Surface-enhanced Raman spectroscopy of bacteria and pollen

A technique for distinguishing biological material based on surface-enhanced Raman scattering (SERS) is reported in this work. Of particular interest is biological material that can be airborne. Silver colloidal particles with diameters in the range 10 to 20 nm and with a characteristic ultraviolet-visible (UV-VIS) absorption band at 400 nm were used to obtain SERS spectra of Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium bacteria and a number of tree and grass pollens (Cupressus arizonica (cypress), Sequoia sempervirens (redwood), Populus deltoides (cottonwood), Poa pratensis (Kentucky bluegrass), and Anthoxanthum odoratum (sweet vernal grass)). While differences in the SERS spectra among the bacteria were small, we found that the pollen spectra we analyzed could readily be distinguished from the bacteria spectra, and there were significant differences between pollen from different families. In order to obtain reproducible results, we studied the parameters controlling the interaction between the analyte and the nanoscale metallic surface. Our results show that the volume ratio of analyte to colloidal particles must be within a narrow range of values to optimize the signal-to-noise ratio of the SERS spectra and minimize the fluorescence from the analyte. Also, we found that the time-dependent behavior of colloidal/bacterial suspensions (or adsorption rate of the silver colloid particles on the bacteria) is strongly dependent on pH, density of bacteria in solution, and even, to some extent, the type of bacteria.     

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 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.     

Electrochemical detection of quorum sensing signaling molecules by dual signal confirmation at microelectrode arrays

n-Acyl homoserine lactones (AHLs) are produced by gram-negative bacteria to regulate gene expression in a cell density dependent manner. For instance, expression of virulence factors by pathogens such as Pseudomonas aeruginosa is induced only when a threshold concentration of AHLs is reached, which indicates that the bacterial population is big enough to promote infection. In this study, the indicator strain Agrobacterium tumefaciens NTL4 (pZLR4), which carries a β-galactosidase (β-gal) reporter gene under the control of a quorum sensing promoter, was used to develop an electrochemical biosensor to detect AHLs using the model n-(3-oxo)-dodecanoyl-L-homoserine lactone (oxo-C12-HSL), an AHL previously detected in cystic fibrosis patients infected with P. aeruginosa. The substrate 4-aminophenyl β-D-galactopyranoside was used to detect β-gal activity by cyclic voltammetry. Furthermore, simultaneous monitoring of substrate consumption and p-aminophenol production by β-gal allowed on-chip result verification by dual-signal confirmation. The sensor exhibited high reproducibility and accurately detected oxo-C12-HSL in a low picomolar to low nanomolar range in spiked liquid cultures and artificial saliva, as well as AHLs naturally released by P. aeruginosa in culture supernatants. Moreover, detection took just 2 h, required no sample pretreatment or preconcentration steps, and was easier and faster than traditional methods.     

Surface-enhanced Raman spectroscopy studies of surfactant adsorption to a hydrophobic interface

Surface-enhanced Raman spectroscopy is used to investigate the kinetics of adsorption of the cationic surfactant cetylpyridinium chloride (CPC) to hydrophobic surfaces from water. A hydrophobic surface, with stable and reproducible SERS activity, is produced by binding gold colloids to an amine-terminated glass slide and then modifying this surface with octadecyltrimethoxysilane. In situ SERS-detected adsorption of CPC from aqueous solution is found to follow a Frumkin isotherm. Interactions between the charged head groups could be detected in frequency shifts in the symmetric ring breathing mode, consistent with an interfacial surfactant environment similar to a CPC micelle. Rates of surfactant adsorption were determined by time-resolved SERS measurements and were found to be much slower than the diffusion-controlled limit, indicating a significant kinetic barrier to adsorption. Desorption kinetics were heterogeneous, consistent with the spectroscopic results. Alkylsilane-modified gold colloids were shown to be useful substrates for investigating amphiphile adsorption from aqueous solutions to hydrophobic surfaces, where the adsorption kinetics could also be used to determine analyte concentrations in solution.     

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.     

Biosensing systems for the detection of bacterial quorum signaling molecules

Bacterial quorum sensing (QS) is a cell-to-cell communication phenomenon that allows bacteria to control the expression of certain specialized genes depending on their cell population size. Signaling molecules such N-acylhomoserine lactones (AHLs) mediate the communication, and their concentration reflects the bacterial population density. Quorum sensing regulates several processes including bacterial pathogenicity. We developed a method for the rapid, sensitive, and quantitative detection of AHLs in biological samples such as saliva and stools. The method is based on whole-cell sensing systems that employ QS regulatory systems as recognition elements and the luxCDABE gene cassette as a reporter. The method proved to be reproducible when applied to real samples and was able to detect low analyte concentrations down to 1 x 10(-9) M without requiring extensive sample preparation. We envision that this novel biosensing system could be employed in the diagnosis and management of various bacteria-related disorders, thus supporting the use of quorum sensing molecules as potential biomarkers of disease. Due to cost-effectiveness and high throughput, these biosensing systems could be successfully employed as a new tool for the screening of novel drugs that target quorum sensing mechanisms.     

