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The three-way junction DNAzyme based probe for label-free colorimetric detection of DNA
Biosensors and Bioelectronics 41 (2013) 397–402
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
The three-way junction DNAzyme based probe for label-free colorimetric detection of DNA Shurong Tang a, Ping Tong a,b, Heng Li a, Fang Gu a, Lan Zhang a,b,n a
Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, college of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, Fujian 350108, China b Analytical and Testing Center, The Sport Science Research Center, Fuzhou University, Fuzhou, Fujian 350002, China
a r t i c l e i n f o
abstract
Article history: Received 26 May 2012 Received in revised form 28 August 2012 Accepted 30 August 2012 Available online 5 September 2012
A novel three-way junction DNAzyme based probe has been designed for the colorimetric sensing of target DNA. Specifically, a DNAzyme-linked hairpin DNA is used as a signal probe. In the presence of target DNA, the signal probe, assistant probe and target DNA can hybridize with each other, resulting in the formation of a three-way junction DNA. At the same time, the signal probe is opened and the DNAzyme sequence in the signal probe is dehybridized. Subsequently, in the presence of hemin, the DNAzyme sequence forms a G-quadruplex–hemin complex, which catalyzes oxidation of 2, 20 -azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by H2O2 to the colored ABTS. radical. The significant color changes can be distinguished visually. By the combination of the hairpin probe and the three-way junction DNA probe, the proposed sensor exhibits high recognition property for single-nucleotide polymorphisms (SNPs). This sensor allows the detection of target DNA at a concentration as low as 0.25 nmol L 1. The proposed sensor is easy to fabricate, which avoids the tedious and expensive labeling procedures, and exhibits high selectivity against single-base mismatched DNA. & 2012 Elsevier B.V. All rights reserved.
Keywords: Three-way junction G-quadruplex–hemin DNAzyme Label free DNA detection
1. Introduction DNAzymes (also called deoxyribozyme or catalytic DNAs) are single stranded DNA molecules of a particular sequence, which possess specific catalytic activities to some chemical reactions. DNAzymes have several advantages over traditional protein enzymes, such as high chemical stability, low cost, simple preparation and easy modification (Breaker, 2000). For these unique properties, DNAzymes have been used in numerous biochemical reactions, such as DNA or RNA cleavage (Burmeister et al., 1997; Carmi et al., 1998), porphyrin metalation (Li and Sen, 1996), and DNA self-modification (Li and Breaker, 1999; Sheppard et al., 2000). Various kinds of artificial DNAzymes have been screened by using in vitro selection methodologies or SELEX (systemic evolution of ligands by exponential enrichment) techniques (Osborne and Andrew, 1997; Tuerk and Gold, 1990). One interesting example of a catalytic DNAzyme that reveals peroxidase-like activity is a complex between hemin and a G-quadruplex (Travascio et al., 1998). G-quadruplexes are unique higher-order
n Corresponding author at: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, Fujian 350108, China. Fax: þ86 591 87893207. E-mail address: [email protected] (L. Zhang).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.08.056
structures, in which G-rich nucleic acid sequences form stacked arrays known as the G-quartets (four guanine bases are connected to each other by hydrogen bonded) (Sen and Gilbert, 1988). Hemin is able to specifically bind to G-quadruplex with high affinity. It has been reported that such G-quadruplex–hemin complexes possess peroxidase-like activity 250 times greater than that of hemin alone (Travascio et al., 1999). This neotype G-quadruplex based DNAzymes exhibit high catalytic activity toward the oxidation of 2,20 -azinobis (3-ethylbenzothiazoline)6-sulfonic acid (ABTS) by H2O2, which causes a color change (Travascio et al., 1998). The use of DNAzymes as catalytic labels for biosensor applications has the advantage of avoiding the tedious and expensive labeling procedures for signal readout. Therefore, the G-quadruplex–hemin DNAzymes have been widely used to colorimetric detection of biomolecules and other small molecules, such as DNA molecules (Nakayama and Sintim, 2009), proteins (Li et al., 2007) and metal ions (Li et al., 2008). Recently, there has been an increasing interest in the detection of single-nucleotide polymorphisms (SNPs). SNPs are singlenucleotide variations that may be substituted, deleted or inserted in a natural DNA sequence. When SNPs occur, both the structure and the function of the encoded protein are changed which often leads to harmful diseases, such as Tay Sachs (Gravel et al., 1995), cystic fibrosis (Cronin et al., 1996), and thalassemia (Muniz et al., 2000). SNPs are the most common variations in human genome, which occur approximately once per 250–1000 bases in a large
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sample of aligned genome sequence (Collins et al., 1998). The high density and mutational stability of SNPs make them particularly useful DNA markers for identification of complex disease genes (Ye et al., 2001). Therefore, the development of a highly selective method for SNPs detection is of great importance for early disease diagnosis and treatment. In an attempt to improve the selectivity of DNA detection, hairpin probes have been used in conjunction with DNAzymes to construct novel colorimetric biosensors (Li et al., 2011; Xiao et al., 2004). Hairpin probe is single-stranded DNA that possesses a stem-loop structure. Conventional hairpin probe-based colorimetric sensor employs a G-quadruplex DNAzyme-linked DNA sequence, which forms a stem-loop structure in the absence of target DNA, and results in inactivation of the DNAzyme. However, in the presence of target DNA, the stem-loop structure is opened to produce a catalytically active DNAzyme that leads to generation of a colorimetric signal. Hairpin probes can distinguish mismatches over a wider temperature range than that of linear probes (Bonnet et al., 1999), but they exhibit poor distinguishing capability for single-base mismatch (Demidov and Frank, 2004; Kolpashchikov, 2010), which limits their application in SNPs analysis. Thus, a more selective strategy with high discrimination ability for single-base mismatched DNA still needs to be explored. The general idea of a junction probe is that two DNA probes are hybridized to one DNA target. A detectable signal can be observed only when the two parts of the probes hybridize to the one DNA target (Kolpashchikov, 2010). Since the two parts of the probes form relatively short (7–10 nucleotides) duplexes with target DNA, the junction probe’s selectivity is better than that of hairpin probe. Junction probe can be used for highly accurate detection of SNPs at room temperature without the precise temperature control (Kolpashchikov, 2005). Up to date, some new junction probes have been introduced for SNPs analysis, such as binary DNA probe (Kolpashchikov, 2006), three-way junction probe (Kong et al., 2011; Nakayama et al., 2008) and four-way junction probe (Lake et al., 2010). In this paper, a new three-way junction DNAzyme based probe has been used for the colorimetric detection of target DNA. A Gquadruplex DNAzyme-linked hairpin probe is specially designed as a signal probe, which avoids the tedious and expensive labeling steps for signal readout. The signal probe is opened after hybridization with target DNA and assistant probe, which is accompanied by the formation of a three-way junction DNA. After addition of hemin, the DNAzyme is activated and then catalyzes the oxidation of ABTS by H2O2 to form the colored product ABTS.for ultraviolet–visible (UV–vis) absorbance detection. The proposed DNA sensor is simple, label free, and exhibits high discrimination ability against single-base mismatched DNA.
