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Magnetic bead-based chemiluminescence detection of sequence-specific DNA by using catalytic nucleic acid labels
Analytica Chimica Acta 557 (2006) 152–158
Magnetic bead-based chemiluminescence detection of sequence-specific DNA by using catalytic nucleic acid labels Zhijuan Cao a , Zengxi Li b , Yujie Zhao a , Yumin Song a , Jianzhong Lu a,∗ a
b
School of Pharmacy, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China Graduate University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
Received 21 August 2005; received in revised form 18 October 2005; accepted 20 October 2005 Available online 28 November 2005
Abstract We wish to report a new chemiluminescence (CL) method for the determination of the sequence-specific DNA by the coupling of catalytic nucleic acid label (DNAzyme) based CL detection route with an efficient magnetic isolation of the hybrid. The assay relies on (i) the immobilization of NH2 -modified capture DNA on the surface of carboxyl-terminated magnetic beads activated by EDC, (ii) first hybridization event occurring between the bead-captured DNA probe and one 15-mer portion (5 -AAT ATT GAT AAG GAT-3 ) of the target sequence, (iii) second hybridization happening between one part of the DNAzyme modified reporter sequence and another 15-mer portion (5 -GAG GGA TTA TTG TTA-3 ) of the target sequence, and then direct detection of CL signal on the surface of the magnetic beads in a basic solution of luminol and H2 O2 . The influence of relevant experimental variables, including the effect of the amount of the magnetic beads, the duration of the hybridization and the parameters affecting CL signal, is examined and optimized. The results showed that this simple and sensitive protocol was suitable for the determination of specific-sequence DNA as exemplified by the 30-base sequence related to the anthrax lethal factor with a good linear correlation in the concentration range of 0.02–2 pmol (R2 = 0.9987) and a detection limit of 1 × 10−10 M. Overall, this new CL protocol coupled the high sensitivity of CL analysis with effective magnetic separation for discriminating against unwanted constituents such as mismatched sequences and proteins, hence, offers great promise for DNA hybridization analysis. © 2005 Elsevier B.V. All rights reserved. Keywords: Catalytic nucleic acid label; Specific sequence DNA; Chemiluminescent; Magnetic beads
1. Introduction The polymerase chain reaction (PCR) and other forms of target amplification have enabled rapid advances in the development of powerful tools for detecting and quantifying sequencespecific DNA of interest for research, forensic and clinical applications [1–4]. Many detection techniques have been developed, and their quantifications are generally achieved by measuring the specific activity of the label linked to the capture probe, in which enzymes are often employed as biocatalytic labels for the amplified detection of DNA-sensing events. In this context, electrochemical detection of hybridization can be accomplished by the application of oligonucleotide-functionalized redox enzymes as bioelectrocatalytic probes for the formation of the double-stranded DNA complex [5] or the selective associ-
∗
Corresponding author. Tel.: +86 21 54237587; fax: +86 21 54237587. E-mail address: [email protected] (J. Lu).
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.10.048
ation of enzymes to the double-stranded oligonucleotide-DNA assembly, the amplified detection of DNA by the application of rotating functional magnetic particles [6] and the subsequent biocatalyzed precipitation of an insoluble product on the transducers for the oligonucleotide-DNA recognition event [7], etc. Although large signal amplification can be achieved, labeling of the oligonucleotide by enzyme is not a straightforward route, and once prepared, the activity of the conjugate must be periodically controlled owing to the inherent poor stability of the enzymes. In addition, the preparation of DNA-based enzymes attracts substantial research efforts directed to the development of novel biocatalysts [8–13], and many catalytic deoxyribozymes for numerous chemical transformations were prepared in recent years [14,15]. An interesting example of a catalytic nucleic acid sequence that reveals peroxidase-like activity includes a guanine-rich nucleic acid (aptamer) that can folds into a Gquadruplex structure and binds hemin into a supramolecular catalytic configuration [16–18]. The use of nucleic acids as cat-
Z. Cao et al. / Analytica Chimica Acta 557 (2006) 152–158
alytic labels may reveal two advantages: (i) the use of noncomplementary negatively charged nucleic acid labels may reduce the nonspecific adsorption caused by proteins to the nucleic acid interfaces; (ii) the base sequence in the nucleic acid label may be tailored to include the catalytic DNAzyme domain and a nucleic acid domain that is complementary and hybridizes with the target DNA. Based on this fact, Xiao et al. [19,20] reported on the use of a catalytic nucleic acid (DNAzyme) as a label for the homogeneous detection of DNA with a detection limit of 2 × 10−7 M and Pavlov et al. [21] developed a DNAzyme-based heterogeneous detection of DNA with a detection limit of 1 × 10−9 M. Further lowering of the detection limit into 1 × 10−10 M could be obtained in connection with a complicated DNAzyme-modified gold nanoparticle amplification system [22], which enabled sensitive detection of telomerase activity originating from 1000 HeLa cells. These DNAzyme-based systems are sensitive. However, they deliver results much slower than previously reported optical and electrochemical techniques [23,24], i.e. the entire assay normally takes around 30 h to reach a detection limit of 1 × 10−9 or 1 × 10−10 M. Still, a desired yet unapproached goal is to develop a simple and sensitive DNAzyme-based method for the detection of DNA hybridization events with significantly shorter assay time. Magnetic nanoparticles as special biomolecule immobilizing carrier offer a promising alternative to conventional methodology. Magnetic nanoparticles have been used in immunoassays, enzyme, DNA and protein immobilization, DNA purification, and magnetically controlled transport of anticancer drugs [25]. Wang et al. [26] developed a new metal nanoparticlebased electrochemical stripping potentiometric detection of DNA hybridization. Recently, we [27,28] used magnetic beads for DNA hybridization detection assay and also reported a new oxidative gold metal dissolution-based CL method for the noncompetitive immunoassay of a human immunoglobulin G by taking advantage of a magnetic separation/mixing process and the amplification feature of colloidal gold label. In this article, we describe a new CL method for the detection of the specific DNA sequence by the coupling of DNAzyme CL detection route with an efficient magnetic isolation of the hybrid. Compared with previous DNAzyme-based methods, our new method has such characteristics as speedy, easy to use, and high sensitivity. The entire assay can be completed within 2–3 h at a concentration 0.02–2 pmol and a detection limit of 1 × 10−10 M for the recognition of 30-base model oligonucleotide.
