Graphene nanosensor for highly sensitive fluorescence turn-on detection of Hg²⁺ based on target recycling amplification

Abstract

The development of sensitive and selective methods for the monitoring of toxic heavy metal ions is highly demanded because of their threats to the environment and human health. Based on a new exonuclease III (Exo III)-assisted target recycling amplification strategy, a highly sensitive fluorescence turn-on nanosensor for Hg²⁺ detection using graphene oxide (GO)-quenched, thymine-rich FAM-ssDNA nanoprobes is developed. The target Hg²⁺ ions bind and fold the GO-adsorbed FAM-ssDNA into duplex structures through the formation of T–Hg²⁺–T base pairing, leading to the release of the FAM-ssDNA from the surface of GO and recovery of the fluorescent signal. Besides, the released and folded duplex can be digested by Exo III to liberate the bound Hg²⁺ ions, which can again associate with the GO-quenched FAM-ssDNA nanoprobes and trigger the target recycling process to cause cyclic cleavage of the GO-adsorbed FAM-ssDNA. This target recycling process therefore results in the release of numerous FAM labels back into the solution and significantly amplified fluorescent signal is obtained for highly sensitive detection of Hg²⁺ down to the sub-nanomolar level. The developed nanosensor also exhibits high selectivity against non-specific ions and can be potentially employed to monitor other toxic heavy metal ions at ultralow levels.

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Introduction

Graphene, first reported by the Geim group in 2004, is a two dimensional single layer sheet of sp²-hybridized carbon atoms perfectly arranged in a hexagonal lattice structure.¹ Despite the fact that graphene and graphene-based composites are relatively new materials, they have found wide applications in various fields such as nanoelectronics,^2,3 energy storage/conversion,^4,5 biomedicine^6,7 and sensors,^8–10 owing to the distinct electronic, thermal, optical and mechanical properties of these materials.^11–13 Indeed, we have witnessed increasing research interest in the employment of graphene-based materials in designing different types of sensors in recent years. Due to their excellent conductivity, graphene-based materials (graphene/reduced graphene) are promising candidates for the construction of modified sensing electrodes for detecting a variety of molecule.^14,15 Moreover, graphene oxide (GO) was reported to be a fluorescence “superquencher” with long-range energy-transfer property.^16,17 In addition, GO has the ability to intimately interact with ssDNA via π–π stacking between the exposed nucleobases of the ssDNA and the surface of GO, while the nucleobases in dsDNA are blocked in the helical structure through base pairing, which prevents the direct interaction of nucleobases with GO surface. The preferentially selective interaction of GO with ssDNA over dsDNA has demonstrated to be very useful in developing simple fluorescent sensors for DNA, RNA, protein and enzyme activities by using fluorescent tag-conjugated oligonucleotide probes.¹⁸ Recently, based on specific interaction between Hg²⁺ and T-rich DNA sequences,¹⁹ several GO-based fluorescent sensors for Hg²⁺ detection have been reported.^20–24 In the design of these sensors, the Hg²⁺ ions associate with the fluorophore-labeled, T-rich ssDNA to form dsDNA with T–Hg²⁺–T structures, which switches the absorbability of the fluorophore-labeled ssDNA and causes change in the fluorescence emission for Hg²⁺ detection. With no doubt, these GO-based fluorescent sensors significantly facilitate the detection of Hg²⁺ compared with other traditional instrumental methods based on atomic absorption/emission spectroscopy^25,26 or inductively coupled plasma mass spectrometry,²⁷ which require tedious sample preparation and pre-concentration procedures, expensive instruments and skilled technicians.²⁸ Although convenient, the detection limits of the reported GO-based methods for Hg²⁺ barely meet the standard of United States Environmental Protection Agency (10 nM of Hg²⁺ in drinkable water).

To further push down the detection limit for Hg²⁺ to achieve highly sensitive monitoring of heavy metal ions, we introduce herein a new enzyme-assisted target recycling amplification strategy for sensitive fluorescence turn-on detection of Hg²⁺ based on the GO-quenched, thymine rich FAM-labeled ssDNA (GO/FAM-ssDNA) nanoprobes. The association of Hg²⁺ with the GO-adsorbed FAM-ssDNA leads to the formation of dsDNA with T–Hg²⁺–T pairing and the release of the FAM-ssDNA from the surface of GO, resulting in the recovery of the fluorescence emission of the FAM labels. Moreover, the folded dsDNA with T–Hg²⁺–T pairing can be degraded by exonuclease III (Exo III) to release the target Hg²⁺ ions, which in turn again bind the GO-adsorbed FAM-ssDNA and cause cyclic release and degradation of the FAM-labeled ssDNA. With this new target recycling strategy, significantly amplified recovery of fluorescence emission can be achieved and sub-nanomolar detection of Hg²⁺ can be realized.

