Fluorescence-Based Detection of Point Mutation in DNA Sequences

J. Phys. Chem. B 2009, 113, 14487–14490

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Fluorescence-Based Detection of Point Mutation in DNA Sequences by CdS Quantum Dot Aggregation Taehoon Kim,† Minho Noh,† Hosub Lee,† Sang-Woo Joo,*,‡ So Yeong Lee,§ and Kangtaek Lee*,† Department of Chemical and Biomolecular Engineering, Yonsei UniVersity, Seoul, Korea; Department of Chemistry, Soongsil UniVersity, Seoul, Korea; and Laboratory of Veterinary Pharmacology, Seoul National UniVersity, Seoul, Korea ReceiVed: June 30, 2009; ReVised Manuscript ReceiVed: September 15, 2009

We present a novel method for the detection of single base mismatch based on fluorescence quenching that unmodified CdS quantum dots exhibit upon aggregation. Target DNA sequences of interest are breast cancer 2 (BRCA2) and signal-induced proliferation-associated gene 1 (Sipa1) sequences. We monitor aggregation of CdS quantum dots upon addition of double-stranded DNAs at different salt concentration using quasielastic light scattering (QELS), transmission electron microscopy (TEM), photoluminescence spectroscopy, and zeta potential measurement. Our results indicate that the double-stranded DNA with a perfectly matched sequence can easily be discerned by naked eye from the single base mismatched one due to the fluorescence quenching phenomenon caused by selective aggregation of the CdS quantum dots. 1. Introduction Quantum dots and metal nanoparticles are a new class of nanomaterials that exhibit unique optical and electronic properties not available from either isolated molecules or bulk materials.1-3 Colors of light that these nanoparticles absorb and emit can be tuned simply by varying their size and morphology,4-6 and so they have shown great promise as imaging probes, biosensors, catalysts, display materials, and biological tagging materials.7-11 For instance, one of the most promising techniques of DNA detection is the use of the colorimetric sensors based on gold nanoparticles.12-17 Mirkin and co-workers have modified the gold nanoparticle surface with the thiol-functionalized DNAs (i.e., probe DNA) for the hybridization with the target DNA sequences, which was followed by the cross-linking and aggregation of nanoparticles and the color change of solution.18 These detection methods, though, have a few drawbacks such as high cost, inefficiency of tagging reactions, and their complexity, which may be alleviated by introducing label-free methods.19 Rothberg and co-workers have recently reported the detection of DNA sequences using unmodified gold nanoparticles. Using the different adsorption propensities of the single- and doublestranded DNAs, they were able to selectively aggregate the unmodified gold nanoparticles under appropriate salt concentration, which enabled a rapid detection of the perfectly matched DNA from the single base mismatched one.20 This method provides a possibility to utilize the nanoparticles whose optical properties change upon aggregation in detection of various DNA sequences. Here we present a novel method for detection of single base mismatch based on fluorescence quenching that unmodified CdS quantum dots exhibit upon aggregation. The target DNA * To whom correspondence should be addressed. E-mail: [email protected] (S.-W.J.); [email protected] (K.L.). † Yonsei University. ‡ Soongsil University. § Seoul National University.

Figure 1. Experimental procedure for detection of dsDNA with the perfectly matched sequence at the optimal salt concentration.

