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Surface-enhanced Raman scattering: a new optical probe in molecular biophysics and biomedicine
(2010)
Sensitive and detailed molecular structural
information plays an increasing role in molecular biophysics
and molecular medicine. Therefore, vibrational
spectroscopic techniques, such as Raman scattering, which
provide high structural information content are of growing
interest in biophysical and biomedical research. Raman
spectroscopy can be revolutionized when the inelastic
scattering process takes place in the very close vicinity of
metal nanostructures. Under these conditions, strongly
increased Raman signals can be obtained due to resonances
between optical fields and the collective oscillations of the
free electrons in the metal. This effect of surface-enhanced
Raman scattering (SERS) allows us to push vibrational
spectroscopy to new limits in detection sensitivity, lateral
resolution, and molecular structural selectivity. This opens
up exciting perspectives also in molecular biospectroscopy.
This article highlights three directions where SERS can offer interesting new capabilities. This includes SERS as a
technique for detecting and tracking a single molecule, a
SERS-based nanosensor for probing the chemical composition
and the pH value in a live cell, and the effect of socalled
surface-enhanced Raman optical activity, which
provides information on the chiral organization of molecules
on surfaces.
Two-photon vibrational spectroscopy for biosciences based on surface-enhanced hyper-Raman scattering
(2006)
In Vivo Molecular Probing of Cellular Compartments with Gold Nanoparticles and Nanoaggregates
(2006)
Surface-enhanced Raman (SERS) signatures were measured from single living cells at different times after the uptake of gold nanoparticles. The spectra are indicative of chemical changes in the environment of the nanostructures over time. The increase of the SERS signal strength and parallel TEM studies indicate the formation of nanoaggregates providing optimum SERS enhancement for ultrasensitive probing inside the endosomal compartment. The results have implications for medical and biotechnology applications of SERS nanosensors in cells.
We demonstrate spatially resolved probing and imaging of pH in live cells by mobile and biocompatible nanosensors using surface-enhanced Raman scattering (SERS) of 4-mercaptobenzoic acid (pMBA) on gold nanoaggregates. Moreover, we also show that this concept of pH nanosensors can be extended to two-photon excitation by using surface-enhanced hyper-Raman scattering (SEHRS). In addition to the advantages of two-photon excitation, the SEHRS sensor enables measurements over a wide pH range without the use of multiple probes.
Nanoaggregates formed by metal spheres of different radii and interparticle distances represent finite, deterministic, self-similar systems that efficiently concentrate optical fields and act as nanolenses. Here we verify experimentally the theoretical concept of nanolenses and explore their potential as enhancing nanostructures in surface enhanced Raman scattering (SERS). Self-similar structures formed by gold nanospheres of different sizes are generated by laser ablation from solid gold into water. These nanolenses exhibit SERS enhancement factors on the order of 109. The chemically clean preparation process provides several advantages over chemically prepared nanoaggregates and makes the stable and biocompatible gold nanolenses potent enhancing structures for various analytical and sensing applications.
During surface-enhanced Raman scattering (SERS), molecules exhibit a significant increase in their Raman signals when attached, or in very close vicinity, to gold or silver nanostructures. This effect is exploited as the basis of a new class of optical labels. Here we demonstrate robust and sensitive SERS labels as probes for imaging live cells. These hybrid labels consist of gold nanoparticles with Rose Bengal or Crystal Violet attached as reporter molecules. These new labels are stable and nontoxic, do not suffer from photobleaching, and can be excited at any excitation wavelength, even in the near infrared. SERS labels can be detected and imaged through the specific Raman signatures of the reporters. In addition, surface-enhanced Raman spectroscopy in the local optical fields of the gold nanoparticles also provides sensitive information on the immediate molecular environment of the label in the cell and allows imaging of the native constituents of the cell. This is demonstrated by images based on a characteristic Raman line of the reporter as well as by displaying lipids based on the SERS signal of the C—H deformation/bending modes at ~ 1470 cm-1.
The surface enhanced Raman scattering (SERS) spectrum of a reporter molecule attached to gold or silver nanostructures, which is pH-sensitive, can deliver information on the local pH in the environment of the nanostructure. Here, we demonstrate the use of a mobile SERS nanosensor made from gold nanaoaggregates and 4-mercaptobenzoic acid (pMBA) attached as a reporter for monitoring changes in local pH of the cellular compartments of living NIH/3T3 cells. We show that SERS nanosensors enable the dynamics of local pH in individual live cells to be followed at subendosomal resolution in a timeline of cellular processes. This information is of basic interest for a better understanding of a broad range of physiological and metabolic processes as well as for a number of biotechnological applications.
This review introduces multifunctional optical nanosensors based on surface-enhanced Raman scattering (SERS) and demonstrates their application in live cells. The novel nanosensors have the potential to improve our understanding of cellular processes on the molecular level. The hybrid sensor consists of gold or silver nanoparticles with an attached reporter species. The sensor can be detected and imaged based on the SERS signature of the reporter. This results in several advantages, such as high spectral specificity, multiplex capabilities, improved contrast, and photostability. SERS sensors not only highlight cellular structures, based on enhanced Raman spectra of intrinsic cellular molecules measured in the local optical fields of the gold nanoparticles, they also provide molecular structural information on their cellular
environment. Moreover, the SERS signature of the reporter can deliver information on the local pH value inside a cell at subendosomal resolution. SERS sensors are suitable for one- and two-photon excitation.
RATIONALE
A fast and reliable online identification of pollen is not yet available. The identification of pollen is based mainly on the evaluation of morphological data obtained by microscopic methods.
METHODS
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) was applied to the analysis of extracts and milled pollen samples. The obtained MALDI data were explored for characteristic peak patterns which could be subjected to a multivariate statistical analysis.
RESULTS
Two sample preparation methods are presented, which require only minimal or no chemical extraction of the pollen. MALDI pollen spectra could be recorded showing various peak patterns. A multivariate statistics approach allowed the classification of pollen into clusters indicating similarities and differences between various species.
