Polydimethylsiloxane Surface Modification of Microfluidic Devices for Blood Plasma Separation.

Over the last decade, researchers have developed a variety of new analytical and clinical diagnostic devices. These devices are predominantly based on microfluidic technologies, where biological samples can be processed and manipulated for the collection and detection of important biomolecules. Polydimethylsiloxane (PDMS) is the most commonly used material in the fabrication of these microfluidic devices. However, it has a hydrophobic nature (contact angle with water of 110°), leading to poor wetting behavior and issues related to the mixing of fluids, difficulties in obtaining uniform coatings, and reduced efficiency in processes such as plasma separation and molecule detection (protein adsorption). This work aimed to consider the fabrication aspects of PDMS microfluidic devices for biological applications, such as surface modification methods. Therefore, we studied and characterized two methods for obtaining hydrophilic PDMS surfaces: surface modification by bulk mixture and the surface immersion method. To modify the PDMS surface properties, three different surfactants were used in both methods (Pluronic® F127, polyethylene glycol (PEG), and polyethylene oxide (PEO)) at different percentages. Water contact angle (WCA) measurements were performed to evaluate the surface wettability. Additionally, capillary flow studies were performed with microchannel molds, which were produced using stereolithography combined with PDMS double casting and replica molding procedures. A PDMS microfluidic device for blood plasma separation was also fabricated by soft lithography with PDMS modified by PEO surfactant at 2.5% (v/v), which proved to be the best method for making the PDMS hydrophilic, as the WCA was lower than 50° for several days without compromising the PDMS's optical properties. Thus, this study indicates that PDMS surface modification shows great potential for enhancing blood plasma separation efficiency in microfluidic devices, as it facilitates fluid flow, reduces cell aggregations and the trapping of air bubbles, and achieves higher levels of sample purity.

Critical Issues on the Surface Functionalization of Plasmonic Au-Ag/TiO2 Thin Films with Thiolated Oligonucleotide-Based Biorecognition Elements.

This work reports on the surface functionalization of a nanomaterial supporting localized surface plasmon resonances (LSPRs) with (synthetic) thiolated oligonucleotide-based biorecognition elements, envisaging the development of selective LSPR-based DNA biosensors. The LSPR thin-film transducers are composed of noble metal nanoparticles (NPs) embedded in a TiO2 dielectric matrix, produced cost-effectively and sustainably by magnetron sputtering. The study focused on the immobilization kinetics of thiolated oligonucleotide probes as biorecognition elements, followed by the evaluation of hybridization events with the target probe. The interaction between the thiolated oligonucleotide probe and the transducer's surface was assessed by monitoring the LSPR signal with successive additions of probe solution through a microfluidic device. The device was specifically designed and fabricated for this work and adapted to a high-resolution LSPR spectroscopy system with portable characteristics. Benefiting from the synergetic characteristics of Ag and Au in the form of bimetallic nanoparticles, the Au-Ag/TiO2 thin film proved to be more sensitive to thiolated oligonucleotide binding events. Despite the successful surface functionalization with the biorecognition element, the detection of complementary oligonucleotides revealed electrostatic repulsion and steric hindrance, which hindered hybridization with the target oligonucleotide. This study points to an effect that is still poorly described in the literature and affects the design of LSPR biosensors based on nanoplasmonic thin films.

Enhancing the Extinction Efficiency and Plasmonic Response of Bimetallic Nanoparticles of Au-Ag in Robust Thin Film Sensing Platforms.

The extinction efficiency of noble metal nanoparticles (NPs), namely gold (Au) and silver (Ag), are dependent on their size and surrounding dielectric. Exploiting the Localized Surface Plasmon Resonance (LSPR) phenomenon, the composition and structure of the NPs might be tailored to achieve a configuration that optimizes their response (sensitivity) to environmental changes. This can be done by preparing a bimetallic system, benefiting from the chemical stability of Au NPs and the higher scattering efficiency of Ag NPs. To enhance the LSPR sensing robustness, incorporating solid supports in the form of nanocomposite thin films is a suitable alternative. In this context, the NPs composed of gold (Au), silver (Ag), and their mixture in bimetallic Au-Ag NPs, were grown in a titanium dioxide (TiO2) matrix using reactive DC magnetron sputtering. Thermal treatment at different temperatures (up to 700 °C) tuned the LSPR response of the films and, consequently, their sensitivity. Notably, the bimetallic film with Au/Ag atomic ratio 1 exhibited the highest refractive index sensitivity (RIS), with a value of 181 nm/RIU, almost one order of magnitude higher than monometallic Au-TiO2. The nanostructural analysis revealed a wide NP size distribution of bimetallic NPs with an average size of 31 nm, covering about 20% of the overall surface area. These findings underscore the significant potential of bimetallic film systems, namely AuAg-TiO2, in LSPR sensing enhancement.

