Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1250–1257
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Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization Jun Peng, Jian Ling ⇑, Xiu-Qing Zhang, Hui-Ping Bai, Liyan Zheng, Qiu-E Cao ⇑, Zhong-Tao Ding Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Designed a new fluorescent DNA/
AgNCs probe for mercury and copper ions detection. The fluorescent AgNCs are stabilized in double-strand DNA with bright fluorescence. The proposed fluorescence method for Hg2+ and Cu2+ detection is very sensitive.
a r t i c l e
i n f o
Article history: Received 21 March 2014 Received in revised form 7 August 2014 Accepted 31 August 2014
Keywords: DNA/Ag nanoclusters Fluorescence Guanine-enhanced Mercury Copper
a b s t r a c t In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/ AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0–160.0 and 6– 240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Heavy metal ions are widespread and severe environmental pollutant exists in different forms of metal, inorganic salts and organic complexes. Mercury can accumulate readily in organisms through the food chain and cause acute and permanent damage ⇑ Corresponding authors. Tel.: +86 871 65033719; fax: +86 871 65033679. E-mail addresses: [email protected] (J. Ling), [email protected] (Q.-E. Cao). http://dx.doi.org/10.1016/j.saa.2014.08.135 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
to the brain, liver and the central nervous system [1,2]. Copper can also lead to serious environmental issues and is potentially toxic for all living organisms if high-dose of copper was exposed [3,4]. Increasing concerns over the potential threat of heavy metal are the prime motivations for the development of sensitive, simple and rapid heavy metal detection methods [5–7]. Traditional and reliable methods for the detection of metal ions are based on atomic absorption/emission spectrometry. However, most of them are time-consuming and require large equipment.
J. Peng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1250–1257
In recent years, great efforts have been devoted to the detection of heavy metal ions in environmental and biological systems using fluorescent metal nanoclusters (NCs) as novel sensing probes [8–11]. Various heavy metal ions, including mercury [12–19] and copper [20–30], can be sensitively and selectively detected by a variety of fluorescent nanoclusters, such as protein-stabilized Au nanoclusters [18,27,31,32] and oligonucleotides-stabilized Ag nanoclusters (DNA/AgNCs) [12,14,29]. In these nanoclusters probes, DNA/AgNCs are novel fluorescence materials with good quantum yield, high photostability, and tunable fluorescence emission by DNA sequence [33–35]. In recent years, DNA/AgNCs attracted greater attention and have been used for detection of metal ions, small molecules [36–38], DNA/ RNA [39–41], proteins [42–44] and also used for fluorescence imaging in live cells [45]. Metal ions such as Hg2+ and Cu2+ had been reported to quench the fluorescence of DNA/AgNCs [12]. However, single-stranded DNA/AgNCs still have some shortcomings such as weak and instable fluorescence. To improve the fluorescence of DNA/AgNCs, new concepts and strategies have been proposed by many researchers. Werner and Martinez’s group has reported that weak DNA/AgNCs could emit bright fluorescence when placed in proximity to guanine bases [46]. Upon hybridization of a guanine-rich sequence with the NCs sequence, strong red fluorescence emission was observed with a significant 500-fold enhancement [46]. Based on this report, we designed a new fluorescent DNA/AgNCs probe for sensitive detection of mercury and copper ions based on the ‘‘guanine-enhanced’’ strategies. AgNCs was firstly synthesized in a cytosine-rich singlestrands DNA, and then, a guanine-rich sequence was hybridized with the DNA/AgNCs for signal enhancement (Scheme 1). We presume that the fluorescence enhanced double-strand DNA/AgNCs could be used as a good fluorescent probe for detecting mercury and copper ions and improve the detection sensitivity and stability of the metal ions.
Materials and methods Reagents and instruments Silver nitrate (99.9999%) was purchased from Alfa Aesar (USA). Sodium borohydride (powder, 98%) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All oligonucleotides used for synthesis of silver nanoclusters were purchased from Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). Phosphate buffer (PB, 20 mM, pH 7.0), was used for synthesis of silver nanoclusters and detection of metal ions. Phosphate buffer saline (PBS, 20 mM, pH 7.0), prepared by dissolving 0.296 g NaH2PO42H2O, 2.90 g Na2HPO412H2O, 8.0 g NaCl, and 1.0 g KCl in one liter ultra-pure water (18.2 MX), was used as a buffer for DNA hybridization. 1.00 lM of Hg2+ and Cu2+ standard solutions prepared by dissolving copper nitrate and mercury
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chloride (99.9%) in ultra-pure water were used as stock solutions. Other chemicals such as chromium chloride, ferric chloride, mercuric chloride and zinc chloride were analytical reagent grade without further purification. Ultrapure water (18.25 MX) was used all through the whole experiment. Fluorescence and absorption spectra of silver nanoclusters were measured with a F-7000 fluorospectrophotometer (Hitachi, Japan) and UV-2550 spectrophotometer (Shimadzu, Japan), respectively. Fluorescence lifetime of DNA/AgNCs was measured by a FluoroLog-3 fluorescence spectrophotometer (Horiba Jobin Yvon Inc., France) capable of time-correlated single-photon counting. Synthesis of DNA/AgNCs For the preparation of the DNA/AgNCs, a designed a nanoclusters sequence (50 -T3AT3AT3AT3AC3AC3AC3GC3AT3AT3AT3AT3), was used as DNA template for preparing AgNCs. The procedure of preparation is similar as the reported methods with some modification [47,48]. Briefly, 48 lL of 50 lM DNA solution prepared in 20 mM phosphate buffer (pH 7.0) was mixed and vortexed with 38.4 lL of 1.0 mM AgNO3 solution. After standing for 20 min, this mixture was reduced by adding 42 lL freshly prepared NaBH4 (1 mM). The reduced DNA/Ag solution was incubated at room temperature in the dark for 18 h. The color of the mixture changing from colorless to pink indicated the formation of NCs in solution. The fluorescence spectra of the DNA/AgNCs were measured by fluorospectrophotometer at 480 nm of excitation. Enhance the fluorescence of DNA/AgNCs by hybridization To enhance the fluorescence of the as-prepared DNA/AgNCs, a guanine-rich DNA sequence (50 -A3TA3TA3TA3AG3AG3AG3AG3AA3TA3TA3TA3) was designed to hybridize on the DNA/AgNCs. Briefly, the as-prepared DNA/AgNCs solution was mixed and vortexed with the guanine-rich DNA sequence solution at molar ratio of 1:1, and then, diluted to 380 lL by 20 mM phosphate buffer saline (PBS, pH 7.0) solution. After hybridization for 1.5 h at room temperature, the hybridized double-strand DNA/AgNCs was then used for metal ion detection. General procedure for Hg2+and Cu2+ detection For metal ions detection, the as-prepared double-strand DNA/ AgNCs was firstly diluted 50-fold before using. Then the properly diluted metal ions solution was mixed with aliquots of the DNA/ AgNCs and diluted to 1.0 mL with 20 mM of PB buffer (pH 7.0). The option of pH and buffer solution was according to reported papers that DNA/AgNCs displayed strong and stable fluorescence emission signals in pH value range from 5 to 7 [11,33]. The mixtures would stay for 1.5 h at room temperature before fluorescence measurement. In the studies of selectivity and interferes, the same procedure was carried.
Scheme 1. Guanine-enhanced fluorescence DNA/AgNCs for sensitive detection of Hg2+ and Cu2+.
