A Novel Single-Labeled Fluorescent Oligonucleotide Probe for Silver(I

May 1, 2014 - A Novel Single-Labeled Fluorescent Oligonucleotide Probe for .... Subhankar Singha , Dokyoung Kim , Hyewon Seo , Seo Won Cho , Kyo Han ...
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A Novel Single-Labeled Fluorescent Oligonucleotide Probe for Silver(I) Ion Detection in Water, Drugs, and Food Liujiao Bian,*,† Xu Ji,† and Wei Hu‡ †

College of Life Science, Northwest University, Xi’an, Shaanxi 710069, China Emergency Department, Shaan’xi Provincial People’s Hospital, Xi’an, Shaanxi 710068, China

‡

S Supporting Information *

ABSTRACT: Due to the high toxicity of silver(I) ions, a method for the rapid, sensitive, and selective detection for silver(I) ions in water, pharmaceutical products, and food is of great importance. Herein, a novel single-labeled fluorescent oligonucleotide (OND) probe based on cytosine−Ag(I)−cytosine coordination and the inherent fluorescence quenching ability of the Gquadruplex is designed to detect silver(I) ions. The formation of a hairpin structure in the OND−Ag(I) complex brings the hexachloro fluorescein (HEX) labeled at the 5′-end of the OND probe close to the G-quadruplex located at the 3′-end of the OND probe, leading to a fluorescence quenching due to photoinduced electron transfer between HEX and the G-quadruplex. Through this method, silver(I) ions can be detected quantitatively, the linear response range is from 1 to 100 nmol/L with a detection limit of 50 pmol/L, and no obvious interference occurs with other metal ions with a 10-fold concentration. This assay is simple, sensitive, and selective, and it can be used to detect silver(I) ions in actual water, drug, and food samples. KEYWORDS: silver(I) ions, detection, fluorescent oligonucleotide probe, hexachloro fluorescein, G-quadruplex

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INTRODUCTION Silver(I) ions are one of the most important bioactive cations. They exhibit low toxicity to living beings within the scope of the low concentration while high toxicity in high concentration range. For example, under a low concentration range, silver(I) ions have long been known to have strong inhibitory and bactericidal effects as well as a broad spectrum of antimicrobial activities. They have a microbicidal (germicidal and bacteriostatic) effect in drinking water, enabling it to be disinfected and conserved. The recommended and maximum quantities of silver(I) ions in drinking water are usually limited to a concentration of 100 μg/L. In both the United States (United States Environmental Protection Agency, secondary drinking water regulation) and Australia (National Health and Medical Research Council), the recommended value is 100 μg/L. In Germany (Current German Drinking Water Ordinance, TrinkwV 2001), the statutory limit is 100 or 80 μg/L depending on the treatment substances and disinfecting processes. Whereas in a high concentration area, a high dose of silver(I) ions has been found to be highly toxic to aquatic organisms and humans owing to the fact that silver(I) ions have a high affinity to sulfhydryl groups and amino groups and thereby form some harmful complexes with amino acids, nucleic acids, and other compounds in the body. For example, exposed to a high dose of silver nitrate, sodium/potassium ATPase, which regulates the level of potassium and sodium ions in fish and zooplankton, was seriously interfered with the silver(I) ions. In addition, silver(I) ions are the most common pollution cations and one of the main toxic substances in the field of agriculture and food. Through polluted water sources, such as river water for irrigation and lake or seawater for aquiculture, they can accumulate to the seed of crops and fruits and the body of aquatic organisms, and thereby enter the food chain of humans. Therefore, it is very important to monitor the © 2014 American Chemical Society

