Understanding tip‐enhanced Raman spectra of biological molecules: a combined Raman,SERS and TERS study

Although conventional Raman, surface‐enhanced Raman (SERS) and tip‐enhanced Raman spectroscopy (TERS) have been known for a long time, a direct, thorough comparison of these three methods has never been carried out. In this paper, spectra that were obtained by conventional Raman, SERS (on gold and silver substrates) and TERS (in ‘gap mode’ with silver tips and gold substrates) are compared to learn from their differences and similarities. Because the investigation of biological samples by TERS has recently become a hot topic, this work focuses on biologically relevant substances. Starting from the TER spectra of bovine serum albumin as an example for a protein, the dipeptides Phe–Phe and Tyr–Tyr and the tripeptide Tyr–Tyr–Tyr were investigated. The major findings were as follows. (1) We show that the widely used assumption that spectral bands do not shift when comparing SER, TER and conventional Raman spectra (except due to binding to the metal surface in SERS or TERS) is valid. However, band intensity ratios can differ significantly between these three methods. (2) Marker bands can be assigned, which should allow one to identify and localize proteins in complex biological environments in future investigations. From our results, general guidelines for the interpretation of TER spectra are proposed. Copyright © 2012 John Wiley & Sons, Ltd.     

Detection of biologically active diterpenoic acids by Raman Spectroscopy

Three poorly detectable, biologically active diterpenoic acids, kaurenoic, abietic, and gibberellic acid, were studied by using different modes of Raman spectroscopy. Because of their structural similarities, in the absence of strongly polarizable groups, conventional Raman spectroscopy is not suitable for their unambiguous identification, especially not in solution. We attempted to increase the sensitivity by applying UV‐resonance Raman spectroscopy and surface‐enhanced Raman spectroscopy (SERS) techniques. The UV‐Raman spectra of the three compounds in ethanol/water 50:50 showed only very few enhanced Raman lines. SERS spectra with 514‐nm excitation with Ag colloids were also relatively weak. The best SERS spectra were obtained with 785‐nm excitation on a novel nanostructured substrate, ‘black silicon’ coated with a 400‐nm gold layer. The spectra showed clear differences, and these ‘fingerprints’ would be suitable for the unambiguous identification of these diterpenoic acids. Copyright © 2009 John Wiley & Sons, Ltd.     

Raman spectroscopy of dipyrrins: nonresonant,resonant and surface‐enhanced cross‐sections and enhancement factors

Detailed studies of the mechanism of surface‐enhanced (resonance) Raman spectroscopy (SE(R)RS), and its applications, place a number of demands on the properties of SERS scatterers. With large Raman cross‐sections, versatile synthetic chemistry and complete lack of fluorescence, free dipyrrins meet these demands but the Raman and SE(R)RS spectroscopy of free dipyrrins is largely unknown. The first study of the Raman spectroscopy of free dipyrrins is therefore presented in this work. The nonresonant Raman, resonant Raman and surface‐enhanced Raman spectra of a typical meso aryl‐substituted‐dipyrrin are reported. Absolute differential cross‐sections are obtained for excitation wavelengths in the near infrared and visible region, in solution phase and for dipyrrin adsorbed on the surface of silver nanoparticles. Raman enhancement factors for SERRS and resonance Raman are calculated from the observed differential cross‐sections. The magnitudes of the resonantly enhanced cross‐sections are similar to those recently reported for strong SERS dyes such as Rhodamine 6G and Crystal Violet. Free dipyrrins offer the advantages of existing SERS dyes but without the drawback of strong fluorescence. Free dipyrrins should therefore find applications in all areas of Raman spectroscopy including fundamental studies of the mechanisms of SERS and bioanalytical and environmental applications. Copyright © 2011 John Wiley & Sons, Ltd.     

Sensitive Raman spectroscopy of lipids based on drop deposition using DCDR and SERS

Raman spectroscopy is widely used for study of lipids and membrane models. A severe limitation of this technique lies in the low Raman cross section requiring high sample concentrations. We report sensitive detection of synthetic 1,2‐dimyristoyl‐3‐trimethylammonium‐propane (DMTAP) lipid employing two Raman techniques with improved sensitivity: drop coating deposition Raman (DCDR) and surface‐enhanced Raman scattering (SERS) spectroscopies. DCDR provided well‐reproducible DMTAP spectra without considerable loss of its solution properties if measured from the ‘coffee ring’ pattern of a drop dried on a SpectRIM^TM plate. DMTAP was detected at ~10 μM initial solution concentration, which is about three orders of magnitude lower than that for conventional Raman spectroscopy. Moreover, SERS spectra from dried ring of Ag hydrosol/DMTAP system were obtained down to ~0.3 μM DMTAP concentration, which means that sensitivity of SERS is about five orders of magnitude higher than that of conventional Raman spectroscopy. In contrast to the DCDR technique, good SERS spectra of DMTAP were obtained only from some spots of the ring containing big nanoparticle aggregates, and the structural properties of DMTAP were significantly perturbed by adsorption on the Ag nanoparticles. Copyright © 2013 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman spectra of dimethoate and omethoate

