Surface Coverage and Structure of Mixed DNA/Alkylthiol Monolayers on Gold: Characterization by XPS, NEXAFS, and Fluorescence Intensity Measurements

Abstract

Self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) on gold has served as an important model system for DNA immobilization at surfaces. Here, we report a detailed study of the surface composition and structure of mixed self-assembled DNA monolayers containing a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)] on gold supports. These mixed DNA monolayers were studied with X-ray photoelectron spectroscopy (XPS), near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements. XPS results on sequentially adsorbed DNA/MCU monolayers on gold indicated that adsorbed MCU molecules first incorporate into the HS-ssDNA monolayer and, upon longer MCU exposures, displace adsorbed HS-ssDNA molecules from the surface. Thus, HS-ssDNA surface coverage steadily decreased with MCU exposure time. Polarization-dependent NEXAFS and fluorescence results both show changes in signals consistent with changes in DNA orientation after only 30 min of MCU exposure. NEXAFS polarization dependence (followed by monitoring the N 1s → π* transition) of the mixed DNA monolayers indicated that the DNA nucleotide base ring structures are oriented more parallel to the gold surface compared to DNA bases in pure HS-ssDNA monolayers. This indicates that HS-ssDNA oligomers reorient toward a more-upright position upon MCU incorporation. Fluorescence intensity results using end-labeled DNA probes on gold show little observable fluorescence on pure HS-ssDNA monolayers, likely due to substrate quenching effects between the fluorophore and the gold. MCU diluent incorporation into HS-ssDNA monolayers initially increases DNA fluorescence signal by densifying the chemisorbed monolayer, prompting an upright orientation of the DNA, and moving the terminal fluorophore away from the substrate. Immobilized DNA probe density and DNA target hybridization in these mixed DNA monolayers, as well as effects of MCU diluent on DNA hybridization in complex milieu (i.e., serum) were characterized by surface plasmon resonance (SPR) and ³²P-radiometric assays and reported in a related study

Methods for surface-immobilizing single-strand nucleic acids that preserve their original hybridization specificity with minimized nonspecific interactions remain an important goal for improving the performance of DNA microarray and biosensor applications. As summarized in a recent review,¹ nucleic acid hybridization behavior observed between complementary probe and target DNA molecules in bulk solution differ from identical hybridization at a solid–liquid interface. In surface hybridization, nonspecific probe–surface interactions, electrostatic forces, and steric issues between adjacent DNA probes influence DNA target hybridization efficiency and capacity. For example, nucleotide primary amines on nonhybridized DNA segments can interact (e.g., covalently² or by acid–base adsorption) with the surface, becoming unavailable to hybridize with target DNA molecules. Effects of immobilized DNA probe density, charge density, and orientation on target capture and methods to reliably control these surface states remain poorly characterized on most surfaces. Therefore, before the full potential of DNA microarrays can be realized, many fundamental issues must be better understood, including how the crowding, conformation, and orientation of immobilized DNA impact DNA target hybridization efficiency.

Tarlov and co-workers pioneered a technique for DNA immobilization that utilizes the chemisorptive self-assembly of thiol-terminated single-stranded DNA (HS-ssDNA) monolayers onto gold surfaces, mixed with a short hydroxyl-terminated alkylthiol surface diluent [e.g., mercaptohexanol (MCH)].^3–5 Based on X-ray photoelectron spectroscopy (XPS),^4,6 neutron reflectivity,³ and surface plasmon resonance (SPR)⁵ studies of this mixed self-assembled monolayer (SAM) system, the MCH has been proposed to prevent the nonspecific attachment of DNA to the surface by nucleotide amine groups and enhance specific attachment by the thiolated group.

Chemisorbed adlayers of alkylthiols on gold provide a high degree of control over surface physical properties, providing useful model systems for examining relationships between HS-ssDNA surface composition and orientation, as well as subsequent hybridization behavior with solution-phase targets. These previous studies have provided some important quantitative information on possible HS-ssDNA conformational changes in self-assembled mixed monolayers resulting from addition of the MCH surface diluent. However, HS-ssDNA oligomers may behave differently when the packing density or other properties (e.g., DNA length) of the film are varied. To develop a fuller understanding of surface properties in these mixed DNA monolayers at the molecular level, the packing density of the HS-ssDNA was systematically changed by backfilling the pure HS-ssDNA monolayer with a short alkylthiol surface diluent [11-mercapto-1-undecanol (MCU)], similar to those used in the original reports.^3–5 The effect of diluent backfill time on mixed adlayer surface composition, density, and orientation of HS-ssDNA oligomers was evaluated with XPS, near-edge X-ray absorption fine structure spectroscopy (NEXAFS), and fluorescence intensity measurements.