Tuning the Refractive Index Sensitivity of LSPR Transducers Based on Nanocomposite Thin Films Composed of Noble Metal Nanoparticles Dispersed in TiO2.

This work reports on the development of nanoplasmonic thin films consisting of Au, Ag, or Au-Ag nanoparticles dispersed in a TiO2 matrix and the optimization of the deposition parameters to tune their optical response. The thin films were produced by reactive DC magnetron sputtering of a Ti target with Au and/or Ag pellets placed on the erosion zone. The thicknesses (50 and 100 nm) of the films, the current density (75 and 100 A/m2) applied to the target (titanium), and the number of pellets placed on its surface were the deposition conditions that were used to tailor the optical (LSPR) response. The total noble metal content varied between 13 and 28 at.% for Au/TiO2 films, between 22 and 30 at.% for Ag/TiO2 films, and 8 to 29 at% for the Au-Ag/TiO2 systems with 1:1, 1:1.5, and 1:2 Au:Ag atomic ratios. After thermal annealing at 400 and 600 °C, LSPR bands were found for all films concerning the Au-TiO2 and Au-Ag/TiO2, while for Ag/TiO2, only for thin films with 28 and 30 at.% of Ag concentration. Refractive index sensitivity (RIS) was evaluated for Au and Au-Ag/TiO2 thin films. It was found that for bimetallic nanoparticles, the sensitivity can increase up to five times when compared to a monometallic nanoplasmonic system. Using Au-Ag/TiO2 thin films can decrease the cost of fabrication of LSPR transducers while improving their sensitivity.

Recent advances in nanomaterial-based optical biosensors for food safety applications: Ochratoxin-A detection, as case study.

Global population growth tremendously impacts the global food industry, endangering food safety and quality. Mycotoxins, particularly Ochratoxin-A (OTA), emerge as a food chain production threat, since it is produced by fungus that contaminates different food species and products. Beyond this, OTA exhibits a possible human toxicological risk that can lead to carcinogenic and neurological diseases. A selective, sensitive, and reliable OTA biodetection approach is essential to ensure food safety. Current detection approaches rely on accurate and time-consuming laboratory techniques performed at the end of the food production process, or lateral-flow technologies that are rapid and on-site, but do not provide quantitative and precise OTA concentration measurements. Nanoengineered optical biosensors arise as an avant-garde solution, providing high sensing performance, and a fast and accurate OTA biodetection screening, which is attractive for the industrial market. This review core presents and discusses the recent advancements in optical OTA biosensing, considering engineered nanomaterials, optical transduction principle and biorecognition methodologies. Finally, the major challenges and future trends are discussed, and current patented OTA optical biosensors are emphasized for a particular promising detection method.

Chitosan Micro-Membranes with Integrated Gold Nanoparticles as an LSPR-Based Sensing Platform.

Currently, there is an increasing need to develop highly sensitive plasmonic sensors able to provide good biocompatibility, flexibility, and optical stability to detect low levels of analytes in biological media. In this study, gold nanoparticles (Au NPs) were dispersed into chitosan membranes by spin coating. It has been demonstrated that these membranes are particularly stable and can be successfully employed as versatile plasmonic platforms for molecular sensing. The optical response of the chitosan/Au NPs interfaces and their capability to sense the medium's refractive index (RI) changes, either in a liquid or gas media, were investigated by high-resolution localized surface plasmon resonance (HR-LSPR) spectroscopy, as a proof of concept for biosensing applications. The results revealed that the lowest polymer concentration (chitosan (0.5%)/Au-NPs membrane) presented the most suitable plasmonic response. An LSPR band redshift was observed as the RI of the surrounding media was incremented, resulting in a sensitivity value of 28 ± 1 nm/RIU. Furthermore, the plasmonic membrane showed an outstanding performance when tested in gaseous atmospheres, being capable of distinguishing inert gases with only a 10-5 RI unit difference. The potential of chitosan/Au-NPs membranes was confirmed for application in LSPR-based sensing applications, despite the fact that further materials optimization should be performed to enhance sensitivity.