content level of silver(I) ions in aquicolous ecosystems and the human food chain.1,2 To date, several methods including colorimetric analysis,3 atomic absorption/emission spectroscopy,4−7 inductively coupled plasma−mass spectrometry, 8,9 surface-enhanced Raman scattering,10 ion-selective electrodes11 and quantum dots,12,13 and so forth14 have been established to detect trace level of silver(I) ions in aqueous media, and a lot of efforts has also been made to develop fluorescent chemosensors, but there are only a few successful examples for silver(I) ion detection.15−18 In view of the interaction between metal ions and nucleic acids, some oligonucleotide (OND)-based metal ion detection method had been established,19−26 for example, on the basis of the fact that silver(I) ions are capable of coordinating cytosine bases (C) to form a stable C−Ag(I)−C complex, leading to the development of a silver(I) ion sensor.27−32 As illustrated in Scheme 1a, in the absence of Ag(I) ions, two separate C-rich ssDNA separately form a random coil. However, in the presence of Ag(I) ions, the Ag(I) ions bind directly to N3 of cytosine in place of the imino proton and bridge two cytosine residues in two separate C-rich ssDNA to form the C−Ag(I)−C complexes. By applying this principle, Tanaka et al. have developed a fluorescent C-rich OND probe for silver(I) ion detection with a fluorescent dye and a quencher at its 3′- and 5′-ends, respectively.28 Qu et al.33 and Fan et al.34 separately developed a single-labeled fluorescent OND probe for silver(I) ion detection by employing a singlewalled carbon nanotube or graphene as an extra nanoquencher. Received: Revised: Accepted: Published: 4870

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quadruplex. In this fluorescent OND probe, a G-quadruplex (5′-GGTTGGTGTGGTTGG-3′) was designed to follow a Crich OND (5′-CCCTCCTTCCCTTCCTTTTCCAACCCAACCACC-3′) and a fluorescein-based dye, hexachloro fluorescein (HEX), was labeled at the 5′-end of the C-rich OND. In the presence of silver(I) ions, the C−Ag(I)−C coordination induced the labeled C-rich OND to fold into a hairpin structure that brings the fluorescent termini HEX close to the G-quadruplex, resulting in a significant fluorescence quenching of the fluorescent termini HEX owning to photoinduced electron transfer between HEX and the Gquadruplex.40

Scheme 1. Schematic Illustration of the Formation of Cytosine−Ag(I)−Cytosine Complexes

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MATERIALS AND METHODS

Chemicals. The single-labeled fluorescent OND probe (5′-HEXCCCTCCTTCCCT-TCCTTTTCCAACCCAACCACCGGTTGGTGTGGTTGG-3′) was chemically synthesized by Takara Biotechnology Co. Ltd. (Dalian, China) and purified by highperformance liquid chromatography. DNA concentration was estimated by measuring the absorbance at 260 nm. 3-(N-Morpholino) propanesulfonic acid (MOPS) was purchased from Sigma−Aldrich Inc. (Shanghai, China). AgNO3, Ni(NO3)2, CoSO4, CdSO4, Cu(NO3)2, Hg(NO3)2, Mn(NO3)2, Ca(NO3)2, ZnSO4, Pb(NO3)2, Mg(NO3)2, FeSO4, Fe2(SO4)3, NaNO3, KNO3, KF, and KClO4 were analytical grade without further purification. All the fluorescent OND probe solutions (20.0 nmol/L) and silver(I) ion solutions were prepared with 10.0 mmol/L MOPS buffer (containing 400.0 mmol/L NaNO3 and 20.0 mmol/L KNO3, pH 7.0) and stored in brown reagent bottles. The ultrapure water (18.2 MΩ·cm) was purified by a Millipore ultrapure water system. Tap water and wastewater samples were separately taken from a household water pipe and wastewater storage tank in our laboratory. River water sample was collected from the Wei River near Xi’an. “YinXin-Shuang” silver−zinc emulsion (approval no.: Henan health and diminish inflammation (2000) 0129, batch no.: 110606) and “HongGao-Yao” red ointment (approval no.: Hubei pharmacy (2001) CX02017, batch no.: 120425) are both typical ointment agents. “Bi-LuoChun” green tea (Tianfu Tea Industry Co., Ltd., batch no.: 20120812) and bean sprout were purchased from Walmart supermarket in Xi’an, China. Fluorescence Emission Spectra. A 10 μL amount of 1.0 mmol/L fluorescent OND probe was first added to 490 μL of MOPS buffer and incubated at 95 °C for 10 min in order that the secondary structure of the OND was completely unwinded. Then, the mixed solution was naturally cooled down to 25 °C and was incubated overnight. The incubated solution was then transferred into a cuvette, and its fluorescent emission was scanned at 25 °C under the wavelength range 540−630 nm. The final solution contains 10.0 mmol/L MOPS, 400.0 mmol/L NaNO3, and 20.0 mmol/L KNO3 (pH 7.0), and its total volume is 500 μL. The concentration of the fluorescent OND probe is 20.0 nmol/L. After that, 1.0 μL of silver(I) ion solution with different concentrations was separately added to the above solution at 25 °C, and their fluorescent emissions were scanned under the same conditions. Fluorescent emission spectra were recorded on a Hitachi F-4500 fluorescence spectrophotometer (Hitachi Corporation, Japan). The instrument parameters were as follows: excitation wavelength, 520 nm; emission wavelength, 556 nm; excitation and emission slits, 10 nm; temperature, 25 °C. Ultraviolet−Visible Absorbance Spectra. All the UV−visible (UV−vis) absorbance spectra were recorded on a Shimadzu UV-2450 spectrophotometer. Detection of Silver(I) Ions. To verify the application of this method in actual samples, we determined the silver(I) ions in a tap water sample, a wastewater sample, a river water sample, two drug samples, and two food samples. For the water sample detection, all the tap water, wastewater, and river water samples were filtered through a 0.22 μm membrane before testing. A 400 μL amount of MOPS buffer containing fluorescent

