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A simple and programmed DNA tweezer probes for one-step and amplified detection of UO22+
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 118017
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A simple and programmed DNA tweezer probes for one-step and amplified detection of UO2+ 2 Zhengwei Xiong a,d, Qiang Wang a, Jiafeng Zhang b, Wen Yun c, Xingmin Wang c,⁎, Xia Ha c,⁎, Lizhu Yang b,⁎ a
School of Biological and Chemical Engineering, Innovation Center of Lipid Resources and Children's Daily Chemicals, Chongqing University of Education, Chongqing 400067, China School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China Chongqing Key Laboratory of Catalysis and New Environmental Materials, College of Environment and Resources, Chongqing Technology and Business University, Chongqing 400067, China d Department of Food Biotechnology, Graduate School, Woosuk University, Samnye-eup, Wanju-gun, Jeonbuk Province 55338, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 14 October 2019 Received in revised form 17 December 2019 Accepted 27 December 2019 Available online 30 December 2019 Keywords: Fluorescence DNAzyme Gold nanoparticles DNA tweezer One-step
a b s t r a c t A simple DNA tweezer was proposed for one-step and amplified detection of UO2+ 2 based on DNAzyme catalytic cleavage. The two arms of DNA tweezers are close in the original form. Thus, the fluorescent signal of fluorophore at the end of arm is dramatically quenched. However, the structure of DNA tweezers can be changed from “close” to “open” in the presence of UO2+ 2 , resulting the strong fluorescent signal. The linear range was obtained in the range of 0.1 nM to 60 nM and the limit of detection was 25 pM with the amplification of DNAzyme catalytic cleavage reaction. Importantly, the whole detection process is very simple and only one operation step is required. In addition, it shows great potential and promising prospects for uranyl detection in practical application. © 2019 Published by Elsevier B.V.
1. Introduction Enriched uranium can be used as fuel for nuclear energy and material for nuclear weapons [1,2]. Worldwide consumption of uranium may cause its release into environment by uranium mining and nuclear waste, resulting serious environmental pollution and human health concerns [3]. The uranium can be enriched in human body through the food chain, which may bring about serious childhood leukemia, lung cancer and other radiation-related diseases [4,5]. Thus, the U.S. Environmental Protection Agency (EPA) has set a maximum contamination level for UO2+ in water (130 nM) [6,7]. So far, many techniques 2 have been developed for uranium detection, including inductively coupled plasma mass spectrometry [8] and atomic emission spectrometry [9] et al. However, they need expensive instruments and complicated operations. Recently, DNAzymes composited with enzyme strands (E-DNA) and substrate strands (S-DNA) are used to design bio2+ sensor for metal ions, such as UO2+ , Cu2+, Pb2+, Zn2+ and Cd2+ 2 , Mg [10–14]. Various DNAzyme based UO2+ strategies have been reported 2 including colorimetry, fluorescence and electrochemistry et al. ⁎ Corresponding authors. E-mail addresses: [email protected] (X. Wang), [email protected] (X. Ha), [email protected] (L. Yang).
https://doi.org/10.1016/j.saa.2019.118017 1386-1425/© 2019 Published by Elsevier B.V.
[15–17]. In addition, the DNAzyme based probe has been used to fluorescent image of UO2+ 2 in living cell [18]. DNA nanomachine is a kind of DNA assembled nanostructure which can achieve nanomechanical movement at nanoscopic scale [19,20]. DNA nanomachine is programmed and constructed by versatile material “DNA” which provides its some unique merits such as easy chemical synthesis, good thermal stability and functional modification [21,22]. Moreover, DNA nanomachine is a promising platform for biosensor design, drug delivery and logical molecular computation with one-, twoand three-dimensional nanostructures [19,23]. A serious of DNA nanomachines have been designed with nanoscale controllability and biocompatibility such as DNA tweezers [24], DNA walker [25], DNA motor [26], DNA gear [27] and DNA nanocages [28]. The DNA tweezers are prototypical nanomachines that enable response to different external stimuli, including nucleic acids, metal ions, protein, enzyme and pH [22,29–33]. So far, few of DNA tweezers have combined with DNAzyme for metal ions detection. Here, a one-step and amplified DNAzyme based catalytic DNA tweezer was constructed for sensitive fluorescent detection of UO2+ 2 . The DNA tweezer is formed with mutual hybridization of DNA sequences. The fluorophore and gold nanoparticles (AuNPs) are fixed at the ends of the two arms of DNA tweezer respectively. The two arms of DNA tweezer are linked closely by a single strand DNA, causing the
2
Z. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 118017
quenching of fluorescent signal. Then, the linker sequence is cleaved by UO2+ 2 -specific DNAzyme in the presence of E-DNA and uranyl, resulting the recovery of fluorescent intensity. The E-DNA can circularly cleave other DNA tweezers to significantly improve the sensitivity. The limit of detection was as low as 25 pM.
then the formed DNA tweezer and E-DNA were added and incubated at 40 °C for 60 min. Finally, the fluorescent signal of sample was detected on fluorospectrophotometer.
