Biosensors and Bioelectronics ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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A facile and sensitive detection of organophosphorus chemicals by rapid aggregation of gold nanoparticles using organic compounds Myung Sun Kim 1, Gi Wook Kim 1, Tae Jung Park n Department of Chemistry, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 6 June 2014 Received in revised form 27 August 2014 Accepted 28 August 2014
Organophosphorus (OP) chemicals are highly effective insecticides and germicides, and are the most widely used in agriculture. Unfortunately, OP compounds are some of the most toxic substances to humans, even at very low doses. Because detecting OP residues in agricultural products is essential, simple, sensitive, and particularly rapid on-site detection methods are required. Gold nanoparticles (AuNPs) have been used as signal-enhancing detection probes in the field of biosensors due to their sizedependent optical properties. When imidazole was added to AuNPs mixed with OP compounds, the AuNPs was aggregated and their color changed to purple. This caused the appearance of a new peak at 660–670 nm, which could be measured within approximately 30 s. Therefore, this method allows the detection of OP compounds, including diazinon, iprobenfos, and edifenphos, on-site at part-per-billion (ppb) concentrations, and also affords a straightforward method. Furthermore, the method was successfully applied in the determination of OP compound in a real sample (river water) with satisfactory results. & 2014 Elsevier B.V. All rights reserved.
Keywords: Organophosphorus chemical detection AuNPs Imidazole Pesticide
1. Introduction In agriculture, farmers use numerous agrichemicals to protect crops and seeds before and after harvesting. Agrichemicals is a broad term used that encompasses the organic toxic compounds used to control insects, bacteria, weeds, nematodes, rodents and other pests (Sassolas et al., 2012). Among these, organophosphorus (OP) chemicals are used worldwide, and so large amounts of OP residue could cause contamination by accumulating in the environment including the air, soil, water, and agricultural products. This could eventually lead to serious health concerns for humans even after exposure to only very low concentrations (Zhang et al., 2014; Yang et al., 1995). The high toxicity of OP compounds is caused by their ability to inhibit acetylcholinesterase (AChE), which is an important enzyme that hydrolyzes acetylcholine. Inhibiting AChE activity allows acetylcholine to accumulate in cholinergic clefts, which overstimulates both the peripheral and central cholinergic nervous systems, and has fatal consequences (Yadav et al., 2012). The symptoms of OP poisoning include headache, dizziness, salivation, lacrimation, sweating, vomiting, diarrhea, abrupt tremor, lung edema, coma, and even death from respiratory or cardiac failure (Meng et al., 2013). The development n
Corresponding author. Tel.: þ 82 2 820 5220; fax: þ 82 2 825 4736. E-mail address: [email protected] (T.J. Park). 1 These two authors contributed equally to this paper.
of a sensitive and inexpensive diagnosis tool for environmental and biological monitoring is a current research area to facilitate the removal of excess OP compounds, to prevent disease, and protect the environment. Moreover, it is essential to develop a method that allows the on-site and real-time detection of OP residues rapidly and easily. Several methods to detect OP compounds have been developed over the past decade. For example, liquid or gas chromatography coupled to mass spectrometry, electrochemical analysis, fluorescent bioprobes, and enzyme-linked immunosorbent assays (ELISAs) have been used to detect OP compounds (Mehrvar and Abdi, 2004; Rao et al., 2002; Trojanowicz, 2009; Shi et al., 2006; Jeanty and Marty, 1998; Chen et al., 2009; Meng et al., 2013). These methods have high selectivity, adequate sensitivity, and reliability; however, most are also associated with disadvantages, such as high costs, the need for sophisticated instruments, the requirement for highly qualified and trained operators possessing master professional skills, tedious sample pretreatment, and expensive biomolecular reagents. Therefore, these methods are not suitable for on-site detection in most settings (Zhang et al., 2014; Yi et al., 2013). AuNPs-based colorimetric assays are effective approaches for quantifying many types of analytes without the need for complex equipment because molecular alterations can cause color changes in the AuNPs (Jiao et al., 2014; Sener et al., 2014; Feng et al., 2013; Wang et al., 2013; Zhang et al., 2010; Li et al., 2010; Huang et al., 2005; Liu et al., 2010; Wang et al., 2006). The color of AuNPs is
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size- and shape-dependent and highly sensitive to aggregation states. It can be easily measured by a UV/vis spectrophotometer (Feng et al., 2013). In general, well-dispersed AuNPs are colored red, whereas aggregated AuNPs are purple- or blue-colored. The AuNPs color change can be measured on the basis of the UV/vis absorption spectrum via a shift in the maximum peak and/or the appearence of a second peak in the visible region (Jiao et al., 2014; Sener et al., 2014; Feng et al., 2013; Wang et al., 2013; Zhang et al., 2010; Li et al., 2010; Huang et al., 2005; Liu et al., 2010; Wang et al., 2006; Chegel et al., 2012). Imidazole has been reported as an alkaloid organic compound in relation to conjugation potential. Due to the coexist of position 1 protonated nitrogen of pKa 14.0 and position 3 nitrogen of pKa 6.5, imidazole has an amphoteric property with dual-function as both an acid and a base (Walba and Isensee, 1961; Holze, 1993). The property, through the formation of weak complexs between N-ligands and O-ligands (Lorenzo et al., 1992), enables the imidazole to serve as precursors for the adsorption of other molecules onto metal surfaces (Xue et al., 1988; Wang et al., 2002; Cao et al., 2003). Furthermore, the strong affinity between imidazole and AuNPs induces the aggregation of the AuNPs, which results in a shift in the surface plasmon resonance into the near-infrared (NIR) wavelengths (Glauco et al., 2006). Therefore, the imidazole can be used as an aggregation promotor of AuNPs. For the optical detection of OP compounds, we herein developed a simple and rapid biosensing method based on the aggregation of AuNPs in a two-step process. The proposed sensing strategy for the optical assay to detect OP compounds is shown in Scheme 1. Different concentrations of diazinon were mixed with the AuNPs, followed by addition of imidazole. Finally, the color of the AuNP suspension was immediately changed from red to dark blue (or purple) because of the aggregation of the AuNPs. We selected diazinon [O,O-diethyl O-(2-isopropyl-4-methyl-6-pyrimiinyl)phosphorothioate] as the model OP, because it is a moderately persistent pesticide that is widly used in agriculture (Banaee et al., 2011). To verify this novel bioassay method, two additional OP compounds, iprobenfos and edifenphos, were tested at the concentration range of 0–10 ppm. This strategy for OP detection
was performed without complex modification processes, enzyme immobilization, and/or the addition of salt. The assay results could be measured as soon as the reaction finished, which was within a few seconds.
