Introduction

Among toxic metals, lead is a well-known anthropogenic contaminant, which is discharged into the environment during the disposal of lead-containing consumer products, metal manufacturing processes, fossil fuel combustion, and sewage treatment and disposal processes1,2,3,4. Although lead contamination has been strictly regulated all over the world, the use of Pb-based products has increased owing to the world-wide development and application of solar cells, Pb–acid batteries, and radioisotope shields, etc5,6,7. It is notable that lead pollution does not only occur locally but also worldwide due to its diverse usages.

It has been reported that lead toxicity on human and animal species can cause various harmful effects including biochemical abnormalities, gastrointestinal diseases, impaired growth, cognition problems, and death8,9,10,11. For the support of the technology to reduce lead poisoning, rapid and simple detection of Pb2+ in laboratory and environmental samples is required to improve the existing time- and labor-consuming techniques such as inductively coupled plasma mass spectrometry (ICP) or atomic absorption spectroscopy.

For the detection of Pb2+, we considered the use of self-reacting guanine-rich quadruplexes (G-QDs) as a simple probe that does not require any sensing platform and labeling reagents. G-QDs are a class of DNA secondary structures composed of four-stranded DNA structures12. For the last few decades, G-QDs have been reported for their biological relevance, including their participation in the protection of chromosomes by telomeres and in the control of gene expression13. For these biological processes of G-QDs, the unfolded DNA with guanine-rich sequences must be stabilized by the binding of cations (typically K+ or Na+) and switched to quadruplex secondary structures (Fig. 1A,B)14. The resulting G-QDs with the four-stranded secondary structure have high affinity and selectivity toward specific analytes such as nucleic acids, proteins, small molecules, and metal ions15,16.

Figure 1
figure 1

Guanine-rich quadruplex structures (G-QDs) investigated in this study. (A) Chemical structure of G-QDs involved with a metal ion. (B) Schematic design of G-QDs for five types of nucleotides. Green circles represent metal ions, such as K+, Na+, or Pb2+.

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Until now, various nucleotides that can be switched to G-QDs by K+ stabilization have been reported, such as thrombin aptamer (TBA), PS2.M, human telomeric DNA (HTG), AGRO 100, and telomeric related sequences (T2)17,18,19,20,21. In addition, it has been reported that Pb2+ can replace K+ during the switching of guanine-rich sequences to G-QDs. TBA, PS2.M, HTG, and T2 exhibited a conversion to G-QDs by Pb2+, which was measured by a circular dichroism (CD) spectroscopy21,22,23,24. As an application of these properties to sensor preparation, HTG induced by Pb2+ is utilized as a radon sensor with organic dye malachite green25. In addition, Pb2+ and hemin were co-stabilized in the AGRO 100 quadruplex structure, and Pb2+ could be detected by fluorescent, colorimetric, and electrochemical methods because of the optical and electrical properties of hemin26. Recently, fluorophore-labeled G-QDs have been reported to detect metal ions including Pb2+. Peng et al. used the T30695 sequence [d(GGGT)4] for Pb2+ detection27. They applied a fluorescent ratiometric method for Pb2+ detection by inserting Zn2+ to nucleotides, with a detection limit of 23.5 nM Pb2+. In addition, a perylene moiety was inserted at different phosphate positions of TBA without a significant effect on the G-QD structure, and their fluorescence anisotropy signals showed good linear relationship to the Pb2+ concentrations, with 24.5 nM detection limit28. The TAQ [d(TAG3T)3TAG3] sequence was also reported as a Ba2+ sensor with excellent tolerance to highly concentrated K+ by addition of a fluorophore, hypericin29.

However, the performance of these nucleotides for the detection of Pb2+ based on CD spectroscopy has not been successfully determined in environmental and laboratory samples as a Pb2+ sensor. The CD spectrum of G-QDs provides distinct binding geometry information and confirms the presence of different stacked bonds because of different orientations of guanine with respect to the glucoside bond30. For example, HTG typically shows a peak wavelength that is centered near 260 nm in CD spectrum exhibiting absorption difference (ΔA) caused by right- and left-handed circularly polarized light. However, if HTG switched to the G-QD structure by the K+ ion, it displays an absorption spectrum with a peak wavelength near 295 nm. CD spectroscopy can sensitively determine nucleic acid secondary structures and is particularly well-suited for monitoring structural changes resulting from folding-unfolding reactions caused by binding of specific ions to each nucleotide.

