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The complex nature and structure of biomolecules and nanoparticles and their interactions make it challenging to achieve a deeper understanding of the dynamics at the nano-bio interface of enzymes and plasmonic nanoparticles subjected to light excitation. In this study, circular dichroism (CD) and Raman spectroscopic experiments and molecular dynamics (MD) simulations were used to investigate the potential changes at the nano-bio interface upon plasmonic excitation. Our data showed that photothermal and thermal heating induced distinct changes in the secondary structure of a model nanobioconjugate composed of lipase fromCandida antarcticafraction B (CALB) and gold nanoparticles (AuNPs). The use of a green laser led to a substantial decrease in the α-helix content of the lipase from 66% to 13% and an increase in the ß-sheet content from 5% to 31% compared to the initial conformation of the nanobioconjugate. In contrast, the differences under similar thermal heating conditions were only 55% and 11%, respectively. This study revealed important differences related to the enzyme secondary structure, enzyme-nanoparticle interactions, and the stability of the enzyme catalytic triad (Ser105-Asp187-His224), influenced by the instantaneous local temperature increase generated from photothermal heating compared to the slower rate of thermal heating of the bulk. These results provide valuable insights into the interactions between biomolecules and plasmonic nanoparticles induced by photothermal heating, advancing plasmonic biocatalysis and related fields.

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This study shows a new SERS (Surface-enhanced Raman Scattering) and SEF (Surface-enhanced Fluorescence) platform approach, in which substrates were constructed from the silver nanoparticles stabilized by alginate polymer (AgALG) and encapsulated in hydrogel calcium alginate beads (AgALGbead). In this regard, the electrostatic repulsion or attraction concerning the charged dyes and the carboxylate groups of the alginate could define the distances between the probe molecules and metallic nanoparticles to determine the SERS or SEF effect. In this sense, the anionic dye named New Indocyanine Green (IR-820) and the cationic dye Rhodamine 6G (Rh6G) were selected to discuss the alginate's ability to quench or enhance the fluorescence and the Raman dyes signals. Furthermore, the SEF effect using the IR-820 dye can be detected for the near-infrared emission (S1 â†’ S0) using the 532 and 633 nm laser lines as well at the visible region (S2 â†’ S0) applying the excitation at 532 nm in the AgALGbead substrates. Nevertheless, the cationic dye provides the Surface-enhanced Resonance Raman Scattering (SERRS) effect and quenching of the fluorescence for the same AgALGbeads substrate at 532 nm laser line.

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Although Surface Enhanced Raman Spectroscopy (SERS) is a widespread technique with applications in several fields, the SERS effect is still not thoroughly understood due to the challenge in describing how the interaction between the analyte and the metallic surface contributes to the Raman signal enhancement. One approach to distinguish the charge transfer contribution from the metal to the molecule is the comparison of the coordination complex resonance Raman spectral features with the SERS spectra of the surface complex excited at different wavelengths. Herein, we investigated the molecule 5-nitroisatin, Nisa, its complex with a silver cation, Ag(Nisa), its anionic form, Nisa-, and the adsorbed species over Ag colloid, Nisa/AgNP, by resonance Raman and SERS, respectively. The data show that the resonance Raman spectrum of the coordination complex Ag(Nisa) is comparable to the SERS spectrum obtained out of resonance condition. However, when the SERS spectra of Nisa/AgNP at resonance condition is obtained, quite distinct chromophores are observed. The SERS enhancement profile suggests a charge transfer from the metal to molecule in the green region of the visible spectrum and evidences the higher complexity of the electronic transitions that take place within the surface complex. To support the experimental data, DFT and TDDFT calculations were performed for Nisa, Ag(Nisa), Nisa- and Nisa@Ag20 cluster.

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In this work, we present some properties and use of a nanocomposite formed by gold nanoparticles (NPs) into poly(methyl methacrylate) (PMMA) and its application as substrates for surface-enhanced Raman spectroscopy (SERS). The nanocomposite was formed using low-energy (49 eV) ion implantation of gold in PMMA using a cathodic arc plasma gun. The gold NPs are formed spontaneously from the implanted ions and they remain isolated from each other by the polymer medium surrounding them, ensuring a spacing between the NPs of less than 10 nm (hot spot places). The NPs form below the surface, protected from the environment, guaranteeing the stability of the composite layer. Moreover, here, we present an interesting approach to concentrate analyte molecules closer to the metal surface using the swelling effect in PMMA. Using absorption of the analyte, the molecules stay in the gaps between NPs, which is a good solution for one of the biggest challenges in SERS, that is, to guide molecules to the hot spot places.

