Evaluation of surface-enhanced resonance Raman scattering for quantitative DNA analysis

The labeling of biological species using dyes has become common practice to aid in their detection, and immediate positive identification of specific dyes in high dilution is a key requirement. Here the detection by surface-enhanced resonance Raman scattering (SERRS) of eight commercially available dye labels (ROX, rhodamine 6G, HEX, FAM, TET, Cy3, Cy5, TAMRA) attached to oligonucleotide strands is reported. Each of the eight labels was easily detected by using the SERRS from silver nanoparticles to produce a unique, molecularly specific spectrum. The conditions were optimized to obtain the best signal enhancement, and linear concentration graphs at low oligonucleotide concentrations were obtained. At higher concentrations (above approximately 10(-)(8) mol dm(-)(3)), curvature was introduced into the concentration graphs with the exception of rhodamine 6G, TET, and FAM, which gave linearity over the entire concentration range studied. Detection limits as low as 0.5 fmol were obtained, with lower possible if a smaller sample was analyzed. Investigation was also carried out into the effect of a Tris-HCl buffer containing the surfactant Tween 20 to aid in the prevention of surface adhesion of the oligonucleotides to the sample vessels at ultralow concentrations. The Tween 20 allowed lower detection limits to be obtained for each of the labels studied. This study shows that the different dyes commonly used with oligonucleotides can give quantitative SERRS at concentration levels not possible when the same dyes are used with fluorescence detection.     

Comparison of surface-enhanced resonance Raman scattering and fluorescence for detection of a labeled antibody

A comparison is made of the quantitative detection of a labeled antibody by surface-enhanced resonance Raman scattering (SERRS) and by fluorescence using the same instrument with the same laser excitation source. The area under the curve for the fluorescence band is greater than for any single peak in the SERRS spectrum, but the broad fluorescence band is more difficult to discriminate from the background at low concentrations. Using the peak height of one SERRS band and the peak height at the fluorescence maximum, the detection limit for SERRS was lower (1.19 x 10-11 mol.dm-3) than that obtained using fluorescence (3.46 x 10-10 mol.dm-3). The SERRS detection limit is calculated for the concentration of the sample added, but compared to fluorescence, there is an additional dilution step due to the addition of the colloid and the extent of this dilution is dependent on assay format. For comparison with the detection limits found earlier with labeled oligonucleotides, SERRS was remeasured with a 10 s accumulation time, and the final concentration in the cuvette after colloid addition and before any adsorption to the silver was used to calculate a detection limit of 2.79 x 10-13 mol.dm-3. This is comparable to the detection limit found using a similar SERRS procedure for an oligonucleotide labeled with the same dye. This experiment is dependent on many parameters that could affect this result, including the nature of the SERRS substrate, the excitation wavelength, and the dye chosen. However, the result indicates that SERRS can give assay sensitivities comparable or better than fluorescence for quantitative direct assay determination, suggesting that the much greater potential for multiple analyte detection could be exploited.     

Quantitative analysis of mitoxantrone by surface-enhanced resonance Raman scattering

Mitoxantrone is an anticancer agent for which it is important to know the concentration in blood during therapy. Current methods of analysis are cumbersome, requiring a pretreatment stage. A method based on surface-enhanced resonance Raman scattering (SERRS) has been developed using a flow cell and silver colloid as the SERRS substrate. It is simple, sensitive, fast, and reliable. Both blood plasma and serum can be analyzed directly, but fresh serum is preferred here due to reduced fluorescence in the clinical samples available. Fluorescence is reduced further by the dilution of the serum in the flow cell and by quenching by the silver of surface-adsorbed material. The effectiveness of the latter process is dependent on the contact time between the serum and the silver. The linear range encompasses the range of concentrations detected previously in patient samples using HPLC methods. In a comparative study of a series of samples taken from a patient at different times, there is good agreement between the results obtained by HPLC and SERRS with no significant difference between them at the 95% limit. The limit of detection in serum using the final optimized procedure for SERRS was 4.0 x 10(-11) M (0.02 ng/mL) mitoxantrone. The ease with which the SERRS analysis can be carried out makes it the preferred choice of technique for mitoxantrone analysis.     