2. Experimental section
3.1. The principle of one-step detection of UO2+ 2
2.1. Chemicals and materials
The AuNPs was synthesized according to previous reported method by reducing the HAuCl4 with sodium citrate [34]. The thiolated DNA sequence 4 was mixed with the AuNPs with molar ratio of 1:1 for 12 h at room temperature. The AuNPs was modified at the end of sequence 4 by Au\\S bond. The concentration of the AuNPs was estimated by the absorption spectrum using the reported extinction coefficient of the respective AuNPs [35].
The rationale of DNA tweezer for detection of UO2+ 2 is illustrated in Scheme 1. The DNA tweezer structure is composited with sequence 1–4. The sequence 2 and 3 are partly complementary with the terminal regions of sequence 1, respectively. They can separately hybridize with the ends of sequence 1 to form the two arms of DNA tweezer. Then sequence 4 modified with FAM and AuNPs at each ends can separately hybridize with the single stand part of sequence 2 and 3 to form an entire DNA tweezer structure. The middle of sequence 4 links the two arms of DNA tweezers closely, causing the heavy fluorescent quenching of FAM. The link region has similar sequence with S-DNA of uranyl specific DNAzyme. It can hybridize with sequence 5 (E-DNA) to form the uranyl specific DNAzyme. The link region can be cleaved with the presence of UO2+ 2 , resulting the separation of FAM and AuNPs. Then, the E-DNA can rebind with other DNA tweezer to form another DNAzyme structure and then catalytically cleave of the linker part of DNA tweezer. Consequently, the fluorescent signal is significantly recovered. The concentration of uranyl can be quantitatively detected by the fluorescent intensity.
2.3. Formation of DNA tweezer
3.2. The stability of DNA tweezers
The DNA tweezer structure was formed by mixing 100 nM of sequence 1–4 with a ratio of 1:1 in 10 mM MES buffer solution (pH 5.5) with 300 mM NaCl. Then the mixture was heated up to 95 °C and slowly cooled down to form designed DNA tweezer structure.
The AuNPs and AuNPs modified sequence 4 were characterized by UV–Visible spectrophotometry. The AuNPs modified sequence 4 shows an extra absorption peak around 260 nm comparing with bare AuNPs, indicating DNA sequences have been successfully modified by Au\\S bond (Fig. S1A). The stability of the formed DNA tweezers has been investigated for 4 h. As shown in Fig. S1B, the fluorescence intensity remains almost unchanged; representing the structure of DNA tweezers is stable in 4 h.
The sequences of all oligonucleotides were listed in Table S1. They were all synthesized and purified by Sangon Biotech Co., Ltd. (China). UO2 (NO3)2·6H2O was brought from the China National Nuclear Corporation (Lanzhou, China). HAuCl4 and sodium citrate were provided by Sinopharm Chemical Reagent Co., Ltd. Ultrapurewater with resistivity of 18.2 MΩ cm was used throughout this experiment. 2.2. Synthesis and modification of AuNPs
2.4. One-step detection of UO2+ 2 30 nM of sequence 5 and UO2+ 2 sample were mixed with DNA tweezer in 10 mM MES buffer solution (pH 5.5) with 300 mM NaCl. Then the solution was incubated at 40 °C for 60 min. Finally, the mixed sample was recorded on fluorescence spectrometer from 500 nm to 600 nm with excitation at 492 nm. 2.5. Detection of UO2+ 2 in water samples All the water samples were centrifuged and filtered with 0.22 μm membrane. After that, the pH of water sample was adjusted to 5.5 and
3. Results and discussion
3.3. The demonstration of the feasibility of this strategy The feasibility of the DNA tweezer strategy was demonstrated by samples with different conditions. The results were shown in Fig. 1. The DNA tweezer is still “close” status in the absence of UO2+ 2 . Thus, the weak fluorescent signal is obtained for blank sample without UO22 + (sample 1). Sample 2 without sequence 5 (E-DNA) has a similar fluorescent intensity with sample 1, indicating the DNAzyme cannot be
Scheme 1. Schematic illustration of DNA tweezer probe for UO2+ 2 detection based on DNAzyme catalytic cleavage
Z. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 118017
3
is significantly recovered. This is because the cleavage reaction has proceeded to a certain extent with half reaction time, causing the opening of partial tweezers. The molar ratio of sequence 5 (E-DNA) to DNA tweezer is turned to 2:10, causing a reduction in fluorescent intensity. This can be explained as the incompletion of the DNAzyme cleavage reaction with less amount of E-DNA. The sample 6 in normal conditions shows strong fluorescent signal, indicating that the DNAzyme cleavage reaction and the structure change of DNA tweezer all carry on as expected. 3.4. Optimization of experiment conditions
Fig. 1. Fluorescent intensity of samples with different conditions: (1) DNA tweezer + E2+ DNA (without UO2+ 2 ), (2) DNA tweezer + UO2 (without E-DNA), (3) DNA tweezer + 2+ E'-DNA + UO2+ 2 , (4) DNA tweezer + E-DNA + UO2 with 30 min incubation time (half incubation time), (5) DNA tweezer + E-DNA + UO2+ 2 (Molar ratio of E-DNA to DNA tweezer is 2:10), (6) DNA tweezer + E-DNA + UO2+ 2 .
