In-situ visual and ultrasensitive detection of phosmet using a fluorescent immunoassay probe

Sensors and Actuators B 241 (2017) 915–922

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

In-situ visual and ultrasensitive detection of phosmet using a fluorescent immunoassay probe Zhou Lina a , Cao Yujuan a , Lin Bixia a , Song Shuhua a , Yu Ying a,∗ , Shui Lingling b,∗ a School of Chemistry and Environment, Guangzhou Key Laboratory of Analytical Chemistry for Biomedicine, South China Normal University, Guangzhou, Guangdong, 510006, PR China b South China Academy of Advanced Optoelectronics, South China Normal University, Guangzhou, Guangdong, 510006, PR China

a r t i c l e

i n f o

Article history: Received 21 March 2016 Received in revised form 11 September 2016 Accepted 13 October 2016 Available online 14 October 2016 Keywords: Fluorescent immunoassay Phosmet Polydimethylsiloxane sheet Polymer dots

a b s t r a c t It is necessary to develop new detection methods of pesticide residues because pesticide residues endanger human health. In this study, a fluorescent immunoassay probe (PDs-Ab) was designed in order to establish an in-situ visual and ultrasensitive detection method of phosmet. PDs-Ab probe which could recognize phosmet was synthesized by coupling phosmet antibody with polymer dots (PDs) based on poly [2-methoxy-5-(2-ethylhexyloxy)-1, 4-(1-cyanovinylene-1, 4-phenylene)]. The experiment results showed PDs-Ab probe was used not only for fluorescent imaging but also for ultrasensitive detection of phosmet. The fluorescent imaging realized in-situ visual and semi quantitative detection of phosmet residues on apple surfaces. The ultrasensitive detection of phosmet utilized a poly (dimethylsiloxane) (PDMS) sheet. Phosmet and PDs-Ab probe were orderly loaded on PDMS sheet, and eluted by absolute ethanol. The fluorescence intensity of absolute ethanol eluant increased in proportion to the concentration of phosmet. The limit of detection for phosmet was 0.4 ng/L with a correlation coefficient of 0.9968. The PDMS sheet was served to concentrate trace phosmet and eliminate the fluorescence interference from excess PDs-Ab. Also, this method was used to monitor phosmet residues in real samples, such as lake water, orange peels and Chinese cabbage leaves, with acceptable recovery range from 97.15% to 102.1%. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The effects of pesticides on food, environment and ecology have been received increasing attention as the living standard of people has been improved. Pesticide residues samples have the characteristics of complex components and low concentration analyses. Therefore the detection of pesticide residues is important and challenging. Pesticide residues are correlative with fruits and vegetables of our daily life. Thus, proposing a quick and sensitive detection method for pesticide residues is one of future developing trends. Due to the wide use of phosmet, one of the organophosphorus pesticides, some methods have been developed in the past, but those methods have different kinds of limitation. The detection of phosmet is usually performed with liquid chromatography com-

∗ Corresponding authors. Present address: No.378 Waihuan West Road, University City, Guangdong, 510006, PR China. E-mail addresses: [email protected], [email protected] (Y. Yu), [email protected] (L. Shui). http://dx.doi.org/10.1016/j.snb.2016.10.058 0925-4005/© 2016 Elsevier B.V. All rights reserved.

bined with mass spectrometry (LC–MS) and liquid-solid extraction followed by gas chromatography (GC) or high performance liquid chromatography (HPLC) coupled with selective detectors. However these methods are time-consuming and expensive. Especially, the procedures of sample preparation are complicated. Except for above methods, enzyme-linked immunosorbent assay (ELISA), which has excellent selectivity, is also developed for phosmet. It has been reported that detection limit of phosmet was respectively 1.9 ␮g/L [1] and 0.6 ␮g/kg[2]. In 2013, Liu et al. developed an enhanced chemiluminescence enzyme linked immunosorbent assay (ECL-ELISA) for the detection of phosmet in vegetables samples, and the detection limit of phosmet was 11.32 ␮g/kg [3]. Whereas, ELISA has some defects, such as numerous steps, easy inactivation of enzyme, and low detection limit. Recently, surfaceenhanced raman spectroscopy (SERS), a new detection method of phosmet, is developed. For example, Fan et al. developed an SERS with gold-coated substrates technology to detect phosmet residues in apples [4]. Pan et al. used SERS substrate based on silver nanoparticle functionalized polymethacrylate monoliths to detect residues of phosmet pesticides on tea leaves, apples and oranges [5]. Nevertheless, SERS method is expensive because silver, gold colloid, and