2. Experimental 2.1. Materials Gold(III) chloride hydrate (HAuCl4), diazinon, iprobenfos, edifenphos, and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, MO). Trisodium citrate dehydrate, imidazole were manufactured by Bio-Basic (Ontario, Canada). Methanol was obtained from Merck chemicals (Darmstadt, Germany). Milli-Q grade distilled (DI) water (18.2 MΩ cm, Millipore, Billerica, MA) was used in all experiments. 2.2. Synthesis of citrate-stabilized AuNPs (Cit-AuNPs) Dispersed AuNPs were prepared using a citrate reduction of HAuCl4 (Kim et al., 2011). One hundred milliliters of DI water containing 1 mM HAuCl4 was refluxed during stirring. When the HAuCl4 solution started to boil, 10 mL of 38.8 mM trisodium citrate dihydrate was added, and the reaction was continued for 15 min to reduce the salt content. The color of the solution changed immediately to dark blue and then to dark red, indicating the formation of dispersed Cit-AuNPs. After a 15-min reaction, the solution was cooled while being stirred at room temperature, and was then stored at 4 °C. The synthesis of Cit-AuNPs was confirmed by recording UV/vis spectrum data and analysis using FE-TEM (Tecnai G2 F30 S-Twin, FEI, Hillsboro, OR). 2.3. Preparation of imidazole and OP solution Imidazole was prepared by diluting the concentrated stocks in PBS solution (pH 7.4). The various concentrations of OP compounds were prepared by dissolving 10 mL of OP in 10 mL of 10%
Scheme 1. Schematic illustration of optical assay used to detect OP chemicals.
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(v/v) methanol to obtain a 1000 ppm (parts-per-million) solution. This solution was further diluted using 10% (v/v) methanol to reach the appropriate concentrations. To determine a real sample, various concentrations (0, 0.2, 0.4, 0.6, 0.8 and 1.0 ppm) of diazinon was prepared by dissolving 10 mL of diazinon in 10 mL of river water (1000 ppm). 2.4. Characterizations of AuNPs using zeta potential For zeta potential and dynamic light scattering (DLS) analysis of the AuNPs, AuNP–diazinon mixtures with and without imidazole were applied to a Zeta Potential & Particle size analyzer (ELSZ1000, Otsuka, Japan). 2.5. Quantification of OP compounds Fifty microliters of Cit-AuNPs were added sequentially to 100 mL of OP solution and 50 mL of 0.3 mM imidazole solution in sequence on 96-well plates. (AuNP:OP:Imidazole volumetric ratio of 1:2:1). UV/vis spectroscopy and fluorescence analysis were then sequentially performed at 400–700 nm using a Synergy H1 Hybrid Reader (BioTek, Winooski, VT). Standard OP chemicals after diluting step was filtrated with syringe filter of 0.2-μm. High-performance liquid chromatography (HPLC, Agilent 1260, Santa Clara, CA) was then used for quantifying the OP chemicals with a C18 column (10-cm length and inside diameter of 4.6 mm, Eclipse Plus, Agilent). The analysis was performed at 40 °C. The UV absorption spectrometer was used as an HPLC detector at a detection wavelength of 254 nm. The mobile phase consisted of acetonitrile–water 65:35 (v/v) that were filtered separately before mixing. A flow rate of 1 mL/min was selected.
