JES F OCUS I SSUE ON B IOSENSORS AND M ICRO -N ANO F ABRICATED E LECTROMECHANICAL S YSTEMS Bamboo Fungus-Derived Porous Nitrogen-Doped Carbon for the Fast, Sensitive Determination of Bisphenol A

Biomass-derived carbon materials have been considered as perfect substitutes for the traditional doped carbon materials, owing to its simple synthesis, rich-doped hetero-atoms, low cost and satisfactory electrochemical properties. In this paper, a rich nitrogen-doped carbon (NDC) material derived from bamboo fungus by simple carbonization was ﬁrstly applied for the electrochemical determination of Bisphenol A (BPA). NDC showed high catalytic activity towards the BPA oxidation for its rich doped nitrogen, high conductivity and porous structure. The morphology and structure features of NDC were characterized by the transmission electron microscopes and Raman spectrum. Meanwhile, the electrochemical properties of NDC modiﬁed glassy carbon electrode (GCE) and the behaviors of BPA on the NDC modiﬁed GCE (NDC/GCE) were estimated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimum conditions, differential pulse voltammetry (DPV) was used for the quantitative determination of BPA. A low detection limit (LOD) of 1.068 μ M (IUPAC S/N = 3) was obtained within the range of 1.0 μ M to 50.0 μ M. And the NDC/GCE was further applied for the practical determination of BPA in soil extract samples successfully. The results revealed that the simple and sensitive NDC based platform could be a potential approach for the practical detection of BPA. ©

Carbon materials have attracted a lot of attention in the electrochemical field during the past ten years, owing to its high conductivity, large specific area and sufficient functional groups, etc. However, many efforts have been made to further improve the electrochemical performance of carbon materials, among which, the doping of heteroatoms was reported as an efficient way. [1][2][3][4] The doped hetero-atoms could significantly change the electrical structure of the nearby carbon atoms and enhance the electronic performance accordingly. For example, the nitrogen-doped graphene (NGE) was widely applied as an electrocatalyst for oxygen reduction reaction, 2,5 electrochemical sensors 6,7 and supercapacitors 3,8 for its numerous active sites and better electron transfer ability compared to the un-doped one. However, the synthesis of the doped carbon materials requires costly reagents, complex procedures and rigorous reaction conditions, which greatly limits its application.
In such circumstances, biomass-derived carbon materials are considered as an attractive alternative for its rich-doped heteroatoms, low-cost and perfect electrochemical properties. Over the past few years, various precursors have been utilized for the synthesis of biomass-derived carbon materials, the preparation methods included direct calcination, 9,10 hydrothermal carbonization, 11,12 microwave irradiation 13 and so on. Biomass-derived carbon materials are widely applied as energy storage device, 11-13 catalyst 9 , pressure 14,15 and florescent sensor 16,17 and recyclable organic sorbent. 18,19 An interesting feature in its synthesis process has been noticed, that is, the carbonization products tend to remain the original framework as the precursors. For example, the porous products 20 could be easily obtained from the porous precursors without the use of pore former. The porous biomass-derived carbon materials have attracted more attention porous structure for its large specific area, good adsorption capacity and conductivity ability. Therefore, the porous biomassderived carbon materials can well perform as oxygen reduction reaction catalysts, 21,22 adsorbents 23,24 and supercapacitors. 25,26 However, to the best of our knowledge, there are still quite a few researches focused on the electrochemical sensing applications of porous biomassderived carbon materials. 20 Bisphenol A (BPA) is a widely used raw material in the industrial production of polycarbonate, epoxy, unsaturated polyester resins and flame retardants. However, BPA is confirmed as a typical toxic contaminant and endocrine disruptor chemical (EDC), 29 and the long-term exposure to BPA can lead to various endocrine diseases, even cancers. In order to avoid the risk of BPA residual and trace it efficiently, the reliable detection methods have become vital demands. The traditional instrument analysis technologies are of high-accuracy and widely used in the trace detection of BPA. Such as high performance liquid chromatography, 30 liquid chromatographytandem mass spectrometry, 31 gas chromatography coupled with mass spectrometry 32 and fluorescence analysis. 33,34 But the expensive investment and the highly-trained operators greatly restrain its practical application. Meanwhile, as BPA is an electrochemically-active molecule, the electrochemical method is considered to be a better choice in the on-site determination for its simplicity, sensitivity and selectivity. Different materials were reported for the electrochemical detection of BPA, including metal composites, 35,36 molecularlyimprinted polymer, 37,38 conducting polymer 39 and carbon materials (carbon-nanotube, 40 graphene, 41 quantum dots composite, 42 carbon black 43 ). However, the sustainable and rich-doped carbon materials derived from biomass have not been reported for the electrodetermination of BPA yet.
