Specifically and visually detect methyl-mercury and ethyl-mercury in

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Specifically and visually detect methyl-mercury and ethyl-mercury in fish sample based on DNA-templated alloy Ag-Au nanoparticles Zhiqiang Chen, Xusheng Wang, Xian Cheng, WeiJuan Yang, Yongning Wu, and FengFu Fu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01100 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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Analytical Chemistry

Specifically and visually detect methyl-mercury and ethyl-mercury in fish sample based on DNA-templated alloy Ag-Au nanoparticles Zhiqiang Chen†, Xusheng Wang†, Xian Cheng†, Weijuan Yang*‡, Yongning Wu§, FengFu Fu*† †

Key Lab of Analysis and Detection for Food Safety of Ministry of Education, Fujian Provincial Key Lab of Analysis and Detection for Food Safety, College of Chemistry, Fuzhou University, Fuzhou, Fujian 350116, China. ‡

State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, 350002, P.R. China § China National Center for Food Safety Risk Assessment, Beijing 100022, China ABSTRACT: Methyl-mercury (CH3Hg+) and ethyl-mercury (C2H5Hg+) have much higher toxicity than Hg2+ and can be more easily accumulated by organisms to form severe bio-amplification. Hence, the specific and on-site detection of CH3Hg+ and C2H5Hg+ in seafood is of great significance and a hard challenge. We herein designed two T-rich aptamers (HT5 and HT7) for specifically recognizing CH3Hg+ and the total of CH3Hg+ and C2H5Hg+, respectively. In the presence of all Au3+, Ag+ and T-rich aptamer, CH3Hg+ and C2H5Hg+ specifically and preferentially bind with aptamer and thus induced the formation alloy Ag-Au nanoparticles after reduction, which lead to the color change in solution. This provided a sensing platform for the instrument-free visual discrimination and detection of CH3Hg+ and C2H5Hg+. By using HT5 as probe, the method can be used to detect as low as 5.0 µM (equivalent to 1.0 µg Hg/g) of CH3Hg+ by bare eye observation and 0.5 µM (equivalent to 100 ng Hg/g) of CH3Hg+ by UV-visible spectrometry. By using HT7 as probe, the method can be used to detect the total concentration of CH3Hg+ and C2H5Hg+ with a visual detection limit of 5.0 µM (equivalent to 1.0 µg Hg/g) and a UV-visible spectrometry detection limit of 0.6 µM (equivalent to 120 ng Hg/g). The proposed method has been successfully used to detect CH3Hg+ and C2H5Hg+ in fish muscle samples with a recovery of 101-109% and a RSD (n=6) < 8%. The success of this study provided a potential method for the specific and on-site detection of CH3Hg+ and C2H5Hg+ in seafood by only bare eye observation.

Mercury and its compounds are a class of highly toxic and ubiquitous pollutants impacting on human and ecosystem health,1 and therefore is one of the most studied pollutants. Although many countries have made great efforts to reduce mercury emission, more than 2000t of mercury are emitted into the environment annually from anthropogenic sources.2 Nowadays, the mercury in the aquatic environments attracted increasing attention since the mercury released into the aquatic environment can be converted into organic mercury especially methyl-mercury (CH3Hg+) and ethyl-mercury (C2H5Hg+), which has much higher toxicity and can be more easily accumulated by the organisms to form severe bio-amplification.3,4 Therefore, seafood especially higher predatory fish can have up to 106-fold higher mercury concentrations than the ambient water, and up to 95% of the mercury is organic mercury.4 Hence, a more strict concentration limitation in the aquatic environment and seafood was established for methyl-mercury than inorganic mercury (Hg2+) by World Health Organization (WHO).4 For above reasons, it is mandatory to respectively determine total mercury and methyl-mercury in seafood in many countries. Currently, the main methods used for the speciation analysis of Hg2+, CH3Hg+ and C2H5Hg+ are all based on the combination of efficient separation technology and sensitive elementselective detectors. For example, high performance liquid chromatography (HPLC) or gas chromatography (GC) or capillary electrophoresis (CE) coupled with inductively coupled