To the best of our knowledge, relatively few DNA characterization experiments using high-resolution surface analytical techniques^3,4,6–8 have been directed at immobilized DNA molecules in mixed monolayer systems. In previous investigations, we reported XPS quantitation of the chemical composition of individual DNA nucleobases, nucleotides, and oligomers⁹ and identification of contaminants in thiolated ssDNA monolayer films on gold.¹⁰ In this work, we have used complementary XPS, NEXAFS, and fluorescence intensity measurements to characterize monolayer composition, density, and orientational changes as a function of MCU backfill time and DNA surface displacement. Quantitative atomic compositions of the mixed DNA monolayers were obtained and compared using XPS. Polarization-dependent NEXAFS, also known as X-ray absorption near-edge structure (XANES),¹¹ was used to probe surface orientation and order in pure DNA and mixed DNA/MCU monolayers. NEXAFS has been used to examine the surface molecular orientation and ordering for a variety of materials including SAMs on gold¹² and polymers.^13–16 In previous DNA studies, NEXAFS has been used to characterize DNA nucleobases and nucleotides,^17–21 as well as the orientation of polydA²¹ and polydT^21,22 oligomers on gold. Finally, end-labeled DNA fluorescence intensity measurements were used to obtain information about the interaction between the DNA oligomers and the gold substrate by comparing the normalized fluorescence intensities from DNA monolayers with varied MCU backfill time.

In our companion article,²³ we have used ³²P-radiometric measurements of immobilized DNA densities (e.g., molecules/cm²) to calibrate the XPS nitrogen and phosphorus signals from DNA monolayers identical to those reported here, creating a convenient basis for XPS-based DNA surface density determinations. Furthermore, the molecular densities of these same DNA monolayers were then correlated with their DNA hybridization efficiencies using both radiometric ³²P-labeling and real-time SPR assays. DNA hybridization and target capture efficiencies from complex milieu (i.e., serum dilutions) were further evaluated for an optimized DNA/MCU mixed monolayer surface using SPR. Corroboration of results between these two studies on identical DNA–gold monolayer surfaces provides a new, more comprehensive picture of HS-ssDNA “molecular disposition” immobilized on a surface that influences hybridization events relevant to microarray capture assays and other biotechnological manipulations on surfaces.

EXPERIMENTAL SECTION

Preparation of Mixed DNA/MCU Self-Assembled Monolayers for XPS and NEXAFS Analysis

Silicon wafers (Silicon Valley Microelectronics, Inc., San Jose), coated with 10-nm Cr and 80-nm Au (99.99%) by electron beam evaporation at pressures below 1 × 10^–6 Torr, were used as substrates. DNA oligomers [5′-HS-(CH₂)₆-CTGAACGGTAGCATCTTGAC-3′] were purchased from TriLink Biotechnologies (HPLC-purified for highest purity;¹⁰ San Diego, CA). Mixed DNA/MCU monolayers of varying DNA surface coverage were assembled by a known, sequential two-step process. First, pure DNA monolayers were prepared by immersing freshly gold-coated substrates in 1 μM HS-ssDNA solutions in 1 M NaCl–TE buffer (1 M NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4; Fisher, Fair Lawn, NJ) for 5 h. After HS-ssDNA assembly, samples were rinsed thoroughly with buffer for 30 s and Millipore grade water for 1 min to remove loosely bound HS-ssDNA. These DNA-adsorbed samples were then immersed in 10 μM MCU (97% purity, Sigma-Aldrich) diluent thiol solution (in water) for backfill times ranging from 30 min to 18 h. After the specified MCU backfill time, samples were removed from the solutions and rinsed thoroughly in Millipore grade water for 1 min. Samples were then blown dry with N₂ and stored under N₂ until analysis.

X-ray Photoelectron Spectroscopy

XPS is a quantitative, surface analytical tool sensitive to the atomic composition of the outermost 100 Å of a sample surface. XPS measurements were performed on a Kratos AXIS Ultra DLD instrument using a monochromatic Al Kα X-ray source and a 0° take-off angle. The take-off angle is defined as the angle between the sample surface normal and the axis of the XPS analyzer lens. Compositional survey and detailed scans (P 2p, C 1s, N 1s, O 1s, S 2p) were acquired using a pass energy of 80 eV. High-resolution spectra (P 2p, C 1s, N 1s, O 1s, S 2p, Au 4f) were acquired for the DNA samples using a pass energy of 20 eV. For the high-resolution spectra, peak binding energies were referenced to the Au 4f peak at 84.0 eV. Three spots on two or more replicates of each DNA sample were analyzed. Reported compositional data were averages of values determined at each spot. Data analysis was performed with Vision Processing data reduction software.

Near-Edge X-ray Absorption Fine Structure Spectroscopy

NEXAFS spectra were taken at the National Synchrotron Light Source (NSLS) U7A beamline at Brookhaven National Laboratory, using an ~85% polarized, high-intensity beam.²⁴ This beam line uses a monochromator and 600 l/mm grating that provides a full-width at half-maximum (fwhm) resolution of ~0.15 eV at the carbon K-edge (~285 eV). The monochromator energy scale was calibrated using the 285.35 eV C 1s → π* transition of adventitious hydrocarbon on a gold-coated 90% transmission grid placed in the path of the X-rays. To eliminate the effect of incident beam intensity fluctuations and monochromator absorption features, all NEXAFS spectra were normalized by the signal from a pure gold (gold deposited in situ) control sample (I₀) and the beam flux (I_ring). Partial electron yield was monitored by a detector with the bias voltage maintained at –150 V for the carbon K-edge spectrum and –350 V for the nitrogen K-edge spectrum. Samples were mounted to allow rotation about the vertical axis to change the angle between the sample surface and the incident X-ray beam. The NEXAFS angle is defined as the angle between the incident X-ray beam and the sample surface. The incident beam normal to the surface is defined as 90° while a glancing incident beam is generally 20° from the surface plane. The electric field vector (E) is perpendicular to the X-ray beam; therefore, when the beam is at normal incidence, the E vector lies parallel to the surface. A disordered system on the sample surface does not show any polarization dependence because of random distribution in molecular orientation. Polarization dependence is indicative of directional alignment of the molecules in the overlayer.¹¹