916

L. Zhou et al. / Sensors and Actuators B 241 (2017) 915–922

expensive instruments are required. In conclusion, those methods mentioned above are difficult to apply to in-situ visual detection. In general, in-situ visual detection needs fluorescent probes[6,7]. Fluorescent quantum dots (QDs) as a kind of excellent probe have attracted considerable interests and are widely applied in detection and imaging [8–10]. Recently, Su group reported the advances in the application of QD-based intracellular sensing system [11]. Zhang group reported the surface coordinationoriginated fluorescence resonance energy transfer of CdTe QDs and a simple ligand-replacement turn-on mechanism for the highly sensitive detection of organophosphorothioate pesticides [12]. But the traditional semiconductor QDs are developed from cadmium or other heavy metals with heavy metal pollution, thus eco-friendly probes have attracted considerable interests. Ouyang group synthesized amine-terminated silicon QDs [13] and folate functionalized carbon dots [14], and applied them for the fluorescent imaging. Wu group used reprecipitation techniques for preparing multicolor conjugated polymer dots, which exhibited small particle diameters, extraordinary fluorescence brightness, and excellent photostability [15]. The fluorescent probes are modified with aptamer, antibody and so on, and have the function of identifying the specific analytes [16,17]. Ju group designed a MoS2 nanoplate labelled ATP aptamer for fluorescence imaging of intracellular ATP [18]. Yang group designed an epidermal growth factor-funtionalized quantum dots-capped magnetic bead probe to integrate the specific recognition of EGFR expressed on MCF-7 cell surfaces [19]. In order to establish an in-situ visual and sensitive detection method for phosmet residues, a fluorescent immunoassay probe (PDs-Ab) was designed in this paper. The luminous component of probe was PDs which had good biocompatibility and excellent fluorescent properties. The identification component was phosmet antibody, which was able to recognize phosmet. The research indicated PDs-Ab probe was used for not only fluorescent imaging but also ultrasensitive detection of phosmet. The fluorescent imaging of phosmet residue on apple surfaces was observed under the UV lamps. The ultrasensitive detection of phosmet employs a poly (dimethylsiloxane) (PDMS) sheet. The PDMS sheet could concentrate trace phosmet and eliminate the fluorescence interference from excess PDs-Ab. The concentration of phosmet was well linear with the fluorescence intensity of the probe on the PDMS sheet, and a new selective and ultrasensitive detection method of phosmet was established. The design strategy of this work was shown in scheme (Scheme 1).

Fluorescence spectra were recorded with an F-2500 spectrofluorometer (Hitachi, Japan) with 400 V of voltage, 5 nm of slit and 300 nm/min of scanning speed. Absorption spectra were measured using UV–vis 1700 spectrophotometer (Tianmei, China). Dynamic light scattering (DLS) of samples were characterized with Malvern Zetasizer Nano ZS nanometer particle size analyzer (Malvern, UK). Fluorescence imaging of phosmet residues on apple surfaces were performed under UV lamps (50W, shanghai, China). The PDMS sheet load rate for phosmet was detected by HPLC SCL-6A (Shimadzu, China). 2.2. Synthesis of phosmet polyclonal antibody According to previous methods [20], Mannich reaction was adopted for constructing the complete antigen (phosmet-cBSA) through coupling phosmet with cationic bovine serum albumin (cBSA). According to the immune protocol [3], five New Zealand white rabbits were immunized by immunogen for five times during sixty-six days and then took blood from the heart, and then the antiserum was obtained by centrifugal separation. The antibody concentration detected were range 0.64-0.78 mg/mL by ultraviolet absorption spectrum. The antibody was stored at −20 ◦ C. 2.3. Synthesis of phosmet probe According to previous methods [21] of PDs prepared, CN-PPV and PSMA was dissolved respectively in tetrahydrofuran (THF) to make 1 mg/mL stock solution. 250 ␮L of the CN-PPV and 50 ␮L of the PSMA stock solutions were put into 5 mL of THF. The mixture was sonicated to form a homogeneous solution. 5 mL of the mixture solution was quickly added into 10 mL of MilliQ water in a ultrasonic bath. The THF was volatilized by nitrogen stripping, and the solution was concentrated to 5 mL on a 90 ◦ C hotplate followed by filtration through a 0.2 micron filter. The final PDs solution was at a concentration of 50 ␮g/mL (according the amount of CN-PPV). To conjugate PDs with phosmet antibody, 5.0 mL of PDs solution was added 100 ␮L of freshly-prepared EDC (10 mg/mL in water) and 1.3 mL of 1 M PBS buffer (pH 7.4), the mixture solution was stirred on a vortex for 5 min. Finally, 400 ␮L of phosmet antibody was added to the solution and mixed well on a vortex. The mixture was left on a rotary shaker at 37 ◦ C for 2 h. The resulting PDs-Ab bioconjugates were separated by centrifugal separation and had a concentration of 100 ␮g/mL according the amount of PDs. 2.4. In-situ visual and semi quantitative detection of phosmet