Sun et al. designed a single-labeled fluorescent OND probe for silver(I) ion detection based on the inherent quenching ability of deoxyguanosines,35 and Luo et al. also developed a dualoutput fluorescent DNA sensor for silver(I) ion and cysteine detection.36 Through intramolecular hydrogen-bonding interaction, a guanine base (G)-rich single-stranded DNA can form a specific secondary structure named the G-quadruplex that can serve as a very good electron donor and an extraordinary quencher for a variety of fluorescent groups due to its high density of guanosine stacking and low oxidation potential (1.25 V vs SCE).37 Therefore, a resonant energy transfer or photoinduced electron transfer may take place between it and the fluorescent groups,35,38 leading to the significant fluorescence quenching of the fluorescent groups in the excited state.39 In this work, we develop a novel single-labeled fluorescent OND probe for the sensitive and selective detection of silver(I) ions in aqueous solution based on the C−Ag(I)−C coordination and the inherent quenching ability of the G4871

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OND probe was first incubated at 95 °C for 10 min in order that the secondary structure of the fluorescent OND probes was completely unwinded. Then, the solution was naturally cooled down to 25 °C and was incubated overnight. After that, 100 μL of different water samples were separately mixed with the incubated solution at 25 °C, and their fluorescence responses were monitored under the experimental conditions as described above. All of the final mixed solutions contain 10.0 mmol/L MOPS, 400.0 mmol/L NaNO3, and 20.0 mmol/L KNO3 (pH 7.0), and their total volumes are 500 μL. The final concentration of the fluorescent OND probe is 20.0 nmol/L, and the dilution factor is 4.0. For the drug sample detection, about 1.0 g of the above silver−zinc cream or red ointment was accurately weighed and put in a small digestion flask, 4 mL of nitric acid was added to the digestion flask, and it was heated in order that the drug sample was fully digested. After complete digestion, the drug sample solution was transferred into a volumetric flask and diluted to 10 mL with MOPS buffer. Then, the digested sample was diluted to a proper proportion with MOPS buffer, and its fluorescence response was monitored under the test conditions as the determination of water samples. For the tea and bean sprout sample detection, a porcelain crucible was first heated and cleaned for about 30 min on an electric furnace with a volume ratio of 1:4 hydrochloric acid solution; then, the porcelain crucible was rinsed three times with pure water and laid in a constant temperature box for drying. About 2.0 g of the tea or bean sprout sample was accurately weighed and put in the above porcelain crucible, the porcelain crucible was laid on an electric furnace making the sample be fully carbonized at low temperature until white smoke disappeared, and then, the porcelain crucible was placed into a muffle furnace at 520 °C for about 6 h in order that the sample was fully ashed. After that, it was cooled, 5 mL of 1:1 nitric acid solution was added to the porcelain crucible, and it was placed on a boiling water bath until the sample solution was fully dried. A 9 mL amount of distilled water and 1 mL of 2.0 mol/L nitric acid were added in the porcelain crucible, and it was heated at about 95 °C for 10 min. The sample solution was cooled and transferred into a 50 mL volumetric flask. The porcelain crucible was washed two times with MOPS buffer, the washes were collected into the volumetric flask, the sample solution was diluted to 50 mL with MOPS buffer, and it was mixed evenly. Finally, the sample solution was diluted to a proper proportion with MOPS buffer, and its fluorescence response was monitored under the experimental conditions as the determination of water samples.

