Surface-enhanced Raman spectroscopy for bacterial discrimination utilizing a scanning electron microscope with a Raman spectroscopy interface

Surface-enhanced Raman scattering (SERS) utilizing colloidal silver has already been shown to provide a rapid means of generating "whole-organism fingerprints" for use in bacterial identification and discrimination. However, one of the main drawbacks of the technique for the analysis of microbiological samples with optical Raman microspectroscopy has been the inability to acquire pre-emptively a region of the sample matrix where both the SERS substrate and biomass are both present. In this study, we introduce a Raman interface for scanning electron microscopy (SEM) and demonstrate the application of this technology to the reproducible and targeted collection of bacterial SERS spectra. In secondary electron mode, the SEM images clearly reveal regions of the sample matrix where the sodium borohydride-reduced silver colloidal particles are present, Stokes spectra collected from these regions are rich in vibrational bands, whereas spectra taken from other areas of the sample elicit a strong fluorescence response. Replicate SERS spectra were collected from two bacterial strains and show excellent reproducibility both by visual inspection and as demonstrated by principal components analysis on the whole SERS spectra.     

Bimetallic Nanostructures as Active Raman Markers: Gold‐Nanoparticle Assembly on 1D and 2D Silver Nanostructure Surfaces

It is demonstrated that bimetallic silver–gold anisotropic nanostructures can be easily assembled from various nanoparticle building blocks with well‐defined geometries by means of electrostatic interactions. One‐dimensional (1D) silver nanowires, two‐dimensional (2D) silver nanoplates, and spherical gold nanoparticles are used as representative building blocks for bottom‐up assembly. The gold nanoparticles are electrostatically bound onto the 1D silver nanowires and the 2D silver nanoplates to give bimetallic nanostructures. The unique feature of the resulting nanostructures is the particle‐to‐particle interaction that subjects absorbed analytes to an enhanced electromagnetic field with strong polarization dependence. The Raman activity of the bimetallic nanostructures is compared with that of the individual nanoparticle blocks by using rhodamine 6G solution as the model analyte. The Raman intensity of the best‐performing silver–gold nanostructure is comparable with the dense array of silver nanowires and silver nanoplates that were prepared by means of the Langmuir–Blodgett technique. An optimized design of a single‐nanostructure substrate for surface‐enhanced Raman spectroscopy (SERS), based on a wet‐assembly technique proposed here, can serve as a compact and low‐cost alternative to fabricated nanoparticle arrays.     

Linearly moving low-volume spectroelectrochemical cell for microliter-scale surface-enhanced resonance Raman spectroscopy of heme proteins

Surface-enhanced resonance Raman spectra of cytochrome c on silver electrodes coated with self-assembled monolayers of mercaptopropionic acid were recorded at different potentials using 50 microL of a micromolar solution. For this purpose, a linearly moving, low-volume, small spectroelectrochemical cell was designed and used together with a Raman microprobe. The quality of the spectra obtained is good, and the spectra show essentially the same features reported by other authors using much larger volumes. The cell described in this paper is shown to be useful for studying the spectroelectrochemistry of photosensitive compounds such as heme proteins, which are available only in very small amounts (nanomoles to picomoles).     

Ultrasensitive detection of 1,4-Bis(4-vinylpyridyl)phenylene in a small volume of low refractive index liquid by surface-enhanced Raman scattering-active light waveguide

This paper reports a novel surface-enhanced Raman scattering (SERS)-active light waveguide method for ultrasensitive detection of a sample dissolved in a small volume of low refractive index liquid. The SERS-active light waveguide demonstrated in this study was constructed via the light-guiding silica capillary. The surface of its inner wall was modified with SERS-active silver nanoparticles that can remarkably enhance Raman signals. The capillary with SERS-active modified layer was filled with the sample solution to form the SERS-active liquid core (LC) fiber. The incident laser beam travels through the waveguide in a totally reflective mode within the fiber wall and penetrates a small distance into the sample solution by the evanescent wave field. The Raman scattering of the analytes adsorbed onto the surface of the SERS-active modified layer can be excited by the laser beam and refracted into the fiber wall. Thus, a sample dissolved in low index liquid, e.g., methanol, can be quantitatively monitored by Raman spectroscopy and detection limit of its concentration is lower than 10(-9) mol/L.     

