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Quantum dots coated with molecularly imprinted polymer as fluorescence probe for detection of cyphenothrin
Biosensors and Bioelectronics 64 (2015) 182–188
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Quantum dots coated with molecularly imprinted polymer as fluorescence probe for detection of cyphenothrin Xiaohui Ren, Ligang Chen n Department of Chemistry, College of Science, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China
art ic l e i nf o
a b s t r a c t
Article history: Received 15 July 2014 Received in revised form 23 August 2014 Accepted 27 August 2014 Available online 6 September 2014
A newly designed molecularly imprinted polymer (MIP) material was fabricated and successfully utilized as recognition element to develop a quantum dots (QDs) based MIP-coated composite for selective recognition of the template cyphenothrin. The MIP-coated QDs were characterized by fluorescence spectrophotometer, Fourier transform infrared spectroscopy, transmission electron microscope, dynamic light scattering and X-ray powder diffraction. The fluorescence of the coated QDs is quenched on loading the MIP with cyphenothrin, and the effect is much stronger for the MIP than for the non-imprinted polymer, which indicates the MIP could as a recognition template composite. This method can detect down to 9.0 nmol L 1 of cyphenothrin in water, and a linear relationship has been obtained covering the concentration range of 0.1–80.0 μmol L 1. The method has been used in the determination of cyphenothrin in water samples and gave recoveries in the range from 88.5% to 97.1% with relative standard deviations in the range of 3.1–6.2%. The present study provides a new and general strategy to fabricate inorganic–organic MIP-coated QDs with highly selective recognition ability in aqueous media and is desirable for chemical probe application. & 2014 Elsevier B.V. All rights reserved.
Keywords: Molecular imprinting Quantum dots Fluorescence quenching Cyphenothrin
1. Introduction In recent years, semiconductor nanoparticles, also known as quantum dots (QDs) gained great attention in the scientific community (Chaoa et al., 2014; Dezhkam and Zakery, 2014; Li et al., 2014; Costas-Mora et al., 2014; Mohammadi and Bahrami, 2014). QDs hold promise as fluorescent sensors, probes, and tags for various applications, owing to the narrow emission and resistance to fluorescence quenching (Jan’czewski et al., 2014; Hodlur and Rabinal, 2014; Kim et al., 2014; Wang et al., 2013). In particular, QDs are good candidates for analytical assays because they exhibit a Stern–Volmer quenching behavior (Durán-Toro et al., 2014). Many studies in this area have been focused on the development of new methods to synthesize high-quality QDs with a high luminescence quantum yield, excellent photostability, and good biocompatibility (Samadi-maybodi et al., 2014; Chu et al., 2012; Korala et al., 2013; Kang et al., 2010). Different strategies for the synthesis of these nanoparticles have been developed, so that their composition, size, shape, and surface properties can be systematically controlled with an exceptionally high degree (Fan et al., 2014; Hodlur and Rabinal, 2014; Liu et al., 2007; Peng and n
Corresponding author. E-mail address: [email protected] (L. Chen).
http://dx.doi.org/10.1016/j.bios.2014.08.086 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Peng, 2001; Haldar et al., 2012). The design of probe devices based on QDs is a topic of great interest. Molecular imprinting technique (MIT) has been known as an attractive method to develop artificial receptors, which was used to prepare molecularly imprinted polymers (MIPs) (Gao et al., 2014). This technique is based on the in situ co-polymerization of cross-linkers and functional monomers to form complexes with template molecules prior to polymerization (Sun and Fung, 2013). MIP is a polymer which formed in the presence of a molecule (called template). After extraction, then it leaves complementary cavities behind (LütfiYola et al., 2014; Sanagi et al., 2013). The cross-link ratios of polymers are usually in excess of 80% (Cormack and Elorza, 2004). By virtue of the super-cross-linked nature, MIPs can put up with extremely physical and chemical treatment, such as high temperature, pressure, extreme pH, organic solvents, acids and bases. MIPs are cheap, robust materials with a high mechanical, chemical stability and reusable in different applications (Wang et al., 2014; Davoodi et al., 2014). MIPs have been widely exploited in diverse fields such as catalysis (Wulff and Liu, 2012), chemical analysis (Garcia et al., 2011), chromatography (Hsu et al., 2011; Blanco et al., 2012), capillary electrophoresis (De-Maleki et al., 2010), hollow fiber separation (Son and Kobayashi, 2011), membrane separation (Takeda et al., 2009), sensor technology (Moreno-Bondi et al., 2008) and solid-phase extraction (Lucci et al., 2011). It is reasonably believed that molecular imprinting
X. Ren, L. Chen / Biosensors and Bioelectronics 64 (2015) 182–188
technique will be a powerful tool to improve the selectivity of the optical detection. Cyphenothrin (C24H25NO3) is a synthetic pyrethroid pesticide. The structure of cyphenothrin is shown in Fig. S1. Pyrethroid pesticides, as a new bionic pesticide, have some characteristics including broad-spectrum insecticide, high efficacy and highly environmental compatibility (Li and Chen, 2013). In recent years, the use of synthetic pyrethroids derived from chrysanthemic acid is increasingly replacing the use of toxic pesticides such as organochlorine and organophosphorus pesticides (Van-Emon and Chuang, 2012; Yoshida, 2009). Nevertheless, their bioaccumulation and residual toxicity are to be of concern to human food safety (Arvand et al., 2013). Studies prove that pyrethroids could cause seizures and disturb consciousness by affecting the central nervous system of humans when exposed to high concentrations (Nagy et al., 2014; Feo et al., 2013). Therefore, the development of new methods for the determination of pyrethroid in environment samples has captured great interest. Analytical methods such as gas chromatography (GC) (Mezcua et al., 2009), high performance liquid chromatography (HPLC) (Hunter et al., 2010) and liquid chromatography–mass spectrophotometry (LC–MS) (Vonderheide et al., 2009) have been developed for the determination of pyrethroid. However, these methods have deficiencies, such as long manipulation time, high cost and requiring a tedious sample pretreatment. Hence, there is an urgent need to establish a simple and effective method to determine pyrethroid pesticides in environmental samples. In this work, an eco-friendly fluorescence probe, MIP-coated QDs, was successfully fabricated by using cyphenothrin as a template molecule. The MIP-coated QDs were characterized by fluorescence spectrophotometer, Fourier transform infrared spectroscopy (FT-IR), transmission electron microscope (TEM), dynamic light scattering (DLS) and X-ray powder diffraction (XRD). The fluorescence quenching relationship between MIP-coated QDs and cyphenothrin was investigated. Then the MIP-coated QDs were used as fluorescence probe for simple, rapid, and selective detection of cyphenothrin in water samples.