Figure 1. Fluorescence quenching of the HEX−OND probes under the induction of silver(I) ions. (a) HEX−OND probe (20.0 nmol/L); (b) HEX−OND probe (20.0 nmol/L) + Ag(I) (0.10 μmol/L); (c) HEX−OND probe (20.0 nmol/L) + Ag(I) (1.0 μmol/L); (d) HEX− OND probe (20.0 nmol/L) + Ag(I) (5.0 μmol/L). All measurements were performed in 10.0 mmol/L MOPS buffer (containing 400.0 mmol/L NaNO3 and 20.0 mmol/L KNO3, pH 7.0).

probe increase from 5:1 (curve b) to 250:1 (curve d), the fluorescence quenching degree of the HEX−OND probes increases from 45% to 82%, indicating that the G-quadruplex at the 3′-end of the HEX−OND probe can quench the fluorescence of the fluorescent dye HEX labeled at the 5′-end of the HEX−OND probe. This observation also provides clear evidence to support the Ag(I)-induced formation of double helical structure.25,33,35,36 In addition, a further experiment shows that there is no further decrease of the fluorescence intensity when the concentration of silver(I) ions is higher than 5 μmol/L, indicating the interaction between the HEX−OND probe and the silver(I) ions reaches a dynamic equilibrium. The analytical methods based on oligonucleotide probes modified with quenchers and fluorescent dyes required efficient quenching ability of quenchers. Although the fluorescence of fluorescent dyes could be quenched by the interaction between the dyes and guanosines, the quenching ability of guanosine itself was not enough to meet the need of a highly sensitive assay.33,36 Therefore, as an effective way, the sensitivity can be improved by enhancing the quenching efficiency of the quenchers. It has been reported that guanosine-rich tracts of nucleic acids could form four-stranded structure under appropriate condition of high sodium or potassium ionic strength. Four guanine bases can associate through Hoogsteen hydrogen bonding to form a square planar structure called a guanine tetrad, and two or more guanine tetrads can stack on top of each other to form a G-quadruplex. The quadruplex structure can be further stabilized by the presence of a cation, especially sodium or potassium, which sits in a central channel between each pair of tetrads.41 As a structure with high density of guanosine stacking, the G-quadruplex has very good electrondonating property and extraordinary quenching ability. Therefore, it is used here as a convenient and robust tool to quench the fluorescence of fluorescent dyes.42,43 Fluorescence Quenching Mechanism. In order to further understand the quenching information in this Ag(I) detection method, the fluorescence spectra of HEX and UV− vis absorption spectra of HEX-labeled OND were investigated

