b College of Science, Jiangxi Agricultural University, Nanchang 330045, China
Poly(3,4-ethylenedioxythiophene) (PEDOT),known as one of the most stable,intrinsically conducting polymers (ICPs) available today,possessing inherent high electrical conductivity,excellent environmental stability,controllable surface properties and good biocompatibility [1, 2, 3]. As such,PEDOT is gaining increasing interest as revealed in Web of Science,and its research and sensing applications have also boomed from 2000 to 2012. Regardless of these advantages,the poor water solubility of 3,4-ethylenedioxythiophene (EDOT) is a major disadvantage,which limits its further application in various fields. However,the functionalized polar derivative of EDOT,hydroxymethylated-3,4-ethylenedioxythiophene (EDOTM),has better water solubility and lower onset oxidation potential,and even more importantly,its polymer film possesses similar excellent physicochemical properties of PEDOT. Moreover,some preceding work in our laboratory indicated that poly(hydroxymethylated-3,4-ethylenedioxythiophene) (PEDOTM) is a promising immobilization matrix of biologically active species and electrode materials for the fabrication of efficient chemo/biosensors [4, 5, 6].
Graphene,a versatile two-dimensional carbon nanomaterial, has experienced exponentially growing interest in preparing and characterizing materials because of its outstanding electrical, physical and chemical properties. Yet,graphene oxide (GO),by analogy with graphene itself,seems to be a more promising precursor for the bulk production of graphene-based materials owing to the relatively low cost of synthesis and the superior solvent processibility of GO in water,which makes it particularly attractive for the ‘‘green’’ construction of composites [7, 8, 9, 10]. The highly oxidized structure,with a large number of oxygen containing functional groups (alkoxy,epoxy,carbonyl,and carboxyl groups),results in a negative surface charge of GO flakes and good dispersibility. This is very beneficial for electrochemical doping and processing of GO in various applications [11, 12]. There have been many reports on the facile electrochemical codeposition of ICPs and GO,and nanocomposites have attracted considerable attention in recent years owing to the advantages of combining different materials [12, 13, 14]. However,to the best of our knowledge,there is no report on co-deposition of EDOTM with any other inorganic materials and their subsequent applications.
As the chief representative of auxins classified by phytohormone, a minor component of the metabolome,indole-3-acetic acid (IAA) has attracted growing attention in recent years due to its crucial role played in physiological processes of environmental response,plant growth and development,and also,for instance, gene expression,cell division and tissue decay [15, 16]. However, traditional methods of detecting IAA,such as liquid-phase extraction,solid-phase extraction,vapor-phase extraction and solid-phase microextraction,require sophisticated and expensive instruments,or radioactive and expensive chemicals,or complex purification and skilled operators [17, 18, 19]. In addition,levels of IAA in plants are very low,and furthermore,the phytohormone can be easily decomposed by heat,light and oxygen . With notable advantages of high sensitivity,good selectivity,low cost,rapid response,low reagent consumption,simple operation and convenient apparatus,the electrochemical method circumvents those limitations and tends to be a promising candidate for phytohormone biosensing and bioengineering applications [21, 22]. As shown in Table 1,many efforts have attempted the detection of IAA with electrochemical methods utilizing different materials,such as graphene ,gold nanoparticles (AuNPs) [16, 17],carbon nanotubes [29, 32, 34],etc. However,few reported on utilizing ICPs as sensing materials. Herein,we report a facile one-step,coelectrodeposition of EDOTM and GO as an expeditious and sensitive modified electrode for the assay of IAA (Scheme 1).
2. Experimental 2.1. Chemicals
IAA (Energy Chemical Co.,Ltd.) stock solutions (10 mmol L-1) were prepared in ethanol due to its low solubility in aqueous media. GO,provided by Nanjing XFNANO Materials Tech Co.,Ltd. (Nanjing,China),and was dispersed in doubly-distilled water followed by ultrasonication for 1 h to form the homogenous suspension. EDOTM was synthesized using the efficient transetherification route based on the previous works in our laboratory [4, 5]. The 0.1 mol L-1 phosphate buffer solutions were prepared with sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) and disodium hydrogen phosphate dodecahydrate (NaH2PO4·2H2O). All reagents and chemicals were used without further purification.
Electrochemical experiments were performed on a CHI660B electrochemical workstation (Shanghai Chenhua Co.,Ltd.,China) coupled with a conventional three-electrode cell at room temperature. The reference electrode was an Ag/AgCl electrode, and a glassy carbon electrode (GCE,F = 3 mm)and a platinum wire (F = 1 mm) served as the working electrode and auxiliary electrode,respectively. The addition of sample was carried out with a microsyringe (Shanghai Gaoge Industry & Trade Co.,Ltd., China). The pH value of phosphate buffered solution (PBS) was measured with a Delta 320 pH meter (Mettler-Toledo Instrument, Shanghai,China).
