A Wettability Contrast SERS Droplet Assay for Multiplexed Analyte Detection.

Droplet assay platforms have emerged as a significant methodology, providing distinct advantages such as sample compartmentalization, high throughput, and minimal analyte consumption. However, inherent complexities, especially in multiplexed detection, remain a challenge. We demonstrate a novel strategy to fabricate a plasmonic droplet assay platform (PDAP) for multiplexed analyte detection, enabling surface-enhanced Raman spectroscopy (SERS). PDAP efficiently splits a microliter droplet into submicroliter to nanoliter droplets under gravity-driven flow by wettability contrast between two distinct regions. The desired hydrophobicity and adhesive contrast between the silicone oil-grafted nonadhesive hydrophilic zone with gold nanoparticles is attained through (3-aminopropyl) triethoxysilane (APTES) functionalization of gold nanoparticles (AuNPs) using a scotch-tape mask. The wettability contrast surface facilitates the splitting of aqueous droplets with various surface tensions (ranging from 39.08 to 72 mN/m) into ultralow volumes of nanoliters. The developed PDAP was used for the multiplexed detection of Rhodamine 6G (Rh6G) and Crystal Violet (CV) dyes. The limit of detection for 120 nL droplet using PDAP was found to be 134 pM and 10.1 nM for Rh6G and CV, respectively. These results align with those from previously reported platforms, highlighting the comparable sensitivity of the developed PDAP. We have also demonstrated the competence of PDAP by testing adulterant spiked milk and obtained very good sensitivity. Thus, PDAP has the potential to be used for the multiplexed screening of food adulterants.

Manipulation of photosynthetic energy transfer by vibrational strong coupling.

Uncovering the mystery of efficient and directional energy transfer in photosynthetic organisms remains a critical challenge in quantum biology. Recent experimental evidence and quantum theory developments indicate the significance of quantum features of molecular vibrations in assisting photosynthetic energy transfer, which provides the possibility of manipulating the process by controlling molecular vibrations. Here, we propose and theoretically demonstrate efficient manipulation of photosynthetic energy transfer by using vibrational strong coupling between the vibrational state of a Fenna-Matthews-Olson (FMO) complex and the vacuum state of an optical cavity. Specifically, based on a full-quantum analytical model to describe the strong coupling effect between the optical cavity and molecular vibration, we realize efficient manipulation of energy transfer efficiency (from 58% to 92%) and energy transfer time (from 20 to 500 ps) in one branch of FMO complex by actively controlling the coupling strength and the quality factor of the optical cavity under both near-resonant and off-resonant conditions, respectively. Our work provides a practical scenario to manipulate photosynthetic energy transfer by externally interfering molecular vibrations via an optical cavity and a comprehensible conceptual framework for researching other similar systems.

Deciphering the Potential of Multidimensional Carbon Materials for Surface-Enhanced Raman Spectroscopy through Density Functional Theory.

Surface-enhanced Raman spectroscopy (SERS) is a potent analytical tool, particularly for molecular identification and structural analysis. Conventional metallic SERS substrates, however, suffer from low reproducibility and compatibility with biological molecules. Recently, metal-free SERS substrates based on chemical enhancement have emerged as a promising alternative with carbon-based materials offering excellent reproducibility and compatibility. Nevertheless, our understanding of carbon materials in SERS remains limited, which hinders their rational design. Here we systematically explore multidimensional carbon materials, including zero-dimensional fullerenes (C60), one-dimensional carbon nanotubes, two-dimensional graphene, and their B-, N-, and O-doped derivatives, for SERS applications. Using density functional theory, we elucidate the nonresonant polarizability-enhanced and resonant charge-transfer-based chemical enhancement mechanisms of these materials by evaluating their static/dynamic polarizability and electron excitation properties. This work provides a critical reference for the future design of carbon-based SERS substrates, opening a new avenue in this field.

Place & Play SERS: sample collection and preparation-free surface-enhanced Raman spectroscopy.