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To develop bioconjugated materials, it is necessary to understand how the various elements present in a conjugate interact with one another. To gain insights into nanoparticle-capping agent-protein interactions, gold nanoparticles (AuNPs) measuring 30 nm in diameter were coated with different molecules bearing a thiol group: 3-mercaptopropionic acid, 6-mercaptohexanoic acid, and 11-mercaptoundecanoic acid. The covalent conjugation of AuNPs to the protein bovine serum albumin (BSA) via a cross-linker reaction with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was systematically investigated under different reaction conditions with variation of the concentrations of the mercaptoalkyl carboxylic acid (MA) and BSA. All the products were analyzed by UV-vis spectroscopy, gel electrophoresis, and Raman spectroscopy in every modification step. From analysis of the UV-vis results, it is possible at low concentrations of MA to see strong coupling among AuNPs, observed when they are aggregated by KCl, which does not happen at higher concentration of MA, indicating an AuNP-to-MA ratio of 1:130,000 is best for bioconjugation purposes. Agarose gel electrophoresis, a classic technique for biomolecule characterization, indicated that BSA is capable of altering the mobility of AuNPs when it modifies completely the surface of AuNPs because of its high molecular mass (around 66 kDa). Principal component analysis of surface-enhanced Raman spectroscopy data was successfully used as a chemometric tool to assist the characterization of the nanoparticle modification with linker molecules in the absence and presence of different BSA concentrations, making it possible to clearly evaluate the gradual substitution/modification of AuNPs (1:13,000 < 1:65,000 < 1:130,000 AuNP-to-MA ratio) and the conjugation with BSA, which is homogenous at a concentration of 0.01 g L-1. Graphical abstract.

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Poly(ionic liquid)s are an interesting class of compounds because of their unique chemical and physical properties gathering the characteristics of ionic liquids and polymers. Pressure and temperature have been demonstrated to be alternative parameters to obtain polymers from monomeric species using only physical tools. In this work, we investigate the reaction under high pressure and room temperature of the ionic liquid 1-allyl-3-methylimidazolium iodide by using the diamond anvil cell technique in combination with synchrotron X-ray diffraction and electronic and vibrational spectroscopies. The results indicate a chemical reaction happening through the terminal double bond of the allyl group both in crystalline and glassy phases with the onset of the reaction around ∼7 GPa. Vibrational spectra present evidence for an oligomerization reaction in both the phases. The reaction occurring both in glassy and crystal phases indicates a mechanism not driven by collective motions and likely related to local topological arrangements. The results presented herein extend our understanding of ionic liquid instability boundaries under high pressure and contribute to the development of alternative synthetic routes to achieve poly(ionic liquids).