2. Experimental 2.1. Samples and reagents The standard of cyphenothrin was purchased from SigmaAldrich (St. Louis, MO, USA). Zinc sulfate heptahydrate (ZnSO4 7H2O) was purchased from Shuangchuan (Tianjin, China). Manganese (II) chloride tetrahydrate (MnCl2 4H2O) was purchased from Bodi (Tianjin, China). Sodium Sulfide (Na2S 9H2O) was purchased from Kaitong (Tianjin, China). Tetraethoxysilane (TEOS), ethanol, methanol, acrylamide (AM), azoisobutyronitrile (AIBN) and sodium hydroxide were obtained from Kermel (Tianjin, China). Ethylene glycol dimethacrylate (EDGMA) and 3-aminopropyltriethoxysilane (APTES) were purchased from Aladdin (Shanghai, China). All chemicals employed in this study were of analytical grade. High-purity water was obtained from a Milli-Q water system (Millipore, Billerica, MA, USA). The standard stock solution of cyphenothrin was prepared by dissolving cyphenothrin in methanol, and the concentration was 3 mmol L 1. It was stored in a refrigerator at 4 °C. Three river water samples were collected from Harbin (China). All water samples were stored in a refrigerator at 4 °C. 2.2. Apparatus Fourier transform infrared (FT-IR) spectrum of the MIP-coated QDs was recorded with a FT-IR360 spectrometer (Nicolet,
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Madison, WI, USA) using the KBr method. The X-ray diffraction (XRD) spectrum was collected on a Shimadzu XRD-600 diffractometer (Kyoto, Japan) with Cu Kα radiation. The morphology of MIP-coated QDs was observed with a transmission electron microscope (TEM) (H7650, Hitachi, Japan) and a high-resolution transmission electron microscope (HRTEM) (2100F, JEOL, Japan). The size of MIP-coated QDs was observed with a Malvern Zetasizer model Nano ZS90 (Malvern, England). The UV spectrum was recorded on a TU-1901 spectrometer (PERSEE, Beijing, China). Fluorescence intensity studies were carried out at room temperature by using a Perkin-Elmer LS-55 fluorescence spectrometer (Maryland, USA) which was equipped with a plotter unit and a quartz cell. A KQ5200E ultrasonic apparatus (Kunshan Instrument, Kunshan, China) was used for making samples dispersed evenly. 2.3. Synthesis of MIP-coated QDs The synthesis process of MIP-coated QDs involves two major steps: the first step is the synthesis of Mn-doped ZnS QDs, and the second one is the surface imprinting of polymers onto modified Mn-doped ZnS QDs. The synthesis method of Mn-doped ZnS QDs was shown as follows. At first, 25 mmol of ZnSO4 7H2O, 2 mmol of MnCl2 4H2O and 80 mL of water were kept stirring for 20 min under the protection of nitrogen gas. Then, 10 mL, 25 mmol Na2S 9H2O solution was added drop into the mixture (Liu et al., 2010). After being stirred for 30 min, 4 mL of TEOS and 5 mL of APTES were added for modifying the Mn-doped ZnS QDs. Finally, the modified Mn-doped ZnS QDs was obtained after centrifugation and being washed with ethanol three times. The synthesis method of MIP-coated QDs was carried out as follows. Cyphenothrin (1 mmol), AM (4 mmol), and modified Mndoped ZnS QDs obtained from above procedure were dispersed into 150 mL ethanol. Then AIBN (0.1 g) and the cross-linker EGDMA (10 mmol) were added. The mixture was heated at 60 °C and stirred at 300 rpm in a water bath for 24 h. Then the template was removed by Soxhlet extraction with methanol:acetic acid (19:1, v/v) until no template molecule was detected. After dried in vacuum, MIP-coated QDs were obtained. The non-imprinted polymer-coated Mn-doped ZnS QDs (NIPcoated QDs) were prepared using the same procedure without addition of the template cyphenothrin. 2.4. Fluorescence analysis Fluorescence analysis was performed on a Perkin-Elmer LS-55 fluorescence spectrometer. The spectra were recorded in the wavelength range of 500–700 nm upon excitation at 330 nm. Slit widths (10 nm), scan speed (200 nm min 1) and excitation voltage (750 V) were kept constant within each data set and each spectrum was the average of three scans. Quartz cell (1 cm path length) was used for all measurements. 2.5. Measurements of fluorescent response to cyphenothrin Thirty milligram MIP-coated QDs or NIP-coated QDs was added into a centrifuge tube, and then the given concentration of cyphenothrin solution was added. The constant volume was 30 mL. The fluorescence intensity was measured after fully mixing. The river water samples were applied to evaluate the practical application. The water samples were filtered by 220 nm microporous membrane. The recovery study was carried out by spiking certain volume of cyphenothrin solution into water samples.
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3. Results and discussion Zinc sulfide (ZnS) is particularly suitable for use as a host material for a large variety of dopants because of its wide band gap. The Mn-doped ZnS QDs is obtained by bivalent manganese (Mn2 þ ) doping into ZnS semi-conductive nanocrystals. Just as demonstrated in the literature (Zhao et al., 2012), the Mn-doped ZnS QDs used herein exhibit novel optical properties, including wide excitation, as well as narrow and symmetric emission spectra. Different Mn-doped ZnS QDs were prepared by changing the ZnSO4/MnCl2 molar ratios (50:1, 50:2, 50:4; and 50:8). The fluorescence emission spectrum is shown in Fig. S2. The highest fluorescence intensity was obtained with the ZnSO4/MnCl2 molar ratio at 50:4. Both increasing and decreasing the molar ratio, the fluorescence intensity was decreased. Therefore, the 50:4 of ZnSO4/MnCl2 molar ratio was used. The design of our MIP-coated QDs sensing material is based on the fact that the Mn-doped ZnS QDs act as probe for recognition signal amplification and optical readout, while the MIP shell provides analyte selectivity and prevents interfering molecules from coming into contact with the QDs. As illustrated in Fig. 1, the QDs were coated with silica by hydrolysis of TEOS. Based on the fact that the stability of QDs is improved and the surface defects of QDs are reduced. APTES was connected to the silica by the silanization reaction. The surface of the QDs was modified with amino group act as assistant monomers to drive template molecules into the formed imprinted polymer shells during imprinting polymerization. Finally, the MIPs shell was fabricated on the surface of the amino modified QDs through self-assembled of functional monomer (AM), cross-linking agent (EGDMA) and template (cyphenothrin). After removing the template molecules, recognition cavities complementary to the template molecule in shape, size, and chemical functionality were formed in the crosslinked polymer matrix.
