Oligonucleotide Adsorption to Gold Nanoparticles: A Surface

Nov 14, 2001 - We have examined the adsorption of short 16 base pair DNA to 14 nm protein-sized gold particles using surface-enhanced Raman spectrosco...
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J. Phys. Chem. B 2001, 105, 12609-12615

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Oligonucleotide Adsorption to Gold Nanoparticles: A Surface-Enhanced Raman Spectroscopy Study of Intrinsically Bent DNA Latha A. Gearheart, Harry J. Ploehn,† and Catherine J. Murphy* Department of Chemistry and Biochemistry, UniVersity of South Carolina, 631 Sumter St., Columbia, South Carolina 29208 ReceiVed: February 19, 2001; In Final Form: October 2, 2001

We have examined the adsorption of short 16 base pair DNA to 14 nm protein-sized gold particles using surface-enhanced Raman spectroscopy and electronic absorption spectroscopy. In the presence of KCl, we find greater Raman enhancement of adenine and guanine ring breathing vibrations (per adenine and guanine present in each sequence) for crystallographically “kinked” oligonucleotide than for the DNA-particle interactions for “bent” A-tract and “straight” oligonucleotides. The calculated binding constants are within the range of nonspecific protein-DNA interactions and are nearly the same for each sequence. The difference in the surface plasmon frequency of the gold particles in the presence of either straight, bent, or kinked DNA is attributed to differences in the DNA-mediated aggregation of the nanoparticles. Specifically, the displacement of the gold’s stabilizing citrate ions seems to vary with DNA sequence, and this controls the residual interparticle electrostatic interaction and average interparticle separation in the aggregate.

Introduction The ability of DNA to wrap and bend about nanometer-scale substrates is critical in its packaging1 and transcription,2-5 and the intrinsic sequence-dependent structure and flexibility of DNA surely influence the wrapping and bending of the double helix.6-15 In our laboratory, we have used colloidal inorganic quantum dots as probes of DNA bending, with the photoluminescence of the nanoparticle as the signal of binding.16-19 We have found that oligonucleotides exhibiting curvature on the ∼3 nm scale in crystallographic20-25 and biochemical20,25-30 experiments do bind more tightly and more quickly to nanoparticles that have curvature on a similar length scale.16,17 The details of the binding modes, however, and the dissection of the binding event into static and dynamic components31-33 are still being explored. One disadvantage of the photoluminescence signal is that it does not provide chemical information per se about the DNA adsorbate. Here, we report our results of the binding of oligonucleotides to nanoscale metallic substrates, as monitored by surface-enhanced Raman spectroscopy (SERS) and electronic absorption spectroscopy. In SERS, the Raman vibrational modes of a molecule located in close proximity to a nanosized metal surface are strongly enhanced, providing structural and orientational information about the adsorbate.34-36 Enhancements on the order of 103-106 are typical, and single molecule detection, with enhancements greater than 1014, have been demonstrated.37-41 Because of its extraordinary signal enhancement over normal Raman scattering, SERS has been widely used in biological systems;38,42-49 however, little work has been done with welldefined oligonucleotides. We show that SERS of low concentrations of 16 base-pair “straight”, “bent”, and “kinked” oligonucleotides on gold particles is experimentally observable and that the intensity of the enhanced vibrational modes of the bases within the sequence is dependent on intrinsic DNA curvature. * To whom correspondence should be addressed. † Rm 2C02, Department of Chemical Engineering, Swearingen Engineering Center, University of South Carolina, Columbia, SC 29208.