3. Results and discussion 3.1. Analytical determination of the novel method The UV/vis spectra of each sample were measured to reveal a possibility of the proposed method. The Cit-AuNPs were dispersed well as shown in Fig. S1 and exhibited typical extinction spectra for such particles, with a maximum peak at 520 nm. After the mixed with 1-ppm diazinon, the maximum extinction was mostly unaffected. In case of the high concentrations of diazinon from 10-ppm, AuNPs were aggregated with diazinon concentration increases (Fig. S2), however, this result was unsatisfactory for a sensitive detection of OP compounds. To facilitate the AuNP aggregation even at low concentration of diazinon, imidazole was introduced as an aggregation promotor. As shown in Fig. 1, when imidazole was added into the AuNPs and 1-ppm diazinon– AuNP mixture, this result leads to the appearance of a second broad peak between 600 and 700 nm in the absorbance spectrum, which indicated that the AuNPs were aggregated even at low concentration of diazinon by introducing imidazole, and more intense peak was appeared in the presence of diazinon. Therefore, we used imidazole to promote the AuNP aggregation for sensitive detection of OP compounds as illustrated in Scheme 1. In order to confirm the optimal assay conditions, several concentrations of the imidazole were fixed at a specific volumetric ratio. As shown in Fig. S3, 0.3 mM of imidazole resulted in a welldispersed spectrum and a higher slope according to the concentration of diazinon. Therefore, we used 0.3 mM concentration of imidazole for further subsequent experiments. Next, the zeta potential was measured to investigate changes of the surface charge of the AuNPs. Before the addition of OP compounds or imidazole, the surface charge on AuNPs was negative because of the presence of citrate ions. In contrast, the
Fig. 1. Optical response of the assay. UV/vis spectrum of the AuNP solutions revealed that a color change occurs in the presence of imidazole (0.3 mM) and diazinon (1.0 ppm) with imidazole.
charge of aggregated AuNPs was neutralized, to some extent, after the addition of imidazole. The zeta potential values of welldispersed AuNPs and AuNPs pretreated with 1 ppm diazinon were 34.98 and 36.40 mV, respectively, whereas those of imidazolestimulated aggregates in the absence and presence of diazinon were 12.18 mV and 11.81, respectively, which were different to that of well-dispersed AuNPs (Fig. S4a). These observations were supported by DLS data (Fig. S4b). The average hydrodynamic diameters of well-dispersed AuNPs and AuNPs pretreated with 1 ppm diazinon were 13.4 and 13.2 nm, respectively. However, the addition of imidazole to the AuNPs and diazinon–AuNP mixture increased the diameter of the aggregates to 201.7 and 235.1 nm, respectively. The DLS results corresponding to UV/vis spectra suggest that the current method is practicable to detect the diazinon. 3.2. Detection of the OP compounds, diazinon, iprobenfos, and edifenphos To assess the sensitivity of the assay, we analyzed various concentrations of diazinon solutions ranging from 10.0 ppb to 10.0 ppm (0, 0.01, 0.05, 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1.0, 3.0, 5.0, 7.0 and 10.0 ppm). As the concentration of diazinon increased, the absorption at 520 nm and 600–700 nm increased gradually. The intensity of the maximum absorption of the new peak at 670 nm was comparable to that of free AuNPs at 520 nm (Fig. 2a). The plots of A670 (absorbance at 670 nm) value vs. various concentrations of diazinon. Fig. 2b shows the A670 value gradually with increasing concentrations of diazinon. The maximum A670 value was reached with a concentration of 3.0 ppm diazinon, and further increases in the concentration of diazinon had no effect on the A670 value. The A670 values were linear with diazinon concentrations from 0 to 1.0 ppm, with a linear regression correlation coefficient of 0.985. The lowest detectable diazinon concentration using this assay was 53.3 ppb according to a 3s rule calculation (LOD ¼3 standard deviation corresponding to the blank sample/slope of calibration curve) (Chen et al., 2010). We confirmed the sensitivity of the sensing platform using two different germicidal OP compounds, iprobenfos and edifenphos, using the same experimental procedures as those used for diazinon. Fig. S5a and b shows the changes in absorbance with increasing concentrations of iprobenfos, and a plot of A670 values vs. various concentrations (0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 3.0, 5.0, 7.0 and 10.0 ppm) of iprobenfos, respectively. As the concentration of iprobenfos increased, the absorption peak between 600 and 700 nm increased gradually until the
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Fig. 2. Sensitivity of the assay for detecting diazinon. (a) The changes in absorbance with increasing concentrations of diazinon from 0 to 10.0 ppm. (b) Plots of the absorbance at 670 nm vs. diazinon concentration. A670 indicates the absorption band that appeared at 670 nm. The standard deviations of measurements were calculated from the results of three independent experiments.
intensity of the maximum absorption peak at 670 nm was comparable to that of free AuNPs at 520 nm. As the concentration of iprobenfos increased, the A670 value also increased gradually (Fig. S5b). The maximum A670 value was reached at a concentration of 5.0 ppm iprobenfos. With iprobenfos concentrations ranging from 0 to 1.0 ppm, the A670 values were highly linear, with a linear regression correlation coefficient of 0.994. The lowest iprobenfos concentration that could be detected using this assay was 53.6 ppb. Fig. S5c and d shows that the absorbance intensity increased progressively with increasing edifenphos concentrations, leading to an increased A670 value. A similar trend was observed to that seen with diazinon and iprobenfos. We tested edifenphos solutions with concentrations between 10.0 ppb and 10.0 ppm (0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 3.0, 5.0, 7.0 and 10.0 ppm). The maximum A670 value was reached with a concentration of 5.0 ppm edifenphos. The A670 values were highly linear with edifenphos concentrations from 0 to 1.0 ppm, with a linear regression correlation coefficient of 0.984. The lowest detectable concentration of edifenphos using this assay was 27.9 ppb (Fig. S5d). The lowest detectable concentrations for diazinon, iprobenfos and edifenphos are much lower than the maximum residue limits (MRLs) as reported in Korean Pesticide Residue Database (Ministry of Food and Drug Safety, 2013, http:// fse.foodnara.go.kr/residue/RS/jsp/menu_02_01_01.jsp). MRLs are 0.1, 0.2, and 0.2 ppm for diazinon, iprobenfos, and edifenphos, respectively.