face-centered cubic structure. The XRD patterns of MIP-coated QDs were assigned to the (111), (220) and (311) reflections of the cubic phase Mn-doped ZnS, further suggesting the successfully fabrication of MIP-coated QDs. 3.2. Fluorescence study The fluorescence intensity of the MIP-coated QDs was recorded by varying the excitation wavelength from 300 to 350 nm. The weak blue peak around 444 nm was generated by the defect related to the emission of the ZnS QDs. The strong orange peak around 595 nm could be attributed to the 4T1-6A1 transition of the Mn2 þ impurity. The maximum emission intensity at 595 nm was observed with 330 nm as the excitation wavelength. The orange fluorescence was very strong and the peak was sharp, indicating that the sizes of MIP-coated QDs were very homogeneous. In the current work, the template cyphenothrin was entrapped in the polymer matrixes through non-covalent binding. For further elucidate the high selectivity of the MIP-coated QDs in aqueous media, we prepared the NIP-coated QDs. As shown in Fig. 3, the fluorescence intensity of MIP-coated QDs was relatively weak (spectrum c) before the removal of templates. While after Soxhlet extraction with methanol: acetic acid (19:1, v/v), the fluorescence intensity of the MIP-coated QDs was restored dramatically. However, no difference in the shape and position of the emission spectrum was observed (spectrum b). The fluorescence intensity was restored almost to that of the NIP-coated QDs (spectrum a), which indicated that the templates were removed completely from the recognition cavities in the MIP-coated QDs. It suggests that the MIP-coated QDs actually facilitate the application for the rapid and simple quantification of analytes in aqueous media without preconcentration. 3.3. The effect of pH
3.1. Characterization of the MIP-coated QDs The FT-IR spectrum of MIP-coated QDs is shown in Fig. 2a. The strong and broad peak around 1100 cm 1 indicates Si–O–Si asymmetric stretching. Other observed bands about 458 and 783 cm 1 also show the Si–O vibrations. The presence of the bands around 1624 cm 1 was the C¼ C absorption peak. The peak around 2936 cm 1 was C–H band. The bands at 3422 cm 1 and 1560 cm 1 were N–H stretching. All those bands showed that the MIP was grafted on the surface of the QDs. TEM and HRTEM of MIP-coated QDs were shown in Fig. 2b and c, respectively. The DLS measurement results in Fig. S3 showed that the mean size of QDs and MIP-coated QDs are 7.9 and 36.3 nm, respectively. The material of the MIP-coated QDs was investigated with XRD. Fig. 2d showed that the as-synthesized MIP-coated QDs had a
As we know, the orange fluorescence of the ZnS:Mn2 þ nanocrystals is the Mn2 þ typical emission located at 595 nm. The pH effect of MIP-coated QDs without cyphenothrin was investigated. The effect of pH in a range between 4 and 12 was researched. As shown in Fig. S4, the fluorescence intensity of MIP-coated QDs in the interval 4.0–10.0 was considerably stable. When pH is over 11, the fluorescence intensity is quenched seriously. 3.4. The stable fluorescence emission measurement of MIP-coated QDs The fluorescence emission of doped ions was chosen for detection because that is more stable and controllable and has a higher quantum yield than the defect one. The relative standard deviation (RSD) of 0.3% was obtained by 11 repeated detections of
Fig. 1. The preparation process of MIP-coated QDs.
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the fluorescence insensitivity in the MIP-coated QDs aqueous solution every 5 or 10 min. The result shown in Fig. S5 indicates the stable emission of the QDs. The main reason for the stable emission is that inner Mn2 þ is protected by the amorphous silica shell. 3.5. The effect of incubation time The influence of incubation time on the fluorescence intensity investigated at different time scales was shown in Fig. 4. It was found that the mixture showed a rapid decrease in fluorescence intensity after the cyphenothrin added. The reaction was completed within 10 min, indicating that certain time was needed to
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Fig. 4. Influence of incubation time on the fluorescence quenching reaction between the MIP-coated QDs and cyphenothrin.
complete the interaction, and the fluorescence intensity remained stable for at least 1.5 h. Therefore, the experiments were carried out after 10 min and the time scale of 10 min was also adopted in the following experiments. 3.6. MIP- and NIP-coated QDs with template molecule of different concentrations In this test, our aim is to demonstrate the recognition ability of the MIP-coated QDs versus that of the NIP-coated QDs (Fig. 5). Fig. 5a shows the spectral response of MIP-coated QDs with template molecule cyphenothrin at different concentrations. The prepared MIP-coated QDs had an emission at 595 nm. The fluorescence intensity of the MIP-coated QDs was quenched gradually with the increasing concentration of cyphenothrin. Generally, the
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Fig. 5. Fluorescence emission spectra of MIP-coated QDs (a) and NIP-coated QDs (b) with addition of the indicated concentrations of cyphenothrin. Fluorescence emission spectra of MIP-coated QDs (c) and NIP-coated QDs (d) with addition of the indicated concentrations of beta-cyfluthrin.
fluorescence quenching depends on the absorptive affinity of the particles with the template. The fluorescence intensity quenching was mainly achieved by the affinity of the imprinted cavities with the template due to the specific interactions. It was clearly seen that the decrease of fluorescence intensity of the MIP-coated QDs was much larger than that of the NIP-coated QDs (Fig. 5b) by the template molecule cyphenothrin at the same concentration. The imprinting factor IF, which is the ratio of the △MIP/△NIP. It was used to evaluate the selectivity of the materials. Under optimum conditions, the IF was 3.0, which indicated that the MIP-coated QDs can greatly enhance the quenching efficiency of fluorescence, enlarging the spectral sensitivity of the MIP-coated QDs to the template molecule cyphenothrin. 3.7. Selective adsorption on MIP-coated QDs The beta-cyfluthrin was involved to evaluate the selectivity adsorption of the MIP-coated QDs. The chemical structure of betacyfluthrin was shown in Fig. S1. The relationships obtained for analogs beta-cyfluthrin interacting with MIP- and NIP-coated QDs are given in Fig. 5c and d, respectively. It can be seen that the fluorescence intensity quenches were little affected by the increase of beta-cyfluthrin. Herein, because of the different structure of the analogs beta-cyfluthrin, they could not get into the recognition cavities of MIP-coated QDs. The results suggested that MIPcoated QDs were specific to cyphenothrin but nonspecific to betacyfluthrin.