sequences of primary interest are breast cancer 2 (BRCA2) and signal-induced proliferation-associated gene 1 (Sipa1) sequences: BRCA2 polymorphism is known to be associated with prenatal viability and breast cancer risk, and Sipa1 polymorphism with metastatic process.21,22 This work is, to the best of our knowledge, the first report on the detection of DNA sequences based on the fluorescence quenching phenomenon caused by the selective aggregation of semiconductor quantum dots. These results provide a rapid, easily discernible, and inexpensive way to detect DNA sequences compared to the previous methods based on noble metal nanoparticles,17-19 and thus have great significance in biological and medical research including tailored medicine treatment and clinical diagnosis and therapy. 2. Experimental Section 2.1. Preparation of Water-Soluble CdS Quantum Dots. Experimental procedure is shown in Figure 1. First, we prepared the mercaptoacetic acid stabilized CdS quantum dots according to Mao et al.24 Mercaptoacetic acid (8.0 µL) was added to a solution of CdCl2 in water (100 mL, 1.12 × 10-4 M). The pH was adjusted to 11 by dropwise addition of concentrated NaOH (0.5 M) solution. Under nitrogen atmosphere, Na2S (50 mL, 7.5 × 10-5 M) in water was slowly added to the resulting

10.1021/jp906096a CCC: $40.75  2009 American Chemical Society Published on Web 10/07/2009

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TABLE 1: Oligonucleotide Sequences of Probe, Target, and Single Base Mismatched DNAsa oligonucleotide probe target mismatch

Sipa1

BRCA2

3′-CGA CGG GTC TAC GAC-5′ 3′-GTC GTA GAC CCG TCG-5′ 3′-GTC GTA GAC CCG TCA-5′

5′-AAT CAG AAG CCC TTT-3′ 5′-AAA GGG CTT CTG ATT-3′ 5′-AAA GGG CTT CTG ATG-3′

a Note that the ssDNA of the 5′ f 3′ direction hybridizes with the complementary ssDNA of the 3′ f 5′ direction. b The underlined base indicates the base mismatch.

alkaline solution with vigorous stirring. The reaction mixture was stirred overnight at room temperature. 2.2. Synthesis of Oligonucleotides. Oligonucleotide sequences were designed from Bioneer Inc. (Daejeon, Korea). The single-stranded oligonucleotide probe, the oligonucleotide with its perfectly complementary sequence, and the sequence with a single mismatched base were designed based on the BRCA2 (GenBank no. NM_000059) or mouse Sipa1 sequence (GenBank no. NM_011379) (Table 1). 2.3. Hybridization and Detection of DNA. For the singlestranded DNA (ssDNA), stock probe DNA (1 mM) was diluted 10 times in NaCl (0.15 M)/phosphate buffer solution (0.1 M) at pH 7. The aliquot of DNA solution (4 µL, 0.1 mM) was added to CdS quantum dot suspension (1.3 mL). Then, distilled water (0.59 mL) and the same buffer (0.1 mL) were added to prevent abrupt aggregation. Finally, the NaCl stock solutions (0.2 M) were added so that final NaCl concentrations were 8 × 10-4, 1.2 × 10-3, and 1.6 × 10-3 M for BRCA2, and 4 × 10-4, 8 × 10-4, and 1.2 × 10-3 M for Sipa1 sequence. The final concentration of DNA was 200 nM. For the double-stranded DNA (dsDNA), the diluted oligonucleotide probes (2 µL, 0.2 mM in 0.15 M NaCl/0.1 M phosphate buffer solution at pH 7) were hybridized with their complementary or single base mismatched oligonucleotide (2 µL, 0.2 mM in 0.15 M NaCl/0.1 M phosphate buffer solution at pH 7). Then, dsDNA (4 µL, 0.1 mM) was added to CdS quantum dot suspension (1.3 mL). The next procedures were identical to the above procedure. 2.4. Stringency Titration of DNA. For a stringency titration of BRCA2 DNA, oligomers at nanomolar concentration were incubated in an aqueous solution with NaCl concentrations of 8 × 10-4, 1.2 × 10-3, and 1.6 × 10-3 M. They were subjected to polyacrylamide gel electrophoresis (15%) at 70 V for approximately 1 h. The gel was stained with ethidium bromide (1 µg/mL) and visualized using a UV trans-illuminator (Vilber Lourmat Model ETX-40.m). 2.5. Characterization. The quasi-elastic light scattering (QELS) and zeta potential measurements were used to monitor the number-average hydrodynamic radius and the surface potential of the particles with a Malvern Nano-ZS instrument. Photoluminescence spectra of CdS quantum dots were taken at room temperature using a PerkinElmer LS50 fluorescence spectrometer with a xenon lamp (equivalent to 20 kW for 8 µs duration) as the excitation source. Transmission electron microscopy (TEM, JEOL JEM-2100) was used to observe the morphology of CdS quantum dots. 3. Results and Discussion To confirm duplex formation at all NaCl concentrations, a stringency titration of BRCA2 DNAs was performed in the absence of CdS quantum dots (Figure S1 in Supporting

