A resonance Rayleigh scattering sensor for detection of Pb2+ ions via cleavage-induced G-wire formation

Journal of Hazardous Materials 336 (2017) 195–201

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Research paper

A resonance Rayleigh scattering sensor for detection of Pb2+ ions via cleavage-induced G-wire formation Wang Ren a,b , Ying Zhang a,b , Yu Zhu Fan a , Jiang Xue Dong a , Nian Bing Li a,∗ , Hong Qun Luo a,∗ a Key Laboratory of Eco-Environments in Three Gorges Reservoir Region (Ministry of Education), School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China b School of Chemistry and Environmental Engineering, Sichuan Provincial Academician (Expert) Workstation, Sichuan University of Science and Engineering, Sichuan, Zigong 643000, PR China

h i g h l i g h t s

g r a p h i c a l

a b s t r a c t

• The strategy is label-free and requires no expensive signal reagent.

• G-wire polymer was induced as a new resonance Rayleigh scattering probe. • The sensor showed outstanding selectivity and sensitivity for Pb2+ detection. • The self-blocking substrate and signal-on mechanism avoid false positive results.

a r t i c l e

i n f o

Article history: Received 2 December 2016 Received in revised form 11 March 2017 Accepted 14 April 2017 Available online 26 April 2017 Keywords: Detection of Pb2+ Resonance Rayleigh scattering DNAzyme G-wire

a b s t r a c t A resonance Rayleigh scattering (RRS) aptasensor was fabricated for detection of Pb2+ via hairpin-like label-free substrate and G-wire for signal amplification. A hairpin-like DNA substrate contains a sequence in the loop labeled with ribonucleobase A and c-myc sequence in the stem. When hybridized with 8–17 DNAzyme in the presence of Pb2+ , the sequence in the loop was activated and cleaved. Hundreds of c-myc sequences departing from the 8–17 DNAzyme yield nanowires superstructure called G-wire in the presence of Mg2+ . The polymer G-wire was demonstrated by the RRS spectrum, polyacrylamide gel electrophoresis, and AFM. The RRS intensity was enhanced by the product G-wires, and the RRS signal at 370 nm was linear with the logarithm of Pb2+ concentration in the range of 2.0 nM to 5.0 ␮M. This method was selective for Pb2+ even coexisting with other metal ions at high concentrations and was successfully applied to the determination of Pb2+ in real samples. The aptasensor holds a great promise for universal RRS sensing platform for sensitive detection of various metal ions just by changing the sequence of the probe in the loop and DNAzyme. © 2017 Elsevier B.V. All rights reserved.

∗ Corresponding authors. E-mail addresses: [email protected] (N.B. Li), [email protected] (H.Q. Luo). http://dx.doi.org/10.1016/j.jhazmat.2017.04.039 0304-3894/© 2017 Elsevier B.V. All rights reserved.

