Highly Sensitive and Selective Colorimetric Detection of Methylmercury Based on DNA Functionalized Gold Nanoparticles

Abstract

A new colorimetric detection of methylmercury (CH₃Hg⁺) was developed, which was based on the surface deposition of Hg enhancing the catalytic activity of gold nanoparticles (AuNPs). The AuNPs were functionalized with a specific DNA strand (H_T7) recognizing CH₃Hg⁺, which was used to capture and separate CH₃Hg⁺ by centrifugation. It was found that the CH₃Hg⁺ reduction resulted in the deposition of Hg onto the surface of AuNPs. As a result, the catalytic activity of the AuNPs toward the chromogenic reaction of 3,3,5,5-tetramethylbenzidine (TMB)-H₂O₂ was remarkably enhanced. Under optimal conditions, a limit of detection of 5.0 nM was obtained for CH₃Hg⁺ with a linear range of 10–200 nM. We demonstrated that the colorimetric method was fairly simple with a low cost and can be conveniently applied to CH₃Hg⁺ detection in environmental samples.

1. Introduction

Mercuric ions widely exist in the environment and have distinct toxic effects on human beings. Organic forms of mercury (Hg) demonstrate much higher toxicity than inorganic Hg due to their higher lipophilicity and easier bioaccumulation through the food chain, such as in the tissue of fish [1,2]. The main organic species of mercury, methylmercury (CH₃Hg⁺), has been recognized as a potent neurotoxin that causes damage to the brain and nervous system [1,3]. Due to the severe effects of mercury, the U.S. Environmental Protection Agency has set a maximum level (10 nM, 2 ppb) for mercury species in drinking water [1]. Usually, complex hyphenated techniques, such as high performance liquid chromatography (HPLC) or gas chromatography (GC), coupled to specific detectors, such as mass spectrometry (MS), inductively coupled plasma mass spectrometry (ICP-MS) or atomic fluorescence spectrometry (AFS), are required for methylmercury detection [4,5,6]. However, these techniques generally require expensive instruments and are time-consuming and costly. To overcome the limitation of the above methods, recently, nanomaterial-based assays have been widely used for developing rapid and cost-effective methods for the detection of various heavy metal ions in environmental and biological samples [7,8,9]. Due to the strong metallophilic interactions between Hg²⁺ and some other metallic atoms, such as gold and silver, numerous metallic nanoparticle-based assays for Hg²⁺ have been developed [10,11,12,13,14,15,16]. However, there are fewer nanomaterial-based assays for CH₃Hg⁺ compared to Hg²⁺ ions [17,18,19], which is probably due to the weak interactions between CH₃Hg⁺ and metal nanomaterials.

Only a few studies have reported the development of nanomaterial-based detection methods for CH₃Hg⁺. For example, Chen at el. developed a colorimetric nanosensor for mercury speciation, which was based on the analyte-induced aggregation of gold nanoparticles (Au NPs) with the assistance of a thiol-containing ligand of diethyldithiocarbamate (DDTC) [18]. Pandeeswar et al. presented a novel optoelectronic approach for detection of Hg²⁺ and CH₃Hg⁺, which was based on nanoarchitectonics that consists of an adenine (A)-conjugated small organic semiconductor (BNA) and deoxyribo-oligothymidine (dTn) [20]. However, this device cannot distinguish Hg²⁺ from CH₃Hg⁺. Recently, Deng et al. reported that a DNA strand, H_T7, can bind to CH₃Hg⁺ with a higher K_b value of (5.57 ± 0.47) × 10⁶ M⁻¹ compared to that of Hg²⁺ ((1.51 ± 0.18) × 10⁶ M⁻¹) [19]. Based on this, they were able to discriminate between CH₃Hg⁺ and Hg²⁺ ions by forming Ag/Hg amalgam with a CH₃Hg⁺-specific fluorophore-labeled DNA probe and fabricated a highly selective fluorescent assay for CH₃Hg⁺. More recently, Yang et al. designed a specific visual detection method for CH₃Hg⁺ and ethylmercury based on DNA-templated alloy Ag/Au NPs [21]. However, this visual detection method for methylmercury and ethylmercury requires subtle temperature adjustments and its sensitivity was above the micromolar level. Thus, developing a simple and selective colorimetric assay for CH₃Hg⁺ is still an important and difficult task.