3.8. Fluorescence quenching analysis After adding the template cyphenothrin, there will be an interaction between the template molecule and the MIP-coated QDs, which is a main reason for the fluorescence quenching. We suggest a charge transfer from QDs to cyphenothrin is responsible for fluorescence quenching of MIP-coated QDs. Such charge transfer mechanism has also been reported by Tu et al. (2008) and Wang et al. (2009). The UV absorption band of cyphenothrin is close to the band gap by the absorption spectra of MIP-coated QDs (Fig. S6). The charges of the conductive bands of the QDs can transfer to the lowest unoccupied molecular orbital of cyphenothrin. We can exclude energy transfer as a possible mechanism for fluorescence quenching because there is no spectral overlap between the absorption spectra of cyphenothrin and the emission spectrum of the MIP-coated QDs (Fig. S6). Typical fluorescence quenching of the MIP-coated QDs materials from 0.1 to 80.0 μmol L 1 cyphenothrin was investigated. It demonstrated that the probe showed obvious responses to different concentrations of cyphenothrin, which was very effective and suitable for practical application. The fluorescence quenching in this system can be quantified by the Stern–Volmer equation as follows:
F0/F = 1 + Ksv cq where F0 is the initial fluorescence intensity in the absence of quencher, F is the fluorescence intensity in the presence of analyte, Ksv is the quenching constant of the quencher, and cq is the
This work 88.5–97.1 9.0 Water Fluorescence spectrophotometer
Solid matrix dispersion-size exclusion chromatography clean-up Vigorous shaking-centrifugal extraction Ultrasound assisted extraction-clean up with magnetic molecularly imprinted polymer Fluorescence probe with molecularly imprinted polymer coated quantum dots
Matrix solid phase dispersion
Solid phase microextraction
Milk Blood Fruit
1 102–8 104
60–119 95–97 82.4–101.7 667 0.3 33.1 0.5 103–5.3 103 2.7–2.7 103 66.7–1.3 104
3.1–6.2
Fernandez-Alvarez et al. (2009) 2.5–14.4 Muccio et al. (1997) 0.8–4.3 Ramesh and Ravi (2004) 2.6–5.6 Ma and Chen. (2014) 1.5–7.3 78–86 0.2 11.0–53.0 Cattle feed
40.5–111 0.1 10 3 5.8 10 3 Water
2.8 10 3
3.9–11.9
Fernandez-Alvarez et al. (2008) Casas et al. (2006) o 15 69–118 0.83 Bovine milk 5–267
GC-microelectron capture detector GC-microelectron capture detector GC-microelectron capture detector GC-electron capture detector GC–MS detector HPLC–UV detector Solid phase microextraction
Samples Detection technique Pretreatment method
Table 1 Comparison of this method with other methods used in the literatures.
Analytical ranges (nmol L 1) LODs (nmol L 1) Recovery (%) RSD (%)
Ref.
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concentration of the quencher. The Stern–Volmer plot of MIPcoated QDs with different amounts of cyphenothrin was shown in Fig. S7. The quenching materials MIP-coated QDs with cyphenothrin satisfied the following equation: F0/F ¼0.0208cq þ1.0766, the correlation coefficient is 0.9993. The linear range of the calibration curve is obtained from 0.1 to 80.0 μmol L 1 with a detection limit of 9.0 nmol L 1. When the concentration is more than 80 μ mol L 1, the fluorescence quenches seriously. At this point, the fluorescence intensity measured is small, and the repeatability of measurement is not satisfactory. At the same time, the detection limit was not influenced by the changing of the amount of template molecule cyphenothrin. Our work demonstrated wide linear range and low detection limit of the prepared fluorescence probe based on MIP-coated QDs. The analytical results obtained by this method were compared with those obtained by the methods reported in the literatures for analyzing cyphenothrin (Table 1). The recovery and precision of this method were comparable or superable to other methods. Fluorescence analysis compared with the chromatography methods, without complicated separation process, has the advantages of simple, convenient and time-saving. At the same time, the selectivity of fluorescence analysis was improved with the using of MIP-coated QDs. 3.9. Application to water sample analysis As a proof of concept, the river water sample was analyzed to evaluate the potential application of the MIP-coated QDs, because river water samples still have some coexisting interference including inorganic salts and even trace amounts of other small organic molecules. The result showed that no response corresponding to cyphenothrin was observed in these water samples. The recovery study was carried out by adding different concentrations of cyphenothrin (0.5, 5.0 and 50.0 μmol L 1) into the water samples. Then the water samples were processed according to the procedures described in section measurements of fluorescent response to cyphenothrin. The quantitative recoveries ranged from 88.5% to 97.1%, and the relative standard deviation (RSD) ranged from 3.1% to 6.2% were obtained. The results showed that the fluorescence probe based on MIP-coated QDs has the potential applicability for cyphenothrin detection in river water with no interfering by the coexisting substance existed in water samples.
4. Conclusion In summary, MIP-coated QDs were synthesized successfully. A novel and convenient method for cyphenothrin analysis has been established based on the fluorescence quenching of MIP-coated QDs. The MIP-coated QDs materials integrate the advantages of the high selectivity of molecular imprinting and strong fluorescence property of QDs. The results indicated that the MIP-coated QDs provided selectivity to cyphenothrin, which was based on the interactions of the size, shape, and functionality of the template. The potential advantage of this method including simple preparation, high stability and low cost will attract more and more investigators for its wide application in the near future.
Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572014EB06) and the National Natural Science Foundation of China (No. 21205010).
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Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.086.
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Quantum dots coated with molecularly imprinted polymer as fluorescence probe for detection of cyphenothrin Xiaohui Ren, Ligang Chen n Department of Chemistry, College of Science, Northeast Forestry University, 26 Hexing Road, Harbin 150040, China
art ic l e i nf o
a b s t r a c t
Article history: Received 15 July 2014 Received in revised form 23 August 2014 Accepted 27 August 2014 Available online 6 September 2014
A newly designed molecularly imprinted polymer (MIP) material was fabricated and successfully utilized as recognition element to develop a quantum dots (QDs) based MIP-coated composite for selective recognition of the template cyphenothrin. The MIP-coated QDs were characterized by fluorescence spectrophotometer, Fourier transform infrared spectroscopy, transmission electron microscope, dynamic light scattering and X-ray powder diffraction. The fluorescence of the coated QDs is quenched on loading the MIP with cyphenothrin, and the effect is much stronger for the MIP than for the non-imprinted polymer, which indicates the MIP could as a recognition template composite. This method can detect down to 9.0 nmol L 1 of cyphenothrin in water, and a linear relationship has been obtained covering the concentration range of 0.1–80.0 μmol L 1. The method has been used in the determination of cyphenothrin in water samples and gave recoveries in the range from 88.5% to 97.1% with relative standard deviations in the range of 3.1–6.2%. The present study provides a new and general strategy to fabricate inorganic–organic MIP-coated QDs with highly selective recognition ability in aqueous media and is desirable for chemical probe application. & 2014 Elsevier B.V. All rights reserved.
Keywords: Molecular imprinting Quantum dots Fluorescence quenching Cyphenothrin
1. Introduction In recent years, semiconductor nanoparticles, also known as quantum dots (QDs) gained great attention in the scientific community (Chaoa et al., 2014; Dezhkam and Zakery, 2014; Li et al., 2014; Costas-Mora et al., 2014; Mohammadi and Bahrami, 2014). QDs hold promise as fluorescent sensors, probes, and tags for various applications, owing to the narrow emission and resistance to fluorescence quenching (Jan’czewski et al., 2014; Hodlur and Rabinal, 2014; Kim et al., 2014; Wang et al., 2013). In particular, QDs are good candidates for analytical assays because they exhibit a Stern–Volmer quenching behavior (Durán-Toro et al., 2014). Many studies in this area have been focused on the development of new methods to synthesize high-quality QDs with a high luminescence quantum yield, excellent photostability, and good biocompatibility (Samadi-maybodi et al., 2014; Chu et al., 2012; Korala et al., 2013; Kang et al., 2010). Different strategies for the synthesis of these nanoparticles have been developed, so that their composition, size, shape, and surface properties can be systematically controlled with an exceptionally high degree (Fan et al., 2014; Hodlur and Rabinal, 2014; Liu et al., 2007; Peng and n
Corresponding author. E-mail address: [email protected] (L. Chen).
http://dx.doi.org/10.1016/j.bios.2014.08.086 0956-5663/& 2014 Elsevier B.V. All rights reserved.
Peng, 2001; Haldar et al., 2012). The design of probe devices based on QDs is a topic of great interest. Molecular imprinting technique (MIT) has been known as an attractive method to develop artificial receptors, which was used to prepare molecularly imprinted polymers (MIPs) (Gao et al., 2014). This technique is based on the in situ co-polymerization of cross-linkers and functional monomers to form complexes with template molecules prior to polymerization (Sun and Fung, 2013). MIP is a polymer which formed in the presence of a molecule (called template). After extraction, then it leaves complementary cavities behind (LütfiYola et al., 2014; Sanagi et al., 2013). The cross-link ratios of polymers are usually in excess of 80% (Cormack and Elorza, 2004). By virtue of the super-cross-linked nature, MIPs can put up with extremely physical and chemical treatment, such as high temperature, pressure, extreme pH, organic solvents, acids and bases. MIPs are cheap, robust materials with a high mechanical, chemical stability and reusable in different applications (Wang et al., 2014; Davoodi et al., 2014). MIPs have been widely exploited in diverse fields such as catalysis (Wulff and Liu, 2012), chemical analysis (Garcia et al., 2011), chromatography (Hsu et al., 2011; Blanco et al., 2012), capillary electrophoresis (De-Maleki et al., 2010), hollow fiber separation (Son and Kobayashi, 2011), membrane separation (Takeda et al., 2009), sensor technology (Moreno-Bondi et al., 2008) and solid-phase extraction (Lucci et al., 2011). It is reasonably believed that molecular imprinting
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technique will be a powerful tool to improve the selectivity of the optical detection. Cyphenothrin (C24H25NO3) is a synthetic pyrethroid pesticide. The structure of cyphenothrin is shown in Fig. S1. Pyrethroid pesticides, as a new bionic pesticide, have some characteristics including broad-spectrum insecticide, high efficacy and highly environmental compatibility (Li and Chen, 2013). In recent years, the use of synthetic pyrethroids derived from chrysanthemic acid is increasingly replacing the use of toxic pesticides such as organochlorine and organophosphorus pesticides (Van-Emon and Chuang, 2012; Yoshida, 2009). Nevertheless, their bioaccumulation and residual toxicity are to be of concern to human food safety (Arvand et al., 2013). Studies prove that pyrethroids could cause seizures and disturb consciousness by affecting the central nervous system of humans when exposed to high concentrations (Nagy et al., 2014; Feo et al., 2013). Therefore, the development of new methods for the determination of pyrethroid in environment samples has captured great interest. Analytical methods such as gas chromatography (GC) (Mezcua et al., 2009), high performance liquid chromatography (HPLC) (Hunter et al., 2010) and liquid chromatography–mass spectrophotometry (LC–MS) (Vonderheide et al., 2009) have been developed for the determination of pyrethroid. However, these methods have deficiencies, such as long manipulation time, high cost and requiring a tedious sample pretreatment. Hence, there is an urgent need to establish a simple and effective method to determine pyrethroid pesticides in environmental samples. In this work, an eco-friendly fluorescence probe, MIP-coated QDs, was successfully fabricated by using cyphenothrin as a template molecule. The MIP-coated QDs were characterized by fluorescence spectrophotometer, Fourier transform infrared spectroscopy (FT-IR), transmission electron microscope (TEM), dynamic light scattering (DLS) and X-ray powder diffraction (XRD). The fluorescence quenching relationship between MIP-coated QDs and cyphenothrin was investigated. Then the MIP-coated QDs were used as fluorescence probe for simple, rapid, and selective detection of cyphenothrin in water samples.