CONCLUSIONS
These results demonstrate the potential and the reliability of MALDI-TOF MS for the identification and, in combination with multivariate statistics, also for the classification of pollen.
The formation and growth of hydroxylamine reduced silver nanoparticles were investigated by simultaneous Raman and UV/Vis spectroscopy coupled to an acoustic levitator as a sample holder. Based on the UV/Vis spectra, a two step particle formation mechanism with fast initial formation and adjacent coalescence can be proposed for the reduction of silver nitrate with hydroxylamine. The presence of the analyte adenine during particle formation resulted in differences in the adenine SERS signature compared to experiments, where adenine was added after particle synthesis. It was possible to monitor the adenine and sodium chloride induced aggregation of the nanoparticles and its dynamics based on both the extinction spectra and the SERS data. Correlating the information from the extinction spectra with the SERS intensity, the maximum SERS signals were observed at maximum extinction of the aggregated nanoparticle solution at the Raman excitation wavelength.
The interaction of nanomaterials with biomolecules, cells, and organisms plays an important role in cell biology, toxicology, and nanotechnology. Spontaneous Raman scattering can be used to probe biomolecules, cells, whole animals, and nanomaterials alike, opening interesting avenues to study the interaction of nanoparticles with complex biological systems. In this review we discuss work in biomedical Raman spectroscopy that has either been concerned directly with nanostructures and biosystems, or that indicates important directions for successful future studies on processes associated with nano-bio-interactions.
Monodisperse, citrate-stabilized gold nanoparticles of sizes ranging from 15 to 40 nm were synthesized and characterized by small angle X-ray scattering and UV-vis experiments. Identical surface properties of nanoparticles of different sizes to avoid variation in the chemical surface-enhanced Raman scattering (SERS) enhancement, as well as selection of experimental conditions so that no aggregation took place, enabled the investigation of enhancement of individual nanospheres. Enhancement factors (EFs) for SERS were determined using the dye crystal violet (CV). EFs for individual gold nanospheres ranged from 102 to 103, in agreement with theoretical predictions. An increase of the EFs of individual spheres with size can be correlated to changes in the extinction spectra of nanoparticle solutions. This confirms that the increase in enhancement with increasing size results from an increase in electromagnetic enhancement. Beyond this dependence of EFs of isolated gold spheres on their size, EFs were shown to vary with analyte concentration as a result of analyte-induced aggregation. This has implications for the application of nanoparticle solutions as SERS substrates in quantitative analytical tasks.
A new laser ablation (LA)-ICP-MS method for single cell and cell nucleus imaging was developed. Therein, iodine was employed as an elemental dye for fibroblast cells and for thin tissue sections. At an incubation time of 60 s, iodine is located mainly within the cell nuclei. This effect was illustrated in fibroblast cells, and iodine signal within the cell nucleus was as high as 5 × 104 cps at 4 µm laser spot size. The surrounding cytoplasm was iodinated as well, but to a lesser extent. The spatial resolution attained was sufficient to detect even smaller cell nuclei within a liver biopsy tissue. Furthermore, iodine was successfully employed for biomolecule labeling and we demonstrated that iodine signal increased with increasing thickness of a palatine tonsil tissue. Thus, the use of iodine as an internal standard to correct for tissue inhomogeneities in LA-ICP-MS was investigated for the simultaneous detection of two tumor markers (Her 2 and CK 7) in breast cancer tissue. Additionally, lanthanide background resulting from glass ablation can be corrected for by Eu standardization.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized for spatially resolved bioimaging of the distribution of silver and gold nanoparticles in individual fibroblast cells upon different incubation experiments. High spatial resolution was achieved by optimization of scan speed, ablation frequency, and laser energy. Nanoparticles are visualized with respect to cellular substructures and are found to accumulate in the perinuclear region with increasing incubation time. On the basis of matrix-matched calibration, we developed a method for quantification of the number of metal nanoparticles at the single-cell level. The results provide insight into nanoparticle/cell interactions and have implications for the development of analytical methods in tissue diagnostics and therapeutics.
one and two-photon excited surface-enhanced Raman scattering of investigations of eukaryotic cells
(2008)
Nanosensors for biological applications based on surface-enhanced hyper Raman scattering (SEHRS)
(2008)
We report on the in situ characterization of tree pollen molecular composition based on Raman spectroscopy. Different from purification-based analysis, the nondestructive approach allows (i) to analyze various classes of molecules simultaneously at microscopic resolution and (ii) to acquire fingerprint-like chemical information that was used for the classification of pollen from different species. Hierarchical cluster analysis of spectra from fresh pollen samples of 15 species partly related at the genus level and family level indicates separation of species based on the complete Raman spectral signature and yields classification in accord with biological systematics. The results have implications for the further elucidation of pollen biochemistry and also for the development of chemistry-based online pollen identification methods.
The increase in information content from bioassays and bioimaging requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement, thereby addressing current health and security concerns. For fluorescence techniques, an attractive alternative to commonly performed spectral or color multiplexing presents lifetime multiplexing and the discrimination between different fluorophores based on their fluorescence decay kinetics. This strategy relies on fluorescent labels with sufficiently different lifetimes that are excitable at the same wavelength and detectable within the same spectral window. Here, we report on lifetime multiplexing and discrimination with a set of nanometer-sized particles loaded with near-infrared emissive organic fluorophores chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics in suspension. Furthermore, as a first proof-of-concept, we describe bioimaging studies with 3T3 fibroblasts and J774 macrophages, incubated with mixtures of these reporters employing fluorescence lifetime imaging microscopy. These proof-of-concept measurements underline the potential of fluorescent nanoparticle reporters in fluorescence lifetime multiplexing, barcoding, and imaging for cellular studies, cell-based assays, and molecular imaging.
