“Off–On” fluorescent sensing of organophosphate pesticides using a carbon dot–Au(iii) complex

Herein, we report novel “off–on” fluorescent sensing of organophosphate pesticides using a carbon dot (CD)–Au(iii) complex/acetylcholinesterase (AChE) system. The above sensor utilizes the quenching of CD fluorescence by Au(iii) and its subsequent recovery by thiocholine, which is generated by AChE-catalyzed hydrolysis of acetylthiocholine (ATCh) and effectively scavenges Au(iii). In the presence of organophosphates, the catalytic activity of AChE is inhibited, allowing these species to be quantified based on the decreased recovery of CD fluorescence intensity. The developed sensor was used to analyze a real pesticide-spiked sample (4.48 μM), achieving a recovery of 99.85% and exhibiting a linear response range of 0.45–44.80 μM.


Introduction
Organophosphate pesticides nd extensive applications worldwide, exhibiting acute toxicity upon ingestion and thus posing a serious risk to human health. 1,2 The above pesticides mainly target acetylcholinesterase (AChE), 3 inhibiting its catalytic activity and thus hindering the hydrolysis of acetylcholine (ACh, a neurotransmitter), with the accumulation of excess ACh leading to lethal overexcitation. 4,5 However, the above activity inhibition can be successfully utilized in organophosphate pesticide sensing.
Carbon dots (CDs) have recently found numerous applications due to exhibiting excellent uorescence, good biocompatibility, and low toxicity and cost. 6,7 Recently, much effort has been directed at utilizing CD uorescence or electrochemiluminescence to fabricate "off-on" or "on-off" sensors, [8][9][10] which exhibit the advantages of fast response, easy operation, and clear signal recognition. 11 Notably, metal ions such as Cu 2+ , Fe 3+ , Hg 2+ , K + , and Ag + (ref. [11][12][13][14][15] are able to quench the uorescence of CDs, 16 allowing the sensing of heavy metals, organic pollutants in water, sugar in blood, and biomarkers in living cells. [17][18][19][20] Herein, we report a novel "off-on" uorescent biosensor for organophosphate pesticides, in which the CD uorescence initially quenched by Au(III) 20 is recovered by thiocholine (TCh, an effective Au(III) scavenger) generated by AChE-catalyzed hydrolysis of acetylthiocholine (ATCh) that is structurally similar to ACh. In the presence of organophosphates, the catalytic activity of AChE is inhibited, allowing these species to be quantied based on the decreased recovery of CD uorescence intensity.

Materials and reagents
Eggs were purchased from the Chaoshifa supermarket. AChE and ACh were purchased from Sigma-Aldrich (USA). H 2 SO 4 , NaOH, and HAuCl 4 were sourced from Sinopharm Co. (China), and all other chemicals were obtained from the Beijing Chemical Plant. Deionized (DI) water was produced using the Milli-Q system (USA). Incubators and magnetic stirrer plates were purchased from IKA Instrument Co. (Germany). Fluorescence spectra were recorded on a HITACHI 2000 spectrometer (Hitachi Co., Japan), and Fluorescence Spectrometer FS5 from Edinburgh Instruments (U.K.). Transmission electron microscopy (TEM) imaging was performed using a JEM-2100 instrument (JEOL Co., Japan). The Fourier transform infrared spectrometer was Tensor 27 from Buruker. Co. etc.(Germany). Xray Photoelectron Spectroscopy (XPS) was tested by using PHI Quantera SXM, from ULVAC-PHI (Japan).

CD synthesis
A procedure previously reported elsewhere 23 was optimized and used to prepare CDs.
Fresh egg white (4 mL) was dissolved in DI water (40 mL) upon 3 min stirring, and a 10 mL aliquot of the obtained solution was transferred into a 50 mL ask, treated with 98 wt% H 2 SO 4 (4 mL, slow addition!), agitated, and placed in a water bath (50 C) for 2 h. Subsequently, the mixture was neutralized with 5 M aqueous NaOH and subjected to 5 min centrifugation at 5000 rpm. The supernatant was transferred into an Eppendorf tube and treated with dichloromethane (3 mL), with another 5 min centrifugation at 5000 rpm resulting in the extraction of the synthesized uorescent carbon dots into the organic phase. The dichloromethane phase was separated and blown dry with nitrogen, and the thus obtained CDs were dispersed in DI water for utilization in the next step.

