- Email: [email protected]
Dual-cyclical nucleic acid strand-displacement polymerization based signal amplification system for highly sensitive determination of p53 gene
Biosensors and Bioelectronics 86 (2016) 1024–1030
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Dual-cyclical nucleic acid strand-displacement polymerization based signal amplification system for highly sensitive determination of p53 gene Jianguo Xu a,b, Zai-Sheng Wu a,b,n, Hongling Li a,b, Zhenmeng Wang a,b, Jingqing Le a,b, Tingting Zheng a,b, Lee Jia a,b,n a Cancer Metastasis Alert and Prevention Center, and Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350002, China b Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou 350002, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 May 2016 Received in revised form 7 July 2016 Accepted 8 July 2016 Available online 9 July 2016
In the present study, we proposed a novel dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) based signal amplification system for highly sensitive determination of tumor suppressor genes. The system primarily consisted of a signaling hairpin probe (SHP), a label-free hairpin probe (LHP) and an initiating primer (IP). The presence of target DNA was able to induce one CNDP through continuous process of ligation, polymerization and nicking, leading to extensively accumulation of two nicked triggers (NT1 and NT2). Intriguingly, the NT1 could directly hybridize SHP, while the NT2 could act as the target analog to induce another CNDP. The resulting dual-CNDP contributed the striking signal amplification, and only a very weak blank noise existed since the ligation template of target was not involved. In this case, the target could be detected in a wide linear range (5 orders of magnitude), and a low detection limit (78 fM) was obtained, which is superior to most of the existing fluorescent methods. Moreover, the dual-CNDP sensing system provided a high selectivity towards target DNA against mismatched target and was successfully applied to analysis of target gene extracted from cancer cells or in human serum-contained samples, indicating its great potential for practical applications. & 2016 Elsevier B.V. All rights reserved.
Keywords: Dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) Hairpin probe Tumor suppressor gene p53
1. Introduction Sensitive and reliable detection of sequence-specific nucleic acids (DNA or RNA) that are relevant to cancer initiation, development and metastasis, is of great importance (Deng et al., 2014; Ho et al., 2002; Niemz et al., 2011). A variety of DNA analysis strategies including rolling circle amplification (RCA) (Cheng et al., 2009b; Li et al., 2010), hybridization chain reaction (HCR) (Chen et al., 2012; Huang et al., 2011), and strand displacement amplification (SDA) (Shi et al., 2013; Xu et al., 2016b) have been explored and could plausibly serve as promising alternatives to polymerase chain reaction (PCR). However, even if somewhat success has been achieved, the unwanted limitations, such as insufficient sensitivity (Xu et al., 2015a) or complex operation (Yang et al., 2016; Zhao n Corresponding authors at: Cancer Metastasis Alert and Prevention Center, and Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350002, China. E-mail addresses: [email protected] (L. Jia), [email protected] (Z.-S. Wu).
http://dx.doi.org/10.1016/j.bios.2016.07.029 0956-5663/& 2016 Elsevier B.V. All rights reserved.
et al., 2009) often confined its practical applications to some extent. In these regards, exploring more powerful DNA sensing approaches in a powerful and more straightforward manner is an unmet need for the development of new diagnostic and therapeutic tools. Actually, the crucial points in designing a new DNA amplification protocol to accomplish the goal of substantially increasing the assay ability should take two aspects into account: one is to suppress completely the background signal; another is to improve the output signal as much as possible. Although current research is ongoing to amplify signal and restrain background, lots of existing DNA-amplification protocols have not yet found a very reasonable way to integrate the two aspects into one system, which suffered from elevated background or poor amplification efficiency (Qiu et al., 2011; Xu et al., 2015b; Yang et al., 2015). Accordingly, the performance is compromised. To overcome the above-mentioned technique bottleneck, in the present study, we proposed a dual-cyclical nucleic acid stranddisplacement polymerization (dual-CNDP) based signal amplification system that functions for highly sensitive detection of p53
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
gene, which has been identified as a biomarker for cancers. For this system, two well-tailored hairpin probes (HPs), a FAM and quencher-attached signaling hairpin probe (SHP) and a label-free hairpin probe (LHP), were well designed. In the absence of target, the two oligonucleotide probes separately coexisted, producing a very weak fluorescence background. Once the precisely-matched target fit into the system, the ternary complex of SHP-p53-LHP was formed and the nick was ligated. With the subsequent polymerization and nicking performed consecutively, the target gene was displaced and one cyclical-nucleic acid strand-displacement polymerization (CNDP) occurred, producing two displaced products (i.e., NT1 and NT2). Notably, the NT1 could contribute directly to the further signal amplification via hybridizing with SHPs, while the NT2 could function as the target analog to trigger another CNDP. Given that two types of individual CNDP reactions were achieved by recycling target gene and NT2, we thus termed it as dual-CNDP based signal amplification system, and the sensitivity was high enough so that the target DNA could be down to 78 fM. More importantly, this method is available for the detection of target gene extracted from cancer cells or in the presence of human serum, suggesting its potential for practical applications. The oligonucleotide design, signaling mechanism, and their sensitivity and specificity for detecting target were reported below.
1025
2. Experimental section 2.1. Materials and reagents The oligonucleotides used in this study were shown in Table 1. SHP was custom-synthesized by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China), and others were from Invitrogen Bio Inc., (Shanghai, China). T4 DNA ligase, and universal Genomic DNA exaction kit were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China), while Klenow Fragment (3′–5′ exo-) polymerase, Nt. BbvCI nicking endonuclease, Taq DNA ligase, and low DNA ladder (namely DNA marker) were ordered from New England Biolabs (USA) Ltd. Deoxynucleotide triphosphates (dNTPs) and SYBR Green I were provided by Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China). All chemicals were of analytical grade obtained from commercial sources, and all aqueous solutions involved were prepared by double-distilled water obtained from a Kerton lab MINI water purification system (UK) (resistance ¼ 18.25 MΩ/cm). 1 NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) supplied by New England Biolabs (USA) Ltd was used as the reaction buffer throughout the experiment.
Table 1 Oligonucleotide sequences designed in the present study.
For both SHP and LHP, the lowercase letters could be self-assembled, and the half recognition sites of nickase are shown in italic. Bold regions in SHP and LHP are able to hybridize with the target DNA. The shaded fragments in IP1 and IP2 represent the sequences complementary to LHP. NT1 and NT2 have the same base sequences as the nicked/displaced oligonucleotide strands (see Scheme 1 for details). T1, T2, and T3 are the randomly-designed sequences, while MT1 and MT2 are the single-base mismatch targets with the mutant points in boxes. Forward primer and reverse primer were used to execute asymmetric PCR amplifications.
1026
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
2.2. Instrumentations The fluorescence measurement was performed on a Hitachi F-7000 fluorescence spectrometer at a 600 V PMT Voltage using a quartz fluorescence cell with an optical path length of 1.0 cm. The excitation was made at 492 nm with recording emission range of 500–600 nm, and the excitation and emission slits were set at 5.0 nm. When investigating the assay performance, the peak value at 518 nm was collected. A 12% native polyacrylamide gel (nativePAGE) was prepared and run at 80 V for 90 min at a gel electrophoresis instrument, and finally photographed with a ChemiDoc XRSþ imaging system (BIO-RAD, USA). Since FAM modification could be visualized, only the samples without SHP, for example, DNA ladders, were fluorescent stained by SYBR Green I. 2.3. Asymmetric PCR amplification The human lung cancer cell line A549 for genomic DNA extraction was prepared according to the universal Genomic DNA exaction kit after the cells reached 80% confluency. Forward primer 1/reverse primer 1 and forward primer 2/reverse primer 2 were used to amplify the p53 amplicons and control amplicons, respectively. The details were performed according to the procedure described previously with minor modification (Xu et al., 2016a). In brief, asymmetric PCR amplification was achieved in 50 μL of 1 PCR buffer containing 1 μg of genomic DNA, 0.5 μM forward primer, 0.1 μM reverse primer, 0.15 mM dNTP mixture, as well as 2.5 U of Taq polymerase. The thermal process was consisted of 30 cycles (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 55 s) after the initial denaturation step (94 °C for 5 min), and a final extension at 72 °C for 7 min. The PCR products were verified by 12% native-PAGE and used for fluorescent sensing. 2.4. Dual-CNDP-based sensing system for p53 gene detection In an eppendorf tube containing 17 μL of H2O and 3 μL of 10 NEBuffer 2, 1.5 μL of 10 μM SHP, 1.5 μL of 10 μM LHP, 1.5 μL of 10 μM IP1, and 3 μL of p53 gene at certain concentration were consecutively added and mixed thoroughly. After the tube was heated to 90 °C for 5 min and cooling down to room temperature,
0.5 μL of T4 DNA ligase (350 U/μL), 0.5 μL of klenow fragment (3′– 5′ exo-, 5 U/μL), 0.5 μL of Nt. BbvCI nicking endonuclease (10 U/ μL), and 1 μL of 10 mM dNTPs were added to the tube, and then incubated at 37 °C for 2 h with gentle shaking. Concentrations of all components were calculated from the resulting solution (30 μL). Finally, the reaction was terminated at 80 °C for 20 min according to the manufacturer instructions, and the fluorescence of the produced mixture was recorded after 170 μL of H2O was added.
