Determination of pyrimidine and purine bases by reversed-phase capillary liquid chromatography with at-line surface-enhanced Raman spectroscopic detection employing a novel SERS substrate based on ZnS/CdSe silver-quantum dots

We have developed a new SERS substrate based on the reduction of silver nitrate in the presence of ZnS-capped CdSe quantum dots. This substrate showed higher sensitivities as compared to a hydroxylamine-reduced silver sol. On the basis of this new substrate, at-line SERS detection was coupled with capillary liquid chromatography (cap-LC) for the separation and selective determination of pyrimidine and purine bases. For this purpose, wells of a dedicated microtiter plate were loaded with 20 μL of the SERS substrate and placed on an automated x,y translation stage. A flow-through microdispenser capable of ejecting 50 pL droplets, at a frequency 100 Hz, was used as the interface to connect the cap-LC system to the wells loaded with SERS substrate. A detailed study of the dependence of both the separation and the surface-enhanced Raman spectra of each base on the pH was performed to optimize the system for maximum sensitivity and selectivity. Highly satisfactory analytical figures of merit were obtained for the six investigated bases (cytosine, xanthine, hypoxanthine, guanine, thymine, and adenine) with detection limits ranging between 0.2 and 0.3 ng injected on the capillary LC column, and the precisions were in the range of 3.0-6.3%.     

Functional Femtoliter Droplets for Ultrafast Nanoextraction and Supersensitive Online Microanalysis

A universal femtoliter surface droplet‐based platform for direct quantification of trace of hydrophobic compounds in aqueous solutions is presented. Formation and functionalization of femtoliter droplets, concentrating the analyte in the solution, are integrated into a simple fluidic chamber, taking advantage of the long‐term stability, large surface‐to‐volume ratio, and tunable chemical composition of these droplets. In situ quantification of the extracted analytes is achieved by surface‐enhanced Raman scattering (SERS) spectroscopy by nanoparticles on the functionalized droplets. Optimized extraction efficiency and SERS enhancement by tuning droplet composition enable quantitative determination of hydrophobic model compounds of rhodamine 6G, methylene blue, and malachite green with the detection limit of 10^?9 to 10^?11 m and a large linear range of SERS signal from 10^?9 to 10^?6 m of the analytes. The approach addresses the current challenges of reproducibility and the lifetime of the substrate in SERS measurements. This novel surface droplet platform combines liquid–liquid extraction and highly sensitive and reproducible SERS detection, providing a promising technique in current chemical analysis related to environment monitoring, biomedical diagnosis, and national security monitoring.     

Surface-enhanced Raman spectra of calf thymus DNA adsorbed on concentrated silver colloid

Raman and surface-enhanced Raman scattering (SERS) spectra of calf thymus DNA were investigated. We have carried out improvements to the silver colloid preparation method of Lee and Meisel in two respects. In one method, the silver sol was boiled with rapid stirring for over two hours. In the second method, the silver sol was concentrated by centrifugation before adding it to the DNA solution. The resulting hydrosol could be stored for 15 months because of its high stabilization. Structural information with respect to the phosphate backbone, deoxyribose, and four bases of DNA could be obtained before and after the DNA solutions were added to the concentrated Ag colloid substrate. The intensities of almost all characteristic bands assigned to various groups of the components of DNA were enhanced to a remarkable degree. The enhancement effect of the DNA solution at neutral pH 7.0 was obviously much better than that at acidic pH 3.4 or at alkaline pH 8.5. Intensity increases of the SERS bands of the DNA solution with time were observed. The SERS signals obtained 16 hours after the interaction of the Ag colloid with the DNA solution were much better than the SERS signals obtained just after the mixed liquid was prepared. This method can be widely used to store the Ag colloid for long times and to obtain the SERS spectra of DNA molecules, and it can further be used to study the adsorption behavior of solute biomacromolecules in different solvents.     

Investigation of coated droplets in an optical trap: Raman-scattering, elastic-light-scattering, and evaporation characteristics

Single optically levitated microparticles were investigated by Raman spectroscopy. The particles were composed of di-octyl-phthalate (DOP) and glycerol; these substances are not mixable and form a two-phase droplet. Measurements of the Raman spectrum confirm the formation of droplets containing both chemical species. The spectra show strong input and output structural resonances as expected. If the particle is in resonance, the field inside the particle is enhanced, and most of the inelastically scattered light is emitted from molecules close to the droplet rim. If the particle does not fulfill the resonance condition, the contribution of an individual molecule to the Raman scattering does not depend strongly on the radial position of this molecule. On this basis, the radial distribution of the two components inside the evaporating droplet was determined by time-dependent measurements of the Raman spectrum. Furthermore, elastic-light scattering and the evaporation characteristics of the particles were investigated.     