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1. Introduction Over the past decades, nucleic acids selected in vitro or designed play important roles in the field of highly selective molecular recognition. A kind of specific single strand DNA sequences (DNAzymes) binding specificity with metal ions, showing high cleavage activity of its RNA substrates, was used as a general platform for metal ions sensing applications [1,2]. RNA-cleaving DNAzymes-based sensors have been extensively reported for determination of metal cations, such as Cu2+ [3,4], Zn2+ [5,6], Co2+ [7,8], Mn2+ [9], UO2 2+ [10], Mg2+ [11], Pb2+ [12–16], Hg2+ [17], and Ag+ [18]. As one of the common and hazardous heavy metal ion pollutants, lead ion (Pb2+ ) receives increasing attention because of its serious toxicity and bioaccumulation [19]. Even at low concentration, lead poisoning is related to various neurotoxin effects, impaired kidney function, and reproductive dysfunction [20]. As a result, DNAzyme-based biosensors for lead detection were constructed with various signal transduction mechanisms, including fluorescent [13,16,21–23], colorimetric [14,15,24–27], electrochemical [28–31], and photoelectrochemical [32–34] methods. In most of these strategies, signal output must depend on either direct modification of the nucleic acids, such as bulky fluorophores and electrochemical indicator. The labeled groups may eliminate the DNAzyme’s catalytic activity and easily lead to high backgrounds. Therefore, developing a high selective and label-free biosensor is highly desirable. When individual molecules combined with each other via intermolecular interactions, such as hydrogen bonds, ␲–␲ conjugation, and stacking modes, the molecular complexes showed some unique optical features, which have been widely used in chemical and biological optical sensors during last few years [35–37]. Resonance Rayleigh scattering (RRS) is an impressive spectral analytical technique for its sensitivity, simplicity, and rapidness. The characteristic of RRS spectrum is sensitively influenced by the change of molecular size, shape, and interfacial properties in solution, which holds a great potential in label-free detection [38]. Conjugated to the aptamer, the label-free specific G-rich sequence released from the target recognition event and can fold up into the parallel stranded G-quadruplex. The G-quadruplexes can be further assembled into long and stiff filamentous G-wire [39–41]. The change of molecular size from unimolecular to long polymer remarkably enhanced the RRS intensity, which provided a label-free and highly sensitive signal output strategy to construct the RRS sensor [42]. Herein, we constructed an amplified label-free RRS aptasensor for sensitive detection of Pb2+ by integrating the Pb2+ -dependent DNAzyme assistant target recycling with self-assembly of Gquadruplex for unique RRS characteristic of G-wire signal amplification. The selective and sensitive aptasensor contains a Pb2+ -dependent DNAzyme and hairpin-like substrate labeled with a ribonucleobase A(S(r)). When cleaved by Pb2+ -DNAzyme, the substrates set free the single strands including c-myc sequence which can fold up into unimolecular parallel stranded G-quadruplexes. These G-quadruplexes further formed long filamentous G-wire with the aid of Mg2+ . The long G-wire configuration greatly amplified RRS readout signal and provided a promising alternative for sensitive and label-free Pb2+ detection. Moreover, the proposed aptasensor is of high selectivity and low cost because of no requirement of any sophisticated chemical modification and expensive organic assistant-reagents.

Magnesium chloride and potassium chloride were purchased from Shanghai Sangon. Other reagents of analytical reagent grade were bought from Chengdu Kelong Chemical Reagents Company (China), and used as received. HEPES buffer 1 (HB1, pH 7.0) was prepared with 25 mM HEPES, 100 mM NaCl, and 5 mM MgCl2 . HEPES buffer 2 (HB2, pH 7.4) was prepared with 20 mM HEPES, 150 mM KCl, and 200 mM MgCl2 . A stock solution of Pb2+ (100 mL, 1 mM) was prepared by dissolving Pb(NO3 )2 with 0.5% HNO3 . Ultrapure water (18.2 M cm) was used in all runs. All the oligomers were synthesized and purified by Sangon (Shanghai, China). The sequences of oligonucleotides are listed in Table S1 (Supplementary material). Each oligonucleotide sample was obtained by dissolving in HB1. The prepared oligonucleotides were heated to 90 ◦ C for 10 min and then allowed to slowly cool down to room temperature for 2 h before use. A Hitachi F-4500 spectrofluorophotometer (Hitachi Ltd., Tokyo, Japan) equipped with a 150-W xenon lamp was used for recording the RRS spectrum. An SD-101-005DB super digital thermostat bath (Sida Experimental Equipment Ltd., Chongqing, China) was used to adjust experiment temperature. The pH values of solutions were measured with a pH meter (PHS-3C, Shanghai Leici Instrument Company, Ltd., China). 2.2. Analytical procedure for Pb2+ The detailed protocol for determination of Pb2+ was as follows: At first, the hybridization reaction was implemented by mixing the substrates (S(r)) (50 ␮L, 2 ␮M) and the single-strand DNAzyme (25 ␮L, 2 ␮M 8–17 DNAzyme) at 40 ◦ C for 30 min. Then 50 ␮L of different concentrations of Pb2+ solution was mixed to the solution and reacted for 50 min at 35 ◦ C. Subsequently, the mixture was diluted to 500 ␮L with HB2, followed by incubation for 2 h at 4 ◦ C to facilitate the parallel G-quadruplexes to assemble into large Gwire nanostructures. Finally, the RRS intensity detection of all the oligomer solutions was performed. The RRS spectra of the optical sensor were recorded by synchronous scanning at em = ex (i.e.,  = 0 nm) in the range of 220–650 nm with the protocol parameters including the PMT voltage of 400 V and the slits (ex/em) of 10/10 nm. The increased RRS intensity (IRRS ) was received as IRRS = IRRS − I◦ RRS , where IRRS and I◦ RRS are the RRS intensity of the solution at 370 nm with and without Pb2+ , respectively. 2.3. Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis (PAGE) was applied to corroborate the Pb2+ -triggered G-wires formation. Samples for PAGE assays were planned as follows: (1) S(r)3 (10 ␮M) was used as sample 1; (2) the mixture of 10 ␮M S(r)3 and 100.0 nM Pb2+ was incubated and used as sample 2; (3) the mixture of 10 ␮M S(r)3 and 5 ␮M DNAzyme was incubated and used as sample 3; (4) the mixture of 10 ␮M S(r)3 , 5 ␮M DNAzyme, and 100.0 nM Pb2+ was incubated and used as sample 4. All mixtures were put in a constant temperature bath at 35 ◦ C for 50 min and then were put in 4 ◦ C refrigerator for 2 h. Lastly, the final four mixtures were investigated using 12% native PAGE. Before loading, the DNA loading buffer was added to the mixtures at a volume ratio of 1:1. A potential of 120 V was used for PAGE for 80 min at 25 ◦ C. After separation, PAGE gels were visualized using Ag staining and imaged with a Gel Doc XR system. 2.4. Atomic force microscopy

