Graphitic-Phase C3N4 Nanosheets Combined with MnO2 Nanosheets for Sensitive Fluorescence Quenching Detection of Parathion-Methyl

Bicheng Liu Nanchang University Qianhu Campus: Nanchang University Sihao Wu Nanchang University Qianhu Campus: Nanchang University Zoujun Peng Nanchang University Qianhu Campus: Nanchang University Jiahan Rui Nanchang University Qianhu Campus: Nanchang University Ping Qiu (  pingqiu@ncu.edu.cn ) Nanchang University Qianhu Campus: Nanchang University https://orcid.org/0000-0003-1680-6378


Introduction
The development of agricultural industrialization makes agricultural products more and more dependent on pesticides. As China is a large agricultural country in development, the consumption of pesticides is increasing day by day. However, the chronically, continuously, and largely absurd application of pesticides results in many typical cases of negative environmental consequences [1,2]. Organophosphorus pesticides (OPs) are anti acetylcholinesterase (AChE) and act via a common mechanism of phosphorylation of AChE, which disables AChE to catalyze acetylcholine and results in over-accumulation of acetylcholine leading into cholinergic toxicity [3,4]. OPs are one of the main causes of acute pesticide poisoning and they are also the key pesticide residues in fruits and vegetables. OPs can cause chronic poisoning, long-term contact, or food containing pesticide residues, and make pesticide accumulation in the body, cause physiological barriers, and also can affect the reproductive system, cause fetal malformation. Some pesticides have obvious carcinogenicity and mutagenicity [5]. More people pay attention to all kinds of problems which are caused by pesticide residues.
There are several analytical techniques reported for the determination of OPs from a variety of samples, including liquid/gas-mass spectrometry (LC/GC-MS), high performance liquid chromatography (HPLC), electrochemical method, and enzyme-linked immunoassay [6][7][8][9]. However, the existing detection methods are time-consuming, costly complicated with related equipment conditions, and require specially well-trained personnel to operate [10]. Fluorescence technology is found to be highly sensitive, simple, low cost, and low detection limit for the determination of OPs by using the uorescent probe [11,12]. Yan et al. developed a novel Fe 3 O 4 magnetic nanoparticle (MNP) peroxidase mimetic-based colorimetric method for the rapid detection of OPs and nerve agents [13]. A highly sensitive, rhodamine B-covered gold nanoparticle (RB-AuNP) -based assay with dual readouts (colorimetric and uorometric) for detecting organophosphorus and carbamate pesticides in complex solutions was reported [14]. A simple and sensitive uorescent sensor based on l-tyrosine methyl ester functionalized carbon dots (Tyr-CDs) and tyrosinase system was proposed for methyl parathion [15]. Some common uorescent materials, such as carbon nanomaterials,were prepared with hydrothermal method and must be extensively dialyzed for three days, which was time consuming and leading to a quite low yield (milligram level) [16]. Graphitic-phase C 3 N 4 nanosheets (g-C 3 N 4 ) is easily-prepared via onestep polymerization of cheap feedstocks like cyanamide, urea, thiourea, melamine, and dicyandiamide. It possesses a unique two-dimensional layered graphite-like structure composed of carbon and nitrogen atoms and has some advantages such as excellent chemical stability and tunable electronic structure [17,18]. g-C 3 N 4 is not only economical and environmentally friendly but also possesses excellent physicochemical properties. The characteristics of g-C 3 N 4 include high uorescence quantum yields, heavy-metal free structure, high biocompatibility, and low toxicity, which made it so hot [19]. g-C 3 N 4 as uorophores has been applied to detect all kinds of anions, metal ions, and bio thiols [20][21][22].
MnO 2 NS is a kind of novel, facile-synthesized 2D nanomaterial with good biocompatibility, single-layer manganese dioxide nanosheet [23]. It was reported MnO 2 NS possess oxidase-like property. They that can react with thiocholine (TCh, a hydrolyzed product of acetylthiocholine) and reduced to Mn 2+ by TCh [24,25]. Furthermore, it is interesting that MnO 2 NS can quench many uorophores including g-C 3 N 4 through inner lter effect (IFE) [26].
As we all know, the activity of acetylcholinesterase is inhibited in the presence of OPs, so the hydrolysis of acetylcholine is inhibited. The reaction of hydrolysis product (TCh) with MnO 2 NS is also limited directly, which determines the degree of uorescence recovery of g-C 3 N 4 . The uorescence intensity of g-C3N4 recovers proportionally with the concentration of PM. So we designed a sensitive uorescent assay to detect parathion-methyl using g-C 3 N 4 probe with MnO 2 nanosheets.