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.     

On-column surface-enhanced Raman spectroscopy detection in capillary electrophoresis using running buffers containing silver colloidal solutions

Direct on-column surface-enhanced Raman spectroscopy (SERS) detection is demonstrated in capillary electrophoresis (CE). Distinctive SERS spectra of two test compounds, riboflavin and Rhodamine 6G, are obtained in 100 microm i.d. fused-silica capillaries under CE conditions using running buffers that contain silver colloidal solutions. Detection is performed using an unmodified commercial Raman spectrometer in a confocal microscope mode of operation. The effects of laser power, wavelength, spectra acquisition time, silver colloidal concentration, and applied voltage (i.e., flow rate) on the quality of SERS spectra are evaluated. Using laser powers of 17 mW (at the sample) at 515 nm and employing 1 s spectral acquisition times, spectra with bands exhibiting signal-to-noise ratios greater than 10 could be obtained for 1.0 x 10(-6) M riboflavin and very low nanomolar concentrations of Rhodamine 6G. This was accomplished without optimization of silver colloidal solution compositions and by using a low-throughput spectrometer. Incorporation of the colloidal solutions into running buffers is shown to have little effect on the separation of the test compounds as monitored using a laser-induced fluorescence instrumental scheme. However, SERS spectra degrade if the capillary is not rinsed between experiments. Riboflavin and Rhodamine 6G spectra are obtained on-the-fly for actual CE separations. In the case of the latter solute, the injected quantity was approximately 90 amol.     

Adsorption of S-S containing proteins on a colloidal silver surface studied by surface-enhanced Raman spectroscopy

We present a Raman and surface-enhanced Raman scattering (SERS) study of the following proteins containing S-S group(s): alpha chymotrypsin (alpha-CHT), insulin, lysozyme, oxytocin (OXT), Streptomyces subtilisin inhibitor (SSI), and trypsin inhibitor (STI). The SERS study is performed in order to understand the adsorption mechanism of the above-mentioned proteins on a colloidal silver surface. The SERS spectra presented here show bands associated mainly with aromatic amino acid vibrations. In addition, two distinct vibrations of the -C-S-S-C- fragment are observed in the Raman and SERS spectra, i.e., nu(SS) and nu(CS). The enhancement of the nu(SS) vibration in the SERS spectra yields evidence that the intact disulfide bridge(s) is (are) located near the silver surface. This finding is supported by the presence of the nu(CS) mode(s). The presence of nus(COO-) and nu(C-COO-) in the SERS spectra in the 1384-1399 cm(-1) and 909-939 cm(-1) regions, respectively, indicate that the negatively charged COO- groups (aspartic and glutamic acids) assist in the binding on the positively charged silver surface. The Raman amide I and III bands observed in the 1621-1633 and 1261-1289 cm(-1) ranges, respectively, indicate that the alpha-helical conformation is favored for binding to the surface over the random coil or beta-sheet conformations. In addition, the presence of the imino group of Trp and/or His indicates that these amino acid residues may also bind to the silver sol.     

The Interaction Between Thiolic Antifoggants and Silver Subcolloid on the Interface

The visible spectral absorption at 390 nm of a dear tight-yellow silver subcolloid partly shifted to 510-550 nm due to the adsorption of phenylmercaptotetrazole (PMT) or mercaptobenzo- thiazole (MBT), which produced an enhanced surface Raman scattering effect.

The normal Raman spectra (NRS) of solid PMT and MBT and their surface enhanced Raman spectra (SERS) on the surface of the silver subcolloidal particles are reported and compared. The adsorption of halide ions competes with that of PMT and MBT. However, halide ions have a completely different influence from PMT and MBT on the spectral absorption of the silver sol.     

Nanowires enabling signal-enhanced nanoscale Raman spectroscopy

Silicon nanowires grown by the vapor-liquid-solid (VLS) mechanism catalyzed by gold show gold caps (droplets) approximately 20-500 nm in diameter with a half spherical towards almost spherical shape. These gold droplets are well suited to exploit the surface-enhanced Raman scattering (SERS) effect and could be used for tip-enhanced Raman spectroscopy (TERS). The gold droplet of a nanowire attached to an atomic force microscopy (AFM) tip could locally enhance the Raman signal and increase the spatial resolution. Used as a SERS template, an ensemble of self-organizing nanowires grown bottom up on a silicon substrate could allow highly sensitive signal-enhanced Raman spectroscopy of materials that show a characteristic Raman signature. A combination of a nanowire-based TERS probe and a nanowire-based SERS substrate promises optimized signal enhancement so that the detection of highly dilute species, even single molecules or single bacteria or DNA strands, and other soft matter is within reach. Potential applications of this novel nanowire-based SERS and TERS solution lie in the fields of biomedical and life sciences, as well as security and solid-state research such as silicon technology.     

