Carbon nanotube-tipped endoscope for in situ intracellular surface-enhanced Raman spectroscopy

Gold nanoparticle-decorated carbon nanotubes (CNTs) are used to study intracellular environments in situ using surface-enhanced Raman spectroscopy (SERS). CNTs are decorated with gold nanoparticles and assembled onto the tips of pulled glass capillaries to form a SERS-enabled endoscope. The sub-micrometer size and high mechanical strength of the endoscope make it possible to penetrate the cell membrane for intracellular probing and remain positioned inside during lengthy SERS measurements without causing damage to the cell. Using the SERS-enabled endoscope, DNA and other biomolecules are detected in situ within the nucleus of a single human cervical carcinoma cell in a minimally invasive manner. The SERS-enabled endoscopes exhibit high selectivity and sensitivity for detecting trace amounts of analytes (≈1 pM) in biofluid environments, highlighting their capabilities as label-free, biological sensors for real-time in situ cellular diagnostics, biological detection, and pharmaceutical research.     

Discrimination of bacteria and bacteriophages by Raman spectroscopy and surface-enhanced Raman spectroscopy

Detection of pathogenic organisms in the environment presents several challenges due to the high cost and long times typically required for identification and quantification. Polymerase chain reaction (PCR) based methods are often hindered by the presence of polymerase inhibiting compounds and so direct methods of quantification that do not require enrichment or amplification are being sought. This work presents an analysis of pathogen detection using Raman spectroscopy to identify and quantify microorganisms without drying. Confocal Raman measurements of the bacterium Escherichia coli and of two bacteriophages, MS2 and PRD1, were analyzed for characteristic peaks and to estimate detection limits using traditional Raman and surface-enhanced Raman spectroscopy (SERS). MS2, PRD1, and E. coli produced differentiable Raman spectra with approximate detection limits for PRD1 and E. coli of 10(9) pfu/mL and 10(6) cells/mL, respectively. These high detection concentration limits are partly due to the small sampling volume of the confocal system but translate to quantification of as little as 100 bacteriophages to generate a reliable spectral signal. SERS increased signal intensity 10(3) fold and presented peaks that were visible using 2-second acquisitions; however, peak locations and intensities were variable, as typical with SERS. These results demonstrate that Raman spectroscopy and SERS have potential as a pathogen monitoring platform.     

New surface-enhanced Raman spectroscopy substrates via self-assembly of silver nanoparticles for perchlorate detection in water

Perchlorate (ClO4-) has recently emerged as a widespread contaminant in drinking water and groundwater supplies in the United States, and a need exists for rapid detection and monitoring of this contaminant. In this study, surface-enhanced Raman spectroscopy (SERS) was studied as a means of ClO4- detection, and new sol-gel-based SERS substrates were developed by self-assembly of silver colloidal nanoparticles with various functionalized silane reagents. These substrate materials were tailored to allow detection of ClO4- in water with improved sorptivity, stability, and sensitivity and with the ability to detect ClO4- at concentrations as low as 10(-6) M (or 100 microg/L) with good reproducibility. Similar techniques were used to fabricate capillary SERS flow cells by assembling functionalized silver nanoparticles capable of attracting ClO4- to the SERS surface or the internal wall of glass capillaries. These capillary flow cells could be readily configured to allow for in situ, nondestructive detection of ClO4- via fiber optics.     

Internal standard in surface-enhanced Raman spectroscopy

A method is presented for the use of SAM layers as internal standards for calibration in surface-enhanced Raman spectroscopy. Three cyano-containing compounds were attached to gold colloids via a metal-sulfur bond and evaluated for spectral stability and normalization capacity. The results show that the analyte, rhodamine 6G, and the internal standard signal enhancement covaried, and it was possible to quantify the analyte with PLS. The fact that the enhancing substrate was chaotic assemblies with large variation in signal enhancement shows the versatility of this method.     

Detection of anions by normal Raman spectroscopy and surface-enhanced Raman spectroscopy of cationic-coated substrates

The use of normal Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) of cationic-coated silver and gold substrates to detect polyatomic anions in aqueous environments is examined. For normal Raman spectroscopy, using near-infrared excitation, linear concentration responses were observed. Detection limits varied from 84 ppm for perchlorate to 2600 ppm for phosphate. In general, detection limits in the ppb to ppm concentration range for the polyatomic anions were achieved using cationic-coated SERS substrates. Adsorption of the polyatomic anions on the cationic-coated SERS substrates was described by a Frumkin isotherm. The SERS technique could not be used to detect dichromate, as this anion reacted with the coatings to form thiol esters. A competitive complexation method was used to evaluate the interaction of chloride ion with the cationic coatings. Hydrogen bonding and pi-pi interactions play significant roles in the selectivity of the cationic coatings.     

