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Thiolated Dendrimers as Multi-Point Binding Headgroups for DNA Immobilization on Gold B. Scott Day,*,† Larry R. Fiegland,‡ Erik S. Vint,† Wanqiu Shen,† John R. Morris,‡ and Michael L. Norton*,† † ‡
Department of Chemistry, Marshall University, Huntington, West Virginia 25755, United States Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, United States
bS Supporting Information ABSTRACT: The synthesis of multithiolated DNA molecules that can be used to produce self-assembled monolayers of single-stranded DNA oligonucleotides on gold substrates is described. Generation 3 polyamidoamine (PAMAM) dendrimers were conjugated to DNA oligomers and functionalized with ∼30 protected thiol groups. The protected thiol groups— thioacetate groups—allowed the dendrimerDNA constructs to be stored in a buffer solution for at least 2 months before deprotection without any observable decrease in their ability to assemble into functional layers. The monolayers formed using these multithiolated DNA probe strands demonstrate target capture efficiencies comparable to those of analogous monolayers assembled with DNA functionalized with single thiol groups. A functional advantage of using dendrimer headgroups is the resistance to probe strand loss in prolonged exposure to buffer solutions at a high temperature (95 °C).
I. INTRODUCTION Since the early development of alkanethiol self-assembly on gold substrates, many applications for alkanethiol self-assembly have been developed that range from the control of basic wetting properties, to substrates for oriented crystal growth, to modeling biological surfaces.1 Because of the ease of functionalizing many biological molecules with free thiol groups, biologically based sensor surfaces have seen widespread application. In particular, the modification of single-stranded DNA (ssDNA) molecules with thiol groups has seen extensive use on gold substrates27 with applications ranging from DNA sequencing, genetic disease diagnostics, and gene profiling8,9 to the directed assembly of proteins10,11 and most recently an explosion of work on DNA aptamers,1215 which have the ability to capture a large array of target analytes other than nucleic acids. Applications and fundamental studies that take advantage of the ability to bind DNA to gold through the thiol bond are varied, mainly because of the many ways in which gold can be fabricated and integrated into sensor surfaces. For example, DNA-coated gold nanoparticles have been used in highly sensitive colorimetric assays,16,17 in making various nanostructures,1820 and in nanomedicines based on the asymmetric nanoparticles’ large absorption cross sections in the NIR.21,22 In addition to gold nanoparticles, DNAcoated gold nanowires with varied geometries2326 have been used for biosensing and have been recently utilized in the fabrication process of 40 nm lines of DNA on polymer surfaces.27 Thiolated DNA attached to gold-coated AFM cantilevers has also been used in single-molecule studies of DNA,28 and nanodots of gold coated with DNA on the tips of cantilevers have been applied to resonant nanomechanical devices.29 r 2011 American Chemical Society
One of the major disadvantages of using the thiolgold bond to assemble biologically relevant monolayers is the limited longterm stability of the molecule attachment.3033 The need to improve the stability of the attachment between DNA and gold has been recognized by many researchers that utilize an array of DNA-to-gold systems. The multiple-point binding of probe ssDNA onto planar gold surfaces has been approached previously by Levicky et al.7,34 In their immobilization strategy, a thin layer of poly(mercaptopropyl)methylsiloxane was used to link maleimide-functionalized DNA to the surface through a network of thiol bonds. Their robust attachment strategy displayed the excellent thermal stability of immobilized DNA strands for 1 h at 95 °C in a sodium citrate buffer showing only 10% strand loss compared to the complete loss of DNA prepared through single-thiol headgroups. Similarly, Sakata et al. addressed the issue of thiol stability by immobilizing DNA onto gold with a novel tripod attachment.6 For comparative purposes, the authors also synthesized a similar linker with a single-thiol group to bind to gold surfaces. As expected, the tripod binder showed superior thermal stability when compared to the singlethiol binder. Plaxco and co-workers recently published a paper using the flexible Letsinger-type trihexylthiol anchor and showed that this attachment strategy improved the properties of an electrochemical DNA sensor compared to those of similar monolayers prepared with a monothiol and a rigid trihexylthiol anchor.35 Specifically, they showed that these flexible trihexylthiol monolayers were more stable in long-term solution-phase Received: June 28, 2011 Revised: August 17, 2011 Published: September 14, 2011 12434
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Scheme 1. Synthesis of Multithiolated ssDNA