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RESULTS AND DISCUSSION Fluorescence Quenching of the Fluorescent OND Probe. On the basis of the C−Ag(I)−C coordination and the inherent quenching ability of the G-quadruplex, a schematic diagram to illustrate the single-labeled fluorescent OND probe for silver(I) ion detection is shown in Scheme 1b. In the absence of silver(I) ions, the fluorescent HEX−OND probes form a random coil, which separates the fluorescent dye HEX and the G-quadruplex from each other, and hence, the fluorescent dye HEX exhibits strong fluorescence emission. However, in the presence of silver(I) ions, the silver-mediated base pairs are formed between cytosine residues in the fluorescent HEX−OND probes, leading to a hairpin structure. The fluorescent dye HEX and the G-quadruplex are close to each other upon formation of the hairpin structure, leading to the significant fluorescence quenching of the fluorescent dye HEX in the fluorescent HEX−OND probes. Under the induction of different concentrations of silver(I) ions, the fluorescence quenching effect of the fluorescent HEX−OND probes was determined (Figure 1). In the absence of silver(I) ions, the fluorescent HEX−OND probes exhibit strong fluorescence emission at 556 nm, which can be attributed to the presence of the fluorescein-based dye HEX (curve a). When the molar ratios of Ag(I) to the HEX−OND 4872

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complex compounds or precipitates in alkaline conditions, when pH is higher than 7.0, the response of the HEX−OND probes to silver(I) ions becomes poorer and poorer with the increased pH. Therefore, all the experiments were performed in neutral condition. (ii) Concentration of sodium nitrate. In the DNA hybridization system, ionic strength has an important influence on the stability and sensitivity of the molecular probes. Therefore, we further investigated the fluorescence quenching of the HEX− OND probes in the presence of 0.0−1.0 mol/L sodium nitrate (Figure 3). The quenching effect was evaluated with

on addition of Ag(I) ions, and the results were listed as follows: First, by investigating the effect of Ag(I) on the HEX, it is found that the Ag(I) ions cannot quench the fluorescence of HEX under the same experimental conditions (Figure S1, Supporting Information). Second, in the presence of Ag(I) ions, no new absorption band occurred in the UV−vis absorption spectra of HEX-labeled OND, and almost no visible shift appeared in its maximum absorption (Figure S2, Supporting Information), which implied that there existed no ground-state complexes formed during the detection process.40,44−46 Third, the absorption band of the HEX-labeled OND probe with the addition of Ag(I) ions ranging from 240 to 300 nm (Figure S2, Supporting Information) does not overlap with the emission spectra of HEX ranging from 530 to 580 nm (Figure 1), indicating the energy transfer could be neglected during the fluorescence quenching procedure. Actually, it has been widely reported that the fluorescence of many fluorescent dyes including fluorescein and HEX that is a derivative of fluorescein are quenched by the interaction between the dyes and guanines, and this phenomenon is demonstrated to involve a photoinduced electron transfer mechanism.35,36,40,47−49 For that reason, the fluorescence quenching in this work by the interaction between HEX and the G-quadruplex that is the guanosine stacking is also caused by the photoinduced electron transfer mechanism like the previously works mentioned. Optimization of Detection Conditions. (i) pH. HEX belongs to a fluorescein-based dye. Although the fluorescence emission of HEX is relatively insensitive to pH compared with that of fluorescein, it can be still influenced by pH within a wider pH range. Therefore, we determined the fluorescence intensities of the HEX−OND probes and the OND−Ag(I) complexes over a pH range 4−10. As shown in Figure 2, a decreased pH leads to a decreased fluorescence intensity of the HEX−OND probes; meanwhile, as the silver(I) ions can form

Figure 3. Fluorescence quenching extent of the HEX−OND probes in the presence of different concentrations of sodium nitrate. Concentrations of the HEX−OND probes and silver(I) ions were 20.0 nmol/L and 0.10 μmol/L, respectively. All measurements were performed in 10.0 mmol/L MOPS buffer (containing 20.0 mmol/L KNO3, pH 7.0) with different concentrations of sodium nitrate ranging from 0.0 to 1.0 mol/L.

