J. Phys. Chem. C 2008, 112, 8629–8633
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Selective Aggregation Mechanism of Unmodified Gold Nanoparticles in Detection of Single Nucleotide Polymorphism Kyungnam Cho,† Yunhee Lee,† Chang-Ha Lee,† Kangtaek Lee,*,† Youngmin Kim,‡ Hyoungwoo Choi,‡ Pan-Dong Ryu,§ So Yeong Lee,§ and Sang-Woo Joo*,‡ Department of Chemical Engineering, Yonsei UniVersity, Seoul 120-749 Korea, Department of Chemistry, Soongsil UniVersity, Seoul 156-743 Korea, and Department of Pharmacology, College of Veterinary Medicine, Seoul National UniVersity, Seoul 151-742 Korea ReceiVed: February 5, 2008; ReVised Manuscript ReceiVed: March 16, 2008
Detection of single point mutation based on the hybridization of oligonucleotides was performed using unmodified gold nanoparticles. The sequences of oligonucleotides were designed to detect the metastatic efficiency modifier signal-induced proliferation-associated gene 1 (Sipa1). The detection step was monitored using UV-vis absorption spectroscopy, quasielastic light scattering, and zeta potential measurement. We observed that addition of DNAs into the suspension of unmodified gold nanoparticles could substantially aggregate the gold nanoparticles and change the color of solution. By changing the salt concentration in the presence of a phosphate buffer solution, we were able to selectively aggregate gold nanoparticles for the perfectly matched DNA, which enabled a detection of perfectly matched DNA from the single point-mutated one. Our results indicate that a change in the electrostatic interaction is responsible for the selective aggregation of gold nanoparticles upon the addition of DNA. This suggests a novel design principle for a rapid detection of the DNA sequence by controlling the electrostatic interactions between gold nanoparticles. TABLE 1: DNA Sequences Tested in the Experimenta
1. Introduction Detecting specific DNA sequences is of great importance in numerous applications, including medical research and clinical diagnosis.1 Intensive effort has been devoted to developing detection methods for both sensitive and low-cost analysis of gene expression and single nucleotide polymorphism (SNP) identification.2–5 In most cases, DNA sensors are based on the hybridization of single-stranded target oligonucleotide probes with their complementary tagging oligonucleotide probes. Gold nanoparticles have been extensively used for the colorimetric detection of DNA hybridization due to their unique optical properties.6–9 Because oligonucleotide-modified gold nanoparticles aggregate to change color more distinctly for the perfectly matched single-stranded DNA than for single-base mismatched DNA, they could be utilized to detect a singlebase mismatch. Disadvantages of such detection methods, however, include their cost, complexity, and the inefficiency of tagging reactions.10–13 Li and Rothberg recently reported that single- and doublestranded DNA (ssDNA and dsDNA, respectively) have different propensities to adsorb onto unmodified gold nanoparticles, due to their dissimilar electrostatic interactions, and that single-basepair mismatched and perfectly matched dsDNAs exhibit different stability against dehybridization.14 These characteristics provide a rapid and simple method for detecting the sequences of various types of DNAs. The fact that the detection step is decoupled from the DNA hybridization step makes this system very useful for studying the detection process. In utilizing gold nanoparticles as biosensors for DNA detection, Sato et al. has shown that a suspension changes color * Author e-mail: K.L.:
[email protected], S.-W. J.:
[email protected]. † Yonsei University. ‡ Soongsil University. § Seoul National University.
oligonucleotides
sequences
probe perfect complementaryb single-base mismatchedb
5′-CAG CAT CTG GGC AGC-3′ 5′-GCT GCC CAG ATG CTG-3′ 5′-ACT GCC CAG ATG CTG-3′
a Note that the ssDNA of the 5′f3′ direction hybridize with the complementary ssDNA of the 3′f5′ direction. b The underlined base indicates the base mismatch.
according to the change in salt concentration.8 Thus, controlling the interactions between particles should play an important role in the detection process, and hence provides a new possibility for detecting DNA sequences.15–21 In this paper, we investigate the selective aggregation mechanism of unmodified gold nanoparticles in detection of single nucleotide polymorphism based on the DNA hybridization. The detection step is monitored using UV-vis absorption, quasielastic light scattering (QELS), and zeta potential measurements. The goals of this paper are to explain the selective aggregation of gold nanoparticles for the perfectly matched DNA and, ultimately, to develop a new method for rapidly detecting single-base-mismatched pairs. 2. Experimental Section 2.1. Preparation of Gold Nanoparticles. Citrate-reduced gold nanoparticles were chemically synthesized according to the recipe of Lee and Meisel.22 The resulting Au ion concentration was ∼10-3 M, and the concentration of the gold nanoparticles and ionic strength were estimated to be ∼9 × 10-9 and ∼2 × 10-2 M, respectively. 2.2. Synthesis of Oligonucleotides. Oligonucleotide sequences were designed to detect the signal-induced proliferationassociated gene 1 (Sipa1) since Sipa1 polymorphism is known to be associated with metastatic process.23 The single-stranded oli-
10.1021/jp801078m CCC: $40.75 2008 American Chemical Society Published on Web 05/15/2008
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Figure 1. Experimental scheme at 0.025 M NaCl.
