Preparative soft and reactive landing of multiply charged protein ions on a plasma-treated metal surface

Soft landing on a plasma-treated metal surface of multiply protonated protein ions from the gas phase results in a substantial retention of protein function, as demonstrated for trypsin and streptavidin. The majority of trypsin ions soft-landed at hyperthermal kinetic energies are undamaged and retain 72-98% of enzymatic activity after being washed into solution. A small fraction of trypsin ions that were landed at nominal kinetic energies of 130-200 eV remain tethered to the surface and show approximately 50% enzymatic activity. The streptavidin tetramer is found to dissociate to monomer units upon multiple charging in electrospray. The majority of soft-landed monomers can be washed into solution where they show affinity to biotin. The layer of streptavidin monomer that is immobilized on the surface can be detected if fluorescence-tagged and retains the ability to reversibly bind biotin. A mechanism is proposed to explain nondestructive protein ion discharge on the surface that considers proton migration from the soft-landed cations to the metal oxide layer and metal ion reduction by electron transfer from the bulk metal.     

Preparative soft and reactive landing of gas-phase ions on plasma-treated metal surfaces

Soft landing of singly charged gas-phase ions on dry metal surfaces that were pretreated in situ by oxygen plasma results in 0.1-2% total yields of recovered intact compounds. Lysine, peptides, crystal violet dye, and a biotin conjugate are found to survive soft landing of hyperthermal ions of up to 50-eV kinetic energy. Soft landing at 40-50-eV ion kinetic energies of a fluorescence-labeled biotin conjugate results in an immobilized fraction that cannot be washed from the surface and is found to contain an intact biotin moiety. The present results represent an approximately 10(4) fold improvement in soft-landing efficiency and indicate that plasma-treated metal surfaces can be useful for preparative separation of organic and biological molecules by mass spectrometry. The substantial improvement in soft-landing yields results from a high transmission of electrosprayed ions into the vacuum system, efficient and nondestructive discharge of ions on the metal oxide surface, and facile analyte recovery in the absence of a matrix.     

Vibrational spectroscopy and mass spectrometry for characterization of soft landed polyatomic molecules

We report implementation of two powerful characterization tools, in situ secondary ion mass spectrometry (SIMS) and ex situ surface enhanced Raman spectroscopy (SERS), in analyzing surfaces modified by ion soft landing (SL). Cations derived from Rhodamine 6G are soft landed onto Raman-active silver colloidal substrates and detected using SERS. Alternatively and more conveniently, high-quality SERS data are obtained by spin coating a silver colloidal solution over the modified surface once SL is complete. Well-defined SERS features are observed for Rhodamine 6G in as little as 15 min of ion deposition. Deposition of ~3 pmo1 gave high-quality SERS spectra with characteristic spectroscopic responses being derived from just ~0.5 fmol of material. Confocal SERS imaging allowed the enhancement to be followed in different parts of deposited dried droplets on surfaces. Characteristic changes in Raman spectral features occur when Rhodamine 6G is deposited under conditions that favor gas-phase ion fragmentation. Simultaneous deposition of both the intact dye and its fragment ion occurs and is confirmed by SIMS analysis. The study was extended to other Raman active surfaces, including Au nanostar and Au coated Ni nanocarpet surfaces and to SL of other molecules including fluorescein and methyl red. Overall, the results suggest that combination of SERS and SIMS measurements are effective in the characterization of surfaces produced by ion SL with significantly enhanced molecular specificity.     

Ambient ion soft landing

Ambient ion soft landing, a process in which polyatomic ions are deposited from air onto a surface at a specified location under atmospheric pressure, is described. Ions generated by electrospray ionization are passed pneumatically through a heated metal drying tube, their ion polarity is selected using ion deflectors, and the dry selected ions are soft-landed onto a selected surface. Unlike the corresponding vacuum soft-landing experiment, where ions are mass-selected and soft-landed within a mass spectrometer, here the ions to be deposited are selected through the choice of a compound that gives predominantly one ionic species upon ambient ionization; no mass analysis is performed during the soft landing experiment. The desired dry ions, after electrical separation from neutrals and counterions, are deposited on a surface. Characterization of the landed material was achieved by dissolution and analysis using mass spectrometry or spectrofluorimetry. The treated surface was also characterized using fluorescence microscopy, which allowed surfaces patterned with fluorescent compounds to be imaged. The pure dry ions were used as reagents in heterogeneous ion/surface reactions including the reaction of pyrylium cations with d-lysine to form the N-substituted pyridinium cation. The charged microdroplets associated with incompletely dried ions could be selected for soft landing or surface reaction by choice of the temperature of a drying tube inserted between the ion source and the electrical ion deflectors.     