fluorescence quenching extent (F0 − F)/F0, where F0 is the initial fluorescence intensity of the HEX−OND probes and F is the fluorescence intensity in the presence of 100.0 nmol/L silver(I) ions. The results show that, in the concentration of sodium nitrate ranging from 0 to 0.40 mol/L, the fluorescence quenching extents of the HEX−OND probes increase with the rising of the sodium nitrate concentrations, whereas under the concentration range 0.40−1.0 mol/L, they decrease as the concentrations of sodium nitrate increase. The highest fluorescence quenching extent (about 50%) occurs at 0.40 mol/L sodium nitrate. Therefore, we selected 0.40 mol/L as the optimum concentration of sodium nitrate. (iii) Reaction time. In all of the above experiments, the measurement procedures were carried out right after the addition of the silver(I) ions into the HEX−OND probe solution, and the involvement of longer incubation times does not result in an observable increase in the fluorescence emission, indicating that the process of detection is very fast. Establishment of Detection Method. (i) Sensitivity. To evaluate the sensitivity of this detection system, as shown in Figure 4, we further determined the fluorescence emission spectra of the HEX−OND probes in the presence of different concentrations of silver(I) ions ranging from 0.002 to 5 μmol/L under the above optimum condition. The results show that the fluorescence intensity of the HEX−OND probes is very

Figure 2. Fluorescence intensities of the HEX−OND probes and OND−Ag(I) complexes at different pH values. (A) HEX−OND probe (20.0 nmol/L); (B) HEX−OND probe (20.0 nmol/L) + Ag(I) (0.10 μmol/L). All measurements were performed in 10.0 mmol/L MOPS buffer (containing 400.0 mmol/L NaNO3 and 20.0 mmol/L KNO3) with different pH values. 4873

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Figure 4. Fluorescence emission spectra of the HEX−OND probes in the presence of different concentrations of silver(I) ions. From top to bottom, the concentrations of silver(I) ions were 0, 0.002, 0.005, 0.008, 0.010, 0.020, 0.040, 0.060, 0.080, and 0.10 μmol/L, respectively. The concentration of the HEX−OND probe was 20.0 nmol/L. All measurements were performed in 10.0 mmol/L MOPS buffer (containing 400.0 mmol/L NaNO3 and 20.0 mmol/L KNO3, pH 7.0).

sensitive to silver(I) ions and steadily decreases as the concentrations of silver(I) ions increase from 0.002 to 5 μmol/L. (ii) Selectivity. As shown in Figure 5a, b, the selectivity of the HEX−OND probes for Ag(I) detection was first examined with a variety of environmentally relevant metal ions including Mg(II), Mn(II), Ca(II), Cd(II), Cu(II), Hg(II), Zn(II), Fe(II), Co(II), Ni(II), Pb(II), and Fe (III) ions. Figure 5a shows the difference in the fluorescence intensity between the blank and solutions containing 1.0 μmol/L silver(I) ions and other metal ions, and Figure 5b shows the influence of these metal ions with a 10-fold concentration on the selectivity of the HEX−OND probes. The results show that, in the latter case, although the HEX−OND probes produce slightly bigger fluorescence difference than that observed at 1.0 μmol/L, the use of silver(I) ions still gives the best result. In addition, from Figure 5a, b, it can be also found that Co(II) ion is the most significant interference metal ion for Ag(I) detection. This may be due to the fact that Co(II) ion has strong binding ability with the guanine bases in DNA.50 Then, the selectivity of the probes was examined with some environmentally relevant acid radical anions. In aqueous solution, free silver(I) ions exist mainly in the form of soluble nitrate. In addition to the limited kinds of anions, such as NO3(I), F(I), and ClO4(I), most of the anions, such as Cl(I), Br(I), I(I), S(II), SO3(II), SO4(II), CO3(II), PO4(III), C2O4(II), Ac(I), AsO4(III), BrO3(II), CN(I), and SCN(I), can combine with the free silver(I) ions to form insoluble or slightly soluble precipitates. Therefore, when the latter anions exist in solution, the fluorescence quenching process established in this work will not be performed due to the strong complexation of these anions to the silver(I) ions. Accordingly, here, we only examine the influence of NO3(I), F(I), and ClO4(I) on the selectivity of the fluorescent probe. Figure S3 (Supporting Information) shows the difference in the fluorescence intensities between the blank (no silver(I) ions) and the solution containing 1.0 μmol/L silver(I) ions and the influence of these anions with 10- and 100-fold concentrations to the silver(I) ions on the selectivity of the HEX−OND probes. The results showed that, in the presence of a high