Figure 2. Pictures of CdS quantum dot suspensions after addition of DNAs at 1.2 × 10-3 M NaCl: after (a) 10 min (BRCA2); (b) 30 min (BRCA2); (c) 10 min (Sipa1); (d) 30 min (Sipa1); (i) ssDNA (probe); (ii) dsDNA (mismatch); (iii) dsDNA (target).

Figure 3. TEM images of CdS quantum dot after the addition of BRCA2 dsDNAs: (a) mismatch; (b) target.

Information). The dsDNAs with the perfectly matched or single base mismatched sequence could be visualized with ethidium bromide staining whereas the ssDNA was not seen because of the staining properties of ethidium bromide. These results were consistent at all NaCl concentrations without showing any significant concentration dependence. Figure 2 shows the pictures of the suspensions under the UV lamp (at the excitation wavelength of 365 nm) after the addition of DNAs at optimal salt concentration (vide infra) for both BRCA2 and Sipa1 sequences. Color change was negligible during the initial 30 min in all cases. Interestingly, 30 min after the addition of DNAs the luminescence intensity decreased substantially with a significant bleaching of color only in the suspension containing the dsDNA with the perfectly matched pair, presumably due to a CdS quantum dot aggregation. This may provide a more easily discernible method than our previous method using gold nanoparticles.17 To visualize aggregation of CdS quantum dots, TEM images were taken after the addition of BRCA2 dsDNAs at 1.2 × 10-3 M NaCl (Figure 3). No significant change in size and morphology was observed for the dsDNA with a single base mismatched pair. After the addition of the dsDNA with the perfectly matched sequence, though, the size and morphology of CdS quantum dots exhibited the change that is a result of CdS quantum dot aggregation. To investigate the aggregation behavior of quantum dots, QELS was used to monitor the aggregation process for BRCA2 sequence (Figure 4). At low salt concentration (8.0 × 10-4 M), the average diameter of nanocrystals did not show any increase whereas a significant increase in the average diameter was found in all cases at high NaCl concentration (1.6 × 10-3 M). Quantum dots appear to be colloidally stable against aggregation at low salt concentration, but they become unstable at high salt concentration because salts screen the electrostatic repulsion

Detection of Point Mutation in DNA Sequences

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Figure 4. Change in the average diameter as a function of time after the addition of probe, single base mismatched, and perfectly matched BRCA2 DNAs at different NaCl concentrations: (a) 8 × 10-4 M; (b) 1.2 × 10-3 M; (c) 1.6 × 10-3 M.

Figure 5. Photoluminescence spectra of CdS quantum dots as a function of time after the addition of dsDNAs at the optimal NaCl concentration: (a) BRCA2 mismatch; (b) BRCA2 target; (c) Sipa1 mismatch; (d) Sipa1 target.