Comparison of surface-enhanced resonance Raman scattering from unaggregated and aggregated nanoparticles

The effect of excitation frequency and state of aggregation on the sensitivity obtained in ultratrace analysis using colloidal suspensions of silver nanoparticles and surface-enhanced resonance Raman scattering (SERRS) detection is explored to define suitable conditions for quantitative analysis. Two structurally similar dyes, only one of which causes aggregation, were used as analytes without the use of external aggregating agents, thus simplifying the surface chemistry and removing a major source of error. Addition of the nonaggregating dye caused no change in particle charge or size and no time-dependent aggregation as measured by zeta potential and particle size analysis. The most intense single-particle scattering was obtained using excitation at the wavelength of the plasmon resonance. Molecular resonance added approximately 2 orders of magnitude in sensitivity. Addition of the aggregating dye caused a reduction in surface charge of the particles and initiated a time-dependent aggregation process. However, constant SERRS with time is obtained at some excitation wavelengths probably because a constant number of clusters active at these wavelengths is maintained in the dynamic aggregation process. The additional enhancement caused by aggregation and molecular resonance is spread over a range of excitation frequencies. However, electronic spectra suggested that plasmon resonance enhancement would be effective at the longest wavelength of excitation used (785 nm), but there was a significant drop in intensity this far away from the absorbance maximum of the dye (429 nm). Thus, sensitive analysis using suspensions of single nanoparticles is feasible provided the excitation frequency used is close to that of the plasmon resonance frequency. Aggregation adds only an enhancement of approximately 6 in the experiments performed since only some particles in aggregates will have an active plasmon at any one wavelength, but the range of excitation wavelengths at which good enhancement is obtained is wider giving more flexibility if more complexity.     

SERRS. In situ substrate formation and improved detection using microfluidics

Surface-enhanced resonance Raman scattering (SERRS) of a model derivative of TNT was detected using a microflow cell designed within the framework of the lab-on-a-chip concept, using only the analyte and readily available reagents. The SERRS substrate, silver colloid, was prepared in situ, on-chip, by borohydride reduction of silver nitrate. The silver colloid was imaged within the chip using a white light microscope in either transmission or, due to the high reflectivity of the colloid, reflection mode. A fine stream of colloid approximately 30 microm in width was formed in a 250-microm-wide channel at the point where the colloid preparation reagents met. The chip was designed to produce a concentrated stream of colloid within a laminar regime, such that particles did not readily disperse into the fluid. One result of this was to reduce the effective volume of analysis. Attempts to deliberately disrupt this stream with microstructured pillars, fabricated in the fluidic channels, were unsuccessful. The chip was also designed to have the appropriate dimensions for detection using a modern Raman microscope system, which collects scattering from a very small volume. A dye derived from TNT was used as a model analyte. Quantitative behavior was obtained over 4 orders of magnitude with a detection limit of 10 fmol. This performance is between 1 and 2 orders of magnitude better than that achieved using a macroflow SERRS cell. The technique has the added advantage that both reagent consumption and effluent production are greatly reduced, leading to reduced operating costs and a decreased environmental impact     

Single-molecule detection using surface-enhanced resonance Raman scattering and Langmuir-Blodgett monolayers

The Langmuir-Blodgett (LB) technique has been used to obtain spatially resolved surface-enhanced resonance Raman scattering (SERRS) spectra of single dye molecules dispersed in the matrix of a fatty acid. The experimental results presented here mimic the original electrochemical surface-enhanced Raman scattering (SERS) work where the background bulk water did not interfere with the detection of the SERS signal of molecules adsorbed onto the rough silver electrode. LB monolayers of the dye in fatty acid have been fabricated on silver island films with a concentration, in average, of one probe molecule per micrometer square. The properties of single-molecule spectroscopy were investigated using micro-Raman including mapping and global images. Blinking of the SERRS signal was also observed.     

Quantitative enhanced Raman scattering of labeled DNA from gold and silver nanoparticles

Surface-enhanced resonance Raman scattering (SERRS) from silver nanoparticles using 514.5-nm excitation has been shown to offer huge potential for applications in highly sensitive multiplexed DNA assays. If the technique is to be applied to real biological samples and integrated with other methods, then the use of gold nanoparticles and longer wavelengths of excitation are desirable. The data presented here demonstrate that dye-labeled oligonucleotide sequences can be directly detected by SERRS using gold nanoparticles in a quantitative manner for the first time. The performance of gold and silver nanoparticles as SERRS substrates was assessed using 514.5-, 632.8-, and 785-nm excitation and a range of 13 commercially available dye-labeled oligonucleotides. The quantitative response allowed the limit of detection to be determined for each case and demonstrates that the technique is highly effective, sensitive, and versatile. The possibility of excitation at multiple wavelengths further enhances the multiplexing potential of the technique. The importance of effectively combining the optical properties of the nanoparticle and the dye label is demonstrated. For example, at 632.8-nm excitation, the dye BODIPY TR-X and gold nanoparticles make a strong SERRS combination with very little background fluorescence. This study allows the choice of nanoparticle and dye label for particular experimental setups, and significantly expands the applicability of enhanced Raman scattering for use in many disciplines.     