Surface‐enhanced Raman scattering (SERS) spectra are presented and analyzed for two important organophosphate pesticides, dimethoate (DMT) and omethoate (OMT). Very detailed SERS spectra were obtained by aggregated Ag hydrosols, both in aqueous suspension and dried on a glass substrate. The SERS and ordinary Raman spectra of DMT do not resemble each other, suggesting that a chemical reaction immediately occurs when DMT is adsorbed onto the metal surface. We propose that the reaction product is OMT, which is the oxygen analog of DMT, on the basis of the Raman and SERS spectra of OMT. Further support is derived from the calculated Raman spectra of DMT and OMT. Minor wavenumber and intensity differences that are observed between the SERS spectra of DMT reaction product and those of OMT could be related to different metal/adsorbate interaction modes. The results can be useful in the development of new analytical methods for the determination of pesticide residues in food. Copyright © 2010 John Wiley & Sons, Ltd.     

Characterization of polycyclic aromatic hydrocarbons using Raman and surface‐enhanced Raman spectroscopy

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous atmospheric pollutants and food contaminants, which exhibit potent carcinogenicity, mutagenicity, and teratogenicity. Vibrational spectroscopy techniques, especially Raman spectroscopy and surface‐enhanced Raman spectroscopy (SERS), can be potentially used as an alternative technique to liquid and gas chromatography in PAH analysis. However, there is limited information on the intrinsic Raman and SERS fingerprints of PAHs. In this study, we have acquired the Raman and SERS spectra of seven PAH compounds and compared their experimental spectra with theoretical Raman spectra calculated by density function theory (DFT). The vibrational modes corresponding to the Raman peaks have also been assigned using DFT. Characteristic Raman and SERS peaks have been identified for five PAH compounds, and the limits of detection were estimated. Such information could be useful for developing SERS assays for simple and rapid PAH identification. Copyright © 2014 John Wiley & Sons, Ltd.     

Raman spectroscopy measurement of levofloxacin lactate in blood using an optical fiber nano‐probe

Liquid chromatography and mass spectrometry were time‐consuming and expensive as the main methods for the drug analysis at present, and the samples must be pretreated. The Raman spectroscopy measurement methods were fast and simple, so the Raman spectroscopy methods for the drug analysis were explored in this paper. An optical fiber nano‐probe coated with gold nanoparticles was fabricated and used with surface‐enhanced Raman spectroscopy (SERS) to measure levofloxacin lactate. The resulting SERS spectra of levofloxacin lactate in mouse blood that was detected by the optical fiber nano‐probe clearly showed the characteristic wave numbers of levofloxacin lactate, indicating that optical fiber nano‐probes can be used with spectral techniques to analyze drugs in vitro or potentially even in vivo. Copyright © 2015 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman scattering of protoberberine alkaloids

Quaternary protoberberine alkaloids are a class of natural dyes characterized by bright colors ranging from yellow to orange. As they present a strong fluorescence emission, their analysis by Raman spectroscopy is limited to specific techniques such as Fourier transform (FT)‐Raman and spectral shift Raman techniques such as shifted subtracted Raman difference spectroscopy (SSRDS) and shifted excitation Raman difference spectroscopy (SERDS). In a previous article, we successfully used surface‐enhanced Raman scattering (SERS) in the analysis of the alkaloid dye berberine in an ancient textile. The examination of the Raman and SERS spectra of berberine in combination with density functional theory (DFT) calculations indicated a flat adsorption geometry of the molecule on the Ag surface. In this article we extend that work to the study of related protoberberine alkaloids, palmatine, jatrorrhizine, and coptisine. The same adsorption geometry as in berberine was deduced. We found that the four alkaloids, although minimally different in their chemical structures, could be differentiated by the position of marker bands. Those bands are the most enhanced ones in the SERS spectra, which appear in the 700–800 cm⁻¹ region. Copyright © 2008 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman spectroscopy for trace‐level detection of explosives