2.3. Preparation of PEDOTM/GO electrode
Prior to electrodeposition,the GCE was carefully polished to mirror-like surface with 0.05 mm alumina/water slurry on the chamois leather,then rinsed with deionized water and sequentially sonicated in deionized water,ethanol and deionized water for 5 min,respectively,and dried in air afterwards. The electrodeposition was performed by cyclic voltammetry (CV) with potential scanning between -0.9 V and 1.1 V for 10 cycles at 100 mV s-1 vs. Ag/AgCl. The uniform mixture was formed when EDOTM (0.02 mol L-1) and LiClO4·3H2O (0.1 mol L-1) were added into the aqueous GO dispersions (1.5 mg mL-1) mentioned previously and sonicated for 15 min. Reference films without GO was prepared using the same monomer concentration in 0.1 mol L-1 LiClO4 solution.
2.4. Electrochemical measurements
SWV and DPV methods were employed for quantitative assay of IAA at the as-prepared PEDOTM/GO electrode under optimum conditions. And both of their potential increases were set as 0.004 V. The limit of detection (LOD) was defined as 3s/S,s is the standard deviation of 20 parallel measurements in the measurable concentration of 0.1 mol L-1 PBS,S is the slope of the calibration curve. All the measurements were carried out at laboratory temperature. 3. Results and discussion 3.1. Fabrication and characteristics of PEDOTM/GO film
Fig. 1A and B presents the typical profile of EDOTM and EDOTM with GO electropolymerized by CV,whereas no notable variations are found in the electropolymerization of EDOTM with GO,which is similar to previous report . The first curve reveals a slightly lower onset oxidation potential during electropolymerization of EDOTM with GO,a possible result of stacking interactions and the electrostatic adsorptions (p-p interactions and hydrogen bonding) between the GO layers and aromatic EDOTM rings . In this case,bearing a negative charge and acting as the counter ion,GO was incorporated into the polymer films during oxidative electropolymerization of EDOTM to balance the positive charge on the polymer backbone. Therefore,a formation mechanism of PEDOTM/GO composite film was put forward (Scheme 1).
|Scheme 1.Schematic illustration for the construction of PEDOTM/GO/GCE and trace detection of IAA.|
|Fig. 1.CVs of GCE in 0.02 mol L-1 EDOTM (A) and in 0.02 mol L-1 EDOTM and 1.5 mg mL-1 GO (B),respectively; (C) CVs and (D) Nyquist diagrams obtained at GCE (a),PEDOTM/GCE (b),PEDOTM/GO/GCE (c) in 5.0 mmol L-1 Fe(CN)63-/Fe(CN)64- (1:1) mixture with 0.1 mol L-1 KCl.|
Impedance plots in the frequency range of 10 kHz to 100 MHz recorded at 0.23 V with a 5 mV amplitude (DE) are illustrated in Fig. 1C and D. For bare GCE,the shape of the low-frequency impedance plot presents the beginning of a semicircle and a 458 Warburg diffusion line,which is typical for an electrochemical reaction that is kinetically controlled at high frequencies and diffusion-controlled at low frequencies . The impedance plots of PEDOTM and PEDOTM/GO film are dominated by almost vertical low-frequency capacitive lines,as expected for ICPs composite electrodes [24, 25],illustrating that not only is the charge transfer process expedited as manifested by the little semicircle resistance, but the ion transport is also facilitated as presented in the muchincreased slope of the tail. In analogy with PEDOTM film,the impedance plot of PEDOTM/GO film features a slight deviation at low frequencies,coinciding with the lower redox capacitance determined according to equation: CLF ¼ 1=ð2p fZ00Þ,which suggests that some of the PEDOTM in PEDOTM/GO film is inactive,or that the PEDOT/GO film has a slight increasing resistive behavior owing to the doping of nonconducting GO. However,the lower peak-to-peak potential separation (DEp) for PEDOTM/GO presents a better reversible system,indicating that a rapid electron transfer (heterogeneous electron transfer rate constants,determined from the anodic/cathodic peak separation using the method of Nicholson ) occurs between the as-prepared electrode surface and the electrolyte.
3.2. Electrochemical behavior of IAA
Electrocatalytic activities of the as-prepared electrodes were tested by CV in Fig. 2A. The broaden voltammetric profile with low background current on GCE reveals sluggish electrochemical kinetics on this surface. On the PEDOTM/GCE,the oxidation peak current increases significantly accompanied with the enhanced background current,nevertheless,with higher overpotential. The dramatically enhanced background current was a result of the excellent conductivity of EDOTM,which increased the effective surface of the as-prepared electrode and improved the electron transfer rate . In terms of lower overpotential,higher response current,better defined peak current,as well as smaller voltammetric peak-width than PEDOTM/GCE,the electrochemical response at PEDOTM/GO/GCE indicates the occurrence of better electrocatalysis and a favorable interaction between the PEDOTM/ GO film and the phytohormone that makes the electron transfer process more effective .