storage, were more thermostable in simulated PCR cycles, and were more robust in their electrochemical interrogation. Researchers investigating DNA bound to gold nanoparticles have also realized the need to increase the stability of the DNA bound to gold. Mirkin and co-workers synthesized DNA probe strands that contain one, two, and three thiolated terminal groups36 to use in their highly sensitive and specific DNA colorimetric detection method.36,37 The authors reported an increasing stability of the gold nanoparticle constructs with an increasing number of thiol bonds36 and that the time the DNA resisted displacement by dithiothreitol (DTT) increased from 2 min for the single-thiolated DNA to over 8 h for the DNA immobilized via three thiol bonds. Perkins et al. used three repetitions of dithiol phosphoramidite to link DNA to gold nanoposts for investigations measuring the DNA elasticity.38 The six thiolgold bonds were necessary to prevent the displacement of DNA with smaller thiol molecules used for biological passivation. Additionally, the robustness of their anchoring method allowed the same DNA molecule to be repeatedly probed. Another major consideration for coating surfaces with DNA is the fabrication methods by which the probe DNA can capture target analytes from solution. For DNA biosensors, the goal is to hybridize target DNA rapidly, with high specificity and as efficiently as possible. Factors that influence duplex formation on surfaces include the probe strand density4/entanglement,39 the probe length,40 and the height of the probe strand above the interface.41 Dendrimers are organic molecules that may allow the control of these factors and are promising molecules that have been used in bio-related surface sensors. Dendrimers are useful candidates for immobilizing molecules because the dendrimers’ large “footprint” on the surface provides control over the packing density, the dendrimers’ general flexibility of the arms supply more dynamic freedom, and the dendrimers’ multiple attachment points to the surface provide robust attachment. Cai et al. synthesized dendritic molecules containing three (G0) and nine (G1) thiol groups with a bromophenyl functionality at the dendrimer apex for controlled
orientation and spacing on the surface of gold.42 The authors found that the first-generation dendrimer is ∼3 times larger on the surface than the zeroth-generation dendrimer. In addition, the multithiolated monolayers had a higher thermal stability than monolayers prepared using octadecanethiol molecules. Park and co-workers synthesized cone-shaped dendrimers that when covalently attached to silicon substrates provided nanoscale-controlled spacing of the terminal functional groups.4345 Probe DNA molecules were immobilized at the apex of the surface-bound dendrimers and showed both high hybridization efficiency (80100%) and a selectivity of target strands as high as that in solution-phase experiments. The authors attribute the enhanced selectivity and high hybridization efficiency to the lack of steric hindrance for incoming target DNA molecules. Polyamidoamine dendrimers have also been used as linker molecules to immobilize DNA onto silicon surfaces46 and gold-coated substrates.47 In both cases, the functionalized surfaces were synthesized directly on the solid substrate and the monolayers formed, showed high stability to repeated hybridization cycling. In this study, we discuss a method to attach DNA probe strands to gold surfaces using multithiolated dendrimers as the headgroup of the DNA, thus increasing the thermal stability of the monolayers. The entire synthesis procedure is performed in the solution phase and allows the constructs to be stored for at least 2 months in a buffer solution by protecting the thiols with acetate functionalities. The headgroups are easily deacetylated using hydroxylamine, providing a versatile probe strand that forms monolayers on gold via self-assembly. Monolayers prepared with these DNA constructs display high target capture abilities and exhibit superior thermal stability compared to analogous monolayers prepared with single thiol bonds.
II. MATERIALS AND METHODS II.1. Materials. Generation 3 polyamidoamine (G3:PAMAM) dendrimers with an ethylenediamine core were purchased from Sigma-Aldrich 12435
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Langmuir as a 20 wt % solution in methanol. Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sSMCC), N-succinimidyl-S-acetylthiopropionate (SATP), 5,50 -dithio-bis-(2-nitrobenzoic acid) (Ellman’s reagent), streptavidin-coated magnetic beads, and 20K MWCO Slide-ALyzer MINI dialysis units were all purchased from Pierce. Size-exclusion columns (PD-10 columns) were purchased from GE Healthcare. The gold substrates used for XPS analysis were prepared by the evaporation of gold (100 nm) onto Cr-coated (5 nm) glass slides (Evaporated Metal Films, Inc.). DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT). Three unique DNA strands were used in this study. The probe strand used for immobilization had a sequence of 50 TAA CCA ATA GGC CGA AAT CGG CAA A 30 and was modified on the 50 end with a mercaptohexyl group. The complementary target strand had the sequence 50 TTT GCC GAT TTC GGC CTA TTG GTT A 30 , and a second, noncomplementary target strand with the sequence 50 TTC AGC ATC TTT TAC TTT CAC CAG C 30 was used. Deionized water (18.2 MΩ 3 cm, Barnstead) was used throughout the experiments. II.2. Multithiolated DNA Synthesis. The multithiolated DNA assembling units were prepared in a four-step synthesis as depicted