Monitoring DNA Hybridization and Thermal Dissociation at the Silica

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Monitoring DNA Hybridization and Thermal Dissociation at the Silica/Water Interface Using Resonantly Enhanced Second Harmonic Generation Spectroscopy Md. Shafiul Azam and Julianne M. Gibbs-Davis* Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada S Supporting Information *

ABSTRACT: The immobilization of oligonucleotide sequences onto glass supports is central to the field of biodiagnostics and molecular biology with the widespread use of DNA microarrays. However, the influence of confinement on the behavior of DNA immobilized on silica is not well understood owing to the difficulties associated with monitoring this buried interface. Second harmonic generation (SHG) is an inherently surface specific technique making it well suited to observe processes at insulator interfaces like silica. Using a universal 3nitropyrolle nucleotide as an SHG-active label, we monitored the hybridization rate and thermal dissociation of a 15-mer of DNA immobilized at the silica/aqueous interface. The immobilized DNA exhibits hybridization rates on the minute time scale, which is much slower than hybridization kinetics in solution but on par with hybridization behavior observed at electrochemical interfaces. In contrast, the thermal dissociation temperature of the DNA immobilized on silica is on average 12 °C lower than the analogous duplex in solution, which is more significant than that observed on other surfaces like gold. We attribute the destabilizing affect of silica to its negatively charged surface at neutral pH that repels the hybridizing complementary DNA.

T

standard TIRF experiments, the surface selectivity is determined by the distance that the evanescent wave penetrates into the bulk, which is typically on the order of hundreds of nanometers. As a result, standard TIRF methods cannot discriminate between fluorescent-labeled molecules bound to the interface or those present within the volume probed by the evanescent wave. For example, Krull and co-workers were able to monitor hybridization and thermal dissociation by TIRF utilizing silica fiber optics modified with oligonucleotides.19 Significant corrections to the resulting fluorescent profiles were required to account for the presence of complementary fluorescein-labeled DNA that was not interacting with the surface but was present within the evanescent field.19 Other real-time techniques have been reported that monitored the kinetics and thermodynamics of DNA hybridization, but these methods were isothermal21 or under nonequilibrium conditions.22−24 Even in the few examples of equilibrium thermal denaturation experiments, an experimental comparison of the melting behavior of solution-phase DNA and silica-supported DNA has not been shown.19,25 Second harmonic generation (SHG)26−28 has much greater surface selectivity than fluorescence or absorbance measurements that utilize total internal reflection configurations owing to the SHG selection rules, which require a break in inversion

he advent of strategies for immobilizing DNA onto simple glass microscope slides has revolutionized molecular biology and biodiagnostics.1−4 By spatially controlling the presentation of different DNA sequences, microarrays can be used to sequence genomes or monitor the gene expression levels in diseased or healthy cells. Chip-based platforms are also gaining interest in the detection of oligonucleotide biomarkers associated with infectious diseases or pathogens.4−6 To facilitate these technologies, numerous methods have been devised for immobilizing DNA to glass (silica) and other solid supports, and many of these strategies can be achieved using commercially available oligonucleotide strands and surface modifying molecules.7,8 However, despite their widespread use, the effects of immobilization on the molecular recognition behavior of DNA bound to glass are not well-known.9−11 In contrast, the ability to monitor the thermodynamic12 and kinetic13 properties of immobilized DNA in real-time is straightforward when the substrate is a metal like gold. Electrochemical methods for monitoring redox active groups covalently or physically associated with DNA have allowed the thermal dissociation of DNA to be monitored in real time.14,15 Other techniques like surface plasmon resonance (SPR) are also amenable to monitoring binding processes of immobilized DNA on gold substrates.16−18 For insulating materials like silica, however, such electrochemical and SPR methods are not available. To achieve some amount of surface selectivity, total internal reflection techniques like total internal reflection fluorescence spectroscopy (TIRF) can be employed.19,20 In © 2013 American Chemical Society