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enhanced 500-fold when placed in proximity to guanine-rich DNA sequence, we designed a guanine-rich DNA sequence (50 -A3TA3TA3TA3AG3AG3AG3AG3AA3TA3TA3TA3) which could hybridize with the DNA/AgNCs sequence. After hybridization, guanine-rich DNA sequence in proximity to the cluster-DNA conjugate greatly enhanced the fluorescence of DNA/AgNCs. As shown in Fig. 1B, the fluorescence excitation and emission peaks of the hybridized double-strand DNA/AgNCs shifted to 510 nm and 565 nm slightly with the fluorescence intensity enhanced 200 times. The fluorescence quantum yields (QYs) of the DNA/AgNCs in aqueous solution were determined to be enhanced from 0.4318% to 10.98% by using Rhodamine 6G (95.0% in water [50]) as a reference (Fig. 1C). The fluorescence decay of the emission (Fig. 1D) showed that the double-strand DNA/AgNCs has two dominant fluorescence lifetimes of 0.375 ns (14.60%) and 2.938 ns (85.40%). The average fluorescence lifetime of double-strand DNA/AgNCs is longer than that of single-strand DNA/AgNCs whose dominant lifetimes were 0.648 ns (41.77%) and 2.637 ns (58.23%). In additionally, fluorescence stability of the DNA/AgNCs was also studied. As shown in Supporting Information Figs. S4 and S5, the fluorescence of hybridized double-strand DNA/AgNCs is stable than singlestrand DNA/AgNCs in different pH range.
Results and discussion Fluorescence of DNA/AgNCs The cytosine-rich DNA sequence for AgNCs preparation has been previously reported by many researchers [10–12]. In this paper, a previously reported DNA template (50 -AC3AC3AC3GC3A) was selected for DNA/AgNCs preparation [47–49]. In order to enhance the fluorescence of the AgNCs by guanine-rich DNA sequence, the sequence was modified by adding two link sequences (50 -T3AT3AT3AT3) on the two tails. After preparation, fluorescence spectrum of the DNA/AgNCs was scanned and showed in Fig. 1A. It can be seen that the as-prepared DNA/AgNCs has a fluorescence maximum emission peak at 560 nm under 480 nm of excitation. According to the reported literature [47–49], AgNCs stabilized in the C3AC3AC3TC3A or C3AC3AC3GC3A sequence would show near-infrared emitting property. The results showed that our slight modification of the sequence and addition of the two tails would intensively affect the fluorescence property of the DNA/AgNCs. Considering the cytosine-rich DNA template used in this paper was different with the reported literature, the experimental conditions of DNA/AgNCs preparation were optimized here. As shown in Supporting Information Figs. S1–S3, the molar ratio of Ag:DNA, reaction temperature and the amount of NaBH4 were optimized. The molar ratio of Ag:DNA is preferred at 16:1 in our experimental condition. That is much higher than reported paper, in which molar ratio of 6:1 was adopted [11]. The reaction temperature for AgNCs growth is also different with usually used 4 °C [12], however, room temperature (25 °C) is suitable at our experimental condition. The difference in the detail of experimental condition may be caused by the modification of DNA sequence, difference of buffer solution or other environmental factors.
Response of the DNA/AgNCs fluorescence toward Hg2+ and Cu2+ The both as-prepared single-strand DNA/AgNCs and hybridized double-strand DNA/AgNCs after fluorescence enhancement were used for studying the fluorescence quenching effects toward metal ions. We tested numbers of metal ions on the effects of DNA/AgNCs emission and found that only two metals, Hg2+ and Cu2+, could effectively quench the emission of the both as-prepared DNA/ AgNCs. The fluorescence response of the DNA/AgNCs to metal ions is similar to recently reported proteins [51] and polymers [52] stabilized AgNCs, however, different with recently reported singlestrand DNA/AgNCs whose fluorescence could only quenched by Hg2+ [12,14]. This phenomenon indicates that the DNA/AgNCs with
Enhancing the fluorescence of the DNA/AgNCs
25
A
480 nm
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
On the basis of the new phenomenon reported by Martinez and Werner’s group that the fluorescence of DNA/AgNCs can be 560 nm Excitation Emission
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Rhodamine 6G ssDNA/AgNCs dsDNA/AgNCs
ssDNA/AgNCs dsDNA/AgNCs
1000 300 200
QRhodamine6G= 95.00 % QssDNA/AgNCs= 0.4318 %
Decay
Fluorescence intensity (a.u.)
400
C
540
Wavelength/nm
Wavelength/nm 500
565 nm
100
τssDNA/Ag NCs= 0.648 ns (41.77%)
QdsDNA/AgNCs= 10.98 %
2.637 ns (58.23%)
100
10
0
1
τdsDNA/Ag NCs= 0.375 ns (14.60%) 2.938 ns (85.40%)
0.020 0.025 0.030 0.035 0.040 0.045
A
0.050
25
30
35
40
45
Time (ns)
Fig. 1. Comparison of DNA/AgNCs fluorescence properties before and after hybridized with a guanine-rich sequence. (A) Fluorescence spectra of DNA/Ag NCs prepared by reducing DNA and AgNO3 mixture in 20 mM of phosphate buffer (pH 7.0). ssDNA/Ag NCs concentration (calculated by DNA), 0.40 lM. (B) Fluorescence spectra of DNA/Ag NCs after hybridizing with the guanine-rich DNA sequence solution at molar ratio of 1:1 in 20 mM phosphate buffer saline (pH 7.0). dsDNA/Ag NCs concentration (calculated by DNA), 0.0898 lM. (C) Fluorescence quantum yield. (D) Fluorescence life time.
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2+
0 μΜ 0.006μΜ 0.020μΜ 0.060μΜ 0.100μΜ 0.160μΜ 0.240μΜ 0.360μΜ 0.480μΜ 0.600μΜ
Hg added:
A 320 240 160 80
1.0
B
0.8 4
0.6 3
I0/I
400
and D. It can be seen that up to 97% and 89% of the fluorescence can be quenched at higher Hg2+ or Cu2+ concentration. The fluorescence responses of the single-strand DNA/AgNCs toward Hg2+ and Cu2+ are similar to that of double-strand DNA/AgNCs, however have lower sensitivity. As shown in Supplementary Material Figs. S6 and S7, the fluorescence quench of the single-strand DNA/AgNCs happened at higher Hg2+ and Cu2+ concentration and the Fluorescence quenching ratio has bad relationship toward the
(I0-I)/I0
Fluorescence intensity (a.u.)
different DNA sequence or preparation by different method may have potential for sensing different kinds of targets. As shown in Fig. 2A and C, the fluorescence emission of the double-strand DNA/AgNCs at 565 nm starts to quench when 6.0 nM of Hg2+ or Cu2+ were added to them, and up to 75% of fluorescence were quenched in the presence of 150.0 nM of Hg2+ or 250.0 nM of Cu2+. Fluorescence quenching ratio [(I0 I)/I0] of the DNA/AgNCs toward the two metal ions concentrations were shown in Fig. 2B
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2+
Cu added:
C
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cHg2+ / μM
(I0-I)/I0
250
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cHg2+ / μM
Wavelength/nm Fluorescence intensity (a.u.)
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0
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cCu2+ / μM
Wavelength/nm
Fig. 2. Fluorescence quenching of double-strand DNA/AgNCs in the presence of different concentration of Hg2+ (A) and Cu2+ (C) and corresponding fluorescence quench ratios toward the two metal ions (B) and (D). The insets in (B) and (D) are Stern–Volmer plot of the quenching. Experimental conditions: DNA/Ag NCs, 0.0898 lmol/L; 20 mM phosphate buffer (pH 7.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
10000
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dsDNA/AgNCs 2+ dsDNA/AgNCs + 0.2 μM Hg
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dsDNA/AgNCs 2+ dsDNA/AgNCs + 0.2 μM Cu
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τNCs= 0.375 ns (14.60%) 2.938 ns (85.40%)
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τNCs= 0.375 ns (14.60%) 2.938 ns (85.40%)
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τNCs+Hg= 0.346 ns (18.63%)
τNCs+Cu= 0.295 ns (19.47%)
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2.931 ns (80.53%)
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0.4 lg(I0-I)/I = 15.32 + 2.18 lgcHg R =0.9618
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45
D
0.4 lg(I0-I)/I = 14.31 + 2.10 lgcCu
2
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35
Time (ns)
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lgcHg2+
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Fig. 3. Fluorescence quenching mechanism studied by fluorescence lifetime (A) and (B) and Stern–Volmer relationship (C) and (D).
