Triggered Polycatenated DNA Scaffolds for DNA Sensors and

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Anal. Chem. 2010, 82, 9447–9454

Triggered Polycatenated DNA Scaffolds for DNA Sensors and Aptasensors by a Combination of Rolling Circle Amplification and DNAzyme Amplification Sai Bi, Li Li, and Shusheng Zhang* State Key Laboratory Base of Eco-chemical Engineering, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, People’s Republic of China The concept of triggered polycatenated DNA scaffolds has been elegantly introduced into ultrasensitive biosensing applications by a combination of rolling circle amplification (RCA) and DNAzyme amplification. As compared to traditional methods in which one target could only initiate the formation of one circular template for RCA reaction, in the present study two species of linear single-stranded DNA (ssDNA) monomers are self-assembled into mechanically interlocked polycatenated nanostructures on capture probetagged magnetic nanoparticles (MNPs) only upon the introduction of one base mutant DNA sequence as initiator for single-nucleotide polymorphisms (SNPs) analysis. The resultant topologically polycatenated DNA ladder is further available for RCA process by using the serially ligated circular DNA as template for the synthesis of hemin/Gquadruplex HRP-mimicking DNAzyme chains, which act as biocatalytic labels for the luminol-H2O2 chemiluminescence (CL) system. Notably, the problem of high background induced by excess hemin itself is circumvented by immobilizing the biotinylated RCA products on streptavidin-modified MNPs via biotin-streptavidin interaction. Similarly, a universal strategy is contrived by substitutedly employing aptamer as initiator for the construction of polycatenated DNA scaffolds to accomplish ultrasensitive detection of proteins based on structure-switching of aptamer upon target binding, which is demonstrated by using thrombin as a model analyte in this study. Overall, with two successive amplification steps and one magnetic separation procedure, this flexible biosensing system exhibits not only high sensitivity and specificity with the detection limits of SNPs and thrombin as low as 71 aM and 6.6 pM, respectively, but also excellent performance in real human serum assay with no PCR preamplification for SNPs assay. Given the unique and attractive characteristics, this study illustrates the potential of DNA nanotechnology in bioanalytical applications for both fundamental and practical research. Taking advantage of DNA’s remarkable molecular recognition properties and structural features, DNA nanotechnology, in which * To whom correspondence should be addressed. Phone: +86-532-84022750. Fax: +86-532-84022750. E-mail: [email protected]. 10.1021/ac1021198  2010 American Chemical Society Published on Web 10/18/2010

long, single-stranded DNA (ssDNA) molecules are folded into predetermined shapes, can be used to organize nanomaterials and self-assembled nanostructures in a programmable way.1-3 Recently, DNA nanostructures serving as templates for building materials with new functional properties have attracted increasing research attention.4-8 For example, linked ssDNA rings were synthesized by simultaneously closing the ends of two linear oligonucleotides entwined through a central complementary region by T4 DNA ligase.9 Moreover, also with the assistant of T4 ligase, a polycatenated DNA scaffold was fabricated with two kinds of linear oligonucleotides containing a domain of ssDNA sequence as the template for the circularization of the other, which were available for further hierarchical self-assembly.10 Although substantial efforts have been achieved in the fabrication of proteinDNA and nanoparticle-DNA nanostructures,10-13 the design of functions that derive from the hybrid architectures for bioanalytical application is still a significant challenge in nanotechnology. As an essential aspect of bioanalysis, amplification has been successfully achieved by employing enzymes, nanoparticles, or nanocontainers as amplifiers for sensitive detection of biorecognition events.14-16 Among these efforts, the amplified detection of DNA, especially single-nucleotide polymorphisms (SNPs), is (1) Aldaye, F. A.; Palmer, A. L.; Sleiman, H. F. Science 2008, 321, 1795–1799. (2) Gu, H.; Chao, J.; Xiao, S.-J.; Seeman, N. C. Nature 2010, 465, 202–205. (3) Lund, K.; Manzo, A. J.; Dabby, N.; Michelotti, N.; Johnson-Buck, A.; Nangreave, J.; Taylor, S.; Pei, R.; Stojanovic, M. N.; Walter, N. G.; Winfree, E.; Yan, H. Nature 2010, 465, 206–210. (4) Li, Y.; Tseng, Y. D.; Kwon, S. Y.; D’Espaux, L.; Bunch, J. S.; Mceuen, P. L.; Luo, D. Nat. Mater. 2004, 3, 38–42. (5) Aldaye, F. A.; Lo, P. K.; Karam, P.; Mclaughlin, C. K.; Cosa, G.; Sleiman, H. F. Nat. Nanotechnol. 2009, 4, 349–352. (6) Ackermann, D.; Schmidt, T. L.; Hannam, J. S.; Purohit, C. S.; Heckel, A.; Famulok, M. Nat. Nanotechnol. 2010, 5, 436–442. (7) Wilner, O. I.; Henning, A.; Shlyahovsky, B.; Willner, I. Nano Lett. 2010, 10, 1458–1465. (8) Wang, C.; Huang, Z.; Lin, Y.; Ren, J.; Qu, X. Adv. Mater. 2010, 22, 2792– 2798. (9) Billen, L. P.; Li, Y. Bioorg. Chem. 2004, 32, 582–598. (10) Weizmann, Y.; Braunschweig, A. B.; Wilner, O. I.; Cheglakov, Z.; Willner, I. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 5289–5294. (11) Wilner, O. I.; Weizmann, Y.; Gill, R.; Lioubashevski, O.; Freeman, R.; Willner, I. Nat. Nanotechnol. 2009, 4, 249–254. (12) Cheng, W.; Campolongo, M. J.; Cha, J. J.; Tan, S. J.; Umbach, C. C.; Muller, D. A.; Luo, D. Nat. Mater. 2009, 8, 519–525. (13) Wang, Z.-G.; Wilner, O. I.; Willner, I. Nano Lett. 2009, 9, 4098–4102. (14) Pavlov, V.; Xiao, Y.; Gill, R.; Dishon, A.; Kotler, M.; Willner, I. Anal. Chem. 2004, 76, 2152–2156. (15) Bi, S.; Hao, S.; Li, L.; Zhang, S. Chem. Commun. 2010, 46, 6093–6095.

