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Carbon dot-based bioplatform for dual colorimetric and fluorometric sensing of organophosphate pesticides
Accepted Manuscript Title: Carbon dot-based bioplatform for dual colorimetric and fluorometric sensing of organophosphate pesticides Authors: Hongxia Li, Xu Yan, Geyu Lu, Xingguang Su PII: DOI: Reference:
S0925-4005(17)32506-6 https://doi.org/10.1016/j.snb.2017.12.170 SNB 23858
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
9-10-2017 9-12-2017 27-12-2017
Please cite this article as: Hongxia Li, Xu Yan, Geyu Lu, Xingguang Su, Carbon dot-based bioplatform for dual colorimetric and fluorometric sensing of organophosphate pesticides, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.12.170 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Carbon dot-based bioplatform for dual colorimetric and fluorometric sensing of organophosphate pesticides
Hongxia Lia,†, Xu Yana,b,†,*, Geyu Lua, and Xingguang Suc,*
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a State Key Laboratory on Integrated Optoelectronics, College of Electron Science and
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Engineering, Jilin University, 2699 Qianjin Street, Changchun, 130012, China
b State Key Laboratory of Supramolecular Structure and Materials, College of
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Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China
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Email: [email protected] (X. Yan)
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Qianjin Street, Changchun, 130012, China
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c Department of Analytical Chemistry, College of Chemistry, Jilin University, 2699
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[email protected] (X.G. Su)
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† These authors contributed equally to this work.
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Graphical Abstract
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Highlights:
► A dual-signal readout biosensor with good sensitivity and high anti-interference capacity
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has been developed for OPs detection.
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► One-step hydrothermal treatment approach was developed for the synthesis of CDs. ► Interaction between CDs and enzyme-controlled product leads to fluorescent quenching.
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► OPs can block enzyme activity and result in fluorescence recovery and absorbance
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decrease.
Abstract Rapid and sensitive monitoring strategy plays a crucial role in food safety and environment protection for the screening of organophosphorous pesticides (OPs) due to their extensive usage and harmful neurotoxicity on mammals. Herein, a label-free bioplatform was designed for sensitive 2
detection of OPs through dual-mode (fluorometric and colorimetric) channels based on acetylcholinesterase (AChE)-controlled quenching of fluorescence carbon dots (CDs). This sensing strategy involves the reaction of acetylthiocholine with AChE to produce thiocholine which specifically triggered the decomposition of 5,5-dithiobis (2-nitrobenzoic acid) to form yellow-
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colored 5-thio-2-nitrobenzoic acid (TNBA). Meanwhile, TNBA with positive charge was capable of functioning as a powerful absorber to quench the fluorescence of CDs through dynamic
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quenching process. With the presence of OPs, the enzyme activity of AChE was blocked, leading to the recovery of fluorescence signal and the decrease of absorbance intensity with color variation.
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The dual-output assay provided good sensitivity for rapid detection of paraoxon (model analyte)
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with a detection limit of 0.4 ng mL-1. Therefore, taking advantage of the excellent optical property
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Key words:
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candidate for OPs detection.
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of CDs and the specificity of enzyme, the dual-readout platform can potentially be a promising
Organophosphate pesticides, Carbon dots, Dual-readout, Dynamic quenching process
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1.Introduction
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Organophosphorous pesticides (OPs), as one of the most common and critical agrochemicals, have been extensively utilized in industrial agriculture because of their excellent ability in preventing, controlling or eradicating insect pests, which boost up the production yield of crops and
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vegetables [1-3]. As a result of the extensive use and improper disposal of OPs, their residues have raised people’s great concerns due to not only their negative impacts on environment and food safety issues [3, 4], but also their serious threat to cycles of sensitive ecosystems [5, 6]. Moreover, these residual compounds with high toxicity can irreversibly inactivate acetylcholinesterase (AChE) a key 3
enzyme in neurotransmission, posing serious danger to human health even at very low concentrations [7, 8]. Considering its hazards to environment and potential toxicological effects towards unintended targets, the development of fast and sensitive strategy for the detection of OPs become more and more urgent to efficiently protect environment, monitor food quality, estimate
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pesticide poisoning and safeguard public health. Typically, OPs have been successfully detected through highly sensitive routine analytical techniques, such as liquid/gas chromatography [9-11],
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mass spectrometry [12, 13], enzyme-linked immunosorbent assays (ELISAs) [14-16] and
electrochemical analysis [17-19]. Unfortunately, these conventional instrument-based techniques
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often need sophisticated equipment coupled with expensive detectors, tedious pretreatment
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procedures of sample and skilled manpower [20]. ELISA require high cost and time-consuming
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antibody preparation procedure [21]. Furthermore, ELISA are susceptible to effects from the
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ambient environment and matrix components [4]. Electrochemical approach requires complex
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electrode decoration procedures and labelling process, as well as suffers from false-positive effect [22]. Thus, the above drawbacks impair their regular monitoring and real-time applications,
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particularly emergency cases.
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To address these deficiencies, the optical platforms have recently been emerged as promising candidates for the detection of OPs owing to their operational simplicity, good sensitivity and low cost [23-28]. Fluorescence strategies and colorimetric assays are the typical sensing techniques of
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optical sensors, which can be easily transformed molecular events into fluorescence intensity or color changes. Recently, vast endeavors towards the design of colorimetric and fluorometric dualsignal sensing have been undertaken to develop a sensitive, selective and accurate manner [29-31]. For a typical multi-signal sensor fabrication, the united application of functional fluorophore and 4
gold nanoparticles (AuNPs) is successfully designed for the detection of pesticide with fluorometric and colorimetric responses. For example, Jiang’s group developed simultaneous exploration of the fluorescence and colorimetric of rhodamine B-covered AuNPs probe for the determination of OPs [2]. Guo et al. proposed a dual-channel strategy for glyphosate detection based on Förster resonance
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energy transfer between quantum dots and AuNPs [32]. And our group utilized the dual-signal properties of AuNPs/ratiometric fluorescent probe system for the monitoring of parathion-methyl
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[1]. The colorimetric signals of above studies were all designed by utilizing the localized surface plasmon resonance characteristics of AuNPs. Meanwhile, AuNPs were often susceptible to be
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influenced by chemicals, such as cyanide, salt and melamine. Therefore, to design a convenient
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multi-signal platform for the detection of OPs still remains a practical challenging task.
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Ellman’s reagent (5,5-dithiobis (2-nitrobenzoic acid), DTNB) has been widely utilized as a
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remarkable colorimetric probe through the specific interaction with thiol groups [33-37]. Inspired
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by preceding works, the combination of pesticide-caused activity inhibition and DTNB-assisted fluorescence (FL) quenching is expected to result in promising sensors for both fluorometrically
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and colorimetrically detecting OPs. In this sensing system, we prepared fluorescent carbon dots
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(CDs) by using one-step hydrothermal treatment of folic acid and p-phenylenediamine. As shown in Scheme 1, AChE can catalyze the hydrolysis of acetylthiocholine (ATCh) to thiocholine (TCh), which specifically reacted with DTNB to yield yellow-colored 5-thio-2-nitrobenzoic acid (TNBA)
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with a characteristic absorption peak around 412 nm. The fluorescence intensity of CDs can be efficiently quenched by TNBA through dynamic quenching mechanism. Upon the addition of OPs, the activity of enzyme was blocked, leading to the recovery of fluorescence signal and the decrease of absorbance intensity at 412 nm. Thus, the dual-mode assay can be utilized for OPs detection 5
based on fluorometric and colorimetric responses. The colorimetric assay can be used for preliminary screen with naked eyes, while the fluorometric strategy exhibited a lower detection limit and was preferable for additional signal correction. The established dual-output sensing platform overcame the disadvantage of relatively poor stability and low anti-interference capacity of metal
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nanoparticle-mediated sensor. Meanwhile, the dual optical signal-response in one sensing platform can not only improve detection performance, but also possess an additional correction of output
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signals. Furthermore, this sensing system provided a new way for probing other thiol groups-related
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enzyme system and screening the inhibition of enzyme.
