Biosensors and Bioelectronics 68 (2015) 550–555
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Hairpin assembly-triggered cyclic activation of a DNA machine for label-free and ultrasensitive chemiluminescence detection of DNA Jia Chen a, Hongdeng Qiu a,n, Mingliang Zhang a, Tongnian Gu a, Shijun Shao a, Yong Huang b, Shulin Zhao b a Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China b Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), College of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 October 2014 Received in revised form 21 January 2015 Accepted 22 January 2015 Available online 23 January 2015
DNA plays important regulatory roles in many life activities. Here, we have developed a novel label-free, ultrasensitive and specific chemiluminescence (CL) assay protocol for DNA detection based on hairpin assembly-triggered cyclic activation of a DNA machine. The system involves two hairpin structures, H1 and H2. Firstly, a target DNA binds with and opens the hairpin structure of H1. Then, H2 hybridizes with H1 and displaces the target DNA, which is used to trigger another new hybridization cycle between H1 and H2, leading to the generation of numerous H1–H2 complexes. The generated H1–H2 complexes are further activated with the help of polymerase and nicking enzyme, continuously yielding a large amount of G-riched DNA fragments. The G-riched DNA fragment products interact with hemin to form the activated HRP-mimicking DNAzymes that can catalyze the oxidation of luminol by H2O2 to produce strong CL signal resulting in an amplified sensing process. Our newly proposed homogeneous assay enables the quantitative measurement of p53 DNA (as a model) with a detection limit of 0.85 fM, which is at least 5 orders of magnitude lower than that of traditional unamplified homogeneous optical approaches. Moreover, this assay exhibits high discrimination ability even against a single base mismatch. In addition, this strategy is also capable of detecting p53 DNA in complex biological samples. The proposed sensing approach might hold a great promise for further applications in biomedical research and early clinical diagnosis. & 2015 Elsevier B.V. All rights reserved.
Keywords: P53 DNA Hairpin assembly DNA machine Label-free Chemiluminescence
1. Introduction The development of simple, sensitive and selective bioassay methods for the detection of trace amount DNA is of great demand in clinical diagnostics, detection of genetic disorders, analysis of food and environmental pollutants, as well as homeland security (Cui et al., 2001; Eastman et al., 2006; Turner et al., 2009). Historically, the polymerase chain reaction (PCR) has been employed extensively to detect target DNA. This method offers high detection sensitivity; however, the relatively complex multi-step process often results in false positives (Saik et al., 1988). Recently, some alternative amplification approaches have been developed for DNA detection. These include the use of the rolling circle amplification (RCA) process (Li et al., 2010a, 2010b; Wen et al., 2012; Xu et al., 2012), the loop-mediated isothermal amplification n
Corresponding author. Fax: þ86 931 8277088. E-mail address: [email protected] (H. Qiu).
http://dx.doi.org/10.1016/j.bios.2015.01.054 0956-5663/& 2015 Elsevier B.V. All rights reserved.
(LAMP) (Fang et al., 2010; Li et al., 2011a, 2011b, 2011c), the ligase chain reaction (Claridge et al., 2008; Shen et al., 2012), the stranddisplacement polymerase reaction (He et al., 2010; Huang et al., 2011), the hybridization chain reaction (HCR) (Liu et al. 2013; Yang et al., 2013), the enzyme-free hairpin assembly circuit (Jiang et al. 2013; Li et al., 2012; Zheng et al., 2012a, 2012b), the application of nucleases as biocatalytic amplifiers (Bi et al., 2012; Chen et al., 2012; Liu et al., 2012), the use of advanced nanomaterials (Huang et al., 2012; Li et al., 2011a, 2011b, 2011c; Song et al., 2009) and biobarcode (Giri et al., 2011) as amplifying labels. Most of these approaches, however, require the chemical modification of the oligonucleotide probes for the labeling of optical or electroactive moieties or nanomaterials, a process that is rather costly, timeconsuming, and sophisticated. Therefore, the development of simple, selective and more sensitive methods for detection of trace amount DNA still remains a challenge. Catalytic nucleic acids (DNAzymes or ribozymes) have attracted increasing research interest as amplifying labels for sensing events
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(Willner et al., 2008). The flexibility in mastering DNAzyme structures by encoding recognition functions into DNAzyme sequences and the reduced nonspecific binding properties of these nucleic acids make DNAzymes ideal candidates for bioanalytical applications (Wang et al., 2011). To date, various ion-dependent DNAzymes have been implemented as catalysts for the optical assay of heavy metal ions (Lu et al., 2012; Zhang et al., 2010; Zhao et al., 2011). Specifically, one of the most extensively studied DNAzymes is a horseradish peroxidase (HRP)-mimicking DNAzyme that contains a special G-quadruplex structure with an intercalated hemin (Travascio et al., 1999). Such artificial enzymes can effectively catalyze the H2O2-mediated oxidation of 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonate disodium salt (ABTS2 ) to the colored product ABTS- (Hao et al., 2014; He et al., 2012) or stimulate the oxidation of luminol upon addition of H2O2 to yield chemiluminescence (CL) (Ma et al., 2014; Zhou et al., 2012). These functions of the HRP-mimicking DNAzyme have been used extensively to develop various amplified DNA-based colorimetric (Elbaz et al., 2009; Teller et al., 2009) or chemiluminescence (Bi et al., 2010; Freeman et al., 2011; Liu et al., 2011) biosensors. Recently, the autonomous replication-scission-displacement process of the HRP-mimicking DNAzyme generated by polymerase, dNTPs, and a nicking enzyme was used as an effective amplified replication system for DNA detection (Weizmann et al., 2006). Similarly, the RCA process was applied to detect target DNA through the autonomous synthesis of an HRP-mimicking DNAzyme repeat unit that acted as the biocatalytic amplifier (Cheglakov et al., 2007). Additionally, the HCR process was used for the amplified detection of DNA through the autonomous assembly of polymers consisting of HRP-mimicking DNAzyme nanowires (Shimron et al., 2012). The activation of HRP-mimicking DNAzyme by target-catalyzed hairpin assembly was also used for an amplified analysis of DNA (Zheng et al., 2012a, 2012b). Although these methods can be quite powerful, greater sensitivity and specificity are often desired, particularly when working with limited amounts of sample material or when target level is extremely low (Lee et al., 2013). With these needs in mind, we have developed a novel labelfree CL biosensor platform based on hairpin assembly-triggered cyclic activation of a DNA machine for ultrasensitive CL detection of DNA. Importantly, this assay protocol does not require any chemical modification of DNA, thus making the system simpler and more cost effective. Moreover, the detection sensitivity of the proposed method can be significantly improved compared with most reported homogeneous approaches. As a proof of concept, this sensing platform could detect p53 DNA with a detection limit of 0.85 fM and a wide detection range over 5 orders of magnitude; it was also shown to exhibit high discrimination ability, even against a single base mismatch. Furthermore, the application of this method for biological sample analysis has been demonstrated. The proposed detection method holds great potential for ultrasensitive and selective detection of DNA.