Figure 2. UV-vis extinction spectra of gold nanoparticle solutions for ssDNA, SNP, and perfectly matched dsDNAs at NaCl concentrations of (a) 0.015, (b) 0.025, and (c) 0.040 M.
gonucleotide probe, the oligonucleotides with its perfectly complementary sequence, and the sequence with a single mismatched base (adenine instead of guanine for the single nucleotide polymorphism (SNP) mutation) were designed based on the mouse Sipa1 sequence (GenBank number: NM_011379) (Table 1). The sequence of oligonucleotides was synthesized from Bioneer Inc. (Daejeon,
Korea) and purified by the polyacrylamide gel electrophoresis method, and their molecular weights were checked by a MALDITOF mass spectrometer. The melting temperature of the oligonucleotides was estimated to be ∼47 °C. 2.3. DNA Hybridization and Detection. For the ssDNA, stock probe DNA (1 mM) was diluted 10 times in 0.15 M NaCl/
Selective Aggregation of Gold Nanoparticles
J. Phys. Chem. C, Vol. 112, No. 23, 2008 8631 hybridized with their complement oligonucleotide or single mismatched oligonucleotide (0.2 mM, 2.5 µL, in 0.15 M NaCl, 0.1 M phosphate buffer at pH 7), respectively. Then, dsDNA (0.1 mM, 1µL) was added to 0.3 mL of gold nanoparticle suspension. The next procedures were identical to the above procedure. Note that increasing the DNA concentration resulted in the aggregation of gold nanoparticles in all cases (results not shown). Figure 1 shows our experimental scheme at 0.025 M NaCl.
Figure 3. Typical images of gold nanoparticle suspensions for (a) ssDNA, (b) SNP, and (c) perfectly matched dsDNA at a salt concentration of 0.025M.
0.1 M phosphate buffer at pH 7. The aliquot of DNA solution (0.1 mM, 1 µL) was added to 0.3 mL of gold nanoparticle suspension. Then, 0.1 mL of distilled water and 0.05 mL of the same buffer were added to prevent abrupt aggregation. Finally, the NaCl solutions with concentrations of 0.015, 0.025, and 0.040 M were added to comparatively examine the aggregation behaviors. The final concentration of DNA solution was 200 nM. For the dsDNA, the diluted oligonucleotide probes (0.2 mM, 2.5 µL, in 0.15 M NaCl/0.1 M phosphate buffer at pH 7) were
For a stringency titration of DNAs alone, oligomers at nanomolar concentration were incubated in an aqueous solution with the NaCl at concentration of 0.015, 0.025, and 0.040 M. They were separated on 15% polyacrylamide gel electrophoresis, and the gel was stained with ethidium bromide (1 µg/mL) and visualized using a UV trans-illuminator (Vilber Lourmat Model ETX-40.m). 2.4. Characterization. UV-visible absorption spectroscopy, QELS, and zeta potential measurements were used to monitor the detection step. UV-vis absorbance spectrum of the gold nanoparticle solution was taken using a Shimadzu 1650 PC spectrophotometer. The data acquisition time of the UV-vis spectrum was approximately 3-4 min. The hydrodynamic radii of particles were monitored by the quasielastic light scattering technique using the Nano-ZS instrument from Malvern Instrument Co. Zeta potential measurements were performed on dilute
Figure 4. Change in the average particle diameter as a function of time for ssDNA, SNP, and perfectly matched dsDNAs at NaCl concentration of (a) 0.015, (b) 0.025, and (c) 0.040 M. Lines were drawn as a guide to the eye.
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Figure 5. Change in the zeta potential as a function of time for ssDNA, SNP, and perfectly matched dsDNAs at NaCl concentration of (a) 0.015, (b) 0.025M, and (c) 0.040 M. Lines were drawn as a guide to the eye.
also be estimated from the integrated extinction between 600 and 800 nm.24,25 Note that the intensity of all absorption spectra decreases after the addition of salt/buffer solution, probably due to the dilution effect.
