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Detection of the conformational changes of <i>Discosoma</i> red fluorescent proteins adhered on silver nanoparticles-based nanocomposites <i>via</i> surface-enhanced Raman scattering
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.
关键词： plasmonic substrate,protein conformation,surface-enhanced Raman scattering,plasma deposition process,Discosoma red-fluorescent protein DsRed,Silver nanoparticles
更新于2025-09-23 15:22:29
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Encyclopedia of Spectroscopy and Spectrometry || Surface-Enhanced Raman Optical Activity (SEROA)
摘要： This review discusses research into the enhancement of the chiroptical spectroscopic technique of Raman optical activity (ROA) through plasmonic resonance effects. The sensitivity of ROA spectroscopy to molecular stereochemistry and conformational dynamics has led to its increasing use in biology and the pharmaceutical sciences. However, the relative weakness of the ROA scattering process, being typically three to five orders of magnitude lower than the parent Raman scattering, has limited its application to conditions of relatively high concentrations and data accumulation times. The potential solution to this limitation in ROA by using the surface plasmon resonances that give rise to surface-enhanced Raman scattering (SERS) in order to boost ROA spectral intensity has been recognized by many researchers. Frustratingly, reliable measurement of this new technique of surface-enhanced Raman optical activity (SEROA) proved challenging to accomplish. A previous review by Abdali and Blanch presented the challenges involved in attempts to measure SEROA, as well as early results. Since then, significant advances have been made in exploring the SEROA phenomenon, and the time is appropriate for an updated account of this work. In order to provide sufficient background for guiding readers into the underlying principles of, and challenges faced in, measuring SEROA, we begin with an introduction to ROA before progressing on to its plasmonically-enhanced form.
关键词： chiroptical spectroscopy,SEROA,ROA,plasmonic resonance,Surface-Enhanced Raman Optical Activity,Raman Optical Activity,SERS,surface-enhanced Raman scattering
更新于2025-09-23 15:21:21
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Invisible-ink-assisted pattern and written surface-enhanced Raman scattering substrates for versatile chem/biosensing platforms
摘要： In recent years, highly sensitive surface-enhanced Raman scattering (SERS) integrated with flexible substrates has drawn increasing attention for label-free detection. In this study, an invisible ink-inspired process was developed for the fabrication of plasmonic Au-based SERS substrates through an on-site redox strategy. Tannic acid (TNA), a common green reagent, was used not only for fabricating various SERS absorbents through a confinement reduction of a Au-TNA complex, but also for supplying an amphiphilic inorganic–organic surface structure for outstanding SERS enhancement at micromolar to nanomolar concentrations for a wide range of compounds. In addition to label-free sensing, this TNA/Au-based SERS substrate provides a versatile analysis platform for studies of chemical and biological reactions. A combination of TNA ink with different metal ions allows for a reliable procedure for the synthesis of a bimetallic AuAg SERS substrate that further enhances the SERS intensity of analyte molecules and extends the lower limit of detection.
关键词： bimetallic AuAg SERS substrate,invisible ink,tannic acid,label-free detection,SERS,plasmonic Au-based SERS substrates,surface-enhanced Raman scattering
更新于2025-09-23 15:21:21
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Sensitive surface-enhanced Raman scattering substrates based on anti-pyramidal gold architectures decorated with silver nanoparticles
摘要： A micro-structured gold surface, consisting of a periodic square–based anti–pyramidal array (Klarite) with a smooth boundary surface on which silver nanoparticles (diameter: 60 nm) were deposited, produced an active surface enhanced Raman scattering substrate. With p-aminothiophenol as a probe molecule, the Raman activity of the micro–structured surface was compared before and after deposition of the silver nanoparticles. Experimental results show that the Raman spectra on the silver/p-aminothiophenol/Klarite structure is stronger than that on the silver/p-aminothiophenol/gold film and the Raman spectra on the silver/p-aminothiophenol/gold film is stronger than that on silver/p-aminothiophenol, p-aminothiophenol/Klarite structure, p-aminothiophenol/gold film, which is confirmed by numerical simulations. A similar result is obtained with crystal violet as test molecule.