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.     

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

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

Surface-enhanced resonance Raman spectroscopy as an identification tool in column liquid chromatography

The compatibility of ion-pair reversed-phase column liquid chromatography and surface-enhanced resonance Raman spectroscopy (SERRS) for separation and identification of anionic dyes has been investigated, with emphasis on the at-line coupling via a thin-layer chromatography (TLC) plate. SERR spectra using silver sols were recorded both for aqueous solutions and for samples deposited on aluminum oxide and silica TLC plates at 514.5- and 457.9-nm laser excitation. For some dyes, the shorter wavelength was needed to diminish the fluorescence background. For aqueous solutions and for samples deposited on aluminum oxide, clear SERR spectra were obtained upon addition of poly(L-lysine); for the silica plates, the addition of nitric acid was required. Upon drying the plates, the SERRS signals decreased in intensity; simply adding a drop of water could largely restore them. At-line coupling of LC and SERRS was successfully achieved when using silica, but not aluminum oxide, plates. The application of a gradient, a high water content, and the presence of ion-pair reagents needed for the separation did not adversely affect the deposition and the recording of SERR spectra. The identification limits were 10-20 ng of deposited material, depending on the dye selected, which corresponded to injected concentrations of 5-10 microg mL(-1).     

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.     

Synthesis of high concentration colloid solution of silica-coated AgI nanoparticles

Methods for high concentration silica-coated silver iodide (AgI/SiO2) particles, which could be practically used as X-ray contrast agent, were examined. The first was a single-step method, which was to prepare AgI nanoparticles at an AgI concentration of 5 x 10(-3) M and coat the AgI nanoparticles with silica shell by a St?ber method. The second was a multiple-step method, which was to repeat a step for preparing a AgI/SiO2 particle colloid solution with 10(-3) M AgI 5 times for adjusting a final AgI concentration to 5 x 10(-3) M. In the two methods, dominant particle aggregation took place, though core-shell particles were also produced. The third was a salting-out method, which was to salt out AgI/SiO2 particles in their colloid solution prepared at an AgI concentration of 10(-3) M, remove supernatant by decantation, and redisperse the particles in a fresh solvent. Consequently, AgI/SiO2 particles with an AgI concentration as high as 0.05 M were successfully prepared with the salting-out method, and their core-shell structure was not damaged during the salting-out.     

Fluorescein isothiocyanate linked immunoabsorbent assay based on surface-enhanced resonance Raman scattering

By using fluorescein isothiocyanate (FITC) as a Raman probe, we have developed a simple and sensitive method for an immunoassay based on surface-enhanced resonance Raman scattering (SERRS). For the first time, a SERRS-based immunoassay on the bottom of a microtiter plate is reported. We have applied the main pretreatment method of enzyme-linked immunoabsorbent assay (ELISA) to the present study. In this method, SERRS spectra of FITC are measured after several continuous steps of antigen coating, blocking, antibody adding, and colloidal silver staining. Human immunoglobulin G (IgG) and FITC-antihuman IgG are used for the immunoreaction. The proposed method has several advantages for immunoassay. First, we can determine the concentration of antigens via the intensity of a SERRS signal of FITC molecules that are attached to antibodies without an enzyme reaction, and thus the process is simple and reagent saving. Second, one can obtain SERRS spectra of FITC directly from silver aggregates on the bottom of a microtiter plate without displacement. Third, by using SERRS of FITC, the present method is sensitive enough to detect antigens at the concentration of 0.2 ng/mL, which is comparable to ELISA. Results are presented to demonstrate that the proposed SERRS-based immunoassay may have great potential as a high-sensitivity and high-throughout immunoassay.     