Materials and reagents
Parathion-methyl (PM) was purchased from the Nation Institute of Metrology (Beijing, China) and was used without further puri cation. The standard solution of PM (0.01 mg/mL) was prepared by dissolving in methanol. Tetramethylammonium hydroxide and Tris (hydroxymethyl) methyl aminomethane were purchased from Adamas-beta Reagent Company (Shanghai, China), and manganese chloride (MnCl 2 ) were obtained from Sinopharm Chemical Reagent Company (Shanghai, China). Melamine and hydrogen peroxide (H 2 O 2 ) were purchased from Aladdin Industrial Corporation (Shanghai, China).
Acetylcholinesterase (AChE, 744 unit/mg) and acetylthiocholine iodide (ATCh) were purchased from Sigma-Aldrich (Shanghai, China). The buffer solutions used in this study were as follows: Phosphate buffer (PB) (pH 5; 25 mM); Phosphate buffer solution (PBS) (pH 8.5; 10 mM); Tris-HCl buffer (pH 7.5; prepared in ultrapure water and used within 3 h. All chemicals were of analytical-reagent grade, and the ultrapure water (18.25 MΩ, Millipore, USA) was used throughout the experiments. Instrumentation UV-vis Absorption spectra were recorded using UV-2450 Shi-madzu Vis-spectrometer (Japan), the uorescence spectra were collected on an F-4600 uorescence spectrometer (Hitachi, Japan). The morphology of the MnO 2 NS was characterized by transmission electron microscopy (TEM, JEM-2100, JEOL Co., Japan) operating at 200 kV acceleration voltage. X-ray photoelectron spectroscopy (XPS) was carried out on Esca Lab 250Xi with Al K α X-ray radiation as the source for excitation. The crystalline structure of the nano akes was investigated by X-ray diffraction (XRD, Bruker, Germany) with the scanning speed of 2°/min. Scanning electron microscopy (SEM) images were obtained with a JSM-6701F microscope system (JEOL Co., Tokyo, Japan). Zeta potentials were obtained using a Nano ZS90 (Malvern, UK). The binding energies were calibrated by C 1 s (284.8 eV), and pass energy was 30.0 eV with a spot size of 650 µm. The sample for TEM characterization was prepared by placing a drop of the MnO 2 NS solution on a carbon-coated copper grid and drying it at room temperature.

