Probing Denaturation of Protein A via Surface-Enhanced Infrared Absorption Spectroscopy

Abstract

We apply surface-enhanced infrared absorption (SEIRA) spectroscopy to monitor the denaturation process of a surface-bound protein A monolayer. Our proposed platform relies on a plasmonic metasurface comprising different spatial subregions ("pixels") that are engineered to exhibit different resonances covering the infrared region of the electromagnetic spectrum that is matched to the vibrational modes of the Amide groups. Specifically, we are able to determine changes in the Amide I and Amide II vibration coupled modes, by comparing the SEIRA reflectance spectra pertaining to the native state and a denatured state induced by a pH variation. In particular, we observe some evident red-shifts in the principal Amide I mode and the Amide II vibration coupled modes (attributable to the breaking of hydrogen bonds), which result in insurmountable barriers for refolding. Thanks to the strong field localization, and consequent enhancement of the light-matter interactions, our proposed sensing platform can operate with extremely small amounts of an analyte, with an estimated detection limit of about 3 femtomoles of molecules.

Keywords: biosensors; label-free detection; metasurfaces; nanoplasmonics; surface-enhanced infrared absorption (SEIRA) spectroscopy.

Figure 1

MS modeling and design. (a,b) Schematic of unit-cell and periodic array (top view), respectively. (c) Numerically computed reflectance responses, for plane-wave illumination with normal incidence and x-polarized electric field, and four representative parameter configurations (pixel #1: p = 2.0 µm, L = 1.117 µm, W = 0.255 µm; pixel #2: p = 1.5 µm, L = 1.247 µm, W = 0.267 µm; pixel #3: p = 1.5 µm, L = 1.027 µm, W = 0.270 µm; pixel #4: p = 1.5 µm, L = 0.843 µm, W = 0.250 µm). (d) Intensity distribution (electric field, normalized with respect to the impinging one) within a unit-cell, for pixel #1, computed 10 nm away from the nanoantenna at the resonant wavenumber 1457 cm⁻¹. The thick green arrow indicates the polarization of the impinging electric field (not aligned to the cross arms, for better visualization).

Figure 2

Schematic illustration of the fabrication process.

Figure 3

(a) Schematic of the proposed MS. Pixels are identified by a unique number; (b) SEM image of pixel #2.

Figure 4

Schematic of PA binding on gold Nas and of denaturation process: (a) Binding of PA on gold surface by using DSP molecule; (b) PA denaturation process by incubation in HCl 0.1 M.

Figure 5

Comparison between numerical (black-solid curve) and experimental (red-dotted) reflectance responses pertaining to pixel #2 for naked NAs (no molecules adsorbed).

Figure 6

Experimental reflectance responses pertaining to all pixels for naked NAs (no molecules adsorbed); each inset shows the corresponding NA SEM image and dimensions.

Figure 7

Red-shift of the resonance after the functionalization of (a) pixel #1 and (b) pixel #2.

Figure 8

Baseline-corrected reflectance SEIRA spectrum pertaining to Pixel #1. (a) NAs functionalized with DSP molecules. (b) NAs with dehydrated monolayer of PA in the native (black curve) and denatured (purple curve) states.

Figure 9

Pixel #1 baseline-corrected reflectance SEIRA spectrum of a dehydrated monolayer of PA (black curve) and of the denatured state (purple curve) after the immersion in HCl at 0.1 M with a PH = 1. Note the shift of the bands pertaining to Amide I (pink area) and Amide II (green area). The spectral region around 1665–1650 cm⁻¹ pertains to the helical structure Amide I vibration, while the one around 1640–1620 cm⁻¹ pertains to σ-sheet. The spectral region around 1555–1545 cm⁻¹ pertains to the helical structure Amide II vibration, while the one around 1535–1525 cm⁻¹ pertains to β-sheets.