Biosensors and Bioelectronics 53 (2014) 245–249
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Detection of lead(II) ions with a DNAzyme and isothermal strand displacement signal ampliﬁcation Wenying Li a,b, Yue Yang a,b,c, Jian Chen a, Qingfeng Zhang a,b, Yan Wang a,b, Fangyuan Wang a,b, Cong Yu a,b,n a
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China University of the Chinese Academy of Sciences, Beijing 100049, PR China c College of Biological and Agricultural Engineering, Jilin University, Changchun 130022, PR China b
art ic l e i nf o
a b s t r a c t
Article history: Received 24 August 2013 Received in revised form 23 September 2013 Accepted 24 September 2013 Available online 8 October 2013
A DNAzyme based method for the sensitive and selective quantiﬁcation of lead(II) ions has been developed. A DNAzyme that requires Pb2 þ for activation was selected. An RNA containing DNA substrate was cleaved by the DNAzyme in the presence of Pb2 þ . The 2′,3′-cyclic phosphate of the cleaved 5′-part of the substrate was efﬁciently removed by Exonuclease III. The remaining part of the single stranded DNA (9 or 13 base long) was subsequently used as the primer for the strand displacement ampliﬁcation reaction (SDAR). The method is highly sensitive, 200 pM lead(II) could be easily detected. A number of interference ions were tested, and the sensor showed good selectivity. Underground water samples were also tested, which demonstrated the feasibility of the current approach for real sample applications. It is feasible that our method could be used for DNAzyme or aptazyme based new sensing method developments for the quantiﬁcation of other target analytes with high sensitivity and selectivity. & 2013 Elsevier B.V. All rights reserved.
Keywords: DNAzyme Lead Strand displacement ampliﬁcation Fluorescence Exonuclease III
1. Introduction Lead (Pb2þ ) is a wide-spread and highly toxic contaminant in the biological environment. Accumulation of Pb2 þ in the human body has diverse detrimental effects on human health (Needleman, 1991, 2004). The detection and quantiﬁcation of Pb2 þ with high sensitivity and selectivity has attracted growing attentions over the years. Various analytical techniques for Pb2þ detection have been developed, such as atomic absorption spectrometry (AAS), atomic emission spectrometry (AES), inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS) (Wu and Boyle, 1997), and X-ray ﬂuorescence spectrometry, etc. However, these traditional analytical methods usually require sophisticated instruments, complicated operation and sample preparation/ pretreatment procedures, and are also quite expensive (Elfering et al., 1998). The development of new sensitive and selective techniques for Pb2þ quantiﬁcation is therefore of great importance. Nucleic acid enzymes include ribozymes (catalytic RNA) and deoxyribozymes (catalytic DNA, or DNAzymes) that can catalyze a variety of reactions, such as RNA or DNA cleavage, RNA or DNA
n Corresponding author at: State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China. Tel./fax: þ 86 431 85262710. E-mail addresses: [email protected]
, [email protected]
, [email protected]
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.09.055
