A supramolecular probe for colorimetric detection of Pb²⁺ based on recognition of G-quadruplex

Abstract

A colorimetric probe of Pb²⁺ has been designed based on the mechanism that a supramolecular probe selectively recognized the Pb²⁺-induced conformational transition of G-quadruplexes. The probe exhibited high sensitivity and selectivity towards Pb²⁺, which enabled it to be practically used in detection of the Pb²⁺ level in a freshwater system.

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Lead (Pb) is a major heavy-metal pollutant in the environment and has a seriously adverse effect on human health. Many problems including chronic mental and physical developmental issues in babies and children, renal disease, and hypertension in adults have been found to be intimately related to the poisoning action of lead.¹ Therefore, the detection of Pb²⁺ in environmental and biological samples is of considerable importance. Accordingly, it has attracted significant attention to detect Pb²⁺ below the defined toxic level. Towards this goal, plenty of techniques have been used in the detection of Pb²⁺.² In the past few years, many highly sensitive and selective Pb²⁺ probes have been designed and constructed, most of which are based on Pb²⁺-specific functional DNA molecules such as RNA-cleaving DNAzyme.³ Nevertheless, these probes depend on complicated equipment and expensive technology, which has limited their practical use.

Recently, there is great interest in using G-quadruplex to design nucleic acid-based Pb²⁺ probes.⁴ G-quadruplex is a four-stranded secondary structure stacked by G-quartets connected by Hoogsteen-type base pairing, which is generally stabilized by monovalent metal cations such as K⁺ and Na⁺ in solution.⁵ However, there are some specific G-quadruplexes like the G-quadruplexes formed by PS2.M, pw17, and T30695 showing specifically affinity to Pb²⁺, which were then utilized to develop Pb²⁺ sensors.⁶ These G-quadruplex-based Pb²⁺ sensors exhibited high selectivity and sensitivity. However, the measurement is also dependent on costly instruments. To conveniently monitor Pb²⁺, a simple and fast approach is still in great demand.

In our previous studies, we have designed a series of ion probes based on the cyanine dye aggregates recognizing G-quadruplex structures,⁷ which exhibited great advantages in simple operation and low requirement for equipment. Encouraged by these performances, further we made great effort to design a Pb²⁺ sensor by using the cyanine dye aggregates of 3,3′-di(3-sulfopropyl)-4,5,4′,5′-dibenzo-9-methylthiacarbocyanine triethylammonium salt (MTC). MTC assembles to form J-aggregates by the short-range noncovalent interaction forces such as van der Waals and π–π stacking interactions in aqueous solution,⁸ which was identified by a strong bathochromic effect in the absorption spectra of MTC (Fig. S1†). The aggregates are extremely sensitive to the PS2.M G-quadruplex structures. With recognition of the PS2.M G-quadruplex structures, MTC shows a significant change in absorption spectra. This feature allows us to utilize MTC as a colorimetric probe to monitor the conformational transition of the PS2.M G-quadruplexes induced by Pb²⁺ and realize the quantitate detection of Pb²⁺ (Scheme 1).

Scheme 1 The structure of MTC and the schematic illustration for the mechanism of sensing Pb²⁺ by using MTC and PS2.M G-quadruplex.

Three G-quadruplexes (PS2.M, pw17, and T30695) are known to specially respond to Pb²⁺.⁶ Their G-quadruplex conformations in the solution with K⁺ or Pb²⁺ are different, all of which exhibited more antiparallel feature in the solution with Pb²⁺ compared with that in the solution with K⁺.⁶ It has been found that MTC showed a much weaker affinity towards the antiparallel G-quadruplex structures.⁹ Thus we speculate that MTC will show a decreased affinity with Pb²⁺ inducing the conformational transition of these three G-quadruplexes. To validate the speculation, the absorption spectra of MTC with increasing amounts of different G-quadruplexes were determined. Without G-quadruplexes present, MTC in buffer solution exhibited a sharp absorption band around 658 nm, which belonged to J-aggregate. With titration of three G-quadruplexes that were induced by K⁺, MTC exhibited a sharp decrease of the absorbance at 658 nm while an increase of the absorbance at 580 nm (Fig. 1a and S2†), corresponding to the transition from J-aggregate to monomer of MTC.¹⁰ The results supported the high affinity of MTC towards the K⁺-induced G-quadruplexes. However, with addition of the Pb²⁺-induced G-quadruplexes, slight changes of the absorbance at 580 nm were observed (Fig. 1b and S2†), meaning a weaker affinity of MTC towards the Pb²⁺-induced G-quadruplexes. Comparing the absorbance at 580 nm of MTC without and with 40 μM Pb²⁺, we found that the absorbance of MTC exhibited the most difference in presence of PS2.M (Fig. 1c), which means that PS2.M–MTC is more sensitive to Pb²⁺.