Recently, some methods for the detection of Hg²⁺ were reported, which were based on the peroxidase-like activity of the AuHg alloy NPs. For example, Long et al. [22] found that AuNPs possess excellent peroxidase-like activity after the deposition of Hg²⁺ onto the surface of AuNPs. The peroxidase-like activity enhancement of AuNPs, after Hg⁰ deposition onto the surface of AuNPs, was suggested to be the result of the accelerated decomposition of H₂O₂ and the stabilization of hydroxyl radicals on the surface of AuNPs. This phenomenon can be applied in the development of colorimetric and fluorescent assays for Hg²⁺ [22,23,24]. Our group also reported that catalytic DNA-AuNPs and DNA-Ag/Pt nanoclusters can be used to detect Hg²⁺ with high selectivity and sensitivity by stimulating or inhibiting their peroxidase-like activity [25,26]. Interestingly, compared with citrate stabilized AuNPs, AuNPs functionalized with a T-rich DNA strand can obviously improve the selectivity and can simplify the sample pretreatment for the colorimetric detection of Hg²⁺ [25]. However, to the best of our knowledge, there is no report on the application of nanomaterial enzyme mimics in CH₃Hg⁺ detection. Herein, we found that CH₃Hg⁺ captured by the AuNPs functionalized with CH₃Hg⁺-specific DNA strands can be reduced by NaBH₄. This results in Hg deposition onto the surface of AuNPs, thus stimulating the peroxidase-like activity of the AuNPs. Based on this finding, a highly sensitive and selective colorimetric assay for CH₃Hg⁺ was developed.

2. Materials and Methods

2.1. Chemicals and Materials

HAuCl₄, CH₃Hg⁺Cl, NaBH₄, 3,3,5,5-Tetramethylbenzidine (TMB) and H₂O₂ (30%) were purchased from Aladdin Reagent (Shanghai, China). The single-strand oligonucleotides were obtained from Sangon Biotech (Shanghai, China) and the sequences of these DNA strands were listed in

Table 1. Oligonucleotide Sequences Used in This Work ^a.

Type	Sequence
HT5	5′-SH-CTTTGTTAAAAATTCTTTG-3′
HT7	5′-SH-GTTCTTTGTTAAAAATTCTTTGTTC-3′
HT9	5′-SH-TTGTTCTTTGTTAAAAATTCTTTGTTCTT-3′
HR	5′-SH-CTGCTGCTGCAAAAAGCAGCAGCAG-3′

^a H_T5, H_T7 and H_T9 represent CH₃Hg⁺-specific DNA with different T bases, while H_R represents random DNA.

. Hg(NO₃)₂ and all the other metal salts were purchased from the National Institute of Metrology (Beijing, China). All of the reagents used were of analytical grade. Ultra-pure water prepared with a Milli-Q Pure system was used for all experiments.

2.2. Synthesis of AuNPs and the Modification by DNA Strands

The AuNPs were prepared through the citrate-mediated reduction of HAuCl₄ [24]. Briefly, HAuCl₄ (0.01%, 100 mL) was added to a flask, which had been washed with aqua regia and ultra-pure water. After the solution was heated to boiling, sodium citrate (1.0%, 2.0 mL) was quickly added with stirring. When we observed a color change in the mixture to wine red, the mixture was further boiled for another 5 min and cooled to room temperature. The diameter of AuNPs was about 15 nM and their concentration was estimated to be 3 nM.

The DNA modification of the AuNPs was achieved by directly incubating thiolated single-strand DNA (H_T7) with the AuNPs. Briefly, the AuNPs (3 nM, 990 μL) and thiolated DNA (100 μM, 5 μL) were mixed together and incubated at an ambient temperature for 24 h. After this, the mixture was centrifuged for 15 min at 10,000× g rpm and excessive DNA strands were removed. After repeating the centrifugation once, the obtained DNA-AuNPs complex was resuspended in phosphate buffer (10 mM, pH of 7.0) and stored at 4 °C.