plasma mass spectrometry (ICP-MS) or atomic fluorescence spectrometry (AFS).5-12 However, all above hyphenated techniques not only required sophisticated and costly instruments but also required skillful operator and long analysis time, and thus incompetent for the rapid and on-site detection of organic mercury in seafood.13 To overcome the limitation of hyphenated techniques, various methods including colorimetric methods, fluorescent methods and electrochemical methods have been developed for the rapid detection of inorganic mercury (Hg2+) or total mercury by using DNA aptamer or organic compound as recognition probes.14-20 However, these methods can only determine inorganic mercury (Hg2+) or total mercury, and thus can't be used for the speciation analysis of CH3Hg+ and C2H5Hg+. So far, the method especially colorimetric method for the specific and rapid detection of CH3Hg+ and C2H5Hg+ has been seldom reported since it is very difficult to obtain the specific recognition probe of organic mercury. DNA sequences containing T bases can combine with Hg2+ to form T-Hg2+-T, and therefore many biosensors have been developed for the rapid and sensitive detection of Hg2+ by using DNA sequences containing T bases as recognition probe.14,15,21,22 Recent studies demonstrated that DNA sequences containing T bases not only can combine with Hg2+ but also can combine with CH3Hg+ and C2H5Hg+, and the binding force of DNA sequences to different mercury species is different and tunable by altering the number and locations of T bases.23 Based on above discovery, a fluorescent method has

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been developed for the specific and rapid detection of CH3Hg+ by using a dye-labeled T-rich DNA as probe.23 Inspired by the discovery mentioned above, we herein develop a colorimetric method for the visual discrimination and detection of CH3Hg+ and C2H5Hg+ by using specific T-rich DNA as probe and alloy Ag-Au nanoparticles as signal, in hope of providing a facile, rapid, specific and cost-effective method for the specific and on-site detection of CH3Hg+ and C2H5Hg+ in seafood.

EXPERIMENTAL SECTION Specific and colorimetric detection of methyl-mercury. Firstly, 10 µL of 0.2 mM Ag+ solution and 10 µL of CH3Hg+ standard solution or sample solution were added into a 200 µL centrifuge tube in which containing 20 µL of 75 µM HT5 DNA solution (the sequences of HT5 see Table S1 in supporting information, SI). The whole was mixed and incubated at 0°C for 60 min to form DNA-CH3Hg+ conjugates. The 150 µL of TrisHNO3 buffer (pH 8.0) and 5 µL of 25 mM NaBH4 was added successively, and the whole was mixed gently. Subsequently, the mixture was incubated at 25 °C for 8 minutes without vortex to reduce Ag+ to Ag0. Then, 5 µL of 60 mM HAuCl4 solution was added to the mixture on the ice box, and the mixture was incubated at 95°C to reduce Au3+ to Au0 to form alloy AgAu nanoparticles. After reacting for 10 min, the color change of the solution was observed with bare eye and the UV-visible absorption spectrum of the solution was determined with microplate reader in the range of 400-800 nm. The concentration of CH3Hg+ was quantified based on the absorption at 550 nm (A550) or bare eye observation. Specific and colorimetric detection of the total of methylmercury and ethyl-mercury. Firstly, 10 µL of 0.2 mM Ag+ solution and 10 µL of CH3Hg+ or C2H5Hg+ standard solution (or sample solution) were added into a 200 µL centrifuge tube in which containing 20 µL of 75 µM HT7 DNA solution (the sequences of HT7 see Table S1 in SI). The whole was mixed and incubated at 0°C for 60 min to form DNA-CH3Hg+ or DNA-C2H5Hg+ conjugates. The 150 µL of Tris-HNO3 buffer (pH 8.0) and 5 µL of 25 mM NaBH4 was added successively, and the whole was mixed gently. Subsequently, the mixture was incubated at 25 °C for 8 minutes without vortex to reduce Ag+ to Ag0. Then, 5 µL of 60 mM HAuCl4 solution was added to the mixture on the ice box, and the mixture was incubated at 95°C to reduce Au3+ to Au0 to form Ag-Au alloy nanoparticles. After reacting for 10 min, the color change of the solution was observed with bare eye and the UV-visible absorption spectrum of the solution was determined in the range of 400-800 nm. The concentration of the total of CH3Hg+ and C2H5Hg+ was quantified based on the A550 or bare eye observation. Determination of dried fish muscle sample. All species of mercury in 0.5g dried fish muscle (Tapertail anchovy), which collected from coastal water of Fujian in China, was extracted and then concentrated to near dryness with the method reported in previous study,8 and finally was diluted to 2.0 mL TrisHNO3 buffer (pH 8.0) (detailed procedure see IS). The CH3Hg+ and the total of CH3Hg+ and C2H5Hg+ in final solution were separately detected with above method by using HT5 and HT7 as probe, respectively. The concentration of C2H5Hg+ was then calculated by subtracting the CH3Hg+ concentration from the total of CH3Hg+ and C2H5Hg+. Meanwhile, the CH3Hg+ and C2H5Hg+ in final solution were also detected with CEICP-MS (capillary electrophoresis-inductively coupled plasma mass spectrometry) according to the method reported in previ-

