Highly sensitive and selective ﬂ uorescence detection of Hg 2+ based on turn-on aptamer DNA silver nanoclusters †

A novel turn-on Hg 2+ sensor was constructed based on ﬂ uorescent C – Hg 2+ -aptamer DNA-stabilized Ag nanoclusters (DNA – AgNCs), and was used to determine the concentration of Hg 2+ over the range 2 – 18 nM with a detection limit as low as 0.25 nM. The sensing assay relied on a target-induced conformational transition of Hg 2+ -aptamer DNA. The conformational change of Hg 2+ -aptamer DNA from a single strand to a hairpin DNA due to the formation of a T – Hg 2+ – T complex in the presence of Hg 2+ made the two darkish DNA – AgNCs approach each other closely and then enhanced the ﬂ uorescence of the AgNCs, which enabled the sensitive and speci ﬁ c detection of Hg 2+ . The proposed sensor was found to be easy to use, and allowed for the sensitive, selective, and turn-on detection of Hg 2+ . Furthermore, this approach has also been successfully applied to the detection of Hg 2+ in real water samples, so this sensor may ﬁ nd application in monitoring Hg 2+ in environmental samples.


Introduction
2][3] Therefore, many methods have been developed for detecting Hg 2+ in recent years, including electrochemical methods, 4,5 atomic absorption/emission spectroscopy, 6,7 uorescence resonance energy transfer, 8 use of electrochemiluminescence sensors, 9 inductively coupled plasma mass spectrometry, 10,11 high-performance liquid chromatography, 12 and atomic uorescence spectroscopy. 13owever, these methods have disadvantages, in particular they either require special instrumentation or take a lot of time to perform.Alternatively, molecular uorescence-based methods have garnered special attention owing to their high sensitivity, good selectivity, and low-cost instruments, particularly in those systems involving DNA, 14 small molecules, 15 peptides, and proteins. 16,17luorescent noble metal nanoclusters, including gold/silver nanoclusters comprising a few to tens of atoms, have attracted great interest due to their good photostability, low toxicity, excellent biocompatibility, and ultrasmall size.They have been applied as uorescent probes and photoluminescent and electroluminescent materials for bio-labeling, bio-imaging, and ultrasensitive biological detection. 18,191][22][23][24] Among them, the uorescent Ag nanoclusters using DNA as the scaffold (DNA-AgNCs) have attracted special attention 25,26 because of their outstanding spectral and photophysical properties and tunable uorescence properties from blue to near-infrared emission that is realized by varying the base sequences or strand lengths of the oligonucleotides. 27,28Ag + ions bind selectively to the heterocyclic bases of the DNA oligonucleotides rather than to their phosphate and sugar groups. 29In particular, Ag + can selectively coordinate with the cytosine base. 30Ag atoms reduced by NaBH 4 bind very tightly to cytosines of oligonucleotide, rendering them chemically stable in biological buffers.Fluorescent metal nanoclusters have been employed for the detection of Hg 2+ , 31,32 but most of them are 'light-off' sensors, 28,31,32 which are undesirable from the sensing point of view due to susceptibility to false signals, large background variation, and the limited room for signal change.To overcome these problems, some 'light-up' sensors to detect Hg 2+ have been designed. 14,33However, it is very challenging to develop low-cost, easy-to-use, highly sensitive, real-time turn-on sensors of Hg 2+ based on metal nanoclusters.
Inspired by the uorescence light-up phenomenon resulting from placing two darkish DNA-AgNCs together to form a probe pair through their complementary linkers, 34 we herein used a similar procedure to construct a novel Hg 2+ sensor, and the working principle of the proposed assay is depicted in Scheme 1.The DNA template included two segments, one was a thymine (T)-containing Hg 2+ aptamer segment (Hg 2+ -aptamer-1 and Hg 2+ -aptamer-2, Table S1 †) in the middle of the DNA template, and the other was AgNC-nucleation segments at the two termini.Hg 2+ has been shown to specically bind to two thymine (T) residues of DNA to form the T-Hg 2+ -T complex. 35herefore, the hairpin structure formed by C-Hg 2+ -aptamer-AgNCs (Table S1 †) in the presence of Hg 2+ made the two darkish DNA-AgNCs approach each other closely and then enhanced the uorescence of the DNA-AgNCs, which enabled the sensitive and specic detection of Hg 2+ .This approach provided for a novel uorescent turn-on chemical assay avoiding the need to design a complicated uorescent sensor and use an organic solvent.
Fluorescence spectra were acquired on a Fluoromax-4 spec-trouorometer (Horiba Jobin Yvon Inc., France), and the slit widths were 10 nm and 5.0 nm for emission and excitation, respectively.UV-Vis absorption measurements were recorded on a Cary 50 Bio spectrophotometer (Varian Inc., CA).Timeresolved uorescence measurements were taken using an FL920 uorescence lifetime spectrometer (Edinburgh Instruments, Livingston, UK) operating in the time-correlated single photon counting (TCSPC) mode.For data analyses, commercial soware by Edinburgh Instruments was used.The average excited-state lifetime was expressed by using the equation The reported spectrum of each sample represented the average of three scans.The average sizes and morphologies of the DNA-AgNCs were characterized by using a JEOL JEM-2100 transmission electron microscope with an acceleration voltage of 200 kV.CD spectra were acquired by using a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., Surrey, UK), and the spectrum was recorded from 220 to 320 nm at 1 nm intervals using a quartz cell with a 1 mm optical path length and an instrument scanning speed of 120 nm min À1 at room temperature.X-ray photoelectron spectroscopy (XPS) (ESCAL-ab 220i-XL, VG Scientic, England) was performed by using monochromic Al K-alpha radiation as a source at 1486.6 eV.

