Enzyme- and label-free amplified fluorescence DNA detection using hairpin probes and SYBR Green I

Sensors and Actuators B 200 (2014) 117–122

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Enzyme- and label-free amplified fluorescence DNA detection using hairpin probes and SYBR Green I Jiahao Huang a , Xuefen Su b , Zhigang Li a,∗ a

Department of Mechanical and Aerospace Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong School of Public Health and Primary Care, Faculty of Medicine, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong b

a r t i c l e

i n f o

Article history: Received 11 November 2013 Received in revised form 25 March 2014 Accepted 11 April 2014 Available online 24 April 2014 Keywords: DNA detection Hairpin probes SYBR Green I Fluorescence amplification BRCA1 gene

a b s t r a c t We report an enzyme- and label-free scheme for amplified fluorescence DNA detection by using two complementary hairpin probes (HPs, HP1 and HP2). SYBR Green I, which fluoresces when binding with double stranded DNA, was used for signal generation. In this scheme, the target DNA is repeatedly used to trigger continuous HP1–HP2 hybridizations, leading to amplified fluorescence after SYBR Green I reacts with the HP1–HP2 duplexes. The detection limit of this approach was experimentally obtained as 80 pM, which is comparable to other methods employing expensive enzymes or requiring complex labeling procedures. The current method also showed excellent selectivity, making it a promising platform for DNA-related molecular diagnosis. © 2014 Elsevier B.V. All rights reserved.

1. Introduction DNA detection has attracted considerable interest due to its broad applications in a variety of areas such as food analysis, environmental monitoring, forensic testing, and early diagnosis of diseases [1]. In the literature, various techniques, including optical and electrochemical methods, have been reported for DNA detections [2–4]. In certain applications, the low-abundant DNA biomarkers in biological samples pose a challenge for effective detection since the signals induced by them are difficult to be recognized. Therefore, it is highly desirable to develop DNA detection methods with satisfactory sensitivity. In the past decades, many strategies using different amplification mechanisms have been proposed for sensitive DNA detection. Employing enzyme to either duplicate the target or amplify the recognition signal is one of the most commonly used principles in many methods, such as the polymerase chain reaction [5,6], ligase chain reaction [7,8], rolling-circle amplification [9–12], cyclic nicking reaction [13–16], and exonuclease-assisted target regeneration methods [17–22]. Although these techniques are highly sensitive, the use of enzymes suffers from many limitations in practical

∗ Corresponding author. Tel.: +852 23587186. E-mail address: [email protected] (Z. Li). http://dx.doi.org/10.1016/j.snb.2014.04.032 0925-4005/© 2014 Elsevier B.V. All rights reserved.

applications because enzymes are expensive and usually sequencespecific. In addition, the detection procedures may involve complicated operations and require sophisticated instrumentation. Enzyme-free detection methods have also been developed [23–26]. Some of them take advantages of advanced materials [27–29], such as cationic conjugated polymers [30–32], magnetic microbeads [33–35], gold nanoparticles [36–39], carbon nanotubes [40,41], graphene [42–45], quantum dots [46–49] and single-layer molybdenum disulfide [50], to improve the detection sensitivity. These methods have unique properties, including excellent capability of carrying large number of signal probes and high quenching ability in suppressing background signals. However, the complex material fabrication and potential toxic effects may hinder the applications of these methods. Other enzyme-free detection strategies use novel ideas to strengthen the recognition signals [51–54]. For instance, the response signal could be amplified by a long DNA concatemers consisting of a series of targets and signal probes [51–53], or the continuous formation of molecular beacon duplexes [54] initiated by the target. All these enzyme-free methods could achieve high sensitivity, however, most of them require labeling relevant probes, which makes the detection relatively complex and expensive. In this work, we propose an enzyme- and label-free strategy for sensitive and selective DNA detection by using the unique signaling mechanism of SYBR Green I and the formation of duplexes triggered by the target. Two complementary hairpin probes (HPs), HP1 and

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HP2, are employed. A segment of BRCA1 gene, which produces proteins to help repair damaged DNA in breast cells, was chosen as the target. HP1 is specially designed such that its stem and part of the loop sequences are complementary to the target. In the absence of the target, HP1 and HP2 could maintain their stable structures and do not react in the buffer solution. When the target is introduced, it hybridizes with and opens HP1. This triggers the formation of HP1–HP2 duplex and its binding with SYBR Green I to generate fluorescent signal. At the moment of HP1–HP2 hybridization, the target is freed and becomes available to initiate the formation of another HP1–HP2 duplex and amplify the fluorescence emission. This method does not involve any enzyme and is label-free. Due to the amplified fluorescence signal ensured by the repeated use of the target, high sensitivity is expected.