153
Amino-modified capture probe: 5 -NH2 -(A)20 ATC CTT ATC AAT ATT-3 ; sequence X: 5 -NH2 -(A)20 GGG TTA GGGTTA GGG TTA GGG-3 ; 30-base target DNA sequence associated with the anthrax lethal factor: 5 -GAG GGA TTA TTG TTA AAT ATT GAT AAG GAT -3 ; A–A single-base mismatched target: 5 -GAG GGA TTA TTG TTA AAT ATT GAA AAG GAT-3 ; A–G single-base mismatched target: 5 -GAG GGA TTA TTG TTA AAT ATT GAG AAG GAT-3 ; A–C single-base mismatched target: 5 -GAG GGA TTA TTG TTA AAT ATT GAC AAG GAT-3 ; Two-base-mismatched target: 5 -GAG GGA TTA TTG TTA AAT ATT GAA TAG GAT-3 ; Noncomplementary sequence: 5 -TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG-3 ; and the reporter sequence: 5 -TAA CAA TAA TCC CTC TTT GGG TAG GGC GGG TTG GG-3 . 2.2. Apparatus CL measurements were performed with a BPCL chemiluminescence analyzer (Beijing, China). 2.3. Preparation of DNAzyme-modified reporter sequence The DNAzyme-modified reporter sequence is composed of two parts. One part is the catalytic nucleic acid label (5 -TTT GGG TAG GGC GGG TTG GG-3 , which complexes with hemin), and another part is 5 -TAA CAA TAA TCC CTC that can hybridize with one 15-mer portion (5 -GAG GGA TTA TTG TTA-3 ) of the target sequence. The DNAzyme-modified reporter sequence was prepared as follows. A solution of 2.5 M reporter sequence was heated at 95 ◦ C for 9 min in 0.01 M Tris buffer, pH 7.4, to dissociate any intermolecular quadruplex, and allowed to cool to room temperature. An identical volume of a buffer solution consisting of 50 mM HEPES, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100, and 2% DMSO, pH 7.4, was added to the reporter sequence solution to allow appropriate folding. Hemin, 1.2 M, was added to the nucleic acid solution, and the system was allowed to form the supramolecular complex for 3 h at room temperature.
2. Experimental 2.4. Assay procedures 2.1. Materials and reagents All chemicals were of analytical grade and were used as received. All stock solutions were prepared using deionized and autoclaved water. The carboxyl-modified magnetic beads were obtained from Bangs Lab. Hemin and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma. Oligonucleotides were acquired from Invitrogen Biotechnology Co., Ltd (Shanghai, China) and had the following sequences:
In a typical experiment, 50 L of the carboxyl-modified magnetic beads were transferred into a microtube, where the beads were washed three times with 100 L of 0.1 M imidazole buffer (pH 7.0) and resuspended in 1 mL of 0.1 M imidazole buffer containing 0.1 M EDC for 30 min with gentle shaking. The activated magnetic beads were divided equally into ten wells (100 L/well) of a 96-well microplate, and then 100 pmol of capture DNA were added into each well and incubated for 30 min at 37 ◦ C with gentle shaking. The bead-captured probes were
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Fig. 1. Schematic representation of the analytical protocol: (a) the immobilization of NH2 -modified capture DNA on the surface of carboxyl terminated magnetic beads activated by EDC; (b) the first hybridization event between capture DNA and target DNA; (c) the second hybridization event with the catalytic DNAzyme-modified reporter sequence, and direct detection of hybrid DNA CL signal on the surface of magnetic beads in a basic luminol-H2 O2 solution.
magnetically separated and the supernatants were aspirated and discarded. Next, the beads were washed three times with 100 L of 10 mM pH 7.0 phosphate buffer containing 0.1 M NaCl and once with buffer A (pH 8.0 of 20 mM Tris–HCl buffer containing 0.5 M NaCl). The desired amount of the target DNA and 5 L of the DNAzyme-modified reporter sequence were added simultaneously into each well and then incubated for 30 min in buffer A at 37 ◦ C. The hybrid-conjugated beads were then washed twice with wash buffer B (7 mM Tris–HCl, pH 8.0, 0.17 M NaCl and 0.05% Tween 20), once with wash buffer C (7 mM Tris–HCl, pH 8.0, 0.17 M NaCl) and then once with buffer D (25 mM HEPES, 20 mM KCl and 200 mM NaCl, pH 9.0) at 37 ◦ C. Afterwards the hybrid-conjugated beads were resuspended in 90 L of buffer D and transferred into 14 mm × 40 mm glass tubes, 5 L of 0.5 mM luminol was added and the tubes were placed in the luminescence reader. Finally, 5 L of 0.1 M H2 O2 was injected and the CL signal was displayed.