The ability to perform sensitive, real-time, in situ, multiplex chemical analysis is indispensable for diverse applications such as human health monitoring, food safety testing, forensic analysis, environmental sensing, and homeland security. Surface-enhanced Raman spectroscopy (SERS) is an effective tool to offer the ability by virtue of its high sensitivity and rapid label-free signal detection as well as the availability of portable Raman spectrometers. Unfortunately, the practical utility of SERS is limited because it generally requires sample collection and preparation, namely, collecting a sample from an object of interest and placing the sample on top of a SERS substrate to perform a SERS measurement. In fact, not all analytes can satisfy this requirement because the sample collection and preparation process may be undesirable, laborious, difficult, dangerous, costly, or time-consuming. Here we introduce "Place & Play SERS" based on an ultrathin, flexible, stretchable, adhesive, biointegratable gold-deposited polyvinyl alcohol (PVA) nanomesh substrate that enables placing the substrate on top of an object of interest and performing a SERS measurement of the object by epi-excitation without the need for touching, destroying, and sampling it. Specifically, we characterized the sensitivity of the gold/PVA nanomesh substrate in the Place & Play SERS measurement scheme and then used the scheme to conduct SERS measurements of both wet and dry objects under nearly real-world conditions. To show the practical utility of Place & Play SERS, we demonstrated two examples of its application: food safety testing and forensic analysis. Our results firmly verified the new measurement scheme of SERS and are expected to extend the potential of SERS by opening up untapped applications of sensitive, real-time, in situ multiplex chemical analysis.

Progress of tip-enhanced Raman scattering for the last two decades and its challenges in very recent years.

Tip-enhanced Raman scattering (TERS) has recently attracted remarkable attention as a novel nano-spectroscopy technique. TERS, which provides site-specific information, can be performed on any material surface regardless of morphology. Moreover, it can be applied in various environments, such as ambient air, ultrahigh vacuum (UHV), solutions, and electrochemical environments. This review reports on one hand progress of TERS for the last two decades, and on the other hand, its challenges in very recent years. Part of the progress of TERS starts with the prehistory and history of TERS, and then, the characteristics and advantages of TERS are described. Significant emphasis is put on the development of TERS instrumentation and equipment such as ultrahigh vacuum TERS, liquid TERS, electrochemical-TERS, and tip-preparations. Applications of TERS, particularly those with nanocarbons, biological materials, and surface and interface analysis, are mentioned in some detail. In the part on challenges, we focus on the very recent advances in TERS; progress in spatial resolution to the angstrom scale is the hottest topic. Recent TERS studies performed under UHV, for example chemical imaging at the angstrom scale and Raman detection of bond breaking and making of a chemisorbed up-standing single molecules at single-bond level, are reviewed. Of course, there is no clear border between the two parts. In the last part the perspective of TERS is discussed.

Half-raspberry-like bimetallic nanoassembly: Interstitial dependent correlated surface plasmon resonances and surface-enhanced Raman spectroscopy.

Correlated localized surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS) and localized electromagnetic (EM) field distributions of pure and modified gold (Au) nanoassemblies have been demonstrated. The Au nanoassemblies were decorated as half-raspberry-like nanostructures by silver (Ag) mists, and the characteristics of their SPR and SERS were observed at the same spatial position with and without decoration. The decoration of Au nanoassemblies was analyzed in-depth and confirmed by atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM). Multifunctional and lab-built microscopy was used to capture correlated SPR and SERS imaging and spectral measurements. Without decoration, strong SPR peaks and enhanced SERS signals were observed, whereas intense plasmon excitation deteriorated with a broadening and diminishing peak and the SERS enhancement dropped at least by 10 fold upon the modification. Preferential enhancement near the edge was observed in the correlated SPR and SERS measurements. The variations in localized SPR, subsequent SERS enhancement, and preferential confinement were speculated concerning localized EM near-field deformation. A typical tetramer with five interstitials was modeled and simulated by finite difference time domain (FDTD) analysis at different incident polarizations. The EM near-field distributions were extracted with and without decoration of constituent interstitials by Ag mists. Without the modification of participating interstitials, the EM near-field distributions were found confined, whereas additional EM near-field confinements were observed in the presence of Ag mists. Such EM near-field deformations due to the modification of constituent interstitials were supposed to broaden and deteriorate SPR characteristics of Au nanoassemblies as observed under this investigation.