Synthesis of MnO 2 nanosheets
MnO 2 NS was synthesized by a simple wet chemical method [27]. For this, 10 mL tetramethylammonium hydroxide (6%) was added to 30 mL of H 2 O 2 (3%), then 20 mL of MnCl 2 (0.3 M) was quickly transferred into the above solution within 15 s. The color of the mixture solution quickly turned dark brown, and the mixture was kept on stirring for 12 h at room temperature. After stirring, the obtained mixture was centrifuged at 4000 rpm for 30 min and washed successively with ultrapure water and methanol. Next, the bulk MnO 2 NS was dried at 60 °C for 72 h. 0.0030 g of MnO 2 NS was dissolved in 60 ml of water, and brown MnO 2 NS dispersion was obtained after sonication for 12 h, which was stored in a refrigerator at 4 °C for further experiments.
Preparation of g-C 3 N 4 g-C 3 N 4 was synthesized by the direct thermal treatment of melamine as reported in previous work [28]. An alumina crucible with a cover, contained melamine (10 g) was heated to 600 °C with a heating rate of 3 °C/min under air conditions, and was kept at that temperature for 2 h. Cooling the product to room temperature, the yellow g-C 3 N 4 powder was obtained. 0.0080 g of powder yellow g-C 3 N 4 product was dissolved in 10 ml of water, which was stored in a refrigerator at 4 °C for further experiments.
Fluorescence method for detection of PM AChE (50 µL,100 U/L) contained a series of different concentrations (10 µL) of PM were added to centrifuge tubes and incubated at 37 °C for 0.5 h. ATCh (5 mM, 40 µL), MnO 2 NS (30 µg/mL, 100 µL) and PBS buffer (200 µL, pH 7) were added successively to centrifuge tubes and incubated at 37 °C for another 0.5 h. Then, g-C 3 N 4 (100 µL, 0.8 mg/mL) was mixed to the reaction system and diluted the nal volume of 2000 µL. The uorescence emission spectrum was measured, and the uorescence intensity at 460 nm was recorded.

Results And Discussion
Characterization of MnO 2 NS and g-C 3 N 4 Firstly, MnO 2 NS was characterized by transmission electron microscopy (TEM), which showed a twodimensional structure, and exhibited multiple folds and crinkles in Fig. 1a. Meanwhile, MnO 2 NS had a wide UV absorption band ranging from 300 nm to 600 nm, and the maximum absorption was concentrated at 360 nm (Fig. 1b). Detailed analysis based on Xray photoelectron spectroscopy (XPS) was further investigated to con rm the features of MnO 2 NS. Figure 1c displayed the XPS spectrum for the MnO 2 NS. The two peaks of the Mn2p 3/2 and Mn2p 1/2 located at 642.0 and 653.9 eV were detected and was proved to be attributed to Mn 2+ [29,30]. Thus, the above characterization indicated that MnO 2 NS had been synthesized successfully.
On the other hand, the morphologies of g-C 3 N 4 nanocomposites was carried out using SEM, XRD and XPS in details. Figure 1d showed the SEM images of g-C 3 N 4 , which investigated the microstructure of g-C 3 N 4 . The surface of g-C 3 N 4 has loose aperture and rough block structure. XRD patterns were used to identify the crystal structure of g-C 3 N 4 and the resultshowed a major diffraction peak occurred at around 27.3 as shown in Fig. 1e. No diffraction peaks of other impurity phases were detected in all the samples. The strong peak at around 27.3 could be indexed as (002), which was due to interplanar stacking of aromatic systems [31] . The typical XPS survey scan is an effective way to investigate the chemical bonding environment and the states of elements in g-C 3 N 4 nanocomposites. As shown in Fig. 1f, the complete survey XPS spectrum reveals that C and N peaks could be found in all samples, and no peaks of other elements were observed, indicating that the g-C 3 N 4 nanocomposites kept the same chemical composition and chemical states [31].
Principle of uorescence sensing strategy In the system, it is interesting that MnO 2 NS acts not onlyas the quencher of g-C3N4, but also as as an oxidizing agent of TCh. g-C 3 N 4 exhibited a strong uorescence emission, but MnO 2 NS could quench the uorescence of g-C 3 N 4 at 460 nm via the inner-lter effect (IFE). As shown in scheme 1, ATCh was catalyzed by AChE and hydrolyze to form TCh and acetic acid. A redox reaction occurred between sulfhydryl group (-SH) of TCh with MnO 2 NS to form disul de (-S-S-) and Mn 2+ , resulting in the uorescence recovery of g-C 3 N 4 . OPs, as inhibitors for AChE activity, can prevent the generation of TCh and decomposition of MnO 2 nanosheets, accompanied by uorescence quenching again. So the AChE-ATCh-MnO 2 -g-C 3 N 4 system can be utilized to detect OPs quantitatively based on the g-C 3 N 4 uorescence.
To verify the detection mechamism of uorescence sensing strategy, Fig. 2a shows UV-vis absorption spectrum of MnO 2 NS and the mixture with AChE and ATCh. MnO 2 NS have a obvious UV absorption peak located at 360 nm (Fig. 2a) [27]. In addion, it was found that the color of MnO 2 NS itself was yellow, but it became pale in the presence of AChE and ATCh (the inset images of Fig. 2a), and the absorption peaks of MnO 2 NS also decreased. As described previously, ATCh is hydrolyzed to form TCh and acetic acid hydrolyzing by AChE. We can conclude that redox reaction occurred between sulfhydryl group (-SH) of TCh with MnO 2 NS to form disul de (-S-S-) and Mn 2+ , which can restore the uorescence of g-C3N4 at 460 nm. Figure 2b show the spectra of g-C3N4 and the mixture with different substances, and it was found that the uorescence intensity of g-C3N4 was different signi cantly. In Fig. b (1), (2), (3), they had similar uorescence intensity, which showed that both AChE and ATCh have no effect on the uorescence intensity of g-C3N4. In Fig. b (5) the weak uorescence was observed, which illustrated that MnO 2 NS would quench the uorescence of g-C3N4 strongly. Compared the curve 3 with curve 5, the uorescence intensity of g-C3N4 be restored in the presence of AChE and ATCh. It is because that TCh reduced MnO 2 NS to Mn 2+ , which caused the decrease of MnO 2 NS in the system and the uorescence intensity of g-C3N4 restored. Compared curve 3 with curve 4, there was less TCh to react with MnO 2 NS with the existence of PM, so the uorescence of g-C3N4 was still quenched again. The mechanism of quenching uorescence by MnO 2 NS includes uorescence resonance energy transfer (FRET) and inner-lter effect (IFE). To further explore the quenching mechanism of MnO 2 NS on g-C3N4, the zeta potential and UV-vis absorption spectrum were carried out. As shown in Fig. 2c, the zeta potentials of g-C3N4, MnO 2 NS, and the mixture are all negatively charged, so there is electrostatic repulsion between g-C3N4 and MnO 2 NS. It demonstrated that g-C3N4 and MnO 2 NS could not be close to produce uorescence resonance energy transfer. On the other hand, Fig. 2d showed the uorescence emission spectra of g-C3N4 and the UV-vis absorption spectra of the MnO 2 NS. It can be seen that there was a strong overlap between the emission spectrum and the absorption spectrum, which provided the necessary condition for the inner lter effect (IFE). The above results show that MnO 2 NS quenches the uorescence of g-C 3 N 4 through IFE.