2. Materials and methods 2.1. Materials and apparatus 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was bought from Aladdin Chemistry Co., Ltd., Shanghai, China.

Atomic force microscopy (AFM) was also applied to examine the formation of long filamentous G-wires. The mixture solution (10 ␮L) of 10.0 ␮M S(r)3 , 5.0 ␮M DNAzyme, and 1.0 ␮M Pb2+ was reacted at 35 ◦ C for 80 min. After that, the mixture solution was diluted in HB2 (40 ␮L) and incubated at 4 ◦ C (2 h). Finally, the sam-

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Scheme 1. Schematic diagram of the label-free RRS aptasensor based on Pb2+ - DNAzyme facilitated target recycling and G-wire formation for Pb2+ detection.

Fig. 1. (A) Typical RRS spectra of some oligomers solutions. (a) S(r)3 , (b) S(r)3 + Pb2+ , (c) S(r)3 + DNAzyme, and (d) S(r)3 + Pb2+ + DNAzyme. The concentrations of Mg2+ , S(r)3 , Pb2+ , and DNAzyme are 0.2 M, 0.2 ␮M, 0.1 ␮M, and 0.1 ␮M, respectively. (B) PAGE analysis: (1) DNA marker (pBR322/MSP I); (2) S(r)3 (10.0 ␮M); (3) S(r)3 (10.0 ␮M) + Pb2+ (100.0 nM); (4) S(r)3 (10.0 ␮M) + DNAzyme (5.0 ␮M); (5) S(r)3 (10.0 ␮M) + Pb2+ (100.0 nM) + DNAzyme (5.0 ␮M). (C) AFM image of the resultant products of 10.0 ␮M S(r)3 , 5.0 ␮M DNAzyme and 100.0 nM Pb2+ and the surface roughness of AFM image.

ple (20 ␮L) was dropped onto a freshly cleaved mica substrate. The G-wires were adsorbed on the mica substrate for 30 min. When washed with 1 mL of HEPES (10 mM, pH 7.0), the sample was rapidly dried by N2 gas. Imaging was carried out with a Nanoscope IIIa with a D-scanner at relative humidity <10%.

2.5. Analysis of environmental water samples River water samples were taken from the Fuxi River (Zigong, China) and Jialing River (Chongqing, China). Tap water samples were obtained straight from our laboratory. All water samples were diluted with 1% HNO3 solution with a volume ratio 1:1 and shaken on a vortex at room temperature for 5 min. The insoluble impurities in the river water samples were then removed by filtering through a 0.22 ␮m membrane. Aliquots of the tap water and river water were spiked with ultrapure water or Pb2+ and then diluted with HB1 containing 2 ␮M S(r)3 . Other detection procedures were the same as described above. The prepared samples were then analyzed successively by using the Hitachi F-4500 spectrofluorophotometer.

