Article pubs.acs.org/ac
“Turn-On” Fluorescent Sensor for Hg2+ Based on Single-Stranded DNA Functionalized Mn:CdS/ZnS Quantum Dots and Gold Nanoparticles by Time-Gated Mode Dawei Huang,† Chenggang Niu,*,† Xiaoyu Wang,†,‡ Xiaoxiao Lv,† and Guangming Zeng*,† †
College of Environmental Science and Engineering, Key Laboratory of Environmental Biology and Pollution Control, Ministry of Education, Hunan University, Changsha 410082, China ‡ College of Chemistry and Chemical Engineering, Xinxiang University, Xinxiang 453003, China ABSTRACT: An ultrasensitive “turn-on” fluorescent sensor was presented for determination of Hg2+. This method is mainly based on Hg2+-induced conformational change of a thymine-rich single-stranded DNA. The water-soluble longlifetime fluorescence quantum dot (Mn:CdS/ZnS) acted as the fluorophore, which was labeled on a 33-mer thymine-rich single-stranded DNA (strand A). The gold nanoparticles (GNPs) functionalized 10-mer single-stranded DNA (strand B) is selected as the quencher to quench the fluorescence of Mn:CdS/ZnS. Without Hg2+ in the sample solution, strands A and B could form hybrid structures, resulting in the fluorescence of Mn:CdS/ZnS being decreased sharply. When Hg2+ is present in the sample solution, Hg2+-mediated base pairs induced the folding of strand A into a hairpin structure, leading to the release of GNPs-tagged strand B from the hybrid structures. The fluorescence signal is then increased obviously compared with that without Hg2+. The sensor exhibits two linear response ranges between fluorescence intensity and Hg2+ concentration. Meanwhile, a detection limit of 0.18 nM is estimated based on 3α/slope. Selectivity experiments reveal that the fluorescent sensor is specific for Hg2+ even with interference by high concentrations of other metal ions. This sensor is successfully applied to determination of Hg2+ in tap water and lake water samples. This sensor offers additional advantage to efficiently reduce background noise using long-lifetime fluorescence quantum dots by a time-gated mode. With excellent sensitivity and selectivity, this sensor is potentially suitable for monitoring of Hg2+ in environmental applications.
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It has been previously reported that Hg2+ can selectively link T−T pairs to form stable T−Hg2+−T base pairs.9,10 The Hg2+mediated T−Hg2+−T pair is even more stable than the Watson−Crick A−T pair.11 Hg2+ can be incorporated into DNA duplex without altering the double-helical structure because the van der Waals radius of mercury (≈1.44 Å) is smaller than the base pair spacing of DNA duplex (≈3.4 Å).9 A large number of fluorescent, colorimetric, and electrochemical sensors were designed for Hg2+ based on T−Hg2+−T coordination chemistry.12−14 As far as fluorescent sensors are concerned, most of them can be suitable for Hg2+ detection in drinking water because detection limits are lower than the toxic level reported by the U.S. Environmental Protection Agency. However, many fluorescent sensors work in a “turn-off” mode.15−17 As we know well, the sensors working in “turnoff” mode may produce false positive results because of a number of other quenchers.18 As a result, a “turn-on” sensor is preferred.12,14,19−21 Recently, researchers have devoted a lot of efforts to the fluorescent approach that involves the use of nanometer
eavy metal ion pollution has become an important worldwide issue for years due to the severe risks in human health and the environment. As one of the most toxic heavy metals, mercury with the feature of strong toxicity and bioaccumulation can cause serious human health problems even at very low concentrations.1,2 The exposure to mercury can cause a number of toxicological effects such as brain damage, kidney failure, and various cognitive and motion disorders.3,4 The solvated Hg2+, one of the most stable inorganic forms of mercury, is well-known to be highly toxic due to its good water solubility.5 Because of these environmental and health problems of Hg2+, it is obviously of great necessity to obtain new and efficient Hg2+ detection methods that are cost-effective, rapid, sensitive, and selective. Traditional methods for Hg2+ quantification include atomic absorption/ emission spectroscopy, selective cold vapor atomic fluorescence spectrometry, inductively coupled plasma mass spectrometry (ICPMS), and so forth. These methods are very sensitive and selective but require complicated sample preparation and sophisticated instruments which limit their application in routine Hg2+ monitoring.6−8 Thus, it is still of great challenge to develop a method which is sensitive, selective, and environmentally friendly for aqueous Hg2+ detection. © XXXX American Chemical Society
