Structural Characterization of Single-Stranded DNA Monolayers Using Two-Dimensional Sum Frequency Generation Spectroscopy

Abstract

DNA-covered materials are important in technological applications such as biosensors and microarrays, but obtaining structural information on surface-bound biomolecules is experimentally challenging. In this paper, we structurally characterize single-stranded DNA monolayers of poly(thymine) from 10 to 25 bases in length with an emerging surface technique called two-dimensional sum frequency generation (2D SFG) spectroscopy. These experiments are carried out by adding a mid-IR pulse shaper to a femtosecond broad-band SFG spectrometer. Cross peaks and 2D line shapes in the 2D SFG spectra provide information about structure and dynamics. Because the 2D SFG spectra are heterodyne detected, the monolayer spectra can be directly compared to 2D infrared (2D IR) spectra of poly(thymine) in solution, which aids interpretation. We simulate the 2D SFG spectra using DFT calculations and an excitonic Hamiltonian that relates the molecular geometry to the vibrational coupling. Intrabase cross peaks help define the orientation of the bases and interbase cross peaks, created by coupling between bases, and resolves features not observed in 1D SFG spectra that constrain the relative geometries of stacked bases. We present a structure for the poly(T) oligomer that is consistent with the 2D SFG data. These experiments provide insight into the DNA monolayer structure and set precedent for studying complex biomolecules on surfaces with 2D SFG spectroscopy.

Figure 1.

2D SFG spectroscopy pulse sequence and beam geometry. Mid-IR probe and visible pulses are aligned in the same yz plane, while mid-IR pump pulses are displaced slightly along the x axis. All pulses are polarized along the z axis (p polarized).

Figure 2.

Hydrated and dehydrated deoxythymidine-optimized geometries and transition dipole moment vectors. Direction of vectors for T1 (red), T2 (green), and T3 (blue) are shown. Vector lengths are scaled in each case to indicate the relative strength of calculated transition dipoles.

Figure 3.

IR spectra of (dT)₂₅-SH. FTIR of (dT)₂₅-SH in D₂O CaCl₂−TE buffer (green), FTIR of dehydrated (dT)₂₅-SH film (blue), and IRRAS of (dT)₂₅-SH monolayer on gold (red). Small residual backgrounds are fit with a linear line and subtracted off each spectrum.

Figure 4.

Spectra of (dT)₂₅-SH monolayers, films, and solutions. (a) IRRAS (red) and Raman (blue) spectra of the dehydrated film. (b) 2D SFG spectrum of a monolayer on gold. (c) 2D IR spectrum of the dehydrated film. (d) 2D IR and (e) FTIR spectra in D₂O CaCl₂−TE buffer. Dashed lines indicate the limits of the probe frequency range in d. Peak labels in gray labels represent approximate positions when peaks are weak or nonexistent. Labels refer to T1, T2, and T3 modes, with C_ij being the corresponding cross peaks. All spectra are normalized to the most intense peak and plotted from −1 to +1 with contour steps of 10%, omitting the zero level contour line.

Figure 5.

2D SFG spectra of (dT)₁₀-SH, (dT)₁₅-SH, and (dT)₂₅-SH monolayers on gold. 2D SFG spectra at t₂ = 0 fs for (a) (dT)₁₀-SH, (b) (dT)₁₅-SH, and (c) (dT)₂₅-SH. All spectra are normalized to the most intense peak and plotted from −1 to +1 with contour steps of 10%. Zero level contours are omitted002E

Figure 6.

Simplified 2D SFG spectrum with two modes A and B. (a) Fundamental peaks of a two mode system. (b) The asymmetric peak pattern matches our experimental observation. The equation below is one of the conditions for this asymmetric pattern.

Figure 7.

Simulated 2D SFG spectra of an uncoupled single thymine base. (a) Coordinate system for the single thymine base scan. The thymine base plane is represented by the purple surface with T1 (red), T2 (green), and T3 (blue) vectors shown. (b) Orientation (ψ,θ) plot with configurations that produce simulated 2D SFG spectra within the outlined criteria shown as blue dots. The two positions marked with red cross marks correspond to the orientations and spectra shown in Figure 6c, d and Figure 6e, f, respectively. (c) Thymine base in (ψ = 10°, θ = 86°) conformation, and (d) corresponding simulated 2D SFG spectrum. (e) Thymine base in (ψ = 90°, θ = 75°) conformation, and (f) corresponding simulated 2D SFG spectrum.

Figure 8.

Simulated 2D SFG spectra of four coupled thymine bases. (a) Configuration of four coupled bases with monomer orientation (ψ_i,θ_i) = (135°, 80°), and (b) corresponding simulated 2D SFG spectrum. (c) Oligo(dT) DNA segment from B-type dsDNA (PDB = 1D98) extending from surface in 5′ to 3′ direction and oriented for comparison to a. Spectra are plotted the same as the experimental spectra above.