ligation, phosphorylation, capping, aldol reaction, porphyrin metalation, etc (Liu et al., 2009; Breaker, 1997; Robertson and Ellington, 1999). A nucleic acid enzyme can have very high catalytic efﬁciency. For sensing applications, DNAzymes are more frequently used because of their easy synthesis, high stability, and cost-effectiveness. DNAzymes in many cases require a cofactor (for example, a metal ion) for activation, which forms the basis for the design of a variety of sensing methods (Elbaz et al., 2008; Liu and Lu, 2007; Xiao et al., 2007; Wang et al., 2010). Many DNAzyme based Pb2 þ detection techniques have been developed in recent years, such as the colorimetric (Mazumdar et al., 2010; Wang et al., 2008; Liu and Lu, 2004, 2005), electrochemical (Xiao et al., 2007; Yang et al., 2010; Tang et al., 2013; Shen et al., 2008), ﬂuorometric (Lan et al., 2010; Xu et al., 2013; Zhang et al., 2011; Zhang et al., 2010; Xiang et al., 2009), surface-enhanced Raman scattering (Wang and Irudayaraj, 2011), microarray (Zuo et al., 2009), femtoliterwell reactor (Wang et al., 2009), and dynamic light scattering techniques (Miao et al., 2011). And many of which utilized advanced materials such as gold nanoparticles (Mazumdar et al. 2010; Wang et al. 2008; Liu and Lu, 2005, 2004; Miao et al., 2011), graphene (Wen et al., 2011; Zhao et al., 2011), and quantum dot (Wu et al., 2010), etc. And some recently reported dynamic light scattering, rolling circle ampliﬁcation, and exonuclease aided recycling ampliﬁcation based methods provide quite good detection sensitivities (Miao et al., 2011; Tang et al., 2013; Xu et al., 2013). However, certain limitations still exist. For example, many of the reported literature methods are less sensitive, such as the gold nanoparticle aggregation based colorimetric
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assays (Liu and Lu, 2004, 2005, detection limit 400 nM, 100 nM, respectively), ﬂuorometric assays (Lan et al., 2010, detection limit 3.7 nM; Zhang et al., 2011, detection limit 10 nM; Xiang et al., 2009, detection limit 4 nM), electrochemical methods (Xiao et al., 2007, detection limit 300 nM), surface-enhanced Raman scattering based assay (Wang and Irudayaraj, 2011, detection limit 20 nM). Some methods require covalent labeling of the substrate strand and the DNAzyme, and immobilization of the DNA strand on gold electrode is time consuming. Herein we report the development of an ampliﬁed DNAzyme based method for the sensing of Pb2 þ . A lead ion dependent DNAzyme was selected (GR-5 DNAzyme) (Lan et al., 2010). Upon binding to Pb2 þ , the DNAzyme can cleave an RNA containing substrate (GR-DS, Table 1) with high efﬁciency. The 2′,3′-cyclic phosphate of the 5′-part of the cleaved substrate was removed by exonuclease III. The remaining part of the substrate was subsequently used as a primer for the strand displacement ampliﬁcation reaction (SDAR). SYBR Green I was used for the detection of the SDAR products. Real-time emission intensity changes were detected, which could be directly related to the concentration of Pb2 þ in the assay solution. The method is very sensitive, 200 pM Pb2 þ could be easily detected. And it is also very selective, and could be used for the assay of complex assay mixtures (underground water samples). It is envisioned that our method could be used for DNAzyme and aptazyme based new biosensing technology developments.
2. Materials and methods 2.1. Materials The RNA containing oligonucleotide GR-DS was obtained from TaKaRa Biotechnology Co., Ltd. (Dalian, China). All other oligonucleotides used in the current investigation were synthesized and ULTRAPAGE puriﬁed by Sangon Biotechnology Co., Ltd. (Shanghai, PR China) (Table 1). The enzymes were obtained from the New England Biolabs (Ipswich, MA, USA). Pb(OAc)2 were from Sinopharm Chemical Reagent Co., Ltd (Beijing, PR China). Ultrapure Acetic acid, NaCl and Tris were from Merck KGaA (Darmstadt, Germany). 10000 SYBR Green I was obtained from Beijing Dingguo Biotechnology Co., Ltd. (Beijing, China). All other chemicals were of analytical grade and used as received. The oligonucleotide stock solutions were stored at 4 1C before use. All stock