Surface-enhanced Raman scattering on single-wall carbon nanotubes

Exploiting the effect of surface-enhanced Raman scattering (SERS), the Raman signal of single-wall carbon nanotubes (SWNTs) can be enhanced by up to 14 orders of magnitude when the tubes are in contact with silver or gold nanostructures and Raman scattering takes place predominantly in the enhanced local optical fields of the nanostructures. Such a level of enhancement offers exciting opportunities for ultrasensitive Raman studies on SWNTs and allows resonant and non-resonant Raman experiments to be done on single SWNTs at relatively high signal levels. Since the optical fields are highly localized within so-called "hot spots" on fractal silver colloidal clusters, lateral confinement of the Raman scattering can be as small as 5 nm, allowing spectroscopic selection of a single nanotube from a larger population. Moreover, since SWNTs are very stable "artificial molecules" with a high aspect ratio and a strong electron-phonon coupling, they are unique "test molecules" for investigating the SERS effect itself and for probing the "electromagnetic field contribution" and "charge transfer contribution" to the effect. SERS is also a powerful tool for monitoring the "chemical" interaction between the nanotube and the metal nanostructure.     

On-column surface-enhanced Raman spectroscopy detection in capillary electrophoresis using running buffers containing silver colloidal solutions

Direct on-column surface-enhanced Raman spectroscopy (SERS) detection is demonstrated in capillary electrophoresis (CE). Distinctive SERS spectra of two test compounds, riboflavin and Rhodamine 6G, are obtained in 100 microm i.d. fused-silica capillaries under CE conditions using running buffers that contain silver colloidal solutions. Detection is performed using an unmodified commercial Raman spectrometer in a confocal microscope mode of operation. The effects of laser power, wavelength, spectra acquisition time, silver colloidal concentration, and applied voltage (i.e., flow rate) on the quality of SERS spectra are evaluated. Using laser powers of 17 mW (at the sample) at 515 nm and employing 1 s spectral acquisition times, spectra with bands exhibiting signal-to-noise ratios greater than 10 could be obtained for 1.0 x 10(-6) M riboflavin and very low nanomolar concentrations of Rhodamine 6G. This was accomplished without optimization of silver colloidal solution compositions and by using a low-throughput spectrometer. Incorporation of the colloidal solutions into running buffers is shown to have little effect on the separation of the test compounds as monitored using a laser-induced fluorescence instrumental scheme. However, SERS spectra degrade if the capillary is not rinsed between experiments. Riboflavin and Rhodamine 6G spectra are obtained on-the-fly for actual CE separations. In the case of the latter solute, the injected quantity was approximately 90 amol.     

Formation of flower-like Ag colloids using pulsed proton beam

A new solution phase method is presented for the synthesis of "flower-like" silver nanomaterials using a pulsed proton beam irradiation process at room temperature. It was observed that the morphology of the prepared silver crystals was easily controlled by varying the number of exposures to the pulsed proton beam. The synthesized flower-like silver nanocrystals exhibited excellent surface enhanced Raman scattering (SERS) signals, which were attributed to the rough microstructures on the surface of the synthesized Ag crystals.     