2. Materials and methods 2.1. Materials 2,20 -Azinobis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and HPLC-purified oligonucleotides used in this experiment were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China), and the detail base sequences were shown in Table 1. TritonX-100, Hemin, N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES), and DMSO were bought from Sigma Aldrich (St. Louis, MO). The stock solution of oligonucleotides (100.00 mmol L 1) were prepared in ultrapure water and stored at -20 1C. The stock solution of hemin (1.00 mmol L 1) was prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 20 1C. Before use, the oligonucleotides and hemin solutions were diluted
Table 1 Sequence of oligonucleotides used in the colorimetric assay Oligonucleotide Sequence (from 50 - 30 ) SP1a SP2b SP3c APd Tae S1f S2g
AAC CCA GTC AGT GTC CTC AGC GTG GGT TGG GCG GGA TGG GT CAA CCC AGT CAG TGT CCT CAG CGT GGG TTG GGC GGG ATG GGT CCA ACC CAG TCA GTG TCC TCA GCG TGG GTT GGG CGG GAT GGG T CGC TGA GGA AAT GGA AAA TCT CTA G GCT AGA GAT TTT CCA CAC TGA CT GCT AGA GAT TTT CCA CAC CGA CT GCT AGA GAT TTT CCA AAC TTA CT
a,b,c
Thermodynamic parameters of all oligonucleotides were calculated using integrated DNA technologies (http://www.idtdna.com/analyzer/Applications/ OligoAnalyzer).
a,b,c Hairpin DNAzyme signal probe: the under line sequences are hybridized to form the stem-loop hairpin structure, the italic letters are G-quadruplex DNAzyme sequence. d Assistant probe. e Perfectly complementary target. f Single-base-mismatched target. g Two-bases-mismatched target (the mismatched bases are underlined).
Fig. 1. Colorimetric sensing protocol for target DNA.
to required concentrations with ultrapure water and dilution buffer (25.00 mmol L 1 Tris–HCl, pH 8.0, 20.00 mmol L 1 KCl, 200.00 mmol L 1 NaCl, 0.05% TritonX-100 and 1% DMSO), respectively. ABTS and H2O2 were freshly prepared before use. The ultrapure water supplied with a Milli-Q system (18.5 MO Millipore, USA) was used throughout the experiment. All reagents were used as received without further purification. 2.2. Colorimetric sensing of target DNA The assay protocol of the colorimetric DNA sensor was depicted in Fig.1. Signal probe was heated to 90 1C for 5 min and then gradually cooled to room temperature to form the hairpin probe before use. First, the assistant probe and target DNA were incubated at 4 1C over night in a hybridization buffer which is containing 20.00 mmol/L Tris–HCl (pH 8.0), 50.00 mmol L 1 NaCl and 10.00 mmol L 1 MgCl2. Then, signal probe was added and the hybridization was allowed to proceed at 37 1C for 2 h. The above hybridization solution volume was 50 mL. Next, 5 mL hemin (25.00 mmol L 1) and 185 mL HEPES/NH4OH buffer (25.00 mmol L 1, pH 7.4, 20.00 mmol/L KCl, 100.00 mmol L 1 NaCl, and 1% DMSO) were added and incubated at 25 1C for 40 min. It would allow the formation of G-quadruplex–hemin complexes. Finally, 5.0 mL 0.10 mol L 1 ABTS and 5.0 mL 0.10 mol L 1H2O2 were added to the above reaction mixture to initiate the catalytic reaction. The final concentration of signal probe and assistant probe were 50.00 nmol L 1. The time-dependent
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absorption spectra of the radical anion ABTS. (the product of ABTS oxidation by H2O2) was collected immediately with Perkin Elmer Lambda 750 UV/vis spectrometer (Shelton, USA) at 414 nm. Under the same above conditions, specificity experiments were performed in which target DNA was replaced by S1 or S2. 2.3. Circular dichromism (CD) measurements CD experiments were performed on a model 420 circular dichroism spectrometer (Lake Wood, NJ, USA) at 25 1C in a 1 mm path length cuvette. Scans from 220 to 350 nm were performed. The bandwidth was 1 nm. Each spectrum was corrected by subtracting the CD of the buffer. The concentration of signal probe, assistant probe and target DNA was 10, 10 and 5 mmol L 1, respectively. 2.4. Gel electrophoresis experiments Polyacrylamide gel electrophoresis (PAGE) was performed in an electrophoresis apparatus (DYY-80, China). Native 12% PAGE gel was prepared by mixing 3.94 mL ultrapure water, 1.6 mL 5 TBE buffer (0.45 mol/L Tris–boric acid–EDTA, pH 8.0), 2.4 mL 40% bis-acrylamide, 55 mL 10% ammonium persulfate and 5 mL N,N,N0 ,N0 -tetramethylethylenediamine (TEMED) together. After vortex mixing thoroughly, the fresh prepared gel was transferred to the electrophoresis apparatus, and then gelation for 1 h at room temperature. Before electrophoresis analysis, the samples were prepared in 10 mL reaction solution which contained 5.00 mmol/L DNA samples and 20.00 mmol/L Tris–HCl buffer (10.00 mmol/L MgCl2, 100.00 mmol/L NaCl, pH 7.9). After the DNA hybridization at 37 1C for 2 h, the resulting DNA samples together with loading dye were applied to the 12% polyacrylamide gel prepared before. The electrophoresis was carried out in a 1 TBE buffer (0.09 mol L 1 Tris–boric acid–EDTA, pH 8.0) at 12 mA for 2 h. The obtained gels were stained with ethidium bromide (EB) for 15 min and the staining results were photographed with a Gel Image System.
