- Email: [email protected]
Amplified fluorescence detection of DNA based on catalyzed dynamic assembly and host–guest interaction between β-cyclodextrin polymer and pyrene
Talanta 144 (2015) 529–534
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Amplified fluorescence detection of DNA based on catalyzed dynamic assembly and host–guest interaction between β-cyclodextrin polymer and pyrene Haihua Huang, Xiaohai Yang n, Kemin Wang n, Qing Wang, Qiuping Guo, Jin Huang, Jianbo Liu, Xiaochen Guo, Wenshan Li, Leiliang He State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China
art ic l e i nf o
a b s t r a c t
Article history: Received 30 April 2015 Received in revised form 25 June 2015 Accepted 28 June 2015 Available online 2 July 2015
The detection of nucleic acids is fundamental for studying their functions and for the development of biological studies and medical diagnostics. Herein, we report a new strategy for nucleic acid amplified detection by combining target-catalyzed dynamic assembly with host–guest interaction between β-cyclodextrin polymer (β-CDP) and pyrene. In this strategy, a metastable pyrene-labeled hairpin DNA probe (probe H1) and a metastable unlabeled hairpin DNA probe (probe H2) were elaborately designed as the assembly components, which were kinetically handicapped from cross-opening in the absence of target DNA. In this state, pyrene labled at the 5ʹ-termini of single-stranded stem of probe H1 would be easily trapped into the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to significant fluorescence enhancement. Once the dynamic assembly was catalyzed by target DNA, a hybridized DNA duplex H1–H2 would be created continuously. In this state, it is difficult for pyrene to enter the cavity of β-CDP due to steric hindrance and weak-binding interaction, leading to a weak fluorescent signal. Thus, target DNA could be detected by this simple mix-and-detect amplification method without the need of expensive and perishable protein enzymes. As low as 10 pM of the target DNA was detected by this assay, which was comparable to that of some reported enzyme-dependent amplification methods. Meanwhile, the proposed method was further successfully applied to detect DNA in cell lysate samples, showing great potential for target detection from complex fluids. In addition, as a novel transformation of dynamic DNA assembly technology into enzyme-free signal-amplification analytical application, the proposed strategy has shown great potential for applications in a wide range of fields, such as aptamerbased non-nucleic acid target sensing, biomedicine and bioimaging. & 2015 Elsevier B.V. All rights reserved.
Keywords: Enzyme-free Pyrene β-cyclodextrin polymer Nucleic acid detection
1. Introduction Sensitive and selective detection of DNA sequences plays a vital role in clinical diagnosis and biomedical studies [1]. The dynamic DNA self-assembly technology as an attractive signal-amplification nucleic acid detection approach has attracted significant attention, and immense advances have been achieved [2–8]. Pierce and coworkers firstly introduced the basic concept of catalyzed selfassembly technology, which is one of the most important approaches for “bottom-up” fabrication of nanostructures and nanodevices [9]. DNA, as an ideal building block for nanotechnology, is programmed and self-assembled in a predictable manner in the n
Corresponding authors. Fax: þ 86 731 88821566. E-mail addresses: [email protected] (X. Yang), [email protected] (K. Wang). http://dx.doi.org/10.1016/j.talanta.2015.06.087 0039-9140/& 2015 Elsevier B.V. All rights reserved.
catalyzed dynamic assembly. In the past few decades, inspired by the simple rules governing hybridization of metastable DNA molecules, researchers have developed many attractive signal-amplification approaches based on the catalyzed dynamic assembly for biochemical analytical applications [10–27]. For example, Pierce group constructed a kind of in situ amplifier for multiplexed imaging of mRNAs in fixed whole mount and sectioned zebrafish embryos [18]. Tan group designed a hairpin DNA cascade reaction for mRNA imaging inside live cells [22]. We have also developed several fluorescent amplification methods successfully for sensitive nucleic acid analysis [23–26]. In short, the catalyzed dynamic assembly based amplification method has two obvious advantages compared with conventional enzyme-based methods [28–33]. First, it only relies on strand displacement reactions, avoiding perishable protein enzymes, complicated instrumentations and complex thermal-cycling procedures. Second, it is inherently scalable and modular, just
530
H. Huang et al. / Talanta 144 (2015) 529–534
requiring the design of base-pairing between metastable DNA molecules. Therefore, we believe that there is a great potential to adapt catalyzed self-assembly to support much more analytical applications. In recent years, many new signal amplifying methods based on nanomaterials and supramolecular polymers have emerged [34,35]. A wide variety of fluorescence quenching based bioanalytical methods have been developed. For example, Graphene and graphene oxide can quench the fluorescence of nearby organic dyes and upconverting nanocrystals, resulting in extensive studies of fluorescent quenching biosensors [36]. Gold nanoparticles can serve as excellent fluorescence quenchers for FRET-based assays due to their extraordinary high molar extinction coefficients and many gold nanoparticles based assays have been used to detect and quantify intracellular analytes [37]. Meanwhile, fluorescence enhancement based detection methods have attracted more attention. Metal nanoparticles, for instance, have attracted wide attention for their good metal-enhanced fluorescence (MEF) activity and many detection methods based on MEF have been developed to further improve the fluorescence technique [38–40]. Aggregation-induced emission enhancement (AIEE) has been observed and open new routes to fluorescence enhancement analytical methods [41]. Particularly, supramolecular systems based on host–guest interactions have attracted significant attention on the horizon of material science due to their unique properties [42]. Thus, a new type of signal amplifying method based on the host– guest interaction of supramolecular polymer might contribute to sensitive nucleic acids detection. We have developed several fluorescence analytical methods based on host–guest interaction between epichlorohydrin cross-linked β-cyclodextrin polymer (βCDP) and pyrene [43–45]. Herein, a new enzyme-free nucleic acid amplified detection strategy has been developed by combining target-catalyzed dynamic assembly with host–guest interaction between β-CDP and pyrene. In this work, we designed two metastable hairpin DNA probes (probe H1 and probe H2), a DNA segment was used as the nucleic acid target. The pyrene-labeled probe H1 was chosen as the signal unit. Therefore, a fluorescent turn-off enzyme-free isothermal amplification strategy based on target-catalyzed dynamic assembly of branched junction was constructed, and this fluorescence amplification strategy showed high sensitivity toward target DNA with a detection of 8 pM. Moreover, the proposed strategy was further successfully applied to detect DNA in cell lysate samples.
2. Experimental 2.1. Chemicals and materials DNA marker and loading buffer were purchased from TaKaRa Bio Inc. (Dalian, China). SYBR Gold was purchased from Invitrogen (USA). β-CDP (Mn 94,400; GPC, waters-515) was synthesized according to the procedure reported previously [43]. The purified β-CDP powder was re-dispersed in aqueous solution and stored at 4 °C for use. All the solutions were prepared with ultrapure water obtained from a Millipore water purification system (4 18.2 MΩ cm). All the DNA sequences and pyrene-labeled probe were synthesized by TaKaRa Bio Inc. (Dalian, China) and purified by HPLC. The sequences design process of hairpin probes is summarized in Fig. S1. The structural schematic diagram of pyrene linked to the 5ʹ-terminus of probe H1 is shown in Fig. S2. The sequences used in this work are listed in Table S1. All other chemicals, if not specified here, were all commercially available and used as received and used without further purification or treatment.
2.2. Apparatus All fluorescence spectra were measured using a Hitachi F-7000 fluorescence spectrometer (Hitachi Ltd., Japan) controlled by FL Solution software equipped with aqueous thermostat (Amersham) accurated to 0.1 °C. Excitation and emission slits were all set for a 5.0 nm band-pass with a 700 V PMT voltage. The excitation wavelength was set at 345 nm and the emission spectra from 365 to 480 nm were collected with a 0.2 1 cm2 quartz cuvette containing 100 μL of solution. The fluorescence intensity at 380 nm was used to evaluate the performance of the proposed assay strategy.