Development of a surface-enhanced Raman technique for biomarker studies on Mars

Raman spectroscopy has been identified as a potentially useful tool to collect evidence of past or present life on extraterrestrial bodies. However, it is limited by its inherently low signal strength. In this investigation, laboratory tests were conducted using surface-enhanced Raman spectroscopy (SERS) in an "inverted" mode to detect the presence of organic compounds that may be similar to possible biomarkers present on Mars. SERS was used to overcome the inherently low signal intensity of Raman spectroscopy and was an effective method for detecting small concentrations of organic compounds on a number of surfaces. For small organic molecules, dissolution of the molecule to be analyzed in a suitable solvent and depositing it on a prepared SERS substrate for analysis is possible. However, for larger molecules, an "inverted" SERS (iSERS) technique was shown to be effective. In iSERS, nanoparticles of silver or gold were deposited on the mineral substrate/organic compound to be analyzed. Benzotriazole, benzoic acid, and phthalic acid were used as test organic analogs and the iSERS technique was able to detect femtomole levels of the analytes. The interference from various mineral substrates was also examined. Different methods of depositing silver particles were evaluated, including ion beam-assisted vapor deposition and deposition from aqueous colloidal suspensions.     

Line-scanning Raman imaging spectroscopy for detection of fingerprints

Fingerprints are the best form of personal identification for criminal investigation purposes. We present a line-scanning Raman imaging system and use it to detect fingerprints composed of β-carotene and fish oil on different substrates. Although the line-scanning Raman system has been used to map the distribution of materials such as polystyrene spheres and minerals within geological samples, this is the first time to our knowledge that the method is used in imaging fingerprints. Two Raman peaks of β-carotene (501.2, 510.3 nm) are detected and the results demonstrate that both peaks can generate excellent images with little difference between them. The system operates at a spectra resolution of about 0.4 nm and can detect β-carotene signals in petroleum ether solution with the limit of detection of 3.4×10(-9) mol/L. The results show that the line-scanning Raman imaging spectroscopy we have built has a high accuracy and can be used in the detection of latent fingerprints in the future.     

Novel method for preparing controllable and stable silver particle films for surface-enhanced Raman scattering spectroscopy

A new surface-enhanced Raman scattering (SERS) active substrate has been developed based on our previous study. Small silver nanoparticles on a quartz slide can be enlarged by using a mixture of commercially available reagents called Silver Enhancer and Initiator. The optical properties and characteristics of the new substrate have been investigated by ultraviolet-visible (UV-Vis) spectroscopy and atomic force microscopy (AFM). The results indicate that the small silver nanoparticles grow and some silver aggregates emerge on the quartz slide after the slide is immersed into the Silver Enhancer and Initiator Mixture (SEIM). The average diameter of the silver nanoparticles on the substrate becomes approximately double after the immersion into SEIM for 20 s. 1,4-bis[2-(4-pyridyl)ethenyl]-benzene (BPENB) was used as a Raman probe to evaluate the enhancement ability of the new silver substrate. It has been found that the SERS intensity can be increased about 10 times by using the substrate treated by SEIM compared with that without being treated by SEIM. Interestingly enough, the SERS enhancement increases with time. This may be due to the reorganization of silver nanoparticles on the quartz surface. The new substrate can remain active for more than 90 days. The adsorption mode of BPENB on the new substrate and the dependence of the BPENB configurations on the concentration of BPENB in methanol solution have also been investigated by SERS or UV-Vis spectroscopy. The SERS spectra of a self-assembled monolayer (SAM) BPENB film adsorbed on a silver substrate treated by SEIM show that BPENB molecules are chemically absorbed through the Ag-N bond. Consequently, a nearly perpendicular orientation of BPENB on the silver surface is proposed. The SERS spectra of BPENB SAMs on the new substrates fabricated from methanol solutions with different concentrations are compared. The concentration dependence of the SERS spectra reveals that the BPENB molecules are adsorbed on the silver film as monomers when the film is prepared from the solution with a lower concentration (<4 x 10(-6) M) and as aggregates when it is prepared from the solution with a higher concentration (>1 x 10(-5) M).     

