LETTER pubs.acs.org/NanoLett
DNA-Functionalized Nanochannels for SNP Detection Seung Yun Yang,†,§ Sejin Son,‡,§ Sangsin Jang,† Hyunwoo Kim,‡ Gumhye Jeon,† Won Jong Kim,*,‡ and Jin Kon Kim*,† †
National Creative Research Center for Block Copolymer Self-Assembly, Department of Chemical Engineering, ‡Department of Chemistry, Pohang University of Science and Technology, Kyungbuk 790-784, Korea
bS Supporting Information ABSTRACT: We have developed ultrahigh density array of functionalized nanochannels by using a block copolymer having end di-COOH group. This approach provides a facile route for direct functionalization of wall surface of the nanochannels and immobilization site for molecular recognition agents (MRAs). By using overhanging single-stranded DNA as MRAs, the DNAfunctionalized nanochannels showed high resolution to detect a single-base mismatch as well as to discriminate single-mismatched sequence at various locations by hybridization preference with MRAs. KEYWORDS: Single nucleotide polymorphisms (SNPs), functional nanochannels, molecular recognition agent, oligonucleotide, block copolymer
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ne of the great challenges in DNA analysis is the discrimination and quantification of a small number of minor sequences including single nucleotide polymorphisms (SNPs) in genomic samples. Since SNPs, resulting from single nucleotide substitution of a genome, are the most abundant genetic polymorphisms and have become popular molecular genetic markers, they are regarded as high-resolution genetic markers for mapping genes, identifying inherited diseases, and developing drug candidates.1,2 Numerous methods have been developed to detect SNPs, for example, allele-specific biochemical reaction assisted by enzyme,3,4 selective ligation with an optical indicator such as fluorescent dye,5-7 electrophoresis with additives,8 and biological or synthetic nanopores containing molecular probes.9-11 Among them, synthetic nanoporous membranes10,11 prepared by using electron or ion beam and track-etching, provide a simple and fast platform since the detection is accomplished by specific interaction such as DNA hybridization with molecular probes immobilized on the nanopore walls. However, the reproducibility of nanopore preparation and specific functionalization of wall surface of the nanopores remain critical issues.12 On the other hand, periodic and uniform nanostructure based on selfassembly of block copolymer has been widely used to fabricate nanoporous materials.13-18 After selective removal of pore-forming block components, nanochannel (or nanopore) was further functionalized by surface modification. This functionalized nanochannel was used as a template for nanomaterial preparation and site for surface modification.13-15 However, a long reaction time and rigorous conditions of chain cleavage for pore generation and surface functionalization limit the versatile uses. In this study, we introduced a new concept of DNA-based molecular recognition agents (MRAs) in detecting the SNP with very high precision and efficiency. For this purpose, we fabricated r 2011 American Chemical Society
ultrahigh density array of functional nanochannels having walls covered with carboxylic acid, which served as highly sophisticated molecular recognition probes. In contrast to the conventional single-stranded DNA (ss DNA) probe, which cannot differentiate the closely mismatched DNA strands with similar stability, the nanochannel system employed in this study showed high resolution to detect a single-base mismatch as well as to discriminate single-mismatched sequence at various locations. An ultrahigh density array of nanochannels was fabricated by using polystyrene-block-poly(methyl methacrylate) copolymer having dicarboxylic acid end group (PS-b-PMMA-diCOOH). PS-b-PMMA-diCOOH was synthesized by hydrolysis of ditertbutyl ester terminated PS-b-PMMA (PS-b-PMMA-diE) (Figure 1a). On addition of PMMA homopolymer to PS-b-PMMA-diCOOH, PMMA cylindrical microdomains become oriented normal to the substrate (Figure 1b). A thin film with a thickness of 80 nm was transferred to a polysulfone microporous membrane support (HT Tuffryn, Pall) to enhance the mechanical stability. PMMA homopolymer was removed by washing with acetic acid, a selective solvent for PMMA, to generate nanopores.19,20 Nanochannels having pore size of ∼15 nm were successfully prepared on the supporting membrane, and vertically oriented cylindrical pores spanned through the entire film (Figure 1c). (Details for the synthesis and fabrication of the nanoporous membranes are given in Supporting Information, Sections 1 and 2) The wall of nanochannel was covered with COOH groups because COOH groups were attached to the end of the PMMA block chains. Thus, COOH-decorated nanochannel could be used for immobilization site of MRAs. Also, due to ultrahigh density (∼6 1011/in2) and Received: November 2, 2010 Published: February 16, 2011 1032
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Figure 1. Chemical structures of the block polymers (a) and schematic illustration for the fabrication of functionalized nanoporous membrane with carboxylic group (b). SEM images for the top surface and side view (inset) of nanoporous layer (c).