Fluorescence Intensity Measurements of DNA–Cy3 Layers on Gold

The 3′-thiolated DNA probe bearing a 5′-fluorescent-Cy3 label [5′-Cy3-CTGAACGGTAGCATCTTGAC-(CH₂)₆-SH-3′] was custom-synthesized and HPLC-purified (TriLink Biotechnologies). Gold substrates were prepared by thermal evaporation of 100 nm of Au onto a 10-nm Cr adhesion layer using an Auto306 coating system (Edwards, Wilmington, MA) on low-fluorescence glass slides (OptArray, Accelr8, Denver, CO). The gold substrates were then cleaned with O₂ plasma (5 min, 100 W, 0.1 Torr) just prior to immersion into DNA probe solutions. DNA probe surface immobilization was performed at room temperature using 1 μM thiolated DNA probe in 1 M NaCl–TE buffer at pH 7.0 for 5 h, followed immediately by various timed exposures of 10 μM MCU backfilling diluent solutions as described above. Probe samples were then rinsed with Millipore grade water for 15 s and dried with N₂. Fluorescence intensity images of the resulting surfaces were collected using a ScanArray Express scanner (Perkin-Elmer, Fremont, CA) and analyzed using ImageQuant software (Amersham Biosciences).

RESULTS AND DISCUSSION

XPS Analysis of Pure DNA and Mixed DNA/MCU Monolayer Compositions

XPS was used to obtain detailed information about the chemical composition of mixed monolayers of HS-ssDNA and MCU. Adlayer compositional change was followed as pure DNA monolayers formed from the first assembly step on gold were exposed to MCU diluent thiol for varied lengths of time (0.5–18 h). Table 1 summarizes the XPS elemental compositions from pure HS-ssDNA and mixed DNA/MCU monolayers. Only those elements expected from the HS-ssDNA (P, C, N, O, S), MCU (C, O, S), and substrate (Au) were detected in XPS survey scans. Elements P and N are unique to DNA and are therefore good indicators of relative amounts of HS-ssDNA present on the surface under each condition.

Table 1.

XPS Compositional Data for Pure DNA and Mixed DNA/MCU Monolayers on Gold^a

		atomic percent						atomic ratio
time in HS-ssDNA (h)	time in MCU (h)	P 2p	N 1s	O 1s	C 1s	S 2p	Au 4f	P/N	O/N	C/N
HS-ssDNA theoretical								0.3	1.7	2.8
5	0	1.4	7.6	15.4	41.9	-	33.6	0.2	2.0	5.5
5	0.5	1.1	5.7	12.5	52.0	1.0	27.8	0.2	2.3	9.8
5	1	1.2	5.2	11.9	52.1	1.3	28.2	0.2	2.3	10.4
5	2	0.8	5.3	11.8	53.9	1.2	26.8	0.2	2.3	10.4
5	5	0.8	3.9	10.4	53.7	1.4	29.8	0.2	2.8	14.6
5	18	0.6	3.2	9.7	55.0	1.5	30.1	0.2	3.1	17.4
0	18	0.0	0.0	7.9	57.6	2.6	31.9

^aAll standard deviations <2%.

Surfaces of pure HS-ssDNA on gold, in the absence of MCU diluent thiol, exhibited a composition of 1.4% P, 7.6% N, 15.4% O, 41.9% C, and 33.6% Au (as summarized in Table 1). The experimental atomic ratios calculated from the atomic percentages (P/N = 0.2, O/N = 2.0, C/N = 5.5) are similar to those predicted by the stoichiometry of the DNA molecule (P/N = 0.3, O/N = 1.7, C/N = 2.8) with the exception of excess carbon. This is likely due to the presence of adventitious hydrocarbon contamination, a presumption supported by the accuracy of the P/N and O/N atomic ratios. After short-term exposure of the DNA monolayer to the MCU diluent thiol (<2 h), relative atomic percents of C and S increased, while relative atomic percents of Au, P, N, and O all decreased (see Table 1). The increased atomic percents of C and S are consistent with the presence of MCU, as the smaller MCU diluent thiols take up unoccupied gold sites surrounding the loosely packed DNA on the surface. The corresponding Au signal decreased during this same short-term exposure of the DNA monolayer to MCU consistent with this interpretation. MCU surface reactions could also displace other weaker, nonspecific interactions between nitrogen-containing DNA bases and gold, promoting single-point tethering of DNA oligomers on gold via thiolate bonds, with greater tendency to orient away from the surface. NEXAFS and fluorescence data further support this contention.

For longer MCU exposure times (>2 h), relative Au substrate atomic percent increased, while the relative atomic percents of P and N decreased further. These results indicate that after gold site-filling, further MCU exposure promotes displacement of DNA molecules from the surface by the more competitive MCU diluent thiols. In the companion article,²³ we present radiolabeling evidence which supports the hypothesis that the DNA is displaced by MCU at longer backfill times in identical adlayer systems.

