Visual and Colorimetric Sensing of Metsulfuron-Methyl by Exploiting Hydrogen Bond-Induced Anti-Aggregation of Gold Nanoparticles in the Presence of Melamine

Various highly sensitive and selective analytical methods have been used to monitor metsulfuron-methyl residue in the environment. However, these methods involve costly instruments and complex, time-consuming operations performed in laboratories. Here, a rapid, convenient, and sensitive colorimetric sensor based on anti-aggregation of gold nanoparticles (AuNPs) is demonstrated for the rapid detection of metsulfuron-methyl in agricultural irrigation water. The AuNPs could be induced to aggregate in the presence of melamine and exhibited a distinct color change from wine-red to blue. The aggregation was suppressed by a strong hydrogen-bonding interaction between metsulfuron-methyl and melamine. The differences of the absorbance at 523 nm (ΔA523) and the color change was linearly related to metsulfuron-methyl concentration over the range 0.1–100 mg/L, as observed visually and by UV-vis (Ultraviolet-visible) spectrometry. The detection limit of the sensor was as low as 0.05 mg/L (signal/noise = 3), and was used to determine metsulfuron-methyl in spiked water and in agricultural irrigation water samples. Recoveries were in the range of 71.2–100.4%, suggesting that the colorimetric sensor was suitable for the determination of metsulfuron-methyl in agricultural water samples.


Introduction
Metsulfuron-methyl is a systemic sulfonylurea pesticide that was introduced in the 1980s and has been widely used to control broad-leaved weeds and grasses in rice, maize, wheat, and barley [1]. Because of its highly efficient herbicidal activity and low mammalian toxicity, metsulfuron-methyl has been applied worldwide [2]. However, its high water solubility, high mobility, and slow degradation may result in contamination of soil, environmental water, and food. In addition, metsulfuron-methyl could bring harm to aquatic plants and animals and the overall ecosystem [3]. It is a great challenging task to monitor metsulfuron-methyl in environmental water and soil [4,5]. Hence, a highly sensitive,

Colorimetric Assay
Unless otherwise noted, the citrate-AuNP solution was used without further functionalization. An acetate buffer solution (10 mM, pH 3.5, 0.2 mL) was added to 0.2 mL of the AuNPs solution and incubated for 5 min (after incubation, the mixture solution provided low pH condition and melamine can be protonated in this situation and conjugated to the negatively-charged ions on the surface of AuNPs to induce its aggregation) [35]. Then, different concentrations of metsulfuron-methyl (0.1 mg/L, 0.2 mg/L, 0.5 mg/L, 1.0 mg/L, 2.0 mg/L, 5.0 mg/L, 10.0 mg/L, 20.0 mg/L, 50.0 mg/L, and 100.0 mg/L; 0.2 mL) were added and the mixtures were equilibrated for 5 min. Finally, melamine solution (2.0 mg/L, 0.1 mL) was added and the mixture was incubated for another 20 min. The color change was observed visually and the absorbance spectrum was monitored with the UV-vis spectrophotometer. The absorbance at 523 nm was linear to the logarithm of the metsulfuron-methyl concentration and then was used to estimate the detection response. The sensor selectivity was tested with atrazine, hexazinone, glucose, L-cysteine, vitamin C, Na + , and Mg 2+ as potential interfering substances at the same concentrations as those of metsulfuron-methyl (20 mg/L, 10 mg/L, 5 mg/L, and 2 mg/L).

Sample Pretreatment
Tap and agricultural irrigation water samples were mixed with ethylenediaminetetraacetic acid (EDTA, 1 mM) to remove heavy metal ions, and the solution was then filtered through a 0.2 µm Millipore syringe filter. The water samples were spiked with different metsulfuron-methyl concentrations, and the colorimetric sensor was used to quantitatively determine the concentration. Figure 1 presents surface plasma resonance absorbance spectra for AuNPs, AuNPs with 2.0 mg/L metsulfuron-methyl, AuNPs with 2.0 mg/L melamine, and AuNPs with 2.0 mg/L metsulfuron-methyl and 2.0 mg/L melamine. The unmodified AuNPs possess a distinct wine-red color and a strong absorption peak at 523 nm. The color of AuNPs remained wine-red after adding metsulfuron-methyl. When the AuNPs were aggregated by melamine, the color changed to blue, the 523-nm adsorption decreased, and a new absorption band appeared at 710 nm. However, when the mixture of AuNPs and metsulfuron-methyl was incubated with melamine, much less aggregation occurred, and the color changed slightly or remained purple if there was a high concentration of metsulfuron-methyl.