Circular dichroism measurements
A concentration of 5.0 mM of each C-Hg 2+ -aptamer was rst mixed with or without 20 nM Hg 2+ in 20 mM PBS buffer (pH 6.6 or 6.3), and then C-Hg 2+ -aptamer-DNA-AgNCs were prepared according to the method described above (see Section 2.2).Subsequently, CD spectra of the DNA-AgNCs were acquired.

Analytical applications
To evaluate the practical application of the probe for the detection of Hg 2+ in real samples such as tap water and lake water (from Shanxi University), the samples were spiked with standard Hg 2+ solutions of various known concentrations, and the actual samples were measured under the same condition as that in the buffer.
Scheme 1 Schematic illustration of the strategy used to detect Hg 2+ .This strategy is based on ssDNA-templated silver nanoclusters combining with an Hg 2+ aptamer.
3 Results and discussion

Characterization of the DNA-AgNCs
Two different DNA sequences, C-Hg 2+ -aptamer-1 and C-Hg 2+aptamer-2 (Table S1 †), were designed as the templates to form the DNA-AgNCs, in which the Hg 2+ aptamer segment was in the middle of the template (italic), while the C-rich segments were at the 5 0 and 3 0 ends, respectively (bold).The optical characterizations of C-Hg 2+ -aptamer-1-AgNCs in the absence and presence of 10 nM Hg 2+ are shown in Fig. 1A.Both UV-Vis absorption spectra of C-Hg 2+ -aptamer-1-AgNCs alone (curve a) and with 10 nM Hg 2+ (curve b) showed two peaks at wavelengths of 430 and 560 nm, and the peak at 430 nm was the characteristic plasmon absorption band of the Ag nanoparticles 37,38 and the peak at 560 nm was likely the AgNC absorption band.Fig. S1A and B † show the uorescent emission spectra of C-Hg 2+ -aptamer-1-AgNCs in the absence and presence of 10 nM Hg 2+ under various excitation wavelengths, respectively, and the DNA-AgNCs displayed excitation wavelength-dependent emission properties.Upon excitation at 560 nm, the C-Hg 2+aptamer-1-AgNCs in the absence and presence of Hg 2+ presented a maximum emission peak at 620 nm, but the uorescence intensity of the latter was about nine times stronger than that of the former (Fig. 1B); this difference may have been due to the Hg 2+ -aptamer-1 DNA being folded into a hairpin-shaped structure in the presence of Hg 2+ and hence making the two darkish DNA-AgNCs approach each other closely, resulting in the relatively strong uorescence (Scheme 1).To conrm this proposal, CD spectra of the C-Hg 2+ -aptamer-1-AgNCs in the absence and presence of Hg 2+ were acquired.As shown in Fig. 1C, the CD spectrum of the probe itself showed a random coil (curve a), whereas the CD spectrum of the probe incubated with 20 nM Hg 2+ (curve b) presented a positive band centered at a wavelength of 280 nm together with a negative band at 250 nm.This result was in accordance with the CD spectrum of Hg 2+ -aptamer-1 in the presence of 8 mM Hg 2+ , 39  AgNCs alone and that with 10 nM Hg 2+ each presented a maximum emission peak at a wavelength of 622 nm.In accordance with the results of C-Hg 2+ -aptamer-1-AgNCs, the uorescence intensity of the latter was 5.6 times stronger than that of the former (Fig. S2D †).The CD spectrum of C-Hg 2+aptamer-2-AgNCs with 20 nM Hg 2+ (Fig. S2E †) demonstrated that Hg 2+ -aptamer-2 also formed the hairpin structure when Hg 2+ was added. 