2. Material and methods 2.1. Materials and instruments Poly [2-methoxy-5-(2-ethylhexyloxy)-1, 4-(1-cyanovinylene-1, 4-phenylene)] (CN-PPV, MW 15,000, polydispersity 5.9) was purchased from ADS Dyes Inc. (Quebec, Canada). The copolymer poly (styrene-co-maleic anhydride) (PSMA, cumene terminated, average Mn 1700, styrene content 68%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetrahydrofuran (THF) was purchased from Sinopharm Chemical Reagent Co. Ltd. 1-Ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and phosmet were obtained from Aladdin. Absolute ethanol was purchased from Damao Reagent (Tianjin, China). Bovine Serum Albumin was purchased from Bio Science & Technology Co. Ltd (Shanghai, China). Poly (dimethylsiloxane) (PDMS) sheets were obtained from South China Academy of Advanced Optoelectronics. All reagents used were analytical grade unless otherwise stated. All solutions were prepared with double-distilled water.

Three apples were marked A, B, C. The letter P (phosmet initial) was written with a brush dipped phosmet solution (20 mg/L) on the surface of A and C apples and air-dried. Then, the P was depicted with a brush dipped PDs-Ab probe solution (100 ␮g/mL) on the surface of B and C apples and air-dried. After washed with double-distilled water, A, B and C apples were observed under UV lamps. The other apples were marked D, E, and F. According to the same way described in above test, except the P was written with different concentrations of phosmet solution (100 ␮g/L, 250 ␮g/L and 500 ␮g/L) on the apple surfaces. 2.5. Quantification of phosmet 2.5.1. Detection method A piece of 2 × 2 (cm) PDMS sheet was put into a clean test tube, 2.0 mL of phosmet solution was added and incubated for 2 h at room temperature. Then the PDMS sheet loaded phosmet was taken out, washed 3 times using redistilled water and sucked dry lightly with filter paper. To repeat above steps, the order of various solutions added and incubation time were as follows: BSA solution (1%) for

L. Zhou et al. / Sensors and Actuators B 241 (2017) 915–922

917

Scheme 1. The schematic diagram of the probe synthesis and phosmet detection. (a) synthesis and structure of PDs. (b) preparation of phosmet antibody. (c) PDs-Ab probe structure and fluorescent imaging of phosmet. (d) quantitative detection of phosmet based on phosmet and PDs-Ab orderly loaded on PDMS sheet.

