Quantitative SERRS for DNA sequence analysis

SERRS is an extremely sensitive and selective technique which when applied to the detection of labelled DNA sequences allows detection limits to be obtained which rival, and in most cases are better than, fluorescence. In this tutorial review the conditions are explored which enable the successful detection of DNA using SERRS. The enhancing surface which is used is crucial and in this case suspensions of nanoparticles were the focus as they allow quantitative behaviour to be achieved in systems analogous to current fluorescence based approaches. The aggregation conditions required to obtain SERRS of DNA affect the sensitivity and the reproducibility and we describe the use of spermine as an effective aggregating agent to achieve excellent reproducibility and sensitivity. The nature of the label which is used, be it fluorescent or non-fluorescent, positively or negatively charged, also affects the SERRS response and these conditions are again discussed. Finally, we show how to detect a specific target DNA sequence in a meaningful diagnostic assay using SERRS and how the approaches described previously in the review are vital to the success of such approaches.     

DNA detection by surface enhanced resonance Raman scattering (SERRS)

This Education article outlines the different ways in which surface enhanced resonance Raman scattering (SERRS) can be used for the detection of DNA. The use of various different SERRS detection strategies that have allowed both sensitive and selective detection to be obtained is covered. Detection of DNA by SERRS involves the use of a dye with the DNA, whether as an intercalator or by direct covalent attachment. This generates strong SERRS signals that indicate the presence of the specific DNA sequence. The SERRS detection of DNA in different molecular biological assays is also discussed.     

Quantitative detection of dye labelled DNA using surface enhanced resonance Raman scattering (SERRS) from silver nanoparticles

The detection of dye labelled DNA by surface enhanced resonance Raman scattering (SERRS) is reported. The dye labels used are commercially available and have not previously been used as SERRS dyes. Detection limits using two excitation frequencies were determined for each label. This expands the range of labels which can be used for surface enhanced resonance Raman scattering with silver nanoparticles.     

Quantitative analysis of methyl green using surface-enhanced resonance Raman scattering

Surface-enhanced resonance Raman scattering (SERRS) spectra of aqueous solutions of the triphenylmethane dye methyl green have been obtained for the first time by use of citrate-reduced silver colloids and a laser excitation wavelength of 632.8 nm. Given the highly fluorescent nature of the analyte, which precluded collection of normal Raman spectra of the dye in solution and powdered state, it was highly encouraging that SERRS spectra showed no fluorescence due to quenching by the silver sol. The pH conditions for SERRS were optimised over the pH range 0.5–10 and the biggest enhancement for SERRS of this charged dye was found to be at pH 2.02, thus this condition was used for quantitative analysis. SERRS was found to be highly sensitive and enabled quantitative determination of methyl green over the range 10⁻⁹ to 10⁻⁷ mol dm⁻³. Good fits to correlation coefficients were obtained over this range using the areas under the vibrational bands at 1615 and 737 cm⁻¹. Finally, a limit of detection of 83 ppb was calculated, demonstrating the sensitivity of the technique.     

Single molecular detection of a perylene dye dispersed in a Langmuir-Blodgett fatty acid monolayer using surface-enhanced resonance Raman scattering

The Langmuir-Blodgett (LB) monolayer technique was used to fabricate single molecule LB monolayer containing bis(phenethylimido)perylene (PhPTCD), a red dye dispersed in arachidic acid (AA) with an average doping of 1 molecule per microm2. The monolayer was transferred onto Ag island films to obtain spatially resolved surface-enhanced resonance Raman scattering (SERRS) spectra. The mixed LB monolayers were fabricated with a concentration, on average, of 1, 6, 19 and 118 PhPTCD molecules per microm2 in AA. The AA provides a two-dimensional host matrix whose background signal does not interfere with the detection of the probe molecule's SERRS signal. The properties of the single molecule detection were investigated using micro-Raman with a 514.5-nm laser line. The Ag island surfaces coated with the LB monolayer were mapped with spatial steps of 3 microm and global chemical imaging of the most intense SERRS band in the spectrum was also recorded. The SERRS and surface-enhanced fluorescence (SEF) of the neat and single molecule LB monolayer were recorded in a temperature range from liquid nitrogen to + 200 degrees C. Neat PhPTCD LB monolayer spectra served as reference for the identification of characteristic signatures of the single molecule behavior. The spatial resolution of Raman-microscopy experiments, the multiplicative effect of resonance Raman and SERRS, and the high sensitivity of the new dispersive Raman instruments, allow SERRS to be part of the family of single molecular spectroscopies.     