Herein, we collectively analyzed and compared the performance of the five types of G-QDs, TBA, PS2.M, AGRO 100, HTG, and T2, which have different DNA sequences, as an easy and rapid Pb2+ detection sensor using CD spectroscopy. The detection of Pb2+ was performed based on the degree of absorption caused by right- and left-handed circularly polarized light (ΔA) for Pb2+-incorporated G-QDs. The specific wavelength of the absorption peak was determined according to the structure of the G-QDs complexed by Pb2+. The sensing of Pb2+ complexed with G-QDs by CD spectroscopy does not require any labeling materials such as fluorescence dyes or other optical probes. It has been reported that fluorescence dye-labelled G-QDs were typically used for the application of G-QDs as sensors31,32,33.

In this study, the sensing performance of G-QDs was compared in terms of the absorption band, ΔA intensity at peak wavelength, association constant (KA), and cross-reactivity with other metal ions. Moreover, Pb2+ was spiked into an effluent of a wastewater treatment plant (WWTP) to demonstrate the applicability of G-QDs for the measurement of Pb2+ concentrations in environmental as well as in laboratory samples. Because environmental samples are usually containing K+, the sensing capacity of G-QDs for Pb2+ was investigated in the presence of K+.

Materials and methods

Materials

The nucleotides listed in Table 1 were purchased from Cosmogenetech (Seoul, Korea). The concentration of oligonucleotides was measured by a Cary 100 UV spectropolarimeter (Australia) using the following extinction coefficients: TBA for ε260nm = 147,300 M−1 cm−134, PS2.M for ε260nm = 184,300 M−1 cm−124, HTG for ε260nm = 228,500 M−1 cm−135, AGRO100 for ε260nm = 250,800 M−1 cm−136, telomeric related sequence (T2) for ε257nm = 213,400 M−1 cm−137. Before conducting the experiments, all oligonucleotides in Tris-buffer were denatured at 90 °C for 10 min and cooled at room temperature. KNO3, Al(NO3)3∙9H2O, Cu(NO3)2∙3H2O, Ni(NO3)3∙6H2O, Fe(NO3)3∙9H2O, Co(NO3)2∙6H2O, Cr(NO3)2∙9H2O, and Pb(NO3)2 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Table 1 Types of guanine-rich quadruplex structures (G-QDs) investigated in this study.
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CD spectroscopy for G-QD formation

The oligonucleotides were dissolved by 3 μM in 20 mM Tris–HCl (pH 7.0) without any Na+ and K+. The CD spectra were obtained from 220 to 400 nm by using a J-715 spectropolarimeter with a 1 × 1 cm quartz cuvette. (Tokyo, Japan); data interval: 1 nm; band width: 2 nm; scan speed: 50 nm/min; response time: 2 s. The base line was collected using a control sample without nucleotides and subtracted from each spectrum for the nucleotide samples.

K+ and Pb2+ were dissolved in deionized water (DW), and the final concentration was met by adding 200 μL of cation solution to 1.8 mL nucleotide solution.

Determination of binding constants for Pb2+

To quantitatively determine the binding affinity, the binding constant of Pb2+ for G-QDs was determined using the Langmuir adsorption model38. The binding affinity was calculated by measuring the signal intensity (ΔA) in terms of milli-degree (mdeg) by CD spectroscopy, which was used as the numeric value of the response at equilibrium. The correlation of the Pb2+ concentration in this binding system and the response value at equilibrium can be expressed by the following equation.

$$\frac{C}{q} = \frac{C}{{q_{m} }} + \frac{1}{{q_{m} \cdot {\text{K}}_{A} }},$$
(1)

where q is the CD signal intensity (mdeg) at the peak wavelength of each G-QDs, and C is the Pb2+ concentration. KA is the apparent binding constant, and qm is the CD signal intensity when C is infinity. The binding constant was determined using data from three repetitive experiments.