in Scheme 1. In the first step of the procedure, sSMCC was mixed with the dendrimer in a 0.1 M phosphate buffer solution, pH 7.2, in a 1.5:1 mol ratio, sSMCC/G3 PAMAM at a total dendrimer concentration of 1.25 mM. After 30 min, excess SATP was added to the crude reaction solution to react with the remaining primary amine groups on the dendrimer and place thioacetate groups on the dendrimer periphery. After an hour of room-temperature incubation, the functionalized dendrimers were isolated from the reaction products, unreacted sSMCC, and unreacted SATP by passing the reaction mixture through a PD-10 column. The probe DNA was received from IDT with a disulfide modification that required reduction before reacting it with the maleimide group on the functionalized dendrimers. DisulfideDNA was reduced by incubation in a 1:500 mol ratio of DTT for at least 1 h. After incubation, the thiolated DNA was isolated from DTT and the thiol-terminated side product using a PD-10 column. The reduced, thiol-modified DNA was then mixed in a 1:10 molar ratio with the maleimide-functionalized dendrimers and allowed to react overnight at 4 °C, which is step 3 in Scheme 1. To remove the excess dendrimers from the solution, the DNA was isolated from the solution using biotinylated cDNA immobilized on streptavidin-coated magnetic beads. The dendrimer-functionalized DNA conjugates were hybridized to the cDNA on the magnetic beads and washed at least five times with buffer by drawing the beads to the side of a centrifuge tube and removing >90% of the solution. These washing steps removed the excess dendrimers from the solution. The DNA conjugates were recovered by heating the solution to ∼75 °C for 45 s to denature the DNA, followed by drawing the magnetic beads to the side and collecting the solution. The magnetic beads were never fully removed from the solution by this process, so an additional centrifugation step was used to remove the beads from the solution further. These DNA conjugates were stored in the multiple thioacetate form until monolayer preparation. When DNA monolayers were prepared, hydroxylamine 3 HCl was added to the solution in a 10:1 hydroxylamine to thioacetate mole ratio to form the free thiols, step 4 in Scheme 1, and allowed to react for at least 1 h prior to exposure to gold surfaces. The moles of thioacetate groups were estimated from the UVvis-determined DNA concentration and by assuming 30 thioacetate groups per DNA molecule.
II.3. Nondenaturing Polyacrylamide Gel Electrophoresis. The gels used in electrophoresis contained 8% acrylamide (19:1 acrylamide/bisacrylamide). The sample buffer was 40% (w/v) sucrose in water. The running buffer was 1 TBE (89 mM Tris base, 89 mM boric acid, 2 mM EDTA, pH 8.3). Gels were run at a constant voltage of 180 V for 2 h (at 4 °C) and were visualized by monitoring the fluorescence emission before and after ethidium bromide staining.
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II.4. ssDNA Monolayer Preparation. To prepare the gold substrates for DNA immobilization, the substrates were cleaned in a piranha solution (70/30 v/v mixture of concentrated H2SO4/35% H2O2) for at least 1 h prior to use. Caution! Piranha solution is an oxidizing mixture that reacts violently when in contact with organic materials. Slides removed from the piranha solution were thoroughly rinsed with deionized water and dried with nitrogen. They were then immediately placed into the DNA probe solution for self-assembly. To assemble multithiolated DNA molecules, the freshly cleaned gold surfaces were submerged in a 1 μM solution of the dendrimerDNA construct in a 0.1 M potassium phosphate buffer for 15 h, unless otherwise stated. Monolayers formed in the presence of hydroxylamine were indistinguishable from monolayers formed with excess hydroxylamine and the reaction product of the deacetylation reaction removed, as observed from online SPR experiments (data not shown). For convenience, the monolayers were prepared in the crude hydroxylamine solution. Following exposure of the gold films to the multithiolated DNA constructs, the surfaces were rinsed with phosphate buffer and distilled water and dried with nitrogen. The single-thiol DNA monolayers were prepared as a 1 μM solution in a 0.1 M potassium phosphate buffer solution. Disulfide reduction of the single-thiol DNA probe strands was performed using DTT as described above to reduce the disulfide bond before the thiol maleimide reaction. Following the reduction of the disulfide group and the isolation of the DNA, the freshly cleaned gold surfaces were placed into the DNA solution overnight. After DNA immobilization, the surfaces were rinsed with a phosphate buffer and distilled water and dried with nitrogen. The surfaces were then submerged in a 1 mM solution of mercaptohexanol (MCH) in water for 1 h, rinsed thoroughly with distilled water, and dried with nitrogen. II.5. Measurements. UVvis spectroscopy was used to determine the concentrations of DNA and to monitor the progress of the reaction between SATP and the dendrimers. Sample spectra were recorded on an ND-1000 spectrophotometer (NanoDrop). 