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2. Experimental
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2.1. Reagents and instruments
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All reagents and solvents were at least analytical grade. Folic acid, p-phenylenediamine,
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paraoxon, acetylcholinesterase (AChE) and acetylthiocholine (ATCh) were obtained from SigmaAldrich Corporation, which were used directly without further treatment. The water utilized in this
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study had a good resistivity (>18 MΩ cm-1).
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The fluorescence spectra and ultraviolet spectra were measured by a spectrofluorophotometer (RF-5301 PC, Japan) and UV-Visible spectrophotometer (Shimadzu UV-1700, Japan). Transmission electron microscopy (TEM) was performed on a Philips Tecnai F20 TEM operating. All pH
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measurements were collected by a pH meter (PHS-3C, China). 2.2 Synthesis of carbon dots (CDs) Folic acid (FA, 88.0 mg) and p-phenylenediamine (PPD, 21.6 mg) were mixed thoroughly in 10.0 mL of sodium hydroxide solution (0.2 mol L-1) and transferred into polytetrafluoroethylene 6
autoclaves. Then the solution was heated at 170 °C for 12 h. The obtained solution was freeze-dried to form a brown solid and dissolved with deionized water. Finally, the as-prepared CDs (10 mg L-1) were stored at 4 °C for further study. 2.3 Assays for AChE activity
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Different concentrations of AChE (50 µL) were mixed with 10 mmol L-1 of ATCh (50 µL) and 100 mmol L-1 of PBS (pH = 7.0, 50 µL). After incubating at 37 °C for 25 min, 200 µg mL-1 of
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DTNB (150 µL) and 10 mg L-1 of CDs solution (20 µL) were added. Then, the mixture was diluted with deionized water to 500 µL. The above solution was mixed thoroughly for 5.0 min and optical
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signals were recorded for the detection of AChE.
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2.4 Procedures for OPs detection
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Paraoxon was selected as a model to suppress the activity of AChE. Different concentrations
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of paraoxon (25 μL) were added to 2.0 µg mL-1 of AChE solution (25 μL) for 30 min under 37 °C.
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Then, 10 mmol L-1 of ATCh (50 µL) and 100 mmol L-1 of PBS (pH = 7.0, 50 µL) were successively introduced into the system. After incubating at 37 °C for 25 min, 200 µg mL-1 of DTNB (150 µL),
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10 mg L-1 of CDs solution (20 µL) and deionized water (180 µL) were added and mixed thoroughly
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for 5.0 min at room temperature. The optical signals were recorded for OPs detection.
3. Results and discussion
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3.1 Synthesis and Characterization In this work, CDs were facilely synthesized through the hydrothermal treatment method. The
preparation of fluorescent CDs could be accomplished by using FA and PPD as precursor for heating 12 h at 170 0C (Figure 1A). The morphology of prepared CDs was first characterized by transmission electron microscopy (TEM). As illustrated in Figure 1B, the CDs were mono-dispersed 7
with uniform spherical structure and revealed an average diameter around 2 nm. Furthermore, highresolution TEM image clearly showed that CDs have high-crystalline structure with lattice parameter of 0.214 nm, in accord with the (100) facet of graphite [38, 39]. The optical properties of CDs were measured by employing UV-vis absorption and fluorescence spectroscopy. The UV-vis
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absorption spectrum shows that the strong absorption peak around 273 nm and 335 nm owing to ππ* transition of C=C of aromatic sp2 domains [40] and n-π* transition of C=O in the prepared CDs
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[41, 42], respectively (Figure 1C). Additionally, similar to most carbon nanomaterials [43-45], our CDs display an excitation-wavelength-dependent emission feature due to surface state and size
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distribution of nanomaterials. As depicted in Figure 1D, the FL emission peak obviously shifted
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from 484 to 512 nm with the excited FL wavelength, changing from 340 to 460 nm. Moreover, CDs
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possessed the maximum FL emission intensity located at 505 nm while being excited at 420 nm,
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which was selected for the following experiments. The quantum yields of the as-prepared CDs were
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calculated to be 8.4 % using Rhodamine B as reference. After CDs were successfully prepared, the pH behavior of CDs was measured in PBS buff er (100 mmol L-1) with different pH values (Figure
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1E). Interestingly, the FL intensity of CDs gradually increased with the pH values from 5.5 to 9.5,
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implying that the prepared CDs possess pH-sensitive behavior. The photostability and salt stability of nanomaterials are critical for their meaningful applications. For photostability, the FL emission intensity of CDs was measured under 420 nm excitation for 60 min in 100 mmol L-1 PBS buff er
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(7.5). It is obviously shown in Figure 1F that CDs preserved ~95 % of the initial FL intensity through the continuous intensive excitation, implying that the CDs exhibited good photostability. Furthermore, the FL emission intensity of CDs was investigated under different concentrations of salt at the certain pH value. As display in Figure 1G, the FL signal of CDs did not change obviously 8
in a wide salt concentration range of 0-10.0 mmol L-1. These results indicated that as-prepared CDs had outstanding photostability and salt stability, ensuring their applications in complicated conditions.
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3.2 The detection strategy of OPs To building up a simple and sensitive dual-readout strategy for OPs detection, a CDs-based
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sensing system combined the specific hydrolysis activity of AChE and DTNB-assisted FL quenching was constructed. To validate the feasibility of strategy, the CDs were firstly incubated
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with ATCh (0-2.0 mmol L-1), AChE (0-1000 ng mL-1), DTNB (0-100 μg mL-1) or paraoxon (0-10
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μg mL-1), respectively. As displayed in Figure S1, the FL intensity remains almost constant,
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indicating that CDs cannot be influenced by ATCh, AChE, DTNB or paraoxon, separately. Under
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efficient catalysis of AChE, ATCh can be hydrolyzed into TCh that specially trigger the
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decomposition of DTNB to TNBA with a characteristic absorption peak around 412 nm (Figure 2A,
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curve 2). As shown in equation (1), DTNB were reduced to produce TNBA.