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Around 20 years ago, the first reports of single-molecule surface-enhanced Raman scattering (SM-SERS) caused a revolution in nanotechnology. Several researchers were quick to recognize the importance of a technique that can provide molecular vibrational fingerprinting at the SM level. Since then, a large amount of work has been devoted to the development of nanostructures capable of SM-SERS detection. A great effort has also been geared toward elucidating the different mechanisms that contribute to the effect. The understanding of the concept of plasmonic SERS hotspots, the role of chemical effects, and the dynamics of atomic and cluster rearrangements in nanometric domains has significantly advanced, driven by new computational and experimental methods used to study SM-SERS. In particular, SERS intensity fluctuations (SIFs) are now recognized as a hallmark of SM-SERS. Interpretation of SM-SERS data must take into consideration temporal and spatial variations as a natural consequence of the extreme localization inherent to surface plasmon resonances. Further analysis of variations in spectral signature, due to either molecular reorientation or photo (or thermal) processes, pointed to a new area that combines the power of SERS fingerprinting at the SM level to modern concepts of catalysis, such as hot-electrons-driven chemistry. This large body of work on the fundamental characteristics of the SM-SERS effect paved the way to the interpretation of other related phenomena, such as tip-enhanced Raman scattering (TERS). Despite all the fundamental progress, there are still very few examples of real applications of SM-SERS. In recent years, our research group has been studying SIFs, focused on different ways to use SM-SERS. The obvious application of SM-SERS is in analytical chemistry, particularly for quantification at ultralow concentrations (below 1 nM). However, quantification using SM-SERS faces a fundamental sampling problem: the analytes (adsorbed in very small amounts, i.e., low surface coverage) must find rare SERS hotspots (areas with intense electric field localization that yields SERS). This limitation leads to strong temporal and spatial variations in SERS intensities, which translates into very large error bars in an experimental calibration curve. We tackled this problem by introducing the concept of "digital SERS". This approach provided a roadmap for SERS quantification at ultralow concentrations and a potential pathway for a better understanding of the "reproducibility problem" associated with SERS. In this Account, we discuss not only the analytical applications but also other implementations of SM-SERS demonstrated by our group. These include the use of SM-SERS as a tool to probe colloidal aggregation, to evaluate the efficiency of SERS substrates, and to characterize the energy of localized resonances. SERS involves a series of random processes: hotspots are rare; surfaces/clusters constantly reconstruct; and molecules diffuse, adsorb, and desorb. All these pathways contribute to strong fluctuations in SERS intensities. Our work indicates that a statistical view of the effect can lead to interesting insights and the potential to fulfill the promise of this SM technique for real-world applications.

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Nanostructured systems, such as nanocomposites, are potential materials for usage in different fields since synergistic effects of their components at the nanoscale domain may improve physical/chemical properties when compared to individual phases. We report here the preparation and characterisation of a new nanocomposite composed of polyaniline (PANI), reduced graphene oxide (rGO) and hexaniobate (hexNb) nanoscrolls. Atomic force microscopy images show an interesting architecture of rGO flakes coated with PANI and decorated by hexNb. Such features are attributed to the high stability of the rGO flakes prepared at room temperature. Detailed characterisation by X-ray photoelectron and Raman spectroscopies indicates an intermediate reduction degree for the rGO component and high doping degree of the PANI chains compared to the neat polymer. The latter feature can be attributed to cooperative effects of PANI chains with rGO flakes and hexNb nanoscrolls, which promote conformational changes of the polymer backbone (secondary doping). Spectroscopic and electrochemistry data indicate a synergetic effect on the ternary nanocomposite, which is attributed to interactions between the components resulting from the morphological aspects. Therefore, the new nanocomposite presents promising properties for development of new materials in the film form on substrates for sensing or corrosion protection for example.

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The nature of the cooperativity effect of hydrogen bonds in Watson and Crick and wobble base pairs formed with thymine, uracil, and its 5-halogenated derivatives (5-fluoro, -chloro, and -bromouracil) has been studied through SERS and by using chemometric tools to process data and extract relevant information. Remarkable differences between the two kinds of pairs were clearly observed, and the behavior correlated to the withdrawing character of different substituents at the 5-position of uracil was verified. Multivariate analyses have also unveiled information about the pair's stability, and a stronger cooperativity effect seems to rule the Watson and Crick pairs when compared to wobble pairs. Defined patterns in the behavior of Watson and Crick pairs allowed the design of an indirect methodology for quantifying 5-bromouracil using a partial least squares (PLS) method with variable selection. Limit of detection (LOD) values of 0.037 and 0.112 mmol L-1 in the absence and presence of structurally similar interferences were reached, while its direct surface-enhanced Raman spectroscopy (SERS) quantification is only possible at ∼45 mmol L-1.

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Ionic liquids are an interesting class of materials due to their distinguished properties, allowing their use in an impressive range of applications, from catalysis to hypergolic fuels. However, the reactivity triggered by the application of high pressure can give rise to a new class of materials, which is not achieved under normal conditions. Here, we report on the high-pressure chemical instability of the ionic liquid 1-allyl-3-methylimidazolium dicyanamide, [allylC1im][N(CN)2], probed by both Raman and IR techniques and supported by quantum chemical calculations. Our results show a reaction occurring above 8 GPa, involving the terminal double bond of the allyl group, giving rise to an oligomeric product. The results presented herein contribute to our understanding of the stability of ionic liquids, which is of paramount interest for engineering applications. Moreover, gaining insight into this peculiar kind of reactivity could lead to the development of new or alternative synthetic routes to achieve, for example, poly(ionic liquids).

