- Email: [email protected]
Ultrasensitive detection of DNA based on target-triggered hairpin assembly and exonuclease-assisted recycling amplification
Accepted Manuscript Title: Ultrasensitive detection of DNA based on target-triggered hairpin assembly and exonuclease-assisted recycling amplification Authors: Xiaofan Sun, Shuling Wang, Yiping Zhang, Yaping Tian, Nandi Zhou PII: DOI: Reference:
S0925-4005(17)31029-8 http://dx.doi.org/doi:10.1016/j.snb.2017.06.014 SNB 22481
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
15-3-2017 23-5-2017 3-6-2017
Please cite this article as: Xiaofan Sun, Shuling Wang, Yiping Zhang, Yaping Tian, Nandi Zhou, Ultrasensitive detection of DNA based on target-triggered hairpin assembly and exonuclease-assisted recycling amplification, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.06.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ultrasensitive detection of DNA based on target-triggered hairpin
assembly
and
exonuclease-assisted
recycling
amplification
Xiaofan Sun, Shuling Wang, Yiping Zhang, Yaping Tian, Nandi Zhou*
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China *Corresponding author. Tel: +86-510-85197831; Fax: +86-510-85197831; E-mail: [email protected]
Abstract An ultrasensitive electrochemical detection of target DNA was developed based on target-triggered hairpin assembly and exonuclease III (Exo III)-assisted recycling quadratic amplification strategy. The detection employed a gold nanoparticles (AuNPs) modified Au electrode and two specially designed hairpin probes P1 and P2. P1 probe contained G-quadruplex-forming sequence and target DNA recognition region, and was immobilized on the electrode. P2 probe was used as a secondary complementary sequence which can displace target DNA and hybridize with P1 probe. In the absence of target DNA, these hairpin structures of P1 and P2 can coexist. While in the presence of target DNA, it can trigger the self-assembly process of P1 and P2 and initiate the Exo III-assisted two recycling process, resulting in the formation of G-quadruplex structure on electrode surface. Finally, with the addition of hemin, numerous G-quadruplex-hemin complexes formed on the electrode surface and gave a pronounced electrochemical response in differential pulse voltammogram (DPV). Taking K-ras proto oncogene as an example, the proposed DNA biosensor exhibited a wide detection range from 10 fM to 20 nM, and an extremely low detection limit of 2.86 fM. Moreover, it can clearly discriminate one-base difference in DNA sequence, thus can identify the mutation of the target gene. The proposed DNA biosensor has potential applications in the fields of clinic diagnosis, biomedicine, food and environment microbial monitoring. Keywords: Electrochemical DNA biosensor; K-ras gene; exonuclease III; target recycling amplification; G-quadruplex
1. Introduction Detection of trace disease-related sequence-specific DNA has significant applications in clinic diagnosis [1], gene therapy [2], sickness prevention and forensic investigations [3, 4] and thus has attracted substantial research efforts. So far, various analytical techniques have been employed for DNA detection. Compared with those colorimetric [5], chemiluminescent [6], or fluorescent [7] assays, the electrochemical DNA biosensors have attracted particular attention owing to its excellent characteristics, such as high sensitivity, low cost, simplicity, high specificity and easy to miniaturization [8, 9]. With the development of synthesis technique of nucleic acid probe, the electrochemical DNA biosensors have been widely applied in the detection of target genes [10, 11], environmental monitoring [12], and food quality control [13], etc. Because of the small concentration of sequence-specific target DNAs in biological samples, the development of signal amplification strategies is essential for DNA biosensors to obtain sufficient sensitivity [14]. Generally, several amplification strategies were involved in the fabrication of DNA biosensors. The traditionally used signal amplification was mostly based on DNA-amplification techniques, such as hybridization chain reaction (HCR) [15], strand displacement amplification (SDA) [16], and rolling circle amplification (RCA) [17]. With the development in nanotechnology, a variety of nanomaterials, including gold nanoparticles (AuNPs) [18], graphene [19], carbon nanotubes [20], and nanocomposites [21, 22] were employed in the fabrication of DNA biosensors as a carrier to increase the loading of
the immobilized biomolecules or active signal-labels. The target-triggered hairpin assembly with target DNA recycling strategy consists of two parts. One is the hybridization reaction triggered by the target. The other is strand displacement reaction. These reactions take place without the use of enzymes and complicated processes [7, 23, 24]. However, extremely high sensitivity and satisfactory detection range are not easy to be achieved by using a single amplification strategy. Thus two or more amplification strategies were sometimes introduced into the fabrication of DNA biosensors [25, 26]. G-quadruplex-hemin complex has been widely used in various biosensor configurations as signal amplification labeling [5, 27, 28]. Owing to its characteristic redox signal and peroxidase-like activity, the formation of G-quadruplex-hemin can provide intensive electrochemical signal in the fabrication of biosensors. K-ras proto oncogene is one of the members of ras family which can regulate cell growth as a molecular switch. The codon 12 mutation will disorder cell growth and cause cancer, such as carcinoma of the pancreas [29]. According to statistics, the frequency of K-ras mutations is around 80-100% in pancreatic cancers [30]. Therefore, the detection of K-ras is of great significance since it can be utilized as a biomarker in the early diagnosis of pancreas cancer [31, 32]. Herein, an ultrasensitive, label-free DNA biosensor for K-ras gene sequence was developed by coupling exonuclease III (Exo III)-assisted target-triggered hairpin assembly with target DNA recycling strategy and employing G-quadruplex-hemin as a signal label.
2. Experimental 2.1. Materials and reagents 6-mercaptohexanol (MCH), hemin, HAuCl4, 1, 6-hexanedithiol (HDT) were purchased from Sigma-Aldrich. Exonuclease III (Exo III) was obtained from Takara Biotechnology Co., Ltd. 4- (2-hydroxyethyl)-1-piperazine ethanesulfonic acid sodium salt (HEPES) and tris (2-carboxyethyl) phosphine (TCEP) were purchased from Sangon Biotech Co., Ltd. Other chemicals were of analytical grade. All solutions were prepared with ultrapure water (18.2 MΩ cm) obtained from a Millipore water purification system. All oligonucleotides were synthesized and HPLC-purified by Sangon Biotech Co., Ltd. And all oligonucleotides (100 µM) were stocked in 20 mM Tris-HCl buffer ( 200 mM NaCl, 2 mM MgCl2 and 20 mM KCl, pH 7.4), and diluted with 10 mM Tris-HCl buffer (1 mM EDTA, 0.1 M NaCl, 10 mM TCEP, pH 7.4). These oligonucleotides were heated to 95 ºC for 5 min, and then slowly cooled down to room temperature. The sequences of the oligonucleotides were as below. Hairpin probe 1 (P1): 5’-HS-(CH2)6-GGGTGGGCGGGATGGGTTACGCCACCAGC TCCAACCCATCCTT-3’ Hairpin probe 2 (P2): 5’-AAGGATGGGTGGAGCTGGTGGCGTAGGCACTCCAC CCATCCAGAC-3’ Target DNA (TD): 5’-TGGAGCTGGTGGCGTAGGCA-3’ Single-base mismatched sequence (M1): 5’-TGGAGCTGATGGCGTAGGCA-3’ Three-base mismatched sequence (M3): 5’-TGGAGCTCCAGGCGTAGGCA-3’
Non-target DNA 1: 5’-GGCAGCAATTTCACCAGTACTA-3’ Non-target DNA 2: 5’-GATTTTCTTCCTTTTGTTC-3’ The italic letters are the sequence of the stem arms, and the underlined letters are the mismatched bases.
2.2. Preparation and modification of Au electrode The bare Au electrode (3 mm in diameter) was carefully polished with wet alumina slurry (0.3 and 0.05 µm) and then sonicated in ultrapure water and ethanol for 3 min, respectively. Then the electrode was electrochemically activated by immersing into 0.5 M H2SO4 and cyclic scanning within the potential range from -0.2 to +1.5 V. The electrode was dried at nitrogen atmosphere. The surface of Au electrode was modified with AuNPs through HDT, which possesses free –SH group at both ends and acts as a connection bridge to immobilize AuNPs on gold electrode [33, 34]. Firstly, the cleaned Au electrode was immersed in 10 mM HDT ethanol solution for 3 h to ensure HDT to be self-assembled on the electrode. The obtained HDT-Au electrode was then rinsed by ethanol and ultrapure water to remove the loosely bound HDT. AuNPs were prepared through the reported procedure [35] and characterized by TEM and UV-vis spectrophotometer. The average diameter of AuNPs is about 12 nm and the UV-vis spectrum shows a typical absorption peak at 520 nm. The HDT-Au electrode was subsequently dipped in AuNPs solution overnight under ambient condition. Finally, the prepared AuNPs-HDT-Au electrode was washed with water and dried with nitrogen.