In addition to elemental composition changes, significant differences in the XPS high-resolution spectra between the pure DNA and mixed DNA/MCU monolayers were also observed. Figure 1 compares high-resolution C 1s, N 1s, P 2p, O 1s, S 2p, and Au 4f spectra for a pure DNA monolayer to that after a 1 h MCU backfill. The pure HS-ssDNA adlayer has a C 1s high-resolution spectrum (Figure 1a) with four peaks attributed to DNA, as previously described.^9,10 The relative concentrations of the different carbon species were 54% C–C and C–H (285 eV), 31% C–N and C–O (287 eV), 11% N–C(=O)–C, N–C(=N)–N, N=C–N, and N–C–O (288 eV), and 4% N–C(=O)-N (289 eV), as summarized in Table 2. When compared with theoretically expected values (Table 2), XPS experimental values exhibit higher C–H species concentrations, supporting the same conclusion (vida infra) that excess %C is from adventitious hydrocarbon contamination. After 1 h exposure of the HS-ssDNA monolayer to the MCU diluent thiol, the hydrocarbon peak (C–C and C–H) intensity at 285 eV increased, supporting the conclusion that the increase in carbon composition (summarized in Table 1) is due primarily to increased hydrocarbon species from the MCU (for high-resolution XPS C 1s spectrum, see Figure 1a; for compiled high-resolution XPS C 1s results, see Table 2). Increased C–H species (due to increased MCU exposure) produced a corresponding decrease in peak intensities for the other DNA-related carbon peaks for species at ~287, ~288, and ~289 eV (see Table 2).

Figure 1.

High-resolution XPS (a) C 1s, (b) N 1s, (c) P 2p, (d) O 1s, (e) S 2p, and (f) Au 4f spectra from pure DNA and mixed DNA/MCU (1 h MCU backfill) monolayers on gold. Peak binding energies for high-resolution spectra were referenced to the Au 4f peak (f) at 84.0 eV. Decreases in the N 1s, P 2p, and O 1s peak intensities are also observed after MCU backfill (b–d). S 2p peaks are observed only after MCU backfill. The BE of the S 2p_3/2 peak (e, 161.9 eV) is consistent with the sulfur bound to the gold surface as a thiolate species.25 Note that the spectra in each figure are on the same scale, offset for clarity.

Table 2.

XPS High-resolution C 1s Chemical Species of Pure DNA and Mixed DNA/MCU Monolayers on Gold^a

		Percentage
Time in HS-ssDNA (h)	Time in MCU (h)	C–C, C–H (285 eV)	C–N, C–O (287 eV)	N–C(=O)–C, N–C(=N)–N, N=C–N, N–C–O (288 eV)	N–C(=O)–N (289 eV)
HS-ssDNA theoretical		20.0	45.0	27.0	8.0
5	0	54.3	30.9	10.7	4.1
5	0.5	61.5	25.8	9.0	3.7
5	1	67.7	22.0	7.3	3.0
5	2	70.6	20.2	7.2	1.9
5	5	72.2	20.0	7.0	0.8
5	18	80.5	16.2	3.3
0	18	88.5	11.5
MCU theoretical		90.9	9.1

^aAll standard deviations <2%.

While the presence of an S 2p doublet structure at 161.9 eV in mixed DNA/MCU monolayers (Figure 1e) is clear evidence of sulfur bound to gold as a thiolate,²⁵ no such features were observed in the S 2p region for the pure HS-ssDNA monolayers. This was expected as the stoichiometric sulfur concentration in the HS-ssDNA molecule (0.2 atom %) was close to the XPS detection limit (~0.1%) and the S signal was likely attenuated by the overlying DNA film. Backfill of MCU molecules increased relative percentages of sulfur species on the surface (7.9 atom %), allowing unambiguous thiolate assignment of the sulfur doublet in mixed DNA/MCU films near 162 eV.

Figure 1 also shows that addition of MCU diluent shifts the binding energies (BE) in all XPS high-resolution spectra. Peak binding energies for all high-resolution spectra were referenced to the Au 4f peak at 84.0 eV (see Figure 1f). As seen in Figure 1a–d, the C 1s, N 1s, P 2p, and O 1s XPS peaks from pure HS-ssDNA monolayers shift to lower BEs than those from mixed DNA/MCU films. While the BE shifts for the C 1s, P 2p, and O 1s peaks were small (~0.3–0.5 eV), the BE shift for the N 1s peaks were of larger magnitude (~1.0 eV). These XPS peak shifts seen in pure DNA monolayers are proposed to arise from non-thiol interactions of the DNA polyanions with the gold substrate. Furthermore, the larger BE shift observed for the N 1s spectrum in pure DNA monolayers indicates that these DNA interactions with the surface may also occur through the nitrogen atoms in the DNA bases, producing the observed shift in pure HS-ssDNA adlayers that is removed when MCU diluent outcompetes these interactions to force DNA into a more upright configuration.^4,8

NEXAFS Orientation Studies of Pure DNA and Mixed DNA/MCU Monolayers

Polarization-dependent NEXAFS was used to further probe the surface orientation and order of the pure DNA and mixed DNA/MCU monolayers. X-ray absorption by molecular orbitals is strongly dependent on the favorable overlap of antibonding orbitals with the electric field (E) vector of the incident X-rays. As a result, the polarized X-ray absorption spectra will show differences with differing incident X-ray angles for oriented and ordered molecules at surfaces. For little long-range alignment in the structure of the surface-bound DNA, the X-ray absorption spectra will not vary with angle of incidence. Polarization dependence observed in the NEXAFS spectrum is therefore indicative of both the orientation and the long-range ordering of the molecules in the overlayer. In this study, C K-edge and N K-edge NEXAFS spectra of DNA monolayers were collected at normal (incident X-ray beam 90° to the surface) and glancing (20°) angles to examine the effect of MCU backfill on the orientation of gold-bound DNA oligomers.