Colorimetric Assay
Unless otherwise noted, the citrate-AuNP solution was used without further functionalization. An acetate buffer solution (10 mM, pH 3.5, 0.2 mL) was added to 0.2 mL of the AuNPs solution and incubated for 5 min (after incubation, the mixture solution provided low pH condition and melamine can be protonated in this situation and conjugated to the negatively-charged ions on the surface of AuNPs to induce its aggregation) [35]. Then, different concentrations of metsulfuron-methyl (0.1 mg/L, 0.2 mg/L, 0.5 mg/L, 1.0 mg/L, 2.0 mg/L, 5.0 mg/L, 10.0 mg/L, 20.0 mg/L, 50.0 mg/L, and 100.0 mg/L; 0.2 mL) were added and the mixtures were equilibrated for 5 min. Finally, melamine solution (2.0 mg/L, 0.1 mL) was added and the mixture was incubated for another 20 min. The color change was observed visually and the absorbance spectrum was monitored with the UV-vis spectrophotometer. The absorbance at 523 nm was linear to the logarithm of the metsulfuron-methyl concentration and then was used to estimate the detection response. The sensor selectivity was tested with atrazine, hexazinone, glucose, L-cysteine, vitamin C, Na + , and Mg 2+ as potential interfering substances at the same concentrations as those of metsulfuron-methyl (20 mg/L, 10 mg/L, 5 mg/L, and 2 mg/L).

Sample Pretreatment
Tap and agricultural irrigation water samples were mixed with ethylenediaminetetraacetic acid (EDTA, 1 mM) to remove heavy metal ions, and the solution was then filtered through a 0.2 µ m Millipore syringe filter. The water samples were spiked with different metsulfuron-methyl concentrations, and the colorimetric sensor was used to quantitatively determine the concentration. Figure 1 presents surface plasma resonance absorbance spectra for AuNPs, AuNPs with 2.0 mg/L metsulfuron-methyl, AuNPs with 2.0 mg/L melamine, and AuNPs with 2.0 mg/L metsulfuron-methyl and 2.0 mg/L melamine. The unmodified AuNPs possess a distinct wine-red color and a strong absorption peak at 523 nm. The color of AuNPs remained wine-red after adding metsulfuron-methyl. When the AuNPs were aggregated by melamine, the color changed to blue, the 523-nm adsorption decreased, and a new absorption band appeared at 710 nm. However, when the mixture of AuNPs and metsulfuron-methyl was incubated with melamine, much less aggregation occurred, and the color changed slightly or remained purple if there was a high concentration of metsulfuron-methyl.    (Figure 2a), which suggested that metsulfuron-methyl could not induce AuNPs to aggregate. Only in the presence of melamine, the AuNPs were induced to aggregate, as shown in Figure 2c. After adding metsulfuron-methyl to the AuNP-melamine mixture, fewer and smaller aggregations were present (Figure 2b) than for AuNPs with 2.0 mg/L melamine, indicating that the complex formation between metsulfuron-methyl and melamine inhibits the aggregation.  (Figure 2a), which suggested that metsulfuron-methyl could not induce AuNPs to aggregate. Only in the presence of melamine, the AuNPs were induced to aggregate, as shown in Figure 2c. After adding metsulfuron-methyl to the AuNP-melamine mixture, fewer and smaller aggregations were present ( Figure 2b) than for AuNPs with 2.0 mg/L melamine, indicating that the complex formation between metsulfuron-methyl and melamine inhibits the aggregation. The hydrogen bonding interaction between metsulfuron-methyl and melamine was examined with FT-IR spectroscopy. As shown in Figure 3, the spectra of metsulfuron-methyl had characteristic peaks for -NH-at 1566 cm −1 and for -R-SO2-stretching at 1443 cm −1 . In the spectrum of melamine, adsorption peaks at 3000-3500 cm −1 for the -NH2 group and 1300-1700 cm −1 for the benzene ring substitution group (-N=CH-) were observed. In the metsulfuron-methyl-melamine complex, the -NH2 peak was shifted and the intensities of the stretching vibrations of -R-SO2-in metsulfuron-methyl and -N=CH-in melamine decreased. These observations indicated that the hydrogen bonding interaction between metsulfuron-methyl and melamine was formed.  