3. Results and discussion 3.1. Design principle of this RRS sensor The design principle of the RRS sensor for determination of free Pb2+ ion is schematically illustrated in Scheme 1. The Pb2+ recognition system consists of the oligomer sequence S(r) labeled with a ribonucleobase A, rA, in the loop and the enzyme strand (DNAzyme). The S(r) oligomer sequence is composed of three parts: the special c-myc sequence (part I) at the 5 end was partially complementary to part III at the 3 end; Part II (part of the loop) including the specific rA was designed to recognize the Pb2+ -DNAzyme. The DNAzyme was activated by Pb2+ ion, giving rise to the cleavage of the S(r). As reported, metal ions could coordinate with part of DNAzyme which was not hybridized with substrate strand, and then cleavage site rA of substrate is readily severed by driving attack of the 2 -hydroxyl group of specific nucleotide strand into the adjacent phosphodiester linkage, turning out 2 , 3 -cyclic phosphate and 5 -hydroxyl termini [43]. The cleavage disrupted the stability of the substrate structure, releasing Pb2+ ion and the c-myc sequence from the double strands. The free Pb2+ initiated the next round of cleav-

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3.2. Feasibility study To verify the validity of the proposed strategy, the RRS spectra of four typical solutions were recorded at first. As shown in Fig. 1A, the RRS intensity of the solution containing S(r)3 is very weak and about 550 at 370 nm (curve a). Either treated with Pb2+ or hybridized with the DNAzyme, the RRS intensities of the S(r)3 solution (curves b and c) were the same as that of curve a. However, when Pb2+ (100.0 nM) and DNAzyme were simultaneously added to the solution containing S(r)3 , the RRS intensity was increased significantly in the range of 250–600 nm (curve d) and it was 7 times of the blank sample (curve c) at 370 nm. According to the contrastive research, it was inferred that the Pb2+ -DNAzyme triggered the substrate cleavage, and then the liberated sequence at the 5 end folded up into parallel-stranded G-quadruplexes which further stacked into a filamentous polymer G-wires. Several factors lead to the enhancement of RRS intensity including the enlargement of molecular volume [44]. The molecular volume of the filamentous polymer G-wires is much larger than that of single hairpin-like DNA aptamer and G-quadruplex [39–42]. In addition, the formation of hydrophobic interface causes the scattering enhancement [45]. When positively charged Mg2+ interacted with negatively charged G-quadruplex to form long filamentous G-wires, their hydrophobicity was enhanced greatly, which was conducive to the enhancement of RRS. Furthermore, the configuration of DNA was changed from single probe molecular to filamentous polymers which may also significantly affect the observed scattering intensity [46]. In the absence of Pb2+ DNAzyme coordination, the hairpin structure of S(r) is stable, and accordingly, the RRS intensity of the system is not changed. Because the maximum RRS peak of the sensor appeared at 370 nm, and thus the RRS intensities are measured at 370 nm in the following study. The practicability of the proposed method was further confirmed by PAGE, and the results are shown in Fig. 1B. The low bands in lanes 2, 3, and 4 corresponded to the S(r)3 mixed with buffer, 100.0 nM Pb2+ , and 5.0 ␮M DNAzyme, respectively. The electrophoretic bands appeared in lanes 2, 3, and 4 are almost at the same position, illustrating that lonely Pb2+ or DNAzyme could not induce the G-wire structure. When 100.0 nM Pb2+ and 5.0 ␮M DNAzyme are simultaneously added to the S(r)3 , several bands appeared in lane 5. The lowest band in lane 5 appears weaker, demonstrating that the S(r)3 was consumed by Pb2+ -DNAzyme. Besides, the appeared several higher bands suggested that the Gwire polymers were produced. AFM was also used to directly characterize the size and morphology of the assembled G-wires. Fig. 1C shows a typical AFM image of the solution containing S(r)3 , Pb2+ , and DNAzyme. The clear axial extension filamentous polymers image is presented, indicating that the part of G-rich sequence sealed in the stem of S(r)3 was liberated and could form the G-wires structure. In addition, the length

