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.     

Optimizing detection sensitivity on surface-enhanced Raman scattering of transition-metal electrodes with confocal Raman microscopy

Some points on how to improve the detection sensitivity of confocal Raman microscopy for the study of surface-enhanced Raman scattering (SERS) of transition-metal electrodes are discussed, including the careful design of the spectroelectrochemical cell, proper selection of the thickness of the solution layer, the binning of charge-coupled device (CCD) pixels, and appropriate setting of the notch filter. Various roughening methods for the Pt, Rh, Fe, Co, and Ni electrode surfaces have been introduced in order to obtain SERS-active surfaces. It has been shown that the appropriate roughening procedure and the optimizing performance of the confocal Raman microscope are the two most important factors to directly generate and observe SERS on net transition-metal electrodes.     

Chemical imaging of iron oxides and oxyhydroxides using near infrared Raman imaging microscopy

Abstract

A new method of identifying and mapping the distribution of both iron oxides and oxyhydroxides using near infrared Raman imaging microscopy NIRIM is reported. This technique offers an important alternative to conventional spectroscopic techniques that provide spatially averaged data. The NIRIM instrument used for these studies combines fibre bundle image compression hardware and multivariate signal processing software to identify and map different iron minerals and corrosion products. The NIRIM images clearly distinguish hematite, magnetite, wustite, goethite, and lepidocrocite microstructures. The first chemical maps of naturally occurring iron minerals and corroded steel surfaces obtained using NIRIM spectral images, classified with the aid of a library of pure compound spectra, are presented.     

Detection of alkaline phosphatase using surface-enhanced Raman spectroscopy

A new approach was developed to detect the activity of alkaline phosphatase (ALP) enzyme at ultralow concentrations using a surface-enhanced Raman scattering (SERS) technique. The approach is based on the use of gold nanoparticles as a SERS material whereas 5-bromo-4-chloro-3-indolyl phosphate (BCIP) is used as a substrate of ALP. The enzymatic hydrolysis of BCIP led to the formation of indigo dye derivatives, which were found to be highly SERS active. For the first time, we were able to detect ALP at a concentration of approximately 4 x 10(-15) M or at single-molecule levels when ALP was incubated with BCIP for 1 h in the Tris-HCl buffer. The same technique also was successfully employed to detect surface-immobilized avidin, and a detection limit of 10 ng/mL was achieved. This new technique allows the detection of both free and labeled ALP as a Raman probe in enzyme immunoassays, immunoblotting, and DNA hybridization assays at ultralow concentrations.     

In situ monitoring of adipogenesis with human-adipose-derived stem cells using surface-enhanced Raman spectroscopy

Methods capable of nondestructively collecting high-quality, real-time chemical information from living human stem cells are of increasing importance given the escalating relevance of stem cells in therapeutic and regenerative medicines. Raman spectroscopy is one such technique that can nondestructively collect real-time chemical information. Living cells uptake gold nanoparticles and transport these particles through an endosomal pathway. Once inside the endosome, nanoparticles aggregate into clusters that give rise to large spectroscopic enhancements that can be used to elucidate local chemical environments through the use of surface-enhanced Raman spectroscopy. This report uses 40-nm colloidal gold nanoparticles to create volumes of surface-enhanced Raman scattering (SERS) within living human-adipose-derived adult stem cells enabling molecular information to be monitored. We exploit this method to spectroscopically observe chemical changes that occur during the adipogenic differentiation of human-adipose-derived stem cells over a period of 22 days. It is shown that intracellular SERS is able to detect the production of lipids as little as one day after the onset of adipogenesis and that a complex interplay between lipids, proteins, and chemical messengers can be observed shortly thereafter. After 22 days of differentiation, the cells show visible and spectroscopic indications of completed adipogenesis yet still share spectral features common to the progenitor stem cells.     

Identification of organic materials in historic oil paintings using correlated extractionless surface-enhanced Raman scattering and fluorescence microscopy

A novel spectroscopic approach, correlated surface-enhanced Raman scattering (SERS) and fluorescence microscopy, is used to identify organic materials in two 18th century oil paintings. The vibrational fingerprint of analyte molecules is revealed using SERS, and corresponding fluorescence measurements provide a probe of local environment as well as an inherent capability to verify material identification. Correlated SERS and fluorescence measurements are performed directly on single pigment particles obtained from historic oil paintings with Ag colloids as the enhancing substrate. We demonstrate the first extractionless nonhydrolysis SERS study of oil paint as well as the potential of correlated SERS and fluorescence microscopy studies for the simultaneous identification of organic colorants and binding media in historic oil paintings.     

Profiling an electrospray plume using surface-enhanced Raman spectroscopy

We report the use of silver nanoparticles to obtain surface-enhanced Raman spectra of Crystal Violet in an electrospray plume. Surface enhancement allowed detection at low concentrations with the high specificity afforded by vibrational spectroscopy. SERS spectra were used to obtain an axial concentration profile closely matching that obtained in previous fluorescence experiments. SERS can provide more analyte structural information than has been obtainable from fluorescence studies of the plume.     