2 h in 37 ◦ C, 100 ␮g/mL PDs-Ab solution for 4 h. After the PDMS sheet was dipped in 2.0 mL of absolute ethanol and extracted for 40 min, the PDMS sheet was took out and the fluorescence intensity of the elution was detected by fluorescence spectra. 2.5.2. Samples preparation The samples of lake water, orange peels and Chinese cabbage leaves were chosen to evaluate this method established. Lake water was taken from Yan Lake in South China Normal University, orange peels and Chinese cabbage leaves were bought from local markets. The three kinds of samples were obtained as follows: Lake Water sample was prepared by taking and filtering 45 mL Yan Lake water, spraying a certain amount of phosmet solution and diluting with double-distilled water to 50 mL. The certain amount of orange peels or Chinese cabbage leaves were sprayed phosmet solution, incubated for 12 h at room temperature, and cut into small pieces. The pieces of orange peels or Chinese cabbage leaves (5 g) were crushed and filtered to remove the residue. The residue was washed and the filtrate was diluted to 50 mL with double-distilled water. 2.5.3. Samples assay 2.0 mL samples of lake water, orange peels or Chinese cabbage leaves was added in a clean test tube respectively, and detected according above method. Besides, 2.0 mL spiked samples of lake water, orange peels or Chinese cabbage leaves were detected. Repeat this operation three times. After deducting initial value of phosmet respectively, the recovery rates of this method were calculated.

3. Results and discussion 3.1. Characterization of the probe The optical properties of PDs made PDs-Ab well-suited for biological detection and imaging. The PDs exhibited bright orange fluorescence with a quantum yield of about 60% by comparison with quinoline sulfate (Fig. S2 in Supporting information). The fluorescence spectra of PDs and PDs-Ab were shown in Fig. 1A. Both the excitation peak of PDs (a) and PDs-Ab (b) were among 330–450 nm. PDs exhibited strong fluorescence around 584 nm, the emission peak of PDs-Ab was blue-shifted and observed at 578 nm. The conjugation could be verified through different emission peaks. The absorption spectra of PDs (a), PDs-Ab (b), antibody(c) and phosmet (d) were showed in Fig. 1B. The characteristic absorption peak of phosmet was at 223 nm and 275 nm. The characteristic peak of PDs located at 461 nm, and the absorption peak of PDs-Ab probe was blue-shifted to 452 nm after the coupling of PDs with antibody. Conjugation is always along with the change of particle size, thus before and after antibody coupled was contrasted by means of dynamic light scattering test. Fig. 1C showed that the hydration diameters of PDs (a) and antibody (b) were 8.97 nm and 69.57 nm respectively. The hydration diameters of PDs-Antibody (d) was 105.7 nm differ from the hydration diameters of PDs and antibody mixture which has two different size of 8.91 nm and 67.84 nm (c). The results indicated that antibody was coupled with PDs.

918

L. Zhou et al. / Sensors and Actuators B 241 (2017) 915–922

Fig. 1. Excitation and emission spectra of PDs and PDs-Ab (A) (a: PDs, b: PDs-Ab); UV-vis spectra (B) (a: PDs, b: PDs-Ab, c: antibody, d: phosmet); DLS characterization (C) (a:PDs, b:antibody, c:PDs and antibody, d: PDs-Ab).

3.2. Semi quantitative in-situ visualization detection of phosmet In this paper, the as-prepared PDs-Ab probe was used for visual detection of phosmet residue in apple surfaces, which was recorded by camera under the UV lamps. Both apple and phosmet showed no obvious fluorescence signal under ultraviolet light (Fig. 2A). The water-solubility PDs-Ab was washed by distilled water; weak orange fluorescence of the PDs-Ab could be observed due to nonspecific binding between the apple surface and the probe (Fig. 2B). The significant orange fluorescence of PDs-Ab probe was observed on the apple surface loaded phosmet (Fig. 2C). The result showed the probe could be used for in-situ vistual detection of phosmet residues. The method was simple and the fluorescence was easily observed by naked eye. The detection of semi quantitative visualization was realized on apple surfaces. The National standards of China (GB16320-1996) for maximum residue limit of phosmet was 500 ␮g/L. The apple surfaces exhibited significant orange fluorescence grades with different concentrations of phosmet in Fig. 2D–F. The significant orange fluorescence from phosmet of 500 ␮g/L and weak visual from phosmet of 100 ␮g/L indicated that the probe could been used for semi quantitative detection of real samples. 3.3. Ultrasensitive detection of phosmet In order to concentrate trace phosmet and eliminate the interference of excess probes in ultratrace detection of phosmet, the following strategies were designed. PDMS sheets were selected to