Measurement of selectivity of Pb2+ over other metal ions

The TBA and PS2.M nucleotides were dissolved by 3 μM in 20 mM Tris–HCl (pH 7.0) to measure the cross reactivity of other metal ions to TBA and PS2.M. Metal ions such as Al3+, Cu2+, Ni2+, Fe3+, Co2+, Cr2+, and Sr2+ were dissolved in DW at various concentrations, and the solution at each concentration was added by 200 μL to 1.8 mL of nucleotide solution. To determine the effect of multiple cations, metal ions used above was collectively dissolved in DW to meet 3 μM respectively, and 200 μL of the multiple cation solution was added to make in total 2 mL mixture with nucleotide solution. The measurement of CD spectroscopy was performed as described above.

Detection of Pb2+ dissolved in effluent from a municipal wastewater treatment facility

Three effluent samples (Sample 1–3) of the WWTP was obtained from Korea Water Cluster (Daegu, Korea). Before detection, each effluent sample was filtered through a 0.2-μm membrane. TBA and PS2.M was dissolved to 3 μM concentration in 20 mM Tris–HCl (pH 7.0). Pb2+-spiked effluent was prepared by dissolving Pb2+ from 0 to 120 μM concentration in an effluent sample (Sample 1). Each Pb2+ solution was added by 200 μL to 1.8 mL of the nucleotide solution. The recovery of the signal intensity by spiked Pb2+ in the Sample 1 was calculated by division of the intensity at 314 nm by the intensity of the standard solution prepared by dissolving Pb2+ in DW. To detect the existence of Pb2+ in three effluent samples without spiking of Pb2+, CD intensity was measured by adding 200 μL of each effluent to 3 μM nucleotide solution dissolved in 20 mM Tris–HCl (pH 7.0).

The concentration of metal ions in the effluent from the WWTP was analyzed through inductively coupled plasma optical emission spectrometry (ICP-OES; iCAP7400, Thermo Scientific, Waltham, MA, USA).

Results and discussion

Characteristics of CD spectrum upon binding of Pb2+ to G-QDs

CD spectroscopy was employed to examine the K+ and Pb2+-induced G-QD structures (Fig. 2). Peak [A] indicates the absorption peak of each G-QD without K+, whereas peak [B] indicates the absorption peak induced by K+. The wavelengths of the peaks [B] for TBA and PS2.M were slightly shifted from peak [A], to − 8 and + 4 nm, respectively. For peak [B], a dramatic increase in the absorption intensity was observed at 100 mM K+. However, a slight increase for TBA and no shift for PS2.M were observed at 100 μM K+ concentration, and new absorption bands near 298 and 294 nm were observed for HTG and T2, respectively, at both 100 μM and 100 mM K+ concentration (Fig. 2A). The absorption band of AGRO 100 near 264 nm was not shifted by the addition of K+; however, the peak intensity at 264 nm was dramatically increased at 100 mM K+ concentration. This result indicated that the switched form of AGRO 100 induced by K+ ions had the same peak wavelength as the denatured AGRO 100, and the CD signal intensity was enhanced by complexation with K+ at 100 mM concentration.

Figure 2
figure 2

Circular dichroism (CD) spectra G-QDs by addition of K+ and Pb2+. (A) Structural changes observed in five types of G-QDs by addition of K+. (B,C) Structural changes observed by incorporation of Pb2+. (B) Effect of Pb2+ at 100 μM K+; (c) effect of Pb2+ at 100 mM K+. Yellow line is the spectra induced by Pb2+ without K+.

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The shift of the peak wavelength ([B]-[A]) for each unfolded nucleotide ([A]) and new peaks by K+ complexation ([B]) are summarized in Table 2. The absorption peaks of these G-QDs by K+ complexation were similar to those reported previously for each G-QD19,20,21,22,23.

