Surface-enhanced Raman spectroscopy of soft-landed polyatomic ions and molecules

Surface-enhanced Raman spectroscopy (SERS) was used to detect and characterize polyatomic cations and molecules that were electrosprayed into the gas phase and soft-landed in vacuum on plasma-treated silver substrates. Organic dyes such as crystal violet and Rhodamine B, the nucleobase cytosine, and nucleosides cytidine and 2'-deoxycytidine were immobilized by soft landing on plasma-treated metal surfaces at kinetic energies ranging from near thermal to 200 eV. While enhancing Raman scattering 10(5)-10(6)-fold, the metal surface effectively quenches the fluorescence that does not interfere with the Raman spectra. SERS spectra from submonolayer amounts of soft-landed compounds were sufficiently intense and reproducible to allow identification of Raman active vibrational modes for structure assignment. Soft-landed species appear to be microsolvated on the surface and bound via ion pairing or pi-complexation to the Ag atoms and ions in the surface oxide layer. Comparison of spectra from soft-landed and solution samples indicates that the molecules survive soft landing without significant chemical damage even when they strike the surface at hyperthermal collision energies.     

Surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS): a review of applications

Surface-enhanced Raman scattering (SERS) and surface-enhanced resonance Raman scattering (SERRS) can provide positive identification of an analyte or an analyte mixture with high sensitivity and selectivity. Better understanding of the theory and advances in the understanding of the practice have led to the development of practical applications in which the unique advantages of SERS/SERRS have been used to provide effective solutions to difficult analytical problems. This review presents a basic theory and illustrates the way in which SERS/SERRS has been developed for practical use.     

Separation of nanoparticles by gel electrophoresis according to size and shape

We demonstrate the separation of gold and silver nanoparticles according to their size and shape by agarose gel electrophoresis after coating them with a charged polymer layer. The separation is monitored optically using the size- and shape-dependent plasmon resonance of noble metal particles and confirmed by transmission electron microscopy (TEM). Electrophoretic mobilities are quantitatively explained by a model based on the Henry formula, providing a theoretical framework for predicting gel mobilities of polymer coated nanoparticles.     

Surface-enhanced Raman spectroscopy for in situ measurements of signaling molecules (autoinducers) relevant to bacteria quorum sensing

Autoinducer (AI) molecules are used by quorum sensing (QS) bacteria to communicate information about their environment and are critical to their ability to coordinate certain physiological activities. Studying how these organisms react to environmental stresses could provide insight into methods to control these activities. To this end, we are investigating spectroscopic methods of analysis that allow in situ measurements of these AI molecules under different environmental conditions. We found that for one class of AIs, N-acyl-homoserine lactones (AHLs), surface-enhanced Raman spectroscopy (SERS) is a method capable of performing such measurements in situ. SERS spectra of seven different AHLs with acyl chain lengths from 4 to 12 carbons were collected for the first time using Ag colloidal nanoparticles synthesized via both citrate and borohydride reduction methods. Strong SERS spectra were obtained in as little as 10 seconds for 80 microM solutions of AI that exhibited the strongest SERS response, whereas 20 seconds was typical for most AI SERS spectra collected during this study. Although all spectra were similar, significant differences were detected in the SERS spectra of C4-AHL and 3-oxo-C6-AHL and more subtle differences were noted between all AHLs. Initial results indicate a detection limit of approximately 10(-6)M for C6-AHL, which is within the limits of biologically relevant concentrations of AI molecules (nM-microM). Based on these results, the SERS method shows promise for monitoring AI molecule concentrations in situ, within biofilms containing QS bacteria. This new capability offers the possibility to "listen in" on chemical communications between bacteria in their natural environment as that environment is stressed.     

Surface-enhanced Raman spectroscopy of self-assembled monolayers: sandwich architecture and nanoparticle shape dependence

Surface enhanced Raman scattering (SERS) spectra of 4-mercaptobenzoic acid (4-MBA) self-assembled monolayers (SAMs) on gold substrates is presented for SAMs onto which gold nanoparticles of various shapes have been electrostatically immobilized. SERS spectra of 4-MBA SAMs are enhanced in the presence of immobilized gold nanocrystals by a factor of 10(7)-10(9) relative to 4-MBA in solution. Large enhancement factors are a likely result of plasmon coupling between the nanoparticles (localized surface plasmon) and the smooth gold substrate (surface plasmon polariton), creating large localized electromagnetic fields at their interface, where 4-MBA molecules reside in this sandwich architecture. Moreover, enhancement factors depend on nanoparticle shape and vary by a factor of 10(2). This SERS geometry offers large surface enhancements for molecules adsorbed onto planar substrates and could be quite useful for determining chemical information for poor Raman scatterers from assays on 2-D substrates.     