Table 1 Oligonucleotides used in the current investigation. Oligonucleotide Sequence (5′-3′) GR-DS GR-E Oligo(X′-Y′)
AGA AGA AGA AAG ACT CAC TAT rA GGA AGA GAT GAT GTC TG*T ACA GAC ATC ATC TCT GAA GTA GCG CCG CCG TAT AGT GAG TCG CTA TCA GTT TCT TGG AAT T
Oligo-4 Oligo-8 Oligo-12 Oligo-14
TCG CTA TCA GTT TCT TGG AAT T
2.2. Instruments UV–vis absorption spectra were obtained with a Cary 50 Bio spectrophotometer (Varian, USA). Oligonucleotides were quantiﬁed by UV–vis absorption at 260 nm. Emission spectra were obtained using a Fluoromax-4 spectroﬂuorometer (Horiba Jobin Yvon Inc., USA) with an excitation wavelength of 497 nm. Excitation and emission bandwidths were both of 3 nm for all the measurements. ICP-MS (X Series 2, Thermo Fisher Scientiﬁc Inc., Germany) was used for real sample analysis. 2.3. Assay procedures The DNAzyme (GR-E, 40 nM), substrate (GR-DS, 10 nM), and Pb2 þ ions of a speciﬁc concentration were mixed in an aqueous buffer solution (50 mM Tris–HAc 100 mM NaCl, pH 7.2) and incubated at 30 1C for 50 min (total sample volume: 11 μL). 10 U
exonuclease III (1 μL) and 25 mM MgCl2 (1 μL) were added, and the sample was incubated at 37 1C for 2 h. Exonuclease III was then
inactivated at 70 1C for 20 min (total sample volume, 13 μL). Two separated sample mixtures were prepared (sample A and sample B). Sample A contained Nt.BstNBI reaction buffer, ThermoPol buffer, ampliﬁcation templates [oligo(X′-Y′) and oligo(Y′-Y′)], dNTPs, SYBR Green I. Sample B contained the nicking endonuclease Nt.BstNBI, and Bst DNA polymerase. Sample A and sample B were freshly prepared for each set of assays. 82 μL of sample A was mixed with the above-mentioned assay
solution (13 μL). The sample solution was transferred into an
emission cell and equilibrated at 55 1C. 5 μL of Sample B (cooled on ice) was added, and the emission intensity changes at 520 nm were monitored in real time with data points taken every 5 s for 400 s. The ﬁnal assay solution (total sample volume, 100 mL) contained 80 nM oligo(X′-Y′), 20 nM oligo(Y′-Y′), dNTPs (dATP, dTTP, dCTP, dGTP) each at 250 mM, Nt.BstNBI (0.2 U mL 1), Bst DNA polymerase (0.04 U mL 1), and 0.5 SYBR Green I. 2.4. Underground water sample analysis Underground water samples were collected from Changchun, Jilin province, PR China. The concentrations of Pb2 þ in the water samples were determined by the above-mentioned assay procedures. Conditions: 40 nM DNAzyme (GR-E,), 10 nM substrate (GR-DS), incubation at 30 1C for 50 min. 10 U exonuclease III and 1.92 mM MgCl2 were added to remove the cyclic phosphate. Exonuclease III was then inactivated. The DNAzyme assay solution was used to trigger the strand displacement ampliﬁcation reaction. Pb2 þ concentrations in the underground water samples were also determined by ICP-MS to validate our assay results.
AA CTG ACT CTT ATA GTG
AGT CTT TCT TCT TCT Oligo(Y′-Y′)
and buffer solutions were prepared using water puriﬁed with a Milli-Q A10 ﬁltration system (Millipore, Billerica, MA, USA) and disinfected in a autoclave (120 1C, 15 min).
3. Results and discussion
AA CAG ACT CTT CGC TAT
CAG TTT CTT GGA ATT AGA AGA AGA AAG ACT CAC AGA AGA AGA AAG AC AGA AGA AGA A AGA AGA AG
3.1. Selection of the DNAzyme
“rA” and “*” denote ribonucleotide and phosphorothioate labels, respectively. -GACTC- is the nicking endonuclease Nt.BstNBI recognition sequence, and
the nicking site of the complementary DNA strand. Oligo-4, oligo-8, oligo-12, and oligo-14 were used as the markers.