2.3. Agarose electrophoresis analysis A 2.5% agarose gel was prepared by using 0.5 Tris-borateEDTA (TBE) (pH 8.0). Different mixtures of target DNA with probe H1 and probe H2 were incubated for 3.5 h at 39 °C, the concentration of each hairpin probe was 500 nM. After preparing samples, 10 μL of each sample was mixed with 2 μL of 6 loading buffer, then 10 μL of mixed solution was added into the gel for electrophoresis. SYBR gold was used as the DNA stain and mixed with the samples. The gel was run at 100 V for 45 min at room temperature with loading of 10 mL of sample into each lane, and then photographed in Gel Imaging (Tanon 2500R, Tianneng Ltd., Shanghai, China).
2.4. DNA detection procedures The DNA detection were performed in 100 μL of a hybridization buffer (20 mM Tris–HCl, 140 mM NaCl, and 5 mM KCl, pH 7.5) consisting of different concentrations of target DNA, 100 nM probe H1, 600 nM probe H2. Initially, the prepared solution was incubated for 3.5 h at 39 °C for the assembly reaction. Then 4 mg mL 1 β-CDP was introduced to the resulting solution and incubated for another 5 min for the host–guest interaction. Finally, the resulted solutions were characterized by fluorescence spectrophotometer and the fluorescence intensity was recorded.
2.5. Preparation of complex samples To test the capability of the proposed strategy for detecting target DNA from complex biological samples, Hela cell lines were cultured in our lab and were grown in RPMI 1640 cell medium with 10% inactivated fetal bovine serum (Hyclone, USA) at 37 °C in 5% CO2. Vigorous growth cells were collected after trypsin digestion. The cell density was determined using a hemocytometer, and this was performed prior to each experiment. A suspension of 1 106 cells was centrifuged at 1000 rpm for 3 min, then washed with PBS buffer(10 mM phosphate, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) three times and at last suspended in Tris–HCl buffer. Finally, the cells were disrupted by sonication for 20 min at 0 °C. To remove the homogenate of cell debris, the lysate was centrifuged at 18,000 rpm for 20 min at 4 °C. The supernatant was ready for DNA detection. The target DNA spiked lysate samples were prepared by adding different concentration of target DNA in the lysate samples. Then, the detection procedure according to the proposed strategy was carried out. Unless noted otherwise, all the experiments for measurements were repeated three times at least in this study.
H. Huang et al. / Talanta 144 (2015) 529–534
531
3. Results and discussion 3.1. Design strategy for nucleic acids detection
Target Probe H1 Probe H2 Flourescence intensity (a.u)
In this proposed method, two metastable hairpin DNA probes (probe H1 and probe H2) were designed, a DNA segment was used as a proof-of-concept nucleic acid target. The pyrene-labeled probe H1 was chosen as the signal unit. In the presence of target DNA, a cascade of assembly reactions were catalyzed to form many branched junctions shown in Fig. 1A. Once the dynamic assembly among probes was catalyzed by target DNA, a hybridized DNA duplex H1–H2 would be created continuously. Each assembly circle was terminated by a disassembly step in which probe H2 displaced target DNA from the branched complex, freeing target DNA to catalyze the next assembly circle. Due to strong steric hindrance and weak-binding interaction, it is difficult for pyrene to enter the cavity of β-CDP, leading to a weak fluorescent signal. In the absence of target DNA, probe H1 and probe H2 were in the closed hairpin formation, and pyrene molecule labeled on probe H1 was spatially separated by the sticky end in a length of extra length of 8 mononucleotides shown in Fig. 1B. In this state, pyrene molecules at the 5ʹ-termini of single-stranded stem of probe H1 would be easily trapped into the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to significant fluorescence enhancement. Therefore, a fluorescent turn-off enzyme-free isothermal nucleic acid amplified detection strategy was constructed by combining the target-catalyzed dynamic assembly with host–guest interaction between β-CDP and pyrene.
1
2
3
+ -
+
+ +
4
+ + +
1600
a b c
1400 1200 1000 800
d
600 400
e
200 0 380
400
420
440
460
480
500
Wavelength (nm) 1
4
3*
3
2*
2
4 1* 2* 3* 1
H1 opened
1
2
3
3
1* H1
1 2 3
1
1
2
3
1*
1
4* 3*
4
β CDP
3*
4
4*
1
1*
3*
3
2*
2
1*
1 4
4
2
3
Low Fluorescence
1
3
2* 1*
2 Without target
H1
4
3
2
3*
3
1*
2* No assembly 1*
2
4* 3*
H1
1 4 β CDP
1
4
3*
3
2* 1*
2
H2
2 High Fluorescence
1*
3.2. Verification of the catalyzed dynamic assembly amplification method
1* 4* 3*
4
3*
3
2
3
H1 H2 assembly
1* 2* 3* 1 4
Fig. 2. Characterization and feasibility investigation of the enzyme-free nucleic acid amplified detection. (A) Gel electrophoretic analysis of the products by targetcatalyzed assembly. The concentrations of pyrene-labeled probe H1, probe H2 and target DNA were 500 nM, 500 nM and 50 nM respectively ( þ means in the presence of,– means in the absence of). The gels were run at 100 V for 45 min. (B) Fluorescence spectra demonstrating the target-catalyzed assembly steps in Fig. 1. Curve a: pyrene-labeled probe H1 þβ-CDP; Curve b: pyrene-labeled probe H1þ target DNA þβ-CDP; Curve c: pyrene-labeled probe H1 þ probe H2þ β-CDP; Curve d: pyrene-labeled probe H1þ probe H2 þtarget DNA þ β-CDP; Curve e: pyrene-labeled probe H1. The concentrations of pyrene-labeled probe H1, probe H2, target DNA and β-CDP were 100 nM, 500 nM, 3 nM and 4 mg mL 1 respectively.
2
2
H2
Target displaced
Target
3
1
4* 4 3* H2 Fig. 1. The schematic illustration of the proposed enzyme-free nucleic acid amplified detection based on catalyzed dynamic assembly and host–guest interaction between β-cyclodextrin polymer and pyrene. Numbers marked with n are complementary to the corresponding unmarked numbers.
To confirm the enzyme-free nucleic acid amplified detection method, we utilized gel electrophoresis to monitor the reaction. As shown in Fig. 2A, it could be observed that there was only one band in lane 1, lane 2 and lane 3 in the absence of target DNA, indicating that no catalyzed dynamic assembly occurs. As a comparison, new band appeared and the old band disappeared in lane 4 when target DNA was added, suggesting the formation of H1–H2 complex. In the system, target DNA was re-used until the supply of probe H1 or probe H2 was exhausted. Thus, these results demonstrated that the dynamic assembly was circularly catalyzed by target DNA. To further explore the amplification effect of different conditions, we evaluated their performance with control experiments under the same target DNA concentration of 3 nM. As shown in Fig. 2B, the fluorescence intensity of probe H1 (Fig. 2B (e)) enhanced greatly with the assistance of β-CDP (Fig. 2B (a)). In the absence of target DNA, probe H1 and probe H2 exhibit a decrease
in fluorescence intensity (Fig. 2B (c)). In the presence of 3 nM target DNA, a mild decrease in fluorescence intensity was observed with the presence of probe H1 (Fig. 2B (b)). However, a significant decrease in fluorescence intensity was obtained by adding probe H2 (Fig. 2B (d)). These results showed that a successful targetcatalyzed dynamic assembly could provide higher sensitivity. 3.3. Optimization of experimental conditions To ensure a better performance, several parameters were investigated to establish optimal conditions for our detection system including concentration ratio of probe H1 and probe H2, concentrations of β-CDP, Na þ , K þ , Tris–HCl, reaction temperature and reaction time. The optimization of the variables of our system was based on the fluorescence decreasing factor. The fluorescence decreasing factor was (F0 F)/F0, where F and F0 were the fluorescence intensities of the detection system with and without target DNA respectively. 3.3.1. Optimization of concentration ratio of H1 and H2 The hairpin probes play important roles in the performance of this assay. On one hand, sufficient amount of probe should be added to carry out enough rounds of cycles; on the other hand, an obvious background signal would be observed at a relatively high concentration of probe H2, which might resulted from a bit of spontaneous hybridization between probe H1 and probe H2. Therefore, concentration ratios of probe H1 and H2 were firstly studied towards the detection of 3 nM target DNA. As shown in Fig. S3 (supporting information), when the concentration ratio of probe H1 and H2 is 1:6, the (F0 F)/F0 value is maximum. So we choose the concentration ratio 1:6 in our following detection system to achieve good performance. 3.3.2. Optimization of concentrations of Tris–HCl, K þ and Na þ It is generally acknowledged that salt ions in solution could neutralize the charge of the nucleic acid and has a promoting effect on DNA hybridization and fabrications of hairpin structure. This means that an appropriate ionic strength might affect sensitivity and specificity. To achieve the optimal response of the detection system, the effect of ion strength was thus investigated. As shown in Fig. S4 (A) (supporting information), it was clear that the (F0 F)/F0 value was dependent on the concentration of Tris–HCl. To achieve good performance, we chose 20 mM Tris–HCl in our following detection system. As shown in Fig. S4 (B) and Fig. S4 (C) (supporting information), when the concentration of Na þ and K þ was 140 mM and 5 mM respectively, the (F0 F)/F0 values reached maximum. Therefore, we chose 20 mM Tris–HCl, 140 mM Na þ and 5 mM K þ in our following detection system to achieve good performance. 3.3.3. Optimization of concentration β-CDP β-CDP serves as the host component for host–guest interaction between β-CDP and pyrene. Adding appropriate amount of β-CDP can gain favorable fluorescence enhancement which is conducive to produce obvious response signals. Excess β-CDP may cause strong background signals, so the concentration of β-CDP was optimized. As shown in Fig. S5 (supporting information), when the concentration of β-CDP is 4 mg mL 1, the (F0 F)/F0 value is maximum. Therefore, 4 mg mL 1 β-CDP was chosen as the optimized concentration in the following experiments. 3.3.4. Optimization of reaction time and temperature Finally, the reaction time and temperature was optimized. As indicated in Fig. S6 (A) (supporting information), with the increasing of reaction time, the (F0 F)/F0 value increased and reached maximum at 3.5 h. Therefore, 3.5 h was chosen as the
Fluorescence intensity (a.u.)
