Sequence-Dependent Stability of DNA-Modified Gold Nanoparticles

The binding affinity of deoxynucleosides (dA, dG, dC, and dT) to gold nanoparticles was studied using a colorimetric assay to determine if nucleobase ...
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Langmuir 2002, 18, 6666-6670

Sequence-Dependent Stability of DNA-Modified Gold Nanoparticles James J. Storhoff,†,‡ Robert Elghanian,‡ Chad A. Mirkin,*,†,‡ and Robert L. Letsinger*,‡ Center for Nanofabrication and Molecular Self-Assembly, and Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113 Received March 11, 2002. In Final Form: May 22, 2002 The binding affinity of deoxynucleosides (dA, dG, dC, and dT) to gold nanoparticles was studied using a colorimetric assay to determine if nucleobase sequence may play a role in binding alkanethiol-capped oligonucleotides to gold nanoparticles. These data indicate that the deoxynucleoside dT has a much lower binding affinity to the gold nanoparticle surface than the deoxynucleosides dG, dC, and dA. These data can be correlated with a previous study in our lab which indicated that alkanethiol-capped oligonucleotides containing (dT)20 spacers have a significantly higher surface coverage than oligonucleotides containing (dA)20 spacers. To probe the physical consequences of this phenomenon, gold nanoparticles were loaded with alkanethiol-capped poly dT, dA, and dC oligonucleotide sequences of 5-20 bases in length, and the stabilities of the particles in electrolytic media were measured by monitoring a colorimetric change associated with particle agglomeration. The gold nanoparticles loaded with alkanethiol-capped poly dT oligonucleotides exhibited a dramatic increase in stability as the oligonucleotide length was increased from 5 to 20 bases. By comparison, gold nanoparticles loaded with poly dC and poly dA sequences exhibited only a slight increase in stability as the oligonucleotide length was increased. The sequence-dependent stability observed for the DNA-modified gold nanoparticles is attributed to the weaker interaction of the deoxynucleoside dT with the gold nanoparticle surface which results in higher surface coverages and consequently enhanced stability as the oligonucleotide length is increased.

Introduction The chemical immobilization of oligonucleotides on Au nanoparticles has led to the development of nanoscale materials that have proven useful in the development of novel biological detection technologies.1,2 With respect to DNA detection, particles that are heavily functionalized with oligonucleotides exhibit unusual hybridization properties that have led to advantages with respect to assay sensitivity and selectivity.3-6 In particular, when the particles are hybridized to complementary oligonucleotides, either in solution or immobilized on a flat surface (e.g., glass), they exhibit extraordinarily sharp melting profiles and higher melting temperatures than the analogous oligonucleotides with molecular fluorophore probes.5,7 Methods have been developed to quantify the average number of oligonucleotides on a nanoparticle surface.8 However, the nature of the interactions between the oligonucleotides and the nanoparticle surface remains a * Corresponding authors. E-mail: [email protected]; [email protected]. † Center for Nanofabrication and Molecular Self-Assembly ‡ Department of Chemistry. (1) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (2) Storhoff, J. J.; Mirkin, C. A. Chem. Rev. 1999, 99, 1849-1862. (3) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (4) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (5) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-1760. (6) He, L.; Musick, M. D.; Nicewarner, S. R.; Salinas, F. G.; Benkovic, S. J.; Natan, M. J.; Keating, C. D. J. Am. Chem. Soc. 2000, 122, 90719077. (7) Taton, T. A.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 2000, 122, 6305-6306. (8) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A., III; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535-5541.

Scheme 1

relatively unexplored area. In general, it is known that the thiol portion of the oligonucleotide covalently binds to the surface of the Au particle,9,10 but secondary interactions between the oligonucleotide and the nanoparticle are not well established. Since all of the nucleosides have functional groups (amines, carbonyls, etc.) that could act as ligands for the gold nanoparticle surface as well as phosphate groups along the backbone that could electrostatically bind, it is plausible that there could be a significant secondary interaction between the nucleosides that comprise the oligonucleotide and the gold surface, Scheme 1.8,11,12 For this reason, the protocol for loading the alkanethiol-capped oligonucleotides onto a gold nanoparticle surface involves a gradual increase in salt concentration, which has been proposed to weaken the electrostatic interaction between the oligonucleotide portion of the adsorbate and the positively charged nano(9) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir 1996, 12, 3763-3772. (10) Brust, M.; Bethell, D.; Schiffrin, D. J.; Kiely, C. J. Adv. Mater. 1995, 7, 795. (11) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 89168920. (12) Weitz, D. A.; Lin, M. Y.; Sandroff, C. J. Surf. Sci. 1985, 158, 147-164.