Optimization of the detection conditions for uorescence analysis
To obtain the optimal uorescence sensing response for the detection of PM using g-C3N4/MnO 2 NS, the experimental parameters affecting sensitivity and selectivity were all optimized, including the concentrations of AChE, MnO 2 NS and g-C3N4, pH, the reaction time, and the concentration of ATCh.
For this, the relative intensity was used to evaluate the optimal reaction system, which can be represented by (F 0 -F)/F 0 . Herein, F 0 is the uorescence intensity of g-C3N4, and F is the uorescence intensity after the redox reaction. As shown in Fig. 3a, the sensitivity increased slowly with the increase of AChE content and the maximum sensitivity was obtained when the concentration of AChE was 250 U/L. The effect of the concentrations of MnO 2 NS (15-40 µg/mL) and g-C3N4 (5-80 µg/mL) on relative intensities were both investigated respectively ( Fig. 3b and 3c). It can be concluded that the optimal concentrations of MnO 2 NS and g-C3N4 were 30 µg/mL and 60 µg/mL, respectively. Next, it was found that the maximum sensitivity was obtained when the pH was 7 (Fig. 3d). Figure 3e showed the in uence of oxidation time of probe pairs and the sensitivity reached the stable performance at 30 minutes. The concentration of ATCh could effect the degree of oxidation reaction of MnO 2 NS (Fig. 3f), and the higher the concentration is, the more complete the reaction was. The relative intensity kept stable after 3 mM ATCh.
Linearity and limit of detection PM and DDVP were chosen as typical OPs for the detection based on g-C3N4/ MnO 2 NS. Under optimum experimental conditions, a range of their concentrations for detection was investigated as shown in Fig. 4. According to the detection principle, a uorescence quenching takes place with the addition of OPs and the degree of uorescence quenching is proportional to their concentrations. The linear range of PM concentration was obtained from 0.1 to 2.1 ng/mL (Fig. 4a). The inset indicated a good t to the linear model, and the obtained linear least square equation was expressed as Y = -246.2X + 1509 (ng/mL) with the correlation coe cient (R 2 ) of 0.993. The limit of detection (LOD) was calculated to be 0.069 ng/mL according to LOD = 3σ/k, where σ stands for standard-error of estimate and k is the slope of linearregression-line. For DDVP, the linear regression equation was Y = -53.9X + 1317.2 (ng/mL) with the correlation coe cient (R 2 ) of 0.995 and the LOD was 0.20 ng/mL (Fig. 4b). LOD and linear range of this assay was compared with the already reported methods, and the comparison is presented in Table 1 [32][33][34][35][36], the uorescent assay for the determination of OPs based on g-C3N4/MnO 2 NS/AChE-ATCh uorescent probes has higher sensitivity and excellent analytical performance.  4 3-), and biomolecules (Vitamin B (V B ), glycine and tyrosine, humic acids, chlorophyll). We added the interfering substances to the reaction system in the presence of PM (1 ng/mL), and their nal concentrations were 100 times the standard concentration of PM. Compared to the control group, the relative change varied from 0.91 to 1.1, so there is no signi cant difference (Fig. 5).Therefore, the developed g-C3N4/ MnO 2 NS based uorescence assay was speci c for OPs detection and can be applied for PM determination in these common substances do not interfere with the assay with high selectivity.