Blank sample 100 nM Pb2+

4000 3000

RRS

age substrate strand with DNAzyme. Eventually, each released Pb2+ can trigger many cycles of S(r) cleavage, releasing part I and part II. These liberated fragments with c-myc sequence (part I) folded up into parallel-stranded G-quadruplexes and further self-assembled into long filamentous G-wires in the presence of Mg2+ . Because these long G-wires dramatically raised the RRS intensity, the RRS spectral signal was used as a readout signal for label-free Pb2+ sensing. Without Pb2+ , the hairpin structure substrate is stable, thus the c-myc sequence at the 5 end lost ability to form the parallel Gquadruplex molecule, much fewer G-wires. The hairpin structure substrates greatly reduce the RRS background signal. Therefore, a signal amplified RRS sensor could be constructed for Pb2+ detection with high sensitivity and selectivity. Meanwhile, the signal reagents-free method makes the sensor simple and cost-effective.

I

198

2000 1000 0 S(r)

1

S(r)

2

S(r)

3

S(r)

4

S(r)

5

Fig. 2. Effect of the five substrates on the RRS intensity in the absence and presence of 100.0 nM Pb2+ . Standard deviations indicated as error bars are obtained from three parallel experiments.

of the G-wires ranged from several tens of nanometers to 500 nm, and the average height of the G-wires observed in this study was 1.3 ± 0.1 nm. In order to recognize the characters of G-wire, the surface roughness of AFM image is also shown in Fig. 1C. The above AFM images revealed that Pb2+ -DNAzyme could cleave the substrates and facilitate the formation of high-order linear G-wires. From the experimental results mentioned above, it was indicated that the label-free and sensitive RRS analytical technique was available for Pb2+ detection based on the signal amplification strategy of DNAzyme-assisted Pb2+ ion recycling. 3.3. Optimization of experimental parameters In order to achieve the best sensing performance, various factors influenced on the RRS aptasensor for Pb2+ were investigated and optimized. Firstly, the stem length of S(r) closely relates to the stability of S(r), which eventually impacts the background and sensitivity of this protocol. Herein, the effect of stem length of S(r) on the sensitivity of this strategy has been optimized at first. Substrates with different base pairs in stem have been designed and investigated for highly sensitive detection (the detailed sequences of S(r) are presented in Table S1). As shown in Fig. 2, after RRS signal background correction, the S(r)3 with 7 base pairs in stem showed the best sensitivity for Pb2+ . The stem length of S(r)1 and S(r)2 is shorter than that of S(r)3 , which leads to lower melting temperatures than that of the S(r)3 . The exposing G-rich sequence (part I) of S(r)1 or S(r)2 easily adopted into the parallel-stranded G-quadruplex structure and further formed long filamentous G-wires which cause the high background of noise for optical sensing. Contrarily, S(r)4 and S(r)5 have longer base pairs of the stem than S(r)3 , leading to more stable double strand structures than S(r)3 . The stable stem would resist the dissociation of cleaved substrates, and thus reduce the sensitivity of this optical aptasensor. Hence, the length of substrates stem played an important role for Pb2+ detection, and the S(r)3 was chosen as an optimum structure. Furthermore, the influence factors of cleavage activity on the sensitivity of RRS aptasensor were optimized, including the concentration ratio of DNAzyme and S(r)3 , reaction temperature, and reaction time. Firstly, the DNAzyme/S(r)3 concentration ratio was significant for the signal amplification. Fig. S1 shows that the RRS value increases with increasing ratio, and then reaches a maximum value in the range of 0.4–0.6, decreasing from 0.6. Therefore, the ratio 0.5 was chosen for further investigation. Subsequently, the effect of cleavage temperature on the RRS response was studied by detecting 100.0 nM Pb2+ at different temperatures (from 20 ◦ C to 50 ◦ C). The control sample (without Pb2+ ) was also inves-