Optimization of Au:CuO Thin Films by Plasma Surface Modification for High-Resolution LSPR Gas Sensing at Room Temperature.

In this study, thin films composed of gold nanoparticles embedded in a copper oxide matrix (Au:CuO), manifesting Localized Surface Plasmon Resonance (LSPR) behavior, were produced by reactive DC magnetron sputtering and post-deposition in-air annealing. The effect of low-power Ar plasma etching on the surface properties of the plasmonic thin films was studied, envisaging its optimization as gas sensors. Thus, this work pretends to attain the maximum sensing response of the thin film system and to demonstrate its potential as a gas sensor. The results show that as Ar plasma treatment time increases, the host CuO matrix is etched while Au nanoparticles are uncovered, which leads to an enhancement of the sensitivity until a certain limit. Above such a time limit for plasma treatment, the CuO bonds are broken, and oxygen is removed from the film's surface, resulting in a decrease in the gas sensing capabilities. Hence, the importance of the host matrix for the design of the LSPR sensor is also demonstrated. CuO not only provides stability and protection to the Au NPs but also promotes interactions between the thin film's surface and the tested gases, thereby improving the nanocomposite film's sensitivity. The optimized sensor sensitivity was estimated at 849 nm/RIU, which demonstrates that the Au-CuO thin films have the potential to be used as an LSPR platform for gas sensors.

Immobilization of Streptavidin on a Plasmonic Au-TiO2 Thin Film towards an LSPR Biosensing Platform.

Optical biosensors based on localized surface plasmon resonance (LSPR) are the future of label-free detection methods. This work reports the development of plasmonic thin films, containing Au nanoparticles dispersed in a TiO2 matrix, as platforms for LSPR biosensors. Post-deposition treatments were employed, namely annealing at 400 °C, to develop an LSPR band, and Ar plasma, to improve the sensitivity of the Au-TiO2 thin film. Streptavidin and biotin conjugated with horseradish peroxidase (HRP) were chosen as the model receptor-analyte, to prove the efficiency of the immobilization method and to demonstrate the potential of the LSPR-based biosensor. The Au-TiO2 thin films were activated with O2 plasma, to promote the streptavidin immobilization as a biorecognition element, by increasing the surface hydrophilicity (contact angle drop to 7°). The interaction between biotin and the immobilized streptavidin was confirmed by the detection of HRP activity (average absorbance 1.9 ± 0.6), following a protocol based on enzyme-linked immunosorbent assay (ELISA). Furthermore, an LSPR wavelength shift was detectable (0.8 ± 0.1 nm), resulting from a plasmonic thin-film platform with a refractive index sensitivity estimated to be 33 nm/RIU. The detection of the analyte using these two different methods proves that the functionalization protocol was successful and the Au-TiO2 thin films have the potential to be used as an LSPR platform for label-free biosensors.

Plasmonic Strain Sensors Based on Au-TiO2 Thin Films on Flexible Substrates.

This study aimed at introducing thin films exhibiting the localized surface plasmon resonance (LSPR) phenomenon with a reversible optical response to repeated uniaxial strain. The sensing platform was prepared by growing gold (Au) nanoparticles throughout a titanium dioxide dielectric matrix. The thin films were deposited on transparent polymeric substrates, using reactive magnetron sputtering, followed by a low temperature thermal treatment to grow the nanoparticles. The microstructural characterization of the thin films' surface revealed Au nanoparticle with an average size of 15.9 nm, an aspect ratio of 1.29 and an average nearest neighbor nanoparticle at 16.3 nm distance. The plasmonic response of the flexible nanoplasmonic transducers was characterized with custom-made mechanical testing equipment using simultaneous optical transmittance measurements. The higher sensitivity that was obtained at a maximum strain of 6.7%, reached the values of 420 nm/ε and 110 pp/ε when measured at the wavelength or transmittance coordinates of the transmittance-LSPR band minimum, respectively. The higher transmittance gauge factor of 4.5 was obtained for a strain of 10.1%. Optical modelling, using discrete dipole approximation, seems to correlate the optical response of the strained thin film sensor to a reduction in the refractive index of the matrix surrounding the gold nanoparticles when uniaxial strain is applied.

Me-Doped Ti-Me Intermetallic Thin Films Used for Dry Biopotential Electrodes: A Comparative Case Study.