formed without E-DNA and the linker of sequence of 4 is still intact. The E-DNA has been replaced by E'-DNA (E-DNA of Pb2+-specific DNAzyme) in sample 3. The low fluorescent intensity of sample 3 is attributed to the inhibition of the forming of UO2+ 2 -specific DNAzyme with E'-DNA. The fluorescent intensity of sample 4 (half reaction time)
To achieve the best sensing sensitivity, some crucial parameters were optimized: (1) pH of buffer solution; (2) reaction time of DNAzyme cleavage reaction; (3) reaction temperature of DNAzyme cleavage reaction; (4) molar ratio of sequence 5 (E-DNA) to DNA tweezer. It is reported that the pH of buffer solution has significant influence to the activity of DNAzyme. As shown in Fig. 2A, the fluorescent signal elevates with the pH of reaction buffer and descends after pH of 5.5. This indicates the activity of UO2+ 2 -specific DNAzyme is higher in weak acidity condition. This can be attributed to the hydrolyzation of UO2+ and then the forming of UO2(OH)+ in weak acidity condition 2 [6]. Thus, buffer solution of pH 5.5 was utilized for DNAzyme cleavage reaction. The time of DNAzyme cleavage reaction has influence to the extent of cleavage reaction and finally the performance of DNA tweezer. Thus, the reaction time was optimized here. Fig. 2B shows the fluorescent signal increases sharply with the reaction time and levels off around 60 min. Moreover, the reaction time is shorter at high concentration of UO2+ and longer at low concentration of UO2+ 2 2 , indicating
Fig. 2. The influence of some crucial parameters to the performance of DNA tweezer: (A) pH of buffer solution; (B) reaction time of DNAzyme cleavage reaction; (C) reaction temperature of DNAzyme; (D) molar ratio of sequence 5 (E-DNA) to DNA tweezer.
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Fig. 3. (A) The fluorescent spectra of the DNA tweezer toward different concentrations of UO2+ 2 : 0.1 nM, 5 nM, 10 nM, 30 nM, 60 nM, 100 nM, 150 nM, 200 nM. (B) The relationship 2+ between fluorescent intensity and UO2+ 2 concentration. Inset: the calibration plot between fluorescent intensity and UO2 from 0.1 nM to 60 nM.
the concentration of UO2+ 2 can accelerate the rate of DNAzyme cleavage reaction. Therefore, the optimum reaction time was 60 min. The reaction temperature affects the efficiency of DNAzyme cleavage reaction. The fluorescent signal rises with the reaction temperature, indicating the positive relationship between DNAzyme's activity and reaction temperature (Fig. 2C). However, the fluorescent intensity decreases at 50 °C. This may be attributed to the weak thermal stability of DNAzyme at 50 °C, causing the low efficiency of DNAzyme and weak fluorescent intensity. Therefore, 40 °C was used for DNAzyme cleavage reaction. The molar ratio of sequence 5 (E-DNA) to DNA tweezer is related to the DNAzyme catalytic cleavage reaction. The molar ratios were changed from 0:10 to 5:10. The fluorescent intensity rises with the molar ratio and reaches a platform after 3:10 (Fig. 2D). The more amount of E-DNA can accelerate the DNAzyme cleavage reaction, resulting to the high fluorescent intensity. And the E-DNA is basically saturated when the molar ration is over 3:10. Thus, the molar ratio of sequence 5 to DNA tweezer (3:10) was chosen for this experiment.
3.6. Detection UO2+ 2 in real water samples To demonstrate the applicability of the proposed UO2+ 2 specific DNA tweezer for practical application, different water samples were determined by this method. The uranyl concentrations of water samples determined by this method are 2.9 nM for tap water and 4.7 nM for river water. Recoveries determined by spiked samples are between 91.0% and 107.0%. In addition, the RSDs are from 5.6% to 9.2%. These results reveal that this DNA tweezer is feasible and applicable in real water analysis (Table 1).
4. Conclusion
3.5. Analytical performance of the DNA tweezer probes
In summary, a simple and sensitive DNAzyme based catalytic DNA tweezer was constructed. The tweezer can be tuned from “close” to “open” in the presence of UO2+ 2 . Importantly, only one operation step was required for the whole detection procedure. The LOD is low to 25 pM with amplification of DNAzyme catalytic cleavage reaction. In addition, it shows great sensitivity, specificity and practical application ability for uranyl detection in water samples.
