Effect of Probe–Probe Distance on the Stability of DNA Hybrids on

Article pubs.acs.org/ac

Effect of Probe−Probe Distance on the Stability of DNA Hybrids on Surfaces Lucyano J. A. Macedo, Erin N. Miller, and Aric Opdahl* Department of Chemistry and Biochemistry, University of WisconsinLa Crosse, La Crosse, Wisconsin 54601, United States S Supporting Information *

ABSTRACT: We have used temperature gradient surface plasmon resonance (SPR) measurements to quantitatively evaluate how the stability of different types of hybrids formed with DNA probes on surfaces is affected by probe spacing. SPR sensors with different average surface densities of probes were prepared by coadsorbing probes with lateral spacers strands comprised of phosphorothioated adenine nucleotides (A15*). Increasing the fraction of A15* spacers in the immobilization solution results in larger distances between probes on the sensor, determined here using a combination of SPR and X-ray photoelectron spectroscopy (XPS) measurements. The hybridization activities of probes were simultaneously measured over a temperature range that spanned the denaturation temperature (Tm) of hybrids by applying a spatial temperature gradient across the sensor surface. The resulting temperature profiles of hybridization activity show how the stability of hybrids increases as either the distance between probes or the ionic strength of the hybridization buffer increase. Additionally, hybridization activity profiles sharpen as the spacing between probes increases, indicating more homogeneous hybridization behavior of probes. The results provide quantitative experimental data for testing theoretical models of stability, supporting models that account for both repulsive interactions between DNA strands and local variability in probe surface density.

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acquiring reliable temperature dependent sensor measurements.11−16 In this contribution, we use a recently described surface plasmon resonance (SPR) method to provide experimental measurement of how probe spacing affects hybrid stability.17 The SPR method applies a spatial temperature gradient across a sensor which allows simultaneous monitoring of sensor activity over a temperature range that include the denaturation “melt” temperature (Tm) of probe-target hybrids (Scheme 1). The resulting sensor response data is analogous to solution thermal denaturation profiles, obtained, for example by temperature dependent spectroscopic measurement (e.g., UV−vis, CD, fluorescence) or thermal methods (DSC),18,19 which are widely used to understand DNA thermodynamics and subsequently predict DNA behavior in solution. The SPR measurements reported here were obtained from sensors prepared with different probe surface densities and analyzed in different salt content solutions. They provide detailed information on how these two variables affect probe-target hybridization, allowing evaluation of models of stability for hybridized DNA on surfaces.

n important aspect of biosensor development is understanding how the attachment of probe molecules to the sensor surface affects their binding interactions with analyte targets. For DNA hybridization sensors, several properties of attachment limit the extent to which single-stranded DNA probes can capture complementary DNA targets, and thus restrict sensor responses.1−4 Among these is the packing density of DNA probes on the sensor.5−8 It is widely understood that the negatively charged backbone of DNA presents an electrostatic barrier between probes and targets, which, when combined with other steric restrictions, results in low capture efficiencies from hybridization sensors where the probes are densely packed. A detailed empirical knowledge, however, of how the packing density of the probe layer affects the strength of probe-target binding is lacking. This information is significant, because it informs thermodynamic models for surface hybridization reactions. These models are less developed than their solution counterparts, and can potentially be used for quantitative sensing applications and to aid the design of DNA structures on surfaces for nanoscale building applications.2,3,9 One of these models, proposed by Vainrub and Pettit,9,10 considers how electrostatic repulsive interactions affect hybridization reactions on surfaces, predicting stabilities of different types of hybrids in a wide range of solution environments and probe surface density. Quantitative comparison of such models against experimental data have been limited, however, by the challenges of systematically varying sensor morphology and © XXXX American Chemical Society

Received: October 14, 2016 Accepted: January 10, 2017

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DOI: 10.1021/acs.analchem.6b04048 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry Scheme 1. Temperature Gradient SPRa

a

Reflectivity is simultaneously monitored across a spatial temperature gradient that is established along the length of the gold sensor using two independent heating elements mounted to opposite sides of the flow cell.