Herein, a sustainable and environmentally friendly biomassderived porous nitrogen-doped carbon (NDC) material was firstly proposed as a fast, sensitive BPA electrosensing platform. The porous structure of NDC can provide abundant reaction sites and promote the adsorption and accumulation of BPA efficiently; meanwhile, the rich-doped nitrogen atoms can create sufficient active defects and highly catalyze the electro-reaction. The NDC modified glassy carbon electrode (GCE) shows significant catalytic activity to the oxidation of BPA with the enhanced peak current and negatively shifted peak potential. The electrochemical properties of the NDC modified GCE (NDC/GCE) and the behaviors of BPA on it were investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and differential pulse voltammetry (DPV). Under the optimum conditions, the linear dependence between the oxidation peak current and the BPA concentration was obtained in the range of 1.0 μM to 50.0 μM with a low limit of detection (LOD)

Experimental
Materials.-The fresh bamboo fungi were purchased from the market and carefully washed by deionized water before further use. Deionized water prepared by Milli-Q water purifying system (18 M cm −3 ) was used in all experiments. All chemical reagents were of analytical grade and used without further purification. N 2 airflow was used before every electrochemical measurement to expel the dissolved oxygen in the electrolyte. Phosphate buffer solution (PBS) was prepared with NaH 2 PO 4 /Na 2 HPO 4 solution (0.2 M). All the experiments were performed under room temperature (25 ± 0.5 • C).
Preparation of NDC.-NDC was prepared by simple hydrothermal and calcination process as reported. 44 In an optimized process, 6 g bamboo fungi and 75 mL deionized water were mixed and transferred into a 100 mL Teflon-lined stainless steel autoclave, and then heated under 180 • C for 24 hours. After cooling down to room temperature, the product was collected by centrifugation and washed with deionized water and ethanol for several times. The precipitate was dried under room temperature and ground for further use. The pretreated solid material was then annealed in the presence of ZnCl 2 at a mass ratio of 1:3 in N 2 atmosphere at 800 • C for 2 h (temperature ramp of 10 • C min −1 ). At last, the NDC was cooled to room temperature and ground before further use.
Fabrication of NDC/GCE.-Before modification, GCE was carefully polished with alumina/water slurry (CH Instruments, Inc.), and then rinsed and cleaned by ultrasonic in deionized water. After that, the electrode was dried in nitrogen atmosphere. NDC was dispersed in deionized water to form an aqueous homogeneous solution (3 mg ml −1 ); 15 μL NDC solution was carefully dropped onto the GCE surface and dried in nitrogen N 2 in 3 portions.
Preparation of real samples.-The soil extracts were collected in Nanjing university of science and technology. The soil suspension was prepared from 0.2 kg field soil and 1 L deionized water. After boiled for 1 h and centrifuged, the supernatant of the suspension was used as a soil extract. Prior to experiments, the soil extracts were adjusted to pH 8.0 using solid NaH 2 PO 4 /Na 2 HPO 4 to obtain 10 mL PBS containing 0.5 mL 0.2 M NaH 2 PO 4 and 9.5 mL 0.2 M Na 2 HPO 4 . Spiking experiments were carried by injecting BPA solution into the soil extract and then homogenizing.
Instrumentation.-The electrochemical measurements were performed on CHI660D electrochemical workstation (CH Instruments, Inc.) with a three-electrode system. A bare or modified GCE was used as the working electrode; meanwhile, a saturated calomel electrode (SCE) and a platinum wire electrode were employed as reference and auxiliary electrodes, respectively. The morphology and structure characterization of NDC was performed by Transmission electron microscopy (TEM) and Raman spectral Instrument. TEM images were studied by JEOL-2100 and Raman spectra were recorded on Renishaw inVia Raman Microprobe using a 514.5 nm argon ion laser.