3. Results and discussion 3.1. Design of the three-way junction DNAzyme based probe for colorimetric sensing The proposed sensor uses a novel three-way junction DNAzyme based probe for the colorimetric detection of DNA from HIV-1 U5 (Lu et al., 2009). The detailed principle of this sensor is illustrated in Fig. 1. We have designed a G-quadruplex DNAzyme-linked hairpin
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probe, which serves as a signal probe. A single-stranded DNA which is partially complementary with the signal probe and target DNA is used as assistant probe. In the presence of target DNA, assistant probe and the target DNA will hybridize together with the signal probe, leading to unwinding the signal probe and formation of a three-way junction DNA. As a result, the DNAzyme sequence in the signal probe is dehybridized. After addition of hemin, a G-quadruplex–hemin DNAzyme complex is formed and then catalyzes oxidation of ABTS by H2O2 to the colored ABTS.- radical, accompanied by a significant increase of the absorbance signal. However, in the absence of target DNA, the signal probe cannot be opened through hybridization with the assistant probe to form a doublestranded DNA under the reaction temperature. The DNAzyme sequence is still blocked in the stem portion of signal probe, which inhibits the formation of any free hemin-containing active DNAzyme, so no remarkable absorbance signal can be detected. 3.2. Feasibility of the sensing system 3.2.1. Colorimetric measurement To test the feasibility of the sensing system, control experiments were performed under different conditions. As shown in Fig. 2A, the absorbance produced by hemin alone was negligible, which indicated that free hemin showed only slight catalytic activity toward the oxidation of ABTS (Fig. 2A, curve a). After the addition of signal probe to the homogeneous solution, the absorbance signal was about 3-fold increased (Fig. 2A, curve b). This was probably due to the fact that a small part of signal probes exist as random coils instead of stem-loop structure, which results in a self-assembled formation of G-quadruplex DNAzyme in the presence of hemin. It was worth to note that after the addition of assistant probe, there was no significant increase in the signal intensity (Fig. 2A, curve c). This result represented that most part of the signal probe cannot be opened by assistant probe alone, so no more G-quadruplex–hemin DNAzyme can be formed to enhance the absorbance signal. In contrast, after the addition of target DNA, a remarkable absorbance increase could be observed (Fig. 2A, curve d). These results suggested that the signal probe can be effectually opened only in the presence of target DNA. Therefore, the proposed method can be used for effective detection of target DNA. 3.2.2. Determination of structure change of DNA by CD measurements Moreover, in order to identify the hybridization of DNA and formation of G-quadruplex structure in this experiment, the CD spectra were used to monitor the structural changes. As shown in
Fig. 2. (A) Absorbance generated by 0.50 mmol/L hemin (a); hemin and 50.00 nmol/L signal probe (b); hemin, 50.00 nmol/L signal probe and assistant probe without (c) or with 20.00 nmol/L target (d). The inset was the corresponding time-dependent absorbance curves. (B) CD spectra of the DNA structure in HEPES/NH4OH buffer (25.00 mmol/L, pH 7.4, 20.00 mmol/L KCl, 100.00 mmol/L NaCl, and 1% DMSO). Signal probe (1); signal probe and assistant probe before (2) and after (3) addition of target DNA. (C) PAGE graphs of the sensing system. (1) Signal probe; (2) signal probe and target DNA; signal probe and assistant probe before (3) and after (5) addition of target DNA; (4) signal probe and assistant probe after addition of single-base mismatch DNA.
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Fig. 2B, the signal probe displayed a negative peak at 240 nm and a positive peak at 260 nm, which were characteristic of a DNA duplex (Miyoshi et al., 2004; Xue et al., 2011). After the addition of assistant probe, the positive peak at 260 nm became stronger. However, no obvious change was observed in the negative peak at 240 nm. Upon incubation with target DNA, the spectrum changes to reveal a positive band at 265–285 nm. The number of base pairs was increased after hybridization, which subsequently induced the rise of ellipticity at 273 nm and the drop at 240 nm; it should be the typical characteristic of B-form DNA double helix (Guo et al., 2010; Zhu et al., 2011). Moreover, a positive CD signal at around 265 nm and a negative signal at around 245 nm could be observed, indicating the formation of parallel G-quadruplex (Jin et al., 1992; Nakayama and Sintim, 2009). 3.2.3. PAGE experiment In order to further verify the proposed sensor with high selectivity for target DNA. Five samples including signal probe (1), signal probe and target DNA (2), signal probe and assistant probe before (3) and after (5) addition of target DNA or single-base mismatch DNA (4) were analyzed by native PAGE (Fig. 2C, lanes 1–5). Without addition of the target DNA, only one band could be seen clearly in the presence of signal probe and assistant probe (lane 3), which was the same as that obtained by signal probe alone (lane 1). These results proved the fact that the signal probe existed predominantly in the stem-loop structure without target DNA. At the same time, a new low-mobility band (migrates slower than the 75 base pair DNA marker) was observed only in the presence of fully complementary target (lane 5) but not in the presence of single-base mismatched target (lane 4). This band can be attributed to the three-way junction DNA depicted in Fig.1. The results of PAGE experiments were coincident with the results observed in colorimetric assay. 3.3. Optimization of experimental conditions In order to achieve the best sensing performance, the stem length and the concentration of the signal probe were optimized. The stem length of signal probe has a remarkable affect on the sensitivity. When the stem is too short, the signal probe is not stable enough and can be easily opened by assistant probe, resulting in high background signal. However, a too long stem will lead to very strong hybridization such that the assistant probe and the target DNA are hard to anneal with the signal probe, and hence reducing the signal and sensitivity. Three signal probes (SP1, SP2, and SP3) of different stabilities were used with stem lengths of 7, 8 and 9 base pairs, respectively. The relative absorbance change ((Abs1–Abs0)/Abs0) which defined as (DAbs/Abs0) was used to evaluate the sensitivity of the assay, where Abs1 and Abs0 were the values of absorbance changes at 414 nm of target DNA and the