To determine the fluorescent response of UO2+ 2 , different concentrations of UO2+ were tested with the formed DNA tweezers probe. As 2 shown in Fig. 3A, the fluorescent signals gradually rise with UO2+ in 2 the range of 0.1 nM to 200 nM. A good linear relationship with a coefficient of correlation of 0.993 is obtained in the range of 0.1 nM to 60 nM between fluorescent intensity and uranyl concentration (Fig. 3B). Based on 3σ blank criterion, the limit of detection of this sensing DNA tweezer was evaluated to be 25 pM. Such limit of detection was comparable with other reported DNAzyme based methods including fluorescence, colorimetry and electrochemistry (Table S2). The RSD of six repetitive measurements of 0.1 nM of UO2+ was 8.8%, indicating a satisfactory 2 reproducibility of this DNA tweezer probes. The selectivity of the DNA tweezer was investigated by some other bivalent metal, such as Ca2+, Mg2+, Pb2+, Sn2+, Hg2+, Zn2+, Cu2+ and Co2+. The result shows the fluorescent intensity increases obviously for the target UO2+ (60 nM), comparing with blank samples (Ultra2 pure water). However, other metal ions exhibit negligible fluorescent signal changes; even the concentration is 10 times of uranyl (600 nM). The same result is also obtained at 100 time higher concentration of interference metal ions (data not shown). These results demonstrate the excellent specificity of this DNA tweezer toward UO2+ 2 , due to the high binding constant between UO2+ 2 and DNAzyme [36] (Fig. 4).
Fig. 4. The fluorescent responses of the DNA tweezer to UO2+ 2 and other interfering metal ions.
Z. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 118017 Table 1 Determination of UO2+ 2 in different samples by proposed strategy. Spiked nM
0 5.0 10.0
Tap water
River water
Found nM
Recovery %
RSD % (N = 6)
Found nM
Recovery %
RSD % (N = 6)
2.9 8.1 13.6
– 104.0 107.0
8.8 5.6 8.1
4.7 9.9 13.8
– 104.0 91.0
9.2 8.4 6.3
Author contribution Conceived and designed the experiments: Zhengwei Xiong, Qiang Wang, Wen Yun, Xingmin Wang. Performed the experiments: Zhengwei Xiong, Qiang Wang, Wen Yun, Lizhu Yang. Analyzed the data: Zhengwei Xiong, Qiang Wang, Jiafeng Zhang, Lizhu Yang. Contributed reagents/materials/analysis tools: Wen Yun, Xingmin Wang, Xia Ha, Lizhu Yang. Wrote the paper: Zhengwei Xiong, Wen Yun, Xia Ha, Lizhu Yang. Declaration of competing interest The authors declared that they have no conflicts of interest to this work. Acknowledgments This work is sponsored by the Program of Innovation Center of Lipid Resources and Children's Daily Chemicals at Chongqing University of Education (Grant No. 2017XJPT01), the China Postdoctoral Science Foundation (Grant No. 2019M653475), National Natural Science Foundation of China (Grant No. 31300819), the Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN201900842), the Science and Technology Innovation Project of Chongqing Science and Technology Commission (Grant No. cstc2017shms-zdyfX0063 and cstc2019jscx-msxmX0334). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.118017. References [1] P. Zweifel, A. Praktiknjo, G. Erdmann, Uranium and nuclear energy, Energy Economics, Springer 2017, pp. 247–267. [2] A. Brown, A. Glaser, On the origins and significance of the limit demarcating lowenriched uranium from highly enriched uranium, Sci. Glob. Secur. 24 (2016) 131–137. [3] M. Bergmann, O. Sobral, J. Pratas, M.A. Graça, Uranium toxicity to aquatic invertebrates: a laboratory assay, Environ. Pollut. 239 (2018) 359–366. [4] M. Kreuzer, N. Fenske, M. Schnelzer, L. Walsh, Lung cancer risk at low radon exposure rates in German uranium miners, Br. J. Cancer 113 (2015) 1367. [5] M. Kreuzer, C. Sobotzki, M. Schnelzer, N. Fenske, Factors modifying the radonrelated lung cancer risk at low exposures and exposure rates among German uranium miners, Radiat. Res. 189 (2017) 165–176. [6] W. Yun, H. Wu, Z. Yang, R. Wang, C. Wang, L. Yang, Y. Tang, A dynamic, ultrasensitive and “turn-on” strategy for fluorescent detection of uranyl based on DNAzyme and entropy-driven amplification initiated circular cleavage amplification, Anal. Chim. Acta 1068 (2019) 104–110. [7] W. Yun, H. Wu, X. Liu, H. Zhong, M. Fu, L. Yang, Y. Huang, Ultra-sensitive fluorescent and colorimetric detection of UO22+ based on dual enzyme-free amplification strategies, Sensors Actuators B Chem. 255 (2018) 1920–1926. [8] J.R. Wood, G.A. Gill, L.-J. Kuo, J.E. Strivens, K.-Y. Choe, Comparison of analytical methods for the determination of uranium in seawater using inductively coupled plasma mass spectrometry, Ind. Eng. Chem. Res. 55 (2016) 4344–4350.