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in continuous demand, which is believed to be a promising tool for early diagnosis, risk assessment of malignancy, and forensic applications.17-19 Polymerase chain reaction (PCR) provides a common technique for amplified detection of DNA.20 Although the PCR-based strategy in principle offers the most versatile method to detect minute amounts of DNA with extremely high sensitivity and wide quantitative dynamic range, the disadvantages such as complicated procedures and timing consuming limit the application of this technique for the diagnostic. Alternatively, rolling circle amplification (RCA) has been demonstrated to be a novel tool for amplified assay.21-24 In a RCA process, long ssDNA products that consisted of several hundred tandem repeats of the complementary sequence to the circular DNA template are yielded, which can serve as the detection sites.25-27 As compared to PCR, this technique exhibits several distinct advantages such as the isothermal amplification procedure and the linear kinetic model. Moreover, because the circular template can be synthesized from a padlock probe, whose 5′ and 3′ ends can hybridize precisely onto the target and then be ligated by a DNA ligase, RCA offers an exquisite strategy for highly sensitive and specific SNPs typing assay due to its stringent requirement of ligation on strand matching and high amplification efficiency.28-30 Meanwhile, on the basis of modern biotechnology and nanotechnology, considerable research activities have been attracted for the development of sensitive methods by using DNAzymes.31 DNAzymes are catalytic nucleic acids, which are isolated from random-sequence nucleic acid libraries by an in vitro selection process.32 Considering the advantages of good stability and easy synthesis over native enzymatic labels, DNAzymes find growing interest as amplifying labels for biosensing events.31,33-35 For example, hemin/G-quadruplex horseradish peroxide (HRP) mimicking DNAzyme was recently used as a catalytic label for the (16) Rissin, D. M.; Kan, C. W.; Campbell, T. G.; Howes, S. C.; Fournier, D. R.; Song, L.; Piech, T.; Patel, P. P.; Chang, L.; Rivnak, A. J.; Ferrell, E. P.; Randall, J. D.; Provuncher, G. K.; Walt, D. R.; Duffy, D. C. Nat. Biotechnol. 2010, 28, 595–599. (17) Patolsky, F.; Lichtenstein, A.; Willner, I. Nat. Biotechnol. 2001, 19, 253– 257. (18) Li, J.; Zhong, W. Anal. Chem. 2007, 79, 9030–9038. (19) Huang, Y.; Zhang, Y.-L.; Xu, X.; Jiang, J.-H.; Shen, G.-L.; Yu, R.-Q. J. Am. Chem. Soc. 2009, 131, 2478–2480. (20) Feng, F.; Liu, L.; Wang, S. Nat. Protoc. 2010, 5, 1255–1264. (21) Zhao, W.; Ali, M. M.; Brook, M. A.; Li, Y. Angew. Chem., Int. Ed. 2008, 47, 6330–6337. (22) Konry, T.; Hayman, R. B.; Walt, D. R. Anal. Chem. 2009, 81, 5777–5782. (23) Cheglakov, Z.; Weizmann, Y.; Basnar, B.; Willner, I. Org. Biomol. Chem. 2007, 5, 223–225. (24) Cheng, Y.; Zhang, X.; Li, Z.; Jiao, X.; Wang, Y.; Zhang, Y. Angew. Chem., Int. Ed. 2009, 48, 3268–3272. (25) Cheng, W.; Yan, F.; Ding, L.; Ju, X.; Yin, Y. Anal. Chem. 2010, 82, 3337– 3342. (26) Ali, M. M.; Li, Y. Angew. Chem., Int. Ed. 2009, 48, 3512–3515. (27) Wilner, O. I.; Shimron, S.; Weizmann, Y.; Wang, Z.-G.; Willner, I. Nano Lett. 2009, 9, 2040–2043. (28) Li, J.; Zhong, W. Anal. Chem. 2007, 79, 9030–9038. (29) Li, J.; Deng, T.; Chu, X.; Yang, R.; Jiang, J.; Shen, G.; Yu, R. Anal. Chem. 2010, 82, 2811–2816. (30) Dahl, F.; Bane´r, J.; Gullberg, M.; Mendel-Hartvig, M.; Landegren, U.; Nilsson, M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 4548–4553. (31) Willner, I.; Shlyahovsky, B.; Zayats, M.; Willner, B. Chem. Soc. Rev. 2008, 37, 1153–1165. (32) Ellington, A. D.; Szostak, J. W. Nature 1990, 346, 818–822. (33) Bi, S.; Zhang, J.; Zhang, S. Chem. Commun. 2010, 46, 5509–5511. (34) Yin, B.-C.; Ye, B.-C.; Tan, W.; Wang, H.; Xie, C.-C. J. Am. Chem. Soc. 2009, 131, 14624–14625. (35) Elbaz, J.; Moshe, M.; Shlyahovsky, B.; Willner, I. Chem.-Eur. J. 2009, 15, 3411–3418.