The difference in SERS intensity appears to be a result of sequence-dependent DNA-controlled aggregation of the nanoparticles, as judged by electron absorption spectroscopy and transmission electron microscopy. When metal nanoparticles aggregate, their surface plasmon resonance shifts to longer wavelengths depending on the size and shape of the aggregate and particle and the interparticle distance within the aggregate.50,51 Exciting the surface plasmon at a wavelength that closely matches the plasmon resonance frequency generates a stronger electromagnetic field in the vicinity of the adsorbate, thus, providing greater enhancement.35,36,52,53 Others have controlled aggregation of gold nanoparticles upon DNAmediated assembly by covalently attaching complementary oligonucleotides to the nanoparticles and annealing the sequences.54-58 Unexpectedly, we have observed that the morphology of double stranded oligonucleotides, physisorbed to the surface of gold nanoparticles, also influences nanoparticle aggregation. Experimental Section Materials. Sodium citrate (Fisher), NaBH4 (Aldrich), and HAuCl4‚3H2O (Sigma) were used as received. Oligonucleotides were synthesized and HPLC-purified by Genemed Synthesis: 5′-GGCAACCTGAGGACCC-3′ and complement as a straight duplex; 5′-GGTCCAAAAAATTGCC-3′ and complement as a bent duplex; and self-complementary 5′-GGTCATGGCCATGACC-3′ as a kinked duplex. Each oligonucleotide was received as a pellet in its single stranded form and dissolved in 5 mM tris, 5 mM NaCl buffer solution (pH 7.2). Straight and bent oligonucleotides were combined with their respective complement in equimolar concentrations (per nucleotide) for annealing. After combining the complementary strands, the straight and bent oligo solutions and the self-complementary kinked solution were annealed by first heating the samples to 94 °C for 5 min and then allowing them to cool, slowly, to room temperature. DNA melting temperature studies were performed using circular dichroism and absorption spectroscopy to confirm the annealed oligonucleotides were double-stranded at room temperature. All

10.1021/jp0106606 CCC: $20.00 © 2001 American Chemical Society Published on Web 11/14/2001

12610 J. Phys. Chem. B, Vol. 105, No. 50, 2001 oligonucleotides were stored at -20 °C. Ultrapure deionized water (Continental Water Systems) was used for all solution preparations and experiments. Instrumentation. All SERS measurements were performed using a Detection Limits Solution 633 Raman system. The system utilizes a filtered fiber-optic probe equipped with a microscope objective to focus radiation from a 633 nm helium neon laser on to the sample and collect the 180° Raman backscatter. Laser power at the sample was 25 mW, and a 180 s integration time was used for each measurement. Spectral acquisition and processing were enabled with DLSPEC and GRAMS/32 (Galactic Industries) software. Electronic absorption spectra were collected with a CARY 500 Scan UV-vis-NIR spectrophotometer. Particle sizing and aggregation studies of the gold colloids were performed using a Hitachi H8000 electron microscope. Circular dichroism measurements were made using an Olis RSM 1000 CD spectrophotometer. Synthesis of Gold Nanoparticles. Solutions of citratereduced gold nanoparticles were prepared according to the Frens method.59 All glassware was rigorously cleaned in aquaregia solution before use. Aqueous stock solutions of 0.01 wt % HAuCl4‚3H2O and 1 wt % sodium citrate were prepared. A total of 100.0 mL of the HAuCl4‚3H2O solution was brought to a boil while stirring, and 3.0 mL of the sodium citrate solution was added. The reaction mixture underwent a series of color changes before finally turning a wine red. The boiling was continued for 30 min after the final color change. After cooling to room temperature, the gold nanoparticle solution was diluted to 100.0 mL using DI water. Prior to addition of salt or analyte, the gold solutions consist of mostly isolated, almost spherical, particles with a mean diameter of 14 ( 2 nm and give rise to a relatively sharp extinction maximum at 517 nm. However, a small percentage of particles varying in diameter from 1 to 10 nm are also present. We estimate that fewer than 5% of the nanoparticles in this preparation are less than 10 nm in diameter. Solutions of borohydride-reduced Au colloids were also prepared. First, 147 µL of a 1% sodium citrate solution was added to 20 mL of 0.01% HAuCl4‚3H2O. (Here, citrate is used as a stabilizer.) Next, a fresh solution of 10 mM NaBH4 was prepared, and 600 µL of it was added to the HAuCl4-citrate solution while stirring. Stirring was stopped within 20 s after the NaBH4 was added. The final color of the solution was red. The solution was allowed to stand 4 h before use to ensure the borohydride completely reacted. The average diameter of the particles was 5 ( 2 nm. Au nanoparticle solutions prepared this way typically remained stable no longer than 24 h. SERS and UV-vis Titration Experiments. Each sample was prepared by first combining 25.0 µL of 0.1 M KCl with 5 mM tris, 5 mM NaCl buffer (pH 7.2) and DNA. The volume of the buffer and DNA were varied by 2 µL increments while maintaining a combined volume of 25.0 µL of DNA and buffer. Next, 50.0 µL of gold nanoparticle solution was added, making the total sample volume 100 µL. The entire sample was vortexed, then transferred to a quartz cuvette for spectroscopic measurements. No SERS signals could be detected in the absence of additional salt under our conditions. The presence of salt assists in particle aggregation which in turn tunes the surface plasmon band of the gold particles to better match the laser excitation frequency. After testing various concentrations of KCl, MgCl2, ZnCl2, and CdCl2, KCl was chosen as the aggregating agent. The order in which salt was added was very important. Adding KCl to the gold solution before or after DNA resulted in a weaker Raman signal compared to combining the salt, buffer,