2. Experimental 2.1. Samples and reagents The standard of cyphenothrin was purchased from SigmaAldrich (St. Louis, MO, USA). Zinc sulfate heptahydrate (ZnSO4 7H2O) was purchased from Shuangchuan (Tianjin, China). Manganese (II) chloride tetrahydrate (MnCl2 4H2O) was purchased from Bodi (Tianjin, China). Sodium Sulfide (Na2S 9H2O) was purchased from Kaitong (Tianjin, China). Tetraethoxysilane (TEOS), ethanol, methanol, acrylamide (AM), azoisobutyronitrile (AIBN) and sodium hydroxide were obtained from Kermel (Tianjin, China). Ethylene glycol dimethacrylate (EDGMA) and 3-aminopropyltriethoxysilane (APTES) were purchased from Aladdin (Shanghai, China). All chemicals employed in this study were of analytical grade. High-purity water was obtained from a Milli-Q water system (Millipore, Billerica, MA, USA). The standard stock solution of cyphenothrin was prepared by dissolving cyphenothrin in methanol, and the concentration was 3 mmol L 1. It was stored in a refrigerator at 4 °C. Three river water samples were collected from Harbin (China). All water samples were stored in a refrigerator at 4 °C. 2.2. Apparatus Fourier transform infrared (FT-IR) spectrum of the MIP-coated QDs was recorded with a FT-IR360 spectrometer (Nicolet,
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Madison, WI, USA) using the KBr method. The X-ray diffraction (XRD) spectrum was collected on a Shimadzu XRD-600 diffractometer (Kyoto, Japan) with Cu Kα radiation. The morphology of MIP-coated QDs was observed with a transmission electron microscope (TEM) (H7650, Hitachi, Japan) and a high-resolution transmission electron microscope (HRTEM) (2100F, JEOL, Japan). The size of MIP-coated QDs was observed with a Malvern Zetasizer model Nano ZS90 (Malvern, England). The UV spectrum was recorded on a TU-1901 spectrometer (PERSEE, Beijing, China). Fluorescence intensity studies were carried out at room temperature by using a Perkin-Elmer LS-55 fluorescence spectrometer (Maryland, USA) which was equipped with a plotter unit and a quartz cell. A KQ5200E ultrasonic apparatus (Kunshan Instrument, Kunshan, China) was used for making samples dispersed evenly. 2.3. Synthesis of MIP-coated QDs The synthesis process of MIP-coated QDs involves two major steps: the first step is the synthesis of Mn-doped ZnS QDs, and the second one is the surface imprinting of polymers onto modified Mn-doped ZnS QDs. The synthesis method of Mn-doped ZnS QDs was shown as follows. At first, 25 mmol of ZnSO4 7H2O, 2 mmol of MnCl2 4H2O and 80 mL of water were kept stirring for 20 min under the protection of nitrogen gas. Then, 10 mL, 25 mmol Na2S 9H2O solution was added drop into the mixture (Liu et al., 2010). After being stirred for 30 min, 4 mL of TEOS and 5 mL of APTES were added for modifying the Mn-doped ZnS QDs. Finally, the modified Mn-doped ZnS QDs was obtained after centrifugation and being washed with ethanol three times. The synthesis method of MIP-coated QDs was carried out as follows. Cyphenothrin (1 mmol), AM (4 mmol), and modified Mndoped ZnS QDs obtained from above procedure were dispersed into 150 mL ethanol. Then AIBN (0.1 g) and the cross-linker EGDMA (10 mmol) were added. The mixture was heated at 60 °C and stirred at 300 rpm in a water bath for 24 h. Then the template was removed by Soxhlet extraction with methanol:acetic acid (19:1, v/v) until no template molecule was detected. After dried in vacuum, MIP-coated QDs were obtained. The non-imprinted polymer-coated Mn-doped ZnS QDs (NIPcoated QDs) were prepared using the same procedure without addition of the template cyphenothrin. 2.4. Fluorescence analysis Fluorescence analysis was performed on a Perkin-Elmer LS-55 fluorescence spectrometer. The spectra were recorded in the wavelength range of 500–700 nm upon excitation at 330 nm. Slit widths (10 nm), scan speed (200 nm min 1) and excitation voltage (750 V) were kept constant within each data set and each spectrum was the average of three scans. Quartz cell (1 cm path length) was used for all measurements. 2.5. Measurements of fluorescent response to cyphenothrin Thirty milligram MIP-coated QDs or NIP-coated QDs was added into a centrifuge tube, and then the given concentration of cyphenothrin solution was added. The constant volume was 30 mL. The fluorescence intensity was measured after fully mixing. The river water samples were applied to evaluate the practical application. The water samples were filtered by 220 nm microporous membrane. The recovery study was carried out by spiking certain volume of cyphenothrin solution into water samples.
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3. Results and discussion Zinc sulfide (ZnS) is particularly suitable for use as a host material for a large variety of dopants because of its wide band gap. The Mn-doped ZnS QDs is obtained by bivalent manganese (Mn2 þ ) doping into ZnS semi-conductive nanocrystals. Just as demonstrated in the literature (Zhao et al., 2012), the Mn-doped ZnS QDs used herein exhibit novel optical properties, including wide excitation, as well as narrow and symmetric emission spectra. Different Mn-doped ZnS QDs were prepared by changing the ZnSO4/MnCl2 molar ratios (50:1, 50:2, 50:4; and 50:8). The fluorescence emission spectrum is shown in Fig. S2. The highest fluorescence intensity was obtained with the ZnSO4/MnCl2 molar ratio at 50:4. Both increasing and decreasing the molar ratio, the fluorescence intensity was decreased. Therefore, the 50:4 of ZnSO4/MnCl2 molar ratio was used. The design of our MIP-coated QDs sensing material is based on the fact that the Mn-doped ZnS QDs act as probe for recognition signal amplification and optical readout, while the MIP shell provides analyte selectivity and prevents interfering molecules from coming into contact with the QDs. As illustrated in Fig. 1, the QDs were coated with silica by hydrolysis of TEOS. Based on the fact that the stability of QDs is improved and the surface defects of QDs are reduced. APTES was connected to the silica by the silanization reaction. The surface of the QDs was modified with amino group act as assistant monomers to drive template molecules into the formed imprinted polymer shells during imprinting polymerization. Finally, the MIPs shell was fabricated on the surface of the amino modified QDs through self-assembled of functional monomer (AM), cross-linking agent (EGDMA) and template (cyphenothrin). After removing the template molecules, recognition cavities complementary to the template molecule in shape, size, and chemical functionality were formed in the crosslinked polymer matrix.