3. Results and discussion The present assay format (illustrated in Fig. 1) involves three steps: (1) capture of NH2 -modified DNA probe on the surface of carboxyl terminated magnetic beads activated by EDC, (2) the first hybridization event occurring between the bead-captured DNA probe and one 15-mer portion (5 -AAT ATT GAT AAG GAT-3 ) of the target sequence; (3) the second hybridization event happening between one part of the catalytic DNAzymemodified reporter sequence and another 15-mer portion (5 -GAG GGA TTA TTG TTA-3 ) of the target sequence and magnetic removal of non-hybridized DNA sequences. Afterwards, direct detection of catalytic DNAzyme CL takes place on the surface of the magnetic beads in a basic luminol-H2 O2 solution. Note that CL emission will be emitted only if the catalytic DNAzymemodified reporter sequences are associated with the surface of magnetic beads, and this will occur only provided that the target
Z. Cao et al. / Analytica Chimica Acta 557 (2006) 152–158
Fig. 2. CL ratio vs. the amount of magnetic beads, 100 pmol of capture DNA, 0.5 pmol of target DNA, 5 L of catalytic DNAzyme modified reporter sequence, 5 L of 0.5 mM luminol, 5 L of 0.1 M H2 O2 and the hybridization time 30 min.
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Fig. 3. CL ratio vs. the amount of capture probe DNA, 100 g of magnetic beads, 0.5 pmol of target DNA, 5 L of catalytic DNAzyme-modified reporter sequence, 5 L of 0.5 mM luminol, 5 L of 0.1 M H2 O2 and the hybridization time 30 min.
DNA is hybridized with the capture DNA immobilized on the surface of magnetic beads through EDC. Several parameters were optimized systematically for the catalytic DNAzyme-based CL detection of specific sequence of DNA, including the amounts of magnetic beads, capture DNA and DNAzyme-modified reporter sequence, and the hybridization time, etc. 3.1. Effect of the amount of magnetic beads As shown in Fig. 2, with the increasing of the amount of magnetic beads, the hybridization CL signal ratio (the target signal/background) increased at first, leveled off between 50 and 200 g and then decreased quickly. Hence, subsequent work employed 100 g beads. 3.2. Effects of the amounts of capture DNA and catalytic reporter sequence The effects of the amounts of capture DNA and reporter sequence were examined and optimized, respectively. First, the CL signal ratio was observed to be increased in the range of 10–100 pmol of capture DNA and then decreased greatly, thus 100 pmol of capture DNA was selected for the following experiments (Fig. 3). Second, the CL and the background signal were increased with the increasing of the DNAzyme-modified reporter sequence, but the CL ratio was the highest by using 5 L of catalytic DNAzyme-modified reporter sequence, which was then selected for the use in further studies. The coverage of capture probes on the magnetic particles was determined by employing guanine-specific TMPG CL reaction [27] and the sequence X (5 -NH2 (A)20 GGGTTAGGGTTAGGGTTAGGG-3 ) was thus used as the capture DNA to react with carboxyl-modified magnetic particles through 0.1 M EDC. CL intensities of five different concentration of sequence X (4, 20, 50, 100, 150 pmol) before and after being coupled on the surface of magnetic beads were
Fig. 4. CL ratio vs. [luminol] (a) and CL ratio vs. [H2 O2 ] (b), 5 L of 0.1 M H2 O2 (a) and 5 L of 0.5 mM luminol (b), 100 g of magnetic beads, 100 pmol of capture DNA, 0.5 pmol of target DNA, 5 L of catalytic DNAzyme-modified reporter sequence and the hybridization time 30 min.
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Table 1 Comparison of sensitivity and assay for DNAzyme-based detection methods Analytical method
No. of target bases
Assay time (h)
Detection limit
Homogeneous UV Homogeneous CL Au surface CL Au surface CL-Au NPs amplification Magnetic beads-CL (this work)
35 36 36 36 30
– – >30 >24 2–3
200 nM [19] 600 nM [20] 1 nM [21] 100 pM [22] 100 pM
compared. If assuming the CL signal of the sequence X in the solution phase as 100%, the coverage of amine-terminated DNA probe on the carboxyl-modified magnetic particles was calculated to be about 11%.
of the detection limit is expected in connection with branched DNAzyme-modified reporter sequences. A series of seven repetitive measurements of 0.5 pmol of target DNA, used for estimating the precision, yielded reproducible signals with a relative standard deviation of 5.0%.