Metal ion dependent DNAzymes have been discovered as a promising class of biomolecules for the selective detection of metal ions since the early 1990s. A variety of metal ion dependent DNAzymes have been obtained through an in vitro selection process (Liu et al., 2009; Breaker, 1997; Robertson and Ellington, 1999). Among them, a lead selective DNAzyme termed as 8-17 has often been used for the selective detection of lead ions
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(Faulhammer and Famulok, 1996; Santoro and Joyce, 1997; Peracchi, 2000; Schlosser et al., 2008). Although selective for Pb2 þ , the 8-17 DNAzyme can also be activated by a number of other metal ions at high concentrations (for example, Mg2 þ , Ca2 þ , Zn2 þ , etc.), which could be problematic since these metal ions often exist in large quantities in biological samples. Yi Lu and coworkers reported a new Pb2 þ selective DNAzyme termed as GR-5 (Table 1) (Lan et al., 2010). The GR-5 DNAzyme showed much higher metal ion selectivity than the 8-17 DNAzyme. We therefore selected the GR-5 DNAzyme instead of the 8-17 DNAzyme for the current study. 3.2. Selection of the digestion enzyme The GR-5 DNAzyme can catalyze the cleavage of an RNA base embedded DNA substrate (GR-DS, Table 1) very efﬁciently just like the 8-17 DNAzyme. However, the 5′-part of the cleaved substrate contains a 2′,3′-cyclic phosphate, which prevents its direct use as a primer for the subsequent strand displacement ampliﬁcation reaction. We tested a number of enzymes to remove the 2′, 3′-cyclic phosphate. Exonuclease I was ﬁrst tested, it is an exonuclease that can remove nucleotides from single stranded DNA in the 3′–5′ direction. We then tested phi29 DNA polymerase and DNA Polymerase I (Large Klenow Fragment), these enzymes besides polymerase activity also have 3′–5′ exonuclease activity (proof reading). The 5′-part of the cleaved DNAzyme substrate after treatment with the above mentioned enzymes could not initiate the strand displacement polymerization, which suggests that the 2′,3′-cyclic phosphate could not be removed by these enzymes. We then tested Exonuclease III, it is a commonly used enzyme for the stepwise removal of mononucleotides from the 3′-end of a duplex DNA. Only a limited number of mononucleotides are removed at each binding event, which suggests that binding between the enzyme and the duplex DNA is not very strong. Besides exonuclease activity, Exonuclease III has also been reported to have 3′-phosphatase, RNase H, and AP-endonuclease activities (Rogers and Weiss, 1980). It can also digest single stranded DNA in the 3′–5′ direction. Its enzyme activity can be very much affected by the assay conditions, such as temperature, ionic strength, and the ratio of enzyme to DNA. We found that Exonucleotide III could successfully remove the 2′,3′-cyclic phosphate, and it was used for the current investigation. 3.3. Strand displacement ampliﬁcation reaction (SDAR) Strand displacement ampliﬁcation reaction is a highly efﬁcient isothermal DNA ampliﬁcation technique. It has been used for a number of applications, such as the detection of microRNA, virus DNA, etc (Van Ness et al., 2003; Jia et al., 2010; Tan et al., 2005). A combination of a thermally stable DNA polymerase (Bst DNA polymerase) and a thermally stable nicking endonuclease (Nt. BstNBI) was used in the current investigation. The polymerase initiates DNA synthesis along a template DNA (strand extension) in the presence of a DNA primer (8–16 base long is usually sufﬁcient). The nicking endonuclease creates a nicking point on one of the DNA strands, which initiates another round of DNA synthesis. The just synthesized DNA was replaced (strand displacement), and used as a new primer to initiate another strand displacement polymerization reaction. SYBR Green I is an efﬁcient double stranded DNA speciﬁc dye and was used to monitor the SDAR reaction process. 3.4. Sensing strategy The overall sensing strategy is as follows (Scheme 1): (1) Pb2 þ selectively binds to the DNAzyme, which activates the DNAzyme
Scheme 1. Schematic illustration of the assay strategy for the selective sensing of lead(II).
