摘要： Description of the relationship between protein structure and function remains a primary focus in molecular biology, biochemistry, protein engineering and bioelectronics. Regardless the targeted application, the current strategies on revealing the relationship between protein structure and function lead to exposure and interaction of proteins with non-biological organic and inorganic solid surfaces. Proper description of the underlying mechanisms will certainly unveil the fundamental protein-adsorption problem and add value to the effort of record and quantification of the conformational changes of the protein native state when interacting with solid surfaces. To that end the application of physics-based diagnostic methods is suitable and highly demanded. Raman spectroscopy appears the most frequently used method for the study of biomolecule recognition, and ultra-sensitive analysis, down to a single molecule. However, to tackle the sensitivity limitations of Raman spectroscopy imposed by the small Raman cross sections, the biological systems should be coupled with metallic nanostructures. The scattering efficiency can be thus increased by several orders of magnitude due to the activation of localized surface plasmon resonance (LSPR) that induces strong enhancement of the electromagnetic (EM) field in the vicinity of the metallic surface. This enables to largely extend the application of Raman spectroscopy in molecular spectroscopy, biomolecule recognition, and ultra-sensitive analysis, down to a single molecule. Besides the sensing properties, the strong EM enhancement can be exploited to probe protein conformational changes under photoexcitation, including real-time monitoring. Therefore, since its discovery in the late 70s, the Surface-Enhanced Raman Scattering (SERS) has proven to be a very powerful and reliable analytical tool for chemo- and bio-sensing, due to the strong enhancement of the vibrational signatures of analytes in different chemical environments. In this context, a lot of resources and time have been employed in the effort to develop plasmonic substrates based on metallic nanostructures aiming at a further increase of the EM enhancement for the realization of noninvasive, highly-sensitive, and large-scale optical sensors. A large variety of metallic nanostructure morphologies and arrangements (nanosphere, nanotriangles, nanodisks, nanorods, nanocubes, etc.) and different coupling geometries (dimers, trimers, arrays, etc.) have been developed up to date for SERS platforms. However, their conversion to macroscopic plasmonic substrates relies generally on the NPs volunteer arrangement on dielectric surfaces (mainly through applying chemical methods), thus often resulting in non-uniform distribution on large areas, without a well-defined control of the spacing between the metallic nanostructures and the probed molecules, high point-to-point variability, scarce reproducibility and stability under irradiation conditions (due to photothermal and photodegradation processes). To overcome the limitations in producing solid SERS substrates various physical approaches, like thermal evaporation, combined nanoimprint lithography-shadow evaporation, gas aggregation source (GAS), pulsed laser deposition (PLD), low-energy ion beam synthesis (LE-IBS), and plasma-based deposition processes, have been proposed in the literature. It is generally acknowledged that the silver nanoparticles (AgNPs) realize the best nanoscale antenna in the visible range for amplifying local electronic and vibrational signals, thus providing unique molecular information in the optical far-field regime. Indeed, compared to gold nanoparticles, the AgNPs offer the advantage of stronger plasmonic enhancement because of lower interference between intraband and interband electronic transitions. Moreover, the use of AgNPs covers another aspect of the relationship between protein structure and function which concerns the biological activity of the AgNPs. Because of their antimicrobial properties, the AgNPs have the potential to impact human health and environment. The biological activity of AgNPs goes both ways, through the activity of ionic silver (Ag+) and through direct contact with the AgNPs resulting in protein denaturation at different cell locations; specifically sensible are those enzymes of the respiratory chain and transport channels. Therefore, there exists a recognized need to address the relationship between protein structure and function from two distinctly different vantage points: (i) quantification of the conformational changes of proteins by using the antenna effect of AgNPs and (ii) analysis of the conformational changes of proteins induced by the AgNPs extreme chemical and biological activities. The intent of this work is to bring additional insight into the mechanisms of adsorption of proteins on solid surfaces through quantification of the conformational