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.     

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.     

Determination of technetium and its speciation by surface-enhanced Raman spectroscopy

Technetium-99 (Tc) is an important radionuclide of concern, and there is a great need for its detection and speciation analysis in the environment. For the first time, we report that surface-enhanced Raman spectroscopy (SERS) is capable of detecting an inorganic radioactive anion, pertechnetate (TcO4-), at approximately 10(-7) M concentration levels. The technique also allows the detection of various species of Tc such as oxidized Tc(VII) and reduced and possibly complexed Tc(IV) species by use of gold nanoparticles as a SERS substrate. The primary Raman scattering band of Tc(VII) occurs at about 904 cm-1, whereas reduced Tc(IV) and its humic and ethylenediaminetetraacetic acid (EDTA) complexes show scattering bands at about 866 and 870 cm-1, respectively. Results also indicate that Tc(IV)-humic complexes are unstable and reoxidize to TcO4- upon exposure to oxygen. This study demonstrates that SERS could potentially offer a new tool and opportunity in studying Tc and its speciation and interactions in the environment at low concentrations.     

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.     

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.     

Borate interference in surface-enhanced Raman spectroscopy of amines.

Interference from borate is observed in surface-enhanced Raman (SER) spectra of lysine and propylamine obtained with borohydride-reduced silver colloids. Borate bands are also observed in the spectra of other basic analytes, as well as when certain variations are made in the silver colloid preparation. The relative intensities of the analyte and borate bands depend on the pH of the colloid, the extent of oxidation of the colloid surface, and the relative adsorptivities of the analyte and borate. Benzylamine adsorbs more readily than propylamine and also competes more effectively with borate for adsorption sites. On the other hand, borate virtually excludes lysine from the surface when the solution pH is greater than or equal to 8. The formation of silver oxide in basified colloids may facilitate borate adsorption. For some basic analytes, eliminating the adsorption of borate ion and the resulting spectral interference may require using alternative SERS substrates.     

Characterization of the Ag mediated surface-enhanced Raman spectroscopy of saxitoxin

The rapid detection and quantification of saxitoxin (STX) is reported using surface-enhanced Raman spectroscopy (SERS) with a colloidal hydrosol of silver nanoparticles. Under the conditions of our experiments, the limit of detection (LD) for STX using SERS is 3 nM, with a limit of quantification (LQ) of 20 nM. It is shown that the SERS method is rapid, with spectra being collected in as little as 5 seconds total integration time for a 40 nM STX sample. In order to improve the signal-to-noise ratio, SERS spectra were generally collected with a total integration time of 1 minute (6 accumulations of 10 seconds each), with no need for extensive sample work-up or substrate preparation. Based on these results, the SERS technique shows great promise for the future detection and quantification of STX molecules in aqueous solutions.     

Molecular recognition in monolayers and species detection by surface-enhanced resonance Raman spectroscopy

The current emphasis in efforts to produce systems capable of highly specific molecular recognition has produced a wide variety of compounds such as crown ethers, cryptands, cyclodextrins and other inclusion systems. A more desirable approach, and one obviating laborious organic synthesis, would be based upon a mechanism more like that seen in the in vivo antigen-antibody reaction. Sites having the capability for specific molecular recognition based on a predetermined template molecule would allow realization of systems of the desired specificity. The technique of cosorption of a silane and a surface-active molecule onto a glass surface has been comprehensively described by Sagiv and by Maoz and Sagiv and has indicated the feasibility of this approach, e.g. with surface-active dyes. In the present study, adsorbed monolayers were produced with sites based on chosen template molecules, using the Sagiv method, and the systems then reconstituted with the original template molecule as well with molecules of closely similar structure (i.e. porphyrins or chlorophylls). A high degree of recognition was evidenced, as shown by the use of surface-enhanced resonance Raman spectroscopy as the detection tool. It was also shown that chemically dissimilar species can be reconstituted into sites formed by other species, provided that the molecular shapes are compatible. The ease of resorption into performed sites is strongly dependent on the presence of amphiphilic character in the molecule re-entering a site.     