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 (SERS) detection of low concentrations of tryptophan amino acid in silver colloid

The surface-enhanced Raman scattering (SERS) spectrum of L-tryptophan has been studied in the concentration range 1.4 × 10(-8) to 5 × 10(-4) M. A borohydride-reduced silver colloid was employed as the nanoparticle enhancing agent and different electrolytes have been tested for activation of the colloid. The optimum conditions have been determined for achieving high sensitivity of detection. The experimental procedure developed, which includes the use of a composite electrolyte (a mixture of NaHCO(3) and NaCl) for colloid activation, results in very high enhancement of the Raman signal (up to 10(8)). This gives the possibility of studying SERS spectra of L-tryptophan at concentrations as low as 10(-8) M, which is several orders of magnitude lower than previously reported in the literature. The observed SERS spectra were very reproducible and were detectable 2 minutes after mixing, reaching maximum strength approximately 15 minutes after mixing. The spectral characteristics were stable over the entire period of observation. We have found that SERS spectra of tryptophan in silver colloid differ in several features at low (below ～10(-5) M) and at high (above ～10(-4) M) concentrations. The most important difference is the absence of the peak near 1000 cm(-1) at low concentrations, which is usually assigned to the indole ring breathing mode. The observed spectra allow us to suggest that at low concentrations Trp molecules bind to the surface through the indole ring, which remains flat on the surface. This is in contrast to the previously reported observation of SERS spectra from Trp performed at concentration levels above 10(-5) M.     

On sample preparation for surface-enhanced raman scattering (SERS) of bacteria and the source of spectral features of the spectra

The characterization, detection, and identification of bacteria using surface-enhanced Raman scattering (SERS) spectroscopy is drawing considerable attention due to its ability to provide rich intrinsic molecular information about molecules and molecular structures in close proximity to noble metal surfaces. However, sample preparation methods and experimental conditions must be carefully evaluated in order to obtain healthy, interpretable, and comparable results. In this study, several bacterial species including E. coli, B. megaterium, S. aureus, and S. cohnii were systematically evaluated to demonstrate the source of the spectral features of bacterial SERS spectra. It was found that the features observed in bacterial SERS spectra originate mostly from the bacteria surface with some contributions from metabolic activity or molecular species detached from the bacteria surface during sample preparation.     

A novel method for fabricating the surface-enhanced Raman scattering substrates and its enhanced properties

We have got large area surface-enhanced Raman scattering (SERS) substrates with uniform high enhancement factors by the so-called moulage method for the first time. A silver film (99.99%) with several millimeters thickness was thermally evaporated on the porous anodic alumina templates and the SERS substrate was got after moving off the templates. Surface-enhanced Raman scattering spectra of pyridine (0.01 Mol/L) were measured under 632.8 nm excitation. The experimental enhancement factors were more than 10(5) and S/N(p-p) around 100 was obtained. We have compared the SERS spectra of pyridine collected from different locations on the same SERS substrate and different substrates, which illustrate the well uniform enhance properties and the reproducibility of this method, respectively. The comparison of the SERS spectra, obtained from the SERS substrates and Ag film evaporated directly on glass slide, have proved that the electromagnetic coupling between two adjacent nanoparticles was important to the SERS effect. We also used rhodamine 6G as the probe molecules and found that the different molecules were very sensitive to the morphology of the SERS substrates.     