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MATERIALS AND METHODS

Materials. The commercial oligonucleotides (IDT-DNA) used in this study are listed in Table 1. The 15 nucleotide probe, P15, is part of the overall sequence, A15*T5-P15. The A15* block contains 15 phosphorothioated adenine nucleotides, where each nucleotide has one nonbridging oxygen atom of the phosphate group substituted by a sulfur atom (Figure 1). The target sequence, P15′, is fully complementary to the P15 probes. Other target sequences: P15′m6, P15′m8, and P15′m10, each contain a single nucleotide that is noncomplementary to the P15 probe sequence. The A15* strand was selected to act as a lateral spacer between probes on the surface. Solutions denoted NaCl-TE and CaCl2-TE contained between 0.10 and 1.0 mol·L−1 NaCl or CaCl2, respectively, 10 mmol·L−1 Tris−HCl, 1 mmol·L−1 EDTA, and were adjusted to pH 7. Gold SPR sensors and the gold coated silicon wafer pieces used for samples analyzed by XPS (Platypus Technologies) were cleaned by UV ozone treatment (Procleaner, Bioforce) prior to use, followed by rinsing with high purity (18.2 MΩ·cm) water. SPR. Measurements were made using a home-built SPR imaging spectrometer consisting of a collimated 780 nm LED illumination source and a CCD camera sensitive to near-infrared light.17 The key feature of this instrument is a sample handling system that has the ability to create a spatial temperature gradient along the length of the sensor surface (Scheme 1). The temperature gradient is generated by independent thermoelectric modules regulated to different temperatures. The temperature of the sensor as a function of position in the gradient was calibrated as described previously, using a procedure that takes advantage of the high sensitivity of SPR reflectivity to change in temperature. The temperature resolution of SPR measurements obtained using the gradient is determined by the temperature span of the gradient and the area imaged by a single CCD pixel, typically 0.1−0.4 °C in the measurements reported here. Probe immobilization was monitored while maintaining the SPR sensor at a uniform 35 °C. Hybridization activity of probes was monitored while a spatial temperature gradient was applied to the sensor, typically 20 °C for the cold side of the gradient and 70 °C for the hot side. The procedure used to quantify SPR data of immobilization and hybridization was based on an established method for fixed angle SPR where, for small changes in the refractive index (Δn), changes in reflected light intensity (ΔI) are proportional to Δn.20,21 The method requires two calibration solutions which have a known difference in refractive index. We used two DNA-free buffer solutions that differed in salt concentration by 0.05 mol·L−1, and had Δncal determined by refractive index measurements. The change in reflection intensity observed during either DNA immobilization or hybridization experiments (IDNA−I0) was converted

Figure 1. SPR and XPS data of sensor functionalization. (a) SPR measurement of the immobilization of A15*T5-P15 (black line), A15* (red line), and an equimolar mixture of A15*T5-P15 and A15* (blue line). Sensors were exposed to solutions containing 4 μmol·L−1 total DNA and 1 mol·L−1 CaCl2-TE at 35 °C for 4 h, followed by a rinse in DNA-free buffer solution. SPR data showing immobilization of other mixtures used in this study is provided in Figure S1. (b) XPS spectra of S 2p and P 2p from gold coated silicon functionalized with A15*T5P15 and A15*. Solid lines show results of fits used to determine peak areas. Diagrams in (a) and (b) depict idealized conformations of A15*T5-P15 and A15* strands on gold surfaces.

Table 1. DNA Probe, Spacer, and Target Sequences A15*T5-P15 A15* P15′ P15′m6 P15′m8 P15′m10

5′-(A*)15TTTTTCAATGCAGATACACT-3′ 5′-(A*)14A-3′ 3′-GTTACGTCTATGTGA-5′ 3′-GTTACGTCTGTGTGA-5′ 3′-GTTACGTTTATGTGA-5′ 3′-GTTACATCTATGTGA-5′

to a refractive index change (ΔnDNA) using eq 1, where ΔIcal is the difference in reflection intensities measured for the two calibration solutions. The ΔnDNA values were then converted into DNA coverage by eq 2, where ld is the sensing depth of the evanescent wave (∼350 nm); nDNA and nb are the refractive indices of DNA (1.7) and the buffer solutions, respectively; ρDNA is the bulk density of DNA (1.7 g· cm−3). For measurements made using the spatial gradient, each row of pixel data in the CCD image of the sensor is at a unique temperature and was thus processed individually prior to assembly of hybridization activity vs temperature plots. B