The detection of explosives and their associated compounds for security screening is an active area of research and a wide variety of detection methods are involved in this very challenging area. Surface‐enhanced Raman scattering (SERS) spectroscopy is one of the most sensitive tools for the detection of molecules adsorbed on nano‐scale roughened metal surface. Moreover, SERS combines high sensitivity with the observation of vibrational spectra of species, giving complete information on the molecular structure of material under study. In this paper, SERS was applied to the detection of very small quantities of explosives adsorbed on industrially made substrates. The spectra were acquired with a compact Raman spectrometer. Usually, a high signal‐to‐noise (S/N) spectrum, suitable for identification of explosive molecules down to few hundreds of picograms, was achieved within 30 s. Our measurements suggest that it is possible to exploit SERS using a practical detection instrument for routine analysis. Copyright © 2010 John Wiley & Sons, Ltd.     

An improved self‐assembly gold colloid film as surface‐enhanced Raman substrate for detection of trace‐level polycyclic aromatic hydrocarbons in aqueous solution

To detect trace‐level polycyclic aromatic hydrocarbons, some investigations of an improved self‐assembly method are carried out using gold colloid films for the preparation of the surface‐enhanced Raman scattering (SERS)‐active substrate. Extinction spectra and scanning electron microscopy images reveal that controllable surface plasmonic metal substrates can be obtained by increasing the temperature of (3‐aminopropyl)trimethoxysilane solution up to 64.5 °C. The SERS‐active substrates have a high enhancement factor, and they can be both easily prepared and reproducible. With the use of these substrates, different concentrations of pyrene and anthracene in aqueous solutions were detected by SERS. A further enhancement can be supported by shifted excitation Raman difference spectroscopy. Raman signals of pyrene and anthracene adsorbed on gold colloid substrates up to limits of detection at 5 and 1 nmol/l, respectively, can be obtained. The quantitative analysis shows the possibility of in situ detection of polycyclic aromatic hydrocarbons while such gold colloid film serves as a SERS‐active substrate. Copyright © 2012 John Wiley & Sons, Ltd.     

Proline and hydroxyproline deposited on silver nanoparticles. A Raman,SERS and theoretical study

The Raman and surface‐enhanced Raman scattering (SERS) spectra of l ‐proline (Pro) and trans‐4‐hydroxy‐ l ‐proline (Hyp) were recorded. SERS spectra were obtained on colloidal Ag prepared by reduction with hydroxylamine. Allowing sufficient time for Pro and Hyp to adjust in the colloidal solution resulted fundamentally in obtaining unique and reproducible SERS spectra. Hyp stabilizes on the surface more rapidly than Pro. The spectral analysis indicates that Pro interacts with the Ag surface through the carboxylate group. The interaction of Hyp with the metal surface occurs through the amino, methylene and carboxylate moieties of the molecule. The spectroscopic results are supported by quantum chemical calculations, performed using extended Hückel theory (EHT) of the title compounds interacting with an Ag cluster model. The assignment of the Raman bands was supported by a normal coordinate analysis performed through Becke, three‐parameter, Lee–Yang–Parr/6‐311 G* + calculations. Copyright © 2011 John Wiley & Sons, Ltd.     

Distinguishable behavior of multiple and individual rhodamine‐6G molecules on spherical Ag nanoparticles examined via time dependence of the SERS spectra

We report a surface‐enhanced Raman spectroscopy (SERS) investigation to probe the adsorption and dynamic behavior of rhodamine 6 G (Rh6G) molecules on spherical Ag nanoparticles which were produced via laser ablation in liquid. Assembly of the colloidal Ag nanoparticles on a cover glass was used to work as SERS substrates on which high‐quality SERS spectra of Rh6G were obtained with interesting time dependence when using low and ultralow concentrations, respectively. The variation of SERS spectra over time was identified with the adsorption behavior of multiple and individual molecules on the Ag nanoparticles. Analysis indicates that the adsorbed Rh6G molecules can desorb away from the initial locations on the substrate under continuous laser excitation; simultaneously, some individual molecules can move and become trapped in the gap between the aggregated Ag nanoparticles. These investigations help to clarify the origins of forming ‘hot‐spots’ which host probe molecules and hence improve the understanding of mechanisms for single‐molecule SERS spectroscopy. Copyright © 2012 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman spectroscopy of DNA from leaves of in vitro grown apple plants