Characterization of silane-modified immobilized gold colloids as a substrate for surface-enhanced Raman spectroscopy

Immobilized gold colloid particles coated with a C-18 alkylsilane layer have been characterized as a substrate for surface-enhanced Raman scattering (SERS) studies of adsorption onto hydrophobic surfaces. Atomic force microscopy images, optical extinction spectra, and SERS measurements are reported as a function of accumulation of gold colloid on glass. As the metal particles become increasingly aggregated on the surface, the SERS enhancement increases until the plasmon resonance shifts to wavelengths longer than the excitation laser. The gold colloid substrates are stable and exhibit reproducible SERS enhancement. When octadecyltrimethoxysilane is self-assembled over the gold, the metal surface is protected from exposure to solution-phase species, as evidenced by the inhibition of chemisorption of a disulfide reagent to the overcoated gold surface. The results show that interactions with gold can be blocked by a silane layer so as not to significantly influence physisorption of molecules at the C-18/solution interface. The SERS enhancement from these C-18-overcoated gold substrates is reproducible for different films prepared from the same colloidal suspension; the substrates are also stable with time and upon exposure to laser irradiation.     

Surface-enhanced Raman scattering inside Au@Ag core/shell nanorods

The design and synthesis of plasmonic nanoparticles with Raman-active molecules embedded inside them are of significant interest for sensing and imaging applications.However,direct synthesis of such nanostructures with controllable shape,size,and plasmonic properties remains extremely challenging.Here we report on the preparation of uniform Au@Ag core/shell nanorods with controllable Ag shells of 1 to 25 nm in thickness.1,4-Aminothiophenol (4-ATP) molecules,used as the Raman reporters,were located between the Au core and the Ag shell.Successful embedding of reporter molecules inside the core/shell nanoparticles was confirmed by the absence of selective oxidation of the amino groups,as measured by Raman spectroscopy.The dependence of Raman intensity on the location of the reporter molecules in the inside and outside of the nanorods was studied.The molecules in the interior showed strong and uniform Raman intensity,at least an order of magnitude higher than that of the molecules on the nanoparticle surface.In contrast to the usual surface-functionalized Raman tags,aggregation and clustering of nanoparticles with embedded molecules decreased the surface-enhanced Raman scattering (SERS) signal.The findings from this study provide the basis for a novel detection technique of low analyte concentration utilizing the high SERS response of molecules inside the core/shell metal nanostructures.As an example,we show robust SERS detection of thiram fungicide as low as 10-9 M in solutions.     

Realization of thermally durable close-packed 2D gold nanoparticle arrays using self-assembly and plasma etching

Realization of thermally and chemically durable, ordered gold nanostructures using bottom-up self-assembly techniques are essential for applications in a wide range of areas including catalysis, energy generation, and sensing. Herein, we describe a modular process for realizing uniform arrays of gold nanoparticles, with interparticle spacings of 2?nm and above, by using RF plasma etching to remove ligands from self-assembled arrays of ligand-coated gold nanoparticles. Both nanoscale imaging and macroscale spectroscopic characterization techniques were used to determine the optimal conditions for plasma etching, namely RF power, operating pressure, duration of treatment, and type of gas. We then studied the effect of nanoparticle size, interparticle spacing, and type of substrate on the thermal durability of plasma-treated and untreated nanoparticle arrays. Plasma-treated arrays showed enhanced chemical and thermal durability, on account of the removal of ligands. To illustrate the application potential of the developed process, robust SERS (surface-enhanced Raman scattering) substrates were formed using plasma-treated arrays of silver-coated gold nanoparticles that had a silicon wafer or photopaper as the underlying support. The measured value of the average SERS enhancement factor (2?×?10(5)) was quantitatively reproducible on both silicon and paper substrates. The silicon substrates gave quantitatively reproducible results even after thermal annealing. The paper-based SERS substrate was also used to swab and detect probe molecules deposited on a solid surface.     

Bright Surface‐Enhanced Raman Scattering with Fluorescence Quenching from Silica Encapsulated J‐Aggregate Coated Gold Nanoparticles

Plexitonic nanoparticles offer variable optical properties through tunable excitations, in addition to electric field enhancements that far exceed molecular resonators. This study demonstrates a way to design an ultrabright surface‐enhanced Raman spectroscopy (SERS) signal while simultaneously quenching the fluorescence background through silica encapsulation of the semiconductor–metal composite nanoparticles. Using a multistep approach, a J‐aggregate‐forming organic dye is assembled on the surface of gold nanoparticles using a cationic linker. Excitonic resonance of the J‐aggregate–metal system shows an enhanced SERS signal at an appropriate excitation wavelength. Further encapsulation of the decorated particles in silica shows a significant reduction in the fluorescence signal of the Raman spectra (5× reduction) and an increase in Raman scattering (7× enhancement) when compared to phospholipid encapsulation. This reduction in fluorescence is important for maximizing the useful SERS enhancement from the particle, which shows a signal increase on the order of 10⁴ times greater than J‐aggregated dye in solution and 24 times greater than Oxonica S421 SERS tag. The silica layer also serves to promote colloidal stability. The combination of reduced fluorescence background, enhanced SERS intensity, and temporal stability makes these particles highly distinguishable with potential to enable high‐throughput applications such as SERS flow cytometry.     