Table 2 CD spectral characteristics for each G-QD owing to addition of K+ and Pb2+.
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Peak [C] indicated the absorption peak upon addition of Pb2+ (Fig. 2B,C). The Pb2+-originated absorption wavelength ([C]) was shifted from the K+-induced peak ([B]) for TBA, PS2.M, HTG, AGRO 100, and T2 by 18, 46, 16, 0/40, and 22 nm, respectively ([C]-[B] in Table 2). The peak wavelength of Pb2+-induced AGRO 100 was observed at two wavelengths—264 and 304 nm. The peak intensity at 264 nm was positively related to the concentration of Pb2+, but it was the same as that for the peak induced by K+ ions. The absorbance at 304 nm gave a unique signal for Pb2+ incorporation into AGRO 100, and the signal intensity decreased with increasing Pb2+ concentration. In this study, we investigated the capability of a G-QD sensor for the specific binding of Pb2+; thus, the intensity of the peak at 304 nm was considered to be only a Pb2+-induced response. In Fig. 2B,C, yellow lines indicate the spectra induced by Pb2+ without the addition of K+. The transition of G-QDs were induced by Pb2+, therefore, the intensity of peaks induced by Pb2+ only was similar or higher to those of Pb2+ with 100 μM K+.

All G-QDs studied herein formed Pb2+-induced structures, and the signals were clearly discriminated from the signals by K+. Therefore, the CD signal could facilitate the detection of Pb2+ in water samples, which contained K+ ions near 100 μM. When K+ concentration was increased to 100 mM, the switched structures of TBA, PS2.M, HTG and AGRO 100 by Pb2+ were also observed distinctly from those of K+, but the CD signal intensity was not high as observed for that at 100 μM K+ concentration (Fig. 2C). Moreover, the T2 absorption band by Pb2+ disappeared at 100 mM K+ concentration. It was attributed to the Pb2+ binding affinity to T2 that was not sufficient to stabilize the G-QD structure at high K+ concentration. The stability of Pb2+ binding, instead of K+, depended on the structural differences of G-QDs, which was originated from the nucleotide sequences.

Taken together, there are specific absorption bands induced by the incorporation of Pb2+ ([C]), which are completely different from those of K+-induced G-QDs ([B]) as well as unfolded G-QDs ([A]). The clear absorption band of G-QDs specifically induced by Pb2+ implied that a unique conformation of G-QDs was constructed by the addition of Pb2+. The results indicated that the intensity of the CD signal at the absorption band could be used for the signal of the Pb2+ sensor, reflecting its concentration in the samples.

Determination of maximum binding and association constants of Pb2+ to G-QDs

Figure 3 shows the relationship between the Pb2+ concentration and the intensity of the CD signal at the peak wavelength [C] in Fig. 2. TBA and PS2.M exhibited higher CD signal intensity at both 100 μM and 100 mM K+. At 100 μM K+ concentration, the signal increased from 100 fM Pb2+, and the binding capacity was saturated from 3 μM (Fig. 3A). However, Pb2+ was detected at 3 μM concentration and saturated from 60 μM Pb2+ at 100 mM K+ concentration (Fig. 3B). TBA showed a delayed saturation compared to other nucleotides. This result indicated the importance of K+ concentration in the Pb2+ sample solution, because Pb2+ should compete with K+ at high K+ concentration. Several studies reported that replacement of K+ by Pb2+ in a quadruplex was observed22,24, which supported that K+ concentration could affect the binding affinity of Pb2+ in sensing process.

Figure 3
figure 3

Concentration-dependent CD spectra. (A) Pb2+-dependent CD response at 100 μM K+ concentration. (B) Pb2+-dependent CD response at 100 mM K+ concentration.

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To quantitatively determine the binding affinity, we calculated the binding constant of Pb2+ for G-QDs using the Langmuir adsorption model38. The plot according to Eq. (1) generated a straight line and the ratio of slope to intercept was KA, and the inverse slope was qm (Fig. 4). In Fig. 4A, the linearity of the plots (R2) for the five G-QDs was 0.961–0.994 at 100 μM K+ concentration. However, the plots for AGRO 100 and T2 at 100 mM K+ concentration were not available to determine the binding constant, due to the negative intercept (AGRO 100) and poor C vs. q relationship (T2) (Fig. 4B).