|Fig. 2.(A) CV response of 0.1 mmol L-1 IAA at GCE (a),PEDOTM/GCE (b),PEDOTM/GO/GCE (c) in 0.1 mol L-1 PBS (pH = 3.0); (B) CVs for 0.1 mmol L-1 IAA in 0.1 mol L-1 PBS(pH = 1.0-7.0) at PEDOTM/GO/GCE; the effect of the amount of GO (C) and electropolymerization cycles (D) of composite films on electrocatalytic oxidation of IAA. Scan rate:100 mV s-1.|
To obtain optimum conditions for electrocatalytic oxidation of IAA,the effect of solution acidity at PEDOTM/GO/GCE was initially investigated (Fig. 2B). As can be seen,the peak potentials shifted slightly to more positive values with increasing acidity,while the most well-defined oxidation peak and the highest anodic current were obtained at pH 3.0. An abrupt current decrease was observed from pH 5.0 to 6.0,which was in good agreement with the first ionization constant of IAA (pK1,4.8) . Meanwhile,the oxidation peak potential showed a good linear relationship with the solution pH (linear regression equations: Ep = 1.0797 - 0.0334 pH, R2 = 0.986),implying that the ratio of electron-to-proton involvement in the oxidation reaction of peak current was 2:1 [20, 27]. A solution of PBS with pH 3.0 was thus chosen as optimal parameter for subsequent experiments. Additionally,the effect of the amount of GO and electropolymerization cycles of composite films on electrocatalytic oxidation of IAA were also discussed during our work (Fig. 2C and D). The responding current increased with increasing amount of GO or electropolymerization cycles,but increased slowly when the amount was above 1.5 mg mL-1 or showed a maximum current response to IAA for 10 cycles. Moreover,when the amount was above 2.5 mg mL-1,poorer water solubility was observed. Hence,the optimum value was established at 1.5 mg mL-1 and 10 cycles.
3.3. IAA detection of PEDOTM/GO film
The response changes of different concentrations for IAA detection were recorded in Fig. 3,in order to evaluate the sensitivity and dynamic range of the as-prepared electrode. The linear regression equation for the analytical curve with correlation coefficient shown in the inset graph,revealed that the LOD was 0.087 mmol L-1,and the sensitivity was 0.24 mA (mmol L-1)1 for DPV,while for SWV,the LOD was 0.033 mmol L-1,and the sensitivity was 0.20 mA (mmol L-1)1. The linearity range was 0.6 mmol L-1 to 10 mmol L-1 and 0.05 mmol L-1 to 40 mmol L-1, respectively. As illustrated from Table 1,our proposed method exhibited high sensitivity and the LOD was relatively low compared to previous reports.
|Fig. 3.DPV (A and B) and SWV (C and D) responses for IAA of various concentrations at PEDOTM/GO/GCE. Inset: the calibration.Z.-L. Feng et al. / Chinese Chemical Letters 514 25 (2014) 511-516|
3.4. Reproducibility and interference studies
Accuracy and precision of the proposed method were tested and evaluated by statistical analysis from 20 parallel assay of 10 mmol L-1 IAA,indicating acceptable reproducibility of the PEDOTM/GO electrode with relative standard deviations (RSD) of 2.20% and 2.14% for DPV and SWV,respectively. Many substances existing in crops,such as common anions,cations, carbohydrates,organic acids,vitamins,hormones,and amino acids,are tested,most of them exhibiting no electrical activity, and the oxidation peak currents of IAA for DPV and SWV were approximately 2.59 mA and 4.48 mA respectively. Moreover,the substances which easily suffered from interference by direct electrochemical oxidation of easily oxidizable species,such as glucose,cysteine and vitamin C,did not cause observable interference as well. Additionally,the electrocatalytic oxidation between vitamin B6 (VB6) or salicylic acid (SA) and IAA on the PEDOTM/GO electrode shows two oxidation peaks with larger peak separation (Fig. 4),indicating that the as-prepared composite electrode could realize the simultaneous detection of these biomolecules in sample mixtures. However,the electrocatalytic oxidation of tyrosine (Tyr) and tryptophan (Trp) revealed obvious interference for the detection of IAA, especially Trp displayed a very strong interference (Fig. 4),which may set a limit on its application for actual sample analysis. Taking these factors into consideration,further studies are needed and following work is in progress.
|Fig. 4.Effects of some compounds (10 mmol L-1) such as Trp,Tyr,VB6,and SA on electrochemical responses of 10 mmol L-1 IAA.|
The GO doped PEDOTM film was prepared via a green,fast,onestep co-electropolymerization on GCE. The as-prepared PEDOTM/ GO electrode combines the advantages of both GO and PEDOTM, exhibiting large surface area,and good biocompatibility,as well as favorable electrochemical properties. It showed good electrochemical reversibility,fast electronic transfer kinetics and favorable electrocatalytic performance relative to IAA. High sensitivity, low detection limits and good stability were achieved on the asprepared composite electrode based on PEDOTM/GO/GCE films, indicating the organic/inorganic composite film could be used as a promising platform for electrochemical studies of IAA.
This work was supported by the National Natural Science Foundation of China (Nos. 51263010,51272096),Jiangxi Provincial Department of Education (No. GJJ11590),and Natural Science Foundation of Jiangxi Province (No. 2010GZH0041).
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