background (without target), respectively. From the inset of Fig. 3A, it could be seen that the Abs0 of SP1 was larger than SP2 and SP3, because the melting temperature (Tm) order: SP1oSP2o SP3, the SP1 was not stable enough that it could be opened by assistant probe, producing high background signal. SP3 was so stable that the assistant probe–target DNA was hard to anneal with it, and hence reducing the sensitivity. As shown in Fig. 3A, the maximum relative absorbance change can be observed by SP2, which performed better than the other two probes, so SP2 was used in the later experiments. The concentration of signal probe in the range from 10.00 to 100.00 nmol L 1 was also investigated. From the inset of Fig. 3B, it can been seen that the Abs0 of signal probe was increased with increase in signal probe concentration because the random coil state signal probe was also increased. As shown in Fig. 3B, the relative absorbance intensities increased gradually with the increase of signal probe concentration, and then reaching a maximum value at the concentration of 50.00 nmol L 1. As the concentration was further increased, however, the DAbs/Abs0 was gradually decreased. Thus, 50.00 nmol L 1 was selected as the optimized signal probe concentration. The hybridization efficiency of signal probe and target DNAassistant probe could be affected by the hybridization time, so the hybridization time between signal probe and target DNAassistant probe was investigated as well. From the inset of Fig. 3C, it could be observed that the Abs1 was increased with the increasing of hybridization time because more and more signal probe had been opened with time extension. However, the hybridization time had no significant effect on Abs0. It could be seen from Fig. 3C that with the extension of hybridization time from 0.5 to 3.0 h, the value of DAbs/Abs0 was increased, and then reached a steady value after 2 h. These results indicated that almost all the target DNA-assistant probe were hybridized with signal probe within 2 h, so 2 h was used as the optimal hybridization time between signal probe and target DNA-assistant probe. 3.4. Sensitivity of the sensing system To study the sensitivity of this sensor for target DNA detection, the absorbance signal of the reaction mixture as a function of target DNA concentration was measured. Time-dependent absorbance curves (at 414 nm) of the sensing system upon introduction of different concentrations of target DNA were shown in Fig. 4A. It could be seen that the absorbance value increased remarkably with the increase of target DNA concentration. A maximum absorbance could be obtained within 3 min in the timedependent absorbance curve, which indicated that the rate of the DNAzyme catalytic reaction was very fast. A good linear relationship was observed between maximum absorbance value and target DNA concentrations in the range of 0.50–50.00 nmol/L
Fig. 3. The effect of the different sequences (A) and concentration (B) of signal probe and the hybridization time (C) on the sensing system. Experimental conditions: the system consisted of the 50.00 nmol L 1 assistant probe, 2.00 mmol/L ABTS, 2.00 mmol L 1H2O2 and 0.50 mmol/L hemin. The DAbs are representing the absorbance changes of the sensing system before and after addition of 20.00 nmol/L target DNA.
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Fig. 4. Time-dependent absorbance signal in the presence of different concentrations of target DNA (A) and the calibration curve corresponding to absorbance changes of the system upon analyzing different concentrations of target DNA (B). (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) corresponding to 0, 0.50, 5.00, 10.00, 20.00, 30.00, 40.00, 50.00, 80.00 and 100.00 nmol/L target, respectively.
and three-way junction DNA probe, the proposed sensor exhibited an excellent discrimination ability of SNPs. To estimate the reproducibility of the sensing system, the inter-assay imprecision at seven different assays for detection of 20.00 nmol L 1 target DNA were examined. The relative standard deviation (RSD) of the obtained absorbance values was 5.49%, indicating that the proposed strategy was reliable and can be used for DNA detection with acceptable reproducibility.
4. Conclusions
Fig. 5. Analysis of the SNP mutations. Absorbance of the sensing system toward with blank, single-base mismatched (S1), two-bases mismatched (S2) and fully complementary DNA targets (40.00 nmol/L). The inset photograph was the different color changes of ABTS–H2O2 solutions under corresponding conditions.
(Fig. 4B), with linear correlation coefficient of 0.997. The limit of detection was 0.25 nmol L 1 (S/N ¼3). The sensitivity of this method was higher than that of other reported DNAzyme based DNA sensors (Table S1). These results demonstrated that the proposed method had high sensitivity for target DNA analysis. 3.5. Selectivity and reproducibility SNPs are single-nucleotide variations in a natural DNA sequence. The discrimination ability (selectivity) of single-base mismatch DNA is very important in the SNP analysis. In order to investigate the discrimination ability of mismatch DNA of the sensing system and the absorbance response of single-base mismatch DNA, two-base mismatch DNA and fully complementary target DNA were monitored, respectively. As shown in Fig. 5, the absorbance value changes triggered by the single-base mismatch DNA and two-base mismatch DNA were similar to those of the target-free mixture. However, the absorbance value was greatly increased in the presence of fully complementary target DNA. As can be expected from UV–vis data, the above recognition process was clearly visible to the naked eye with a color change from colorless to green upon addition of perfectly matched target DNA, and no detectable color changes were observed upon addition of mismatched DNA (inset in Fig. 5). These results indicated that the proposed sensor showed high sequence specificity for DNA detection. With an integration of the hairpin probe
In summary, a novel three-way junction DNAzyme based probe has been developed for colorimetric detection of DNA. By the use of a DNAzyme-linked hairpin probe, tedious and expensive oligonucleotides covalent labeling procedures are avoided. Through the catalytic reaction of DNAzyme, the color changes caused by target DNA can be observed with naked eye. More significantly, with the combination of the hairpin probe and the three-way junction DNA probe, the proposed sensor shows a strong SNPs recognition capability. This homogeneous sensing strategy has the advantages of simple operation, low cost, high selectivity and good reproducibility, thus creating a great potential for the clinical diagnosis of genetic diseases.