H. Huang et al. / Talanta 144 (2015) 529–534
1400 1200 1000 800 600 400 200 0 380
400
420
440
460
480
500
20
25
Wavelength (nm) 0.8 0.6
(F0-F)/F0
532
0.4 0.2 0.0
0
5
10
15
Concentration of target DNA (nM) Fig. 3. (A) Fluorescence spectra of the amplification method over a range of target DNA concentrations. The curves from up to bottom contain target DNA with 0 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM and 20 nM respectively. (B) Plot of target DNA vs fluorescence intensity. Inset is the calibration curve for the concentrations of target DNA from 0 nM to 1 nM. The concentrations of pyrene-labeled probe H1, probe H2, β-CDP, Tris–HCl, Na þ and K þ were 100 nM, 600 nM, 4 mg mL 1, 20 mM, 140 mM and 5 mM, respectively. Error bars indicated the standard deviations of three experiments.
optimized time in following experiments. Temperature is a key parameter of reaction kinetics and determines the probability of collisions between the molecules. At low temperature, the hairpin probes could not get a sufficient collision probability that greatly reduced the formation of H1–H2 complexes. Inversely, the specificity of this platform would be debased by high reaction temperature, because temperature is interrelated with stability and integrality of hairpin probes. As shown in Fig. S6 (B) (supporting information), with the increasing reaction temperature, the (F0 F)/F0 value increased and reached maximum at 39 °C. Therefore, 39 °C was chosen as the optimized temperature in the following experiments. 3.4. Sensitivity of DNA detection Under the above optimal conditions and according to the DNA detection procedures described in the previous section, we evaluate the response range and detection limit by measuring fluorescence intensity upon the addition of different concentrations of target DNA. As shown in Fig. 3A, a gradual decrease in the fluorescence intensity was clearly observed in the concentration of
target DNA ranged from 0 nM to 20 nM. The relationship between the change of fluorescence intensity and the concentration of target DNA was demonstrated in Fig. 3B. The results showed that the fluorescence intensity decreased with the increase of target concentration in the range from 10 pM to 1 nM with a detection limit of 8 pM as estimated by the 3s rule. This detection capability was comparable to that of some reported enzyme-dependent colorimetric or electrochemical amplification methods (Table S2 in supporting information). The calibration curve is shown in the inset in Fig. 3B. The regression equation was (F0–F)/ F0 ¼0.0245þ0.1844(C nM 1) during the range from 10 pM to 1 nM (F was the fluorescence intensity of the mixture of probe H1, probe H2 and target DNA; F0 was the fluorescence intensity in the mixture of probe H1 and probe H2; C was the concentration of target DNA; R2 ¼0.9902). We also investigated three prolonged DNA sequences (Prolonged DNA a, b and c) to demonstrate the usefulness of the assay for longer target sequences detection. The result is shown in Fig. S7 (supporting information).
Fluorescence intensity (a.u.)
H. Huang et al. / Talanta 144 (2015) 529–534
533
1200
0 nM
1000 800
20 nM
600 400 200 0 380
400
420
440
460
480
500
Wavelength (nm) 0.7 0.6
The selectivity of the proposed DNA detection strategy was carefully evaluated. Several control DNAs, including target DNA, mismatched DNA and random DNA, were detected by this method. As shown in Fig. 4, the fluorescence intensity change was enormous in the presence of target DNA, while negligible change was observed in the presence of random DNA, which indicated that the proposed strategy was capable of detecting target DNA from random nucleic acid library. Furthermore, at the same detected concentration, although mismatched DNA could result in a minor change in fluorescence intensity, much more fluorescence decrease was observed in the presence of target DNA than that in the presence of any mutant DNA, which indicated that our proposed strategy also had the potential to distinguish single nucleotide polymorphism (SNP). Therefore, based on the above results, this constructed enzyme-free isothermal amplification strategy could be used to detect target nucleic acid with good specificity.
0.5
0.7
(F0-F)/F0
3.5. Selectivity of DNA detection
0.4 0.3 0.2 0.1 0.0 1
2
4
8
12
24
Concentration of target DNA (nM) Fig. 5. (A) Fluorescence spectra of the detection system for various concentrations of target DNA in 50% cell lysate. The curves from up to bottom contain target DNA with 0 nM, 1 nM, 2 nM, 4 nM, 8 nM, 12 nM and 24 nM respectively. (B) Fluorescence intensity at λex/em ¼ 345/380 nm of the system as a function of the various concentrations of target DNA in 50% cell lysate. The concentrations of probe H1, probe H2, β-CDP, Tris–HCl, Na þ and K þ were 100 nM, 600 nM, 4 mg mL 1, 20 mM, 140 mM and 5 mM, respectively. Error bars indicated the standard deviations of three experiments.
0.6 3.6. Target DNA detection in complex samples
(F0-F)/F0
0.5 0.4 0.3 0.2 0.1
c R an do m
is m
at
ch M
at m M is
ch
ed
ed ch at
is m
ed
a
t rg e Ta M
b
0.0
Fig. 4. Specificity evaluation of the proposed method for target DNA detection (3 nM), mismatched DNA a (3 nM), mismatched DNA b (3 nM), mismatched DNA c (3 nM) and random DNA (3 nM). The concentrations of pyrene-labeled probe H1, probe H2, β-CDP, Tris–HCl, Na þ and K þ were 100 nM, 600 nM, 4 mg mL 1, 20 mM, 140 mM and 5 mM, respectively. Error bars indicated the standard deviations of three experiments.
Detection of target DNA in complex samples was carried out to test the practical application capability of the proposed strategy. In this work, diluted Hela cell lines extract was selected as the complex fluid to investigate the feasibility of this strategy in biological samples. Target DNA-containing cell extract samples were prepared by adding target DNA in 50% cell extract contained reaction buffer. Then target DNA-spiked complex fluids were detected by the procedure according to the proposed strategy. The calibration curve was obtained using the 50% cell lysate with the addition of DNA in various concentrations. As shown in Fig. 5, a plot of the fluorescence intensity versus the concentration of DNA revealed a dynamic correlation between the fluorescence intensity and concentration of DNA in the range from 0 nM to 24 nM. As for demonstrating the usefulness of the assay for longer target sequences detection we use cell lysates spiked with prolonged DNA sequences and the result was shown in Fig. S8 (supporting information). The results suggested that the signal change of this method is dependent on the concentration of DNA in cell lysate, which showed that the strategy held great potential for practical detection of target DNA from complex fluids.
