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Cascade signal amplification for sensitive detection of cancer cell based on self-assembly of DNA scaffold and rolling circle amplification
Sensors and Actuators B 171–172 (2012) 361–366
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Cascade signal amplification for sensitive detection of cancer cell based on self-assembly of DNA scaffold and rolling circle amplification Ying Li, Yan Zeng, Xiaoting Ji, Xia Li, Rui Ren ∗ Shandong Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
a r t i c l e
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Article history: Received 18 October 2011 Received in revised form 18 April 2012 Accepted 20 April 2012 Available online 13 June 2012 Keywords: Aptamer Cancer cell Signal amplification DNAzyme Chemiluminescence
a b s t r a c t Combined with a DNA aptamer recognition element, a cascade signal amplification strategy for sensitive detection of cancer cell has been developed by coupling target triggered self-assembly of polycatenated DNA scaffold and rolling circle amplified synthesis of DNAzymes. The aptamers for Ramos cells and the complementary oligonucleotides are firstly hybridized to form duplex structure. When the target Ramos cells are introduced, the complementary oligonucleotides of the cell-specific aptamers are released and trigger the assembly of circular DNA to form polycatenated DNA scaffold. Upon recognition by a nicking endonuclease (NEase), the circular DNA templates are dissociated to form the DNA scaffold and further initiate the rolling circle amplification reaction (RCA) to form a linear array of DNAzymes which can catalyze the oxidation of luminol by H2 O2 to generate chemiluminescence signal upon binding of hemin. Using this cascade signal amplification strategy, Ramos cells can be sensitively and selectively detected. This proposed method offers a great promise for rapid, simple and sensitive analysis of biological samples. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a leading cause of death throughout the world and the critical issue for cancer diagnosis is to develop new diagnostic methods allowing one to detect the disease in an early stage [1]. So far, different techniques have been proposed for cancer diagnosis, and one of the most promising approaches is to detect biomarkers [2]. In this strategy, genetic variations as well as alterations in certain protein levels are mainly used as reporters of the cancer disease. Based on DNA hybridization [3–6], antigen–antibody binding [7–11] and protein–ligand interaction, a variety of methods for biomarker assay have been developed [12,13]. However, the performance of these approaches is often restricted by the variation in genetic patterns among patients and the difficulty of finding new biomarkers for certain cancer types [14]. Aptamers are single-stranded DNA or RNA oligonucleotides obtained through SELEX (systematic evolution of ligands by exponential enrichment) [15,16]. In comparison with traditional molecular recognition elements, aptamers offer several advantages such as simple synthesis, easy labeling and good stability. Additionally, they can bind to a wide range of targets with high affinity and specificity, including small molecules, proteins, and even whole
∗ Corresponding author. Tel.: +86 532 84022946; fax: +86 532 84022750. E-mail address: [email protected] (R. Ren). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.04.060
cells [17–20]. Several aptamers with high specificity toward target cancer cell lines have been selected using tumor cell SELEX and used to detect cancer cells by optical imaging [21,22]. Tan’s group developed an aptamer–nanoparticle strip biosensor for cancer cells detection. The biosensor was capable of detecting 4000 Ramos cells by visual judgment and 800 Ramos cells with a portable strip reader [23]. Because the interaction between aptamer and its target is stronger than that between the aptamer and its complementary oligonucleotide [24,25], replacement of the complementary oligonucleotide by the target cell is facile, which leads to the transduction of aptamer–cancer cell interaction into DNA-based signal amplification assay. Comparing to cells and other biological samples, DNA is more stable and can easily participate into the amplification reaction such as PCR, RCA (rolling circle amplification) [26–31], strand-displacement polymerization, etc. Thus the detected signals can be significantly enhanced and minute amount of target can be detected. By exploiting DNA’s structural features and powerful basepairing rules, the self-assembly of one-, two- and threedimensional DNA nanostructures has attracted substantial research efforts [32,33]. Based on self-assembly of aptamers and a large amount of circular DNAs, Willner and co-workers constructed DNA nanowires and achieved enhanced biocatalytic cascade on the DNA scaffold [34]. Bi et al. introduced a method for single-nucleotide polymorphisms (SNPs) analysis and thrombin assay using mechanically interlocked DNA nanostructures [35].