The processes associated with pollen germination were studied in vitro for two tree species, Salix caprea and Fraxinus excelsior under different nutrient conditions. The results provide evidence of changes in chemical composition of the pollen grains during germination. From the comparison of spectra of the pollen grain body and the growing pollen tube, it can be concluded that there are major chemical differences between these two morphological units. Comparison of germinated and ungerminated pollen grains reveals alterations in the metabolism. Composition of the germinating pollen grain and its morphological units depends on the plant species, but also on the nutrient conditions. The results suggest species-specific utilization of metabolite storage, and potential alterations of the pollen outer coat. Furthermore, discharge of molecules into the nutrient medium may depend on the nutrient conditions in the germination experiments. This has implications for further experiments on dynamic processes in pollen and related plant materials.
Surface-enhanced Raman scattering hybrid nanoprobe multiplexing and imaging in biological systems
(2010)
Surface-enhanced Raman scattering (SERS) labels and probes consisting of gold and silver nanoaggregates and attached reporter molecules can be identified by the Raman signature of the reporter molecule. At the same time, SERS hybrid probes deliver sensitive molecular structural information on their nanoenvironment. Here we demonstrate full exploitation of the multifunctional and multiplexing capabilities inherent to such nanoprobes by applying cluster methods and principal components approaches for discrimination beyond the visual inspection of individual spectra that has been practiced so far. The reported results indicate that fast, multivariate evaluation of whole sets of multiple probes is feasible. Spectra of five different reporters were shown to be separable by hierarchical clustering and by principal components analysis (PCA). In a duplex imaging approach in live cells, hierarchical cluster analysis, K-means clustering, and PCA were used for imaging the positions of different types of SERS probes along with the spectral information from cellular constituents. Parallel to cellular imaging experiments, cytotoxicity of the SERS hybrid probes containing aromatic thiols as reporters is assessed. The reported results suggest multiplexing applications of the nontoxic SERS nanoprobes in high density sensing and imaging in complex biological structures.
Surface-enhanced Raman scattering (SERS) hybrid probes are characterized by the typical spectrum of a reporter molecule. In addition, they deliver information from their biological environment. Here, we report SERS hybrid probes generated by conjugating different reporter molecules to bovine serum albumin (BSA) and using gold nanoparticles as plasmonic core. Advantages of the BSA-conjugate hybrid nanoprobes over other SERS nanoprobes are a high biocompatibility, stabilization of the gold nanoparticles in the biological environment, stable reporter signals, and easy preparation. The coupling efficiencies of the BSA–reporter conjugates were determined by MALDI-TOF-MS. The conjugates' characteristic SERS spectra differ from the spectra of unbound reporter molecules. This is a consequence of the covalent coupling, which leads to altered SERS enhancement and changes in the chemical structures of the reporter and of BSA. The application of the BSA–reporter conjugate hybrid probes in 3T3 cells, including duplex imaging, is demonstrated. Hierarchical cluster analysis and principal components analysis were applied for multivariate imaging using the SERS signatures of the incorporated SERS hybrid nanoprobes along with the spectral information from biomolecules in endosomal structures of cells. The results suggest more successful applications of the SERS hybrid probes in cellular imaging and other unordered high-density bioanalytical sensing.
Plant tissues are complex composite structures of organic and inorganic components whose function relies on molecular heterogeneity at the nanometer scale. Scattering-type near-field optical microscopy (s-SNOM) in the mid-infrared (IR) region is used here to collect IR nanospectra from both fixed and native plant samples. We compared structures of chemically extracted silica bodies (phytoliths) to silicified and nonsilicified cell walls prepared as a flat block of epoxy-embedded awns of wheat (Triticum turgidum), thin sections of native epidermis cells from sorghum (Sorghum bicolor) comprising silica phytoliths, and isolated cells from awns of oats (Avena sterilis). The correlation of the scanning-probe IR images and the mechanical phase image enables a combined probing of mechanical material properties together with the chemical composition and structure of both the cell walls and the phytolith structures. The data reveal a structural heterogeneity of the different silica bodies in situ, as well as different compositions and crystallinities of cell wall components. In conclusion, IR nanospectroscopy is suggested as an ideal tool for studies of native plant materials of varied origins and preparations and could be applied to other inorganic–organic hybrid materials.
Self-assembled monolayers (SAMs) on plasmonic substrates play a significant role applications of surface-enhanced Raman scattering (SERS). At the same time, localized surface plasmon resonances (LSPRs) can be employed for a broad range of plasmon-supported chemical modifications. Here, micropatterning using the derivatization of SAMs on gold nanosubstrates for rewritable SERS-based security labels or as the basis for sensing arrays functionalized with biomolecules is demonstrated using different plasmon-catalyzed reactions. The formation of 4,4′-dimercaptoazobenzene (DMAB) from p-aminothiophenol (PATP) as well as from p-nitrothiophenol (PNTP) and the reduction of PNTP to PATP are used to change the functionality of the substrate in specified positions. Employing LSPR, the reactions are started by illumination using visible laser light at a high intensity in a focal spot of a microscope objective and yield microscopic patterns of the reaction product. The obtained molecular patterns can be erased by other reactions, enabling different strategies for rewriting, encryption, or stepwise functionalization.
By adding a gold core to silica nanoparticles (BrightSilica), silica-like nanoparticles are generated that, unlike unmodified silica nanoparticles, provide three types of complementary information to investigate the silica nano-biointeraction inside eukaryotic cells in situ. Firstly, organic molecules in proximity of and penetrating into the silica shell in live cells are monitored by surface-enhanced Raman scattering (SERS). The SERS data show interaction of the hybrid silica particles with tyrosine, cysteine and phenylalanine side chains of adsorbed proteins. Composition of the biomolecular corona of BrightSilica nanoparticles differs in fibroblast and macrophage cells. Secondly, quantification of the BrightSilica nanoparticles using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) micromapping indicates a different interaction of silica nanoparticles compared to gold nanoparticles under the same experimental conditions. Thirdly, the metal cores allow the investigation of particle distribution and interaction in the cellular ultrastructure by cryo nanoscale X-ray tomography (cryo-XT). In 3D reconstructions the assumption is confirmed that BrightSilica nanoparticles enter cells by an endocytotic mechanism. The high SERS intensities are explained by the beneficial plasmonic properties due to agglomeration of BrightSilica. The results have implications for the development of multi-modal qualitative and quantitative characterization in comparative nanotoxicology and bionanotechnology.