2.3. Fabrication of the electrochemical DNA biosensor The AuNPs-HDT-Au electrode was first incubated in 100 μL of 10 mM Tris-HCl working solution with 350 nM P1 probe for 12 h at room temperature. And then the electrode was immersed in 2 mM MCH for 2 h, followed by rinsing with water and drying with nitrogen. To detect the concentration of target DNA, the P1 probe modified AuNPs-HDT-Au electrode was dipped into 100 μL 10 mM Tris-HCl working solution (100 mM NaCl, 1 mM MgCl2 and 10 mM KCl, pH 7.4), containing 500 nM P2 probe, 50 U Exo III, and different concentration of TD, for 2 h at 37ºC (the optimal reaction temperature of Exo III). Subsequently, 2 μL of 20 mM hemin and 98 μL of 20 mM HEPES buffer (50 mM KCl, 1% DMSO, pH 7.4) were added into the hybridization system mentioned above. The electrode was incubated for another 1 h at room temperature to induce the liberated part I to fold into a G-quadruplex-hemin complex. After rinsing for 30 s, the resultant electrode was used for the electrochemical measurement.
2.4. Electrochemical measurements The
electrochemical
measurements
were
conducted
on
a
CHI
660e
electrochemical workstation. Differential pulse voltammetry (DPV) was performed in 20 mM HEPES testing buffer (20 mM KCl, pH 7.4) in the potential range from -0.1 to -0.6 V. Cyclic voltammetry (CV) and electrochemical impedence spectroscopy (EIS)
were carried out in 0.1 mM [Fe(CN)6]3-/4- (containing 0.1 M KCl) to characterize the different interfacial processes during the modification of the electrode. For CV measurements, the potential range was from -0.2 to -0.6 V at a scan rate of 50 mV/s. For EIS measurements, the frequency range was from 0.1 Hz to 10 kHz.
3. Results and discussion 3.1. The principle of the electrochemical DNA biosensor The principle of the electrochemical DNA biosensor is depicted in Scheme 1. The DNA biosensor is primarily constructed with two hairpin structures and Exo III, where the target DNA can induce hairpin assembly leading to Exo III-assisted autocatalytic recycling reaction. Two special hairpin probes termed P1 and P2 were rationally designed, which can stably coexist in the solution in the absence of target DNA. Both hairpin probes contain three major parts (part I, II and III locate in hairpin probe P1, and part IV, V and VI locate in hairpin probe P2). Part I is a G-quadruplex-forming sequence which is blocked via hybridization with part III; and part II is the target DNA recognition region. Part IV is complementary to part III, and is caged via hybridization with part VI. Part V has the same sequence with target DNA, which can serve as a secondary target to displace target DNA and trigger the cleavage of probes by Exo III. Thus, P1 probe can firstly be immobilized on AuNPs-HDT-Au electrode through Au-S interaction. It can coexist with P2 probe in the detection solution containing Exo III. However, the existence of the target DNA can trigger two independent cycles of reactions: (1) The target DNA first interacts
with part II and opens the hairpin structure of P1. Through a branch migration process, part III can hybridizes with part IV, leading to the displacement of target DNA by P2. The displaced target DNA can then hybridize with another P1 probe in the next cycle. (2) Exo III can catalyze the cleavage of the P1-P2 duplex formed above, which releases P2 as a secondary target and leaves part I on the electrode surface. In the presence of hemin, part I can combine with hemin to form G-quadruplex-hemin complex, which gives a remarkable electrochemical signal.
3.2. Characterization of electrode modification and verification of the electrochemical assay The introduction of AuNPs on the surface of the electrode can increase the immobilization of P1 probe and accelerate the electron transfer between the G-quadruplex-hemin complex and the electrode, which can improve DPV response during the target DNA detection. A control experiment was carried out in the presence of 0.5 μM K-ras DNA by using bare Au electrode and AuNPs-HDT-Au electrode respectively. Compared to that of bare Au electrode, the peak current in DPV of AuNPs-HDT-Au electrode increased 16.49%. Therefore AuNPs modified electrode was chosen for the following experiments. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were carried out using [Fe(CN)6]3-/4- as electrochemical probe to characterize the modification of the electrode. As shown in Fig. 1A, the peak current in CV curves changed intensively during the modification. Compared with the bare Au electrode,
the redox peaks of the HDT-Au electrode almost disappeared due to the HDT layer self-assembled on the surface of Au electrode. The HDT layer hinders the electron transfer between [Fe(CN)6]3-/4- and the Au electrode. However, as AuNPs occupied the other end of HDT through Au-S bond, the peak current showed an obvious increase, indicating that the electron transfer was restored and the AuNPs-HDT-Au electrode had a good electronic communication with solution species. The stepwise modification process of Au electrode was also characterized by EIS, whose results were just in accordance with those of CV, as shown in Fig. 1B. The semicircle diameter in EIS indicates the electron-transfer resistance. Obviously, the bare Au electrode showed negligible resistance (curve a) in contrast to that of the HDT-Au electrode (curve b). After the modification of AuNPs, a large decrement of the resistance was observed (curve c), indicating an increasing of the electron-transfer efficiency due to the excellent conductivity of AuNPs. The feasibility of the electrochemical DNA biosensor was investigated by comparing the DPV responses of the electrode under different conditions. As shown in Fig. 2, when probe P1 modified AuNPs-HDT-Au electrode was dipped with working solution containing only P2 (without Exo III and the target K-ras gene sequence), only low peak current can be observed in DPV after the incubation with hemin (curve a). The electrochemically active G-quadruplex-hemin complex cannot be formed because the G-quadruplex-forming sequence (part I) was blocked at this stage. It is worthy of notice that hemin itself can produce an observable peak in DPV due to its electrochemical reduction on the electrode. The DPV response was scarcely
influenced by the addition of Exo III to the working solution (curve b). However, in the presence of K-ras sequence (without Exo III), it can open the hairpin structure of P1 and be subsequently displaced by P2. Then the G-quadruplex-forming sequence (part I of P1) was unlocked and the G-quadruplex-hemin complex was formed, although the formation of the complex may somehow be hindered by the duplex between P1 and P2. The current response in DPV was obviously increased due to the unlocking of part I and the formation of the G-quadruplex-hemin complex (curve c). Furthermore, after the introduction of Exo III, the current response showed remarkable increase (curve d). Exo III catalyzed the cleavage of P1-P2 duplex DNA and released P2 probe for the next cleavage cycle, which retained a large number of part I on the electrode surface. Moreover, the cleavage of P1 and the release of P2 removed the spatial hindrance for the formation of G-quadruplex. With the help of hemin, a large number of G-quadruplex-hemin complexes formed and signal amplification was achieved. Therefore, the proposed Exo III-assisted target DNA-recycling signal amplification strategy is reliable for sensitive detection of sequence-specific DNA.
3.3. Optimization of experiment conditions The conditions during the construction of the biosensor and the detection will influence the performance of the DNA sensor, such as the concentration of probe P1 and P2, the amount of Exo III and the reaction time of Exo III. These factors were optimized to achieve the best sensing performance.
Firstly, the concentration of probe P1 used to modify the working electrode was investigated. P1 contains the G-quadruplex-forming sequence which can influence the intensity of the electrochemical signal. Different concentrations of P1 ranged from 100 nM to 500 nM were used to modify AuNPs-HDT-Au electrode, and then the obtained electrodes were incubated with probe P2 (500 nM), TD (500 nM) and Exo III (50 U). After the incubation with hemin, DPV curves were recorded. As shown in Fig. 3A, the peak current increases with the increased concentration of P1 until the concentration reaches 350 nM. Then 350 nM was chosen as the optimal concentration of P1 for the following experiments. Secondly, the influence of the concentration of probe P2 added in the test solution was studied. P2 can hybridize with P1, leading to the displacement of target DNA and the cleavage reaction of P1-P2 duplex catalyzed by Exo III, thus acts as an important role in the biosensor configuration. Different concentration of P2 ranging from 100 nM to 600 nM together with 500 nM TD and 50 U Exo III were used to incubate with the optimized P1 modified AuNPs-HDT-Au electrode. As shown in Fig. 3B, the DPV response increases rapidly with the increasing concentration of P2 and reaches a plateau phase after 500 nM P2 was added. Thus, the optimal concentration of P2 is 500 nM. Thirdly, the influence of the amount of Exo III was investigated. The cleavage of P1-P2 duplex depends on the amount of Exo III. The cleavage reaction catalyzed by Exo III can assists P2 recycling process and realizes the signal amplification strategy. As such, different amount of Exo III was introduced into the test solution, and the
result was shown in Fig. 3C. The peak current increases with the increment of Exo III from 10 U to 50 U, and reaches a plateau at 50 U. Thus 50 U was chosen as the optimal amount of Exo III to catalyze the reaction. Finally, the reaction time of Exo III plays an important role in the detection. So it is essential to optimize. It could be seen from Fig. 3D (P1 350 nM, P2 500 nM, TD 500 nM, Exo III 50 U) that the peak current increases with the increase of the incubation time. Meanwhile, the peak current reaches equilibrium when the incubation time is more than 60 min. Thus, 60 min was chosen as the optimized incubation time of Exo III.