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Fragment exo- buffer (10 KF buffer, pH 8.0) were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). The nicking endonuclease Nt.BbvCI and 10 cutsmart buffers were purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Agarose G-10 was obtained from Biowest. Ultra low range molecular weight DNA ladder was purchased from Fermentas, Inc. (Vilnius, Lithuania). 5 TBE buffer (pH 7.9) was provided by Sangon Biotechnology Co. Ltd. (Shanghai, China). hemin, Hydrogen peroxide (H2O2), luminol and sodium 4-(2-hy-droxyethyl) piperazine-1ethanesulfonate (HEPES) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and used without further purification. A hemin stock solution was prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 20 °C. Water was purified with a Milli-Q plus 185 equip from Millipore (Bedford, MA, USA) and used throughout this work. All other reagents used in this work were of analytical grade. Buffer solution for CL reaction was HEPES buffer (pH 9.0, 25 mM HEPES, 0.05% (w/v) Triton X-100, 1% (v/v) DMSO, 20 mM KCl, 200 mM NaCl) solution. 2.2. Cell culture The human HepG2 cells were kindly provided by the First Hospital of Lanzhou University. Cells were maintained routinely in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, USA) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Minghai Biochem, Lanzhou, China). Cells were cultured at 37 °C and 5% CO2 in humid atmosphere. 2.3. Preparation of cell lysate samples Human HepG2 cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined with Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The lysate was stored at 20 °C until analysis, and diluted 10-fold with HEPES buffer before analysis. 2.4. Procedure for target p53 DNA assay
2. Experimental section
After testing various conditions, the following procedure was used to study the concentration-dependent changes in the CL experiments: H1 (10 μL, 10 mM) and H2 (12 μL, 10 mM) were mixed with 10 mL of different concentrations of target p53 DNA for 2 h at 37 °C. The final concentrations of the target in the samples varied from 2.0 fM to 1.0 nM. And then, 10 KF buffer (6.5 μL), 10 mM dNTPs (10 μL), 5 u/μL KF polymerase (5 μL), 10 cutsmart buffer (6.5 μL), 5 u/μL Nt.BbvCI (5 μL) were successively injected into the resulting solution and allowed to incubate for 1 h at 37 °C. Subsequently, hemin (10 μL) was added to the mixture at a final concentration of 0.2 mM hemin and incubated for 1 h at 37 °C to form the hemin/G-quadruplex structures. After incubation, the solution was diluted with 725 μL of HEPES buffer. The obtained sample solutions were then used for the CL measurements.
2.1. Materials and reagents
2.5. Chemiluminescence measurements
All oligonucleotide sequences used in this study were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China), and the sequences were given in Table S1. The oligonucleotides were used as provided and diluted in 20 mM Tris–HCl buffer solution, pH 7.4, to give stock solutions of 100 μM. And each oligonucleotide was heated to 95 °C for 5 min, and slowly cooled down to room temperature for at least 2 h before use. Deoxynucleotide mixture solution (dNTPs), Klenow Fragment polymerase (3′-5′ exo , KF polymerase, 5 u/μL), and 10 Klenow
The CL spectra of the sample solutions obtained above were measured using a LS-55 luminescence spectrometer (Perkin-Elmer, USA) with a 3 mL quartz cuvette (1 cm optical path). The light source of the spectrometer was turned off. The emission wavelength was collected from 360 to 600 nm. The excitation and emission slits were set at 12.0 nm. To measure the CL spectra, 100 μL of luminol (0.01 M) and 100 μL of H2O2 (0.25 M) substrates were quickly added to the quartz cuvette containing the sample solutions obtained above, allowing the biocatalyzed oxidation of
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luminol to yield CL. The CL spectra were measured immediately. The emission intensity at 430 nm was recorded, and the increase of CL intensity (I–I0, I and I0 were the CL intensities at 430 nm in the presence and absence of target DNA, respectively) was used for the quantification of target DNA. 2.6. Gel electrophoresis Gel electrophoresis was used to confirm the activation of the DNA machine by hairpin assembly. Analysis by electrophoresis was carried out on 5% agarose gels by gene green nucleic acid dye staining, cast and ran in 0.5 TBE buffer (pH 7.9) at room temperature. The electrophoresis experiments were performed at a 90 V constant voltage for 1.5 h with loading of 8 mL of each prepared sample into the lanes. The resulting gel was photographed under UV light using a Quantum ST5 gel imaging system (Vilber, France).