The analysis of single cells is a growing research field in many disciplines such as toxicology, medical diagnosis, drug and cancer research or metallomics, and different methods based on microscopic, mass spectrometric, and spectroscopic techniques are under investigation. This review focuses on the most recent trends in which inductively coupled plasma mass spectrometry (ICP-MS) and ICP optical emission spectrometry (ICP-OES) are applied for single-cell analysis using metal atoms being intrinsically present in cells, taken up by cells (e.g., nanoparticles), or which are artificially bound to a cell. For the latter, especially element tagged antibodies are of high interest and are discussed in the review. The application of different sample introduction systems for liquid analysis (pneumatic nebulization, droplet generation) and elemental imaging by laser ablation ICP-MS (LA-ICP-MS) of single cells are highlighted. Because of the high complexity of biological systems and for a better understanding of processes and dynamics of biologically or medically relevant cells, the authors discuss the idea of 'multimodal spectroscopies.'
The cellular response to nanoparticle exposure is essential in various contexts, especially in nanotoxicity and nanomedicine. Here, 14-nm gold nanoparticles in 3T3 fibroblast cells are investigated in a series of pulse-chase experiments with a 30-min incubation pulse and chase times ranging from 15 min to 48 h. The gold nanoparticles and their aggregates are quantified inside the cellular ultrastructure by laser ablation inductively coupled plasma mass spectrometry micromapping and evaluated regarding the surface-enhanced Raman scattering (SERS) signals. In this way, both information about their localization at the micrometre scale and their molecular nanoenvironment, respectively, is obtained and can be related. Thus, the nanoparticle pathway from endocytotic uptake, intracellular processing, to cell division can be followed. It is shown that the ability of the intracellular nanoparticles and their accumulations and aggregates to support high SERS signals is neither directly related to nanoparticle amount nor to high local nanoparticle densities. The SERS data indicate that aggregate geometry and interparticle distances in the cell must change in the course of endosomal maturation and play a critical role for a specific gold nanoparticle type in order to act as efficient SERS nanoprobe. This finding is supported by TEM images, showing only a minor portion of aggregates that present small interparticle spacing. The SERS spectra obtained after different chase times show a changing composition and/or structure of the biomolecule corona of the gold nanoparticles as a consequence of endosomal processing.
We report on the formation of silver subsurface ion-exchanged metal oxide (silver SIMO) glasses and their surface-enhanced Raman scattering (SERS) activity. The samples were prepared by a combined thermal and chemical three-step methodology and characterized by transmission electron microscopy (TEM), atomic force microscopy (AFM), environmental electron scanning microscopy (ESEM), and UV/Vis spectroscopy. This unique method provides SERS substrates with protection against contamination and strong, reliable and reproducible SERS enhancement. The Raman enhancement factors of the long-term stable SIMO glasses were estimated to approximately 107.
Silver nanoparticles were generated based on citrate reduction in the ultrastructure of the sporopollenin biopolymer of Ambrosia artemisiifolia (ragweed) and Secale cereale (rye). The nanoparticles enable the acquisition of SERS spectra and thereby a vibrational characterization of the local molecular structure of sporopollenin.
Increasing the information content from bioassays which requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement is an important field of research, especially in the context of meeting current security and health concerns. An attractive alternative to spectral multiplexing, which relies on fluorescent labels excitable at the same wavelength, yet sufficiently differing in their emission spectra or color presents lifetime multiplexing. For this purpose, we recently introduced a new strategy based on 'pattern-matching' in the lifetime domain, which was exemplary exploited for the discrimination between organic dyes and quantum dot labels revealing multi-exponential decay kinetics and allowed quantification of these labels. Meanwhile, we have succeeded in extending this lifetime multiplexing approach to nanometer-sized particle labels and probes absorbing and emitting in the visible (vis) and near-infrared (NIR) spectral region. Here, we present a first proof-of-principle of this approach for a pair of NIR-fluorescent particles. Each particle is loaded with a single organic dye chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics. Examples for the lifetime-based distinction between pairs of these fluorescent nanoparticles in solution and in cells are presented. The results underline the potential of fluorescenc lifetime multiplexing in life science and bioanalysis.
Raman signatures of the carotenoid component are studied in individual pollen grains from different species of trees. The information is obtained as differences in the strong pre-resonant Raman spectra measured before and after photodepletion of the carotenoid molecules. The results provide the first in situ evidence of interspecies differences in pollen carotenoid content, structure, and/or assembly between plant species without prior purification. The analysis of carotenoids in situ is confirmed by high-performance thin-layer chromatography (HPTLC)-supported resonance Raman data measured directly on the HPTLC plates after separation of carotenoids in pollen extracts. Utilization of the in situ, extraction-free procedure in carotenoid analysis will improve sensitivity and structural selectivity and provides insight into carotenoid structure and composition in single pollen grains.
A combination of two analytical methods, time-resolved X-ray diffraction (XRD) and Raman spectroscopy, is presented as a novel tool for crystallization studies. An acoustic levitator was employed as sample environment. This setup enables the acquisition of XRD and Raman data in situ simultaneously within a 20 s period and hence permits investigation of polymorphic phase transitions during the crystallization process in different solvents (methanol, ethanol, acetone, dichloromethane, acetonitrile). These real time measurements allow the determination of the phase content from the onset of the first crystalline molecular assemblies to the stable system. To evaluate the capability of this approach, the setup was applied to elucidate the crystallization process of the polymorphic compound nifedipine. The results indicate the existence of solvent-dependent transient phases during the crystallization process. The quality of the data allowed the assignment of the lattice constants of the hitherto unknown crystal structure of the β-polymorph.
Cell cultures form the basis of most biological assays conducted to assess the cytotoxicity of nanomaterials.
Since the molecular environment of nanoparticles exerts influence on their physicochemical properties, it can have an impact on nanotoxicity. Here, toxicity of silica nanoparticles upon delivery by fluid-phase uptake is studied in a 3T3 fibroblast cell line. Based on XTT viability assay, cytotoxicity is shown to be a function of (1) particle concentration and (2) of fetal calf serum (FCS) content in the cell culture medium.