Surface-enhanced Raman spectra of calf thymus DNA adsorbed on concentrated silver colloid

Raman and surface-enhanced Raman scattering (SERS) spectra of calf thymus DNA were investigated. We have carried out improvements to the silver colloid preparation method of Lee and Meisel in two respects. In one method, the silver sol was boiled with rapid stirring for over two hours. In the second method, the silver sol was concentrated by centrifugation before adding it to the DNA solution. The resulting hydrosol could be stored for 15 months because of its high stabilization. Structural information with respect to the phosphate backbone, deoxyribose, and four bases of DNA could be obtained before and after the DNA solutions were added to the concentrated Ag colloid substrate. The intensities of almost all characteristic bands assigned to various groups of the components of DNA were enhanced to a remarkable degree. The enhancement effect of the DNA solution at neutral pH 7.0 was obviously much better than that at acidic pH 3.4 or at alkaline pH 8.5. Intensity increases of the SERS bands of the DNA solution with time were observed. The SERS signals obtained 16 hours after the interaction of the Ag colloid with the DNA solution were much better than the SERS signals obtained just after the mixed liquid was prepared. This method can be widely used to store the Ag colloid for long times and to obtain the SERS spectra of DNA molecules, and it can further be used to study the adsorption behavior of solute biomacromolecules in different solvents.     

Degradation of curcumin dye in aqueous solution and on ag nanoparticles studied by ultraviolet-visible absorption and surface-enhanced Raman spectroscopy

The chemical degradation of curcumin (CU) in aqueous solution and on silver nanoparticles was studied by means of ultraviolet (UV)-visible absorption and surface-enhanced Raman (SERS) spectroscopy at different pH levels and upon light irradiation. CU undergoes a chemical degradation in aqueous solution mainly when the pH is increased. The CU degradation is catalytically enhanced in the presence of Ag nanoparticles. In general, CU degradation implies two steps: (1) the breakdown of the interring chain connecting the two CU aromatic side rings, producing smaller phenolic compounds rich in carboxylate groups, and (2) polymerization of the resulting phenolic products, giving rise to phenolic polymeric products. The degradation-polymerization mechanism can be modulated depending on the experimental conditions. The chemical photoproducts resulting from the visible light irradiation are similar to the polycatechol products obtained when catechol is adsorbed on Ag nanoparticles.     

Vibrational properties of silicene and germanene

The structural and vibrational properties of two-dimensional hexagonal silicon (silicene) and germanium (germanene) are investigated by means of first-principles calculations. It is predict that the silicene (germanene) structure with a small buckling of 0.44 Å (0.7 Å) and bond lengths of 2.28 Å (2.44 Å) is energetically the most favorable, and it does not exhibit imaginary phonon mode. The calculated non-resonance Raman spectra of silicene is characterized by a main peak at about 575 cm^?1, namely the G-like peak. For germanene, the highest peak is at about 290 cm^?1. Extensive calculations on armchair silicene nanoribbons and armchair germanene nanoribbons are also performed, with and without hydrogenation of the edges. The studies reveal other Raman peaks mainly distributed at lower frequencies than the G-like peak which could be attributed to the defects at the edges of the ribbons, thus not present in the Raman spectra of non-defective silicene and germanene. Particularly the Raman peak corresponding to the D mode is found to be located at around 515 cm^?1 for silicene and 270 cm^?1 for germanene. The calculated G-like and the D peaks are likely the fingerprints of the Raman spectra of the low-buckled structures of silicene and germanene.      