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6000

199

without Pb2+ 2+ add 100.0 nM Pb

5000

3000

I

RRS

4000

2000 1000

tigated under the same conditions. As shown in Fig. S2, in the presence of Pb2+ , the RRS intensity gets an abrupt increase till 35 ◦ C and then reaches a plateau in the range of 35 ◦ C–50 ◦ C. However, the structure of S(r)3 can be destroyed as increasing temperature, which elevated the background signal. Considering the best signal–to–noise (S/N) ratio, 35 ◦ C was chosen to be the optimum reaction temperature. The optical signal was also influenced by the reaction time of cleavage. Fig. S3 depicts the time-dependent RRS intensity changes. The RRS intensity increased gradually with increasing the reaction time at first and reached a plateau at 50 min. Thus, 50 min was selected as the optimum cleavage time. Accordingly, the 0.5, 35 ◦ C, and 50 min were chosen as the optimal concentration ratio, cleavage temperature, and reaction time, respectively. To further explore whether the other divalent cations affect the growth of G-wire in this protocol or not, the effects of Ca2+ , Zn2+ , Cu2+ , and Ni2+ on the RRS system were investigated instead of Mg2+ . The results indicate that these metal ions are not effective in promoting aggregate formation by the parallel stranded G-quadruplex in solutions (Fig. S4). To test the stability of the RRS sensor, the RRS intensities of the samples (100.0 nM Pb2+ ) as a function of time were recorded and the results are shown in Fig. S5. The intensities of the RRS system ascend with incubation time and reached to a stable platform after 2 h. Then, the intensity of assay system can maintain a stable level. Thus, the intensity of the sensor was detected after 2 h throughout the study. 3.4. Analytical performance of Pb2+ aptasensor To investigate the sensitivity of the proposed RRS aptasensor for Pb2+ , a series of concentrations of Pb2+ standards were detected under selected conditions. Fig. 3 depicts that the RRS intensity value at 370 nm exhibited positive correlation with increasing concentration of Pb2+ , and displays a good linear relationship between the RRS intensities and the logarithm of Pb2+ concentrations from 2 to 5000 nM. The regression equation is I = 2035.7 + 17704.4 log C (R = 0.9970), where I represents the RRS intensity and C is the Pb2+ concentration. The limit of detection was 500.0 pM estimated at a signal-to-noise ratio of 3␴ (where ␴ represents the standard deviation of a blank solution, n = 11). The detection limit was lower than 72 nM defined by the U.S. Environmental Protection Agency (EPA) as the maximum contamination level of lead ions. The repeatability of the proposed aptasensor was also investigated by analyzing 1000.0, 100.0 and 10.0 nM Pb2+ standard solutions for nine times,

Bu f

Fig. 3. Typical RRS spectra of the aptasensor with different concentrations of Pb2+ (nM): (a) 0; (b) 2.0; (c) 5.0; (d) 10.0; (e) 20.0; (f) 50.0; (g) 100.0; (h) 200.0; (i) 500.0; (j) 1000.0; (k) 5000.0. The inset is the relationship between the RRS intensity changes at 370 nm and the logarithm of Pb2+ concentrations.

fe Hg 2 r + A + g Cd 2 + Cu 2 + Zn 2 + Sn 2 + Co 2 + M n 2+ Ca 2 + Fe 3 + Cr 3 + H A

0

Fig. 4. Selectivity of the RRS aptasensor for 100.0 nM Pb2+ towards 10 times of Zn2+ , Sn2+ , Ca2+ , Co2+ , Mn2+ , Hg2+ , Cd2+ , and Cu2+ , 50 times of Fe3+ , Ag+ , Cr3+ , and 1 mg/L HA.