3. Results and discussion 3.1. Working principle of the dual-CNDP amplification To minimize the background noise and to maximize the response signal, we developed the novel dual-CNDP amplification system, which was capable of to execute highly sensitive detection of p53 gene. The corresponding signaling principle was schematically illustrated in Scheme 1. The SHP modified with a fluorophore FAM and a quencher DABCYL consisted of a half recognition site for Nt. BbvCI nicking endonuclease and a recognizing portion at the wobbling 3′ end complementary to target gene, while the LHP also consisted of a half recognition site for Nt. BbvCI nicking endonuclease and the wobbling 5′ end was phosphorylated that partly hybridizes with target gene. The hairpin structure of SHP could force the FAM and DABCYL together to make the fluorescence of FAM almost entirely quenched by DABCYL due to the effect of fluorescence resonance energy transfer (FRET) (Cardullo et al., 1988; Tyagi and Kramer, 1996). In the absence of the target, the two oligonucleotide probes were separately coexisted. Thereby, separation of the HPs produced a very weak fluorescence background. Once the target molecule entered the system, however, the ternary complex of SHP-p53-LHP was formed to open the hairpin structure of LHP for further hybridization with IP simultaneously. Meanwhile, the nick was ligated by the formation of a phosphodiester bond, resulting in the extension of the 3′ end of IP by polymerase over the ligated SHPLHP complex. This enabled formation of the recognition sites for Nt. BbvCI nickase as well as the target recycling via displacement.
Scheme 1. Schematic Diagram of the Dual-CNDP Mediated Signal Amplification. Step ①: Formation of the ternary Complex of SHP-p53-LHP together with further hybridization with IP; Step ②: T4 DNA ligase mediated ligation; Step ③: Polymerization to promote the target-recycling and nicking sites formation, as well as fluorescence recovery; step ④: Continuous reaction of polymerization/nicking/displacement responsible for primary accumulations of NT1 and NT2; Step ⑤: Hybridization between NTI and SHP to contribute the further amplification of the fluorescence signal; Step ⑥: NT2 acts as the target-like template to initiate the more amplification reactions.
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
Of special note, the Nt. BbvCI is one kind of the restriction endonucleases capable of recognizing the specific double-stranded DNA of “5′-CCTCAGC-3′/3′-GGAGTCG-5′” and specifically cleaves the upper strand (Wen et al., 2012). Detailed nicking sites in the formed new duplex can be seen in Fig. S5. Therefore, the subsequent displacements of NT1 and NT2 based on the polymerization/nicking process along the new-formed duplex were realized. Actually, the full recycling events would not be possible without the nickase. Moreover, the molecules of FAM and DABCYL were moved away from each other, resulting in the restoration of fluorescence. Additionally, it is worthwhile pointing out that the resultant NT1 could positively hybridize with SHP to form a rigid duplex and amplify further the fluorescent signal, while the NT2 could serve as the target analog since part of the NT2 bases can hybridize with the 5′ end of LHP and the rest can hybridize with the 3′ end of SHP. Herein, both the target gene and NT2 can be repeatedly used to induce their own CNDP to continually generate more NT1 and NT2 molecules, which in return causes more CNDP reactions. These reactions and the species involved can work with high cooperativity, achieving the synergetic effect of ligase,
1027
polymerase and nickase and accomplishing the highly sensitive detection of p53 gene. As the consequence, the established dualCNDP system was able to amplify the fluorescence signal in a cataract fashion. In addition, since the 3′ end of NT2 could perfectly hybridize with SHP, the successful extension of NT2 over SHP could result in the fluorescence emission increase even in the absence of the ligase and nickase. This amplification strategy maximizes the fluorescence recovery and achieves the multiple amplification effect on signal enhancement. Clearly, one target molecule can induce the production of many NT1 and many NT2. Moreover, each NT2 can execute the target function to initiate the amplification reaction. Additionally, each of hybridized NT2 or hybridized target DNA can be displaced by nicking after polymerization on ligated LHP-SHP probe and repeatedly implement the amplification process, leading to the dramatic accumulation of NT1 that can open SHP. Herein, the concept of “multiple amplification” was used to describe the molecular mechanism for signal transduction. In fact, even when the target gene was only at 5 nM, the system could run in high efficiency and dramatically increase the fluorescence signal following many rounds of isothermal cycles to enable the sensitive detection of the target p53 gene (see below). 3.2. Feasibility demonstration of the dual-CNDP
Fig. 1. Comparison in fluorescent intensity generated from different mixtures: (a) SHP, (b) SHP þLHP þ IP1, (c) SHPþ LHP þIP1 þLigase, (d) SHPþ LHP þIP1 þ p53 þLigase, (e) SHPþ LHP þIP1 þLigase þ Polymerase, (f) SHPþ LHP þIP1 þ p53 þLigase þ Polymerase, (g) SHP þ LHPþ IP1þ Ligase þPolymerase þNickase, (h) SHPþ LHP þIP1 þp53 þ Ligaseþ Polymerase þ Nickase. The concentrations of SHP, LHP, and IP1 were all 500 nM, while the p53 gene was 5 nM.
To investigate whether the dual-CNDP amplification was able to report the target DNA, different samples were prepared in optimized conditions (see Fig. S1) and the measured results were depicted in Fig. 1. We observed extremely weak fluorescence for SHP (curve a). The coexistence of LHP and IP1 could not affect the fluorescence emission of SHP (curve b). Similar results were obtained when ligase was added to the samples regardless of the absence or presence of target (curve c and d). However, upon incubation with the polymerase (curve f), a detectable signal increase was recorded compared with curve e, indicating the successful ligation of SHP and LHP when the target was used as the template, as well as the resultant polymerization of IP1 over the ligated SHP-LHP complex followed by target recycling. Excitingly, as shown in the considerable difference between curve h and g, a remarkable signal enhancement was observed when nickase was added. The corresponding target-to-blank ratio of fluorescent peak value was more than 10. The results clearly demonstrated the utility of designed signal amplification strategy. Furthermore, the background fluorescence in curve g was very close to the inherent fluorescence of SHP (curve a), suggesting the background was still
Fig. 2. Extremely significant differences in fluorescence intensity between samples with and without NT1 or NT2. (A), fluorescence intensity difference between samples with 500 nM SHP (a), or with 500 nM SHP plus 100 nM NT1 (b). (B), substantial fluorescent enhancement induced by NT2 (5 nM; h curve). The experiments were conducted where NT2 was used to replace the target stimulus following the same experimental procedures: (a) SHP, (b) SHP þLHP þ IP1, (c) SHP þLHP þ IP1þ Ligase, (d) SHPþ LHP þIP1 þNT2 þ Ligase, (e) SHP þLHP þ IP1þ Ligase þPolymerase, (f) SHP þLHP þ IP1þ NT2 þLigase þ Polymerase, (g) SHP þLHP þ IP1þ Ligaseþ Polymerase þ Nickase, (h) SHPþ LHP þIP1 þ NT2 þLigase þ Polymerase þ Nickase.