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As shown in Figure 2B, the produced TNBA can obviously quench the FL intensity of CDs (b
line). Interestingly, paraoxon as a typical inhibitor of AChE, decreased the decomposition of DTNB
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(Figure 2A, curve 3), and recovered the FL intensity (Figure 2B, c line). The above results successfully proved that the sensing principle is feasible. In order to elucidate the underlying mechanism of TNBA-induced FL quenching, the zeta potential of CDs and TNBA were firstly investigated. As depicted in Figure 2C, the CDs were negatively charged (ζ = 18.9) while TNBA was positively charged (ζ = -29.6). There exists intensive 9
electrostatic attraction between CDs and TNBA, which shortens the distance of CDs and TNBA. Meanwhile, the FL lifetime of CDs/DTNB/ATCh system in the presence and absence of AChE were studied (Figure 2D). The FL lifetime of CDs/DTNB/ATCh/AChE system was shorter than that of CDs/DTNB/ATCh system, implying that the quenching sensing mechanism was attributed to
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dynamic quenching process [46]. Additionally, with the increase of temperature, an obvious enhance of the quenching eff ect of TNBA on the fluorescence of CDs was observed, further indicating
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dynamic quenching (Figure 2E). To further confirm the existence of dynamic quenching process
between CDs and TNBA, CDs were fully mixed with different concentrations of TNBA and the FL
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emission spectra of the system were collected. As depict in Figure S2, the FL intensity of CDs at
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505 nm were gradually decreased with the increase of TNBA (0-15 μg mL-1), accompanying the
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absorbance intensity at 412 nm change of the TNBA solution. While the FL intensity of CDs still
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remains unchanged in the presence of DTNB (Figure S3), indicating that the quenching ability of
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DTNB was much lower than that of TNBA at the same concentration level. Considering the above results, we can conclude that the quenched FL intensity of system was based on dynamic quenching
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process, which originates from the interaction of CDs and TNBA.
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Via the efficient dynamic quenching process, dual-readout signal in the sensing system were closely related to the amount of AChE. We mixed CDs/DTNB/ATCh system with different concentrations of AChE and monitored the FL signal of system. It was obviously demonstrated from
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Figure 2F that the absorbance intensity of CDs/DTNB/ATCh system at 412 nm increase gradually with the increasing concentration of AChE in the range of 0-100 ng mL-1. The inset was the change trend of absorbance intensity with different AChE concentrations. Owing to the variation in the absorbance intensities of the CDs/DTNB/ATCh system, the noticeable color change from colorless 10
to yellow could be clearly observed with naked eyes (Figure 2G). Then a sensitive assay for AChE was constructed based on the dynamic quenching process. As depicted in Figure 2H, the FL intensity of CDs/DTNB/ATCh system at 505 nm was quenched obviously with the increase of AChE concentration (0-100 μg mL-1). It can be easily observed from Figure 2I that a good linear
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relationship was obtained between the FL intensities ratio FQ/FQ0 and AChE concentration in the range from 0.5 to 100 μg mL-1 (FQ and FQ0 were the FL intensities of CDs/DTNB/ATCh system in
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the presence and absence of AChE, respectively). Therefore, the FL intensity of the
CDs/DTNB/ATCh system can be modulated by the enzyme activity of AChE. To pursue low
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background and high sensitivity, 100 μg mL-1 of AChE was selected for further study.
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3.3 Optimization of the sensing system
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The performance of established platform can be influenced by some related factors, such as
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reaction pH, reaction temperature and incubation time, so these experimental conditions were systematically optimized for OPs detection. The influence of pH value on inhibition efficiency (IE)
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of CDs/DNTB/ATCh/AChE system in the presence 0.2 μg mL-1 of paraoxon is investigated in
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Figure 3A. We used the IE of AChE for the analysis of OPs (equation shown in supporting information). It can be seen that the IE of system gradually increased when pH changed from 5.5 to 7.0, and the obvious decreasing of IE was observed when pH further increased to 8.5. Thus, pH 7.0
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PBS buffer (100 mmol L-1) was chosen for paraoxon detection. Reaction temperature, as a critical factor, could obviously effect the AChE activity. Thus, we investigated the influence of temperature on IE of CDs/DNTB/ATCh/AChE/paraoxon system in Figure 3B. The results shown that the IE of system obviously increased with the increasing of temperature and reached maximum at 37 0C, So 11
37 0C was selected as the optimal reaction temperature for paraoxon detection. The incubation time of paraoxon and AChE was also studied. As shown in Figure 3C, we can see that incubation time had significant influence on IE of CDs/DNTB/ATCh/AChE/paraoxon system. The inhibition occurred immediately with the addition of paraoxon and the reaction can finish within 25 min. Thus,
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the reaction time of 25 min was chosen to ensure completely reaction.
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3.4 Determination of OPs
To evaluate the applicability of the established dual-readout analytical platform for OPs
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detection, the IE of CDs/DNTB/ATCh/AChE system with different concentrations of paraoxon was
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measured. As shown in Figure 4A, the absorbance intensity of system at 412 nm continuously
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decreased along with the paraoxon concentration increasing from 0 to 0.5 μg mL-1. Inset of Figure
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4A showed the good linear relationship between the IE of enzyme and the logarithm of paraoxon
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concentration (R2=0.993). The linear regression equation was: IE = 1.06158+ 0.39804 log [paraoxon], μg mL-1. Moreover, a series of noticeable color changes from yellow to colorless was
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observed with the increase of paraoxon concentration (Figure 4B). By means of the dynamic
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quenching process of TNBA on CDs, an fluorometric output for the sensitive detection of paraoxon was achieved in this study. As shown in Figure 4C, with the increasing of paraoxon concentration, the FL intensities of CDs/DNTB/ATCh/AChE system at 505 nm was continuously restored,
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indicating that the dynamic quenching process was performed in a dose-dependent manner relative to paraoxon. The inset of Figure 4C showed the change trend of FL intensities with various concentrations of paraoxon. Meanwhile, Figure 4D illustrated an excellent linear relationship between the IE and the logarithm concentration of paraoxon in the range from 0.001 to 1.0 μg mL12
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. The regression equation is IE=1.00335 + 0.31602 Log [Paraoxon], with a correlation coefficient
(R2) of 0.993. The detection limit (LOD) defined by 3σ rule (signal-to-noise ratio of 3) was calculated to be 0.4 ng mL-1 for paraoxon, which is comparable to or even lower than those of reported methods (Table S1). In previous studies, gold nanoparticle [7] and silver nanoparticle [47]
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were served as nanoquencher for OPs detection. Those strategies can be easily influenced by chemicals, such as cyanide, salt and melamine. Compared with reported nanosensors, our proposed
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sensing platform exhibits well sensitivity and higher anti-interference ability, suggesting that the dual-output system can be utilized for the detection of OPs. To illustrate that the proposed
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CDs/DNTB/ATCh/AChE system was functional not only for paraoxon, but also for other OPs, we
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investigated the sensing platform for four common OPs: parathion, phosalone, azinphos-methyl,
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malathione. The influence of OPs on CDs/DNTB system were investigated firstly. It can be observed
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from Figure S4A that the FL intensity of CDs/DNTB system remains almost constant, indicating
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that OPs cannot trigger the decomposition of DNTB and also cannot affect the FL intensity of CDs. Figure S4B showed that the IE were 0.682, 0.510, 0.458, 0.444 and 0.414 for paraoxon, parathion,
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phosalone, azinphos-methyl and malathione at the concentration of 0.1 μg mL-1, demonstrating that
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this platform could be employed for monitoring many kinds of OPs.