Real samples detection
OPs often processes into the lake with watering, rainfall, and other. So Lake water samples were collected as real samples from Longteng Lake and Qian Lake in Nanchang city, China. Water samples were spiked with PM to get that desired concentration and then were pretreated according to the method reported in the previous literature [37]. The collected samples were ltered through a 0.45 µm membrane syringe lter to remove any particulate matter. As summarized in Table 1, the recovery (recovery rate (%) = 100×(C detection amount /C addition amount )) for PM of the proposed methods for two samples was in the range of 85.2-116.9% and 82.2-109.6%, with RSD (n = 3) for less than 3.4% and 4.2%, respectively. The results demonstrated that the proposed methods could be applied to detect PM in practical samples.

Conclusions
In this study, we developed a convenient, reliable, and sensitive assay for OPs detection based on MnO 2 NS/g-C 3 N 4 . The g-C 3 N 4 were employed as the uorescence units, and the MnO 2 nanosheets were used as the dual-functional nanoquencher and oxidizing agent in this developed platform. The strategy depends on TCh decomposition of MnO 2 nanosheets and OPs-caused inhibition of the enzyme. The proposed method shows sensitivity and high-selectivity towards PM, and the limit of detection reaches as low as 0.069 ng/mL. The present assay shows many advantages including environmental-friendliness, costeffectiveness, and ease-of-use, demonstrating that the convenient strategy could serve as a promising platform for detecting OPs.

Declarations
Authors' Contributions All authors contributed in the present study. All authors commented on this version of the manuscript and they read and approved the nal manuscript.
Funding This work is founded by the National Natural Science Foundation of China (21765015, 21808099) and the Science and Technology Innovation Platform of Jiangxi Province (20192BCD40001), China.
Availability of Data and Materials Not applicable.

Compliance with Ethical Standards
Competing Interests Authors declare that they have no con ict of interst.    The selectivity of proposed method.

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