and the relative standard deviations (RSDs) were found to be 3.2, 4.9, and 5.8%, respectively. 3.5. Selectivity of the RRS aptasensor for Pb2+ detection To study the selectivity of the aptasensor, the RRS responses of the buffer solutions and the samples containing 100.0 nM Pb2+ were compared with high concentration metal ions (Ag+ , Cd2+ , Hg2+ , Cu2+ , Zn2+ , Sn2+ , Ca2+ , Co2+ , Mn2+ , Fe3+ , Cr3+ ) and organic molecule humic acid (HA). The tolerance limit was obtained when the maximum concentration of the coexistent metal ions caused a ± 5% relative error in the determination. The experimental results showed that 10 times of Zn2+ , Sn2+ , Ca2+ , Co2+ , Mn2+ , Hg2+ , Cd2+ , and Cu2+ , 50 times of Fe3+ , Ag+ , and Cr3+ , 1 mg/L HA did not interfere with the Pb2+ determination. As can be seen from Fig. 4, the target metal ion Pb2+ caused an increase in the RRS intensity, while all the other metal ions had no obvious variation of RRS intensity. These results distinctly demonstrated the good selectivity of the resultant RRS aptasensor arising from highly specific Pb2+ -dependent DNAzyme and stable structure of the substrate. The analytical performance of our proposed strategy has been compared with other reported methods for determination of Pb2+ in Table S2. The sensitivity and selectivity of the proposed strategy are better compared to those of some previously reported protocols. Most importantly, our strategy was cheap and convenient due to avoid complicated probe modification or immobilization, complex protease-enzymatic procedure, expensive signal reagents and sophisticated signal transduction. Due to the removal of signal reagents and labeling, the proposed strategy costs about 3 h for detection, which is longer than that of previously reported protocols. 3.6. Analysis of real samples To evaluate the reliability and applicability of the aptasensor for detection of Pb2+ , the real river water samples which were taken from Fuxi River (Zigong, China) and Jialing River water (Chongqing, China) and tap water from our lab were investigated. The water samples spiked with different concentrations of Pb2+ standards were tested on the basis of the foregoing procedure with three RRS spectra detections. The measurement results are presented in Table 1. Satisfactory values ranged from 94.0% to 107.5% were obtained for the recovery assays, indicating that the possible interference from different background compositions of water samples was negligible. The results showed that the developed optical

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Table 1 Determination of Pb2+ in real water samples using the proposed approach. Sample

Tap water 1 Tap water 2 Tap water 3 Fuxi River water 1 Fuxi River water 2 Fuxi River water 3 Jialing River water 1 Jialing River water 2 Jialing River water 3 a b c

Added (nM)

0 20.0 100.0 0 20.0 100.0 0 20.0 100.0

Found (meana ± SDb ) (nM) NDc 18.8 ± 3.2 97.0 ± 2.8 NDc 19.7 ± 2.1 102.0 ± 1.8 NDc 21.5 ± 2.0 105.0 ± 1.7

Recovery (%)

– 94.0 97.0 – 98.5 102.0 – 107.5 105.0

Mean of three determinations. SD, standard deviation. Not detected.

aptasensor can be successfully used to analyze Pb2+ in real environmental water samples. 4. Conclusions A new label-free RRS aptasensor for Pb2+ detection was developed via DNAzyme-assisted the metal ion recycling and recognizing the conformational transition of G-wires. Without external signal reagents and labeling, the new optical sensing way concerning G-wire was constructed and applied to the RRS spectroscopy. Based on the RRS spectral analytical method, the sensitivity of this aptasensor reached 2 nM, and the detection limit for lead ions in real environmental water is lower than the EPA regulation limit. The method also has a high specificity and can realize accurate measurement of the Pb2+ pollution as coexisting with other metal ions at high concentrations. The other merits of the strategy avoided complicated probe modification or immobilization, complex protease-enzymatic procedure, expensive signal reagents, and sophisticated signal transduction. The designed RRS aptasensor not only can be used for the detection of Pb2+ but also provides a label-free optical sensor for highly sensitive detection of other metal ions and non-metal targets by using aptamers and aptazymes. Acknowledgments We gratefully acknowledge the financial support by the National Natural Science Foundation of China (No. 21273174, 21675131), the Municipal Science Foundation of Chongqing City (No. cstc2015jcyjB50001), the Fund Project of Sichuan Provincial Academician (Expert) Workstation (2015YSGZZ03), the Undergraduate Innovation Foundation of Sichuan Province (201410622032), and the Opening Project of Key Laboratory of Green Catalysis of Sichuan Institutes of High Education (LYJ1501, LYJ1402). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2017.04. 039. References [1] H.K. Kim, J.W. Liu, J. Li, N. Nandini, M.X. Li, C.M. Pavot, Y. Lu, Metal- dependent global folding and activity of the 8–17 DNAzyme studied by fluorescence resonance energy transfer, J. Am. Chem. Soc. 129 (2007) 6896–6902. [2] J. Liu, Z. Cao, Y. Lu, Functional nucleic acid sensors, Chem. Rev. 109 (2009) 1948–1998.

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