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3.5 Selectivity for OPs Specific recognition property is a vital parameter to assess the performance of proposed
strategy, especially for sensor in real-time visual applications. The selectivity of the dual-output nanosensor was investigated by measuring the spectral responses toward common coexistence substances in agricultural or biological samples, including Na+, K+, Ca2+, Mg2+, glucose, lactose, 13
aspartic acid (ASP), glycine (GLY), ascorbic acid (Vc), laccase (LAC), bovine serum albumin (BSA), invertase (INV), and pepsin (PEP). As illustrated in Figure 5A, the CDs/DNTB/ATCh/AChE system possessed a remarkable response toward paraoxon (0.2 μg mL-1), while FL intensity displayed no obvious changes after the addition of 2 μg mL-1 of those common substances (10-fold),
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demonstrating that the proposed strategy shown high selectivity to paraoxon. To investigate the ability of resist interference, we further studied the FL response of CDs/DNTB/ATCh/AChE system
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to paraoxon (0.2 μg mL-1) in the presence of interfering substances. It can be obviously found that, even interfering substance (2 μg mL-1) exists, CDs/DNTB/ATCh/AChE system still possessed the
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same response to paraoxon (Figure 5B). So the established system hold excellent specificity and
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promising anti-interference capability for paraoxon detection, implying that the system was suitable
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3.6 Real samples detection
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for paraoxon analysis.
To estimate the practical applications in real samples detection, our method and gas
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chromatography (GC) were used to detect paraoxon concentration in environmental and agricultural
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samples (water, rice, and cabbage). The results demonstrated that no paraoxon exists in the test samples (Table S2). The results obtained by standard addition method were listed in Table S2, the average recovery of paraoxon ranges from 90 % to 102 % and the relative standard deviation (RSD)
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was lower than 4.17 %. Furthermore, the obtained results were well consistent with those of GC, implying that the sensing platform hold highly accurate and reproducible for paraoxon detection in real samples and possess potential applicability.
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In summary, by combining AChE-induced the decomposition of DTNB and TNBA-triggered the dynamic quenching process, we developed a dual-readout methodology that allows a simple optical (colorimetric or fluorometric) detection of OPs in highly selective and sensitive manner. The colorimetric and fluorescent responds were based on the formation of yellow-colored TNBA that
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served as color reporter and FL quencher. The dual-readout signal in the sensing system was proportional to the amount of OPs in the range of 0.001 to 1.0 μg mL-1 with a LOD of 0.4 ng mL-1.
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Moreover, benefiting from the changes of absorbance intensity, a visual recognition for paraoxon by color changes was achieved in a concentration-dependent manner. This proposed dual-readout
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platform could thereby be a reliable option to quantitatively detect pesticide in environmental and
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agricultural samples due to additional signal correction from each other, which validated its
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efficiency in on-site application. The dual-signal strategy not only improved detection sensitivity,
Acknowledgments
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but also held an additional correction of output signals.
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This work was financially supported by the National Natural Science Foundation of China (Nos. 21275063 and No. 21575048), the Science and Technology Development project of Jilin
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province, China (No. 20150204010GX). X. Yan is thankful for support from the National
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Postdoctoral Program for Innovative Talents (BX201700096).
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Hongxia Li received her M.S. degree in 2013 from Nanjing Agricultural University. and received her Ph.D. degree in 2016 at Jilin University. Since then, she did postdoctoral work with Prof. Geyu Lu. Currently, her research interests mainly focus
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on the development of the functional nanomaterials for chem/bio sensors.
Xu Yan received his M.S. degree in 2013 from Nanjing Agricultural University. He
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joined the group of Prof. Xingguang Su at Jilin University and received his Ph.D. degree
in June 2017. Since then, he did postdoctoral work with Prof. Geyu Lu. Currently, his
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research interests mainly focus on the development of the functional nanomaterials for
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chem/bio sensors.
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Geyu Lu received the B. Sci. degree in electronic sciences in 1985 and the M. Sci.
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degree in 1988 from Jilin University in China and the Dr. Eng. degree in 1998 from Kyushu University in Japan. Now he is a professor of Jilin University, China. His
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current research interests include the development of chemical sensors and the
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application of the function materials.
Xingguang Su is a professor at the Department of Analytical Chemistry at the College
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of Chemistry, Jilin University. She received her MS degree from Jilin University (China) in 1992 and her PhD degree from Jilin University (China) in 1999. Her research focuses on the synthesis, characterization, functionalization and application of quantum dots and quantum dots-tagged microspheres in biomedicine. 22
Captions:
Scheme 1 The schematic illustration of the dual-signal optical system for OPs detection. Figure 1 (A) One-pot synthesis for CDs. (B) The TEM image of the CDs. (C) The UV-Vis absorption spectrum of CDs. (D) FL excitation and emission spectra of the CDs. (E) FL spectrum
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of CDs in PBS buffer with diff erent pH values range from 5.5 to 9.5. Inset is FL trends of CDs with the variation of pH. (F) The photobleaching experiment of the CDs with 420 nm excitation source
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for continuous intensive excitation. (G) The effect of NaCl concentration on FL intensity of CDs.
Figure 2 (A) Absorbance spectrum and (B) FL spectrum of CDs+DTNB+ATCh,
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CDs+DTNB+ATCh+AChE and CDs+DTNB+ATCh+AChE+OPs system. (C) The zeta potential of
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CDs and TNBA. (D) The fluorescence lifetime measurement of CDs+DNTB+ATCh system in
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absence and presence of AChE. (E) The effect of temperature on the FL quenching of
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CDs+DNTB+ATCh system in the presence of 50 ng mL-1 AChE. (F) Absorption spectrum of
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CDs+DNTB+ATCh system upon the addition of AChE at different concentrations from 0 to 100 ng mL-1 (0, 0.5, 1.0, 2.0, 5.0, 10, 20, 50 and 100 ng mL-1). (G) The corresponding photographs of
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CDs+DNTB+ATCh system with different concentration of AChE taken under daylight. (H) The
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fluorescence spectra of CDs+DNTB+ATCh system in the presence of different concentration of AChE (0, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100 and 200 ng mL-1). (I) The linear plot of FL intensity ratio FQ/FQ0 versus the logarithm of AChE concentration.
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Figure 3 Effect of pH (A), incubation temperature (B) and inhibition time (C) on IE in the presence of 0.2 μg mL-1 paraoxon. Figure 4 (A) Absorption spectra of CDs/DNTB/ATCh/AChE system upon the addition of paraoxon at different concentrations from 0 to 0.5 μg mL-1 (0, 0.005, 0.01, 0.05, 0.1 and 0.5 μg mL-1). (B) The 23
corresponding photographs of CDs/DNTB/ATCh/AChE system in the presence of different concentration
of
OPs
taken
under
daylight.
(C)
The
fluorescence
spectra
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CDs/DNTB/ATCh/AChE system in the presence of different concentration of paraoxon (0, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1.0 μg mL-1). (D) The linear plot of (IE)
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versus the logarithm of paraoxon concentration. Figure 5 (A) The FL intensity of the CDs/DNTB/ATCh/AChE system with the interfering
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substances (2 μg mL-1) or paraoxon (0.2 μg mL-1), (D) The FL intensity of the
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CDs/DNTB/ATCh/AChE /paraoxon system with the interfering substances (2 μg mL-1).