Received: October 23, 2012 Accepted: December 20, 2012
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Japan). Atomic fluorescence measurements were performed on an atomic fluorescence spectrometer (AFS-9700) (Beijing, China). PBS buffer (100 mM) was prepared by mixing an appropriate content of 200 mM Na2HPO4 and 200 mM NaH2PO4. The composition of hybridization buffer, incubation buffer, and washing buffer was 10 mM PBS buffer (pH = 7.4), 100 mM NaNO3. In addition, 2 M NaCl was also prepared. Synthesis of Mn-Doped CdS/ZnS Core/Shell QDs. Mndoped CdS/ZnS core/shell QDs (Mn:CdS/ZnS QDs) were prepared according to a three-step synthesis method.33,34 The resulting Mn:CdS/ZnS QDs were dispersed in hexane. The lack of water solubility of the prepared QDs hindered their reaction with the water-soluble alkylthiol-capped oligonucleotides. Therefore, the authors used 3-mercaptopropionic acid to prepare the water-soluble QDs according to the literature.38 The resulting water-soluble QDs also have long-lifetime fluorescence (∼4.8 ms) and exhibit high stability in aqueous solutions. The water-soluble QDs should be an excellent fluorescence label and could play an important role in a number of QDs-based biochemical and biomedical applications. Synthesis of GNPs. All glassware and mechanical stirrers used for the synthesis were thoroughly cleaned in aqua regia (3 parts HCl, 1 part HNO3), rinsed with ultrapure water, and then oven-dried prior to use. The colloidal solution of GNPs was synthesized by means of citrate reduction of AuCl3·HCl·4H2O.39 An amount of 5 mL of 38.8 mM sodium citrate was rapidly added to a boiled 50 mL of 1 mM HAuCl4 solution with vigorous stirring in a 250 mL round-bottom flask equipped with a condenser. The color changed from light yellow to wine red. Boiling was continued for 10 min; the heating mantle was then removed, and stirring was continued for an additional 15 min. After the solution was cooled to room temperature, the prepared GNPs solution was stored in the 4 °C refrigerator before use. The size of the nanoparticles was typically ∼13 nm in average diameter. The concentration of the GNPs was ∼17 nM, which was determined according to the Beer’s law by using UV−vis spectroscopy, based on the extinction coefficient of 2.7 × 108 M−1 cm−1 at λ = 520 nm for 13 nm particles.40 Preparation of DNA-Functionalized GNPs. According to literature with slight modifications,41 8.0 mL of 17 nM GNPs were incubated with 20 μL of 0.1 mM oligonucleotides overnight. After standing for 16 h at 50 °C, the mixed solution was changed into 0.1 M NaCl, 10 mM phosphate buffer (pH = 7.4) by addition of the necessary salts and was kept at 50 °C for 40 h. To remove unreacted oligonucleotides, the oligonucleotide-conjugated GNPs were purified three times by centrifugation at 13 200 rpm for 30 min. The final product was redispersed into 1.2 mL of PBS buffer (10 mM, pH = 7.4) to make a stock solution and stored at 4 °C for future usage. The number of oligonucleotides probes immobilized on the GNPs was estimated by measuring the absorbance difference at 260 nm before and after modification with oligonucleotides. The average oligonucleotide loadings were about 10 oligonucleotides per GNP, and the final concentration of GNPs was 95 nM. Preparation of DNA-Functionalized Mn:CdS/ZnS QDs. DNA-functionalized QDs were prepared according to a previously published protocol with minor modifications.38 The QDs solution and oligonucleotides solution were mixed together at a ratio of 11 oligonucleotides per QD (100 μL of 1.8 μM QDs mixed with 20 μL of 0.1 mM oligonucleotides).