NEXAFS Orientation Studies: N K-Edge

Figure 2a presents the NEXAFS N K-edge spectra for the pure DNA and mixed DNA/MCU (0–18 h MCU backfill) monolayers measured at normal and glancing X-ray incident angles. All NEXAFS N K-edge spectra from pure DNA and mixed DNA/MCU monolayers contained a doublet feature in the 399–402 eV region. Previous studies on nucleobases,^17–21 DNA oligomers,^21,26 and other polymers with similar nitrogen-containing ring structures^27–29 found similar splitting of the two π* orbitals (ΔE = 1.7 eV). We hypothesize that the π* doublet observed in the nitrogen region represents an average signal over the four different nucleotide bases. While the higher energy π* peak of ~401 eV is attributed to the nitrogen atoms in the nucleobases located next to carbonyl groups,^21,26 the lower energy π* peak near 399–400 eV is consistent with the location of the “aromatic” nitrogen π* peak in molecules that have nitrogen atoms present in a ring structure.^27,29 The broader peak above 405 eV is attributed to the N 1s → σ* transition.^21,26

Figure 2.

Nitrogen (a) and carbon (b) K-edge NEXAFS spectra from pure DNA and mixed DNA/MCU monolayers on gold at normal (90°) and glancing (20°) incident X-ray angles (t = MCU backfill time in hours). The increase in polarization dependence of nitrogen K-edge NEXAFS spectra (a) indicates that DNA bases are oriented more parallel to the surface than bases in the pure DNA monolayer and that ssDNA oligomers reorient on average toward a more upright orientation on the surface upon MCU addition. The decrease in intensity of the π*_C=C and σ*_C–NH peaks in the carbon K-edge NEXAFS spectra (b) with longer MCU backfill time is consistent with DNA displacement from the surface. From the polarization dependence of the mixed monolayer spectra (t > 0), MCU alkyl chains orient in an upright position away from the surface. The increase in σ*_C–H and σ*_C–C peak intensities with increasing MCU backfill time is consistent with increasing MCU surface density with prolonged exposure to the DNA monolayer.

For the pure HS-ssDNA monolayer (Figure 2a, t = 0), the intensity of the π* peaks was slightly higher when the X-ray beam was at a glancing angle of incidence compared to that at normal incidence. At glancing incidence, the E vector of the polarized X-ray source is nearly perpendicular to the surface; therefore, the overlap of this E vector with the aromatic nitrogen bonds that cause the 1s → π* transition indicates that the DNA bases in a pure DNA film were, on average, oriented nominally parallel to the surface. Similar trends were observed with surface-bound double-stranded DNA oligomers on gold.²⁶ This indicates that on average the HS-ssDNA chains had a slightly perpendicular orientation to the substrate. It is believed that the electrostatic repulsive forces between the ssDNA chains may cause the chains to stand on average slightly upright on the surface, although it is likely they remain disordered overall. Upon incorporation of the MCU diluent thiol into the DNA monolayer, this polarization dependence increased significantly (also Figure 2a, t ≥ 0.5), indicating that MCU-induced changes to the ssDNA oligomers forces some degree of reorientation, on average more toward an upright conformation on the surface, with DNA bases oriented more parallel to the surface. This change in orientation is further evidence that the MCU diluent thiol removes unintended nucleo-base amine groups from interactions with the gold surface.

To compare the change in orientation of these DNA monolayers as a function of backfill time, the dichroic ratio, ΔN_π*,^30,31

$$\mathrm{\Delta }{N}_{\pi *}=\frac{N_{\pi *,20°}-N_{\pi *,90°}}{N_{\pi *,20°}+N_{\pi *,90°}}$$

was calculated. These ΔN_π* values are summarized in Figure 3. Note that the dichroic ratios calculated here are relative and cannot be directly compared to the values from different experimental setups. Comparison of dichroic ratios derived from different experiments requires a correction factor 1/(2P – 1), where P is the polarization degree of the synchrotron light.^30,31 ΔN_π* initially increased rapidly with MCU backfill time, reaching a maximum at 0.5–1 h. Beyond 1 h of MCU backfill, ΔN_π* began to decrease slightly. All ΔN_π* values for the mixed DNA/MCU monolayers were greater than the ΔN_π* value for the pure HS-ssDNA monolayer, indicating that, on average, the DNA chains in the MCU backfilled surfaces are more perpendicular to the surface than those in pure DNA adlayers. The trend toward ΔN_π* decreasing at longer MCU backfill times may be due to loss of ssDNA (seen with the XPS results shown above as well as ³²P-labeling results in the companion paper²³). With less DNA on the surface, the electrostatic repulsive interactions between ssDNA chains would be less effective at holding the DNA molecules perpendicular to the surface, permit more disorder among the DNA chains, and facilitate some nucleotide–surface re-engagement. Despite the reduction in N 1s nitrogen signal at longer MCU backfill times, ΔN_π* at t = 18 h was still greater than that of the pure HS-ssDNA surface, leading to the conclusion that the change in polarization dependence was due to relative orientational changes in the DNA layer. For MCU-backfilled DNA samples where the greatest polarization dependence was observed for the N 1s → π* peaks (i.e., 0.5–1 h MCU backfill times), the N 1s → σ* peak also showed a slight polarization dependence (Figure 2a).