1028
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
very low. These outcomes were in great accordance with the principle designed and experimentally confirmed the feasibility of our dual-CNDP sensing system for the amplification detection of target DNA. Gel electrophoresis results were also carefully tested and corroborated the expected protocol (Fig. S2). 3.3. NTs-induced signal amplification In this section, the discussion pointed to verify the reactions that the NT1 caused fluorescence recovery and NT2 induced signal amplification. As witnessed in Fig. 2A, a significant increase of fluorescent intensity was achieved in curve b compared with curve a, indicating that NT1 could efficiently open the hairpin structure of SHP. Fig. 2B displayed the similar trend as shown in Fig. 1, where NT2 was used instead of the target under identical conditions (see the experimental section). In addition, a visible increase in fluorescent response was detected for sample f, suggesting that the fluorescence response induced by NT2 was relatively higher than that by the target DNA due to that NT2 was easier to induce the polymerization than the target DNA. It seems reasonable because the 3′ end of NT2 is activated and the target is passivated. Moreover, NT2 is able to induce the remarkable signal amplification as demonstrated by the results obtained from Fig. 2B in the presence of nickase (curve h versus g). More supporting data demonstrated the NT2 amplification effect by native-PAGE was given in Fig. S4. 3.4. Capability to sense target A number of genes have been discovered that associate with the increased susceptibility to a variety of disease states due to their functions in encoding, transmitting, and expressing genetic information. Hence, reliable analysis of the given DNA is necessary for diagnosis and monitoring of diseases. After establishing the dual-CNDP amplification strategy, we assessed the dynamic response of our system to different target concentrations under identical experimental conditions. As shown in Fig. 3A, the fluorescence intensity increased linearly with the increment of target concentration ranging from 0 to 5 nM, and there was no linear relationship between fluorescence intensity and target concentrations beyond 5 nM (Fig. 3B) in view of the exhaustion of SHP molecules. Fig. 3B displayed an impressive calibration curve responding to the fluorescent intensity against target concentrations ranging from 100 fM to 5 nM. The regression equation was fitted to Y¼ 185.66 þ391.48X with a correlation coefficient R2 of 0.9900, where Y and X represent the peak value of fluorescence at 518 nm (the maximum emission wavelength) and target concentration,
respectively. The limit of detection was calculated as 78 fM according to the 3s rule. Additionally, the relative standard deviation of the mean data was within 5.0%, indicating an acceptable repeatability. Detailed comparison of the current study with reported ones for gene detection was given in Table S1. The present dual-CNDP amplification sensing system is obviously improved with respect to the sensitivity and dynamic range with some literature reports (Gao et al., 2014; Li et al., 2014; Wang and Zhang, 2012; Zhou et al., 2013). However, the analytical performance of the dual-CNDP amplification strategy should be further improved to meet unique public demands when compared with some transducing techniques (Shen et al., 2012; Wang et al., 2013). Nevertheless, via circumventing the intrinsic drawbacks of the electrical and colorimetric sensing strategies, such as complex electrical interface construction (Wang et al., 2013) and time-consuming oligonucleotide-conjugated golden nanoparticles synthesis (Shen et al., 2012), an inspiring fact is that the dual-CNDP amplification process only involved several simple mixing steps, and it is very easy to manipulate the target detection. Moreover, it is still a great challenge to construct powerful amplification platforms to produce significant signal upon a trace amount of target DNAs. We addressed the technical limitation via proposing the dual-CNDP based sensing system that provided very weak background noise but offered the multiple amplification of the target signal. Generally, as a standard method for gene amplification, fluorescence-based real-time PCR has been widely used as it combines the excellent experimental amplification effect with quantitative detection of the amplified product and possesses ultrahigh sensitivity and a wide dynamic range. However, as reported, the PCR technique has some fundamental limitations of use. For instance, the requirement of an expensive electrically powered thermal cycler and sometimes skilled technician. This may be the reason that increasing concern has been devoted for exploiting alternative amplification strategies methodologically (Shen et al., 2014; Xia et al., 2010). In our case, although the fluorescence labeling is involved (just like the TaqMan probe used in PCR), the goal is to present a new isothermal amplification protocol, which is expected to encourage the following studies. 3.5. Detection selectivity of dual-CNDP amplification system To evaluate the selectivity of the dual-CNDP sensing method, several samples of the same concentration of either the target gene (T) or mismatched targets were prepared and tested using a sample without any target as the blank control. T1, T2, and T3 are
Fig. 3. (A) Typical fluorescence spectra of the sensing system upon addition of different concentrations of p53 gene. The inset amplified the fluorescence spectra of the targets at the low concentration. (B) Dynamic relationship between the fluorescence intensity and p53 gene concentration. The error bars represented the standard deviation of three replicate measurements.
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
1029
Fig. 4. High selectivity of the dual-CNDP system. Only the target (T) that precisely matched the system produced substantial fluorescent intensity, while the target analogs (T1, T2, and T3) and single-base mismatched target (MT1 and MT2) produced insignificant fluorescence. The relative signal response is calculated as [(F-F0)/(Ft-F0)]100%, where F0, Ft, and F are the fluorescence values for the control sample, target gene (T), and mismatched ones (T1, T2, T3, MT1, and MT2), respectively. Concentrations of all target analytes were 20 nM. Error bars were the standard deviation of three replicate experiments.
Fig. 5. Comparison in asymmetric PCR amplifications between p53 amplicons and control amplicons. (A), asymmetric amplicons demonstrated by native-PAGE. Lane a and lane b indicated the p53 amplicons and control amplicons, respectively; (B), changes in fluorescent intensity of p53 amplicons and control amplicons. Error bars were the standard deviation of three replicate experiments.
three randomly-designed target, while MT1 and MT2 were projected with single-base mismatch at the ligation sites. As shown in Fig. 4, only the perfect complementary target induces a substantially signal increase, while the responses from poorly-matched oligonucleotide sequences (T1, T2, and T3) are very low (Fig. 4A). This should be attributed to the stringent match ability between oligonucleotide strands. Besides, since the T4 DNA ligase has been demonstrated with a poor specificity to discriminate a single-base mutation (Cheng et al., 2009a), the selectivity study for single-base mismatched targets was performed by using Taq DNA ligase. We can see that the signal achieved by the single-base mismatched targets (MT1 and MT2) substantially decreases when compared with the perfectly-matched target (seen in Fig. 4B), indicating the satisfactory selectivity. 3.6. Target detection from real biological samples Practical applicability of the dual-CNDP sensing system for target gene detection was demonstrated through the detection of genomic DNA from cancer cells. Prior to detection, an asymmetric PCR should be employed based on the use of different amount of forward primer and reverse primer to generate the single-stranded counterpart since the conventional PCR by using equal
amount of forward primer and reverse primer only produces double-stranded DNAs, which were not suitable for directly detection. In our study, the genomic p53 gene was amplified by asymmetric PCR to produce the single-stranded target amplicons suitable for the subsequent detection. It should be noted that both the target amplicons and control amplicons were obtained via amplifying the extracted genomic p53 gene. But, the target amplicons contained the expected target sequence, while the control amplicons were a random sequence with the similar length as target amplicons. As shown in Fig. 5A, two bright bands correspond individually to the designed p53 amplicons in lane a and control amplicons in lane b, confirming the asymmetric amplifications. By the way, the single-stranded counterpart moved faster than the double-stranded amplicons because the migration rate was inversely proportional to the molecule weight. However, the resultant single-stranded DNAs (ssDNA) were not visible due to the relatively poor intercalating ability of SYBR green I to the ssDNA. Fig. 5B shows the measured fluorescent difference between the p53 amplicons and the control amplicons. We can see that the control amplicons only induce a very weak fluorescence change, which was significantly lower than that produced by the p53 amplicons, indicating the great potential for real sample applications. In addition, potential application of the strategy to complex
1030
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
biological samples was demonstrated by detecting the target gene in the presence of human serum (Fig. S3).