3. Results and discussion 3.1. Principle of hairpin assembly-triggered cyclic activation of a DNA machine This study aims to construct a hairpin assembly-triggered cyclic activation of DNA machine toward target p53 DNA detection. The overall steps of this strategy are depicted in Fig. 1. The system mainly consists of two DNA hairpins (H1 and H2, their sequences are shown in Table S1). H1 contains four domains termed as I, II, III, and IV according to their different functions as shown in Fig. 1. The domain I consists of the complementary sequence of HRPmimicking DNAzyme. The domain II is partly complementary to domain IV. The domain III is the loop of H1. Domain IV is complementary to the target p53 DNA. H2 contains three domains termed as V, VI and VII. The domain V is complementary to the 3′ part of H1 and is blocked by hybridization with domain VII. It is worth noted that both H1 and H2 have a single-stranded sequences at their 3′ ends. In the absence of target, H1 and H2 can coexist in solution. Upon the addition of p53 DNA (a), p53 DNA
hybridizes with domain IV of H1 (b), and the hairpin structure of H1 is opened, forming a p53 DNA-H1 complex. Meanwhile, the unfolded domain II of H1 can serve as a toehold to hybridize to the domain VII of H2 (c), leading to the displacement of target p53 DNA through a branch migration process (Li et al., 2011a, 2011b, 2011c) and the formation of a H1–H2 complex. The displaced target p53 DNA can then hybridize to another H1, and the cycle starts anew, which leads to formation of numerous H1–H2 complexes. The generated H1–H2 complexes with the 3′ sticky end of H2 can act as DNA primer to trigger the polymerization reaction in the presence of KF polymerase and dNTPs, generating stable dsDNA structures with the recognition site for Nt.BbvCI. The cleavage of the single strand in the dsDNA structures by Nt.BbvCI generates new sites for the initiation of replication. Thus, the polymerase completes the replication of the HRP-mimicking DNAzyme units, and the reactivated replication at the scission site displaces the already synthesized HRP-mimicking DNAzyme units. As a result, an autonomous operation of the DNA machine can be realized through the replication. By following this mechanism, abundant HRP-mimicking DNAzyme units are synthetized. The HRP-mimicking DNAzyme unit products can assemble with hemin to form the hemin/G-quadruplexes that exhibit HRP-mimicking DNAzyme activity, which can catalyze the oxidation of luminol by H2O2 to generate CL (Lee et al., 2011) that provides a readout signal for amplified sensing of target p53 DNA. Importantly, the amplification strategy described here is isothermal, label-free, and does not require separation and washing steps, which is very simple and cost-effective. Moreover, because the proposed assay protocol combines the cyclic DNA assembly reaction with the amplification of DNA machine and HRP-mimicking DNAzyme, it can substantially improve the sensitivity of detection compared with previously reported homogeneous optical DNA assays. 3.2. Feasibility study In order to demonstrate the feasibility of our proposed assay strategy, the CL response of the sensing system under different conditions was investigated as shown in Fig. 2. The system containing only hemin exhibits low CL intensity (curve a). Upon the
Fig. 1. Schematic illustrating the principle of hairpin assembly-triggered cyclic activation of a DNA machine for ultrasensitive CL detection of target p53 DNA.
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Fig. 2. CL spectra of sample solutions under different conditions: (a) only hemin (200 nM); (b) H1 (100 nM)/H2 (120 nM)/dNTPs (0.1 mM)/KF polymerase (25 U)/Nt. BbvCI (25 U)/hemin (200 nM); (c) H1 (100 nM)/H2 (120 nM)/p53 DNA (1 nM)/ hemin (200 nM); (d) H1 (100 nM)/H2 (120 nM)/p53 DNA (1 nM)/dNTPs (0.1 mM)/ KF polymerase (25 U)/Nt.BbvCI (25 U)/hemin (200 nM).
addition of H1/H2/KF polymerase/dNTPs/Nt.BbvCI (curve b) and H1/H2/p53 DNA (curve c), the CL intensity of the system was slightly increased and almost equal. However, it had significant enhancement upon simultaneous addition of H1/H2/KF polymerase/dNTPs/Nt.BbvCI and p53 DNA (curve c), these phenomena indicated that the two hairpins H1 and H2 can maintain the sufficiently stable stem-loop structure in the solution. After addition of p53 DNA and KF polymerase/dNTPs/Nt.BbvCI, a single target can generate many H1–H2 complexes and thus yield many HRP-mimicking DNAzyme units. Thus, the proposed DNAzyme sensor can be used for the amplified detection of p53 DNA. 3.3. Gel electrophoresis characterization The hairpin assembly-triggered DNA machine amplification reactions were further verified using agarose gel electrophoresis, and the results were in agreement with the proposed mechanism (Fig. 3). In the absence of p53 DNA, the hairpin structures of H1
Fig. 3. Gene green nucleic acid dye stained agarose gel (5.0%) showing different mobilities of (M) DNA Marker; (Lane a) p53 DNA only; (Lane b) H1 only; (Lane c) H2 only; (Lane d) H1 incubated with H2 for 2 h at 37 °C; (Lane e) H1 incubated with p53 DNA for 2 h at 37 °C; (Lane f) H1 and H2 incubated with p53 DNA for 2 h at 37 °C; (Lane g) H1 and H2 incubated with p53 DNA for 2 h at 37 °C, followed by the addition of KF buffer (5 μL), 10 mM dNTPs (5 μL), and 5 u/μL KF polymerase (3 μL) to the mixture for another 1 h incubation at 37 °C; (Lane h) the products of hairpin assembly-triggered cyclic activation of a DNA machine. The final concentration of p53 DNA, H1 and H2 are all set as 0.5 μM.
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and H2 were maintained (lanes b, c and d), indicating no occurrence of hairpin assembly reaction. When p53 DNA was mixed with H1, the H1 band disappeared, and a new band with lower mobility compared with lane b (lane e) appeared, suggesting that the hybridization between p53 DNA and H1 had taken place and generated duplex DNA with a higher molecular weight. Once p53 DNA was incubated with H1 and H2, a new band with a slightly lower mobility compared with lane e appeared, and the bands of both H1 and H2 disappeared compared with lane d (lane f), indicating that the hairpin assembly reaction between H1 and H2 had been initiated by the p53 DNA. Upon the addition of the polymerase and dNTPs to the mixture of p53 DNA, H1 and H2, a new band with lower mobility compared with lane f (lane g) was observed, demonstrating that the replication reaction had been initiated by KF polymerase and dNTPs; however, when p53 DNA was mixed with H1, H2, KF polymerase, dNTPs and Nt.BbvCI, a new band with fast mobility compared with lane g (lane h) was observed, suggesting that the replication-scission-displacement process by KF polymerase, dNTPs, and Nt.BbvCI had taken place and generated the HRP-mimicking DNAzyme units. These gel electrophoresis results indicate the successful proceeding of the hairpin assembly-triggered DNA machine amplification reactions. 3.4. Analytical performance of the DNAzyme sensor Under the optimum conditions (Fig. S1 in Supporting Information), we evaluated the CL spectra of the proposed amplified assay for p53 DNA at varying concentrations. Fig. 4 displayed the CL response to the target DNA concentrations in the range from 0 fM to 1.0 nM. As seen in the figure, the CL intensities gradually increased upon increasing the concentration of p53 DNA. The increase of CL intensity showed a good linear correlation to the logarithm of p53 DNA concentration within the range from 2.0 fM to 1.0 nM (inset in Fig. 4). The regression equation was ΔI¼546.3þ36.12log C with a correlation coefficient of 0.9981, where ΔI is the increase of CL intensity and C is the concentration of p53 DNA. Based on 3s/S (s is the standard deviation for the blank solution, n¼10, and S is the slope of the calibration curve), the detection limit was estimated to be 0.85 fM. Supporting Information Table S2 compares the performance of the proposed method to previous homogeneous optical methods for
Fig. 4. CL response of the proposed DNA machine to varying concentrations of target DNA. The concentration of target DNA: (a) 0 fM; (b) 2.0 fM; (c) 8.0 fM; (d) 20 fM; (e) 0.1 pM; (f) 0.5 pM; (g) 2.0 pM; (g) 10 pM; (h) 0.1 nM; (i) 1.0 nM. The inset shows a linear relationship between the increase of CL intensity (ΔI¼ I I0) at 430 nm and the logarithm of the target p53 DNA concentration. Error bars show the standard deviation of three independent experiments. The system also includes 100 nM H1, 120 nM H2, 200 nM hemin, 25 U Nt.BbvCI, 0.1 mM dNTPs, 25 U KF polymerase, 1 mM luminol, 25 mM H2O2.