Application of dynamic light scattering shows that both parameters affect particle agglomeration. The DLS Experiments verify the stability of the nanoparticles in culture medium without FCS over a wide range of particle concentrations.
The related toxicity can be mainly accounted for by single silica nanoparticles and small agglomerates. In contrast, agglomeration of silica nanoparticles in all FCS-containing media is observed, resulting in a decrease of the associated toxicity. This result has implications for the evaluation of the cytotoxic potential of silica nanoparticles and possibly also other nanomaterials in standard cell culture.
Throughout the history of preparation of biological samples for microscopy the choice of the mounting medium was sometimes dictated merely by availability of the used media. Thus, a plethora of resins and other organic polymers as well as complex mixtures are found to serve as mounting agents in microscope slide collections of museums of natural history, impeding the work for both curators and conservators. Dramatically, in some cases the used mounting media can already be observed to have undergone crystallization and other decomposition processes within few years of mounting demanding immediate action in restoring as well as an imminent precaution in conservation. Therefore, an unambiguous chemical identification of the used agent as well as its current aging stage is of great interest for the biologist community. The technical demands on the analytical approach to obtain this information can be straightforwardly identified. Any used technique has to be non-destructive, yield in molecular information allowing for a chemical identification of the used mounting agents and allow for a spatially well-defined interrogation in a thin sample slice, typically through a transparent cover slip. In this contribution we present a thorough study of the applicability of Raman spectroscopy for the described task. The obtained results clearly demonstrate the successful feasibility of the chosen method for a) a clear distinction between different media, b) the elucidation of the chemical composition of a multicomponent medium and c) an unambiguous identification of real unknown samples by a distinct assignment to a previously recorded spectral library. This library database was built up by recording pure mounting agents and will be provided to the general public. In combination with a Raman spectrometer, it can be an invaluable tool for future curation and conservation endeavors devoted to microscope slide collections at natural history museums.
Aqueous pollen extracts of varying taxonomic relations were analyzed with surface enhanced Raman scattering (SERS) by using gold nanoparticles in aqueous suspensions as SERS substrate. This enables a selective vibrational characterization of the pollen water soluble fraction (mostly cellular components) devoid of the spectral contributions from the insoluble sporopollenin outer layer. The spectra of the pollen extracts are species-specific, and the chemical fingerprints can be exploited to achieve a classification that can distinguish between different species of the same genus. In the simple experimental procedure, several thousands of spectra per species are generated. Using an artificial neural network (ANN), it is demonstrated that analysis of the intrinsic biochemical information of the pollen cells in the SERS data enables the identification of pollen from different plant species at high accuracy. The ANN extracts the taxonomically-relevant information from the data in spite of high intra-species spectral variation caused by signal fluctuations and preparation specifics. The results show that SERS can be used for the reliable characterization and identification of pollen samples. They have implications for improved investigation of pollen physiology and for allergy warning.
The spectroscopic characterization by surface-enhanced Raman scattering (SERS) has shown great potential in studies of heterogeneous catalysis. We describe a plug-in multifunctional optofluidic platform that can be tailored to serve both as a variable catalyst material and for sensitive optical characterization of the respective reactions using SERS in microfluidic systems. The platform enables the characterization of reactions under a controlled gas atmosphere and does not present with limitations due to nanoparticle adsorption or memory effects. Spectra of the gold-catalyzed reduction of p-nitrothiophenol by sodium borohydride using the plug-in probe provide evidence that the borohydride is the direct source of hydrogen on the gold surface, and that a radical anion is formed as an intermediate. The in situ monitoring of the photoinduced dimerization of p-aminothiophenol indicates that the activation of oxygen is essential for the plasmon-catalyzed oxidation on gold nanoparticles and strongly supports the central role of metal oxide species.
Multivariate analysis of MALDI imaging mass spectrometry data of mixtures of single pollen grains
(2018)
Mixtures of pollen grains of three different species (Corylus avellana, Alnus cordata, and Pinus sylvestris) were investigated by matrixassisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF imaging MS). The amount of pollen grains was reduced stepwise from > 10 to single pollen grains. For sample pretreatment, we modified a previously applied approach, where any additional extraction steps were omitted.
Our results show that characteristic pollen MALDI mass spectra can be obtained from a single pollen grain, which is the prerequisite for a reliable pollen classification in practical applications. MALDI imaging of laterally resolved pollen grains provides additional information by reducing the complexity of the MS spectra of mixtures, where frequently peak discrimination is observed. Combined with multivariate statistical analyses, such as principal component analysis (PCA), our approach offers the chance for a fast and reliable identification of individual pollen grains by mass spectrometry.
This study demonstrates the bottom-up synthesis of
silver nanolenses.Arobust coating protocol enabled the
functionalization of differently sized silver nanoparticles with
DNAsingle strands of orthogonal sequence.Coated particles
10 nm, 20 nm, and 60 nm in diameter were self-assembled by
DNAorigami scaffolds to form silver nanolenses.Single
molecules of the protein streptavidin were selectively placed in
the gap of highest electric field enhancement. Streptavidin
labelled with alkyne groups served as model analyte in surface-
enhanced Raman scattering (SERS) experiments.Bycorre-
lated Raman mapping and atomic force microscopy, SERS
signals of the alkyne labels of asingle streptavidin molecule,
from asingle silver nanolens,were detected. The discrete,self-
similar aggregates of solid silver nanoparticles are promising
for plasmonic applications.