1H NMR spectra were obtained using a Varian Unity+ 500 MHz NMR instrument for the G3:PAMAM starting material, dendrimers fully functionalized with thioacetate groups, and dendrimers fully functionalized with the free thiol groups. All samples were run in D2O. X-ray photoelectron spectroscopy (XPS) was used to characterize monolayers formed from thiolated dendrimers, multithiolated ssDNA, and single-thiolated ssDNA. Achromatic Al Kα (1486.6 eV) X-ray radiation was generated from an X-ray source operating at 250 W. The kinetic energy of the ejected photoelectrons was detected using an 18 in. hemispherical energy analyzer (Perkin-Elmer PHI 5300) operating with a pass energy of 71.5 eV and a takeoff angle normal to the surface. First, a survey scan of each surface was performed with a step size of 0.5 eV to verify the alignment. Next, high-resolution spectra of the following elements were recorded in the order S 2p, C 1s, O 1s, P 2p, N 1s, and Au 4f. All high-resolution spectra were recorded with a step size of 0.1 eV. The binding energies for all spectra were referenced to the Au 4f7/2 peak at 83.8 eV.48 The XPS data were analyzed using a customwritten nonlinear least-squares fitting procedure; the S 2p region was fit with 100% Gaussian peaks and a linear baseline. Surface plasmon resonance (SPR) experiments were performed on a Reichert SR7000 single-channel SPR spectrometer. Typically, the DNA interaction experiments were performed in 0.1 M phosphate buffer solution at 25 °C at a flow rate of 15 μL min1. To regenerate ssDNA surfaces after hybridization, the monolayers were exposed to 50 mM KOH solutions for 5 min.
III. RESULTS AND DISCUSSION III.1. Dendrimer Synthesis and Characterization. 1H NMR
spectra were obtained for the G3:PAMAM starting material, 12436
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Figure 1. (a) Polyacrylamide gel visualized by the presence of fluorescein attached to the generation 3 dendrimers. The inset shows an overexposed image enlarged around the bands in lanes 24. (b) The same gel as in image a, stained with ethidium bromide to visualize the DNA. Lane 1: free disulfide ssDNA. Lane 2: fluorescein-labeled G3 dendrimers. Lane 3: fluorescein-labeled G3 dendrimers conjugated to ssDNA. Lane 4: fluorescein-labeled G3 dendrimers and ssDNA mixed without conjugation. Lane 5: free disulfide ssDNA.
dendrimers fully functionalized with thioacetate groups, and dendrimers fully functionalized with free thiol groups. Fully functionalized dendrimers, rather than dendrimers with sSMCC, were used in the NMR analysis to simplify the spectral interpretation. The 1H NMR spectrum (Supporting Information) for the G3:PAMAM starting material was interpreted on the basis of work by Davis et al., who assigned the 1H NMR spectra of G0 and G1 PAMAM dendrimers.49 Peaks were observed at 3.22 and 2.57 ppm (CONHCH2CH2NR2), 3.17 and 2.67 ppm (CONHCH2CH2NH2), and 2.75 and 2.37 ppm (NCH2CH2CONH). For the dendrimers fully functionalized with thioacetate groups, peaks were observed at 3.20 ppm (CONHCH2CH2NHCO and CONHCH2CH2NR2), 2.57 ppm (CONH CH2CH2NR2), 2.75 and 2.37 ppm (NCH2CH2 CONH), 2.43 and 2.99 ppm (HNCOCH2CH2 SCOCH3), and 2.26 ppm (HNCOCH2CH2S COCH3). Finally, the peak assignments for the dendrimers fully functionalized with thiol groups were 3.20 ppm (CONH CH2CH2NHCO and CONHCH2CH2NR2),
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2.57 ppm (CONHCH2CH2NR2), 2.75 and 2.37 ppm (NCH2CH2CONH), and 2.54 and 2.86 ppm (HN COCH2CH2SH). Further verification that the synthesis yielded dendrimer molecules with thiol peripheral groups was determined using Ellman’s reagent test. The thioacetate-terminated dendrimers were mixed with hydroxylamine and allowed to react for 30 min. After the deacetylation reaction, Ellman’s reagent was added and allowed to react for 15 min. The absorbance was measured at 412 nm using the ND-1000 spectrophotometer and used to calculate the free thiol concentration by assuming that the free thiols in solution yield a stoichiometric quantity of 2-nitro-5thiobenzoic acid. The concentration of thiols in solution was determined to be 98 ( 4% of the expected value for these dendrimers fully functionalized with free thiol groups. III.2. Multithiolated DNA Preparation. Confirmation of the covalent attachment between DNA molecules and the functionalized dendrimers was verified through gel electrophoresis. A slight modification to the synthesis was used to visualize the dendrimers and DNA separately on the gel. In step 1 of the synthesis, NHSfluorescein was added to the dendrimer/ sSMCC solution in a 2:1 ratio of fluorescein to dendrimer. Following the addition of NHSfluorescein and sSMCC, the synthesis and purification of the DNA dendrimers were followed as outlined in section II.2 of Materials and Methods. Figure 1 shows (a) unstained and (b) stained (ethidium bromide, EtBr) polyacrylamide gel images. The probe DNA used in the experiments (after reduction with DTT) was loaded into lanes 1 and 5. The fluorescently labeled dendrimers were loaded into lane 2. Lane 3 contains the conjugated DNA and dendrimers, and lane 4 contains a control experiment where the maleimide group on the dendrimer was inactivated with mercaptoethanol prior to mixing the dendrimers with the thiolated DNA. Figure 1a shows that the dendrimers barely migrate out of the well (lanes 2 and 4) except in lane 3. In comparing the unstained gel to the same gel stained with EtBr, the band in lane 3 of the unstained gel