materials, especially semiconductor quantum dots (QDs) or/ and gold nanoparticles (GNPs), for the detection of Hg2+ based on this T−Hg2+−T coordination chemistry.22,23 QDs have many unique photophysical properties such as high fluorescence quantum yields, narrow emission bands, high Stokes shifts, and stability against photobleaching. Because of these excellent properties, QDs were used as fluorescence labels to develop fluorescent sensors.24−26 GNPs have been of great interest because of their high extinction coefficient and a broad absorption spectrum in a visible light. GNPs are unique quenchers for organic dyes or QDs through both energytransfer and electron-transfer processes.27,28 A lot of sensors have been fabricated based on GNPs as quenchers for DNA, small molecules, or protein detection.29−32 Up to now, fluorescent sensors based on QDs and GNPs are still a good choice for the detection of different analytes. No matter whether it is “turn-on” or “turn-off” mode, most fluorescent sensors for Hg2+ are based on organic dyes or QDs that usually have short-lifetime fluorescence. The background signals might interfere with the fluorescence of organic dyes or QDs, affecting the sensitivity of the fluorescent sensors. Therefore, it should be desirable to develop a novel method, which uses a long-lifetime fluorophore to decrease the background noises on the basis of time-gated fluorescence assay. The authors found that Mn-doped QDs are good choices which have high quantum yield and long-lifetime fluorescence.33−37 In this work, the authors designed a simple “turn-on” fluorescent sensor for Hg2+ in aqueous solution that utilized the T−Hg2+−T coordination chemistry and its induced displacement of the quencher-labeled oligonucleotides. Water-soluble Mn-doped CdS/ZnS core/shell QDs [the QDs have longlifetime fluorescence (∼4.8 ms) and excellent stability in aqueous solution] and GNPs are selected as the fluorophore and quencher, respectively. This fluorescent sensor has a detection limit below the U.S. Environmental Protection Agency limit of acceptable Hg2+ concentration in drinking water. The sensor also exhibits superior selectivity toward Hg2+ even in the presence of other competitive metal ions. Furthermore, the sensor was employed to detect Hg2+ spiked in tap water and lake water samples to demonstrate its potential for practical applications.
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EXPERIMENTAL SECTION Chemicals and Apparatus. All oligonucleotides used in the present study were synthesized and HPLC-purified by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China) and dissolved in ultrapure water (18.3 MΩ·cm) produced by a Millpore water purification system. The sequences were shown as follows: 5′SH−C6− TGAAA CTGTA-3′; 5′SH−C6−TACAG TTTCA CCTTT TCCCC CGTTT TGGTG TTT-3′. AuCl3·HCl·4H2O was purchased from Shanghai Institute of Fine Chemical Materials (Shanghai, China). 3-Mercaptopropionic acid (MPA, 99+%) was purchased from Sigma-Aldrich. The chemicals were used as received without further purification. All other chemicals used were of analytical grade and were used without further purification. Ultrapure water was used throughout the experiments. The time-gated fluorescence intensities were measured and recorded with a Perkin-Elmer LS-55 spectrofluorimeter (United Kingdom). UV−vis absorption spectra were recorded by using a Shimadzu UV spectrophotometer (UV-2550, Kyoto, B
dx.doi.org/10.1021/ac303084d | Anal. Chem. XXXX, XXX, XXX−XXX
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Scheme 1. Schematic Description of the “Turn-On” Fluorescent Sensor for Hg2+ Based on the Hg2+-Mediated Formation in DNA Duplexesa
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The 33-mer single-stranded DNA (strand A) with a Mn:CdS/ZnS quantum dot attached at the 5′ end was hybridized with a 10-mer single-stranded DNA (strand B) with a gold nanoparticle attached at the 5′ end, which resulted in energy transfer from the Mn:CdS/ZnS quantum dot to the gold nanoparticle, leading to a decrease in the time-gated fluorescence intensity of the Mn:CdS/ZnS quantum dot. In the presence of Hg2+, the folding of strand A releases strand B and increases the fluorescence of the Mn:CdS/ZnS quantum dot. The drawing of QDs and GNPs modified singlestranded DNA is only a graphic presentation.