关键词： Anti-pyramidal architectures,surface enhanced Raman scattering,p-aminothiophenol,silver nanoparticles
更新于2025-09-23 15:21:21
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Dual-mode Au nanoprobe based on surface enhancement Raman scattering and colorimetry for sensitive determination of telomerase activity both in cell extracts and the urine of patients
摘要： Telomerase is a valuable biomarker, which is highly correlated to cancer diseases. However, single-mode probe for telomerase detection cannot satisfy the challenge of detection of telomerase activity rapidly, simply with high selectivity, sensitivity and accuracy both in preliminary diagnosis and point of care (POC) testing. Therefore, there is an urgent need to develop a robust approach with controllable assembly and high accuracy to consider both the simplification of preliminary diagnosis and POC testing and the quantification requirement for early clinical diagnosis and treatment. Herein, a novel dual-mode Au NPs probe was developed for determination of telomerase activity with controllable assembly and aggregation statement based on surface enhancement Raman scattering (SERS) and colorimetry. In this strategy, an Au dimer-based probe with high uniformity was assembled successfully, telomerase activity was reflected according to the color variations of solution and the Raman intensity of Raman reporter. Taking advantage of the uniformity of Au dimers and the combination of colorimetry and SERS techniques, our strategy determined the telomerase activity with high accuracy, sensitivity and wide range. The established probe possessed of high selectivity, sensitivity and accuracy, which was approved as a reliable, intuitional and convenient approach for detecting telomerase activity.
关键词： colorimetry,bladder cancer,surface enhanced Raman scattering,Au nanoparticles,telomerase activity
更新于2025-09-23 15:21:21
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Photonic Crystal Enhanced SERS Detection of Analytes Separated by Ultrathin Layer Chromatography Using a Diatom Frustule Monolayer
摘要： Diatoms are single-celled algae that biologically fabricate nanostructured silica shells with ordered pore arrays called frustules that resemble a 2D photonic crystal. A monolayer of Pinnularia frustules isolated from cell culture is deposited on a glass substrate and then conformally coated with silver nanoparticles (AgNPs) to serve as a nanostructured thin film for ultrathin layer chromatography (UTLC). Malachite green and Nile red are resolved in toluene mobile phase and the separated analytes are profiled micro-Raman spectroscopy, where plasmonic AgNPs provide surface-enhanced Raman scattering (SERS). The AgNP-diatom frustule monolayer improves SERS detection of malachite green by an average factor of 1.8 ± 0.1 over the plasmonic AgNP layer on glass. Analysis of hot spots on the AgNP-diatom frustule monolayer reveals that nearly 20% of the SERS active area intensifies the SERS signal at least tenfold over the SERS signal for AgNP on glass. Diatom-SERS enhancement is attributed to guided-mode resonances of the Raman laser source, which in turn further enhances the localized surface plasmonic resonance from AgNPs. Overall, the AgNP-diatom frustule monolayer thin film is a new functional material that uniquely enables separation of analytes by UTLC, quantitative SERS detection of separated analytes, and photonic enhancement of the SERS signals.
关键词： ultrathin layer chromatography,Raman,diatoms,surface-enhanced Raman scattering (SERS),photonic crystals
更新于2025-09-23 15:21:01
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Superficial-Layer-Enhanced Raman Scattering (SLERS) for Depth Detection of Noncontact Molecules
摘要： Although the strength of Raman signals can be increased by many orders of magnitude on noble metal nanoparticles, this enhancement is confined to an extremely short distance from the Raman-active surface. The key to the development of Raman spectroscopy for applications in diagnosis and detection of cancer and inflammatory diseases, and in pharmacology, relies on the capability of detecting analytes that are noninteractive with Raman-active surfaces. Here, a new Raman enhancement system is constructed, superficial-layer-enhanced Raman scattering (SLERS), by covering elongated tetrahexahedral gold nanoparticle arrays with a superficial perovskite (CH3NH3PbBr3) film. Plasmonic decay is depressed along the vertical direction away from the noble metal surface and the penetration depth is increased in the perovskite media. The vertical penetration of SLERS is verified by the spatial distribution of the analytes via Raman imaging in layer-scanning mode.