Figure 4
figure 4

Plot of Langmuir isotherm equation for the calculation of association constant (KA) of G-QDs for Pb2+. The plot was prepared by c vs c/q. (A) Association relationship for each G-QD at 100 μM K+ concentration. (B) Association relationship for each G-QD at 100 mM K+ concentration. c, Pb concentration; q, CD absorption response.

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The value of KA for Pb2+ binding to G-QDs was determined as shown in Table 3. The binding constant of G-QDs at 100 μM K+ concentration was several orders of magnitude (10–100 fold) higher than that measured at 100 mM K+ concentration. The binding constants of G-QDs at 100 μM K+ concentration were also higher than those of other Pb2+ sensors, such as the surface plasmon resonance biosensor (2.4 × 102–7 × 105/M)39. In a recent study, a fluorophore was synthesized and the Pb2+ concentration was measured by fluorescence spectroscopy, and the binding constant was 5 × 105/M40, comparable or lower than that calculated in this study. Taken together, the PS2.M structure had the highest association constant for Pb2+ (1.09 × 105/M) even when there was a high concentration of competitive K+ up to 100 mM. TBA showed the highest qm among the five G-QDs at both 100 μM and 100 mM K+ concentrations, which indicated that TBA could have the largest binding capacity for Pb2+ by forming a stable G-QD structure among the five G-QDs used in this study. Thus, TBA and PS2.M are considered as more suitable sensing materials for Pb2+ detection as an additional labeling probe and a platform are not required to immobilize them.

Table 3 Association constants of G-QDs for the binding of Pb2+.
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Selectivity of Pb2+ over other metal ions

The specificity of Pb2+ for TBA and PS2.M, which were selected as the most efficient sensing materials among the five G-QDs investigated in this study, was investigated with other metal ions. As demonstrated in Fig. 5A (TBA) and 5B (PS2.M), neither G-QDs responded to other metal ions such as Al3+, Cu2+, Ni2+, Fe3+, Co2+, Cr2+, and Sr2+. The result exhibited a highly selective structural conversion by Pb2+ in contrast to the above-mentioned metal ions. The peak [B] indicates the peak induced by K+ ([B] in Fig. 2), and the peak [C] indicates the wavelength of absorption by Pb2+-induced G-QDs ([C] in Fig. 2).

Figure 5
figure 5

Cross-reactivity of G-QDs to metal ions. (A) Cross-reactivity of metal ions to TBA. (B) Cross-reactivity of metal ions to PS2.M. Black arrows indicate the K+-induced peak. Blue-dot arrows indicate the wavelength of Pb2+-induced G-QDs (314 nm for both G-QDs).

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For TBA, the specific wavelength of the CD peak caused by K+ complexation was 294 nm, and the peak height at 294 nm was decreased with increasing concentrations of Al3+ and Fe3+ (Fig. 5A). This was attributed to the deconstruction of the K+-specific structure owing to the addition of Al3+ and Fe3+ ions. However, the CD signal intensity at Pb2+-specific wavelength (314 nm) was not observed, which indicated that Al3+ and Fe3+ could not induce the structural conversion, as done by Pb2+, even if they could affect the binding of K+. All other ions such as Cu2+, Ni2+, Co2+, Cr2+, and Sr2+ could not affect the K+-specific binding of TBA and did not induce structural conversion as Pb2+ did.

For PS2.M, the main peak at 266 nm originated from the structural conversion by K+ (Fig. 5B). The addition of Al3+, Ni2+, and Fe3+ to PS2.M decreased the peak height with increasing ion concentration. Although slight destruction of K+-induced G-QDs was observed by Al3+, Ni2+, and Fe3+, a Pb2+-specific peak that should be observed at 314 nm did not appear. This result indicated that PS2.M also had high specificity to Pb2+. Figure 6 shows the spectra of TBA and PS2.M induced by Pb2+ under the co-existence of seven ions. The result indicates that multiple ions did not interfere the transition of G-QDs by Pb2+.

Figure 6
figure 6

CD spectra of TBA and PS2.M by Pb2+ in the presence of multiple ions, Al3+, Cu2+, Ni2+, Fe3+, Co2+, Cr2+ and Sr2+. Each ion concentration including Pb2+ is 3 μM, without K+. Peak C indicated the 314 nm peaks of two G-QDs induced by Pb2+.

