the analysis of various materials as voltammetric sensors [28-31]. Nowadays, composite materials combining CNTs and poly amino acid have increased attentions due to the synergistic contribution of two or more functional components and many potential applications [32-34]. In the current work, a novel voltammetric sensor, composite materials combining SWCNTs and poly(L-Citrulline) modified glassy carbon electrode (poly(L-Citrulline)/SWCNTs/GCE), was fabricated and used for investigating the electrochemical characters of picroside II. At the proposed sensor, picroside II had sensitive electrochemical response. The redox machenism and dynamics parameters of the electrode process were investigated systematically by various electrochemical techniques. Moreover, a sensitive and reliable electroanalytical method for determination of picroside II was established with detection linear range and low detection limit. This is the first report about the electrochemical informations and electroanalytical method for 4

picroside II. Finally, the proposed method was applied to determine picroside II in real samples with satisfactory results.

2. Experimental 2.1 Apparatus and Reagents

Model CHI650A electrochemical system (Chenhua Instrument Company, Shanghai, China) was employed for electrochemical techniques. A standard three-electrode system was used for electrochemical measurement with a bare GCE or modified GCE (d=3 mm) used as working electrode; A platinum (Pt) wire used as an auxiliary electrode and a saturated calomel electrode (SCE) used as a reference electrode, respectively. L-Citrulline and the standard reagent of picroside II were obtained from Aladdin Co. Ltd. (Shanghai, China). A stock solution of picroside II (1×10-3 mol L-1) was prepared using ethyl alcohol and stored under 4 ºC. It was diluted to suitable concentration when used. SWCNTs were purchased from Shenzhen Nanotech Port Co., Ltd. Phosphate buffer solution (PBS, 0.1 mol L-1) were prepared using the mixture of the stock solutions (0.1 mol L-1 NaH2PO4 and Na2HPO4). All other reagents were of analytical grade and were used without any further purification. Doubly distilled water was used for all preparations.

2.2 Preparation of carboxylic SWCNTs

Carboxylic acid groups were grafted onto the SWCNTs by means of ultrasound for 4 hours in acid mixture (98% concentrated sulfuric and 70% nitric acid, 3:1). The resulting SWCNTs were washed with water until the pH of the cleaning fluid was about 7.0 [35]. The 5

carboxylation-functionalized SWCNTs were ready after drying at 120 ºC. Then the functionalized SWCNTs were dispersed in Dimethylformamide (DMF) and sonicated for 24 h to obtain a suspension of 0.1 mg mL-1.

2.3 Fabricated of poly(L-Citrulline)/SWCNTs/GCE

The GCE was polished with 0.1 μm aluminum slurry, then rinsed ultrasonically with ethanol and double distilled water, each for 3 min. Afterwards, The fresh GCE surface was coated with 1 μL SWCNTs suspension and dried under IR-lamp. Then the poly(L-Citrulline)/SWCNTs modified GCE (poly(L-Citrulline)/SWCNTs/GCE) was obtained by cyclic sweeping between -1.8 V and 2.0 V at the rate of 0.1 V s-1 for 8 cycles in a PBS (pH 8.0) containing 2.5×10-3 mol L-1 L-Citrulline. This was the optimal depositional condition for fabricating the poly(L-Citrulline)/SWCNTs/GCE from test. As shown in Fig. 1, an oxidation peak (+1.6 V) and a reduction peak (-0.65 V) were observed. In subsequent scans, a new oxidation peak appeared at 0.35 V and the peak currents increased with the scanning cycles, indicating the continuous growth of the film. When scanning at high positive potential, L-Citrulline monomer is oxidized to form α-amino free radical, which can be linked on the electrode surface. Then poly(L-Citrulline) films can be formed. As is well known, amino acid monomer could be electro-polymerized onto the carboxylic SWCNTs surface by forming a carbon-nitrogen linkage at GCE surface [32-34, 36]. The proposed schematic representation of the poly(L-Citrulline)/SWCNTs/GCE was shown in Scheme 3. For comparison, a poly(L-Citrulline) modified GCE (denoted as poly(L-Citrulline)/GCE) was prepared using the same electro-polymerizing method and a SWCNTs modified GCE (named as SWCNTs/GCE)was established by deposited the above SWCNTs suspension (1 μL) on the fresh GCE surface. 6

Here is for Fig. 1 Here is for Scheme 3

2.4 Treatment of herbal samples

Picrorhiza kurroa Royle ex Benth (Zhengzhou Ruikang Pharmaceutical Co., Ltd., China), was employed to determine the picroside II content and evaluate the performance in practical applicability of the proposed voltammetric sensor. 2 g Picrorhiza kurroa was weighed and ground into powder. Then the powder was dispersed in 10 mL methanol and soaked for 2 h under the condition of ultrasonic [37]. After that, the solution was centrifuged for 5 min at 5000 rpm and the supernatant was taken for further use. The residue was leaching in another 10 mL methanol, repeated the above operation for four times. Lastly, the filtrates was merged and concentrated to 8 mL, which was used as detection sample.