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Scheme 1
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S0925-4005(17)32506-6 https://doi.org/10.1016/j.snb.2017.12.170 SNB 23858
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
9-10-2017 9-12-2017 27-12-2017
Please cite this article as: Hongxia Li, Xu Yan, Geyu Lu, Xingguang Su, Carbon dot-based bioplatform for dual colorimetric and fluorometric sensing of organophosphate pesticides, Sensors and Actuators B: Chemical https://doi.org/10.1016/j.snb.2017.12.170 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Carbon dot-based bioplatform for dual colorimetric and fluorometric sensing of organophosphate pesticides
Hongxia Lia,†, Xu Yana,b,†,*, Geyu Lua, and Xingguang Suc,*
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a State Key Laboratory on Integrated Optoelectronics, College of Electron Science and
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Engineering, Jilin University, 2699 Qianjin Street, Changchun, 130012, China
b State Key Laboratory of Supramolecular Structure and Materials, College of
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Chemistry, Jilin University, 2699 Qianjin Street, Changchun 130012, China
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Email: [email protected] (X. Yan)
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Qianjin Street, Changchun, 130012, China
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c Department of Analytical Chemistry, College of Chemistry, Jilin University, 2699
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[email protected] (X.G. Su)
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† These authors contributed equally to this work.
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Graphical Abstract
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Highlights:
► A dual-signal readout biosensor with good sensitivity and high anti-interference capacity
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has been developed for OPs detection.
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► One-step hydrothermal treatment approach was developed for the synthesis of CDs. ► Interaction between CDs and enzyme-controlled product leads to fluorescent quenching.
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► OPs can block enzyme activity and result in fluorescence recovery and absorbance
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decrease.
Abstract Rapid and sensitive monitoring strategy plays a crucial role in food safety and environment protection for the screening of organophosphorous pesticides (OPs) due to their extensive usage and harmful neurotoxicity on mammals. Herein, a label-free bioplatform was designed for sensitive 2
detection of OPs through dual-mode (fluorometric and colorimetric) channels based on acetylcholinesterase (AChE)-controlled quenching of fluorescence carbon dots (CDs). This sensing strategy involves the reaction of acetylthiocholine with AChE to produce thiocholine which specifically triggered the decomposition of 5,5-dithiobis (2-nitrobenzoic acid) to form yellow-
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colored 5-thio-2-nitrobenzoic acid (TNBA). Meanwhile, TNBA with positive charge was capable of functioning as a powerful absorber to quench the fluorescence of CDs through dynamic
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quenching process. With the presence of OPs, the enzyme activity of AChE was blocked, leading to the recovery of fluorescence signal and the decrease of absorbance intensity with color variation.
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The dual-output assay provided good sensitivity for rapid detection of paraoxon (model analyte)
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with a detection limit of 0.4 ng mL-1. Therefore, taking advantage of the excellent optical property
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Key words:
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candidate for OPs detection.
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of CDs and the specificity of enzyme, the dual-readout platform can potentially be a promising
Organophosphate pesticides, Carbon dots, Dual-readout, Dynamic quenching process
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1.Introduction
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Organophosphorous pesticides (OPs), as one of the most common and critical agrochemicals, have been extensively utilized in industrial agriculture because of their excellent ability in preventing, controlling or eradicating insect pests, which boost up the production yield of crops and
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vegetables [1-3]. As a result of the extensive use and improper disposal of OPs, their residues have raised people’s great concerns due to not only their negative impacts on environment and food safety issues [3, 4], but also their serious threat to cycles of sensitive ecosystems [5, 6]. Moreover, these residual compounds with high toxicity can irreversibly inactivate acetylcholinesterase (AChE) a key 3
enzyme in neurotransmission, posing serious danger to human health even at very low concentrations [7, 8]. Considering its hazards to environment and potential toxicological effects towards unintended targets, the development of fast and sensitive strategy for the detection of OPs become more and more urgent to efficiently protect environment, monitor food quality, estimate
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pesticide poisoning and safeguard public health. Typically, OPs have been successfully detected through highly sensitive routine analytical techniques, such as liquid/gas chromatography [9-11],
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mass spectrometry [12, 13], enzyme-linked immunosorbent assays (ELISAs) [14-16] and
electrochemical analysis [17-19]. Unfortunately, these conventional instrument-based techniques
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often need sophisticated equipment coupled with expensive detectors, tedious pretreatment
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procedures of sample and skilled manpower [20]. ELISA require high cost and time-consuming
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antibody preparation procedure [21]. Furthermore, ELISA are susceptible to effects from the
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ambient environment and matrix components [4]. Electrochemical approach requires complex
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electrode decoration procedures and labelling process, as well as suffers from false-positive effect [22]. Thus, the above drawbacks impair their regular monitoring and real-time applications,
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particularly emergency cases.
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To address these deficiencies, the optical platforms have recently been emerged as promising candidates for the detection of OPs owing to their operational simplicity, good sensitivity and low cost [23-28]. Fluorescence strategies and colorimetric assays are the typical sensing techniques of
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optical sensors, which can be easily transformed molecular events into fluorescence intensity or color changes. Recently, vast endeavors towards the design of colorimetric and fluorometric dualsignal sensing have been undertaken to develop a sensitive, selective and accurate manner [29-31]. For a typical multi-signal sensor fabrication, the united application of functional fluorophore and 4
gold nanoparticles (AuNPs) is successfully designed for the detection of pesticide with fluorometric and colorimetric responses. For example, Jiang’s group developed simultaneous exploration of the fluorescence and colorimetric of rhodamine B-covered AuNPs probe for the determination of OPs [2]. Guo et al. proposed a dual-channel strategy for glyphosate detection based on Förster resonance
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energy transfer between quantum dots and AuNPs [32]. And our group utilized the dual-signal properties of AuNPs/ratiometric fluorescent probe system for the monitoring of parathion-methyl
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[1]. The colorimetric signals of above studies were all designed by utilizing the localized surface plasmon resonance characteristics of AuNPs. Meanwhile, AuNPs were often susceptible to be
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influenced by chemicals, such as cyanide, salt and melamine. Therefore, to design a convenient
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multi-signal platform for the detection of OPs still remains a practical challenging task.
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Ellman’s reagent (5,5-dithiobis (2-nitrobenzoic acid), DTNB) has been widely utilized as a
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remarkable colorimetric probe through the specific interaction with thiol groups [33-37]. Inspired
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by preceding works, the combination of pesticide-caused activity inhibition and DTNB-assisted fluorescence (FL) quenching is expected to result in promising sensors for both fluorometrically
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and colorimetrically detecting OPs. In this sensing system, we prepared fluorescent carbon dots
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(CDs) by using one-step hydrothermal treatment of folic acid and p-phenylenediamine. As shown in Scheme 1, AChE can catalyze the hydrolysis of acetylthiocholine (ATCh) to thiocholine (TCh), which specifically reacted with DTNB to yield yellow-colored 5-thio-2-nitrobenzoic acid (TNBA)
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with a characteristic absorption peak around 412 nm. The fluorescence intensity of CDs can be efficiently quenched by TNBA through dynamic quenching mechanism. Upon the addition of OPs, the activity of enzyme was blocked, leading to the recovery of fluorescence signal and the decrease of absorbance intensity at 412 nm. Thus, the dual-mode assay can be utilized for OPs detection 5
based on fluorometric and colorimetric responses. The colorimetric assay can be used for preliminary screen with naked eyes, while the fluorometric strategy exhibited a lower detection limit and was preferable for additional signal correction. The established dual-output sensing platform overcame the disadvantage of relatively poor stability and low anti-interference capacity of metal
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nanoparticle-mediated sensor. Meanwhile, the dual optical signal-response in one sensing platform can not only improve detection performance, but also possess an additional correction of output
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signals. Furthermore, this sensing system provided a new way for probing other thiol groups-related
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enzyme system and screening the inhibition of enzyme.