Mg2+, Zn2+, Al3+, Fe3+, Pb2+, Ag+, and Au3+. The lake water samples were taken from Taozi lake in Hunan University. The time-gated fluorescence signal was measured and recorded by a Perkin-Elmer LS-55 spectrofluorimeter. The parameters of the spectrofluorimeter are set as λex = 400 nm; λem = 609 nm; delay time, 0.1 ms; gate time, 1.0 ms; excitation slit, 15 nm; emission slit, 20 nm.
After standing for 12 h, the mixed solution was brought to 0.15 M NaCl and the particles were aged for an additional 12 h. The NaCl concentration was then raised to 0.3 M, and the mixture was allowed to stand for a further 40 h before centrifugalization using centrifugal filter devices (Amicon Ultra-0.5). Finally, the QDs were redispersed into 4.0 mL of PBS buffer (10 mM, pH = 7.4) by vortex and stored at 4 °C for future usage. The number of oligonucleotides probes immobilized on the QDs was also estimated by measuring the absorbance difference at 260 nm before and after modification with oligonucleotides. The average oligonucleotide loadings were about six oligonucleotides per QD, and the final concentration of QDs was 45 nM. Procedures for Hg2+ Detection. To detect Hg2+ or other metal ions in buffer or real environmental water samples, 30 μL of 95 nM DNA/GNPs, 70 μL of 45 nM DNA/QDs, and 140 μL of 0.01 M PBS buffer were mixed uniformly by vortex and hybridized for 35 min first. Then, various concentrations of Hg2+ (20 μL) were added into the mixture solution and incubated for 16 min to form T−Hg2+−T coordination chemistry. Finally, the time-gated fluorescence spectra of different concentrations of Hg2+ were monitored after the completion of the reaction. For the sensitivity experiment, the concentrations of Hg2+ were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0 nM, respectively. For the optimizing experiment, 1.0 μM was selected as the Hg2+ concentration to determine the optimum hybridization time and incubation time. Various metal ions of 10 μM were used in the selectivity experiments. The metal ions are as follows: Mn2+, Ba2+, Ni2+, Cu2+, Ca2+, Cr2+, Co2+, Cd2+,
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RESULTS AND DISCUSSION
Experimental Principle of the Proposed Sensor. The “turn-on” fluorescent sensor for Hg2+ is outlined in Scheme 1. The sensor system comprises two single-stranded DNAs (strand A and strand B) with an alkanethiol moiety at their 5′-terminus. Strand A is a 33-mer thymine-rich oligonucleotide, and strand B is a 10-mer oligonucleotide complementary with strand A. Strand A contains two major parts: the first part (in red) is a five-base segment close to the 5′-terminal that could hybridize with strand B close to the 3′-terminal; five selfcomplementary base pairs separated by seven thymine− thymine mismatches are introduced to the second part (in blue). There are five-base segments in the second part which could hybridize with five-base segments of strand B close to the 5′-terminal. Strand A was functionalized with ∼5 nm sized Mn:CdS/ZnS QDs, and strand B was prepared by functionalization of ∼13 nm diameter GNPs, according to the 5′terminal −SH reaction. The QDs with ∼5 nm in diameter showed the fluorescence emission at 609 nm under light excitation at 400 nm. In our experiment, the average distance between QD and GNP in hybridized structures was estimated to be 5.25 nm. In the absence of Hg2+, strands A and B could C
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operational temperature, and media pH played crucial roles in the detection sensitivity. The hybridization time between strands A and B was investigated as it may influence the hybridization efficiency. Although longer hybridization time may yield more stable fluorescence signal, it is unnecessary if the reaction attained the equilibrium. The fluorescence signal was recorded along with the hybridization time increasing (Figure 2). It is found that the fluorescence signal decreased
hybridize each other because of the 10 complementary base pairs. Under these conditions, the QDs and GNPs are close to each other, resulting in fluorescence quenching due to fluorescence resonance energy transfer. On the contrary, when Hg2+ was present in the sensor solution, mercurymediated base pairs (T−Hg2+−T) induce the folding of strand A into a hairpin structure. Consequently, there are only five base pairs remaining between strands A and B, which is not stable enough under the conditions we used in the proposed method. As a result, strand B will be released from the hybrid structure, and the time-gated fluorescence of QDs will be observed upon light excitation. The fluorescence spectra of the sensor before and after the addition of 1.0 μM Hg2+ is shown in Figure 1A. Furthermore, the authors used a control experiment with the results of no fluorescence intensity changed, in which Hg2+ was added to the solution only containing strand A. The results indicated that Hg2+ makes negligible contribution to quench the fluorescence of QDs (Figure 1B). Optimization of the Experimental Conditions. In the present strategy, the hybridization and incubation times,
Figure 2. Optimization experiments of hybridization and incubation time; 1.0 μM was selected as the Hg2+ concentration to determine the optimum hybridization and incubation time.