between particles. Note that a significant aggregation was observed exclusively for the perfectly matched dsDNA at 1.2 × 10-3 M which is the optimal salt concentration for selective aggregation. It has been suggested that ssDNA can rapidly adsorb onto the nanoparticle surface to act as a stabilizer whereas dsDNA that has a stable double-helix geometry with the negatively charged phosphate backbones does not easily adsorb on the surface, but screens the electrostatic repulsion.20,23 In addition, the dsDNA with the single base mismatch was shown to exhibit a significant dehybridization, followed by the stabilization from dehybridized ssDNA (Figure 1).20 At the optimal salt concentration (1.2 × 10-3 M), therefore, quantum dots could be stabilized in the presence of ssDNA or dsDNA with the single base mismatch. To test this hypothesis, we monitored the zeta potential as a function of time at different NaCl concentrations for BRCA2

sequence (Figure S2 in Supporting Information). The zeta potential value of the unmodified CdS quantum dots was initially -51.6 mV. At low salt concentration (8 × 10-4 M NaCl), all samples showed a negligible change in zeta potential, meaning that the colloidal stability of CdS suspension was not affected by the addition of electrolyte or DNA. At high salt concentration (1.6 × 10-3 M NaCl), however, all samples showed a significant decrease in the magnitude of zeta potential (ca. -30 mV), leading to a reduced electrostatic repulsion and subsequent aggregation of CdS quantum dots. At the optimal salt concentration (1.2 × 10-3 M NaCl), only the sample with the perfectly matched dsDNA showed a significant decrease in the magnitude of zeta potential because dsDNA did not adsorb on the particle surface, but probably screened the electrostatic repulsion. Note that this was consistent with the QELS and zeta potential results from Sipa1 sequence (Figures S3 and S4 in Supporting Information). This confirms the selective aggregation of quantum

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dots in the presence of the perfectly matched dsDNA due to the reduced colloidal stability. Thus, by changing the salt concentration it was possible to control the colloidal stability and induce selective aggregation of unmodified quantum dots exclusively for the perfectly matched dsDNA. Figure 5 compares the photoluminescence (PL) spectra of CdS quantum dots after the addition of dsDNAs at the optimal NaCl concentration. For both BRCA2 and Sipa1 sequences, addition of dsDNA caused a significant decrease in the PL intensity after 30 min for the dsDNA with perfectly matched sequence while the PL intensity change was negligible for the dsDNA with a single base mismatched pair. In all cases, decrease in the photoluminescence intensity was negligible at lower salt concentration, whereas the significant decrease in intensity was observed at higher salt concentration (Figure S5 in Supporting Information). We could not, therefore, discriminate the perfectly matched sequence from the single base mismatched pair at other NaCl concentrations. The fluorescence quenching phenomenon results from aggregation of the CdS quantum dots, which agrees well with the QELS and TEM results. In addition, the fluorescence peak maximum shifted to longer wavelengths (i.e., from 570 to 590 nm for BRCA2 and from 550 to 570 nm for Sipa1) with increasing aggregate size.25,26 To determine the sensitivity limit of our method, we simply reduced the reactor volume and found that the single base mismatch could be discerned up to ca. 5 pmol DNA. We believe that sensitivity limit can further be improved by optimizing the reactor composition. 4. Conclusions In this paper, we have attempted to detect DNA sequences using the selective aggregation of CdS quantum dots. Our results indicate that the dsDNA with a perfectly matched sequence can easily be discerned by naked eye from the single base mismatched one using the fluorescence quenching phenomenon caused by selective aggregation of the CdS quantum dots. To design a robust assay based on this method, though, it is still necessary to make a few improvements. For instance, the narrow range of optimal salt concentration for selective aggregation can be improved by fine-tuning the composition and colloidal stability of CdS quantum dots for practical assay. This method is advantageous to other methods in detection of DNA sequences due to its speed, simplicity, and low cost. This work will help the development of sensors which are ubiquitous in many science and engineering fields including chemistry, physics, material science, and biological and medical community. Acknowledgment. K.L. thanks the NRF for financial support through Grant no. M10755020001-08N5502-00110 and National