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2. Experimental
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2.1. Reagents and instruments
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All reagents and solvents were at least analytical grade. Folic acid, p-phenylenediamine,
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paraoxon, acetylcholinesterase (AChE) and acetylthiocholine (ATCh) were obtained from SigmaAldrich Corporation, which were used directly without further treatment. The water utilized in this
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study had a good resistivity (>18 MΩ cm-1).
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The fluorescence spectra and ultraviolet spectra were measured by a spectrofluorophotometer (RF-5301 PC, Japan) and UV-Visible spectrophotometer (Shimadzu UV-1700, Japan). Transmission electron microscopy (TEM) was performed on a Philips Tecnai F20 TEM operating. All pH
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measurements were collected by a pH meter (PHS-3C, China). 2.2 Synthesis of carbon dots (CDs) Folic acid (FA, 88.0 mg) and p-phenylenediamine (PPD, 21.6 mg) were mixed thoroughly in 10.0 mL of sodium hydroxide solution (0.2 mol L-1) and transferred into polytetrafluoroethylene 6
autoclaves. Then the solution was heated at 170 °C for 12 h. The obtained solution was freeze-dried to form a brown solid and dissolved with deionized water. Finally, the as-prepared CDs (10 mg L-1) were stored at 4 °C for further study. 2.3 Assays for AChE activity
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Different concentrations of AChE (50 µL) were mixed with 10 mmol L-1 of ATCh (50 µL) and 100 mmol L-1 of PBS (pH = 7.0, 50 µL). After incubating at 37 °C for 25 min, 200 µg mL-1 of
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DTNB (150 µL) and 10 mg L-1 of CDs solution (20 µL) were added. Then, the mixture was diluted with deionized water to 500 µL. The above solution was mixed thoroughly for 5.0 min and optical
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signals were recorded for the detection of AChE.
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2.4 Procedures for OPs detection
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Paraoxon was selected as a model to suppress the activity of AChE. Different concentrations
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of paraoxon (25 μL) were added to 2.0 µg mL-1 of AChE solution (25 μL) for 30 min under 37 °C.
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Then, 10 mmol L-1 of ATCh (50 µL) and 100 mmol L-1 of PBS (pH = 7.0, 50 µL) were successively introduced into the system. After incubating at 37 °C for 25 min, 200 µg mL-1 of DTNB (150 µL),
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10 mg L-1 of CDs solution (20 µL) and deionized water (180 µL) were added and mixed thoroughly
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for 5.0 min at room temperature. The optical signals were recorded for OPs detection.
3. Results and discussion
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3.1 Synthesis and Characterization In this work, CDs were facilely synthesized through the hydrothermal treatment method. The
preparation of fluorescent CDs could be accomplished by using FA and PPD as precursor for heating 12 h at 170 0C (Figure 1A). The morphology of prepared CDs was first characterized by transmission electron microscopy (TEM). As illustrated in Figure 1B, the CDs were mono-dispersed 7
with uniform spherical structure and revealed an average diameter around 2 nm. Furthermore, highresolution TEM image clearly showed that CDs have high-crystalline structure with lattice parameter of 0.214 nm, in accord with the (100) facet of graphite [38, 39]. The optical properties of CDs were measured by employing UV-vis absorption and fluorescence spectroscopy. The UV-vis
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absorption spectrum shows that the strong absorption peak around 273 nm and 335 nm owing to ππ* transition of C=C of aromatic sp2 domains [40] and n-π* transition of C=O in the prepared CDs
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[41, 42], respectively (Figure 1C). Additionally, similar to most carbon nanomaterials [43-45], our CDs display an excitation-wavelength-dependent emission feature due to surface state and size
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distribution of nanomaterials. As depicted in Figure 1D, the FL emission peak obviously shifted
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from 484 to 512 nm with the excited FL wavelength, changing from 340 to 460 nm. Moreover, CDs
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possessed the maximum FL emission intensity located at 505 nm while being excited at 420 nm,
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which was selected for the following experiments. The quantum yields of the as-prepared CDs were
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calculated to be 8.4 % using Rhodamine B as reference. After CDs were successfully prepared, the pH behavior of CDs was measured in PBS buff er (100 mmol L-1) with different pH values (Figure
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1E). Interestingly, the FL intensity of CDs gradually increased with the pH values from 5.5 to 9.5,
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implying that the prepared CDs possess pH-sensitive behavior. The photostability and salt stability of nanomaterials are critical for their meaningful applications. For photostability, the FL emission intensity of CDs was measured under 420 nm excitation for 60 min in 100 mmol L-1 PBS buff er
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(7.5). It is obviously shown in Figure 1F that CDs preserved ~95 % of the initial FL intensity through the continuous intensive excitation, implying that the CDs exhibited good photostability. Furthermore, the FL emission intensity of CDs was investigated under different concentrations of salt at the certain pH value. As display in Figure 1G, the FL signal of CDs did not change obviously 8
in a wide salt concentration range of 0-10.0 mmol L-1. These results indicated that as-prepared CDs had outstanding photostability and salt stability, ensuring their applications in complicated conditions.
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3.2 The detection strategy of OPs To building up a simple and sensitive dual-readout strategy for OPs detection, a CDs-based
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sensing system combined the specific hydrolysis activity of AChE and DTNB-assisted FL quenching was constructed. To validate the feasibility of strategy, the CDs were firstly incubated
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with ATCh (0-2.0 mmol L-1), AChE (0-1000 ng mL-1), DTNB (0-100 μg mL-1) or paraoxon (0-10
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μg mL-1), respectively. As displayed in Figure S1, the FL intensity remains almost constant,
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indicating that CDs cannot be influenced by ATCh, AChE, DTNB or paraoxon, separately. Under
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efficient catalysis of AChE, ATCh can be hydrolyzed into TCh that specially trigger the
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decomposition of DTNB to TNBA with a characteristic absorption peak around 412 nm (Figure 2A,
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curve 2). As shown in equation (1), DTNB were reduced to produce TNBA.