Mechanism of cellular uptake of graphene oxide studied by surface-enhanced Raman spectroscopy

The last few years have witnessed rapid development of biological and medical applications of graphene oxide (GO), such as drug/gene delivery, biosensing, and bioimaging. However, little is known about the cellular uptake mechanism and pathway of GO. In this work, surface-enhanced Raman scattering (SERS) spectroscopy is employed to investigate the cellular internalization of GO loaded with Au nanoparticles (NPs) by Ca Ski cells. The presence of Au NPs on the surface of GO enables detection of enhanced intrinsic Raman signals of GO inside the cell. The SERS results reveal that GO is distributed inhomogeneously inside the cell. Furthermore, internalization of Au-GO into Ca Ski cells is mainly via clathrin-mediated endocytosis, and is an energy-dependent process.     

Semi-quantitative analysis of gentian violet by surface-enhanced Raman spectroscopy using silver colloids

The viability of the application of surface-enhanced Raman spectroscopy (SERS) to the semi-quantitative analysis of the triphenylmethane dye gentian violet was examined by using activated borohydride-reduced silver colloids. Raman and SERS spectra of aqueous solutions of gentian violet at different pH values were acquired for the first time and equally intense SERS signals were obtained at both acidic and alkaline pH values. Two maxima intensities observed in the pH profile revealed the presence of different ionization states of the dye. The pH conditions for SERS were optimized over the pH range 1 to 12 and the biggest enhancement for SERS of this charged dye was found to be at pH 2.0; thus, this condition was used for semi-quantitative analysis. A good linear correlation was observed for the dependence of the signal intensities of the SERS bands at 1620 cm(-1) (R = 0.999) and 1370 cm(-1) (R = 0.952) on dye concentration over the range 10(-6) to 10(-4) mol/L, using laser excitation at 514.5 nm. At concentrations of dye above 10(-2) mol/L, the concentration dependence of the SERS signals is nonlinear. This is explained as due to the precipitation of metallic silver as well as due to saturation caused by complete coverage of the SERS substrate. A series of intensities of the band at 1620 cm(-1) measured from dye molecules proved that the single-molecule limit of gentian violet is attained at the concentration of 10(-9) mol/L.     

First steps of in situ surface-enhanced Raman scattering during shipboard experiments

It is shown that the surface-enhanced Raman scattering (SERS) technique can be applied to detect organic molecules during in situ experiments. To this purpose, we used trans-1,2-bis(4-pyridyl)ethylene (BPE) as a target molecule. Adsorbed on the SERS chemosensor surface and excited under laser, the vibration modes of the molecules can be identified. SERS chemosensors are based on quartz substrates functionalized by silanization and partially coated with gold nanoparticles. SERS measurements during shipboard experiments were made with a home-made in situ Raman spectrometer connected to a marinized micro-fluidic system. The device was designed to host chemosensors in order to ensure measurements with a flow cell. A theoretical limit of detection was estimated in the range of picomolar (pM) concentrations based on Freundlich isotherm calculations.     

Detection of drugs of abuse in saliva by surface-enhanced Raman spectroscopy (SERS)

Eighty drugs of abuse and metabolites were successfully measured by surface-enhanced Raman spectroscopy (SERS) using gold- and silver-doped sol-gels immobilized in glass capillaries. A method was developed that provided consistent detection of 50 ppb cocaine in saliva in a focused study. This general method was successfully applied to the detection of a number of additional drugs in saliva, such as amphetamine, diazepam, and methadone.     

Adsorption of a cholesteric liquid crystal polyester on silver nanoparticles studied by surface-enhanced Raman scattering and micro Raman spectroscopy

A Raman study of the adsorption of thermotropic cholesteric liquid crystal polyester PTOBDME ([C(34)H(36)O(8)](n)) on Ag surfaces is presented in this work. The affinity and adsorption mechanism of this polymer was tested on Ag metal colloids and on Ag films obtained by direct immobilization of the colloidal nanoparticles. We have first studied the structure of PTOBDME suspended in several solvents in order to identify the Raman bands used as structural markers. The adsorption of the polymer leads to a deep conformational change involving both the main chain and the aliphatic side chain. The interaction of polymers like PTOBDME with metals could be interesting in the formation of functionalized surfaces, providing them with specific physicochemical properties with possible applications in recognition phenomena, which can be easily characterized by Raman spectroscopy.