Figure 2. Schematic illustration for SNP detection based on molecular recognition using DNA-functionalized nanochannels.
short length (∼80 nm) of nanochannels, molecular separation with very high resolution is easily achieved. The oligodeoxynucleotide (ODN-A) labeled with carboxyfluorescein (FAM) was immobilized on nanochannel walls. ODN-A consists of 14 bases and an amine substituent at the 30 end that could attach itself covalently to the wall of nanochannel containing COOH group (Figure 2a). Carboxy-tetramethyl- rhodamine (TAMRA)-labeled overhanging ODN-B contains 19 bases including 14 bases perfectly matched to ODN-A (Figure 2b). ODN-B was hybridized to ODN-A to form 14-basepair (bp)
double-stranded (ds) DNA and thus this ODN-B can act as an MRA which contains five unhybridized bases. As the target ss DNAs (ODN-C, C1, C2) having perfectly matched or singlemismatched sequences with overhanging ODN-B pass through the nanochannel, the ODN-B becomes dissociated from ODN-A and undergoes hybridization with target ss DNA to form more stable 19-bp ds DNA (Figure 2c). However, the displacement amount of ODN-B from the nanochannel varies depending on the thermodynamic stability of newly formed ds DNA having fullmatched or single-mismatched sequence.3 Furthermore, since the 1033
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Figure 3. Displacement profile of ODN-B from the ODN-A on the nanochannels for four different target ss DNAs. Full-matched target ODN-C displayed the highest displacement whereas scramble ODN-D exhibited no displacement.
location of the mismatched sequence also induces a subtle change in the stability of ds DNAs, the variation in the location of the single-base mismatched sequence could be discriminated by simple measurement of the flux of ds DNAs through the nanochannels. SNP detection was conducted in a module containing nanoporous membrane after immobilization of ODN-A (Supporting Information, Section 3). Prior to the SNP detection, gold with 2 nm thickness was deposited onto the top surface of nanoporous membrane to effectively prevent nonspecific adsorption and thus the possibility of hybridization between ODN-A and ODN-B at the top surface of the membrane was eliminated (Supporting Information, Section 4). As a result, the hybridization reaction leading to single-mismatch selectivity took place only at the nanochannel wall, not on the film surface. Furthermore, chemical conjugation of ODN-A on the nanopore wall was proved by flowing 0.05% (v/v) Triton X-100 solution (Sigma) and, thus stability of probe DNA could offer the versitile use in harsh conditoin (Supporting Information, Section 5). After ODN-B solution (1.25 nmol) dissolved in potassium phosphate buffer (20 mM, 100 mM KCl, pH 7.4) passed through the nanochannel, three different target ss DNAs (ODN-C, ODN-C1, ODN-C2) were fed into nanochannels. We found that the extent of hybridization between ODN-A and ODN-B taking place inside the nanochannels was very high (more than 90%) and reproducible (Supporting Information, Section 6). The detection of SNP was achieved by passing target ss DNAs through the nanochannels followed by the quanitification of the eluted resultant ds DNA (ODN-B and target ss DNA). Displacement amount of ODN-B is governed by the difference in hybridization stability between the preformed ds DNA (ODN-A and ODN-B) and resultant ds DNA. The amount of resultant ds DNA was analyzed by measuring the fluorescence intensity observed for TAMRA-labeled ODN-B, which is directly proportional to the amount of displaced ODN-B. As shown in Figure 3, perfectly matched DNA (ODN-C) showed 60% higher displacement of ODN-B compared to that of ODN-C1 with single-mismatched sequence located close to the end. The selectivity coefficient, which is defined as the ratio of the
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Figure 4. Displacement profile of ODN-B0 from the ODN-A0 on the nanochannels for two different target ss DNAs (ALDH2*2 and ALDH2).