Figure 3.

Dichroic ratio (ΔN_π*) for pure DNA and mixed DNA/MCU monolayers on gold as a function of MCU backfill time. ΔN_π* reaches its maximum at ~0.5–1 h of MCU exposure, after which ΔN_π* decreases due to the loss of DNA from surface.

NEXAFS Orientation Studies: C K-Edge

The C K-edge spectra contained a series of features originating from 1s → π* and 1s → σ* transitions in organic molecules (see Figure 2b). The major peaks in the C K-edge NEXAFS spectra were assigned to functional groups present in HS-ssDNA and MCU using results from previous studies of alkylthiol monolayers, amino acids, and polymers. For example, the peak at 285 eV was assigned to π*_C=C species in DNA based on the features found in NEXAFS spectra of polymers¹¹ and amino acids³² containing double-bonded and aromatic carbon atoms. Upon addition of MCU diluent thiols to the DNA adlayer surface, the π*_C=C peak intensity decreased due to displacement of DNA oligomers by MCU molecules. The peak at ~289 eV was attributed to the σ*_CNH peak in DNA, based on previous assignments for amino acids.³² The σ*_CNH peak was also more pronounced in the C K-edge spectra of pure HS-ssDNA than that in mixed DNA/MCU (0–18 h MCU backfill) monolayers due to the higher concentration of C–N species in pure DNA, as shown by XPS analysis in the previous section. The peak at 287.4 eV was attributed to a transition to antibonding orbitals involving the C–H group.^12,33 With the addition of MCU diluent thiol, this peak was enhanced when the X-ray beam was at normal (90°) incidence to the sample surface, indicating that the C–H bonds in the MCU molecules were oriented nominally parallel to the surface. Meanwhile, the peak at 293 eV, assigned to the C–C species (σ*_C–C),12,33 was enhanced when the X-rays were at glancing (20°) incidence. This indicates that the C–C σ* orbital had more of a perpendicular orientation. The polarization dependences of the σ*_C–H and σ*_C–C peaks were similar to those for spectra previously reported for hydrocarbon adlayers¹² and Langmuir–Blodgett films.³³ These results indicate that the MCU alkyl chain is oriented in an upright position away from the surface, further suggesting that the MCU diluent thiol structurally orders the mixed DNA/MCU surface.

Fluorescence Studies of Pure DNA and Mixed DNA/MCU Monolayers

Fluorescence signal intensities of analogous, fluorophore-labeled HS-ssDNA probes on gold were used to study orientation changes in pure DNA and mixed DNA/MCU monolayers on gold of known densities of 1.7 × 10¹³–4.4 × 10¹³ probes/cm² (based on ³²P-DNA radiolabel work from Table 2 in the companion paper²³). Normalized relative fluorescence intensity of surface-bound Cy3-labeled DNA probes as a function of MCU backfilling time is shown in Figure 4a. Very little fluorescence signal was observed from pure fluorophore HS-ssDNA adlayers when MCU diluent molecules were absent. This could be explained using two different quenching mechanisms: lateral nonradiative energy transfer (self-quenching) between dyes and nonradiative gold surface–dye energy transfer.

Figure 4.

3′-Thiol–DNA–Cy3–5′ 20-mer probe surface fluorescence intensity in pure DNA and mixed DNA/MCU adlayers on gold. (a) Normalized immobilized DNA fluorescence signal relative to the 2 h MCU backfill data point (100%). Little fluorescence is observed from the pure DNA layer indicating a “lying down” conformation producing Cy3 fluorophore–gold nonradiant quenching without MCU diluent. Relative adlayer DNA–Cy3 fluorescence increases as MCU diluent molecules were incorporated into the adlayer, reaching a maximum at the 2 h MCU backfill condition, then decreasing as additional MCU molecules are assembled into the adlayer, resulting in displacement of DNA-Cy3 from the surface. (b) Rates of change in normalized fluorescence intensity and normalized DNA surface probe densities (data taken from ³²P DNA probe density results in ref 23) are plotted against MCU backfill time. More dramatic rate change is observed for the normalized fluorescence signal, indicating the initial fluorescence intensity increase is a result of change in DNA probe conformation, not surface displacement. (c) Relative fluorescence per probe DNA molecule is derived by dividing the normalized fluorescence signal by corresponding DNA surface probe densities. Fluorescence signal increases dramatically as soon as MCU diluent is introduced into the DNA monolayer, reaching a maximum near 5 h of MCU backfill (density, 2.3 × 10¹³ probes/cm²).