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The formation of cooperative hydrogen bonds between the thymine-adenine pair was used to indirectly determine thymine in aqueous solution by surface-enhanced Raman spectroscopy (SERS), therefore improving the limit of detection (LOD) values up to two orders of magnitude. The concentration of adenine was held constant and SERS spectra over gold nanoparticles were acquired through variable concentrations of thymine. The overall methodology followed a multivariate approach leading us to find the most suitable adenine concentrations to determine thymine and evidence the formation of new species whose response maintains a linear correlation with thymine nominal concentrations. Partial Least Squares (PLS) regression has been applied for modelling the data and close values of the root mean square error of cross-validation (RMSECV) and root mean square error of prediction (RMSEP) were obtained as indicators of model quality for modelling and prediction. The LOD for the thymine determination has been lowered from ∼20 to 0.278 mmol L(-1) with a mean prediction error of 3.3%. It was verified that the improvement in LOD is only possible if the base pair is formed prior to the addition of the plasmonic nanoparticles. Finally, the assessment of the effect of an interference species introducing uracil to the system showed that it was possible to isolate the analyte response from the overall signals.

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Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.

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The SERS enhancement factor (SERS-EF) is one of the most important parameters that characterizes the ability of a given substrate to enhance the Raman signal for SERS applications. The comparison of SERS intensities and SERS-EF values across different substrates is a common practice to unravel the performance of a given substrate. In this study, it is shown that such a comparison may lack significance if we compare substrates of very distinct nature and optical properties. It is specifically shown that the SERS-EF values for static substrates (e.g. immobilized metallic nanostructures) cannot be compared to those of dynamic ones (e.g. colloidal metal nanoparticle solutions), and that the optical properties for the latter show strong dependence on the metal-molecule interaction dynamics. The most representative experimental results concerning the dynamic substrates have been supported by generalized Mie theory simulations, which are tools used to describe the substrate complexity and the microscopic information not usually taken into account.

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This work deals with the spectroscopic (supported by quantum chemistry calculations), structural, and morphological characterization of mefenamic acid (2-[(2,3-(dimethylphenyl)amino] benzoic acid) polymorphs, known as forms I and II. Polymorph I was obtained by recrystallization in ethanol, while form II was reached by heating form I up to 175 °C, to promote the solid phase transition. Experimental and theoretical vibrational band assignments were performed considering the presence of centrosymmetric dimers. Besides band shifts in the 3345-3310 cm(-1) range, important vibrational modes to distinguish the polymorphs are related to out-of-phase and in-phase N-H bending at 1582 (Raman)/1577 (IR) cm(-1) and 1575 (Raman)/1568 (IR) cm(-1) for forms I and II, respectively. In IR spectra, bands assigned to N-H bending out of plane are observed at 626 and 575 cm(-1) for polymorphs I and II, respectively. Solid-state (13)C NMR spectra pointed out distinct chemical shifts for the dimethylphenyl group: 135.8 to 127.6 ppm (carbon bonded to N) and 139.4 to 143.3 ppm (carbon bonded to methyl group) for forms I and II, respectively.

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Hybrid organic-inorganic material containing Laponite clay and ionic liquids forming cations have been prepared and characterized by FT-Raman spectroscopy, X-ray diffraction, and thermal analysis. The effect of varying the length of the alkyl side chain and conformations of cations has been investigated by using different ionic liquids based on piperidinium and imidazolium cations. The structure of the N,N-butyl-methyl-piperidinium cation and the assignment of its vibrational spectrum have been further elucidated by quantum chemistry calculations. The X-ray data indicate that the organic cations are intercalated parallel to the layers of the clay. Comparison of Raman spectra of pure ionic liquids with different anions and the resulting solid hybrid materials in which the organic cations have been intercalated into the clay characterizes the local environment experienced by the cations in the hybrid materials. The Raman spectra of hybrid materials suggest that the local environment of all confined cations, in spite of this diversity in properties, resembles the liquid state of ionic liquids with a relatively disordered structure.