3.3. Effects of luminol and H2 O2 concentrations In addition, the concentrations of luminol and H2 O2 also affected the CL signal greatly. So these parameters were also examined and optimized (see Fig. 4). First, it was found that both signal and background were increased with increasing luminol concentration. However, signal/noise ratio was the highest at 0.5 mM luminol. Next, for H2 O2 , signal/noise ratio was increased from 0 to 0.05 M, almost constant between 0.05 and 0.1 M and then decreased sharply. Hence, 0.5 mM luminol and 0.1 M H2 O2 were selected for the following experiments. 3.4. Other parameters affecting CL signal The CL signal was almost the same when hybridizing target DNA and catalytic DNAzyme-modified reporter sequence separately or together. For convenience, the two solutions of target and reporter DNA were added simultaneously for the first and second hybridization reactions. The hybridization times have been examined. Four hybridization times of 15, 30, 60 and 90 min were studied. It was found that the CL signal ratio was almost constant by using a longer hybridization time than 30 min (see Fig. 5). Hence, 30 min of hybridization time was selected for the following experiments.
3.6. Discrimination of complementary DNA sequence from mismatched ones The new protocol provided a good capability in discriminating two target DNA sequences that differ by one base mismatch such as A–A mismatch, A–G mismatch, A–C mismatch; two base mismatch and noncomplementary sequence upon DNA hybridization. After that, the resultants were washed with wash buffer A twice, wash buffer B once and buffer C once at 37, 45 or 55 ◦ C, respectively. It was found that the wash temperature has a great effect on the discrimination efficiency of single base-mismatch. Single base-mismatch could not be discriminated at 37 ◦ C whereas perfectly complementary target DNA was also denatured at 55 ◦ C, and thus 45 ◦ C was found to be a suitable wash temperature. As shown in Fig. 6, the CL signal of the complementary sequence was much higher than those of one base mismatched, two base mismatched and noncomplementary DNA sequence. Therefore, the proposed CL technique is particularly attractive for the CL detection of specific DNA sequences. In addition, this novel CL approach could provide a good capability in detecting selectively and sensitively target DNA in synthetic samples containing target DNA, foreign
3.5. Quantification of the target DNA The quantitative behavior was assessed by monitoring the dependence of the CL intensity upon the concentration of the target DNA. A calibration graph in the concentration range of 0.02–2.0 pmol showed a linear correlation (r2 = 0.9987) between the amount of target DNA and the CL intensity, represented by I = 798803C − 12961. In addition, at an amount of higher than 2.0 pmol, the CL intensity was slightly increased and deviated from the calibration curve. The detection limit was estimated to be 0.01 pmol, i.e., 1 × 10−10 M (0.01 pmol in the 100 L sample), which is equal or better than previous DNAzyme-based techniques but with significantly shorter assay time (Table 1). Moreover, this sensitivity is also comparable with several gold nanoparticle-based hybridization assays as reported recently for shorter DNA sequences as shown in Table 2. Further lowering
Fig. 5. CL ratio vs. the hybridization time, 100 g of magnetic beads, 100 pmol of capture DNA, 0.5 pmol of target DNA, 5 L of catalytic DNAzyme-modified reporter sequence, 5 L of 0.5 mM luminol and 5 L of 0.1 M H2 O2 .
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Table 2 Comparison of sensitivity for DNA assay methods Analytical method
Label
No. of target bases
Detection limit
Colorimetric detection Colorimetric detection Flatbed scanner Surface plasma resonance Quartz crystal microbalance Magnetic beads-electrochemical Magnetic beads-CL (this work) Screen-printed electrode
Gold nanoparticle Gold nanoparticle Gold nanoparticle Gold nanoparticle Gold nanoparticle Gold nanoparticle Catalytic DNAzyme HRP
30 24 27 24 24 19 30 38
50 pM [29] 500 pM [30] 50 fM [31] 10 pM [32] 32 pM [33] 15 nM [26] 100 pM 20 pM [34]
the concept to the monitoring of the breast cancer BRCA1 gene and telomere, etc. Acknowledgements The authors acknowledge National Natural Science Foundation of China (20575014), Shanghai Key Basic Research Program (05JC14010) and “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. References
Fig. 6. CL intensity vs. different DNA sequences, 100 g of magnetic beads, 100 pmol of capture DNA, 0.5 pmol of target DNA, 5 L of catalytic DNAzymemodified reporter sequence, 5 L of 0.5 mM luminol, 5 L of 0.1 M H2 O2 , the hybridization time 30 min and wash temperature set at 45 ◦ C.