Experimental

Chemicals and materials

Morpholinopropanesulfonic acid (MOPS) was purchased from J&K Scientific Ltd. (Beijing, China). The metal salts (Hg(NO₃)₂, Pb(NO₃)₂, Ni(NO₃)₂, Cd(NO₃)₂, CaCl₂, CoCl₂, ZnCl₂, CuCl₂, ZnCl₂ and BaCl₂) were received from Kelong Chemicals Ltd. (Chengdu, China). Exo III with 10× reaction buffer (660 mM Tris–HCl, 6.6 mM MgCl₂, pH 8.0) and the FAM-ssDNA were obtained from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The sequence of the FAM-ssDNA was as follows: 5′-CATTCTTTCTTCCCCTTGTTTGTTT-(FAM)-G-3′ with FAM modification at the italic T position. All reagents were of analytical grade and ultrapure water (specific resistance of 18.25 MΩ cm) was used to prepare all solutions during the experimental process.

Preparation of GO

GO was prepared by following the modified Hummers' methods.^29,30 In brief, graphite powder (10 g) was added to a solution containing concentrated H₂SO₄ (15 mL), K₂S₂O₈ (5 g), and P₂O₅ (5 g) at 80 °C. The resulting dark blue mixture was thermally isolated and cooled to room temperature over a period of 6 h, followed by dilution with water, filtration and washing with water until pH = 7.0. The pellet was dried under ambient conditions overnight to obtain the oxidized graphite powder. Subsequently, the oxidized graphite powder (10 g) was mixed with cold (0 °C) concentrated H₂SO₄ (230 mL). Then, KMnO₄ (30 g) was gradually added to the mixture and the temperature of the mixture was kept below 20 °C by cooling. Afterwards, the mixture was stirred at 35 °C for 2 h, and then diluted with water (460 mL). In next 15 min, water (1.4 L) and 30% H₂O₂ (25 mL) was added to the mixture to produce a bright yellow solution. Finally, the mixture was purified by repeated filtration and rinsing with 1 : 10 HCl solution (2.5 L) to remove the metal ions and acids to obtain GO.

Target recycling amplification for fluorescence turn-on detection of Hg²⁺

GO (10 μg mL⁻¹) was first mixed with the FAM-ssDNA (0.05 μM) for 5 min in the reaction buffer (66 mM Tris–HCl, 6.6 mM MgCl₂, 10 mM MOPS, pH 7.0). Next, Hg²⁺ at various concentrations and Exo III (10 U) were added to the nanoprobe solution to a total volume of 200 μL. The mixture was incubated at 37 °C for 30 min and fluorescence measurement was performed to collect the data.

Fluorescence measurements

Fluorescence measurements were preformed on a RF-5301-PC spectrophotometer (Shimadzu, Tokyo, Japan). The emission spectra were obtained by exciting the samples at 490 nm and fluorescence data were collected from 480 nm to 650 nm. The excitation and emission slit widths were set at 5 nm.

Results and discussion

Our Exo III-assisted target recycling amplification strategy for sensitive fluorescence turn-on detection of Hg²⁺ using the GO/FAM-ssDNA nanoprobes is depicted in Scheme 1. The T-rich, FAM-ssDNA probes are first adsorbed on the surface of GO via π–π stacking and the fluorescence emission of the FAM labels is quenched due to the excellent fluorescence quenching capability of GO. In the absence of the target Hg²⁺ ions, the FAM-ssDNA probes are resistant to Exo III digestion due to two reasons. First, Exo III shows activity for the stepwise removal of mononucleotides from the 3′-hydroxyl termini of duplex DNA with 3′-blunt or recessing termini and its activity on ssDNA is limited.^31–33 Second, the π–π stacking adsorption of the ssDNA probes on the surface of GO prevents Exo III from accessing the nucleobases of the ssDNA and the enzymatic activity of Exo III is therefore inhibited.³⁴ Owing to these two facts, the fluorescence emission of the FAM-ssDNA is effectively quenched by GO in the absence of the target Hg²⁺ ions even with the addition of Exo III. On the contrary, when the target Hg²⁺ ions are added, they associate with the GO-adsorbed FAM-ssDNA through the formation of stable T–Hg²⁺–T base pairing, which subsequently folds and releases the FAM-ssDNA from the GO surface and further leads to a fluorescence turn-on state of the sensor. The folding of the FAM-ssDNA generates duplex DNA with 3′-blunt termini, which can be digested by Exo III from the 3′-hydroxyl termini to liberate the target Hg²⁺ ions and to produce mononucleotides and short ssDNA segments. The liberated Hg²⁺ ions can again bind the GO-adsorbed FAM-ssDNA to initiate the target recycling process to cause cyclic and effective cleavage of the FAM-ssDNA, which further results in the release of numerous fluorophore (FAM) into the solution and significantly amplified fluorescence turn-on signal for highly sensitive Hg²⁺ detection.