The hydrogen bonding interaction between metsulfuron-methyl and melamine was examined with FT-IR spectroscopy. As shown in Figure 3, the spectra of metsulfuron-methyl had characteristic peaks for -NH-at 1566 cm −1 and for -R-SO 2 -stretching at 1443 cm −1 . In the spectrum of melamine, adsorption peaks at 3000-3500 cm −1 for the -NH 2 group and 1300-1700 cm −1 for the benzene ring substitution group (-N=CH-) were observed. In the metsulfuron-methyl-melamine complex, the -NH 2 peak was shifted and the intensities of the stretching vibrations of -R-SO 2 -in metsulfuron-methyl and -N=CH-in melamine decreased. These observations indicated that the hydrogen bonding interaction between metsulfuron-methyl and melamine was formed.  (Figure 2a), which suggested that metsulfuron-methyl could not induce AuNPs to aggregate. Only in the presence of melamine, the AuNPs were induced to aggregate, as shown in Figure 2c. After adding metsulfuron-methyl to the AuNP-melamine mixture, fewer and smaller aggregations were present (Figure 2b) than for AuNPs with 2.0 mg/L melamine, indicating that the complex formation between metsulfuron-methyl and melamine inhibits the aggregation. The hydrogen bonding interaction between metsulfuron-methyl and melamine was examined with FT-IR spectroscopy. As shown in Figure 3, the spectra of metsulfuron-methyl had characteristic peaks for -NH-at 1566 cm −1 and for -R-SO2-stretching at 1443 cm −1 . In the spectrum of melamine, adsorption peaks at 3000-3500 cm −1 for the -NH2 group and 1300-1700 cm −1 for the benzene ring substitution group (-N=CH-) were observed. In the metsulfuron-methyl-melamine complex, the -NH2 peak was shifted and the intensities of the stretching vibrations of -R-SO2-in metsulfuron-methyl and -N=CH-in melamine decreased. These observations indicated that the hydrogen bonding interaction between metsulfuron-methyl and melamine was formed.   Figure 4 illustrates the mechanism of the colorimetric sensor for metsulfuron-methyl detection based on anti-aggregation of AuNPs induced by melamine. The wine-red AuNP solution exhibited a strong SPR absorption peak at 523 nm. The AuNPs could be aggregated by melamine, with an accompanying color change to blue. However, the combination between metsulfuron-methyl and melamine will generate the strong hydrogen bonding interaction to prevent the aggregation of AuNPs. Thus, melamine and metsulfuron-methyl were used as the aggregation and anti-aggregation reagents, respectively, in this colorimetric sensing assay. Because there was a linear relationship between the 523-nm absorbance and the metsulfuron-methyl concentration, the concentration of metsulfuron-methyl could be qualitatively determined.  Figure 4 illustrates the mechanism of the colorimetric sensor for metsulfuron-methyl detection based on anti-aggregation of AuNPs induced by melamine. The wine-red AuNP solution exhibited a strong SPR absorption peak at 523 nm. The AuNPs could be aggregated by melamine, with an accompanying color change to blue. However, the combination between metsulfuron-methyl and melamine will generate the strong hydrogen bonding interaction to prevent the aggregation of AuNPs. Thus, melamine and metsulfuron-methyl were used as the aggregation and anti-aggregation reagents, respectively, in this colorimetric sensing assay. Because there was a linear relationship between the 523-nm absorbance and the metsulfuron-methyl concentration, the concentration of metsulfuron-methyl could be qualitatively determined.