compounds (benzene, phenol, toluene, xylene, dichlorobenzene, and phosphoric acid) with similar chemical structures were tested using the same experimental conditions as those used for the diazinon assay. UV/vis absorption spectra showed that none of these organic compounds affected the absorbance at 670 nm and interfered with the assay at concentrations of 1.0 ppm (Fig. 3). This result suggests that the absorbance signals observed with diazinon were generated by specific interactions between the AuNP–imidazole complex and diazinon. Therefore, this optical assay is highly selective for diazinon.
3.3. Effect of pH
3.5. Comparison of the proposed assay with liquid chromatography
We next assessed whether pH could affect the reaction between AuNPs, diazinon, and imidazole. Specifically, we investigated the effects of pHs from 3.4 to 10.4 while maintaining a constant imidazole concentration (Fig. S6). The difference value of A670 between 1 ppm and 0 ppm increased gradually with increasing pH at 1 ppm diazinon. The result shows that this assay was saturated toward pH changes from 7.4 to 10.4. The highest absorption (A670) was obtained at pH 8.4. However, A670 value at pH 7.4 was comparable to that at pH 8.4. Thus, pH 7.4 was chosen as the optimal reaction pH in the experiments to avoid the inconvenience of increasing to pH 8.4.
The proposed method was used to quantify the concentrations of diazinon, as shown in Table 1. To verify the accuracy of the proposed method, HPLC was used to assay several diazinon samples. The results obtained by the method were high accurate than those achieved using the HPLC method at various concentrations of diazinon. In addition, the method is more rapid, straightforward, and acceptable, with mean recovery of 100.1%. Therefore, it is obvious that the proposed method has great potentials for analyzing OP compounds in practical field samples.
3.4. Selectivity of the biosensing system
To evaluate the possibility of the detection of real sample, standard curve was prepared in river water, which was applied to determine the concentrations of diazinon (0, 0.2, 0.4, 0.6, 0.8 and 1.0 ppm) (Fig. 4). Detection limit of diazinon in river water was
Selectivity is another critical aspect of a potential assay system. To evaluate the selectivity of the novel model, six organic
Fig. 3. Selectivity of the assay. Benzene, phenol, toluene, xylene, dichlorobenzene, phosphoric acid, and diazinon were each tested at the concentrations of 1.0 ppm.
3.6. Determination of diazinon in river water
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Table 1 Analytical results of the determination of diazinon sample using the proposed optical sensor and HPLC. Diazinon conc. added (ppm)
In deionized water mean 7 SD (ppm)a
Recovery (%) In river water mean7 SD (ppm)a
Recovery (%) HPLC mean7 SD (ppm)a
0.080 0.170 0.300 Average
0.080 7 0.024 0.1737 0.056 0.295 7 0.042 –
100.4 101.6 98.3 100.1
98.5 86.3 113.5 99.4
a
0.0797 0.012 0.1467 0.012 0.340 70.001 –
0.082 7 0.002 0.149 7 0.004 0.263 7 0.064 –
Recovery (%)
96.2 87.6 87.7 90.5
The standard deviations (SD) of measurements are calculated from three independent experiments.
Acknowledgments This work was supported by the Advanced Production Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (312066-3) and a grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare (HI13C0862), Republic of Korea.
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.2014.08.073.
References Fig. 4. Plots of A670 values vs. diazinon concentrations in river water. The standard deviations of the samples were calculated from the results 3 times.
calculated at 36.3 ppb by 3s rule. Table 1 shows the results for analysis of spiked river water samples. A recovery of 86.3% and 113.5% was observed in river water samples, which indicated the accuracy and reliability of the proposed method for the determination of OP pesticide in water samples. The results demostrate that the method have been successfully applied to water samples and can be applied to other samples.
4. Conclusion We successfully developed a simple, rapid, and highly sensitive assay for the detection of OP chemicals using imidazole to promote the aggregation of AuNPs. The assay uses common CitAuNPs without the need for tedious surface modification steps, such as DNA oligomers and peptides, or the addition of an excess amount of salt, making the method practical and cost-effective. Data revealed that the detection limits of the method were 53.3 ppb for diazinon, 53.6 ppb for iprobenfos, and 27.9 ppb for edifenphos, respectively. This assay strategy gives highly linear data with concentrations ranging from 10.0 ppb to 1.0 ppm. Furthermore, the novel method has provided an effective reliable detection of diazinon in a real sample with a detection limit of 36.3 ppb. In addition, the response of the optical assay was very rapid, occurring within 30 s, and all analyses including the sample preparation steps could be completed within a few minutes. We anticipate that this method will facilitate the detection of many OP compounds in the field. Importantly, this is the first study to reveal that AuNP–imidazole complexes could be used to detect OP compounds. We propose that the novel method has advantages over several current AuNPs-based methods, and that its use in practical applications is feasible.
