Ingenieurwissenschaften und zugeordnete Tätigkeiten
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- 2016 (7) (entfernen)
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- Dye (3)
- Assay (2)
- Fluorescence (2)
- Activatable probe (1)
- Aggregation (1)
- Aldrithiol (1)
- Cellular uptake (1)
- Characterization (1)
- Chiral (1)
- Cyanine (1)
Quality control requirements imposed on assays used in clinical diagnostics and point-of-care-diagnostic testing (POCT), utilizing amplification reactions performed at elevated temperatures of 35 to 95 °C are very stringent. As the temperature of a reaction vessel has a large impact on the specificity and sensitivity of the amplification reaction, simple tools for local in situ temperature sensing and monitoring are required for reaction and assay control. We describe here a platform of stem-and-loop structured DNA hairpins (molecular beacons, MBs), absorbing and emitting in the visible and red spectral region, rationally designed for precise temperature measurements in microfluidic assays for POCT, and their ap-plication for temperature measurements in a common DNA-based molecular biological assay utilizing thermophilic helicase-dependent amplification (tHDA). Spectroscopic studies of these MBs, rationally designed from DNA se-quences of different thermal stabilities, chosen not to interact with the DNA probes applied in the nucleic acid amplification assay, and temperature-dependent fluorescence measurements of MB-assay mixtures revealed the suitability of these MBs for temperature measurements directly in such an assay with a temperature resolution of about 0.5 °C without interferences from assay components. Combining two spectrally distinguishable MBs provides a broader response range and an increase in temperature sensitivity up to 0.1 °C. This approach will find future application for temperature monitoring and quality control in commercialized diagnostics assays using dried reagents and microfluidic chips as well as assays read out with tube and microplate readers and PCR detection systems for temperature measurements in the range of 35 to 95 °C.
3-Aminovinylquinoxalines are readily accessible from (hetero)aryl glyoxylic acids or heterocyclic π-nucleophiles by consecutive four- and fivecomponent syntheses in the sense of an activation-alkynylation-cyclocondensation-addition sequence or glyoxylation-alkynylation-cyclocondensation-addition sequence in good yields. The title compounds are highly fluorescent with pronounced emission solvatochromicity and protochromic fluorescence quenching. Time-resolved fluorescence spectroscopy furnishes radiative and nonradiative fluorescence decay rates in various solvent polarities. The electronic structure is corroborated by DFT and TD-DFT calculations rationalizing the observed spectroscopic effects.
Simple, fast, and versatile methods for the quantification of thiol groups are of considerable interest not only for protein analysis but also for the characterization of the surface chemistry of nanomaterials stabilized with thiol ligands or bearing thiol groups for the subsequent (bio-) functionalization via maleimide−thiol chemistry. Here, we compare two simple colorimetric assays, the widely used Ellman’s assay performed at alkaline pH and the aldrithiol assay executed at acidic and neutral pH, with respect to their potential for the quantification of thiol groups and thiol ligands on different types of nanoparticles like polystyrene nanoparticles, semiconductor nanocrystals (SC NC), and noble metal particles, and we derive criteria for their use. In order to assess the underlying reaction mechanisms and to obtain stoichiometry factors mandatory for reliable thiol quantification, both methods were studied photometrically and with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), thereby demonstrating the influence of different thiols on the reaction mechanism. Our results underline the suitability of both methods for the quantification of directly accessible thiol groups or ligands on the surface of 2D- and 3D-supports, here exemplarily polystyrene nanoparticles. Moreover, we could derive strategies for the use of these simple assays for the determination of masked (i.e., not directly accessible) thiol groups like disulfides such as lipoic acid and thiol stabilizing ligands coordinatively bound to Cd and/or Hg surface atoms of II/VI and ternary SC NC and to gold and silver nanoparticles.
Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach.
With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.
Nanoclays like laponites, which are commercially avaible in large quantities for a very moderate price, provide a facile solubilization strategy for hydrophobic dyes without the need for chemical functionalization and can act as a carrier for a high number of dye molecules. This does not require reactive dyes, amplifies fluorescence signals from individual emitters due to the high number of dyes molecules per laponite disk, and renders hydrophobic emitters applicable in aqueous environments. Aiming at the rational design of bright dye-loaded nanoclays as a new class of fluorescent reporters for bioanalysis and material sciences and the identification of dye structure−property relationships, we screened a series of commercial fluorescent dyes, differing in dye class, charge, and character of the optical transitions involved, and studied the changes of their optical properties caused by clay adsorption at different dye loading concentrations. Upon the basis of our dye loading density-dependent absorption and fluorescence measurements with S2105 and Lumogen F Yellow 083, we could identify two promising dye−nanoclay hybrid materials that reveal high fluorescence quantum yields of the nanoclay-adsorbed dyes of at least 0.20 and low dye self-quenching even at high dye-loading densities of up to 50 dye molecules per laponite platelet.
Enzyme-activatable optical probes are important for future advances in cancer imaging, but may easily suffer from low signal-to-background ratios unless not optimized. To address this shortcoming, numerous mechanisms to modulate the fluorescence signal have been explored.
We report herein newly synthesized probes based on selfimmolative linkers containing chiral J-aggregate-forming dyes.
Signal modulation by formation of chiral J-aggregates is yet unexplored in optical enzyme probe design. The comprehensive characterization of the probes by absorption, CD, fluorescence, and time-resolved fluorescence spectroscopy revealed dye−dye interactions not observed for the free dyes in solution as well as dye−protein interactions with the enzyme. This suggested
that J-aggregate formation is challenging to achieve with current probe design and that interactions of the dyes with the Enzyme may interfere with achieving high signal-to-background ratios. The detailed understanding of the interactions provided herein provides valuable guidelines for the future design of similar probes.
We present colloidally stable and highly luminescent ZnxCd1_xS:Mn/ZnS core–shell nanocrystals (NCs) synthesized via a simple non-injection one-pot, two-step synthetic route, which can be easily upscaled. A systematic variation of the reaction component, parameters and thickness of the ZnS shell yielded doped nanocrystals with a very high photoluminescence quantum yield (pl) of 70%, which is the highest value yet reported for these Mn-doped sulfide-semiconductor NCs. These materials can be synthesized with high reproducibility in large quantities of the same high quality, i.e., the same pl using accordingly optimized reaction conditions. The application of these zero-reabsorption high quality NCs in the light conversion layers, deposited on top of a commercial monocrystalline silicon (mono-Si) solar cell, led to a significant enhancement of the external quantum efficiency (EQE) of this device in the ultraviolet spectral region between 300 and 400 nm up to ca. 12%. EQE enhancement is reflected by an increase in the power conversion efficiency (PCE) by nearly 0.5 percentage points and approached the theoretical limit (0.6%) expected from down-shifting for this Si solar cell. The resulting PCE may result in a BoM (bill of materials) cost reduction of app. 3% for mono-Si photovoltaic modules. Such small but distinct improvements are expected to pave the road for an industrial application of doped semiconductor NCs as cost-effective light converters for silicon photovoltaic (PV) and other optoelectronic applications.