Surface-enhanced spectra on D-gluconic acid coated silver nanoparticles

Coated silver (Ag) colloids synthesized with D-glucose permit the observation of surface-enhanced fluorescence (SEF) and surface-enhanced resonance Raman scattering (SERRS) of the rhodamine B (RhB) molecule. The organic coating formed during the synthesis of the Ag nanostructures was identified by its surface-enhanced Raman scattering (SERS) spectrum as D-gluconic acid. The RhB molecule is used to exemplify the distance dependence of SEF and SERRS on the coated Ag nanostructures. The fluorescence enhancement factor for RhB on D-gluconic acid coated silver nanoparticles was determined experimentally and estimated using a simple model. Further support for the plasmon enhancement is obtained from the fact that the measured fluorescence lifetime of RhB on the silver coated with D-gluconic acid is shorter than that found on a glass surface. A very modest enhancement factor is obtained, as expected for very short distance between RhB and the metal surface. Given the very thin metal-fluorophore separation, estimated from the size of the D-gluconic acid, the energy transfer or fluorescence quenching is still efficient and the SEF enhancement is just overcoming the energy transfer. Therefore, both SEF and SERRS are observed. Notably, the aggregation of coated nanoparticles also increases the enhancement factor for SEF.     

Analytical technique for label-free multi-protein detection based on Western blot and surface-enhanced Raman scattering

We have developed a new analytical procedure for label-free protein detection designated "Western SERS", consisting of protein electrophoresis, Western blot, colloidal silver staining, and surface-enhanced Raman scattering (SERS) detection. A novel method of silver staining for Western blot that uses a silver colloid, an excellent SERS-active substrate, is first proposed in the present study. During the process of silver staining, interactions between proteins and silver nanoparticles result in the emergence of SERS of proteins. In the present study, we use myoglobin (Mb) and bovine serum albumin (BSA) as model proteins. From different protein bands on a nitrocellulose (NC) membrane, we have observed surface-enhanced resonance Raman scattering (SERRS) spectra of Mb and SERS spectra of BSA. The proposed technique offers dual advantages of simplicity and high sensitivity. On one hand, after the colloidal silver staining, we can detect label-free multi-proteins directly on a NC membrane without digestion, extraction, and other pretreatments. On the other hand, the detection limit of the Western SERS is almost consistent with the detection limit of colloidal silver staining, and the SERRS detection limit of Mb is found to be 4 ng/band. This analytical method, which combines the technique of protein separation with SERS, may be a powerful protocol for label-free protein detection in proteomic research.     

8-hydroxyquinolinyl azo dyes: a class of surface-enhanced resonance Raman scattering-based probes for ultrasensitive monitoring of enzymatic activity

A series of surface-enhanced resonance Raman scattering (SERRS) based probes for the detection of lipase activity are reported. A number of novel SERRS-active 8-hydroxylquinolinyl azo dyes have been prepared and via synthetic esterification or subsequent enzymatic hydrolysis at the 8-hydroxyl position the SERRS signal can be "switched" on or off. In the first instance, the technique has been demonstrated for the successful detection of lipase from Pseudomonas cepacia, and these new compounds offer a limit of detection of 0.2 ng mL-1 enzyme, up to a 100-fold lower limit than observed for benzotriazolyl dyes used in previous studies. The chemical synthesis is straightforward and allows for facile introduction of a wide range of different masking groups, using commonly known synthetic methodologies. The potential for multiplexing analysis of enzyme activity using this technology is presented within.     

From micro to nano: analysis of surface-enhanced resonance Raman spectroscopy active sites via multiscale correlations

Effective correlation of data from a number of analytical techniques over length scales spanning several orders of magnitude is required to more fully investigate the active sites on silver nanoparticles that are responsible for surface-enhanced resonance Raman scattering (SERRS). In this paper, a method is presented that uses fluorescent beads as optical markers to allow direct correlation between a SERRS/fluorescence map and a transmission electron microscope (TEM) collage of the same area. Factors influencing the accuracy of the technique include the flatness of the substrate, the size of the fluorescent beads, and the strength of the signal from the fluorescent beads. When the effect of each of these factors on the technique is addressed, a simple and accurate correlation between the optical spectroscopy and the electron microscopy is achieved. A statistically significant number of particles can then be easily and reliably located and characterized at both optical limits, by SERRS, and with subnanometer resolution in the high-resolution TEM. Examples of HRTEM images and the locations of these particles within the SERRS map/TEM collage are presented. Our findings reveal that the relative SERRS activity of single particles is very low compared to dimers and larger aggregates of particles. The relative activity of dimers is estimated to be 12.4 times greater than single particles, and as the number of particles in the aggregate increase, the relative SERRS activity also increases. The relative SERRS activities of single particles/dimers/trimers/aggregates of 4-9 particles/aggregates of 10-20 are estimated to be 1/12.4/15.6/23.2/43.     