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A facile and sensitive detection of organophosphorus chemicals by rapid aggregation of gold nanoparticles using organic compounds Myung Sun Kim 1, Gi Wook Kim 1, Tae Jung Park n Department of Chemistry, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 156-756, Republic of Korea
art ic l e i nf o
a b s t r a c t
Article history: Received 6 June 2014 Received in revised form 27 August 2014 Accepted 28 August 2014
Organophosphorus (OP) chemicals are highly effective insecticides and germicides, and are the most widely used in agriculture. Unfortunately, OP compounds are some of the most toxic substances to humans, even at very low doses. Because detecting OP residues in agricultural products is essential, simple, sensitive, and particularly rapid on-site detection methods are required. Gold nanoparticles (AuNPs) have been used as signal-enhancing detection probes in the field of biosensors due to their sizedependent optical properties. When imidazole was added to AuNPs mixed with OP compounds, the AuNPs was aggregated and their color changed to purple. This caused the appearance of a new peak at 660–670 nm, which could be measured within approximately 30 s. Therefore, this method allows the detection of OP compounds, including diazinon, iprobenfos, and edifenphos, on-site at part-per-billion (ppb) concentrations, and also affords a straightforward method. Furthermore, the method was successfully applied in the determination of OP compound in a real sample (river water) with satisfactory results. & 2014 Elsevier B.V. All rights reserved.
Keywords: Organophosphorus chemical detection AuNPs Imidazole Pesticide
1. Introduction In agriculture, farmers use numerous agrichemicals to protect crops and seeds before and after harvesting. Agrichemicals is a broad term used that encompasses the organic toxic compounds used to control insects, bacteria, weeds, nematodes, rodents and other pests (Sassolas et al., 2012). Among these, organophosphorus (OP) chemicals are used worldwide, and so large amounts of OP residue could cause contamination by accumulating in the environment including the air, soil, water, and agricultural products. This could eventually lead to serious health concerns for humans even after exposure to only very low concentrations (Zhang et al., 2014; Yang et al., 1995). The high toxicity of OP compounds is caused by their ability to inhibit acetylcholinesterase (AChE), which is an important enzyme that hydrolyzes acetylcholine. Inhibiting AChE activity allows acetylcholine to accumulate in cholinergic clefts, which overstimulates both the peripheral and central cholinergic nervous systems, and has fatal consequences (Yadav et al., 2012). The symptoms of OP poisoning include headache, dizziness, salivation, lacrimation, sweating, vomiting, diarrhea, abrupt tremor, lung edema, coma, and even death from respiratory or cardiac failure (Meng et al., 2013). The development n
Corresponding author. Tel.: þ 82 2 820 5220; fax: þ 82 2 825 4736. E-mail address: [email protected] (T.J. Park). 1 These two authors contributed equally to this paper.
of a sensitive and inexpensive diagnosis tool for environmental and biological monitoring is a current research area to facilitate the removal of excess OP compounds, to prevent disease, and protect the environment. Moreover, it is essential to develop a method that allows the on-site and real-time detection of OP residues rapidly and easily. Several methods to detect OP compounds have been developed over the past decade. For example, liquid or gas chromatography coupled to mass spectrometry, electrochemical analysis, fluorescent bioprobes, and enzyme-linked immunosorbent assays (ELISAs) have been used to detect OP compounds (Mehrvar and Abdi, 2004; Rao et al., 2002; Trojanowicz, 2009; Shi et al., 2006; Jeanty and Marty, 1998; Chen et al., 2009; Meng et al., 2013). These methods have high selectivity, adequate sensitivity, and reliability; however, most are also associated with disadvantages, such as high costs, the need for sophisticated instruments, the requirement for highly qualified and trained operators possessing master professional skills, tedious sample pretreatment, and expensive biomolecular reagents. Therefore, these methods are not suitable for on-site detection in most settings (Zhang et al., 2014; Yi et al., 2013). AuNPs-based colorimetric assays are effective approaches for quantifying many types of analytes without the need for complex equipment because molecular alterations can cause color changes in the AuNPs (Jiao et al., 2014; Sener et al., 2014; Feng et al., 2013; Wang et al., 2013; Zhang et al., 2010; Li et al., 2010; Huang et al., 2005; Liu et al., 2010; Wang et al., 2006). The color of AuNPs is
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size- and shape-dependent and highly sensitive to aggregation states. It can be easily measured by a UV/vis spectrophotometer (Feng et al., 2013). In general, well-dispersed AuNPs are colored red, whereas aggregated AuNPs are purple- or blue-colored. The AuNPs color change can be measured on the basis of the UV/vis absorption spectrum via a shift in the maximum peak and/or the appearence of a second peak in the visible region (Jiao et al., 2014; Sener et al., 2014; Feng et al., 2013; Wang et al., 2013; Zhang et al., 2010; Li et al., 2010; Huang et al., 2005; Liu et al., 2010; Wang et al., 2006; Chegel et al., 2012). Imidazole has been reported as an alkaloid organic compound in relation to conjugation potential. Due to the coexist of position 1 protonated nitrogen of pKa 14.0 and position 3 nitrogen of pKa 6.5, imidazole has an amphoteric property with dual-function as both an acid and a base (Walba and Isensee, 1961; Holze, 1993). The property, through the formation of weak complexs between N-ligands and O-ligands (Lorenzo et al., 1992), enables the imidazole to serve as precursors for the adsorption of other molecules onto metal surfaces (Xue et al., 1988; Wang et al., 2002; Cao et al., 2003). Furthermore, the strong affinity between imidazole and AuNPs induces the aggregation of the AuNPs, which results in a shift in the surface plasmon resonance into the near-infrared (NIR) wavelengths (Glauco et al., 2006). Therefore, the imidazole can be used as an aggregation promotor of AuNPs. For the optical detection of OP compounds, we herein developed a simple and rapid biosensing method based on the aggregation of AuNPs in a two-step process. The proposed sensing strategy for the optical assay to detect OP compounds is shown in Scheme 1. Different concentrations of diazinon were mixed with the AuNPs, followed by addition of imidazole. Finally, the color of the AuNP suspension was immediately changed from red to dark blue (or purple) because of the aggregation of the AuNPs. We selected diazinon [O,O-diethyl O-(2-isopropyl-4-methyl-6-pyrimiinyl)phosphorothioate] as the model OP, because it is a moderately persistent pesticide that is widly used in agriculture (Banaee et al., 2011). To verify this novel bioassay method, two additional OP compounds, iprobenfos and edifenphos, were tested at the concentration range of 0–10 ppm. This strategy for OP detection
was performed without complex modification processes, enzyme immobilization, and/or the addition of salt. The assay results could be measured as soon as the reaction finished, which was within a few seconds.