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Figure 1. SERS of (a) 0.1 mM double-stranded bent DNA, 5′GGTCCAAAAAATTGCC-3′ and complement and (b) 0.1 mM singlestranded 5′-GGTCCAAAAAATTGCC-3′ in gold nanoparticle solution. Both solutions contain 0.025 mM KCl and 0.0013 mM tris + NaCl buffer.

and DNA together first, and then adding the gold nanoparticle solution. Combining KCl with nanoparticles before adding DNA resulted in the weakest signal. Without the initial presence of DNA, the KCl caused the particles to immediately aggregate and fall out of solution within 5 min. The gold surface plasmon band of the KCl-induced aggregate was relatively broad and weak and did not correspond well with the frequency of the excitation source. Adding KCl after combining the DNA with gold nanoparticles resulted in only a slightly weaker SERS signal compared to combining DNA and KCl before nanoparticle addition. The absorption spectrum of each sample was measured ∼2 min after combining the DNA, salt, buffer, and gold nanoparticles, and the SERS measurements were measured immediately after the UV-vis measurements. Neither the absorbance of the solutions nor their SERS spectra changed from t ) 2 min to t ) 24 h. Titrations performed a few minutes after mixing gave the same results as titrations performed 24 h after mixing. Results and Discussion DNAs containing A-tracts, with 5′-An-3′ (where n ) 3-7) phased one full double-helical twist apart, appear to induce curvature in the context of a long strand (∼102 bp) of DNA in gel mobility, enzyme cyclization kinetics, and transmission electron/scanning probe microscopy experiments.28,29,60-64 The overall curvature observed in these experiments is due to individual ∼18° bends per A-tract summed over the entire strand. Another sequence of DNA that seems to be intrinsically bent is 5′-GGCC-3′.24-27 The inner 10 base pairs of the selfcomplementary DNA sequence 5′-GGTCATGGCCATGACC3′ have been shown crystallographically,25 with multivalent counterions, to be kinked by 23° across the central GGCC and show anomalous gel mobility26,65 in the presence of divalent metal ions, indicating curvature. In solution, this GGCC sequence appears to be bent ∼70° according to fluorescence resonance energy transfer experiments.33 Using gold nanoparticles as protein-sized substrates, we were able to obtain SERS of double-stranded straight, bent, and kinked oligonucleotides. Figure 1a shows a SERS spectrum of bent DNA adsorbed to gold and is representative of all three sequences. The intense peak at 735 cm-1 is assigned to the ring-breathing vibration of adenine,66,67 and the weaker peak at 661 cm-1 corresponds to the ring-breathing mode of the other purine base, guanine.66,68,69 Other groups have shown the Raman enhancement of the adenine ring-breathing mode for single-stranded or denatured calf-thymus DNA to be much larger than that of double-stranded calf-thymus or Escherichia coli DNA adsorbed to silver

Oligonucleotide Adsorption to Au Nanoparticles

Figure 2. SERS titrations of 50 µL of gold particles with 0.0, 0.053, 0.11, 0.16, 0.21, 0.28, 0.32, 0.37, 0.43, 0.48, and 0.53 mM straight DNA (top panel); 0.0, 0.044, 0.087, 0.13, 0.17, 0.22, and 0.26 mM bent DNA (middle panel); and 0.0, 0.052, 0.10, 0.16, 0.21, 0.26, 0.31, and 0.36 mM kinked DNA (bottom panel). The bands increase in intensity as DNA is added. The concentration of KCl and tris + NaCl buffer was held constant at 0.025 mM and 0.0013 mM, respectively.