and results in cleavage of the RNA embedded DNA substrate. (2) The 2′,3′-cyclic phosphate of the 5′-part of the cleaved substrate was removed by Exonuclease III. (3) The remaining 5′part of the substrate was subsequently used as a primer for the strand displacement ampliﬁcation reaction. 3.5. Assay conditions and optimization In order to prevent the unreacted substrate GR-DS from digestion by Exonuclease III, 3′-end of GR-DS was protected by a phosphorothioate label. Any other part of the substrate was not protected. It was therefore quite important to ensure that Exonuclease III did not digest the intact substrate GR-DS. Fig. 1 shows that in the presence of only the substrate strand, no digestion by Exonuclease III was observed (lines 1 and 2). However, when the DNAzyme GR-E, the substrate strand GR-DS, and Pb2 þ were mixed, a new band of shorter length appeared, which indicates that the substrate strand was efﬁciently cleaved by the DNAzyme. When the reaction product was mixed with Exonuclease III for a certain period of time (2 h), new bands of shorter length appeared (line 4). The results suggest that the 2′,3′-cyclic phosphate of the cleaved substrate was efﬁciently removed. And in addition, the DNA strand was further digested by Exonuclease III. The results suggest that oligonucleotids of 9 and 13 base long were the major remaining products after the Exonuclease III digestion. Prolonged incubation with Exonuclease III did not reduce the length of the digested oligonucleotides. It seems that since Exonuclease III is mainly used for binding duplex DNA, its binding to single stranded DNA is not very strong. When the oligonucleotide was reduced to a certain length, Exonuclease III was unable to bind, and therefore the oligonucleotide could not be further digested. Oligonucleotids of 9 and 13 base long were well enough to be used as the primer for the subsequent strand displacement ampliﬁcation reaction. It is worth noting that the 3′-part of the cleaved substrate did not appear on the gel (not digested by Exonuclease III since the 3′-end was labeled with a phosphorothioate). This is because that at a concentration below 500 nM, it could not be stained by silver staining (Fig. S1, supplementary material). The assay conditions were optimized. It was found that when 100 nM template [80 nM oligo(X′-Y′)þ20 nM oligo(Y′-Y′)], 0.2 U mL 1 Nt.BstNBI, and 0.04 U mL 1 Bst DNA polymerase were used, best SDAR emission enhancement (with 500 pM Pb2þ added) against the background was observed (Fig. S2, supplementary material). Fig. 2(a) shows the real-time SYBR Green I emission intensity changes at 520 nm. The results clearly show that with the addition of increasing concentrations of Pb2 þ , emission started to increase
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Fig. 1. Polyacrylamide (20%) gel electrophoresis analysis of the DNA digestion by Exonuclease III and the removal of the 2′,3′-cyclic phosphate. Line 1: 300 nM GR-DS (the substrate); line 2: 300 nM GR-DS þ10 U exonuclease III; line 3: 500 nM GRDS þ 1.5 mM GR-E (the DNAzyme) þ 500 nM Pb2 þ ; line 4: 500 nM GR-DSþ 1.5 mM GR-Eþ 500 nM Pb2 þ þ 10 U exonuclease III; line 5: the markers, 200 nM oligo-4 (18 base long) þ 400 nM oligo-8 (14 base long)þ 800 nM oligo-12 (10 base long) þ 1 mM oligo-14 (8 base long); line 6: 4 mM GR-E. Gel conditions: 80 V, 30 min. Relatively large quantities of DNA were used for better staining (silver staining).
at a shorter reaction time. A saturation point was reached at approximately 20 nM Pb2 þ concentration. Further increase of the Pb2 þ concentration caused little changes of the emission curve. The POI value (the SDAR reaction time at the maximum slope of the emission curve) was plotted against the Pb2 þ concentration, and a linear relationship was obtained at a Pb2 þ concentration range of 200 pM–20 nM. [Fig. 2(b)]. The linear correlation equation is POI ¼198.18 68.71 log C (correlation coefﬁcient R2 ¼0.99), where “C” was the concentration of Pb2 þ in nM. The assay is highly sensitive, our results show that 200 pM Pb2 þ could be easily detected.
Fig. 2. (a) Real-time emission intensity changes of SYBR Green I at 520 nm. The SDAR reactions were triggered by the addition of different concentrations of Pb2 þ . (b) The POI value was plotted against the Pb2 þ concentration.