sequence and BSA. Even in the presence of 10 pmol foreign DNA and 10 pmol BSA, 0.5 pmol target DNA could be selectively and sensitively detected. 4. Conclusion In conclusion, a novel and simple CL approach to DNA detection has been exemplified for a 30-base model oligonucleotide. The new method couples the high sensitivity of catalytic DNAzyme CL methods with effective discrimination against closely related nucleotide sequences. It has such characteristics as speedy, easy to use, and high sensitivity. For example, the entire assay can be completed within 2–3 h at a concentration 0.02–2 pmol target DNA, its sensitivity is equal or better than previous DNAzyme-based techniques (Table 1), and also comparable with several recently reported nanoparticle-based approaches, but the coupling of magnetic hybridization surfaces with catalytic DNAzyme CL detection eliminates complex reporter molecular labeling, and offers great promise for centralized and decentralized genetic testing. Current work is aimed at optimizing the reporter sequence and exploring the extension of
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Magnetic bead-based chemiluminescence detection of sequence-specific DNA by using catalytic nucleic acid labels Zhijuan Cao a , Zengxi Li b , Yujie Zhao a , Yumin Song a , Jianzhong Lu a,∗ a
b
School of Pharmacy, Fudan University, 138 Yixueyuan Road, Shanghai 200032, China Graduate University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China
Received 21 August 2005; received in revised form 18 October 2005; accepted 20 October 2005 Available online 28 November 2005
Abstract We wish to report a new chemiluminescence (CL) method for the determination of the sequence-specific DNA by the coupling of catalytic nucleic acid label (DNAzyme) based CL detection route with an efficient magnetic isolation of the hybrid. The assay relies on (i) the immobilization of NH2 -modified capture DNA on the surface of carboxyl-terminated magnetic beads activated by EDC, (ii) first hybridization event occurring between the bead-captured DNA probe and one 15-mer portion (5 -AAT ATT GAT AAG GAT-3 ) of the target sequence, (iii) second hybridization happening between one part of the DNAzyme modified reporter sequence and another 15-mer portion (5 -GAG GGA TTA TTG TTA-3 ) of the target sequence, and then direct detection of CL signal on the surface of the magnetic beads in a basic solution of luminol and H2 O2 . The influence of relevant experimental variables, including the effect of the amount of the magnetic beads, the duration of the hybridization and the parameters affecting CL signal, is examined and optimized. The results showed that this simple and sensitive protocol was suitable for the determination of specific-sequence DNA as exemplified by the 30-base sequence related to the anthrax lethal factor with a good linear correlation in the concentration range of 0.02–2 pmol (R2 = 0.9987) and a detection limit of 1 × 10−10 M. Overall, this new CL protocol coupled the high sensitivity of CL analysis with effective magnetic separation for discriminating against unwanted constituents such as mismatched sequences and proteins, hence, offers great promise for DNA hybridization analysis. © 2005 Elsevier B.V. All rights reserved. Keywords: Catalytic nucleic acid label; Specific sequence DNA; Chemiluminescent; Magnetic beads
1. Introduction The polymerase chain reaction (PCR) and other forms of target amplification have enabled rapid advances in the development of powerful tools for detecting and quantifying sequencespecific DNA of interest for research, forensic and clinical applications [1–4]. Many detection techniques have been developed, and their quantifications are generally achieved by measuring the specific activity of the label linked to the capture probe, in which enzymes are often employed as biocatalytic labels for the amplified detection of DNA-sensing events. In this context, electrochemical detection of hybridization can be accomplished by the application of oligonucleotide-functionalized redox enzymes as bioelectrocatalytic probes for the formation of the double-stranded DNA complex [5] or the selective associ-
∗
Corresponding author. Tel.: +86 21 54237587; fax: +86 21 54237587. E-mail address: [email protected] (J. Lu).
0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.10.048
ation of enzymes to the double-stranded oligonucleotide-DNA assembly, the amplified detection of DNA by the application of rotating functional magnetic particles [6] and the subsequent biocatalyzed precipitation of an insoluble product on the transducers for the oligonucleotide-DNA recognition event [7], etc. Although large signal amplification can be achieved, labeling of the oligonucleotide by enzyme is not a straightforward route, and once prepared, the activity of the conjugate must be periodically controlled owing to the inherent poor stability of the enzymes. In addition, the preparation of DNA-based enzymes attracts substantial research efforts directed to the development of novel biocatalysts [8–13], and many catalytic deoxyribozymes for numerous chemical transformations were prepared in recent years [14,15]. An interesting example of a catalytic nucleic acid sequence that reveals peroxidase-like activity includes a guanine-rich nucleic acid (aptamer) that can folds into a Gquadruplex structure and binds hemin into a supramolecular catalytic configuration [16–18]. The use of nucleic acids as cat-
Z. Cao et al. / Analytica Chimica Acta 557 (2006) 152–158
alytic labels may reveal two advantages: (i) the use of noncomplementary negatively charged nucleic acid labels may reduce the nonspecific adsorption caused by proteins to the nucleic acid interfaces; (ii) the base sequence in the nucleic acid label may be tailored to include the catalytic DNAzyme domain and a nucleic acid domain that is complementary and hybridizes with the target DNA. Based on this fact, Xiao et al. [19,20] reported on the use of a catalytic nucleic acid (DNAzyme) as a label for the homogeneous detection of DNA with a detection limit of 2 × 10−7 M and Pavlov et al. [21] developed a DNAzyme-based heterogeneous detection of DNA with a detection limit of 1 × 10−9 M. Further lowering of the detection limit into 1 × 10−10 M could be obtained in connection with a complicated DNAzyme-modified gold nanoparticle amplification system [22], which enabled sensitive detection of telomerase activity originating from 1000 HeLa cells. These DNAzyme-based systems are sensitive. However, they deliver results much slower than previously reported optical and electrochemical techniques [23,24], i.e. the entire assay normally takes around 30 h to reach a detection limit of 1 × 10−9 or 1 × 10−10 M. Still, a desired yet unapproached goal is to develop a simple and sensitive DNAzyme-based method for the detection of DNA hybridization events with significantly shorter assay time. Magnetic nanoparticles as special biomolecule immobilizing carrier offer a promising alternative to conventional methodology. Magnetic nanoparticles have been used in immunoassays, enzyme, DNA and protein immobilization, DNA purification, and magnetically controlled transport of anticancer drugs [25]. Wang et al. [26] developed a new metal nanoparticlebased electrochemical stripping potentiometric detection of DNA hybridization. Recently, we [27,28] used magnetic beads for DNA hybridization detection assay and also reported a new oxidative gold metal dissolution-based CL method for the noncompetitive immunoassay of a human immunoglobulin G by taking advantage of a magnetic separation/mixing process and the amplification feature of colloidal gold label. In this article, we describe a new CL method for the detection of the specific DNA sequence by the coupling of DNAzyme CL detection route with an efficient magnetic isolation of the hybrid. Compared with previous DNAzyme-based methods, our new method has such characteristics as speedy, easy to use, and high sensitivity. The entire assay can be completed within 2–3 h at a concentration 0.02–2 pmol and a detection limit of 1 × 10−10 M for the recognition of 30-base model oligonucleotide.