colloids.46,70,71 Surprisingly, we found that for short oligonucleotides under the given experimental conditions, SERS is possible only after annealing the DNA to make a double helix, and samples containing single-stranded oligonucleotides, dissolved in buffer at room temperature, showed no SERS (Figure 1). The single-stranded A-rich oligonucleotide of Figure 1 does not display a SERS signal for the adenine ring-breathing mode. The large enhancement of the ring-breathing mode suggests, according to some workers, that the bases (at least adenine and guanine) align perpendicularly to the surface of the particle.36,67 Thus, the DNA appears to adsorb to the surface without unwinding to lay the bases flat on the surface. We note that recent scanning probe microscopy experiments have shown that oligonucleotide duplexes that are covalently attached to gold surfaces via the 3′ end (which might be expected to stand endup on the surface) lie down on the surface as a duplex, similar to our model.88 DNA duplex formation, compared to single strands, is also favored at high salt; thus, the observation that both salt and DNA need to be premixed before exposure to the gold nanoparticles also suggests that the DNA is double-stranded in the presence of nanoparticles. We also note, however, that there is still disagreement in the literature on the interpretation of DNA base orientation from SERS data.89

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Figure 3. Extinction spectra of 50 µL of gold particles titrated with 0.0, 0.044, 0.18, 0.31, 0.44, 0.57, 0.70, and 0.84 mM straight DNA (top panel); 0.0, 0.047, 0.19, 0.33, 0.47, 0.61, 0.75, 0.89, and 1.0 mM bent DNA (middle panel); and 0.0, 0.047, 0.19, 0.33, 0.47, 0.66, and 0.75 mM kinked DNA (bottom panel). The absorption red-shifts and increases as DNA is added.

Figure 4. FSE plot for straight (diamond), bent (square), and kinked (triangle) oligonucleotides adsorbed to 14 nm gold particles (from SERS titrations). A good fit was obtained with K1 ) 0.5.

The vibrational frequency for the adenine and guanine ringbreathing modes is independent of the sequence; however, the intensity of a specific band depends on the content and order of bases in the duplex. Titrations of gold nanoparticles with the three different sequences of DNA indicate increasing SERS intensity with increasing DNA concentration (Figure 2) accompanied by a change in the extinction spectrum of the gold solution (Figure 3). We monitored the intensity increase of the 735 cm-1 band in the SERS spectrum and fit the FrischSimha-Erich (FSE) isotherm to our data72 (Figure 4):

[θ exp(2K1θ)]/(1 - θ) ) (KC)1/ν

(1)

where θ is the fractional surface coverage, which is assumed to be directly proportional to fractional change in SERS

12612 J. Phys. Chem. B, Vol. 105, No. 50, 2001 intensity; θ ) (I - I0)/(If - I0), where I is the integrated intensity of the 735 cm-1 band at different points in the titration, I0 is the initial intensity before DNA is added, and If is the intensity at which no further increase takes place as DNA is added; C is the DNA concentration in molar nucleotides; K1 is a constant that is a function of the interaction of adsorbed polymer segments and has empirically been found to give best fits when set to 0.5, as it is here;16 K is the equilibrium binding constant; and ν is related to the number of contact points between the adsorbate and the surface. Our definition of θ implicitly assumes a two-state model for the nanoparticle-DNA interaction; the nanoparticle is either bound to DNA (and SERS intensity of DNA is I to If depending on DNA concentration) or free of DNA and I0 ) 0. We previously applied the FSE adsorption isotherm to luminescence data in order to explore the interactions between protein-sized CdS quantum-dot particles with straight, bent, and kinked DNA and other sequences of DNA.16-19 From the isotherm, the binding constants (K) of duplex DNA adsorbed to 14 nm gold nanoparticles are 11 000 ( 1300, 13 000 ( 1400, and 14 000 ( 350 M-1 for straight, bent, and kinked DNA, respectively. These values are the average of three independent titrations and are in the range of nonspecific protein-DNA interactions73 and similar to those determined for DNA bound to 4 nm Cd2+-rich CdS quantum dots.16 Unlike the CdS quantum dot experiments,16,17 the calculated binding constants indicate only a weak dependence of sequencedependent affinity for the surfaces of 14 nm gold particles. This can be explained by the different sizes of the two types of nanoparticles. Relative to the length and curvature of our DNA (length 5 nm), the surface of the 14 nm gold nanoparticle appears more flat, from the DNA’s perspective, compared to the smaller 4 nm quantum dot CdS. Therefore, the curvature of the DNA does not match well with the curvature of the gold particle.