3.6. Selectivity of the assay Our method shows good selectivity. A number of potential interference metal ions were tested, which included Hg2 þ , Cu2 þ , Zn2 þ , Ca2 þ , Fe2 þ , Cd2 þ . Fig. 3 shows that 5 nM Pb2 þ gave a very signiﬁcant change in POI value, and the other ions did not provide much interference. A mixture of these possible interference ions were also tested, and not much interference was observed. The results clearly show that the other ions do not have the capability to activate the DNAzyme, which is a result of the stringent in vitro DNAzyme selection process (Faulhammer and Famulok, 1996; Santoro and Joyce, 1997; Peracchi, 2000; Schlosser et al., 2008). 3.7. Real sample analysis Our assay method was also tested for the detection of Pb2 þ in real samples (Fig. S3). Underground water samples were taken from Changchun, Jilin Province. The samples were centrifuged brieﬂy to get rid of the insoluble materials, and the supernatant was used for the quantitative analysis. The underground water samples were diluted in the ﬁnal assay mixtures, and the obtained mean Pb2 þ content was about 1.017 0.11 nM (average value of three independent measurements). The calculated average Pb2 þ concentration in the underground water sample was about 11.11 71.21 nM (2.30 70.25 ppb), which is well below the allowed concentrations deﬁned by the United States Environmental Protection Agency for drinking water (72 nM). The underground water sample Pb2 þ content was also determined by ICP-MS. The obtained Pb2 þ content was 2.597 0.11 ppb, which is in agreement with our assay results. The results clearly show that our assay
Fig. 3. Selectivity study. Conditions: Pb2 þ concentration: 5 nM; other metal ions: 5 nM each; the mixture contained all six interference ions, and each at a concentration of 5 nM. Experimental conditions: the same as those described for Fig. 2.
method can be used for the detection of Pb2 þ in real samples (Zen and Wu, 1996; Leung and Jiao, 2006; Martins et al., 2009).
4. Conclusions In conclusion, a highly sensitive and selective method for the quantiﬁcation of Pb2 þ has been developed. A Pb2 þ selective DNAzyme was used for the cleavage of the RNA embedded substrate. Exonuclease III was used to remove the 2′,3′-cyclic phosphate, and the remaining part of the substrate (5′-part) was subsequently used as the primer for the strand displacement ampliﬁcation reaction. Since the DNAzyme is selective for Pb2 þ ,
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the method is selective for the quantiﬁcation of Pb2 þ . In addition, because of the efﬁcient DNA ampliﬁcation by the strand displacement ampliﬁcation reaction, high sensitivity was obtained. 200 pM Pb2 þ could be easily detected. We envision that our assay could be used for the development of highly sensitive and selective DNAzyme and aptazyme based new sensing techniques for the quantiﬁcation of various target molecules. Acknowledgments This work was supported by the “100 Talents” program of the Chinese Academy of Sciences, the National Basic Research Program of China (973 Program, No. 2011CB911002), the National Natural Science Foundation of China (21075119, 91027036, 21275139), the Pillar Program of Changchun Municipal Bureau of Science and Technology (No. 2011225). Appendix A. supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.09.055. References Breaker, R.R., 1997. Chemical Reviews 97, 371–390. Elbaz, J., Shlyahovsky, B., Willner, I., 2008. Chemical Communications 13, 1569–1571. Elfering, H., Andersson, J.T., Poll, K.G., 1998. Analyst 123, 669–674. Faulhammer, D., Famulok, M., 1996. Angewandte Chemie International Edition in English 35, 2837–2841. Jia, H., Li, Z., Liu, C., Cheng, Y., 2010. Angewandte Chemie International Edition 49, 5498–5501. Lan, T., Furuya, K., Lu, Y., 2010. Chemical Communications 46, 3896–3898. Leung, C.M., Jiao, J.J., 2006. Water Research 40, 753–767. Liu, J., Cao, Z., Lu, Y., 2009. Chemical Reviews 109, 1948–1998. Liu, J., Lu, Y., 2004. Journal of the American Chemical Society 126, 12298–12305. Liu, J., Lu, Y., 2005. Journal of the American Chemical Society 127, 12677–12683. Liu, J., Lu, Y., 2007. Journal of the American Chemical Society 129, 9838–9839.
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