with increasing hybridization time and became stable over 32 min, then continued at an almost constant value. To ensure the completeness of hybridization, the authors chose 35 min as the optimum hybridization time. The incubation time after the introduction of Hg2+ into the solution to prompt the T−Hg2+−T formation was also investigated, and the results are shown in Figure 2. The fluorescence intensity increased when Hg2+ was introduced and then tended to stabilize after more than 16 min. On the basis of this, the authors chose 16 min as the optimum incubation time. Furthermore, taking into account operational convenience, room temperature (25−28 °C) was selected as the operational temperature for all experiments. In order to facilitate the hybridization reaction, the media pH for all the experimental steps was 7.4. Herein, 1.0 μM was used as the Hg 2+ concentration to optimize the experimental conditions. Sensitivity of the Sensor. On the basis of the above standard procedures and optimized assay conditions, various concentrations of Hg2+ were introduced to the buffer to evaluate the sensitivity of the “turn-on” fluorescent sensor. The various concentrations of Hg2+ were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 20.0, 50.0, 100.0, 200.0, 500.0, and 1000.0 nM. As shown in Figure 3 parts A and B, higher concentrations of Hg2+ resulted in more fluorescence intensity enhancement. It is worth noting that, even at very low concentration of Hg2+, the time-gated fluorescence intensity exhibited perceptible change which indicated that the Hg2+ could be detected with high sensitivity in this proposed fluorescent sensor. The time-gated fluorescence intensity was found to be linear with the concentration of Hg2+ in the range from 0 to 1 × 10−9 M and from 1 × 10−9 to 1 × 10−8 M (Figure 3C). The equations for the resulting calibration plot were y = 5.75x + 0.43 (eq 1) and y = 1.71x + 5.40 (eq 2) (x was the
Figure 1. (A) Time-gated fluorescence emission spectra of the sensor without and with 1.0 μM Hg2+. The measure conditions are given below: hybridization and incubation times, operational temperature, and media pH are 60 min, 30 min, 25−28 °C, and 7.4, respectively. The parameters of the spectrofluorimeter are set as λex = 400 nm; λem = 609 nm; delay time, 0.1 ms; gate time, 1.0 ms; excitation slit, 15 nm; emission slit, 20 nm. (B) Fluorescence emission spectra of Mn:CdS/ ZnS quantum dots modified strand A in the absence and presence of 10, 100, and 500 nM Hg2+, indicating that Hg2+ has negligible effect on the quenching of fluorescence. D
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Hg2+ detection using fluorescent methods, which is lower than the standards of the U.S. Environmental Protection Agency (EPA). The EPA regulates the maximum allowable level of Hg2+ in drinking water to be 10 nM. In the present method, higher concentrations of Hg2+ resulted in more free GNPs in the sample solution, and free GNPs have a quenching effect on the fluorescence of QDs. As shown in Figure 3, parts B and C, the proposed method exhibits nonlinear character in the range from 0 to 1000 nM; such behavior may be caused by the free GNPs. For examination of the repeatability of this sensor, three sample solutions with different concentrations of Hg2+ were prepared, and the relative standard deviations (RSDs) are about 6.2%, 3.8%, and 2.5% for three independent determinations of 0.8, 6.0, and 50 nM Hg2+ under the optimum conditions, respectively. Selectivity of the Sensor. To determine the selectivity of this protocol, two control experiments were conducted. First, 1.0 μM of Hg2+ and 10 μM of other metal ions including Mn2+, Ba2+, Ni2+, Cu2+, Ca2+, Cr2+, Co2+, Cd2+, Mg2+, Zn2+, Al3+, Fe3+, Pb2+, Ag+, and Au3+ were added to the sample solution, and then the time-gated fluorescence intensity was recorded. As indicated in Figure 4, only Hg2+ exhibits significant response.