Acknowledgments The authors are grateful for the National Nature Sciences Foundation of China (21075016, 21275029), National Basic Research Program of China (no. 2010CB732403), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (708056), the Program for Changjiang Scholars and Innovative Research Team in University (no. IRT1116), China. The National Key Technologies R & D Program of China during the 12th Five-Year Plan Period (Key technology of quality and safety control during aquatic product processing, No. 2012BAD29B06).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.08.056.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
The three-way junction DNAzyme based probe for label-free colorimetric detection of DNA Shurong Tang a, Ping Tong a,b, Heng Li a, Fang Gu a, Lan Zhang a,b,n a
Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, college of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, Fujian 350108, China b Analytical and Testing Center, The Sport Science Research Center, Fuzhou University, Fuzhou, Fujian 350002, China
a r t i c l e i n f o
abstract
Article history: Received 26 May 2012 Received in revised form 28 August 2012 Accepted 30 August 2012 Available online 5 September 2012
A novel three-way junction DNAzyme based probe has been designed for the colorimetric sensing of target DNA. Specifically, a DNAzyme-linked hairpin DNA is used as a signal probe. In the presence of target DNA, the signal probe, assistant probe and target DNA can hybridize with each other, resulting in the formation of a three-way junction DNA. At the same time, the signal probe is opened and the DNAzyme sequence in the signal probe is dehybridized. Subsequently, in the presence of hemin, the DNAzyme sequence forms a G-quadruplex–hemin complex, which catalyzes oxidation of 2, 20 -azinobis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) by H2O2 to the colored ABTS. radical. The significant color changes can be distinguished visually. By the combination of the hairpin probe and the three-way junction DNA probe, the proposed sensor exhibits high recognition property for single-nucleotide polymorphisms (SNPs). This sensor allows the detection of target DNA at a concentration as low as 0.25 nmol L 1. The proposed sensor is easy to fabricate, which avoids the tedious and expensive labeling procedures, and exhibits high selectivity against single-base mismatched DNA. & 2012 Elsevier B.V. All rights reserved.
Keywords: Three-way junction G-quadruplex–hemin DNAzyme Label free DNA detection
1. Introduction DNAzymes (also called deoxyribozyme or catalytic DNAs) are single stranded DNA molecules of a particular sequence, which possess specific catalytic activities to some chemical reactions. DNAzymes have several advantages over traditional protein enzymes, such as high chemical stability, low cost, simple preparation and easy modification (Breaker, 2000). For these unique properties, DNAzymes have been used in numerous biochemical reactions, such as DNA or RNA cleavage (Burmeister et al., 1997; Carmi et al., 1998), porphyrin metalation (Li and Sen, 1996), and DNA self-modification (Li and Breaker, 1999; Sheppard et al., 2000). Various kinds of artificial DNAzymes have been screened by using in vitro selection methodologies or SELEX (systemic evolution of ligands by exponential enrichment) techniques (Osborne and Andrew, 1997; Tuerk and Gold, 1990). One interesting example of a catalytic DNAzyme that reveals peroxidase-like activity is a complex between hemin and a G-quadruplex (Travascio et al., 1998). G-quadruplexes are unique higher-order
n Corresponding author at: Ministry of Education Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, College of Chemistry and Chemical Engineering, Fuzhou University, Fuzhou, Fujian 350108, China. Fax: þ86 591 87893207. E-mail address: [email protected] (L. Zhang).
0956-5663/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2012.08.056
structures, in which G-rich nucleic acid sequences form stacked arrays known as the G-quartets (four guanine bases are connected to each other by hydrogen bonded) (Sen and Gilbert, 1988). Hemin is able to specifically bind to G-quadruplex with high affinity. It has been reported that such G-quadruplex–hemin complexes possess peroxidase-like activity 250 times greater than that of hemin alone (Travascio et al., 1999). This neotype G-quadruplex based DNAzymes exhibit high catalytic activity toward the oxidation of 2,20 -azinobis (3-ethylbenzothiazoline)6-sulfonic acid (ABTS) by H2O2, which causes a color change (Travascio et al., 1998). The use of DNAzymes as catalytic labels for biosensor applications has the advantage of avoiding the tedious and expensive labeling procedures for signal readout. Therefore, the G-quadruplex–hemin DNAzymes have been widely used to colorimetric detection of biomolecules and other small molecules, such as DNA molecules (Nakayama and Sintim, 2009), proteins (Li et al., 2007) and metal ions (Li et al., 2008). Recently, there has been an increasing interest in the detection of single-nucleotide polymorphisms (SNPs). SNPs are singlenucleotide variations that may be substituted, deleted or inserted in a natural DNA sequence. When SNPs occur, both the structure and the function of the encoded protein are changed which often leads to harmful diseases, such as Tay Sachs (Gravel et al., 1995), cystic fibrosis (Cronin et al., 1996), and thalassemia (Muniz et al., 2000). SNPs are the most common variations in human genome, which occur approximately once per 250–1000 bases in a large
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sample of aligned genome sequence (Collins et al., 1998). The high density and mutational stability of SNPs make them particularly useful DNA markers for identification of complex disease genes (Ye et al., 2001). Therefore, the development of a highly selective method for SNPs detection is of great importance for early disease diagnosis and treatment. In an attempt to improve the selectivity of DNA detection, hairpin probes have been used in conjunction with DNAzymes to construct novel colorimetric biosensors (Li et al., 2011; Xiao et al., 2004). Hairpin probe is single-stranded DNA that possesses a stem-loop structure. Conventional hairpin probe-based colorimetric sensor employs a G-quadruplex DNAzyme-linked DNA sequence, which forms a stem-loop structure in the absence of target DNA, and results in inactivation of the DNAzyme. However, in the presence of target DNA, the stem-loop structure is opened to produce a catalytically active DNAzyme that leads to generation of a colorimetric signal. Hairpin probes can distinguish mismatches over a wider temperature range than that of linear probes (Bonnet et al., 1999), but they exhibit poor distinguishing capability for single-base mismatch (Demidov and Frank, 2004; Kolpashchikov, 2010), which limits their application in SNPs analysis. Thus, a more selective strategy with high discrimination ability for single-base mismatched DNA still needs to be explored. The general idea of a junction probe is that two DNA probes are hybridized to one DNA target. A detectable signal can be observed only when the two parts of the probes hybridize to the one DNA target (Kolpashchikov, 2010). Since the two parts of the probes form relatively short (7–10 nucleotides) duplexes with target DNA, the junction probe’s selectivity is better than that of hairpin probe. Junction probe can be used for highly accurate detection of SNPs at room temperature without the precise temperature control (Kolpashchikov, 2005). Up to date, some new junction probes have been introduced for SNPs analysis, such as binary DNA probe (Kolpashchikov, 2006), three-way junction probe (Kong et al., 2011; Nakayama et al., 2008) and four-way junction probe (Lake et al., 2010). In this paper, a new three-way junction DNAzyme based probe has been used for the colorimetric detection of target DNA. A Gquadruplex DNAzyme-linked hairpin probe is specially designed as a signal probe, which avoids the tedious and expensive labeling steps for signal readout. The signal probe is opened after hybridization with target DNA and assistant probe, which is accompanied by the formation of a three-way junction DNA. After addition of hemin, the DNAzyme is activated and then catalyzes the oxidation of ABTS by H2O2 to form the colored product ABTS.for ultraviolet–visible (UV–vis) absorbance detection. The proposed DNA sensor is simple, label free, and exhibits high discrimination ability against single-base mismatched DNA.