load phosmet, which was based on the hydrophobic force between PDMS sheet and phosmet. To block any remaining active sites of PDMS sheet, BSA solution was added. The PDs-Ab probe could bind specificly with phosmet loaded on PDMS sheet due to antigenantibody immunoreaction. So phosmet and PDs-Ab probe were orderly loaded on the PDMS sheet by layer assembly. The concentrations of phosmet were well linear with the fluorescence intensity of eluants from the loaded PDMS sheet. Thus, a sensitive and selective detection method of phosmet was established. 3.3.1. Condition experiments In order to elute phosmet or PDs-Ab-phosmet loaded on PDMS sheets, various elution solvents were explored. As showed in Fig. 3A, both the fluorescence intensities (black) from phosmetPDs-Ab eluants and the UV absorbance (gray) from phosmet eluants were different. The elution effectivity of DMF, acetonitrile, absolute ethanol were alike. But in view of safety and toxicity, absolute ethanol was chosen as the elution solvent. The adsorption time of PDMS sheet for phosmet or PDs-Ab was studied. As showed in Fig. 3B, the UV absorbance of eluant form PDMS sheet loaded phosmet was detected at 275 nm, enhanced with adsorption time increasing and stabilized after loading 12 h (3B-a). Adsorption capacity and efficiency considered, 12 h was chosen for the optimal adsorption time. The adsorption time of PDsAb was investigated before and after PDMS sheet sealed up using BSA. The result showed PDMS sheet could adsorb PDs-Ab probe directly and the adsorption amount of PDs-Ab enhanced with prolong adsorption time (3B-b). However, after PDMS sheet was sealed

L. Zhou et al. / Sensors and Actuators B 241 (2017) 915–922

919

Fig. 2. In-situ semi quantitative visualization detection of phosmet residue on apple surfaces under ultraviolet light (A: phosmet; B: PDs-Ab; C, D, E, F: phosmet (20 mg/L, 500 ␮g/L, 250 ␮g/L and 100 ␮g/L) and PDs-Ab). Table 1 Recoveries of phosmet in real samples. Samples

Initial Detected (ng/L) n=3

Spilked (ng/L)

Detected (ng/L) n=3

Recovery (%) n=3

lake water

9.81 ± 0.11

5 15

14.68 ± 0.21 24.50 ± 0.33

99.12 98.75

Chinese cabbage leaves

7.94 ± 0.10

5 15

12.71 ± 0.20 22.32 ± 0.29

98.22 97.30

orange peels

6.87 ± 0.19

5 15

11.54 ± 0.31 22.52 ± 0.40

97.22 103.0

Table 2 Comparison with reported methods. Test Method 1 2 3 4 5 6 a

This method SERS SPR ELISA ECL-ELISA MSPD and GC/MS

Analytes apple, cabbage, lake water apple pear, apple, cabbage, barley pear, apple vegetables olive

LOD 0.36 ng/L 1.44 mg/kg 1.6 ng/L 0.6 ␮g/ kg 11.32 ␮g/kg 10 ␮g/ kg

Detection Range

references

a

2.5–40 ng/L 0.5–10 mg/kg 8.0–60.0 ng/L 0.1–10000 ␮g/kg 0.05–10000 ␮g/L 0.25 mg/kg–2 mg/kg

[4] [22] [2] [3] [23]

In theory, the linear range can be increased by means of enlargement scale of PDMS.

up using BSA, the fluorescence intensity of the eluant was low and stabilized with prolong time (3B-c). Thus free PDs-Ab loaded on PDMS sheet was suppressed after PDMS sheet was sealed up using BSA. The binding time between PDs-Ab and phosmet was researched. It was found in Fig. 3C that the fluorescence intensity of eluant form PDMS sheet loaded phosmet-PDs-Ab was different with various binding time of PDs-Ab and phosmet. The fluorescence intensity of eluant enhanced with binding time increasing and stabilized after phosmet binded PDs-Ab for 3.5 h. Thus 4 h was chosen for the optimal binding time of PDs-Ab with phosmet. The elution time was studied. As showed in Fig. 3D, the fluorescence intensity of eluant from PDMS sheet loaded phosmet-PDs-Ab was different with various elution time. The fluorescence intensity enhanced with elution time increasing from 10 to 30 min and stabilized after 30 min 30 min was chosen for the optimal elution time.