2. Experimental 2.1. Materials Gold(III) chloride hydrate (HAuCl4), diazinon, iprobenfos, edifenphos, and phosphate-buffered saline (PBS, pH 7.4) were purchased from Sigma-Aldrich (St. Louis, MO). Trisodium citrate dehydrate, imidazole were manufactured by Bio-Basic (Ontario, Canada). Methanol was obtained from Merck chemicals (Darmstadt, Germany). Milli-Q grade distilled (DI) water (18.2 MΩ cm, Millipore, Billerica, MA) was used in all experiments. 2.2. Synthesis of citrate-stabilized AuNPs (Cit-AuNPs) Dispersed AuNPs were prepared using a citrate reduction of HAuCl4 (Kim et al., 2011). One hundred milliliters of DI water containing 1 mM HAuCl4 was refluxed during stirring. When the HAuCl4 solution started to boil, 10 mL of 38.8 mM trisodium citrate dihydrate was added, and the reaction was continued for 15 min to reduce the salt content. The color of the solution changed immediately to dark blue and then to dark red, indicating the formation of dispersed Cit-AuNPs. After a 15-min reaction, the solution was cooled while being stirred at room temperature, and was then stored at 4 °C. The synthesis of Cit-AuNPs was confirmed by recording UV/vis spectrum data and analysis using FE-TEM (Tecnai G2 F30 S-Twin, FEI, Hillsboro, OR). 2.3. Preparation of imidazole and OP solution Imidazole was prepared by diluting the concentrated stocks in PBS solution (pH 7.4). The various concentrations of OP compounds were prepared by dissolving 10 mL of OP in 10 mL of 10%
Scheme 1. Schematic illustration of optical assay used to detect OP chemicals.
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(v/v) methanol to obtain a 1000 ppm (parts-per-million) solution. This solution was further diluted using 10% (v/v) methanol to reach the appropriate concentrations. To determine a real sample, various concentrations (0, 0.2, 0.4, 0.6, 0.8 and 1.0 ppm) of diazinon was prepared by dissolving 10 mL of diazinon in 10 mL of river water (1000 ppm). 2.4. Characterizations of AuNPs using zeta potential For zeta potential and dynamic light scattering (DLS) analysis of the AuNPs, AuNP–diazinon mixtures with and without imidazole were applied to a Zeta Potential & Particle size analyzer (ELSZ1000, Otsuka, Japan). 2.5. Quantification of OP compounds Fifty microliters of Cit-AuNPs were added sequentially to 100 mL of OP solution and 50 mL of 0.3 mM imidazole solution in sequence on 96-well plates. (AuNP:OP:Imidazole volumetric ratio of 1:2:1). UV/vis spectroscopy and fluorescence analysis were then sequentially performed at 400–700 nm using a Synergy H1 Hybrid Reader (BioTek, Winooski, VT). Standard OP chemicals after diluting step was filtrated with syringe filter of 0.2-μm. High-performance liquid chromatography (HPLC, Agilent 1260, Santa Clara, CA) was then used for quantifying the OP chemicals with a C18 column (10-cm length and inside diameter of 4.6 mm, Eclipse Plus, Agilent). The analysis was performed at 40 °C. The UV absorption spectrometer was used as an HPLC detector at a detection wavelength of 254 nm. The mobile phase consisted of acetonitrile–water 65:35 (v/v) that were filtered separately before mixing. A flow rate of 1 mL/min was selected.