Semi-quantitative trace analysis of nuclear fast red by surface enhanced resonance Raman scattering

The dye nuclear fast red has been detected and determined semi-quantitatively by means of surface enhanced resonance Raman scattering (SERRS) and surface enhanced Raman scattering (SERS), using laser exciting wavelengths of 514.5 and 632.8 nm, respectively, by employing a citrate-reduced silver colloid. A good linear correlation is observed for the dependence of the intensities of the SERRS bands at 989 cm⁻¹ (R=0.9897) and 1278 cm⁻¹ (R=0.9872) on dye concentration over the range 10⁻⁹ to 10⁻⁷ M, when using an exciting wavelength of 514.5 nm. At dye concentrations above 10⁻⁷ M, the concentration dependence of the SERRS signals is non-linear. This is almost certainly due to the coverage of the colloidal silver particles being in excess of a full monolayer of the dye. A linear correlation is also observed for the dependence of the intensities of the SERS bands at 989 cm⁻¹ (R=0.9739) and 1278 cm⁻¹ (R=0.9838) on the dye concentration over the range 10⁻⁸ to 10⁻⁶ M when using an exciting wavelength of 632.8 nm. Strong fluorescence prevented collection of resonance Raman scattering (RRS) spectra from powdered samples or aqueous solutions of the dye using an exciting wavelength of 514.5 nm, but weak bands were observed in the spectra obtained from both powdered and aqueous samples of the dye using an exciting wavelength of 632.8 nm. A study of the pH dependence of SERRS/SERS and UV–VIS absorption spectra revealed the presence of different ionisation states of the dye. The limits of detection for nuclear fast red by SERRS (514.5 nm), SERS (632.8 nm) and visible spectroscopy (535 nm) are 9, 89 and 1000 ng ml⁻¹, respectively.     

Surface enhanced resonance Raman scattering imaging of Langmuir-Blodgett monolayers of bis (benzimidazo) thioperylene.

The synthesis, spectroscopic characterization and surface-enhanced spectroscopy of a new electro active organic material bis (benzimidazo) thioperylene (Monothio BZP) are reported. Langmuir monolayers of Monothio BZP were successfully formed on water subphase and characterized by the pi-A surface-pressure area isotherm. Langmuir-Blodgett (LB) monomolecular layers of Monothio BZP were fabricated onto glass substrates and onto silver island films for surface-enhanced spectroscopic studies. The results of surface-enhanced resonance Raman scattering (SERRS), SERRS imaging and surface-enhanced fluorescence (SEF) studies for Monothio BZP LB monolayers are reported. Raman imaging (global imaging and point-by-point mapping) of the SERRS signal for a single monomolecular layer on silver islands were obtained using the 514.5 nm laser line. The SERRS imaging permits a visualization of the variation of the SERRS intensity across of the rough metal surface. The SEF was recorded for the excimer emission of aggregates in the LB film. The distance dependence and the enhancement factor of SEF were determined using fatty acid spacing layers. A temperature dependence study of the LB monolayer SERRS and SEF spectra was carried out between -190 degrees and + 200 degrees C confirming the thermal stability of the LB monolayer on silver. The specificity and the sensitivity of SERRS signal on metal island films was probed using mixed LB films with 0.01% molecular ratio of Monothio BZP in Arachidic Acid (AA). The micro-Raman SERRS spectra from ca. 10(-3) attomole of the dye were recorded.     

