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Eingeladener Vortrag
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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.
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.
Entwicklung neuer Sonden für bioanalytische Anwendungen der oberflächenverstärkten Raman-Streuung
(2011)
Surface-enhanced Raman scattering (SERS) has been established as a versatile tool for probing and labeling in analytical applications, based on the vibrational spectra of samples as well as label molecules in the proximity of noble metal nanostructures. The aim of this work was the construction of novel SERS hybrid probes. The hybrid probes consisted of Au and Ag nanoparticles and reporter molecules, as well as a targeting unit. The concept for the SERS hybrid probe design was followed by experiments comprising characterization techniques such as UV/Vis- spectroscopy (UV/Vis), Transmission electron microscopy (TEM) and Dynamic Light Scattering (DLS), respectively. SERS experiments were per- formed for studying and optimizing the plasmonic properties of nanoparticles with respect to their enhancement capabilities. The SERS-probes had to meet following requirements: biocompatibility, stability in physiological media, and enhancement of Raman-signals from Raman reporter molecules enabling the identification of different probes even in a complex biological environment. Au and Ag nanoaggregates were found to be the most appropriate SERS substrates for the hybrid probe design. The utilization of Raman reporters enabled the identification of different SERS probes in multiplexing experiments. In particular, the multiplexing capability of ten various reporter molecules para-aminobenzenethiol, 2-naphthalenethiol, crystal violet, rhodamine (B) isothiocyanate, fiuorescein isothiocyanate, 5,5'dithiobis(2-nitrobenzoic acid), para- mercaptobenzoic acid, acridine orange, safranine O und nile blue was studied using NIR-SERS excitation. As demonstrated by the results the reporters could be identified through their specific Raman signature even in the case of high structural similarity. Chemical separation analysis of the reporter signatures was performed in a trivariate approach, enabling the discrimination through an automated calculation of specific band ratios. The trivariate identification could be a promising method for SERS-multiplexing in analytical applications. Multivariate methods such as Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were as well applied for discrimination and imaging of the reporter signatures. With the help of multivariate imaging methods based on cluster analysis, it could be for the first time demonstrated that such methods provided a fast identification of various SERS hybrid probes inside the biological matrix, this was demonstrated using living 3T3 cells. Further, in a duplex imaging approach, the probes fulfill the requirements for the sensitive detection of both the specific reporter signatures and intrinsic information coming from eukaryotic cells. The results of cluster methods and principal components approaches for discrimination indicate that fast, multivariate evaluation of whole sets of multiple probes is feasible, beyond the visual inspection of individual spectra that has been practiced so far. This suggests multiplexing applications with SERS hybrid nanoprobes and SERS tags in very high density sensing and biological imaging applications, where fast read-out is required. The pH-sensitivity of SERS-Tags that consisted of different reporter molecules attached to aggregated Au and Ag nanoparticles in the range between pH∼3-10 was studied. It could be demonstrated that the reporter molecules provided pH-dependent SERS signatures and could therefore be suitable for the sensitive pH-detection, e.g., inside cellular compartments. The construction of targeted SERS probes was based on the integration of a goat-anti- mouse antibody as targeting element Antibodies were coupled to Au and Ag nanoprobes surrounded by a Bovine Serum Albumin (BSA) coating which served as a carrier for the covalent linkage of a Raman reporter molecule and the targeting units at the same time In experiments with BSA and a conjugated reporter the spectra of the BSA-coupled re- porters provided an indication of the secondary structure of BSA which is related to the BSA-reporter coupling procedure In in v i t ro -experiments with BSA-coupled nanoprobes inside 3T3 cells reporter signatures and intrinsic information from the cellular matrix could be delivered BSA enabled the coupling of reporter molecules as well as the targeting of antibodies and served as stabilizer of the gold nanoaggregates The functionality of the coupled antibodies after their integration into the SERS probe was retained This was verified by the results of a direct Enzyme-Linked Immunosorbent Assay (ELISA) Conjugates with implemented reporter molecules could be characterized using SERS The application of the complete probes suggested a use of these novel biocompatible stable and targeted SERS probes that can be excited out-of- resonance also for other bioanalytical applications. On the basis of the constructed SERS hybrid probes comprising a large number of BSA- coupled reporters could e g be implemented for automated high-througput immuno- assays where they are arranged on a microstructured device for the simultaneous and multilevel SERS-readout in one step .