39ccording to the above results, the enhancement of the uorescence intensity of the C-Hg 2+ -aptamer-1-AgNCs aer incubating with 10 nM Hg 2+ was greater than that of the C-Hg 2+ -aptamer-2-AgNCs in the same condition.Thus, we further investigated the properties of the C-Hg 2+ -aptamer-1-AgNCs probe alone and with Hg 2+ .The uorescence lifetimes at a wavelength of 620 nm of the C-Hg 2+ -aptamer-1-Ag NCs incubated with and without the target Hg 2+ were rst measured (Fig. S3 †).The uorescence transients of C-Hg 2+ -aptamer-1-Ag NCs presented the tri-exponential time constants as tabulated in Table S2.† The results illustrated that there were no obvious differences between the average uorescence lifetimes of C-Hg 2+ -Aptamer-1-AgNCs in the absence and presence of different concentrations of Hg 2+ , further demonstrating a static interaction mechanism.Furthermore, the acquired TEM image of C-Hg 2+ -aptamer-1-AgNCs in the presence of 10 nM Hg 2+ illustrated the formation of uniformly dispersed and nearly spherical DNA-AgNCs with an average diameter of nearly 2 nm (Fig. 1D).XPS was performed to determine whether various elements were present and to determine the valence state of the Ag element in the C-Hg 2+ -aptamer-1-AgNCs.As shown in Fig. 1E, the presence of B, C, N, O, P, Na and Ag was conrmed.The percentage of the AgNCs consisting of Ag was calculated from the peak areas of the elements to be 0.65%.As shown in the magnied view of the spectrum in its Ag 3d region (Fig. 1F), binding energy values at 368.2 eV for Ag 3d 5/2 and 374.2 eV for Ag 3d 3/2 were observed, conrming the presence of elemental Ag(0) in the C-Hg 2+ -aptamer-1-AgNCs. 402 Optimization of experimental conditions for Hg 2+ determination 3.2.1 Optimization of pH.The uorescence of AgNCs has been shown to be dependent on pH, and changing the pH would lead to obvious uorescence quenching.31 Fig.S4A † shows a plot of F/F 0 of C-Hg 2+ -aptamer-1-AgNCs as a function of pH, where F 0 and F are the maximum emission intensities of the DNA-AgNCs without and with 10 nM Hg 2+ , respectively.Here, F/ F 0 of C-Hg 2+ -aptamer-1-AgNCs increased as the pH was increased from 5 to 6.6 and then decreased as the pH was further increased from 6.6 to 9, indicating that the performance of this sensor was the best at pH 6.6.Similarly, the relative uorescence intensity of C-Hg 2+ -aptamer-2-AgNCs with 10 nM Hg 2+ was maximum for a pH value of 6.3 (Fig. S4B †).
3.2.2Optimization of detection time.As shown in Fig. S5A, † the uorescence of the C-Hg 2+ -aptamer-1-AgNCs incubated with 10 nM Hg 2+ reached a maximum, and sustained it for nearly one hour, aer the AgNCs were also incubated with NaBH 4 for 2.5 hours.However, for the C-Hg 2+aptamer-2-AgNCs with 10 nM Hg 2+ , the uorescence increased to the maximum aer one hour and then decreased rapidly to a minimum aer 1.75 hours (Fig. S5B †).Test of the stability of the probe itself also gave a similar result (Fig. S6 †).The strongest uorescence intensity of the C-Hg 2+ -aptamer-1-AgNC probe was observed to be maintained for about 1.5 hours (Fig. S6A †), but the uorescence intensity of the C-Hg 2+aptamer-2-AgNC probe decreased rapidly aer reaching the maximum value (Fig. S6B †).So C-Hg 2+ -aptamer-1-AgNCs and C-Hg 2+ -aptamer-2-AgNCs were used to perform the experiments described below aer being prepared for 2.5 hours and 1 hour, respectively.