Surface-enhanced Raman scattering spectroscopy as a sensitive and selective technique for the detection of folic acid in water and human serum

Surface-enhanced Raman scattering (SERS) is shown to give linear and sensitive concentration-dependent detection of folic acid using silver nanoparticles created via ethylene-diaminetetraacetic acid (EDTA) reduction. Optical detection by SERS overcomes the primary limitation of photodissociation encountered during the application of other shorter wavelength ultraviolet (UV)/near-UV techniques such as fluorescence based microscopy. The SERS approach in water-based samples was demonstrated and optimized using several longer wavelengths of excitation (514.5, 632.8, and 785 nm). Excitation in the green (514.5 nm) was found to achieve the best balance between photodissociation and SERS efficiency. Linear concentration dependence was observed in the range of 0.018 to 1 microM. The importance of folic acid in a clinical setting and the potential applications of this technique in a biological environment are highlighted. We demonstrate the potential to transfer this technique to real biological samples by the detection of folic acid in human serum samples by SERS.     

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.     

Identification and characterization of artists' red dyes and their mixtures by surface-enhanced Raman spectroscopy

Silver film over nanospheres (AgFONs) were successfully employed as surface-enhanced Raman spectroscopy (SERS) substrates to characterize several artists' red dyes including: alizarin, purpurin, carminic acid, cochineal, and lac dye. Spectra were collected on sample volumes (1 x 10(-6) M or 15 ng/microL) similar to those that would be found in a museum setting and were found to be higher in resolution and consistency than those collected on silver island films (AgIFs). In fact, to the best of the authors' knowledge, this work presents the highest resolution spectrum of the artists' material cochineal to date. In order to determine an optimized SERS system for dye identification, experiments were conducted in which laser excitation wavelengths were matched with correlating AgFON localized surface plasmon resonance (LSPR) maxima. Enhancements of approximately two orders of magnitude were seen when resonance SERS conditions were met in comparison to non-resonance SERS conditions. Finally, because most samples collected in a museum contain multiple dyestuffs, AgFONs were employed to simultaneously identify individual dyes within several dye mixtures. These results indicate that AgFONs have great potential to be used to identify not only real artwork samples containing a single dye but also samples containing dyes mixtures.     

Specific base recognition of oligodeoxynucleotides by capillary affinity gel electrophoresis using polyacrylamide-poly(9-vinyladenine) conjugated gel.

Poly(9-vinyladenine) was synthesized and utilized as an affinity macroligand entrapped within the gel matrix. Base-specific separation of oligodeoxynucleotides was achieved with high resolution and high speed by electrophoresis, using capillaries filled with conjugated polyacrylamide-poly(9-vinyladenine) gel. Oligothymidylic acids were selectively separated from the mixture of oligothymidylic and oligodeoxyadenylic acids by utilizing a specific hydrogen bonding between poly(9-vinyladenine) and oligothymidylic acids. Migration time and resolution of oligodeoxynucleotides were influenced by several parameters, such as the size of poly(9-vinyladenine), capillary temperature, and concentrations of poly(9-vinyladenine) and urea. Some guidelines are presented, based on the theoretical formulation of the effect of these parameters, in order to find optimum electrophoretic conditions. Analytical capillary affinity gel electrophoresis was developed for the selective and sensitive base recognition of oligodeoxynucleotides with efficiencies as high as several 10(6) plates/m by using a urea-gel capillary with poly(9-vinyladenine) and temperature-programming.     

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.     

Resonance Raman spectroscopy characterization of single-wall carbon nanotube separation by their metallicity and diameter

Several techniques were recently reported for the bulk separation of metallic (M) and semiconducting (S) single wall carbon nanotubes (SWNTs), using optical absorption and resonance Raman spectroscopy (RRS) as a proof of the separation. In the present work, we develop a method for the quantitative evaluation of the M to S separation ratio, and also for the SWNT diameter selectivity of the separation process, based on RRS. The relative changes in the integrated intensities of the radial-breathing mode (RBM) features, with respect to the starting material, yield the diameter probability distribution functions for M and S SWNTs in the separated fractions, accounting for the different resonance conditions of individual SWNTs, while the diameter distribution of the starting material is obtained following the fitting procedure developed by Kuzmany and coworkers. Features other than the RBM are generally less effective for characterization of the separation process for SWNTs.     