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metal ion concentration. Thus, we believed that the double-strand DNA/AgNCs whose fluorescence is enhanced by guanine-rich DNA are good for Hg2+ and Cu2+ sensing. The fluorescence quenching mechanism of double-strand DNA/ AgNCs by Hg2+ and Cu2+ was also studied. We compared the fluorescence lifetime of double-strand DNA/AgNCs before and after quenched by metal ions. As shown in Fig. 3A and B, no change in fluorescence lifetime indicated a static fluorescence quenching mechanism. However, the Stern–Volmer plots shown in Fig. 2B and D (inset figures) are not linear with the concentration of metal ions. Considering DNA/AgNCs is a biomolecule stabilized fluorescence probe, we inferred that the reaction ratio of the fluorophore
to quencher is not 1:1, and supposed 1 mol of DNA/AgNCs would combine n mole of metal ions. Thus, the static quenching equation should be:
(I0-I)/I0
0.6 0.5 0.4 2+
0.2
0.2μM of Cu
2+
0.1 0.0 0
20
40
60
80
100
ð2Þ
I0 =I ¼ 1 þ K a ½Q n
ð3Þ
lg½ðI0 IÞ=I ¼ lg K a þ n lg½Q
ð4Þ
According to Eq. (4), the association constant (Ka) and combination ratio (n) of DNA/AgNCs to Hg2+ and Cu2+ could be obtained by fitting relationship between lg(I0 I)/I and lg[Q]. As shown in Fig. 3C and D, by repeating the experiments, the value of lg Ka and n was calculated to be 15.32 and 2.18 for Hg2+, and 14.31 and 2.10 for Cu2+, respectively. These parameters indicate that every DNA/AgNCs would combine two metal ions and the combination ability of DNA/AgNCs to Hg2+ is larger than that of Cu2+. The fluorescence quenching kinetics of double-strand DNA/ AgNCs by Hg2+ and Cu2+ were investigated by recording the fluorescence spectrum of them and certain concentrations of Hg2+ and Cu2+ in two hours. As shown in Fig. 4, in the presence of 0.2 lM Hg2+, the fluorescence intensity of the DNA/AgNCs decreased sharply in the first 60 min, and then decreased slowly and remained in the latter 60 min. The quenching kinetics of the DNA/AgNCs under Cu2+ ion shows similar results with Hg2+. Thus, a reliable detection of Hg2+ and Cu2+ need an hour of waiting.
0.7
0.2μM of Hg
ð1Þ
K a ¼ ½FQ n =½F½Q n
where [F], [Q] and [FQn] is concentration of fluorophore, quencher and formed complex, respectively. Ka is association constant of the complex. Thus,
0.8
0.3
½F þ n½Q ¼ ½FQ n
120
t / min Fig. 4. Fluorescence quenching kinetics of the double-strand DNA/AgNCs toward 0.2 lM of Hg2+ and Cu2+. Experimental conditions: DNA/Ag NCs, 0.0898 lM; 20 mM phosphate buffer (pH 7.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
Selectivity and interference The selectivity and interference of double-stand DNA/AgNCs fluorescence for metal ions were also investigated. As indicated 0.8
A
1.0
B 0.6
(I0-I)/I0
(I0-I)/I0
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+
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Fig. 5. Selectivity and interferes for Hg2+ detection. (A) Metal ion concentrations: Hg2+ and Cu2+, 0.8 lM; others, 40 lM. (B) Fluorescence quenching ratios toward 0.1 lM Hg2+ with coexistence of 1.25 lM other metal ions. Experimental conditions: dsDNA/Ag NCs, 0.0898 lM; 20 mM phosphate buffer (pH 7.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
0.8
0.8
A
0.6
(I0-I)/I0
(I0-I)/I0
(I0-I)/I0=0.00659 + 4.769 cHg 0.6 R2=0.9985 0.4
0.0
0.0 0.08
cHg2+ / μΜ
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0.16
2
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0.04
(I0-I)/I0=0.00835 + 3.018 cCu
R =0.9953
0.2
0.00
B
0.00
0.05
0.10
0.15
0.20
0.25
cCu2+ / μΜ
Fig. 6. Linear relationship between double-strand DNA/AgNCs fluorescence quenching ratio and Hg2+ (A) and Cu2+ (B) concentration. Experimental conditions: DNA/Ag NCs, 0.0898 lM; 20 mM phosphate buffer (pH 7.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
J. Peng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1250–1257 Table 1 Determination of Hg2+ by fluorescent metal nanoclusters probes in recent papers. Probes
Linear range
LOD
References
DNA–AgNCs Denatured BSA–AgNCs Lys–AgNCs GSH–AgNCs PMAA–AgNCs G-enhanced DNA–AgNCs
2.5–50 nM 10 nM–5 lM 1–15 lM 5–125 nM 10 nM–20 lM 6–260 nM
0.9 nM 10 nM 0.6 lM 5 nM 10 nM 2.1 nM
[53] [18] [54] [13] [52] This work
Table 2 Determination of Cu2+ by fluorescent metal nanoclusters probes in recent papers. Probes
Linear range
LOD
References
PMAA–AgNCs PMAA–AgNCs MPAA–AgNCs DNA–AgNCs MUA–AuNPs G-enhanced DNA–AgNCs
10 nM–30 lM 10 nM–6 lM 5 lM–15 mM 15–200 nM 10 nM–1 lM 6–240 nM
10 nM 8 nM 5 lM 8 nM 87 nM 3.4 nM
[52] [26] [55] [28] [56] This work
Table 3 Analytical results of mercury and copper ions detection in real water samples. Samplesa
Metal ions
Added (lM)
Detected (lM)
Recovery (%)
RSD (n = 3 (%))
1
Hg2+ Hg2+
0.030 0.060
0.035 0.059
116.67 98.33
8.01 4.92
2
Hg2+ Hg2+
0.040 0.080
0.045 0.087
112.50 108.75
5.58 8.04
3
Cu2+ Cu2+
0.040 0.080
0.041 0.076
102.50 95.00
8.25 2.70
4
Cu2+ Cu2+
0.050 0.100
0.048 0.094
96.00 94.00
4.73 4.27
a The four samples were taken from different areas of Panlong River at Kunming City.
in Fig. 5A, the DNA/AgNCs exhibited high selectivity for Hg2+ and Cu2+ ions over a variety of other metal ions. The interference of other metals (except Cu2+) for Hg2+ detection is also very slight. As shown in Fig. 5B, the fluorescence of the hybridized DNA/AgNCs partially quenched by 50 nM Hg2+ was not affected by coexistence of 1.25 mM competing metal ions. Calibration curve for Hg2+ and Cu2+ detection
300 250 200
was possible to develop a new fluorescence sensing system for Hg2+ and Cu2+ based on AgNCs. Fluorescence quenching ratio [(I0 I)/I0] was used for establishing calibration curve for Hg2+ and Cu2+ detection. As shown in Fig. 6 a good linear relationship between (I0 I)/I0 and the concentration of Hg2+ and Cu2+ can be observed in the Hg2+ and Cu2+ concentration range from 6.0 nM to 160.0 and 6 nM to 240 nM, respectively. The linear regression equation (c, lM) can be expressed as (I0 I)/I0 = 0.00659 + 4.769c for Hg2+ and (I0 I)/I0 = 0.00835 + 3.018c for Cu2+ with correlation , coefficients (R2) of 0.9916 and 0.9952, respectively. The limits of detection (LOD, 3r) are 2.1 nM for Hg2+ and 3.4 nM for Cu2+, respectively. We compared the analytical parameters of our method with the recently published Ref. [13,18,26,28,52–56], and found that the selectivity is better than others (see Tables 1 and 2).