ous study to confirm the reliability of the proposed method.8 The samples spiked with different concentrations of CH3Hg+ and C2H5Hg+ were detected with the same manner to obtain recovery.

RESULTS AND DISCUSSION The principle for visually detecting methyl-mercury and ethyl-mercury. As we mentioned above, the DNA sequences containing T bases can bind mercury species with higher affinity, and the binding affinity of T-rich DNA to different mercury species is different and tunable by altering the number and locations of T bases.23,24 In general, the binding affinity of mercury species to T-rich DNA increases in order of Hg2+ 100 nm) (Fig. 2-c, d), indicating that the alloy Ag-Au nanoparticles were formed in solution after reduction since DNA preferentially bind with CH3Hg+ or C2H5Hg+ and keep Ag+ and Au3+ as free ions in solution. The EDX analysis reveals that the alloy Ag-Au nanoparticles contained trace Hg, this may be attributed to that a little of CH3Hg+ or C2H5Hg+ was adsorbed or wrapped in the alloy Ag-Au nanoparticles. Above deduction can be also demonstrated by the EDX mapping results (see Fig. S1 in SI). Optimization of the DNA sequence. As we mentioned above, the binding affinity of T-rich DNA to different mercury species is different and tunable by altering the number and locations of T bases.23,24 To realize the specific detection of CH3Hg+ and C2H5Hg+, we designed rationally five different Trich aptamers (Table S1 in SI) according to the principles of metallic ion-mediated stable base pairs.26-30 As the results shown in Fig. 3, among these sequences, when HT5 was used

Fig. 2. The TEM images and EDX analysis of nanoparticles observed in the assay system for detecting blank (a); Hg2+ (b), CH3Hg+ (c), C2H5Hg+ (d) by using HT7 as probe.

Optimization of other experimental conditions. To achieve the best analytical performance, other experimental conditions including the concentration of DNA aptamer, reducing agent, the pH of Tris-HNO3 buffer and Ag+ concentration were also optimized. To ascertain the optimal concentration of aptamer, we used HT7 as representative to optimize the


Analytical Chemistry DNA concentration in the range of 10 µM to 100 µM. As observed in Fig. S2 (see SI), CH3Hg+ and C2H5Hg+ cannot be specifically discriminated and detected from the color of solution when the DNA concentration is too low or too high. This is because that excess DNA will not only bind mercury species but also bind Au3+ and Ag+, whereas inadequate DNA cannot completely bind mercury species. When the DNA concentration is 75 µM, CH3Hg+ and C2H5Hg+ can be specifically detected by bare eye observation with the similar sensitivity. Therefore, 75 µM of DNA was the optimal selection and used in the experiment.

pH 8.0, the solution showed obvious difference in solution color between Hg2+ (colorless) and CH3Hg+ or C2H5Hg+ (purple). Thus, pH 8.0 was deemed as the optimum pH. The Ag+ concentration directly affect the formation of alloy Ag-Au nanoparticles, and thus affect the performance of the method. As observed in Fig. S5 (see SI), lower concentration of Ag+ prevented the formation of alloy Ag-Au nanoparticles or Ag/Au/Hg amalgam, and thus lead to a thin solution color. Whereas, higher concentration of Ag+ lead to the excess of Ag0 in alloy Ag-Au nanoparticles, which results in unconspicuous color change between Hg2+ and CH3Hg+ or C2H5Hg+. When Ag+ concentration is 0.2 mM, the solution showed obvious difference in color between Hg2+ (colorless) and CH3Hg+ or C2H5Hg+ (purple). Thus, 0.2 mM of Ag+ was used in this study.

