From Bulk to Single Molecules: Surface-Enhanced Raman Scattering of Cytochrome C Using Plasmonic DNA Origami Nanoantennas

Abstract

Cytochrome C, an evolutionarily conserved protein, plays pivotal roles in cellular respiration and apoptosis. Understanding its molecular intricacies is essential for both academic inquiry and potential biomedical applications. This study introduces an advanced single-molecule surface-enhanced Raman scattering (SM-SERS) system based on DNA origami nanoantennas (DONAs), optimized to provide unparalleled insights into protein structure and interactions. Our system effectively detects shifts in the Amide III band, thereby elucidating protein dynamics and conformational changes. Additionally, the system permits concurrent observations of oxidation processes and Amide bands, offering an integrated view of protein structural and chemical modifications. Notably, our approach diverges from traditional SM-SERS techniques by de-emphasizing resonance conditions for SERS excitation, aiming to mitigate challenges like peak oversaturation. Our findings underscore the capability of our DONAs to illuminate single-molecule behaviors, even within aggregate systems, providing clarity on molecular interactions and behaviors.

Keywords: Amide III; Cytochrome C; DNA origami; SERS; Single molecules.

Figure 1

(A) Schematic model of CytC with the heme unit visible in the center. Underneath is a model of the heme unit. (B) UV–visible absorption spectrum of powder CytC. The vertical lines show the laser positions used in our study. (C) A schematic model of the DNA Nanofork, showing the attachment of CytC to the DNA Nanofork and the subsequent attachment of the AuNPs to form the DONA structure. The structure of CytC is based on the crystal structure 2b4z deposited at the protein data bank (PDB): https://www.rcsb.org/structure/2b4z.

Figure 2

(A) Normal Raman (NR) spectra of powder CytC using different excitation lasers (blue zones highlight heme unit peaks, the red zone indicates peptide backbone peaks, and the purple zone shows spectral contributions from both). (B) SERS spectra of powder CytC mixed with 60 nm AuNPs using different lasers (blue zones highlight heme unit peaks, the red zone indicates peptide backbone peaks, and the purple zone shows spectral contributions from both). More detailed band assignments are summarized in Tables SI1 and SI2.

Figure 3

(A) Heat map of a single DONA measurement (left) with corresponding SERS signals at different time points (right). Inset: AFM image (325 nm × 240 nm, W × H) showing a single DONA with CytC. (B) SERS spectra showing measurements of individual DONAs averaged over time as well as the total average calculated from all single-DONA measurements, compared to reference NR and SERS of CytC (vertical orange lines represent bands observed in the NR and SERS references and literature, summarized in Table SI1 and Table SI2; purple lines represent bands observed in our SERS reference measurements and not assigned in the literature). Each spectrum represents the time average of a measurement from a single DONA (blue zones highlight heme unit peaks, the red zone indicates peptide backbone peaks, and the purple zone shows spectral contributions from both). (C) SERS spectra showing different time points from a single DONA measurement, highlighting the shift of the Amide III band (blue zones highlight heme unit peaks, the red zone indicates peptide backbone peaks, and the purple zone shows spectral contributions from both).

Figure 4

(A) Heat map of a DONA aggregate measurement (left) with corresponding SERS signals at different time points (right). Inset: AFM image (290 nm × 310 nm, W×H) showing a DONA aggregate. (B) SERS spectra showing measurements of individual DONA aggregates averaged over time as well as the total average calculated from all DONA aggregate measurements, compared to reference NR and SERS of CytC (vertical orange lines represent bands observed in the NR and SERS references and literature, summarized in Table SI1 and Table SI2; purple lines represent bands observed in our SERS reference measurements and not assigned in the literature). Each spectrum represents the time average of a measurement from a single DONA aggregate (blue zones highlight heme unit peaks, the red zone indicates peptide backbone peaks, and the purple zone shows spectral contributions from both). (C) SERS spectra showing different time points from a single DONA aggregate measurement, highlighting the shift of the Amide III band (blue zones highlight heme unit peaks, the red zone indicates peptide backbone peaks, and the purple zone shows spectral contributions from both).

Figure 5

(A) Schematic of the CytC in the hotspot of the DONA and the possible reorientation or diffusion of the CytC inside the hotspot. (B) Spectra showing the shift from 1269 to 1281 cm^–1 and back. (C) The time map visualizes the 1269 cm^–1 peak (top, dark violet) and the 1281 cm^–1 peak (bottom, light violet). A notable decrease in the 1269 cm^–1 peak intensity around 160 s coincides with the emergence of the 1281 cm^–1 peak. This pattern reverses around 200 s, indicating a transient shift between these two spectral positions. The structure of CytC is based on the crystal structure 2b4z deposited at the protein data bank (PDB): https://www.rcsb.org/structure/2b4z.