flux for the perfectly matched target ss DNA to the singlemismatched target ss DNA, is 1.6 for ODN-C1, while it is 2.1 for ODN-C2, which contains single-mismatched base at the middle. On the other hand, scramble ODN-D without genetic similarity to ODN-B showed fluorescence intensity of base level. Despite the subtle difference in melting temperature (Tm) of 0.3 °C calculated by IDT SciTools OligoAnalyzer 3.1, the selectivity difference between ODN-C1 and ODN-C2 was very significant. To investigate the effect of the feeding concentration of target ODNs on the displacement of ODN-B, we performed displacement experiment of ODN-B using buffer solution of ODN-C and ODN-C2 at a reduced concentration (1.25 nmol). Perfectly matched DNA (ODN-C) demonstrated ∼50% higher displacement of ODN-B than that of ODN-C2 (middle one-mismatch). Although the selectivity coefficient decreased from 2.1 to 1.5 by changing the feed concentration from 2.5 to 1.25 nmol, the displacement profile of ODN-B was still distinguishable and effective for SNP detection (Supporting Information, Section 7). The highly distinguishable displacement profile even for negligible differences in the hybridization stability might be attributed to kinetic effect arising from the flowing of DNAs through the nanochannels. Because of the confinement within the nanopores and the presence of highly populated MRAs in the walls, target ss DNAs passing through the narrow nanochannels could hybridize with MRAs more easily and frequently compared with the conventional microfluidic-based DNA chips having twodimensional micrometer-sized channels. Thus, both thermodynamic stability and kinetic effects play important roles in governing the resolution. Finally, we used the nanochannels to evaluate the detection of SNP in alcohol dehydrogenase 2 (ALDH2). This SNP sequence can be used for the evaluation of the susceptibility of human organs to damage induced by alcohol.21 We designed a probe DNA consisting of template ODN (ODN-A0 ) and MRA (ODNB0 ) whose base sequences are given in Figure 4. Two target ss DNAs (ALDH2*2 and ALDH2 having perfectly matched or single-mismatched sequences with overhanging ODN-B0 ) were fed inside the nanochannels. The resultant ds DNAs (ODN-B0 and target ss DNA) were analyzed by measuring the fluorescence intensity and given in Figure 4. The perfectly matched DNA (ALDH2*2) showed 50% higher displacement of ODN-B0 1034
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Nano Letters compared to that of ALDH2 (wild-type) with single-mismatched sequence located at the middle. This indicates that ss target ODN without or with SNP was promptly and accurately discerned. In summary, we have introduced a simple and straightforward strategy to functionalize the nanochannels based on self-assembly of block copolymer. Functional groups inside the nanopores were used as immobilization site for MRAs. The nanochannel was exploited to evaluate the potential of SNP detection. The biggest advantage of this nanochannel system is that unlike other prevalent SNP detection devices3-11 it does not require any enzyme or electrical/chemical signal. Additionally, the system in this study detects precisely the SNP during continuous flowing. It could be employed in genetic diagnosis and separation related to molecular recognition.
’ ASSOCIATED CONTENT
bS
Materials and method for fabrication of nanochannels and DNA experiments and Figures S1S6. This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Information.