Surface-enhanced Raman spectroscopy biosensors: excitation spectroscopy for optimisation of substrates fabricated by nanosphere lithography

In the 28 years since its discovery, surface-enhanced Raman scattering (SERS) has progressed from model system studies of pyridine on a roughened silver electrode to state-of-the-art surface science studies and real-world sensing applications. Each year, the number of SERS publications increases as nanoscale material design techniques advance and the importance of trace analyte detection increases. To achieve the lowest limits of detection, both the relationship between surface nanostructure and laser excitation wavelength and the analyte-surface binding chemistry must be carefully optimised. This work exploits the highly tunable nature of nanoparticle optical properties to establish the optimisation conditions. Two methods are used to study the optimised conditions of the SERS substrate: plasmon-sampled and wavelength-scanned surfaced Raman excitation spectroscopy (SERES). The SERS enhancement condition is optimised when the energy of the localised surface plasmon resonance of the nanostructures lies between the energy of the excitation wavelength and the energy of the vibration band of interest. These optimised conditions enabled the development of SERS-based sensors for the detection of a Bacillus anthracis biomarker and glucose in a serum-protein matrix.     

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.     

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.     

Surface-enhanced Raman spectroscopy of chernozem humic acid and their fractions obtained by coupled size exclusion chromatography-polyacrylamide gel electrophoresis (SEC-PAGE)

A humic acid extracted from a chernozem soil was fractionated combining size exclusion chromatography and polyacrylamide electrophoresis (SEC-PAGE). Three fractions named A, B, and C+D, with different electrophoretic mobilities and molecular sizes (MS), were obtained and subsequently characterized by thermochemolysis and surface-enhanced Raman spectroscopy (SERS). The data confirmed that fraction A, with the higher MS, was more aliphatic than fractions B and C+D and, in turn, fractions with lower MS (B and C+D) denoted an enrichment in lignin residues. These structural features explain conformational changes when varying the pH in the humic fraction A and indicated that combination of the two techniques is a good approach for characterizing humic substances.     

Surface-enhanced resonance Raman scattering of black inkjet dyes in solution and in situ printed onto paper

Understanding the changes that occur when dyes are absorbed onto paper is crucial for the design of new inkjet dyes. This problem is particularly difficult for black dyes that have complex chromophores, and as a result, spectroscopic information on electronic and structural changes can be of importance. Surface-enhanced resonance Raman scattering (SERRS) and electronic structure calculations were used to probe in situ changes in the chromophore in black di-azo dyes printed onto paper. The data indicate that the low-energy chromophore is due mainly to the hydrazone group and the high-energy chromophore to both the azo and hydrazone groups. A comparison of SERRS from the dyes adsorbed onto silver particles in suspension and from the dyes on paper demonstrated a broadening of the chromophore into the red for both dyes and evidence of a structural change in one dye.     

In situ measurements of Raman scattering in clear ocean water

We have further developed and improved the prototype oceanic Fraunhofer line discriminator by using a well-protected fiber-optic-wire cable and in-water electronic housing. We conducted a series of in situ measurements in clear ocean water in the Florida Straits. By comparing the reduced data with the Monte Carlo simulation results, we verify the Raman scattering coefficient B(r) with an excitation wavelength at 488 nm to be 2.6 x 10(-4)m(-1) [Appl. Opt. 29, 71-84 (1990)], as opposed to 14.4 x 10(-4) m(-1) [Appl. Opt.14, 2116-2120 (1975)]. The wavelength dependence of the Raman scattering coefficient is found to have an insignificant effect on the in-water light field. We also discuss factors that lead to errors. This study can be used as a basis for inelastic light scattering in the radiative transfer theory and will allow other inelastic light, e.g., fluorescence, to be detected with in situ measurements.     

Modified coin cells for in situ Raman spectroelectrochemical measurements of Li(x)V2O5 for lithium rechargeable batteries

In situ Raman spectroscopy is an extremely valuable technique for investigating fundamental reactions that occur inside lithium rechargeable batteries. However, specialized in situ Raman spectroelectrochemical cells must be constructed to perform these experiments. These cells are often quite different from the cells used in normal electrochemical investigations. More importantly, the number of cells is usually limited by construction costs; thus, routine usage of in situ Raman spectroscopy is hampered for most laboratories. This paper describes a modification to industrially available coin cells that facilitates routine in situ Raman spectroelectrochemical measurements of lithium batteries. To test this strategy, in situ Raman spectroelectrochemical measurements are performed on Li//V2O5 cells. Various phases of Li(x)V2O5 could be identified in the modified coin cells with Raman spectroscopy, and the electrochemical cycling performance between in situ and unmodified cells is nearly identical.     