Masking of Cu2+ using EDTA The above results revealed that this assay approach had high sensitivity toward mercury and copper ions. As mercury is a severe environmental pollutant which is more harmful to human beings than copper, establishing a sensitive and selective method for Hg2+ detection is important for monitoring environmental pollutants. In order to achieve this, an efficient Cu2+ chelating reagent, ethylene diamine tetraacetate (EDTA) was added to minimize the interference of the Cu2+. Similar to the recently reported paper [51,52], a proper amount of EDTA was added into the double-strand DNA/AgNCs and 1.0 lM of Cu2+ mixed solution and then applied for the Hg2+ detection. The results showed that the fluorescence intensity decreased with the increase of Hg2+ concentration (Fig. 7). The Fluorescence quenching ratio [(I0 I)/I0] toward Hg2+ concentration has a good linear relation in the range from 20.0 to 400.0 nM and the LOD was estimated about 8 nM (3r). Therefore, Cu2+ can be successfully masked in this system with little effect on the detection of Hg2+, allowing for the sensitive detection of a single ion. It should be noticed that Cu2+ could also be detected selectivity by masking of Hg2+ using similar methods that had been intensively studied by previous reports [51,52]. In this paper, no more repetition has been done here.
Real sample detection To evaluate the availability of the proposed analytical method for natural samples, three bottles of natural water samples collected from a local river were analyzed by the standard addition method. Quantification results shown in Table 3 indicated that our method is reliable for detection real water samples. 1.0
A
B
2+
Hg added: 2+
Cu 1.0 μM EDTA 1.0 μM
150 100 50
0μM 0.005μM 0.010μM 0.020μM 0.040μM 0.080μM 0.240μM 0.320μM 0.400μM 0.500μM 0.600μM
0.8
(I0-I)/I0 = 0.11353 + 1.74758 cHg 2
R = 0.99414
(I0-I)/I0
Fluorescence intensity (a.u.)
In view of the intensive quenching of the fluorescence emission of double-strand DNA/AgNCs in the presence of Hg2+ and Cu2+, it
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Fig. 7. (A) Fluorescence changes of double-strand DNA/Ag NCs in the presence of different concentrations of Hg2+ after Cu2+ was masked. (B) Corresponding linear relationship between DNA/Ag NCs fluorescence quenching ratio and Hg2+ concentration. Experimental conditions: DNA/Ag NCs, 0.0898 lM; 20 mM phosphate buffer (pH 5.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
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Conclusion In summary, we designed a new hybridized double-strand DNA/ AgNCs for fluorescence detection of mercury and copper ions. Compared with the single-strand DNA/AgNCs before hybridization, the hybridized double-strand DNA/AgNCs probe is brighter and stable in fluorescence. Experimental results showed that mercury and copper ions in the range of 6.0–160.0 and 6–240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. The analytical parameters of the method for mercury and copper ions detection are better than that using a single-strand DNA/AgNCs. By using EDTA as a masking reagent, Hg2+ could be selectively detected coexisted with Cu2+ and other metal ions. Mercury and copper ions in nature water were also successfully detected by the standard addition method. Overall, our work offers an important strategy for DNA/AgNCs fluorescence improvement and its further application. Acknowledgments We thank Dr. Lin-Ling Zheng and Ming-Xuan Gao from Southwest University, Chongqing, China, for fluorescence lifetime measurement and data analysis. We also show our great appreciation for the financial support of the National Natural Science Foundation of China (21105087, 21465025), Applied and Basic Research Program of Yunnan Province (2011FB014) and Cultivation Program for Key Young Teachers of Yunnan University. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.08.135. References [1] P.B. Tchounwou, W.K. Ayensu, N. Ninashvili, D. Sutton, Environmental exposure to mercury and its toxicopathologic implications for public health, Environ. Toxicol. 18 (2003) 149–175. [2] I. Onyido, A.R. Norris, E. Buncel, Biomolecule–mercury interactions: modalities of DNA base-mercury binding mechanisms. Remediation strategies, Chem. Rev. 104 (2004) 5911–5929. [3] P.G. Georgopoulos, A. Roy, M.J. Yonone-Lioy, R.E. Opiekun, P.J. Lioy, Environmental copper: its dynamics and human exposure issues, J. Toxicol. Environ. Health, B 4 (2001) 341–394. [4] E.L. Que, D.W. Domaille, C.J. Chang, Metals in neurobiology: probing their chemistry and biology with molecular imaging, Chem. Rev. 108 (2008) 1517– 1549. [5] J.-M. Liu, H.-F. Wang, X.-P. Yan, A gold nanorod based colorimetric probe for the rapid and selective detection of Cu2+ ions, Analyst 136 (2011) 3904–3910. [6] Y.J. Long, Y.F. Li, Y. Liu, J.J. Zheng, J. Tang, C.Z. Huang, Visual observation of the mercury-stimulated peroxidase mimetic activity of gold nanoparticles, Chem. Commun. 47 (2011) 11939–11941. [7] P. Zhang, S. Chen, Y. Kang, Y. Long, Trace mercury ion determination based on the highly selective redox reaction between stannous ion and mercury ion enhanced by gold nanoparticles, Spectrochim. Acta, A 99 (2012) 347–352. [8] H. Xu, K.S. Suslick, Sonochemical synthesis of highly fluorescent Ag nanoclusters, ACS Nano 4 (2010) 3209–3214. [9] L. Shang, S. Dong, G.U. Nienhaus, Ultra-small fluorescent metal nanoclusters: synthesis and biological applications, Nano Today 6 (2011) 401–418. [10] J.T. Petty, J. Zheng, N.V. Hud, R.M. Dickson, DNA-templated Ag nanocluster formation, J. Am. Chem. Soc. 126 (2004) 5207–5212. [11] C.I. Richards, S. Choi, J.-C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y.-L. Tzeng, R.M. Dickson, Oligonucleotide-stabilized Ag nanocluster fluorophores, J. Am. Chem. Soc. 130 (2008) 5038–5039. [12] W. Gue, J. Yuan, E. Wang, Oligonucleotide-stabilized Ag nanoclusters as novel fluorescence probes for the highly selective and sensitive detection of the Hg2+ ion, Chem. Commun. (2009) 3395–3397. [13] X. Yuan, T.J. Yeow, Q. Zhang, J.Y. Lee, J. Xie, Highly luminescent Ag+ nanoclusters for Hg2+ ion detection, Nanoscale 4 (2012) 1968–1971. [14] J.L. MacLean, K. Morishita, J. Liu, DNA stabilized silver nanoclusters for ratiometric and visual detection of Hg2+ and its immobilization in hydrogels, Biosens. Bioelectron. 48 (2013) 82–86. [15] B. Adhikari, A. Banerjee, Facile synthesis of water-soluble fluorescent silver nanoclusters and Hg-II sensing, Chem. Mater. 22 (2010) 4364–4371.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Sensitive detection of mercury and copper ions by fluorescent DNA/Ag nanoclusters in guanine-rich DNA hybridization Jun Peng, Jian Ling ⇑, Xiu-Qing Zhang, Hui-Ping Bai, Liyan Zheng, Qiu-E Cao ⇑, Zhong-Tao Ding Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Designed a new fluorescent DNA/
AgNCs probe for mercury and copper ions detection. The fluorescent AgNCs are stabilized in double-strand DNA with bright fluorescence. The proposed fluorescence method for Hg2+ and Cu2+ detection is very sensitive.
