Simple, rapid and label-free colorimetric assay for arsenic based on unmodified gold nanoparticles and a phytochelatin-like peptide

Abstract

In this paper, we report a simple, rapid and selective colorimetric visualization of arsenic using unmodified gold nanoparticles (AuNPs) and a phytochelatin-like peptide (γ-Glu-Cys)₃-Gly-Arg (denoted as PC₃R). Arsenic prevented the peptide from attaching to the surface of AuNPs by coordinating to all the three cysteine residues of PC₃R, thus preventing the PC₃R-triggered AuNPs aggregation and color change. The present approach is selective to arsenic detection and is much faster and simpler than the conventional analytical methods. The detection limit is 20 nM, which is lower than the World Health Organization's (WHO) standard for drinking water. The feasibility for the detection of arsenic in groundwater has also been demonstrated. This method will be valuable for the design of new types of metal ions sensors and will likely lead to many colorimetric detection applications in environmental monitoring.

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1. Introduction

Because of its toxicity to insects, bacteria and fungi, arsenic has been used in agricultural insecticide, wood preservative and animal food for disease prevention and growth stimulation.^1,2 However, the excessive use of arsenic will be a threat to global health.^3–5 For example, arsenic contamination has led to a massive epidemic of arsenic poisoning in Bangladesh and neighbouring countries. Thus, it is of utmost importance to develop analytical methods for arsenic detection in water and food. For the accurate quantification of arsenic, the currently used methods, such as atomic absorption spectrometry (AAS), inductively coupled plasma (ICP), ICP/mass spectrometry and electrochemistry, are usually time-consuming, lack sensitivity or require complicated instruments.^3,4,6–8 There remains significant room for the development of a theoretically and technically simple approach for arsenic detection.

In recent years, gold nanoparticles (AuNPs) based colorimetric assays have been widely applied in a variety of research fields due to the high extinction coefficients and the unique size-dependent optical property of AuNPs. Such methods are very promising in that they require very simple sample handling procedures and minimum instrumental investment and can be conducted in the field with portable devices. The rational design of the surface chemistry of AuNPs promotes specific interactions between the receptors and analytes and, as a result, renders the measurements highly selective.^9–23 Most AuNPs-based assays rely on the modification of AuNPs with specific binding-ligands. In comparison, a modification-free AuNPs assay is more convenient and cost-effective for its absence of the elaborate and expensive synthesis of ligand-modified AuNPs.²⁴ Recently, unmodified AuNPs have been applied to the detection of DNA, enzymes, proteins, metal ions and other small molecules.^25–30

Metal ions can interact with specifically sequenced peptides, and peptides can induce or prevent the aggregation of AuNPs.^28,31–34 Based on AuNPs and specifically sequenced peptides, colorimetric assays have been developed for the detection of metal ions (e.g., Cd²⁺, Ni²⁺, Co²⁺, Hg²⁺, Pb²⁺, Cu²⁺, Al³⁺ and Zn²⁺).^28,31,33,34 In this work, we reported a simple, fast, and label-free arsenic colorimetric assay based on unmodified AuNPs and a phytochelatin-like peptide (γ-Glu-Cys)₃-Gly-Arg (denoted as PC₃R). The thiol groups of PC₃R tend to readily adsorb onto the surface of AuNPs via Au–S bonds; then the aggregation and color change of AuNPs suspension were anticipated to occur through the electrostatic interaction between the positively charged guanidine groups on the arginine side chain and the negatively charged groups on the AuNPs surface.^28,32,35 In contrast, due to the strong and unique coordination between arsenic and all three cysteine residues of PC₃R,^36–38 the peptide could not adsorb onto the AuNPs surface in the presence of arsenic. Thus, the aggregation of AuNPs did not occur. The present approach is selective to arsenic and is much faster and simpler than the existing methods without the requirement of expensive and complicated instruments.

2. Experimental

2.1 Reagents and materials

Peptides (γ-Glu-Cys)₃-Gly-Arg and (γ-Glu-Cys)₃-Gly were synthesized and purified by ChinaPeptides Co., Ltd (Shanghai, China). Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), trisodium citrate and HAuCl₄ were obtained from Sigma-Aldrich. 2,6-Pyridinedicarboxylic acid (PDCA) and other chemicals were analytical-grade reagents and were used without further purification. The stock solution of 60 μM peptide was prepared by dissolving an appropriate amount of peptide in 500 μM TCEP solution. Deionized water was purified by a Millipore system (Simplicity Plus, Millipore Corp.). Unless otherwise noted, the reactions were conducted at room temperature.