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In this article, we developed a novel polycatenated DNA scaffold for amplified detection of cancer cell. When the cancer cells were recognized by the DNA aptamer, the complementary oligonucleotides of the aptamer could be released and trigger the self-assembly of the polycatenated DNA scaffold by hybridization in the presence of linker DNA and circular DNA. In the subsequent process of RCA, a lot of circular DNA molecules could be used as the templates compared to traditional methods in which one target could only initiate the formation of one circular template. As a result, the chemiluminescence signals could be greatly enhanced due to the large amount of DNAzymes formed by RCA. By the self-assembly of the polycatenated DNA scaffold and rolling circle amplification, a cascaded signal amplification method has been developed, indicating the wide applicability of the new paradigm for DNA assembly, biocatalytic cascade and sensitive biomolecule detection. 2. Experimental 2.1. Materials and apparatus 2.1.1. Materials Microbeads coated with thiol groups (SH-MBs, particle size: 3.0–4.0 m) were purchased from Tianjin BaseLine ChroTechResearch Center (China). NEase (Nt.BstNBI), T4 DNA ligase, Phi29 DNA polymerase, Rsa I restriction enzyme and dNTPs were obtained from New England Biolabs. Hydrogen tetrachloroaurate (III) tetrahydrate (HAuCl4 ·4H2 O), tri(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), luminol standard powder and hemin were ordered from Sigma–Aldrich. Analytical grade H2 O2 was purchased from Shanghai Chemical Reagent Company (Shanghai, China). A luminol stock solution (1.0 × 10−2 M) was prepared by dissolving it in NaOH solution (0.1 M) and stored in dark. A hemin stock solution (5.0 × 10−3 M) was prepared in DMSO and stored in dark at −20 ◦ C. Other chemicals employed were of analytical reagent grade and were used without further purification. Double distilled water was used throughout the experiments. All oligonucleotides used in the present study were synthesized and purified by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (China), and the sequences were listed in Table S1 (see supplementary materials). 2.1.2. Apparatus The CL measurements were performed with a BPCL ultraweak luminescence analyzer (Institute of Biophysics Academic Sinica, Beijing, China). A Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan) equipped with 1 cm quartz cells was used for fluorescence measurements. Gel electrophoresis was carried out on the EPS300 electrophoretic apparatus (Shanghai Tanon Science & Technology Co., Ltd). The CL spectrum was measured on a model FL 4600 spectrofluorometer (HITACHI) with the excitation light source being turned off.
2.3. Cancer cell culture The cells (Ramos cells and HeLa cells) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin–streptomycin, followed by culturing cells in a humidified atmosphere (95% air and 5% CO2 ) at 37 ◦ C. The cell density was counted on a hemocytometer prior to each experiment. After that, 1.0 mL suspension of ∼8.0 × 106 cells dispersed in RPMI 1640 cell media buffer was centrifuged at 3500 rpm for 5 min, then it was washed with phosphate buffer solution (0.1 M, pH 7.4) for three times and resuspended in 1.0 mL cell media buffer [38,39].
2.4. Preparation of capture probe modified MB-AuNPs The capture DNA (S1 or S7) was immobilized on the surface of MB-AuNPs through Au S bonds. Briefly, thiol group modified capture DNA (10 nmol) was added to 100 L of the prepared MB-AuNPs and incubated for 24 h. The prepared capture DNA modified MBAuNPs were washed thrice with PBS (200 L, 0.01 M), resuspended in PBS (200 L) and stored at 4 ◦ C for future use.
2.5. Circularization of DNA template The padlock probe (S4, 10 nmol) and its complementary oligonucleotide (S5, 10 nmol) were mixed in ligation buffer (20 L, 50 mM Tris–HCl, pH 7.5, 10 mM MgCl2 and 1 mM ATP) and incubated at 55 ◦ C for 30 min. Then, 0.5 L T4 DNA ligase (40 U L−1 ) was added and incubated at 22 ◦ C for 1 h. After ligation, T4 DNA ligase was inactivated by heating the reaction mixture at 65 ◦ C for 10 min. The resulting mixture could be used directly or stored at 4 ◦ C [40].
2.6. Cancer cell assay For cancer cell assay, a duplex probe was prepared by hybridizing Ramos cell aptamer (S1, 1 nmol) and its complementary oligonucleotide (S2, 1 nmol) for 1 h at 37 ◦ C. In the presence of target cells, S2 was released and reacted with the capture probe (S3), circular DNA templates (10 nmol, prepared by S4 and S5) and linker strands (S6, 10 nmol) to form polycatenated DNA scaffold. After magnetic separation, the NEBuffer (50 L) and NEase (1 L, 10 U L−1 ) were added. After standing for 1 h at 55 ◦ C, the resulting mixtures were heated at 80 ◦ C for 20 min to inactivate the NEase. The released circular DNA templates were captured by S7 (1 nmol) modified on MB-AuNPs and used for RCA. In detail, after magnetic separation, phi29 DNA polymerase (0.5 L, 10 U L−1 ) and dNTPs (5 L, 1 mM) were added, then the complex was incubated at 37 ◦ C for 60 min and the resulting mixture was incubated at 65 ◦ C for 10 min to inactivate the Phi29 polymerase, followed by the addition of hemin (1.0 × 10−7 M) to form DNAzymes.
2.7. CL measurements 2.2. Preparation of MB-AuNPs Au nanoparticles (AuNPs) were synthesized by the reduction of tetrachloroauric acid (HAuCl4 ) with trisodium citrate which has been reported previously [36]. The final AuNPs have an average diameter of ∼20 nm as measured by TEM. Gold nanoparticles functionalized magnetic microbeads (MB-AuNPs) were obtained by capping the synthesized AuNPs on the surface of SH-MBs through Au S bonds according to our previously reported method [37].