Received: April 9, 2013 Accepted: August 8, 2013 Published: August 8, 2013 8031

dx.doi.org/10.1021/ac401009u | Anal. Chem. 2013, 85, 8031−8038

Analytical Chemistry

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Scheme 1. Hybridization and Dissociation of SHG-Active 3-Nitropyrrole-Modified Complementary DNA

excitation. When 2ω approaches ωca, the molecular hyperpolarizability and consequently χ(2) become large. This resonant enhancement allows one to monitor the presence and organization of specific molecules at the interface, which is another major advantage of second harmonic generation over techniques like SPR that do not report on the nature of the interfacial species.28 DNA possesses electronic resonances in the ultraviolet region of the electromagnetic spectrum. Thus, resonantly enhanced second harmonic generation has been employed by Geiger and co-workers to monitor the association of DNA, without the addition of any labels.38 These measurements, however, are complicated by the similar resonant wavelengths of both single-stranded and double-stranded DNA at 260 nm. Therefore, hybridization could only be discerned by comparing the signal intensities for different incident linear polarizations. The linear dichroic ratio based on these different signal intensities and the fraction of duplexes at the interface are not directly proportional,40 which adds further complexity to determining the extent of hybridization from the signal changes. Consequently, this method has only been used to monitor hybridization over hours under a given set of experimental conditions.38 We were interested in developing a complementary SHG method for monitoring hybridization and dissociation utilizing a synthetic nucleotide that possessed a resonant frequency distinct from that of DNA. In our strategy, the synthetic nucleotide is only present in the solution-phase strand that is complementary to the surface-bound strand. Consequently the number density of the synthetic nucleotide is proportional to the number of duplexes at the interface, allowing hybridization and dissociation to be directly monitored by changes in the second harmonic electric field (Scheme 1). We selected the commercially available universal base 3-nitropyrrole deoxyribonucleotide as an SHG label. Although 3-nitropyrrole is not a naturally occurring nucleobase, it forms stable interactions with all of the canonical bases. Moreover, it has a similar hydrophobicity and size to that of the pyrimidine bases. In acetonitrile, the phosphoramidite precursor of our 3-nitropyrrole nucleotide absorbs light in the UV, with a local maximum at 285 nm red-shifted from the canonical nucleobases (λmax ∼ 260 nm) (Supporting Information, Figure

symmetry. Consequently, SHG has been widely used to study important biological interfaces.29−37 For noncentrosymmetric materials like fused silica or water, inversion symmetry is only broken at the interface. Therefore, any signal arising from the aqueous phase originates from molecular assemblies that possess some net orientation. The SHG intensity (I2ω) emanating from the interface is proportional to the square modulus of the second harmonic electric field (E2ω), which depends on the incident electric field (Eω) and the secondorder susceptibility (χ(2)) according to I2ω = E2ω 2 ∝ χ (2) EωEω

2

(1)

The second-order susceptibility χ(2) is very small for most interfacial systems. Consequently, ultrafast pulsed laser assemblies are necessary for achieving high electric fields at ω that generate a measurable second harmonic response. χ(2) can be decomposed into a resonant and nonresonant contribution as shown in eq 2: (2) (2) χ (2) = χNR + χRes

(2)

The resonant contribution can be related to the molecular hyperpolarizability of species present at the interface. Specifically, χ(2) depends linearly on the number density of molecules in resonance N and the orientational average of their corresponding hyperpolarizability tensor β(2): (2) χRes = N ⟨β (2)⟩

(3)

Within the electric dipole approximation, only molecules oriented at the interface contribute to χ(2).39 Consequently, even if the molecules in resonance are present within the bulk volume penetrated by the evanescent field generated by the incident laser field at ω, they will not lead to SHG if they are isotropically distributed within this volume. Equation 4 illustrates the resonant enhancement of the molecular hyperpolarizability β(2) β (2) ∝

A ωca − 2ω + i Γ

(4)

where A is the oscillator strength, Γ is the spectral line width, and ωca is the resonant frequency of a particular electronic 8032



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Figure 1. (A) Schematic illustration of the covalent attachment of DNA to the silica surface by reaction of a benzyl azido monolayer with alkynemodified oligonucleotides. (B) The high-resolution XPS nitrogen spectra before and after the reaction with alkyne-terminated DNA. The 401/405 eV peak ratio increased after the reaction as a result of the conversion of azides to triazoles and the incorporation of the N-containing nucleotides. (C) The low-resolution fluorine XPS spectra before and after the reaction using F-labeled alkyne-terminated DNA.