2.2 Synthesis of AuNPs

All glassware used in the following procedures was cleaned in a bath of freshly prepared 1 : 3 HNO₃–HCl, rinsed thoroughly with water and dried in air prior to use. The citrated-stabilized AuNPs were prepared using a trisodium citrate reduction method as reported previously.³⁹ Briefly, trisodium citrate (5 mL, 38.8 mM) was rapidly added to a boiling solution of HAuCl₄ (50 mL, 1 mM), and the solution was boiled continually for an additional 30 min to yield a wine-red solution. The solution was filtered by a 0.45 μm membrane filter to remove the precipitate. The filtrate was stored in a refrigerator at 4 °C for use.

2.3 Detection of As^III

20 μL solution of peptide (60 μM) and 10 μL solution of As^III with different concentrations were added successively to 970 μL of 5 mM borate buffer. Then 500 μL dispersion of AuNPs was introduced to the peptide-containing mixed solution. The incubation time for the AuNPs with peptide was 1 min. The color change and absorption spectra were observed by the naked eye and recorded with a UV/Vis spectrometer, respectively.

For a real sample assay, tap water and pond water were collected and filtered with a 0.45 μm membrane filter. After that, 500 μL of tap/pond water and 500 μL dispersion of AuNPs were added successively to 500 μL of 10 mM borate buffer containing 2.4 μM PC₃R probe. The amounts of arsenic determined by AuNPs sensors were further verified by graphite furnace AAS.

2.4 Characterizations

Photographs were taken with a Sony Cyber-shot digital camera. The UV/Vis spectra were recorded using a Cary 50 spectrophotometer with a 1 cm quartz spectrophotometer cell. The morphology of AuNPs was observed using a FEI Tecnai G2 T20 transmission electron microscope (TEM).

3. Results and discussion

3.1 Mechanism of colorimetric assay for arsenic

The surface adsorption of thiol-containing ligands on AuNPs via Au–S bonds, such as dithiothreitol (DTT), mercaptoacetic acid (MPA), cysteine (Cys), DNA and peptides, has been well documented in the literature.^{10,13,20,32,40} As^III has a high binding affinity to thiol groups. For example, the stability constant of the As^III–DTT complex is about 30 orders of magnitude higher than that of other interfering metal ions, such as Cd, Cu, Hg, Ni, Pb and Zn.¹³ Phytochelatins (PCs) are oligomers of glutathione with the general structure (γ-Glu-Cys)_n-Gly abbreviated PC_n (n = 2–11). They are found in plants, fungi, nematodes as well as all groups of algae including cyanobacteria and exert their function in detoxifying heavy metals, such as As^III, Cd^II, Hg^II and Cu^I.^{37,38,41–43} Among these metal ions, As^III typically forms a three-coordinate trigonal-pyramidal complex by binding to three thiol groups.^36–38,44 We first attempted to use a multithiol peptide PC₃ as a cross-linker for AuNPs. As a result, no obvious color change was observed in the presence of micromolar concentration of PC₃. Hence, the peptide probe PC₃R was designed and employed for the assay of As^III in this work. This peptide consists of two important parts: one is the Au/As^III binding part containing the (γ-Glu-Cys)₃ residues with tricysteine; the other is the cross-linking part containing an arginine residue. In the absence of As^III, the peptide probe can be anchored on the citrate-stabilized AuNPs surface via three Au–S bonds. The positively charged guanidine group of the arginine residue interacts with the negatively charged carboxyl group of the glutamate residue on the AuNPs surface, resulting in AuNPs aggregation and a corresponding red-to-blue color change (Fig. 1). In the presence of As^III, the peptide probe binds to As^III through the strong coordination of all three cysteine residues with As^III, preventing the peptide from anchoring on the AuNPs surface via Au–S bonds. Consequently, AuNPs are stable and the solution remains red. We demonstrated that arsenic can be detected using the color change of AuNPs suspension.

Fig. 1 Schematic illustration of the strategy of As^III detection based on a dual-functional peptide probe and unmodified AuNPs.