coincides with the upper band in lane 3 of the stained gel, indicating that both DNA and dendrimers are present within that band. In lane 4, the dendrimers did not migrate far down the lane and the migration of DNA has been unaltered as compared to that in lanes 1 and 5 where no dendrimers were present. This suggests that the coincident band of DNA and dendrimers in lane 3 comes from the covalent attachment of DNA and dendrimers, not from entanglement or electrostatic binding. Even though the tertiary amines within the dendrimer are likely to be protonated under the conditions of the gel, the electrostatic binding strength between the negatively charged DNA backbone and the positively charged core of the dendrimers is insignificant. The synthesis method used to make dendrimerDNA conjugates seems to be valid for producing the conjugates in a small distribution. The upper band containing both dendrimers and DNA in lane 3 most likely contains dendrimers covalently bound to DNA in a 1:1 ratio. During synthesis, dendrimers and thiolated DNA were mixed in a 10:1 ratio to push the stoichiometry toward one DNA per dendrimer. The absence of dendrimers close to the well in lane 3 suggests that the purification step of DNA from dendrimers using magnetic beads with cDNA sufficiently removes the excess dendrimers. (See the Figure 1a inset for an overexposed image.) However, the faint band close to the well in lane 3 that appears upon ethidium bromide staining is likely dendrimers with two or more DNA molecules attached. On 12437
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Table 1. Summary of the Elemental Compositions Calculated from XPS Data for Monolayers Prepared from Dendrimers, Multithiolated ssDNA, and Single-Thiolated ssDNA % elemental composition (observed/theoretical)a monolaver carbon nitrogen oxygen phosphorus dendrimer
68/62
18/19
11/14
0/0
MT ssDNA 56/55
19/19
23/22
2.6/2.1
STssDNA
12/
20/
2.7/
62/
sulfur N/P ratio 3.5/4.9 /2.7 3.5/
7.3/9.0 4.4/4.2
a
Theoretical values for MT ssDNA were calculated by assuming a 1:1 conjugation with each dendrimer having 31 terminal thiol groups.
the basis of a comparison of the intensities of the band referred to as the 1:1 band to the band near the well and considering that there are more DNA molecules to stain per construct in the upper band, the distribution is very small. The yield of placing a dendrimer onto the DNA probe strands can be estimated by comparing the intensities of the two bottom bands in lane 3 to the upper band referred to as the 1:1 conjugation band. The two lower bands of DNA correspond to thiol-terminated DNA (25 bp) and disulfide DNA (50 bp), respectively.50 Assuming that all DNA is accounted for by the intensities in lane 3 and ignoring background fluorescence from the dendrimers, the relative percent yield of the 1:1 conjugation product is ∼70%. The multithiolated DNA probe strands were synthesized according to the sequence outlined in Scheme 1 to maximize the product yield and provide a stable surfactant for long-term storage. The thioacetate groups could be stored for up to 2 months (the longest time tested) in aqueous buffer solutions, a convenient advantage when working with DNA. Chechik and Crooks synthesized generation 4 (G4) PAMAM dendrimers with terminal groups functionalized with thiols and noted that dry solvents and an inert atmosphere were essential to prevent the gelation of the dendrimers.51 They attributed the gelation to either the dendrimers cross-linking by the oxidation of thiols into disulfides or the rearrangement of the dendrimers into an insoluble configuration. The long-term storage of our multithiolated DNA constructs was apparently facilitated by the protecting acetate groups. III.3. DNA Monolayer Characterization. III.3.a. X-ray Photoelectron Spectroscopy. XPS was used to obtain detailed information on the compositions of monolayers formed from multithiolated dendrimers, multithiolated (MT) ssDNA, and singlethiolated (ST) ssDNA. Table 1 is a summary of the observed elemental composition of each of these monolayers along with the relative nitrogen to phosphorus ratios for the DNA monolayers. Chemical compositions were calculated from the integrated intensities of high-resolution spectra for each of the five elements contained in these monolayers: C, N, O, P, and S. Figure 2 shows the spectra for the five elements studied, along with the Au spectra for each of the three monolayers. As an illustration of fitting the spectra for integration, the fitted distribution and baseline used to integrate the carbon signal are included in the spectrum of the dendrimer monolayer in Figure 2b. The percent elemental composition for the dendrimer monolayer and the multithiolated ssDNA monolayers is in good agreement with the theoretical predicted compositions. The dendrimers used in the XPS studies are the same as those used
Figure 2. XPS spectra of (a) N 1s, (b) C 1s, (c) S 2p, (d) O 1s, (e) P 2p, and (f) Au 4f for monolayers prepared from multithiolated dendrimers (+), ssDNA molecules with dendrimer headgroups (O), and ssDNA molecules with single-thiol headgroups (4). The fitting of the spectra is illustrated in the C 1s spectrum of the dendrimer monolayer. All spectra were referenced to the Au 4f7/2 peak at 83.8 eV as indicated by the vertical line in f.