3. Results and discussion 3.1 Electrochemical characterization of poly(L-Citrulline)/SWCNTs/GCE

The [Fe(CN)6]3- (1×10-3 mol L-1 containing 0.1 mol L-1 KCl) was used as electrochemical probe to investigate properties of modified electrodes by cyclic voltammetry. Fig. 2 displayed voltammograms with different electrodes. At bare GCE, a reversible redox voltammogram was obtained with iPa=-17.03 μA, iPc=17.43 μA (Fig. 2a). The redox currents increased a little at SWCNTs/GCE (Fig. 2b). Meanwhile, when L-Citrulline was electrodeposited on the electrode surface (Fig. 2c), the currents also became larger than that at bare GCE. It might be due to the large active surface of positive charged poly(L-Citrulline) film. And more, when L-citrulline 7

modified on the SWCNTs (Fig. 2d), the response currents reached the maximum among the four electrodes, which benefited from combined effect of both SWCNTs and L-citrulline. In order to further characterize the modified electrode, electrochemical impedance spectroscopy (EIS) was carried out in the electrolyte containing 5×10-3 mol L-1 [Fe(CN)6]3-/4- and 0.1 mol L-1 KCl. The Nyquist plot of EIS has two parts, the linear segment at lower frequency and the semicircle part at higher frequency, which mark the diffusion controlled process and the electron transfer limited process, respectively. The charge transfer resistance (Rct) can draw from the semicircle diamter. Fig. 3 was the enlargement of the Nyquist plots (Z’ vs. -Z’’) at bare GCE and the modified electrodes in high frequency region. The Rct of the four electrodes were 48.01 Ω at bare GCE (Fig. 3a), 26.06 Ω at SWCNTs/GCE (Fig. 3b), 27.3 Ω at poly(L-Citrulline)/GCE (Fig. 3c), and 22.45 Ω at poly(L-Citrulline)/SWCNTs/GCE (Fig. 3d). These data demonstrated that impedance would decrease when SWCNTs added on GCE or L-Citrulline polymerized on GCE. It was the result of conductibility of SWCNTs and high electrochemical activity of L-Citrulline. At poly(L-Citrulline)/SWCNTs/GCE, it reduced sharply of resistance than bare GCE, attributing to the function together of SWCNTs and poly(L-Citrulline). These results were consistent with that expressed in cyclic voltammetry. Here is for Fig. 2 Here is for Fig. 3

3.2 Cyclic Voltammetric Behaviors of picroside II

The electrochemical behavior of picroside II (5.0×10-6 mol L-1) at different electrodes was investigated by CVs in 0.1 mol L-1 PBS buffer solution (pH 2.0) with scan rate of 0.1 V s-1. Fig. 3 8

displayed the superimposed voltammograms. Each scan was performed two cycles to observe complete phenomena thoroughly. After each measurement, the electrode was put in a 0.05 mol L-1 NaOH for 1 cyclic scan to obtain a fresh electrode surface. At the four electrodes, the voltammogram outline and the present peak order of picroside II were the same. During the first cyclic scan, picroside II showed one oxidation peak and one reduction peak (P1 and P2) with EP1 about 0.84 V and EP2 about 0.53 V, respectively. In the 2nd cycle, a new anodic peak at about 0.56 V (EP3) appeared and P1 disappeared. But the P2 was no change. After two cycles, only P2 and P3 were left. From this observation, we preliminarily estimated that P1 was an irreversible anodic peak, and P2/P3 was a pair of redox peaks. The active group of P2/P3 came from the product of the P1 oxidation. In spite of the same voltammogram outline, the response currents of picroside II were large difference at the four electrodes (Fig. 4, a-d). In order to facilitate observation, a histogram (Fig. 5) was created that visually demonstrated the peak current difference of picroside II at different electrodes.