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matched target DNA (p53 DNA) led to an increase in the CL intensity, while the other DNAs did not lead to an obvious variation of the CL intensity. These results clearly demonstrate the high specificity of the developed hairpin assembly-triggered cyclic activation of a DNA machine. 3.6. Real sample analysis
Fig. 5. Detection specificity of the proposed DNA machine. The concentration of target p53 DNA (TD) was 1 nM, non-complementary oligonucleotide (NC), singlebase mismatched oligonucleotide (1 MT) and three-base mismatched oligonucleotide (3 MT) were 10 nM. Other experimental conditions were the same as those given in Fig. 4. Error bars show the standard deviation of three independent experiments.
To be useful in bioassays, the proposed detection method should be able to tolerate any interference from the biological samples. Therefore, we first prepared a series of samples by adding different concentrations of p53 DNA in different diluted ratios of human HepG2 cell lysate samples and analyzed the mixtures using the proposed assay method. When the ratio of the human HepG2 cell lysate sample and assay buffer reached 1:10, comparable responses were found for p53 DNA in both the buffer and cell lysate. Thus, 10-fold diluted human HepG2 cell lysate samples were selected and used to analyse the various concentrations of p53 DNA. Fig. 6 shows the CL responses with different concentrations of p53 DNA in both the buffer and cell lysate. A slight signal change in the cell lysate sample could be observed compared to the CL intensity in the buffer under the same experimental conditions, which may be related to the interference of the sample matrix during the process of hairpin assembly-triggered DNA machine amplification. Despite the slight response differences, the fabricated CL DNAzyme sensor still showed a distinguishable performance at different p53 DNA concentrations in the cell lysate samples. Then, three independent spiked human HepG2 cell lysate samples were tested by this assay, and the concentration of p53 DNA in the spiked human HepG2 cell lysate samples was calculated by using the calibration curve of standard target p53. The analytical results are shown in Table S3. The recoveries were found to be in the range of 93.1–98.6%, and the RSDs were between 2.5% and 4.6%. These results indicated the potential of the proposed assay method for detecting target DNA in complex biological samples.
4. Conclusions
Fig. 6. Results obtained upon testing 10-fold diluted human HepG2 cell lysate samples spiked with different concentrations of p53 DNA. The same reaction mixtures without p53 DNA were used as blanks. Other experimental conditions were the same as those given in Fig. 4. Error bars show the standard deviation of three independent experiments.
sensing DNA. The results revealed that the sensitivity of the present assay was at least 5 orders of magnitude higher compared to an unamplified homogeneous DNA assay (Li et al., 2011a, 2011b, 2011c) and was also higher sensitivity than those of other amplified homogeneous optical assays (Jiang et al., 2013; Li et al., 2013; Liu et al., 2012, 2013; Shimron et al., 2012; Xu et al., 2012; Zou et al., 2011). In addition, assay reproducibility was also investigated by analysing 5.0 pM and 500 pM p53 DNA standard solutions nine times. The results showed that the relative standard deviations (RSDs) for p53 DNA quantity less than 4.5%. 3.5. Detection specificity To investigate the specificity of the proposed assay, different DNA targets (such as target p53 DNA (TD), a single-base mismatched DNA target (1MT), a three-base mismatched DNA target (3MT)) and a non-complementary oligonucleotide (NC); the sequences are shown in Table S1) were analysed. The results obtained are presented in Fig. 5. As seen in this figure, the perfectly
In summary, we have developed a novel, label-free CL amplification strategy based on hairpin assembly-triggered cyclic activation of a DNA machine. As a proof-of-concept, the proposed strategy can be successfully used for p53 DNA detection both in aqueous buffer and in human HepG2 cell lysate samples. This assay has several important features. First, by using hairpin assembly-triggered cyclic activation of a DNA machine, we could detect the p53 DNA as low as 0.85 fM, which is significantly improved by at least five orders of magnitude over traditional homogeneous DNA assays. Second, this method is capable of discriminating mismatched DNA from perfect matched target DNA with a high selectivity. Third, the method does not require any chemical modification of the DNA, resulting in a very simple and cost-effective process. Finally, this assay protocol can be easily extended to the analysis of other DNAs or RNAs molecules by simply changing the sequences of the hairpin structures. These characteristics of our proposed assay protocol make it a potential platform for further applications in the early diagnosis of gene-related diseases, biological process researches, and drug discovery.
Acknowledgements This work was supported by the “Hundred Talents Program” of the Chinese Academy of Science, the National Natural Science Foundation of China (Nos. 21405163 and 21475142) and the
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Foundation for Sci & Tech Research Project of Gansu Province (145RJYA271).