2. Experimental 2.1. Materials The hairpin DNA probes and oligonucleotides were synthesized by TaKaRa Bio Inc. (Dalian, China). Their sequences are listed in Table 1. Both HP1 and HP2 had a stem of 10 bp and a loop of 20 nt without any special labeling. The sequence of the target (oligo 1) was perfectly matched to the stem and part of the loop sequence of HP1. The sequences of HP1 and HP2 were completely complementary. Oligos 2 and 3 contained one and three mismatched bases, respectively, while oligo 4 was a random DNA sequence. SYBR Green I (10,000×), which can bind to double-stranded DNA (dsDNA) through both electrostatic intercalation and minor groove binding [55], was purchased from Invitrogen (Hong Kong). All the other reagents were of analytical grade and were used without further purification and modification. All the solutions were prepared with 10 × TAE/12.5 mM Mg2+ buffer solution (pH 8.0).

2.2. Fluorescence measurement All the fluorescence measurements were conducted using a F4500 fluorometer (Hitachi, Japan) at 24 ◦ C. According to the fluorescent properties of SYBR Green I, the excitation and emission wavelengths were set at 497 and 525 nm, respectively. The slit widths for both excitation and emission were set at 5 nm. To obtain the emission spectra, the samples were excited by a 497 nm-light and the fluorescence emission was scanned from 510 to 600 nm with a step of 1 nm. To confirm the sensing principle, samples that contained HP1 with and without HP2 were prepared. All the samples were of 600 ␮L and made by mixing the corresponding hairpin DNA probes, SYBR Green I, and buffer solution containing 10 × TAE/12.5 mM Mg2+ (pH 8.0). All the samples were incubated at 24 ◦ C for at least 10 min before the experiments.

Table 1 Hairpin DNA probes and other oligonucleotides used in the experiments. Name

Sequence (5 to 3 )

HP1 HP2 Oligo 1 (target) Oligo 2 (one-base mismatched) Oligo 3 (three-base mismatched) Oligo 4 (random)

GAACAAAAGGTTTTTTTTTTTGATTTTCTTCCTTTTGTTC GAACAAAAGGAAGAAAATCAAAAAAAAAAACCTTTTGTTC GAACAAAAGGAAGAAAATC GAACAAAAGGAATAAAATC

§

CAACAACAGGAATAAAATC CAACAATAACAACAAGGTA

The bold letters indicate the stem sequences of the hairpin DNA probes. The italic letters in HP1 show the sequences complementary to the target. The underlined letters represent the mismatched sites.

3. Results and discussion 3.1. Detection mechanism The detection mechanism is demonstrated in Fig. 1. In the absence of the target, the hairpin DNA probes, HP1 and HP2 (Table 1) adapt the stem-loop structure individually due to the hybridization of the complementary sequences at their ends. Although HP1 and HP2 are totally complementary to each other, the stem-loop structures are quite stable and their hybridization is not favored. Since the sequences in the stem and part of the loop of HP1 are complementary to the target, the target hybridizes with and opens the hairpin structure of HP1 when it is introduced into the solution. This, simultaneously, exposes the complementary sequence of HP1 to HP2, leading to the binding of HP2 with HP1. As HP1–HP2 duplex is more stable than that of the targetHP1 complex, HP2 will replace and free the target, and hybridize with HP1 completely (Fig. 1a). After the formation of HP1–HP2 duplex, SYBR Green I will bind to the dsDNA and generate strong fluorescence signal (Fig. 1b). The freed target then becomes available to react with a new HP1 and trigger the formation of another HP1–HP2 complex, amplifying the fluorescence emission. Theoretically, such circular reactions (Fig. 1a) will continue till all the possible HP1–HP2 hybridizations are formed. Therefore, in this enzyme- and label-free strategy, a small amount of target can excite substantial fluorescence emission, leading to high sensitivity. In the detection mechanism, the continuous circular reactions triggered by the target (Fig. 1a) and the SYBR Green I-dsDNA binding for fluorescence generation (Fig. 1b) play essential roles. To confirm the working principle of SYBR Green I, the fluorescence responses were monitored when SYBR Green I reacted with the target DNA (single-stranded DNA (ssDNA)) and HP1 (partial dsDNA), as shown in Fig. S1 (Supporting information). It is seen that SYBR Green I barely fluoresces when it bound with ssDNA, while the fluorescence intensity was significant when binding with dsDNA (the stem of HP1). This is consistent with the tests performed in the literature [56,57]. It is also noted that the stem-loop structure of the HPs makes them thermodynamically stable and their hybridization is not favored without the presence of the target although they are totally complementary to each other [23,24]. 3.2. Detection feasibility To examine the feasibility of the proposed strategy, the fluorescence response of the sensing platform was recorded before and after 3 nM target DNA was introduced, as shown in Fig. 2, where the HP and SYBR Green I concentrations were 15 nM and 0.75×, respectively. After the addition of the target DNA, the fluorescence intensity of the sensing system (solution containing HP1 and HP2 probes and SYBR Green I) increased significantly. Compared with the case containing HP1 only, the current sensing platform led to 8.6-fold increase in the signal gain after 1 h reaction (Fig. 2), which is calculated as (FHP1+HP2+Target − FHP1+HP2 )/(FHP1+Target − FHP1 ) with F being the fluorescence intensity. Evidently, the signal enhancement was induced by the recycling use of the target DNA and the continuous production of HP1–HP2 complexes as shown in Fig. 1. It is also found that the signal of the sensing platform did not change in the absence of the target DNA. This is because HP1 and HP2 were sufficiently stable and their hybridization was not favored. Hence, the background signal of the sensing method only came from the weak fluorescence emission of SYBR Green I when binding with the stems of the HPs. It is noted that the reaction time depends on the target concentration. For target concentration of 10 nM or higher, 1 h was sufficiently long for the signal to slowly level off. The performance of the detection scheme could be affected by the concentrations of HPs and SYBR Green I. By varying the HP