Immunoassay for P38 MAPK using surface enhanced resonance Raman spectroscopy (SERRS)

A micro-bead sandwich assay for P38 mitogen-activated protein kinase using surface enhanced resonance Raman spectroscopy (SERRS) detection is reported. Monoclonal capture antibodies were immobilised on a solid phase of magnetic micro-beads with secondary detection using a rhodamine-labelled antibody. Quantitative SERRS detection of the secondary antibody was possible with a limit of detection of 9.5 x 10(-12) mol dm(-3). The sandwich assay was quantitative and sensitive to 6 ng ml(-1). The mechanism of the SERRS detection in the immunoassay was investigated. The addition of SERRS aggregating agents causes the dissociation of the immuno-complex from the magnetic beads. Scanning electron microscopy images indicate that the colloidal suspension rather than adsorbed silver nanoparticles on the beads provide the SERRS signals, that the aggregate size is partially controlled and that there is some inhomogeneity in the distribution of organic matter on the nanoscale.     

Single molecule level detection of allophycocyanin by surface enhanced resonance Raman scattering

Single molecule level detection of the near-infrared fluorescent protein allophycocyanin (APC) has been achieved using surface enhanced resonance Raman scattering (SERRS). The detection limit using the peak height of the 440 cm(-1) band was 1 x 10(-13) mol l(-1), compared to 2 x 10(-12) mol l(-1) for the fluorescence peak at 660 nm.     

SERRS-based enzymatic probes for the detection of protease activity

Measurement of protease activity, for the first time using SERRS as a detection method, is reported herein. Synthetic introduction of phenylalanine to a benzotriazole azo dye allows the SERRS response to be "switched off" and subsequent exposure to protease restores the SERRS response. The substrates exhibit varying reactivity for a range of proteases and allow for in situ, real-time analysis of protease reactivity. A limit of detection for one protease, Subtilisin carlsberg, was investigated and was established to be 50 ng ml-1.     

Sample preparation for evaluation of detection limits in X-ray fluorescence spectrometry.

The influence of analyte mass concentration on determination of detection limits in X-ray fluorescence spectrometry has been investigated experimentally. Both the total reflection X-ray fluorescence (TXRF) and the conventional energy-dispersive X-ray fluorescence techniques have been used to derive the dependence of analyte mass concentration on the values of detection limits. Results obtained indicate that values of detection limits are optimum, or in other words, they are closer to the true detection limit of the technique, when analyte concentrations are in the range of 10 times of the detection limit.     

Reproducible SERRS from structured gold surfaces

Metallic substrates with ordered spherical cavities have been shown to be very effective for surface-enhanced Raman scattering (SERS) and can be fabricated reproducibly using electrodeposition. The sensitivity of detection is increased by several orders of magnitude by using surface-enhanced resonance Raman scattering (SERRS). In this report we demonstrate SERRS for the first time on electrodeposited gold films templated with colloidal spheres and demonstrate the reproducibility of the response. We also obtain a direct comparison between SERRS and SERS by choosing two dyes, Cy5 and Cy3, which are similar in structure but differ in their excitation maxima, such that one is resonant and the other non-resonant with our laser excitation. As expected, the resonant enhancement is found to be of the order of 10(3) over and above that for SERS. The net SERRS enhancements are shown to be of the order of 10(9). We also find that the resonant enhancement profile of the different peaks for the chromophore follows the plasmonic resonance absorption spectrum obtained for the structured surface.     

SERRS immunoassay for quantitative human CRP analysis

SERRS has been used for the first time for the measurement of C-reactive protein (CRP) in an immunoassay. CRP, a biological marker for the diagnosis of infection and inflammation, is quantified in an ELISA using conventional reagents, but the usual colorimetric detection step is replaced by SERRS detection, offering improved sensitivity and potential for multiplexing analysis.     

Construction and performance of a time-multiplex multiple-slit flame atomic fluorescence spectrometer for multi-element analysis

The design and performance characteristics of a new multi-element flame atomic fluorescence spectrometer are presented. Radiation from four hollow-cathode tubes is directed onto an unsheathed air—hydrogen flame. The resulting atomic fluorescence is viewed by a special monochromator with a separate exit slit for each element. The light exiting from all slits is directed to a single photomultiplier tube. The fluorescence signals from different elements are distinguished by a time multiplex approach. Single-element detection limits for ten elements and multi-element detection limits for four elements are presented. The degradation of detection limits by flame background emission noise and effect of flame composition on performance are discussed. Better than 1% precision is obtained for moderate analyte concentrations.     