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colorimetric or chemiluminescent detection of DNA or enzyme activities.33-35 In addition, aptamers, a novel class of artificial DNA/RNA oligonucleotides, are selected by SELEX (systematic evolution of ligands by exponential enrichment).36 As a new-found recognition element for a variety of targets, aptamers possess numerous practical advantages over traditional protein reagents like antibodies and enzymes, such as high affinity and specificity, simple selection in vitro, convenient handling, and flexible application, making them attractive as molecular receptors and sensing elements for protein analysis, diagnostic assays, and targeted therapy applications. More importantly, because the aptamer-ligand recognition event can be easily amplified via nucleic acid sequence-based amplification strategy, a remarkably low detection limit could be obtained, which paves a new avenue for the development of aptasensors.37,38 In the present study, triggered polycatenated DNA scaffolds combining with RCA and HRP-mimicking DNAzyme as coupled amplification steps are developed for diverse amplified bioanalytical strategies. For the detection of SNPs (Scheme 1A), after “sandwiching” the target DNA between the capture DNA immobilized on carboxylated MNPs and the padlock probe, ligation reaction is carried out only when the padlock probe perfectly anneals to the target, inducing the ligase to link the 5′ and 3′ ends of the padlock probe together to form a circular. Subsequently, an ABAB-type DNA copolymer is one-step self-assembled on the surface of MNPs via hybridizing with a ssDNA domain of padlock probe. The polycatenated ladder is formed by mechanically interlocking two kinds of linear monomers with ligase, in which the “rungs” of the ladder are used to bring together the individual rings of the mechanically interlocked structure, while two kinds of “rails” on each ring (one is complementary to HRP-mimicking DNAzyme, and the other is complementary to primer) are facilitated to RCA reaction by using individual ligated circular DNA as template in the presence of Phi29 DNA polymerase and dNTPs. The RCA products contain thousands of repeated HRP-mimicking DNAzyme units, which stimulate the generation of enhanced chemiluminescence (CL) signal upon complexing with hemin in the presence of luminol and H2O2. In this case, each analyte will trigger a one-step self-assembly of hierarchical nanostructures, which further generate many copies of DNAzymes by a RCA process, and each DNAzyme will generate many copies of chemiluminescent products. Thus, it is conceivable that the detection sensitivity could be significantly improved. For the aptameric sensing system (Scheme 1B), aptamertagged MNPs are prepared that trigger the fabrication of mechanically interlocked DNA scaffolds as above. By utilizing the structure switching of aptamer upon binding to target, polycatenated DNA scaffolds are forced to release from the aptamers that can initiate RCA and DNAzyme amplification. As compared to the traditional methods that are usually limited to the requirement of two or more binding sites per target protein to form a sandwich structure to amplify signals, the proposed scheme undoubtedly provided a promising protocol for the development of diverse aptameric systems. More importantly, this strategy is suitable for the detection not only of small molecules (e.g., ATP) (36) Liu, J.; Cao, Z.; Lu, Y. Chem. Rev. 2009, 109, 1948–1998. (37) Ren, R.; Leng, C.; Zhang, S. Chem. Commun. 2010, 46, 5758–5760. (38) Wu, Z.-S.; Zhou, H.; Zhang, S.; Shen, G.; Yu, R. Anal. Chem. 2010, 82, 2281–2289.

