Simultaneous measurements of electronic conduction and Raman response in molecular junctions

Electronic conduction through single molecules is affected by the molecular electronic structure as well as by other information that is extremely difficult to assess, such as bonding geometry and chemical environment. The lack of an independent diagnostic technique has long hampered single-molecule conductance studies. We report simultaneous measurement of the conductance and the Raman spectra of nanoscale junctions used for single-molecule electronic experiments. Blinking and spectral diffusion in the Raman response of both p-mercaptoaniline and a fluorinated oligophenylyne ethynylene correlate in time with changes in the electronic conductance. Finite difference time domain calculations confirm that these correlations do not result from the conductance modifying the Raman enhancement. Therefore, these observations strongly imply that multimodal sensing of individual molecules is possible in these mass-producible nanostructures.     

Characterization of carbon/nitroazobenzene/titanium molecular electronic junctions with photoelectron and Raman spectroscopy

Molecular junctions consisting of nitroazobenzene (NAB) chemisorbed to a substrate of pyrolyzed photoresist film (PPF) and a top contact of vapor-deposited titanium were examined with Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). The thickness of the NAB layer varied from submonolayer to 4.5 nm, and a thin (1-3 nm) overlayer of Ti was deposited by electron beam deposition. Without Ti, the NAB surface Raman spectrum was sufficiently strong to observe previously unreported modes in the 500-1000-cm(-1) region, and the 1000-1700-cm(-1) region was sufficiently strong to observe the effects of metal deposition. Upon Ti deposition, the intensities of NAB modes associated with the nitro group decreased significantly, and the XPS indicated formation of a Ti-N bond. For the thicker NAB layers (1.9 and 4.5 nm), the intensities of the NO(2) Raman modes partially recovered over a several-day period, but they remain depressed or absent in the submonolayer sample. The results indicate a reaction between condensing Ti atoms and the terminal NO(2) group, probably to form a Ti-nitroso linkage between NAB and Ti. The result is a molecular junction with covalent bonding at both ends in the form of a C-C bond between PPF and NAB and a Ti-N bond to the top contact. The structural implications of the current results are interpreted in the context of recently reported functioning PPF/NAB/Ti molecular electronic junctions. In particular, the reaction between Ti and the nitro group appears to prevent short circuits resulting from incursion of Ti into the NAB layer.     

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.     

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.     

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.     

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.     

Complex line shapes in surface-enhanced coherent Raman spectroscopy

Peaks, dips, and intermediate line shapes have been observed in surface-enhanced coherent Raman spectroscopy. Here, we report an experimental observation of a peculiar line shape revealing both a peak and a dip as two vibrational transitions of pyridazine in the presence of aggregated gold nanoparticles. We propose a simple model based on plasmonic phase effects and quantum chemistry calculations, and compare the simulated coherent (SECARS) and incoherent (SERS) Raman signals from several complexes. Complex SECARS line shapes provide additional information compared to SERS and can be used as a tool in nanoscale sensing and spectroscopy.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Stable and unique graphitic Raman internal standard nanocapsules for surface-enhanced Raman spectroscopy quantitative analysis

Graphitic nanomaterials have unique, strong, and stable Raman vibrations that have been widely applied in chemistry and biomedicine. However, utilizing them as internal standards (ISs) to improve the accuracy of surface-enhanced Raman spectroscopy (SERS) analysis has not been attempted. Herein, we report the design of a unique IS nanostructure consisting of a large number of gold nanoparticles (AuNPs) decorated on multilayered graphitic magnetic nanocapsules (AGNs) to quantify the analyte and eliminate the problems associated with traditional ISs. The AGNs demonstrated a unique Raman band from the graphitic component, which was localized in the Raman silent region of the biomolecules, making them an ideal IS for quantitative Raman analysis without any background interference. The IS signal from the AGNs also indicated superior stability, even under harsh conditions. With the enhancement of the decorated AuNPs, the AGN nanostructures greatly improved the quantitative accuracy of SERS, in particular the exclusion of quantitative errors resulting from collection loss and non-uniform distribution of the analytes. The AGNs were further utilized for cell staining and Raman imaging, and they showed great promise for applications in biomedicine.

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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.     

Accurate concentration measurements using surface-enhanced Raman and deuterium exchanged dye pairs

Quantitative applications of surface-enhanced resonance Raman scattering (SERRS) are often limited by the reproducibility of SERRS intensities, given the difficulty of controlling analyte-substrate interactions and the associated local field enhancement. As demonstrated here, SERRS from dye molecules even within the same structural class that compete with similar substrates display distinct spectral intensities that are not proportional to analyte concentrations, which limits their use as internal standardization probes and/or for multiplex analysis. Recently, we demonstrated that isotopic variants of rhodamine 6G (R6G), namely R6G-d0 and R6G-d4, can be used for internal standards in SERRS experiments with a linear optical response from picomolar to micromolar concentrations (of total analytes). Here we extend these results by describing a straightforward method for obtaining isotopomeric pairs of other Raman active dyes by hydrogen-deuterium exchange conditions for substitution at electron rich aromatic heterocycles. Most of the known SERRS active probes can be converted into the corresponding isotopomeric molecule by this exchange method, which significantly expands the scope of the isotopic edited internal standard (IEIS) approach. The relative quantification using IEIS enables accurate, reproducible (residual standard deviation +/-2.2%) concentration measurements over a range of 200 pM to 2 muM. These studies enable easy access to a variety of isotopically substituted Raman active dyes and establish the generality of the methodology for quantitative SERRS measurements. For the first time, three rhodamine 6G isotopomers have been created and show distinct Raman spectra, demonstrating the principle of the approach for application as a multiplex technique in biomolecular detection/quantification.     

Bacillus spore classification via surface-enhanced Raman spectroscopy and principal component analysis

Surface-enhanced Raman spectroscopy (SERS) can provide rapid fingerprinting of biomaterial in a nondestructive manner. The adsorption of colloidal silver to biological material suppresses native biofluorescence while providing electromagnetic surface enhancement of the normal Raman signal. This work validates the applicability of qualitative SER spectroscopy for analysis of bacterial species by utilizing principal component analysis (PCA) to show discrimination of biological threat simulants, based upon multivariate statistical confidence limits bounding known data clusters. Gram-positive Bacillus spores (Bacillus atrophaeus, Bacillus anthracis, and Bacillus thuringiensis) are investigated along with the Gram-negative bacterium Pantoea agglomerans.