After luminol (5.0 × 10−4 M) was added to the above MBAuNPs–DNAzymes complex, the CL reaction was triggered by injecting 100 L of H2 O2 (5.0 × 10−3 M) with a syringe through a septum after the CL analyzer began to record at 10 s. The kinetics curve of CL signals between 0 and 60 s was recorded and the peak heights of the emission curves were measured as the chemiluminescence intensities. The CL spectrum was shown in Supplementary materials (Fig. S5).
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Scheme 1. Schematic representation of cascade signal amplification strategy for the chemiluminescence detection of cancer cell.
3. Results and discussion
3.2. Verification of the formation of the polycatenated DNA scaffold
3.1. The overall concept of strategy As shown in Scheme 1, equivalent aptamers (S1) for Ramos cell and the partially complementary oligonucleotides (S2) were first hybridized to form rigid duplexes. In the presence of the target cells, the aptamers (S1) bound to the cancer cells and the single-stranded DNAs (S2) were released and then hybridized with the capture DNA (S3) immobilized on the surface of gold nanoparticles functionalized magnetic microbeads (MB-AuNPs). When the circular DNA templates (formed by the padlock DNA S4 and its partly complementary sequence S5) and the linker strands (S6) were introduced, a polycatenated DNA scaffold could be formed and recognized by a nicking endonuclease (NEase). Unlike restriction enzymes, the NEase can recognize specific nucleotide sequences in doublestranded DNA and cleave only one of the two strands. After the nicking on the polycatenated DNA scaffold, the circular DNAs were released and could be used as the templates of RCA. The circular DNA contained three enzyme information sequences, each complementary to the DNAzyme, so a linear array of DNAzymes could be generated by polymerase-mediated RCA process on MB-AuNPs. The DNAzyme is a G-quadruplex nucleic acid sequence, mimicking the functions of peroxidase upon binding of hemin and can stimulate the generation of chemiluminescence (CL) in the presence of luminol and H2 O2 . Thus, each cancer cell can induce the assembly of multiple circular DNA molecules and the generation of a large number of DNAzymes. This cascade signal amplification strategy leads to an extremely low limit of detection for cancer cell and creates positive influence for the detection of disease in an early stage.
In order to verify the formation of the polycatenated DNA scaffold, we investigated the flow chemiluminescence of the system in the absence and presence of the linker strand (S6). For simplification, the complementary sequence (S2) of the Ramos cell aptamer (S1) was used as the target DNA. As shown in Fig. 1A, in the absence of S2, the circular DNA templates could not hybridize with the capture DNA (S3) immobilized on the surface of MB-AuNPs and the DNAzymes could not be produced, so the CL intensity was very weak (Fig. 1A, curve a). In the presence of S2 (1.0 × 10−14 M) and in the absence of linker strand (S6), the circular DNA templates could be captured by S3 but the polycatenated DNA scaffold could not be formed. The CL intensity was slightly higher than above (Fig. 1A, curve b). When the target DNA (S2) and the linker strand (S6) were both introduced, the CL intensity was greatly increased (Fig. 1A, curve c), indicating the formation of the polycatenated DNA scaffold. As further evidence, the fluorescence response was investigated using FAM-labeled linker strand (S8). From Fig. 1B, it could be seen that the fluorescence intensity of the solution containing 1.0 × 10−7 M S8 at 520 nm was very high when excited at 490 nm (Fig. 1B, curve c). After being used to form the polycatenated DNA scaffold by hybridization of 1.0 × 10−8 M S3 immobilized on MB-AuNPs, 1.0 × 10−8 M S2, 1.0 × 10−7 M circular DNA templates and 1.0 × 10−7 M S8, the fluorescence intensity of the supernatant was decreased greatly (Fig. 1B, curve b). The results meant that most of the linker strands were consumed to form the DNA scaffold.
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Fig. 2. Agrose gel electrophoresis images of the RCA products in cancer cell detection with the concentration of Ramos cell at 10,000 cells mL−1 (lane 1). Lane 2 is the control experiment in the absence of target cells. The marker is indicated by M.
substrates or inactivation of the phi29 DNA polymerase. Thus, 60 min was selected as the optimum time for the RCA reaction. Fig. 1. (A) CL intensities of luminol–H2 O2 system (a) in the absence of S2; (b) in the presence of 1.0 × 10−14 M S2 and in the absence of S6; (c) in the presence of 1.0 × 10−14 M S2 and 1.0 × 10−7 M S6. Experimental conditions: luminol, 5.0 × 10−4 M; H2 O2 , 5 × 10−3 M; hemin, 1.0 × 10−7 M. (B) Fluorescence intensities of (a) PBS buffer; (b) the supernatant separated from the mixture of 1.0 × 10−8 M S3 modified MB-AuNPs, 1.0 × 10−8 M S2, 1.0 × 10−8 M circular DNA templates and 1.0 × 10−7 M S8 (after hybridized for 1 h to form the polycatenated DNA scaffold); (c) 1.0 × 10−7 M FAM-labeled linker strand (S8).