for the NMR analysis (i.e., all 32 primary amines were reacted with SATP and deprotected to form free thiols). The relative elemental composition observed for this monolayer is very similar to their theoretical values, which further confirm the NMR analysis that the molecules used to make the monolayers have a high degree of functionalization. In calculating the theoretical yield for the multithiolated ssDNA monolayers, two assumptions were used. First, it was assumed that each DNA molecule was attached to the dendrimer by the sSMCC crosslinker and that the remaining 31 peripheral groups on the dendrimer were all thiolated. Second, it was assumed that the monolayer is completely composed of dendrimerDNA conjugates in a 1:1 ratio with no unconjugated dendrimers or unconjugated bound DNA. Theoretical compositions for the single-thiol DNA monolayers could not be calculated because they were generated in a two-step synthesis and the relative number of DNA molecules to mercaptohexanol molecules is unknown. However, the nitrogen to phosphorus ratio for the two DNA monolayers could be used to further compare the compositions of the monolayers to their expected values. Nitrogen and phosphorus were chosen because they are the only two elements unique to DNA for the single-thiol DNA monolayer. The observed N/P ratio from XPS analysis is 4.4, which is very close to the expected ratio of 4.2, indicating that this elemental ratio is a good predictor of composition estimations within thin DNA 12438
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Figure 3. Online SPR traces of (a) single-thiol DNA monolayers and (b) multithiolated DNA monolayers.
films. For the multithiolated DNA monolayer, the ratio observed, 7.3, is lower than the expected value of 9.0. The disagreement between the predicted and expected N/P ratio is probably due to single-thiolated DNA without the conjugated headgroup or nonspecifically bound DNA. As shown in Figure 2f, the binding energies for all spectra were referenced to the Au 4f7/2 peak at 83.8 eV,48 so any bindingenergy shifts between monolayers could be examined. Most differences among the three monolayers are expected. For example, the O 1s peak has a higher binding energy (peak maximum about 1.1 eV higher) in the two DNA monolayers than in the dendrimer-only monolayer because of the large number of oxygen atoms present in the DNA phosphate backbone. Additionally, no phosphorus signal is observed for the dendrimer monolayer as expected. The largest surprise is the lack of a sulfur signal in the MT ssDNA monolayer. The sulfur signal is low in the dendrimer-only monolayers, as the comparison between the observed and theoretical elemental compositions indicates. This is most likely due to a lack of sensitivity from the instrument rather than the extent of dendrimer functionalization because the NMR analysis shows a nearly complete conversion of the terminal amines to free thiols. Even though the sulfur signal is large enough to observe for the dendrimer-only surface, there is no guarantee that the dendrimer number density is the same for these two monolayers. Additionally, the sulfur signal is almost certainly attenuated by the DNA molecules in the MT ssDNA monolayer. The sulfur atom density for the ST DNA monolayer is about 5 1014 molecules/cm2 assuming a well-packed monolayer of the mercaptohexanol.1 A typical DNA number density for DNA monolayers is about 5 1012 molecules/cm2,2,4,52 and because each headgroup in the MT ssDNA monolayer has 32 sulfur atoms, the sulfur density would be about 3 times lower than the
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thiol density in the ST ssDNA monolayers. Considering that the signal-to-noise value is only about 3:1 for the ST ssDNA monolayer, it is reasonable not to observe the sulfur signal for the MT ssDNA sample in our experiments. III.3.b. Surface Plasmon Resonance. To characterize these multithiolated DNA monolayers further, we investigated the heterogeneous hybridization using SPR and compared them to sensors formed from ST DNA probe strands. Figure 3 shows the SPR traces for the online assembly of both (a) ST DNA and (b) MT DNA monolayers. Exposing the bare gold surface to a 1.0 μM solution of ST probe strands resulted in an approximate 10.1 pixel unit increase in the signal. Following the standard protocol by Tarlov et al.,2 the surface was then exposed to a 1 mM solution of mercaptohexanol to orient the remaining, immobilized probe strands away from the surface for proper target strand capture. The signal change upon exposure of the surface to mercaptohexanol rapidly increased to about 9.0 pixel units as the small molecules passivated the bare gold surface, followed by a slow decrease over the remaining exposure time. This decrease in signal is likely due to the mercaptohexanol molecules displacing nonspecifically bound DNA and some DNA bound through a thiol linkage.2 The surface was then exposed to 2.0 μM solutions of complementary and noncomplementary DNA. The baseline increase for the exposure of cDNA was approximately 15 times larger than for the noncomplementary strands, indicating