Diese Studie demonstriert die Bottom-up-Synthese
von Silbernanolinsen. Ein robustes Beschichtungsprotokoll
ermçglichte die Funktionalisierung unterschiedlich großer
Silbernanopartikel mit einzelstr-ngiger DNAunterschiedlicher
Sequenz. Derartig beschichtete Partikel mit Durchmessern von
10 nm, 20 nm und 60 nm wurden mithilfe von DNA-Origami-
Gergsten zu Silbernanolinsen angeordnet. Ein einzelnes Mo-
lekgldes Proteins Streptavidin ist in demjenigen Spalt zwi-
schenden Partikeln immobilisiert worden, der die hçchste
Feldverst-rkung zur Verfggung stellt. Streptavidin war dabei
mit Alkin-Gruppen modifiziert und fungierte als Modellanalyt
in Experimenten zur oberfl-chenverst-rkten Raman-Streuung
(SERS). Korrelierte Raman- und Rasterkraftmikroskopie-
Messungen ermçglichten die Aufnahme von SERS-Signalen
der Alkingruppen eines einzelnen Streptavidins,von einer
einzelnen Silbernanolinse.Diese diskreten, selbst-hnlichen
Silbernanopartikelaggregate versprechen vielf-ltige Anwen-
dungen im Feld der Plasmonik
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been suggested as a promising tool for the investigation of pollen, but the usefulness of this approach for classification and identification of pollen species has to be proven by an application to samples of varying taxonomic relations.
Hot carriers (electrons and holes) generated from the decay of localized surface plasmon resonances can take a major role in catalytic reactions on metal nanoparticles. By obtaining surface enhanced Raman scattering (SERS) spectra of p-aminothiophenol as product of the reduction of p-nitrothiophenol by hot electrons, different catalytic activity is revealed here for nanoparticles of silver, gold, and copper. As a main finding, a series of different ligands, comprising halide and non-halide species, are found to enhance product formation in the reduction reaction on nanoparticles of all three metals. A comparison with the standard electrode potentials of the metals with and without the ligands and SERS data obtained at different electrode potential indicate that the higher catalytic activity can be associated with a higher Fermi level, thereby resulting in an improved efficiency of hot carrier generation. The concept of such a ligand-enhanced hot electron reduction provides a way to make light-to-chemical energy conversion more efficient due to improved electron harvesting.
The analysis of pollen chemical composition is important to many fields, including agriculture, plant physiology, ecology, allergology, and climate studies. Here, the potential of a combination of different spectroscopic and spectrometric methods regarding the characterization of small biochemical differences between pollen samples was evaluated using multivariate statistical approaches. Pollen samples, collected from three populations of the grass Poa alpina, were analyzed using Fourier-transform infrared (FTIR) spectroscopy, Raman spectroscopy, surface enhanced Raman scattering (SERS), and matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS). The variation in the sample set can be described in a hierarchical framework comprising three populations of the same grass species and four different growth conditions of the parent plants for each of the populations. Therefore, the data set can work here as a model system to evaluate the classification and characterization ability of the different spectroscopic and spectrometric methods. ANOVA Simultaneous Component Analysis (ASCA) was applied to achieve a separation of different sources of variance in the complex sample set. Since the chosen methods and sample preparations probe different parts and/or molecular constituents of the pollen grains, complementary information about the chemical composition of the pollen can be obtained. By using consensus principal component analysis (CPCA), data from the different methods are linked together. This enables an investigation of the underlying global information, since complementary chemical data are combined. The molecular information from four spectroscopies was combined with phenotypical information gathered from the parent plants, thereby helping to potentially link pollen chemistry to other biotic and abiotic parameters.
Grasses accumulate silicon in the form of silicic acid, which is precipitated as amorphous silica in microscopic particles termed phytoliths. These particles comprise a variety of morphologies according to the cell type in which the silica was deposited. Despite the evident morphological differences, phytolith chemistry has mostly been analysed in bulk samples, neglecting differences between the varied types formed in the same species. In this work, we extracted leaf phytoliths from mature plants of Sorghum bicolor (L.) Moench. Using solid state NMR and thermogravimetric analysis, we show that the extraction methods alter greatly the silica molecular structure, its condensation degree and the trapped organic matter. Measurements of individual phytoliths by Raman and synchrotron FTIR microspectroscopies in combination with multivariate analysis separated bilobate silica cells from prickles and long cells, based on the silica molecular structures and the fraction and composition of occluded organic matter. The variations in structure and composition of sorghum phytoliths suggest that the biological pathways leading to silica deposition vary between these cell types.
Background signals from in situ-formed amorphous carbon, despite not being fully understood, are known to be a common issue in few-molecule surface-enhanced Raman scattering (SERS). Here, discrete gold and silver nanoparticle aggregates assembled by DNA origami were used to study the conditions for the formation of amorphous carbon during SERS measurements. Gold and silver dimers were exposed to laser light of varied power densities and wavelengths. Amorphous carbon prevalently formed on silver aggregates and at high power densities. Time-resolved measurements enabled us to follow the formation of amorphous carbon. Silver nanolenses consisting of three differently-sized silver nanoparticles were used to follow the generation of amorphous carbon at the single-nanostructure level. This allowed observation of the many sharp peaks that constitute the broad amorphous carbon signal found in ensemble measurements. In conclusion, we highlight strategies to prevent amorphous carbon formation, especially for DNA-assembled SERS substrates.
Biopolymers are very efficient for significant applications ranging from tissue engineering, biological devices to water purification. There is a tremendous potential value of cellulose because of ist being the most abundant biopolymer on earth, swellability, and functional groups to be modified. A novel, highly efficient route for the fabrication of mechanically stable and natural hydrogels is described in which cellulose and glycine are dissolved in an alkaline solution of NaOH and neutralized in an acidic solution. The dissolving temperature and the glycine amount are essential parameters for the self-assembly of cellulose chains and for Tuning the morphology and the aggregate structures of the resulting hydrogels. Glycine plays the role of a physical crosslinker based on the Information obtained from FTIR and Raman spectra. Among the prepared set of hydrogels, CL5Gly30 hydrogels have the highest capacity to absorb water. The prepared CL5Gly30 gels can absorb up to seven times their dry weight due to its porous 3-D network structure. CL5Gly10 hydrogel exhibits 80% deformation under 21 N force executed. The method developed in this article can contribute to the application of heavy metal adsorption in aqueous solutions for water purification and waste management.