3.4. Analytical performance of the DNA biosensor Under the optimized conditions, the sensitivity and dynamic response range of the electrochemical DNA biosensor were investigated. For the detection of K-ras gene, different concentration of the target DNA was introduced into 10 mM Tris-HCl working solution containing 500 nM P2 and 50 U Exo III. After incubation of P1 modified AuNPs-HDT-Au electrode in the test solution and subsequent addition of hemin, DPV curves were recorded. As shown in Fig. 4A, the peak current in DPV increases with the increased concentration of K-ras in the range from 10 fM to 20 nM. A linear relationship can be derived between the peak current and the logarithm of the concentration of the target DNA in the range from 50 fM to 10 nM (Fig. 5B). The linear regression equation is y=3.147+logx, R2=0.997, where x is the concentration of target DNA (pM), and y is the peak current (μA). The detection limit was estimated to
be 2.86 fM (3 x standard deviation of the blank signal/sensitivity). Since diverse signal amplification strategies have been reported to fabricate DNA biosensors, a comparison of the analytical performance between the reported DNA biosensors [7, 15, 25, 31, 32, 36-38] and that of this work was listed in Table 1. The quadratic signal amplification assay exhibited not only a wide detection range over six orders of magnitude, but also a very low detection limit down to fM level. Since the detection of K-ras mutation can be utilized for early diagnosis and monitoring of pancreatic cancer, the ability of the DNA sensor to discriminate mutant sequence from the wild type is significant. Then the specificity of the constructed DNA biosensor was checked by comparing the current response of the target DNA to those of mismatched sequences, such as single-base and three-base mismatched sequences. The results are shown in Fig. 5. Compared with the wild type K-ras gene sequence, the current responses are greatly reduced in the presence of mutant sequences. Obviously, the fabricated DNA biosensor can clearly discriminate one-base difference in DNA sequence, indicating very high specificity. Furthermore, the interferent-resistance of the DNA sensor was checked by comparing the current response in the presence of 10 pM target DNA only and in the presence of 10 pM target DNA and high concentration of two non-target DNA sequences (0.5 μM non-target DNA1 and 0.5 μM non-target DNA2). As a result, the peak current increased only 4.75% in co-existence of much higher concentration of other DNAs, indicating excellent interferent-resistance. The inter-electrode reproducibility was investigated by using five different
modified electrodes. The current responses were determined in the presence of 10 pM K-ras DNA. And the relative standard deviation (RSD) of 7.31% was derived, which suggested good reproducibility of the proposed DNA biosensor.
4. Conclusion In conclusion, a novel electrochemical DNA biosensor for ultrasensitive detection of K-ras gene was developed. The DNA biosensor exhibited excellent sensitivity as well as wide detection range, which can be attributed to the configuration design of the biosensor. On the one hand, the AuNPs modified on Au electrode can not only facilitate electron transfer, but also greatly increase the assembly area of P1 probe [39, 40], resulting in the wide response range towards the target DNA. On the other hand, numerous of G-quadruplex-hemin complexes can be formed on the electrode by coupling target-triggered self-assembly of hairpin probe P1 and P2 with Exo III-assisted cleavage process, resulting in very high sensitivity. Such target-triggered hairpin assembly with target recycling strategy shows powerful signal amplification ability without the need of labeling DNA probes. Meanwhile, the assay exhibits a high specificity to discriminate single-base mutation of K-ras gene sequence. However, the multi-step assay increases the complexity of the experiment and extends the operation time. For practical application, the scheme can be simplified by transferring the assay to the solution and utilizing UV-vis spectroscopic method based on the peroxidase activity of G-quadruplex-hemin complex. Moreover, by changing the sequence design of probe P1 and P2, the assay can be used for the detection of other sequence-specific
fragments and mutants, thus hold great potential for early diagnosis in gene-related diseases, as well as environmental monitoring or microbial-related food quality control.
Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 31271860) and the Fundamental Research Funds for the Central Universities (JUSRP51402A).
References [1] J. Wang, Electrochemical biosensors: towards point-of-care cancer diagnostics, Biosens. Bioelectron. 21 (2006) 1887-92. [2] Q.C. Liu, Q. Yi, W.N. Wu, Y. Wang, L.P. Lin, C.F. Zhao, X.H. Lin, DNA electrochemical sensor for detection of PRSS1 point mutation based on restriction endonuclease technique, Prep. Biochem. Biotech. 45 (2015) 430-7. [3] A. Rasooly, J. Jacobson, Development of biosensors for cancer clinical testing, Biosens. Bioelectron. 21 (2006) 1851-8. [4] I.E. Tothill, Biosensors for cancer markers diagnosis, Semin. Cell. Dev. Biol. 20 (2009) 55-62. [5] M.G. Deng, D. Zhang, Y.Y. Zhou, X. Zhou, Highly effective colorimetric and visual detection of nucleic acids using an asymmetrically split peroxidase DNAzyme, J. Am. Chem. Soc. 130 (2008) 13095-102. [6] M. Luo, X. Chen, G.H. Zhou, X. Xiang, L. Chen, X.H. Ji, Z.K. He, Chemiluminescence biosensors for DNA detection using graphene oxide and a horseradish peroxidase-mimicking DNAzyme, Chem. Commun. 48 (2012) 1126-8. [7] Z.Z. Jiang, H. Wang, X.B. Zhang, C.H. Liu, Z.P. Li, An enzyme-free signal
amplification strategy for sensitive detection of microRNA via catalyzed hairpin assembly, Anal. Methods 6 (2014) 9477-82. [8] T.G. Drummond, M.G. Hill, J.K. Barton, Electrochemical DNA sensors, Nat. Biotechnol. 21 (2003) 1192-9. [9] Y. Xiao, X.G. Qu, K.W. Plaxco, A.J. Heeger, Label-free electrochemical detection of DNA in blood serum via target-induced resolution of an electrode-bound DNA pseudoknot, J. Am. Chem. Soc. 129 (2007) 11896-7. [10] S.L. Wang, Y. Liu, X.F. Sun, Y.P. Tian, N.D. Zhou, Ultrasensitive electrochemical detection of dual DNA targets based on G-quadruplex-mediated amplification, Rsc Adv. 5 (2015) 57532-7. [11] H.Y. Xiong, Y. Chen, X.H. Zhang, H.S. Gu, S.F. Wang, An electrochemical biosensor for the rapid detection of DNA damage induced by xanthine oxidase-catalyzed Fenton reaction, Sens. Actuators B Chem.181 (2013) 85-91. [12] G. Marrazza, I. Chianella, M. Mascini, Disposable DNA electrochemical biosensors for environmental monitoring, Anal. Chim. Acta 387 (1999) 297-307. [13] N.D. Zhou, J.B. Luo, J. Zhang, Y.D. You, Y.P. Tian, A label-free electrochemical aptasensor for the detection of kanamycin in milk, Anal. Methods 7 (2015) 1991-6. [14] S.H. Zhou, L. Yuan, X. Hua, L.L. Xu, S.Q. Liu, Signal amplification strategies for DNA and protein detection based on polymeric nanocomposites and polymerization: a review, Anal. Chim. Acta 877 (2015) 19-32. [15] Y. Chen, J. Xu, J. Su, Y. Xiang, R. Yuan, Y.Q. Chai, In situ hybridization chain reaction amplification for universal and highly sensitive electrochemiluminescent detection of DNA, Anal. Chem. 84 (2012) 7750-5. [16] C. Wang, H. Zhou, W.P. Zhu, H.B. Li, J.H. Jiang, G.L. Shen, R.Q. Yu, Ultrasensitive electrochemical DNA detection based on dual amplification of circular strand-displacement polymerase reaction and hybridization chain reaction, Biosens. Bioelectron. 47 (2013) 324-8. [17] H.X. Ji, F. Yan, J.P. Lei, H.X. Ju, Ultrasensitive electrochemical detection of nucleic acids by template enhanced hybridization followed with rolling circle amplification, Anal. Chem. 84 (2012) 7166-71.