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Analytical Chemistry Table 2. Summary of Probe Immobilization and Hybridization Data sensor hybridization

A15*T5-P15a (μmol·L−1)

A15*a (μmol·L−1)

4 3 2 1 0

0 1 2 3 4

nucleotidesb (×1014 cm−2) 3.8 2.5 2.0 1.5 1.1

± ± ± ± ±

0.5 0.2 0.2 0.2 0.3

max. valuef (×1012 cm−2)

Tm (°C)e

probe immobilization XPS P 2p/ S 2p 2.8 2.1 1.6 1.3 1.0

± ± ± ± ±

probesc (×1012 cm−2)

dd (nm)

± ± ± ±

2.7 3.7 5.0 7.1

0.1 0.1 0.2 0.1 0.1

11 5.6 3.2 1.5 0

2 0.4 0.3 0.3

0.10 mol·L−1 NaCl 34.5 39.7 42.8 44.6

± ± ± ±

0.2 0.5 0.5 0.7

0.25 mol·L−1 NaCl 43.7 48.2 49.2 50.8

± ± ± ±

0.2 0.7 0.6 0.4

0.10 mol·L−1 NaCl 2.7 2.5 1.5 0.7

± ± ± ±

0.1 0.2 0.2 0.3

0.25 mol·L−1 NaCl 3.1 2.5 1.8 0.9

± ± ± ±

0.2 0.2 0.2 0.1

a

Concentrations of probe and lateral spacer DNA used during immobilization procedure. bSurface coverage values obtained by SPR after 4 h immobilization followed by a rinse in DNA-free buffer solution. For the mixed composition sensors, calculated values used a nucleotide molar mass of 320 g·mol−1, which is the average mass of nucleotides in A15* (324 g·mol−1) strands and A15*T5-P15 (316 g·mol−1) strands. cCalculated from SPR and XPS data using procedures presented in the Supporting Information. dAverage distance between probes, assuming a uniform distribution on the sensor. eReported values are the temperatures at which sensor responses were 50% of maximum observed values. fSensor response observed at 25 °C. Error bars for all values in Table 2 are standard deviations from replicate samples.

⎛I − I0 ⎞ ΔnDNA = ⎜ DNA ⎟ × (Δncal) ⎝ ΔIcal ⎠

(1)

⎛ l ⎞ ⎛ ΔnDNA ⎞ ⎛ ρDNA × NA ⎞ DNA·cm−2 = ⎜ d ⎟ × ⎜ ⎟ ⎟×⎜ ⎝ 2 ⎠ ⎝ nDNA − nb ⎠ ⎝ MW ⎠

(2)

values for its nonphosphorothioated analog, consistent with a flat conformation on sensors and their intended use as nonhybridizing lateral spacers. AFM images obtained from samples prepared using template-stripped gold surfaces displayed no evidence of aggregation of DNA (Figure S2), indicating uniform coating of sensors. Probe coverages less than 1013 cm−2 were realized by using different ratios of A15* and A15*T5-P15 in the immobilization solution (Table 2). SPR measurements (Figure 1a, Figure S1, Table 2) cannot distinguish the two components, providing here simply the total nucleotide coverage of the adsorbed layer, for example, the sum of both A15*T5-P15 and A15*. This quantity systematically decreases as the relative amount of A15* used in the immobilization solution increases, trending toward the nucleotide coverage observed from the sensors coated exclusively with A15*, providing evidence that the fraction of A15*T5-P15 present on sensor surfaces decreases as the amount of A15* used in the immobilization solution is increased. XPS measurements were used to provide the relative amounts of A15*T5-P15 and A15* immobilized on each sensor. Reference spectra of the P 2p and S 2p regions obtained from samples prepared using just A15*T5-P15 and just A15* are shown in Figure 1b. Since only the phosphorothioated nucleotides contain sulfur, the two DNA have different P/S stoichiometric ratios, 2.3 and 1.1, respectively (Figure 1b). The difference in the fitted XPS P/S peak area ratios for these samples, 2.8 and 1.0 respectively, is consistent with the different compositions. The mixed A15*T5-P15/A15* samples produced peak area ratios (Table 2) bracketed by these two values, decreasing as the amount of A15* used during immobilization is increased−further evidence that probe coverage decreases as the amount of A15* is increased. We note that the assumptions made in our treatment neglect any organizational properties of the adsorbed layer which may affect XPS peak areas. Quantitatively (Table 2), the P15 probe coverages calculated using the combined SPR and XPS information span roughly an order of magnitude. The highest P15 probe coverage, 1.1 × 1013 cm−2, correlates with an average distance, d, between probes of roughly 2.7 nm, and the lowest coverage of 1.5 × 1012 cm−2, to roughly 7 nm. This range falls within a regime where electrostatic repulsive interactions are predicted to influence the hybridization activity of probes.2,15 Additionally, assuming a length of 0.71 nm per nucleotide for unhybridized P15 probes,