The discussion of the surface-enhanced Raman scattering (SERS) spectra of p-aminothiophenol (PATP) and of ist photocatalytic reaction product 4,4′-dimercaptoazobenzene (DMAB) is important for understanding plasmon-supported spectroscopy and catalysis. Here, SERS spectra indicate that DMAB forms also in a nonphotocatalytic reaction on silver nanoparticles. Spectra measured at low pH, in the presence of the acids HCl, H2SO4, HNO3, and H3PO4, show that DMAB is reduced to PATP when both protons and chloride ions are present. Moreover, the successful reduction of DMAB in the presence of other, halide and nonhalide, ligands suggests a central role of these species in the reduction. As discussed, the ligands increase the efficiency of hot-electron harvesting. The pH-associated reversibility of the SERS spectrum of PATP is established as an Observation of the DMAB dimer at high pH and of PATP as a product of its hot-electron reduction at low pH, in the presence of the appropriate ligand.
Role of metal cations in plasmon-catalyzed oxidation: A case study of p-aminothiophenol dimerization
(2017)
The mechanism of the plasmon-catalyzed reaction of p-aminothiophenol (PATP) to 4,4′-dimercaptoazobenzene (DMAB) on the surface of metal nanoparticles has been discussed using data from surface-enhanced Raman scattering of DMAB. Oxides and hydroxides formed in a plasmon-catalyzed process were proposed to play a central role in the reaction. Here, we report DMAB formation on gold nanoparticles occurring in the presence of the metal cations Ag+, Au3+, Pt4+, and Hg2+. The experiments were carried out under conditions where formation of gold oxide or hydroxide from the nanoparticles can be excluded and at high pH where the formation of the corresponding oxidic species from the metal ions is favored. On the basis of our results, we conclude that, under these conditions, the selective oxidation of PATP to DMAB takes place via formation of a metal oxide from the ionic species in a plasmon-catalyzed process. By evidencing the necessity of the presence of the metal cations, the reported results underpin the importance of metal oxides in the reaction.
The process of silicification in plants and the biochemical
effects of silica in plant tissues are largely unknown. To
study the molecular changes occurring in growing cells
that are exposed to higher than normal concentration of
silicic acid, Raman spectra of germinating pollen grains of
three species (Pinus nigra, Picea omorika, and Camellia
japonica) were analyzed in a multivariate classification
approach that takes into account the variation of biochemical
composition due to species, plant tissue structure,
and germination condition. The results of principal
component analyses of the Raman spectra indicate differences
in the utilization of stored lipids, a changed mobilization
of storage carbohydrates in the pollen grain bodies,
and altered composition and/or structure of cellulose of
the developing pollen tube cell walls. These biochemical
changes vary in the different species.
Nanolenses are self-similar chains of metal nanoparticles, which can theoretically provide extremely high field enhancements. Yet, the complex structure renders their synthesis challenging and has hampered closer analyses so far. Here, DNA origami is used to self-assemble 10, 20, and 60 nm gold nanoparticles as plasmonic gold nanolenses (AuNLs) in solution and in billions of copies. Three different geometrical arrangements are assembled, and for each of the three designs, surface-enhanced Raman scattering (SERS) capabilities of single AuNLs are assessed. For the design which shows the best properties, SERS signals from the two different internal gaps are compared by selectively placing probe dyes. The highest Raman enhancement is found for the gap between the small and medium nanoparticle, which is indicative of a cascaded field enhancement.
Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a well-implemented analytical technique for the investigation of complex biological samples. In MS, the sample preparation strategy is decisive for the success of the measurements. Here, sample preparation processes and target materials for the investigation of different pollen grains are compared. A reduced and optimized sample preparation process prior to MALDI-TOF measurement is presented using conductive carbon tape as target. The application of conductive tape yields in enhanced absolute signal intensities and mass spectral pattern information, which leads to a clear separation in subsequent pattern analysis. The results will be used to improve the taxonomic differentiation and identification, and might be useful for the development of a simple routine method to identify pollen based on mass spectrometry.
Raman microspectra combine information on chemical composition of plant tissues with spatial information. The contributions from the building blocks of the cell walls in the Raman spectra of plant tissues can vary in the microscopic sub-structures of the tissue. Here, we discuss the analysis of 55 Raman maps of root, stem, and leaf tissues of Cucumis sativus, using different spectral contributions from cellulose and lignin in both univariate and multivariate imaging methods. Imaging based on hierarchical cluster analysis (HCA) and principal component analysis (PCA) indicates different substructures in the xylem cell walls of the different tissues. Using specific signals from the cell wall spectra, analysis of the whole set of different tissue sections based on the Raman images reveals differences in xylem tissue morphology. Due to the specifics of excitation of the Raman spectra in the visible wavelength range (532 nm), which is, e.g., in resonance with carotenoid species, effects of photobleaching and the possibility of exploiting depletion difference spectra for molecular characterization in Raman imaging of plants are discussed. The reported results provide both, specific information on the molecular composition of cucumber tissue Raman spectra, and general directions for future imaging studies in plant tissues.
Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) is a well-implemented analytical technique for the investigation of complex biological samples. In MS, the sample preparation strategy is decisive for the success of the measurements. Here, sample preparation processes and target materials for the investigation of different pollen grains are compared. A reduced and optimized sample preparation process prior to MALDI-TOF measurement is presented using conductive carbon tape as target. The application of conductive tape yields in enhanced absolute signal intensities and mass spectral pattern information, which leads to a clear separation in subsequent pattern analysis. The results will be used to improve the taxonomic differentiation and identification, and might be useful for the development of a simple routine method to identify pollen based on mass spectrometry.