Figure 6. Polyacrylamide gel electrophoresis results for stringency test of oligomers without gold nanoparticles. Sample 1: ssDNA at 0.015 M NaCl; Sample 2: dsDNA for perfect match at 0.015 M NaCl; Sample 3: dsDNA for single-base mismatch at 0.015 M NaCl; Sample 4: ssDNA at 0.025 M NaCl; Sample 5: dsDNA for perfect match at 0.025 M NaCl; Sample 6: dsDNA for single-base mismatch at 0.025 M NaCl; Sample 7: ssDNA at 0.040 M NaCl; Sample 8: dsDNA for perfect match at 0.040 M NaCl; Sample 9: dsDNA for single-base mismatch at 0.040 M NaCl.
dispersions of the aggregates using the Nano-ZS instrument from Malvern Instrument Co. 3. Results and Discussion UV-vis absorption spectra were obtained to examine the spectral changes of surface plasmon bands of gold nanoparticles after the addition of oligonucleotides at different NaCl concentrations (Figure 2). Aggregation of gold particles is known to be accompanied by a red shift in a UV-vis absorption spectrum due to a decrease in the interparticle distance during a selfassembly process.6 The extent of aggregation (flocculation) can
A negligible change in UV-vis spectra was observed at 0.015 M NaCl in all cases (ssDNA and dsDNA with perfect match and single-base mismatch), which suggests that gold nanoparticles do not aggregate at this concentration (Figure 2a). At 0.04 M NaCl, though, there was a red shift in the surface plasmon band and an increased extinction between 600 and 800 nm, indicating that aggregation occurs in all cases (Figure 2c). It was the 0.025 M NaCl at which only the dsDNA with the perfectly matched sequence produced a pronounced increase in the UV-vis absorption band between 600 and 800 nm (i.e., aggregation). This suggests a possibility for a detection of singlebase mismatch in DNA at this concentration. Figure 3 shows the color change of gold nanoparticle solutions at 0.025 M NaCl upon the addition of oligonuceotides. After the addition of the ssDNA or the dsDNA with a single-base mismatched pair, the color change was negligible compared to that of the citrate-reduced gold solution as shown in Figures 3a-b. After the addition of the dsDNA with the perfectly matched sequence; however, the solution exhibited a significant color change as shown in Figure 3c, which is a result of gold nanoparticle aggregation. This confirms that the dsDNA with a perfect sequence can be discerned from the single-base mismatched strand at the optimal NaCl concentration (0.025 M
Selective Aggregation of Gold Nanoparticles NaCl). This is also consistent with the previous report by Li and Rothberg.14 To monitor the different aggregation behavior of gold nanoparticles, QELS was used in Figure 4. Note that particles aggregated to some extent in all cases because addition of salt/ buffer solution destabilizes gold nanoparticles by screening electrostatic repulsion between nanoparticles. The aggregation behaviors were almost identical for three DNA strands at the NaCl concentrations of 0.015 and 0.040 M, as shown in Figure 4, panels a and c, respectively. The aggregate size was larger at 0.040 M than at 0.015 M NaCl because higher salt concentration reduces the electrostatic repulsion between particles and more efficiently induces aggregation. Note that the aggregate size for the perfectly matched DNA was much larger than the others at the intermediate concentration (0.025 M) as shown in Figure 4b. This suggests that by changing the salt concentration it is possible to control the electrostatic repulsion between gold nanoparticles to induce selective aggregation for the perfectly matched DNA. In a recent study,14 it has been suggested that single- and double-stranded oligonucleotides (ssDNA and dsDNA) have different electrostatic properties. The ssDNA can sufficiently uncoil to expose its bases, but the dsDNA has stable doublehelix geometry with the negatively charged phosphate backbones. Thus, the ssDNA could preferentially adsorb onto the gold nanoparticle surface and could be used to stabilize particles, whereas the dsDNA remained in the solution and screened the electrostatic repulsion to induce aggregation. In addition, the dsDNA with the single-base mismatch exhibited a significant dehybridization, resulting in adsorption of ssDNA on the particle surface and stabilization. These characteristics may explain the presence of the optimal salt concentration that can selectively destabilize and aggregate gold nanoparticles for the perfectly matched dsDNA. To confirm this hypothesis, we have monitored the zeta potential of aggregates during aggregation. Figure 5 shows the change in zeta potential as a function of time at different NaCl concentration. Zeta potential for the unmodified gold nanoparticles was -38.2 mV, which is consistent with the previous reports.15,16,20 In all cases, the zeta potential quickly (