2.3. Colorimetric Detection of CH₃Hg⁺

To 25 μL of DNA-AuNPs complex (0.6 nM), 175 μL of Tris-HNO₃ buffer (5.0 mM, pH 7.0) and 500 μL of CH₃Hg⁺ solution at different concentrations were added. After being incubated for 10 min, the mixtures were centrifuged at 10,000 rpm for 15 min and the supernatants were discarded. To the 50 μL of retained mixture, we added 50 μL of NaBH₄ (1.0 mM). After being incubated for another 10 min, 90 μL of citrate buffer (100 mM, pH 4.5), 100 μL of TMB (1.5 mM) and 60 μL of H₂O₂ (1.5 M) were transferred into the solution. The catalytic reaction was subsequently recorded at 650 nm by a microplate reader (PowerWave XS₂, Bio-Tek, Winooski, VT, USA) after 10 min. For detection of CH₃Hg⁺ in lake water, the samples were filtered through microfiltration membranes and measured by the above method.

3. Results and Discussion

3.1. Characterization of AuNPs and DNA-AuNPs Complex

Figure 1 shows that the UV–vis absorption spectra of the AuNPs has a maximum absorption peak (λ_max) at 520 nm. After the AuNPs were modified with H_T7, which is a CH₃Hg⁺ recognition DNA strand, the λ_max of the AuNPs experienced a red shift to 522 nm. This result suggested that the DNA-AuNPs complex (H_T7-AuNPs) was obtained. The H_T7-AuNPs complex was stable in 0.15 M NaCl (the inset in Figure 1), which also indicated the successful preparation of the H_T7-AuNPs.

3.2. Colorimetric Detection of CH₃Hg⁺

As shown in Figure 2, AuNPs-H_T7 demonstrated weak catalytic activity and we only found a weak signal with a peak at 650 nm. After being captured by the H_T7 strand, CH₃Hg⁺ species can be deposited onto the surface of AuNPs through Au/Hg amalgamation since they can be reduced to Hg⁰ by NaBH₄ [19,27,28]. In the above process, the catalytic activity of AuNPs-HT₇ was obviously increased, which was supported by the appearance of a strong signal at 650 nm. This was due to the oxidation of TMB by the hydroxyl radical that is stabilized on the surface of AuNPs, which produces a blue one-electron oxidation product (i.e., cation free-radical, TMB⁺) [29]. The reaction is shown in Figure S1. This change in peroxidase-like activity of the AuNPs suggests the deposition of Hg onto the surface of AuNPs [22,25]. The CH₃Hg⁺ sensing mechanism is depicted in Scheme 1.

Since the number of T-T pairs may affect the response of the AuNPs-ssDNA complex to CH₃Hg⁺, H_T5, H_T7 and H_T9 strands were used to modify the AuNPs, respectively, before we carried out a comparison of these AuNPs-ssDNA complexes. These AuNPs-ssDNA complexes were also characterized by UV–vis spectra, which demonstrated the same change compared with the AuNPs. As shown in Figure 3, H_T7 modified AuNPs demonstrated more sensitive responses to CH₃Hg⁺ compared to H_T5, H_T9 or H_R modified AuNPs. This result suggested that the higher affinity of DNA strand to CH₃Hg⁺ over Hg²⁺ was still the main factor determining the selectivity of this probe [19].

The enhanced peroxidase-like activity of AuNPs caused by CH₃Hg⁺ was further applied in the development of a colorimetric assay for CH₃Hg⁺. As shown in Figure 4, the absorbance increased as the CH₃Hg⁺ concentration increased in the range of 0–1000 nM. A good linear relationship between CH₃Hg⁺ concentration and absorbance values can be obtained in the range of 10–200 nM. The limit of detection (3-fold signal to noise, S/N = 3) was evaluated to be 5.0 nM.