534
H. Huang et al. / Talanta 144 (2015) 529–534
4. Conclusions In summary, the present study introduced a new path for enzyme-free DNA amplified detection by combining the isothermal signal-amplification capability of target-catalyzed dynamic assembly with host–guest interaction between β-CDP and pyrene. First, the hairpin probes in this detection system is easy to design, synthesize, purify and convenient for usage. Second, it was a simple mix-and-detect amplification method, which only relied on hybridization and strand displacement reactions, without requiring perishable protein enzymes, complicated instrumentations and complex thermal-cycling procedures. In addition, our strategy achieved a good detection capability with the detection of 8 pM and successfully application in complex samples. Overall, as a novel transformation of catalyzed dynamic assembly technology into enzyme-free signal-amplification analytical application, we infer that this strategy might be applicable for a wide range of fields, such as aptamer-based non-nucleic acid target sensing, biomedicine and bioimaging.
[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
[23] [24] [25]
Acknowledgments This work was supported by the National Natural Science Foundation of China (21190044 and 21175035), National Basic Research Program of China (2011CB911002), and International Science & Technology Cooperation Program of China (2010DFB30300).
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.talanta.2015.06.087.
References [1] H. Zhang, F. Li, B. Dever, X. Li, X.C. Le, Chem. Rev. 113 (2013) 2812–2841. [2] F. Wang, X. Liu, I. Willner, Angew. Chem. Int. Ed. 54 (2015) 1098–1129. [3] S. Venkataraman, R.M. Dirks, P.W.K. Rothemund, E. Winfree, N.A. Pierce, Nat. Nanotechnol. 2 (2007) 490–494. [4] D. Zhang, A.J. Turberfield, B. Yurke, E. Winfree, Science 318 (2007) 1121–1125. [5] Y. Zang, J. Lei, H. Ju, Anal. Chem. 87 (2015) 5430–5436, http://dx.doi.org/ 10.1021/acs.analchem.5b00888. [6] G. Seelig, B. Yurke, E. Winfree, J. Am. Chem. Soc. 128 (2006) 12211–12220. [7] D. Soloveichika, G. Seeliga, E. Winfree, Proc. Natl. Acad. Sci. USA 107 (2010) 5393–5398. [8] Y. Qian, C. Wang, F. Gao, Talanta 130 (2014) 33–38.
[26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45]
P. Yin, H.M.T. Choi, C.R. Calvert, N.A. Pierce, Nature 451 (2008) 318–322. C. Geary, P.W.K. Rothemund, E.S. Andersen, Science 345 (2014) 799–804. K.V. Gothelf, T.H. LaBean, Org. Biomol. Chem. 3 (2005) 4023–4037. B.L. Li, A.D. Ellington, X. Chen, Nucleic Acids Res. 39 (2011) e110. S. Shimron, F. Wang, R. Orbach, I. Willner, Anal. Chem. 84 (2012) 1042–1048. B. Zhang, B. Liu, D. Tang, R. Niessner, G. Chen, D. Knopp, Anal. Chem. 84 (2012) 5392–5399. J. Huang, X. Su, Z. Li, Anal. Chem. 84 (2012) 5939–5943. Y. Chen, J. Xu, J. Su, Y. Xiang, R. Yuan, Y. Chai, Anal. Chem. 84 (2012) 7750–7755. C. Lin, J.K. Nangreave, Z. Li, Y. Liu, H. Yan, Nanomedicine 3 (2008) 521–528. H.M.T. Choi, J.Y. Chang, L.A. Trinh, J.E. Padilla, S.E. Fraser, N.A. Pierce, Nat. Biotechnol. 28 (2010) 1208–1212. G. Zhu, J. Zheng, E. Song, M. Donovan, K. Zhang, C. Liu, W. Tan, Proc. Natl. Acad. Sci. USA 110 (2013) 7998–8003. G. Zhu, S. Zhang, E. Song, J. Zheng, R. Hu, X. Fang, W. Tan, Angew. Chem. Int. Ed. 52 (2013) 5490–5496. J. Zheng, G. Zhu, Y. Li, C. Li, M. You, T. Chen, E. Song, R. Yang, W. Tan, ACS Nano 7 (2013) 6545–6554. C. Wu, S. Cansiz, L. Zhang, I. Teng, L. Qiu, J. Li, Y. Liu, C. Zhou, R. Hu, T. Zhang, C. Cui, W. Tan, J. Am. Chem. Soc. 137 (2015) 4900–4903, http://dx.doi.org/ 10.1021/jacs.5b00542. K. Quan, J. Huang, X. Yang, Y. Yang, L. Ying, H. Wang, K. Wang, Chem. Commun. 5 (2014) 937–940. P. Liu, X. Yang, S. Sun, Q. Wang, K. Wang, J. Huang, J. Liu, L. He, Anal. Chem. 85 (2013) 7689–7695. J. Huang, Y. Wu, Y. Chen, Z. Zhu, X. Yang, C. Yang, K. Wang, W. Tan, Angew. Chem. Int. Ed. 50 (2011) 401–404. Z. Qing, X. He, J. Huang, K. Wang, Z. Zou, T. Qing, Z. Mao, H. Shi, D. He, Anal. Chem. 86 (2014) 4934–4939. Y.S. Jiang, S. Bhadra, B. Li, A. Ellington, Angew. Chem. Int. Ed. 53 (2014) 1–5. A. Fire, S. Xu, Proc. Natl. Acad. Sci. USA 92 (1995) 4641–4645. J.V. Ness, L.K. Ness, D.J. Galas, Proc. Natl. Acad. Sci. USA 100 (2003) 4504–4509. Y. Weizmann, M. Beissenhirtz, Z. Cheglakov, R. Nowarski, M. Kotler, I. Willner, Angew. Chem. Int. Ed. 45 (2006) 7384–7388. B. Zou, Y. Ma, H. Wu, G. Zhou, Angew. Chem. Int. Ed. 50 (2011) 7395–7398. Q. Wang, L. Yang, X. Yang, K. Wang, L. He, J. Zhu, T. Su, Chem. Commun. 48 (2012) 2982–2984. R. Deng, L. Tang, Q. Tian, Y. Wang, L. Lin, J. Li, Angew. Chem. Int. Ed. 53 (2014) 2389–2393. M.J. Serpe, S.L. Craig, Langmuir 23 (2007) 1626–1634. L. Cheng, C. Wang, L. Feng, K. Yang, Z. Liu, Chem. Rev. 114 (2014) 10869–10939. P. Yin, S. Shah, M. Chhowalla, K. Lee, Chem. Rev. 115 (2015) 2483–2531. K. Saha, S. Agasti, C. Kim, X. Li, V. Rotello, Chem. Rev. 112 (2012) 2739–2779. J. Zhang, N. Ma, F. Tang, Q. Cui, F. He, L. Li, ACS Appl. Mater. Interfaces 4 (2012) 1747–1751. D. Cheng, Q. Xu, Chem. Commun. 43 (2007) 248–250. Y. Li, L. Guan, H. Zhang, J. Chen, S. Lin, Z. Ma, Y. Zhao, Anal. Chem. 83 (2011) 4103–4109. Y. Hong, J. Lam, B. Tang, Chem. Soc. Rev. 40 (2011) 5361–5388. X. Ma, Y. Zhao, Chem. Rev. (2014), http://dx.doi.org/10.1021/cr500392w. X. Guo, P. Liu, X. Yang, K. Wang, Q. Wang, Q. Guo, J. Huang, J. Liu, C. Song, W. S. Li, Analyst 140 (2015) 2016–2022. L. He, X. Yang, F. Zhao, K. Wang, Q. Wang, J. Liu, J. Huang, W. Li, M. Yang, Anal. Chem. 87 (2015) 2459–2465. C. Song, X. Yang, K. Wang, Q. Wang, J. Liu, J. Huang, L. He, P. Liu, Z. Qing, W. Liu, Chem. Commun. 51 (2015) 1815–1819.