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As shown in Figure 2B, the produced TNBA can obviously quench the FL intensity of CDs (b
line). Interestingly, paraoxon as a typical inhibitor of AChE, decreased the decomposition of DTNB
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(Figure 2A, curve 3), and recovered the FL intensity (Figure 2B, c line). The above results successfully proved that the sensing principle is feasible. In order to elucidate the underlying mechanism of TNBA-induced FL quenching, the zeta potential of CDs and TNBA were firstly investigated. As depicted in Figure 2C, the CDs were negatively charged (ζ = 18.9) while TNBA was positively charged (ζ = -29.6). There exists intensive 9
electrostatic attraction between CDs and TNBA, which shortens the distance of CDs and TNBA. Meanwhile, the FL lifetime of CDs/DTNB/ATCh system in the presence and absence of AChE were studied (Figure 2D). The FL lifetime of CDs/DTNB/ATCh/AChE system was shorter than that of CDs/DTNB/ATCh system, implying that the quenching sensing mechanism was attributed to
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dynamic quenching process [46]. Additionally, with the increase of temperature, an obvious enhance of the quenching eff ect of TNBA on the fluorescence of CDs was observed, further indicating
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dynamic quenching (Figure 2E). To further confirm the existence of dynamic quenching process
between CDs and TNBA, CDs were fully mixed with different concentrations of TNBA and the FL
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emission spectra of the system were collected. As depict in Figure S2, the FL intensity of CDs at
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505 nm were gradually decreased with the increase of TNBA (0-15 μg mL-1), accompanying the
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absorbance intensity at 412 nm change of the TNBA solution. While the FL intensity of CDs still
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remains unchanged in the presence of DTNB (Figure S3), indicating that the quenching ability of
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DTNB was much lower than that of TNBA at the same concentration level. Considering the above results, we can conclude that the quenched FL intensity of system was based on dynamic quenching
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process, which originates from the interaction of CDs and TNBA.
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Via the efficient dynamic quenching process, dual-readout signal in the sensing system were closely related to the amount of AChE. We mixed CDs/DTNB/ATCh system with different concentrations of AChE and monitored the FL signal of system. It was obviously demonstrated from
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Figure 2F that the absorbance intensity of CDs/DTNB/ATCh system at 412 nm increase gradually with the increasing concentration of AChE in the range of 0-100 ng mL-1. The inset was the change trend of absorbance intensity with different AChE concentrations. Owing to the variation in the absorbance intensities of the CDs/DTNB/ATCh system, the noticeable color change from colorless 10
to yellow could be clearly observed with naked eyes (Figure 2G). Then a sensitive assay for AChE was constructed based on the dynamic quenching process. As depicted in Figure 2H, the FL intensity of CDs/DTNB/ATCh system at 505 nm was quenched obviously with the increase of AChE concentration (0-100 μg mL-1). It can be easily observed from Figure 2I that a good linear
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relationship was obtained between the FL intensities ratio FQ/FQ0 and AChE concentration in the range from 0.5 to 100 μg mL-1 (FQ and FQ0 were the FL intensities of CDs/DTNB/ATCh system in
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the presence and absence of AChE, respectively). Therefore, the FL intensity of the
CDs/DTNB/ATCh system can be modulated by the enzyme activity of AChE. To pursue low
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background and high sensitivity, 100 μg mL-1 of AChE was selected for further study.
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3.3 Optimization of the sensing system
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The performance of established platform can be influenced by some related factors, such as
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reaction pH, reaction temperature and incubation time, so these experimental conditions were systematically optimized for OPs detection. The influence of pH value on inhibition efficiency (IE)
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of CDs/DNTB/ATCh/AChE system in the presence 0.2 μg mL-1 of paraoxon is investigated in
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Figure 3A. We used the IE of AChE for the analysis of OPs (equation shown in supporting information). It can be seen that the IE of system gradually increased when pH changed from 5.5 to 7.0, and the obvious decreasing of IE was observed when pH further increased to 8.5. Thus, pH 7.0
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PBS buffer (100 mmol L-1) was chosen for paraoxon detection. Reaction temperature, as a critical factor, could obviously effect the AChE activity. Thus, we investigated the influence of temperature on IE of CDs/DNTB/ATCh/AChE/paraoxon system in Figure 3B. The results shown that the IE of system obviously increased with the increasing of temperature and reached maximum at 37 0C, So 11
37 0C was selected as the optimal reaction temperature for paraoxon detection. The incubation time of paraoxon and AChE was also studied. As shown in Figure 3C, we can see that incubation time had significant influence on IE of CDs/DNTB/ATCh/AChE/paraoxon system. The inhibition occurred immediately with the addition of paraoxon and the reaction can finish within 25 min. Thus,
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the reaction time of 25 min was chosen to ensure completely reaction.
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3.4 Determination of OPs
To evaluate the applicability of the established dual-readout analytical platform for OPs
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detection, the IE of CDs/DNTB/ATCh/AChE system with different concentrations of paraoxon was
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measured. As shown in Figure 4A, the absorbance intensity of system at 412 nm continuously
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decreased along with the paraoxon concentration increasing from 0 to 0.5 μg mL-1. Inset of Figure
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4A showed the good linear relationship between the IE of enzyme and the logarithm of paraoxon
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concentration (R2=0.993). The linear regression equation was: IE = 1.06158+ 0.39804 log [paraoxon], μg mL-1. Moreover, a series of noticeable color changes from yellow to colorless was
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observed with the increase of paraoxon concentration (Figure 4B). By means of the dynamic
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quenching process of TNBA on CDs, an fluorometric output for the sensitive detection of paraoxon was achieved in this study. As shown in Figure 4C, with the increasing of paraoxon concentration, the FL intensities of CDs/DNTB/ATCh/AChE system at 505 nm was continuously restored,
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indicating that the dynamic quenching process was performed in a dose-dependent manner relative to paraoxon. The inset of Figure 4C showed the change trend of FL intensities with various concentrations of paraoxon. Meanwhile, Figure 4D illustrated an excellent linear relationship between the IE and the logarithm concentration of paraoxon in the range from 0.001 to 1.0 μg mL12
1
. The regression equation is IE=1.00335 + 0.31602 Log [Paraoxon], with a correlation coefficient
(R2) of 0.993. The detection limit (LOD) defined by 3σ rule (signal-to-noise ratio of 3) was calculated to be 0.4 ng mL-1 for paraoxon, which is comparable to or even lower than those of reported methods (Table S1). In previous studies, gold nanoparticle [7] and silver nanoparticle [47]
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were served as nanoquencher for OPs detection. Those strategies can be easily influenced by chemicals, such as cyanide, salt and melamine. Compared with reported nanosensors, our proposed
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sensing platform exhibits well sensitivity and higher anti-interference ability, suggesting that the dual-output system can be utilized for the detection of OPs. To illustrate that the proposed
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CDs/DNTB/ATCh/AChE system was functional not only for paraoxon, but also for other OPs, we
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investigated the sensing platform for four common OPs: parathion, phosalone, azinphos-methyl,
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malathione. The influence of OPs on CDs/DNTB system were investigated firstly. It can be observed
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from Figure S4A that the FL intensity of CDs/DNTB system remains almost constant, indicating
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that OPs cannot trigger the decomposition of DNTB and also cannot affect the FL intensity of CDs. Figure S4B showed that the IE were 0.682, 0.510, 0.458, 0.444 and 0.414 for paraoxon, parathion,
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phosalone, azinphos-methyl and malathione at the concentration of 0.1 μg mL-1, demonstrating that
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this platform could be employed for monitoring many kinds of OPs.