H2 O Cd2+ Mg2+

Hg2+ CH 3Hg+ C2H 5Hg+

Zn2+ Co2+ Cu2+ Pb2+ Fe3+ Cr3+ Ni2+ 0.5

H T5

H2O + CH3Hg

3+

Fe

2+

Cu

+

C2H5Hg

0.4

2+

Hg

0.3

2+

Zn

2+

Mg

2+

Cd 3+ Cr

2+

Pb

2+

2+

Co

Abs

Ni

0.2 0.1 0.0 400

Fig. 3. The effect of aptamer sequences on the specific recognition for mercury species. (a) HT5; (b) HT7; (c) HT9; (d) HT10; (e) HT12. Other conditions are all under the optimal selection described in text.

The reductant used to reduce Ag+, Au3+ and Hg2+ to form Ag0, Au0 and Hg0 is another key consideration in this study. Three different reductants, NaBH4, trisodium citrate and ascorbic acid, were used to reduce Ag+, Au3+ and Hg2+ respectively. The experimental results (Fig. S3 in SI) clearly indicated that CH3Hg+ and C2H5Hg+ cannot be specifically discriminated and visually detected when trisodium citrate and ascorbic acid was used as reductant. This is because trisodium citrate and ascorbic acid have relatively low reducibility and thus cannot rapidly reduce Ag+ and Au3+ to form alloy Ag-Au nanoparticles. Only NaBH4 can lead to the notable distinction in solution color between CH3Hg+ or C2H5Hg+ and Hg2+, and thus NaBH4 was selected as reductant in this study. The pH of solution will affect the stability and redox potential of NaBH4, and thus affect the reduction of Ag+, Au3+ and Hg2+. The results (Fig. S4 in SI) revealed that the pH had an obvious effect on the formation of alloy Ag-Au nanoparticles, and thus affect the color change of solution and UV-visible absorption spectra. When the pH was 7.0 or 7.5, all the solutions were colorless in spite of the presence of mercury species or not. When the pH was 8.5 or 9.0, the solutions showed the same claret color between blank and organic mercury. Only at

500

600 700 Wavelength/nm

H2 O Cd2+ Mg2+

800

Hg2+ CH 3Hg+ C2H 5Hg+

Zn2+ Co2+ Cu 2+ Pb2+ Fe3+ Cr3+ Ni2+ 0.5

H T7

+

C2H5Hg

+

CH3Hg

0.4

2+

2+

Mg

2+

Cd

2+

Hg

Zn

H2O

Pb

2+

0.3

2+

3+

Co

2+

Cr

Fe Ni

Abs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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3+ 2+

Cu

0.2 0.1 0.0 400

500

600 700 Wavelength/nm

800

Fig. 4. The photographs and UV-visible absorption spectra for detecting various metal ions with HT5 and HT7 as probe, respectively. The concentration of Fe3+, Mg2+, Zn2+, K+, Pb2+, Co2+, Cr3+, Cd2+, Ni2+ and Cu2+ were all 10 mM, and that of Hg2+, CH3Hg+ and C2H5Hg+ is 100 µM.

The selectivity of the proposed method. Many metallic ions coexisting in seafood might interfere with the detection of CH3Hg+ and C2H5Hg+, so the selectivity of our method was