azide linker and the DNA strand, the amounts of N and F from the low-resolution spectra were used to determine the conversion efficiency of the reaction according to

S1). The presence of the lone pair on the nitrogen within the heterocycle and the nitro withdrawing group at the 3-position suggested that 3-nitropyrrole might possess the push−pull electronic structure of classic SHG chromophores like 4nitrophenol.28

F

#DNA 8 % yield = × 100% = × 100% 40F ⎛ ⎞ N − #Azide ⎜ 38 ⎟ ⎝ ⎠

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EXPERIMENTAL SECTION Preparation of Immobilized DNA Samples. DNA sequences are listed in Table S1 in the Supporting Information. The DNA was synthesized on an Applied Biosystems model 392 DNA/RNA Synthesizer using Glen Research reagents. The 4-azidomethylphenyl trimethoxysilane used to make the benzyl azido monolayer was synthesized as described in our previous work.45 Using a modification of the procedure described by Sun and co-workers,39 we performed the [3 + 2] cycloaddition reactions between the benzyl azide-modified surface and DNA strands terminated by an alkyne-modified thymine (Talkyne) using a 1:1:1 catalyst−ligand mixture of CuSO4−TBTA−TCEP in a mixture of DMSO and water. Details can be found in the Supporting Information. XPS Analysis. Experiments were performed on three different samples using fluorine-labeled cytidine instead of the native nucleotide. Owing to the nitrogen present in both the

(5)

Similarly, the area of the peaks at 401 and 405 eV from the high-resolution N-1s spectra were utilized to determine the conversion efficiency as we described previously, as the peak at 405 eV is unique to the azide group.40 Each triazole-linked DNA contained 43 nitrogen atoms; therefore, the conversion yield was determined from Yield = =

#DNA × 100% #Azide N401eV − 3.07N405eV 43 N401eV − 3.07N405eV 3.07N405eV + 43 3

× 100% (6)

The value of 3.07 is based on the ratio of N401 eV/N405 eV observed for the unreacted benzyl azide monolayer. The 8033



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oligonucleotide and determined the conversion efficiency by the fluorine/nitrogen ratio in the low-resolution XPS spectra (Figure 1C). This analysis yielded a conversion efficiency of 30(10)%. As a point of comparison, using phenyl azido monolayers results in the conversion of only 1% of the azides to triazole-linked DNA strands, which may correspond to differences in reactivity for phenyl vs benzyl azides.47 If we assume that our benzyl azide monolayer contained 3 × 1013 sites per cm2, which is ∼10% the density of silanols on silica,48 then our modification method based on 20% conversion resulted in 6 × 1012 DNA strands per cm2 or interstrand distances of ∼4 nm. As the polyanionic DNA strands repel one another, the density of DNA monolayers is expected to be much lower than monolayers composed of neutral small molecules, which is consistent with our conversion efficiencies. Monitoring Hybridization with SHG. After verifying that DNA was immobilized at the surface by XPS, we performed a similar immobilization chemistry on 1-in. diameter fused silica hemispheres. These DNA-modified hemispheres were placed into our custom-built Teflon cell, which allowed us to modulate both the temperature and composition of the aqueous phase while simultaneously monitoring the SHG signal (Supporting Information, Figure S2). Figure 2A illustrates the change in SHG signal at 290 nm upon adding 0.61 μM of the complementary oligonucleotide bearing the three terminal 3nitropyrrole universal nucleotides, (NP)3. The significant increase in signal indicated that the 3-nitropyrrole was