Contents lists available at ScienceDirect
Talanta journal homepage: www.elsevier.com/locate/talanta
Amplified fluorescence detection of DNA based on catalyzed dynamic assembly and host–guest interaction between β-cyclodextrin polymer and pyrene Haihua Huang, Xiaohai Yang n, Kemin Wang n, Qing Wang, Qiuping Guo, Jin Huang, Jianbo Liu, Xiaochen Guo, Wenshan Li, Leiliang He State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, Hunan University, Changsha 410082, China
art ic l e i nf o
a b s t r a c t
Article history: Received 30 April 2015 Received in revised form 25 June 2015 Accepted 28 June 2015 Available online 2 July 2015
The detection of nucleic acids is fundamental for studying their functions and for the development of biological studies and medical diagnostics. Herein, we report a new strategy for nucleic acid amplified detection by combining target-catalyzed dynamic assembly with host–guest interaction between β-cyclodextrin polymer (β-CDP) and pyrene. In this strategy, a metastable pyrene-labeled hairpin DNA probe (probe H1) and a metastable unlabeled hairpin DNA probe (probe H2) were elaborately designed as the assembly components, which were kinetically handicapped from cross-opening in the absence of target DNA. In this state, pyrene labled at the 5ʹ-termini of single-stranded stem of probe H1 would be easily trapped into the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to significant fluorescence enhancement. Once the dynamic assembly was catalyzed by target DNA, a hybridized DNA duplex H1–H2 would be created continuously. In this state, it is difficult for pyrene to enter the cavity of β-CDP due to steric hindrance and weak-binding interaction, leading to a weak fluorescent signal. Thus, target DNA could be detected by this simple mix-and-detect amplification method without the need of expensive and perishable protein enzymes. As low as 10 pM of the target DNA was detected by this assay, which was comparable to that of some reported enzyme-dependent amplification methods. Meanwhile, the proposed method was further successfully applied to detect DNA in cell lysate samples, showing great potential for target detection from complex fluids. In addition, as a novel transformation of dynamic DNA assembly technology into enzyme-free signal-amplification analytical application, the proposed strategy has shown great potential for applications in a wide range of fields, such as aptamerbased non-nucleic acid target sensing, biomedicine and bioimaging. & 2015 Elsevier B.V. All rights reserved.
Keywords: Enzyme-free Pyrene β-cyclodextrin polymer Nucleic acid detection
1. Introduction Sensitive and selective detection of DNA sequences plays a vital role in clinical diagnosis and biomedical studies [1]. The dynamic DNA self-assembly technology as an attractive signal-amplification nucleic acid detection approach has attracted significant attention, and immense advances have been achieved [2–8]. Pierce and coworkers firstly introduced the basic concept of catalyzed selfassembly technology, which is one of the most important approaches for “bottom-up” fabrication of nanostructures and nanodevices [9]. DNA, as an ideal building block for nanotechnology, is programmed and self-assembled in a predictable manner in the n
Corresponding authors. Fax: þ 86 731 88821566. E-mail addresses: [email protected] (X. Yang), [email protected] (K. Wang). http://dx.doi.org/10.1016/j.talanta.2015.06.087 0039-9140/& 2015 Elsevier B.V. All rights reserved.
catalyzed dynamic assembly. In the past few decades, inspired by the simple rules governing hybridization of metastable DNA molecules, researchers have developed many attractive signal-amplification approaches based on the catalyzed dynamic assembly for biochemical analytical applications [10–27]. For example, Pierce group constructed a kind of in situ amplifier for multiplexed imaging of mRNAs in fixed whole mount and sectioned zebrafish embryos [18]. Tan group designed a hairpin DNA cascade reaction for mRNA imaging inside live cells [22]. We have also developed several fluorescent amplification methods successfully for sensitive nucleic acid analysis [23–26]. In short, the catalyzed dynamic assembly based amplification method has two obvious advantages compared with conventional enzyme-based methods [28–33]. First, it only relies on strand displacement reactions, avoiding perishable protein enzymes, complicated instrumentations and complex thermal-cycling procedures. Second, it is inherently scalable and modular, just
530
H. Huang et al. / Talanta 144 (2015) 529–534
requiring the design of base-pairing between metastable DNA molecules. Therefore, we believe that there is a great potential to adapt catalyzed self-assembly to support much more analytical applications. In recent years, many new signal amplifying methods based on nanomaterials and supramolecular polymers have emerged [34,35]. A wide variety of fluorescence quenching based bioanalytical methods have been developed. For example, Graphene and graphene oxide can quench the fluorescence of nearby organic dyes and upconverting nanocrystals, resulting in extensive studies of fluorescent quenching biosensors [36]. Gold nanoparticles can serve as excellent fluorescence quenchers for FRET-based assays due to their extraordinary high molar extinction coefficients and many gold nanoparticles based assays have been used to detect and quantify intracellular analytes [37]. Meanwhile, fluorescence enhancement based detection methods have attracted more attention. Metal nanoparticles, for instance, have attracted wide attention for their good metal-enhanced fluorescence (MEF) activity and many detection methods based on MEF have been developed to further improve the fluorescence technique [38–40]. Aggregation-induced emission enhancement (AIEE) has been observed and open new routes to fluorescence enhancement analytical methods [41]. Particularly, supramolecular systems based on host–guest interactions have attracted significant attention on the horizon of material science due to their unique properties [42]. Thus, a new type of signal amplifying method based on the host– guest interaction of supramolecular polymer might contribute to sensitive nucleic acids detection. We have developed several fluorescence analytical methods based on host–guest interaction between epichlorohydrin cross-linked β-cyclodextrin polymer (βCDP) and pyrene [43–45]. Herein, a new enzyme-free nucleic acid amplified detection strategy has been developed by combining target-catalyzed dynamic assembly with host–guest interaction between β-CDP and pyrene. In this work, we designed two metastable hairpin DNA probes (probe H1 and probe H2), a DNA segment was used as the nucleic acid target. The pyrene-labeled probe H1 was chosen as the signal unit. Therefore, a fluorescent turn-off enzyme-free isothermal amplification strategy based on target-catalyzed dynamic assembly of branched junction was constructed, and this fluorescence amplification strategy showed high sensitivity toward target DNA with a detection of 8 pM. Moreover, the proposed strategy was further successfully applied to detect DNA in cell lysate samples.
2. Experimental 2.1. Chemicals and materials DNA marker and loading buffer were purchased from TaKaRa Bio Inc. (Dalian, China). SYBR Gold was purchased from Invitrogen (USA). β-CDP (Mn 94,400; GPC, waters-515) was synthesized according to the procedure reported previously [43]. The purified β-CDP powder was re-dispersed in aqueous solution and stored at 4 °C for use. All the solutions were prepared with ultrapure water obtained from a Millipore water purification system (4 18.2 MΩ cm). All the DNA sequences and pyrene-labeled probe were synthesized by TaKaRa Bio Inc. (Dalian, China) and purified by HPLC. The sequences design process of hairpin probes is summarized in Fig. S1. The structural schematic diagram of pyrene linked to the 5ʹ-terminus of probe H1 is shown in Fig. S2. The sequences used in this work are listed in Table S1. All other chemicals, if not specified here, were all commercially available and used as received and used without further purification or treatment.
2.2. Apparatus All fluorescence spectra were measured using a Hitachi F-7000 fluorescence spectrometer (Hitachi Ltd., Japan) controlled by FL Solution software equipped with aqueous thermostat (Amersham) accurated to 0.1 °C. Excitation and emission slits were all set for a 5.0 nm band-pass with a 700 V PMT voltage. The excitation wavelength was set at 345 nm and the emission spectra from 365 to 480 nm were collected with a 0.2 1 cm2 quartz cuvette containing 100 μL of solution. The fluorescence intensity at 380 nm was used to evaluate the performance of the proposed assay strategy.
2.3. Agarose electrophoresis analysis A 2.5% agarose gel was prepared by using 0.5 Tris-borateEDTA (TBE) (pH 8.0). Different mixtures of target DNA with probe H1 and probe H2 were incubated for 3.5 h at 39 °C, the concentration of each hairpin probe was 500 nM. After preparing samples, 10 μL of each sample was mixed with 2 μL of 6 loading buffer, then 10 μL of mixed solution was added into the gel for electrophoresis. SYBR gold was used as the DNA stain and mixed with the samples. The gel was run at 100 V for 45 min at room temperature with loading of 10 mL of sample into each lane, and then photographed in Gel Imaging (Tanon 2500R, Tianneng Ltd., Shanghai, China).