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Fig. 1. Principles for DNA detection using hairpin DNA probes and SYBR Green I. (a) Working mechanism of the target-induced hybridizations between HP1 and HP2. HP1 and HP2 assume stem-loop structures, which are thermodynamically stable in solutions without the presence of the target. When the target appears, it binds and opens HP1. Then HP2 hybridizes with HP1 to form HP1–HP2 duplex and liberates the target, which becomes available to trigger another reaction cycle for the formation of HP1–HP2 complex. (b) Schematic of the signaling principle of the sensing platform, where SYBR Green I binds with HP1–HP2 complexes (dsDNA) and generates a very strong fluorescence signal.

and SYBR Green I concentrations, the background and response signals of the sensing platform were investigated. As depicted in Fig. S2, the signal-to-background (S/B) ratio changed nonlinearly and assumed optimal values when the concentrations of HPs and SYBR Green I were increased from 1 to 45 nM and from 0.125× to 0.625×, respectively. Further experiments showed that the optimal S/B ratio reached about 3.02 at 15 nM and 0.375× concentrations for HPs and SYBR Green I, which were used for the experiments of testing the sensitivity and selectivity of the detection strategy. 3.3. Detection sensitivity and selectivity The sensitivity of the detection method was investigated by changing the target concentration from 80 pM to 10 nM. The emission spectra after one hour are shown in Fig. 3a, where HP1, HP2, and SYBR Green I were mixed for 2 h first, and then the target was introduced. In Fig. 3a, it is seen that the spectra could be clearly identified, especially in the vicinity of 525 nm, as the target concentration was varied. The dependence of the fluorescence intensity on the target concentration at the wavelength of 525 nm is