Fitting the FSE isotherm to the SERS intensity data also yields values of ν, a parameter related to adsorbate configuration. For the straight, bent, and kinked sequences, the best fit of the data gives ν values of 0.378, 0.380, and 0.287, respectively. These values suggest that the kinked DNA assumes a more extended configuration on the gold surface compared to either the straight or bent sequences (see below). The Langmuir isotherm was also fit to the SERS data (not shown). The binding constants (K) calculated from this simpler isotherm were approximately 10 000 M-1, 14 000, and 20 000 M-1 for straight, bent, and kinked DNA, respectively, indicating more prominent differences in binding between the three sequences compared to the FSE isotherm. However, the Langmuir isotherm did not fit the data as well as the FSE isotherm. We attempted to determine how much DNA was bound to the nanoparticles at the end of a titration. After separating the supernatant from particles by centrifugation, absorption at a wavelength of 260 nm failed to show any change in the solution concentration. The total adsorbed mass was too low to be measured due to the low concentration of gold particles and the relatively high DNA concentration in solution. The aggregated nanoparticle precipitates were redispersed in 100 µL of DI water, and the Raman spectrum of each sample was

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Figure 5. Extinction spectra of 50 µL of gold particles containing (a) no DNA, (b) 0.026 mM kinked DNA, (c) 0.48 mM straight DNA, (d) 0.39 mM bent DNA, and (e) 0.36 mM “kinked” DNA. All solutions contain 0.025 mM KCl and 0.0013 mM tris + NaCl buffer.

measured. The same DNA vibrational bands were observed; however, the SERS intensity decreased by ∼20% compared to the original spectra. The decrease in intensity may be due to our inefficient separation of the solid and liquid portions before redispersing the nanoparticles in DI water, or our difficulty in redispersing all of the nanoparticles after centrifugation and separation. The absorption spectra of the nanoparticles taken after separation and redispersion showed similar shape to the original solutions but ∼40% lower absorbance, consistent with inefficient redispersion of the nanoparticle-DNA aggregates. We have support for our notion that double-stranded DNAs adsorb as a duplex. In addition to the observed SERS bands corresponding to modes with base orientations consistent with duplex DNA (at least according to some authors), melting temperature experiments of solutions of DNA bound to gold indicate that the DNA is double-helical at room temperature. (The bound DNA was concentrated by combining multiple sets of particles after centrifuging and removing the unbound DNA contained in the supernatant. The combined aggregated particles plus bound DNA were redispersed in buffer.) Though the binding constants were similar for the different oligonucleotides (FSE isotherm), considerable differences in both the SERS and extinction spectra were observed. The differences appear to be due to how the nanoparticles aggregate in the presence of DNAs with different curvature. The control sample, containing no DNA, showed a broadening of the conventional 520 nm plasmon band for gold as the KCl and buffer induced particle aggregation (Figure 5a), and the color of the solution changed from red to gray-blue. These DNAfree aggregates settled out of solution within 15 min. For all samples, at relatively low DNA concentration, but also containing KCl and buffer, the gold particles appeared as individual spheres (Figure 6a), and the conventional 520 nm plasmon band remained unchanged (Figures 5b). As the concentration of DNA increased, a plasmon band emerged at lower energy between 600 and 700 nm as the particles formed larger aggregate structures (Figures 5a-e and 6b). Aggregates assembled in the presence of these oligonucleotides formed immediately (as indicated by an immediate color change) and were stable for at least 24 h. Both straight and bent DNA interacted with the gold nanoparticles to induce a nanoparticle aggregate with a plasmon band maximum around 612 nm. The interaction of kinked DNA with the particles produced aggregates with a peak maximum around 632 nm, approximately the same wavelength as the excitation source in the SERS experiments. Also, the extinction of the 632 nm plasmon band was large compared to the red shifted gold plasmon band of the straight and bent DNA samples. The