a r t i c l e
i n f o
Article history: Received 21 March 2014 Received in revised form 7 August 2014 Accepted 31 August 2014
Keywords: DNA/Ag nanoclusters Fluorescence Guanine-enhanced Mercury Copper
a b s t r a c t In this work, we designed a new fluorescent oligonucleotides-stabilized silver nanoclusters (DNA/AgNCs) probe for sensitive detection of mercury and copper ions. This probe contains two tailored DNA sequence. One is a signal probe contains a cytosine-rich sequence template for AgNCs synthesis and link sequence at both ends. The other is a guanine-rich sequence for signal enhancement and link sequence complementary to the link sequence of the signal probe. After hybridization, the fluorescence of hybridized double-strand DNA/AgNCs is 200-fold enhanced based on the fluorescence enhancement effect of DNA/ AgNCs in proximity of guanine-rich DNA sequence. The double-strand DNA/AgNCs probe is brighter and stable than that of single-strand DNA/AgNCs, and more importantly, can be used as novel fluorescent probes for detecting mercury and copper ions. Mercury and copper ions in the range of 6.0–160.0 and 6– 240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. Our results indicated that the analytical parameters of the method for mercury and copper ions detection are much better than which using a single-strand DNA/AgNCs. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Heavy metal ions are widespread and severe environmental pollutant exists in different forms of metal, inorganic salts and organic complexes. Mercury can accumulate readily in organisms through the food chain and cause acute and permanent damage ⇑ Corresponding authors. Tel.: +86 871 65033719; fax: +86 871 65033679. E-mail addresses: [email protected] (J. Ling), [email protected] (Q.-E. Cao). http://dx.doi.org/10.1016/j.saa.2014.08.135 1386-1425/Ó 2014 Elsevier B.V. All rights reserved.
to the brain, liver and the central nervous system [1,2]. Copper can also lead to serious environmental issues and is potentially toxic for all living organisms if high-dose of copper was exposed [3,4]. Increasing concerns over the potential threat of heavy metal are the prime motivations for the development of sensitive, simple and rapid heavy metal detection methods [5–7]. Traditional and reliable methods for the detection of metal ions are based on atomic absorption/emission spectrometry. However, most of them are time-consuming and require large equipment.
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In recent years, great efforts have been devoted to the detection of heavy metal ions in environmental and biological systems using fluorescent metal nanoclusters (NCs) as novel sensing probes [8–11]. Various heavy metal ions, including mercury [12–19] and copper [20–30], can be sensitively and selectively detected by a variety of fluorescent nanoclusters, such as protein-stabilized Au nanoclusters [18,27,31,32] and oligonucleotides-stabilized Ag nanoclusters (DNA/AgNCs) [12,14,29]. In these nanoclusters probes, DNA/AgNCs are novel fluorescence materials with good quantum yield, high photostability, and tunable fluorescence emission by DNA sequence [33–35]. In recent years, DNA/AgNCs attracted greater attention and have been used for detection of metal ions, small molecules [36–38], DNA/ RNA [39–41], proteins [42–44] and also used for fluorescence imaging in live cells [45]. Metal ions such as Hg2+ and Cu2+ had been reported to quench the fluorescence of DNA/AgNCs [12]. However, single-stranded DNA/AgNCs still have some shortcomings such as weak and instable fluorescence. To improve the fluorescence of DNA/AgNCs, new concepts and strategies have been proposed by many researchers. Werner and Martinez’s group has reported that weak DNA/AgNCs could emit bright fluorescence when placed in proximity to guanine bases [46]. Upon hybridization of a guanine-rich sequence with the NCs sequence, strong red fluorescence emission was observed with a significant 500-fold enhancement [46]. Based on this report, we designed a new fluorescent DNA/AgNCs probe for sensitive detection of mercury and copper ions based on the ‘‘guanine-enhanced’’ strategies. AgNCs was firstly synthesized in a cytosine-rich singlestrands DNA, and then, a guanine-rich sequence was hybridized with the DNA/AgNCs for signal enhancement (Scheme 1). We presume that the fluorescence enhanced double-strand DNA/AgNCs could be used as a good fluorescent probe for detecting mercury and copper ions and improve the detection sensitivity and stability of the metal ions.
Materials and methods Reagents and instruments Silver nitrate (99.9999%) was purchased from Alfa Aesar (USA). Sodium borohydride (powder, 98%) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All oligonucleotides used for synthesis of silver nanoclusters were purchased from Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China). Phosphate buffer (PB, 20 mM, pH 7.0), was used for synthesis of silver nanoclusters and detection of metal ions. Phosphate buffer saline (PBS, 20 mM, pH 7.0), prepared by dissolving 0.296 g NaH2PO42H2O, 2.90 g Na2HPO412H2O, 8.0 g NaCl, and 1.0 g KCl in one liter ultra-pure water (18.2 MX), was used as a buffer for DNA hybridization. 1.00 lM of Hg2+ and Cu2+ standard solutions prepared by dissolving copper nitrate and mercury
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chloride (99.9%) in ultra-pure water were used as stock solutions. Other chemicals such as chromium chloride, ferric chloride, mercuric chloride and zinc chloride were analytical reagent grade without further purification. Ultrapure water (18.25 MX) was used all through the whole experiment. Fluorescence and absorption spectra of silver nanoclusters were measured with a F-7000 fluorospectrophotometer (Hitachi, Japan) and UV-2550 spectrophotometer (Shimadzu, Japan), respectively. Fluorescence lifetime of DNA/AgNCs was measured by a FluoroLog-3 fluorescence spectrophotometer (Horiba Jobin Yvon Inc., France) capable of time-correlated single-photon counting. Synthesis of DNA/AgNCs For the preparation of the DNA/AgNCs, a designed a nanoclusters sequence (50 -T3AT3AT3AT3AC3AC3AC3GC3AT3AT3AT3AT3), was used as DNA template for preparing AgNCs. The procedure of preparation is similar as the reported methods with some modification [47,48]. Briefly, 48 lL of 50 lM DNA solution prepared in 20 mM phosphate buffer (pH 7.0) was mixed and vortexed with 38.4 lL of 1.0 mM AgNO3 solution. After standing for 20 min, this mixture was reduced by adding 42 lL freshly prepared NaBH4 (1 mM). The reduced DNA/Ag solution was incubated at room temperature in the dark for 18 h. The color of the mixture changing from colorless to pink indicated the formation of NCs in solution. The fluorescence spectra of the DNA/AgNCs were measured by fluorospectrophotometer at 480 nm of excitation. Enhance the fluorescence of DNA/AgNCs by hybridization To enhance the fluorescence of the as-prepared DNA/AgNCs, a guanine-rich DNA sequence (50 -A3TA3TA3TA3AG3AG3AG3AG3AA3TA3TA3TA3) was designed to hybridize on the DNA/AgNCs. Briefly, the as-prepared DNA/AgNCs solution was mixed and vortexed with the guanine-rich DNA sequence solution at molar ratio of 1:1, and then, diluted to 380 lL by 20 mM phosphate buffer saline (PBS, pH 7.0) solution. After hybridization for 1.5 h at room temperature, the hybridized double-strand DNA/AgNCs was then used for metal ion detection. General procedure for Hg2+and Cu2+ detection For metal ions detection, the as-prepared double-strand DNA/ AgNCs was firstly diluted 50-fold before using. Then the properly diluted metal ions solution was mixed with aliquots of the DNA/ AgNCs and diluted to 1.0 mL with 20 mM of PB buffer (pH 7.0). The option of pH and buffer solution was according to reported papers that DNA/AgNCs displayed strong and stable fluorescence emission signals in pH value range from 5 to 7 [11,33]. The mixtures would stay for 1.5 h at room temperature before fluorescence measurement. In the studies of selectivity and interferes, the same procedure was carried.
Scheme 1. Guanine-enhanced fluorescence DNA/AgNCs for sensitive detection of Hg2+ and Cu2+.