Figure 5. Difference in the fluorescence intensities between the blank and solutions containing different metal ions. (a) Concentration of all metal ions was 1.0 μmol/L; (b) concentrations of silver(I) ions and other metal ions were 1.0 and 10.0 μmol/L, respectively. The concentration of the HEX−OND probes was 20.0 nmol/L. All measurements were performed in 10.0 mmol/L MOPS buffer (containing 400.0 mmol/L NaNO3 and 20.0 mmol/L KNO3, pH 7.0).

concentration of NO3(I), F(I), and ClO4(I), the changes of the fluorescent signals were within 5%, indicating that the fluorescent probe has good selectivity to silver(I) ions too in this case. (iii) Calibration curve. According to the fluorescence emission spectra of the HEX−OND probes in the presence of different concentrations of silver(I) ions, the plot of the fluorescence quenching extents (F0 − F)/F0 of the HEX−OND probes was created as the function of the concentrations of silver(I) ions (Figure 6) where F0 and F are the fluorescence intensities of the HEX−OND probes at 556 nm in the absence and presence of different concentrations of silver(I) ions, respectively. The results show that a good linear relationship exists between the fluorescence quenching extents and the concentrations of silver(I) ions ranging from 1 to 100 nmol/L, and the equation of the calibration curve is shown as eq 1: F0 − F = 5.17·CAg(I) + 0.0025, F0

R2 = 0.9999

(1)

Through the parallel tests, it is known that the detection limit (3σ) is 50 pmol/L that is much higher than that of previous reported methods. 4874

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Table 1. Determination Results of Silver(I) Ions in Tap Water and Wastewater Samples (n = 4) cAg(I) (nmol/L) sample tap water

wastewater

detected

added

found

recovery (%)

24.0 ± 1.6 24.0 ± 1.6 24.0 ± 1.6 284.0 ± 6.4 284.0 ± 6.4 284.0 ± 6.4

20.0 50.0 100.0 200.0 300.0 400.0

43.8 ± 2.0 72.0 ± 2.6 126.4 ± 3.4 486.0 ± 10.8 578.4 ± 14.2 682.2 ± 26.4

95−104 94−101 99−105 98−103 97−101 96−104

Table 2. Determination Results of Silver(I) Ions in the River Water Sample (n = 4) sample

GB/T119081989 (μmol/L)

ICP-AES (μmol/L)

this method (μmol/L)

relative deviation (%)a

0.30 ± 0.06

0.32 ± 0.06

0.32 ± 0.04

3.2

river water a

The mean value determined by GB/T11908-1989 and ICP-AES.