In situ surface-enhanced Raman scattering analysis of biofilm

Biofilms represent the predominant form of microbial life on Earth. They are aggregates of microorganisms embedded in a matrix formed by extracellular polymeric substances (EPS). Detailed information about chemical composition and structure of the EPS matrix is relevant e.g. for the optimization of biocides, of antifouling strategies and for biological wastewater treatment. Raman microscopy (RM) is a capable tool that can provide detailed chemical information about biofilm constituents with spatial resolution of optical microscope. However, the sensitivity of RM is limited. Surface-enhanced Raman scattering (SERS), which enables investigations of biomolecules at very low concentration levels, allows overcoming this drawback. To our knowledge, this paper is the first report on reproducible SERS spectra from different constituents of a multispecies biofilm. We believe that the reproducibility is partly owed to the in situ measurement of the biofilm, while up to now SERS measurements of microbiological samples by RM were carried out after sample drying. We employed colloidal silver nanoparticles for in situ SERS measurements by RM. The achieved enhancement factor of up to 2 orders of magnitude illustrates a high potential of SERS for ultrasensitive chemical analysis of biofilms, including the detection of different components and the determination of their relative abundance in the complex biofilm matrix.     

Airborne cell analysis

A miniaturized analysis system for the study of living cells and biochemical reactions in microdroplets was developed. The technique utilizes an in-house-developed piezoelectric flow-through droplet dispenser for precise reagent supply and an ultrasonic levitator for contactless sample handling. A few-cell study was performed with living primary adipocytes. Droplets (500 nL) containing 3-15 individual cells were acoustically levitated. The addition of beta-adrenergic agonists into the levitated droplet using the droplet dispenser stimulated adipocyte lipolysis, leading to free fatty acid release and a consequent pH decrease of the surrounding buffer. The addition of insulin antagonized lipolysis and hence also the decrease in pH. The changes in pH, i.e., the cell response in the droplet, were followed using a pH-dependent fluorophore continuously monitored by fluorescence imaging detection. An image analysis computer program was employed to calculate the droplet intensities. To counteract droplet evaporation, found to affect the fluorescence intensities, a separate dispenser was used to continually add water, thus keeping the droplet volume constant.     

Experimental indication of chemical effects in surface enhanced hyper Rayleigh scattering using dye-adsorbed Ag nanoparticles

We report on the observation of chemical effects in surface enhanced hyper Rayleigh scattering (SEHRS), using silver nanoparticles adsorbed with stilbazolium dyes. The experimental results show that the enhancement factors varied with different donor structures of the dyes, which cannot be explained only according to the electromagnetic effect. Using the extinction spectra and surface enhanced Raman scattering spectra (SERS) to provide the complementary results, we deduce that chemical effects also play an important role in SEHRS in addition to the electromagnetic effect. The chemical effects may be related with the Ag-adsorbate charge transfer transition, similar to the mechanism in SERS.     

A Droplet‐Based High‐Throughput SERS Platform on a Droplet‐Guiding‐Track‐Engraved Superhydrophobic Substrate

A novel droplet‐based surface‐enhanced Raman scattering (SERS) sensor for high‐throughput real‐time SERS monitoring is presented. The developed sensors are based on a droplet‐guiding‐track‐engraved superhydrophobic substrate covered with hierarchical SERS‐active Ag dendrites. The droplet‐guiding track with a droplet stopper is designed to manipulate the movement of a droplet on the superhydrophobic substrate. The superhydrophobic Ag dendritic substrates are fabricated through a galvanic displacement reaction and subsequent self‐assembled monolayer coating. The optimal galvanic reaction time to fabricate a SERS‐active Ag dendritic substrate for effective SERS detection is determined, with the optimized substrate exhibiting an enhancement factor of 6.3 × 10⁵. The height of the droplet stopper is optimized to control droplet motion, including moving and stopping. Based on the manipulation of individual droplets, the optimized droplet‐based real‐time SERS sensor shows high resistance to surface contaminants, and droplets containing rhodamine 6G, Nile blue A, and malachite green are successively controlled and detected without spectral interference. This noble droplet‐based SERS sensor reduces sample preparation time to a few seconds and increased detection rate to 0.5 µ L s^?1 through the simple operation mechanism of the sensor. Accordingly, our sensor enables high‐throughput real‐time molecular detection of various target analytes for real‐time chemical and biological monitoring.     