Obviously,

the

peak

current

achieved

the

maximum

at

poly(L-Citrulline)/SWCNTs/GCE (Fig. 4d), which could be reasonably ascribed to the synergistic effect of poly(L-Citrulline) and SWCNTs. Here is for Fig. 4 Here is for Fig. 5 In order to study the relationship between the several peaks of picroside II, potential window was

controlled

in

different

range

to

investigate

the

redox

of

picroside

II

at

poly(L-Citrulline)/SWCNTs/GCE by cyclic voltammetry. Firstly, the potential window was set between 0.25 V and 0.68 V for two cyclic scan (Fig. 6a) and there was no redox peak appearance. 9

That is, P2/P3 could not be obtained because of P1 absent. Secondly, potential window was set between 0.68 V and 0.95 V (Fig. 6b) for two cyclic scan. The oxidation peak (P1) was observed in the first cycle and disappeared in the second cycle. Thirdly, when the potential window was set between 0.25 V and 0.95 V going initially from 0.68 V to 0.25 V (Fig. 6c), the pair redox peaks (P2/P3) was in sight not before but after the appearance of P1. These phenomena illustrated that the pair of redox peaks (P2/P3) were in relation to P1 and the active group of P2/P3 was the product of P1 oxidation. The CVs at the whole potential window (0.25 V-0.95 V) also explained this point (Fig. 6d). Here is for Fig. 6

3.3 Effect of solution pH

The voltammetric response of picroside II (5.0×10-6 mol L-1) at proposed sensor were examined in PBS solution with different pH value from 2.0 to 7.0 by CVs. In order to facilitate observation, the superimposed voltammograms of the first segment in 1st cycle and the whole 2nd cycle were shown respectively and displayed in Fig. 7A and B. The three peak currents decreased with the pH increased and the P2 and P3 disappeared at pH 7.0, suggesting that the pH value obviously affected the peak currents of picroside II and the maximum currents response were obtained at pH 2.0. Thus pH 2.0 of PBS was selected for detection of picroside II. Besides, the peak potential of the three peaks shifted towards negative potential when the pH increased, indicating that protons were directly involved in the electrode reaction. Fig. 7C showed that the peak potentials were shifted linearly negative with solution pH from 2.0 to 7.0 (6.0 for P2, P3). The equations relating EP with pH was listed in Table 1. The slope of -51.2, -56.4, -55.6 mV 10

pH-1was close to the theoretical value of -59 mV pH-1, explaining that the numbers of electron and proton taking part in the three peak redox were equal. Here is for Fig. 7 Here is for Table 1

3.4 The effect of scan rate

The influence of scan rate (ν) on the three peaks of picroside II (5.0×10-6 mol L-1) was examined in the range of 0.06-0.8 V s-1.With the increasing of scan rate, the oxidation peak potential (P1) moved to positive direction (Fig. 8A) and the peak current increased simultaneously. In addition, the peak potential and the logarithm of scan rate showed a linear relationship (the inset in Fig. 8A) with the equation: EP1(mV)=(24.59±1.21)ln(ν/Vs-1)+(903.0±2.12) (R=0.986), which suggesting that the oxidation process was irreversible. According to Laviron’s model [38] (Eqn 1), the number of the electron (n) transfer in P1 was calculated 2 by assuming an electron transfer coefficient of α=0.5. That is, there were two protons and two electrons involved in the oxidation process of P1. Then, the heterogeneous electron transfer rate constant (ks) and the charge transfer coefficient (α) could be calculated reversely by Eqn 1. The results were that ks1=3.404, α1=0.52 for P1.

EP  E 0' 

RTks RT RT ln  ln  (1) nF nF nF

where E0 is the formal standard potential, and ν, R, F and T represent their usual meaning. A superposed voltammogram of 2nd cycle was represented in Fig. 8B. The redox peak potentials were change inconspicuously when the scan rate increased. The peak-to-peak separation (ΔEP) was very small, indicating a fast electron transfer rate. The peak currents and scan rates was 11

good linear relationships of P2 and P3 with regression equations of iP2(μA)=(0.0316±0.001)ν(V s-1)+(0.369±0.295) (R=0.997) (Fig. 8C) and iP3(μA)=(0.0294±0.001)ν(Vs-1)+(1.249±0.658) (R=0.990) (Fig. 8D). It illustrated that the electrode process of P2/P3 should be a quasi-reversible and controlled by adsorption. Based on Laviron's theory of adsorption-controlled process, it should satisfy the Eqn 2: ip 

n 2 F 2 vAΓ nFQv (2)  4 RT 4 RT

Where n is the number of electron transferred, F is Faraday’s constant, A is the geometric surface area of the electrode and Q is the peak area of CV at different scan rates, R and T have their usual meanings. So, the electron transfer number n contained in P2 and P3 redox can also be calculated to be 2. In addition, the heterogeneous electron transfer rate constant ks was 3.003 s-1 obtained from Eqn 3.