Ultrasensitive Raman measurements of nucleic acids are possible by exploiting the effect of surface‐enhanced Raman scattering (SERS). In this work, the vibrational spectra of eight genomic DNAs from in vitro grown apple leaf tissues (Malus domestica Borkh., Fam Rosaceae, cvs. Florina, Idared, Rebra, Goldrush, Romus 3, Romus 4 and the rootstocks M9 and M26) were analyzed using surface‐enhanced Raman spectroscopy, in the wavenumber range 200–1800 cm⁻¹. SERS signatures, spectroscopic band assignments and structural interpretations of these plant genomic DNAs are reported. Strong dependences of the SERS spectra on genomic DNA amount in the measured sample volume and on time were observed. Similarities of the SERS signals of DNAs from Rebra and Romus 3 leaves were detected. To our knowledge, this is the first SERS study on genomic DNA from leaf tissues. The present work provides a basis for future use of surface‐enhanced Raman spectroscopy to analyze specific plant DNA–ligand interactions or DNA structural changes induced by plants' stress conditions associated with their natural environment. Besides, this study will generate information that is valuable in the development of low‐level plant DNA‐based analytical sensors. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of self‐repair mechanisms of Phaseolus vulgaris var. saxa using near‐infrared surface‐enhanced Raman spectroscopy

We used surface‐enhanced Raman spectroscopy (SERS) to investigate ultrastructural changes in cell‐wall composition during the self‐repair of lacerated hypocotyls of Phaseolus vulgaris var. saxa. A detailed study of self‐repair mechanisms requires localized information about cell‐wall structure and morphology in addition to the chemical cell‐wall composition. Characteristic Raman and SER spectra yielded two‐dimensional maps of cross sections of P. vulgaris var. saxa visualizing chemical compositions in the walls of different cell types and during various repair phases. SERS substrate particles were produced by the reduction of gold chloride on the plant tissue surface and characterized with absorption spectroscopy, scanning electron microscopy and energy‐dispersive X‐ray spectroscopy. The SERS results were compared with stained cross sections of the same plant using dark‐field microscopy with focus on lignin and suberin contents in repairing cells. In addition, SERS measurements revealed Au cyanide compounds on the cell surface, indicating the formation of hydrogen cyanide during the self‐repair phase. Copyright © 2009 John Wiley & Sons, Ltd.     

Surface‐enhanced Raman scattering study of human serum on PVA?Ag nanofilm prepared by using electrostatic self‐assembly

Polyvinyl alcohol (PVA)‐protected silver nanoarchitecture (PVA Ag nanofilm) on the surface of the glass substrate was prepared by using electrostatic self‐assembly at a proper voltage. The two‐dimensional morphology of the PVA Ag nanofilm has been examined by scanning electron microscopy (SEM). The surface‐enhanced Raman scattering (SERS) spectra of human serum (HS) on PVA Ag nanofilms were recorded. The results show that the Raman scattering of HS can be enhanced efficiently based on these PVA Ag nanofilms. However, it also can be seen that the effect of sodium citrate (SC) acting as anticoagulant on the SERS spectrum of HS is unnegligible, which has not been discussed adequately in the previous reports. To discuss the effect of SC on the SERS spectrum of HS, we have studied the normal Raman spectra of solid SC and the SERS spectra of 1.0 × 10⁻³ mol/l aqueous solution of SC adsorbed on the PVA–Ag nanofilms. Meanwhile, Raman wavenumbers of the SC molecule were calculated by using the method of DFT‐B3LYP/6‐31G*, and the dominant assignations of the calculated wavenumbers were performed. It was found that the density functional theory (DFT) calculation of SC Raman spectrum matches well with the experimental results. With the perfect reproducibility and high SERS activity, this method will be useful in the development of HS detection methods. Copyright © 2010 John Wiley & Sons, Ltd.     

Biocompatible gold/silver nanostars for surface‐enhanced Raman scattering

Raman‐enhancing properties of chitosan (CS)‐coated gold/silver nanostars (Au/AgNSs) were demonstrated by using them as a surface‐enhanced Raman scattering (SERS) probe. Based on the energy‐dispersive X‐ray spectroscopy element distribution maps and highly enhanced SERS spectra, we suggest that the incorporation of silver into the NS tips leads to a stronger SERS behavior. The SERS spectra of the proteins adsorbed on the NS surface greatly differ from their respective Raman spectra in both the band positions and relative intensities, indicating that the protein molecules penetrate through the CS coating layer and interact closely with the NS surface. Raman and SERS spectra of Chlamydia trachomatis protease/proteasomelike activity factor are reported for the first time, demonstrating the potential of these NSs for the development of a diagnosis method for Chlamydia based on SERS. The results showed a good SERS performance of the Au/AgNSs and their potential for SERS detection of biomolecules. Copyright © 2016 John Wiley & Sons, Ltd.     

