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Directing nanoparticles to the nucleus by attachment of nuclear localization sequences (NLS) is an aim in many applications. Gold nanoparticles modified with two different NLS were studied while crossing barriers of intact cells, including uptake, endosomal escape, and nuclear translocation. By imaging of the nanoparticles and by characterization of their molecular interactions with surface-enhanced Raman scattering (SERS), it is shown that nuclear translocation strongly depends on the particular incubation conditions. After an 1 h of incubation followed by a 24 h chase time, 14 nm gold particles carrying an adenoviral NLS are localized in endosomes, in the cytoplasm, and in the nucleus of fibroblast cells. In contrast, the cells display no nanoparticles in the cytoplasm or nucleus when continuously incubated with the nanoparticles for 24 h. The ultrastructural and spectroscopic data indicate different processing of NLS-functionalized particles in endosomes compared to unmodified particles. NLS functionalized nanoparticles form larger intraendosomal aggregates than unmodified gold nanoparticles. SERS spectra of cells with NLS-functionalized gold nanoparticles contain bands assigned to DNA and were clearly different from those with unmodified gold nanoparticles. The different processing in the presence of an NLS is influenced by a continuous exposure of the cells to nanoparticles and an ongoing nanoparticle uptake. This is supported by mass-spectrometry-based quantification that indicates enhanced uptake of NLS-functionalized nanoparticles compared to unmodified particles under the same conditions. The results contribute to the optimization of nanoparticle analysis in cells in a variety of applications, e.g., in theranostics, biotechnology, and bioanalytics.
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.
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.
The field of epigenetics describes the relationship between genotype and phenotype, by regulating gene expression without changing the canonical base sequence of DNA. It deals with molecular genomic information that is encoded by a rich repertoire of chemical modifications and molecular interactions. This regulation involves DNA, RNA and proteins that are enzymatically tagged with small molecular groups that alter their physical and chemical properties. It is now clear that epigenetic alterations are involved in development and disease, and thus, are the focus of intensive research. The ability to record epigenetic changes and quantify them in rare medical samples is critical for next generation diagnostics. Optical detection offers the ultimate single-molecule sensitivity and the potential for spectral multiplexing. Here we review recent progress in ultrasensitive optical detection of DNA and histone modifications.
Microfluidic paper-based analytical devices (μPADs) in combination with surface enhanced Raman scattering (SERS) provide a way for analyses of complex mixtures. The μPADs can be used for the chromatographic separation of different compounds of mixtures in combination with the separate detection of the analytes in different zones on the paper by SERS. SERS allows to observe analytes directly without labelling in low concentrations in aqueous solutions and to identify them by their spectral fingerprint.
SERS substrates on the μPADs were created by drying standard silver nanoparticle (AgNP) solution on the paper. The microfluidic structure of the μPADs was prepared by wax printing. As a model system, an aqueous solution of the non-fluorescent analyte adenine and two fluorescent dyes tris(2,2’-bipyridyl)dichlororuthenium(II) and sulforhodamine B was tested. The dependency of the SERS signal intensity on the analyte concentration can be fitted using a Langmuir isotherm curve progression. With this approach, a semi-quantitative analysis of the components is possible. The reproducibility and stability of the measurement procedure was tested with several measurements over time, different NP batches, and with different analytes in different concentrations and resulted in an average relative standard deviation of 16 %.
The SERS spectra of the mixture of the model system are dominated by one compound depending on the concentration ratio. For the detection and identification of all components of the mixture, the compounds were therefore separated on the μPADs and measured at different positions. The position of adenine on the μPADs is dependent on the AgNP coverage of the paper. Due to this effect, it possible to detect adenine on a defined point on the μPADs and to get an information on the concentration in a mixture of three components.
The growing need to implement sensors such as NIR or Raman spectroscopy for the in-situ monitoring of bioprocesses which follows the standards of Quality by Design is either restricted by the impact of the huge water signal or by a disturbing fluorescence background originating from compounds in the culture media. Furthermore, the characterization of the bioprocess samples is challenging due to changing conditions in course of cultivation.