Appendix A. Supplementary material The supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015. 01.054.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Hairpin assembly-triggered cyclic activation of a DNA machine for label-free and ultrasensitive chemiluminescence detection of DNA Jia Chen a, Hongdeng Qiu a,n, Mingliang Zhang a, Tongnian Gu a, Shijun Shao a, Yong Huang b, Shulin Zhao b a Key Laboratory of Chemistry of Northwestern Plant Resources and Key Laboratory for Natural Medicine of Gansu Province, Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences, Lanzhou 730000, China b Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Education of China), College of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
art ic l e i nf o
a b s t r a c t
Article history: Received 24 October 2014 Received in revised form 21 January 2015 Accepted 22 January 2015 Available online 23 January 2015
DNA plays important regulatory roles in many life activities. Here, we have developed a novel label-free, ultrasensitive and specific chemiluminescence (CL) assay protocol for DNA detection based on hairpin assembly-triggered cyclic activation of a DNA machine. The system involves two hairpin structures, H1 and H2. Firstly, a target DNA binds with and opens the hairpin structure of H1. Then, H2 hybridizes with H1 and displaces the target DNA, which is used to trigger another new hybridization cycle between H1 and H2, leading to the generation of numerous H1–H2 complexes. The generated H1–H2 complexes are further activated with the help of polymerase and nicking enzyme, continuously yielding a large amount of G-riched DNA fragments. The G-riched DNA fragment products interact with hemin to form the activated HRP-mimicking DNAzymes that can catalyze the oxidation of luminol by H2O2 to produce strong CL signal resulting in an amplified sensing process. Our newly proposed homogeneous assay enables the quantitative measurement of p53 DNA (as a model) with a detection limit of 0.85 fM, which is at least 5 orders of magnitude lower than that of traditional unamplified homogeneous optical approaches. Moreover, this assay exhibits high discrimination ability even against a single base mismatch. In addition, this strategy is also capable of detecting p53 DNA in complex biological samples. The proposed sensing approach might hold a great promise for further applications in biomedical research and early clinical diagnosis. & 2015 Elsevier B.V. All rights reserved.
Keywords: P53 DNA Hairpin assembly DNA machine Label-free Chemiluminescence
1. Introduction The development of simple, sensitive and selective bioassay methods for the detection of trace amount DNA is of great demand in clinical diagnostics, detection of genetic disorders, analysis of food and environmental pollutants, as well as homeland security (Cui et al., 2001; Eastman et al., 2006; Turner et al., 2009). Historically, the polymerase chain reaction (PCR) has been employed extensively to detect target DNA. This method offers high detection sensitivity; however, the relatively complex multi-step process often results in false positives (Saik et al., 1988). Recently, some alternative amplification approaches have been developed for DNA detection. These include the use of the rolling circle amplification (RCA) process (Li et al., 2010a, 2010b; Wen et al., 2012; Xu et al., 2012), the loop-mediated isothermal amplification n
Corresponding author. Fax: þ86 931 8277088. E-mail address: [email protected] (H. Qiu).
http://dx.doi.org/10.1016/j.bios.2015.01.054 0956-5663/& 2015 Elsevier B.V. All rights reserved.
(LAMP) (Fang et al., 2010; Li et al., 2011a, 2011b, 2011c), the ligase chain reaction (Claridge et al., 2008; Shen et al., 2012), the stranddisplacement polymerase reaction (He et al., 2010; Huang et al., 2011), the hybridization chain reaction (HCR) (Liu et al. 2013; Yang et al., 2013), the enzyme-free hairpin assembly circuit (Jiang et al. 2013; Li et al., 2012; Zheng et al., 2012a, 2012b), the application of nucleases as biocatalytic amplifiers (Bi et al., 2012; Chen et al., 2012; Liu et al., 2012), the use of advanced nanomaterials (Huang et al., 2012; Li et al., 2011a, 2011b, 2011c; Song et al., 2009) and biobarcode (Giri et al., 2011) as amplifying labels. Most of these approaches, however, require the chemical modification of the oligonucleotide probes for the labeling of optical or electroactive moieties or nanomaterials, a process that is rather costly, timeconsuming, and sophisticated. Therefore, the development of simple, selective and more sensitive methods for detection of trace amount DNA still remains a challenge. Catalytic nucleic acids (DNAzymes or ribozymes) have attracted increasing research interest as amplifying labels for sensing events
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(Willner et al., 2008). The flexibility in mastering DNAzyme structures by encoding recognition functions into DNAzyme sequences and the reduced nonspecific binding properties of these nucleic acids make DNAzymes ideal candidates for bioanalytical applications (Wang et al., 2011). To date, various ion-dependent DNAzymes have been implemented as catalysts for the optical assay of heavy metal ions (Lu et al., 2012; Zhang et al., 2010; Zhao et al., 2011). Specifically, one of the most extensively studied DNAzymes is a horseradish peroxidase (HRP)-mimicking DNAzyme that contains a special G-quadruplex structure with an intercalated hemin (Travascio et al., 1999). Such artificial enzymes can effectively catalyze the H2O2-mediated oxidation of 2,2′-azinobis(3-ethylbenzothiazoline)-6-sulfonate disodium salt (ABTS2 ) to the colored product ABTS- (Hao et al., 2014; He et al., 2012) or stimulate the oxidation of luminol upon addition of H2O2 to yield chemiluminescence (CL) (Ma et al., 2014; Zhou et al., 2012). These functions of the HRP-mimicking DNAzyme have been used extensively to develop various amplified DNA-based colorimetric (Elbaz et al., 2009; Teller et al., 2009) or chemiluminescence (Bi et al., 2010; Freeman et al., 2011; Liu et al., 2011) biosensors. Recently, the autonomous replication-scission-displacement process of the HRP-mimicking DNAzyme generated by polymerase, dNTPs, and a nicking enzyme was used as an effective amplified replication system for DNA detection (Weizmann et al., 2006). Similarly, the RCA process was applied to detect target DNA through the autonomous synthesis of an HRP-mimicking DNAzyme repeat unit that acted as the biocatalytic amplifier (Cheglakov et al., 2007). Additionally, the HCR process was used for the amplified detection of DNA through the autonomous assembly of polymers consisting of HRP-mimicking DNAzyme nanowires (Shimron et al., 2012). The activation of HRP-mimicking DNAzyme by target-catalyzed hairpin assembly was also used for an amplified analysis of DNA (Zheng et al., 2012a, 2012b). Although these methods can be quite powerful, greater sensitivity and specificity are often desired, particularly when working with limited amounts of sample material or when target level is extremely low (Lee et al., 2013). With these needs in mind, we have developed a novel labelfree CL biosensor platform based on hairpin assembly-triggered cyclic activation of a DNA machine for ultrasensitive CL detection of DNA. Importantly, this assay protocol does not require any chemical modification of DNA, thus making the system simpler and more cost effective. Moreover, the detection sensitivity of the proposed method can be significantly improved compared with most reported homogeneous approaches. As a proof of concept, this sensing platform could detect p53 DNA with a detection limit of 0.85 fM and a wide detection range over 5 orders of magnitude; it was also shown to exhibit high discrimination ability, even against a single base mismatch. Furthermore, the application of this method for biological sample analysis has been demonstrated. The proposed detection method holds great potential for ultrasensitive and selective detection of DNA.