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(14) Rzayev, J.; Hillmyer, M. A. J. Am. Chem. Soc. 2005, 127, 13373–13379. (15) Ryu, J.-H.; Park, S.; Kim, B.; Klaikherd, A.; Russell, T. P.; Thayumanavan, S. J. Am. Chem. Soc. 2009, 131, 9870–9871. (16) Kim, J. K.; Lee, J. I.; Lee, D. H. Macromol. Res. 2008, 16, 267–292. (17) Kim, J. K.; Yang, S. Y.; Lee, Y. M.; Kim, Y. S. Prog. Polym. Sci. 2010, 35, 1325–1349. (18) Kim, Y. S.; Han, Y. S.; Lee, W.; Alexe, M.; Baik, S. G.; Kim, J. K. Nano Lett. 2010, 10, 2141–2146. (19) Yang, S. Y.; Ryu, I.; Kim, H. Y.; Kim, J. K.; Jang, S. K.; Russell, T. P. Adv. Mater. 2006, 18, 709–712. (20) Yang, S. Y.; Yang, J.-A; Kim, E. S.; Jeon, G.; Oh, E. J.; Choi, K. Y.; Hahn, S. K.; Kim, J. K. ACS Nano 2010, 4, 3817–3822. (21) Chen, C.; Lu, R.; Chen, Y.; Wang, M.; Chang, Y.; Li, T.; Yin, S. Am. J. Hum. Genet. 1999, 65, 795–807.
’ NOTE ADDED AFTER ASAP PUBLICATION This article was published ASAP on February 16, 2011. Figure 2 has been modified. The correct version was published on February 23, 2011.
’ AUTHOR INFORMATION Corresponding Author
*E-mail: (J.K.K.)
[email protected]; (W.J.K.) wjkim@ postech.ac.kr. Author Contributions §
These authors contributed equally to this work.
’ ACKNOWLEDGMENT This work was supported by the National Creative Research Initiative Program of by the National Research Foundation of Korea (NRF) and by NRF Grant (20100019914) funded by the Korea government (MEST). ’ REFERENCES (1) Kolpashchikov, D. M. J. Am. Chem. Soc. 2008, 130, 2934–2935. (2) Kathiresan, S.; Voight, B.; Purcell, S.; Musunuru, K.; Ardissino, D.; Mannucci, P.; Anand, S.; Engert, J.; Samani, N.; Schunkert, H. Nat. Genet. 2009, 41, 334–341. (3) Kim, W. J.; Sato, Y.; Akaike, T.; Maruyama, A. Nat. Mater. 2003, 2, 815–820. (4) Kim, S.; Misra, A. Annu. Rev. Biomed. Eng. 2007, 9, 289–320. (5) Huh, Y. S.; Lowe, A. J.; Strickland, A. D.; Batt, C. A.; Erickson, D. J. Am. Chem. Soc. 2009, 131, 2208–2213. (6) Wang, X.; Lou, X.; Wang, Y.; Guo, Q.; Fang, Z.; Zhong, X.; Mao, H.; Jin, Q.; Wu, L.; Zhao, H. Biosens. Bioelectron. 2010, 255, 1934–1940. (7) Gunnarsson, A.; Jonsson, P.; Marie, R.; Tegenfeldt, J. O.; Hook, F. Nano Lett. 2008, 8, 183–188. (8) Kanayama, N.; Takarada, T.; Shibata, H.; Kimura, A.; Maeda, M. Anal. Chim. Acta 2008, 619, 101–109. (9) Howorka, S.; Cheley, S.; Bayley, H. Nat. Biotechnol. 2001, 19, 636–639. (10) Kohli, P.; Harrell, C. C.; Cao, Z.; Gasparac, R.; Tan, W.; Martin, C. R. Science 2004, 305, 984–986. (11) Wanunu, M.; Sutin, J.; Meller, A. Nano Lett. 2009, 9, 3498–3502. (12) Martin, C. R.; Siwy, Z. S. Science 2007, 317, 331–332. (13) Ha, J.-M.; Wolf, J. H.; Hillmyer, M. A.; Ward, M. D. J. Am. Chem. Soc. 2004, 126, 3382–3383. 1035
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