3.3. Characterization of the RCA products Moreover, the products with high molecular weight generated through the RCA process were verified by agarose gel electrophoresis experiments as shown in Fig. 2. In the presence of 500 L of 10,000 cells mL−1 targets yielded RCA products with extremely low mobility (lane a), indicating the high efficiency of RCA reaction. When S10 (5 -CCC AAC CCG ATC TAC-3 ) was introduced and hybridized with the RCA products, multiple equivalent duplex structures were formed. These duplex regions were designed to contain an Rsa I recognition site, and the RCA product was further confirmed by the presence of correctly sized fragments upon restriction digestion of assay products (lane b). Whereas no RCA product was obtained in the absence of Ramos cell (lane c).
3.5. Sensitivity and selectivity The sensitivity of the assay for cancer cell was determined using cultured Ramos cell samples. As shown in Fig. 3, the CL intensity of the luminol–H2 O2 CL system catalyzed by HRP-mimicking DNAzyme increased with the increase of the target cell concentrations in the range of 200–10,000 cells mL−1 . The liner range was from 200 to 1000 cells mL−1 with a correlation coefficient of 0.991. These results indicated that this method could detect low amounts of target cells from a given sample. The detection limit of 137 cells mL−1 was estimated using 3. A series of eleven repetitive measurements of 1000 cells mL−1 target cells were used for
3.4. Optimization of the reaction time for RCA Considering that the CL response was dependent upon the amount of the DNAzymes formed by the RCA products, a long RCA reaction time was expected to generate more complementary copies of the circular template for yielding enhanced signal. Fig. S1 (see supplementary materials) depicted the effect of RCA reaction time on the CL intensity. It could be observed that the CL intensity increased rapidly with the RCA reaction time up to 60 min and became saturated over 60 min due to the exhaustion of RCA
Fig. 3. The calibration curve of peak height versus the concentration of target cells from 200 to 10,000 cells mL−1 . Inset is the amplification of the linear range from 200 to 1000 cells mL−1 for target cells determination.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2012.04.060. References
Fig. 4. CL intensities for the specificity analysis (a) with no cells; (b) with 1000 cells mL−1 K562 cells; (c) with 1000 cells mL−1 HeLa cells; (d) with 200 cells mL−1 Ramos cells; (e) with 1000 cells mL−1 Ramos cells.
estimating the precision, and the relative standard deviation (RSD) was 6.3%, showing good reproducibility. In a further set of experiments, the selectivity of the above designed strategy was investigated. As shown in Fig. 4, a significant increase of the CL intensity induced by the target cell (200 cells mL−1 and 1000 cells mL−1 of Ramos cell, Fig. 4, bars d and e) was observed compared to that of 1000 cells mL−1 of K562 cells and HeLa cells (Fig. 4, bars b and c), which was almost identical to the blank sample (Fig. 4, bar a). The excellent selectivity of the assay stems from the high specificity and affinity of cell aptamer utilized in the experiment. Finally, the cell assay in real blood samples was performed to show the applicability of the method. Ramos cells were spiked into whole blood samples from healthy adults and then detected with the assay in this study. The ratio of the measured value against the added amount of target cells was calculated as recovery rate. The recovery was found to vary from 87.5 to 97.0% (see supplementary materials, Table S2), indicating the application potential of this assay for real samples. 4. Conclusions In conclusion, the present study has introduced a new cascade signal amplification strategy for detection of cancer cell and a detection limit of 137 cells mL−1 could be achieved. This proposed method possesses several significant advantages. Firstly, aptamers can be easily selected by SELEX and synthesized conveniently. In our study, there is no need to modify or immobilize the aptamers, which has no effect on the affinity and specificity of the original aptamers. Secondly, one cancer cell can induce the generation of numerous DNAzymes by coupling the target triggered self-assembly of polycatenated DNA scaffold and rolling circle amplification, so the sensitivity can be improved significantly. Thirdly, as the RCA process is carried out on the surface of MBAuNPs, excess hemin can be removed by magnetic separation, and the high background of luminol–H2 O2 CL system can be successfully circumvented. Finally, this strategy can be readily employed to detect different types of targets (small molecule drugs, proteins) by using different aptamers, thus offering a new approach for biomolecule analysis and vitro diagnostics tests. Acknowledgment This work was supported by the Education Administration Foundation of Shandong Province (No. J09LB04).