Figure 4. Selectivity of the “turn-on” fluorescent Hg2+ sensor. The concentrations of Hg2+ and other metal ions are 1.0 and 10 μM. Every data point was the mean of three measurements. The error bars are the standard deviation.
Second, 1.0 μM of Hg2+ and 10 μM of other metal ions were mixed together to form a mixture solution as a sample for selectivity testing (Figure 4). The fluorescence intensity was obviously higher than that of other samples without Hg2+. These results clearly indicated that the approach is not only insensitive to other metal ions but also selective toward Hg2+ in their presence. Assay of Hg2+ Concentrations in Environmental Water Samples. With excellent sensitivity and selectivity in buffer solution, the proposed method was further tested with real environmental water samples to demonstrate its practical application. The environmental water samples used in the study were tap water and lake water samples. These samples spiked with Hg2+, with concentrations of 0, 1.0, 2.0, and 10.0 nM, were tested using the proposed method and AFS (atomic fluorescence spectrometer). The lake water samples were
Figure 3. (A) Time-gated fluorescence emission spectra of the sample solution after addition of various concentrations (0−1000 nM) of Hg2+ in buffer. (B) Plot of time-gated fluorescence intensity as a function of the concentration of Hg2+. (C) Linear region of panel B. The concentrations of Hg2+ were 0, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, and 10 nM (C). Every data point was the mean of three measurements. The error bars are the standard deviation.
concentration of Hg2+, y was the time-gated fluorescence intensity) with correlation coefficient of 0.9939 and 0.9967, respectively. According to the standard deviation of 0.35 for the blank signal with 20 parallel measurements and eq 1, a detection limit of approximately 0.18 nM was estimated based on a 3α/slope. This is an exceptionally low detection limit for E
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filtered by qualitative filter paper and then centrifuged for 15 min at 12 000 rpm. The concentrations of total mercury in tap water and lake water samples were 0.045 and 0.056 nM which were measured by AFS. The results are summarized in Table 1 and show good agreement with those achieved by AFS, indicating that the present sensor can also work in environmental water samples.
Hg2+ (nM) added
tap water 1 tap water 2 tap water 3 tap water 4 lake water 1 lake water 2 lake water 3 lake water 4
0 1.0 2.0 10.0 0 1.0 2.0 10.0
proposed method meana ± SDb AFS mean ± SD c 1.07 2.06 10.12 c 1.06 2.06 10.15
± 0.10 ± 0.14 ± 0.20 ± 0.14 ± 0.15 ± 0.29
0.045 0.995 2.16 10.10 0.056 0.987 2.10 10.01
± ± ± ± ± ± ± ±
0.003 0.075 0.10 0.17 0.005 0.068 0.15 0.055
a
Mean of four determinations. bSD, standard deviation. cNo Hg2+ concentration could be detected.
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CONCLUSIONS The authors have developed a “turn-on” fluorescent sensor for determination of Hg2+ in aqueous media with excellent sensitivity and selectivity by using Hg2+-mediated T−Hg2+−T pairs, long-lifetime fluorescence QDs, and GNPs. It combines the advantages of specific and stable binding interactions between Hg2+ and thymine, the unique photophysical properties and long-lifetime fluorescence of QDs, and the excellent quenching performance of GNPs. The detection limit (LOD, 0.18 nM) is much lower than the EPA limit of Hg2+ in drinkable water (