logks  log (1   )  (1   )log  log

nFEp RT (3)   (1   ) nF 2.3RT

Thus, the numbers of electron and proton taking part in the electrode reaction of the three peaks were all of 2. Here is for Fig. 8

3.5 Chronocoulometric studies

The diffusion coefficient and adsorption capacity were indispensable parameters for adsorption or diffusion controlled electrode process. These parameters of picroside II were investigated by thrice potential step chronoamperometry. The step potentials were from 0.65 V to 0.95 V, 0.95 V to 0.25 V and 0.25 V to 0.65 V successionally. For this system, experiments were performed in a picroside II solution (2.0×10-5 mol L-1) and blank PBS respectively. The Q~t curves 12

were shown in Fig. 9A: curves a, b and c in picroside II solution and curves a', b' and c' in blank PBS. The corresponding Q~t1/2 curves were shown in Fig. 9B-D. The marker a1, b1 and c1 correspond with P1, P2 and P3. The linear equations of Q~t1/2 were listed in Table 2. From the Fig. 9B, the difference of intercepts and slopes were obtained from curves a and a'1, demonstrating that the oxidation process of picroside II (P1) was controlled by both adsorption and diffusion. According to Eqn 4 given by Anson [39]: 2nFAC Dt 

1/ 2

Q

 1/ 2

 Qdl  Qads

(4)

Where Qdl is the double-layer charge; Qads is the Faradaic charge due to the oxidation of adsorbed picroside II. Other symbols have their conventional significance. The adsorption capacity (Γ*) of picroside II (P1) on poly(L-Citrulline)/SWCNTs/GCE was calculated to be 1.19×10-9 mol cm-2 using Laviron’s theory of Q=nFAΓ* by substituting n=2 into the equation. Meanwhile, the diffusion coefficient (D) was obtained to be 5.72×10-10 cm2 s-1 at P1 process according the slope of curve a1. Obviously in Fig. 9C and D, the two slope values of the Q~t1/2 plots were almost equal when performed in the picroside II solution (2.0×10-5mol L-1) and in blank PBS, but the intercepts were quite different with each other, which was an additional evidence for an adsorption-driven electrode process just as mention above. The values of saturating adsorption capacity were calculated to be 2.14×10-9 cm2 s-1 of P2 and 1.51×10-9 cm2 s-1 of P3. Here is for Fig. 9 Here is for Table 2

3.6 Electrode reaction mechanism 13

On the basis of experimental phenomenon and the data obtained above, a reasonable electrode reaction process was presumed and presented in Fig. 10. The electrochemical oxidation of picroside II in the first scan (Fig. 4) is coupled to an oxidation peak, which results in the hydrolysis of the 2-methoxy group to form an o-benzoquinone unit [40, 41] (P1). Then the o-benzoquinone part of the picroside II falls in a redox electrochemical loop with catechol (B and C) [42] which are observed as P2 and P3. Here is for Fig. 10

3.7 Calibration curve, detection limit, repeatability, reproducibility and stability

For establishing the calibration curve, the peak of P1 was employed as a detect signal for its better peak shape and larger peak current. Linear sweep voltammetry (LSV) was adopted to doing so, and the potential was scanned from 0.65 V to 0.95 V after accumulation of 30 s under open circle. A superposed voltammogram was displayed in Fig. 11A. Fig. 11B was the linear relationship between the peak currents of P1 and the concentrations of picroside II. The linear regression equation was expressed as iP1(μA)=(1.996±0.088)c(μM)+(8.040±0.248) (R=0.9884) in the range from 8×10-8 mol L-1 to 5×10-6 mol L-1. The limit of detection was 5×10-8 mol L-1. The comparison of the proposed method with other techniques for picroside II determination was listed in Table 3. Here is for Fig. 11 Here is for Table 3 The obtained relative standard deviation (R.S.D.) for five successive determination of a picroside II solution (5.0×10-6 mol L-1) was 2.25%, suggesting good reproducibility of the 14

proposed method. Five respective modified electrodes were used for the determination of picroside II solution (5.0×10-6 mol L-1) and the relative standard deviation was 3.1%, explaining excellent repeatability of the modified electrode. After the electrode was stored in pH 7.0 PBS for seven days, the peak current of P1 decreased to 95.8%, illustrating that the modified electrode had long-term stability.

