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Home > Chemical pre-reduction and electro-reduction guided preparation of a porous graphene bionanocomposite for indole-3-acetic acid detection
Chemical pre-reduction and electro-reduction guided preparation of a porous graphene bionanocomposite for indole-3-acetic acid detection
Zhaohong Su, a Xiaolin Xu,†a Yongbing Cheng,†a Yueming Tan, b Langtao Xiao,c Daili Tang,a Hongmei Jiang, a Xiaoli Qin*a and Huixian Wang
Introduction
Indole-3-acetic acid (IAA), as the primary natural phytohormone in plants, exists extensively in species at various stages of the evolutionary cycle. The content of IAA in most plant issues and seeds is very low (ng g−1 level, even pg g−1 level), and it is sensitive to outside conditions such as light, tempera- ture, and oxidants. The detection methods for IAA need high sensitivity and specificity due to the interference by the complex background of metabolites in the plant tissue.1,2 To date, a variety of techniques have been developed for IAA detection, including colorimetric,2 chromatography,3 fluorescence,4 chemiluminescence,5 and enzyme linked immuno- sorbent assay.6 Unfortunately, some of these methods require sophisticated and expensive instruments, or expensive chemi- cals, or involve a tedious procedure and are time-consuming, but the electrochemical detection is usually simple, rapid and sensitive. Thus, the development of a sensitive and convenient measurement procedure for IAA assay has become important for constructing high-performance electrochemical sensors.7
Porous graphene (PG) can improve the mass transfer and increase the loading by virtue of its good conductivity and large specific surface area, and then improve the sensitivity and detection limit in electrochemical sensors. The three dimensional structure of PG not only retains the excellent properties of graphene, but also presents plentiful pores which can promote the transport efficiency of the material compared with the surface of smooth graphene. In addition, it is rapidly becoming the key of chemical sensors,8 electrode materials,9 molecular sieves,10,11 field effect transistors12 and other aspects of research. At present, the methods of preparing porous graphene include etching,13 carbothermal reduction,14 solvothermal method,15 and free radical attack method.
However, most of these methods are too complicated and difficult to control precisely. In this work, we will adopt an innovative method to synthesize PG by pre-reduction/electric reduction with simple operation and good performance.
In this work, in order to improve the sensitivity and detec- tion limit of IAA, a label-free electrochemical immunosensor was developed for the selective detection based on the PG com- posite, AuNPs and anti-IAA. This immunosensor was prepared by simple chemical pre-reduction and electrochemical methods (Scheme 1), which can produce a AuNPs modified PG composite film with surface properties similar to those of gra- phene oxide (GO)-based nanocomposites.7 The decrease of the reduction peak current of Fe(CN)63− was used to monitor the immunoreaction. This immunosensor exhibited a lower detec- tion limit and a higher sensitivity for the determination of IAA compared with those reported in most of the previous studies (Table 1). In addition, for confirming the applicability of the fabricated immunosensor, IAA extracted from different plant seeds was detected.
Experimental methods
Materials
IAA, gibberellin (Gb), abscisic acid (ABA), salicylic acid (SA), potassium ferricyanide (K3[Fe(CN)6]), trisodium citrate and chloroauric acid (HAuCl4) were purchased from Aladdin (Shanghai, China). Rat monoclonal antibody against IAA was purchased from Sigma (U.S.). Phosphate buffer solutions (PBS) were prepared by mixing the stock solution of 0.1 M NaH2PO4 and 0.1 M Na2HPO4, and the pH was adjusted by using NaOH or HCl. Double-distilled deionized water was autoclaved and used throughout the experiments and the other reagents were of analytical grade or better.
AuNPs were synthesized according to a previous report.17
Apparatus
All electrochemical experiments were conducted on a CHI660E electrochemical workstation (CH Instrument Co., USA). A conven- tional three-electrode system consisted of a glassy carbon disk electrode (GCE) with a diameter of 3 mm as the working elec- trode, a KCl-saturated calomel electrode (SCE) as the reference electrode and a platinum plate as the counter electrode. All potentials in this work are referenced to the SCE. Scanning elec- tron microscopy (SEM, JEM-6700F field emission scanning elec- tron microscope) and transmission electron microscopy (TEM, JEM-3010, Jeol, Japan) were applied to characterize the structure of composites. The elemental analysis results were tested by EDS. Raman spectroscopy were obtained in the Labram-010 (France). Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet Nexus 670 FT-IR spectrophotometer. A UV-2450 spectro- photometer was used to characterize the UV-vis spectra.