Oligonucleotide Adsorption to Au Nanoparticles

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Figure 7. SERS spectra of 0.37 mM straight DNA, 0.39 mM bent DNA, and 0.31 mM kinked DNA on 14 nm gold particles.

Figure 6. (top panel) Transmission electron micrographs of gold colloids containing 0.047 mM kinked DNA (magnification ) ×19000) and (bottom panel) 4.7 mM “kinked” DNA (magnification ) ×7200). Both solutions also contain 0.025 mM KCl and 0.0013 mM tris + NaCl buffer.

differences in absorption spectra of the particles was manifested in visible color differences in the three samples containing straight, bent, or kinked DNA, appearing reddish-pink, bluered, and blue-gray, respectively (see the Supplementary Information, Figure 1). On the basis of this spectroscopic evidence and the TEM data, it appears that the kinked DNA promotes more nanoparticle aggregation than the other two oligonucleotides. Figure 7 shows the adenine and guanine ring breathing peaks at maximum intensity for all three sequences. The intensity of the adenine ring-breathing band for straight DNA is roughly half of that for bent DNA, whereas the guanine band for straight is more intense than the same band for bent. Some of the observed differences in intensity are due to the number of purine bases present in each duplex. The bent duplex contains nine adenines and seven guanines, and the straight duplex contains only five adenines and 11 guanines. However, even for a slightly lower concentration of DNA, the adenine band for kinked DNA

is as intense as that of bent despite containing three fewer adenines per duplex. Also, the guanine band of kinked DNA is twice as intense as that of straight even though it contains the same number of guanines per duplex. The greater enhancement is due to the more red-shifted plasmon band of the gold particles complexed with the kinked duplex. The experimental conditions present a number of complicating factors that may influence our interpretation of the dependence of the gold surface plasmon frequency on DNA sequence or curvature. Lecomte and Baron have demonstrated that the presence of different cations in solution with DNA and silver colloids notably influences how the Raman signal of DNA is enhanced.71 They showed that the Raman bands of 2 × 10-5 M single-stranded calf-thymus DNA were weakly enhanced in the presence of Na+ but greatly enhanced in the presence of Mg2+. Also, the concentration of Mg2+ affected the enhancement. Samples containing double-stranded E. coli DNA required the presence of Mg2+ before SERS was observed, and solutions containing Na+ showed no enhancement. Other studies also suggest that inorganic cations, both monovalent and divalent, can locate within the solvent region and grooves of DNA and influence the structural nature of the duplex.74-83 The site at which the cations will position is likely to be base specific. Aside from the influence of specific cation-DNA interactions that may influence the SERS data, it is clear that the presence of DNA (together with salt and buffer) is required to induce rapid particle aggregation as well as a surface-enhanced Raman signal. Furthermore, the specific DNA sequence influences the nature of the aggregation process as manifested in the details of the gold particle plasmon frequency (Figure 5). These observations can be rationalized in terms of the known aggregation behavior of colloidal gold stabilized by citrate ions,59,84 especially the use of pyridine to control the aggregation rate.85 In the latter studies, neutral pyridine is used to displace charged citrate ions from the colloidal gold surface, leading to aggregation in the presence of salt. A similar phenomenon probably occurs here. The single-stranded DNAs do not adsorb because their molecular weights are below the critical threshold for adsorption.86 The double-stranded DNAs are above the critical MW threshold and adsorb, displacing citrate ions from the gold surface. In the presence of salt, rapid diffusion-limited aggregation (DLA) leads to ramified fractal structures87 (Figure 6) that settle from solution slowly because of hydrodynamic considerations. In the presence of sufficient salt but no DNA, the gold may undergo reaction-limited aggregation87 because of sufficient screening of the particles’ mutual electrostatic repulsion. This leads initially to slower formation of more compact aggregates that ultimately settle more quickly (compared to the settling rate of more ramified aggregates produced by DLA). This picture can also account for the effect of DNA sequence. For higher molecular weight adsorbates, it is well-known86 that