In a new era for digital health, dry electrodes for biopotential measurement enable the monitoring of essential vital functions outside of specialized healthcare centers. In this paper, a new type of nanostructured titanium-based thin film is proposed, revealing improved biopotential sensing performance and overcoming several of the limitations of conventional gel-based electrodes such as reusability, durability, biocompatibility, and comfort. The thin films were deposited on stainless steel (SS) discs and polyurethane (PU) substrates to be used as dry electrodes, for non-invasive monitoring of body surface biopotentials. Four different Ti-Me (Me = Al, Cu, Ag, or Au) metallic binary systems were prepared by magnetron sputtering. The morphology of the resulting Ti-Me systems was found to be dependent on the chemical composition of the films, specifically on the type and amount of Me. The existence of crystalline intermetallic phases or glassy amorphous structures also revealed a strong influence on the morphological features developed by the different systems. The electrodes were tested in an in-vivo study on 20 volunteers during sports activity, allowing study of the application-specific characteristics of the dry electrodes, based on Ti-Me intermetallic thin films, and evaluation of the impact of the electrode-skin impedance on biopotential sensing. The electrode-skin impedance results support the reusability and the high degree of reliability of the Ti-Me dry electrodes. The Ti-Al films revealed the least performance as biopotential electrodes, while the Ti-Au system provided excellent results very close to the Ag/AgCl reference electrodes.

Un caso atípico de neoplasia de pulmón / An atypical case of lung neoplasia

Introducción: El cáncer de pulmón se considera el tumor maligno que más muertes produce en el mundo, por encima de otros cánceres y en el 90 por ciento de los casos se diagnostica en estadios avanzados. Objetivos: Describir una forma atípica de presentación de una neoplasia de pulmón. Caso clínico: Paciente masculino de 68 años, fumador de más de 37 años, con antecedentes de insuficiencia cardíaca para lo cual llevó tratamiento con nitrosorbide (10 mg) 1 tableta cada 8 horas y aspirina (125 mg) 1 tableta al día. Refirió dolor en miembro inferior izquierdo, intenso que no se aliviaba durante todo el día y se exacerbaba con los movimientos y esfuerzos físicos. Además, presentó aumento de volumen en la región anterior media del tórax, de aproximadamente 15 cm de longitud, dura, dolorosa, de bordes regulares, superficie lisa. No tuvo tos, disnea, fiebre u otra sintomatología. Conclusiones: El paciente tuvo una forma diferente de presentación de la neoplasia de pulmón. No presentó manifestaciones respiratorias que orientaran hacia la identificación de una causa pulmonar. Es necesaria la identificación del patrón cancerígeno, así como su estadiaje para prescribir el tratamiento y conducta adecuada (AU)

Introduction: Lung cancer is considered the malignant tumor that produces more deaths in the world over other cancers and in 90 percent of cases they are diagnosed in advanced stages. Objectives: To describe an atypical form of presentation of lung neoplasia. Clinical case: A 68-year-old male patient, a smoker over 37 years of age, with a history of heart failure for which he was treated with nitrosorbide (10 mg) 1 tablet every 8 hours and aspirin (125 mg) 1 tablet per day. He reported pain in the lower left limb, which was not relieved throughout the day and was exacerbated by physical movements and efforts. In addition, there was an increase in volume in the middle anterior region of the chest, approximately 15 cm long, hard, painful, with regular edges, smooth surface. He had no cough, dyspnea, fever or other symptoms. Conclusions: The patient had a different form of lung neoplasia presentation. He did not present respiratory manifestations that oriented towards the identification of a pulmonary cause. The identification of the carcinogenic pattern is necessary, as well as its staging to prescribe the appropriate treatment and management(AU)

Molybdenum Oxide Thin Films Grown on Flexible ITO-Coated PET Substrates.

Molybdenum oxide thin films were deposited on stiff and flexible substrates by reactive DC magnetron sputtering. Two sets of samples were prepared. The first with different O2/Ar flow rate ratios and the second, fixing the oxygen content, with different time of deposition. As the O2/Ar flow rate ratio varies from 0 up to 0.56, a threshold was found, ranging from crystalline to amorphous nature, and from a nontransparent appearance with metallic-like electrical conductivity to transparent and dielectric behaviour. From the second set, all transparent, the MoOx films present a compact/dense and featureless morphology with thickness from 190 up to 910 nm, depending on the time of deposition. Their structure was corroborated by XPS and Rutherford Backscattering Spectrometry (RBS) and density measurements were performed by RBS and X-ray reflectivity (XRR), revealing a value of 2.4 g/cm3. The surface roughness is in the order of a few nanometers and the maxima optical transmission, in the visible range, is around 89%. Electrochemical cyclic voltammograms showed noticeable color reversibility and reproducibility on the flexible substrates opening new framework possibilities for new electrochomic devices.