3.8 Interference studies

As a sensitive analytical method, it is necessary to investigate some possible interference in spite of no interference in our target sample, picrorhizae (herbal medicine). Herein, some normal inorganic compounds and small organic compounds, which might coexist in other samples, were introduced under the same conditions to test the selectivity of poly(L-Citrulline)/SWCNTs/GCE to picroside II by LSV. The decrease of the current was all less than 8% when each 100-fold Fe3+, Mg2+, Zn2+, Al3+, SO42-, Cl- and 50-flod glucose, starch, calcium benzoate, sodium citrate, glutamic acid, ascorbic acid were used, which indicated these common impurities barely interfere in the determination of picroside II. These results showed that the present method had remarkable anti-interference ability.

3.9 Analysis of herbal samples

The LSV curves of 1 μL Picrorhiza kurroa sample solution and the added different amount of standard solution in pH 2.0 PBS. As a comparison, HPLC method was also employed for detection sample. The results were listed in Table 4. The concentration of picroside II in sample solution determined by LSV (1.91×10-6 mol L-1) was almost the same with that by HPLC (2.07×10-6 mol 15

L-1). Conversely, the content of picroside II in Picrorhiza kurroa was calculated to be 3.915 mg/g in this determination (LSV). Here is for Table 4

4. Conclusions

In conclusion, a simple and novel electrochemical sensor, combining SWCNTs and electro-polymerizing poly(L-Citrulline) film modified GCE was fabricated and used for sensitive determination of picroside II. This was the first report about the poly(L-Citrulline) used in modified electrode and the electroanalytical method for picroside II. Because of the synergistic effect of poly(L-Citrulline) and SWCNTs, the resulting poly(L-Citrulline)/SWCNTs/GCE showed a remarkably response towards the determination of picroside II with high sensitivity, selectivity, wide linear range, low detection limit and long-term stability. Then, based on the data in the experimentation, the reaction mechanism was obtained reliably. Lastly, the proposed sensor was well used for the determination of picroside II in real sample. Therefore, L-Citrulline was a promising modified material for fabricating electrochemical sensor.

Acknowledgements The authors were really grateful to the financial support from the National Natural Science Foundation of China (Grant Nos. 21275132).

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D. Bouchta, L.M.C. Aguilera, Talanta 79 (2009) 22-26. [21] F.Y. Zhang, Z.H. Wang, Y.Z. Zhang, Z.X. Zheng, C.M. Wang, Y.L. Du, W.C. Ye, Talanta 93 (2012) 320-325. [22] F.C. Pereira, A.G. Fogg, M.V.B. Zanoni, Talanta 60 (2003) 1023-1032. [23] E. Curis, I. Nicolis, C. Moinard, S. Osowska, N. Zerrouk, S. Benazeth, L. Cynober, Amino Acids 29 (2005) 177-205. [24] A. Barroso, L. Oliveira, E. Campesatto-Mella, C. Silva, M.A. Timoteo, M.T. Magalhaes-Cardoso, W. Alves-do-Prado, P. Correia-de-Sa, Br. J. Pharmacol. 151 (2007) 541-550. [25] A. Mori, M. Morita, K. Morishita, K. Sakamoto, T. Nakahara, K. Ishii, J. Pharmacol. Sci. 127 (2015) 419-423. [26] S. Iijima, Nature 354 (1991) 56-58. [27] G.G. Wildgoose, C.E. Banks, H.C. Leventis, R.G. Compton, Microchim. Acta 152 (2006) 187-214. [28] L.S.T. Alamo, T. Tangkuaram, S. Satienperakul, Talanta 81 (2010) 1793-1799. [29] P. Luo, J. Liu, Y. Li, Y. Miao, B. Ye, Anal. Lett. 45 (2012) 2445-2454. [30] J. Zhou, F. Wang, K. Zhang, G. Song, J. Liu, B. Ye, Microchim. Acta 178 (2012) 179-186. [31] M. Amatatongchai, S. Laosing, O. Chailapakul, D. Nacapricha, Talanta 97 (2012) 267-272. [32] C. Jiang, T. Yang, K. Jiao, H. Gao, Electrochim. Acta 53 (2008) 2917-2924. [33] X. Liu, L. Luo, Y. Ding, D. Ye, Bioelectrochemistry 82 (2011) 38-45. [34] A.T. Ezhil Vilian, S. Chen, B. Lou, Biosens. Bioelectron. 61 (2014) 639-647. [35] J. Liu, A.G. Rinzler, H. Dai, J.H. Hafner, R.K. Bradley, P.J. Boul, A. Lu, T. Iverson, K. 18

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Figures caption Table 1 The linear relationship parameters between EP (mV) and pH.

Table 2 The corresponding Q~t1/2 curves.