[18] L. Lin, Y. Liu, L.H. Tang, J.H. Li, Electrochemical DNA sensor by the assembly of graphene and DNA-conjugated gold nanoparticles with silver enhancement strategy, Analyst 136 (2011) 4732-7. [19] W. Yang, K.R. Ratinac, S.P. Ringer, P. Thordarson, J.J. Gooding, F. Braet, Carbon nanomaterials in biosensors: should you use nanotubes or graphene?, Angew. Chem. Int. Ed. 49 (2010) 2114-38. [20] W. Zhang, T. Yang, D.M. Huang, K. Jiao, Electrochemical sensing of DNA immobilization
and
hybridization
based
on
carbon
nanotubes/nano
zinc
oxide/chitosan composite film, Chinese Chem. Lett. 19 (2008) 589-91. [21] Z.H. Zhang, X.M. Fu, K.Z. Li, R.X. Liu, D.L. Peng, L.H. He, M.H. Wang, H.Z. Zhang, L.M. Zhou, One-step fabrication of electrochemical biosensor based on DNA-modified
three-dimensional
reduced
graphene
oxide
and
chitosan
nanocomposite for highly sensitive detection of Hg(II), Sens. Actuators B Chem. 225 (2016) 453-62. [22] K.J. Huang, Y.J. Liu, H.B. Wang, T. Gan, Y.M. Liu, L.L. Wang, Signal amplification for electrochemical DNA biosensor based on two-dimensional graphene analogue tungsten sulfide–graphene composites and gold nanoparticles, Sens. Actuators B Chem. 191 (2014) 828-36. [23] Z.H. Qing, X.X. He, J. Huang, K.M. Wang, Z. Zou, T.P. Qing, Z.G. Mao, H. Shi, D.G. He, Target-catalyzed dynamic assembly-based pyrene excimer switching for enzyme-free nucleic acid amplified detection, Anal. Chem. 86 (2014) 4934-9. [24] B. Fu, J.C. Cao, W. Jiang, L. Wang, A novel enzyme-free and label-free fluorescence aptasensor for amplified detection of adenosine, Biosens. Bioelectron. 44 (2013) 52-6. [25] Y.H. Hu, X.Q. Xu, Q.H. Liu, L. Wang, Z.Y. Lin, G.N. Chen, Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification, Anal. Chem. 86 (2014) 8785-90. [26] X.C. Dong, C.M. Lau, A. Lohani, S.G. Mhaisalkar, J. Kasim, Z.X. Shen, X.N. Ho, J.A. Rogers, L.J. Li, Electrical detection of femtomolar DNA via gold-nanoparticle
enhancement in carbon-nanotube-network field-effect transistors, Adv. Mater. 20 (2008) 2389-93. [27] P. Liu, M. Liu, H.S. Yin, Y.L. Zhou, S.Y. Ai, Electrochemical biosensor for DNA methyltransferase detection based on DpnI digestion triggering the formation of G-quadruplex DNAzymes, Sens. Actuators B Chem. 220 (2015) 101-6. [28] M.D. Xu, J.Y. Zhuang, X. Chen, G.N. Chen, D.P. Tang, A difunctional DNA-AuNP dendrimer coupling DNAzyme with intercalators for femtomolar detection of nucleic acids, Chem. Commun. 49 (2013) 7304-6. [29] R.C. Ebersole, J.A. Miller, J.R. Moran, M.D. Ward, Spontaneously formed functionally active avidin monolayers on metal surfaces: a strategy for immobilizing biological reagents and design of piezoelectric biosensors, J. Am. Chem. Soc. 112 (1990) 3239-41. [30] T. Uemura, K. Hibi, T. Kaneko, S. Takeda, S. Inoue, O. Okochi, T. Nagasaka, A. Nakao, Detection of K-ras mutations in the plasma DNA of pancreatic cancer patients, J. Gastroenterol. 39 (2004) 56-60. [31] X. Fang, L.J. Bai, X.W. Han, J. Wang, A.Q. Shi, Y.Z. Zhang, Ultra-sensitive biosensor for K-ras gene detection using enzyme capped gold nanoparticles conjugates for signal amplification, Anal. Biochem. 460 (2014) 47-53. [32] X.Y. Wang, G.F. Shu, C.C. Gao, Y. Yang, Q. Xu, M. Tang, Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy, Anal. Biochem. 466 (2014) 51-8. [33] Y.J. Liu, F. Yin, Y.M. Long, Z.H. Zhang, S.Z. Yao, Study of the immobilization of alcohol dehydrogenase on Au-colloid modified gold electrode by piezoelectric quartz crystal sensor, cyclic voltammetry, and electrochemical impedance techniques, J. Colloid. Int. Sci. 258 (2003) 75-81. [34] L.X. Cao, Q.C. Li, J.S. Ji, P.S. Yan, T. Wang, Development and rvaluation of ultramicro electrode ensembles based on 1.6-Hexanedithiol self-assembled modified layer and its application for HRP detection, Int. J. Electrochem. Sci. 8 (2013) 3074-83. [35] A. Sivanesan, P. Kannan, S.A. John, Electrocatalytic oxidation of ascorbic acid
using a single layer of gold nanoparticles immobilized on 1, 6-hexanedithiol modified gold electrode, Electrochim. Acta 52 (2007) 8118-24. [36] C.H. Luo, H. Tang, W. Cheng, L. Yan, D.C. Zhang, H.X. Ju, S.J. Ding, A sensitive electrochemical DNA biosensor for specific detection of Enterobacteriaceae bacteria by Exonuclease III-assisted signal amplification, Biosens. Bioelectron. 48 (2013) 132-7. [37] R.M. Kong, Z.L. Song, H.M. Meng, X.B. Zhang, G.L. Shen, R.Q. Yu, A label-free electrochemical biosensor for highly sensitive and selective detection of DNA via a dual-amplified strategy, Biosens. Bioelectron. 54 (2014) 442-7. [38] Y.L. Huang, Z.F. Gao, H.Q. Luo, N.B. Li, Sensitive detection of HIV gene by coupling exonuclease III-assisted target recycling and guanine nanowire amplification, Sens. Actuators B Chem. 238 (2017) 1017-23. [39] W. Wang, L.N. Song, Q. Gao, H.L. Qi, C.X. Zhang, Highly sensitive detection of DNA using an electrochemical DNA sensor with thionine-capped DNA/gold nanoparticle conjugates as signal tags, Electrochem. Commun. 34 (2013) 18-21. [40] T. Liu, J.A. Tang, L. Jiang, The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity, Biochem. Biophys. Res. Commun. 313 (2004) 3-7.
Author Biographies Xiaofan Sun is a graduate student at School of Biotechnology, Jiangnan University, China. Her research interests mainly focus on the fabrication of electrochemical DNA biosensor. Shuling Wang received her M.S. degree in biochemistry and molecular biology from Jiangnan University, China in 2016. She mainly engaged in the fabrication of electrochemical DNA biosensor. Yiping Zhang is an undergraduate student at School of Biotechnology, Jiangnan University, China. Her current research focuses on the electrochemical DNA biosensor. Yaping Tian received her Ph.D. degree in fermentation engineering from Jiangnan University, China in 2006. She is currently a professor at School of Biotechnology, Jiangnan University. Her research focuses on the analysis and production of bio-active molecules. Nandi Zhou received his Ph.D. degree in biochemistry and molecular biology from Nanjing University, China in 2007. He is currently a professor at School of Biotechnology, Jiangnan University. His research focuses on aptamer-based bioanalysis, fabrication of novel nano-biosensors, monitoring and control of harmful residues in food, and real-time analysis of metabolites in fermentation process.
Figure Captions Fig. 1. (A) Cyclic voltammograms of bare Au (a), HDT-Au (b) and AuNPs-HDT-Au (c) electrodes in 0.1 mM [Fe(CN)6]3-/4-; (B) Electrochemical impedance spectra (EIS) of bare Au (a), HDT-Au (b), and AuNPs-HDT-Au (c) electrodes.
Fig. 2. The DPV curves recorded under different conditions: (a) 350 nM P1 and 500 nM P2, (b) 350 nM P1, 500 nM P2 and 50 U Exo III, (c) 350 nM P1, 500 nM P2 and 500 nM TD, (d) 350 nM P1, 500 nM P2, 500 nM TD and 50 U Exo III.
Fig. 3. The influence of (A) the concentration of P1, (B) the concentration of P2, (C) the amount of Exo III, and (D) the incubation time of Exo III on the peak current in DPV.
Fig. 4. (A) DPV curves recorded in the presence of various concentration of target DNA; (B) Calibration curve corresponding to the peak current measured at -0.32 V in DPV and the concentration of target DNA; Inset shows the linear relationship between the peak current and the logarithm of the concentration of the target DNA. Error bars represent standard deviations of measurements (n=3).
Fig. 5. Histograms of relative current changes of the DNA sensor towards 20 nM target DNA and the mismatched sequences.
Scheme 1. Principle of the electrochemical detection of K-ras DNA based on target-triggered hairpin assembly and Exo III-assisted target recycling amplification.