Fluorophore Cy3 lateral self-quenching at most fabricated DNA densities will likely be insignificant when compared to quenching by Cy3–gold interactions since self-quenching does not typically occur until much higher fluorophore densities (~1.5 × 10¹⁴ molecules/cm²).^34–36 Tsukanova and co-workers showed that fluorescence quenching from 7-nitro-2–1,3-benzoxadiazol-4-yl dye aggregate stacking in compressed dye–lipid monolayers at the air–water interface did not occur at a surface density of 1.1 × 10¹⁴ molecules/cm² (0.9 nm²/molecule) but was eventually observed at 2.5 × 10¹⁴ molecules/cm² (0.4 nm²/molecule) upon further lateral monolayer compression and resulting in-plane molecular ordering.³⁴ Schmitt et al. showed that, for two cyanine dyes studied as adsorbed planar films on fatty acid monolayers at the air–water interface, the first evidence for optical transitions associated with monomer → dimer → aggregate in-plane in these compressed films occur between 0.6 and 0.7 nm²/molecule. This translates dye density requirements of 1.4 × 10¹⁴–1.7 × 10¹⁴ molecules/cm² in these compressed but initially fluid monolayer systems to produce consistent, observable energy-transfer effects.³⁵ Both molecular density estimates for in-plane quenching are 1 order of magnitude greater than the planar immobilized HS-ssDNA Cy3 surface densities produced in this study (1.7 × 10¹³–4.4 × 10¹³ molecules/cm²), underscoring the unlikely probability that Cy3–Cy3 quenching is the major cause for the low fluorescence signals observed from pure DNA monolayers. Dye self-quenching via fluorescence resonance energy transfer (FRET) is a viable mechanism for decreased fluorescence intensity in high-density systems with highly oriented fluorophores.³⁷ The fluorophore orientational dependence of FRET quenching, as described by the orientation factor, K², indicates the significance of fluorophore dipole orientation in efficient energy-transfer quenching.³⁸ However, given the nearly random orientation of the immobilized DNA probes observed in the pure HS-ssDNA monolayer (cf. NEXAFS evidence), their low lateral densities compared to known fluorophore Förster distance dependence^37,41 shown in other systems,^33–35 and results from the lateral spacing fluorophore dilution experiments (see below, Figure 4b), self-quenching is not perceived to be a major cause of the low DNA Cy3 fluorescence intensity emanating from the pure DNA monolayer. Cy3 dye self-quenching is therefore not significant under desiccated, DNA surface-immobilized conditions that limit effective dynamic chain motion, rearrangement, or dye mobility (e.g., rotational, translational) within each layer except possibly during MCU diluent addition.

Alternatively, low fluorescence intensity observed for pure HS-ssDNA adlayers on gold is primarily attributed to the substantial fraction of non-upright probe DNA molecules at these lower immobilization densities. Gold surfaces in proximity to a fluorophore are well known to strongly quench fluorescence from excited dipoles, primarily from nonradiative energy transfer of dye to metal.^39–41 Lack of vertical DNA chain orientation permits sufficient Cy3–gold surface contact as an energy-transfer and dye-quenching mechanism in these adlayers. This quenching efficiency decreases as the gold–fluorophore separation distance (Förster distance, generally up to a few nanometers) increases, diminishing to zero beyond this radius.⁴² Nonvertical DNA conformation is consistent with both poor fluorescence yield from these surfaces and the relatively poor hybridization efficiency observed for this pure DNA adlayer as well (i.e., Figure 3 in the companion paper²³). Also consistent with both XPS and NEXAFS results, DNA fluorescence intensity increases dramatically upon MCU exposure, reflecting changes in either DNA surface density or adlayer conformational or orientational changes that remove the terminal Cy3 dye away from the gold, reducing nonradiative energy transfer, increasing signal. Figure 4b shows relative rates of fluorescence intensity change and loss of surface probe DNA, both as functions of MCU exposure in DNA–gold adlayers. Importantly, as in NEXAFS results, most fluorescence signal increase occurs at relatively short MCU backfill times when only moderate changes in DNA molecular density occur (i.e., low probe displacement). These results indicate that adlayer DNA conformational and orientational changes are the primary contributors to the observed significant increases in fluorescence signal at short MCU backfill times. These interpretations are consistent with NEXAFS data and ³²P density measurements.²³

Orientational effects between proximal fluorophores also contribute to quenching probability within the Förster distance.⁴³ A fully extended 20-mer DNA molecule measures ~6–7 nm in length, comparable to the largest effective quenching distance. At highest molecular densities (pure DNA monolayers, 4.4 × 10¹³ probes/cm²), average probe–probe separation distance is ~1.5 nm, increasing to 2.5 nm as MCU diluent concentration increases. As all DNA densities in this study are well below an ideally close-packed layer⁴⁴ (estimated to be 8 × 10¹³ probes/cm², 1.1 nm average lateral spacing), fluorophore Cy3 dye labels on 5′-ends of 3′-thiol gold-immobilized DNA molecules have sufficient spacing to reside in proximity to the substrate gold surface, allowing fluorescence energy transfer resulting in fluorescence quenching of the dye labels. XPS evidence (vida infra) also shows some DNA–gold interactions with nucleobases that might also stabilize such interactions. Thus, the lack of fluorescence intensity from the pure DNA monolayer is consistent with an average lying-down conformational tendency for thiol–DNA on the gold surface at lower surface densities. As MCU backfill molecules increase, the relative adlayer fluorescence increases, reaching a maximum at the 2-h backfill condition and then decreasing as even more MCU molecules are assembled into the adlayer (see Figure 4a). This relative fluorescence signal observed is a combined result of both fluorophore surface density and the distance between dye and gold substrate. MCU addition to the DNA monolayer initially increases the fluorescence intensity by reorienting the DNA chains to a more upright configuration, removing the fluorophore from gold-quenching interactions, and eventually decreases the fluorescence intensity as MCU displaces DNA molecules from the surface at longer MCU backfill times. This interpretation is consistent with the XPS and NEXAFS data presented above.