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Fragment exo- buffer (10 KF buffer, pH 8.0) were obtained from Sangon Biotech Co. Ltd. (Shanghai, China). The nicking endonuclease Nt.BbvCI and 10 cutsmart buffers were purchased from New England Biolabs, Inc. (Ipswich, MA, USA). Agarose G-10 was obtained from Biowest. Ultra low range molecular weight DNA ladder was purchased from Fermentas, Inc. (Vilnius, Lithuania). 5 TBE buffer (pH 7.9) was provided by Sangon Biotechnology Co. Ltd. (Shanghai, China). hemin, Hydrogen peroxide (H2O2), luminol and sodium 4-(2-hy-droxyethyl) piperazine-1ethanesulfonate (HEPES) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and used without further purification. A hemin stock solution was prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 20 °C. Water was purified with a Milli-Q plus 185 equip from Millipore (Bedford, MA, USA) and used throughout this work. All other reagents used in this work were of analytical grade. Buffer solution for CL reaction was HEPES buffer (pH 9.0, 25 mM HEPES, 0.05% (w/v) Triton X-100, 1% (v/v) DMSO, 20 mM KCl, 200 mM NaCl) solution. 2.2. Cell culture The human HepG2 cells were kindly provided by the First Hospital of Lanzhou University. Cells were maintained routinely in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, USA) supplemented with 10% (v/v) heat-inactivated fetal calf serum (Minghai Biochem, Lanzhou, China). Cells were cultured at 37 °C and 5% CO2 in humid atmosphere. 2.3. Preparation of cell lysate samples Human HepG2 cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined with Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The lysate was stored at 20 °C until analysis, and diluted 10-fold with HEPES buffer before analysis. 2.4. Procedure for target p53 DNA assay
2. Experimental section
After testing various conditions, the following procedure was used to study the concentration-dependent changes in the CL experiments: H1 (10 μL, 10 mM) and H2 (12 μL, 10 mM) were mixed with 10 mL of different concentrations of target p53 DNA for 2 h at 37 °C. The final concentrations of the target in the samples varied from 2.0 fM to 1.0 nM. And then, 10 KF buffer (6.5 μL), 10 mM dNTPs (10 μL), 5 u/μL KF polymerase (5 μL), 10 cutsmart buffer (6.5 μL), 5 u/μL Nt.BbvCI (5 μL) were successively injected into the resulting solution and allowed to incubate for 1 h at 37 °C. Subsequently, hemin (10 μL) was added to the mixture at a final concentration of 0.2 mM hemin and incubated for 1 h at 37 °C to form the hemin/G-quadruplex structures. After incubation, the solution was diluted with 725 μL of HEPES buffer. The obtained sample solutions were then used for the CL measurements.
2.1. Materials and reagents
2.5. Chemiluminescence measurements
All oligonucleotide sequences used in this study were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China), and the sequences were given in Table S1. The oligonucleotides were used as provided and diluted in 20 mM Tris–HCl buffer solution, pH 7.4, to give stock solutions of 100 μM. And each oligonucleotide was heated to 95 °C for 5 min, and slowly cooled down to room temperature for at least 2 h before use. Deoxynucleotide mixture solution (dNTPs), Klenow Fragment polymerase (3′-5′ exo , KF polymerase, 5 u/μL), and 10 Klenow
The CL spectra of the sample solutions obtained above were measured using a LS-55 luminescence spectrometer (Perkin-Elmer, USA) with a 3 mL quartz cuvette (1 cm optical path). The light source of the spectrometer was turned off. The emission wavelength was collected from 360 to 600 nm. The excitation and emission slits were set at 12.0 nm. To measure the CL spectra, 100 μL of luminol (0.01 M) and 100 μL of H2O2 (0.25 M) substrates were quickly added to the quartz cuvette containing the sample solutions obtained above, allowing the biocatalyzed oxidation of
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luminol to yield CL. The CL spectra were measured immediately. The emission intensity at 430 nm was recorded, and the increase of CL intensity (I–I0, I and I0 were the CL intensities at 430 nm in the presence and absence of target DNA, respectively) was used for the quantification of target DNA. 2.6. Gel electrophoresis Gel electrophoresis was used to confirm the activation of the DNA machine by hairpin assembly. Analysis by electrophoresis was carried out on 5% agarose gels by gene green nucleic acid dye staining, cast and ran in 0.5 TBE buffer (pH 7.9) at room temperature. The electrophoresis experiments were performed at a 90 V constant voltage for 1.5 h with loading of 8 mL of each prepared sample into the lanes. The resulting gel was photographed under UV light using a Quantum ST5 gel imaging system (Vilber, France).