Trinitrotoluene explosive lights up ultrahigh Raman scattering of nonresonant molecule on a top-closed silver nanotube array

The highest Raman enhancement factors are obtained in a double resonance: molecular electronic resonance and plasmon resonance with a "hot spot" in surface-enhanced Raman scattering (SERS). However, for most molecules of interest the double resonance is not realized with the excitation frequencies normally used in Raman. The latter may limit the practical applications of SERS for trace analysis. Here, we report that Raman-inactive trinitrotoluene (TNT) lights up the ultrahigh Raman scattering of off-resonated p-aminobenzenethiol (PABT) through the formation of charge-transfer TNT-PABT complex on the top-closed flexible silver nanotube array. Raman hot spots can spontaneously form in a reversible way by the self-approaching of flexible nanotubes driven through the capillary force of solvent evaporation. Meanwhile, the PABT-TNT-PABT bridges between self-approaching silver nanotubes possibly form by the specific complexing and zwitterion interactions, and the resultant chromophores can absorb the visible light that matches with the incident laser and the localized surface plasmon of a silver nanotube array. The multiple spectral resonances lead to the huge enhancement of Raman signals of PABT molecules due to the presence of ultratrace TNT. The enhancement effect is repeatedly renewable by the reconstruction of molecular bridges and can selectively detect TNT with a limit of 1.5 × 10(-17) M. The results in this report provide the simple and supersensitive approach to the detection of TNT explosives and the possibility of building a robust Raman-based assay platform.     

Remote Raman spectroscopic detection of minerals and organics under illuminated conditions from a distance of 10 m using a single 532 nm laser pulse

Raman spectra of several minerals and organics were obtained from a small portable instrument at a distance of 10 m in a well-illuminated laboratory with a single 532 nm laser pulse with energy of 35 mJ/pulse. Remote Raman spectra of common minerals (dolomite, calcite, marble, barite, gypsum, quartz, anatase, fluorapatite, etc.) obtained in a short period of time (1.1 mus) clearly show Raman features that can be used as fingerprints for mineral identification. Raman features of organics (benzene, cyclohexane, 2-propanol, naphthalene, etc.) and other chemicals such as oxides, silicates, sulfates, nitrates, phosphates, and carbonates were also easily detected. The ability to identify minerals from their Raman spectra obtained from a single laser pulse has promise for future space missions where power consumption is critical. Such a system could be reduced in size by minimizing the cooling requirements for the laser unit. The remote Raman system is also capable of performing time-resolved measurements. Data indicate that further improvement in the performance of the system is possible by reducing the gate width of the detector (ICCD) from 1.1 mus to approximately 20 ns, which would significantly reduce the background signal from daylight or a well-illuminated laboratory. The 1.1 mus signal gating was effective in removing nearly all background fluorescence with 532 nm excitation, indicating that the fluorescence in most minerals is probably from long-lifetime inorganic phosphorescence.     

High-density silver nanoparticle film with temperature-controllable interparticle spacing for a tunable surface enhanced Raman scattering substrate

The formation of high-density silver nanoparticles and a novel method to precisely control the spacing between nanoparticles by temperature are demonstrated for a tunable surface enhanced Raman scattering substrates. The high-density nanoparticle thin film is accomplished by self-assembling through the Langmuir-Blodgett (LB) technique on a water surface and transferring the particle monolayer to a temperature-responsive polymer membrane. The temperature-responsive polymer membrane allows producing a dynamic surface enhanced Raman scattering substrate. The plasmon peak of the silver nanoparticle film red shifts up to 110 nm with increasing temperature. The high-density particle film serves as an excellent substrate for surface-enhanced Raman spectroscopy (SERS), and the scattering signal enhancement factor can be dynamically tuned by the thermally activated SERS substrate. The SERS spectra of Rhodamine 6G on a high-density silver particle film at various temperatures is characterized to demonstrate the tunable plasmon coupling between high-density nanoparticles.     

Semi-automated detection of trace explosives in fingerprints on strongly interfering surfaces with Raman chemical imaging

We have previously demonstrated the use of wide-field Raman chemical imaging (RCI) to detect and identify the presence of trace explosives in contaminated fingerprints. In this current work we demonstrate the detection of trace explosives in contaminated fingerprints on strongly Raman scattering surfaces such as plastics and painted metals using an automated background subtraction routine. We demonstrate the use of partial least squares subtraction to minimize the interfering surface spectral signatures, allowing the detection and identification of explosive materials in the corrected Raman images. The resulting analyses are then visually superimposed on the corresponding bright field images to physically locate traces of explosives. Additionally, we attempt to address the question of whether a complete RCI of a fingerprint is required for trace explosive detection or whether a simple non-imaging Raman spectrum is sufficient. This investigation further demonstrates the ability to nondestructively identify explosives on fingerprints present on commonly found surfaces such that the fingerprint remains intact for further biometric analysis.     