3.2.3Detection of Hg 2+ .To evaluate, under optimal conditions, the analytical performance of the method, including its detection limit and the linearity of its plots, the uorescence of C-Hg 2+ -aptamer-1-AgNCs was monitored upon adding various concentrations of Hg 2+ .As shown in Fig. 2A, the uorescence intensity of C-Hg 2+ -aptamer-1-AgNCs continually increased as the concentration of Hg 2+ was increased from 2 to 30 nM.A good linear relationship (R ¼ 0.9985) was observed within the range 2 to 18 nM (Fig. 2B) with a detection limit (LOD) of 0.25 nM (S/N ¼ 3) based on 3s 0 /k, where s 0 is the standard deviation of background and k is the slope of the calibration line.This detection limit is much lower than the maximum allowable level of inorganic Hg 2+ in drinking water (10 nM) according to the U.S. Environmental Protection Agency (EPA) standard 41 and is also below the maximum permissible limit of mercury in drinking water (5 nM) permitted by the European Union. 15For comparison, the detection limits and linear ranges for Hg 2+ detection by other sensors are listed in Table S3.3][44][45][46] These results demonstrated that the proposed sensor we developed for the quantitative analysis of Hg 2+ provided a good linear plot of signal to Hg 2+ concentration and a high sensitivity for Hg 2+ .We also tested C-Hg 2+ -aptamer-2-AgNCs for the detection of Hg 2+ .As shown in Fig. S7A, † the change in the uorescence of C-Hg-aptamer-2-AgNCs as a result of changes in the concentrations of added Hg 2+ was in accordance with that of C-Hg 2+ -aptamer-1-AgNCs.As shown in Fig. S7B, † C-Hg 2+ -aptamer-1-AgNCs also yielded a good linear plot from 2 to 18 nM (R ¼ 0.9972) with a detection limit (LOD) of 0.8 nM.Comparing the sensitivity and stability of the two kinds of DNA-AgNCs indicated the C-Hg 2+ -aptamer-1-AgNC probe to be better, so we used it for the subsequent experiments.
3.2.4Selectivity of C-Hg 2+ -aptamer-1-Ag NCs for detecting Hg 2+ .To investigate the specicity of C-Hg 2+ -aptamer-1-Ag NC sensing system, other metal ions were also investigated using the above strategy.As shown in Fig. 3, only Hg 2+ (10 nM) caused a signicant increase in the relative uorescence intensity (F/ F 0 ), and even a high concentration of any of the other metal ions (100 nM), specically including Cu 2+ , Cr 3+ , Cd 2+ , Zn 2+ , Fe 3+ , K + , Pb 2+ , Mg 2+ , Ca 2+ , Co 2+ , Na + , Tb 3+ , and Mn 2+ , only caused a negligible increase in the uorescence of C-Hg 2+ -aptamer-1-AgNCs.We attributed this specicity for Hg 2+ to the presence of the thymine (T)-rich sequence segment in the middle of the DNA portion of the C-Hg 2+ -aptamer-1-AgNC probe, as Hg 2+ can specically bind to two thymine (T) residues of DNA to form a T-Hg 2+ -T complex. 35The results clearly demonstrated this sensor to be highly selective for Hg 2+ , and the proposed approach could thus be used to specically detect Hg 2+ in biological and environmental samples.
3.2.5 Analytical applications.To evaluate the practical application of the proposed sensor, tap water and lake water (from Shanxi University) were monitored by using this method.The water samples were ltered three times through qualitative lter paper and centrifuged, and the supernatants were used for the quantitative analysis.Hg 2+ was not detected in these real samples because the concentration of Hg 2+ was much lower than the detection limit of the C-Hg 2+ -aptamer-1-AgNCs sensing system.So these samples were spiked with different standard Hg 2+ solutions, and the average values of the results  for three replicate determinations are listed in Table 1.The recoveries varied from 99.27% to 106.3%, and standard deviations were all less than 0.32.These results revealed the developed method to be reliable for assaying Hg 2+ in environmental water samples.