Table 3 The comparable analytical methods for the determination of picroside II.

Table 4 Results of the determination of Picroside II in real sample.

Scheme 1 The chemical structure of picroside II.

Scheme 2 The structure of L-Citrulline. 19

Scheme 3 Schematic illustration for the stepwise preparation of poly(L-Citrulline)/SWCNTs/GCE.

Fig. 1. Cyclic voltammograms for the electropolymerization of L-Citrulline. Supporting electrolyte: PBS buffer solution (pH 8.0); scan rate: 0.1 V s-1; L-Citrulline concentration: 2.5×10-3 mol L-1.

Fig. 2. Cyclic voltammograms of K[Fe3(CN)6] (2.0×10-3 mol L-1) at different electrodes, form a to d: bare GCE, SWCNT/GCE, poly(L-Citrulline)/GCE, poly(L-Citrulline)/SWCNTs/GCE. Supporting electrolyte: 0.1 mol L-1 KCl; Scan rate: 0.1 V s-1.

Fig. 3. The Nyquist plots of EIS at high frequency region at: (a) bare GCE, (b) SWCNT/GCE, (c) poly(L-Citrulline)/GCE, (d) poly(L-Citrulline)/SWCNTs/GCE. Supporting electrolyte: 5×10 -3 mol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) with 0.1 mol L-1 KCl solution; Working potential: 0.285 V; Frequency: 1 M Hz to 0.01 Hz.

Fig. 4. Cyclic voltammograms of Picroside II (5.0×10 −6 mol L−1) at bare GCE (a), SWCNTs/GCE (b), poly(L-Citrulline)/GCE (c) and poly(L-Citrulline)/SWCNTs/GCE (d). Supporting electrolyte: 0.1 mol L−1 PBS (pH 2.0); Scan rate: 0.1 V s−1.

Fig. 5. Histogram of the peak currents of P1 on different electrodes.

Fig. 6. Voltammograms of Picroside II (5.0×10−6 mol L−1) within different potential window. (A) 20

between 0.25 V and 0.68 V; (B) between 0.68 V and 0.95 V, (C) initial from 0.68 V negative going; (D) between 0.25 V and 0.95 V. Other conditions the same as in Fig. 3.

Fig. 7. The superposed voltammograms of Picroside II (5.0×10 −6 mol L−1) in different pH PBS. (A) the first segment of 1st cycle; (B) the 2nd cycle. pH (curves a to f in A): 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, (curves a to e in B): 2.0, 3.0, 4.0, 5.0, 6.0. Scan rate: 0.1 V s-1, accumulation time: 30 s. (C) The relation between peak potentials and solution pH: (a) P1, (b) P2, (c) P3.

Fig. 8. Superimposed voltammograms of Picroside II (5.0×10-6 mol L-1) with different scan rates of (A) 1st cycle; (B) the 2nd cycle. Scan rates (from a to g): 0.06, 0.1, 0.15, 0.2, 0.3, 0.5, 0.8 V s -1. Supporting electrolyte: 0.1 mol L−1 PBS (pH 2.0); Scan rate: 0.1 V s−1; accumulation time: 30 s. Inset in Fig. 7 A: Linear relationships of EP1 and ln ν. (C) Linear relationship of the peak currents and scan rates about P2. (D) Linear relationship of the peak currents and scan rates about P3.

Fig. 9. (A) Chronoamperograms obtained under presence (a, b, c) and absence (a1, b1, c1) of Picroside II (2.0×10-5 mol L-1). P1, P2, P3 corresponding to a, b, c. (B) The dependency of charge Q vs. t1/2, corresponding data were derived from Fig. 8A a and a1. (C) The dependency of charge Q vs. t1/2, corresponding data were derived from Fig. 8A b and b1. (D) The dependency of charge Q vs. t1/2, corresponding data were derived from Fig. 8A c and c1.

Fig. 10. The proposed reaction mechanism of picroside II at the poly(L-Citrulline)/SWCNTs/GCE. 21

Fig. 11. (A) Superimposed LSV curves of different Picroside II concentrations obtained in 0.1 mol L -1 PBS (pH 2.0). Picroside II concentration: (from a to h): 0, 8×10 -8, 5×10-7, 1×10-6, 2×10-6, 3×10-6, 4×10-6, 5×10-6 mol L-1. (B) Calibration plot of peak current versus picroside II concentrations.