Coded aperture Raman spectroscopy for quantitative measurements of ethanol in a tissue phantom

Coded aperture spectroscopy allows for sources of large étendue to be efficiently coupled into dispersive spectrometers by replacing the traditional input slit with a patterned mask. We describe a coded aperture spectrometer optimized for Raman spectroscopy of diffuse sources, (e.g., tissue). We provide design details of the Raman system, along with quantitative estimation results for ethanol at non-toxic levels in a lipid tissue phantom. With 60 mW of excitation power at 808 nm, leave-one-out and blind cross-validation of partial least squares (PLS) regression models achieve r(2) > 0.98. Leave-one-out cross-validation demonstrates prediction errors of <15% at the common legal limit for intoxication (17.4 mmol/L = 0.08% by vol) and the best blind cross-validation achieves <12% error at this concentration.     

Specific chemical effects on surface-enhanced Raman spectroscopy for ultra-sensitive detection of biological molecules

Achieving high signal amplification in surface-enhanced Raman scattering (SERS) is important for reaching single molecule level sensitivity and has been the focus of intense research efforts. We introduce a novel chemical enhancer, lithium chloride, that provides an additional order of magnitude increase in SERS relative to previously reported enhancement results. We have duplicated single molecule detection of the DNA base adenine that has previously been reported, thereby providing independent validation of this important result. Building upon this work, we show that the chemical enhancer LiCl produces strong SERS signal under a wide range of experimental conditions, including multiple laser excitation wavelengths and target molecule concentrations, for nucleotides, nucleosides, bases, and dye molecules. This is significant because while selection of anions used in chemical enhancement is well known to affect the degree of amplification attained, cation selection has previously been reported to have no major effect on the magnitude of SERS enhancement. Our findings indicate that cation selection is quite important in ultra-sensitive SERS detection, opening the door to further discussion and theory development involving the role of cations in SERS.     

Noise and artifact characterization of in vivo Raman spectroscopy skin measurements

In this work principal component analysis (PCA), a multivariate pattern recognition technique, is used to characterize the noise contribution of the experimental apparatus and two commonly used methods for fluorescence removal used in biomedical Raman spectroscopy measurements. These two methods are a fifth degree polynomial fitting and an iterative variation of it commonly known as the Vancouver method. The results show that the noise in Raman spectroscopy measurements is related to the spectral resolution of the measurement equipment, the intrinsic variability of the biological measurements, and the fluorescence removal algorithm used.     

Guided-mode-resonance-coupled plasmonic-active SiO(2) nanotubes for surface enhanced Raman spectroscopy

We demonstrate a surface enhanced Raman scattering (SERS) substrate by integrating plasmonic-active SiO(2) nanotubes into Si(3)N(4) gratings. First, the dielectric grating that is working under guided mode resonance (GMR) provides enhanced electric field for localized surface plasmon polaritons on the surface of metallic nanoparticles. Second, we use SiO(2) nanotubes with densely assembled silver nanoparticles to provide a large amount of "hot spots" without significantly damping the GMR mode of the grating. Experimental measurement on Rhodamine-6G shows a constant enhancement factor of 8?～?10 in addition to the existing SERS effect across the entire surface of the SiO(2) nanotubes.     

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.     

Surface-enhanced Raman scattering (SERS) characterization of trace organoarsenic antimicrobials using silver/polydimethylsiloxane nanocomposites

Organoarsenic drugs such as roxarsone and 4-arsanilic acid are poultry feed additives widely used in US broilers to prevent coccidosis and to enhance growth and pigmentation. Despite their veterinary benefits there has been growing concern about their use because over 90% of these drugs are released intact into litter, which is often sold as a fertilizing supplement. The biochemical degradation of these antimicrobials in the litter matrix can release significant amounts of soluble As(III) and As(V) to the environment, representing a potential environmental risk. Silver/polydimethylsiloxane (Ag/PDMS) nanocomposites are a class of surfaceenhanced Raman scattering (SERS) substrates that have proven effective for the sensitive, reproducible, and field-adaptable detection of aromatic acids in water. The work presented herein uses for the first time Ag/PDMS nanocomposites as substrates for the detection and characterization of trace amounts of roxarsone, 4-arsanilic acid, and acetarsone in water. The results gathered in this study show that organoarsenic species are distributed into the PDMS surface where the arsonic acid binds onto the embedded silver nanoparticles, enhancing its characteristic 792 cm(-1) stretching band. The chemisorption of the drugs to the metal facilitates its detection and characterization in the parts per million to parts per billion range. An extensive analysis of the distinct spectroscopic features of each drug is presented with emphasis on the interactions of the arsonic acid, amino, and nitro groups with the metal surface. The benefits of SERS based methods for the study of arsenic drugs are also discussed.