10.1021/la0202428 CCC: $22.00 © 2002 American Chemical Society Published on Web 07/18/2002

Stability of DNA-Modified Gold Nanoparticles

particle surface.8 In addition to the electrostatic interaction, loading is strongly dependent on oligonucleotide base sequence. For example, oligonucleotides rich in thymidine (dT) exhibit higher surface coverages than ones rich in adenine (dA) under identical loading conditions.8 Herein, we explore the interactions between the nucleosides (dA, dT, dC, and dG) and the gold nanoparticles via a novel colorimetric assay. In addition, we present a study aimed at examining the stability of nanoparticles modified with alkanethiol-capped poly dT, poly dA, and poly dC, of 5-20 bases in length. Alkanethiol-capped poly dG was not studied because it is capable of forming secondary structures through Hoogsteen base pairing, which complicates the interpretation of such studies.13 These studies provide evidence that the deoxynucleoside binding affinity (dA, dG, dT, and dC) for the Au nanoparticle surface is dependent on the nucleobase present, which can be correlated with the sequence-dependent stability and oligonucleotide surface coverage8 observed for the DNAmodified gold nanoparticles.

Langmuir, Vol. 18, No. 17, 2002 6667 Scheme 2

Experimental Section Reagents. HAuCl4‚3H2O and trisodium citrate were purchased from Aldrich. NAP-5 columns (Sephadex G-25 Medium, DNA grade) were purchased from Pharmacia Biotech (Uppsala, Sweden). 2′-Deoxyadenosine (dA) and 2′-deoxycytidine (dC) were purchased from Sigma, 2′-deoxyguanosine (dG) was purchased from Merck, and thymidine (dT) was purchased from American Bionuclear. Magnesium chloride solution (4.9 M) and sodium chloride (biological grade) were purchased from Sigma Diagnostics. 5′ HO-(CH2)6S-S(CH2)6-modified oligonucleotides were provided by Perkin-Elmer Corp. Instrumentation. Electronic absorption spectra and melting analyses were recorded using a HP 8453 diode array spectrophotometer equipped with a HP89090a Peltier temperature controller. High-performance liquid chromatography (HPLC) was performed with a HP series 1100 HPLC. An Eppendorf 5415C centrifuge was used for centrifugation of Au nanoparticle solutions. Nanopure H2O (18.1 MΩ) purified with a Barnstead NANOpure ultrapure water system was used for all experiments. Transmission electron microscopy (TEM) images of Au nanoparticle solutions were recorded with a Hitachi 8100 TEM. Preparation of Au Nanoparticles. Au particles (13-17 nm diameter) were prepared by the citrate reduction of HAuCl4.14,15 The average particle size and distribution were determined by measuring at least 200 particles by TEM. The Au nanoparticles prepared for this study were 16.2 ( 1.9 nm in diameter. The approximate extinction coefficient at 520 nm for 16.2 nm diameter particles was determined to be 520 ≈ 5.72 × 108 M-1 cm-1 using a method reported previously.16 The concentrations of the Au nanoparticle solutions were determined using the absorbance values in conjunction with the calculated extinction coefficient (∼6.4 nM final particle concentration for this preparation). Preparation of DNA-Modified Au Nanoparticles. The 5′ disulfide bond of the 5′ HO-(CH2)6S-S(CH2)6-modified oligonucleotides was cleaved, and the oligonucleotides were purified as reported previously.4 The DNA-modified Au nanoparticle solutions were prepared by adding 3 nmol of DNA per 1 mL of 16.2 nm Au particles (∼3 µM DNA concentration, ∼6.4 nM Au particle concentration). After standing for g16 h at room temperature, the solutions were diluted to 0.1 M NaCl, 10 mM phosphate (pH 7) (will be referred to as 0.1 M PBS) and allowed to stand for 40 h, followed by centrifugation at 25 000 rpm for g15 min to remove excess reagents. Following removal of the supernatant, the DNA-modified Au nanoparticles were redispersed in 0.1 M PBS. (13) Marsh, T. C.; Vesenka, J.; Henderson, E. Nucleic Acids Res. 1995, 23, 696-700. (14) Grabar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743. (15) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22. (16) Mucic, R. C.; Storhoff, J. J.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 12674-12675.