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enhanced 500-fold when placed in proximity to guanine-rich DNA sequence, we designed a guanine-rich DNA sequence (50 -A3TA3TA3TA3AG3AG3AG3AG3AA3TA3TA3TA3) which could hybridize with the DNA/AgNCs sequence. After hybridization, guanine-rich DNA sequence in proximity to the cluster-DNA conjugate greatly enhanced the fluorescence of DNA/AgNCs. As shown in Fig. 1B, the fluorescence excitation and emission peaks of the hybridized double-strand DNA/AgNCs shifted to 510 nm and 565 nm slightly with the fluorescence intensity enhanced 200 times. The fluorescence quantum yields (QYs) of the DNA/AgNCs in aqueous solution were determined to be enhanced from 0.4318% to 10.98% by using Rhodamine 6G (95.0% in water [50]) as a reference (Fig. 1C). The fluorescence decay of the emission (Fig. 1D) showed that the double-strand DNA/AgNCs has two dominant fluorescence lifetimes of 0.375 ns (14.60%) and 2.938 ns (85.40%). The average fluorescence lifetime of double-strand DNA/AgNCs is longer than that of single-strand DNA/AgNCs whose dominant lifetimes were 0.648 ns (41.77%) and 2.637 ns (58.23%). In additionally, fluorescence stability of the DNA/AgNCs was also studied. As shown in Supporting Information Figs. S4 and S5, the fluorescence of hybridized double-strand DNA/AgNCs is stable than singlestrand DNA/AgNCs in different pH range.
Results and discussion Fluorescence of DNA/AgNCs The cytosine-rich DNA sequence for AgNCs preparation has been previously reported by many researchers [10–12]. In this paper, a previously reported DNA template (50 -AC3AC3AC3GC3A) was selected for DNA/AgNCs preparation [47–49]. In order to enhance the fluorescence of the AgNCs by guanine-rich DNA sequence, the sequence was modified by adding two link sequences (50 -T3AT3AT3AT3) on the two tails. After preparation, fluorescence spectrum of the DNA/AgNCs was scanned and showed in Fig. 1A. It can be seen that the as-prepared DNA/AgNCs has a fluorescence maximum emission peak at 560 nm under 480 nm of excitation. According to the reported literature [47–49], AgNCs stabilized in the C3AC3AC3TC3A or C3AC3AC3GC3A sequence would show near-infrared emitting property. The results showed that our slight modification of the sequence and addition of the two tails would intensively affect the fluorescence property of the DNA/AgNCs. Considering the cytosine-rich DNA template used in this paper was different with the reported literature, the experimental conditions of DNA/AgNCs preparation were optimized here. As shown in Supporting Information Figs. S1–S3, the molar ratio of Ag:DNA, reaction temperature and the amount of NaBH4 were optimized. The molar ratio of Ag:DNA is preferred at 16:1 in our experimental condition. That is much higher than reported paper, in which molar ratio of 6:1 was adopted [11]. The reaction temperature for AgNCs growth is also different with usually used 4 °C [12], however, room temperature (25 °C) is suitable at our experimental condition. The difference in the detail of experimental condition may be caused by the modification of DNA sequence, difference of buffer solution or other environmental factors.
Response of the DNA/AgNCs fluorescence toward Hg2+ and Cu2+ The both as-prepared single-strand DNA/AgNCs and hybridized double-strand DNA/AgNCs after fluorescence enhancement were used for studying the fluorescence quenching effects toward metal ions. We tested numbers of metal ions on the effects of DNA/AgNCs emission and found that only two metals, Hg2+ and Cu2+, could effectively quench the emission of the both as-prepared DNA/ AgNCs. The fluorescence response of the DNA/AgNCs to metal ions is similar to recently reported proteins [51] and polymers [52] stabilized AgNCs, however, different with recently reported singlestrand DNA/AgNCs whose fluorescence could only quenched by Hg2+ [12,14]. This phenomenon indicates that the DNA/AgNCs with
Enhancing the fluorescence of the DNA/AgNCs
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Fig. 1. Comparison of DNA/AgNCs fluorescence properties before and after hybridized with a guanine-rich sequence. (A) Fluorescence spectra of DNA/Ag NCs prepared by reducing DNA and AgNO3 mixture in 20 mM of phosphate buffer (pH 7.0). ssDNA/Ag NCs concentration (calculated by DNA), 0.40 lM. (B) Fluorescence spectra of DNA/Ag NCs after hybridizing with the guanine-rich DNA sequence solution at molar ratio of 1:1 in 20 mM phosphate buffer saline (pH 7.0). dsDNA/Ag NCs concentration (calculated by DNA), 0.0898 lM. (C) Fluorescence quantum yield. (D) Fluorescence life time.
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2+
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I0/I
400
and D. It can be seen that up to 97% and 89% of the fluorescence can be quenched at higher Hg2+ or Cu2+ concentration. The fluorescence responses of the single-strand DNA/AgNCs toward Hg2+ and Cu2+ are similar to that of double-strand DNA/AgNCs, however have lower sensitivity. As shown in Supplementary Material Figs. S6 and S7, the fluorescence quench of the single-strand DNA/AgNCs happened at higher Hg2+ and Cu2+ concentration and the Fluorescence quenching ratio has bad relationship toward the
(I0-I)/I0
Fluorescence intensity (a.u.)
different DNA sequence or preparation by different method may have potential for sensing different kinds of targets. As shown in Fig. 2A and C, the fluorescence emission of the double-strand DNA/AgNCs at 565 nm starts to quench when 6.0 nM of Hg2+ or Cu2+ were added to them, and up to 75% of fluorescence were quenched in the presence of 150.0 nM of Hg2+ or 250.0 nM of Cu2+. Fluorescence quenching ratio [(I0 I)/I0] of the DNA/AgNCs toward the two metal ions concentrations were shown in Fig. 2B
0.4
2 1
0.2
0.00
525
550
575
600
625
650
675
0.0
0.1
0.2
0.3
200 150 100 50
1.0
575
600
0.5
0.6
0.7
4
0.6
3
0.4 0.2
625
650
0.0
0.1
0.2
0.3
cCu2+ / μM
0.0
675
2 1
0.0 550
0.4
0.8
0 525
0.20
D
I0/I
0μΜ 0.006μΜ 0.020μΜ 0.060μΜ 0.100μΜ 0.160μΜ 0.240μΜ 0.360μΜ 0.480μΜ 0.600μΜ
2+
Cu added:
C
0.15
cHg2+ / μM
(I0-I)/I0
250
0.10
cHg2+ / μM
Wavelength/nm Fluorescence intensity (a.u.)
0.05
0.0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
cCu2+ / μM
Wavelength/nm
Fig. 2. Fluorescence quenching of double-strand DNA/AgNCs in the presence of different concentration of Hg2+ (A) and Cu2+ (C) and corresponding fluorescence quench ratios toward the two metal ions (B) and (D). The insets in (B) and (D) are Stern–Volmer plot of the quenching. Experimental conditions: DNA/Ag NCs, 0.0898 lmol/L; 20 mM phosphate buffer (pH 7.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
10000
A
10000
dsDNA/AgNCs 2+ dsDNA/AgNCs + 0.2 μM Hg
1000
Decay
Decay
1000
dsDNA/AgNCs 2+ dsDNA/AgNCs + 0.2 μM Cu
B
τNCs= 0.375 ns (14.60%) 2.938 ns (85.40%)
100
τNCs= 0.375 ns (14.60%) 2.938 ns (85.40%)
100
τNCs+Hg= 0.346 ns (18.63%)
τNCs+Cu= 0.295 ns (19.47%)
2.936 ns (81.37%)
10
2.931 ns (80.53%)
10 25
30
35
40
45
25
30
Time (ns) 0.8
0.8
C
0.4 lg(I0-I)/I = 15.32 + 2.18 lgcHg R =0.9618
lg(I0-I)/I
lg(I0-I)/I
40
45
D
0.4 lg(I0-I)/I = 14.31 + 2.10 lgcCu
2
0.0
35
Time (ns)
-0.4
2
R =0.9705
0.0 -0.4
-0.8 -0.8 -1.2 -1.2
-1.6 -7.8
-7.6
-7.4
-7.2
lgcHg2+
-7.0
-6.8
-6.6
-7.4
-7.2
-7.0
-6.8
-6.6
lgcCu2+
Fig. 3. Fluorescence quenching mechanism studied by fluorescence lifetime (A) and (B) and Stern–Volmer relationship (C) and (D).