4. Conclusion In conclusion, by using cancer-related gene as the model analyte, we successfully constructed a dual-CNDP signal amplification system, in which a pair of HPs was admirably designed and applied to realize the robust sensing. The critical importance of the work is that: (1) the participation of ligase, polymerase, and nickase, as well as the continuous reactions of ligation, polymerization, and nicking, contributing to the dual-CNDP amplification; (2) the target DNA-caused CNDP and NT2-caused CNDP worked with high cooperativity. This enables the detection strategy that exhibits multiple amplification effect and very weak background, possessing the merits of low detection limit, wide linear range, and desirable selectivity; (3) the genomic DNA after asymmetric amplifications and the target DNA existing in human serum-contained samples can be encouragingly detected, indicating it a great promise for practical application. Owing to the above distinctive advantages and unique capabilities, the established assay is expected to provide valuable reference for design of more versatile strategies.
Acknowledgments This work was supported by Ministry of Science and Technology of China (2015CB931804), National Natural Science Foundation of China (NSFC) (Grant# 81273548, 21275002, and U1505225), and Independent Research Project of State Key Laboratory of Photocatalysis on Energy and Environment (No. 2014CO1).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.07.029.
References Cardullo, R.A., Agrawal, S., Flores, C., Zamecnik, P.C., Wolf, D.E., 1988. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 85 (23), 8790–8794. Chen, Y., Xu, J., Su, J., Xiang, Y., Yuan, R., Chai, Y., 2012. In situ hybridization chain reaction amplification for universal and highly sensitive electrochemiluminescent detection of DNA. Anal. Chem. 84 (18), 7750–7755. Cheng, Y., Zhang, X., Li, Z., Jiao, X., Wang, Y., Zhang, Y., 2009a. Highly sensitive determination of microrna using target-primed and branched rolling-circle amplification. Angew. Chem. 121 (18), 3318–3322. Cheng, Y., Zhang, X., Li, Z., Jiao, X., Wang, Y., Zhang, Y., 2009b. Highly sensitive determination of microrna using target-primed and branched rolling-circle amplification. Angew. Chem. 121 (18), 3318–3322. Deng, R., Tang, L., Tian, Q., Wang, Y., Lin, L., Li, J., 2014. Toehold-initiated rolling circle amplification for visualizing individual micrornas in situ in single cells.
Angew. Chem. Int. Ed. 53 (9), 2389–2393. Gao, Z.F., Ling, Y., Lu, L., Chen, N.Y., Luo, H.Q., Li, N.B., 2014. Detection of singlenucleotide polymorphisms using an on–off switching of regenerated biosensor based on a locked nucleic acid-integrated and toehold-mediated strand displacement reaction. Anal. Chem. 86 (5), 2543–2548. Ho, H.A., Boissinot, M., Bergeron, M.G., Corbeil, G., Doré, K., Boudreau, D., Leclerc, M., 2002. Colorimetric and fluorometric detection of nucleic acids using cationic polythiophene derivatives. Angew. Chem. 114 (9), 1618–1621. Huang, J., Wu, Y., Chen, Y., Zhu, Z., Yang, X., Yang, C.J., Wang, K., Tan, W., 2011. Pyrene-excimer probes based on the hybridization chain reaction for the detection of nucleic acids in complex biological fluids. Angew. Chem. Int. Ed. 50 (2), 401–404. Li, H., Wu, Z.-S., Shen, Z., Shen, G., Yu, R., 2014. Architecture based on the integration of intermolecular G-quadruplex structure with sticky-end pairing and colorimetric detection of DNA hybridization. Nanoscale 6 (4), 2218–2227. Li, J., Deng, T., Chu, X., Yang, R., Jiang, J., Shen, G., Yu, R., 2010. Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms. Anal. Chem. 82 (7), 2811–2816. Niemz, A., Ferguson, T.M., Boyle, D.S., 2011. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 29 (5), 240–250. Qiu, L.-P., Wu, Z.-S., Shen, G.-L., Yu, R.-Q., 2011. Highly sensitive and selective bifunctional oligonucleotide probe for homogeneous parallel fluorescence detection of protein and nucleotide sequence. Anal. Chem. 83 (8), 3050–3057. Shen, J., Li, Y., Gu, H., Xia, F., Zuo, X., 2014. Recent development of sandwich assay based on the nanobiotechnologies for proteins, nucleic acids, small molecules, and ions. Chem. Rev. 114 (15), 7631–7677. Shen, W., Deng, H., Gao, Z., 2012. Gold nanoparticle-enabled real-time ligation chain reaction for ultrasensitive detection of DNA. J. Am. Chem. Soc. 134 (36), 14678–14681. Shi, C., Liu, Q., Ma, C., Zhong, W., 2013. Exponential strand-displacement amplification for detection of microRNAs. Anal. Chem. 86 (1), 336–339. Tyagi, S., Kramer, F.R., 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14 (3), 303–308. Wang, G.-l, Zhang, C.-y, 2012. Sensitive detection of microRNAs with hairpin probebased circular exponential amplification assay. Anal. Chem. 84 (16), 7037–7042. Wang, X., Wang, X., Wang, X., Chen, F., Zhu, K., Xu, Q., Tang, M., 2013. Novel electrochemical biosensor based on functional composite nanofibers for sensitive detection of p53 tumor suppressor gene. Anal. Chim. Acta 765, 63–69. Wen, Y., Xu, Y., Mao, X., Wei, Y., Song, H., Chen, N., Huang, Q., Fan, C., Li, D., 2012. DNAzyme-based rolling-circle amplification DNA machine for ultrasensitive analysis of MicroRNA in Drosophila larva. Anal. Chem. 84 (18), 7664–7669. Xia, F., White, R.J., Zuo, X., Patterson, A., Xiao, Y., Kang, D., Gong, X., Plaxco, K.W., Heeger, A.J., 2010. An Electrochemical supersandwich assay for sensitive and selective DNA detection in complex matrices. J. Am. Chem. Soc. 132 (41), 14346–14348. Xu, J., Wu, Z.-S., Shen, W., Xu, H., Li, H., Jia, L., 2015a. Cascade DNA nanomachine and exponential amplification biosensing. Biosens. Bioelectron. 73, 19–25. Xu, J., Li, H., Wu, Z.-S., Qian, J., Xue, C., Jia, L., 2016a. Double-stem hairpin probe and ultrasensitive colorimetric detection of cancer-related nucleic acids. Theranostics 6 (3), 319. Xu, J., Wu, Z.-S., Wang, Z., Li, H., Le, J., Jia, L., 2016b. Two-wheel drive-based DNA nanomachine and its sensing potential for highly sensitive analysis of cancerrelated gene. Biomaterials 100, 110–117. Xu, Y., Zhou, W., Zhou, M., Xiang, Y., Yuan, R., Chai, Y., 2015b. Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and nonlabel sensitive fluorescent detection of thrombin. Biosens. Bioelectron. 64, 306–310. Yang, X., Tang, Y., Mason, S.D., Chen, J., Li, F., 2016. An enzyme-powered three dimensional DNA nanomachine for DNA walking, payload release, and biosensing. ACS Nano. Yang, Y., Huang, J., Yang, X., Quan, K., Wang, H., Ying, L., Xie, N., Ou, M., Wang, K., 2015. FRET nanoflares for intracellular mRNA detection: avoiding false positive signals and minimizing effects of system fluctuations. J. Am. Chem. Soc. 137 (26), 8340–8343. Zhao, C., Song, Y., Ren, J., Qu, X., 2009. A DNA nanomachine induced by singlewalled carbon nanotubes on gold surface. Biomaterials 30 (9), 1739–1745. Zhou, H., Wu, Z.-S., Shen, G.-L., Yu, R.-Q., 2013. Intermolecular G-quadruplex structure-based fluorescent DNA detection system. Biosens. Bioelectron. 41, 262–267.