Scheme 1 Principle for highly sensitive fluorescence turn-on graphene nanosensor for Hg²⁺ based on Exo III-assisted target recycling signal amplification.

As the first step toward the sensor development, the cleavage of the FAM-ssDNA by Exo III in the presence of Hg²⁺ was verified by native polyacrylamide gel electrophoresis (PAGE). As shown in Fig. 1A, the FAM-ssDNA (2 μM) exhibits a clear band (lane a), and the addition of the target Hg²⁺ (2 μM) or Exo III (30 U) alone causes no visual band shift (lane b and c vs. a), which indicates that the addition of Hg²⁺ has no effect on the length of the FAM-ssDNA and the FAM-ssDNA is resistant to enzymatic digestion by Exo III in the absence of Hg²⁺ due to the inactivity of Exo III on ssDNA. However, after the incubation of the FAM-ssDNA with Hg²⁺ and Exo III, the band of FAM-ssDNA becomes invisible (lane d). The disappearance of this band is basically due to the cyclic digestion of the folded FAM-ssDNA with blunt termini by Exo III from the 3′ termini in the presence of Hg²⁺, and the enzyme-digested FAM-T mononucleotide has relatively high mobility and migrates out of the band edge under the electrophoretic conditions. The results of the PAGE analysis demonstrate that the presence of Hg²⁺ can associate with and fold the FAM-ssDNA into hairpin structures with blunt termini through specific T–Hg²⁺–T base pairing to result in Exo III-catalyzed, cyclic digestion of the FAM-ssDNA.

Fig. 1 (A) PAGE analysis of different reaction mixtures: (a) FAM-ssDNA (2 μM), (b) FAM-ssDNA (2 μM) and Hg²⁺ (2 μM), (c) FAM-ssDNA (2 μM) and Exo III (30 U); (d) FAM-ssDNA (2 μM), Hg²⁺ (2 μM) and Exo III (30 U). (B) Typical florescence spectra of (a) FAM-ssDNA (0.05 μM), (b) GO (10 μg mL⁻¹)/FAM-ssDNA (0.05 μM), (c), GO (10 μg mL⁻¹)/FAM-ssDNA (0.05 μM) and Hg²⁺ (2 μM); (d) GO (10 μg mL⁻¹)/FAM-ssDNA (0.05 μM), Hg²⁺ (2 μM) and Exo III (5 U). The enzymatic reaction was performed at 37 °C for 40 min.

For proof-of-concept demonstration of the proposed method for amplified detection of Hg²⁺, the fluorescence intensity of the probe solutions were monitored. According to Fig. 1B, the FAM-ssDNA (0.05 μM) shows strong fluorescence emission at 520 nm (curve a) while the addition of GO (10 μg mL⁻¹) can efficiently quench the fluorescence emission due to the adsorption of the FAM-ssDNA on the surface of GO (curve b). The addition of the target Hg²⁺ to the GO/FAM-ssDNA nanoprobe solution leads to the association of Hg²⁺ with the FAM-ssDNA through T–Hg²⁺–T pairing and the folding of the FAM-ssDNA, which in turn results in the desorption of the folded FAM-ssDNA from the GO surface and restoration of the fluorescence emission (curve c). Importantly, when the GO/FAM-ssDNA nanoprobe solution is incubated with the target Hg²⁺ in the presence of Exo III (5 U), increased restoration of fluorescence emission is observed (curve d vs. c). Such increase is owing to the Exo III-assisted target recycling amplification of the fluorescence restoration as discussed previously. The preliminary results here suggest that the proposed nanosensor can potentially offer highly sensitive monitoring of Hg²⁺ at low levels.

The amplified, fluorescence turn-on signal for Hg²⁺ monitoring in our method is based on Exo III-catalyzed, cyclic digestion of the GO-adsorbed FAM-ssDNA. In order to achieve optimal performance of the sensing system, the effect of the enzymatic reaction time and the amount of enzyme on the restoration of the fluorescence emission intensity was evaluated. The enzymatic reaction time was optimized by incubating the GO/FAM-ssDNA probes with Hg²⁺ (2 μM) and Exo III (5 U) at 37 °C from 10 to 50 at a time interval of 10 min. As depicted in Fig. 2A, the fluorescence intensity increases with prolonged incubation time from 10 to 30 min and levels off thereafter, indicating an optimal enzymatic reaction time of 30 min. The dependence of the fluorescence intensity upon the amount of Exo III was examined by elevating the amount of Exo III from 1 to 15 U at 37 °C for 30 min. From Fig. 2B, we can see that increasing amount of Exo III from 1 to 10 U leads to increasing restoration of fluorescence intensity and further increase in the amount of Exo III shows insignificant effect on the signal output. Therefore, 10 U of Exo III was used in subsequent experiments.