Figures

HO

OH O

O

HO

OH

HO

O

OCH3 OH

H

H O O

H O

Scheme 1 The chemical structure of picroside II. Chemical name: h-D-glucopyranoside,1a,1b,2,5a,6,6a-hexa-hydro-6-[(4-hydroxy-3methoxybenzoyl)oxy]-1a(hydroxymethyl)oxireno[4,5]cyclopenta[1,2-c]pyran-2-yl.

O

H2N

O

OH

N H NH2

Scheme 2 The structure of L-Citrulline.

22

Scheme 3 Schematic illustration for the stepwise preparation of poly(L-Citrulline)/SWCNTs/GCE.

23

Fig. 1. Cyclic voltammograms for the electropolymerization of L-Citrulline. Supporting electrolyte: PBS buffer solution (pH 8.0); scan rate: 0.1 V s-1; L-Citrulline concentration: 2.5×10-3 mol L-1.

24

Fig. 2. Cyclic voltammograms of K[Fe3(CN)6] (2.0×10-3 mol L-1) at different electrodes, form a to d: bare GCE, SWCNT/GCE, poly(L-Citrulline)/GCE, poly(L-Citrulline)/SWCNTs/GCE. Supporting electrolyte: 0.1 mol L-1 KCl; Scan rate: 0.1 V s-1.

25

Fig. 3. The Nyquist plots of EIS at high frequency region at: (a) bare GCE, (b) SWCNT/GCE, (c) poly(L-Citrulline)/GCE, (d) poly(L-Citrulline)/SWCNTs/GCE. Supporting electrolyte: 5×10-3 mol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) with 0.1 mol L-1 KCl solution; Working potential: 0.285 V; Frequency: 1 M Hz to 0.01 Hz.

26

Fig. 4. Cyclic voltammograms of Picroside II (5.0×10−6 mol L−1) at bare GCE (a), SWCNTs/GCE (b), poly(L-Citrulline)/GCE (c) and poly(L-Citrulline)/SWCNTs/GCE (d). Supporting electrolyte: 0.1 mol L−1 PBS (pH 2.0); Scan rate: 0.1 V s−1.

27

Fig. 5. Histogram of the peak currents of P1 on different electrodes.

28

Fig. 6. Voltammograms of Picroside II (5.0×10−6 mol L−1) within different potential window. (A) between 0.25 V and 0.68 V; (B) between 0.68 V and 0.95 V, (C) initial from 0.68 V negative going; (D) between 0.25 V and 0.95 V. Other conditions the same as in Fig. 3.

29

Fig. 7. The superposed voltammograms of Picroside II (5.0×10−6 mol L−1) in different pH PBS. (A) the first segment of 1st cycle; (B) the 2nd cycle. pH (curves a to f in A): 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, (curves a to e in B): 2.0, 3.0, 4.0, 5.0, 6.0. Scan rate: 0.1 V s-1, accumulation time: 30 s. (C) The relation between peak potentials and solution pH: (a) P1, (b) P2, (c) P3.

30

Fig. 8. Superimposed voltammograms of Picroside II (5.0×10 -6 mol L-1) with different scan rates of (A) 1st cycle; (B) the 2nd cycle. Scan rates (from a to g): 0.06, 0.1, 0.15, 0.2, 0.3, 0.5, 0.8 V s -1. Supporting electrolyte: 0.1 mol L−1 PBS (pH 2.0); Scan rate: 0.1 V s−1; accumulation time: 30 s. Inset in Fig. 7 A: Linear relationships of EP1 and ln ν. (C) Linear relationship of the peak currents and scan rates about P2. (D) Linear relationship of the peak currents and scan rates about P3.

31

Fig. 9. (A) Chronoamperograms obtained under presence (a, b, c) and absence (a1, b1, c1) of Picroside II (2.0×10-5 mol L-1). P1, P2, P3 corresponding to a, b, c. (B) The dependency of charge Q vs. t1/2, corresponding data were derived from Fig. 8A a and a1. (C) The dependency of charge Q vs. t1/2, corresponding data were derived from Fig. 8A b and b1. (D) The dependency of charge Q vs. t1/2, corresponding data were derived from Fig. 8A c and c1.

32

HO

OH O

O

HO OH HO

O

OCH3

H

OH

H O O

H O

HO

OH -

-2e

P1

+

-2H

O

OH

O

+2e

-

O

B

-2e-

H O

HO

OH +

+2H

O

O

HO

A

P2

O

H

H

O

HO

O

-2H+ P3

O

HO OH

O

OH

O O

HO

C

Fig. 10. The proposed reaction mechanism of picroside II at the poly(L-Citrulline)/SWCNTs/GCE.