MCR-ALS with sample insertion constraint to enhance the sensitivity of surface-enhanced Raman scattering detection.

The multivariate curve resolution-alternative least squares (MCR-ALS) algorithm was modified with sample insertion constraint to deconvolute the overlapping peaks in SERS spectra. The developed method was evaluated by the spectral data simulated using a Gaussian distribution function to generate two independent peaks corresponding to a capping agent and an analyte. The spectra were generated with different overlapping levels and various intensity ratios of the analyte to the capping agent. By using MCR-ALS with the sample insertion constraint, the peak of the capping agent was completely excluded to obtain a calibration model of the analyte with R2 > 0.95 under all conditions. Furthermore, our developed method was later applied to a real SERS measurement to quantify carbofuran (analyte) using the azo-coupling reaction with p-ATP (capping agent) on silver nanoparticles as a SERS substrate. A calibration model of derivative carbofuran phenol was generated with R2 = 0.99 and LOD = 28.19 ppm. To assess the performance of the calibration model, the model was used to estimate the concentration of carbofuran in an external validation set. It was found that the RMSE of prediction was only 2.109 with a promising R2 = 0.97.

Porous carbon nanowire array for surface-enhanced Raman spectroscopy.

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for vibrational spectroscopy as it provides several orders of magnitude higher sensitivity than inherently weak spontaneous Raman scattering by exciting localized surface plasmon resonance (LSPR) on metal substrates. However, SERS can be unreliable for biomedical use since it sacrifices reproducibility, uniformity, biocompatibility, and durability due to its strong dependence on "hot spots", large photothermal heat generation, and easy oxidization. Here, we demonstrate the design, fabrication, and use of a metal-free (i.e., LSPR-free), topologically tailored nanostructure composed of porous carbon nanowires in an array as a SERS substrate to overcome all these problems. Specifically, it offers not only high signal enhancement (~106) due to its strong broadband charge-transfer resonance, but also extraordinarily high reproducibility due to the absence of hot spots, high durability due to no oxidization, and high compatibility to biomolecules due to its fluorescence quenching capability.

Raman image-activated cell sorting.

The advent of image-activated cell sorting and imaging-based cell picking has advanced our knowledge and exploitation of biological systems in the last decade. Unfortunately, they generally rely on fluorescent labeling for cellular phenotyping, an indirect measure of the molecular landscape in the cell, which has critical limitations. Here we demonstrate Raman image-activated cell sorting by directly probing chemically specific intracellular molecular vibrations via ultrafast multicolor stimulated Raman scattering (SRS) microscopy for cellular phenotyping. Specifically, the technology enables real-time SRS-image-based sorting of single live cells with a throughput of up to ~100 events per second without the need for fluorescent labeling. To show the broad utility of the technology, we show its applicability to diverse cell types and sizes. The technology is highly versatile and holds promise for numerous applications that are previously difficult or undesirable with fluorescence-based technologies.

Interactions Between Epitaxial Graphene Grown on the Si- and C-Faces of 4H-SiC Investigated Using Raman Imaging and Tip-Enhanced Raman Scattering.

Interactions between epitaxial graphene grown on Si- and C-faces were investigated using Raman imaging and tip-enhanced Raman scattering (TERS). In the TERS spectrum, which has a spatial resolution exceeding the diffraction limit, a D band was observed not from graphene surface, but from the edges of the epitaxial graphene ribbons without a buffer layer, which interacts with SiC on the Si-face. In contrast, for a graphene micro-island on the C-face, the D band disappeared even on the edges where the C atoms were arranged in armchair configurations. The disappearance of the edge chirality via combination between the C atoms and SiC on the C-face is responsible for this phenomenon. The TERS signals from the C-face were weaker than those from the Si-face without the buffer layer. On the Si-face with a buffer layer, the graphene TERS signal was hardly observed. TERS enhancement was suppressed by interactions on the edges or by the buffer layer between the SiC and graphene on the C- or Si-face, respectively.