Dry Electrodes for Surface Electromyography Based on Architectured Titanium Thin Films.

Electrodes of silver/silver chloride (Ag/AgCl) are dominant in clinical settings for surface electromyography (sEMG) recordings. These electrodes need a conductive electrolyte gel to ensure proper performance, which dries during long-term measurements inhibiting the immediate electrode's reuse and is often linked to skin irritation episodes. To overcome these drawbacks, a new type of dry electrodes based on architectured titanium (Ti) thin films were proposed in this work. The architectured microstructures were zigzags, obtained with different sputtering incidence angles (α), which have been shown to directly influence the films' porosity and electrical conductivity. The electrodes were prepared using thermoplastic polyurethane (TPU) and stainless-steel (SS) substrates, and their performance was tested in male volunteers (athletes) by recording electromyography (EMG) signals, preceded by electrode-skin impedance measurements. In general, the results showed that both SS and TPU dry electrodes can be used for sEMG recordings. While SS electrodes almost match the signal quality parameters of reference electrodes of Ag/AgCl, the performance of electrodes based on TPU functionalized with a Ti thin film still requires further improvements. Noteworthy was the clear increase of the signal to noise ratios when the thin films' microstructure evolved from normal growth towards zigzag microstructures, meaning that further tailoring of the thin film microstructure is a possible route to achieve optimized performances. Finally, the developed dry electrodes are reusable and allow for multiple EMG recordings without being replaced.

Enhancing the Sensitivity of Nanoplasmonic Thin Films for Ethanol Vapor Detection.

Nanoplasmonic thin films, composed of noble metal nanoparticles (gold) embedded in an oxide matrix, have been a subject of considerable interest for Localized Surface Plasmon Resonance (LSPR) sensing. Ethanol is one of the promising materials for fuel cells, and there is an urgent need of a new generation of safe optical sensors for its detection. In this work, we propose the development of sensitive plasmonic platforms to detect molecular analytes (ethanol) through changes of the LSPR band. The thin films were deposited by sputtering followed by a heat treatment to promote the growth of the gold nanoparticles. To enhance the sensitivity of the thin films and the signal-to-noise ratio (SNR) of the transmittance-LSPR sensing system, physical plasma etching was used, resulting in a six-fold increase of the exposed gold nanoparticle area. The transmittance signal at the LSPR peak position increased nine-fold after plasma treatment, and the quality of the signal increased six times (SNR up to 16.5). The optimized thin films seem to be promising candidates to be used for ethanol vapor detection. This conclusion is based not only on the current sensitivity response but also on its enhancement resulting from the optimization routines of thin films' architectures, which are still under investigation.

Optimization of Au:CuO Nanocomposite Thin Films for Gas Sensing with High-Resolution Localized Surface Plasmon Resonance Spectroscopy.

Gas sensing based on bulk refractive index (RI) changes, has been a challenging task for localized surface plasmon resonance (LSPR) spectroscopy, presenting only a limited number of reports in this field. In this work, it is demonstrated that a plasmonic thin film composed of Au nanoparticles embedded in a CuO matrix can be used to detect small changes (as low as 6 × 10-5 RIU) in bulk RI of gases at room temperature, using a high-resolution LSPR spectroscopy system. To optimize the film's surface, a simple Ar plasma treatment revealed to be enough to remove the top layers of the film and to partially expose the embedded nanoparticles, and thus enhance the film's gas sensing capabilities. The treated sample exhibits high sensitivity to inert gases (Ar, N2), presenting a refractive index sensitivity (RIS) to bulk RI changes of 425 nm/RIU. Furthermore, a 2-fold signal increase is observed for O2, showing that the film is clearly more sensitive to this gas due to its oxidizing nature. The results showed that the Au:CuO thin film system is a RI sensitive platform able to detect inert gases, which can be more sensitive to detect noninert gases as O2 or even other reactive species.