Spectroscopic tags using dye-embedded nanoparticles and surface-enhanced Raman scattering

Surface-enhanced Raman scattering is capable of providing rich vibrational information at the level of single molecules and single nanoparticles, but the practical applications of this enormous enhancement effect are still a challenge. Here we report a new class of dye-embedded core-shell nanoparticles that are highly efficient for surface Raman enhancement and could be used as spectroscopic tags for multiplexed detection and spectroscopy. The core-shell particles contain a metallic core for optical enhancement, a reporter molecule for spectroscopic signature, and an encapsulating silica shell for protection and conjugation. A surprising finding is that organic molecules with an isothiocyanate (-N=C=S) group or multiple sulfur atoms are compatible with silica encapsulation. In comparison with fluorescent dyes and quantum dots, enhanced Raman probes contain a built-in mechanism for signal amplification and provide rich spectroscopic information under ambient experimental conditions.     

Lactate and sequential lactate-glucose sensing using surface-enhanced Raman spectroscopy

Lactate production under anaerobic conditions is indicative of human performance levels, fatigue, and hydration. Elevated lactate levels result from several medical conditions including congestive heart failure, hypoxia, and diabetic ketoacidosis. Real-time detection of lactate can therefore be useful for monitoring these medical conditions, posttrauma situations, and in evaluating the physical condition of a person engaged in strenuous activity. This paper represents a proof-of-concept demonstration of a lactate sensor based on surface-enhanced Raman spectroscopy (SERS). Furthermore, it points the direction toward a multianalyte sensing platform. A mixed decanethiol/mercaptohexanol partition layer is used herein to demonstrate SERS lactate sensing. The reversibility of the sensor surface is characterized by exposing it alternately to aqueous lactate solutions and buffer without lactate. The partitioning and departitioning time constants were both found to be approximately 30 s. In addition, physiological lactate levels (i.e., 6-240 mg/dL) were quantified in phosphate-buffered saline medium using multivariate analysis with a root-mean-square error of prediction of 39.6 mg/dL. Finally, reversibility was tested for sequential glucose and lactate exposures. Complete partitioning and departitioning of both analytes was demonstrated.     

One- and two-photon excited optical ph probing for cells using surface-enhanced Raman and hyper-Raman nanosensors

We demonstrate spatially resolved probing and imaging of pH in live cells by mobile and biocompatible nanosensors using surface-enhanced Raman scattering (SERS) of 4-mercaptobenzoic acid (pMBA) on gold nanoaggregates. Moreover, we also show that this concept of pH nanosensors can be extended to two-photon excitation by using surface-enhanced hyper-Raman scattering (SEHRS). In addition to the advantages of two-photon excitation, the SEHRS sensor enables measurements over a wide pH range without the use of multiple probes.     

Single-molecule detection using surface-enhanced resonance Raman scattering and Langmuir-Blodgett monolayers

The Langmuir-Blodgett (LB) technique has been used to obtain spatially resolved surface-enhanced resonance Raman scattering (SERRS) spectra of single dye molecules dispersed in the matrix of a fatty acid. The experimental results presented here mimic the original electrochemical surface-enhanced Raman scattering (SERS) work where the background bulk water did not interfere with the detection of the SERS signal of molecules adsorbed onto the rough silver electrode. LB monolayers of the dye in fatty acid have been fabricated on silver island films with a concentration, in average, of one probe molecule per micrometer square. The properties of single-molecule spectroscopy were investigated using micro-Raman including mapping and global images. Blinking of the SERRS signal was also observed.     

Highly controlled surface-enhanced Raman scattering chips using nanoengineered gold blocks

Well defined gold nanostructures of various sizes are fabricated on glass substrates using high-resolution electron-beam lithography/lift-off techniques and detailed surface-enhanced Raman scattering (SERS) properties of crystal violet molecules are studied in order to elucidate electromagnetic (EM) field enhancement effects on the fabricated structures. SERS measurements are performed with high reproducibility using in situ Raman microspectroscopy in aqueous solution. An analysis based on EM theory is performed using field-enhancement factors obtained from finite-difference time-domain (FDTD) simulations and the analysis reproduces experimental results very well. It is noteworthy, furthermore, that the proposed analytic method of EM effects on SERS allows the estimate of the ideal local temperature of gold nanostructures by canceling out the difference in EM field factors at Stokes and anti-Stokes Raman scattering wavelengths. Thus, these experimental results demonstrate that quantitative analysis based on EM theory can be obtained using highly controlled gold nanostructures for SERS measurements with high reproducibility, a result that is promising for the construction of a SERS analysis chip. Although no SERS chip reported so far has been usable for quantitative analysis, this study opens the door for construction of a quantitative SERS chip.     