The relationship between load capacity and scale of PDMS sheet was studied. As showed in Fig. 3E, the load capacity of phosmet enhanced with PDMS sheet scale increasing. The fluorescence intensity plateaued when the amount of phosmet reached the maximum load capacity. The scale of 1 × 1 (cm) could meet load capacity of 10.0 ng phosmet, which was confirmed by the same fluorescence intensity with scale form 1 × 1 (cm) to 2 × 2 (cm). Similarly, the experiment result showed the scale of 1 × 2 (cm) could meet load capacity of 20.0 ng. Thus, load capacity (ng) was equal to 10 × length × width (cm). The scale of 2 × 2 (cm) could meet the load capacity of 40.0 ng in theory. The PDMS sheet load rate for phosmet was detected by HPLC when the amount of phosmet was lower than the maximum load capacity, 98.5% of phosmet in samples could been loaded.

920

L. Zhou et al. / Sensors and Actuators B 241 (2017) 915–922

Fig. 3. Condition optimization of the phosmet detection and the linear relation curve. A: elution effect of solvents (black: fluorescence intensity of PDs-Ab eluents, gray: absorbance of phosmet eluents); B: adsorption time (a: absorbance of phosmet eluents; b: fluorescence intensity of PDs-Ab eluents; c: fluorescence intensity of PDs-Ab eluents after sealing up); C: binding time of phosmet and PDs-Ab; D: elution time of PDs-Ab; E: PDMS sheet scales versus amount (ng) of phosmet (a: 10, b: 20, c: 30); F: The linear relation curve of the fluorescence intensity of PDs-Ab versus phosmet concentration.

3.3.2. The linear relation curve The linear relation curve was obtained using PDMS sheet of 2 × 2 (cm) scale according to the experiment method 2.5. As shown in Fig. 3F, the linear relationship between the fluorescence intensity of eluant and the concentration of phosmet was obtained in the range from 2.5 to 40 ng/L. The regression equation could be expressed as F = −21.84 + 26.88C (ng/L), with a correlation coefficient of 0.9968 and the limit of detection for phosmet was 0.4 ng/L. In theory, the linear range could be increased by means of enlargement scale of PDMS.

3.3.3. The selectivity of the probe The selectivity of the method established was verified. The structural similar such as DPMMP, some pesticides such as chlorpyrifos et al. were employed for control studies. As showed in Fig. 4, when the seven samples were detected using the established method respectively, only phosmet could achieve strong fluorescence responses, whereas the fluorescence intensity in the presence of the six controls showed little change. The above results indicated high selectivity of the PDs-Ab probe to phosmet. Fig. 4B showed the interference of various common ions on the fluorescence intensity of detecting phosmet, including Ca2+ , Na+ , Ba2+ , Cu2+ , Cr3+ , Fe3+ , Mg2+ , Ni2+ , Fe2+ , K+ with a concentration of

L. Zhou et al. / Sensors and Actuators B 241 (2017) 915–922

921

Fig. 4. Effects of common pesticides (A) and metal ions (B) on PDs-Ab probe for phosmet detection.

20 ␮g/mL. No obvious fluorescence intensity change was observed in the coexistence of phosmet with these metal ions respectively. 3.3.4. Quantification detection of real samples The real samples and recovery rates were detected according to the experiment method 2.5 and the results were given in Table 1. It could be seen that the average recoveries of phosmet in lake water, orange peels, Chinese cabbage leaves were in the range of 98.75% ∼ 99.12%, 97.30% ∼ 98.22%, 97.22% ∼ 103.0%. Thus, the method could meet the requirements of sample detection and could been applied to detect phosmet residues in environment and food samples. 3.4. Comparison with reported methods The detection methods of phosmet reported by the major analytical chemistry publications were compared. SPR and SERS showed high sensitivity [21,22]; ELISA displayed wide linear range [23]. The detection sensitivity of matrix solid phase dispersionGC/MS was similar to ELISA,but the linear range for the detection of phosmet was less than ELISA. In this paper, the detection method established had the characteristic of higher sensitivity than other methods, and the prior complex separation of samples was avoided. Besides, the visual and semi quantitation detection of phosmet residues could be achieved, which had potential for daily life detection of pesticide residues Table 2. 4. Conclusion In this paper, PDs-Ab probe was constructed for specific recognition of phosmet. The PDs-Ab probe could be usedP for in-situ