Multidimensional analysis of poly(ethylene glycols) by size exclusion chromatography and dynamic surface tension detection

Substantial improvements in a multidimensional dynamic surface tension detector (DSTD) are presented. Rapid, online calibration and measurement of the dynamic surface tension for high-performance liquid chromatography separations is achieved. Dynamic surface tension is determined by measuring the differential pressure across the liquid-air interface of repeatedly growing and detaching drops. Continuous surface tension measurement throughout the entire drop growth (50 ms to 2 s) is achieved, for each eluting drop, providing insight into the kinetic behavior of molecular orientation processes at the liquid-air interface. Three-dimensional data are obtained, with surface tension first converted to surface pressure, which is plotted as a function of elution time axis versus drop time axis. Two key innovations will be reported. First, a novel calibration procedure is described and implemented. Differential pressure signals from three drops (mobile phase, standard in mobile phase, and analyte in mobile phase) are utilized to make the dynamic surface tension measurement, thereby eliminating the need for optical imaging, and viscosity and hydrostatic pressure corrections, as required by other methods. Only pressure signals from one mobile-phase drop and one standard drop pressure signal are required, while the analyte drop pressure signal is measured along the chromatographic time axis. Second, corrections for drop elongation are not required, because the drops are precisely detached by an air burst actuation method in a regime were the surface tension forces significantly dominate gravitational forces. Drops that would fall with a volume of approximately 10 microL due to gravity are precisely and repeatedly detached earlier at a volume of 2 microL. The sensitivity and unique selectivity of the DSTD opens up new possibilities in the analysis of small molecular weight polymers of varying degrees of surface activity, as illustrated for the size-exclusion chromatography analyses of complex poly(ethylene glycol) (PEG) samples. Using partial least squares for data analysis, polydispersity of complex PEG samples is determined at a relative precision of approximately 1%.     

Effect of metal ions on the molecular weight distribution of humic substances derived from municipal compost: ultrafiltration and size exclusion chromatography with spectrophotometric and inductively coupled plasma-MS detection

The effect of metal ions (Co, Cu, Ni, Pb, Zn) on the molecular weight distribution of humic substances (HSs) obtained from compost is studied. We believe this is the first of this type of study applied in this way to humic substances. Size exclusion chromatography is coupled with two on-line detection systems (spectrophotometric and ICPMS) to study the binding of metal ions by humic substances leached from compost. ICPMS provided highly specific, sensitive, and multielement analytical information that enabled obtaining direct experimental evidence for the participation of metal ions in molecular size distributions of humic compounds. The compost extract or its high molecular weight fraction (>5,000) was put in contact with EDTA or citrate ions, thereby competing with HSs for binding metals. The experiments were carried out by varying the pH maintained by Tris-HCl or CAPS buffer (pH 8.0 and 10.3) and keeping the ionic strength constant. The elution profile of humic substances using UV/ visible detection was compared with those from ICPMS detection of Co, Cu, Ni, Pb, and Zn in the same chromatographic runs. The results obtained suggested that both bridging between small molecules and complexation/ chelation by individual molecules are involved in metal ion binding to humic substances. The use of ICPMS to study the role of metal ions in aggregation/disassociation of humic substances proposed in this work is promising. Coupling element-specific detection with SEC or other separation systems allows better understanding of the mobility and bioaccessibility of elemental species in the environment and further elucidation of the dissolved humic structure.     

Part III: Surface-enhanced Raman scattering of amino acids and their homodipeptide monolayers deposited onto colloidal gold surface

Surface-enhanced Raman scattering (SERS) spectra were measured for monolayers of various amino acids: L-methionine (Met), L-cysteine (Cys), L-glycine (Gly), L-leucine (Leu), L-phenylalanine (Phe), and L-proline (Pro) and their homodipeptides (Met-Met, Cys-Cys, Gly-Gly, Leu-Leu, Phe-Phe, and Pro-Pro) deposited onto a colloidal gold surface. Orientation of amino acids and their homodipeptides, as well as specific-competitive interactions of their functional groups with the gold surface, were predicted by detailed spectral analysis of the obtained SERS spectra. The analysis performed allowed us to propose a particular surface geometry for each amino acid and homodipeptide on the gold surface. In addition, we compared the structures of these molecules adsorbed on colloidal gold and silver surfaces.     

Sensitive detection of selenoproteins in gel electrophoresis by high repetition rate femtosecond laser ablation-inductively coupled plasma mass spectrometry

A laser ablation-ICPMS method using an infrared (1030 nm), low-energy (39 microJ/pulse), high repetition rate (10 kHz), femtosecond laser was developed to improve the sensitivity of detection of heteroatom-containing proteins in 1D polyacrylamide gels. A 2-mm-wide lane was ablated by ultrafast (10 cm s(-1)) back-and-forth movement of a 20-microm laser beam parallel to the protein bands while the gel advanced perpendicularly. This procedure resulted in a considerable increase in detection sensitivity (>40-fold) compared to the nanosecond 266-nm laser ablation-ICPMS, mainly because of the much larger amount of ablated material introduced into the plasma on the time scale of the dwell time of the mass spectrometer. The method was applied to the specific detection in the gel of formate dehydrogenase expressed in Escherichia coli and of selenoproteins in Desulfococcus multivorans with detection limits at the low-femtomolar levels.     