Preparation of Au Nanoparticle/Deoxynucleoside Solutions. Stock solutions (5 mM) of the deoxynucleosides (dA, dG, dC, and dT) were prepared in water. Deoxynucleoside solution (1 µL, 5 µM final concentration) was added to 1 mL of Au nanoparticle solution (∼6.4 nM final concentration) and monitored spectroscopically.

Results and Discussion Deoxynucleoside Binding to Au Nanoparticles. Previous reports have demonstrated that the binding of uncharged molecules to Au particles (i.e., pyridine) leads to particle agglomeration accompanied by a plasmon frequency red-shift.12,17 This colorimetric change has been used to monitor the binding of uncharged molecules such as alkanethiols to a gold nanoparticle surface.18 We designed a simple experiment to probe the binding of the deoxynucleosides (dA, dC, dG, and dT) to the Au nanoparticle surface by monitoring changes in the surface plasmon band associated with nanoparticle aggregation, Scheme 2. In a typical experiment, separate solutions of ∼16 nm diameter Au particles (∼6.4 nM final concentration) containing one of the deoxynucleosides (5 µM final concentration) were prepared and monitored spectroscopically, Scheme 2 and Figure 1. Figure 1A shows the UV-vis spectrum for each Au nanoparticle/deoxynucleoside solution after standing for 1 h at room temperature. Almost no change in the surface plasmon band (λmax ) 520 nm) was observed for the Au nanoparticle/dT solution. In contrast, large changes in the surface plasmon band frequency and intensity were observed for the Au nanoparticle solutions containing dA, dG, or dC, which exhibited surface plasmon band red-shifts to λmax ) 650, 667, and 693 nm, respectively. After a period of greater than 4 h, precipitates were observed for the Au nanoparticle solutions containing dA, dG, or dC, while no precipitation was observed for the Au nanoparticle solution containing dT. On the basis of these initial data, we monitored the kinetics of particle agglomeration for each Au nanoparticle/deoxynucleoside solution by following the (17) Blatchford, C. G.; Campbell, J. R.; Creighton, J. A. Surf. Sci. 1982, 120, 435-455. (18) Weisbecker, C. S.; Merritt, M. V.; Whitesides, G. M. Langmuir 1996, 12, 3763-3772.

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Figure 1. (A) UV-vis spectra of separate Au nanoparticle solutions containing one of the deoxynucleosides (dA, dC, dG, or dT). The spectra were recorded 57.5 min after deoxynucleoside addition. The unmodified Au nanoparticles (prepared by citrate reduction) are shown for reference. (B) Kinetics of nanoparticle agglomeration of Au nanoparticle solutions containing dA, dC, dG, or dT followed by monitoring the extinction changes at 650 nm as a function of time.

extinction at 650 nm as a function of time (Figure 1B), which is close to the plasmon band maximum of the aggregated Au nanoparticle/deoxynucleoside solutions after 1 h. The nanoparticle solutions containing dC or dG agglomerated at the fastest rate (maximum intensity at 650 nm in e10 min), while the nanoparticle solution containing dA agglomerated at a slightly slower rate. However, the nanoparticle solution containing dT exhibited only slight changes in the plasmon band over the 1 h time period indicating that almost no aggregation takes place under these conditions. To ensure that the adsorption of dT to the Au nanoparticle surface would result in particle aggregation, a 20-fold higher concentration of dT (100 µM final concentration) was added to the Au nanoparticles (∼6.4 nM final concentration) and monitored spectroscopically. Under these conditions, the dT/Au nanoparticle solution exhibited spectral changes that were similar to those of the Au nanoparticle solutions containing dA, dG, or dC and also precipitated after a period of greater than 4 h. This experiment demonstrates that the binding of dT to the Au nanoparticle surface will result in particle agglomeration as observed for the other deoxynucleosides, but only at dT concentrations that are over an order of magnitude higher than that observed for dA, dG, or dC. On the basis of these data, we concluded that dA, dG, and dC have a significantly higher binding affinity for the Au nanoparticle surface than dT under these conditions. Previous surface coverage measurements of alkanethiol oligonucleotides on gold nanoparticles concluded that (alkanethiol)oligonucleotides with (dT)20 spacers have a significantly higher surface coverage (∼1.5 times) on gold nanoparticles than the same oligonucleotides with (dA)20