Here we evaluate two different process-suitable Raman spectroscopic approaches, namely time-gated Raman which bears the potential to extract the Raman signal from the fluorescence background, and cw- Raman with NIR excitation in combination with Surface Enhanced Raman Spectroscopy- (SERS) to investigate cell-free supernatants of Escherichia coli sampled over the course of a cultivation. A confocal Raman microscope was used as a reference for the process devices. The concentration of the analytes, glucose, acetate as well as metabolites such as cAMP, AMP and amino-acids were determined by offline by High-Performance Liquid Chromatography (HPLC) to serve as reference for the calibration of the Raman and SERS spectral data.
Multivariate evaluation of the Raman and SERS spectra by Partial Least Squares Regression (PLSR) yielded for most of the analytes robust correlations at each sampling point. Repeated investigation of the off-line samples over a larger experimental period suggested not only a high reliability of the Raman data in general but also a high repeatability of the SERS experiments. Similar spectral features in different quality and signal/noise ratios were measured with all three set-ups. Major results of the comparison of the different Raman spectroscopic approaches and their combination with SERS are summarized and conclusions are drawn on which approach provides the most accurate concentration data among the target analytes.
Acknowledgement
The authors kindly thank Mario Birkholz (IHP, Frankfurt (Oder), Germany) for the opportunity to use a confocal Raman microscope, Alex Bunker and Tapani Viitala (Division of Pharmaceutical Biosciences, Centre for Drug Research, University of Helsinki, Finland).
Gold nanostructures that serve as probes for nanospectroscopic analysis of eukaryotic cell cultures can be obtained by the in situ reduction of tetrachloroauric acid (HAuCl4). To understand the formation process of such intracellularly grown particles depending on the incubation medium, the reaction was carried out with 3T3 fibroblast cells in three different incubation media, phosphate buffer, Dulbecco's Modified Eagle Medium (DMEM), and standard cell culture medium (DMEM with fetal calf serum). The size, the optical properties, the biomolecular corona, and the localization of the gold nanoparticles formed in situ vary for the different conditions. The combination of surface-enhanced Raman scattering (SERS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) microscopic mapping and transmission electron microscopy (TEM) provides complementary perspectives on plasmonic nanoparticles and non-plasmonic gold compounds inside the cells. While for the incubation with HAuCl4 in PBS, gold particles provide optical signals from the nucleus, the incubation in standard cell culture medium leads to scavenging of the toxic molecules and the formation of spots of high gold concentration in the cytoplasm without formation of SERS-active particles inside the cells. The biomolecular corona of nanoparticles formed in situ after incubation in buffer and DMEM differs, suggesting that different intracellular molecular species serve for reduction and stabilization. Comparison with data obtained from ready-made gold nanoparticles suggests complementary application of in situ and ex situ generated nanostructures for optical probing.
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.
The excitation of localized surface plasmons in noble metal nanoparticles (NPs) results in different nanoscale effects such as electric field enhancement, the generation of hot electrons and a temperature increase close to the NP surface. These effects are typically exploited in diverse fields such as surface-enhanced Raman scattering (SERS), NP catalysis and photothermal therapy (PTT). Halogenated nucleobases are applied as radiosensitizers in conventional radiation cancer therapy due to their high reactivity towards secondary electrons. Here, we use SERS to study the transformation of 8-bromoadenine (8BrA) into adenine on the surface of Au and AgNPs upon irradiation with a low-power continuous wave laser at 532, 633 and 785 nm, respectively. The dissociation of 8BrA is ascribed to a hot-electron transfer reaction and the underlying kinetics are carefully explored. The reaction proceeds within seconds or even milliseconds. Similar dissociation reactions might also occur with other electrophilic molecules, which must be considered in the interpretation of respective SERS spectra. Furthermore, we suggest that hot-electron transfer induced dissociation of radiosensitizers such as 8BrA can be applied in the future in PTT to enhance the damage of tumor tissue upon irradiation.