X-ray Photoelectron Spectroscopy (XPS). Measurements were made using a PHI 5400 XPS equipped with a hemispherical analyzer. Spectra were obtained using an aluminum anode source operated at 250 W. Spectra of the P 2p and S 2p regions were obtained at normal emission angle using 15−20 eV windows and pass energy of 71 eV. Spectra were fit using a Shirley background and splitting of 0.84 and 1.18 eV, respectively, for the P 2p and S 2p doublets.22,23

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RESULTS AND DISCUSSION Sensor Functionalization and Determination of Probe Coverage. Hybridization sensors functionalized with P15 probes at coverages ranging between 1012 and 1013 probes·cm−2 were prepared by coimmobilizing A15*T5-P15 and A15* strands from solution onto clean gold sensors. The procedures followed those developed for nonphosphorothioated A15T5P15 and A15.6,24 Those strands have previously been demonstrated, by a combination of SPR and XPS measurements, to immobilize as a single layer of adsorbed DNA. The A15 block of nucleotides of A15T5-P15 preferentially adsorbs to the gold surface, effectively functioning as a surface attachment group. When the surface is saturated, the P15 probe portions of the strands are end-tethered, a conformation suited for hybridization with DNA targets. Co-immobilization with A15 strands dilutes the A15T5-P15, increasing the separation between P15 probes.6 Samples prepared here used the affinity of thioates (*) for gold surfaces to promote additional stability at elevated temperature, through multipoint attachment, and are stable for multiple hybridization cycles.6,13,17,25 Attachment of A15*T5-P15 and A15* was quantified using in situ SPR measurements of immobilization (Figure 1a, Table 2, Figure S1) and ex situ XPS measurements of the phosphorus and sulfur content of the resulting DNA layers (Figure 1b, Table 2). SPR measurements of sensors prepared using exclusively A15*T5-P15 exhibited the highest coverage, 3.8 × 1014 nucleotides·cm−2 (1.1 × 1013 probes·cm−2), very similar to the previously reported value of 1.3 × 1013 probes·cm−2 for nonphosphorothioated A15T5-P15.6 Sensors prepared using exclusively A15* strands had much lower nucleotide coverage (Figure 1a, Table 2) also similar to our previously reported C