Scheme 1. Schematic Representation of Polycatenated DNA Scaffold-Mediated RCA Reaction and DNAzyme Amplification for Amplified Assay of (A) SNPs Identification of G12C Mutation in the K-ras Gene and (B) Thrombin Detection by Employing Streptavidin-MNPs to Reduce Backgrounda

a

The detailed principles are described in the main text.

but also of macromolecules (e.g., thrombin), as long as they have a selected aptamer sequence. In the present study, thrombin is used as a model analyte to demonstrate the generality of the strategy. In this work, a subtle difference between the strategies for the detection of SNPs and thrombin lies in the ligated circulars directly dissociating from the surface of MNPs individually into solution during a RCA process in the former, while first releasing into solution as polycatenated entirety and later departing from each other during a RCA reaction in the latter. It is noteworthy that considering hemin itself without intercalating in the Gquadruplex units could also generate CL signals in the luminolH2O2 system,33,39 the primers of RCA reaction are biotinylated, and the resultant RCA products can be easily binded to streptavidinmodified MNPs via biotin-streptavidin specific recognition to perform the following amplified CL detection. Because free hemin could be facilely removed through magnetic separation, the detection sensitivity of the protocol would be further improved. EXPERIMENTAL SECTION Conjugation of Capture Probes on MNPs. Coupling the capture probes onto the surface of MNPs was performed as (39) Elbaz, J.; Moshe, M.; Shlyahovsky, B.; Willner, I. Chem.-Eur. J. 2009, 15, 3411–3418.

follows. First, 100 µL of carboxylated MNPs suspension was previously washed with 400 µL of 0.1 M imidazol-HCl buffer (pH 7.0) three times, followed by activation in a mixture of 200 µL of 0.2 M NHS solution and 200 µL of 0.8 M EDC solution at 37 °C for 30 min. After the mixture was washed three times with 400 µL of 0.01 M PBS buffer and resuspended to a final volume of 200 µL, 500 µL of 20 nM amino-modified capture probes was added to the above resulting MNPs solution. Finally, the excess capture probes were removed by magnetic separation. The resulting capture probes conjugated MNPs were washed with 200 µL of 0.01 M PBS buffer (pH 7.0) three times and then resuspended in 200 µL of PBS buffer and stored at 4 °C for further use. From the results of our previous study, the number of capture probes immobilized on each MNPs was estimated to be ∼1.39 × 106 per particle.40 Fabrication of Polycatenated DNA Scaffolds on MNPs. 50 µL of capture probe-modified MNPs suspension was incubated for 60 min with 30 µL of target sequences, single-base mutated DNA in SNPs analysis or aptamers in thrombin detection, followed by washing twice using 200 µL of 0.01 M PBS buffer solution (pH 7.0). Next, 30 µL of 1.0 nM padlock probe solution was added and hybridized with target sequences in Quick Ligation Kit buffer, (40) Bi, S.; Zhou, H.; Zhang, S. Chem. Commun. 2009, 5567–5569.

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Figure 1. (A) FI-CL signals for luminol-H2O2 (a), and SNPs detection in the absence (b) and presence (c) of single-base mutant target DNA. (B) The comparison of FI-CL signals for luminol-H2O2 (a), luminol-H2O2-hemin (d), and blank sample (without target DNA) by employing streptavidin-MNPs (b) and without employing streptavidin-MNPs (c) to reduce the background induced from free hemin in the HRP-mimicking DNAzymes-catalyzed luminol-H2O2 CL system. Experimental conditions: luminol, 5.0 × 10-4 M; H2O2, 7.5 × 10-3 M; hemin, 1.0 × 10-6 M; target DNA, 1.0 fM.