To explicitly test the proposed effect of MCU diluent in changing probe DNA orientation and lateral spacing, identical experiments were conducted using 10- and 100-fold diluted Cy3–probe–HS-ssDNA (i.e., diluted with identical thiolated probe DNA lacking a dye label). At a 10-fold dye probe DNA dilution, identical increasing fluorescence trends with MCU exposure observed in Figure 4a for pure dye probe DNA layers were also observed, although the absolute fluorescence intensity was reduced by the dye dilution. At a 100-fold dye probe dilution, fluorescence was indistinguishable from background (dilution data not shown).

Relative fluorescence signal per probe DNA molecule was derived by dividing the normalized fluorescence signal by the corresponding surface DNA probe density derived from ³²P measurements on identical systems (from Table 2 in the companion paper²³). As shown in Figure 4c, a large increase in fluorescence intensity was observed as soon as the diluent MCU molecules were introduced into the adlayer, even at low concentrations (0.5 h MCU backfill), suggesting that adsorbed DNA conformation changes readily under this influence, displacing dye away from the surface as MCU occupies space between probe molecules. In Figure 4c, the normalized fluorescence curve reaches a maximum near 5 h of MCU backfill (corresponding to 2.3 × 10¹³ probes/cm² from the companion article²³). With increased MCU backfill times (1–5 h), the MCU displaces some probe HS-ssDNA from the adlayer as seen by XPS. The small increase in fluorescence intensity in this time period is likely due to loss of Cy3–Cy3 quenching. Beyond 5 h of MCU backfill, relative fluorescence signal remained constant. Relative rates of change in fluorescence intensity as a function of MCU exposure and loss of probe DNA from the surface as a function of MCU exposure support this interpretation (Figure 4b).

CONCLUSIONS

In this work, the composition and orientation of mixed self-assembled DNA/MCU monolayers designed for the specific capture of DNA targets have been characterized in detail using several high-resolution surface analytical methods. XPS results indicate that, during short-term backfill of MCU diluent thiols, the MCU molecules incorporate into the unoccupied surface sites surrounding the loosely packed low-density HS-ssDNA. While DNA displacement by MCU is initially slow (vacant site filling preferred), upon extended MCU backfill time, the HS-ssDNA surface coverage steadily decreased. The observed increase in NEXAFS polarization dependence with MCU diluent addition into DNA monolayers indicates that the immobilized ssDNA oligomers reorient toward a more upright orientation on the surface. MCU addition, yielding a strong thiol–gold bond (XPS evidence), displaces weaker nucleobase nitrogen–gold interactions, producing an overall change in ssDNA chain orientation. The NEXFAS nitrogen polarization dependence reaches a maximum at ~0.5–1 h of MCU exposure, beyond which the polarization dependence decreases slightly due to DNA displacement. Despite reduction in HS-ssDNA surface density with longer MCU backfill times, evidence suggests that the upright orientation of remaining ssDNA is generally retained. Fluorescence intensity results show little fluorescence signal in pure fluorophore-labeled DNA monolayers. This is mainly attributed to substantial interactions between DNA oligomers and the gold substrate, resulting in fluorescence quenching of the terminal fluorophore. Addition of MCU surface diluent into the DNA adlayers initially increases the relative fluorescence signal due to MCU-induced reorientation of the DNA and relocation of the terminal fluorophore away from the gold substrate. At longer MCU backfill times, fluorescence intensity decreases due to DNA probe displacement from the gold surface and some loss of Cy3–Cy3 interactions. Experimental differences observed in kinetics of maximum HS-ssDNA orientational changes determined from fluorescence compared to NEXAFS measurements could be explained by the different measurement reporting capabilities of these two methods. NEX-AFS measures average bond orientation throughout the oligo-nucleotide chain ensemble, while fluorescence intensity measurements are simply indicative of the local environment around the fluorophore. In this case, the fluorescence intensity measurements are primarily affected by the position of the end-labeled fluorescent molecule relative to the gold substrate. In the fluorescence measurements, Cy3–gold quenching is rapidly eliminated at short MCU backfill times, in agreement with the NEXAFS results. The Cy3–gold quenching explains the majority of the MCU response detected by fluorescence intensity increase. At longer MCU backfill times, MCU displaces HS-ssDNA chains from the gold substrate, diluting the surface-immobilized fluorescent labeled DNA molecules, thereby reducing the dye–dye interactions in any subpopulations on the surface capable of proximity (within Forster radius) Cy3–Cy3 interactions. Supporting evidence from a different set of methods analyzing identical DNA adlayers in a companion paper²³ suggests that immobilized DNA molecular orientations are correlated with their surface densities and can be changed using MCU diluent addition to affect hybridization efficiencies with complementary target. Hence, full understanding and control of both DNA probe density and orientation on surfaces seem very significant to DNA’s capture efficiency and capture assay performance in buffer and complex milieu.