2.4. DNA detection procedures The DNA detection were performed in 100 μL of a hybridization buffer (20 mM Tris–HCl, 140 mM NaCl, and 5 mM KCl, pH 7.5) consisting of different concentrations of target DNA, 100 nM probe H1, 600 nM probe H2. Initially, the prepared solution was incubated for 3.5 h at 39 °C for the assembly reaction. Then 4 mg mL 1 β-CDP was introduced to the resulting solution and incubated for another 5 min for the host–guest interaction. Finally, the resulted solutions were characterized by fluorescence spectrophotometer and the fluorescence intensity was recorded.
2.5. Preparation of complex samples To test the capability of the proposed strategy for detecting target DNA from complex biological samples, Hela cell lines were cultured in our lab and were grown in RPMI 1640 cell medium with 10% inactivated fetal bovine serum (Hyclone, USA) at 37 °C in 5% CO2. Vigorous growth cells were collected after trypsin digestion. The cell density was determined using a hemocytometer, and this was performed prior to each experiment. A suspension of 1 106 cells was centrifuged at 1000 rpm for 3 min, then washed with PBS buffer(10 mM phosphate, 137 mM NaCl, and 2.7 mM KCl, pH 7.4) three times and at last suspended in Tris–HCl buffer. Finally, the cells were disrupted by sonication for 20 min at 0 °C. To remove the homogenate of cell debris, the lysate was centrifuged at 18,000 rpm for 20 min at 4 °C. The supernatant was ready for DNA detection. The target DNA spiked lysate samples were prepared by adding different concentration of target DNA in the lysate samples. Then, the detection procedure according to the proposed strategy was carried out. Unless noted otherwise, all the experiments for measurements were repeated three times at least in this study.
H. Huang et al. / Talanta 144 (2015) 529–534
531
3. Results and discussion 3.1. Design strategy for nucleic acids detection
Target Probe H1 Probe H2 Flourescence intensity (a.u)
In this proposed method, two metastable hairpin DNA probes (probe H1 and probe H2) were designed, a DNA segment was used as a proof-of-concept nucleic acid target. The pyrene-labeled probe H1 was chosen as the signal unit. In the presence of target DNA, a cascade of assembly reactions were catalyzed to form many branched junctions shown in Fig. 1A. Once the dynamic assembly among probes was catalyzed by target DNA, a hybridized DNA duplex H1–H2 would be created continuously. Each assembly circle was terminated by a disassembly step in which probe H2 displaced target DNA from the branched complex, freeing target DNA to catalyze the next assembly circle. Due to strong steric hindrance and weak-binding interaction, it is difficult for pyrene to enter the cavity of β-CDP, leading to a weak fluorescent signal. In the absence of target DNA, probe H1 and probe H2 were in the closed hairpin formation, and pyrene molecule labeled on probe H1 was spatially separated by the sticky end in a length of extra length of 8 mononucleotides shown in Fig. 1B. In this state, pyrene molecules at the 5ʹ-termini of single-stranded stem of probe H1 would be easily trapped into the hydrophobic cavity of β-CDP because of weak steric hindrance, leading to significant fluorescence enhancement. Therefore, a fluorescent turn-off enzyme-free isothermal nucleic acid amplified detection strategy was constructed by combining the target-catalyzed dynamic assembly with host–guest interaction between β-CDP and pyrene.
1
2
3
+ -
+
+ +
4
+ + +
1600
a b c
1400 1200 1000 800
d
600 400
e
200 0 380
400
420
440
460
480
500
Wavelength (nm) 1
4
3*
3
2*
2
4 1* 2* 3* 1
H1 opened
1
2
3
3
1* H1
1 2 3
1
1
2
3
1*
1
4* 3*
4
β CDP
3*
4
4*
1
1*
3*
3
2*
2
1*
1 4
4
2
3
Low Fluorescence
1
3
2* 1*
2 Without target
H1
4
3
2
3*
3
1*
2* No assembly 1*
2
4* 3*
H1
1 4 β CDP
1
4
3*
3
2* 1*
2
H2
2 High Fluorescence
1*
3.2. Verification of the catalyzed dynamic assembly amplification method
1* 4* 3*
4
3*
3
2
3
H1 H2 assembly
1* 2* 3* 1 4
Fig. 2. Characterization and feasibility investigation of the enzyme-free nucleic acid amplified detection. (A) Gel electrophoretic analysis of the products by targetcatalyzed assembly. The concentrations of pyrene-labeled probe H1, probe H2 and target DNA were 500 nM, 500 nM and 50 nM respectively ( þ means in the presence of,– means in the absence of). The gels were run at 100 V for 45 min. (B) Fluorescence spectra demonstrating the target-catalyzed assembly steps in Fig. 1. Curve a: pyrene-labeled probe H1 þβ-CDP; Curve b: pyrene-labeled probe H1þ target DNA þβ-CDP; Curve c: pyrene-labeled probe H1 þ probe H2þ β-CDP; Curve d: pyrene-labeled probe H1þ probe H2 þtarget DNA þ β-CDP; Curve e: pyrene-labeled probe H1. The concentrations of pyrene-labeled probe H1, probe H2, target DNA and β-CDP were 100 nM, 500 nM, 3 nM and 4 mg mL 1 respectively.
2
2
H2
Target displaced
Target
3
1
4* 4 3* H2 Fig. 1. The schematic illustration of the proposed enzyme-free nucleic acid amplified detection based on catalyzed dynamic assembly and host–guest interaction between β-cyclodextrin polymer and pyrene. Numbers marked with n are complementary to the corresponding unmarked numbers.
To confirm the enzyme-free nucleic acid amplified detection method, we utilized gel electrophoresis to monitor the reaction. As shown in Fig. 2A, it could be observed that there was only one band in lane 1, lane 2 and lane 3 in the absence of target DNA, indicating that no catalyzed dynamic assembly occurs. As a comparison, new band appeared and the old band disappeared in lane 4 when target DNA was added, suggesting the formation of H1–H2 complex. In the system, target DNA was re-used until the supply of probe H1 or probe H2 was exhausted. Thus, these results demonstrated that the dynamic assembly was circularly catalyzed by target DNA. To further explore the amplification effect of different conditions, we evaluated their performance with control experiments under the same target DNA concentration of 3 nM. As shown in Fig. 2B, the fluorescence intensity of probe H1 (Fig. 2B (e)) enhanced greatly with the assistance of β-CDP (Fig. 2B (a)). In the absence of target DNA, probe H1 and probe H2 exhibit a decrease
in fluorescence intensity (Fig. 2B (c)). In the presence of 3 nM target DNA, a mild decrease in fluorescence intensity was observed with the presence of probe H1 (Fig. 2B (b)). However, a significant decrease in fluorescence intensity was obtained by adding probe H2 (Fig. 2B (d)). These results showed that a successful targetcatalyzed dynamic assembly could provide higher sensitivity. 3.3. Optimization of experimental conditions To ensure a better performance, several parameters were investigated to establish optimal conditions for our detection system including concentration ratio of probe H1 and probe H2, concentrations of β-CDP, Na þ , K þ , Tris–HCl, reaction temperature and reaction time. The optimization of the variables of our system was based on the fluorescence decreasing factor. The fluorescence decreasing factor was (F0 F)/F0, where F and F0 were the fluorescence intensities of the detection system with and without target DNA respectively. 3.3.1. Optimization of concentration ratio of H1 and H2 The hairpin probes play important roles in the performance of this assay. On one hand, sufficient amount of probe should be added to carry out enough rounds of cycles; on the other hand, an obvious background signal would be observed at a relatively high concentration of probe H2, which might resulted from a bit of spontaneous hybridization between probe H1 and probe H2. Therefore, concentration ratios of probe H1 and H2 were firstly studied towards the detection of 3 nM target DNA. As shown in Fig. S3 (supporting information), when the concentration ratio of probe H1 and H2 is 1:6, the (F0 F)/F0 value is maximum. So we choose the concentration ratio 1:6 in our following detection system to achieve good performance. 3.3.2. Optimization of concentrations of Tris–HCl, K þ and Na þ It is generally acknowledged that salt ions in solution could neutralize the charge of the nucleic acid and has a promoting effect on DNA hybridization and fabrications of hairpin structure. This means that an appropriate ionic strength might affect sensitivity and specificity. To achieve the optimal response of the detection system, the effect of ion strength was thus investigated. As shown in Fig. S4 (A) (supporting information), it was clear that the (F0 F)/F0 value was dependent on the concentration of Tris–HCl. To achieve good performance, we chose 20 mM Tris–HCl in our following detection system. As shown in Fig. S4 (B) and Fig. S4 (C) (supporting information), when the concentration of Na þ and K þ was 140 mM and 5 mM respectively, the (F0 F)/F0 values reached maximum. Therefore, we chose 20 mM Tris–HCl, 140 mM Na þ and 5 mM K þ in our following detection system to achieve good performance. 3.3.3. Optimization of concentration β-CDP β-CDP serves as the host component for host–guest interaction between β-CDP and pyrene. Adding appropriate amount of β-CDP can gain favorable fluorescence enhancement which is conducive to produce obvious response signals. Excess β-CDP may cause strong background signals, so the concentration of β-CDP was optimized. As shown in Fig. S5 (supporting information), when the concentration of β-CDP is 4 mg mL 1, the (F0 F)/F0 value is maximum. Therefore, 4 mg mL 1 β-CDP was chosen as the optimized concentration in the following experiments. 3.3.4. Optimization of reaction time and temperature Finally, the reaction time and temperature was optimized. As indicated in Fig. S6 (A) (supporting information), with the increasing of reaction time, the (F0 F)/F0 value increased and reached maximum at 3.5 h. Therefore, 3.5 h was chosen as the
Fluorescence intensity (a.u.)