Distinguishing Enantiomers by Tip-Enhanced Raman Scattering: Chemically Modified Silver Tip with an Asymmetric Atomic Arrangement.

Discrimination between enantiomers is achieved by tip-enhanced Raman scattering (TERS) using a silver tip that is chemically modified by an achiral para-mercaptopyridine (pMPY) probe molecule. Differences in the relative intensities of the pMPY spectra were monitored for three pairs of enantiomers containing hydroxy (-OH) and/or amino (-NH2 ) groups. The N: or N+ -H functionality of the pMPY-modified tip participates in hydrogen-bond interactions with a particular molecular orientation of each chiral isomer. The asymmetric arrangement of silver atoms at the apex of the tip induces an asymmetric electric field, which causes the tip to become a chiral center. Differences in the charge-transfer (CT) states of the metal-achiral probe system in conjunction with the asymmetric electric field produce different enhancements in the Raman signals of the two enantiomers. The near-field effect of the asymmetric electric field, which depends on the number of analyte functional groups capable of hydrogen-bond formation, improves the degree of discrimination.

Local structural changes in graphene oxide layers induced by silver nanoparticles.

Structural changes of graphene oxide (GO) in silver/graphene oxide (AGO) nanocomposites are investigated using tip-enhanced Raman scattering (TERS). Because of markedly high spatial resolution of the TERS technique, the measurements of molecular information at specific nano-scaled positions can be achieved by constructing line-profile TERS spectra straight from the center of silver nanoparticles (AgNPs) on GO layers. The results show evidences that AgNPs cause shortening of C-C bonds beneath AgNPs, flattening of GO layers, and critical bending on GO layers. Additionally, a connection of carbon atoms via a C-C network subsequently expands structural changes with a distance of 200-250 nm from the center of AgNPs, even though this distance is larger than the size of AgNPs. The proposed model of GO structural changes unveils new understanding about changes in properties from GO to AGO nanocomposites, which will contribute to a development of advanced nanostructures/nanocomposites in the near future.

Excitation wavelength selection for quantitative analysis of carotenoids in tomatoes using Raman spectroscopy.

The difference in Raman spectra for different excitation wavelengths (532 nm, 785 nm, and 1064 nm) was investigated to identify an appropriate wavelength for the quantitative analysis of carotenoids in tomatoes. For the 532 nm-excited Raman spectra, the intensity of the peak assigned to the carotenoid has no correlation with carotenoid concentration, and the peak shift reflects carotenoid composition changing from lycopene to ß-carotene and lutein. Thus, 532 nm-excited Raman spectra are useful for the qualitative analysis of carotenoids. For the 785 nm- and 1064 nm-excited Raman spectra, the peak intensity of the carotenoid showed good correlation with carotenoid concentration; thus, regression models for carotenoid concentration were developed using these Raman spectra and partial least squares regression. A regression model designed using the 785 nm-excited Raman spectra showed a better result than the 532 nm- and 1064 nm-excited Raman spectra. Therefore, it can be concluded that 785 nm is the most suitable excitation wavelength for the quantitative analysis of carotenoid concentration in tomatoes.

Calculated shape dependence of electromagnetic field in tip-enhanced Raman scattering by using a monopole antenna model.

To evaluate the shape of an Ag tip with regard to tip-enhanced Raman scattering (TERS) signal, the enhanced electromagnetic (EM) field and scattering spectrum, arising from surface plasmon resonance at the apex of the tip, were calculated using a finite-difference time domain (FDTD) method. In the calculated forward scattering spectra from the smooth Ag tip, the band appeared within the visible region, similar to the experimental results and calculation for a corrugated Ag cone. In the FDTD calculation of TERS, the Ag tip acting as a monopole antenna was adopted by insertion of a perfect electric conductor between the root of the tip and a top boundary surface of the calculation space. As a result, the EM field was only enhanced at the apex. The shape dependence i.e. the EM field calculated at the apex with various curvatures on the different tapered tips, obtained using the monopole antenna model, was different from that simulated using a conventional dipole antenna model.

Observation and Analysis of Blinking Surface-enhanced Raman Scattering.