Fig. 1 The absorption spectra of 4 μM MTC with increasing PS2.M (a) in absence of Pb²⁺ and (b) in the presence of 40 μM Pb²⁺ in 10 mM Tris buffer solution (pH 7.2) with 10 mM K⁺. (c) Plots of the absorbance at 580 nm versus the concentrations of different G-quadruplexes.

It is reported that Pb²⁺-stabilized G-quadruplexes have shorter M–O and O–O bonds than K⁺-stabilized G-quadruplexes,¹¹ and Pb²⁺ can competitively bind to the K⁺-stabilized G-quadruplexes and then replace K⁺, resulting in transition of the G-quadruplex conformations. As shown in Fig. 2a, PS2.M exhibited the CD spectral feature of parallel G-quadruplex structure in the presence of K⁺.¹² The parallel G-quadruplex binds to MTC with high affinity, making MTC to exhibit a strong monomer band in absorption spectra (Fig. 2b). On addition of Pb²⁺, PS2.M was induced to form antiparallel G-quadruplex according to the CD spectra,¹³ which had weak affinity towards MTC, and thus the absorbance of MTC monomers obviously decreased. The absorbance of MTC monomers with Pb²⁺ has a good linear correlation within the range of 0 to 5 μM Pb²⁺. These results provide a rationale for utilizing MTC as a probe to analyze Pb²⁺ quantitatively using colorimetric means.

Fig. 2 (a) The CD spectra of 2 μM PS2.M G-quadruplex with increasing [Pb²⁺] in 10 mM Tris buffer solution (pH 7.2) with 10 mM K⁺ with 4 μM MTC present. (b) The absorption spectra of 4 μM MTC with increasing [Pb²⁺] in 10 mM Tris buffer solution (pH 7.2) with 10 mM K⁺ and 2 μM PS2.M. The insert is the plots of the absorbance at 580 nm versus [Pb²⁺].

To find the optimal condition for Pb²⁺ detection, the concentration of K⁺ as well as the molar concentration ratio between MTC and PS2.M has been further studied. The absorption spectra of MTC in the presence of PS2.M were first measured with titration of K⁺. In the absence of K⁺, G-quadruplex structures should not form and MTC will exist in the aggregate state owing to the weak affinity between MTC and single-stranded DNA. This speculation can be supported by the spectra of Fig. 3a, which exhibited a maximum absorbance at 530 nm, belonging to MTC H-aggregate.^9,10 With increasing the concentration of K⁺, there is an obvious enhancement of the absorbance at 580 nm (Fig. 3a), meaning the transition of MTC from H-aggregate to monomer. The reason for this transition is due to the formation of PS2.M G-quadruplex structure which shows a high affinity to MTC monomer. The enhancement of the absorbance at 580 nm was saturated when the concentration of K⁺ was more than 1 mM which was in accordance the complete formation of G-quadruplex structure.¹⁴ Based on the above results, more than 1 mM K⁺ was used to construct the Pb²⁺ probe.

Fig. 3 (a) The absorption spectra of 4 μM MTC with increasing K⁺ in the buffer solution containing 2 μM PS2.M, and the insert shows the relationship between absorbance at 580 nm and [K⁺]. (b) The absorption spectra of 4 μM MTC with different concentrations of PS2.M at 580 nm in 10 mM Tris–K⁺ (pH 7.2).

The effect of the molar concentration ratio between MTC and PS2.M was evaluated by measuring the absorbance at 580 nm of MTC with increasing Pb²⁺ in the presence of variant concentrations of PS2.M. Fig. 3b depicts a larger change of MTC absorbance versus trace Pb²⁺ under the condition with a lower [PS2.M]/[MTC], meaning a higher sensitivity will be obtained when the probe has a lower [PS2.M]/[MTC]. However, we should note that a much lower [PS2.M]/[MTC] will also cause a narrow detection range.

The sensitivity of the Pb²⁺ sensor has been evaluated according to the above optimal condition. In the low concentration of PS2.M, the probe is more sensitive to Pb²⁺. The state of MTC is mainly monomers when the concentration of PS2.M is 0.5 μM, responding to the positive band at 580 nm. The absorption intensity has a sharp decrease accompany with the increasing concentration of Pb²⁺ at 580 nm. Fig. 4 shows the intensity change of against the concentration of Pb²⁺ from 0 μM to 5 μM, showing a perfect linear response towards Pb²⁺. There is a clear trend that the spectra intensity decreases with increasing concentration of Pb²⁺ at 580 nm. The limit of quantitative determination for Pb²⁺ should be 1.4 nM according to the 3σ/slope rule (σ represents the standard deviation of the blank samples, which is 0.0648, while the slope is 0.139 according to the inset figure in Fig. 4a.), which provided a comparable sensitivity towards Pb²⁺ relative to other fluorescence sensors of Pb²⁺.¹⁵

Fig. 4 The absorption spectra of 0.5 μM PS2.M and 4 μM MTC in 10 mM Tris buffer solution with 1 mM K⁺ (pH 7.2) to analyze different concentrations of Pb²⁺. The insert shows linear correlation of the absorbance change with logarithmic concentrations of Pb²⁺.