Laser-excited fluorescence line-narrowing: an analytical study

The limits of detection obtained by fluorescence line-narrowing spectroscopy for 6 polynuclear aromatic hydrocarbons are compared with those obtained by low-temperature and room-temperature fluorescence, and the merits of the technique are discussed.     

Semi-quantitative analysis of indigo by surface enhanced resonance Raman spectroscopy (SERRS) using silver colloids

In this paper we report for the first time semi-quantitative analysis of indigo using surface enhanced Raman spectroscopy (SERS) and surface enhance resonance Raman spectroscopy (SERRS). Indigo, a dye widely used today in the textile industry, has been used, historically, both as a dye and as a pigment; the latter in both paintings and in printed material. The molecule is uncharged and largely insoluble in most solvents. The application of SERS/SERRS to the semi-quantitative analysis of indigo has been examined using aggregated citrate-reduced silver colloids with appropriate modifications to experimental protocols to both obtain and maximise SERRS signal intensities. Good linear correlations are observed for the dependence of the intensities of the SERRS band at 1151 cm(-1) using laser exciting wavelengths of 514.5 nm (R=0.9985) and 632.8 nm (R=0.9963) on the indigo concentration over the range 10(-7)-10(-5) and 10(-8)-10(-5) mol dm(-3), respectively. Band intensities were normalised against an internal standard (silver sol band at 243 cm(-1)). Resonance Raman spectra (RRS) of aqueous solutions of indigo could not be collected because of its low solubility and the presence of strong fluorescence. It was, however, possible to obtain RS and RRS spectra of the solid at each laser excitation wavelength. The limits of detection (L.O.D.) of indigo by SERS and SERRS using 514.5 and 632.8 nm were 9 ppm at both exciting wavelengths. Signal enhancement by SERS and SERRS was highly pH dependent due to the formation of singly protonated and possibly doubly protonated forms of the molecule at acidic pH. The SERS and SERRS data provide evidence to suggest that an excess of monolayer coverage of the dye at the surface of silver colloids is observed at concentrations greater than 7.85x10(-6) mol dm(-3) for each exciting wavelength. The data reported herein also strongly suggest the presence of multiple species of the indigo molecule.     

Quantitative evaluation of blinking in surface enhanced resonance Raman scattering and fluorescence by electromagnetic mechanism

We analyze blinking in surface enhanced resonance Raman scattering (SERRS) and surface enhanced fluorescence (SEF) of rhodamine 6G molecules as intensity and spectral instability by electromagnetic (EM) mechanism. We find that irradiation of intense NIR laser pulses induces blinking in SERRS and SEF. Thanks to the finding, we systematically analyze SERRS and SEF from stable to unstable using single Ag nanoparticle (NP) dimers. The analysis reveals two physical insights into blinking as follows. (1) The intensity instability is inversely proportional to the enhancement factors of decay rate of molecules. The estimation using the proportionality suggests that separation of the molecules from Ag NP surfaces is several angstroms. (2) The spectral instability is induced by blueshifts in EM enhancement factors, which have spectral shapes similar to the plasmon resonance. This analysis provides us with a quantitative picture for intensity and spectral instability in SERRS and SEF within the framework of EM mechanism.     

STRUCTURAL AND FUNCTIONAL INTEGRITY OF THE PHOTOSYSTEM II REACTION CENTER ON SILVER ELECTRODES: FLUORESCENCE AND REDOX PROBES

Two simple and sensitive methods have been developed to assess the structural and functional integrity of isolated photosystem II reaction centers deposited on a roughened Ag electrode. Surface-enhanced resonance Raman scattering (SERRS) spectra useful for ascertaining structural information can be obtained from biological materials with this technique. The first method presented is based on observing differences in the fluorescence emission properties of reaction centers; these depend on the activity of the material. The second is based on the observation of changes in Raman bands that are sensitive to the redox state of cytochrome b₅₅₉ present in the reaction center complex. It is concluded that the conditions used here to obtain SERRS spectra do not affect the structural or functional integrity of the isolated photosystem II reaction center complex. In principle these approaches also could be used with other chromoproteins.     