3. Results and discussion 3.1. Principle of hairpin assembly-triggered cyclic activation of a DNA machine This study aims to construct a hairpin assembly-triggered cyclic activation of DNA machine toward target p53 DNA detection. The overall steps of this strategy are depicted in Fig. 1. The system mainly consists of two DNA hairpins (H1 and H2, their sequences are shown in Table S1). H1 contains four domains termed as I, II, III, and IV according to their different functions as shown in Fig. 1. The domain I consists of the complementary sequence of HRPmimicking DNAzyme. The domain II is partly complementary to domain IV. The domain III is the loop of H1. Domain IV is complementary to the target p53 DNA. H2 contains three domains termed as V, VI and VII. The domain V is complementary to the 3′ part of H1 and is blocked by hybridization with domain VII. It is worth noted that both H1 and H2 have a single-stranded sequences at their 3′ ends. In the absence of target, H1 and H2 can coexist in solution. Upon the addition of p53 DNA (a), p53 DNA
hybridizes with domain IV of H1 (b), and the hairpin structure of H1 is opened, forming a p53 DNA-H1 complex. Meanwhile, the unfolded domain II of H1 can serve as a toehold to hybridize to the domain VII of H2 (c), leading to the displacement of target p53 DNA through a branch migration process (Li et al., 2011a, 2011b, 2011c) and the formation of a H1–H2 complex. The displaced target p53 DNA can then hybridize to another H1, and the cycle starts anew, which leads to formation of numerous H1–H2 complexes. The generated H1–H2 complexes with the 3′ sticky end of H2 can act as DNA primer to trigger the polymerization reaction in the presence of KF polymerase and dNTPs, generating stable dsDNA structures with the recognition site for Nt.BbvCI. The cleavage of the single strand in the dsDNA structures by Nt.BbvCI generates new sites for the initiation of replication. Thus, the polymerase completes the replication of the HRP-mimicking DNAzyme units, and the reactivated replication at the scission site displaces the already synthesized HRP-mimicking DNAzyme units. As a result, an autonomous operation of the DNA machine can be realized through the replication. By following this mechanism, abundant HRP-mimicking DNAzyme units are synthetized. The HRP-mimicking DNAzyme unit products can assemble with hemin to form the hemin/G-quadruplexes that exhibit HRP-mimicking DNAzyme activity, which can catalyze the oxidation of luminol by H2O2 to generate CL (Lee et al., 2011) that provides a readout signal for amplified sensing of target p53 DNA. Importantly, the amplification strategy described here is isothermal, label-free, and does not require separation and washing steps, which is very simple and cost-effective. Moreover, because the proposed assay protocol combines the cyclic DNA assembly reaction with the amplification of DNA machine and HRP-mimicking DNAzyme, it can substantially improve the sensitivity of detection compared with previously reported homogeneous optical DNA assays. 3.2. Feasibility study In order to demonstrate the feasibility of our proposed assay strategy, the CL response of the sensing system under different conditions was investigated as shown in Fig. 2. The system containing only hemin exhibits low CL intensity (curve a). Upon the
Fig. 1. Schematic illustrating the principle of hairpin assembly-triggered cyclic activation of a DNA machine for ultrasensitive CL detection of target p53 DNA.
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Fig. 2. CL spectra of sample solutions under different conditions: (a) only hemin (200 nM); (b) H1 (100 nM)/H2 (120 nM)/dNTPs (0.1 mM)/KF polymerase (25 U)/Nt. BbvCI (25 U)/hemin (200 nM); (c) H1 (100 nM)/H2 (120 nM)/p53 DNA (1 nM)/ hemin (200 nM); (d) H1 (100 nM)/H2 (120 nM)/p53 DNA (1 nM)/dNTPs (0.1 mM)/ KF polymerase (25 U)/Nt.BbvCI (25 U)/hemin (200 nM).
addition of H1/H2/KF polymerase/dNTPs/Nt.BbvCI (curve b) and H1/H2/p53 DNA (curve c), the CL intensity of the system was slightly increased and almost equal. However, it had significant enhancement upon simultaneous addition of H1/H2/KF polymerase/dNTPs/Nt.BbvCI and p53 DNA (curve c), these phenomena indicated that the two hairpins H1 and H2 can maintain the sufficiently stable stem-loop structure in the solution. After addition of p53 DNA and KF polymerase/dNTPs/Nt.BbvCI, a single target can generate many H1–H2 complexes and thus yield many HRP-mimicking DNAzyme units. Thus, the proposed DNAzyme sensor can be used for the amplified detection of p53 DNA. 3.3. Gel electrophoresis characterization The hairpin assembly-triggered DNA machine amplification reactions were further verified using agarose gel electrophoresis, and the results were in agreement with the proposed mechanism (Fig. 3). In the absence of p53 DNA, the hairpin structures of H1
Fig. 3. Gene green nucleic acid dye stained agarose gel (5.0%) showing different mobilities of (M) DNA Marker; (Lane a) p53 DNA only; (Lane b) H1 only; (Lane c) H2 only; (Lane d) H1 incubated with H2 for 2 h at 37 °C; (Lane e) H1 incubated with p53 DNA for 2 h at 37 °C; (Lane f) H1 and H2 incubated with p53 DNA for 2 h at 37 °C; (Lane g) H1 and H2 incubated with p53 DNA for 2 h at 37 °C, followed by the addition of KF buffer (5 μL), 10 mM dNTPs (5 μL), and 5 u/μL KF polymerase (3 μL) to the mixture for another 1 h incubation at 37 °C; (Lane h) the products of hairpin assembly-triggered cyclic activation of a DNA machine. The final concentration of p53 DNA, H1 and H2 are all set as 0.5 μM.