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metal ion concentration. Thus, we believed that the double-strand DNA/AgNCs whose fluorescence is enhanced by guanine-rich DNA are good for Hg2+ and Cu2+ sensing. The fluorescence quenching mechanism of double-strand DNA/ AgNCs by Hg2+ and Cu2+ was also studied. We compared the fluorescence lifetime of double-strand DNA/AgNCs before and after quenched by metal ions. As shown in Fig. 3A and B, no change in fluorescence lifetime indicated a static fluorescence quenching mechanism. However, the Stern–Volmer plots shown in Fig. 2B and D (inset figures) are not linear with the concentration of metal ions. Considering DNA/AgNCs is a biomolecule stabilized fluorescence probe, we inferred that the reaction ratio of the fluorophore
to quencher is not 1:1, and supposed 1 mol of DNA/AgNCs would combine n mole of metal ions. Thus, the static quenching equation should be:
(I0-I)/I0
0.6 0.5 0.4 2+
0.2
0.2μM of Cu
2+
0.1 0.0 0
20
40
60
80
100
ð2Þ
I0 =I ¼ 1 þ K a ½Q n
ð3Þ
lg½ðI0 IÞ=I ¼ lg K a þ n lg½Q
ð4Þ
According to Eq. (4), the association constant (Ka) and combination ratio (n) of DNA/AgNCs to Hg2+ and Cu2+ could be obtained by fitting relationship between lg(I0 I)/I and lg[Q]. As shown in Fig. 3C and D, by repeating the experiments, the value of lg Ka and n was calculated to be 15.32 and 2.18 for Hg2+, and 14.31 and 2.10 for Cu2+, respectively. These parameters indicate that every DNA/AgNCs would combine two metal ions and the combination ability of DNA/AgNCs to Hg2+ is larger than that of Cu2+. The fluorescence quenching kinetics of double-strand DNA/ AgNCs by Hg2+ and Cu2+ were investigated by recording the fluorescence spectrum of them and certain concentrations of Hg2+ and Cu2+ in two hours. As shown in Fig. 4, in the presence of 0.2 lM Hg2+, the fluorescence intensity of the DNA/AgNCs decreased sharply in the first 60 min, and then decreased slowly and remained in the latter 60 min. The quenching kinetics of the DNA/AgNCs under Cu2+ ion shows similar results with Hg2+. Thus, a reliable detection of Hg2+ and Cu2+ need an hour of waiting.
0.7
0.2μM of Hg
ð1Þ
K a ¼ ½FQ n =½F½Q n
where [F], [Q] and [FQn] is concentration of fluorophore, quencher and formed complex, respectively. Ka is association constant of the complex. Thus,
0.8
0.3
½F þ n½Q ¼ ½FQ n
120
t / min Fig. 4. Fluorescence quenching kinetics of the double-strand DNA/AgNCs toward 0.2 lM of Hg2+ and Cu2+. Experimental conditions: DNA/Ag NCs, 0.0898 lM; 20 mM phosphate buffer (pH 7.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
Selectivity and interference The selectivity and interference of double-stand DNA/AgNCs fluorescence for metal ions were also investigated. As indicated 0.8
A
1.0
B 0.6
(I0-I)/I0
(I0-I)/I0
0.8 0.6 0.4 0.2
0.4 0.2
0.0 2+
2+
2+
3+
2+
2+
3+
2+
2+
3+
2+
2+
2+
+
0.0
2+
Blank Cu Hg Fe Cr Mg Cd Al Mn Ca Fe Cd Zn Co Ag Pd
BlankHg
2+
2+
3+
2+
2+
3+
2+
2+
Fe Cr Mg Pb Al Mn Ca Fe
Metal ions
3+
2+
2+
2+
+
Cd Zn Co Ag Pd
2+
Metal ions
Fig. 5. Selectivity and interferes for Hg2+ detection. (A) Metal ion concentrations: Hg2+ and Cu2+, 0.8 lM; others, 40 lM. (B) Fluorescence quenching ratios toward 0.1 lM Hg2+ with coexistence of 1.25 lM other metal ions. Experimental conditions: dsDNA/Ag NCs, 0.0898 lM; 20 mM phosphate buffer (pH 7.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
0.8
0.8
A
0.6
(I0-I)/I0
(I0-I)/I0
(I0-I)/I0=0.00659 + 4.769 cHg 0.6 R2=0.9985 0.4
0.0
0.0 0.08
cHg2+ / μΜ
0.12
0.16
2
0.4 0.2
0.04
(I0-I)/I0=0.00835 + 3.018 cCu
R =0.9953
0.2
0.00
B
0.00
0.05
0.10
0.15
0.20
0.25
cCu2+ / μΜ
Fig. 6. Linear relationship between double-strand DNA/AgNCs fluorescence quenching ratio and Hg2+ (A) and Cu2+ (B) concentration. Experimental conditions: DNA/Ag NCs, 0.0898 lM; 20 mM phosphate buffer (pH 7.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
J. Peng et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 137 (2015) 1250–1257 Table 1 Determination of Hg2+ by fluorescent metal nanoclusters probes in recent papers. Probes
Linear range
LOD
References
DNA–AgNCs Denatured BSA–AgNCs Lys–AgNCs GSH–AgNCs PMAA–AgNCs G-enhanced DNA–AgNCs
2.5–50 nM 10 nM–5 lM 1–15 lM 5–125 nM 10 nM–20 lM 6–260 nM
0.9 nM 10 nM 0.6 lM 5 nM 10 nM 2.1 nM
[53] [18] [54] [13] [52] This work
Table 2 Determination of Cu2+ by fluorescent metal nanoclusters probes in recent papers. Probes
Linear range
LOD
References
PMAA–AgNCs PMAA–AgNCs MPAA–AgNCs DNA–AgNCs MUA–AuNPs G-enhanced DNA–AgNCs
10 nM–30 lM 10 nM–6 lM 5 lM–15 mM 15–200 nM 10 nM–1 lM 6–240 nM
10 nM 8 nM 5 lM 8 nM 87 nM 3.4 nM
[52] [26] [55] [28] [56] This work
Table 3 Analytical results of mercury and copper ions detection in real water samples. Samplesa
Metal ions
Added (lM)
Detected (lM)
Recovery (%)
RSD (n = 3 (%))
1
Hg2+ Hg2+
0.030 0.060
0.035 0.059
116.67 98.33
8.01 4.92
2
Hg2+ Hg2+
0.040 0.080
0.045 0.087
112.50 108.75
5.58 8.04
3
Cu2+ Cu2+
0.040 0.080
0.041 0.076
102.50 95.00
8.25 2.70
4
Cu2+ Cu2+
0.050 0.100
0.048 0.094
96.00 94.00
4.73 4.27
a The four samples were taken from different areas of Panlong River at Kunming City.
in Fig. 5A, the DNA/AgNCs exhibited high selectivity for Hg2+ and Cu2+ ions over a variety of other metal ions. The interference of other metals (except Cu2+) for Hg2+ detection is also very slight. As shown in Fig. 5B, the fluorescence of the hybridized DNA/AgNCs partially quenched by 50 nM Hg2+ was not affected by coexistence of 1.25 mM competing metal ions. Calibration curve for Hg2+ and Cu2+ detection
300 250 200
was possible to develop a new fluorescence sensing system for Hg2+ and Cu2+ based on AgNCs. Fluorescence quenching ratio [(I0 I)/I0] was used for establishing calibration curve for Hg2+ and Cu2+ detection. As shown in Fig. 6 a good linear relationship between (I0 I)/I0 and the concentration of Hg2+ and Cu2+ can be observed in the Hg2+ and Cu2+ concentration range from 6.0 nM to 160.0 and 6 nM to 240 nM, respectively. The linear regression equation (c, lM) can be expressed as (I0 I)/I0 = 0.00659 + 4.769c for Hg2+ and (I0 I)/I0 = 0.00835 + 3.018c for Cu2+ with correlation , coefficients (R2) of 0.9916 and 0.9952, respectively. The limits of detection (LOD, 3r) are 2.1 nM for Hg2+ and 3.4 nM for Cu2+, respectively. We compared the analytical parameters of our method with the recently published Ref. [13,18,26,28,52–56], and found that the selectivity is better than others (see Tables 1 and 2).