33

OH

H

H H O

Fig. 11. (A) Superimposed LSV curves of different Picroside II concentrations obtained in 0.1 mol L-1 PBS (pH 2.0). Picroside II concentration: (from a to h): 0, 8×10 -8, 5×10-7, 1×10-6, 2×10-6, 3×10-6, 4×10-6, 5×10-6 mol L-1. (B) Calibration plot of peak current versus picroside II concentrations.

Graphical abstract

Schematic illustration for the stepwise preparation of poly(L-Citrulline)/SWCNTs/GCE.

Cyclic voltammograms of Picroside II at

The superimposed LSV curves of P1 in

different

Picroside II with different concentrations

electrodes:

bare

GCE

(a),

-1

SWCNTs/GCE (b), poly(L-Citrulline)/GCE (c)

in 0.1mol L PBS (pH=2.0).

and poly(L-Citrulline)/SWCNTs/GCE (d). 34

HO

OH O

O

HO OH

O

OCH3

H

OH

H O O

H O

HO

HO

OH

-2e- -2H+ P1

O

OH

O

+2e

-

HO

OH +

+2H

O

O O

HO

A

P2

O

H

H

O

HO

O

H O

-2e- -2H+ P3

O

HO OH

O

O O

H

C

The proposed reaction mechanism of picroside II on the poly(L-Citrulline)/SWCNTs/GCE.

Figure: A novel sensor for high-sensitive determination of picroside II was constructed by simple dripping single-walled carbon nanotubes (SWCNTs) onto the glass carbon electrode (GCE) firstly and electro-polymerizing L-Citrulline film subsequently. Compared with bare GCE, SWCNTs/GCE and poly(L-Citrulline)/GCE, there were dramatic increase of the peak currents. Then the electrochemical behavior of picroside II was carefully and systematically investigated and some kinetics parameters were calculated. The reaction mechanism of picroside II at poly(L-Citrulline)/SWCNTs/GCE was also discussed. A simple and high-sensitive electroanalytical method for picroside II was established using LSV and applied in the drug sample analysis.

Tables

Table 1 The linear relationship parameters between EP (mV) and pH. pH range

Slope (mV pH-1)

Intercept (mV)

P1

2.0-7.0

-51.2

957.0

0.9913

P2

2.0-6.0

-56.4

669.4

0.9959

P3

2.0-6.0

-55.6

685.6

0.9955

35

R

OH

H

HO

B

OH

H

O

Table 2 The corresponding Q~t1/2 curves.

Slope

Adsorption capacity

Diffusion

(Γ*)

coefficient

(mol cm-2)

(D) (cm2 s-1)

Intercept

Picroside II (a1)

-0.736

-1.977

Blank (a′1)

-0.249

-0.464

Picroside II (b1)

0.667

-4.044

Blank (b′1)

0.514

-1.128

Picroside II (c1)

-0.412

-1.305

Blank (c′1)

-0.195

P1

P2

P3

1.19×10-9

5.72×10-10

2.14×10-9

_

1.51×10-9

_

0.748

Table 3 The comparable analytical methods for the determination of picroside II. Analytical methods

Linear ranges (mol L-1)

Detection limit (mol L-1)

Ref.

LC-ESI-MS

2.0×10-7 to 9.8×10-5

3.9×10-8

10

LC-MS

5.8×10-8 to 1.2×10-6

5.8×10-8

11

LC-Tandem-MS

2.0×10-10 to 7.8×10-7

2.0×10-10

13

HPLC

4.9×10-6 to 9.8×10-4

9.8×10-8

14

HPLC-ELSD

5.9×10-5 to 5.9×10-4

-

16

LC-ESI-MS/MS

9.8×10-10 to 9.8×10-7

9.8×10-10

17

Voltammetry

8.0×10-8 to 5.0×10-6

3.0×10-8

This work

Table 4 Results of the determination of Picroside II in real sample. LSV

HPLC

Original

R.S.D.

Standard

Total

R.S.D.

Recovery

Amount

R.S.D.

found (μM)

(%)

added (μM)

found (μM)

(%)

(%)

found (μM)

(%)

36

(n=3) 1.910

1.13

(n=3)

(n=3)

0.1

0.105

0.73

105.0

0.2

0.215

0.36

107.5

0.6

0.592

0.45

98.67

2.071

0.86

Research Highlights:

►This is the first report about the electrochemical properties and electroanalytical method for picroside II. ►The L-Citrulline was used as the modified materials of electrode for the first time. ►The resulting sensor showed a significant voltammetric response to picroside II and used in real sample with satisfactory results. ►This work promoted the potential applications of amino acid materials and SWCNTs in electro-chemical sensors.

37