and semi quantitative visualization detection of phosmet. Phosmet and PDs-Ab probe were orderly loaded on the PDMS sheet by layer assembly, which based on the hydrophobic force and antigenantibody immunoreactions. This strategy concentrated trace of phosmet and eliminated the fluorescence interference of excess probes. This method could be applied to real samples and the results showed the method was reliable, high sensitive, specific and easy to operate. Not only semi quantitative in-situ visualization but also ultrasensitive detection of phosmet was realized by this method. Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21275056, 21575043, 61574065), IRT13064, the Platform Construction Project of Guangzhou Science Technology and Innovation Commission (No. 15180001) and the cultivation foundation of South China Normal University for young teachers (No.14KJ08). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2016.10.058. References [1] Y. Liang, X.J. Liu, Y. Liu, X.Y. Yu, M.T. Fan, Synthesis of three haptens for the class-specific immunoassay of O,O-dimethyl organophosphorus pesticides and effect of hapten heterology on immunoassay sensitivity, Anal. Chim. Acta 615 (2008) 174–183. [2] Y. Song, Y. Ge, Y. Zhang, B. Liu, Y. Lu, T. Dong, et al., Hapten synthesis and enzyme-linked immunosorbent assay for phosmet residues: assay

922

[3]

[4]

[5]

[6] [7]

[8]

[9]

[10]

[11] [12]

[13]

[14]

[15]

[16]

[17]

L. Zhou et al. / Sensors and Actuators B 241 (2017) 915–922 optimization and investigation of matrix effects from different food samples, Anal. Bioanal. Chem. 393 (2009) 2001–2008. B. Liu, Y. Ge, Y. Zhang, Y. Song, Y.R. Chen, S. Wang, Development of a simplified enhanced chemiluminescence enzyme linked immunosorbent assay (ECL-ELISA) for the detection of phosmet, azinphos-methyl and azinphos-ethyl residues in vegetable samples, Anal. Methods 5 (2013) 5938–5943. Y.X. Fan, K.Q. Lai, B.A. Rasco, Y.Q. Huang, Analyses of phosmet residues in apples with surface-enhanced Raman spectroscopy, Food Control 37 (2014) 153–157. Y.C. Pan, X. Wang, H. Zhang, Y. Kang, T. Wu, Y.P. Du, Gold-nanoparticle, functionalized-porous-polymer monolith enclosed in capillary for on-column SERS detection, Anal. Methods 7 (2015) 1349–1357. Y. Gao, Y. Yu, X. Hu, Y. Cao, J. Wu, Imaging of jasmonic acid binding sites in tissue, Anal. Biochem. 440 (2013) 205–211. F. Liu, Y. Yu, B. Lin, X. Hu, Y. Cao, J. Wu, Visualization of hormone binding proteins in vivo based on Mn-doped CdTe QDs, Spectrochimica acta Part A, Mol. Biomol. Spectrosc. 131 (2014) 9–16. T. Kang, K. Um, J. Park, H. Chang, D.C. Lee, C.-K. Kim, et al., Minimizing the fluorescence quenching caused by uncontrolled aggregation of CdSe/CdS core/shell quantum dots for biosensor applications, Sens. Actuators B 222 (2016) 871–878. B. Lin, Y. Yu, R. Li, Y. Cao, M. Guo, Turn-on sensor for quantification and imaging of acetamiprid residues based on quantum dots functionalized with aptamer, Sens. Actuators B 229 (2016) 100–109. Y.-P. Dong, Y. Zhou, J. Wang, J.-J. Zhu, Electrogenerated chemiluminescence resonance energy transfer between lucigenin and CdSe quantum dots in the presence of bromide and its sensing application, Sens. Actuators B 226 (2016) 444–449. Q. Ma, X. Su, Advances in the application of QD-based intracellular sensing systems, Appl. Spectrosc. Rev. 51 (2015) 162–181. K. Zhang, Q. Mei, G. Guan, B. Liu, S. Wang, Z. Zhang, Ligand replacement-induced fluorescence switch of quantum dots for ultrasensitive detection of organophosphorothioate pesticides, Anal. Chem. 82 (2010) 9579–9586. P. Liu, N. Na, L. Huang, D. He, C. Huang, J. Ouyang, The application of amine-terminated silicon quantum dots on the imaging of human serum proteins after polyacrylamide gel electrophoresis (PAGE), Chemistry 18 (2012) 1438–1443. Q. Liu, S. Xu, C. Niu, M. Li, D. He, Z. Lu, et al., Distinguish cancer cells based on targeting turn-on fluorescence imaging by folate functionalized green emitting carbon dots, Biosens. Bioelectron. 64 (2015) 119–125. C. Wu, B. Bull, C. Szymanski, K. Christensen, J. McNeill, Multicolor conjugated polymer dots for biological fluorescence imaging, ACS nano 2 (2008) 2415–2423. Y.S. Li, Y. Zhou, X.Y. Meng, Y.Y. Zhang, J.Q. Liu, Y. Zhang, et al., Enzyme-antibody dual labeled gold nanoparticles probe for ultrasensitive detection of kappa-casein in bovine milk samples, Biosens. Bioelectron. 61 (2014) 241–244. Q. Zhao, Q. Lv, H. Wang, Aptamer fluorescence anisotropy sensors for adenosine triphosphate by comprehensive screening tetramethylrhodamine labeled nucleotides, Biosens. Bioelectron. 70 (2015) 188–193.