Results and Discussion
Morphology characterization of NDC.-The morphology of NDC was analyzed by TEM, as demonstrated in Figure 1. The typical NDC is of layer structure and loosely packed by porous carbon sheets with a size about several hundred nanometers. As presented in larger-magnification images ( Figures 1B and 1C), the nanopores are uniformly-distributed in the surface of the nanosheet with an average size about 20-30 nm. As the molecular size of BPA is 0.94 nm × 0.59 nm × 0.48 nm, 45 in view of this, the nanopores of NDC are absolutely capable for the adsorption and transfer process of BPA molecules. Moreover, the surface area of NDC is 1895.5 m 2 g −1 as reported in our previous work. 44 Therefore, the transport and adsorption process of BPA on the NDC modified electrode would be greatly promoted. Additionally, the porous structure could further offer large interaction area and plenty active sites during the BPA oxidation. As a result, the sensitivity of the electrochemical sensor will be efficiently enhanced.
Raman analysis was performed to further investigate the structural defects and the graphitization degree of NDC. As shown in Figure 2, just like other carbon materials, NDC exhibits a pair of typical peaks at 1340 cm −1 and 1587 cm −1 corresponding to the D band and G  band, respectively. Generally, D band is related to the carbon defects while G band signifies the in-plane vibration of the sp 2 atoms. 46 The Raman data indicate that there are defects and the partially disordered graphitic structure in NDC. The intensity ratio between D and G bands (I D /I G ) was calculated to be 1.12, which is close to that of the reduced graphene oxide (1.10), 47 suggesting that NDC is of the similar graphitization degree to the graphene oxide.  Figure 3B). The semicircle of the Nyquist plot at the higher frequencies corresponds to the electron transfer limited process, while the diameter of the semicircle is proportional to the electron transfer resistance (R ct ). As illustrated in Figure 3B, after the electrode was modified with NDC, the semicircle diameter of the NDC/GCE decreased distinctly, demonstrating that the modification of NDC could greatly improve the conductivity of the electrode, and accelerate the electron transfer process between the electrode surface and the electrolyte. Figure 4 shows the CVs of 20 μM BPA at GCE (curve a) and NDC/GCE (curve b) in 0.1 M PBS (pH 8.0), besides, the CV of NDC/GCE in 0.1 M PBS (pH 8.0) without BPA is presented as curve c (scan rate: 100 mV s −1 ). Obviously, the absence of the oxidation peak of curve c proves that the peak at 0.44 V (a and b) exactly corresponds to the oxidation of BPA. Also, as there is only one oxidation peak at GCE and NDC/GCE, the electrochemical oxidation of BPA is defined to be an irreversible process. The irreversibility results from the inactivation of the electrode surface caused by the deposited BPA oxydate film. 48 Compared with the bare GCE (curve a), the oxidation potential on NDC/GCE (curve b) negatively shifted from 0.485 V to 0.44 V; meanwhile, the anodic peak current at NDC/GCE (17.82 μA) was about 10 times higher than that at bare GCE (1.614 μA). The NDC/GCE shows the distinct enhancement of catalytic activity and significant amplification of the response intensity during the oxidation of BPA. The improvements can owe to the following points.
Firstly, the porous structure of NDC could efficiently promote the BPA adsorption and the electron transfer process; secondly, the abundant defects and the pyridine-like nitrogen 44 can act as active sites and greatly catalyze the oxidation of BPA. Therefore, the proposed NDC/GCE presented an excellent sensitivity, making it possible to be applied further as a sensing platform for the determination of BPA.
The kinetic mechanism of the BPA oxidation process was investigated by CV method with different scan rates. Figure 5 depicts the CVs of 20 μM BPA at NDC/GCE in 0.1 M PBS (pH 8.0) under different scan rates (ν). It is obvious that the oxidation peak currents increases proportionally as the scan rate gradually is increased from 20 to 200 mV s −1 (Figure 5A), the linear calibration ( Figure 5B) between them can be derived as: i (μA) = 0.1016 ν (mV s −1 ) + 5.7464 (R 2 = 0.998). The result reveals that the oxidation of BPA on the NDC /GCE is an adsorption-confined process. 49 Optimization of experiment conditions.-The effects of the pH condition on the peak potential and current were measured in 0.1 M PBS containing 20 μM BPA (pH: 6.0-10.0) with a scan rate of 100 mV s −1 . As observed in Figure 6A, when the pH value was adjusted from 6.0 to 8.0, the peak currents increased successively. However, as the pH further grew to 10.0, the peak current conversely decreased. In order to obtain the best electro-response, pH 8.0 was applied in the experiments. Meanwhile, the peak potentials shifted  negatively as the pH value changed from the acidic environment to alkaline environment ( Figure 6B). The linear dependence between the anodic peak potential and pH can be expressed as: E (V) = −0.072 pH + 1.0342 (R 2 = 0.992). The slope value of −72 mV per pH unit is reasonably close to the theoretical value of −57.6 mV per pH, indicating that there are equal numbers of electrons and protons involved in the BPA oxidation at NDC/GCE. According to literature, 50 in the BPA oxidation reaction, the electron transfer number is 2, so the number of the involved protons is defined as 2. In summary, the oxidation of BPA at NDC/GCE is a two-electron and two-proton process, and the reaction equation is shown in Figure 6C.