2. Materials and methods 2.1. Materials 2,20 -Azinobis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) and HPLC-purified oligonucleotides used in this experiment were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China), and the detail base sequences were shown in Table 1. TritonX-100, Hemin, N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (HEPES), and DMSO were bought from Sigma Aldrich (St. Louis, MO). The stock solution of oligonucleotides (100.00 mmol L 1) were prepared in ultrapure water and stored at -20 1C. The stock solution of hemin (1.00 mmol L 1) was prepared in dimethyl sulfoxide (DMSO) and stored in the dark at 20 1C. Before use, the oligonucleotides and hemin solutions were diluted
Table 1 Sequence of oligonucleotides used in the colorimetric assay Oligonucleotide Sequence (from 50 - 30 ) SP1a SP2b SP3c APd Tae S1f S2g
AAC CCA GTC AGT GTC CTC AGC GTG GGT TGG GCG GGA TGG GT CAA CCC AGT CAG TGT CCT CAG CGT GGG TTG GGC GGG ATG GGT CCA ACC CAG TCA GTG TCC TCA GCG TGG GTT GGG CGG GAT GGG T CGC TGA GGA AAT GGA AAA TCT CTA G GCT AGA GAT TTT CCA CAC TGA CT GCT AGA GAT TTT CCA CAC CGA CT GCT AGA GAT TTT CCA AAC TTA CT
a,b,c
Thermodynamic parameters of all oligonucleotides were calculated using integrated DNA technologies (http://www.idtdna.com/analyzer/Applications/ OligoAnalyzer).
a,b,c Hairpin DNAzyme signal probe: the under line sequences are hybridized to form the stem-loop hairpin structure, the italic letters are G-quadruplex DNAzyme sequence. d Assistant probe. e Perfectly complementary target. f Single-base-mismatched target. g Two-bases-mismatched target (the mismatched bases are underlined).
Fig. 1. Colorimetric sensing protocol for target DNA.
to required concentrations with ultrapure water and dilution buffer (25.00 mmol L 1 Tris–HCl, pH 8.0, 20.00 mmol L 1 KCl, 200.00 mmol L 1 NaCl, 0.05% TritonX-100 and 1% DMSO), respectively. ABTS and H2O2 were freshly prepared before use. The ultrapure water supplied with a Milli-Q system (18.5 MO Millipore, USA) was used throughout the experiment. All reagents were used as received without further purification. 2.2. Colorimetric sensing of target DNA The assay protocol of the colorimetric DNA sensor was depicted in Fig.1. Signal probe was heated to 90 1C for 5 min and then gradually cooled to room temperature to form the hairpin probe before use. First, the assistant probe and target DNA were incubated at 4 1C over night in a hybridization buffer which is containing 20.00 mmol/L Tris–HCl (pH 8.0), 50.00 mmol L 1 NaCl and 10.00 mmol L 1 MgCl2. Then, signal probe was added and the hybridization was allowed to proceed at 37 1C for 2 h. The above hybridization solution volume was 50 mL. Next, 5 mL hemin (25.00 mmol L 1) and 185 mL HEPES/NH4OH buffer (25.00 mmol L 1, pH 7.4, 20.00 mmol/L KCl, 100.00 mmol L 1 NaCl, and 1% DMSO) were added and incubated at 25 1C for 40 min. It would allow the formation of G-quadruplex–hemin complexes. Finally, 5.0 mL 0.10 mol L 1 ABTS and 5.0 mL 0.10 mol L 1H2O2 were added to the above reaction mixture to initiate the catalytic reaction. The final concentration of signal probe and assistant probe were 50.00 nmol L 1. The time-dependent
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absorption spectra of the radical anion ABTS. (the product of ABTS oxidation by H2O2) was collected immediately with Perkin Elmer Lambda 750 UV/vis spectrometer (Shelton, USA) at 414 nm. Under the same above conditions, specificity experiments were performed in which target DNA was replaced by S1 or S2. 2.3. Circular dichromism (CD) measurements CD experiments were performed on a model 420 circular dichroism spectrometer (Lake Wood, NJ, USA) at 25 1C in a 1 mm path length cuvette. Scans from 220 to 350 nm were performed. The bandwidth was 1 nm. Each spectrum was corrected by subtracting the CD of the buffer. The concentration of signal probe, assistant probe and target DNA was 10, 10 and 5 mmol L 1, respectively. 2.4. Gel electrophoresis experiments Polyacrylamide gel electrophoresis (PAGE) was performed in an electrophoresis apparatus (DYY-80, China). Native 12% PAGE gel was prepared by mixing 3.94 mL ultrapure water, 1.6 mL 5 TBE buffer (0.45 mol/L Tris–boric acid–EDTA, pH 8.0), 2.4 mL 40% bis-acrylamide, 55 mL 10% ammonium persulfate and 5 mL N,N,N0 ,N0 -tetramethylethylenediamine (TEMED) together. After vortex mixing thoroughly, the fresh prepared gel was transferred to the electrophoresis apparatus, and then gelation for 1 h at room temperature. Before electrophoresis analysis, the samples were prepared in 10 mL reaction solution which contained 5.00 mmol/L DNA samples and 20.00 mmol/L Tris–HCl buffer (10.00 mmol/L MgCl2, 100.00 mmol/L NaCl, pH 7.9). After the DNA hybridization at 37 1C for 2 h, the resulting DNA samples together with loading dye were applied to the 12% polyacrylamide gel prepared before. The electrophoresis was carried out in a 1 TBE buffer (0.09 mol L 1 Tris–boric acid–EDTA, pH 8.0) at 12 mA for 2 h. The obtained gels were stained with ethidium bromide (EB) for 15 min and the staining results were photographed with a Gel Image System.