[18] L. Jia, L. Ding, J. Tian, L. Bao, Y. Hu, H. Ju, et al., Aptamer loaded MoS2 nanoplates as nanoprobes for detection of intracellular ATP and controllable photodynamic therapy, Nanoscale 7 (2015) 15953–15961. [19] Y. Tang, S. Zhang, Q. Wen, H. Huang, P. Yang, A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces, Anal. Chim. Acta 881 (2015) 148–154. [20] X.Y, Studies on the Immunological Method of the Oganophosphorous Pesticides Dichlorvos and Phomet, Huazhong Agricultural University, 2009 (Master thesis). [21] F. Ye, C. Wu, Y. Jin, M. Wang, Y.H. Chan, J. Yu, et al., A compact and highly fluorescent orange-emitting polymer dot for specific subcellular imaging, Chem. Commun. 48 (2012) 1778–1780. [22] Y. Song, M. Liu, S. Wang, Surface plasmon resonance sensor for phosmet of agricultural products at the ppt detection level, J. Agric. Food Chem. 61 (2013) 2625–2630. [23] S.C. Cunha, J.O. Fernandes, M. Beatriz, P.P. Oliveira, Determination of phosmet and its metabolites in olives by matrix solid-phase dispersion and gas chromatography-mass spectrometry, Talanta 73 (2007) 514–522.

Biographies Lina Zhou is a student under the supervision of Professor Ying Yu in South China Normal University. Her research interests focus on the design, synthesis and application of fluorescent probes. Yujuan Cao is currently an associate Professor of Analytical Chemistry at South China Normal University. She obtained Ph.D degree (2005) at the Chinese Academy of Science. Her research interests focus on the design, synthesis and application of probes. Bixia Lin received her master degree in Analytical Chemistry from South China Normal University in 2011. She started her doctoral work under the supervision of Professor Ying Yu at South China Normal University in 2015. Her research interests focus on the design, synthesis and application of fluorescent probes. Shuhua Song is a student under the supervision of Professor Ying Yu in South China Normal University. Her research interests focus on the design, synthesis and application of fluorescent probes. Ying Yu is currently a Professor of Analytical Chemistry at South China Normal University. She received Bachelor degrer (1986), Master degree (1989) from Jiangxi University and Ph.D degree (2005) from South China University of Technology. Her research interests focus on the design, synthesis and application of fluorescent probes. Lingling Shui is currently a Professor of South China Academy of Advanced Optoelectronics at South China Normal University. She obtained Ph.D degree (2009) from the Netherlands, University of Twente. Her research interests focus on the design, synthesis and application of microfluidic chip.