followed by the circularization in the presence of 40 units/µL ligase. After another incubation at 45 °C for 1 h, the sandwich hybridized MNPs were cleaned twice using the above PBS buffer solution, followed by the addition of 300 µL of 1.0 nM linear DNA monomers, 4 and 5. Self-assembly of polycatenated DNA scaffolds on MNPs was carried out by heating the above resultant suspension to 90 °C for 10 min, and fast cooling to 50 °C and holding for 30 min, and subsequent fast cooling to 25 °C, followed by adding 40 units/µL ligase for 30 min. The fabricated polycatenated DNA scaffolds-MNPs were washed twice with the above PBS buffer solution through magnetically controlled separation. Detection of SNPs. To evaluate the ability of the developed biosensor for SNPs assay, a single-base mutant target DNA sequence was used for sandwich hybridization. The fabrication procedures of polycatenated DNA scaffolds were carried out as described above. Subsequently, 2 µL of RCA-reaction buffer containing 10 units/µL Phi29 DNA polymerase and 10 mM dNTPs, and 300 µL of 1.0 nM biotinylated primers, was mixed with the above resultant polycatenated DNA scaffolds-MNPs conjugates. The RCA reaction was performed by hybridizing the biotinylated primers with the circularized templates to proceed at 37 °C for 60 min, which was terminated by heating to 65 °C for 10 min to inactivate the polymerase. To reduce the background of the detection system, the supernatant containing the biotinylated RCA products was transferred and reacted with 100 µL of streptavidin-modified MNPs. The specific recognition process was carried out for 30 min. After being washed with PBS buffer, the resulting MNPs were incubated in 1.0 µM hemin solution to be intercalated with the synthesized DNAzyme chains. Finally, the FI-CL detection was carried out after the MNPs were washed with PBS twice and dispersed in 1.2 mL of 0.01 M PBS buffer (pH ) 7.0). The mode of the FI-CL detection system is presented in Figure S1. Detection of Thrombin. To assess the feasibility of the proposed protocol to aptameric systems, an aptamer sequence of thrombin was used for sandwich hybridization. The fabrication procedures of polycatenated DNA scaffolds were carried out as described above. Next, 10 µL of thrombin sample at a specific concentration was added and incubated at 37 °C for 30 min to 9450

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make the aptamer change its structure to bind target. After magnetic separation, the supernatant was transferred, and we performed the following RCA reaction and FI-CL detection as the operations of SNPs detection. Transmission Electron Microscopy (TEM). A TEM image of ∼3 nm AuNP-functionalized polycatenane released in solution triggered by the addition of thrombin was taken on a JEOL JEM-2000EX at an accelerating voltage of 160 kV. A small amount of sample solution was placed on a 3 mm diameter, 200 mesh copper grid and allowed to evaporate to dryness, followed by directly placing in the TEM for imaging. RESULTS AND DISCUSSION SNPs Detection. Feasibility. As demonstrated in Scheme 1A, in the absence of target DNA, the 3′ and 5′ ends of padlock probe could not be brought into close proximity without the ligation template; thus, no circularized template could be gained even with the assistance of ligase, let alone the following fabrication of polycatenated DNA scaffolds on MNPs. On the contrary, in the presence of a fixed concentration of target DNA, terminal regions of padlock probe were hybridized with half part of target DNA, forming a ligation junction. Ligation of the adjacent ends by DNA ligase led to the circularization of the padlock probe. The circularized template was subsequently followed by self-assembly of polycatenated DNA nanostructure in an ABAB-type. The RCA products with biotinylation at 5′-end were then immobilized on streptavidin-modified MNPs and intercalated with hemin, resulting in the formation of the HRP-mimicking DNAzyme under the RCA synthesized thousands of G-quadruplex self-assembly. From the results, 1.0 fM target DNA induced a CL signal of ∼340, while a control experiment without target only exhibited ∼61 that was equivalent to the CL background of the luminol-H2O2 system (Figure 1A). Additionally, it should be noted that utilizing the last procedure of magnetic separation, the background inherent from the luminol-H2O2 system catalyzed by free hemin was circumvented because the excess hemin could be easily removed by magnetic separation (Figure 1B) and the detection sensitivity was expected to substantially improve (vide infra). Further, a series of control experiments revealed that only mutant

Figure 2. (A) FI-CL signals for the HRP-mimicking DNAzyme-catalyzed luminol-H2O2 system corresponding to different concentrations of single-base mutant target DNA: (a) 0; (b) 0.1; (c) 0.2; (d) 0.4; (e) 0.6; (f) 0.8; (g) 1.0; (h) 2.0; (i) 4.0; (j) 6.0; (k) 8.0; (l) 10.0 fM. (B) The corresponding calibration curve of peak height versus the concentration of target DNA.

target could trigger the assembly of topologically ladder-shaped polycatenane followed by double amplification steps of RCA reaction and synthesized DNAzyme units as catalyzer for the luminol-H2O2 CL system (Figure S4). Sensitivity. The assay was subsequently used in the detection of G12C mutation in the K-ras gene with the substitution of an adenine (A) for a cytosine (C) in the wide-type sequence. Figure 2 shows the change in FI-CL intensity caused by the HRPmimicking DNAzyme-catalyzed luminol-H2O2 system as a result of the RCA process, which employed polycatenated DNA scaffolds as circular templates for different concentrations of target analyte. FI-CL intensity increased as the concentration of target DNA increased, while no detectable signal was observed when the primary hybridization was performed in the absence of target (blank solution, curve a). The analysis of target DNA covered the concentration range extending 2 orders of magnitude from 0.1 to 10 fM showed a good linear relationship between peak height of FI-CL signal intensity and target DNA concentration in the range of 0.1-1.0 fM. A relative standard deviation (RSD) was 7.8% obtained by 11 replicate measurements of 0.4 fM target DNA, indicating a good reproducibility of the assay. The theoretical 3σ limit of detection (LOD) was calculated to be 71 aM, which is better than or comparable to the reported assays for SNPs detection.17-19,28-30 In previous reports, the detection limits for DNA detection were only 1.0 nM by just employing single HRP-mimicking DNAzyme as catalytic label,14 and 10 fM by using one circular as template for RCA that synthesized HRP-mimicking DNAzyme chains.23 The ultrahigh sensitivity achieved by this proposed strategy could be attributed to the amplification of the polycatenated DNA scaffold-mediated RCA reaction and DNAzyme amplification, and the conquest of high background, which resulted from free hemin in the HRP-mimicking DNAzymecatalyzed luminol-H2O2 CL system by employing streptavidinmodified MNPs as immobilizers. Coming up to the expectations, the detection limit is 1 order of magnitude higher than that without employing streptavidin-modified MNPs to reduce background, while approximately 10-fold lower than that just taking the padlock probe as the circular template for RCA reaction (detailed results are shown in the Supporting Information). It is worth commenting that after the polycatenane fabricat-