Chitosan-coated anisotropic silver nanoparticles as a SERS substrate for single-molecule detection

Surface-enhanced Raman spectroscopy (SERS) is a technique that has become widely used for identifying and providing structural information about molecular species in low concentration. There is an ongoing interest in finding optimum particle size, shape and spatial distribution for optimizing the SERS substrates and pushing the sensitivity toward the single-molecule detection limit. This work reports the design of a novel, biocompatible SERS substrate based on small clusters of anisotropic silver nanoparticles embedded in a film of chitosan biopolymer. The SERS efficiency of the biocompatible film is assessed by employing Raman imaging and spectroscopy of adenine, a significant biological molecule. By combining atomic force microscopy with SERS imaging we find that the chitosan matrix enables the formation of small clusters of silver nanoparticles, with junctions and gaps that greatly enhance the Raman intensities of the adsorbed molecules. The study demonstrates that chitosan-coated anisotropic silver nanoparticle clusters are sensitive enough to be implemented as effective plasmonic substrates for SERS detection of nonresonant analytes at the single-molecule level.     

Discrimination of bacteria using surface-enhanced Raman spectroscopy

Raman spectroscopy has recently been shown to be a potentially powerful whole-organism fingerprinting technique and is attracting interest within microbial systematics for the rapid identification of bacteria and fungi. However, while the Raman effect is so weak that only approximately 1 in 10(8) incident photons are Raman scattered (so that collection times are in the order of minutes), it can be greatly enhanced (by some 10(3)-10(6)-fold) if the molecules are attached to, or microscopically close to, a suitably roughened surface, a technique known as surface-enhanced Raman scattering (SERS). In this study, SERS, employing an aggregated silver colloid substrate, was used to analyze a collection of clinical bacterial isolates associated with urinary tract infections. While each spectrum took 10 s to collect, to acquire reproducible data, 50 spectra were collected making the spectral acquisition times per bacterium approximately 8 min. The multivariate statistical techniques of discriminant function analysis (DFA) and hierarchical cluster analysis (HCA) were applied in order to group these organisms based on their spectral fingerprints. The resultant ordination plots and dendrograms showed correct groupings for these organisms, including discrimination to strain level for a sample group of Escherichia coli, which was validated by projection of test spectra into DFA and HCA space. We believe this to be the first report showing bacterial discrimination using SERS.     

Surface-enhanced Raman optical activity on adenine in silver colloidal solution

We report the collection of Raman optical activity (ROA) spectra of adenine in silver colloidal solution, that is, surface-enhanced Raman optical activity (SEROA) using considerably shorter data acquisition times, reduced excitation power, and lower concentration, as compared to classical ROA measurements on molecules of biological interest so far reported in the literature. These improvements in experimental parameters for ROA measurements can be explained by enhanced Raman signals in the local optical fields of the silver nanoparticles and by at least 1 order of magnitude higher values for circular intensity differences (CIDs), as compared to classical ROA that has been suggested before and theoretically discussed in terms of large field gradients near a metal surface. The measured ROA effect for adenine can be understood in terms of adsorption-induced chirality in the prochiral molecules on the silver nanoparticles. Surface-enhanced Raman optical activity offers potential capabilities for sensitive, rapid, stereochemical characterization of basic building blocks of biopolymers, such as amino acids and nucleosides, as well as biologically active molecules, in particular, also for probing organization and self-assembling of such molecules on metal surfaces.