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rank the relative stabilities of hybrids that formed in the different sensor environments explored here. Considering first the hybrids formed from sensors using the 0.10 mol·L−1 NaCl-TE hybridization buffer solution, the Tm values noticeably shift to higher temperature as the spacing between probes on the sensors increases, approximately 10 °C for the range of probe coverages explored here. This shift is a clear indication that the stability of hybrids increases as the distance between probes increases. Additionally, the width of the melt transitions becomes narrower as the probe coverage decreases. The trends are similar for the data obtained from sensors using higher salt content 0.25 mol·L−1 NaCl-TE, however the entire set of plots is shifted higher by 5−10 °C, indicating greater stability of these hybrids. The higher stability in this solution is due to the additional salt counteracting the repulsive interactions of the phosphate backbone, as commonly observed for DNA in solution.18 For comparison to solution behavior, melt curves obtained from UV absorbance measurements of P15:P15′ hybrids in the same salt content solutions (Figure S3) indicate Tm values of 49.0 °C (0.10 mol·L−1 NaCl-TE) and 55.5 °C (0.25 mol·L−1 NaCl-TE). Both solution-based values are higher than the highest values obtained from sensors in the same environments, indicating hybrid formation is less favorable on these sensors as compared to solution. The solution Tm values are just a few degrees higher than those observed from the lowest coverage sensors, which can therefore be concluded to provide the most solution-like environment for hybridization. Figure 2 compares normalized responses from sensors. In absolute terms, the highest hybridization signals, (hybrids· cm−2) observed from each type of sensor at 25 °C, are reported in Table 2. These values decrease as probe coverage decreases, and are slightly higher for the hybridization set performed in 0.25 mol·L−1 NaCl-TE. Hybridization efficiencies, the fraction of probes hybridized, increase as probes become more spaced out on the sensor. The data in Table 2 indicate an increase from roughly 25% of probes hybridized at 25 °C on sensors with the highest probe coverage to over 50% for the lowest coverage sensors. Both trends have been reported in many other studies of DNA hybridization on surfaces,5−8 and are widely attributed to steric effects and repulsive interactions between adjacent probes−effects which rapidly diminish as the separation between probes increases. Together with the Tm data, these observations illustrate that in addition to exhibiting low hybridization efficiencies, that sensors with high probe density produce hybrids that are destabilized, relative to the same hybrid in solution. From a practical standpoint, data such as in Figure 2 and Table 2 can be used to evaluate optimal hybridization conditions for a probe-target pair. For this specific probe−target combination, a probe coverage of approximately 3 × 1012 cm−2 presents a favorable compromise of hybridization activity, efficiency, and stability. Comparison to Theory. Basic evaluation of the hybridization data of Figure 2 follows van’t Hoff analysis,18 fitting each curve to eq 3. Variable c is the concentration of P15′ targets, θ the fraction of the sensor hybridized, ΔH° and ΔS° are enthalpy and entropy terms for the hybridization reaction and are the key fit parameters. The fitted values of ΔH° and ΔS° allow calculation of ΔG° for the surface hybridization reaction at a specified temperature (e.g., 37 °C), which can then be used to compare relative stabilities of different hybrids in different environments. For our data set, this analysis would qualitatively result in less favorable values of ΔG37 ° for hybridization as either

Figure 2. Temperature profiles of SPR measured hybridization activity from sensors with different coverages of P15 probes. Hybridization is with P15′ targets in (a) 0.10 mol·L−1 NaCl-TE buffer solution and (b) 0.25 mol·L−1 NaCl-TE buffer solution for approximately 20 min. Scatter plots are the normalized averages of experimental data from multiple replicate sensors, and have a temperature resolution of approximately 0.3 °C, for which error bars are displayed for every fifth data point. Solid lines show the results of fitting the experimental to the electrostatic model described by eq 4.

physical interactions between adjacent probes are possible at these coverages, which may influence hybridization behavior.26 Hybridization Activity and Hybrid Stability. The ability of the P15 probes to form hybrids with complementary P15′ targets was quantified by SPR, using the temperature gradient to simultaneously monitor sensor response over a range of temperatures that span the expected melt temperature (Tm) of P15:P15′. Measurements are shown in Figure 2 for sensors that have the four probe coverages described in Table 2 and that used two sets of hybridization conditions, 0.10 and 0.25 mol· L−1 NaCl-TE. Each plot is the averaged result of data obtained from multiple replicate sensors and has been normalized, using its maximum observed hybridization signal, for ease of comparison. This data set provides a means to directly compare the stabilities of P15:P15′ hybrids in different environments. Hybridization activities of each sensor decrease as temperature increases, and plots have the characteristic sigmoid shape of a DNA melt curve obtained from solution measurements. Melt curves from solution measurements are widely used to evaluate the stability of DNA structures, often using the Tm, the temperature where roughly 50% of the DNA is hybridized, as a convenient measure for comparing the stabilities of different hybrids. By analogy, we report the Tm from sensor data (Table 2) as the temperature at which the hybridization response is 50% of its observed saturation value.17 We used those values to D