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Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
A simple and programmed DNA tweezer probes for one-step and amplified detection of UO2+ 2 Zhengwei Xiong a,d, Qiang Wang a, Jiafeng Zhang b, Wen Yun c, Xingmin Wang c,⁎, Xia Ha c,⁎, Lizhu Yang b,⁎ a
School of Biological and Chemical Engineering, Innovation Center of Lipid Resources and Children's Daily Chemicals, Chongqing University of Education, Chongqing 400067, China School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang 325035, China Chongqing Key Laboratory of Catalysis and New Environmental Materials, College of Environment and Resources, Chongqing Technology and Business University, Chongqing 400067, China d Department of Food Biotechnology, Graduate School, Woosuk University, Samnye-eup, Wanju-gun, Jeonbuk Province 55338, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 14 October 2019 Received in revised form 17 December 2019 Accepted 27 December 2019 Available online 30 December 2019 Keywords: Fluorescence DNAzyme Gold nanoparticles DNA tweezer One-step
a b s t r a c t A simple DNA tweezer was proposed for one-step and amplified detection of UO2+ 2 based on DNAzyme catalytic cleavage. The two arms of DNA tweezers are close in the original form. Thus, the fluorescent signal of fluorophore at the end of arm is dramatically quenched. However, the structure of DNA tweezers can be changed from “close” to “open” in the presence of UO2+ 2 , resulting the strong fluorescent signal. The linear range was obtained in the range of 0.1 nM to 60 nM and the limit of detection was 25 pM with the amplification of DNAzyme catalytic cleavage reaction. Importantly, the whole detection process is very simple and only one operation step is required. In addition, it shows great potential and promising prospects for uranyl detection in practical application. © 2019 Published by Elsevier B.V.
1. Introduction Enriched uranium can be used as fuel for nuclear energy and material for nuclear weapons [1,2]. Worldwide consumption of uranium may cause its release into environment by uranium mining and nuclear waste, resulting serious environmental pollution and human health concerns [3]. The uranium can be enriched in human body through the food chain, which may bring about serious childhood leukemia, lung cancer and other radiation-related diseases [4,5]. Thus, the U.S. Environmental Protection Agency (EPA) has set a maximum contamination level for UO2+ in water (130 nM) [6,7]. So far, many techniques 2 have been developed for uranium detection, including inductively coupled plasma mass spectrometry [8] and atomic emission spectrometry [9] et al. However, they need expensive instruments and complicated operations. Recently, DNAzymes composited with enzyme strands (E-DNA) and substrate strands (S-DNA) are used to design bio2+ sensor for metal ions, such as UO2+ , Cu2+, Pb2+, Zn2+ and Cd2+ 2 , Mg [10–14]. Various DNAzyme based UO2+ strategies have been reported 2 including colorimetry, fluorescence and electrochemistry et al. ⁎ Corresponding authors. E-mail addresses: [email protected] (X. Wang), [email protected] (X. Ha), [email protected] (L. Yang).
https://doi.org/10.1016/j.saa.2019.118017 1386-1425/© 2019 Published by Elsevier B.V.
[15–17]. In addition, the DNAzyme based probe has been used to fluorescent image of UO2+ 2 in living cell [18]. DNA nanomachine is a kind of DNA assembled nanostructure which can achieve nanomechanical movement at nanoscopic scale [19,20]. DNA nanomachine is programmed and constructed by versatile material “DNA” which provides its some unique merits such as easy chemical synthesis, good thermal stability and functional modification [21,22]. Moreover, DNA nanomachine is a promising platform for biosensor design, drug delivery and logical molecular computation with one-, twoand three-dimensional nanostructures [19,23]. A serious of DNA nanomachines have been designed with nanoscale controllability and biocompatibility such as DNA tweezers [24], DNA walker [25], DNA motor [26], DNA gear [27] and DNA nanocages [28]. The DNA tweezers are prototypical nanomachines that enable response to different external stimuli, including nucleic acids, metal ions, protein, enzyme and pH [22,29–33]. So far, few of DNA tweezers have combined with DNAzyme for metal ions detection. Here, a one-step and amplified DNAzyme based catalytic DNA tweezer was constructed for sensitive fluorescent detection of UO2+ 2 . The DNA tweezer is formed with mutual hybridization of DNA sequences. The fluorophore and gold nanoparticles (AuNPs) are fixed at the ends of the two arms of DNA tweezer respectively. The two arms of DNA tweezer are linked closely by a single strand DNA, causing the
2
Z. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 118017
quenching of fluorescent signal. Then, the linker sequence is cleaved by UO2+ 2 -specific DNAzyme in the presence of E-DNA and uranyl, resulting the recovery of fluorescent intensity. The E-DNA can circularly cleave other DNA tweezers to significantly improve the sensitivity. The limit of detection was as low as 25 pM.
then the formed DNA tweezer and E-DNA were added and incubated at 40 °C for 60 min. Finally, the fluorescent signal of sample was detected on fluorospectrophotometer.