derivation of these equations is given in the Supporting Information. SHG Hybridization and SHG Spectroscopy Experiments. A detailed description of the laser system used in these experiments can be found in our previously published work.41,42 All experiments were performed with a freshly prepared DNA modified hemisphere, which was placed into a custom-made steel sample cell connected to a heater and temperature controller (Supporting Information, Figure S2). The sample cell was then filled with buffer and the appropriate [NaCl] via syringe or calibrated pipet (1.2 mL, 10 mM phosphate buffer saline, pH 7). After optimizing the SHG signal, a solution of the complementary DNA (20.7 μM, 36 μL) was added and mixed using a 1-mL plastic syringe by quickly pulling and dispensing ∼0.5 mL of solution 4−5 times. To monitor resonant enhancement, we collected SHG as a function of incident wavelength (λ = 590−560 nm) after observing hybridization for 15 min and then blocking the beam for 1 h. The data from two separate samples was normalized, combined, and smoothed with a 2-point box smoothing function (Igor Pro). The smoothed data and the fit with a Lorentzian function are shown in Figure 2B. The reported SHG λmax and the error are from the Lorentzian fit. SHG Melting Experiments. After watching the hybridization for about 15 min, the beam was blocked to avoid any chance of burning the sample. One hour later, the heater was turned on and SHG signal was collected for 1 min at 1−2 °C intervals after a 1-min hold time at each temperature. The resonantly enhanced E2ω proportional to the number density of (NP)3-DNA strands was determined based on a 90° phase difference between the resonant and nonresonant E2ω at the silica/water interface.43 The fraction of the DNA duplexes dissociated was then ascertained by dividing the temperaturedependent resonant E2ω values by the maximum resonant E2ω value.

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RESULTS AND DISCUSSION Immobilization of DNA on Fused Silica. To immobilize the DNA to the silica surface, we selected the copper-catalyzed Huisgen-Meldal-Sharpless [3 + 2] cycloaddition, commonly referred to as a click reaction, which is a high yielding strategy for immobilizing biomolecules to a variety of substrates based on the reaction between an azide and a terminal alkyne.44−46 Using our recently reported method for generating well-defined benzyl azide-functionalized monolayers on silica from a benzyl azido trimethoxysilane precursor, we performed the immobilization reaction between our monolayer and an alkyneterminated strand using standard click conditions for biomolecules in aqueous solutions (Figure 1A).40 To determine how much of the azide was converted to the DNA-immobilized triazole, we took advantage of the characteristic azide signature in the high-resolution X-ray photoelectron spectra. As shown in Figure 1B, the azide functional group resulted in two peaks in the N-region of the XPS spectra; as the azides were converted to triazoles, the peak at 401 eV grew in magnitude while the peak at 405 eV disappeared. From the change in the peak ratio, the surface conversion was determined according to our published method.40 Specifically, upon reacting the alkynemodified DNA overnight with our benzyl azido monolayer, the 401 eV/405 eV ratio increased from 3.1(1) to 11(3), indicating that 15(4)% of the azides had been converted. Using an alternative method for monitoring surface conversion with XPS, we introduced a fluorine label into our alkyne-modified

Figure 2. (A) Change in E2ω with time upon adding nitropyrollelabeled complementary DNA to the DNA-functionalized surface. The signal leveled off in approximately 500 s indicating that hybridization was complete. (B) Resonant enhancement of the SHG signal by the nitropyrrole nucleotides was verified by varying the wavelength of the incident laser field and monitoring the corresponding signal intensity at the second harmonic wavelength (I2ω). A Lorentzian fit to the data is shown, which yielded a λmax value of 291.1(2) nm and a half-width half-maximum (HWHM) value of 4(1) nm. The nitropyrrole resonant peak is more narrow than that of single and double-stranded DNA at the silica/water interface (HWHM = 6(1) nm),38 which suggests that the 3-nitropyrrole has a shorter excited state lifetime than that of the standard nucleobases. 8034