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Dual-cyclical nucleic acid strand-displacement polymerization based signal amplification system for highly sensitive determination of p53 gene Jianguo Xu a,b, Zai-Sheng Wu a,b,n, Hongling Li a,b, Zhenmeng Wang a,b, Jingqing Le a,b, Tingting Zheng a,b, Lee Jia a,b,n a Cancer Metastasis Alert and Prevention Center, and Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350002, China b Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou 350002, China
art ic l e i nf o
a b s t r a c t
Article history: Received 8 May 2016 Received in revised form 7 July 2016 Accepted 8 July 2016 Available online 9 July 2016
In the present study, we proposed a novel dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) based signal amplification system for highly sensitive determination of tumor suppressor genes. The system primarily consisted of a signaling hairpin probe (SHP), a label-free hairpin probe (LHP) and an initiating primer (IP). The presence of target DNA was able to induce one CNDP through continuous process of ligation, polymerization and nicking, leading to extensively accumulation of two nicked triggers (NT1 and NT2). Intriguingly, the NT1 could directly hybridize SHP, while the NT2 could act as the target analog to induce another CNDP. The resulting dual-CNDP contributed the striking signal amplification, and only a very weak blank noise existed since the ligation template of target was not involved. In this case, the target could be detected in a wide linear range (5 orders of magnitude), and a low detection limit (78 fM) was obtained, which is superior to most of the existing fluorescent methods. Moreover, the dual-CNDP sensing system provided a high selectivity towards target DNA against mismatched target and was successfully applied to analysis of target gene extracted from cancer cells or in human serum-contained samples, indicating its great potential for practical applications. & 2016 Elsevier B.V. All rights reserved.
Keywords: Dual-cyclical nucleic acid strand-displacement polymerization (dual-CNDP) Hairpin probe Tumor suppressor gene p53
1. Introduction Sensitive and reliable detection of sequence-specific nucleic acids (DNA or RNA) that are relevant to cancer initiation, development and metastasis, is of great importance (Deng et al., 2014; Ho et al., 2002; Niemz et al., 2011). A variety of DNA analysis strategies including rolling circle amplification (RCA) (Cheng et al., 2009b; Li et al., 2010), hybridization chain reaction (HCR) (Chen et al., 2012; Huang et al., 2011), and strand displacement amplification (SDA) (Shi et al., 2013; Xu et al., 2016b) have been explored and could plausibly serve as promising alternatives to polymerase chain reaction (PCR). However, even if somewhat success has been achieved, the unwanted limitations, such as insufficient sensitivity (Xu et al., 2015a) or complex operation (Yang et al., 2016; Zhao n Corresponding authors at: Cancer Metastasis Alert and Prevention Center, and Pharmaceutical Photocatalysis of State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou 350002, China. E-mail addresses: [email protected] (L. Jia), [email protected] (Z.-S. Wu).
http://dx.doi.org/10.1016/j.bios.2016.07.029 0956-5663/& 2016 Elsevier B.V. All rights reserved.
et al., 2009) often confined its practical applications to some extent. In these regards, exploring more powerful DNA sensing approaches in a powerful and more straightforward manner is an unmet need for the development of new diagnostic and therapeutic tools. Actually, the crucial points in designing a new DNA amplification protocol to accomplish the goal of substantially increasing the assay ability should take two aspects into account: one is to suppress completely the background signal; another is to improve the output signal as much as possible. Although current research is ongoing to amplify signal and restrain background, lots of existing DNA-amplification protocols have not yet found a very reasonable way to integrate the two aspects into one system, which suffered from elevated background or poor amplification efficiency (Qiu et al., 2011; Xu et al., 2015b; Yang et al., 2015). Accordingly, the performance is compromised. To overcome the above-mentioned technique bottleneck, in the present study, we proposed a dual-cyclical nucleic acid stranddisplacement polymerization (dual-CNDP) based signal amplification system that functions for highly sensitive detection of p53
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
gene, which has been identified as a biomarker for cancers. For this system, two well-tailored hairpin probes (HPs), a FAM and quencher-attached signaling hairpin probe (SHP) and a label-free hairpin probe (LHP), were well designed. In the absence of target, the two oligonucleotide probes separately coexisted, producing a very weak fluorescence background. Once the precisely-matched target fit into the system, the ternary complex of SHP-p53-LHP was formed and the nick was ligated. With the subsequent polymerization and nicking performed consecutively, the target gene was displaced and one cyclical-nucleic acid strand-displacement polymerization (CNDP) occurred, producing two displaced products (i.e., NT1 and NT2). Notably, the NT1 could contribute directly to the further signal amplification via hybridizing with SHPs, while the NT2 could function as the target analog to trigger another CNDP. Given that two types of individual CNDP reactions were achieved by recycling target gene and NT2, we thus termed it as dual-CNDP based signal amplification system, and the sensitivity was high enough so that the target DNA could be down to 78 fM. More importantly, this method is available for the detection of target gene extracted from cancer cells or in the presence of human serum, suggesting its potential for practical applications. The oligonucleotide design, signaling mechanism, and their sensitivity and specificity for detecting target were reported below.
1025
2. Experimental section 2.1. Materials and reagents The oligonucleotides used in this study were shown in Table 1. SHP was custom-synthesized by Shanghai Sangon Biological Engineering Technology and Services Co. Ltd. (Shanghai, China), and others were from Invitrogen Bio Inc., (Shanghai, China). T4 DNA ligase, and universal Genomic DNA exaction kit were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China), while Klenow Fragment (3′–5′ exo-) polymerase, Nt. BbvCI nicking endonuclease, Taq DNA ligase, and low DNA ladder (namely DNA marker) were ordered from New England Biolabs (USA) Ltd. Deoxynucleotide triphosphates (dNTPs) and SYBR Green I were provided by Dingguo Changsheng Biotechnology Co., Ltd (Beijing, China). All chemicals were of analytical grade obtained from commercial sources, and all aqueous solutions involved were prepared by double-distilled water obtained from a Kerton lab MINI water purification system (UK) (resistance ¼ 18.25 MΩ/cm). 1 NEBuffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9) supplied by New England Biolabs (USA) Ltd was used as the reaction buffer throughout the experiment.
Table 1 Oligonucleotide sequences designed in the present study.
For both SHP and LHP, the lowercase letters could be self-assembled, and the half recognition sites of nickase are shown in italic. Bold regions in SHP and LHP are able to hybridize with the target DNA. The shaded fragments in IP1 and IP2 represent the sequences complementary to LHP. NT1 and NT2 have the same base sequences as the nicked/displaced oligonucleotide strands (see Scheme 1 for details). T1, T2, and T3 are the randomly-designed sequences, while MT1 and MT2 are the single-base mismatch targets with the mutant points in boxes. Forward primer and reverse primer were used to execute asymmetric PCR amplifications.
1026
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
2.2. Instrumentations The fluorescence measurement was performed on a Hitachi F-7000 fluorescence spectrometer at a 600 V PMT Voltage using a quartz fluorescence cell with an optical path length of 1.0 cm. The excitation was made at 492 nm with recording emission range of 500–600 nm, and the excitation and emission slits were set at 5.0 nm. When investigating the assay performance, the peak value at 518 nm was collected. A 12% native polyacrylamide gel (nativePAGE) was prepared and run at 80 V for 90 min at a gel electrophoresis instrument, and finally photographed with a ChemiDoc XRSþ imaging system (BIO-RAD, USA). Since FAM modification could be visualized, only the samples without SHP, for example, DNA ladders, were fluorescent stained by SYBR Green I. 2.3. Asymmetric PCR amplification The human lung cancer cell line A549 for genomic DNA extraction was prepared according to the universal Genomic DNA exaction kit after the cells reached 80% confluency. Forward primer 1/reverse primer 1 and forward primer 2/reverse primer 2 were used to amplify the p53 amplicons and control amplicons, respectively. The details were performed according to the procedure described previously with minor modification (Xu et al., 2016a). In brief, asymmetric PCR amplification was achieved in 50 μL of 1 PCR buffer containing 1 μg of genomic DNA, 0.5 μM forward primer, 0.1 μM reverse primer, 0.15 mM dNTP mixture, as well as 2.5 U of Taq polymerase. The thermal process was consisted of 30 cycles (94 °C for 30 s, 55 °C for 30 s, and 72 °C for 55 s) after the initial denaturation step (94 °C for 5 min), and a final extension at 72 °C for 7 min. The PCR products were verified by 12% native-PAGE and used for fluorescent sensing. 2.4. Dual-CNDP-based sensing system for p53 gene detection In an eppendorf tube containing 17 μL of H2O and 3 μL of 10 NEBuffer 2, 1.5 μL of 10 μM SHP, 1.5 μL of 10 μM LHP, 1.5 μL of 10 μM IP1, and 3 μL of p53 gene at certain concentration were consecutively added and mixed thoroughly. After the tube was heated to 90 °C for 5 min and cooling down to room temperature,
0.5 μL of T4 DNA ligase (350 U/μL), 0.5 μL of klenow fragment (3′– 5′ exo-, 5 U/μL), 0.5 μL of Nt. BbvCI nicking endonuclease (10 U/ μL), and 1 μL of 10 mM dNTPs were added to the tube, and then incubated at 37 °C for 2 h with gentle shaking. Concentrations of all components were calculated from the resulting solution (30 μL). Finally, the reaction was terminated at 80 °C for 20 min according to the manufacturer instructions, and the fluorescence of the produced mixture was recorded after 170 μL of H2O was added.