2. Experimental section
3.1. The principle of one-step detection of UO2+ 2
2.1. Chemicals and materials
The AuNPs was synthesized according to previous reported method by reducing the HAuCl4 with sodium citrate [34]. The thiolated DNA sequence 4 was mixed with the AuNPs with molar ratio of 1:1 for 12 h at room temperature. The AuNPs was modified at the end of sequence 4 by Au\\S bond. The concentration of the AuNPs was estimated by the absorption spectrum using the reported extinction coefficient of the respective AuNPs [35].
The rationale of DNA tweezer for detection of UO2+ 2 is illustrated in Scheme 1. The DNA tweezer structure is composited with sequence 1–4. The sequence 2 and 3 are partly complementary with the terminal regions of sequence 1, respectively. They can separately hybridize with the ends of sequence 1 to form the two arms of DNA tweezer. Then sequence 4 modified with FAM and AuNPs at each ends can separately hybridize with the single stand part of sequence 2 and 3 to form an entire DNA tweezer structure. The middle of sequence 4 links the two arms of DNA tweezers closely, causing the heavy fluorescent quenching of FAM. The link region has similar sequence with S-DNA of uranyl specific DNAzyme. It can hybridize with sequence 5 (E-DNA) to form the uranyl specific DNAzyme. The link region can be cleaved with the presence of UO2+ 2 , resulting the separation of FAM and AuNPs. Then, the E-DNA can rebind with other DNA tweezer to form another DNAzyme structure and then catalytically cleave of the linker part of DNA tweezer. Consequently, the fluorescent signal is significantly recovered. The concentration of uranyl can be quantitatively detected by the fluorescent intensity.
2.3. Formation of DNA tweezer
3.2. The stability of DNA tweezers
The DNA tweezer structure was formed by mixing 100 nM of sequence 1–4 with a ratio of 1:1 in 10 mM MES buffer solution (pH 5.5) with 300 mM NaCl. Then the mixture was heated up to 95 °C and slowly cooled down to form designed DNA tweezer structure.
The AuNPs and AuNPs modified sequence 4 were characterized by UV–Visible spectrophotometry. The AuNPs modified sequence 4 shows an extra absorption peak around 260 nm comparing with bare AuNPs, indicating DNA sequences have been successfully modified by Au\\S bond (Fig. S1A). The stability of the formed DNA tweezers has been investigated for 4 h. As shown in Fig. S1B, the fluorescence intensity remains almost unchanged; representing the structure of DNA tweezers is stable in 4 h.
The sequences of all oligonucleotides were listed in Table S1. They were all synthesized and purified by Sangon Biotech Co., Ltd. (China). UO2 (NO3)2·6H2O was brought from the China National Nuclear Corporation (Lanzhou, China). HAuCl4 and sodium citrate were provided by Sinopharm Chemical Reagent Co., Ltd. Ultrapurewater with resistivity of 18.2 MΩ cm was used throughout this experiment. 2.2. Synthesis and modification of AuNPs
2.4. One-step detection of UO2+ 2 30 nM of sequence 5 and UO2+ 2 sample were mixed with DNA tweezer in 10 mM MES buffer solution (pH 5.5) with 300 mM NaCl. Then the solution was incubated at 40 °C for 60 min. Finally, the mixed sample was recorded on fluorescence spectrometer from 500 nm to 600 nm with excitation at 492 nm. 2.5. Detection of UO2+ 2 in water samples All the water samples were centrifuged and filtered with 0.22 μm membrane. After that, the pH of water sample was adjusted to 5.5 and
3. Results and discussion
3.3. The demonstration of the feasibility of this strategy The feasibility of the DNA tweezer strategy was demonstrated by samples with different conditions. The results were shown in Fig. 1. The DNA tweezer is still “close” status in the absence of UO2+ 2 . Thus, the weak fluorescent signal is obtained for blank sample without UO22 + (sample 1). Sample 2 without sequence 5 (E-DNA) has a similar fluorescent intensity with sample 1, indicating the DNAzyme cannot be
Scheme 1. Schematic illustration of DNA tweezer probe for UO2+ 2 detection based on DNAzyme catalytic cleavage
Z. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 118017
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is significantly recovered. This is because the cleavage reaction has proceeded to a certain extent with half reaction time, causing the opening of partial tweezers. The molar ratio of sequence 5 (E-DNA) to DNA tweezer is turned to 2:10, causing a reduction in fluorescent intensity. This can be explained as the incompletion of the DNAzyme cleavage reaction with less amount of E-DNA. The sample 6 in normal conditions shows strong fluorescent signal, indicating that the DNAzyme cleavage reaction and the structure change of DNA tweezer all carry on as expected. 3.4. Optimization of experiment conditions
Fig. 1. Fluorescent intensity of samples with different conditions: (1) DNA tweezer + E2+ DNA (without UO2+ 2 ), (2) DNA tweezer + UO2 (without E-DNA), (3) DNA tweezer + 2+ E'-DNA + UO2+ 2 , (4) DNA tweezer + E-DNA + UO2 with 30 min incubation time (half incubation time), (5) DNA tweezer + E-DNA + UO2+ 2 (Molar ratio of E-DNA to DNA tweezer is 2:10), (6) DNA tweezer + E-DNA + UO2+ 2 .