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time scale, which is consistent with our observations on silica. In contrast, the hybridization of free DNA in solution requires 10−100 s depending on the sequence length and concentration,17,52 while hybridization of DNA-modified materials that form three-dimensional assemblies requires hours.53,54 This comparison indicates that the restricted degrees of freedom of the immobilized strand significantly impact the rate of hybridization, but that the “two-dimensional” environment of the interface is not as constrained as that of materials forming three-dimentional aggregates through DNA hybridization. Quantifying DNA Duplex Stability with SHG. The thermodynamics of hybridization at surfaces is often determined from measuring the isotherm of the fraction surface coverage versus the concentration of complementary DNA in solution using models like the Langmuir model.21 However, the Langmuir model only holds for reversible interactions. As shown in Figure 3, the hybridization of DNA

responsible for the signal increase through resonant enhancement. Indeed, upon varying the SHG wavelength, we observed a maximum at 291.1(2) nm, attributed to the resonant wavelength for 3-nitropyrrole at the silica/aqueous interface (Figure 2B), which was slightly red-shifted based on the solution spectra of NP in acetonitrile (Supporting Information, Figure S1). We note that adding the same complementary strand lacking the (NP)3 terminus did not lead to discernible changes in signal. After verifying that the presence of the nitropyrrole nucleotides resulted in resonant enhancement, the second harmonic electric field was directly correlated with the fraction hybridized according to eqs 1−3. To verify that the signal enhancement was due to specific interactions between the complementary DNA strands, we monitored E2ω when the same concentration of (NP)3-DNA was added to bare silica. The signal enhancement was so small that it could only be observed upon focusing the incident beam beyond our typical experimental setup (Supporting Information, Figure S3). Similarly, adding (NP)3-DNA to a surface modified with a noncomplementary polyadenosine sequence led to a signal increase that was much smaller than that observed for the complementary surface (Figures S6 and S7, Supporting Information). These experiments support that the main source of the signal enhancement stems from the formation of duplex at the interface. Regarding the time required for hybridization, the profile of E2ω versus time indicated that the complementary DNA had hybridized with the interface within 10 min (Figure 2A). Allowing the sample to hybridize for another 2 h led to a small increase in signal (∼10% increase), which suggested a few possibilities (Figure S8, Supporting Information). The first is that the duplexes reorganized at the interface over time, which led to a subtle change in net orientation of the nitropyrrole groups and consequently the magnitude of χ(2). Similarly, it is possible that the strands formed a bound species that rearranged to the duplex over time. To confirm this reorganization or rearrangement required determining the hyperpolarizability tensor of 3-nitropyrrole, which is currently unknown. Another possibility is that two hybridization processes occurred at the interface. In this scenario the first event involved hybridization of DNA in solution with the very accessible surface strands, which comprised most of the hybridization processes. Next, strands that more closely neighbored other immobilized strands hybridized with the DNA in solution. Because of the high charge density around these sites, the second process would be much slower owing to repulsive interactions. Regardless of the origin of the small increase, with this functionalization strategy we achieved rapid hybridization or captured of most (or all) of the accessible immobilized DNA. Typical hybridization protocols using DNAfunctionalized chips wait hours to allow for complete hybridization.49 Our result indicates that only minutes are required to capture the DNA at these probe densities. The observed rate of hybridization is very consistent with the kinetic model of heterogeneous hybridization developed by Krull and co-workers.20 Indeed, for an immobilized DNA density of 10−7 mol/m2 (6 × 1012 molecule/cm2), their model predicts that at 100 s 55−60% of hybridization will occur, which we observed experimentally. As previously mentioned, the influence of immobilization on the kinetics of DNA has been extensively explored on gold surfaces utilizing primarily electrochemical methods.50,51 These studies have found that hybridization occurs on the minute

Figure 3. The change in signal upon hybridizing the DNA-modified surface with (NP)3-labeled DNA (orange). The decrease in signal upon removing the hybridization solution and replacing it multiple times with water and finally buffer (Wash-1). This process was repeated twice (W-2 and W-3), but the signal did not drop to the original starting point indicating that the binding of many of the DNA strands was essentially irreversible at room temperature. The beam was blocked while the sample was washed, which is why the intensity dropped to zero counts.