face-centered cubic structure. The XRD patterns of MIP-coated QDs were assigned to the (111), (220) and (311) reflections of the cubic phase Mn-doped ZnS, further suggesting the successfully fabrication of MIP-coated QDs. 3.2. Fluorescence study The fluorescence intensity of the MIP-coated QDs was recorded by varying the excitation wavelength from 300 to 350 nm. The weak blue peak around 444 nm was generated by the defect related to the emission of the ZnS QDs. The strong orange peak around 595 nm could be attributed to the 4T1-6A1 transition of the Mn2 þ impurity. The maximum emission intensity at 595 nm was observed with 330 nm as the excitation wavelength. The orange fluorescence was very strong and the peak was sharp, indicating that the sizes of MIP-coated QDs were very homogeneous. In the current work, the template cyphenothrin was entrapped in the polymer matrixes through non-covalent binding. For further elucidate the high selectivity of the MIP-coated QDs in aqueous media, we prepared the NIP-coated QDs. As shown in Fig. 3, the fluorescence intensity of MIP-coated QDs was relatively weak (spectrum c) before the removal of templates. While after Soxhlet extraction with methanol: acetic acid (19:1, v/v), the fluorescence intensity of the MIP-coated QDs was restored dramatically. However, no difference in the shape and position of the emission spectrum was observed (spectrum b). The fluorescence intensity was restored almost to that of the NIP-coated QDs (spectrum a), which indicated that the templates were removed completely from the recognition cavities in the MIP-coated QDs. It suggests that the MIP-coated QDs actually facilitate the application for the rapid and simple quantification of analytes in aqueous media without preconcentration. 3.3. The effect of pH
3.1. Characterization of the MIP-coated QDs The FT-IR spectrum of MIP-coated QDs is shown in Fig. 2a. The strong and broad peak around 1100 cm 1 indicates Si–O–Si asymmetric stretching. Other observed bands about 458 and 783 cm 1 also show the Si–O vibrations. The presence of the bands around 1624 cm 1 was the C¼ C absorption peak. The peak around 2936 cm 1 was C–H band. The bands at 3422 cm 1 and 1560 cm 1 were N–H stretching. All those bands showed that the MIP was grafted on the surface of the QDs. TEM and HRTEM of MIP-coated QDs were shown in Fig. 2b and c, respectively. The DLS measurement results in Fig. S3 showed that the mean size of QDs and MIP-coated QDs are 7.9 and 36.3 nm, respectively. The material of the MIP-coated QDs was investigated with XRD. Fig. 2d showed that the as-synthesized MIP-coated QDs had a
As we know, the orange fluorescence of the ZnS:Mn2 þ nanocrystals is the Mn2 þ typical emission located at 595 nm. The pH effect of MIP-coated QDs without cyphenothrin was investigated. The effect of pH in a range between 4 and 12 was researched. As shown in Fig. S4, the fluorescence intensity of MIP-coated QDs in the interval 4.0–10.0 was considerably stable. When pH is over 11, the fluorescence intensity is quenched seriously. 3.4. The stable fluorescence emission measurement of MIP-coated QDs The fluorescence emission of doped ions was chosen for detection because that is more stable and controllable and has a higher quantum yield than the defect one. The relative standard deviation (RSD) of 0.3% was obtained by 11 repeated detections of
Fig. 1. The preparation process of MIP-coated QDs.
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the fluorescence insensitivity in the MIP-coated QDs aqueous solution every 5 or 10 min. The result shown in Fig. S5 indicates the stable emission of the QDs. The main reason for the stable emission is that inner Mn2 þ is protected by the amorphous silica shell. 3.5. The effect of incubation time The influence of incubation time on the fluorescence intensity investigated at different time scales was shown in Fig. 4. It was found that the mixture showed a rapid decrease in fluorescence intensity after the cyphenothrin added. The reaction was completed within 10 min, indicating that certain time was needed to
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complete the interaction, and the fluorescence intensity remained stable for at least 1.5 h. Therefore, the experiments were carried out after 10 min and the time scale of 10 min was also adopted in the following experiments. 3.6. MIP- and NIP-coated QDs with template molecule of different concentrations In this test, our aim is to demonstrate the recognition ability of the MIP-coated QDs versus that of the NIP-coated QDs (Fig. 5). Fig. 5a shows the spectral response of MIP-coated QDs with template molecule cyphenothrin at different concentrations. The prepared MIP-coated QDs had an emission at 595 nm. The fluorescence intensity of the MIP-coated QDs was quenched gradually with the increasing concentration of cyphenothrin. Generally, the
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Fig. 5. Fluorescence emission spectra of MIP-coated QDs (a) and NIP-coated QDs (b) with addition of the indicated concentrations of cyphenothrin. Fluorescence emission spectra of MIP-coated QDs (c) and NIP-coated QDs (d) with addition of the indicated concentrations of beta-cyfluthrin.
fluorescence quenching depends on the absorptive affinity of the particles with the template. The fluorescence intensity quenching was mainly achieved by the affinity of the imprinted cavities with the template due to the specific interactions. It was clearly seen that the decrease of fluorescence intensity of the MIP-coated QDs was much larger than that of the NIP-coated QDs (Fig. 5b) by the template molecule cyphenothrin at the same concentration. The imprinting factor IF, which is the ratio of the △MIP/△NIP. It was used to evaluate the selectivity of the materials. Under optimum conditions, the IF was 3.0, which indicated that the MIP-coated QDs can greatly enhance the quenching efficiency of fluorescence, enlarging the spectral sensitivity of the MIP-coated QDs to the template molecule cyphenothrin. 3.7. Selective adsorption on MIP-coated QDs The beta-cyfluthrin was involved to evaluate the selectivity adsorption of the MIP-coated QDs. The chemical structure of betacyfluthrin was shown in Fig. S1. The relationships obtained for analogs beta-cyfluthrin interacting with MIP- and NIP-coated QDs are given in Fig. 5c and d, respectively. It can be seen that the fluorescence intensity quenches were little affected by the increase of beta-cyfluthrin. Herein, because of the different structure of the analogs beta-cyfluthrin, they could not get into the recognition cavities of MIP-coated QDs. The results suggested that MIPcoated QDs were specific to cyphenothrin but nonspecific to betacyfluthrin.