12614 J. Phys. Chem. B, Vol. 105, No. 50, 2001 stronger adsorption actually leads to more extended adsorbate configurations that enable more adsorbate molecules to pack onto a unit area of surface. The picture may be similar for our short double-stranded DNA adsorbates. Results from the fit of the SERS intensity data using the FSE and Langmuir isotherms (Figure 4) suggest that the kinked sequence may have a somewhat greater affinity for the gold surface and that its adsorbed configuration may be more extended. The straight DNA, adsorbed in a flatter configuration, would have lower specific adsorption (moles/area) and less efficient displacement of citrate ions. The kinked DNA, perhaps adsorbed in a more extended configuration, may have greater specific adsorption, leading to more efficient displacement of citrate ions. The bent DNA probably adsorbs in a configuration similar to that of the straight DNA. Because each sequence displaces a different fraction of the stabilizing citrate ion, the electrostatic repulsion differs, leading to different aggregation behavior as well as the strength of interparticle attraction in the aggregate. If the kinked DNA is most effective in displacing citrate ions, we expect these gold particles to have the strongest mutual attraction in the aggregated state, explaining the greater red shift and height of the corresponding peak in the extinction spectrum (Figure 5e). Less efficient citrate displacement in the straight and bent samples leads to aggregates with somewhat weaker interparticle attraction, accounting for the smaller shifts in their extinction spectra (Figure 5c,d). A small percentage of particles with diameters between 1 and 10 nm accompanied the 14 nm nanoparticles in solution. (These small particles are approximately the same diameter of the CdS particles used to preferentially bind the kinked DNA16,17). It is possible that the kinked sequence would preferentially bind to the small particles compared to straight and bent and that the differences in DNA-particle interactions for the three sequences would change the nature of particle aggregation. We attempted SERS of the three oligonucleotides using borohydride-reduced Au nanoparticles, with average diameter of 5 nm as the substrate but saw no SERS enhancement or shift in the absorption band. Absorption and SERS spectra of different ratio mixtures of citrate-reduced and borohydridereduced Au nanoparticles in equimolar concentrations of straight, bent, and kinked DNA were also measured. For each sequence, the SERS intensity decreased as the ratio of borohydride-reduced particles increased. The gold nanoparticle surface plasmon band blue shifted as the ratio of borohydride-reduced particles increased and the SERS signal all but vanished. The borohydride-reduced nanoparticles evidently competed with the citratereduced nanoparticles for DNA binding, suggesting a possible preference of 16 base-pair DNA to bind to the smaller particles. However, because smaller Au nanoparticles do not give SERS, we were unable to directly compare oligonucleotide binding to 4-5 nm CdS16,17 and Au nanoparticles. Conclusions Our experimental results show that the Raman signals of different sequences of 16 base pair double-stranded DNA adsorbed to 14 nm gold particles are enhanced because of proximity to a metallic nanosurface. Surprisingly, singlestranded DNA showed no enhancement. Binding constants calculated from the FSE isotherm are in the same range as nonspecific protein-DNA interactions; however, intrinsic curvature does not appear to affect adsorption to the much larger (compared to DNA curvature) 14 nm particles. Although strong preferential binding is not observed, kinked DNA shows greater Raman enhancement than bent, which is more enhanced than