3. Results and discussion 3.1. Design of the three-way junction DNAzyme based probe for colorimetric sensing The proposed sensor uses a novel three-way junction DNAzyme based probe for the colorimetric detection of DNA from HIV-1 U5 (Lu et al., 2009). The detailed principle of this sensor is illustrated in Fig. 1. We have designed a G-quadruplex DNAzyme-linked hairpin
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probe, which serves as a signal probe. A single-stranded DNA which is partially complementary with the signal probe and target DNA is used as assistant probe. In the presence of target DNA, assistant probe and the target DNA will hybridize together with the signal probe, leading to unwinding the signal probe and formation of a three-way junction DNA. As a result, the DNAzyme sequence in the signal probe is dehybridized. After addition of hemin, a G-quadruplex–hemin DNAzyme complex is formed and then catalyzes oxidation of ABTS by H2O2 to the colored ABTS.- radical, accompanied by a significant increase of the absorbance signal. However, in the absence of target DNA, the signal probe cannot be opened through hybridization with the assistant probe to form a doublestranded DNA under the reaction temperature. The DNAzyme sequence is still blocked in the stem portion of signal probe, which inhibits the formation of any free hemin-containing active DNAzyme, so no remarkable absorbance signal can be detected. 3.2. Feasibility of the sensing system 3.2.1. Colorimetric measurement To test the feasibility of the sensing system, control experiments were performed under different conditions. As shown in Fig. 2A, the absorbance produced by hemin alone was negligible, which indicated that free hemin showed only slight catalytic activity toward the oxidation of ABTS (Fig. 2A, curve a). After the addition of signal probe to the homogeneous solution, the absorbance signal was about 3-fold increased (Fig. 2A, curve b). This was probably due to the fact that a small part of signal probes exist as random coils instead of stem-loop structure, which results in a self-assembled formation of G-quadruplex DNAzyme in the presence of hemin. It was worth to note that after the addition of assistant probe, there was no significant increase in the signal intensity (Fig. 2A, curve c). This result represented that most part of the signal probe cannot be opened by assistant probe alone, so no more G-quadruplex–hemin DNAzyme can be formed to enhance the absorbance signal. In contrast, after the addition of target DNA, a remarkable absorbance increase could be observed (Fig. 2A, curve d). These results suggested that the signal probe can be effectually opened only in the presence of target DNA. Therefore, the proposed method can be used for effective detection of target DNA. 3.2.2. Determination of structure change of DNA by CD measurements Moreover, in order to identify the hybridization of DNA and formation of G-quadruplex structure in this experiment, the CD spectra were used to monitor the structural changes. As shown in
Fig. 2. (A) Absorbance generated by 0.50 mmol/L hemin (a); hemin and 50.00 nmol/L signal probe (b); hemin, 50.00 nmol/L signal probe and assistant probe without (c) or with 20.00 nmol/L target (d). The inset was the corresponding time-dependent absorbance curves. (B) CD spectra of the DNA structure in HEPES/NH4OH buffer (25.00 mmol/L, pH 7.4, 20.00 mmol/L KCl, 100.00 mmol/L NaCl, and 1% DMSO). Signal probe (1); signal probe and assistant probe before (2) and after (3) addition of target DNA. (C) PAGE graphs of the sensing system. (1) Signal probe; (2) signal probe and target DNA; signal probe and assistant probe before (3) and after (5) addition of target DNA; (4) signal probe and assistant probe after addition of single-base mismatch DNA.
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Fig. 2B, the signal probe displayed a negative peak at 240 nm and a positive peak at 260 nm, which were characteristic of a DNA duplex (Miyoshi et al., 2004; Xue et al., 2011). After the addition of assistant probe, the positive peak at 260 nm became stronger. However, no obvious change was observed in the negative peak at 240 nm. Upon incubation with target DNA, the spectrum changes to reveal a positive band at 265–285 nm. The number of base pairs was increased after hybridization, which subsequently induced the rise of ellipticity at 273 nm and the drop at 240 nm; it should be the typical characteristic of B-form DNA double helix (Guo et al., 2010; Zhu et al., 2011). Moreover, a positive CD signal at around 265 nm and a negative signal at around 245 nm could be observed, indicating the formation of parallel G-quadruplex (Jin et al., 1992; Nakayama and Sintim, 2009). 3.2.3. PAGE experiment In order to further verify the proposed sensor with high selectivity for target DNA. Five samples including signal probe (1), signal probe and target DNA (2), signal probe and assistant probe before (3) and after (5) addition of target DNA or single-base mismatch DNA (4) were analyzed by native PAGE (Fig. 2C, lanes 1–5). Without addition of the target DNA, only one band could be seen clearly in the presence of signal probe and assistant probe (lane 3), which was the same as that obtained by signal probe alone (lane 1). These results proved the fact that the signal probe existed predominantly in the stem-loop structure without target DNA. At the same time, a new low-mobility band (migrates slower than the 75 base pair DNA marker) was observed only in the presence of fully complementary target (lane 5) but not in the presence of single-base mismatched target (lane 4). This band can be attributed to the three-way junction DNA depicted in Fig.1. The results of PAGE experiments were coincident with the results observed in colorimetric assay. 3.3. Optimization of experimental conditions In order to achieve the best sensing performance, the stem length and the concentration of the signal probe were optimized. The stem length of signal probe has a remarkable affect on the sensitivity. When the stem is too short, the signal probe is not stable enough and can be easily opened by assistant probe, resulting in high background signal. However, a too long stem will lead to very strong hybridization such that the assistant probe and the target DNA are hard to anneal with the signal probe, and hence reducing the signal and sensitivity. Three signal probes (SP1, SP2, and SP3) of different stabilities were used with stem lengths of 7, 8 and 9 base pairs, respectively. The relative absorbance change ((Abs1–Abs0)/Abs0) which defined as (DAbs/Abs0) was used to evaluate the sensitivity of the assay, where Abs1 and Abs0 were the values of absorbance changes at 414 nm of target DNA and the