Characterization of CDs and CD/Au(III) systems
The emission spectrum of carbon dots was scanned with different excitation wavelength (slit width ¼ 10 nm). And the morphology of carbon dots was characterized by transmission electron microscope (TEM). The surface groups and elemental speciations were tested by FTIR and XPS respectively.
A series of Eppendorf tubes was charged with the abovementioned aqueous CD dispersion (20 mL), which was followed by the addition of 0.2 M phosphate-buffered saline (PBS, pH 7.0, 100 mL) and solutions containing different concentrations of Au(III) ions. The resulting mixtures were diluted with DI water to 2 mL, incubated at room temperature for 8 min, and probed by uorescence spectroscopy.

Organophosphate pesticide sensing
AChE-catalyzed hydrolysis of ATCh afforded TCh that could bind Au(III) ions and thus recover CD uorescence. Since organophosphate pesticides inhibit AChE, their presence resulted in slower TCh generation and thus decreased CD uorescence recovery. A series of organophosphate pesticide solutions of different concentrations was added to the CD/ Au(III)/(ATCh + AChE) system to construct a calibration curve.

Results and discussion
3.1 Principle of "off-on" uorescent sensing AChE catalyzes the hydrolysis of S-acetylthiocholine iodide to TCh, 22 which can effectively capture Au(III) and thus recover the CD uorescence quenched by the same. 20 As mentioned above, organophosphate pesticides inhibit AChE, thus decreasing the rate of TCh generation and hindering uorescence recovery, which was used for their detection (Scheme 1).

CD synthesis and properties
The synthesized CDs exhibited nearly spherical shapes (diameter z 20-40 nm) and were well dispersible in DI water (Fig. 1a). With the different excitation wavelength, the emission wavelengths of bare CDs vary from 480 to 530 nm, respectively (Fig. 1b). According to the FITR spectrum (S.1 †), the broad absorption bands near 3400 cm À1 was the character peak of n(O-H) and n(N-H). The peak at 1720 cm À1 should be n(C]O). And the peak at 1600 cm À1 should be scissor bending vibration of H 2 O. The peaks of 1395 cm À1 and 1150 cm À1 refer to the bending vibration of d(CH 2 ) and n(C-O-H). Composition of carbon dots synthesized was analyses by using XPS. While insensitive to H, the result (S.2 †) showed that the C, O, N and S were at present of 62.84, 12.53, 22.56 and 2.07%, respectively. The uorescence quantum yield (QY) of carbon dot was 7.06%, tested by Fluorescence Spectrometer FS5 (Edinburgh Instruments, U.K.). However, with excitation light with different wavelength, the emission light wavelength also varies. Regarding the steady of testing and the harm of UV light to AChE, we choose 470 nm as the excitation wavelength in following experiments.

Fluorescence quenching by different metal ions
Metal ions exhibit a well-documented ability to quench the uorescence of CDs. The results obtained for selected ions (Fig. 2) revealed that the most signicant quenching was observed for Au(III), with increasing Au(III) concentration thus causing a decrease of CD uorescence.
When excitation was performed at 365 nm, the uorescence changes could be observed by the naked eye, as shown in Fig. 3a. Fig. 3b shows that Au(III) could effectively quench CD uorescence, with the uorescence intensity at the maximum emission wavelength linearly decreasing with increasing Au(III) concentration.