formed without E-DNA and the linker of sequence of 4 is still intact. The E-DNA has been replaced by E'-DNA (E-DNA of Pb2+-specific DNAzyme) in sample 3. The low fluorescent intensity of sample 3 is attributed to the inhibition of the forming of UO2+ 2 -specific DNAzyme with E'-DNA. The fluorescent intensity of sample 4 (half reaction time)
To achieve the best sensing sensitivity, some crucial parameters were optimized: (1) pH of buffer solution; (2) reaction time of DNAzyme cleavage reaction; (3) reaction temperature of DNAzyme cleavage reaction; (4) molar ratio of sequence 5 (E-DNA) to DNA tweezer. It is reported that the pH of buffer solution has significant influence to the activity of DNAzyme. As shown in Fig. 2A, the fluorescent signal elevates with the pH of reaction buffer and descends after pH of 5.5. This indicates the activity of UO2+ 2 -specific DNAzyme is higher in weak acidity condition. This can be attributed to the hydrolyzation of UO2+ and then the forming of UO2(OH)+ in weak acidity condition 2 [6]. Thus, buffer solution of pH 5.5 was utilized for DNAzyme cleavage reaction. The time of DNAzyme cleavage reaction has influence to the extent of cleavage reaction and finally the performance of DNA tweezer. Thus, the reaction time was optimized here. Fig. 2B shows the fluorescent signal increases sharply with the reaction time and levels off around 60 min. Moreover, the reaction time is shorter at high concentration of UO2+ and longer at low concentration of UO2+ 2 2 , indicating
Fig. 2. The influence of some crucial parameters to the performance of DNA tweezer: (A) pH of buffer solution; (B) reaction time of DNAzyme cleavage reaction; (C) reaction temperature of DNAzyme; (D) molar ratio of sequence 5 (E-DNA) to DNA tweezer.
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Z. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 118017
Fig. 3. (A) The fluorescent spectra of the DNA tweezer toward different concentrations of UO2+ 2 : 0.1 nM, 5 nM, 10 nM, 30 nM, 60 nM, 100 nM, 150 nM, 200 nM. (B) The relationship 2+ between fluorescent intensity and UO2+ 2 concentration. Inset: the calibration plot between fluorescent intensity and UO2 from 0.1 nM to 60 nM.
the concentration of UO2+ 2 can accelerate the rate of DNAzyme cleavage reaction. Therefore, the optimum reaction time was 60 min. The reaction temperature affects the efficiency of DNAzyme cleavage reaction. The fluorescent signal rises with the reaction temperature, indicating the positive relationship between DNAzyme's activity and reaction temperature (Fig. 2C). However, the fluorescent intensity decreases at 50 °C. This may be attributed to the weak thermal stability of DNAzyme at 50 °C, causing the low efficiency of DNAzyme and weak fluorescent intensity. Therefore, 40 °C was used for DNAzyme cleavage reaction. The molar ratio of sequence 5 (E-DNA) to DNA tweezer is related to the DNAzyme catalytic cleavage reaction. The molar ratios were changed from 0:10 to 5:10. The fluorescent intensity rises with the molar ratio and reaches a platform after 3:10 (Fig. 2D). The more amount of E-DNA can accelerate the DNAzyme cleavage reaction, resulting to the high fluorescent intensity. And the E-DNA is basically saturated when the molar ration is over 3:10. Thus, the molar ratio of sequence 5 to DNA tweezer (3:10) was chosen for this experiment.
3.6. Detection UO2+ 2 in real water samples To demonstrate the applicability of the proposed UO2+ 2 specific DNA tweezer for practical application, different water samples were determined by this method. The uranyl concentrations of water samples determined by this method are 2.9 nM for tap water and 4.7 nM for river water. Recoveries determined by spiked samples are between 91.0% and 107.0%. In addition, the RSDs are from 5.6% to 9.2%. These results reveal that this DNA tweezer is feasible and applicable in real water analysis (Table 1).
4. Conclusion
3.5. Analytical performance of the DNA tweezer probes
In summary, a simple and sensitive DNAzyme based catalytic DNA tweezer was constructed. The tweezer can be tuned from “close” to “open” in the presence of UO2+ 2 . Importantly, only one operation step was required for the whole detection procedure. The LOD is low to 25 pM with amplification of DNAzyme catalytic cleavage reaction. In addition, it shows great sensitivity, specificity and practical application ability for uranyl detection in water samples.