ing on MNPs was triggered by the target, the extraneous structures in solution could be easily washed by magnetic separation. In this case, only the target triggered polycatenated DNAs were assembled on the surface of MNPs, further acting as the templates for the following RCA reaction. Moreover, comparing the results by employing polycatenated DNA scaffolds and one padlock probe as template for RCA reaction in target detection under the same reaction conditions, the signals were much more higher by taking polycatenane structure as templates than just one padlock probe, which could also confirm that the RCA reaction occurred on the ABAB-type DNA copolymers. Additionally, the high sensitivity could also be attributed to the high polymerization efficiency of the DNA polymerase Phi29, and the excellent detection sensitivity of FI-CL. However, considering the steric effect induced by MNPs, the interlocked polycatenated DNA scaffolds assembled on the surface of MNPs could not enhance the signals as much as expected. Specificity. Sensitivity and specificity are the two critical factors to evaluate the success of an SNPs detection assay for clinical applications. As a promising amplification method, RCA possesses not only high sensitivity but also excellent specificity due to the stringent strand matching requirement for the ligation reaction. To assess the specificity of our assay, the FI-CL detection corresponding a similar experiment performed with the wide-type sequence is shown as column a in the inset of Figure 3. It was evident that no increase in the FI-CL intensity was observed upon the treatment of the resulting RCA products immobilized on streptavidin-MNPs with hemin. This good specificity of the proposed SNPs assay depended in general on the fidelity of the DNA ligase-mediated coupling of padlock to the mutant assembly, which further resulted in the interlocked polycatenated scaffolds for RCA reaction and the DNAzyme biocatalytic luminol-H2O2 system. In addition, the target oligonucleotides of wide-type and mutated sequences were mixed at different mole ratios with a total concentration of 10.0 pM, which subsequently reacted to the padlock probe with complementary sequence to the mutant target. From the results of Figure 3, the FI-CL intensity increased substantially when only 1.0 fM mutant target existed in the mixture, a wide-type strands to mutant ratio of 10 000:1. The results exhibited an excellent ability of our assay in detecting a small quantity of mutation among a large quantity Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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Scheme 2. Aptameric System by Binding the Aptamer (Magenta) to Target Exposing a Sticky End (Blue) That Could Not Trigger the Fabrication of Polycatenated DNA Scaffolds Due to the Steric Effect Induced by the Formation of Target/Aptamer Complex

Figure 3. Effect of normal sequence to mutant ratio on the detection of mutant with a total concentration of 10.0 pM. Inset depicts the intensity of the FI-CL peak height recorded for pure wild-type (a) and one-base mismatched samples (b), respectively, with the concentrations of both samples as 10.0 pM, respectively.

Figure 4. FI-CL signals for the detection of Tay-Sachs mutant and normal gene in real human serum samples. The FI-CL signal for blank sample in this assay is ∼61. Each sample was repeated three times.

of wild types, which allowed the strategy great potential in clinical applications of disease early diagnosis and prognosis along with the high sensitivity and wide dynamic range. The specific detection was further supported by the reaction between the padlock probe with G-base at the 3′-end and the wide-type sequence. The joint reaction followed by the assembly of polycatenated DNA scaffolds for RCA reaction resulted in the formation of thousands of DNAzyme on streptavidin-MNPs, which was reflected in a high FI-CL signal, while it did not yield any increase in FI-CL for the mutant, implying that no DNAzyme was generated (Figure S7). Moreover, the proposed SNPs detection strategy was demonstrated to be a general method to identify single-point mutation sites and was not influenced by DNA contaminants (see the Supporting Information). Real Sample Analysis of SNPs. One major challenge of the proposed protocol is to apply the method for the selective detection of respective mutations in real biological samples. To accomplish this end, two serum samples, Tay-Sachs mutant and normal gene, were analyzed, respectively. The amino-group modified capture probe (1′′), which is complementary to the genes, was immobilized on carboxylated MNPs. As shown in Figure 4, only the mutant sample yielded to a high FI-CL signal, whereas there was just a little change in FI-CL intensity for normal 9452