For an adsorption-confined process, both the modification amount of the NDC and enrichment time would influence the adsorption of BPA, so the effects of the NDC modification amount and the enrichment time were optimized. As exhibited in Figure 7A, the measurements towards 20 μM BPA were performed by using the electrodes with different NDC modification amounts (3-90 μg) in 0.1 M PBS (pH 8.0) at a scan rate of 100 mV s −1 . When the NDC modification amount increased from 3 to 45 μg, the peak current increased accordingly, however, as the modification amount further added, the peak current decreased. Apparently, certain amount of the NDC modification can enhance the conductivity of the electrode as well as the BPA adsorption, as the result, the peak current is amplified. However, the excessive modification would lead to the dense package of NDC and hinder the transfer process of electrons and BPA molecules instead. So 45 μg was determined as the modification amount in the subsequent experiments.
The influence of enrichment time was also estimated in 0.1 M PBS (pH 8.0) with 20 μM BPA (scan rate: 100 mV s −1 ) at NDC/GCE. Obviously, as demonstrated in Figure 7B, the peak current kept increasing as the enrichment time was extended from 10 s to 40 s, when the enrichment time was longer than 40 s, the electro-response signal conversely decreased. Even though the enrichment process could promote the exchange and adsorption of BPA, but the over-enrichment would result in the saturated BPA exchange and adsorption and further weaken the peak current. 51 Considering both the sensitivity and working efficiency of NDC/GCE, 40 s was determined as the enrichment time in the further experiments.
Determination of BPA on NDC/GCE.-Under the optimal conditions, DPV method was used for the quantity determination of BPA at NDC/GCE. As shown in Figure 8A, the anodic peak current at NDC/GCE increased linearly as the BPA concentration was changed from 0.5 to 30 μM in 0.1 M PBS buffer (pH 8.0). The linear relationship between the oxidation peak current and the concentration of BPA is presented in Figure 8B. In the range of 1.0 μM -50.0 μM, a low    Table I. method with soil extract. According to the experiments, in the soil extracts without BPA, there was no electro-response. After different concentrations of BPA (10, 30, 50 μM) were spiked into the soil extracts, the analytical data were recorded as shown in Table II. The high recovery and low RSD data suggest that the NDC modified electrode can act as a potential approach for the determination of BPA.

Real samples determination.-The potential practical application of the proposed NDC/GCE was investigate by standard-additions
Stability, repeatability and selectivity performance.-The stability, repeatability and selectivity performance of NDC/GCE were investigated by CV method in 0.1 M PBS (pH 8.0) containing 20 μM BPA under the optimized conditions. The relative standard deviation (RSD) of ten successive detections of 20 μM BPA was 1.46% for the same NDC/GCE, indicating the good repeatability of the proposed NDC/GCE. As for the stability measurement, the NDC/GCE was kept at room temperature. After three days, the peak current retained 94.7% of the initial response intensity; after ten days, the peak current still remained 87.2%, suggesting the satisfactory stability performance.  In addition, various potential interfering species were evaluated in PBS (pH 8.0) in the presence of 20 μM BPA (Figure 9), including 10-fold concentration of p-nitrophenol (PNP), pyrocatechol (CT), hydroquinone (HQ), acetaminophen (AP) and 500-fold concentration of inorganic ions, such as K + , Cu 2+ , Ni 2+ , Zn 2+ , SO 4 2− , Cl − . The interfering species showed no obvious interference to the oxidation peak current of BPA, demonstrating the perfect selectivity of the NDC modified electrode.

Conclusions
A novel sustainable, biomass-derived rich nitrogen-doped porous carbon material was proposed as a sensitive, selective and stable sensing platform for the electrochemical determination of BPA. The unique porous structure can highly enhance the adsorption and accumulation of BPA, while the rich-doped pyridine-like nitrogen can act as active sites and greatly catalyze the oxidation of BPA. The constructed NDC/GCE presented the high sensitivity, low detection limit and perfect stability and was successfully applied for the BPA determination in the real samples. The simple and easy-controlled material synthesis and electrode fabrication process make NDC/GCE a potential approach for the field detection of BPA.