From a single molecule at a silver nanoaggregate junction, blinking surface-enhanced Raman scattering (SERS) is observed. Here, a protocol is presented on how to prepare the SERS-active silver nanoaggregate, record a video of certain blinking spots in the microscopic image, and analyze the blinking statistics. In this analysis, a power law reproduces the probability distributions for bright events relative to their duration. The probability distributions for dark events are fitted by a power law with an exponential function. The parameters of the power law represent molecular behavior in both bright and dark states. The random walk model and the speed of the molecule across the entire silver surface can be estimated. It is difficult to estimate even when using averages, autocorrelation functions, and super-resolution SERS imaging. In the future, power law analyses should be combined with spectral imaging, because the origins of blinking cannot be confirmed by this analysis method alone.

Use of the product of mean intensity ratio (PMIR) technique for discriminant analysis of lycopene-rich vegetable juice using a portable NIR-excited Raman spectrometer.

In this study, a lycopene-content-based discriminant analysis was performed using a portable near-infrared-excited Raman spectrometer. In the vegetable-juice Raman spectra, the peak intensity of the lycopene band increased with increasing lycopene concentration, but scattering decreased the repeatability of the peak intensity. Consequently, developing a lycopene-concentration regression model using peak intensity is not straightforward. Therefore, a new method known as the product of mean intensity ratio (PMIR) analysis was developed to rapidly identify lycopene-rich samples on-site. In the PMIR analysis, Raman spectra are measured with short exposure times, confirming only the peaks of carotenoids with high concentrations, and thus the lycopene concentrations of vegetable juice samples could be determined successfully. Exposure times of 20ms and 100ms could detect lycopene concentrations of ≥5mg/100g and ≥2mg/100g with 93.2% and 97.7% accuracy, respectively; thus, lycopene-content-based discriminant analysis using the PMIR and a portable Raman spectrometer is feasible.

Measurement of pH-dependent surface-enhanced hyper-Raman scattering at desired positions on yeast cells via optical trapping.

Surface-enhanced hyper-Raman scattering (SEHRS) spectra were obtained at desired positions on yeast by focusing a continuous wave near-infrared laser beam while silver nanoparticles (AgNPs) were simultaneously optically trapped. However, the optically trapped colloidal AgNP suspension bubbled up at the focusing point, preventing spectral measurement. In the case of optically trapped AgNPs functionalized with 4-mercaptobenzoic acid (p-MBA), surface-enhanced hyper-Rayleigh scattering was considerably strong, indicating the suppression of the photothermal conversion to form the bubble. Interestingly, the SEHRS peaks that are attributed not only to p-MBA, but also to other species, were very occasionally observed. They may be partly assigned to the ß1,3 glucan and protein amide II band. The SEHRS peak at 1366 cm-1 was barely visible in the measurements of conventional baker's yeast even in the suspension (pH 9) despite the effects of high pH on p-MBA. In contrast, the SEHRS peak in the measurements of yeast for biological applications was occasionally observed at 1366 cm-1. This suggests that acidity is correlated with fermentation efficiency. At different positions on single yeast cells, the intensity of the SEHRS peak at 1366 cm-1 varied. This result represents the pH distribution on yeast.

Unveiling the Aggregation of Lycopene in Vitro and in Vivo: UV-Vis, Resonance Raman, and Raman Imaging Studies.

The present study investigates the structure of lycopene aggregates both in vitro and in vivo using ultraviolet-visible (UV-vis) and Raman spectroscopies. The electronic absorption bands of the J- and H-aggregates in vitro shift to lower and higher energies, respectively, compared to that of the lycopene monomer. Along with these results, the frequencies of the ν1 Raman bands were shifted to lower and higher frequencies, respectively. By plotting the frequencies of the ν1 Raman band against the S0 → S2 transition energy, a linear relationship between the data set with different aggregation conformations can be obtained. Therefore, the band positions depending on the different conformations can be explained based on the idea that the effective conjugated C═C chain lengths within lycopene molecules are different due to the environmental effect (site-shift effect) caused by the aggregation conformation. Applying this knowledge to the in vivo measurement of a tomato fruit sample, the relationship between the aggregation conformation of lycopene and the spectral patterns observed in the UV-vis as well as Raman spectra in different parts of tomato fruits was discussed in detail. The results showed that the concentration of lycopene (particularly that of the J-aggregate) specifically increased, whereas that of chlorophyll decreased, with ripening. Furthermore, Raman imaging indicated that lycopene with different aggregate conformations was distributed inhomogeneously, even within one sample. The layer formation in tomato tissues with high concentrations of J- and H-aggregates was successfully visualized. In this manner, the presence of lycopene distributions with different aggregate conformations was unveiled in vivo.