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Figure 7 shows the intensity of the CD signal according to the metal ion concentration, at which TBA (Fig. 7A) and PS2.M (Fig. 7B) were switched to G-QD structures (314 nm) by Pb2+. As shown in Fig. 7, the structural transition of TBA and PS2.M induced by Pb2+ was clearly observed, and seven metal ions—Al3+, Cu2+, Ni2+, Fe3+, Co2+, Cr2+, and Sr2+—could not induce a structural transition. The selective transition at the Pb2+-specific wavelength could be a critical property of TBA and PS2.M as specific detection sensors of Pb2+.

Figure 7
figure 7

Cross-reactivity of TBA and PS2.M to other metal ions at 314 nm. (a) Cross-reactivity of metal ions to TBA. (b) Cross-reactivity of metal ions to PS2.M.

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Analysis of Pb2+ spiked into wastewater samples

In order to test whether the proposed strategy for Pb2+ detection could be applied to environmental samples, the concentration of Pb2+ was analyzed in effluent samples of a WWTP located in Daegu, Korea. For our reference, the concentrations of metal ions in the sample were measured by ICP analysis including Pb2+ and K+ (Table 4). As shown in Table 4, the concentration of K+ in wastewater was 616 μM (Sample 1), 484 (Sample 2), and 214 μM (Sample 3), which could facilitate the slight transition of TBA to K+-dependent structures, as shown in Fig. 2A. For PS2.M, it was not expected that the transition of the K+-specific structure was occurred in the effluent samples, because the transition by 100 μM K+ concentration was not observed in Fig. 2A.

Table 4 Metal concentrations determined by ICP-OES analysis in the wastewater samples.
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Pb2+ in the Sample 1 and Sample 2 was as low as 0.0159 μM (3.30 ppb) and 0.00676 μM (1.40 ppb), and in Sample 3, Pb2+ was not detected. To investigate the peak transition and recovery of CD signals in the effluent samples, Pb2+ was spiked into the Sample 1 from 0 to 120 μM. When Pb2+-spiked Sample 1 was added to the TBA solution, the transition of the TBA structure by Pb2+ was occurred at 314 nm, and the CD intensity was increased according to the Pb2+ concentration from 3 μM (Fig. 8A). The peak wavelength at 0 μM Pb2+ was 296 nm, which was identical to peak [B], the K+-induced transition. Thus, TBA could not detect the background Pb2+ concentration in the effluent sample (0.0159 μM). However, if the Pb2+ existed over 3 μM, it would be possible to detect Pb2+ dissolved in complex environmental samples.

Figure 8
figure 8

CD spectra of Pb2+-spiked effluent samples discharged from a local wastewater treatment plant (WWTP). Pb2+ detection using TBA (A) and PS2.M (B). Comparison of CD spectra of TBA (C) and PS2.M (D) added by Pb2+-spiked effluent samples and Pb2+in 100 μM and 100 mM K+ solution.

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On the other hand, the switching of PS2.M by K+ did not occur in the effluent Sample 1, as expected by the result in Fig. 2 (Fig. 8B). However, the CD spectrum shifted toward a Pb2+-specific transition wavelength (314 nm) due to the background Pb2+ in the effluent was observed (spiked [Pb2+] = 0). In addition, the CD signal intensity at 314 nm displayed a positive relationship with the spiked Pb2+ concentration.