3.8. Fluorescence quenching analysis After adding the template cyphenothrin, there will be an interaction between the template molecule and the MIP-coated QDs, which is a main reason for the fluorescence quenching. We suggest a charge transfer from QDs to cyphenothrin is responsible for fluorescence quenching of MIP-coated QDs. Such charge transfer mechanism has also been reported by Tu et al. (2008) and Wang et al. (2009). The UV absorption band of cyphenothrin is close to the band gap by the absorption spectra of MIP-coated QDs (Fig. S6). The charges of the conductive bands of the QDs can transfer to the lowest unoccupied molecular orbital of cyphenothrin. We can exclude energy transfer as a possible mechanism for fluorescence quenching because there is no spectral overlap between the absorption spectra of cyphenothrin and the emission spectrum of the MIP-coated QDs (Fig. S6). Typical fluorescence quenching of the MIP-coated QDs materials from 0.1 to 80.0 μmol L 1 cyphenothrin was investigated. It demonstrated that the probe showed obvious responses to different concentrations of cyphenothrin, which was very effective and suitable for practical application. The fluorescence quenching in this system can be quantified by the Stern–Volmer equation as follows:
F0/F = 1 + Ksv cq where F0 is the initial fluorescence intensity in the absence of quencher, F is the fluorescence intensity in the presence of analyte, Ksv is the quenching constant of the quencher, and cq is the
This work 88.5–97.1 9.0 Water Fluorescence spectrophotometer
Solid matrix dispersion-size exclusion chromatography clean-up Vigorous shaking-centrifugal extraction Ultrasound assisted extraction-clean up with magnetic molecularly imprinted polymer Fluorescence probe with molecularly imprinted polymer coated quantum dots
Matrix solid phase dispersion
Solid phase microextraction
Milk Blood Fruit
1 102–8 104
60–119 95–97 82.4–101.7 667 0.3 33.1 0.5 103–5.3 103 2.7–2.7 103 66.7–1.3 104
3.1–6.2
Fernandez-Alvarez et al. (2009) 2.5–14.4 Muccio et al. (1997) 0.8–4.3 Ramesh and Ravi (2004) 2.6–5.6 Ma and Chen. (2014) 1.5–7.3 78–86 0.2 11.0–53.0 Cattle feed
40.5–111 0.1 10 3 5.8 10 3 Water
2.8 10 3
3.9–11.9
Fernandez-Alvarez et al. (2008) Casas et al. (2006) o 15 69–118 0.83 Bovine milk 5–267
GC-microelectron capture detector GC-microelectron capture detector GC-microelectron capture detector GC-electron capture detector GC–MS detector HPLC–UV detector Solid phase microextraction
Samples Detection technique Pretreatment method
Table 1 Comparison of this method with other methods used in the literatures.
Analytical ranges (nmol L 1) LODs (nmol L 1) Recovery (%) RSD (%)
Ref.
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concentration of the quencher. The Stern–Volmer plot of MIPcoated QDs with different amounts of cyphenothrin was shown in Fig. S7. The quenching materials MIP-coated QDs with cyphenothrin satisfied the following equation: F0/F ¼0.0208cq þ1.0766, the correlation coefficient is 0.9993. The linear range of the calibration curve is obtained from 0.1 to 80.0 μmol L 1 with a detection limit of 9.0 nmol L 1. When the concentration is more than 80 μ mol L 1, the fluorescence quenches seriously. At this point, the fluorescence intensity measured is small, and the repeatability of measurement is not satisfactory. At the same time, the detection limit was not influenced by the changing of the amount of template molecule cyphenothrin. Our work demonstrated wide linear range and low detection limit of the prepared fluorescence probe based on MIP-coated QDs. The analytical results obtained by this method were compared with those obtained by the methods reported in the literatures for analyzing cyphenothrin (Table 1). The recovery and precision of this method were comparable or superable to other methods. Fluorescence analysis compared with the chromatography methods, without complicated separation process, has the advantages of simple, convenient and time-saving. At the same time, the selectivity of fluorescence analysis was improved with the using of MIP-coated QDs. 3.9. Application to water sample analysis As a proof of concept, the river water sample was analyzed to evaluate the potential application of the MIP-coated QDs, because river water samples still have some coexisting interference including inorganic salts and even trace amounts of other small organic molecules. The result showed that no response corresponding to cyphenothrin was observed in these water samples. The recovery study was carried out by adding different concentrations of cyphenothrin (0.5, 5.0 and 50.0 μmol L 1) into the water samples. Then the water samples were processed according to the procedures described in section measurements of fluorescent response to cyphenothrin. The quantitative recoveries ranged from 88.5% to 97.1%, and the relative standard deviation (RSD) ranged from 3.1% to 6.2% were obtained. The results showed that the fluorescence probe based on MIP-coated QDs has the potential applicability for cyphenothrin detection in river water with no interfering by the coexisting substance existed in water samples.
4. Conclusion In summary, MIP-coated QDs were synthesized successfully. A novel and convenient method for cyphenothrin analysis has been established based on the fluorescence quenching of MIP-coated QDs. The MIP-coated QDs materials integrate the advantages of the high selectivity of molecular imprinting and strong fluorescence property of QDs. The results indicated that the MIP-coated QDs provided selectivity to cyphenothrin, which was based on the interactions of the size, shape, and functionality of the template. The potential advantage of this method including simple preparation, high stability and low cost will attract more and more investigators for its wide application in the near future.
Acknowledgments This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572014EB06) and the National Natural Science Foundation of China (No. 21205010).
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Appendix A. Supplementary information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.08.086.
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