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Amino-modified capture probe: 5 -NH2 -(A)20 ATC CTT ATC AAT ATT-3 ; sequence X: 5 -NH2 -(A)20 GGG TTA GGGTTA GGG TTA GGG-3 ; 30-base target DNA sequence associated with the anthrax lethal factor: 5 -GAG GGA TTA TTG TTA AAT ATT GAT AAG GAT -3 ; A–A single-base mismatched target: 5 -GAG GGA TTA TTG TTA AAT ATT GAA AAG GAT-3 ; A–G single-base mismatched target: 5 -GAG GGA TTA TTG TTA AAT ATT GAG AAG GAT-3 ; A–C single-base mismatched target: 5 -GAG GGA TTA TTG TTA AAT ATT GAC AAG GAT-3 ; Two-base-mismatched target: 5 -GAG GGA TTA TTG TTA AAT ATT GAA TAG GAT-3 ; Noncomplementary sequence: 5 -TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG TTA GGG-3 ; and the reporter sequence: 5 -TAA CAA TAA TCC CTC TTT GGG TAG GGC GGG TTG GG-3 . 2.2. Apparatus CL measurements were performed with a BPCL chemiluminescence analyzer (Beijing, China). 2.3. Preparation of DNAzyme-modified reporter sequence The DNAzyme-modified reporter sequence is composed of two parts. One part is the catalytic nucleic acid label (5 -TTT GGG TAG GGC GGG TTG GG-3 , which complexes with hemin), and another part is 5 -TAA CAA TAA TCC CTC that can hybridize with one 15-mer portion (5 -GAG GGA TTA TTG TTA-3 ) of the target sequence. The DNAzyme-modified reporter sequence was prepared as follows. A solution of 2.5 M reporter sequence was heated at 95 ◦ C for 9 min in 0.01 M Tris buffer, pH 7.4, to dissociate any intermolecular quadruplex, and allowed to cool to room temperature. An identical volume of a buffer solution consisting of 50 mM HEPES, 40 mM KCl, 400 mM NaCl, 0.1% Triton X-100, and 2% DMSO, pH 7.4, was added to the reporter sequence solution to allow appropriate folding. Hemin, 1.2 M, was added to the nucleic acid solution, and the system was allowed to form the supramolecular complex for 3 h at room temperature.
2. Experimental 2.4. Assay procedures 2.1. Materials and reagents All chemicals were of analytical grade and were used as received. All stock solutions were prepared using deionized and autoclaved water. The carboxyl-modified magnetic beads were obtained from Bangs Lab. Hemin and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) were purchased from Sigma. Oligonucleotides were acquired from Invitrogen Biotechnology Co., Ltd (Shanghai, China) and had the following sequences:
In a typical experiment, 50 L of the carboxyl-modified magnetic beads were transferred into a microtube, where the beads were washed three times with 100 L of 0.1 M imidazole buffer (pH 7.0) and resuspended in 1 mL of 0.1 M imidazole buffer containing 0.1 M EDC for 30 min with gentle shaking. The activated magnetic beads were divided equally into ten wells (100 L/well) of a 96-well microplate, and then 100 pmol of capture DNA were added into each well and incubated for 30 min at 37 ◦ C with gentle shaking. The bead-captured probes were
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Fig. 1. Schematic representation of the analytical protocol: (a) the immobilization of NH2 -modified capture DNA on the surface of carboxyl terminated magnetic beads activated by EDC; (b) the first hybridization event between capture DNA and target DNA; (c) the second hybridization event with the catalytic DNAzyme-modified reporter sequence, and direct detection of hybrid DNA CL signal on the surface of magnetic beads in a basic luminol-H2 O2 solution.
magnetically separated and the supernatants were aspirated and discarded. Next, the beads were washed three times with 100 L of 10 mM pH 7.0 phosphate buffer containing 0.1 M NaCl and once with buffer A (pH 8.0 of 20 mM Tris–HCl buffer containing 0.5 M NaCl). The desired amount of the target DNA and 5 L of the DNAzyme-modified reporter sequence were added simultaneously into each well and then incubated for 30 min in buffer A at 37 ◦ C. The hybrid-conjugated beads were then washed twice with wash buffer B (7 mM Tris–HCl, pH 8.0, 0.17 M NaCl and 0.05% Tween 20), once with wash buffer C (7 mM Tris–HCl, pH 8.0, 0.17 M NaCl) and then once with buffer D (25 mM HEPES, 20 mM KCl and 200 mM NaCl, pH 9.0) at 37 ◦ C. Afterwards the hybrid-conjugated beads were resuspended in 90 L of buffer D and transferred into 14 mm × 40 mm glass tubes, 5 L of 0.5 mM luminol was added and the tubes were placed in the luminescence reader. Finally, 5 L of 0.1 M H2 O2 was injected and the CL signal was displayed.