Elucidation of interaction between metal-free tetraphenylporphine and surface Ag atoms through temporal fluctuation of surface-enhanced resonance Raman scattering and background-light emission

We have observed simultaneously temporal fluctuation of surface-enhanced resonance Raman scattering (SERRS) and its background-light emission from single Ag nanoaggregates that were adsorbed with metal-free tetraphenylporphine (H(2)TPP) molecules. We found that temporally stable SERRS spectra showed clearly a SERRS band that is attributed to a stretching mode of a chemical bond between a carbon atom and a non-hydrogenated nitrogen atom (C(alpha)-N). This stretching mode was not observed in regular resonance Raman spectra which are free from surface enhancement. On the other hand, we also found that temporally unstable SERRS spectra did not clearly show a C(alpha)-N stretching mode in SERRS bands. Furthermore, temporally stable SERRS spectra were accompanied by temporally stable background-light emission. Kobayashi et al. [J. Phys. Chem. 1985, 89, 5174] reported that formation of an Ag-N bond between surface Ag atoms and non-hydrogenated N atoms in a pyrrole ring enhances the intensity of a C(alpha)-N stretching mode. Thus, the observed relationship between clear appearance of a C(alpha)-N stretching mode and temporal stability of SERRS plus background-light emission strongly suggests that formation of a stable Ag-N bond suppresses fluctuation of both SERRS and background-light emission. Furthermore, the observed relationship implies that chemical contribution to SERRS is stabilization of H(2)TPP molecules that are adsorbed on SERRS-active sites by formation of Ag-N bonds. Additionally, we attributed background-light emission to luminescence of complexes between H(2)TPP molecules and surface Ag atoms considering possible formation of Ag-N bonds, synchronized SERRS intensity with background-light emission intensity, blue-shifted background-light emission maxima from normal fluorescence maxima, and previous reports related to electronic structures of H(2)TPP molecules on Ag surfaces.     

Interfacing capillary electrophoresis and surface-enhanced resonance Raman spectroscopy for the determination of dye compounds

The at-line coupling of capillary electrophoresis (CE) and surface-enhanced resonance Raman spectroscopy (SERRS) was optimized for the separation and subsequent spectroscopic identification of charged analytes (dye compounds). Raman spectra were recorded following deposition of the electropherogram onto a moving substrate. To this end a new interface was developed using a stainless steel needle as a (grounded) cathode. The outlet end of the CE capillary was inserted into this metal needle; CE buffer touching the needle tip served as the electrical connection for the CE separation. A translation table was used to move the TLC plate at a constant speed during the deposition. The distance between the tip of the fused silica column and the TLC plate was kept as small as possible in order to establish a constant bridge-flow, while avoiding direct contact. The dyes Basic Red 9 (BR9), Acid Orange 7 (AO7) and Food Yellow 3 (FY3) were used as test compounds. After CE separation in a 20 mM borate buffer at pH 10, after deposition, concentrated silver colloid was added to each analyte spot, followed by irradiation with 514.5 nm light from an argon ion laser to record the SERRS signal using a Raman microscope. Different types of silver colloids were tested: Lee–Meisel type (citrate), borate, and gold-coated silver. BR9 (positively charged) gave much more intense SERRS spectra than the two negatively charged dyes. For BR9 and AO7 the citrate-coated Lee–Meisel colloid yielded the most intense SERRS spectra. The CE–SERRS system was used to separate and detect the negatively charged dyes. Silver colloid and nitric acid (to improve adsorption) were added post-deposition. Even though their chemical structures are very similar, AO7 and FY3 could be readily distinguished based on their SERRS spectra. The limits of detection (S/N=3) of the CE–SERRS system ranged from 6.7×10^–5 M (2.6×10^–12 mol injected) for FY3 down to 1.8×10^–6 M (7.0×10^–14 mol injected) for BR9.D. Arráez Román and E. Efremov contributed equally to this work.