3.2 Colorimetric assay for arsenic

As shown in Fig. 2, the AuNPs solution appeared red in color and exhibited an absorption peak at 520 nm (A₅₂₀) (red curve), which was ascribed to the surface plasmon resonance of the AuNPs. Upon the addition of PC₃R, the color of the solution changed from red to blue. Meanwhile, the original absorbance of AuNPs at 520 nm decreased while a new absorbance peak at ∼640 nm (A₆₄₀) increased obviously (blue curve). This indicates that the aggregation of AuNPs was induced by PC₃R. We also found that the A₆₄₀ increased and reached a plateau value within 1 min after addition of AuNPs suspension to PC₃R solution. In the case of As^III detection, PC₃R was first mixed with As^III and then the AuNPs were added to the mixed solution. As a result, the color of AuNPs remained red and only one absorption peak at 520 nm was observed (green curve), demonstrating the good dispersion of AuNPs in the As^III–PC₃R solution. Note that As^III alone did not cause the changes in the color and absorbance of the AuNPs (black curve). These results were further confirmed by the TEM observations: the significant aggregation of AuNPs in the presence of PC₃R alone and the monodisperse AuNPs in the presence of PC₃R and As^III. Moreover, the dependence of absorbance change of AuNPs on the PC₃R concentration was also examined. As shown in Fig. 3A, ΔA^′₆₄₀ (the absolute value of absorbance change at 640 nm) increased with an increase in the PC₃R concentration, which is indicative of the dependence of the aggregation of AuNPs on PC₃R. A linear relationship was found between ΔA₆₄₀ and PC₃R concentration over the range 0.05–0.8 μM. Therefore, the concentration of PC₃R was kept at 0.8 μM for the quantitative analysis of As^III.

Fig. 2 Visual color change (A), UV/Vis absorption spectra (B) and TEM images (C) of AuNPs with the PC₃R probe in the absence and presence of As^III. The concentrations of PC₃R and As^III are both 1 μM.

Fig. 3 The effect of PC₃R concentration (A), pH (B) and iron strength from NaNO₃ (C) on the absorbance change at 640 nm. In panel (A), the concentrations of PC₃R are 0.05, 0.10, 0.20, 0.40, 0.60, 0.80, 1.00, 1.20 and 1.50 μM. In panels (B) and (C), the concentrations of PC₃R and As^III are 0.8 and 1 μM, respectively.

Solution pH affects not only the stability of AuNPs and the binding of As^III to PC₃R, but also the electrostatic interactions between guanidine groups and carboxyl groups. Therefore, the effect of pH on the A₆₄₀ was carried out ranging from pH 6.0 to 8.0. ΔA₆₄₀ (the absorbance change at 640 nm (A⁰₆₄₀ − A^′₆₄₀), where A⁰₆₄₀ and A^′₆₄₀ represent the absorbance of AuNPs treated with PC₃R alone and treated with PC₃R and As^III, respectively) was used here to evaluate the performances of the sensor. As shown in Fig. 3B, ΔA₆₄₀ reaches maximum at pH 6.8. Thus, we chose a pH 6.8 borate buffer solution as the reaction media. Moreover, the instability of AuNPs in high-salt environments prevents their use in some applications. We found that the As^III–PC₃R solution caused a significant decrease in ΔA₆₄₀ when it contained 40 mM NaNO₃ due to the spontaneous aggregation of AuNPs at high NaNO₃ concentrations.

3.3 Selectivity of AuNPs to arsenic

To further confirm that the above mentioned changes were caused by the formation of PC₃R–As^III, we studied the effect of other ions. As shown in Fig. 4, no other ions, except for Pb^II, prevented the PC₃R-induced color change from red to blue. Although phytochelatin can also bind to other metal ions, such as Cd^II, Cu^I, Hg^II and Zn^II, the corresponding absorbance data indicate that these metal ions caused a negligible absorbance response. There are two reasons for these results: (1) the stability constant of thiol–As^III is much higher than that of these interfering metal ions,^13,37 and (2) these interfering metal ions cannot bind to all three thiol groups of PC₃R to form a stable metal–PC₃R complex in 1 : 1 stochiometric ratio,^37,38,41,42 allowing PC₃R to locate on the AuNPs surface via Au–S bonds. Moreover, we found that the interference of Pb^II could be masked by using PDCA as a complexation reagent (Fig. 4B), whereas PDCA showed no interference for the assay of As^III.^13,28 The mixtures of different metal ions did not interfere in the detection of As^III, further indicating that the present colorimetric method is selective for As^III detection. We also found that this method can be used to detect As^V. This is understandable because thiol species can reduce As^V to As^III and the resulting disulfide product can be reduced to cysteine residues by TCEP in the solution.^44–47 Thus, the present strategy developed in this study can realize the simultaneous detection of As^III as well as As^V and is promising to detect total As in groundwater.