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illustrated in Fig. 3b, which indicated that the number of HP1–HP2 duplexes triggered by the target increased with increasing target concentration. The detection limit was experimentally determined as 80 pM, which was the lowest target concentration for producing distinguishable fluorescence from the background signal. It is close to the theoretical value 62 pM, which is calculated using 3/S, where  is the standard deviation of the background signal and S is the slope of the fluorescence-logarithmic target concentration line shown in the inset of Fig. 3b. This detection limit is comparable to many other sensing methods for BRCA1 gene detection [19,34]. Table SI (Supporting information) lists the detection limit of other sensing systems for BRCA1 gene related DNA detections. To confirm that the high sensitivity of the current method is the consequence of the target-catalyzed circular reactions shown in Fig. 1a, control experiments involving HP1 only were performed at different target concentrations. Fig. 4a shows the time response of the fluorescence intensity at the wavelength of 525 nm when the target concentration was varied from 2.5 to 20 nM. A careful comparison between Figs. 3a and 4a shows that the amount of the target needed for the proposed sensing platform (Fig. 3) was much smaller than that without the aid of HP2 (Fig. 4) in order to generate comparable fluorescence intensity. The detection limit of the scheme without HP2 was about 2.5 nM (Fig. 4b), which is about 30-fold poorer than that of the current sensing method. The selectivity of the sensing platform was also evaluated by using one base-, three base-mismatched, and random DNA sequences (Table 1). The fluorescence intensities after the addition of 5 nM different DNA sequences are depicted in Fig. 5. At the emission wavelength of 525 nm, the fluorescence intensities for the perfectly matched, single-base-mismatched, three-basemismatched, and random DNA were about 2.48, 1.34, 1.07, and 1.01 times the background signal, respectively. Therefore, the proposed method possessed the capability for sequence-specific DNA detection. This might be due to the relatively long stems of the HPs which make the hairpin structures thermodynamically stable and difficult to be opened by mismatched sequences. However, it should be noted that the interactions between the stems of the HPs and SYBR Green I could generate a relatively high background signal if the stem is too long, which will reduce the selectivity of the sensing method. For enzyme-free methods, a potential technique to reduce the background signal could be using new nanomaterials, such as graphene oxide (GO), which has excellent fluorescence quenching ability and has been used for various detections [58]. GO can be employed in the current method by slightly modifying the

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structure of the HPs [58]. With the reduced background signal, the selectivity can be greatly improved. This will be investigated in the future work. Finally, it is worth mentioning that the stem length of the HPs in the sensing platform needs to be carefully designed. If the stem length is too short, the HPs may not be sufficiently stable and their hybridization could cause a high background signal and deteriorate the detection sensitivity. On the other hand, if the stem length is too long, the target may not be able to open HP1 and the current detection principle will not work. We studied the effect of the stem length of the HPs and 10 bp employed in this work was found the most feasible configuration in terms of S/B ratio and signal gain, as shown in Fig. S3. This might be the limitation of the current method. Other than this, the proposed strategy is an operationally simple and low-cost approach for highly sensitive and selective DNA detection. Moreover, if the lengths and sequences of the hairpin probes are well designed, the proposed method can be applied to other DNA targets.

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Fig. 4. Fluorescence responses of the control experiment containing 15 nM HP1 and 0.375× SYBR Green I. (a) Fluorescence responses of a control involving HP1 only upon incubation with different target concentrations for half an hour. (b) The relationship of the fluorescence intensity with the target concentration for the control experiments. The error bars represent the standard deviations of three independent measurements.

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nl y DN A DN A NA es o DNA Prob Random matched atched atched D m s m i s e mi erfectly se m e-ba ngle-bas P Thr e Si Fig. 5. Fluorescence intensity of the sensing platform upon incubation with perfectly matched and mismatched DNA for 1 h at 525 nm wavelength. The samples contained 15 nM HP1 and HP2 and 0.375× SYBR Green I.