at this buried interface did not appear to be reversible at room temperature. Specifically, removing the aqueous solution that contained 0.61 μM complementary DNA, rinsing it several times with Millipore water, and replacing it with the same buffer that lacked any DNA led to a decrease in the signal. Yet even after several minutes this signal was much greater than that of the original DNA-modified surface before the introduction of complementary (NP)3-labeled DNA. Even with multiple rinses, the higher signal was maintained, which indicated that equilibrium was very slow to establish at the interface. This observed irreversibility can be used to rationalize the harsh treatments employed to regenerate microarrays, which involve heating the microarray to 70 °C in water to remove any hybridized DNA.55 Another method for quantifying the affinity of DNA duplexes in solution or at surfaces involves monitoring the fraction hybridized as a function of temperature. These thermal dissociation or melting curves can be utilized to determine the temperature where half the duplexes have dissociated. Using SHG, we measured such melting curves from the changes in resonantly enhanced E2ω for DNA duplexes formed 8035



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immobilization is much larger than that observed for DNA immobilized at the gold/water interface, which exhibited Tm values that were 3−7 °C lower than those in solution.15,56 This difference between the effect of the two interfaces indicates that immobilization on silica has a more detrimental influence on the stability of the DNA duplex than that of gold, which has important implications for assays using glass chips. To quantify the thermodynamic parameters associated with these melting events and gain insight into the reason behind the destabilizing affect of silica, we utilized the following sigmoidal function that relates the fraction dissociated to the temperature according to

at the silica/buffer interface by incorporating temperature control into our experimental setup (Figure 4). The

fraction dissociated 1

=

( (

1 + exp

−ΔH R

1 T + 273.14

−

1 Tm + 273.14

))

(7)

where T is the temperature of the solution in Celsius, Tm is the melting temperature in Celsius, ΔH is the dissociation enthalpy, and R is the ideal gas constant.57 The values listed in Table 1 represent the average of values determined from two Table 1. Immobilized vs Solution DNA Dissociation Parameters system

[NaCl] (M)

ΔH (kcal/mol)

Tm (°C)

immobilized

0.05 0.1 0.3 0.05 0.1 0.3

45.0(9) 49(5) 42(8) 47.9(2) 52.75(5) 54.3(3)

41(2) 44(1) 48(2) 53.0(5) 56.7(3) 60.4(1)

solution

or more independent measurements, and the reported error represents the standard deviation. We compared the melting behavior of solution and surface-bound duplexes for three different salt concentrations. For both systems, the melting temperatures increased with increasing salt concentration, which stems from the decrease in charge repulsion between the hybridizing strands with greater concentrations of NaCl. According to the condensed cation theory,58,59 the plot of Tm versus ln[salt] should be linear, which we indeed observed for both the solution and surface systems (Figure 4C). The slope of both lines was very similar revealing that both the solution and the surface systems exhibited similar salt dependence, which has also been observed at the gold/aqueous interface.15 The trends in ΔH, however, varied between the surface and solution phase systems (Table 1). For the solution phase system, we found that the ΔH of dissociation increased with increasing salt concentration, which has been observed and attributed to the stabilizing effect of salt on the duplex.60 In contrast, the ΔH for the surface-bound DNA did not increase with increasing salt concentration, although the significant error in the ΔH values make the trend inconclusive. We note that Krull and co-workers observed that the ΔH of dissociation decreased with increasing salt concentration in their TIRF measurements utilizing DNA immobilized on silica fiber optics, although the melting temperature was found to increase.19 The opposite salt dependence of the ΔH and Tm is possible as the salt-dependent stabilization of DNA duplexes is considered to be primarily an entropic effect.61 Krull and co-workers attributed the decrease in ΔH for the DNA immobilized on

Figure 4. (A) Comparison of the melting curves between solution and immobilized DNA in the presence of a PBS buffer solution containing 0.1 M NaCl. The dashed lines are sigmoidal fits to the experimental data. (B) Melting curves for the immobilized DNA at the silica/buffer interface with 0.05 M, 0.1 M, and 0.3 M NaCl. (C) Variation of Tm as a function of the natural logarithm of the salt concentration. The lines represent a linear fit of the data.

dissociation, or melting, temperature (Tm) corresponds to a normalized E2ω value of 0.5, which was determined from a sigmoidal fit to the data (vide infra). For the 15-mer sequence utilized in this investigation, we observed a Tm of 44(1) °C for the immobilized system in the presence of 0.1 M NaCl (Figure 4A). In contrast, the solution-phase experiment performed with 0.61 μM of each complementary strand yielded a Tm of 56.7(3) °C (Figure 4A). According to these results, immobilizing one of the strands onto the surface decreased the melting temperature by approximately 12 °C. This suppression of the Tm upon 8036