The selectivity of this probe of Pb²⁺ detection is evaluated by testing the absorbance of MTC in the presence of other common metal ions including Hg²⁺, Ni²⁺, Mn²⁺, Co²⁺, Cd²⁺, Cu²⁺, Ca²⁺, Mg²⁺, Fe³⁺ and Zn²⁺. It is reported that Hg²⁺ has potential influence on the precise detection of Pb²⁺ because Hg²⁺ can combine to thymine into the formation of T–Hg²⁺–T base pairs.¹⁶ PS2.M contains a few of T residues, thereby it is possible that Hg²⁺ will affect the PS2.M G-quadruplex structures. To overcome this problem, we choose SCN⁻ as a chelator to avoid the interference of Hg²⁺ according to the strategy that previous reported.¹⁷ Fig. 5 shows the change of absorbance of MTC for analyzing different metal ions in the presence of 5 mM SCN⁻, the concentration of which is high enough to eliminate the possible interference of Hg²⁺. The concentration of metal ions is 5 μM, only Pb²⁺ causes a sharp decrease. The result indicates that the probe has a better selectivity towards Pb²⁺ over other common metal ions.

Fig. 5 Selectivity assays based on the MTC/PS2.M complex in 10 mM Tris buffer solution (pH 7.4) with the assistance of masking agent KSCN (5 mM). All the metal ions were used at the concentration of 5 μM (A₀ and A stand for the absorbance at 580 nm in the absence and presence of metal ions).

The response of the MTC–DNA probe system to Pb²⁺ is quick. The time-dependent absorbance measurement was done once Pb²⁺ was injected in. MTC exhibited a sharp decrease of the absorbance at 580 nm after Pb²⁺ was added in (Fig. 6). The absorbance reached the minimum value within 8 minutes according to the time appeared in the inflection points, meaning the probe has the performance of quick measurement.

Fig. 6 The absorbance at 580 nm of MTC with time after addition of Pb²⁺ in the presence of PS2.M.

To demonstrate the application of the probe in real example, we apply MTC to analyze the lake water. The water samples are centrifuged two times through centrifugal machine. We use the recovery of Pb²⁺ added in lake water to evaluate the accuracy of the probe. 10% lake water was titrated into the probe solution to detect the recovery. The results are shown in Table 1. It is found that the recovery is high with the Pb²⁺ concentrations from 0.45 to 9.95 μM, which suggests that our designed G-quadruplexes-based probe can be applied to the detection of Pb²⁺ in lake water with a high accuracy.

Table 1 The recoveries for the detection of Pb²⁺ in lake by the proposed probe

Sample	[Pb^2+]added (μM)	[Pb^2+]measured (μM)	Recovery^a (%)
a Recovery (%) = (1−|1 − [Pb^2+]measured/[Pb^2+]added|) × 100%.
1	0.45	0.459	98
2	0.95	0.953	97
3	1.95	2.262	84
4	9.95	8.535	86

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Conclusions

In summary, we have design a G-quadruplex-based supramolecular colorimetric probe with high sensitivity and selectivity for Pb²⁺ detection. In the presence of K⁺, PS2.M was induced to form parallel G-quadruplex structure, whereas Pb²⁺ could competitively bind to PS2.M and induce PS2.M to form an antiparallel G-quadruplex structure. MTC exhibited great changes in its absorption spectra following the conformational transition of PS2.M G-quadruplexes owing to the different affinity of MTC towards the parallel and antiparallel G-quadruplex structures. The mechanism made colorimetric detection of Pb²⁺ be feasible by using MTC and PS2.M G-quadruplexes. The probe has been proved to show a high sensitivity with the detection limit of 1.4 nM and a high selectivity towards Pb²⁺ over other common metal ions. The excellent performance enabled the probe to be applied to analyze the Pb²⁺ in lake water. Compared to those fluorescence and electrochemical probes, the MTC supramolecular colorimetric probe exhibited great advantages in simple operation and low requirement for equipment, implying a promising application.

Acknowledgements

This research was supported under Major National Basic Research Projects (973) (Grant no. 2011CB606104), the National Natural Science Foundation of China (grant numbers 31200576 and 21443011), Chinese Academy of Sciences (Grant no. KJCX2-EW-N06-01), open project program of Beijing National Laboratory for Molecule Sciences, and National Natural Science Foundation of Hebei Province (no. B2012401026).

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Footnote

† Electronic supplementary information (ESI) available: Details of materials, methods, and Fig. S1–S3. See DOI: 10.1039/c4ra11395k

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