3. Results and discussion 3.1. Analytical determination of the novel method The UV/vis spectra of each sample were measured to reveal a possibility of the proposed method. The Cit-AuNPs were dispersed well as shown in Fig. S1 and exhibited typical extinction spectra for such particles, with a maximum peak at 520 nm. After the mixed with 1-ppm diazinon, the maximum extinction was mostly unaffected. In case of the high concentrations of diazinon from 10-ppm, AuNPs were aggregated with diazinon concentration increases (Fig. S2), however, this result was unsatisfactory for a sensitive detection of OP compounds. To facilitate the AuNP aggregation even at low concentration of diazinon, imidazole was introduced as an aggregation promotor. As shown in Fig. 1, when imidazole was added into the AuNPs and 1-ppm diazinon– AuNP mixture, this result leads to the appearance of a second broad peak between 600 and 700 nm in the absorbance spectrum, which indicated that the AuNPs were aggregated even at low concentration of diazinon by introducing imidazole, and more intense peak was appeared in the presence of diazinon. Therefore, we used imidazole to promote the AuNP aggregation for sensitive detection of OP compounds as illustrated in Scheme 1. In order to confirm the optimal assay conditions, several concentrations of the imidazole were fixed at a specific volumetric ratio. As shown in Fig. S3, 0.3 mM of imidazole resulted in a welldispersed spectrum and a higher slope according to the concentration of diazinon. Therefore, we used 0.3 mM concentration of imidazole for further subsequent experiments. Next, the zeta potential was measured to investigate changes of the surface charge of the AuNPs. Before the addition of OP compounds or imidazole, the surface charge on AuNPs was negative because of the presence of citrate ions. In contrast, the
Fig. 1. Optical response of the assay. UV/vis spectrum of the AuNP solutions revealed that a color change occurs in the presence of imidazole (0.3 mM) and diazinon (1.0 ppm) with imidazole.
charge of aggregated AuNPs was neutralized, to some extent, after the addition of imidazole. The zeta potential values of welldispersed AuNPs and AuNPs pretreated with 1 ppm diazinon were 34.98 and 36.40 mV, respectively, whereas those of imidazolestimulated aggregates in the absence and presence of diazinon were 12.18 mV and 11.81, respectively, which were different to that of well-dispersed AuNPs (Fig. S4a). These observations were supported by DLS data (Fig. S4b). The average hydrodynamic diameters of well-dispersed AuNPs and AuNPs pretreated with 1 ppm diazinon were 13.4 and 13.2 nm, respectively. However, the addition of imidazole to the AuNPs and diazinon–AuNP mixture increased the diameter of the aggregates to 201.7 and 235.1 nm, respectively. The DLS results corresponding to UV/vis spectra suggest that the current method is practicable to detect the diazinon. 3.2. Detection of the OP compounds, diazinon, iprobenfos, and edifenphos To assess the sensitivity of the assay, we analyzed various concentrations of diazinon solutions ranging from 10.0 ppb to 10.0 ppm (0, 0.01, 0.05, 0.1, 0.3, 0.4, 0.5, 0.6, 0.7, 0.9, 1.0, 3.0, 5.0, 7.0 and 10.0 ppm). As the concentration of diazinon increased, the absorption at 520 nm and 600–700 nm increased gradually. The intensity of the maximum absorption of the new peak at 670 nm was comparable to that of free AuNPs at 520 nm (Fig. 2a). The plots of A670 (absorbance at 670 nm) value vs. various concentrations of diazinon. Fig. 2b shows the A670 value gradually with increasing concentrations of diazinon. The maximum A670 value was reached with a concentration of 3.0 ppm diazinon, and further increases in the concentration of diazinon had no effect on the A670 value. The A670 values were linear with diazinon concentrations from 0 to 1.0 ppm, with a linear regression correlation coefficient of 0.985. The lowest detectable diazinon concentration using this assay was 53.3 ppb according to a 3s rule calculation (LOD ¼3 standard deviation corresponding to the blank sample/slope of calibration curve) (Chen et al., 2010). We confirmed the sensitivity of the sensing platform using two different germicidal OP compounds, iprobenfos and edifenphos, using the same experimental procedures as those used for diazinon. Fig. S5a and b shows the changes in absorbance with increasing concentrations of iprobenfos, and a plot of A670 values vs. various concentrations (0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 3.0, 5.0, 7.0 and 10.0 ppm) of iprobenfos, respectively. As the concentration of iprobenfos increased, the absorption peak between 600 and 700 nm increased gradually until the
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Fig. 2. Sensitivity of the assay for detecting diazinon. (a) The changes in absorbance with increasing concentrations of diazinon from 0 to 10.0 ppm. (b) Plots of the absorbance at 670 nm vs. diazinon concentration. A670 indicates the absorption band that appeared at 670 nm. The standard deviations of measurements were calculated from the results of three independent experiments.
intensity of the maximum absorption peak at 670 nm was comparable to that of free AuNPs at 520 nm. As the concentration of iprobenfos increased, the A670 value also increased gradually (Fig. S5b). The maximum A670 value was reached at a concentration of 5.0 ppm iprobenfos. With iprobenfos concentrations ranging from 0 to 1.0 ppm, the A670 values were highly linear, with a linear regression correlation coefficient of 0.994. The lowest iprobenfos concentration that could be detected using this assay was 53.6 ppb. Fig. S5c and d shows that the absorbance intensity increased progressively with increasing edifenphos concentrations, leading to an increased A670 value. A similar trend was observed to that seen with diazinon and iprobenfos. We tested edifenphos solutions with concentrations between 10.0 ppb and 10.0 ppm (0, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 3.0, 5.0, 7.0 and 10.0 ppm). The maximum A670 value was reached with a concentration of 5.0 ppm edifenphos. The A670 values were highly linear with edifenphos concentrations from 0 to 1.0 ppm, with a linear regression correlation coefficient of 0.984. The lowest detectable concentration of edifenphos using this assay was 27.9 ppb (Fig. S5d). The lowest detectable concentrations for diazinon, iprobenfos and edifenphos are much lower than the maximum residue limits (MRLs) as reported in Korean Pesticide Residue Database (Ministry of Food and Drug Safety, 2013, http:// fse.foodnara.go.kr/residue/RS/jsp/menu_02_01_01.jsp). MRLs are 0.1, 0.2, and 0.2 ppm for diazinon, iprobenfos, and edifenphos, respectively.