关键词： perovskites,self-assembly,Raman imaging,superficial-layer-enhanced Raman scattering
更新于2025-09-23 15:21:01
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In situ monitoring silver adsorption on assembled gold nanorods by surface-enhanced Raman scattering
摘要： Self-assembly of metal nanocrystals is capable to create a gap of sub-nanometer distance for concentrating the incoming light by the strong coupling of surface plasmon resonance, known as “hot spot”. Although the plasmonic property of silver is better than the other metals in the visible range, the superior Raman enhancement of silver comparing to gold is still under debate. To provide direct evidence, in this work, we studied the silver adsorption on assembled gold nanorods (AuNRs) by in situ surface-enhanced Raman scattering (SERS) measurement. The self-assembled AuNR multimers were used as the SERS substrate, where the 4-mercaptophenol (MPh) molecules in our experiment played dual roles as both probe molecules for the Raman scattering and linking molecules for the AuNR assembly in a basic environment. Silver atoms were adsorbed on the surface of gold nanorod assemblies by reduction of Ag+ anions. The stability of the adsorbed silver was guaranteed by the basic environment. We monitored the SERS signal during the silver adsorption with a home-built in situ Raman spectroscopy, which was synchronized by recording the UV-vis absorption spectra of the reaction solution to instantly quantify the plasmonic effect of the silver adsorption. Although a minor change was found in plasmonic resonance wavelength or intensity, the measured SERS signal at specific modes faced a sudden increase by 2.1 folds during the silver adsorption. The Finite element method (FEM) simulation confirmed that the silver adsorption corresponding to the plasmonic resonance variation gave little change to the electric field enhancement. We attributed the mode-specific enhancement mechanism of the adsorption of silver to the chemical enhancement from charge transfer (CT) for targeting molecules with a specific orientation. Our findings provided new insights to construct SERS substrates with higher enhancement factor (EF), which hopefully would encourage new applications in the field of surface-enhanced optical spectroscopies.
关键词： Surface-enhanced Raman scattering,In situ measurement,Enhancement mechanism
更新于2025-09-23 15:21:01
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Xenobiotic Contamination of Water by Plastics and Pesticides Revealed Through Real-time, Ultrasensitive and Reliable Surface Enhanced Raman Scattering
摘要： Uncontrolled utilization and consequent ubiquitous percolation of carcinogenic and xenobiotic contaminants, such as plasticizers and pesticides, into ecosystem has created an immediate demand for robust analytical detection techniques to identify their presence in water. Addressing this demand, we uncover the presence of xenobiotic contaminants such as Bisphenol A (BPA), Triclosan (TC), and Dimethoate (DM) through a robust, ultrasensitive and reliable Surface Enhanced Raman Scattering (SERS) platform. Thereby, conclusive real-time evidence of degradation of polyethylene terephthalate (PET) leading to release of BPA in water is presented. Worryingly, the release of BPA occurs at ambient temperature (40 0C) and within realistic timescales (12 hours) that are regularly encountered during the handling, transport and storage of PET-based water containers. Complementary mass-spectrometric, surface-specific atomic force microscopy and surface selective X-ray Photoelectron spectroscopy confirms the nanoscale surface degradation of PET through loss of C=O and C-O surface functionalities. Such ultra-sensitive (ppm-level), spectroscopic detection is enabled by the bottom-up assemblies of metal nanoparticles (Soret Colloids, SCs) acting as SERS platform to provide high analytical enhancement factor (108) with high reliability (relative standard deviation, RSD <5%). Effective and rapid detection (30 s) of several other potential xenobiotic contaminants such as Triclosan (TC) and Dimethoate (DM) over a wide range of concentrations (10-5 to 10-1 M) has also been demonstrated. Finally, non-destructive real-time spectroscopic “sniffing” of organophosphorous pesticides from the surface of fruits is achieved, illustrating the multi-phasic versatility of this label-free, non-lithography-based SERS platform.
关键词： plastic degradation,Soret colloids,water and food contamination,real-time detection,nanoparticle assembly,surface enhanced Raman scattering,Xenobiotics
更新于2025-09-23 15:21:01
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Periodic ZnO-Elevated Gold Dimer Nanostructures for Surface-Enhanced Raman Scattering Applications
摘要： The electron beam lithography-defined periodic ZnO-elevated gold dimer nanostructures for surface-enhanced Raman spectroscopy with strong electromagnetic field enhancements were fabricated in the present work. The ZnO nanorods (NRs) were grown on the patterned substrate using the hydrothermal process and Au dimers were deposited on the top surface of ZnO NRs, forming suspended gold dimers and providing plasmonic hot-spots with nanocavity effect. The effects of dimer radius, gap size and ZnO NR height on the dark-field scattering spectra and surface-enhanced Raman scattering (SERS) were investigated experimentally and theoretically. The SERS response was enhanced with increasing dimer radius and decreasing gap size. The SERS enhancement factor (EF) due to the localized surface plasmon resonance was calculated from simulation results. The calculated EF as a function of ZnO NR height showed the periodic trend of electric field intensity with the periodicity of about half of the exciting laser wavelength, and was in agreement with SERS measurements. Our hybrid substrates combining plasmonic nanocavity effect with charge transfer across the heterojunction denoted the potential candidate for SERS applications.
关键词： Plasmonics,Surface-Enhanced Raman Scattering,ZnO-Elevated Gold Dimer Nanostructures,Magnetic,Optical,and Hybrid Materials
更新于2025-09-23 15:21:01