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ref 10. This deviation may be attributed to the cumulative uncertainties of probe density, temperature, and hybridization activity in our experimental measurements. In this context, the electrostatic model satisfactorily describes many of the hybridization behaviors of this system. We note that our fitting neglected any effect that the A15* spacer strands may have on the overall electrostatic environment that probes experience. To evaluate the impact of this component, we prepared a sensor that had a low coverage of A15*T5-P15 probes (3.9 × 1012 cm−2), but without additional A15* spacers (Figure S4). Temperature profiles of hybridization activity measured from this sample in 0.10 mol·L−1 NaCl-TE were slightly sharper, but otherwise similar to those obtained from the sensor prepared using 0.50 mol. fraction A15* in the immobilization solution (Table 2, probe coverage 3.2 × 1012 cm−2). Accordingly, suitable fits were obtained using the same values of w and ZP, suggesting that the major influence on hybridization behavior is the salt content of the solution and the spacing of the probes, and that the additional A15* strands have only a small impact on the overall stability of these hybrids. Hybridization with Mismatched DNA Targets. Finally, we evaluated the temperature gradient SPR method for its potential to differentiate hybrids that contain single nucleotide mismatches, and to characterize how these hybrids are affected by probe coverage. In solution, hybrids containing nucleotide mismatches are less stable than perfectly matched hybrids, having Tm shifted to lower temperature by an amount that depends on the composition and location of the defect. Figure 3 summarizes results of temperature gradient SPR measurements of P15 probe hybridization with three different mismatched targets: P15′m10, P15′m8, P15′m6. Each of these targets contains a nucleotide that is noncomplementary to the P15 probe sequence (Table 1), and that is located in the central portion of the strand. The hybridization measurements in Figure 3 were performed in 0.25 mol·L−1 NaCl-TE using sensors prepared with two probe densities, the presumed optimal probe coverage from Figure 2 (3.2 × 1012 cm−2, Figure 3a) and higher probe coverage (1.1 × 1013 cm−2 Figure 3b). For each of the three mismatch hybrids in Figure 3, the temperature profiles of hybridization activity follow the same stability trends as observed for fully complementary P15:P15′ hybrids (Figure 2). Specifically, profiles observed using the higher probe density sensor are shifted to lower temperature and are noticeably broader than those from the lower coverage sensor. The rank order of stability for the three hybrid types does not change with probe density, and follows the expectations of solutionbased models for these hybrids: P15:P15′m10 < P15:P15′m8 < P15:P15′m6. We used the data of Figure 3 to further evaluate the effectiveness of the Vainrub and Pettitt model for predicting stability. In our evaluation, we used solution models to obtain nominal values of ΔH° and ΔS° for each of mismatched hybrid structures (P15′m10: ΔH° = −378 kJ·mol−1, ΔS° = −1080 J· mol−1·K−1, P15′m8: ΔH° = −400 kJ·mol−1, ΔS° = −1130 J· mol−1·K−1, and P15′m6: ΔH° = −417 kJ·mol−1, ΔS° = −1160 J·mol−1·K−1), which were allowed to vary by less than two percent. Additionally, since the w electrostatic parameter is a function of the solution environment and not the type of hybrid being investigated, we used the value that was determined from the data set in Figure 2 (in 0.25 mol·L−1 NaCl, 7.0 × 10−17 J· m2·mol−1). The resulting predictions of hybridization activity vs

the probe spacing or salt content of the solution decrease, a trend that correlates with the observed Tm values in Table 2. Although eq 3 does not account for the additional variables present in surface hybridization reactions, it can be used to identify and quantify the differences between surface and solution hybridization behaviors.13,15,16 ⎛ ΔH ° − T ΔS° ⎞ c(1 − θ ) ⎟ = exp⎜ ⎝ ⎠ θ RT

(3)

Since the collective results of Figure 2 and Table 2 illustrate how probe coverage influences the stability of hybrids, we focused on using our data set to evaluate a model developed by Vainrub and Pettit that includes an electrostatic component.9,10 This destabilizing component, described by the second exponential of eq 4., includes the numbers of nucleotides in the probe (ZP) and target (ZT) sequences, probe density (Nm), and an electrostatic interaction parameter (w) whose value depends on the ionic strength of the hybridization solution and is proportional to the Debye screening length of the solution. The model predicts how θ, should vary as a function of temperature, for different probe coverages, solution salinity, and nucleotide compositions of probes and targets. Local variations in the probe density are accounted for by considering differences in the number of nearest neighbors, m (0−6), that each probe has in a hexagon lattice on the surface.10 These variations are significant because the nearest neighboring strands have the largest impact on repulsive interactions experienced by probes. For each sensor, the full predicted melt curve θ(T), is obtained from the summation of a set of individual melting curves (θm(T)), each weighted by a corresponding probability, pm, that an individual probe has m neighbors (eq 5). ⎛ ΔH ° − T ΔS° ⎞ ⎛ wNmZ P(Z P + θmZT ) ⎞ c(1 − θm) ⎟exp⎜ = exp⎜ ⎟ ⎝ ⎠ ⎝ ⎠ θm RT RT (4) 6