compounds (benzene, phenol, toluene, xylene, dichlorobenzene, and phosphoric acid) with similar chemical structures were tested using the same experimental conditions as those used for the diazinon assay. UV/vis absorption spectra showed that none of these organic compounds affected the absorbance at 670 nm and interfered with the assay at concentrations of 1.0 ppm (Fig. 3). This result suggests that the absorbance signals observed with diazinon were generated by specific interactions between the AuNP–imidazole complex and diazinon. Therefore, this optical assay is highly selective for diazinon.
3.3. Effect of pH
3.5. Comparison of the proposed assay with liquid chromatography
We next assessed whether pH could affect the reaction between AuNPs, diazinon, and imidazole. Specifically, we investigated the effects of pHs from 3.4 to 10.4 while maintaining a constant imidazole concentration (Fig. S6). The difference value of A670 between 1 ppm and 0 ppm increased gradually with increasing pH at 1 ppm diazinon. The result shows that this assay was saturated toward pH changes from 7.4 to 10.4. The highest absorption (A670) was obtained at pH 8.4. However, A670 value at pH 7.4 was comparable to that at pH 8.4. Thus, pH 7.4 was chosen as the optimal reaction pH in the experiments to avoid the inconvenience of increasing to pH 8.4.
The proposed method was used to quantify the concentrations of diazinon, as shown in Table 1. To verify the accuracy of the proposed method, HPLC was used to assay several diazinon samples. The results obtained by the method were high accurate than those achieved using the HPLC method at various concentrations of diazinon. In addition, the method is more rapid, straightforward, and acceptable, with mean recovery of 100.1%. Therefore, it is obvious that the proposed method has great potentials for analyzing OP compounds in practical field samples.
3.4. Selectivity of the biosensing system
To evaluate the possibility of the detection of real sample, standard curve was prepared in river water, which was applied to determine the concentrations of diazinon (0, 0.2, 0.4, 0.6, 0.8 and 1.0 ppm) (Fig. 4). Detection limit of diazinon in river water was
Selectivity is another critical aspect of a potential assay system. To evaluate the selectivity of the novel model, six organic
Fig. 3. Selectivity of the assay. Benzene, phenol, toluene, xylene, dichlorobenzene, phosphoric acid, and diazinon were each tested at the concentrations of 1.0 ppm.
3.6. Determination of diazinon in river water
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Table 1 Analytical results of the determination of diazinon sample using the proposed optical sensor and HPLC. Diazinon conc. added (ppm)
In deionized water mean 7 SD (ppm)a
Recovery (%) In river water mean7 SD (ppm)a
Recovery (%) HPLC mean7 SD (ppm)a
0.080 0.170 0.300 Average
0.080 7 0.024 0.1737 0.056 0.295 7 0.042 –
100.4 101.6 98.3 100.1
98.5 86.3 113.5 99.4
a
0.0797 0.012 0.1467 0.012 0.340 70.001 –
0.082 7 0.002 0.149 7 0.004 0.263 7 0.064 –
Recovery (%)
96.2 87.6 87.7 90.5
The standard deviations (SD) of measurements are calculated from three independent experiments.
Acknowledgments This work was supported by the Advanced Production Technology Development Program, Ministry of Agriculture, Food and Rural Affairs (312066-3) and a grant of the Korean Health Technology R&D Project, Ministry of Health and Welfare (HI13C0862), Republic of Korea.
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.2014.08.073.
References Fig. 4. Plots of A670 values vs. diazinon concentrations in river water. The standard deviations of the samples were calculated from the results 3 times.
calculated at 36.3 ppb by 3s rule. Table 1 shows the results for analysis of spiked river water samples. A recovery of 86.3% and 113.5% was observed in river water samples, which indicated the accuracy and reliability of the proposed method for the determination of OP pesticide in water samples. The results demostrate that the method have been successfully applied to water samples and can be applied to other samples.
4. Conclusion We successfully developed a simple, rapid, and highly sensitive assay for the detection of OP chemicals using imidazole to promote the aggregation of AuNPs. The assay uses common CitAuNPs without the need for tedious surface modification steps, such as DNA oligomers and peptides, or the addition of an excess amount of salt, making the method practical and cost-effective. Data revealed that the detection limits of the method were 53.3 ppb for diazinon, 53.6 ppb for iprobenfos, and 27.9 ppb for edifenphos, respectively. This assay strategy gives highly linear data with concentrations ranging from 10.0 ppb to 1.0 ppm. Furthermore, the novel method has provided an effective reliable detection of diazinon in a real sample with a detection limit of 36.3 ppb. In addition, the response of the optical assay was very rapid, occurring within 30 s, and all analyses including the sample preparation steps could be completed within a few minutes. We anticipate that this method will facilitate the detection of many OP compounds in the field. Importantly, this is the first study to reveal that AuNP–imidazole complexes could be used to detect OP compounds. We propose that the novel method has advantages over several current AuNPs-based methods, and that its use in practical applications is feasible.
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