MALDI time-of-flight mass spectrometry (MALDI-TOF MS) has become a widely used tool for the classification of biological samples. The complex chemical composition of pollen grains leads to highly specific, fingerprint-like mass spectra, with respect to the pollen species. Beyond the species-specific composition, the variances in pollen chemistry can be hierarchically structured, including the level of different populations, of environmental conditions or different genotypes. We demonstrate here the sensitivity of MALDI-TOF MS regarding the adaption of the chemical composition of three Poaceae (grass) pollen for different populations of parent plants by analyzing the mass spectra with partial least squares discriminant analysis (PLS-DA) and principal component analysis (PCA). Thereby, variances in species, population and specific growth conditions of the plants were observed simultaneously. In particular, the chemical pattern revealed by the MALDI spectra enabled discrimination of the different populations of one species. Specifically, the role of environmental changes and their effect on the pollen chemistry of three different grass species is discussed. Analysis of the Group formation within the respective populations showed a varying influence of plant genotype on the classification, depending on the species, and permits conclusions regarding the respective rigidity or plasticity towards environmental changes.
The characterization of a catalyst often occurs by averaging over large areas of the catalyst material. On the other hand, optical probing is easily achieved at a resolution at the micrometer scale, specifically in microspectroscopy. Here, using surface-enhanced Raman scattering (SERS) mapping of larger areas with micrometer-sized spots that contain tens to hundreds of supported gold nanoparticles each, the photoinduced dimerization of p-aminothiophenol (PATP) to 4,4′-dimercaptoazobenzene (DMAB) was monitored. The mapping data reveal an inhomogeneous distribution of catalytic activity in the plasmon-catalyzed reaction in spite of a very homogeneous plasmonic enhancement of the optical signals in SERS. The results lead to the conclusion that only a fraction of the nanostructures may be responsible for a high catalytic activity. The high spot-to-spot variation in catalytic activity is also demonstrated for DMAB formation by the plasmon-catalyzed reduction from p-nitrothiophenol (PNTP) and confirms that an improvement of the accuracy and reproducibility in the characterization of catalytic reactions can be achieved by microspectroscopic probing of many positions. Using SERS micromapping during the incubation of PATP, we demonstrate that the reaction occurs during the incubation process and is influenced by different parameters, leading to the conclusion of dimerization in a gold-catalyzed, nonphotochemical reaction as an alternative to the plasmon-catalyzed process. The results have implications for the future characterization of new catalyst materials as well as for optical sensing using plasmonic materials.
1. Fourier transform infrar ed (FTIR) spectroscopy enables chemical analysis of pollen samples for plant phenotyping to stud y plant–environment interactions, such as influence of climate change or pathogens. However, current approach, such as microspectrosc opy and attenuated total refle ction spectrosco py, doe s not allow fo r high-throughput protocols. This st udy at hand suggests a new sp ectroscopic method for high-throughp ut characterization of pollen.
2. Samples were measured as thin films of pollen fragments using a Bruker FTIR spe ctrometer with a high-throughput eXTension (HTS-XT) unit employing 384-well plates. In total, 146 pollen samples, belonging to 31 different pollen species of Fagaceae and Betulaceae and collected during three consecutive years (2012–2014) at locations in Croatia, Germany and Norway, were analysed. Critical steps in the sample preparation and measurement, such as variabilities between technical replicates, between microplates and between spectrometers, were studied.
3. Measurement variations due to sample preparation, microplate holders and instrumentation were low, and thus allowed differentiation of samples with respect to phylogeny and biogeography. The spectral variability for a ran ge of Fagales spec ies (Fagus, Quercus, Betula, Corylus, Alnus and Ostrya) showed high-species-specific differences in pollen’s chemical composition due to eithe r location or year. Statistically significant inter-annual and locational differences in the pollen spectra indicate that pollen chemical composition has high phenotypic plasticity and is influenced by local climate conditions. The variations in composition are connected to lipids, proteins, carbohydrates and sporopollenins that play crucial ro les in cold and desiccation tolerance, protection against UV radiation and as material and energy reserves.
4. The results of this study demonstrate the value of high-throughput FTIR approach for the systematic collection of data on ecosystems. The novel FTIR approach offers fast, reliable and economical screening of large number of samples by semi-automated methodology. The high-throughput approach could provide crucial understanding on plant–climate interactions with respect to biochemical variation within genera, species and populations.
ICP-MS is a well-established analytical method which excels by high accuracy, high dynamic range and extremely low limits of detection for most metals. Furthermore ICP-MS offers a very high multi-element coverage so that many elements of the periodic table can be detected simultaneously. In this series of lectures, we want to focus on the historical developments, fundamentals, instrumentation and novel applications of ICP-MS in the life and material sciences.
1. Fourier transform infrared (FTIR) spectroscopy enables chemical analysis of pollen samples for plant phenotyping to study plant–environment interactions, such as influence of climate change or pathogens.However, current approach, such as microspectroscopy and attenuated total reflection spectroscopy, does not allow for highthroughput protocols. This study at hand suggests a new spectroscopic method for high-throughput characterization of pollen.
2. Samples were measured as thin films of pollen fragments using a Bruker FTIR spectrometer with a highthroughput eXTension (HTS-XT) unit employing 384-well plates. In total, 146 pollen samples, belonging to 31 different pollen species of Fagaceae and Betulaceae and collected during three consecutive years (2012–2014) at locations in Croatia, Germany and Norway, were analysed. Critical steps in the sample preparation and measurement, such as variabilities between technical replicates, between microplates and between spectrometers, were studied.
3. Measurement variations due to sample preparation, microplate holders and instrumentation were low, and thus allowed differentiation of samples with respect to phylogeny and biogeography. The spectral variability for a range of Fagales species (Fagus, Quercus, Betula, Corylus, Alnus and Ostrya) showed high-species-specific differences in pollen’s chemical composition due to either location or year. Statistically significant inter-annual and locational differences in the pollen spectra indicate that pollen chemical composition has high phenotypic plasticity and is influenced by local climate conditions. The variations in composition are connected to lipids, proteins, carbohydrates and sporopollenins that play crucial roles in cold and desiccation tolerance, protection against UVradiation and asmaterial and energy reserves.
4. The results of this study demonstrate the value of high-throughput FTIR approach for the systematic collection of data on ecosystems. The novel FTIR approach offers fast, reliable and economical screening of large number of samples by semi-automated methodology. The high-throughput approach could provide crucial understanding on plant–climate interactions with respect to biochemical variation within genera, species and populations.