3. Results and discussion 3.1. Working principle of the dual-CNDP amplification To minimize the background noise and to maximize the response signal, we developed the novel dual-CNDP amplification system, which was capable of to execute highly sensitive detection of p53 gene. The corresponding signaling principle was schematically illustrated in Scheme 1. The SHP modified with a fluorophore FAM and a quencher DABCYL consisted of a half recognition site for Nt. BbvCI nicking endonuclease and a recognizing portion at the wobbling 3′ end complementary to target gene, while the LHP also consisted of a half recognition site for Nt. BbvCI nicking endonuclease and the wobbling 5′ end was phosphorylated that partly hybridizes with target gene. The hairpin structure of SHP could force the FAM and DABCYL together to make the fluorescence of FAM almost entirely quenched by DABCYL due to the effect of fluorescence resonance energy transfer (FRET) (Cardullo et al., 1988; Tyagi and Kramer, 1996). In the absence of the target, the two oligonucleotide probes were separately coexisted. Thereby, separation of the HPs produced a very weak fluorescence background. Once the target molecule entered the system, however, the ternary complex of SHP-p53-LHP was formed to open the hairpin structure of LHP for further hybridization with IP simultaneously. Meanwhile, the nick was ligated by the formation of a phosphodiester bond, resulting in the extension of the 3′ end of IP by polymerase over the ligated SHPLHP complex. This enabled formation of the recognition sites for Nt. BbvCI nickase as well as the target recycling via displacement.
Scheme 1. Schematic Diagram of the Dual-CNDP Mediated Signal Amplification. Step ①: Formation of the ternary Complex of SHP-p53-LHP together with further hybridization with IP; Step ②: T4 DNA ligase mediated ligation; Step ③: Polymerization to promote the target-recycling and nicking sites formation, as well as fluorescence recovery; step ④: Continuous reaction of polymerization/nicking/displacement responsible for primary accumulations of NT1 and NT2; Step ⑤: Hybridization between NTI and SHP to contribute the further amplification of the fluorescence signal; Step ⑥: NT2 acts as the target-like template to initiate the more amplification reactions.
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
Of special note, the Nt. BbvCI is one kind of the restriction endonucleases capable of recognizing the specific double-stranded DNA of “5′-CCTCAGC-3′/3′-GGAGTCG-5′” and specifically cleaves the upper strand (Wen et al., 2012). Detailed nicking sites in the formed new duplex can be seen in Fig. S5. Therefore, the subsequent displacements of NT1 and NT2 based on the polymerization/nicking process along the new-formed duplex were realized. Actually, the full recycling events would not be possible without the nickase. Moreover, the molecules of FAM and DABCYL were moved away from each other, resulting in the restoration of fluorescence. Additionally, it is worthwhile pointing out that the resultant NT1 could positively hybridize with SHP to form a rigid duplex and amplify further the fluorescent signal, while the NT2 could serve as the target analog since part of the NT2 bases can hybridize with the 5′ end of LHP and the rest can hybridize with the 3′ end of SHP. Herein, both the target gene and NT2 can be repeatedly used to induce their own CNDP to continually generate more NT1 and NT2 molecules, which in return causes more CNDP reactions. These reactions and the species involved can work with high cooperativity, achieving the synergetic effect of ligase,
1027
polymerase and nickase and accomplishing the highly sensitive detection of p53 gene. As the consequence, the established dualCNDP system was able to amplify the fluorescence signal in a cataract fashion. In addition, since the 3′ end of NT2 could perfectly hybridize with SHP, the successful extension of NT2 over SHP could result in the fluorescence emission increase even in the absence of the ligase and nickase. This amplification strategy maximizes the fluorescence recovery and achieves the multiple amplification effect on signal enhancement. Clearly, one target molecule can induce the production of many NT1 and many NT2. Moreover, each NT2 can execute the target function to initiate the amplification reaction. Additionally, each of hybridized NT2 or hybridized target DNA can be displaced by nicking after polymerization on ligated LHP-SHP probe and repeatedly implement the amplification process, leading to the dramatic accumulation of NT1 that can open SHP. Herein, the concept of “multiple amplification” was used to describe the molecular mechanism for signal transduction. In fact, even when the target gene was only at 5 nM, the system could run in high efficiency and dramatically increase the fluorescence signal following many rounds of isothermal cycles to enable the sensitive detection of the target p53 gene (see below). 3.2. Feasibility demonstration of the dual-CNDP
Fig. 1. Comparison in fluorescent intensity generated from different mixtures: (a) SHP, (b) SHP þLHP þ IP1, (c) SHPþ LHP þIP1 þLigase, (d) SHPþ LHP þIP1 þ p53 þLigase, (e) SHPþ LHP þIP1 þLigase þ Polymerase, (f) SHPþ LHP þIP1 þ p53 þLigase þ Polymerase, (g) SHP þ LHPþ IP1þ Ligase þPolymerase þNickase, (h) SHPþ LHP þIP1 þp53 þ Ligaseþ Polymerase þ Nickase. The concentrations of SHP, LHP, and IP1 were all 500 nM, while the p53 gene was 5 nM.
To investigate whether the dual-CNDP amplification was able to report the target DNA, different samples were prepared in optimized conditions (see Fig. S1) and the measured results were depicted in Fig. 1. We observed extremely weak fluorescence for SHP (curve a). The coexistence of LHP and IP1 could not affect the fluorescence emission of SHP (curve b). Similar results were obtained when ligase was added to the samples regardless of the absence or presence of target (curve c and d). However, upon incubation with the polymerase (curve f), a detectable signal increase was recorded compared with curve e, indicating the successful ligation of SHP and LHP when the target was used as the template, as well as the resultant polymerization of IP1 over the ligated SHP-LHP complex followed by target recycling. Excitingly, as shown in the considerable difference between curve h and g, a remarkable signal enhancement was observed when nickase was added. The corresponding target-to-blank ratio of fluorescent peak value was more than 10. The results clearly demonstrated the utility of designed signal amplification strategy. Furthermore, the background fluorescence in curve g was very close to the inherent fluorescence of SHP (curve a), suggesting the background was still
Fig. 2. Extremely significant differences in fluorescence intensity between samples with and without NT1 or NT2. (A), fluorescence intensity difference between samples with 500 nM SHP (a), or with 500 nM SHP plus 100 nM NT1 (b). (B), substantial fluorescent enhancement induced by NT2 (5 nM; h curve). The experiments were conducted where NT2 was used to replace the target stimulus following the same experimental procedures: (a) SHP, (b) SHP þLHP þ IP1, (c) SHP þLHP þ IP1þ Ligase, (d) SHPþ LHP þIP1 þNT2 þ Ligase, (e) SHP þLHP þ IP1þ Ligase þPolymerase, (f) SHP þLHP þ IP1þ NT2 þLigase þ Polymerase, (g) SHP þLHP þ IP1þ Ligaseþ Polymerase þ Nickase, (h) SHPþ LHP þIP1 þ NT2 þLigase þ Polymerase þ Nickase.