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Biographies Ying Li received the Ph.D. degree from Nanjing University of Technology in 2011. She is also now a lecturer at Qingdao University of Science and Technology. Her research is now focused on chemiluminescence sensors for biomolecules. Yan Zeng is a postgraduate at Qingdao University of Science and Technology. Her research is now focused on chemiluminescence detection of biomolecules. Xiaoting Ji is a postgraduate at Qingdao University of Science and Technology. Her research is now focused on chemiluminescence detection of biomolecules. Xia Li is a postgraduate at Qingdao University of Science and Technology. Her research is now focused on chemiluminescence detection of biomolecules. Rui Ren received the Ph.D. degree from Nanjing University of Technology in 2011. He is also now a lecturer at Qingdao University of Science and Technology. His research is now focused on electrochemical sensors for biomolecules.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Cascade signal amplification for sensitive detection of cancer cell based on self-assembly of DNA scaffold and rolling circle amplification Ying Li, Yan Zeng, Xiaoting Ji, Xia Li, Rui Ren ∗ Shandong Provincial Key Laboratory of Biochemical Analysis, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
a r t i c l e
i n f o
Article history: Received 18 October 2011 Received in revised form 18 April 2012 Accepted 20 April 2012 Available online 13 June 2012 Keywords: Aptamer Cancer cell Signal amplification DNAzyme Chemiluminescence
a b s t r a c t Combined with a DNA aptamer recognition element, a cascade signal amplification strategy for sensitive detection of cancer cell has been developed by coupling target triggered self-assembly of polycatenated DNA scaffold and rolling circle amplified synthesis of DNAzymes. The aptamers for Ramos cells and the complementary oligonucleotides are firstly hybridized to form duplex structure. When the target Ramos cells are introduced, the complementary oligonucleotides of the cell-specific aptamers are released and trigger the assembly of circular DNA to form polycatenated DNA scaffold. Upon recognition by a nicking endonuclease (NEase), the circular DNA templates are dissociated to form the DNA scaffold and further initiate the rolling circle amplification reaction (RCA) to form a linear array of DNAzymes which can catalyze the oxidation of luminol by H2 O2 to generate chemiluminescence signal upon binding of hemin. Using this cascade signal amplification strategy, Ramos cells can be sensitively and selectively detected. This proposed method offers a great promise for rapid, simple and sensitive analysis of biological samples. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Cancer is a leading cause of death throughout the world and the critical issue for cancer diagnosis is to develop new diagnostic methods allowing one to detect the disease in an early stage [1]. So far, different techniques have been proposed for cancer diagnosis, and one of the most promising approaches is to detect biomarkers [2]. In this strategy, genetic variations as well as alterations in certain protein levels are mainly used as reporters of the cancer disease. Based on DNA hybridization [3–6], antigen–antibody binding [7–11] and protein–ligand interaction, a variety of methods for biomarker assay have been developed [12,13]. However, the performance of these approaches is often restricted by the variation in genetic patterns among patients and the difficulty of finding new biomarkers for certain cancer types [14]. Aptamers are single-stranded DNA or RNA oligonucleotides obtained through SELEX (systematic evolution of ligands by exponential enrichment) [15,16]. In comparison with traditional molecular recognition elements, aptamers offer several advantages such as simple synthesis, easy labeling and good stability. Additionally, they can bind to a wide range of targets with high affinity and specificity, including small molecules, proteins, and even whole
∗ Corresponding author. Tel.: +86 532 84022946; fax: +86 532 84022750. E-mail address: [email protected] (R. Ren). 0925-4005/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.04.060
cells [17–20]. Several aptamers with high specificity toward target cancer cell lines have been selected using tumor cell SELEX and used to detect cancer cells by optical imaging [21,22]. Tan’s group developed an aptamer–nanoparticle strip biosensor for cancer cells detection. The biosensor was capable of detecting 4000 Ramos cells by visual judgment and 800 Ramos cells with a portable strip reader [23]. Because the interaction between aptamer and its target is stronger than that between the aptamer and its complementary oligonucleotide [24,25], replacement of the complementary oligonucleotide by the target cell is facile, which leads to the transduction of aptamer–cancer cell interaction into DNA-based signal amplification assay. Comparing to cells and other biological samples, DNA is more stable and can easily participate into the amplification reaction such as PCR, RCA (rolling circle amplification) [26–31], strand-displacement polymerization, etc. Thus the detected signals can be significantly enhanced and minute amount of target can be detected. By exploiting DNA’s structural features and powerful basepairing rules, the self-assembly of one-, two- and threedimensional DNA nanostructures has attracted substantial research efforts [32,33]. Based on self-assembly of aptamers and a large amount of circular DNAs, Willner and co-workers constructed DNA nanowires and achieved enhanced biocatalytic cascade on the DNA scaffold [34]. Bi et al. introduced a method for single-nucleotide polymorphisms (SNPs) analysis and thrombin assay using mechanically interlocked DNA nanostructures [35].