good selectivity of the sensor surface to the target strands. We also studied the online assembly of MT DNA monolayers using SPR as indicated in Figure 3b. The net difference in pixel units for the multithiolated probe strand was ∼9.6 over the exposure time in this experiment for a 1.0 μM solution of DNA. Mercaptohexanol was not used for these surfaces because the amount of target DNA captured was independent of MCH exposure. This is expected for these surfaces because the dendrimer underlayer is probably thicker than the MCH molecules adsorbed through their porous structure. These MT ssDNA monolayers showed a similar specificity to the target strand, as did the ST DNA monolayer upon exposure to noncomplementary and complementary DNA, approximately 15 times the response to the target strand. Although making quantitative comparisons between the probe densities of the ST DNA monolayer compared to the MT monolayer is difficult because their local refractive indices are likely different, we can make some general observations and comparisons. Assuming that the SPR response signal is directly proportional to the mass density only, the probe density of the MT ssDNA monolayer is lower upon initial immobilization than the ST ssDNA monolayer given the approximate 2.2-fold larger mass and the nearly equivalent pixel change. Approximately 10% of the ST probe strands are lost upon exposure to MCH on the basis of the decreasing portion of the curve. The decrease in signal is not large enough to reduce the density to that of the MT DNA surface. Given the higher density of probe strands on the ST DNA surface, it is interesting that the response of these two surfaces to target DNA is nearly the same. In fact, upon exposure of bare gold to the probe strands overnight and exposure times for these monolayers to target DNA for 1 h, we found that the MT DNA monolayer was able to capture nearly 15% more target DNA than the ST DNA monolayer. (See the Stability Measurements section below.) We infer from this data that the large dendrimer headgroup controls the lateral spacing of the probe strands, and we suggest that the optimum probe density for capturing target strands may be more easily manipulated by the 12439
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Figure 5. Summary plot comparing the thermal stabilities of the singlethiol and multithiol DNA monolayers by their ability to capture target DNA. Target capture is depicted by filled symbols for single-thiol DNA (triangles) and multithiol DNA (circles). NoncDNA is depicted by open symbols for single-thiol DNA (triangles) and multithiol DNA (circles). The y axis depicts the net pixel change following equilibrium from target strand assembly onto DNA monolayers that were heated in 95 °C PBS for 04 h. The error bars represent (1 standard deviation for at least five independent measurements. Figure 4. SPR traces of target DNA capture after 0, 1, 2, 3, and 4 h of heating (a) the multithiolated ssDNA sensor surface and (b) the singlethiol ssDNA surface.
size of the headgroup than kinetically controlling probe DNA and MCH exposure times. Park and co-workers have shown a similar size effect of the headgroup by using a cone-shaped dendrimer to immobilize DNA onto silicon substrates.4345 In their experiments, the probe strands were mesospaced ∼3.2 nm apart and were proven to discriminate single nucleotide polymorphisms as well as probe strands reported in the solution phase. Elicia et al. have also shown an increase in hybridization efficiencies when the DNA probe strand is elevated above diluent molecules,41 as may be the case with the DNA bound to gold substrates through dendrimer headgroups. For our conjugate monolayers, it appears that the dendrimers’ size plays a substantial role in controlling the probe density upon assembly and the general flexibility may help in capturing target strands. III.4. Stability Measurements. Polydentate binding to gold surfaces should provide increased long-term stability of the monolayers compared to monodentate binding when the attachment is the labile part of the system.42 The 31 thiol groups on the dendrimers should provide exceptional binding stability for DNA probe strands. However, given the spherical symmetry of dendrimers in solution, only a fraction of the thiols will bind to the gold. Chechik and Crooks showed that about 30% of the thiol groups bind to planar gold surfaces for multithiolated G4 PAMAM molecules.51 We expect a similar fraction of thiol groups to bind to the gold surfaces for our MT DNA monolayers. To determine the thermal stability of the multipoint binding to the gold surfaces, we conducted experiments in which immobilized ssDNA was exposed to 95 °C phosphate buffer from 0 to 4 h. For these studies, DNA probe strands were exposed to the surface for at least 18 h before any heat treatment to form the best quality monolayers. For the single-thiol monolayers, the probe assembly was followed by a 1 h exposure to a 1 mM solution of mercaptohexanol. To determine the effect of heating on these monolayers, we recorded their ability to capture target DNA