1028
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
very low. These outcomes were in great accordance with the principle designed and experimentally confirmed the feasibility of our dual-CNDP sensing system for the amplification detection of target DNA. Gel electrophoresis results were also carefully tested and corroborated the expected protocol (Fig. S2). 3.3. NTs-induced signal amplification In this section, the discussion pointed to verify the reactions that the NT1 caused fluorescence recovery and NT2 induced signal amplification. As witnessed in Fig. 2A, a significant increase of fluorescent intensity was achieved in curve b compared with curve a, indicating that NT1 could efficiently open the hairpin structure of SHP. Fig. 2B displayed the similar trend as shown in Fig. 1, where NT2 was used instead of the target under identical conditions (see the experimental section). In addition, a visible increase in fluorescent response was detected for sample f, suggesting that the fluorescence response induced by NT2 was relatively higher than that by the target DNA due to that NT2 was easier to induce the polymerization than the target DNA. It seems reasonable because the 3′ end of NT2 is activated and the target is passivated. Moreover, NT2 is able to induce the remarkable signal amplification as demonstrated by the results obtained from Fig. 2B in the presence of nickase (curve h versus g). More supporting data demonstrated the NT2 amplification effect by native-PAGE was given in Fig. S4. 3.4. Capability to sense target A number of genes have been discovered that associate with the increased susceptibility to a variety of disease states due to their functions in encoding, transmitting, and expressing genetic information. Hence, reliable analysis of the given DNA is necessary for diagnosis and monitoring of diseases. After establishing the dual-CNDP amplification strategy, we assessed the dynamic response of our system to different target concentrations under identical experimental conditions. As shown in Fig. 3A, the fluorescence intensity increased linearly with the increment of target concentration ranging from 0 to 5 nM, and there was no linear relationship between fluorescence intensity and target concentrations beyond 5 nM (Fig. 3B) in view of the exhaustion of SHP molecules. Fig. 3B displayed an impressive calibration curve responding to the fluorescent intensity against target concentrations ranging from 100 fM to 5 nM. The regression equation was fitted to Y¼ 185.66 þ391.48X with a correlation coefficient R2 of 0.9900, where Y and X represent the peak value of fluorescence at 518 nm (the maximum emission wavelength) and target concentration,
respectively. The limit of detection was calculated as 78 fM according to the 3s rule. Additionally, the relative standard deviation of the mean data was within 5.0%, indicating an acceptable repeatability. Detailed comparison of the current study with reported ones for gene detection was given in Table S1. The present dual-CNDP amplification sensing system is obviously improved with respect to the sensitivity and dynamic range with some literature reports (Gao et al., 2014; Li et al., 2014; Wang and Zhang, 2012; Zhou et al., 2013). However, the analytical performance of the dual-CNDP amplification strategy should be further improved to meet unique public demands when compared with some transducing techniques (Shen et al., 2012; Wang et al., 2013). Nevertheless, via circumventing the intrinsic drawbacks of the electrical and colorimetric sensing strategies, such as complex electrical interface construction (Wang et al., 2013) and time-consuming oligonucleotide-conjugated golden nanoparticles synthesis (Shen et al., 2012), an inspiring fact is that the dual-CNDP amplification process only involved several simple mixing steps, and it is very easy to manipulate the target detection. Moreover, it is still a great challenge to construct powerful amplification platforms to produce significant signal upon a trace amount of target DNAs. We addressed the technical limitation via proposing the dual-CNDP based sensing system that provided very weak background noise but offered the multiple amplification of the target signal. Generally, as a standard method for gene amplification, fluorescence-based real-time PCR has been widely used as it combines the excellent experimental amplification effect with quantitative detection of the amplified product and possesses ultrahigh sensitivity and a wide dynamic range. However, as reported, the PCR technique has some fundamental limitations of use. For instance, the requirement of an expensive electrically powered thermal cycler and sometimes skilled technician. This may be the reason that increasing concern has been devoted for exploiting alternative amplification strategies methodologically (Shen et al., 2014; Xia et al., 2010). In our case, although the fluorescence labeling is involved (just like the TaqMan probe used in PCR), the goal is to present a new isothermal amplification protocol, which is expected to encourage the following studies. 3.5. Detection selectivity of dual-CNDP amplification system To evaluate the selectivity of the dual-CNDP sensing method, several samples of the same concentration of either the target gene (T) or mismatched targets were prepared and tested using a sample without any target as the blank control. T1, T2, and T3 are
Fig. 3. (A) Typical fluorescence spectra of the sensing system upon addition of different concentrations of p53 gene. The inset amplified the fluorescence spectra of the targets at the low concentration. (B) Dynamic relationship between the fluorescence intensity and p53 gene concentration. The error bars represented the standard deviation of three replicate measurements.
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
1029
Fig. 4. High selectivity of the dual-CNDP system. Only the target (T) that precisely matched the system produced substantial fluorescent intensity, while the target analogs (T1, T2, and T3) and single-base mismatched target (MT1 and MT2) produced insignificant fluorescence. The relative signal response is calculated as [(F-F0)/(Ft-F0)]100%, where F0, Ft, and F are the fluorescence values for the control sample, target gene (T), and mismatched ones (T1, T2, T3, MT1, and MT2), respectively. Concentrations of all target analytes were 20 nM. Error bars were the standard deviation of three replicate experiments.
Fig. 5. Comparison in asymmetric PCR amplifications between p53 amplicons and control amplicons. (A), asymmetric amplicons demonstrated by native-PAGE. Lane a and lane b indicated the p53 amplicons and control amplicons, respectively; (B), changes in fluorescent intensity of p53 amplicons and control amplicons. Error bars were the standard deviation of three replicate experiments.
three randomly-designed target, while MT1 and MT2 were projected with single-base mismatch at the ligation sites. As shown in Fig. 4, only the perfect complementary target induces a substantially signal increase, while the responses from poorly-matched oligonucleotide sequences (T1, T2, and T3) are very low (Fig. 4A). This should be attributed to the stringent match ability between oligonucleotide strands. Besides, since the T4 DNA ligase has been demonstrated with a poor specificity to discriminate a single-base mutation (Cheng et al., 2009a), the selectivity study for single-base mismatched targets was performed by using Taq DNA ligase. We can see that the signal achieved by the single-base mismatched targets (MT1 and MT2) substantially decreases when compared with the perfectly-matched target (seen in Fig. 4B), indicating the satisfactory selectivity. 3.6. Target detection from real biological samples Practical applicability of the dual-CNDP sensing system for target gene detection was demonstrated through the detection of genomic DNA from cancer cells. Prior to detection, an asymmetric PCR should be employed based on the use of different amount of forward primer and reverse primer to generate the single-stranded counterpart since the conventional PCR by using equal
amount of forward primer and reverse primer only produces double-stranded DNAs, which were not suitable for directly detection. In our study, the genomic p53 gene was amplified by asymmetric PCR to produce the single-stranded target amplicons suitable for the subsequent detection. It should be noted that both the target amplicons and control amplicons were obtained via amplifying the extracted genomic p53 gene. But, the target amplicons contained the expected target sequence, while the control amplicons were a random sequence with the similar length as target amplicons. As shown in Fig. 5A, two bright bands correspond individually to the designed p53 amplicons in lane a and control amplicons in lane b, confirming the asymmetric amplifications. By the way, the single-stranded counterpart moved faster than the double-stranded amplicons because the migration rate was inversely proportional to the molecule weight. However, the resultant single-stranded DNAs (ssDNA) were not visible due to the relatively poor intercalating ability of SYBR green I to the ssDNA. Fig. 5B shows the measured fluorescent difference between the p53 amplicons and the control amplicons. We can see that the control amplicons only induce a very weak fluorescence change, which was significantly lower than that produced by the p53 amplicons, indicating the great potential for real sample applications. In addition, potential application of the strategy to complex
1030
J. Xu et al. / Biosensors and Bioelectronics 86 (2016) 1024–1030
biological samples was demonstrated by detecting the target gene in the presence of human serum (Fig. S3).