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3.5 Selectivity for OPs Specific recognition property is a vital parameter to assess the performance of proposed
strategy, especially for sensor in real-time visual applications. The selectivity of the dual-output nanosensor was investigated by measuring the spectral responses toward common coexistence substances in agricultural or biological samples, including Na+, K+, Ca2+, Mg2+, glucose, lactose, 13
aspartic acid (ASP), glycine (GLY), ascorbic acid (Vc), laccase (LAC), bovine serum albumin (BSA), invertase (INV), and pepsin (PEP). As illustrated in Figure 5A, the CDs/DNTB/ATCh/AChE system possessed a remarkable response toward paraoxon (0.2 μg mL-1), while FL intensity displayed no obvious changes after the addition of 2 μg mL-1 of those common substances (10-fold),
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demonstrating that the proposed strategy shown high selectivity to paraoxon. To investigate the ability of resist interference, we further studied the FL response of CDs/DNTB/ATCh/AChE system
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to paraoxon (0.2 μg mL-1) in the presence of interfering substances. It can be obviously found that, even interfering substance (2 μg mL-1) exists, CDs/DNTB/ATCh/AChE system still possessed the
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same response to paraoxon (Figure 5B). So the established system hold excellent specificity and
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promising anti-interference capability for paraoxon detection, implying that the system was suitable
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3.6 Real samples detection
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for paraoxon analysis.
To estimate the practical applications in real samples detection, our method and gas
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chromatography (GC) were used to detect paraoxon concentration in environmental and agricultural
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samples (water, rice, and cabbage). The results demonstrated that no paraoxon exists in the test samples (Table S2). The results obtained by standard addition method were listed in Table S2, the average recovery of paraoxon ranges from 90 % to 102 % and the relative standard deviation (RSD)
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was lower than 4.17 %. Furthermore, the obtained results were well consistent with those of GC, implying that the sensing platform hold highly accurate and reproducible for paraoxon detection in real samples and possess potential applicability.
Conclusion 14
In summary, by combining AChE-induced the decomposition of DTNB and TNBA-triggered the dynamic quenching process, we developed a dual-readout methodology that allows a simple optical (colorimetric or fluorometric) detection of OPs in highly selective and sensitive manner. The colorimetric and fluorescent responds were based on the formation of yellow-colored TNBA that
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served as color reporter and FL quencher. The dual-readout signal in the sensing system was proportional to the amount of OPs in the range of 0.001 to 1.0 μg mL-1 with a LOD of 0.4 ng mL-1.
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Moreover, benefiting from the changes of absorbance intensity, a visual recognition for paraoxon by color changes was achieved in a concentration-dependent manner. This proposed dual-readout
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platform could thereby be a reliable option to quantitatively detect pesticide in environmental and
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agricultural samples due to additional signal correction from each other, which validated its
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efficiency in on-site application. The dual-signal strategy not only improved detection sensitivity,
Acknowledgments
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but also held an additional correction of output signals.
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This work was financially supported by the National Natural Science Foundation of China (Nos. 21275063 and No. 21575048), the Science and Technology Development project of Jilin
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province, China (No. 20150204010GX). X. Yan is thankful for support from the National
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Postdoctoral Program for Innovative Talents (BX201700096).
15
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Hongxia Li received her M.S. degree in 2013 from Nanjing Agricultural University. and received her Ph.D. degree in 2016 at Jilin University. Since then, she did postdoctoral work with Prof. Geyu Lu. Currently, her research interests mainly focus
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on the development of the functional nanomaterials for chem/bio sensors.
Xu Yan received his M.S. degree in 2013 from Nanjing Agricultural University. He
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joined the group of Prof. Xingguang Su at Jilin University and received his Ph.D. degree
in June 2017. Since then, he did postdoctoral work with Prof. Geyu Lu. Currently, his
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research interests mainly focus on the development of the functional nanomaterials for
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chem/bio sensors.
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Geyu Lu received the B. Sci. degree in electronic sciences in 1985 and the M. Sci.
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degree in 1988 from Jilin University in China and the Dr. Eng. degree in 1998 from Kyushu University in Japan. Now he is a professor of Jilin University, China. His
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current research interests include the development of chemical sensors and the
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application of the function materials.
Xingguang Su is a professor at the Department of Analytical Chemistry at the College
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of Chemistry, Jilin University. She received her MS degree from Jilin University (China) in 1992 and her PhD degree from Jilin University (China) in 1999. Her research focuses on the synthesis, characterization, functionalization and application of quantum dots and quantum dots-tagged microspheres in biomedicine. 22
Captions:
Scheme 1 The schematic illustration of the dual-signal optical system for OPs detection. Figure 1 (A) One-pot synthesis for CDs. (B) The TEM image of the CDs. (C) The UV-Vis absorption spectrum of CDs. (D) FL excitation and emission spectra of the CDs. (E) FL spectrum
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of CDs in PBS buffer with diff erent pH values range from 5.5 to 9.5. Inset is FL trends of CDs with the variation of pH. (F) The photobleaching experiment of the CDs with 420 nm excitation source
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for continuous intensive excitation. (G) The effect of NaCl concentration on FL intensity of CDs.
Figure 2 (A) Absorbance spectrum and (B) FL spectrum of CDs+DTNB+ATCh,
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CDs+DTNB+ATCh+AChE and CDs+DTNB+ATCh+AChE+OPs system. (C) The zeta potential of
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CDs and TNBA. (D) The fluorescence lifetime measurement of CDs+DNTB+ATCh system in
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absence and presence of AChE. (E) The effect of temperature on the FL quenching of
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CDs+DNTB+ATCh system in the presence of 50 ng mL-1 AChE. (F) Absorption spectrum of
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CDs+DNTB+ATCh system upon the addition of AChE at different concentrations from 0 to 100 ng mL-1 (0, 0.5, 1.0, 2.0, 5.0, 10, 20, 50 and 100 ng mL-1). (G) The corresponding photographs of
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CDs+DNTB+ATCh system with different concentration of AChE taken under daylight. (H) The
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fluorescence spectra of CDs+DNTB+ATCh system in the presence of different concentration of AChE (0, 0.5, 1.0, 2.0, 5.0, 10, 20, 50, 100 and 200 ng mL-1). (I) The linear plot of FL intensity ratio FQ/FQ0 versus the logarithm of AChE concentration.
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Figure 3 Effect of pH (A), incubation temperature (B) and inhibition time (C) on IE in the presence of 0.2 μg mL-1 paraoxon. Figure 4 (A) Absorption spectra of CDs/DNTB/ATCh/AChE system upon the addition of paraoxon at different concentrations from 0 to 0.5 μg mL-1 (0, 0.005, 0.01, 0.05, 0.1 and 0.5 μg mL-1). (B) The 23
corresponding photographs of CDs/DNTB/ATCh/AChE system in the presence of different concentration
of
OPs
taken
under
daylight.
(C)
The
fluorescence
spectra
of
CDs/DNTB/ATCh/AChE system in the presence of different concentration of paraoxon (0, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 and 1.0 μg mL-1). (D) The linear plot of (IE)
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versus the logarithm of paraoxon concentration. Figure 5 (A) The FL intensity of the CDs/DNTB/ATCh/AChE system with the interfering
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substances (2 μg mL-1) or paraoxon (0.2 μg mL-1), (D) The FL intensity of the
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CDs/DNTB/ATCh/AChE /paraoxon system with the interfering substances (2 μg mL-1).
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Scheme 1
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Figure 1
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Figure 4
29
A ED
PT
CC E
IP T
SC R
U
N
A
M
Figure 5
30