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sample by using (3′′-M) as padlock probe, which was opposite of using (3′′-W) as padlock probe. The treatment of serum samples was done according to the proposed method of Willner’s (see the Supporting Information for details).7 It should be noted that the analyzed genes were isolated from 0.5 mL serum samples without PCR amplification. This can be explained as the capture probetagged MNPs and specific padlock probe made a contribution to the impressive selectivity in analyzing the target mutants within an enormous mixture of genomic fragments. Thus, the sensitivity of the method achieved the detection of mutations in real clinical genomic samples requiring no PCR amplification. Thrombin Detection. Encouraged by the above principle of SNPs assay, an aptasensor could be directly designed as Scheme 2 by using an aptamer construct that exposed an initiator strand upon binding with target. However, the main disadvantage of this design is that for the detection of biomacromolecules, the sensitivity must be limited because the following fabrication of the triggered polycatenated architecture would be significantly limited by the steric effect induced by the formation of target/ aptamer complex. Thus, considering the universality of the strategy, an aptasensor was flexibly devised as Scheme 1B by previously fabricating the DNA polycatenane on MNPs via the aptamer sequences, and entirely dissociated into solution upon target binding followed by RCA and DNAzyme amplification. In this case, the self-assembly of polycatenated nanostructure was independent of the aptamer/target binding event and could further adopt as amplifier for the detection of any aptamer of interest. The analytical characteristics of the proposed aptameric sensing system are discussed as follows. Feasibility. To demonstrate the versatility of the present protocol, a strategy for protein analysis, taking thrombin as a model analyte, was designed as displayed in Scheme 1B based on aptamer-protein recognition. As compared to the strategy for SNPs detection (Scheme 1A), the only difference in thrombin detection was that the polycatenated DNA scaffolds were previously assembled on carboxylated MNPs and entirely released into solution by the introduction of thrombin. The released high molecular weight, interlocked nanowires were further characterized by agarose gel electrophoresis and TEM via functionalizing the released polycatenane with ∼3 nm AuNP-labeled thiolmodified ssDNA (Figure 5). From the gel image, a broad band with higher molecular weight than that corresponding to the 1 kb marker of 1000 bp was observed (lane 2) when the concentration of thrombin was at 0.1 nM, while no band appeared in the absence of thrombin (lane 1), determining the desirable detection specificity (vide infra). Moreover, a denaturing polyacrylamide gel was also run to explore mechanical bonding in the polycatenane (Figure S10), in which a wide band was observed triggered by thrombin, reflecting both high molecular weight and polydispersity characteristic of a polymeric material. Thus, it was well-grounded

Figure 5. Agarose gel electrophoresis of the polycatenated DNA scaffolds released into the solution triggered by the addition of thrombin. TEM image showing the ∼3 nm AuNP-functionalized polycatenane released in solution.

to demonstrate that a mechanical bond existed in the polycatenated DNA scaffolds, which made the polymeric material retain its high molecular weight even under denaturing conditions.9,10 Sensitivity. The detection signal was also amplified by the RCA process and DNAzyme amplification, which was further enhanced by employing streptavidin-modified MNPs to reduce background. As the target concentration increased, the amount of released circularized templates gradually increased due to the high affinity and specific structure-switching of aptamers upon target binding. As expected, the FI-CL intensity increased with the increase of target concentration, indicating the feasibility of the signal-on and further double signal-amplification mechanism of the strategy. As shown in Figure 6, when the peak height of FI-CL signal intensity was plotted versus the concentration of thrombin, the present CL aptasensor offered a linear response to the concentration of target analyte in the range from 0.01 M to 0.1 nM. Further increasing the concentration of target generated a nonlinear change in peak height, while decreasing could not distinguish with the blank. Thus, the response data of the two cases were beyond the linear response range with a RSD of 7.2% and LOD of 6.6 pM (3σ). As compared to the aptasensors without employing streptavidinmodified MNPs to reduce background and just taking the padlock