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spacers.8 These data can be explained by the binding affinity measurements reported here which support a weaker interaction between the gold nanoparticle surface and dT which results in significantly higher surface coverages. Stability of DNA-Modified Au Nanoparticles as a Function of Electrolyte. The next set of experiments was designed to probe the stability of DNA-modified Au nanoparticles in electrolyte media as a function of oligonucleotide sequence and length. The 16 nm diameter Au particles prepared by the citrate method are very unstable toward electrolyte since they are predominantly electrostatically stabilized,19 and the particles aggregate in NaCl solutions g50 mM as evidenced by a solution color change from red to blue and eventual precipitation (data not shown). By comparison, we qualitatively observed previously that Au nanoparticles functionalized with alkanethiol-modified 8 base or 12 base oligonucleotide sequences were stable in electrolyte solutions with concentrations as high as 1 M NaCl, which indicated that the alkanethiolmodified oligonucleotides provide significant stabilization for the gold nanoparticles in electrolytic media.4,20 In this study, the sequence-dependent stability of DNA-modified Au nanoparticles was measured to determine the physical consequences of nucleobase sequence. A series of Au nanoparticles functionalized with (hexanethiol)oligonucleotides containing homobase sequences of A, C, or T were prepared for the stability measurements. The oligonucleotides are abbreviated according to modification, sequence, and length, such as 5S(A)20 for a 5′ thiol-modified oligonucleotide containing a 20 base A sequence. For each thiol-modified homooligonucleotide sequence, 5S(A)x, 5S(T)x, and 5S(C)x, a homologous series of different lengths (x ) 5, 10, 15, and 20) was prepared. In a typical experiment, the DNA-modified gold nanoparticle stability was measured by monitoring changes in the surface plasmon band as a function of ionic strength of the medium. We initially tested the stability of a 5S(T)10-Au particle solution as a representative sample of the other DNA-modified particles. The particle concentration of this solution was first normalized to 3.1 nM (absorbance at 520 nm ) 1.8 absorbance units) by dilution with solvent (0.1 M PBS). The DNA-modified particles (0.1 M PBS) were subsequently mixed with the appropriate electrolyte solution in a 1:1 ratio to test the particle stability at varying ionic strengths (1.55 nM final particle concentration, 10 mM phosphate (pH 7)). This procedure was used to test the stability of all the DNA-modified particles discussed herein. MgCl2 was used as an electrolyte in this study since it is present in many common DNA hybridization buffers and associates with high affinity to the phosphate backbone but not to the specific nucleobases.21 The 5S(T)10-Au particles aggregate quickly (e1 min) in MgCl2 solutions of high ionic strength as evidenced by significant colorimetric shifts after electrolyte addition. Figure 2A shows a comparison of the UV-vis spectra of 1.55 nM 5S(T)10-Au particles dispersed in 0.1 M PBS and in 0.75 M MgCl2, 10 mM phosphate pH 7 (µ (MgCl2) ) 2.25) recorded 4 min after electrolyte addition. The aggregated 5S(T)10-Au nanoparticles in 0.75 M MgCl2 solution exhibit a broad, red-shifted plasmon band centered at 620 nm, which is the wavelength used in this study to monitor the ag(19) Enustun, B. V.; Turkevich, J. J. Am. Chem. Soc. 1963, 85, 33173328. (20) Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609. (21) Kazakov, S. A. Bioorganic Chemistry: Nucleic Acids; Oxford University Press: New York, 1996.

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Figure 2. (A) UV-vis spectra of ∼16.2 nm diameter Au particles functionalized with 5′ SH-(CH2)6-T10 in 0.1 M PBS and after addition of MgCl2 electrolyte (0.75 M final concentration). The UV-vis spectrum of the functionalized particles in MgCl2 was recorded 4 min after the addition of the MgCl2 electrolyte. (B) Kinetics of nanoparticle agglomeration of the same 5S(T)10-functionalized Au particles as in (A) followed by monitoring the extinction changes at 620 nm as a function of time at various MgCl2 concentrations (0.25, 0.5, and 0.75 M).

gregation of DNA-modified particles. To determine an appropriate time for reproducibly measuring the extinction changes associated with nanoparticle aggregation, the extinction at 620 nm was monitored as a function of time for the 5S(T)10-Au particle solutions at three different ionic strengths, Figure 2B. In all three cases, there is a sharp increase in extinction at 620 nm over the first 2-4 min due to nanoparticle aggregation, followed by a gradual decrease in extinction at 620 nm due to the formation of larger aggregates and precipitation. On the basis of the aggregation kinetics of these solutions, the nanoparticle extinction values at 620 nm were recorded after 4 min for the other DNA-modified particles studied herein to monitor aggregation, which provided reproducible extinction values. The MgCl2 stability data for the Au nanoparticles functionalized with each homologous series of homooligonucleotides (5S(A)x, 5S(C)x, and 5S(T)x, where x ) 5, 10, 15, and 20) are shown in Figure 3. The 5S(T)x-Au particles exhibit a dramatic stability increase as the oligonucleotide length is increased in the 5-15 base range, Figure 3A. The Au particles functionalized with 5S(A)x or 5S(C)x oligomers also exhibit an increase in stability due to increased oligonucleotide length, although the effect is much less dramatic than that observed for the 5S(T)x-Au particles, Figure 3B,C. It should be noted that the 5S(T)x series of oligonucleotides is plotted on a log scale while the 5S(A)x and 5S(C)x series of oligonucleotides are plotted on a linear scale. From the stability data, we have defined a critical ionic strength parameter (µc) which allows us to compare the relative stabilities of the DNA-modified