Fig. 2 Effect of (A) enzymatic reaction time and (B) amount of Exo III on the fluorescence intensity of the GO/FAM-ssDNA nanoprobe solution.

The developed graphene nanosensor was further applied to different concentrations of Hg²⁺ to evaluate the dependence of the fluorescence intensity of the sensor upon the concentration of Hg²⁺. Based on the results in Fig. 3A, the presence of increasing concentration of Hg²⁺ from 0.001 to 2 μM leads to gradual increase of the fluorescence intensity, and further increase in the concentration of Hg²⁺ causes no apparent change in fluorescence intensity (data not shown). By plotting fluorescence intensity vs. the concentration of Hg²⁺, a linear correlation (R² = 0.998) was obtained over the range from 0.001 to 1 μM (Fig. 3B). The limit of detection was estimated to be 0.3 nM based on three times the standard deviation of the blank tests. Such detection limit is much lower than the standard of United States Environmental Protection Agency (10 nM of Hg²⁺ in drinkable water) and shows about 9.3 to 50-fold improvement compared to other reported fluorescent graphene sensors for Hg²⁺ detection.^20–24

Fig. 3 (A) Typical fluorescence responses of the developed sensor for different concentrations of Hg²⁺. From bottom to top: 0, 0.001, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 2 μM. (B) The corresponding calibration plot of fluorescence intensity vs. the concentration of Hg²⁺ from 0 to 1 μM. Error bars, SD, n = 3. The samples were incubated at 37 °C for 30 min.

The selectivity of the proposed graphene nanosensor for Hg²⁺ was investigated by comparing the fluorescence response of the sensor for Hg²⁺ against those environmentally relevant metal ions, including Ca²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ni²⁺, Pb²⁺, Cd²⁺, Zn²⁺, Ba²⁺. As shown in Fig. 4, the presences of the control ions at even high concentrations (10 μM) show close fluorescence intensity to that of the blank test (in the absence of Hg²⁺), due to the lack of the capability of these ions to bind the T-rich, FAM-ssDNA probes. However, the addition of a 10-fold lower concentration of the target Hg²⁺ (1 μM) results in a significant increase in the fluorescence intensity, indicating the high selectivity of the sensor for Hg²⁺ that is associated with the formation of specific T–Hg²⁺–T pairing. To check real application of the proposed sensor, recovery tests of Hg²⁺ in both river and tap water samples with the stand addition method were performed on the nanosensor. According to the results summarized in Table 1, the sensor shows good recovery values (97.0–104%) for the tested water samples, which suggests that the developed sensor is suitable for monitoring Hg²⁺ in real samples.

Fig. 4 Selectivity analysis of the developed sensor for Hg²⁺ (1 μM) against other control ions (each at 10 μM).

Table 1 Determination of the concentration of Hg²⁺ in water samples using the proposed nanosensor

Sample	Spiked Hg^2+ (nM)	Our proposed sensor (mean ± SD, nM)	Recovery (%)
River water	1	1.01 ± 0.14	101
	5	4.89 ± 0.28	97.8
	10	9.82 ± 1.3	98.2
Tap water	1	1.04 ± 0.12	104
	5	4.93 ± 0.34	98.6
	10	9.70 ± 1.6	97.0

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In conclusion, we have demonstrated a highly sensitive fluorescent graphene nanosensor for the detection of Hg²⁺ based on Exo III-assisted target recycling signal amplification. The presence of Hg²⁺ folds and releases the GO-adsorbed FAM-ssDNA through T–Hg²⁺–T pairing and creates nicking sites for Exo III, which cyclically cleaves the folded FAM-ssDNA and generates significantly amplified fluorescent signal for ultrasensitive detection of Hg²⁺ down to 0.3 nM. Besides, the developed sensor is highly selective toward Hg²⁺ against other control ions. The demonstration of the enzyme-assisted signal amplification strategy for amplified detection of Hg²⁺ thus offers a useful addition to the monitoring of trace amounts of heavy metal ions.

Acknowledgements

This work was supported by NSFC (no. 21275004, 20905062, 21075100 and 21275119), the New Century Excellent Talent Program of MOE (NCET-12-0932) and Fundamental Research Funds for the Central Universities (XDJK2014A012).

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