Near-Field Surface-Enhanced Raman Imaging of Dye-Labeled DNA with 100-nm Resolution

Raman chemical imaging on a scale of 100 nm is demonstrated for the first time. This is made possible by the combination of scanning near-field optical microscopy (SNOM or NSOM) and surface-enhanced Raman scattering (SERS), using brilliant cresyl blue (BCB)-labeled DNA as a sample. SERS substrates were produced by evaporating silver layers on Teflon nanospheres. The near-field SERS spectra were measured with an exposure time of 60 s and yielded good signal-to-noise ratios (25:1). The distinction between reflected light from the excitation laser and Raman scattered light allows the local sample reflectivity to be separated from the signal of the adsorbed DNA molecules. This is of general importance to correct for topographic coupling that often occurs in near-field optical imaging. The presented data show a lateral dependence of the Raman signals that points to special surface sites with particularly high SERS enhancement.     

Preparation of gold nano-cones as surface-enhanced Raman scattering sensors for molecule detection

Surface-enhanced Raman spectroscopy (SERS) is a powerful novel analytical tool which integrates high levels of sensitivity for trace analysis of chemical and biomolecular species due to the massive enhancement of Raman signals by using nanometre-sized metal particles. However, SERS can be envisaged as an analytical tool only if substrates with strong, predictable and reproducible SERS enhancement can be produced. Here we have developed one simple Ar+ ions sputtering technology to prepare gold nano-cones array on silicon substrates as surface-enhanced Raman scattering (SERS)-active substrates. The tip of the gold cone-structures exhibited an extremely sharp curvature with an apex diameter of 20 nm and the interior apex angle of the nanocones was around 20 degrees. These samples were evaluated as potential SERS substrates using Rhodamine 6G molecules as molecule probe and exhibited SERS enhancement factor of greater than 10, originated from the localized electron field enhancement around the apex of cones and the surface plasmon coupling of periodic structures.     

Competitive Reaction Pathway for Site‐Selective Conjugation of Raman Dyes to Hotspots on Gold Nanorods for Greatly Enhanced SERS Performance

Common methods to prepare SERS (surface‐enhanced Raman scattering) probes rely on random conjugation of Raman dyes onto metal nanostructures, but most of the Raman dyes are not located at Raman‐intense electromagnetic hotspots thus not contributing to SERS enhancement substantially. Herein, a competitive reaction between transverse gold overgrowth and dye conjugation is described to achieve site selective conjugation of Raman dyes to the hotspots (ends) on gold nanorods (GNRs). The preferential overgrowth on the nanorod side surface creates a barrier to prevent the Raman dyes from binding to the side surface except the ends of the GNRs, where the highest SERS enhancement factors are expected. The SERS enhancement observed from this special structure is dozens of times larger than that from conjugates synthesized by conventional methods. This simple and powerful strategy to prepare SERS probes can be extended to different anisotropic metal nanostructures with electromagnetic hotspots and has immense potential in in‐depth SERS‐based biological imaging and single‐molecule detection.     

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).     

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 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 to probe reversibly photoswitchable azobenzene in controlled nanoscale environments

We apply in situ surface-enhanced Raman spectroscopy (SERS) to probe the reversible photoswitching of azobenzene-functionalized molecules inserted in self-assembled monolayers that serve as controlled nanoscale environments. Nanohole arrays are fabricated in Au thin films to enable SERS measurements associated with excitation of surface plasmons. A series of SERS spectra are recorded for azobenzene upon cycling exposure to UV (365 nm) and blue (450 nm) light. Experimental spectra match theoretical calculations. On the basis of both the simulations and the experimental data analysis, SERS provides quantitative information on the reversible photoswitching of azobenzene in controlled nanoscale environments.