Masking of Cu2+ using EDTA The above results revealed that this assay approach had high sensitivity toward mercury and copper ions. As mercury is a severe environmental pollutant which is more harmful to human beings than copper, establishing a sensitive and selective method for Hg2+ detection is important for monitoring environmental pollutants. In order to achieve this, an efficient Cu2+ chelating reagent, ethylene diamine tetraacetate (EDTA) was added to minimize the interference of the Cu2+. Similar to the recently reported paper [51,52], a proper amount of EDTA was added into the double-strand DNA/AgNCs and 1.0 lM of Cu2+ mixed solution and then applied for the Hg2+ detection. The results showed that the fluorescence intensity decreased with the increase of Hg2+ concentration (Fig. 7). The Fluorescence quenching ratio [(I0 I)/I0] toward Hg2+ concentration has a good linear relation in the range from 20.0 to 400.0 nM and the LOD was estimated about 8 nM (3r). Therefore, Cu2+ can be successfully masked in this system with little effect on the detection of Hg2+, allowing for the sensitive detection of a single ion. It should be noticed that Cu2+ could also be detected selectivity by masking of Hg2+ using similar methods that had been intensively studied by previous reports [51,52]. In this paper, no more repetition has been done here.
Real sample detection To evaluate the availability of the proposed analytical method for natural samples, three bottles of natural water samples collected from a local river were analyzed by the standard addition method. Quantification results shown in Table 3 indicated that our method is reliable for detection real water samples. 1.0
A
B
2+
Hg added: 2+
Cu 1.0 μM EDTA 1.0 μM
150 100 50
0μM 0.005μM 0.010μM 0.020μM 0.040μM 0.080μM 0.240μM 0.320μM 0.400μM 0.500μM 0.600μM
0.8
(I0-I)/I0 = 0.11353 + 1.74758 cHg 2
R = 0.99414
(I0-I)/I0
Fluorescence intensity (a.u.)
In view of the intensive quenching of the fluorescence emission of double-strand DNA/AgNCs in the presence of Hg2+ and Cu2+, it
1255
0.6 0.4 0.2
0 510
540
570
600
Wavelength/nm
630
660
0.0
0.1
0.2
0.3
0.4
cHg2+ / μM
Fig. 7. (A) Fluorescence changes of double-strand DNA/Ag NCs in the presence of different concentrations of Hg2+ after Cu2+ was masked. (B) Corresponding linear relationship between DNA/Ag NCs fluorescence quenching ratio and Hg2+ concentration. Experimental conditions: DNA/Ag NCs, 0.0898 lM; 20 mM phosphate buffer (pH 5.0); kex at 510 nm and kem at 565 nm; reaction time, 90 min.
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Conclusion In summary, we designed a new hybridized double-strand DNA/ AgNCs for fluorescence detection of mercury and copper ions. Compared with the single-strand DNA/AgNCs before hybridization, the hybridized double-strand DNA/AgNCs probe is brighter and stable in fluorescence. Experimental results showed that mercury and copper ions in the range of 6.0–160.0 and 6–240 nM, can be linearly detected with the detection limits of 2.1 and 3.4 nM, respectively. The analytical parameters of the method for mercury and copper ions detection are better than that using a single-strand DNA/AgNCs. By using EDTA as a masking reagent, Hg2+ could be selectively detected coexisted with Cu2+ and other metal ions. Mercury and copper ions in nature water were also successfully detected by the standard addition method. Overall, our work offers an important strategy for DNA/AgNCs fluorescence improvement and its further application. Acknowledgments We thank Dr. Lin-Ling Zheng and Ming-Xuan Gao from Southwest University, Chongqing, China, for fluorescence lifetime measurement and data analysis. We also show our great appreciation for the financial support of the National Natural Science Foundation of China (21105087, 21465025), Applied and Basic Research Program of Yunnan Province (2011FB014) and Cultivation Program for Key Young Teachers of Yunnan University. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2014.08.135. References [1] P.B. Tchounwou, W.K. Ayensu, N. Ninashvili, D. Sutton, Environmental exposure to mercury and its toxicopathologic implications for public health, Environ. Toxicol. 18 (2003) 149–175. [2] I. Onyido, A.R. Norris, E. Buncel, Biomolecule–mercury interactions: modalities of DNA base-mercury binding mechanisms. Remediation strategies, Chem. Rev. 104 (2004) 5911–5929. [3] P.G. Georgopoulos, A. Roy, M.J. Yonone-Lioy, R.E. Opiekun, P.J. Lioy, Environmental copper: its dynamics and human exposure issues, J. Toxicol. Environ. Health, B 4 (2001) 341–394. [4] E.L. Que, D.W. Domaille, C.J. Chang, Metals in neurobiology: probing their chemistry and biology with molecular imaging, Chem. Rev. 108 (2008) 1517– 1549. [5] J.-M. Liu, H.-F. Wang, X.-P. Yan, A gold nanorod based colorimetric probe for the rapid and selective detection of Cu2+ ions, Analyst 136 (2011) 3904–3910. [6] Y.J. Long, Y.F. Li, Y. Liu, J.J. Zheng, J. Tang, C.Z. Huang, Visual observation of the mercury-stimulated peroxidase mimetic activity of gold nanoparticles, Chem. Commun. 47 (2011) 11939–11941. [7] P. Zhang, S. Chen, Y. Kang, Y. Long, Trace mercury ion determination based on the highly selective redox reaction between stannous ion and mercury ion enhanced by gold nanoparticles, Spectrochim. Acta, A 99 (2012) 347–352. [8] H. Xu, K.S. Suslick, Sonochemical synthesis of highly fluorescent Ag nanoclusters, ACS Nano 4 (2010) 3209–3214. [9] L. Shang, S. Dong, G.U. Nienhaus, Ultra-small fluorescent metal nanoclusters: synthesis and biological applications, Nano Today 6 (2011) 401–418. [10] J.T. Petty, J. Zheng, N.V. Hud, R.M. Dickson, DNA-templated Ag nanocluster formation, J. Am. Chem. Soc. 126 (2004) 5207–5212. [11] C.I. Richards, S. Choi, J.-C. Hsiang, Y. Antoku, T. Vosch, A. Bongiorno, Y.-L. Tzeng, R.M. Dickson, Oligonucleotide-stabilized Ag nanocluster fluorophores, J. Am. Chem. Soc. 130 (2008) 5038–5039. [12] W. Gue, J. Yuan, E. Wang, Oligonucleotide-stabilized Ag nanoclusters as novel fluorescence probes for the highly selective and sensitive detection of the Hg2+ ion, Chem. Commun. (2009) 3395–3397. [13] X. Yuan, T.J. Yeow, Q. Zhang, J.Y. Lee, J. Xie, Highly luminescent Ag+ nanoclusters for Hg2+ ion detection, Nanoscale 4 (2012) 1968–1971. [14] J.L. MacLean, K. Morishita, J. Liu, DNA stabilized silver nanoclusters for ratiometric and visual detection of Hg2+ and its immobilization in hydrogels, Biosens. Bioelectron. 48 (2013) 82–86. [15] B. Adhikari, A. Banerjee, Facile synthesis of water-soluble fluorescent silver nanoclusters and Hg-II sensing, Chem. Mater. 22 (2010) 4364–4371.
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