results show that the detection result by this method is accurate and close to those determined by using the China National Standard and ICP-AES methods, and the relative deviation is 3.2% (n = 4). (iii) Drug samples. “Yin-Xin-Shuang” silver−zinc emulsion and “Hong-Gao-Yao” red ointment are separately typical ointment agents. The former is made of 2% silver sulfadiazine and zinc sulfadiazine while the latter is a composite of silver sulfadiazine and the herbal ingredient salvia miltiorrhiza. Both of them have strong anti-inflammatory and antiseptic effects. By using atomic absorption spectroscopy (AAS) and the singlelabeled fluorescent OND probe established in this work, we separately determined the concentrations of silver(I) ions in silver−zinc emulsion and red ointment samples (Table 3). The results again show that the detection result by this method is accurate and close to that determined by AAS, and the relative deviations are lower than 5% (n = 3). Figure 6. Fluorescence quenching extent (F0 − F)/F0 of the HEX− OND probes in the presence of different concentrations of silver(I) ions. (a) Concentration range of silver(I) ions was from 0.002 to 5.0 μmol/L; (b) concentration range of silver(I) ions was from 0.002 to 0.1 μmol/L. The concentration of the HEX−OND probe was 20.0 nmol/L. All measurements were performed in 10.0 mmol/L MOPS buffer (containing 400.0 mmol/L NaNO3 and 20.0 mmol/L KNO3, pH 7.0).

Table 3. Determination Results of Silver(I) Ions in Drug Samples (n = 3) sample silver−zinc emulsion red ointment

Detection of Silver(I) Ions. (i) Tap water and wastewater samples. For the silver(I) ion detection in tap water and wastewater samples, we carried out a standard addition experiment (Table 1). The results show that the recoveries are between 95% and 105% and all the relative standard deviations (RSD) are lower than 5% (n = 4), showing that this method can be used to detect silver(I) ions in tap water and wastewater samples with satisfaction. (ii) River water sample. Wei River is a seriously polluted river near Xi ’an, and the content of silver(I) ions in it is at the level of micromoles. By means of the China National Standard (GB/ T11908-1989, Cadion 2B spectrophotometry), inductively coupled plasma−atomic emission spectrometry (ICP-AES) and the single-labeled fluorescent OND probe established in this work, we separately determined the concentration of silver(I) ions in the Wei River water sample (Table 2). The

AAS (μmol/g)

this method (μmol/g)

relative deviation (%)

55.8 ± 4.2

53.6 ± 5.4

−3.9

60.4 ± 4.6

63.2 ± 5.6

+4.6

(iv) Tea and bean sprout samples. Through using ICP-AES and the single-labeled fluorescent OND probe designed in this work, we separately determined the concentrations of silver(I) ions in tea and bean sprout samples (Table 4). The results further demonstrated that the detection result by this method is accurate and close to that determined by ICP-AES, and the relative deviations are lower than 5% (n = 3). Table 4. Determination Results of Silver(I) Ions in Tea and Bean Sprout Samples (n = 3)

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sample

ICP-AES (μg/g)

this method (μg/g)

relative deviation (%)

tea bean sprouts

0.52 ± 0.08 0.84 ± 1.02

0.51 ± 0.10 0.81 ± 1.24

−3.7 −3.6

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In conclusion, based on cytosine−Ag(I)−cytosine coordination and the inherent quenching ability of the G-quadruplex, a novel single-labeled HEX−OND probe is developed for Ag(I) detection. Compared with previous studies, this detection method provided several advantages, including simplicity, rapidity, and relative insensitivity to pH. In addition, the linear response range is 1−100 nmol/L with a detection limit of 50 pmol/L, and no obvious interference occurs with other metal ions with a 10-fold concentration increase, which indicates the sensitivity and selectivity are much higher than those of reported methods. Our design will provide a new method to help people to improve Ag(I) detection in water samples, pharmaceutical products, biological products, and food without the interference from other coexisting metal ions.

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ASSOCIATED CONTENT

* Supporting Information S

Fluorescence spectra of HEX solution on addition of different concentrations of silver(I) ions; UV absorption spectra of the HEX−OND probes on addition of different concentrations of silver(I) ions; and difference in the fluorescence intensities in the presence of 10- and 100-fold concentrations of anions to the silver(I) ions. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +0086-29-88303446-221; e-mail: bianliujiao@ sohu.com. Funding

The study was financially supported by the National Natural Science Foundation of China (no. 21075097) and Key Program for Science and Technology Innovative Research Team of Shaanxi Province (no. 2013KCT-24). Notes

The authors declare no competing financial interest.

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REFERENCES

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