Fig. 4 The selectivity of the As sensing system. (A) Color change of AuNPs with the PC₃R probe in the presence of different metal ions. (B) The absorbance change at 640 nm for metal ions. The concentrations of As and interfering metal ions are 1 and 20 μM, respectively.

3.4 Sensitivity of AuNPs to arsenic

To further evaluate the analytical performance of the developed method for As^III assay, different amounts of As^III were added to the PC₃R solution before the addition of AuNPs. Fig. 5A shows the color change of AuNPs–PC₃R samples with the increase of As^III concentration. The colorimetric assay allows a detection concentration as low as 0.2 μM for a rapid and reliable visualization of As^III by comparison with the blank sample. The concomitant change of the absorbance was monitored by UV/Vis spectroscopy (Fig. 5B). It is clearly seen that an increase in the As^III concentration induces a decrease of the absorbance at 640 nm. The dependence of ΔA₆₄₀ on the As^III concentration is presented in Fig. 5C. ΔA₆₄₀ increases linearly with [As^III] between 0.04 μM and 0.8 μM, which can be expressed using ΔA₆₄₀ = 0.675[As^III](μM) − 0.023 (R = 0.99). The detection limit (3σ) of the method was estimated to be 20 nM (n = 11), which is comparable to (or even lower than) that obtained by conventional analytical methods.⁷ The World Health Organization's (WHO) standard limit for As^III is 10 ppb (0.13 μM).⁴⁸ Thus, the present strategy developed in this study is promising to determine the levels of arsenic in groundwater. Moreover, ΔA₆₄₀ becomes constant beyond 0.8 μM As^III which is equivalent to that of PC₃R (Fig. 3), indicating a 1 : 1 stoichiometry between As^III and PC₃R.

Fig. 5 (A) Color change with an increase in the As^III concentration. (B) Absorbance response for different concentrations of As^III. (C) Calibration curve for the detection of As^III over the range of 0.04–1.2 μM.

3.5 Detection of As^III in real samples

To demonstrate the viability of the assay to measure the arsenic content in groundwater, we measured the amount of arsenic in tap water and pond water. As shown in Table 1, no detectable arsenic was found in tap water; thus, As^III was added into the tap water and then analyzed. The amount of arsenic in pond water was found to be 268 nM. To compare the data with well-established techniques, we also measured the arsenic content using graphite furnace AAS. The sensitivity of AAS is still not sufficient to measure the arsenic content in tap water, whereas the amount of arsenic in pond water was found to be 275 nM, which is comparable with the colorimetric assay data. These results confirm that the present method is applicable for arsenic detection in real samples.

Table 1 Determination of arsenic in tap water and pond water using AuNPs sensors and AAS

Sample	Amount^a (nM)	Amount^b (nM)	Added (nM)	Amount^a (nM)	Amount^b (nM)
a Detected by AuNPs sensors. b Detected by AAS.
Tap water	—	—	100	99.2 ± 5.8	98.7 ± 6.7
Pond water	268 ± 18.5	275 ± 27.8	200	466 ± 24.5	473 ± 29.8

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4. Conclusions

In summary, we described a simple, fast and selective colorimetric assay for arsenic based on a dual-functional peptide probe and unmodified AuNPs. The peptide probe selectively coordinated to arsenic, thus preventing the peptide-triggered AuNPs aggregation and color change from red to blue. The analytical merits of the developed approach (e.g., dynamic range, selectivity, and detection level) were also evaluated, and a detection limit of 20 nM was achieved. The theoretical simplicity and high selectivity of this method facilitated the analysis of arsenic in tap water and pond water. This method will be valuable for the design of new types of metal ion sensors and likely lead to many colorimetric detection applications in environmental monitoring.

Acknowledgements

Partial support of this work by the National Natural Science Foundation of China (21205003), the China Scholarship Council (2009637056), the Graduate Degree Thesis Innovation Foundation of Hunan Province (1960-71131110015) and the Science & Technology Foundation of Henan Province (122102310517) is gratefully acknowledged.

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