To determine the fluorescent response of UO2+ 2 , different concentrations of UO2+ were tested with the formed DNA tweezers probe. As 2 shown in Fig. 3A, the fluorescent signals gradually rise with UO2+ in 2 the range of 0.1 nM to 200 nM. A good linear relationship with a coefficient of correlation of 0.993 is obtained in the range of 0.1 nM to 60 nM between fluorescent intensity and uranyl concentration (Fig. 3B). Based on 3σ blank criterion, the limit of detection of this sensing DNA tweezer was evaluated to be 25 pM. Such limit of detection was comparable with other reported DNAzyme based methods including fluorescence, colorimetry and electrochemistry (Table S2). The RSD of six repetitive measurements of 0.1 nM of UO2+ was 8.8%, indicating a satisfactory 2 reproducibility of this DNA tweezer probes. The selectivity of the DNA tweezer was investigated by some other bivalent metal, such as Ca2+, Mg2+, Pb2+, Sn2+, Hg2+, Zn2+, Cu2+ and Co2+. The result shows the fluorescent intensity increases obviously for the target UO2+ (60 nM), comparing with blank samples (Ultra2 pure water). However, other metal ions exhibit negligible fluorescent signal changes; even the concentration is 10 times of uranyl (600 nM). The same result is also obtained at 100 time higher concentration of interference metal ions (data not shown). These results demonstrate the excellent specificity of this DNA tweezer toward UO2+ 2 , due to the high binding constant between UO2+ 2 and DNAzyme [36] (Fig. 4).
Fig. 4. The fluorescent responses of the DNA tweezer to UO2+ 2 and other interfering metal ions.
Z. Xiong et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 229 (2020) 118017 Table 1 Determination of UO2+ 2 in different samples by proposed strategy. Spiked nM
0 5.0 10.0
Tap water
River water
Found nM
Recovery %
RSD % (N = 6)
Found nM
Recovery %
RSD % (N = 6)
2.9 8.1 13.6
– 104.0 107.0
8.8 5.6 8.1
4.7 9.9 13.8
– 104.0 91.0
9.2 8.4 6.3
Author contribution Conceived and designed the experiments: Zhengwei Xiong, Qiang Wang, Wen Yun, Xingmin Wang. Performed the experiments: Zhengwei Xiong, Qiang Wang, Wen Yun, Lizhu Yang. Analyzed the data: Zhengwei Xiong, Qiang Wang, Jiafeng Zhang, Lizhu Yang. Contributed reagents/materials/analysis tools: Wen Yun, Xingmin Wang, Xia Ha, Lizhu Yang. Wrote the paper: Zhengwei Xiong, Wen Yun, Xia Ha, Lizhu Yang. Declaration of competing interest The authors declared that they have no conflicts of interest to this work. Acknowledgments This work is sponsored by the Program of Innovation Center of Lipid Resources and Children's Daily Chemicals at Chongqing University of Education (Grant No. 2017XJPT01), the China Postdoctoral Science Foundation (Grant No. 2019M653475), National Natural Science Foundation of China (Grant No. 31300819), the Science and Technology Research Program of Chongqing Municipal Education Commission (Grant No. KJQN201900842), the Science and Technology Innovation Project of Chongqing Science and Technology Commission (Grant No. cstc2017shms-zdyfX0063 and cstc2019jscx-msxmX0334). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.saa.2019.118017. References [1] P. Zweifel, A. Praktiknjo, G. Erdmann, Uranium and nuclear energy, Energy Economics, Springer 2017, pp. 247–267. [2] A. Brown, A. Glaser, On the origins and significance of the limit demarcating lowenriched uranium from highly enriched uranium, Sci. Glob. Secur. 24 (2016) 131–137. [3] M. Bergmann, O. Sobral, J. Pratas, M.A. Graça, Uranium toxicity to aquatic invertebrates: a laboratory assay, Environ. Pollut. 239 (2018) 359–366. [4] M. Kreuzer, N. Fenske, M. Schnelzer, L. Walsh, Lung cancer risk at low radon exposure rates in German uranium miners, Br. J. Cancer 113 (2015) 1367. [5] M. Kreuzer, C. Sobotzki, M. Schnelzer, N. Fenske, Factors modifying the radonrelated lung cancer risk at low exposures and exposure rates among German uranium miners, Radiat. Res. 189 (2017) 165–176. [6] W. Yun, H. Wu, Z. Yang, R. Wang, C. Wang, L. Yang, Y. Tang, A dynamic, ultrasensitive and “turn-on” strategy for fluorescent detection of uranyl based on DNAzyme and entropy-driven amplification initiated circular cleavage amplification, Anal. Chim. Acta 1068 (2019) 104–110. [7] W. Yun, H. Wu, X. Liu, H. Zhong, M. Fu, L. Yang, Y. Huang, Ultra-sensitive fluorescent and colorimetric detection of UO22+ based on dual enzyme-free amplification strategies, Sensors Actuators B Chem. 255 (2018) 1920–1926. [8] J.R. Wood, G.A. Gill, L.-J. Kuo, J.E. Strivens, K.-Y. Choe, Comparison of analytical methods for the determination of uranium in seawater using inductively coupled plasma mass spectrometry, Ind. Eng. Chem. Res. 55 (2016) 4344–4350.
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