Effect of Melamine Concentration
The optimal concentration of melamine was an important factor for the AuNP aggregation and to affect the sensitivity and linear range of the colorimetric sensor. In Figure 5a, with the concentration of melamine increasing, an increase of the absorbance ratio in the presence of different concentrations of metsulfuron-methyl is observed. The absorbance difference ΔA523 was equal to (Ablank − Asample), where Ablank was the absorbance without metsulfuron-methyl and Asample was the absorbance with metsulfuron-methyl. When the melamine concentration was above 2.0 mg/L, the level of AuNP aggregation was too high and the analytical signals decreased and narrowed the linear range of the sensor. When the concentration was below 2.0 mg/L, the analytical signals became less sensitive. Therefore, 2.0 mg/L was the optimum melamine concentration used in experiments discussed below.

Effect of Melamine Concentration
The optimal concentration of melamine was an important factor for the AuNP aggregation and to affect the sensitivity and linear range of the colorimetric sensor. In Figure 5a, with the concentration of melamine increasing, an increase of the absorbance ratio in the presence of different concentrations of metsulfuron-methyl is observed. The absorbance difference ∆A523 was equal to (A blank − A sample ), where A blank was the absorbance without metsulfuron-methyl and A sample was the absorbance with metsulfuron-methyl. When the melamine concentration was above 2.0 mg/L, the level of AuNP aggregation was too high and the analytical signals decreased and narrowed the linear range of the sensor. When the concentration was below 2.0 mg/L, the analytical signals became less sensitive. Therefore, 2.0 mg/L was the optimum melamine concentration used in experiments discussed below.  Figure 4 illustrates the mechanism of the colorimetric sensor for metsulfuron-methyl detection based on anti-aggregation of AuNPs induced by melamine. The wine-red AuNP solution exhibited a strong SPR absorption peak at 523 nm. The AuNPs could be aggregated by melamine, with an accompanying color change to blue. However, the combination between metsulfuron-methyl and melamine will generate the strong hydrogen bonding interaction to prevent the aggregation of AuNPs. Thus, melamine and metsulfuron-methyl were used as the aggregation and anti-aggregation reagents, respectively, in this colorimetric sensing assay. Because there was a linear relationship between the 523-nm absorbance and the metsulfuron-methyl concentration, the concentration of metsulfuron-methyl could be qualitatively determined.

Effect of Melamine Concentration
The optimal concentration of melamine was an important factor for the AuNP aggregation and to affect the sensitivity and linear range of the colorimetric sensor. In Figure 5a, with the concentration of melamine increasing, an increase of the absorbance ratio in the presence of different concentrations of metsulfuron-methyl is observed. The absorbance difference ΔA523 was equal to (Ablank − Asample), where Ablank was the absorbance without metsulfuron-methyl and Asample was the absorbance with metsulfuron-methyl. When the melamine concentration was above 2.0 mg/L, the level of AuNP aggregation was too high and the analytical signals decreased and narrowed the linear range of the sensor. When the concentration was below 2.0 mg/L, the analytical signals became less sensitive. Therefore, 2.0 mg/L was the optimum melamine concentration used in experiments discussed below.

Effect of pH
Because the pH can affect the stability and the sensitivity of this sensor, an acetate buffer solution was varied over pH 3.0-6.5. As shown in Figure 5b, when the pH was above 3.5, the AuNPs were less sensitive to melamine and the limit of detection increased. When the pH was less than 3.5, the AuNPs were easily aggregated, which decreased the anti-aggregation performance of metsulfuron-methyl. Therefore, pH 4.5 was chosen as the optimum pH for experiments discussed below.

Colorimetric Sensing of Metsulfuron-Methyl
Metsulfuron-methyl concentrations over the range 0.1-100 mg/L were used to investigate the limit of detection and the linear range of the colorimetric sensor. Figure 6A revealed that the wine-red AuNPs solution was aggregated by melamine, producing a new adsorption peak at 640 nm and reducing the intensity of the 523-nm surface plasmon resonance adsorption. The aggregation was inhibited when metsulfuron-methyl was initially added to the AuNPs solution. The color changed from blue (purple) to wine-red, the 640-nm peak intensity decreased, and the 523-nm peak intensity increased.

Effect of pH
Because the pH can affect the stability and the sensitivity of this sensor, an acetate buffer solution was varied over pH 3.0-6.5. As shown in Figure 5b, when the pH was above 3.5, the AuNPs were less sensitive to melamine and the limit of detection increased. When the pH was less than 3.5, the AuNPs were easily aggregated, which decreased the anti-aggregation performance of metsulfuron-methyl. Therefore, pH 4.5 was chosen as the optimum pH for experiments discussed below.

Colorimetric Sensing of Metsulfuron-Methyl
Metsulfuron-methyl concentrations over the range 0.1-100 mg/L were used to investigate the limit of detection and the linear range of the colorimetric sensor. Figure 6A revealed that the wine-red AuNPs solution was aggregated by melamine, producing a new adsorption peak at 640 nm and reducing the intensity of the 523-nm surface plasmon resonance adsorption. The aggregation was inhibited when metsulfuron-methyl was initially added to the AuNPs solution. The color changed from blue (purple) to wine-red, the 640-nm peak intensity decreased, and the 523-nm peak intensity increased. The calibration linearity was evaluated by measuring ΔA523 vs. the log of the metsulfuron-methyl concentration. The curve fit in Figure 6B indicated a linear relationship over the range 0.1-100 mg/L. The calibration fit was ΔA523 = 0.305x + 0.4105 (x = log (conc.)) with a correlation coefficient R 2 = 0.9568. The detection limit (signal/noise (S/N) = 3) was 0.05 mg/L. In Table 1, the analytical performance of the sensor was compared with previously reported metsulfuron-methyl detection methods. Relative to these methods, the colorimetric sensor was more rapid, convenient, and sensitive without requiring sophisticated instruments or time-consuming treatment procedures.  The calibration linearity was evaluated by measuring ∆A523 vs. the log of the metsulfuron-methyl concentration. The curve fit in Figure 6B indicated a linear relationship over the range 0.1-100 mg/L. The calibration fit was ∆A523 = 0.305x + 0.4105 (x = log (conc.)) with a correlation coefficient R 2 = 0.9568. The detection limit (signal/noise (S/N) = 3) was 0.05 mg/L. In Table 1, the analytical performance of the sensor was compared with previously reported metsulfuron-methyl detection methods. Relative to these methods, the colorimetric sensor was more rapid, convenient, and sensitive without requiring sophisticated instruments or time-consuming treatment procedures.