Table 1. The comparison of the analytical performance between the reported DNA sensors and this work. Signal amplification strategy
Detection
Linear range
Detection
Reference
method
(pM)
limit (fM)
Catalyzed hairpin assembly
Fluorescence
1-2000
1000
[7]
Hybridization chain reaction
ECL
0.025-100
15
[15]
Nicking endonuclease
DPV
0.0001-0.02
0.08
[25]
Gold nanoparticles and horseradish peroxidase
DPV
0.0001-1000
0.035
[31]
Functional composite nanofibers
DPV
0.1-100
30
[32]
Exonuclease III
DPV
0.01-1000
8.7
[36]
Nanoparticle and enzyme
DPV
1-10000
600
[37]
Guanine nanowire
Amperometry
10-100000
3600
[38]
Gold nanoparticles and exonuclease III
DPV
0.05-10000
2.86
This work
S0925-4005(17)31029-8 http://dx.doi.org/doi:10.1016/j.snb.2017.06.014 SNB 22481
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
15-3-2017 23-5-2017 3-6-2017
Please cite this article as: Xiaofan Sun, Shuling Wang, Yiping Zhang, Yaping Tian, Nandi Zhou, Ultrasensitive detection of DNA based on target-triggered hairpin assembly and exonuclease-assisted recycling amplification, Sensors and Actuators B: Chemicalhttp://dx.doi.org/10.1016/j.snb.2017.06.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ultrasensitive detection of DNA based on target-triggered hairpin
assembly
and
exonuclease-assisted
recycling
amplification
Xiaofan Sun, Shuling Wang, Yiping Zhang, Yaping Tian, Nandi Zhou*
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China *Corresponding author. Tel: +86-510-85197831; Fax: +86-510-85197831; E-mail: [email protected]
Abstract An ultrasensitive electrochemical detection of target DNA was developed based on target-triggered hairpin assembly and exonuclease III (Exo III)-assisted recycling quadratic amplification strategy. The detection employed a gold nanoparticles (AuNPs) modified Au electrode and two specially designed hairpin probes P1 and P2. P1 probe contained G-quadruplex-forming sequence and target DNA recognition region, and was immobilized on the electrode. P2 probe was used as a secondary complementary sequence which can displace target DNA and hybridize with P1 probe. In the absence of target DNA, these hairpin structures of P1 and P2 can coexist. While in the presence of target DNA, it can trigger the self-assembly process of P1 and P2 and initiate the Exo III-assisted two recycling process, resulting in the formation of G-quadruplex structure on electrode surface. Finally, with the addition of hemin, numerous G-quadruplex-hemin complexes formed on the electrode surface and gave a pronounced electrochemical response in differential pulse voltammogram (DPV). Taking K-ras proto oncogene as an example, the proposed DNA biosensor exhibited a wide detection range from 10 fM to 20 nM, and an extremely low detection limit of 2.86 fM. Moreover, it can clearly discriminate one-base difference in DNA sequence, thus can identify the mutation of the target gene. The proposed DNA biosensor has potential applications in the fields of clinic diagnosis, biomedicine, food and environment microbial monitoring. Keywords: Electrochemical DNA biosensor; K-ras gene; exonuclease III; target recycling amplification; G-quadruplex
1. Introduction Detection of trace disease-related sequence-specific DNA has significant applications in clinic diagnosis [1], gene therapy [2], sickness prevention and forensic investigations [3, 4] and thus has attracted substantial research efforts. So far, various analytical techniques have been employed for DNA detection. Compared with those colorimetric [5], chemiluminescent [6], or fluorescent [7] assays, the electrochemical DNA biosensors have attracted particular attention owing to its excellent characteristics, such as high sensitivity, low cost, simplicity, high specificity and easy to miniaturization [8, 9]. With the development of synthesis technique of nucleic acid probe, the electrochemical DNA biosensors have been widely applied in the detection of target genes [10, 11], environmental monitoring [12], and food quality control [13], etc. Because of the small concentration of sequence-specific target DNAs in biological samples, the development of signal amplification strategies is essential for DNA biosensors to obtain sufficient sensitivity [14]. Generally, several amplification strategies were involved in the fabrication of DNA biosensors. The traditionally used signal amplification was mostly based on DNA-amplification techniques, such as hybridization chain reaction (HCR) [15], strand displacement amplification (SDA) [16], and rolling circle amplification (RCA) [17]. With the development in nanotechnology, a variety of nanomaterials, including gold nanoparticles (AuNPs) [18], graphene [19], carbon nanotubes [20], and nanocomposites [21, 22] were employed in the fabrication of DNA biosensors as a carrier to increase the loading of
the immobilized biomolecules or active signal-labels. The target-triggered hairpin assembly with target DNA recycling strategy consists of two parts. One is the hybridization reaction triggered by the target. The other is strand displacement reaction. These reactions take place without the use of enzymes and complicated processes [7, 23, 24]. However, extremely high sensitivity and satisfactory detection range are not easy to be achieved by using a single amplification strategy. Thus two or more amplification strategies were sometimes introduced into the fabrication of DNA biosensors [25, 26]. G-quadruplex-hemin complex has been widely used in various biosensor configurations as signal amplification labeling [5, 27, 28]. Owing to its characteristic redox signal and peroxidase-like activity, the formation of G-quadruplex-hemin can provide intensive electrochemical signal in the fabrication of biosensors. K-ras proto oncogene is one of the members of ras family which can regulate cell growth as a molecular switch. The codon 12 mutation will disorder cell growth and cause cancer, such as carcinoma of the pancreas [29]. According to statistics, the frequency of K-ras mutations is around 80-100% in pancreatic cancers [30]. Therefore, the detection of K-ras is of great significance since it can be utilized as a biomarker in the early diagnosis of pancreas cancer [31, 32]. Herein, an ultrasensitive, label-free DNA biosensor for K-ras gene sequence was developed by coupling exonuclease III (Exo III)-assisted target-triggered hairpin assembly with target DNA recycling strategy and employing G-quadruplex-hemin as a signal label.
2. Experimental 2.1. Materials and reagents 6-mercaptohexanol (MCH), hemin, HAuCl4, 1, 6-hexanedithiol (HDT) were purchased from Sigma-Aldrich. Exonuclease III (Exo III) was obtained from Takara Biotechnology Co., Ltd. 4- (2-hydroxyethyl)-1-piperazine ethanesulfonic acid sodium salt (HEPES) and tris (2-carboxyethyl) phosphine (TCEP) were purchased from Sangon Biotech Co., Ltd. Other chemicals were of analytical grade. All solutions were prepared with ultrapure water (18.2 MΩ cm) obtained from a Millipore water purification system. All oligonucleotides were synthesized and HPLC-purified by Sangon Biotech Co., Ltd. And all oligonucleotides (100 µM) were stocked in 20 mM Tris-HCl buffer ( 200 mM NaCl, 2 mM MgCl2 and 20 mM KCl, pH 7.4), and diluted with 10 mM Tris-HCl buffer (1 mM EDTA, 0.1 M NaCl, 10 mM TCEP, pH 7.4). These oligonucleotides were heated to 95 ºC for 5 min, and then slowly cooled down to room temperature. The sequences of the oligonucleotides were as below. Hairpin probe 1 (P1): 5’-HS-(CH2)6-GGGTGGGCGGGATGGGTTACGCCACCAGC TCCAACCCATCCTT-3’ Hairpin probe 2 (P2): 5’-AAGGATGGGTGGAGCTGGTGGCGTAGGCACTCCAC CCATCCAGAC-3’ Target DNA (TD): 5’-TGGAGCTGGTGGCGTAGGCA-3’ Single-base mismatched sequence (M1): 5’-TGGAGCTGATGGCGTAGGCA-3’ Three-base mismatched sequence (M3): 5’-TGGAGCTCCAGGCGTAGGCA-3’
Non-target DNA 1: 5’-GGCAGCAATTTCACCAGTACTA-3’ Non-target DNA 2: 5’-GATTTTCTTCCTTTTGTTC-3’ The italic letters are the sequence of the stem arms, and the underlined letters are the mismatched bases.
2.2. Preparation and modification of Au electrode The bare Au electrode (3 mm in diameter) was carefully polished with wet alumina slurry (0.3 and 0.05 µm) and then sonicated in ultrapure water and ethanol for 3 min, respectively. Then the electrode was electrochemically activated by immersing into 0.5 M H2SO4 and cyclic scanning within the potential range from -0.2 to +1.5 V. The electrode was dried at nitrogen atmosphere. The surface of Au electrode was modified with AuNPs through HDT, which possesses free –SH group at both ends and acts as a connection bridge to immobilize AuNPs on gold electrode [33, 34]. Firstly, the cleaned Au electrode was immersed in 10 mM HDT ethanol solution for 3 h to ensure HDT to be self-assembled on the electrode. The obtained HDT-Au electrode was then rinsed by ethanol and ultrapure water to remove the loosely bound HDT. AuNPs were prepared through the reported procedure [35] and characterized by TEM and UV-vis spectrophotometer. The average diameter of AuNPs is about 12 nm and the UV-vis spectrum shows a typical absorption peak at 520 nm. The HDT-Au electrode was subsequently dipped in AuNPs solution overnight under ambient condition. Finally, the prepared AuNPs-HDT-Au electrode was washed with water and dried with nitrogen.