Development of label-free plasmonic Au-TiO2 thin film immunosensor devices.

This work reports on the development of a label-free immunosensor technology, based on nanoplasmonic Au-TiO2 thin films. The Au-TiO2 thin films were prepared by cost-effective reactive DC magnetron sputtering, followed by a thermal annealing procedure. The latter promoted the growth of the Au nanoparticles throughout the TiO2 matrix and induced some morphological changes, which are the base for the immunosensor device functionality. A posterior plasma etching treatment was required to partially expose the nanoparticles to the biological environment. It gave rise to a 6-fold increase of the total area of gold exposed, allowing further possibilities for the sensor sensitivity enhancement. Experimental results demonstrated the successful functionalization of the films' surface with antibodies, with the immobilization occurring preferentially in the exposed nanoparticles and negligibly on the TiO2 matrix. Antibody adsorption surface coverage studies revealed antibody low affinity to the film's surface. Nevertheless, immunoassay development experiments showed a strong and active immobilized antibody monolayer at an optimized antibody concentration. This allowed a 236 signal-to-noise-ratio in a confocal microscope, using mouse IgG and 100 ng/ml of Fab-specific anti-mouse IgG-FITC conjugated. Label-free detection of the optimized antibody monolayer on Au-TiO2 thin films was also tested, revealing an expected redshift in the LSPR band, which demonstrates the suitability for the development of cost-effective, label-free LSPR based immunosensor devices.

Nanoplasmonic response of porous Au-TiO2 thin films prepared by oblique angle deposition.

In this work, a versatile method is proposed to increase the sensitivity of optical sensors based on the localized surface plasmon resonance (LSPR) phenomenon. It combines a physical deposition method with the oblique angle deposition technique, allowing the preparation of plasmonic thin films with tailored porosity. Thin films of Au-TiO2 were deposited by reactive magnetron sputtering in a 3D nanostructure (zigzag growth), at different incidence angles (0° ≤ α ≤ 80°), followed by in-air thermal annealing at 400 °C to induce the growth of the Au nanoparticles. The roughness and surface porosity suffered a gradual increment by increasing the incidence angle. The resulting porous zigzag nanostructures that were obtained also decreased the principal refractive indexes (RIs) of the matrix and favoured the diffusion of Au through grain boundaries, originating broader nanoparticle size distributions. The transmittance minimum of the LSPR band appeared at around 600 nm, leading to a red-shift to about 626 nm for the highest incidence angle α = 80°, due to the presence of larger (scattering) nanoparticles. It is demonstrated that zigzag nanostructures can enhance adsorption sites for LSPR sensing by tailoring the porosity of the thin films. Atmosphere controlled transmittance-LSPR measurements showed that the RI sensitivity of the films is improved for higher incidence angles.

Electron Tomography of Plasmonic Au Nanoparticles Dispersed in a TiO2 Dielectric Matrix.

Plasmonic Au nanoparticles (AuNPs) embedded into a TiO2 dielectric matrix were analyzed by combining two-dimensional and three-dimensional electron microscopy techniques. The preparation method was reactive magnetron sputtering, followed by thermal annealing treatments at 400 and 600 °C. The goal was to assess the nanostructural characteristics and correlate them with the optical properties of the AuNPs, particularly the localized surface plasmon resonance (LSPR) behavior. High-angle annular dark field-scanning transmission electron microscopy results showed the presence of small-sized AuNPs (quantum size regime) in the as-deposited Au-TiO2 film, resulting in a negligible LSPR response. The in-vacuum thermal annealing at 400 °C induced the formation of intermediate-sized nanoparticles (NPs), in the range of 10-40 nm, which led to the appearance of a well-defined LSPR band, positioned at 636 nm. Electron tomography revealed that most of the NPs are small-sized and are embedded into the TiO2 matrix, whereas the larger NPs are located at the surface. Annealing at 600 °C promotes a bimodal size distribution with intermediate-sized NPs embedded in the matrix and big-sized NPs, up to 100 nm, appearing at the surface. The latter are responsible for a broadening and a redshift, to 645 nm, in the LSPR band because of increase of scattering-to-absorption ratio. Beyond differentiating and quantifying the surface and embedded NPs, electron tomography also provided the identification of "hot-spots". The presence of NPs at the surface, individual or in dimers, permits adsorption sites for LSPR sensing and for surface-enhanced spectroscopies, such as surface-enhanced Raman scattering.