from solution using SPR. Figure 4 shows representative SPR response curves for (a) MT ssDNA monolayers and (b) ST ssDNA monolayers exposed to target DNA following the thermal treatment. From Figure 4, we see that the relative amount of target DNA captured after 1 h of heating decreases by about 10% for the MT DNA monolayer, whereas a nearly 35% loss in signal is observed for the ST DNA monolayer. Further heating for 2 h shows losses of 15 and 60% for the multithiolated and single-thiolated monolayers, respectively. The decrease in the measured target strand response is interpreted as the probe strand loss from the surface; therefore, fewer target strands were captured by the surface. The more robust attachment of the MT ssDNA monolayer to the surface through the multiple thiol headgroup implies that these monolayers can withstand multiple thermal cycles such as in polymerase chain reaction protocols. Figure 5 summarizes the data for these heating studies and also includes the response of the surfaces to the noncomplementary sequences. We do not observe any changes in the response of our surfaces to heating for noncDNA strands. An interesting observation appears in the single-thiolated DNA monolayers upon heating that is not observed for the multithiolated DNA monolayers. The target capture trace for the surface that was not subjected to heating shows a very different kinetic profile than does each of the heated surfaces. Peterson et al. reported a difference in the kinetic profiles for DNA monolayers having different probe densities in which monolayers with densities greater than 5 1012 molecules/cm2 had slower kinetics than monolayers having densities of less than 3 1012 molecules/cm2.4 The change we observe in the kinetic profile is consistent with a loss of probe strands upon heating as the probe strand density decreases below this threshold region. As noted in the analysis of the online SPR profiles, we believe that the ST monolayers formed in this study have a much higher packing density than the MT monolayers. The lack of change in the MT monolayer profiles suggests that either the initial probe density is below the threshold region of the kinetic change or the flexibility of the dendrimer headgroups allows the DNA to hybridize to target strands more rapidly. 12440
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IV. SUMMARY We have described the synthesis of multithiolated headgroups for DNA immobilization on gold that show superior properties to analogous monolayers assembled with single-thiolate headgroups. Protecting the thiols with acetate groups was integral in both synthesis and storage. An X-ray photoelectron analysis of monolayers prepared from the multithiol DNA constructs showed that the relative elemental composition is consistent with the expected empirical formula of 1:1 DNA to dendrimer conjugates. The large number of goldthiol bonds provided resistance to probe desorption in a 0.1 M potassium phosphate buffer at 95 °C compared to monolayers formed through single thiol bonds to the gold. In addition to the superior thermal stability of these multipoint binders, the size and flexibility of the dendrimers appeared to order the monolayers into highly efficient films to capture cDNA. We believe that these conjugates could be used in many applications of DNA immobilized on planar gold surfaces and gold nanoparticles to provide both the robust attachment and lateral spacing needed in those methods. Control over the molecular-level spacing of biomolecules is particularly important for DNA aptamer sensors because the 3D structure of the aptamer and target molecules with a variety of sizes and shapes can be detected on these platforms. Additionally, the chemistry that is utilized in preparing these conjugates is common to many researchers in biologically oriented research. The multithiolated dendrimer molecule could easily be added to any biological molecule to increase the stability of its surface adhesion. ’ ASSOCIATED CONTENT
bS
Supporting Information. UVvis data monitoring the reaction between SATP and primary amines on dendrimers. 1H NMR spectra for the dendrimer starting material, fully functionalized dendrimers with thioacetate groups, and fully functionalized dendrimers with thiol groups along with additional discussion. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*(B.S.D.) Phone: (304) 696-7054. Fax: (304) 696-3243. E-mail:
[email protected]. (M.L.N.) Phone: (304) 696-6627. Fax: (304) 696-3243. E-mail:
[email protected].
’ ACKNOWLEDGMENT B.S.D., E.S.V., W.S., and M.L.N. acknowledge support under West Virginia State Challenge Grant 0304 and ARO award G: W911NF-05-1-0309;P00002. Funding for L.R.F. and J.R.M. was provided by the National Science Foundation (career award no. CHE-0948293). B.S.D. thanks Tom Ryan at Reichert Instruments for providing the use of the SPR spectrometer and for providing technical assistance. ’ REFERENCES (1) Love, J. C.; Estroff, L. A.; Kriebel, J. K.; Nuzzo, R. G.; Whitesides, G. M. Chem. Rev. 2005, 105, 1103–1169. (2) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916–8920.
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