Preparation of PG by pre-reduction/electrochemical reduction
The pre-reduction/electrical reduction method was first used to prepare porous graphene materials (Scheme 1). The steps are as follows: 0.088 g of ascorbic acid was added to 2 mL (5 mg mL−1) solution of GO and stirred for 2 h at room temp- erature, then 2 mL of absolute ethanol was added to the solu- tion and the mixture was sonicated at room temperature for 30 min to obtain a mixed solution of reduced GO (rGO) and GO. Subsequently, 4 μL of the mixed solution was added onto a GCE, and then immersed in 0.1 mol L−1 ( pH = 5) PBS for 15 cycles of cyclic voltammetry scanning; the scan range was from −1.5 to 0 V with 50 mV s−1. Finally, the PG modified elec- trode was obtained, that is, the PG material is prepared on the working electrode. After characterization by FT-IR, CV, EIS, SEM, TEM, Raman, EDS and DPV, the PG modified electrodes show good structural and electrochemical properties.
Preparation of the AuNPs/PG modified electrode
The electrochemical deposition of a porous nanostructured gold film was performed in the 20 mL mixed solution (0.25 mol L−1 H2SO4 +0.5 mol L−1 ethylene glycol +0.2 mmol L−1 HAuCl4). The porous nanostructured gold film grew on the PG modified electrode surface under an applied potential of −0.35 V (vs. SCE) for 300 s.
Preparation of the immunosensor
Briefly, the obtained electrode (AuNPs/PG/GCE) was rinsed with double distilled deionized water and dried with nitrogen blowing before incubation in 1 mg mL−1 anti-IAA solution ( pH 7.4) for 2.5 h at room temperature and under humid con- ditions. After the electrode had been washed with water, the modified electrode was then immersed in 16 μL of 1.0 mg mL−1 BSA for 30 min to eliminate the nonspecific binding effect.
Electrochemical measurements and IAA immunodetection
All electrochemical measurements were performed in 0.1 M PBS ( pH 7.4) solution containing 5 mM Fe(CN)63− at room temperature. The reduced peak current of Fe(CN)63− obtained by DPV was used to characterize the immunosensor and con- struct a calibration curve. DPV parameters are as follows: initial potential, 0.45 V, final potential, −0.15 V, stepping potential, 0.004 V, amplitude, 0.05 V, pulse width, 0.05 s, pulse period, 0.2 s, and quiet time, 2 s. For IAA detection, 10 μL of different concentrations of IAA standard solution were dropped onto the surface of the modified electrodes and incu- bated for 1 h under humid conditions. The immunosensor was then rinsed with 10 mM PBS ( pH 7.4) to remove unreacted IAA. Thereafter, the electrochemical signals are recorded and the concentration of the IAA is reflected by the change of the reduction peak current (ΔI).
Preparation of plant material samples
The extraction and purification of IAA were conducted accord- ing to a previous report with some modifications.5 To prevent light exposure, the samples were kept in the dark throughout the process. Firstly, a grinding rod was used to grind the seeds (corn, brown rice, soybeans) into coarse powder. Then, the powder was collected in a vessel and mixed with 20 mL of 80% (V/V) methanol for overnight extraction in a refrigerator. Subsequently, the supernatant was collected after centrifugation at 15 000 rpm for 20 min and then passed through a Sep-Pak C18 column (Sigma, USA) in which the Sep-Pak C18 column was pre-washed with ethanol, double distilled deionized water and 80% methanol solution. The effluent was collected and the solvent was blown with nitrogen at ambient temperature. Finally, the powder was dissolved in 10 mL of 10 mM NaOH solution and diluted with 990 mL of 10 mM PBS ( pH 7.4).
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