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In this article, we developed a novel polycatenated DNA scaffold for amplified detection of cancer cell. When the cancer cells were recognized by the DNA aptamer, the complementary oligonucleotides of the aptamer could be released and trigger the self-assembly of the polycatenated DNA scaffold by hybridization in the presence of linker DNA and circular DNA. In the subsequent process of RCA, a lot of circular DNA molecules could be used as the templates compared to traditional methods in which one target could only initiate the formation of one circular template. As a result, the chemiluminescence signals could be greatly enhanced due to the large amount of DNAzymes formed by RCA. By the self-assembly of the polycatenated DNA scaffold and rolling circle amplification, a cascaded signal amplification method has been developed, indicating the wide applicability of the new paradigm for DNA assembly, biocatalytic cascade and sensitive biomolecule detection. 2. Experimental 2.1. Materials and apparatus 2.1.1. Materials Microbeads coated with thiol groups (SH-MBs, particle size: 3.0–4.0 m) were purchased from Tianjin BaseLine ChroTechResearch Center (China). NEase (Nt.BstNBI), T4 DNA ligase, Phi29 DNA polymerase, Rsa I restriction enzyme and dNTPs were obtained from New England Biolabs. Hydrogen tetrachloroaurate (III) tetrahydrate (HAuCl4 ·4H2 O), tri(2-carboxyethyl)phosphine hydrochloride (TCEP, 98%), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), luminol standard powder and hemin were ordered from Sigma–Aldrich. Analytical grade H2 O2 was purchased from Shanghai Chemical Reagent Company (Shanghai, China). A luminol stock solution (1.0 × 10−2 M) was prepared by dissolving it in NaOH solution (0.1 M) and stored in dark. A hemin stock solution (5.0 × 10−3 M) was prepared in DMSO and stored in dark at −20 ◦ C. Other chemicals employed were of analytical reagent grade and were used without further purification. Double distilled water was used throughout the experiments. All oligonucleotides used in the present study were synthesized and purified by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (China), and the sequences were listed in Table S1 (see supplementary materials). 2.1.2. Apparatus The CL measurements were performed with a BPCL ultraweak luminescence analyzer (Institute of Biophysics Academic Sinica, Beijing, China). A Hitachi F-4500 fluorescence spectrophotometer (Tokyo, Japan) equipped with 1 cm quartz cells was used for fluorescence measurements. Gel electrophoresis was carried out on the EPS300 electrophoretic apparatus (Shanghai Tanon Science & Technology Co., Ltd). The CL spectrum was measured on a model FL 4600 spectrofluorometer (HITACHI) with the excitation light source being turned off.
2.3. Cancer cell culture The cells (Ramos cells and HeLa cells) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin–streptomycin, followed by culturing cells in a humidified atmosphere (95% air and 5% CO2 ) at 37 ◦ C. The cell density was counted on a hemocytometer prior to each experiment. After that, 1.0 mL suspension of ∼8.0 × 106 cells dispersed in RPMI 1640 cell media buffer was centrifuged at 3500 rpm for 5 min, then it was washed with phosphate buffer solution (0.1 M, pH 7.4) for three times and resuspended in 1.0 mL cell media buffer [38,39].
2.4. Preparation of capture probe modified MB-AuNPs The capture DNA (S1 or S7) was immobilized on the surface of MB-AuNPs through Au S bonds. Briefly, thiol group modified capture DNA (10 nmol) was added to 100 L of the prepared MB-AuNPs and incubated for 24 h. The prepared capture DNA modified MBAuNPs were washed thrice with PBS (200 L, 0.01 M), resuspended in PBS (200 L) and stored at 4 ◦ C for future use.
2.5. Circularization of DNA template The padlock probe (S4, 10 nmol) and its complementary oligonucleotide (S5, 10 nmol) were mixed in ligation buffer (20 L, 50 mM Tris–HCl, pH 7.5, 10 mM MgCl2 and 1 mM ATP) and incubated at 55 ◦ C for 30 min. Then, 0.5 L T4 DNA ligase (40 U L−1 ) was added and incubated at 22 ◦ C for 1 h. After ligation, T4 DNA ligase was inactivated by heating the reaction mixture at 65 ◦ C for 10 min. The resulting mixture could be used directly or stored at 4 ◦ C [40].
2.6. Cancer cell assay For cancer cell assay, a duplex probe was prepared by hybridizing Ramos cell aptamer (S1, 1 nmol) and its complementary oligonucleotide (S2, 1 nmol) for 1 h at 37 ◦ C. In the presence of target cells, S2 was released and reacted with the capture probe (S3), circular DNA templates (10 nmol, prepared by S4 and S5) and linker strands (S6, 10 nmol) to form polycatenated DNA scaffold. After magnetic separation, the NEBuffer (50 L) and NEase (1 L, 10 U L−1 ) were added. After standing for 1 h at 55 ◦ C, the resulting mixtures were heated at 80 ◦ C for 20 min to inactivate the NEase. The released circular DNA templates were captured by S7 (1 nmol) modified on MB-AuNPs and used for RCA. In detail, after magnetic separation, phi29 DNA polymerase (0.5 L, 10 U L−1 ) and dNTPs (5 L, 1 mM) were added, then the complex was incubated at 37 ◦ C for 60 min and the resulting mixture was incubated at 65 ◦ C for 10 min to inactivate the Phi29 polymerase, followed by the addition of hemin (1.0 × 10−7 M) to form DNAzymes.
2.7. CL measurements 2.2. Preparation of MB-AuNPs Au nanoparticles (AuNPs) were synthesized by the reduction of tetrachloroauric acid (HAuCl4 ) with trisodium citrate which has been reported previously [36]. The final AuNPs have an average diameter of ∼20 nm as measured by TEM. Gold nanoparticles functionalized magnetic microbeads (MB-AuNPs) were obtained by capping the synthesized AuNPs on the surface of SH-MBs through Au S bonds according to our previously reported method [37].