Observation of sudden temperature jumps in optically levitated microdroplets due to morphology-dependent input resonances

During the slow evaporation of an optically levitated microdroplet of a glycerol-water mixture (3:1) (approximately 12.44 μm in radius) several morphology-dependent input resonances have been observed in its Raman spectrum. These resonances yield sudden temperature jumps of approximately 10 °C in the microdroplet as evidenced by sudden shifts in the output (Raman) resonance spectra. The latter effects could be explained by a simple energy balance calculation and the dependence of droplet refractive index and density on temperature.     

Effect of conformation and drop properties on surface-enhanced Raman spectroscopy of dried biopolymer drops

Biofluids are complex solutions consisting of small ions and large biopolymers such as DNA, proteins, or proteoglycans. Biopolymers affect fluid properties but their effect on drop deposition has not been examined. Hyaluronic acid (HA), an important component in synovial fluid, was chosen as a model biopolymer, and examined using surface-enhanced Raman spectroscopy (SERS). Nanoliter volumes of HA solutions were dried onto a patterned SERS substrate and spectra were collected from the dried hyaluronic acid drops with a near-infrared Raman microscope. Characteristic hyaluronic acid bands were examined. Capillary viscometry measured properties of HA solutions, and entanglement behavior was also modeled using scaling theory principles. Viscosity measurements were incorporated into models of suspended particle droplets to account for the effect of inter-chain attraction on droplet formation. Microscope images were used to evaluate the shape of the dried drop. Relative drop thickness was estimated from concentric rings found at drop edges using established models of light interference by thin films. We found SERS spectra were sensitive not only to polymer conformation, but also to type of deposition (ring versus uniform), and the thickness of the resulting deposition. These data suggest an approach to elucidate the effects of biopolymers and dehydrated biofluids on SERS analysis.     

Uniform Microgels Containing Agglomerates of Silver Nanocubes for Molecular Size‐Selectivity and High SERS Activity

Surface‐enhanced Raman scattering (SERS) is a promising technique for molecular analysis as the molecular fingerprints (Raman spectra) are amplified to detectable levels compared with common spectroscopy. Metal nanostructures localize electromagnetic field on their surfaces, which can lead to dramatic increase of Raman intensity of molecules adsorbed. However, the metal surfaces are prone to contamination, thereby requiring pretreatment of samples to remove adhesive molecules. To avoid the pretreatment and potentially achieve point‐of‐care (POC) analysis, we have developed SERS‐active microgels using the droplet‐microfluidic system. As the microgels are composed of water‐swollen network with consistent mesh size, they selectively allow diffusion of molecules smaller than the mesh, thereby excluding large adhesives. To render the microgels highly SERS‐active, we destabilize silver nanocubes to form agglomerates, which are embedded in the matrix of microgels. The nanogaps in the agglomerates provide high sensitivity in Raman measurement and size‐selective permeability of the microgel matrix obviates the pretreatment of samples. To validate the functions, we demonstrate the direct detection of Aspirin dissolved in whole blood without any pretreatment.     

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 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.     

Synthesis of silver leaves and their potential application for analysis and degradation of phenolic pollutants

A one‐pot bottom‐up synthesis method was used to synthesise multi‐level leaf‐like nano‐silver (silver leaf) by simply mixing AgNO₃, L‐ascorbic acid, Sodium sodium citrate, and polyvinylpyrrolidone (PVP) in the ethanol‐water mixed solvents. Scanning electron microscopy (SEM) characterisations show that the silver leaves have tertiary structures and their sizes are controllable. In addition, silver leaves exhibit excellent Raman enhancement effect (SERS) and chemical catalytic activities for phenolic molecules. Interestingly, the SERS and catalytic activities increase as the size of the silver leaves decrease within a certain range, but when the size is too small, both of these performances weaken. The nanometre size and interstitial structure have a common amplification effect and influence on these activities. The present work not only showed a new method for the synthesis of silver leaves but also could be generalised to find other metallic leaves that could be used as promising heterogeneous catalysts for various reactions. The production of such small‐sized silver leaves will facilitate the analysis of phenolic pollutants through Raman enhancement and treat these pollutants through catalytic degradation.     

Surface-enhanced Raman spectroscopy as a tool for probing specific biochemical components in bacteria

Treatment of bacteria with silver yields intense and highly specific surface-enhanced Raman spectroscopy (SERS) spectra from various cellular chemical components located in the vicinity of the silver colloids. In particular, we demonstrate an extreme sensitivity to flavin components associated with the cell envelope and to their state of oxidation. Different spectra, possibly associated with DNA, carboxylates, and perhaps phosphates, are obtained from the soluble interior fraction of the cell.