Potential‐induced Raman behavior of individual (R)‐di‐2‐naphthylprolinol molecules on a Ag‐modified Ag electrode

The applicability of surface‐enhanced Raman spectroscopy is demonstrated to probe the adsorption behavior of individual molecules on a Ag electrode. High‐quality SERS spectra of (R)‐di‐2‐naphthylprolinol (DNP) were obtained from ultradilute solutions (10⁻¹² M ) on the Ag‐nanoparticle‐modified Ag electrode, which is attributed to the high electromagnetic (EM) effect of the SERS‐active system as well as to the strong adsorption and interaction of DNP molecules with Ag. The stable SERS spectra present remarkable potential dependence, which gives evidence for the behavior of individual DNP molecules on the Ag surface. Based on statistical analysis for the probability of DNP molecules located in ‘hot spots’, we propose an SERS mechanism for individual molecules in the electrode system, in combination with the hot‐spot model and orientation of the probe molecules. Copyright © 2010 John Wiley & Sons, Ltd.     

Adsorption of 1,2,4‐triazole on a silver electrode: surface‐enhanced Raman spectroscopy and density functional theory studies

The pH‐dependent surface‐enhanced Raman scattering (SERS) of 1,2,4‐triazole adsorbed on silver electrode and normal Raman (NR) spectra of this compound in the aqueous solutions were investigated. The observed bands in the NR and SERS spectra were assigned with the help of density functional theory calculations for model molecules in the neutral, anionic, and cationic forms and their complexes with silver. The Raman wavenumbers and intensities were computed at the optimized molecular geometry. Vibrational assignments of the SERS and NR spectra are provided by calculated potential energy distributions. The combination of experimental SERS results and density functional theory calculations provide an insight into the molecular structure of adlayers formed by 1,2,4‐triazole on a silver surface at varying pH values and enable the determination of molecular orientation with respect to the surface. Copyright © 2012 John Wiley & Sons, Ltd.     

A hybrid substrate for surface‐enhanced Raman scattering spectroscopy: coupling metal nanoparticles to strong localised fields on a micro‐structured surface

Electromagnetic coupling between localised plasmons on metal nanoparticles and the strong localised fields on a micro‐structured surface is demonstrated as a means to increase the enhancement factor in surface‐enhanced Raman scattering (SERS) spectroscopy. Au nanoparticles of diameter 20 nm were deposited on a micro‐structured Au surface consisting of a periodic array of square‐based pyramidal pits (Klarite). The spectra of 4‐aminothiophenol (4‐ATP) were compared before and after deposition of Au nanoparticles on the micro‐structured surface. The addition of Au nanoparticles is shown to provide significantly higher signal intensities, with improvements of the order of ∼10³ per molecule compared with spectra obtained from the micro‐structured substrate alone. This hybrid approach offers promise for combining nanoparticles with micro‐ and nano‐structured surfaces in order to design SERS substrates with higher sensitivities. Copyright © 2011 John Wiley & Sons, Ltd.     

Quick detection of contaminants leaching from polypropylene centrifuge tubes with surface‐enhanced Raman spectroscopy and ultraviolet absorption spectroscopy

Anomalous surface‐enhanced Raman scattering (SERS) peaks were identified for liquid sample stored in polypropylene (PP) centrifuge tubes for months. We observed unexpected Raman peaks during experiments with thiamine hydrochloride aqueous solutions stored in PP tubes for 2 months. In order to identify the contaminants, we have performed SERS experiments on deionized (DI) water stored in PP centrifuge tubes for 2 months and compared them with those from fresh DI water sample. We have also carried out ultraviolet (UV) absorption spectra for both fresh and contaminated water. We believe that the water is contaminated because of chemicals leaching from the PP tube. From the gas chromatography‐mass spectrometry data, the main contaminants were found to be phthalic acid (PA) and its derivatives. Further SERS and UV absorption experiment for PA correlated well with the anomalous peaks identified earlier. We qualitatively confirmed the identification and quantitatively estimated the concentration of the suspect contaminants as between 1 and 10 µM with both SERS and UV absorption spectroscopy. With UV absorption spectroscopy, we precisely estimated the concentration as 2.1 µM . We have shown that the sample in PP tube can be contaminated by the leaching chemicals upon long‐term storage, and suggest SERS and UV absorption spectroscopy as two quick and simple techniques to detect the contamination. Copyright © 2011 John Wiley & Sons, Ltd.