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and H2 were maintained (lanes b, c and d), indicating no occurrence of hairpin assembly reaction. When p53 DNA was mixed with H1, the H1 band disappeared, and a new band with lower mobility compared with lane b (lane e) appeared, suggesting that the hybridization between p53 DNA and H1 had taken place and generated duplex DNA with a higher molecular weight. Once p53 DNA was incubated with H1 and H2, a new band with a slightly lower mobility compared with lane e appeared, and the bands of both H1 and H2 disappeared compared with lane d (lane f), indicating that the hairpin assembly reaction between H1 and H2 had been initiated by the p53 DNA. Upon the addition of the polymerase and dNTPs to the mixture of p53 DNA, H1 and H2, a new band with lower mobility compared with lane f (lane g) was observed, demonstrating that the replication reaction had been initiated by KF polymerase and dNTPs; however, when p53 DNA was mixed with H1, H2, KF polymerase, dNTPs and Nt.BbvCI, a new band with fast mobility compared with lane g (lane h) was observed, suggesting that the replication-scission-displacement process by KF polymerase, dNTPs, and Nt.BbvCI had taken place and generated the HRP-mimicking DNAzyme units. These gel electrophoresis results indicate the successful proceeding of the hairpin assembly-triggered DNA machine amplification reactions. 3.4. Analytical performance of the DNAzyme sensor Under the optimum conditions (Fig. S1 in Supporting Information), we evaluated the CL spectra of the proposed amplified assay for p53 DNA at varying concentrations. Fig. 4 displayed the CL response to the target DNA concentrations in the range from 0 fM to 1.0 nM. As seen in the figure, the CL intensities gradually increased upon increasing the concentration of p53 DNA. The increase of CL intensity showed a good linear correlation to the logarithm of p53 DNA concentration within the range from 2.0 fM to 1.0 nM (inset in Fig. 4). The regression equation was ΔI¼546.3þ36.12log C with a correlation coefficient of 0.9981, where ΔI is the increase of CL intensity and C is the concentration of p53 DNA. Based on 3s/S (s is the standard deviation for the blank solution, n¼10, and S is the slope of the calibration curve), the detection limit was estimated to be 0.85 fM. Supporting Information Table S2 compares the performance of the proposed method to previous homogeneous optical methods for
Fig. 4. CL response of the proposed DNA machine to varying concentrations of target DNA. The concentration of target DNA: (a) 0 fM; (b) 2.0 fM; (c) 8.0 fM; (d) 20 fM; (e) 0.1 pM; (f) 0.5 pM; (g) 2.0 pM; (g) 10 pM; (h) 0.1 nM; (i) 1.0 nM. The inset shows a linear relationship between the increase of CL intensity (ΔI¼ I I0) at 430 nm and the logarithm of the target p53 DNA concentration. Error bars show the standard deviation of three independent experiments. The system also includes 100 nM H1, 120 nM H2, 200 nM hemin, 25 U Nt.BbvCI, 0.1 mM dNTPs, 25 U KF polymerase, 1 mM luminol, 25 mM H2O2.
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matched target DNA (p53 DNA) led to an increase in the CL intensity, while the other DNAs did not lead to an obvious variation of the CL intensity. These results clearly demonstrate the high specificity of the developed hairpin assembly-triggered cyclic activation of a DNA machine. 3.6. Real sample analysis
Fig. 5. Detection specificity of the proposed DNA machine. The concentration of target p53 DNA (TD) was 1 nM, non-complementary oligonucleotide (NC), singlebase mismatched oligonucleotide (1 MT) and three-base mismatched oligonucleotide (3 MT) were 10 nM. Other experimental conditions were the same as those given in Fig. 4. Error bars show the standard deviation of three independent experiments.
To be useful in bioassays, the proposed detection method should be able to tolerate any interference from the biological samples. Therefore, we first prepared a series of samples by adding different concentrations of p53 DNA in different diluted ratios of human HepG2 cell lysate samples and analyzed the mixtures using the proposed assay method. When the ratio of the human HepG2 cell lysate sample and assay buffer reached 1:10, comparable responses were found for p53 DNA in both the buffer and cell lysate. Thus, 10-fold diluted human HepG2 cell lysate samples were selected and used to analyse the various concentrations of p53 DNA. Fig. 6 shows the CL responses with different concentrations of p53 DNA in both the buffer and cell lysate. A slight signal change in the cell lysate sample could be observed compared to the CL intensity in the buffer under the same experimental conditions, which may be related to the interference of the sample matrix during the process of hairpin assembly-triggered DNA machine amplification. Despite the slight response differences, the fabricated CL DNAzyme sensor still showed a distinguishable performance at different p53 DNA concentrations in the cell lysate samples. Then, three independent spiked human HepG2 cell lysate samples were tested by this assay, and the concentration of p53 DNA in the spiked human HepG2 cell lysate samples was calculated by using the calibration curve of standard target p53. The analytical results are shown in Table S3. The recoveries were found to be in the range of 93.1–98.6%, and the RSDs were between 2.5% and 4.6%. These results indicated the potential of the proposed assay method for detecting target DNA in complex biological samples.
4. Conclusions
Fig. 6. Results obtained upon testing 10-fold diluted human HepG2 cell lysate samples spiked with different concentrations of p53 DNA. The same reaction mixtures without p53 DNA were used as blanks. Other experimental conditions were the same as those given in Fig. 4. Error bars show the standard deviation of three independent experiments.
sensing DNA. The results revealed that the sensitivity of the present assay was at least 5 orders of magnitude higher compared to an unamplified homogeneous DNA assay (Li et al., 2011a, 2011b, 2011c) and was also higher sensitivity than those of other amplified homogeneous optical assays (Jiang et al., 2013; Li et al., 2013; Liu et al., 2012, 2013; Shimron et al., 2012; Xu et al., 2012; Zou et al., 2011). In addition, assay reproducibility was also investigated by analysing 5.0 pM and 500 pM p53 DNA standard solutions nine times. The results showed that the relative standard deviations (RSDs) for p53 DNA quantity less than 4.5%. 3.5. Detection specificity To investigate the specificity of the proposed assay, different DNA targets (such as target p53 DNA (TD), a single-base mismatched DNA target (1MT), a three-base mismatched DNA target (3MT)) and a non-complementary oligonucleotide (NC); the sequences are shown in Table S1) were analysed. The results obtained are presented in Fig. 5. As seen in this figure, the perfectly
In summary, we have developed a novel, label-free CL amplification strategy based on hairpin assembly-triggered cyclic activation of a DNA machine. As a proof-of-concept, the proposed strategy can be successfully used for p53 DNA detection both in aqueous buffer and in human HepG2 cell lysate samples. This assay has several important features. First, by using hairpin assembly-triggered cyclic activation of a DNA machine, we could detect the p53 DNA as low as 0.85 fM, which is significantly improved by at least five orders of magnitude over traditional homogeneous DNA assays. Second, this method is capable of discriminating mismatched DNA from perfect matched target DNA with a high selectivity. Third, the method does not require any chemical modification of the DNA, resulting in a very simple and cost-effective process. Finally, this assay protocol can be easily extended to the analysis of other DNAs or RNAs molecules by simply changing the sequences of the hairpin structures. These characteristics of our proposed assay protocol make it a potential platform for further applications in the early diagnosis of gene-related diseases, biological process researches, and drug discovery.
Acknowledgements This work was supported by the “Hundred Talents Program” of the Chinese Academy of Science, the National Natural Science Foundation of China (Nos. 21405163 and 21475142) and the
J. Chen et al. / Biosensors and Bioelectronics 68 (2015) 550–555
Foundation for Sci & Tech Research Project of Gansu Province (145RJYA271).
Appendix A. Supplementary material The supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015. 01.054.
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