4. Conclusion In conclusion, by using cancer-related gene as the model analyte, we successfully constructed a dual-CNDP signal amplification system, in which a pair of HPs was admirably designed and applied to realize the robust sensing. The critical importance of the work is that: (1) the participation of ligase, polymerase, and nickase, as well as the continuous reactions of ligation, polymerization, and nicking, contributing to the dual-CNDP amplification; (2) the target DNA-caused CNDP and NT2-caused CNDP worked with high cooperativity. This enables the detection strategy that exhibits multiple amplification effect and very weak background, possessing the merits of low detection limit, wide linear range, and desirable selectivity; (3) the genomic DNA after asymmetric amplifications and the target DNA existing in human serum-contained samples can be encouragingly detected, indicating it a great promise for practical application. Owing to the above distinctive advantages and unique capabilities, the established assay is expected to provide valuable reference for design of more versatile strategies.
Acknowledgments This work was supported by Ministry of Science and Technology of China (2015CB931804), National Natural Science Foundation of China (NSFC) (Grant# 81273548, 21275002, and U1505225), and Independent Research Project of State Key Laboratory of Photocatalysis on Energy and Environment (No. 2014CO1).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2016.07.029.
References Cardullo, R.A., Agrawal, S., Flores, C., Zamecnik, P.C., Wolf, D.E., 1988. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 85 (23), 8790–8794. Chen, Y., Xu, J., Su, J., Xiang, Y., Yuan, R., Chai, Y., 2012. In situ hybridization chain reaction amplification for universal and highly sensitive electrochemiluminescent detection of DNA. Anal. Chem. 84 (18), 7750–7755. Cheng, Y., Zhang, X., Li, Z., Jiao, X., Wang, Y., Zhang, Y., 2009a. Highly sensitive determination of microrna using target-primed and branched rolling-circle amplification. Angew. Chem. 121 (18), 3318–3322. Cheng, Y., Zhang, X., Li, Z., Jiao, X., Wang, Y., Zhang, Y., 2009b. Highly sensitive determination of microrna using target-primed and branched rolling-circle amplification. Angew. Chem. 121 (18), 3318–3322. Deng, R., Tang, L., Tian, Q., Wang, Y., Lin, L., Li, J., 2014. Toehold-initiated rolling circle amplification for visualizing individual micrornas in situ in single cells.
Angew. Chem. Int. Ed. 53 (9), 2389–2393. Gao, Z.F., Ling, Y., Lu, L., Chen, N.Y., Luo, H.Q., Li, N.B., 2014. Detection of singlenucleotide polymorphisms using an on–off switching of regenerated biosensor based on a locked nucleic acid-integrated and toehold-mediated strand displacement reaction. Anal. Chem. 86 (5), 2543–2548. Ho, H.A., Boissinot, M., Bergeron, M.G., Corbeil, G., Doré, K., Boudreau, D., Leclerc, M., 2002. Colorimetric and fluorometric detection of nucleic acids using cationic polythiophene derivatives. Angew. Chem. 114 (9), 1618–1621. Huang, J., Wu, Y., Chen, Y., Zhu, Z., Yang, X., Yang, C.J., Wang, K., Tan, W., 2011. Pyrene-excimer probes based on the hybridization chain reaction for the detection of nucleic acids in complex biological fluids. Angew. Chem. Int. Ed. 50 (2), 401–404. Li, H., Wu, Z.-S., Shen, Z., Shen, G., Yu, R., 2014. Architecture based on the integration of intermolecular G-quadruplex structure with sticky-end pairing and colorimetric detection of DNA hybridization. Nanoscale 6 (4), 2218–2227. Li, J., Deng, T., Chu, X., Yang, R., Jiang, J., Shen, G., Yu, R., 2010. Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms. Anal. Chem. 82 (7), 2811–2816. Niemz, A., Ferguson, T.M., Boyle, D.S., 2011. Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 29 (5), 240–250. Qiu, L.-P., Wu, Z.-S., Shen, G.-L., Yu, R.-Q., 2011. Highly sensitive and selective bifunctional oligonucleotide probe for homogeneous parallel fluorescence detection of protein and nucleotide sequence. Anal. Chem. 83 (8), 3050–3057. Shen, J., Li, Y., Gu, H., Xia, F., Zuo, X., 2014. Recent development of sandwich assay based on the nanobiotechnologies for proteins, nucleic acids, small molecules, and ions. Chem. Rev. 114 (15), 7631–7677. Shen, W., Deng, H., Gao, Z., 2012. Gold nanoparticle-enabled real-time ligation chain reaction for ultrasensitive detection of DNA. J. Am. Chem. Soc. 134 (36), 14678–14681. Shi, C., Liu, Q., Ma, C., Zhong, W., 2013. Exponential strand-displacement amplification for detection of microRNAs. Anal. Chem. 86 (1), 336–339. Tyagi, S., Kramer, F.R., 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14 (3), 303–308. Wang, G.-l, Zhang, C.-y, 2012. Sensitive detection of microRNAs with hairpin probebased circular exponential amplification assay. Anal. Chem. 84 (16), 7037–7042. Wang, X., Wang, X., Wang, X., Chen, F., Zhu, K., Xu, Q., Tang, M., 2013. Novel electrochemical biosensor based on functional composite nanofibers for sensitive detection of p53 tumor suppressor gene. Anal. Chim. Acta 765, 63–69. Wen, Y., Xu, Y., Mao, X., Wei, Y., Song, H., Chen, N., Huang, Q., Fan, C., Li, D., 2012. DNAzyme-based rolling-circle amplification DNA machine for ultrasensitive analysis of MicroRNA in Drosophila larva. Anal. Chem. 84 (18), 7664–7669. Xia, F., White, R.J., Zuo, X., Patterson, A., Xiao, Y., Kang, D., Gong, X., Plaxco, K.W., Heeger, A.J., 2010. An Electrochemical supersandwich assay for sensitive and selective DNA detection in complex matrices. J. Am. Chem. Soc. 132 (41), 14346–14348. Xu, J., Wu, Z.-S., Shen, W., Xu, H., Li, H., Jia, L., 2015a. Cascade DNA nanomachine and exponential amplification biosensing. Biosens. Bioelectron. 73, 19–25. Xu, J., Li, H., Wu, Z.-S., Qian, J., Xue, C., Jia, L., 2016a. Double-stem hairpin probe and ultrasensitive colorimetric detection of cancer-related nucleic acids. Theranostics 6 (3), 319. Xu, J., Wu, Z.-S., Wang, Z., Li, H., Le, J., Jia, L., 2016b. Two-wheel drive-based DNA nanomachine and its sensing potential for highly sensitive analysis of cancerrelated gene. Biomaterials 100, 110–117. Xu, Y., Zhou, W., Zhou, M., Xiang, Y., Yuan, R., Chai, Y., 2015b. Toehold strand displacement-driven assembly of G-quadruplex DNA for enzyme-free and nonlabel sensitive fluorescent detection of thrombin. Biosens. Bioelectron. 64, 306–310. Yang, X., Tang, Y., Mason, S.D., Chen, J., Li, F., 2016. An enzyme-powered three dimensional DNA nanomachine for DNA walking, payload release, and biosensing. ACS Nano. Yang, Y., Huang, J., Yang, X., Quan, K., Wang, H., Ying, L., Xie, N., Ou, M., Wang, K., 2015. FRET nanoflares for intracellular mRNA detection: avoiding false positive signals and minimizing effects of system fluctuations. J. Am. Chem. Soc. 137 (26), 8340–8343. Zhao, C., Song, Y., Ren, J., Qu, X., 2009. A DNA nanomachine induced by singlewalled carbon nanotubes on gold surface. Biomaterials 30 (9), 1739–1745. Zhou, H., Wu, Z.-S., Shen, G.-L., Yu, R.-Q., 2013. Intermolecular G-quadruplex structure-based fluorescent DNA detection system. Biosens. Bioelectron. 41, 262–267.