3. Results and discussion The present assay format (illustrated in Fig. 1) involves three steps: (1) capture of NH2 -modified DNA probe on the surface of carboxyl terminated magnetic beads activated by EDC, (2) the first hybridization event occurring between the bead-captured DNA probe and one 15-mer portion (5 -AAT ATT GAT AAG GAT-3 ) of the target sequence; (3) the second hybridization event happening between one part of the catalytic DNAzymemodified reporter sequence and another 15-mer portion (5 -GAG GGA TTA TTG TTA-3 ) of the target sequence and magnetic removal of non-hybridized DNA sequences. Afterwards, direct detection of catalytic DNAzyme CL takes place on the surface of the magnetic beads in a basic luminol-H2 O2 solution. Note that CL emission will be emitted only if the catalytic DNAzymemodified reporter sequences are associated with the surface of magnetic beads, and this will occur only provided that the target
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Fig. 2. CL ratio vs. the amount of magnetic beads, 100 pmol of capture DNA, 0.5 pmol of target DNA, 5 L of catalytic DNAzyme modified reporter sequence, 5 L of 0.5 mM luminol, 5 L of 0.1 M H2 O2 and the hybridization time 30 min.
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Fig. 3. CL ratio vs. the amount of capture probe DNA, 100 g of magnetic beads, 0.5 pmol of target DNA, 5 L of catalytic DNAzyme-modified reporter sequence, 5 L of 0.5 mM luminol, 5 L of 0.1 M H2 O2 and the hybridization time 30 min.
DNA is hybridized with the capture DNA immobilized on the surface of magnetic beads through EDC. Several parameters were optimized systematically for the catalytic DNAzyme-based CL detection of specific sequence of DNA, including the amounts of magnetic beads, capture DNA and DNAzyme-modified reporter sequence, and the hybridization time, etc. 3.1. Effect of the amount of magnetic beads As shown in Fig. 2, with the increasing of the amount of magnetic beads, the hybridization CL signal ratio (the target signal/background) increased at first, leveled off between 50 and 200 g and then decreased quickly. Hence, subsequent work employed 100 g beads. 3.2. Effects of the amounts of capture DNA and catalytic reporter sequence The effects of the amounts of capture DNA and reporter sequence were examined and optimized, respectively. First, the CL signal ratio was observed to be increased in the range of 10–100 pmol of capture DNA and then decreased greatly, thus 100 pmol of capture DNA was selected for the following experiments (Fig. 3). Second, the CL and the background signal were increased with the increasing of the DNAzyme-modified reporter sequence, but the CL ratio was the highest by using 5 L of catalytic DNAzyme-modified reporter sequence, which was then selected for the use in further studies. The coverage of capture probes on the magnetic particles was determined by employing guanine-specific TMPG CL reaction [27] and the sequence X (5 -NH2 (A)20 GGGTTAGGGTTAGGGTTAGGG-3 ) was thus used as the capture DNA to react with carboxyl-modified magnetic particles through 0.1 M EDC. CL intensities of five different concentration of sequence X (4, 20, 50, 100, 150 pmol) before and after being coupled on the surface of magnetic beads were
Fig. 4. CL ratio vs. [luminol] (a) and CL ratio vs. [H2 O2 ] (b), 5 L of 0.1 M H2 O2 (a) and 5 L of 0.5 mM luminol (b), 100 g of magnetic beads, 100 pmol of capture DNA, 0.5 pmol of target DNA, 5 L of catalytic DNAzyme-modified reporter sequence and the hybridization time 30 min.
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Table 1 Comparison of sensitivity and assay for DNAzyme-based detection methods Analytical method
No. of target bases
Assay time (h)
Detection limit
Homogeneous UV Homogeneous CL Au surface CL Au surface CL-Au NPs amplification Magnetic beads-CL (this work)
35 36 36 36 30
– – >30 >24 2–3
200 nM [19] 600 nM [20] 1 nM [21] 100 pM [22] 100 pM
compared. If assuming the CL signal of the sequence X in the solution phase as 100%, the coverage of amine-terminated DNA probe on the carboxyl-modified magnetic particles was calculated to be about 11%.
of the detection limit is expected in connection with branched DNAzyme-modified reporter sequences. A series of seven repetitive measurements of 0.5 pmol of target DNA, used for estimating the precision, yielded reproducible signals with a relative standard deviation of 5.0%.