probe as the circular template for RCA reaction, the detection capability of the present strategy demonstrated an improvement by factors both of 10 for the detection of thrombin (see the Supporting Information for details), which was consistent with the results of agarose gel electrophoresis and TEM as in Figure 5. Thus, we estimated that approximately 10 circular DNAs in one polycatenated DNA scaffold were acting as templates for the RCA reaction to synthesize the HRP-mimicking DNAzyme chains. Moreover, as compared to the previous studies, the amplification strategy triggered by binding the target to one aptamer rather than two different aptamers made our signaling scheme more attractive and convenient. Besides the properties of target/aptamer binding, such analytical capabilities should also be attributed to no modification on the original aptamer, except for an amino group at the spacer end to attach the aptamer onto carboxylated MNPs, which made the aptamer preserve the intrinsic bioactivity and desirable specificity comparable to the original. Specificity. The detection specificity of the method mainly depended on the aptameric recognition properties. To investigate the detection specificity of the present detection system, five nontarget proteins in place of thrombin were tested according to the same experimental procedures as those for thrombin. Noteworthy is that the selected proteins for specific testing are either abundant in serum (e.g., IgG, fibrinogen, and transferrin) or similar to thrombin participating in blood coagulation (e.g., fibrinogen). In addition, as a worst-case scenario for its potential nonspecific interaction with the negatively charged oligonucleotide, lysozyme was also analyzed, which is a positively charged protein at neutral pH. From Figure S13, the CL signals corresponding to 10-fold excess of BSA, IgG, transferrin, lysozyme, and fibrinogen were very low, which were equal to that of blank sample, indicating those proteins did not interfere with the assay for a trace level of thrombin (0.1 nM). These experimental results demonstrated that the proposed amplification assay for thrombin exhibited a good specificity originating from the specific binding between target and aptamer. Real Sample Analysis of Thrombin. The practical applicability of the proposed aptasensor was investigated by detecting thrombin in a human real serum sample. As expected, no thrombin in the serum sample was detected because no coagulation factors were contained in healthy human serum sample. Thus, we spiked

Figure 6. (A) FI-CL signals for the HRP-mimicking DNAzyme-catalyzed luminol-H2O2 system corresponding to different concentrations of thrombin: (a) 0; (b) 0.01; (c) 0.02; (d) 0.04; (e) 0.06; (f) 0.08; (g) 0.1; (h) 0.2; (i) 0.4; (j) 0.6; (k) 0.8; (l) 1.0 nM. (B) The corresponding calibration curve of peak height versus the concentration of thrombin. Analytical Chemistry, Vol. 82, No. 22, November 15, 2010

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thrombin into 10-fold-diluted serum samples to examine the applicability of this aptasensor in real serum sample. More to the point, the addition of thrombin did not affect the samples.41 According to previous studies for the analysis of thrombin, dilution of serum samples by 10-fold prior to assay is in common use by taking aptamers or antibodies as affinity probes, because the 10fold or more diluted serum samples have little matrix effects on the analysis of thrombin.42 The serum samples were spiked with 5.0 × 10-11, 2.0 × 10-10, and 8.0 × 10-10 M thrombin, respectively. After FI-CL measurement, the calibration method was used to determine thrombin concentrations, and the recoveries were obtained by comparing the measured amounts to that of added human R-thrombin varied from 88.0% to 107.0%, which apparently provides the potentiality of the proposed aptasensor for thrombin detection in real clinical samples (Table S2). It was worthy to note that because the blood samples (4 and 5) employed were patient serums that might suffer from diseases known to be associated with coagulation abnormalities, some activation of the blood-clotting cascade may have occurred,43 thereby producing detectable levels of thrombin with the recoveries ranging from 103.3% to 110.0%.

assembly of polycatenated DNA nanostructures for amplified assay of SNPs identification and thrombin detection. The high sensitivity of the method is attributed not only to the polycatenated DNA scaffolds combining with the RCA process for the synthesis of DNAzyme units that act as the catalytic units, but also to the low background chemiluminescence associated with the specific recognition of streptavidin-modified MNPs with biotinylated RCA products, which enables the quantitative analysis of real human serum samples with no PCR preamplification. Moreover, the design of the strategy is straightforward: Only the sequences of the capture probe and terminal regions of padlock probe are changed with the new analyte, while the same ssDNA monomers are used to fabricate the polycatenated DNA scaffolds. This work promises to open an exciting new avenue for future development of DNA nanotechnology in bioanalytical applications.

CONCLUSIONS The present study introduces the concept of triggered double amplification strategies (RCA and DNAzyme amplification) by self-

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. The material is available free of charge via the Internet at http://pubs.acs.org.

(41) Bini, A.; Minunni, M.; Tombelli, S.; Centi, S.; Mascini, M. Anal. Chem. 2007, 79, 3016–3019. (42) Zhao, Q.; Lu, X.; Yuan, C.-G.; Li, X.-F.; Le, X. C. Anal. Chem. 2009, 81, 7484–7489. (43) Zeymer, U.; Mateblowski, M.; Neuhaus, K.-L. J. Thromb. Thrombolysis 1998, 5, 203–207.

Received for review August 13, 2010. Accepted October 6, 2010.

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ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21025523), the National Basic Research Program of China (2010CB732404), and the Excellent Young Scientists Foundation of Shandong Province (JQ200805).

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