2.3. Fabrication of the electrochemical DNA biosensor The AuNPs-HDT-Au electrode was first incubated in 100 μL of 10 mM Tris-HCl working solution with 350 nM P1 probe for 12 h at room temperature. And then the electrode was immersed in 2 mM MCH for 2 h, followed by rinsing with water and drying with nitrogen. To detect the concentration of target DNA, the P1 probe modified AuNPs-HDT-Au electrode was dipped into 100 μL 10 mM Tris-HCl working solution (100 mM NaCl, 1 mM MgCl2 and 10 mM KCl, pH 7.4), containing 500 nM P2 probe, 50 U Exo III, and different concentration of TD, for 2 h at 37ºC (the optimal reaction temperature of Exo III). Subsequently, 2 μL of 20 mM hemin and 98 μL of 20 mM HEPES buffer (50 mM KCl, 1% DMSO, pH 7.4) were added into the hybridization system mentioned above. The electrode was incubated for another 1 h at room temperature to induce the liberated part I to fold into a G-quadruplex-hemin complex. After rinsing for 30 s, the resultant electrode was used for the electrochemical measurement.
2.4. Electrochemical measurements The
electrochemical
measurements
were
conducted
on
a
CHI
660e
electrochemical workstation. Differential pulse voltammetry (DPV) was performed in 20 mM HEPES testing buffer (20 mM KCl, pH 7.4) in the potential range from -0.1 to -0.6 V. Cyclic voltammetry (CV) and electrochemical impedence spectroscopy (EIS)
were carried out in 0.1 mM [Fe(CN)6]3-/4- (containing 0.1 M KCl) to characterize the different interfacial processes during the modification of the electrode. For CV measurements, the potential range was from -0.2 to -0.6 V at a scan rate of 50 mV/s. For EIS measurements, the frequency range was from 0.1 Hz to 10 kHz.
3. Results and discussion 3.1. The principle of the electrochemical DNA biosensor The principle of the electrochemical DNA biosensor is depicted in Scheme 1. The DNA biosensor is primarily constructed with two hairpin structures and Exo III, where the target DNA can induce hairpin assembly leading to Exo III-assisted autocatalytic recycling reaction. Two special hairpin probes termed P1 and P2 were rationally designed, which can stably coexist in the solution in the absence of target DNA. Both hairpin probes contain three major parts (part I, II and III locate in hairpin probe P1, and part IV, V and VI locate in hairpin probe P2). Part I is a G-quadruplex-forming sequence which is blocked via hybridization with part III; and part II is the target DNA recognition region. Part IV is complementary to part III, and is caged via hybridization with part VI. Part V has the same sequence with target DNA, which can serve as a secondary target to displace target DNA and trigger the cleavage of probes by Exo III. Thus, P1 probe can firstly be immobilized on AuNPs-HDT-Au electrode through Au-S interaction. It can coexist with P2 probe in the detection solution containing Exo III. However, the existence of the target DNA can trigger two independent cycles of reactions: (1) The target DNA first interacts
with part II and opens the hairpin structure of P1. Through a branch migration process, part III can hybridizes with part IV, leading to the displacement of target DNA by P2. The displaced target DNA can then hybridize with another P1 probe in the next cycle. (2) Exo III can catalyze the cleavage of the P1-P2 duplex formed above, which releases P2 as a secondary target and leaves part I on the electrode surface. In the presence of hemin, part I can combine with hemin to form G-quadruplex-hemin complex, which gives a remarkable electrochemical signal.
3.2. Characterization of electrode modification and verification of the electrochemical assay The introduction of AuNPs on the surface of the electrode can increase the immobilization of P1 probe and accelerate the electron transfer between the G-quadruplex-hemin complex and the electrode, which can improve DPV response during the target DNA detection. A control experiment was carried out in the presence of 0.5 μM K-ras DNA by using bare Au electrode and AuNPs-HDT-Au electrode respectively. Compared to that of bare Au electrode, the peak current in DPV of AuNPs-HDT-Au electrode increased 16.49%. Therefore AuNPs modified electrode was chosen for the following experiments. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were carried out using [Fe(CN)6]3-/4- as electrochemical probe to characterize the modification of the electrode. As shown in Fig. 1A, the peak current in CV curves changed intensively during the modification. Compared with the bare Au electrode,
the redox peaks of the HDT-Au electrode almost disappeared due to the HDT layer self-assembled on the surface of Au electrode. The HDT layer hinders the electron transfer between [Fe(CN)6]3-/4- and the Au electrode. However, as AuNPs occupied the other end of HDT through Au-S bond, the peak current showed an obvious increase, indicating that the electron transfer was restored and the AuNPs-HDT-Au electrode had a good electronic communication with solution species. The stepwise modification process of Au electrode was also characterized by EIS, whose results were just in accordance with those of CV, as shown in Fig. 1B. The semicircle diameter in EIS indicates the electron-transfer resistance. Obviously, the bare Au electrode showed negligible resistance (curve a) in contrast to that of the HDT-Au electrode (curve b). After the modification of AuNPs, a large decrement of the resistance was observed (curve c), indicating an increasing of the electron-transfer efficiency due to the excellent conductivity of AuNPs. The feasibility of the electrochemical DNA biosensor was investigated by comparing the DPV responses of the electrode under different conditions. As shown in Fig. 2, when probe P1 modified AuNPs-HDT-Au electrode was dipped with working solution containing only P2 (without Exo III and the target K-ras gene sequence), only low peak current can be observed in DPV after the incubation with hemin (curve a). The electrochemically active G-quadruplex-hemin complex cannot be formed because the G-quadruplex-forming sequence (part I) was blocked at this stage. It is worthy of notice that hemin itself can produce an observable peak in DPV due to its electrochemical reduction on the electrode. The DPV response was scarcely
influenced by the addition of Exo III to the working solution (curve b). However, in the presence of K-ras sequence (without Exo III), it can open the hairpin structure of P1 and be subsequently displaced by P2. Then the G-quadruplex-forming sequence (part I of P1) was unlocked and the G-quadruplex-hemin complex was formed, although the formation of the complex may somehow be hindered by the duplex between P1 and P2. The current response in DPV was obviously increased due to the unlocking of part I and the formation of the G-quadruplex-hemin complex (curve c). Furthermore, after the introduction of Exo III, the current response showed remarkable increase (curve d). Exo III catalyzed the cleavage of P1-P2 duplex DNA and released P2 probe for the next cleavage cycle, which retained a large number of part I on the electrode surface. Moreover, the cleavage of P1 and the release of P2 removed the spatial hindrance for the formation of G-quadruplex. With the help of hemin, a large number of G-quadruplex-hemin complexes formed and signal amplification was achieved. Therefore, the proposed Exo III-assisted target DNA-recycling signal amplification strategy is reliable for sensitive detection of sequence-specific DNA.
3.3. Optimization of experiment conditions The conditions during the construction of the biosensor and the detection will influence the performance of the DNA sensor, such as the concentration of probe P1 and P2, the amount of Exo III and the reaction time of Exo III. These factors were optimized to achieve the best sensing performance.
Firstly, the concentration of probe P1 used to modify the working electrode was investigated. P1 contains the G-quadruplex-forming sequence which can influence the intensity of the electrochemical signal. Different concentrations of P1 ranged from 100 nM to 500 nM were used to modify AuNPs-HDT-Au electrode, and then the obtained electrodes were incubated with probe P2 (500 nM), TD (500 nM) and Exo III (50 U). After the incubation with hemin, DPV curves were recorded. As shown in Fig. 3A, the peak current increases with the increased concentration of P1 until the concentration reaches 350 nM. Then 350 nM was chosen as the optimal concentration of P1 for the following experiments. Secondly, the influence of the concentration of probe P2 added in the test solution was studied. P2 can hybridize with P1, leading to the displacement of target DNA and the cleavage reaction of P1-P2 duplex catalyzed by Exo III, thus acts as an important role in the biosensor configuration. Different concentration of P2 ranging from 100 nM to 600 nM together with 500 nM TD and 50 U Exo III were used to incubate with the optimized P1 modified AuNPs-HDT-Au electrode. As shown in Fig. 3B, the DPV response increases rapidly with the increasing concentration of P2 and reaches a plateau phase after 500 nM P2 was added. Thus, the optimal concentration of P2 is 500 nM. Thirdly, the influence of the amount of Exo III was investigated. The cleavage of P1-P2 duplex depends on the amount of Exo III. The cleavage reaction catalyzed by Exo III can assists P2 recycling process and realizes the signal amplification strategy. As such, different amount of Exo III was introduced into the test solution, and the
result was shown in Fig. 3C. The peak current increases with the increment of Exo III from 10 U to 50 U, and reaches a plateau at 50 U. Thus 50 U was chosen as the optimal amount of Exo III to catalyze the reaction. Finally, the reaction time of Exo III plays an important role in the detection. So it is essential to optimize. It could be seen from Fig. 3D (P1 350 nM, P2 500 nM, TD 500 nM, Exo III 50 U) that the peak current increases with the increase of the incubation time. Meanwhile, the peak current reaches equilibrium when the incubation time is more than 60 min. Thus, 60 min was chosen as the optimized incubation time of Exo III.