θ=

∑ pm θm m=0

(5)

The results of applying this model to our experimental data are shown by the solid lines in Figure 2. In our analysis we used: C = 4 μmol·L−1, ZP = 35 (15 hybridizing nucleotides +5 nonhybridizing T nucleotides +15 A15* nucleotides), ZT = 15. The ΔH° and ΔS° were obtained from solution-based models for the hybridization reaction, and allowed to vary by a few percent during the fitting procedure (ΔH° = −460 kJ·mol−1, ΔS° = −1320 J·mol−1·K−1). The pm values were calculated as described in ref 10 using the average probe densities reported in Table 2. We used the electrostatic interaction parameter as the primary fit parameter, obtaining a single value for the data from each salt content solution. For the 0.10 mol·L−1 NaCl-TE hybridization data, best fits were found using w of 9.9 × 10−17 J· m2·mol−1. For the 0.25 mol·L−1 NaCl-TE hybridization data, best fits were obtained using a w of 7.0 × 10−17 J·m2·mol−1. Visually, the model provides reasonable matches to our full data set. In particular, curves from the model display the shift of the hybridization activity curves to lower temperature and broadening of the curves as the probe surface density increases. The fitted w values for the two data sets are roughly proportional to the difference in Debye screening lengths for 0.10 mol·L−1 NaCl and 0.25 mol·L−1 NaCl solutions, although smaller than expected if calculated directly by the methods of E

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interactions are affected by properties of the sensor and solution environment. Such measurements facilitate the development of accurate modeling of kinetic and thermodynamic parameters for sensor binding and are important information for the continued development of surface-based technologies relying on DNA hybridization.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04048. Procedure for calculation of probe coverage; immobilization data from additional samples; AFM images of sensors; melt curves for P15:P15′ hybrids obtained from solution measurements; and immobilization and hybridization data from low coverage sensor (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Aric Opdahl: 0000-0003-4200-6200 Author Contributions

The manuscript was written through contributions of all authors.

Figure 3. Temperature profiles of SPR measured hybridization activity of P15 probes with DNA targets containing single nucleotide mismatches. Hybridization is with P15′m10 (black), P15′m8 (blue), and P15′m6 (red) targets on sensors with probe coverages of (a) 3.2 × 1012 cm−2 and (b) 1.1 × 1013 cm−2. Hybridization measurements were performed in 0.25 mol·L−1 NaCl-TE buffer solution. Scatter plots are the normalized averages of experimental data from multiple replicate sensors. Solid lines show the results of applying the theoretical model described by eq 4, using solution values for ΔH° and ΔS° and the w (7.0 × 10−17 J·m2·mol−1) that was determined from the fitting of the experimental data shown in Figure 2.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation under Grant No. NSF-RUI 1152042. REFERENCES

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temperature are shown by the solid lines in Figure 3. These predictions are reasonably matched to the experimental data, in particular, describing the shapes and shifts of experimental data on both low and high probe coverage sensors. Thus, the model would seem generally acceptable for predicting hybrid stabilities, provided the morphology of the probe layer is known. Ongoing investigations include evaluation of temperature gradient SPR measurements from a larger set of mismatch hybrids and correlation of hybridization activity (e.g., sensor response) with hybrid stability.

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CONCLUSION We have used temperature gradient SPR measurements to experimentally determine how the probe density on a hybridization sensor impacts the stability of hybrids formed with target DNA strands. Specifically, the experimental results presented here provide quantitative evidence of how DNA hybrids on surfaces are affected by electrostatic interactions and were used to provide support for a predictive model of stability that incorporates these repulsive interactions. More generally, the results demonstrate the utility of the temperature gradient SPR method as a technique that can be used to better understand biosensor binding interactions, by providing a route to efficiently generate a large empirical data set of how these F

DOI: 10.1021/acs.analchem.6b04048 Anal. Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.analchem.6b04048 Anal. Chem. XXXX, XXX, XXX−XXX