3.3. Effects of luminol and H2 O2 concentrations In addition, the concentrations of luminol and H2 O2 also affected the CL signal greatly. So these parameters were also examined and optimized (see Fig. 4). First, it was found that both signal and background were increased with increasing luminol concentration. However, signal/noise ratio was the highest at 0.5 mM luminol. Next, for H2 O2 , signal/noise ratio was increased from 0 to 0.05 M, almost constant between 0.05 and 0.1 M and then decreased sharply. Hence, 0.5 mM luminol and 0.1 M H2 O2 were selected for the following experiments. 3.4. Other parameters affecting CL signal The CL signal was almost the same when hybridizing target DNA and catalytic DNAzyme-modified reporter sequence separately or together. For convenience, the two solutions of target and reporter DNA were added simultaneously for the first and second hybridization reactions. The hybridization times have been examined. Four hybridization times of 15, 30, 60 and 90 min were studied. It was found that the CL signal ratio was almost constant by using a longer hybridization time than 30 min (see Fig. 5). Hence, 30 min of hybridization time was selected for the following experiments.
3.6. Discrimination of complementary DNA sequence from mismatched ones The new protocol provided a good capability in discriminating two target DNA sequences that differ by one base mismatch such as A–A mismatch, A–G mismatch, A–C mismatch; two base mismatch and noncomplementary sequence upon DNA hybridization. After that, the resultants were washed with wash buffer A twice, wash buffer B once and buffer C once at 37, 45 or 55 ◦ C, respectively. It was found that the wash temperature has a great effect on the discrimination efficiency of single base-mismatch. Single base-mismatch could not be discriminated at 37 ◦ C whereas perfectly complementary target DNA was also denatured at 55 ◦ C, and thus 45 ◦ C was found to be a suitable wash temperature. As shown in Fig. 6, the CL signal of the complementary sequence was much higher than those of one base mismatched, two base mismatched and noncomplementary DNA sequence. Therefore, the proposed CL technique is particularly attractive for the CL detection of specific DNA sequences. In addition, this novel CL approach could provide a good capability in detecting selectively and sensitively target DNA in synthetic samples containing target DNA, foreign
3.5. Quantification of the target DNA The quantitative behavior was assessed by monitoring the dependence of the CL intensity upon the concentration of the target DNA. A calibration graph in the concentration range of 0.02–2.0 pmol showed a linear correlation (r2 = 0.9987) between the amount of target DNA and the CL intensity, represented by I = 798803C − 12961. In addition, at an amount of higher than 2.0 pmol, the CL intensity was slightly increased and deviated from the calibration curve. The detection limit was estimated to be 0.01 pmol, i.e., 1 × 10−10 M (0.01 pmol in the 100 L sample), which is equal or better than previous DNAzyme-based techniques but with significantly shorter assay time (Table 1). Moreover, this sensitivity is also comparable with several gold nanoparticle-based hybridization assays as reported recently for shorter DNA sequences as shown in Table 2. Further lowering
Fig. 5. CL ratio vs. the hybridization time, 100 g of magnetic beads, 100 pmol of capture DNA, 0.5 pmol of target DNA, 5 L of catalytic DNAzyme-modified reporter sequence, 5 L of 0.5 mM luminol and 5 L of 0.1 M H2 O2 .
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Table 2 Comparison of sensitivity for DNA assay methods Analytical method
Label
No. of target bases
Detection limit
Colorimetric detection Colorimetric detection Flatbed scanner Surface plasma resonance Quartz crystal microbalance Magnetic beads-electrochemical Magnetic beads-CL (this work) Screen-printed electrode
Gold nanoparticle Gold nanoparticle Gold nanoparticle Gold nanoparticle Gold nanoparticle Gold nanoparticle Catalytic DNAzyme HRP
30 24 27 24 24 19 30 38
50 pM [29] 500 pM [30] 50 fM [31] 10 pM [32] 32 pM [33] 15 nM [26] 100 pM 20 pM [34]
the concept to the monitoring of the breast cancer BRCA1 gene and telomere, etc. Acknowledgements The authors acknowledge National Natural Science Foundation of China (20575014), Shanghai Key Basic Research Program (05JC14010) and “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation. References
Fig. 6. CL intensity vs. different DNA sequences, 100 g of magnetic beads, 100 pmol of capture DNA, 0.5 pmol of target DNA, 5 L of catalytic DNAzymemodified reporter sequence, 5 L of 0.5 mM luminol, 5 L of 0.1 M H2 O2 , the hybridization time 30 min and wash temperature set at 45 ◦ C.
sequence and BSA. Even in the presence of 10 pmol foreign DNA and 10 pmol BSA, 0.5 pmol target DNA could be selectively and sensitively detected. 4. Conclusion In conclusion, a novel and simple CL approach to DNA detection has been exemplified for a 30-base model oligonucleotide. The new method couples the high sensitivity of catalytic DNAzyme CL methods with effective discrimination against closely related nucleotide sequences. It has such characteristics as speedy, easy to use, and high sensitivity. For example, the entire assay can be completed within 2–3 h at a concentration 0.02–2 pmol target DNA, its sensitivity is equal or better than previous DNAzyme-based techniques (Table 1), and also comparable with several recently reported nanoparticle-based approaches, but the coupling of magnetic hybridization surfaces with catalytic DNAzyme CL detection eliminates complex reporter molecular labeling, and offers great promise for centralized and decentralized genetic testing. Current work is aimed at optimizing the reporter sequence and exploring the extension of
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