Some common metal ions were tested in this colorimetric assay. As shown in Figure 5, most of common metal ions at a 20-fold higher concentration and the same concentration of Hg²⁺ showed very weak responses. On the contrary, when citrate-stabilized AuNPs were incubated with Hg²⁺ or CH₃Hg⁺ ions, almost the same catalytic enhancement of AuNPs was observed (Figure S2). The above results clearly demonstrated the good selectivity of this colorimetric assay for CH₃Hg⁺, which was mainly due to the two aspects: (1) CH₃Hg⁺-specific DNA scaffold has much higher affinity to CH₃Hg⁺ (a K_b value of (5.57 ± 0.47) × 10⁶ M⁻¹) compared to Hg²⁺ ((1.51 ± 0.18) × 10⁶ M⁻¹); and (2) the centrifugation and separation of AuNPs-H_T7 enriched CH₃Hg⁺ over Hg²⁺. It also should be pointed out that Hg²⁺ has good affinity with AuNPs. However, the DNA strand on the AuNPs will interact with Hg²⁺ and thus, will eventually affect the deposition of Hg⁰. In this case, the H_T7 strands probably hindered the deposition of Hg²⁺.

The sensitivity of the proposed method was higher than the two typical colorimetric methods [18,21] and comparable with some typical nanosensors or chemosensors (

Table 2. Summarize of some typical method of CH₃Hg⁺.

Method	Probe	Limit of Detection	Linear Range	Selectivity to Hg2+	Sample	Ref.
Fluorescent	Lys VI-AuNCs	CH3Hg+: 3 pM Hg2+: 4 nM	CH3Hg+: 15–500 nM; Hg2+: 10−5000 pM		seawater	[1]
Upconversion fluorescence	hCy7-UCNPs	0.8 ppb	0–7 μM;	Not clear	cells	[17]
Colorimetric	Diethyldithiocarbamate-AuNPs	CH3Hg+: 15 nM Hg2+: 10 nM	CH3Hg+: 0.03–0.8 μM; Hg2+: 0.01–0.1 μM	EDTA can mask Hg2+	drinking water	[18]
Fluorescent sensing by in-situ synthesis	carbon dots	5.9 nM	23–278 nM	tolerate with 250-fold Hg2+	River/sea water a	[30]
Fluorescent sensing by in-situ synthesis	Silver nanocluster	0.4 nM	2.0 nM–12.0 μM	tolerate with 50-fold Hg2+	Fish sample	[19]
chiro-optical	adenine -small organic semiconductor and oligothymidine	CH3Hg+/Hg2+: 0.1 nM	1–1000 nM	-	water	[20]
AIE-based fluorescence	tetraphenylethylene–monoboronic acid	CH3Hg+/Hg2+: 0.12 ppm	0.6–30 ppm	-	Fish muscle	[31]
Colorimetric	DNA-Templated Ag–Au nanoparticles synthesis	0.5 μM	0–200 μM	tolerate with 50-fold Hg2+	Fish muscle	[21]
Colorimetric	DNA-AuNPs	5 nM	20–500 nM	tolerate with 1-fold Hg2+	Lake water	This work

^a cleanup using C18 cartridges.

) [1,17,30,31]. However, the selectivity of this method needs to be further improved when compared with the established colorimetric [21] and fluorescent methods [19,30].

The real water samples from the Li Lake in Wuxi, Jiangsu Province were obtained and spiked with different concentrations of CH₃Hg⁺ (20 nM, 50 nM and 100 nM). As shown in

Table 3. Detection of CH₃Hg⁺ in real water samples (n = 3).

Water Sample	Added (nM)	Mean Found (nM)	Mean Recovery (%)
1	20	19.1 ± 0.9	95.5%
2	50	46.8 ± 2.3	93.6%
3	100	102.1 ± 3.7	102.1%

, the recovery of the added CH₃Hg⁺ with the colorimetric method was in the range of 93.6–102.1%, which demonstrated the reliability of this assay for the detection of CH₃Hg⁺ in real samples.

4. Conclusions

In summary, we developed a highly sensitive and selective colorimetric method for the detection of CH₃Hg⁺, which was based on the surface deposition of Hg enhancing the catalytic activity of AuNPs. The limit of detection was 5.0 nM with a linear range of 10–200 nM. This colorimetric method has potential in the detection of CH₃Hg⁺ in environmental samples since it also demonstrated other advantages of being simple, rapid and cost-effective. However, this method needs to be further improved with respect to its selectivity to Hg²⁺. This probably can be further improved through adopting magnetic core gold shell nanocomposites due to their more convenient separation and enrichment capability.