H. Huang et al. / Talanta 144 (2015) 529–534
1400 1200 1000 800 600 400 200 0 380
400
420
440
460
480
500
20
25
Wavelength (nm) 0.8 0.6
(F0-F)/F0
532
0.4 0.2 0.0
0
5
10
15
Concentration of target DNA (nM) Fig. 3. (A) Fluorescence spectra of the amplification method over a range of target DNA concentrations. The curves from up to bottom contain target DNA with 0 nM, 0.01 nM, 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 10 nM and 20 nM respectively. (B) Plot of target DNA vs fluorescence intensity. Inset is the calibration curve for the concentrations of target DNA from 0 nM to 1 nM. The concentrations of pyrene-labeled probe H1, probe H2, β-CDP, Tris–HCl, Na þ and K þ were 100 nM, 600 nM, 4 mg mL 1, 20 mM, 140 mM and 5 mM, respectively. Error bars indicated the standard deviations of three experiments.
optimized time in following experiments. Temperature is a key parameter of reaction kinetics and determines the probability of collisions between the molecules. At low temperature, the hairpin probes could not get a sufficient collision probability that greatly reduced the formation of H1–H2 complexes. Inversely, the specificity of this platform would be debased by high reaction temperature, because temperature is interrelated with stability and integrality of hairpin probes. As shown in Fig. S6 (B) (supporting information), with the increasing reaction temperature, the (F0 F)/F0 value increased and reached maximum at 39 °C. Therefore, 39 °C was chosen as the optimized temperature in the following experiments. 3.4. Sensitivity of DNA detection Under the above optimal conditions and according to the DNA detection procedures described in the previous section, we evaluate the response range and detection limit by measuring fluorescence intensity upon the addition of different concentrations of target DNA. As shown in Fig. 3A, a gradual decrease in the fluorescence intensity was clearly observed in the concentration of
target DNA ranged from 0 nM to 20 nM. The relationship between the change of fluorescence intensity and the concentration of target DNA was demonstrated in Fig. 3B. The results showed that the fluorescence intensity decreased with the increase of target concentration in the range from 10 pM to 1 nM with a detection limit of 8 pM as estimated by the 3s rule. This detection capability was comparable to that of some reported enzyme-dependent colorimetric or electrochemical amplification methods (Table S2 in supporting information). The calibration curve is shown in the inset in Fig. 3B. The regression equation was (F0–F)/ F0 ¼0.0245þ0.1844(C nM 1) during the range from 10 pM to 1 nM (F was the fluorescence intensity of the mixture of probe H1, probe H2 and target DNA; F0 was the fluorescence intensity in the mixture of probe H1 and probe H2; C was the concentration of target DNA; R2 ¼0.9902). We also investigated three prolonged DNA sequences (Prolonged DNA a, b and c) to demonstrate the usefulness of the assay for longer target sequences detection. The result is shown in Fig. S7 (supporting information).
Fluorescence intensity (a.u.)
H. Huang et al. / Talanta 144 (2015) 529–534
533
1200
0 nM
1000 800
20 nM
600 400 200 0 380
400
420
440
460
480
500
Wavelength (nm) 0.7 0.6
The selectivity of the proposed DNA detection strategy was carefully evaluated. Several control DNAs, including target DNA, mismatched DNA and random DNA, were detected by this method. As shown in Fig. 4, the fluorescence intensity change was enormous in the presence of target DNA, while negligible change was observed in the presence of random DNA, which indicated that the proposed strategy was capable of detecting target DNA from random nucleic acid library. Furthermore, at the same detected concentration, although mismatched DNA could result in a minor change in fluorescence intensity, much more fluorescence decrease was observed in the presence of target DNA than that in the presence of any mutant DNA, which indicated that our proposed strategy also had the potential to distinguish single nucleotide polymorphism (SNP). Therefore, based on the above results, this constructed enzyme-free isothermal amplification strategy could be used to detect target nucleic acid with good specificity.
0.5
0.7
(F0-F)/F0
3.5. Selectivity of DNA detection
0.4 0.3 0.2 0.1 0.0 1
2
4
8
12
24
Concentration of target DNA (nM) Fig. 5. (A) Fluorescence spectra of the detection system for various concentrations of target DNA in 50% cell lysate. The curves from up to bottom contain target DNA with 0 nM, 1 nM, 2 nM, 4 nM, 8 nM, 12 nM and 24 nM respectively. (B) Fluorescence intensity at λex/em ¼ 345/380 nm of the system as a function of the various concentrations of target DNA in 50% cell lysate. The concentrations of probe H1, probe H2, β-CDP, Tris–HCl, Na þ and K þ were 100 nM, 600 nM, 4 mg mL 1, 20 mM, 140 mM and 5 mM, respectively. Error bars indicated the standard deviations of three experiments.
0.6 3.6. Target DNA detection in complex samples
(F0-F)/F0
0.5 0.4 0.3 0.2 0.1
c R an do m
is m
at
ch M
at m M is
ch
ed
ed ch at
is m
ed
a
t rg e Ta M
b
0.0
Fig. 4. Specificity evaluation of the proposed method for target DNA detection (3 nM), mismatched DNA a (3 nM), mismatched DNA b (3 nM), mismatched DNA c (3 nM) and random DNA (3 nM). The concentrations of pyrene-labeled probe H1, probe H2, β-CDP, Tris–HCl, Na þ and K þ were 100 nM, 600 nM, 4 mg mL 1, 20 mM, 140 mM and 5 mM, respectively. Error bars indicated the standard deviations of three experiments.
Detection of target DNA in complex samples was carried out to test the practical application capability of the proposed strategy. In this work, diluted Hela cell lines extract was selected as the complex fluid to investigate the feasibility of this strategy in biological samples. Target DNA-containing cell extract samples were prepared by adding target DNA in 50% cell extract contained reaction buffer. Then target DNA-spiked complex fluids were detected by the procedure according to the proposed strategy. The calibration curve was obtained using the 50% cell lysate with the addition of DNA in various concentrations. As shown in Fig. 5, a plot of the fluorescence intensity versus the concentration of DNA revealed a dynamic correlation between the fluorescence intensity and concentration of DNA in the range from 0 nM to 24 nM. As for demonstrating the usefulness of the assay for longer target sequences detection we use cell lysates spiked with prolonged DNA sequences and the result was shown in Fig. S8 (supporting information). The results suggested that the signal change of this method is dependent on the concentration of DNA in cell lysate, which showed that the strategy held great potential for practical detection of target DNA from complex fluids.