Gearheart et al. straight, at similar concentrations. The difference in enhancement is due to differences in the position of the surface plasmon bands of the gold nanoparticles containing the different sequences of DNA. The surface plasmon frequency for gold nanoparticles complexed with kinked DNA is red-shifted to match the frequency of the excitation source. These observations can be rationalized in terms of DNA displacement of stabilizing citrate ions from the gold surface. This leads to rapid particle aggregation and ultimately to a SERS signal. Different adsorption behavior of the various sequences modifies the strength of the interparticle attraction in the aggregates, perhaps explaining the effect of sequence on the extinction spectra. Clearly, additional experiments are required to confirm this picture. In particular, experiments are needed to probe the electrostatic environment around the colloidal gold particles, characterize more directly the adsorption of short DNAs, and quantify the effect of DNA adsorption on colloidal gold aggregation. Acknowledgment. We thank the National Science Foundation for funding. Supporting Information Available: Supplementary Figure 1 shows a photograph of gold nanoparticle solutions mixed with straight, bent, and kinked oligonucleotides. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Luger, K.; Mader, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J. Nature 1997, 389, 251. (2) Schultz, S. C.; Sheilds, G. C.; Steitz, T. A. Science 1991, 253, 1001. (3) Kim, J. L.; Nikolov, D. B.; Burley, S. K. Nature 1993, 365, 520. (4) Kim, Y.; Geiger, J. H.; Hahn, S.; Sigler, P. B. Nature 1993, 365, 512. (5) Juo, Z. S.; Chiu, T. K.; Leiberman, P. M.; Baikalov, I.; Berk, A. J.; Dickerson, R. E. J. Mol. Biol. 1996, 261, 239. (6) Robinson, H.; Gao, Y.-G.; McCrary, B. S.; Edmondson, S. P.; Shriver, J. W.; Wang, A. H.-J. Nature 1998, 392, 202. (7) Ferrari, S.; Harley, V. R.; Pontiggia, A.; Goodfellow, P. N.; LovellBadge, R.; Bianchi, M. E. EMBO J. 1992, 11, 4497. (8) Lilley, D. M. Nature 1992, 357, 282. (9) Fisher, R. P.; Lisowsky, T.; Parisi, M. A.; Clayton, D. A. J. Biol. Chem. 1992, 267, 3358. (10) Raumann, B. E.; Rould, M. A.; Pabo, C. O.; Sauer, R. T. Nature 1994, 367, 754. (11) Erie, D. A.; Yang, G. L.; Schultz, H. C.; Bustamante, C. Science 1994, 266, 1562. (12) Sun, D.; Hurley, L. H. Biochemistry 1994, 33, 9578. (13) Meierhans, D.; Sieber, M.; Allemann, R. K. Nucleic Acids Res. 1997, 25, 4537. (14) Becker, S.; Groner, B.; Muller, C. W. Nature 1998, 394, 145. (15) Dickerson, R. E. Nucleic Acids Res. 1998, 26, 1906. (16) Mahtab, R.; Rogers, J. P.; Murphy, C. J. J. Am. Chem. Soc. 1995, 117, 9099. (17) Mahtab, R.; Rogers, J. P.; Singleton, C. P.; Murphy, C. J. J. Am. Chem. Soc. 1996, 118, 7028. (18) Mahtab, R.; Harden, H. H.; Murphy, C. J. J. Am. Chem. Soc. 2000, 122, 14. (19) Murphy, C. J.; Brauns, E. B.; Gearheart, L. Mater. Res. Soc. Synp. Proc. 1997, 452, 597. (20) Crothers, D. M.; Haran, T. E.; Nadeau, J. G. J. Biol. Chem. 1990, 265, 7093. (21) DiGabriele, A. D.; Steitz, T. A. J. Mol. Biol. 1993, 231, 1024. (22) Dickerson, R. E.; Goodsell, D.; Kopka, M. L. J. Mol. Biol. 1996, 256, 108. (23) Young, M. A.; Srinivasan, J.; Golier, I.; Kumar, S.; Beveridge, D. L.; Bolton, P. H. Methods Enzymol. 1995, 261, 121. (24) Dornberger, U.; Flemming, J.; Fritzshe, H. J. Mol. Biol. 1998, 284, 1453. (25) Goodsell, D. S.; Kopka, M. L.; Cascio, D.; Dickerson, R. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 2930. (26) Brukner, I.; Susic, S.; Dlakic, M.; Savic, A.; Pongor, S. J. Mol. Biol. 1994, 236, 26. (27) Brukner, I.; Dlakic, M.; Savic, A.; Susic, S.; Pongor, S.; Suck, D. Nucleic Acids Res. 1993, 21, 1025.

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