An enhanced degree of charge transfer in dye-sensitized solar cells with a ZnO-TiO2/N3/Ag structure as revealed by surface-enhanced Raman scattering.

A number of recent studies have focused on improving the performance of dye-sensitized solar cells (DSSCs). Cells with a ZnO-TiO2/N3/Ag structure have attracted particular attention because of their excellent power conversion efficiencies. Using a dendritic crystal ZnO-TiO2 composite semiconductor and Ag in conjunction leads to different charge-transfer (CT) processes, and this is the main theoretical basis for the improvement of DSSC performances. Thus, in the present study, TiO2/N3, ZnO/N3, ZnO-TiO2/N3, TiO2/N3/Ag, ZnO/N3/Ag, and ZnO-TiO2/N3/Ag assemblies have been fabricated and their CT processes have been monitored by using surface-enhanced Raman scattering (SERS) spectra, with particular focus on the differences caused by the synergistic effect of the ZnO-TiO2 component. The dye loading capacity of the dendritic crystal ZnO-TiO2 is much larger than that of TiO2. There are extra enhancements in the SERS intensity and degree of CT (ρCT) in ZnO-TiO2/N3 compared to ZnO + TiO2/N3 (based on a simulation curve for the physically mixed TiO2 and ZnO semiconductors) with 476.5 nm excitation due to the synergistic effect of the ZnO-TiO2 component. And these enhancements in ZnO-TiO2/N3/Ag compared to ZnO + TiO2/N3/Ag appear with 476.5 and 532 nm excitation, which are particularly large with 532 nm excitation. Accordingly, the participation of Ag in this synergistic effect can reduce its energy threshold, which will make it easier to appear. Finally, to rationalize these extra enhancements, the models describing the CT mechanism have been proposed. Thus, the use of the dendritic crystal ZnO-TiO2 composite semiconductor in the semiconductor/N3/Ag system can improve the adsorption capacity of N3 compared to that with TiO2. Meanwhile, the synergistic effect of ZnO-TiO2 and Ag can promote the CT process, demonstrating that ZnO-TiO2/N3/Ag is an excellent structure for DSSCs.

Surface-enhanced resonance Raman scattering of hemoproteins and those in complicated biological systems.

In this review article, we discuss surface-enhanced resonance Raman scattering (SERRS) studies of hemeproteins such as myoglobin, hemoglobin, and cytochrome on various metal substrates; for example, colloidal silver nanoparticles coated with and without self-assembled monolayers (SAM), a roughened silver electrode protected with and without SAM, a sharp silver tip, and colloidal gold nanoparticles coated with and without SAM. Moreover, we classify the studies in terms of an excitation wavelength; namely, excitation at the B- (Soret) band, Q- (α and ß) band, and in the near infrared (NIR) range. In the SERRS studies with B band excitation, it has been shown that the hemeprotein on a silver surface takes a non-native form through detachment from the heme pocket in the protein. With Q band excitation, on the other hand, the change in SERRS has been explained by the orientation of the hemeprotein on the surface. Even by excitation in the NIR range, the peak positions are consistent with the assignment of the major vibrational modes of heme despite there being no resonance Raman effect. Thus, the SERRS of hemeproteins is influenced by a resonance Raman effect, LSPR, and interactions with the metal surface such as structural changes, orientation, and selective adsorption. Moreover, we discuss how SERRS has been applied to complicated biological systems such as living cells containing hemeprotein. For mitochondria, a change of the oxidation-state was observed by the electron transport chain in the cell and at different positions. As an example of a biomedical application of SERRS, the sensitive detection of malaria is presented.