After luminol (5.0 × 10−4 M) was added to the above MBAuNPs–DNAzymes complex, the CL reaction was triggered by injecting 100 L of H2 O2 (5.0 × 10−3 M) with a syringe through a septum after the CL analyzer began to record at 10 s. The kinetics curve of CL signals between 0 and 60 s was recorded and the peak heights of the emission curves were measured as the chemiluminescence intensities. The CL spectrum was shown in Supplementary materials (Fig. S5).
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Scheme 1. Schematic representation of cascade signal amplification strategy for the chemiluminescence detection of cancer cell.
3. Results and discussion
3.2. Verification of the formation of the polycatenated DNA scaffold
3.1. The overall concept of strategy As shown in Scheme 1, equivalent aptamers (S1) for Ramos cell and the partially complementary oligonucleotides (S2) were first hybridized to form rigid duplexes. In the presence of the target cells, the aptamers (S1) bound to the cancer cells and the single-stranded DNAs (S2) were released and then hybridized with the capture DNA (S3) immobilized on the surface of gold nanoparticles functionalized magnetic microbeads (MB-AuNPs). When the circular DNA templates (formed by the padlock DNA S4 and its partly complementary sequence S5) and the linker strands (S6) were introduced, a polycatenated DNA scaffold could be formed and recognized by a nicking endonuclease (NEase). Unlike restriction enzymes, the NEase can recognize specific nucleotide sequences in doublestranded DNA and cleave only one of the two strands. After the nicking on the polycatenated DNA scaffold, the circular DNAs were released and could be used as the templates of RCA. The circular DNA contained three enzyme information sequences, each complementary to the DNAzyme, so a linear array of DNAzymes could be generated by polymerase-mediated RCA process on MB-AuNPs. The DNAzyme is a G-quadruplex nucleic acid sequence, mimicking the functions of peroxidase upon binding of hemin and can stimulate the generation of chemiluminescence (CL) in the presence of luminol and H2 O2 . Thus, each cancer cell can induce the assembly of multiple circular DNA molecules and the generation of a large number of DNAzymes. This cascade signal amplification strategy leads to an extremely low limit of detection for cancer cell and creates positive influence for the detection of disease in an early stage.
In order to verify the formation of the polycatenated DNA scaffold, we investigated the flow chemiluminescence of the system in the absence and presence of the linker strand (S6). For simplification, the complementary sequence (S2) of the Ramos cell aptamer (S1) was used as the target DNA. As shown in Fig. 1A, in the absence of S2, the circular DNA templates could not hybridize with the capture DNA (S3) immobilized on the surface of MB-AuNPs and the DNAzymes could not be produced, so the CL intensity was very weak (Fig. 1A, curve a). In the presence of S2 (1.0 × 10−14 M) and in the absence of linker strand (S6), the circular DNA templates could be captured by S3 but the polycatenated DNA scaffold could not be formed. The CL intensity was slightly higher than above (Fig. 1A, curve b). When the target DNA (S2) and the linker strand (S6) were both introduced, the CL intensity was greatly increased (Fig. 1A, curve c), indicating the formation of the polycatenated DNA scaffold. As further evidence, the fluorescence response was investigated using FAM-labeled linker strand (S8). From Fig. 1B, it could be seen that the fluorescence intensity of the solution containing 1.0 × 10−7 M S8 at 520 nm was very high when excited at 490 nm (Fig. 1B, curve c). After being used to form the polycatenated DNA scaffold by hybridization of 1.0 × 10−8 M S3 immobilized on MB-AuNPs, 1.0 × 10−8 M S2, 1.0 × 10−7 M circular DNA templates and 1.0 × 10−7 M S8, the fluorescence intensity of the supernatant was decreased greatly (Fig. 1B, curve b). The results meant that most of the linker strands were consumed to form the DNA scaffold.
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Fig. 2. Agrose gel electrophoresis images of the RCA products in cancer cell detection with the concentration of Ramos cell at 10,000 cells mL−1 (lane 1). Lane 2 is the control experiment in the absence of target cells. The marker is indicated by M.
substrates or inactivation of the phi29 DNA polymerase. Thus, 60 min was selected as the optimum time for the RCA reaction. Fig. 1. (A) CL intensities of luminol–H2 O2 system (a) in the absence of S2; (b) in the presence of 1.0 × 10−14 M S2 and in the absence of S6; (c) in the presence of 1.0 × 10−14 M S2 and 1.0 × 10−7 M S6. Experimental conditions: luminol, 5.0 × 10−4 M; H2 O2 , 5 × 10−3 M; hemin, 1.0 × 10−7 M. (B) Fluorescence intensities of (a) PBS buffer; (b) the supernatant separated from the mixture of 1.0 × 10−8 M S3 modified MB-AuNPs, 1.0 × 10−8 M S2, 1.0 × 10−8 M circular DNA templates and 1.0 × 10−7 M S8 (after hybridized for 1 h to form the polycatenated DNA scaffold); (c) 1.0 × 10−7 M FAM-labeled linker strand (S8).