assessed by detecting various metallic ions including Fe3+, Mg2+, Zn2+, K+, Pb2+, Co2+, Cr3+, Cd2+, Ni2+ and Cu2+ in 100fold concentration versus mercury species. As results shown in Fig. 4, when HT5 was used as probe, only CH3Hg+ can lead to the formation of alloy Ag-Au nanoparticles, which results in a remarkable color change from colorless to purple in solution and a high absorption at 550 nm. The C2H5Hg+ showed a very thin purple color and a negligible absorption at 550 nm in comparison with CH3Hg+, indicating that the interference of C2H5Hg+ can be ignored since C2H5Hg+ generally is lower than CH3Hg+ in seafood and aqueous environment. No obvious color change and approximately no absorption at 550 nm were observed for other metallic ions even if their concentrations are higher than CH3Hg+. Above results indicated that HT5 has excellent specificity to CH3Hg+, and other metallic ions including Hg2+ and C2H5Hg+ do not interfere with the detection of CH3Hg+. When HT7 was used as probe, the obvious color change from colorless to purple in solution and a similar high absorption at 550 nm was observed for both CH3Hg+ and C2H5Hg+. No obvious color change and approximately no absorption at 550 nm were observed for other metallic ions including Hg2+ although their concentrations are higher than CH3Hg+ and C2H5Hg+, indicating that HT7 can specifically recognize CH3Hg+ and C2H5Hg+ and thus can be used for colorimetric detection of total of CH3Hg+ and C2H5Hg+. Thus, the concentration of C2H5Hg+ can be calculated by subtracting CH3Hg+ concentration obtained with HT5 from the total concentration of CH3Hg+ and C2H5Hg+ obtained with HT7, and thus realized the speciation analysis of CH3Hg+ and C2H5Hg+. The analytical performance of the proposed method. The color change and the A550 of the solution induced by CH3Hg+ and C2H5Hg+ were monitored with UV-visible spectrometry by using HT5 and HT7 as probe, respectively. As results in Fig. 5 showed, in the case of HT5, the color of the solution changed from colorless to deep purple step by step and correspondingly the A550 increased when the CH3Hg+ concentration increased from 0.0 to 200 µM. When the concentration of CH3Hg+ is 5.0 µM, the color change in solution can be definitely identified by bare eye observation, i.e. the visual detection limit of CH3Hg+ is 5.0 µM (equivalent to 1.0 µg Hg/g). The A550 showed a linear relationship with the CH3Hg+ concentration in the range of 0.0 to 200 µM, with an equation of A550=0.0015×C+0.0219, where C is the CH3Hg+ concentration with unit of µM. The detection limit (3σ/S) was calculated to be 0.5µM (equivalent to 100 ng Hg/g) for CH3Hg+ with UVvisible spectrometry. By using HT7 as probe, the color change in solution can be definitely identified by bare eye observation when CH3Hg+ or C2H5Hg+ concentration is 5.0 µM, i.e. the visual detection limit is 5.0 µM (equivalent to 1.0 µg Hg/g) for both CH3Hg+ and C2H5Hg+. Correspondingly, the A550 showed a linear relationship with the CH3Hg+ and C2H5Hg+ concentrations in the range of 0.0 to 200 µM, with an equation of A550=0.0014×C+0.0572 and A550=0.0013×C+0.0493, respectively (where C is the CH3Hg+ or C2H5Hg+ concentration with unit of µM). Above results indicated that the method has approximately the same sensitivity for CH3Hg+ and C2H5Hg+ when HT7 was used as probe, i.e. HT7 can be used to detect the total concentration of CH3Hg+ and C2H5Hg+. The detection limit (3σ/S) was calculated to be 0.6 µM (equivalent to 120 ng Hg/g) for the total of CH3Hg+ and C2H5Hg+ with UV-visible spectrometry.

By using HT5 and HT7 DNA sequence as probe, we can realize the specific and visual detection of CH3Hg+ and C2H5Hg+, respectively. CH 3Hg+

10

5

0

20

50

100

200

C2H 5Hg+ 0.5 CH3Hg+ y = 0.0015x + 0.0219 R2 = 0.9959 C2H5Hg+ y = 0.0004x + 0.0193 R2 = 0.987

0.4

0.3 A5 5 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.2

0.1

H T5 0

0

50

CH 3Hg+

100 150 Concentration (µM)

0

5

10

20

50

200

100

250

200

C 2H 5Hg+ 0.5 CH3Hg+ y = 0.0013x + 0.0572 +

C2H5Hg y = 0.0014x + 0.0493

0.4

R2= 0.992 R2 = 0.995

0.3 A5 5 0

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0.2

0.1

H T7 0 0

50

100 150 Concentration (µM)

200

250

Fig. 5. The photographs and calibration curves for detecting CH3Hg+ and C2H5Hg+ with HT5 and HT7 as probe, respectively.

Determination of dried fish muscle sample. The CH3Hg+ and C2H5Hg+ in dried fish muscle (Tapertail anchovy) collected from coastal water of Fujian in China were detected to investigate the reliability and applicability of the proposed method. The results obtained were compared with the results obtained by CE-ICP-MS, a reliable hyphenated technique for CH3Hg+ and C2H5Hg+ determination.8 The sample spiked with different concentrations of CH3Hg+ and C2H5Hg+ was also detected with the same manner to obtain the recovery. As results shown in Table 1, the CH3Hg+ in dried fish muscles can be specifically detected with bare eye observation or UVvisible spectrometry by using HT5 as probe with a recovery of 107-108% and a relative standard deviation (RSD, n=6) < 7%. Whereas, the total of CH3Hg+ and C2H5Hg+ in dried fish muscles can be detected with bare eye observation or UV-visible spectrometry by using HT7 as probe with a recovery of 101109% and a RSD (n=6) < 8%. The results obtained with our