Adsorption of reactive dyes from aqueous solutions by fly ash: kinetic and equilibrium studies

Adsorption kinetic and equilibrium studies of three reactive dyes namely, Remazol Brillant Blue (RB), Remazol Red 133 (RR) and Rifacion Yellow HED (RY) from aqueous solutions at various initial dye concentration (100–500 mg/l), pH (2–8), particle size (45–112.5 μm) and temperature (293–323 K) on fly ash (FA) were studied in a batch mode operation. The adsorbent was characterized with using several methods such as SEM, XRD and FTIR. Adsorption of RB reactive dye was found to be pH dependent but both RR and RY reactive dyes were not. The result showed that the amount adsorbed of the reactive dyes increased with increasing initial dye concentration and contact time. Batch kinetic data from experimental investigations on the removal of reactive dyes from aqueous solutions using FA have been well described by external mass transfer and intraparticle diffusion models. It was found that external mass transfer and intraparticle diffusion had rate limiting affects on the removal process. This was attributed to the relatively simple macropore structure of FA particles. The adsorption data fitted well with Langmuir and Freundlich isotherm models. The optimum conditions for removal of the reactive dyes were 100 mg/l initial dye concentration, 0.6 g/100 ml adsorbent dose, temperature of 293 K, 45 μm particle size, pH 6 and agitation speed of 250 rpm, respectively. The values of Langmuir and Freundlich constants were found to increase with increasing temperature in the range 135–180 and 15–34 mg/g for RB, 47–86 and 1.9–3.7 mg/g for RR and 37–61 and 3.0–3.6 mg/g for RY reactive dyes, respectively. Different thermodynamic parameters viz., changes in standard free energy, enthalpy and entropy were evaluated and it was found that the reaction was spontaneous and endothermic in nature.     

Reproducible surface-enhanced Raman scattering spectra of bacteria on aggregated silver nanoparticles

Surface-enhanced Raman scattering (SERS) is proven to be a powerful technique for rapid identification and discrimination of microorganisms. However, due to the heterogeneous nature of the samples, the acquisition of reproducible spectra hinders the further development of the technique. In this study, we demonstrate the influence of the experimental conditions on SERS spectra. Then, we report a simple sample preparation method coupled with a light microscope attached to a Raman spectrometer to find a proper spot on the sample to acquire reproducible SERS spectra. This method utilizes the excited surface plasmons of the aggregated silver nanoparticles to visualize the spots on the sample. The samples are prepared using the concentrated silver colloidal solutions. The collection time for one spectrum is 10 s and each spectrum is a very good representative of the other spectra acquired from the same sample. The nature of the surface charge of the silver nanoparticles influences the spectral features by determining the strength of the interactions between nanoparticles and bacteria and the aggregation properties of the nanoparticles. Although increasing the colloid concentration in the sample resulted in reproducible spectra from arbitrary points on the sample, a great variation from sample to sample prepared with the different colloidal solution concentrations is observed.     

New Approach for the Surface Enhanced Resonance Raman Scattering (SERRS) Detection of Dopamine at Picomolar (pM) Levels in the Presence of Ascorbic Acid

The development of a novel surface-enhanced resonance Raman scattering (SERRS) platform that allows fast and sensitive detection of dopamine (DA) has been reported. The iron-nitrilotriacetic acid attached silver nanoparticle (Ag-Fe(NTA)) substrate provides remarkable sensitivity and reliable repeatability. The advantages of both the surface functionalization for specific analytes and the SERRS are integrated into a single functional unit. While the silver core gives the necessary enhancing properties, the Fe-NTA receptors can trap DA adjacent the silver core and the NTA-Fe-DA complex formed provides resonance enhancement with a 632.8 nm laser. DA could be detected in pM level without any pretreatment with a reliable discrimination against AA, by utilizing low laser power (10 mW) and short data acquisition time (10 s). The high sensitivity along with the improved selectivity of this sensing approach is a significant step toward molecular diagnosis of Parkinson's disease.