Conclusions
In summary, a novel strategy for the detecting Hg 2+ has been developed using C-Hg 2+ -aptamer-DNA-stabilized AgNCs as a turn-on uorescent probe based on an Hg 2+ -induced conformational transition of Hg 2+ -aptamer-DNA.This sensor presented high sensitivity and selectivity with an LOD of 0.25 nM that met the requirements of industrial and environmental monitoring applications.This novel sensing assay was a lowcost, turn-on, and sensitive assay and could be easily performed by carrying out simple mixing.In addition, it was also successfully applied to practical situation, specically the detection of Hg 2+ in real water samples.So the proposed sensing system is considered to have the potential to be a useful tool for detection of Hg 2+ in environmental water.
3 for C-Hg 2+ -aptamer-2-AgNCs) and then incubated for 10 min.Then, an AgNO 3 aqueous solution was added to the mixture under vigorous shaking for 30 s, and the resulting mixture was kept in the dark at 4 C for 20 min, followed by being reduced with freshly prepared NaBH 4 with vortexing for 1 min.The nal concentrations of DNA, AgNO 3 , and NaBH 4 were 0.3, 1.8, and 1.8 mM, respectively.The nal C-Hg 2+ -aptamer-1-AgNC and Hg 2+aptamer-2-AgNC mixtures were stored in the dark at 4 C for 2.5 hours and one hour, respectively, and these incubation times were calculated based on the amount of NaBH 4 added.The uorescence spectrum of each sample was monitored at room temperature.

Fig. 2 (
Fig. 2 (A) Fluorescence emission spectra (l ex ¼ 560 nm) of C-Hg 2+ -aptamer-1-AgNCs resulting from the addition of various concentrations of Hg 2+ .(B) Fluorescence emission intensity at a wavelength of 620 nm as a function of Hg 2+ concentration.The inset shows the linear range of the plot in the main figure; this linear region encompassed concentrations of 2-18 nM (R ¼ 0.9985).Each error bar represents the standard deviation of three independent measurements.

Fig. 3
Fig. 3 Selectivity of C-Hg 2+ -aptamer-1-AgNCs to different metal cations.Each bar shows the relative fluorescence emission intensity (F/ F 0 ) of C-Hg 2+ -aptamer-1-Ag NCs upon the addition of 10 nM Hg 2+ and 100 nM of another ionic metal.F 0 and F represent the maximum emission intensities of the DNA-AgNCs, respectively, before and after addition of the ionic metal.

Table 1
Recoveries of Hg 2+ in water samples detected using the proposed Hg 2+ assay (N ¼ 3) a The mean of three determinations.b SD ¼ standard deviation.