Ag nanorod arrays tailored for surface-enhanced Raman imaging in the near-infrared region

Using a dynamic oblique angle deposition technique, we demonstrate the direct formation of Ag nanorods with quasi-parallel major axes on a template layer of oxide having a strongly anisotropic surface morphology. The optical properties of the nanorods are tuned by varying the deposition conditions without any pre-?or post-treatment, and the resulting Ag nanorod arrays exhibit high surface-enhanced Raman scattering (SERS) activity. In addition to high macroscopic uniformity over a large area, our nanorod arrays contain a high density of isolated nanorods. Using the optimum Ag nanorod arrays, the SERS imaging of the microdroplets of a rhodamine 6G solution is successfully demonstrated. The space resolution of the imaging is of the order of at least a few μm. These features are suitable for the SERS imaging of biomaterials.     

Detection and identification of aqueous saccharides by using surface-enhanced Raman spectroscopy

The sensitive detection and characterization of carbohydrates by means of a strategy based on surface-enhanced Raman spectroscopy is demonstrated. Spectra are obtained after injecting a small amount of saccharide solution onto a roughened silver substrate, with subsequent deposition of silver colloid. The sensitivity achieved by this two-step approach enables high-quality Raman spectra to be obtained for small amounts of aqueous saccharides (5 microL of a 10(-2) M solution) utilizing minimal laser power and small signal acquisition times (a few seconds). Spectral "fingerprints" obtained for seven structurally similar monosaccharides demonstrate clearly an effective means by which each sugar can be identified. The application to more complex analyses is demonstrated for monosaccharide mixtures and a disaccharide, whereby the SERS fingerprints aid in the determination of components.     

Ultrasensitive detection and characterization of posttranslational modifications using surface-enhanced Raman spectroscopy

Posttranslational modification (PTM) of proteins is likely to be the most common mechanism of altering the expression of genetic information. It is essential to characterize PTMs to establish a complete understanding of the activities of proteins. Here, we present a sensitive detection method using surface-enhanced Raman spectroscopy (SERS) that can detect PTMs from as little as zeptomoles of peptide. We demonstrate, using model peptides, the ability of SERS to detect a variety of protein modifications, such as acetylation, trimethylation, phosphorylation, and ubiquitination. In addition, we show the capability to obtain positional information for modifications such as trimethylation and phosphorylation using SERS and wavelet decomposition data analysis techniques. We further show that it is possible to apply SERS to detect PTMs from biological samples such as histones. We envision that this detection method might be a valuable technique that is complementary to mass spectrometry in obtaining orthogonal chemical and modification-specific information from biological samples at sensitive levels.     

Quantitative characterization of individual microdroplets using surface-enhanced resonance Raman scattering spectroscopy

Surface-enhanced resonance Raman scattering (SERRS) spectroscopy is a highly sensitive optical technique capable of detecting multiple analytes rapidly and simultaneously. There is significant interest in SERRS detection in micro- and nanotechnologies, as it can be used to detect extremely low analyte concentrations in small volumes of fluids, particularly in microfluidic systems. There is also rapidly growing interest in the field of microdroplets, which promises to offer the analyst many potential advantages over existing technologies for both design and control of microfluidic assays. While there have been rapid advances in both fields in recent years, the literature on SERRS-based detection of individual microdroplets remains lacking. In this paper, we demonstrate the ability to quantitatively detect multiple variable analyte concentrations from within individual microdroplets in real time using SERRS spectroscopy. We also demonstrate the use of a programmable pump control algorithm to generate concentration gradients across a chain of droplets.     

Analysis of thin film coatings on poly(ethylene terephthalate) by confocal Raman microscopy and surface-enhanced Raman scattering

The characterization of thin coatings on polymers such as poly(ethylene terephthalate) (PET) is required in order to study chemical composition and coating continuity. Two different methods of applying Raman spectroscopy for this purpose are compared in this paper. Using confocal Raman microscopy, thick coatings (> 10 microns) are relatively easily identified; however, the Raman scattering from the acrylic coatings commonly used is much weaker than that of PET and consequently, there is a background due to the substrate. Thin acrylic coatings (< 1 micron) usually cannot be detected. Surface-enhanced Raman scattering (SERS) of uncoated PET gives intense signals and if the spectra are taken from the metal-coated side, there is no evidence of the underlying Raman scattering from the bulk. Acrylic coatings do not give sufficiently strong or reproducible SERS to be reliably identified, but even thin (20 nm) coatings completely block the SERS from the substrate. Only where gaps appear in the coating is the SERS of the underlying PET seen. To detect a positive signal from the coating, SERS active labels were incorporated into the acrylic at low concentrations either as a physical mixture or as reactive co-monomers. This uniquely labels the coating and allows detection and, in principle, mapping of the coverage. Thus, for thick (> > 1 micron) coatings, normal Raman spectroscopy is an effective technique for detecting the presence of the surface coating. However, it is ineffective with thin (< 1 micron) coatings, and SERS alone only indicates where the coating is incomplete or defective. However, when a SERS label is added, spectra can be detected from very thin coatings (20 nm). The concentration of the labels is sufficiently low for the coating to remain colorless.