Kim et al. Core Research Center for Nanomedical Technology (R15-2004024-00000-0). Supporting Information Available: Stringency titration data; QELS and zeta potential as a function of time for BRCA2 and Sipa1 DNAs; PL spectra at nonoptimal salt concentrations. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Alivisatos, A. P. J. Phys. Chem. 1996, 100, 13226–13239. (2) Alivisatos, A. P. Science 1996, 271, 933–937. (3) Yin, Y.; Alivisatos, A. P. Nature 2005, 437, 664–670. (4) Peng, X.; Manna, L.; Yang, W.; Wickham, J.; Scher, E.; Kadavanich, A.; Alivisatos, A. P. Nature 2000, 404, 59–61. (5) Hu, J.; Li, L.; Yang, W.; Manna, L.; Wang, L.; Alivisatos, A. P. Science 2001, 292, 2060–2063. (6) Pinaud, F.; Michalet, X.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Iyer, G.; Weiss, S. Biomaterials 2006, 27, 1679–1687. (7) Larson, D. R.; Zipfel, W. R.; Williams, R. M.; Clark, S. W.; Bruchez, M. P.; Wise, F. W.; Webb, W. W. Science 2003, 300, 1434– 1436. (8) Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759–1762. (9) Gill, R.; Zayats, M.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 7602–7625. (10) Daniel, M.-C.; Astruc, D. Chem. ReV. 2004, 104, 293–346. (11) Kang, S. H.; Bozhilov, K. N.; Myung, N. V.; Mulchandani, A.; Chen, W. Angew. Chem., Int. Ed. 2008, 47, 5186–5189. (12) Sato, K.; Hosokawa, K.; Maeda, M. J. Am. Chem. Soc. 2003, 125, 8102–8103. (13) Liu, J.; Cao, Z.; Lu, Y. Chem. ReV. 2009, 109, 1948–1998. (14) Liu, J.; Lu, Y. J. Am. Chem. Soc. 2005, 127, 12677–12683. (15) Kim, T.; Lee, K.; Gong, M.-S.; Joo, S.-W. Langmuir 2005, 21, 9524–9528. (16) Kim, T.; Lee, C.-H.; Joo, S.-W.; Lee, K. J. Colloid Interface Sci. 2008, 318, 238–243. (17) Cho, K.; Lee, Y.; Lee, C.-H.; Lee, K.; Kim, Y.; Choi, H.; Ryu, P.-D.; Lee, S. Y.; Joo, S.-W. J. Phys. Chem. C. 2008, 112, 8629–8633. (18) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607–609. (19) Peng, H.; Soeller, C.; Vigar, N.; Kilmartin, P. A.; Cannell, M. B.; Bowmaker, G. A.; Cooney, R. P.; Travas-Sejdic, J. Biosens. Bioelectron. 2005, 20, 1821–1828. (20) Li, H.; Rothberg, L. Proc. Natl. Acad. Sci. 2004, 101, 14036–14039. (21) Healey, C. S.; Dunning, A. M.; Teare, M. D.; Chase, D.; Parker, L.; Burn, J.; Chang-Claude, J.; Mannermaa, A.; Kataja, V.; Huntsman, D. G.; Pharoah, P. D. P.; Luben, R. N.; Easton, D. F.; Ponder, B. A. J. Nat. Genet. 2000, 26, 362–364. (22) Park, Y. G.; Zhao, X.; Lesueur, F.; Lowy, D. R.; Lancaster, M.; Pharoah, P.; Qian, X.; Hunter, K. W. Nat. Genet. 2005, 37, 1055–1062. (23) Li, H.; Nelson, E.; Pentland, A.; Van Buskirk, J.; Rothberg, L. Plasmonics 2007, 2, 165–171. (24) Mao, J.; Yao, J.-N.; Wang, L.-N.; Liu, W.-S. J. Colloid Interface Sci. 2008, 319, 353–356. (25) Chan, W. C. W.; Nie, S. Science 1998, 281, 2016–2018. (26) Mattoussi, H.; Mauro, J. M.; Goldman, E. R.; Anderson, G. P.; Sundar, V. C.; Mikulec, F. V.; Bawendi, M. G. J. Am. Chem. Soc. 2000, 122, 12142–12150.

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