Selectivity
In the selectivity experiments, atrazine and hexazinone were chosen as the structure analogue for metsulfuron-methyl to study the anti-interference capacity of this sensor. Additionally, glucose, vitamin C, Na + , and Mg 2+ were used as potential organic and inorganic interference compounds. Their concentrations (20 mg/L, 10 mg/L, 5 mg/L, and 2 mg/L) were the same as those used above for metsulfuron-methyl. As shown in Figure 7, only metsulfuron-methyl inhibited the aggregation of AuNPs by melamine and prevented the color change to blue. None of the above compounds exhibited anti-aggregation and no spectral changes were observed. Therefore, the colorimetric sensor had high selectivity and responsiveness to metsulfuron-methyl.

Selectivity
In the selectivity experiments, atrazine and hexazinone were chosen as the structure analogue for metsulfuron-methyl to study the anti-interference capacity of this sensor. Additionally, glucose, vitamin C, Na + , and Mg 2+ were used as potential organic and inorganic interference compounds. Their concentrations (20 mg/L, 10 mg/L, 5 mg/L, and 2 mg/L) were the same as those used above for metsulfuron-methyl. As shown in Figure 7, only metsulfuron-methyl inhibited the aggregation of AuNPs by melamine and prevented the color change to blue. None of the above compounds exhibited anti-aggregation and no spectral changes were observed. Therefore, the colorimetric sensor had high selectivity and responsiveness to metsulfuron-methyl.

Application to Agricultural Irrigation Water
To validate the analytical performance of the colorimetric sensor, metsulfuron-methyl was added to tap water and to agricultural irrigation water samples (containing 1 mM EDTA), and was then analyzed under the optimized procedure. The results in Table 2 reveal metsulfuron-methyl recoveries of 0.5 mg/L, 1.0 mg/L, and 5.0 mg/L from spiked tap water samples that ranged from 74.3-93.1%, respectively, with relative standard deviations (RSDs) that ranged from 4.73-6.37%. Metsulfuron-methyl recoveries of 0.5 mg/L, 1.0 mg/L, and 5.0 mg/L from spiked well water samples ranged from 71.2-100.4%, and RSDs ranged from 3.81-6.52%. These results suggest that the colorimetric sensor was suitable for the determination of metsulfuron-methyl in agricultural water samples. Table 2. Recoveries of metsulfuron-methyl in water samples (n = 3).

Application to Agricultural Irrigation Water
To validate the analytical performance of the colorimetric sensor, metsulfuron-methyl was added to tap water and to agricultural irrigation water samples (containing 1 mM EDTA), and was then analyzed under the optimized procedure. The results in Table 2 reveal metsulfuron-methyl recoveries of 0.5 mg/L, 1.0 mg/L, and 5.0 mg/L from spiked tap water samples that ranged from 74.3-93.1%, respectively, with relative standard deviations (RSDs) that ranged from 4.73-6.37%. Metsulfuron-methyl recoveries of 0.5 mg/L, 1.0 mg/L, and 5.0 mg/L from spiked well water samples ranged from 71.2-100.4%, and RSDs ranged from 3.81-6.52%. These results suggest that the colorimetric sensor was suitable for the determination of metsulfuron-methyl in agricultural water samples.

Conclusions
A rapid, highly sensitive, and selective colorimetric sensor to monitor metsulfuron-methyl residue in agricultural irrigation water samples was demonstrated. The sensing mechanism was based on preventing melamine-induced aggregation of AuNPs by strong hydrogen bonding between metsulfuron-methyl and the melamine. The mechanism was verified via FT-IR and UV-Vis spectra, and TEM imaging. Under optimum experiments conditions, the ∆A523 were found to be linearly related to different metsulfuron-methyl concentration (0.1 to 100 mg/L). The detection limit was as low as 0.05 mg/L (S/N = 3). The colorimetric sensor was performed easily and economically with no need for complex experimental procedures or instruments. All experimental steps took no more than 30 min to finish. Its rapid response, high sensitivity, and good test sample recoveries indicate that it has applicability for metsulfuron-methyl residue detection in aqueous water samples.