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4. Conclusions In summary, we have demonstrated a target-activated HP-based highly sensitive and selective DNA biosensor with SYBR Green I as the signal-generation entity. It is enzyme- and label-free. The detection limit of the approach was 80 pM, which is about 30-fold lower than that of the conventional method without any signal amplification principle. Therefore, it is a simple, sensitive, and selective strategy for picomolar DNA detection and could be applied in the field of DNA-related molecular diagnosis. Finally, we would like to point out that the proposed sensing method can be used to monitor a wide range of DNA targets if the HPs are well designed. Acknowledgements This work was supported by the Research Grants Council of the Hong Kong Special Administrative Region under Grant nos. 615710 and 615312. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2014.04.032. References [1] E. Farjami, L. Clima, K. Gothelf, E.E. Ferapontova, “Off-On” electrochemical hairpin-DNA-based genosensor for cancer diagnostics, Anal. Chem. 83 (2011) 1594–1602. [2] J.E.N. Dolatabadi, O. Mashinchian, B. Ayoubi, A.A. Jamali, A. Mobed, D. Losic, Y. Omidi, M. de la Guardia, Optical and electrochemical DNA nanobiosensors, TrAC, Trends Anal. Chem. 30 (2011) 459–472. [3] K.M. Wang, Z.W. Tang, C.Y.J. Yang, Y.M. Kim, X.H. Fang, W. Li, Y.R. Wu, C.D. Medley, Z.H. Cao, J. Li, P. Colon, H. Lin, W.H. Tan, Molecular engineering of DNA: molecular beacons, Angew. Chem. Int. Ed. 48 (2009) 856–870. [4] A. Sassolas, B.D. Leca-Bouvier, L.J. Blum, DNA biosensors and microarrays, Chem. Rev. 108 (2008) 109–139. [5] Z. Cheglakov, Y. Weizmann, M.K. Beissenhirtz, I. Willner, Ultrasensitive detection of DNA by the PCR-induced generation of DNAzymes: the DNAzyme primer approach, Chem. Commun. 30 (2006) 3205–3207. [6] M. Hashimoto, F. Barany, S.A. Soper, Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations, Biosens. Bioelectron. 21 (2006) 1915–1923. [7] Y.Q. Cheng, Q. Du, L.Y. Wang, H.L. Jia, Z.P. Li, Fluorescently cationic conjugated polymer as an indicator of ligase chain reaction for sensitive and homogeneous detection of single nucleotide polymorphism, Anal. Chem. 84 (2012) 3739–3744. [8] Y.Q. Cheng, E.J. Wee, M.J. Shiddiky, M.A. Brown, M. Trau, eLCR: electrochemical detection of single DNA base changes via Ligase Chain Reaction, Chem. Commun. 48 (2012) 12014–12016. [9] S.B. Zhang, Z.S. Wu, G.L. Shen, R.Q. Yu, A label-free strategy for SNP detection with high fidelity and sensitivity based on ligation-rolling circle amplification and intercalating of methylene blue, Biosens. Bioelectron. 24 (2009) 3201–3207. [10] P.M. Lizardi, X. Huang, Z. Zhu, P. Bray-Ward, D.C. Thomas, D.C. Ward, Mutation detection and single-molecule counting using isothermal rolling-circle amplification, Nat. Genet. 19 (1998) 225–232. [11] G. Nallur, C.H. Luo, L.H. Fang, S. Cooley, V. Dave, J. Lambert, K. Kukanskis, S. Kingsmore, R. Lasken, B. Schweitzer, Signal amplification by rolling circle amplification on DNA microarrays, Nucleic Acids Res. 29 (2001) e118. [12] W. Zhao, M.M. Ali, M.A. Brook, Y. Li, Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids, Angew. Chem. Int. Ed. 47 (2008) 6330–6337. [13] Y. Weizmann, M.K. Beissenhirtz, Z. Cheglakov, R. Nowarski, M. Kotler, I. Willner, A virus spotlighted by an autonomous DNA machine, Angew. Chem. Int. Ed. 45 (2006) 7384–7388. [14] J.W.J. Li, Y.Z. Chu, B.Y.H. Lee, X.L.S. Xie, Enzymatic signal amplification of molecular beacons for sensitive DNA detection, Nucleic Acids Res. 36 (2008) e36. [15] C.H. Lu, F.A. Wang, I. Willner, Zn2+ -ligation DNAzyme-driven enzymatic and nonenzymatic cascades for the amplified detection of DNA, J. Am. Chem. Soc. 134 (2012) 10651–10658. [16] B.J. Zou, Y.J. Ma, H.P. Wu, G.H. Zhou, Ultrasensitive DNA detection by cascade enzymatic signal amplification based on Afu flap endonuclease coupled with nicking endonuclease, Angew. Chem. Int. Ed. 50 (2011) 7395–7398. [17] X.L. Zuo, F. Xia, Y. Xiao, K.W. Plaxco, Sensitive, selective amplified fluorescence DNA detection based on exonuclease III-aided target recycling, J. Am. Chem. Soc. 132 (2010) 1816–1818.

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Biographies Jiahao Huang received his M.S. degree (2010) in biomedical engineering and B.S. degree (2007) in biotechnology at Hunan University. And now he is a Ph.D. student in the Department of Mechanical and Aerospace Engineering at The Hong Kong University of Science and Technology. His research interests are in the field of DNAbased optical biosensors. Xuefen Su received her Doctor of Science degree (2009) in Epidemiology from Harvard University School of Public Health and Master of Public Health (2004) in Community Health Education from Temple University, USA. Currently she is an Assistant Professor in the School of Public Health and Primary Care at The Chinese University of Hong Kong. Her research interests cover the area of breast cancer etiology and prevention, molecular epidemiology, and nutritional epidemiology. Zhigang Li received his Ph.D. (2005) in Mechanical Engineering from the University of Delaware, USA. and his M. Eng. degree (1999) in Thermal Engineering from Tsinghua University, PR China. He is now an Associate Professor in the Department of Mechanical and Aerospace Engineering at The Hong Kong University of Science and Technology. His research interests mainly focus on Micro/nano fluidics, biomolecule transport in micro/nano confinements, and bio-detection.