3.4. Analytical performance of the DNA biosensor Under the optimized conditions, the sensitivity and dynamic response range of the electrochemical DNA biosensor were investigated. For the detection of K-ras gene, different concentration of the target DNA was introduced into 10 mM Tris-HCl working solution containing 500 nM P2 and 50 U Exo III. After incubation of P1 modified AuNPs-HDT-Au electrode in the test solution and subsequent addition of hemin, DPV curves were recorded. As shown in Fig. 4A, the peak current in DPV increases with the increased concentration of K-ras in the range from 10 fM to 20 nM. A linear relationship can be derived between the peak current and the logarithm of the concentration of the target DNA in the range from 50 fM to 10 nM (Fig. 5B). The linear regression equation is y=3.147+logx, R2=0.997, where x is the concentration of target DNA (pM), and y is the peak current (μA). The detection limit was estimated to
be 2.86 fM (3 x standard deviation of the blank signal/sensitivity). Since diverse signal amplification strategies have been reported to fabricate DNA biosensors, a comparison of the analytical performance between the reported DNA biosensors [7, 15, 25, 31, 32, 36-38] and that of this work was listed in Table 1. The quadratic signal amplification assay exhibited not only a wide detection range over six orders of magnitude, but also a very low detection limit down to fM level. Since the detection of K-ras mutation can be utilized for early diagnosis and monitoring of pancreatic cancer, the ability of the DNA sensor to discriminate mutant sequence from the wild type is significant. Then the specificity of the constructed DNA biosensor was checked by comparing the current response of the target DNA to those of mismatched sequences, such as single-base and three-base mismatched sequences. The results are shown in Fig. 5. Compared with the wild type K-ras gene sequence, the current responses are greatly reduced in the presence of mutant sequences. Obviously, the fabricated DNA biosensor can clearly discriminate one-base difference in DNA sequence, indicating very high specificity. Furthermore, the interferent-resistance of the DNA sensor was checked by comparing the current response in the presence of 10 pM target DNA only and in the presence of 10 pM target DNA and high concentration of two non-target DNA sequences (0.5 μM non-target DNA1 and 0.5 μM non-target DNA2). As a result, the peak current increased only 4.75% in co-existence of much higher concentration of other DNAs, indicating excellent interferent-resistance. The inter-electrode reproducibility was investigated by using five different
modified electrodes. The current responses were determined in the presence of 10 pM K-ras DNA. And the relative standard deviation (RSD) of 7.31% was derived, which suggested good reproducibility of the proposed DNA biosensor.
4. Conclusion In conclusion, a novel electrochemical DNA biosensor for ultrasensitive detection of K-ras gene was developed. The DNA biosensor exhibited excellent sensitivity as well as wide detection range, which can be attributed to the configuration design of the biosensor. On the one hand, the AuNPs modified on Au electrode can not only facilitate electron transfer, but also greatly increase the assembly area of P1 probe [39, 40], resulting in the wide response range towards the target DNA. On the other hand, numerous of G-quadruplex-hemin complexes can be formed on the electrode by coupling target-triggered self-assembly of hairpin probe P1 and P2 with Exo III-assisted cleavage process, resulting in very high sensitivity. Such target-triggered hairpin assembly with target recycling strategy shows powerful signal amplification ability without the need of labeling DNA probes. Meanwhile, the assay exhibits a high specificity to discriminate single-base mutation of K-ras gene sequence. However, the multi-step assay increases the complexity of the experiment and extends the operation time. For practical application, the scheme can be simplified by transferring the assay to the solution and utilizing UV-vis spectroscopic method based on the peroxidase activity of G-quadruplex-hemin complex. Moreover, by changing the sequence design of probe P1 and P2, the assay can be used for the detection of other sequence-specific
fragments and mutants, thus hold great potential for early diagnosis in gene-related diseases, as well as environmental monitoring or microbial-related food quality control.
Acknowledgements This work was supported by the National Natural Science Foundation of China (no. 31271860) and the Fundamental Research Funds for the Central Universities (JUSRP51402A).
References [1] J. Wang, Electrochemical biosensors: towards point-of-care cancer diagnostics, Biosens. Bioelectron. 21 (2006) 1887-92. [2] Q.C. Liu, Q. Yi, W.N. Wu, Y. Wang, L.P. Lin, C.F. Zhao, X.H. Lin, DNA electrochemical sensor for detection of PRSS1 point mutation based on restriction endonuclease technique, Prep. Biochem. Biotech. 45 (2015) 430-7. [3] A. Rasooly, J. Jacobson, Development of biosensors for cancer clinical testing, Biosens. Bioelectron. 21 (2006) 1851-8. [4] I.E. Tothill, Biosensors for cancer markers diagnosis, Semin. Cell. Dev. Biol. 20 (2009) 55-62. [5] M.G. Deng, D. Zhang, Y.Y. Zhou, X. Zhou, Highly effective colorimetric and visual detection of nucleic acids using an asymmetrically split peroxidase DNAzyme, J. Am. Chem. Soc. 130 (2008) 13095-102. [6] M. Luo, X. Chen, G.H. Zhou, X. Xiang, L. Chen, X.H. Ji, Z.K. He, Chemiluminescence biosensors for DNA detection using graphene oxide and a horseradish peroxidase-mimicking DNAzyme, Chem. Commun. 48 (2012) 1126-8. [7] Z.Z. Jiang, H. Wang, X.B. Zhang, C.H. Liu, Z.P. Li, An enzyme-free signal
amplification strategy for sensitive detection of microRNA via catalyzed hairpin assembly, Anal. Methods 6 (2014) 9477-82. [8] T.G. Drummond, M.G. Hill, J.K. Barton, Electrochemical DNA sensors, Nat. Biotechnol. 21 (2003) 1192-9. [9] Y. Xiao, X.G. Qu, K.W. Plaxco, A.J. Heeger, Label-free electrochemical detection of DNA in blood serum via target-induced resolution of an electrode-bound DNA pseudoknot, J. Am. Chem. Soc. 129 (2007) 11896-7. [10] S.L. Wang, Y. Liu, X.F. Sun, Y.P. Tian, N.D. Zhou, Ultrasensitive electrochemical detection of dual DNA targets based on G-quadruplex-mediated amplification, Rsc Adv. 5 (2015) 57532-7. [11] H.Y. Xiong, Y. Chen, X.H. Zhang, H.S. Gu, S.F. Wang, An electrochemical biosensor for the rapid detection of DNA damage induced by xanthine oxidase-catalyzed Fenton reaction, Sens. Actuators B Chem.181 (2013) 85-91. [12] G. Marrazza, I. Chianella, M. Mascini, Disposable DNA electrochemical biosensors for environmental monitoring, Anal. Chim. Acta 387 (1999) 297-307. [13] N.D. Zhou, J.B. Luo, J. Zhang, Y.D. You, Y.P. Tian, A label-free electrochemical aptasensor for the detection of kanamycin in milk, Anal. Methods 7 (2015) 1991-6. [14] S.H. Zhou, L. Yuan, X. Hua, L.L. Xu, S.Q. Liu, Signal amplification strategies for DNA and protein detection based on polymeric nanocomposites and polymerization: a review, Anal. Chim. Acta 877 (2015) 19-32. [15] Y. Chen, J. Xu, J. Su, Y. Xiang, R. Yuan, Y.Q. Chai, In situ hybridization chain reaction amplification for universal and highly sensitive electrochemiluminescent detection of DNA, Anal. Chem. 84 (2012) 7750-5. [16] C. Wang, H. Zhou, W.P. Zhu, H.B. Li, J.H. Jiang, G.L. Shen, R.Q. Yu, Ultrasensitive electrochemical DNA detection based on dual amplification of circular strand-displacement polymerase reaction and hybridization chain reaction, Biosens. Bioelectron. 47 (2013) 324-8. [17] H.X. Ji, F. Yan, J.P. Lei, H.X. Ju, Ultrasensitive electrochemical detection of nucleic acids by template enhanced hybridization followed with rolling circle amplification, Anal. Chem. 84 (2012) 7166-71.