534
H. Huang et al. / Talanta 144 (2015) 529–534
4. Conclusions In summary, the present study introduced a new path for enzyme-free DNA amplified detection by combining the isothermal signal-amplification capability of target-catalyzed dynamic assembly with host–guest interaction between β-CDP and pyrene. First, the hairpin probes in this detection system is easy to design, synthesize, purify and convenient for usage. Second, it was a simple mix-and-detect amplification method, which only relied on hybridization and strand displacement reactions, without requiring perishable protein enzymes, complicated instrumentations and complex thermal-cycling procedures. In addition, our strategy achieved a good detection capability with the detection of 8 pM and successfully application in complex samples. Overall, as a novel transformation of catalyzed dynamic assembly technology into enzyme-free signal-amplification analytical application, we infer that this strategy might be applicable for a wide range of fields, such as aptamer-based non-nucleic acid target sensing, biomedicine and bioimaging.
[9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22]
[23] [24] [25]
Acknowledgments This work was supported by the National Natural Science Foundation of China (21190044 and 21175035), National Basic Research Program of China (2011CB911002), and International Science & Technology Cooperation Program of China (2010DFB30300).
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.talanta.2015.06.087.
References [1] H. Zhang, F. Li, B. Dever, X. Li, X.C. Le, Chem. Rev. 113 (2013) 2812–2841. [2] F. Wang, X. Liu, I. Willner, Angew. Chem. Int. Ed. 54 (2015) 1098–1129. [3] S. Venkataraman, R.M. Dirks, P.W.K. Rothemund, E. Winfree, N.A. Pierce, Nat. Nanotechnol. 2 (2007) 490–494. [4] D. Zhang, A.J. Turberfield, B. Yurke, E. Winfree, Science 318 (2007) 1121–1125. [5] Y. Zang, J. Lei, H. Ju, Anal. Chem. 87 (2015) 5430–5436, http://dx.doi.org/ 10.1021/acs.analchem.5b00888. [6] G. Seelig, B. Yurke, E. Winfree, J. Am. Chem. Soc. 128 (2006) 12211–12220. [7] D. Soloveichika, G. Seeliga, E. Winfree, Proc. Natl. Acad. Sci. USA 107 (2010) 5393–5398. [8] Y. Qian, C. Wang, F. Gao, Talanta 130 (2014) 33–38.
[26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45]
P. Yin, H.M.T. Choi, C.R. Calvert, N.A. Pierce, Nature 451 (2008) 318–322. C. Geary, P.W.K. Rothemund, E.S. Andersen, Science 345 (2014) 799–804. K.V. Gothelf, T.H. LaBean, Org. Biomol. Chem. 3 (2005) 4023–4037. B.L. Li, A.D. Ellington, X. Chen, Nucleic Acids Res. 39 (2011) e110. S. Shimron, F. Wang, R. Orbach, I. Willner, Anal. Chem. 84 (2012) 1042–1048. B. Zhang, B. Liu, D. Tang, R. Niessner, G. Chen, D. Knopp, Anal. Chem. 84 (2012) 5392–5399. J. Huang, X. Su, Z. Li, Anal. Chem. 84 (2012) 5939–5943. Y. Chen, J. Xu, J. Su, Y. Xiang, R. Yuan, Y. Chai, Anal. Chem. 84 (2012) 7750–7755. C. Lin, J.K. Nangreave, Z. Li, Y. Liu, H. Yan, Nanomedicine 3 (2008) 521–528. H.M.T. Choi, J.Y. Chang, L.A. Trinh, J.E. Padilla, S.E. Fraser, N.A. Pierce, Nat. Biotechnol. 28 (2010) 1208–1212. G. Zhu, J. Zheng, E. Song, M. Donovan, K. Zhang, C. Liu, W. Tan, Proc. Natl. Acad. Sci. USA 110 (2013) 7998–8003. G. Zhu, S. Zhang, E. Song, J. Zheng, R. Hu, X. Fang, W. Tan, Angew. Chem. Int. Ed. 52 (2013) 5490–5496. J. Zheng, G. Zhu, Y. Li, C. Li, M. You, T. Chen, E. Song, R. Yang, W. Tan, ACS Nano 7 (2013) 6545–6554. C. Wu, S. Cansiz, L. Zhang, I. Teng, L. Qiu, J. Li, Y. Liu, C. Zhou, R. Hu, T. Zhang, C. Cui, W. Tan, J. Am. Chem. Soc. 137 (2015) 4900–4903, http://dx.doi.org/ 10.1021/jacs.5b00542. K. Quan, J. Huang, X. Yang, Y. Yang, L. Ying, H. Wang, K. Wang, Chem. Commun. 5 (2014) 937–940. P. Liu, X. Yang, S. Sun, Q. Wang, K. Wang, J. Huang, J. Liu, L. He, Anal. Chem. 85 (2013) 7689–7695. J. Huang, Y. Wu, Y. Chen, Z. Zhu, X. Yang, C. Yang, K. Wang, W. Tan, Angew. Chem. Int. Ed. 50 (2011) 401–404. Z. Qing, X. He, J. Huang, K. Wang, Z. Zou, T. Qing, Z. Mao, H. Shi, D. He, Anal. Chem. 86 (2014) 4934–4939. Y.S. Jiang, S. Bhadra, B. Li, A. Ellington, Angew. Chem. Int. Ed. 53 (2014) 1–5. A. Fire, S. Xu, Proc. Natl. Acad. Sci. USA 92 (1995) 4641–4645. J.V. Ness, L.K. Ness, D.J. Galas, Proc. Natl. Acad. Sci. USA 100 (2003) 4504–4509. Y. Weizmann, M. Beissenhirtz, Z. Cheglakov, R. Nowarski, M. Kotler, I. Willner, Angew. Chem. Int. Ed. 45 (2006) 7384–7388. B. Zou, Y. Ma, H. Wu, G. Zhou, Angew. Chem. Int. Ed. 50 (2011) 7395–7398. Q. Wang, L. Yang, X. Yang, K. Wang, L. He, J. Zhu, T. Su, Chem. Commun. 48 (2012) 2982–2984. R. Deng, L. Tang, Q. Tian, Y. Wang, L. Lin, J. Li, Angew. Chem. Int. Ed. 53 (2014) 2389–2393. M.J. Serpe, S.L. Craig, Langmuir 23 (2007) 1626–1634. L. Cheng, C. Wang, L. Feng, K. Yang, Z. Liu, Chem. Rev. 114 (2014) 10869–10939. P. Yin, S. Shah, M. Chhowalla, K. Lee, Chem. Rev. 115 (2015) 2483–2531. K. Saha, S. Agasti, C. Kim, X. Li, V. Rotello, Chem. Rev. 112 (2012) 2739–2779. J. Zhang, N. Ma, F. Tang, Q. Cui, F. He, L. Li, ACS Appl. Mater. Interfaces 4 (2012) 1747–1751. D. Cheng, Q. Xu, Chem. Commun. 43 (2007) 248–250. Y. Li, L. Guan, H. Zhang, J. Chen, S. Lin, Z. Ma, Y. Zhao, Anal. Chem. 83 (2011) 4103–4109. Y. Hong, J. Lam, B. Tang, Chem. Soc. Rev. 40 (2011) 5361–5388. X. Ma, Y. Zhao, Chem. Rev. (2014), http://dx.doi.org/10.1021/cr500392w. X. Guo, P. Liu, X. Yang, K. Wang, Q. Wang, Q. Guo, J. Huang, J. Liu, C. Song, W. S. Li, Analyst 140 (2015) 2016–2022. L. He, X. Yang, F. Zhao, K. Wang, Q. Wang, J. Liu, J. Huang, W. Li, M. Yang, Anal. Chem. 87 (2015) 2459–2465. C. Song, X. Yang, K. Wang, Q. Wang, J. Liu, J. Huang, L. He, P. Liu, Z. Qing, W. Liu, Chem. Commun. 51 (2015) 1815–1819.