Differentiation of microcystin, nodularin, and their component amino acids by drop-coating deposition Raman spectroscopy

Raman spectra of microcystin-LR (MC-LR), MC-RR, MC-LA, MC-LF, MC-LY, MC-LW, MC-YR, and nodularin collected by drop-coating deposition Raman (DCDR) spectroscopy are sufficiently unique for variant identification. Amino acid spectra of L-phenylalanine, L-leucine, L-alanine, D-alanine, L-glutamic acid, L-arginine, L-tryptophan, L-tyrosine, and N-methyl-D-aspartic acid were collected in crystalline, DCDR, and aqueous forms to aid in cyanotoxin Raman peak assignments. Both peak ratio analysis and principal component analysis (PCA) properly classified 72 DCDR spectra belonging to the eight toxins. Loading plots for the first three principal components (PCs) most heavily weighted the peaks highlighted in the peak ratio analysis, specifically the 760 cm(-1) tryptophan peak, 853 cm(-1) tyrosine peak, and 1006 cm(-1) phenylalanine peak. Peak ratio analyses may be preferred under some circumstances because of the ease and speed with which the ratios can be computed, even by untrained lab technicians. A set of rules was created to mathematically classify toxins using the peak ratios. DCDR methods hold great potential for future application in routine monitoring because portable and hand-held Raman spectrometers are commercially available, DCDR spectra can be collected in seconds for biomolecule mixtures as well as samples containing impurities, and the method requires far fewer consumables than conventional cyanotoxin detection methods.     

Synthesis and characterisation of cellulose/silica hybrids obtained by heteropoly acid catalysed sol–gel process

Cellulose/silica hybrids (CSHs) were synthesized by a sol–gel method using eucalyptus bleached kraft pulp as cellulose source and tetraethyl orthosilicate (TEOS) as the silica precursor in the presence of heteropoly acids (HPAs) as catalysts. HPAs, and especially tungstophosphoric acid H₃PW₁₂O₄₀, showed better catalytic efficiency than conventional mineral acids. Silica was deposited on fibres in the form of a thin film or mesoparticles as revealed by SEM/EDS and AFM analyses. Roughly 40–60% of silica was incorporated into cellulosic material considerably diminishing its hydrophilicity and improving thermal stability. CSHs were structurally characterised by FTIR, ¹³C and ²⁹Si solid state NMR. It was suggested that proportions of Q², Q³ and Q⁴ structures in silica counterpart depended on the synthesis conditions (H₂O/TEOS molar ratio and catalyst concentration among others). A clear relationship between the thermal stability of CSH and the degree of silica crosslinking in hybrids has been observed.     

On-line coupling of size exclusion chromatography with electrospray ionization-tandem mass spectrometry for the analysis of aquatic fulvic and humic acids

A method was developed for the analysis of humic and fulvic acids by size-exclusion chromatography-electrospray ionization-tandem mass spectrometry using a completely volatile eluent. Humic and fulvic acids were separated into three peaks. These fractions occupied different mass ranges and showed differences in the fine structure of their mass spectra. The low-molecular-weight (LMW) fraction of fulvic acids is most sensitively determined by ESI-MS, and it appears that previous results obtained by infusion-ESI-MS were primarily determined by this fulvic acid fraction. The average molecular weight of this fractions turned out to be lower than that reported from infusion-ESI-MS measurements. Its scan spectra and the product ion spectra of some of its molecular anions perfectly match those previously obtained from whole fulvic acid mixtures. Obviously, a class of well-defined polycarboxylated molecules exist that occurs in all fulvic acid fractions thus far investigated. With decreasing elution time and increasing molecular weight, detection by ESI-MS loses sensitivity as compared to the parallel UV recording, and the fine structure of the scan spectra becomes increasingly uniform for both fulvic and humic acids. The average molecular weight of the HMW fraction exceeds those values calculated from infusion experiments. Scan spectra and product ion spectra of the high-molecular-weight (HMW) fraction of both the humic and the fulvic acids suggest that the HMW fraction consists of several subunits that originate from the LMW fraction.     

Evaluation of two different gas temperatures and their volumetric fraction from broadband N(2) coherent anti-Stokes Raman spectroscopy spectra

Technical flames often contain regions of high temperature gradients at a length scale that is smaller than or comparable with the size of the probe volume used. In these situations the coherent anti-Stokes Raman spectroscopy (CARS) signal is composed of contributions of gases of different temperatures. Here a fitting routine is presented that allows the evaluation of the different temperatures that occur in the CARS spectra by splitting the temperature mixed spectra into contributions of two main temperatures. Additionally, the volumetric fraction of both gas components with those two different temperatures can be determined. The evaluation procedure has been tested by the use of measured temperature mixed CARS spectra that have been taken in two gas samples with different gas temperatures.