changes of proteins adhered on AgNPs-based nanocomposites via SERS. We focus on the wild-type Discosoma recombinant red fluorescent protein (DsRed), belonging to the family of naturally fluorescent proteins (FPs). The strong interest toward the FP family originates from their application in molecular biology as reporters of gene expression, as noninvasive markers in molecular biology and other singular events of cell activity. Potential use of the FPs extends toward therapeutics, tissue regeneration, bioelectronics and protein engineering. The most widely characterized member of this family is the green fluorescent protein (GFP). The lately cloned from reef coral Discosoma sp. DsRed protein possesses the longest yet reported, for a wild-type spontaneously fluorescent protein, excitation and emission maxima at 558 nm and 583 nm, respectively. Owing to its high fluorescence yield the red fluorescent DsRed protein has become important both as a model for understanding fluorescent proteins and as a tool for biomedical research. The DsRed protein and its engineered derivatives have found broad use in cell and molecular biology including fluorescence microscopy as a marker, fluorescence correlation spectroscopy (FCS) and fluorescence activated cell sorting (FACS). Recently, the DsRed was found suitable for rational design of ultra-stable and reversible photoswitches for super-resolution imaging. Moreover, it has been hypothesized that FPs from reef-building corals operate as part of an adaptive mechanism to optically interact and to regulate the symbiotic relationship between corals and photosynthetic algae. Structural rearrangements near the chromophore influence the maturation speed and brightness of the DsRed variants. It is therefore essential to examine the conformational transitions that affect the protein’s ability to transfer optical excitation energy. Studies of the conformational changes of DsRed protein have been reported in the literature but the DsRed Raman fingerprints were investigated only by recurring to chemically synthesized model chromophores. However, the later differ from the wild-type DsRed protein for the absence of the α-helix and β-sheets that naturally surround the chromophore and for the different extensions of the conjugated π-system. The choice of chemically synthesized model chromophores is explained by the complications brought by the presence of immature green species in the solution created as a photoproduct of the red ones, thus often resulting in an unclear or incomplete band assignment. The novelty of this work lays down the point that we work with the wild-type DsRed protein in its native state and not with DsRed model chromophore. All reported experimental studies in the literature were performed in solution. No information on the DsRed protein structural and conformational changes can be found when the DsRed protein is adhered on a solid substrate and irradiated by light. The lack of information on the above discussed issues motivated this study focusing on the investigation of the interaction of wild-type DsRed proteins with AgNPs-based plasmonic substrates. Our approach involves analysis of dehydrated DsRed protein layers in link with natural conditions during drying. To perform the SERS study on the conformational changes of DsRed proteins adhered on AgNPs-based nanocomposites we have elaborated, by plasma process, highly uniform and reproducible plasmonic substrates composed of a single layer of AgNPs coated by a silica layer. Focus was made on the possibility to well control, on a large scale, the AgNPs size distribution and interparticle distances. The resulting uniformity of hot-spot distribution guarantees the reproducibility and stability of this plasmonic sensor. Subsequently, we show how the enhanced EM field in the vicinity of the AgNPs could be employed to detect the presence and identify the conformational changes of proteins, adsorbed and adhered to the plasmonic substrate, during optical irradiation. The achieved enhancement of the electromagnetic field in the vicinity of the AgNPs is as high as 105. This very strong enhancement factor allowed detecting Raman signals from discontinuous layers of DsRed issued from solution with protein concentration of only 80 nM. Three different conformations of the DsRed proteins after adhesion and dehydration on the plasmonic substrates were identified. It was found that the DsRed chromophore structure of the adsorbed proteins undergoes optically assisted chemical transformations when interacting with the optical beam, which leads to reversible transitions between the three different conformations. The proposed time-evolution scenario endorses the dynamical character of the relationship between protein structure and function. It also confirms that the conformational changes of proteins with strong internal coherence, like DsRed proteins, are reversible.