background (without target), respectively. From the inset of Fig. 3A, it could be seen that the Abs0 of SP1 was larger than SP2 and SP3, because the melting temperature (Tm) order: SP1oSP2o SP3, the SP1 was not stable enough that it could be opened by assistant probe, producing high background signal. SP3 was so stable that the assistant probe–target DNA was hard to anneal with it, and hence reducing the sensitivity. As shown in Fig. 3A, the maximum relative absorbance change can be observed by SP2, which performed better than the other two probes, so SP2 was used in the later experiments. The concentration of signal probe in the range from 10.00 to 100.00 nmol L 1 was also investigated. From the inset of Fig. 3B, it can been seen that the Abs0 of signal probe was increased with increase in signal probe concentration because the random coil state signal probe was also increased. As shown in Fig. 3B, the relative absorbance intensities increased gradually with the increase of signal probe concentration, and then reaching a maximum value at the concentration of 50.00 nmol L 1. As the concentration was further increased, however, the DAbs/Abs0 was gradually decreased. Thus, 50.00 nmol L 1 was selected as the optimized signal probe concentration. The hybridization efficiency of signal probe and target DNAassistant probe could be affected by the hybridization time, so the hybridization time between signal probe and target DNAassistant probe was investigated as well. From the inset of Fig. 3C, it could be observed that the Abs1 was increased with the increasing of hybridization time because more and more signal probe had been opened with time extension. However, the hybridization time had no significant effect on Abs0. It could be seen from Fig. 3C that with the extension of hybridization time from 0.5 to 3.0 h, the value of DAbs/Abs0 was increased, and then reached a steady value after 2 h. These results indicated that almost all the target DNA-assistant probe were hybridized with signal probe within 2 h, so 2 h was used as the optimal hybridization time between signal probe and target DNA-assistant probe. 3.4. Sensitivity of the sensing system To study the sensitivity of this sensor for target DNA detection, the absorbance signal of the reaction mixture as a function of target DNA concentration was measured. Time-dependent absorbance curves (at 414 nm) of the sensing system upon introduction of different concentrations of target DNA were shown in Fig. 4A. It could be seen that the absorbance value increased remarkably with the increase of target DNA concentration. A maximum absorbance could be obtained within 3 min in the timedependent absorbance curve, which indicated that the rate of the DNAzyme catalytic reaction was very fast. A good linear relationship was observed between maximum absorbance value and target DNA concentrations in the range of 0.50–50.00 nmol/L
Fig. 3. The effect of the different sequences (A) and concentration (B) of signal probe and the hybridization time (C) on the sensing system. Experimental conditions: the system consisted of the 50.00 nmol L 1 assistant probe, 2.00 mmol/L ABTS, 2.00 mmol L 1H2O2 and 0.50 mmol/L hemin. The DAbs are representing the absorbance changes of the sensing system before and after addition of 20.00 nmol/L target DNA.
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Fig. 4. Time-dependent absorbance signal in the presence of different concentrations of target DNA (A) and the calibration curve corresponding to absorbance changes of the system upon analyzing different concentrations of target DNA (B). (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) corresponding to 0, 0.50, 5.00, 10.00, 20.00, 30.00, 40.00, 50.00, 80.00 and 100.00 nmol/L target, respectively.
and three-way junction DNA probe, the proposed sensor exhibited an excellent discrimination ability of SNPs. To estimate the reproducibility of the sensing system, the inter-assay imprecision at seven different assays for detection of 20.00 nmol L 1 target DNA were examined. The relative standard deviation (RSD) of the obtained absorbance values was 5.49%, indicating that the proposed strategy was reliable and can be used for DNA detection with acceptable reproducibility.
4. Conclusions
Fig. 5. Analysis of the SNP mutations. Absorbance of the sensing system toward with blank, single-base mismatched (S1), two-bases mismatched (S2) and fully complementary DNA targets (40.00 nmol/L). The inset photograph was the different color changes of ABTS–H2O2 solutions under corresponding conditions.
(Fig. 4B), with linear correlation coefficient of 0.997. The limit of detection was 0.25 nmol L 1 (S/N ¼3). The sensitivity of this method was higher than that of other reported DNAzyme based DNA sensors (Table S1). These results demonstrated that the proposed method had high sensitivity for target DNA analysis. 3.5. Selectivity and reproducibility SNPs are single-nucleotide variations in a natural DNA sequence. The discrimination ability (selectivity) of single-base mismatch DNA is very important in the SNP analysis. In order to investigate the discrimination ability of mismatch DNA of the sensing system and the absorbance response of single-base mismatch DNA, two-base mismatch DNA and fully complementary target DNA were monitored, respectively. As shown in Fig. 5, the absorbance value changes triggered by the single-base mismatch DNA and two-base mismatch DNA were similar to those of the target-free mixture. However, the absorbance value was greatly increased in the presence of fully complementary target DNA. As can be expected from UV–vis data, the above recognition process was clearly visible to the naked eye with a color change from colorless to green upon addition of perfectly matched target DNA, and no detectable color changes were observed upon addition of mismatched DNA (inset in Fig. 5). These results indicated that the proposed sensor showed high sequence specificity for DNA detection. With an integration of the hairpin probe
In summary, a novel three-way junction DNAzyme based probe has been developed for colorimetric detection of DNA. By the use of a DNAzyme-linked hairpin probe, tedious and expensive oligonucleotides covalent labeling procedures are avoided. Through the catalytic reaction of DNAzyme, the color changes caused by target DNA can be observed with naked eye. More significantly, with the combination of the hairpin probe and the three-way junction DNA probe, the proposed sensor shows a strong SNPs recognition capability. This homogeneous sensing strategy has the advantages of simple operation, low cost, high selectivity and good reproducibility, thus creating a great potential for the clinical diagnosis of genetic diseases.
Acknowledgments The authors are grateful for the National Nature Sciences Foundation of China (21075016, 21275029), National Basic Research Program of China (no. 2010CB732403), the Cultivation Fund of the Key Scientific and Technical Innovation Project, Ministry of Education of China (708056), the Program for Changjiang Scholars and Innovative Research Team in University (no. IRT1116), China. The National Key Technologies R & D Program of China during the 12th Five-Year Plan Period (Key technology of quality and safety control during aquatic product processing, No. 2012BAD29B06).
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2012.08.056.
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