[18] L. Lin, Y. Liu, L.H. Tang, J.H. Li, Electrochemical DNA sensor by the assembly of graphene and DNA-conjugated gold nanoparticles with silver enhancement strategy, Analyst 136 (2011) 4732-7. [19] W. Yang, K.R. Ratinac, S.P. Ringer, P. Thordarson, J.J. Gooding, F. Braet, Carbon nanomaterials in biosensors: should you use nanotubes or graphene?, Angew. Chem. Int. Ed. 49 (2010) 2114-38. [20] W. Zhang, T. Yang, D.M. Huang, K. Jiao, Electrochemical sensing of DNA immobilization
and
hybridization
based
on
carbon
nanotubes/nano
zinc
oxide/chitosan composite film, Chinese Chem. Lett. 19 (2008) 589-91. [21] Z.H. Zhang, X.M. Fu, K.Z. Li, R.X. Liu, D.L. Peng, L.H. He, M.H. Wang, H.Z. Zhang, L.M. Zhou, One-step fabrication of electrochemical biosensor based on DNA-modified
three-dimensional
reduced
graphene
oxide
and
chitosan
nanocomposite for highly sensitive detection of Hg(II), Sens. Actuators B Chem. 225 (2016) 453-62. [22] K.J. Huang, Y.J. Liu, H.B. Wang, T. Gan, Y.M. Liu, L.L. Wang, Signal amplification for electrochemical DNA biosensor based on two-dimensional graphene analogue tungsten sulfide–graphene composites and gold nanoparticles, Sens. Actuators B Chem. 191 (2014) 828-36. [23] Z.H. Qing, X.X. He, J. Huang, K.M. Wang, Z. Zou, T.P. Qing, Z.G. Mao, H. Shi, D.G. He, Target-catalyzed dynamic assembly-based pyrene excimer switching for enzyme-free nucleic acid amplified detection, Anal. Chem. 86 (2014) 4934-9. [24] B. Fu, J.C. Cao, W. Jiang, L. Wang, A novel enzyme-free and label-free fluorescence aptasensor for amplified detection of adenosine, Biosens. Bioelectron. 44 (2013) 52-6. [25] Y.H. Hu, X.Q. Xu, Q.H. Liu, L. Wang, Z.Y. Lin, G.N. Chen, Ultrasensitive electrochemical biosensor for detection of DNA from Bacillus subtilis by coupling target-induced strand displacement and nicking endonuclease signal amplification, Anal. Chem. 86 (2014) 8785-90. [26] X.C. Dong, C.M. Lau, A. Lohani, S.G. Mhaisalkar, J. Kasim, Z.X. Shen, X.N. Ho, J.A. Rogers, L.J. Li, Electrical detection of femtomolar DNA via gold-nanoparticle
enhancement in carbon-nanotube-network field-effect transistors, Adv. Mater. 20 (2008) 2389-93. [27] P. Liu, M. Liu, H.S. Yin, Y.L. Zhou, S.Y. Ai, Electrochemical biosensor for DNA methyltransferase detection based on DpnI digestion triggering the formation of G-quadruplex DNAzymes, Sens. Actuators B Chem. 220 (2015) 101-6. [28] M.D. Xu, J.Y. Zhuang, X. Chen, G.N. Chen, D.P. Tang, A difunctional DNA-AuNP dendrimer coupling DNAzyme with intercalators for femtomolar detection of nucleic acids, Chem. Commun. 49 (2013) 7304-6. [29] R.C. Ebersole, J.A. Miller, J.R. Moran, M.D. Ward, Spontaneously formed functionally active avidin monolayers on metal surfaces: a strategy for immobilizing biological reagents and design of piezoelectric biosensors, J. Am. Chem. Soc. 112 (1990) 3239-41. [30] T. Uemura, K. Hibi, T. Kaneko, S. Takeda, S. Inoue, O. Okochi, T. Nagasaka, A. Nakao, Detection of K-ras mutations in the plasma DNA of pancreatic cancer patients, J. Gastroenterol. 39 (2004) 56-60. [31] X. Fang, L.J. Bai, X.W. Han, J. Wang, A.Q. Shi, Y.Z. Zhang, Ultra-sensitive biosensor for K-ras gene detection using enzyme capped gold nanoparticles conjugates for signal amplification, Anal. Biochem. 460 (2014) 47-53. [32] X.Y. Wang, G.F. Shu, C.C. Gao, Y. Yang, Q. Xu, M. Tang, Electrochemical biosensor based on functional composite nanofibers for detection of K-ras gene via multiple signal amplification strategy, Anal. Biochem. 466 (2014) 51-8. [33] Y.J. Liu, F. Yin, Y.M. Long, Z.H. Zhang, S.Z. Yao, Study of the immobilization of alcohol dehydrogenase on Au-colloid modified gold electrode by piezoelectric quartz crystal sensor, cyclic voltammetry, and electrochemical impedance techniques, J. Colloid. Int. Sci. 258 (2003) 75-81. [34] L.X. Cao, Q.C. Li, J.S. Ji, P.S. Yan, T. Wang, Development and rvaluation of ultramicro electrode ensembles based on 1.6-Hexanedithiol self-assembled modified layer and its application for HRP detection, Int. J. Electrochem. Sci. 8 (2013) 3074-83. [35] A. Sivanesan, P. Kannan, S.A. John, Electrocatalytic oxidation of ascorbic acid
using a single layer of gold nanoparticles immobilized on 1, 6-hexanedithiol modified gold electrode, Electrochim. Acta 52 (2007) 8118-24. [36] C.H. Luo, H. Tang, W. Cheng, L. Yan, D.C. Zhang, H.X. Ju, S.J. Ding, A sensitive electrochemical DNA biosensor for specific detection of Enterobacteriaceae bacteria by Exonuclease III-assisted signal amplification, Biosens. Bioelectron. 48 (2013) 132-7. [37] R.M. Kong, Z.L. Song, H.M. Meng, X.B. Zhang, G.L. Shen, R.Q. Yu, A label-free electrochemical biosensor for highly sensitive and selective detection of DNA via a dual-amplified strategy, Biosens. Bioelectron. 54 (2014) 442-7. [38] Y.L. Huang, Z.F. Gao, H.Q. Luo, N.B. Li, Sensitive detection of HIV gene by coupling exonuclease III-assisted target recycling and guanine nanowire amplification, Sens. Actuators B Chem. 238 (2017) 1017-23. [39] W. Wang, L.N. Song, Q. Gao, H.L. Qi, C.X. Zhang, Highly sensitive detection of DNA using an electrochemical DNA sensor with thionine-capped DNA/gold nanoparticle conjugates as signal tags, Electrochem. Commun. 34 (2013) 18-21. [40] T. Liu, J.A. Tang, L. Jiang, The enhancement effect of gold nanoparticles as a surface modifier on DNA sensor sensitivity, Biochem. Biophys. Res. Commun. 313 (2004) 3-7.
Author Biographies Xiaofan Sun is a graduate student at School of Biotechnology, Jiangnan University, China. Her research interests mainly focus on the fabrication of electrochemical DNA biosensor. Shuling Wang received her M.S. degree in biochemistry and molecular biology from Jiangnan University, China in 2016. She mainly engaged in the fabrication of electrochemical DNA biosensor. Yiping Zhang is an undergraduate student at School of Biotechnology, Jiangnan University, China. Her current research focuses on the electrochemical DNA biosensor. Yaping Tian received her Ph.D. degree in fermentation engineering from Jiangnan University, China in 2006. She is currently a professor at School of Biotechnology, Jiangnan University. Her research focuses on the analysis and production of bio-active molecules. Nandi Zhou received his Ph.D. degree in biochemistry and molecular biology from Nanjing University, China in 2007. He is currently a professor at School of Biotechnology, Jiangnan University. His research focuses on aptamer-based bioanalysis, fabrication of novel nano-biosensors, monitoring and control of harmful residues in food, and real-time analysis of metabolites in fermentation process.
Figure Captions Fig. 1. (A) Cyclic voltammograms of bare Au (a), HDT-Au (b) and AuNPs-HDT-Au (c) electrodes in 0.1 mM [Fe(CN)6]3-/4-; (B) Electrochemical impedance spectra (EIS) of bare Au (a), HDT-Au (b), and AuNPs-HDT-Au (c) electrodes.
Fig. 2. The DPV curves recorded under different conditions: (a) 350 nM P1 and 500 nM P2, (b) 350 nM P1, 500 nM P2 and 50 U Exo III, (c) 350 nM P1, 500 nM P2 and 500 nM TD, (d) 350 nM P1, 500 nM P2, 500 nM TD and 50 U Exo III.
Fig. 3. The influence of (A) the concentration of P1, (B) the concentration of P2, (C) the amount of Exo III, and (D) the incubation time of Exo III on the peak current in DPV.
Fig. 4. (A) DPV curves recorded in the presence of various concentration of target DNA; (B) Calibration curve corresponding to the peak current measured at -0.32 V in DPV and the concentration of target DNA; Inset shows the linear relationship between the peak current and the logarithm of the concentration of the target DNA. Error bars represent standard deviations of measurements (n=3).
Fig. 5. Histograms of relative current changes of the DNA sensor towards 20 nM target DNA and the mismatched sequences.
Scheme 1. Principle of the electrochemical detection of K-ras DNA based on target-triggered hairpin assembly and Exo III-assisted target recycling amplification.
Table 1. The comparison of the analytical performance between the reported DNA sensors and this work. Signal amplification strategy
Detection
Linear range
Detection
Reference
method
(pM)
limit (fM)
Catalyzed hairpin assembly
Fluorescence
1-2000
1000
[7]
Hybridization chain reaction
ECL
0.025-100
15
[15]
Nicking endonuclease
DPV
0.0001-0.02
0.08
[25]
Gold nanoparticles and horseradish peroxidase
DPV
0.0001-1000
0.035
[31]
Functional composite nanofibers
DPV
0.1-100
30
[32]
Exonuclease III
DPV
0.01-1000
8.7
[36]
Nanoparticle and enzyme
DPV
1-10000
600
[37]
Guanine nanowire
Amperometry
10-100000
3600
[38]
Gold nanoparticles and exonuclease III
DPV
0.05-10000
2.86
This work