3.3. Characterization of the RCA products Moreover, the products with high molecular weight generated through the RCA process were verified by agarose gel electrophoresis experiments as shown in Fig. 2. In the presence of 500 L of 10,000 cells mL−1 targets yielded RCA products with extremely low mobility (lane a), indicating the high efficiency of RCA reaction. When S10 (5 -CCC AAC CCG ATC TAC-3 ) was introduced and hybridized with the RCA products, multiple equivalent duplex structures were formed. These duplex regions were designed to contain an Rsa I recognition site, and the RCA product was further confirmed by the presence of correctly sized fragments upon restriction digestion of assay products (lane b). Whereas no RCA product was obtained in the absence of Ramos cell (lane c).
3.5. Sensitivity and selectivity The sensitivity of the assay for cancer cell was determined using cultured Ramos cell samples. As shown in Fig. 3, the CL intensity of the luminol–H2 O2 CL system catalyzed by HRP-mimicking DNAzyme increased with the increase of the target cell concentrations in the range of 200–10,000 cells mL−1 . The liner range was from 200 to 1000 cells mL−1 with a correlation coefficient of 0.991. These results indicated that this method could detect low amounts of target cells from a given sample. The detection limit of 137 cells mL−1 was estimated using 3. A series of eleven repetitive measurements of 1000 cells mL−1 target cells were used for
3.4. Optimization of the reaction time for RCA Considering that the CL response was dependent upon the amount of the DNAzymes formed by the RCA products, a long RCA reaction time was expected to generate more complementary copies of the circular template for yielding enhanced signal. Fig. S1 (see supplementary materials) depicted the effect of RCA reaction time on the CL intensity. It could be observed that the CL intensity increased rapidly with the RCA reaction time up to 60 min and became saturated over 60 min due to the exhaustion of RCA
Fig. 3. The calibration curve of peak height versus the concentration of target cells from 200 to 10,000 cells mL−1 . Inset is the amplification of the linear range from 200 to 1000 cells mL−1 for target cells determination.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.snb.2012.04.060. References
Fig. 4. CL intensities for the specificity analysis (a) with no cells; (b) with 1000 cells mL−1 K562 cells; (c) with 1000 cells mL−1 HeLa cells; (d) with 200 cells mL−1 Ramos cells; (e) with 1000 cells mL−1 Ramos cells.
estimating the precision, and the relative standard deviation (RSD) was 6.3%, showing good reproducibility. In a further set of experiments, the selectivity of the above designed strategy was investigated. As shown in Fig. 4, a significant increase of the CL intensity induced by the target cell (200 cells mL−1 and 1000 cells mL−1 of Ramos cell, Fig. 4, bars d and e) was observed compared to that of 1000 cells mL−1 of K562 cells and HeLa cells (Fig. 4, bars b and c), which was almost identical to the blank sample (Fig. 4, bar a). The excellent selectivity of the assay stems from the high specificity and affinity of cell aptamer utilized in the experiment. Finally, the cell assay in real blood samples was performed to show the applicability of the method. Ramos cells were spiked into whole blood samples from healthy adults and then detected with the assay in this study. The ratio of the measured value against the added amount of target cells was calculated as recovery rate. The recovery was found to vary from 87.5 to 97.0% (see supplementary materials, Table S2), indicating the application potential of this assay for real samples. 4. Conclusions In conclusion, the present study has introduced a new cascade signal amplification strategy for detection of cancer cell and a detection limit of 137 cells mL−1 could be achieved. This proposed method possesses several significant advantages. Firstly, aptamers can be easily selected by SELEX and synthesized conveniently. In our study, there is no need to modify or immobilize the aptamers, which has no effect on the affinity and specificity of the original aptamers. Secondly, one cancer cell can induce the generation of numerous DNAzymes by coupling the target triggered self-assembly of polycatenated DNA scaffold and rolling circle amplification, so the sensitivity can be improved significantly. Thirdly, as the RCA process is carried out on the surface of MBAuNPs, excess hemin can be removed by magnetic separation, and the high background of luminol–H2 O2 CL system can be successfully circumvented. Finally, this strategy can be readily employed to detect different types of targets (small molecule drugs, proteins) by using different aptamers, thus offering a new approach for biomolecule analysis and vitro diagnostics tests. Acknowledgment This work was supported by the Education Administration Foundation of Shandong Province (No. J09LB04).
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Biographies Ying Li received the Ph.D. degree from Nanjing University of Technology in 2011. She is also now a lecturer at Qingdao University of Science and Technology. Her research is now focused on chemiluminescence sensors for biomolecules. Yan Zeng is a postgraduate at Qingdao University of Science and Technology. Her research is now focused on chemiluminescence detection of biomolecules. Xiaoting Ji is a postgraduate at Qingdao University of Science and Technology. Her research is now focused on chemiluminescence detection of biomolecules. Xia Li is a postgraduate at Qingdao University of Science and Technology. Her research is now focused on chemiluminescence detection of biomolecules. Rui Ren received the Ph.D. degree from Nanjing University of Technology in 2011. He is also now a lecturer at Qingdao University of Science and Technology. His research is now focused on electrochemical sensors for biomolecules.