Figure 3. Stability of (alkanethiol)oligonucleotide-modified Au particles in MgCl2 solutions of various ionic strengths at pH 7. The extinction at 620 nm was recorded 4 min after MgCl2 addition for each solution and plotted as a function of MgCl2 ionic strength. (A) 5S(T)x-Au particles (x ) 5, 10, 15, or 20); the ionic strength is plotted on a log scale. (B) 5S(C)x-Au particles (x ) 5, 10, 15, or 20). (C) 5S(A)x-Au particles (x ) 5, 10, 15, or 20). Smoothed lines have been drawn to guide the eye.

particles of varying sequence and length. This parameter is determined by taking the midpoint in the extinction change at 620 nm as a function of ionic strength for each set of DNA-modified Au nanoparticles measured. The critical ionic strength values for the DNA-modified particles of varying sequence have been plotted as a function of the number of bases, Figure 4. These data clearly demonstrate significant stability differences related to oligonucleotide sequence. Most notably, the 5S(T)x-Au particles in the 15-20 base length region exhibit over an order of magnitude greater stability when compared to the 5S(A)x-Au or 5S(C)x-Au particles of

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Figure 4. Critical ionic strength (µc) for nanoparticle aggregation plotted as a function of the number of oligonucleotide bases (x) for (alkanethiol)oligonucleotide-Au particles of varying sequence (5S(A)x, 5S(T)x, or 5S(C)x) in MgCl2 solutions. The critical ionic strength parameter (µc) is defined in the text.

comparable oligonucleotide length. The dramatic stability enhancement observed for the 5S(T)x-Au particles (x ) 15 or 20) can be explained by the higher surface coverage for 5S(T)x-Au particles (vide supra),8 which enhances surface charge and steric stability at these oligonucleotide lengths. Conclusions Previous surface coverage measurements indicated that oligonucleotides with dT spacers exhibit significantly higher surface coverages than oligonucleotides with dA spacers.8 A study of deoxynucleoside binding to the Au nanoparticle surface presented here has demonstrated that the nucleoside dT has a much lower binding affinity than nucleosides dA, dC, or dG for the nanoparticle surface.

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The deoxynucleoside data support the proposed oligonucleotide binding model (Scheme 1) where the strength of the interaction between the oligonucleotide and the nanoparticle surface is dictated by nucleobase sequence, which affects oligonucleotide surface coverage. Stability measurements on DNA-modified Au nanoparticles of varying sequence were performed to probe the changes in physical properties due to this phenomenon. These data indicate that Au particles functionalized with 5S(T)x oligomers exhibit dramatically enhanced stability toward electrolyte when compared with particles functionalized with oligomers of 5S(A)x or 5S(C)x. The 5S(T)x-Au particles exhibit a dramatic stability increase when the oligonucleotide length (x) is increased from 5 to 15 bases. By comparison, the Au particles functionalized with 5S(A)x or 5S(C)x oligonucleotides exhibit stabilities that are relatively insensitive to oligonucleotide length and are approximately an order of magnitude less stable than the corresponding 5S(T)x-Au particles of comparable length in the x ) 10-20 base region. The enhanced stability observed for the 5S(T)x-Au particles toward electrolyte is attributed to an increase in surface coverage which enhances surface charge and steric stability. This work has significant ramifications for the design of DNAmodified Au nanoparticle probes for detection assays that utilize these probes. Probe stability is essential for realizing high sensitivity and selectivity in such assays.22 Further work is aimed at understanding the structural characteristics of these adsorbed oligonucleotide layers and designing gold nanoparticle probes with optimized stability and hybridization characteristics. LA0202428 (22) Taton, T. A.; Lu, G.; Mirkin, C. A. J. Am. Chem. Soc. 2001, 123, 5164-5165.