Fluorescence recovery in the presence of ATCh and selected anions
As mentioned above, AChE can catalyze the hydrolysis of Sacetylthiocholine iodide into TCh 23 that can effectively capture Au(III) and thus recover CD uorescence. 21 The amount of produced TCh, and thus, the extent of CD uorescence recovery, was expected to be proportional to the quantity of added ATCh. A preliminary experiment was carried out to prove feasibility of the mechanism. The naked CDs, CDs quenched by Au(III), CDs with Au(III) and ATCh, CDs with Au(III) and ATCh hydrolyzed by AChE were compared by using Scheme 1 Mechanism of organophosphate pesticide sensing by the developed CD/Au(III) sensor. uorescent spectrum scanning. As Fig. 4 shown, the uorescence of CDs can be signicantly quenched by adding 50 mM Au(III). However, the CDs Au(III) ATCh hydrolyzed by AChE got stronger uorescence as the TCh caught most of the Au(III). And it makes the uorescence of system just a little bit lower than naked CDs. Therefore, this experiment proved the assembled scheme was correct.
Next, we investigated the inuence of potential interferants on uorescence quenching in the CD/Au(III) system, showing that most inorganic ions and amino acids exhibited no apparent effects (Fig. 5). However, the addition of cysteine signicantly increased the uorescence of the above system due to this compound possessing an SH group capable of scavenging Au(III), as outlined in Scheme 1.

Optimization of ATCh concentration
With the increasing of TCh coming from hydrolyzed ATCh, more and more Au(III) were captured. However, this linear dependence was only observed in a certain range. It can be observed in Fig. 6a that the certain range was 0-25 mM. Thus, the optimal ATCh concentration in the reaction system was determined as 25 mM.

Effects of pH
Optimal concentrations of Au(III) and CDs were selected to investigate the effect of pH, which was adjusted by PBS. Subsequently, the products of AChE-catalyzed ATCh (1 mL, 50 mM) hydrolysis performed at 37 C for 8 min were added to the CD/Au(III) system, and uorescence spectra were recorded aer 8 min (Fig. 6b). As a result, optimum performance was observed at pH 6.98, which is similar to the pH of maximal AChE activity. 24

Effects of hydrolysis time
The effect of hydrolysis time was investigated at pH 6.98. As described above, the products of AChE-catalyzed ATCh (1 mL, 50 mM) hydrolysis performed at 37 C for 1, 3, 5, 8, 10, and 15 min were added to the CD/Au(III) system, and uorescence spectra were recorded aer 8 min (Fig. 6c). The optimal hydrolysis time was determined as 8 min and was used in subsequent experiments.

Determination of organophosphate pesticides
The relationship between uorescence recovery and organophosphate pesticide concentration (Fig. 6d) was used to construct a calibration curve (Fig. 7).

Organophosphate pesticide sensing in real samples
An apple was homogenized and spiked with an organophosphate pesticide (4.48 mM) to afford a positive sample. The equal volume of methanol was added into the spiked sample and stirred violently for sufficiently extraction. Then the samples with extraction solution were centrifuged for 5 min, and the precipitation was discarded. Aer nitrogen blowing with Termovap Sample Concentrator about 10 min, the methanol was nearly removed. And equal volume of 10% ethanol-PBS complex solution was added into tubes as redissolution liquid. Subsequently, AChE and ATCh were added, and the mixture was incubated for 8 min and treated with CD/Au(III), followed by uorescence recovery measurements. Then according to the correction curve established in 3.8, the concentration of organophosphate can be obtained by calculation.
The obtained data were used to determine the pesticide concentration and recovery as 4.47 mM and 99.85%, respectively, using the abovementioned calibration curve.
The obtained recovery met the requirements of organophosphate pesticide determination, lying in the stipulated range of 80-120%.

Conclusions
Herein, CDs prepared from egg white by a hydrothermal method were used to fabricate a biosensor for detecting organophosphate pesticides. Initially, the uorescence of CDs was quenched by a certain amount of Au(III) ions, which were subsequently scavenged by TCh generated by AChE-catalyzed hydrolysis of ATCh, which resulted in CD uorescence recovery. The activity of AChE was reduced in the presence of organophosphates, which decreased uorescence recovery and allowed the pesticide concentration to be determined using a pre-established calibration curve. Tests performed for a real