The CD signal at 314 nm in the effluent Sample 1 was compared to the CD intensity for Pb2+-induced G-QDs with 100 μM and 100 mM K+(Fig. 8C,D), respectively. For both TBA and PS2.M, the CD intensity for Pb2+ in the effluent sample was decreased compared to that of Pb2+ in 100 μM K+, and increased to that of Pb2+ in 100 mM K+. The CD intensity at 314 nm by TBA and PS2.M in the effluent sample was 0.52- and 0.78-fold decreased than that of 100 μM K+ solution, respectively. However, they were increased to 3.1 and 2.9 folds to that of 100 mM K+ solution for TBA and PS2.M, respectively. The high recovery ratio of Pb2+ signal in effluent sample when comparing in 100 mM K+ solution was attributed to the low concentration of K+ in effluent sample, because K+ competes to Pb2+ as shown in Fig. 2B,C. However, the ratio of CD intensity of Pb2+-induced TBA in the effluent sample to 100 μM K+ solution (0.52) was more reduced than that of PS2.M (0.78). These results suggest that PS2.M could determine the Pb2+ concentration more efficiently, even though the samples were composed of complex materials released to municipal wastewater. In Supplementary Fig. S1, the Pb2+-induced spectra of TBA and PS2.M in 616 μM K+ was compared to those of 100 μM K+. The peak intensity and the shape of spectrum was almost the same between them.

Figure 9 shows that the CD spectra of TBA and PS2.M added in effluent samples. The concentration of Pb2+ in Sample 1 and Sample 2 was 3.30 ppb and 1.40 ppb, respectively, but not detected in Sample 3. In case of PS2.M, the three effluent samples without spiked Pb2+ caused a discrete shift of the absorption band to the Pb2+-specific wavelength, 314 nm. This result indicated that the Pb2+ in the effluent sample as low as 3.3 ppb (0.0159 μM) could convert PS2.M to a G-QD structure, and be simply measured by CD spectroscopy without any other platform and labeling reagents. Therefore, PS2.M could be more promising as a detection probes for Pb2+ in environmental samples, because they caused a suitable absorption signal in CD spectroscopy.

Figure 9
figure 9

CD spectra of TBA and PS2.M added by three effluent samples (Sample 1–3), DW and 100 μM K+.

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The limits of permissible concentration of Pb2+ in surface water are as follows: 50 ppb for drinking water supply41; 15 ppb for drinking water42; 50–100 ppb for permissible effluent water43; and 200–500 ppm industrial wastewater36. In Fig. 9, the CD intensity at 314 nm by PS2.M could be related to the concentration of Pb2+, measured by ICP-OES. Furthermore, the PS2.M was applicable to detect the level of Pb2+ concentration permissible for drinking water supply (50 ppb) and for drinking water (15 ppb).

Comparing to other methods to determine the low concentration of Pb2+, ICP-OES and Inductively Coupled Plasma Mass Spectrometer (ICP-MS) could be applied. The detection limit of Pb2+ by ICP-OES and ICP-MS is known nearly as 5 ppb and 5 ppt, respectively, but the required volume of samples is 50–100 mL for each instrument. In addition, the operation of both instruments is difficult compared to CD spectroscopy. One of the G-QDs studied in this study, PS2.M, could detect the Pb2+ in environmental samples below 5 ppb using 2 mL volume.

Along with K+, Na+ could be also contaminated in environmental samples, which is known to be intercalated into the nucleotides to convert to G-QDs. However, PS2.M was reported to show no response to Na+24, and TBA could be converted to G-QDs by Na+ but the spectra converted by Na+ was similar to that of K+44.

Conclusion

To determine the performance of G-QDs as a probe for Pb2+ detection, five types of nucleotides were compared for their binding affinity. Two of them, TBA and PS2.M, which showed higher binding constants, were analyzed for their specificity and applicability for environmental sensors to detect Pb2+. Although K+ has been reported to have a strong binding affinity to G-QDs, we found that the coexistence of Pb2+ with K+ concentrations up to 100 μM has a negligible effect on Pb2+ binding. The binding constants of TBA and PS2.M were comparable to those of the antigen–antibody reaction, and the Pb2+ detection property of PS2.M was suitable for the analysis of environmental water.

In this study, several G-QDs were thoroughly examined for the binding constant for Pb2+ and cross-reactivity to other metal ions, which were crucial data for the application of them to platform- and labeling-free detection of Pb2+. The results demonstrated the substantial role of the structural transition of TBA and PS2.M in sensing the Pb2+ concentration. Furthermore, in a complex sample such as an effluent discharged from WWTP, solubilized Pb2+ was detected below 5 ppb levels. Therefore, the proposed method has promising applications in environmental and laboratory sample analysis, because of the simple and cost-effective preparation of G-QDs and detection procedures.