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method were consistent with those of CE-ICP-MS. In addition, a shellfish sample, dried abalone, was also detected with the same manner to confirm the applicability of our method to different seafood (see Table S2 in SI). All above results indicated that our method is reliable and then can be applied for the instrument-free visual analysis of CH3Hg+ and C2H5Hg+ in seafood. Table 1. The analytical results of CH3Hg+ and C2H5Hg+ in dried fish muscle sample †

Sample

DNA sequence

Added CH3Hg+ (µM)

Added C2H5Hg+ (µM)

0.0

CH3Hg+ and C2H5Hg+ detection

‡

UV-visible spectrometry Bare eye observation

CE-ICP-MS

Con. (µM)

Con. (µM)

Con. (µM)

Rec. (%)

RSD (n=6)

CH3Hg+

CH3Hg+

0.0

1.35

-

7%

1.30

0.0

5.0

0.0

6.75

108%

5%

6.34

0.0

3

8.0

0.0

9.92

107%

5%

9.41

0.0

1

0.0

0.0

1.38

-

8%

1.27

0.0

2

5.0

0.0

6.85

109%

5%

6.33

0.0

3

0.0

5.0

6.63

105%

6%

1.31

4.96

4

5.0

5.0

11.5

101%

5%

6.29

4.93

1

2

HT5

HT7

†

The concentration in the extract of dried fish muscle obtained with our method. ‡The concentration in the extract of dried fish muscle obtained with CE-ICP-MS method reported in reference (8).

CONCLUSSION In summary, we herein developed a novel colorimetric method for the instrument-free visual discrimination and detection of CH3Hg+ and C2H5Hg+ in seafood based on DNAtemplated alloy Ag-Au nanoparticles. We designed two T-rich aptamers, HT5 and HT7, for specifically recognizing CH3Hg+ and the total of CH3Hg+ and C2H5Hg+, respectively. In the presence of all Au3+, Ag+ and aptamer, CH3Hg+ or C2H5Hg+ preferentially and specifically bind with aptamer and thus induced the formation alloy Ag-Au nanoparticles after reduction, which leads to the color change from colorless to purple in solution. By using HT5 as recognition probe, the method can be used to detect as low as 5.0 µM (equivalent to 1.0 µg Hg/g) of CH3Hg+ by bare eye observation and 0.5 µM (equivalent to 100 ng Hg/g) of CH3Hg+ by UV-visible spectrometry. By using HT7 as recognition probe, the method can be used to detect the total concentration of CH3Hg+ and C2H5Hg+ with a visual detection limit of 5.0 µM (equivalent to 1.0 µg Hg/g) and a

UV-visible spectrometry detection limit of 0.6 µM (equivalent to 120 ng Hg/g). The proposed method has been successfully used to detect CH3Hg+ and C2H5Hg+ in fish muscle samples with a recovery of 101-109% and a RSD (n=6) < 8%. The success of this study provided a potential method for specific, simple, rapid and cost-effective detection of CH3Hg+ and C2H5Hg+ in seafood by bare eye observation.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Chemicals, apparatus and DNA sequences (Table S1) used in the experiments; detailed procedure for the pre-treatment of fish muscle sample (including the analytical results of other seafood sample, Table S2), the EDX mapping images of alloy Ag-Au nanoparticles in the case of CH3Hg+ and C2H5Hg+ (Fig. S1) and conditions optimization (Fig. S2-S5).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]

Author Contributions Prof. F.-F. Fu and Dr W.-J. Yang perform the experimental design, data analysis and interpretation, and manuscript writing. Z.Q. Chen, X. S. Wang and X. Cheng performed the experiments. Prof. Y.-N. Wu co-performed data analysis and interpretation. The manuscript was written through contributions of all authors, and all authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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The authors gratefully acknowledge The National Key Research and Development Program of China (2017YFC1600500), NSFC (21677034), Fujian Provincial Department of Science and Technology (2016Y0005) and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT-15R11) for financial support.

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Specifically and visually detect methyl-mercury and ethyl-mercury in

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