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This paper reports the spectroscopic study on the structural differences of thermally induced cross-linking segments in polyaniline in its emeraldine salt (PANI-ES) and base (PANI-EB) forms. Casting films of PANI-ES (ES-film) and PANI-EB (EB-film) were prepared and heated at 150 °C under atmospheric air for 30 min. Raman spectra excited at 632.8 nm of heated ES-film presented the characteristic bands of phenazine-like structures at 1638, 1392, and 575 cm(-1), whereas EB-film showed lower relative intensities for these bands. The lower content of phenazine-like segments in heated EB-film is related to residual polaronic segments from preparation procedures, as revealed by Raman. This statement was confirmed by a sequence of thermal and doping experiments in both films. Quantum-chemical calculations by density functional theory (DFT) and time-dependent density functional theory (TD-DFT) showed that the phenazine-like structure presents the intense Raman band at 1350 cm(-1) due to heterocycle breathing mode, and the non-phenazine-like structure (substituted hydrophenazine-type) presents higher energy for HOMO-LUMO transition, indicating the lack of conjugation in the heterocycle compared with the phenazine-like structure. According to experimental and theoretical data reported here, it is proposed that only thermally treated PANI-ES presents phenazine-like rings, whereas PANI-EB presents heterocyclic non-aromatic structures.

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The anomalies in the anti-Stokes to Stokes intensity ratios in single-molecule surface-enhanced resonance Raman scattering were investigated. Brilliant green and crystal violet dyes were the molecular probes, and the experiments were carried out on an electrochemically activated Ag surface. The results allowed new insights into the origin of these anomalies and led to a new method to confirm the single-molecule regime in surface-enhanced Raman scattering. Moreover, a methodology to estimate the distribution of resonance energies that contributed to the imbalance in the anti-Stokes to Stokes intensity ratios at the electromagnetic hot spots was proposed. This method allowed the local plasmonic resonance energies on the metallic surface to be spatially mapped.

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This paper investigates the structure of the products obtained from the polymerization of aniline with ammonium persulfate in a citrate/phosphate buffer solution at pH 3 by resonance Raman, NMR, FTIR, and UV-vis-NIR spectroscopies. All the spectroscopic data showed that the major product presented segments that were formed by a 1,4-Michael reaction between aniline and p-benzoquinone monoimine, ruling out the formation of polyazane structure that has been recently proposed. The characterization of samples obtained at different stages of the reaction indicated that, as the reaction progressed, phenazine units were formed and 1,4-Michael-type adducts were hydrolyzed/oxidized to yield benzoquinone. Raman mapping data suggested that phenazine-like segments could be related to the formation of the microspheres morphology.

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Polycarbonate membranes (PCM) of various pores sizes (400, 200, 100 and 50 nm) were used as templates for gold deposition. The electrodeposition from gold ions resulted in the formation of gold nanotubes when large pores size PCMs (400 and 200 nm) were used. On the other hand, gold nanowires were predominant for the PCMs with smaller pores size (100 and 50 nm). Surface-enhanced Raman scattering (SERS) from the probe molecule 4-mercaptopyridine (4-MPy) was obtained from all these nanostructures. The SERS efficiency of the substrates produced using the PCM templates were compared to two commonly used SERS platforms: a roughened gold electrode and gold nanostructures electrodeposited through organized polystyrene spheres (PSS). The SERS signal of the probe molecule increased as the pore diameter of the PCM template decreased. Moreover, the SERS efficiency from the nanostructures produced using 50 nm PCM templates was four and two times better than the signal from the roughened gold electrode and the PSS template, respectively. The SERS substrates prepared using PCM templates were more homogenous over a larger area (ca. 1 cm2), presented better spatial and sample to sample reproducibility than the other substrates. These results show that SERS substrates prepared using PCM templates are promising for the fabrication of planar SERS platforms for analytical/bioanalytical applications.

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Glycine-rich proteins (GRPs) serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs' glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113)GGGGYGGGYG(123) GGY(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was made when CLEAR amide resin (Fmoc chemistry) was used, as it allowed for obtaining fragment G(113)-K(132). NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon, as proven by NIR-FT-Raman analysis of the growing peptide-resin, allowing the total synthesis of desired fragment Gly(101)-K(132). In summary, this work describes a new difficult sequence, contributes to understanding stepwise solid-phase synthesis of this type of peptide and shows that, at least while protected and linked to a resin, this GRP's glycine-rich motif presents an early tendency to assume beta-sheet structures.

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