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silica to the improved screening of the negative charges of the single stranded DNA with increasing salt, which allowed for other interactions besides hybridization between neighboring surface strands or DNA and silica. However, another effect of increasing salt concentration that may influence the ΔH of DNA dissociation is the increased ionization of surface silanol sites. Indeed, different techniques including SHG have found that increasing the NaCl concentration led to deprotonation of the surface and, therefore, an increase in the number of negatively charged sites.62,63 Consequently, the higher salt concentrations may lead to a greater density of negative surface charges, which in turn destabilizes the polyanionic duplex and decreases the ΔH of dissociation. Although the amount of silanol groups might be expected to be small in our system as we utilized a benzyl azido monolayer, Kopelman and coworkers observed that significant deprotonation of silica occurred even with monolayers present due to incomplete condensation between the monolayer and the surface silanols.64 Thus, it is feasible that our observed lack of trend for ΔH stemmed from two opposing effects of increasing salt concentration: minimizing repulsion between the hybridizing strands and promoting ionization of the silanol groups. We have found that ionization of the silica surface is very ion specific.41,42 Therefore, future work will involve monitoring specific ion effects on DNA duplex stability at the silica/water interface, which may allow us to differentiate the effect of ions on duplex stabilization and silica ionization. Finally, we also note that the silica/water interface is negatively charged at neutral pH even in the absence of salt owing to its low point of zero charge (pzc < 3).65 Repulsive interactions between the silica surface and the hybridizing DNA at neutral pH also explain why the Tm is much lower for the silica interface in contrast to gold. For designing assays using capture DNA strands immobilized on glass, it is important that researchers consider the effect of the aqueous composition on the surface charge41,42 and the corresponding destabilizing effect on the Tm of the surface-bound duplex.

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ASSOCIATED CONTENT

S Supporting Information *

Details concerning the surface preparation, the XPS experiments and analysis, the UV−vis absorbance spectrum of the nitropyrolle phosphoramidite, the SHG spectrum for the benzyl azido monolayer, and control experiments monitoring the signal enhancement upon introducing (NP)3-DNA to bare silica and an A15-modified surface. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Champika Weeraman and Mr. Abu Kausar for helpful discussions, the Machine Shop and Electronics Shop (UAlberta) for building our temperature-variable SHG cell and the ACSES Center (UAlberta) for the XPS measurements. We also gratefully acknowledge the Canada Foundation for the Innovation-Leaders Opportunity Fund for funding the laser and surface functionalization infrastructure. We also acknowledge the Natural Sciences and Engineering Council-Discovery Grant for operating funds and an NSERC Research Tools and Instruments Grant for the temperature variable UV−vis spectrophotometer.

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REFERENCES

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CONCLUSIONS In conclusion, we have utilized SHG as an inherently surface specific technique to observe processes at the DNAimmobilized silica/water interface. Using a universal 3-nitropyrolle nucleotide as an SHG-active label, we monitored the hybridization rate and thermal dissociation of a 15-mer of DNA immobilized at the silica/aqueous interface. The immobilized DNA exhibited hybridization rates on the minute time scale, which is much slower than hybridization kinetics in solution but on par with hybridization observed at electrochemical interfaces. Additionally, the thermal dissociation temperature of the DNA immobilized on silica was on average 12 °C lower than the analogous duplex in solution. This decrease in melting temperature is much more significant than that observed on other surfaces like gold. We attribute the destabilizing effect of silica to its negatively charged surface at neutral pH that repels the hybridizing complementary DNA. Our results also reveal that SHG is the ideal method for studying DNA hybridization at the buried silica/water interfaces because it can distinguish bound DNA from DNA in solution. As silica is the most widely used support for DNA, identifying the effects of immobilization at this interface are critical for optimizing assay performances. 8037



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Monitoring DNA Hybridization and Thermal Dissociation at the Silica

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