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Biophotonics and analytics - Photoluminescence properties of nanocrystals and surface group analysis
(2018)
Correlating the photoluminescence (PL) properties of nanomaterials like semiconductor nanocrystals (QDs) and upconversion nanocrystals (UCNPs) assessed in ensemble studies and at the single particle level and studying their surface chemistry is increasingly relevant for applications of these nanomaterials in the life and material sciences. Here we present a comparison of the spectroscopic properties of ensembles and single nanocrystalline emitters and simple methods for the quantification of functional groups and ligands on particle surfaces. The overall goal of this study was to derive particle architectures and surface chemistries well suited for spectroscopic and microscopic applications.
Optical spectroscopic studies of the influence of size, particle architecture, and surface chemistry of different types of photoluminescent nanocrystals with emission in the vis/NIR will be presented including semiconductor quantum dots and lanthanide-based upconversion nanoparticles. This will include the photophysics of these materials assessed with steady state and time-resolved fluorometry on the ensemble and single particle level and concepts for the quantification of surface groups at nanomaterials with optical methods using cleavable probes and catch-and-release assays.
The comparison of different emitter classes and the rational design of the next generation of molecular and nanoscale reporters require accurate and quantitative photo-luminescence measurements. This is of special importance for all photoluminescence applications in the life and material sciences and nanobiophotonics. In the following, procedures for the determination of the spectroscopic key parameter photoluminescence quantum yield, i.e., the number of emitted per absorbed photons, in the UV/vis/NIR/SWIR are presented including pitfalls and achievable uncertainties and material-specific effects related to certain emitter classes are addressed.
Quantum dot (QD) based nanomaterials are very promising materials for the fabrication of optoelectronic devices like solar cells, light emitting diodes (LEDs), and photodetectors as well as as reporters for chemo- and biosensing and bioimaging. Many of These applications involve the monitoring of changes in photoluminescence intensity and energy transfer processes which can strongly depend on excitation wavelength or energy. In this work, we analyzed the excitation energy dependence (EED) of the photoluminescence quantum yields (PL QYs) and decay kinetics and the circular dichroism (CD) spectra of CdSe/CdS core/shell QDs with different thicknesses of the surface passivation shell. Our results demonstrate a strong correlation between the spectral position of local maxima observed in the EED of PL QY and the zero-crossing points of the CD profiles. Theoretical analysis of the energy band structure of the QDs with effective mass approximation suggests that these structures could correspond to exciton energy levels. This underlines the potential of CD spectroscopy for the study of electronic energy structure of chiroptically active nanocrystals which reveal quantum confinement effects.
The potential of semiconducting nanocrystals or so-called quantum dots (QDs) for lifetime multiplexing has not been investigated yet, despite the increasing use of QDs in (bio)analytical detection, biosensing, and fluorescence imaging and the obvious need for simple and cost-effective tools and strategies for the simultaneous detection of multiple analytes or events. This is most likely related to their multiexponential decay behavior as for multiplex chromophores, typically monoexponential decay kinetics are requested. The fluorescence decay kinetics of various mixtures of a long-lived, multiexponentially decaying CdSe QD and a short-lived organic dye were analyzed, and a model was developed for the quantification of these labels from the measured complex decay kinetics as a first proof-of-concept for the huge potential of these labels for lifetime multiplexing. In a second step, we evaluated the potential of mixtures of two types of QDs, varying in constituent material to realize distinguishable, yet multiexponential decay kinetics and similar absorption and emission spectra. Strategies for lifetime multiplexing with nanocrystalline labels were derived on the basis of these measurements.
The implementation of fluorescent methods is of outstanding importance in the field of optical chemical sensor Technology and biosciences. Their bioanalytical applications are manifold including fluorescence microscopy, fluorescence in situ hybridization, DNA sequencing, fluorescence-activated cell sorting, immunoassays, analysis of DNA and Protein microarrays, and quantitative PCR, just to name a few examples. Particularly, fluorescence microscopy is a valuable method in the versatile field of biomedical imaging methods which nowadays utilizes different fluorescence Parameters like emission wavelength/Color and lifetime for the discrimination between different targets. Sectional Images are available with confocal microscopes. Tissue, cells or single cellular compartments can be stained and visualized with fluorescent dyes and biomolecules can be selectively labeled with fluorescent dyes to Monitor biomolecular interactions inside cells or at Membrane bound receptors.
On the other hand , fluorophores can act as indicator (or "molecular probe") to visualize intrinsically colorless and non-fluorescent ionic and neutral analytes such as pH, Oxygen (pO2), metal ions, anions, hydrogen peroxide or bioactive small organic molecules such as Sugars or nucleotides. Thereby, their photoluminescent properties (fluorescence or phoporescence intensity, exitation and/or Emission wavelength, emission lifetime or anisotropy) respond to the presence of these species in their immediate Environment. In general, the use of luminescent probes has the advantage that they can be delivered directly into the sample, and detected in a contactless remote mode. By now, these probes are often encapsulated in different types of nanoparticles (NPs) made from (biodegradable) organic polymers, biopolymers or inorganic materials like silica or bound to their surface.
Mandatory for the comparison of different emitter classes and the rational design of the next generation of molecular and nanoscale reporters are reliable and quantitative photoluminescence measurements. This is of special relevance for all fluorescence applications in the life and material sciences. In the following, procedures for the determination of this spectroscopic key parameter are presented including pitfalls and achievable uncertainties and material-specific effects related to certain emitter classes are addressed.
Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is gaining importance for bioimaging cytometry to answer analytical, biological and biomedical questions. High sensitivity and spatial resolution make it an excellent tool for imaging of metal and heteroelement distribution in single cells. Comparable to CyTOF imaging mass cytometry, metal coded antibodies can be used for multiparametric analysis as well as quantification.
In this project, nanocrystals are investigated as new highly sensitive metal tags for identification and quantification of biomarkers, like Alzheimer’s or breast cancer, in clinical cell assays and tissue samples. Of high significance is the simultaneous analysis of several biomarkers at once, which is possible by special coding of lanthanide tags on the biomarker associated antibody. Nanocrystals show potential for sensitive measurement in MS due to high stability and signal amplification compared to tags with fewer metal atoms. For proof of principle, synthesis and characterization of lanthanide doped nanocrystals was performed by a nanoPET pharma GmbH with great reproducibility and homogenous size. In A549 cell cultures, the uptake and distribution of these nanocrystals within the monolayered cells was investigated by LA-ICP-MS measurements using subcellular resolution. The nanocrystals showed high sensitivity and the possibility of multiparametric analysis by doting different lanthanides. Additionally, stability of the bioconjugation of the nanocrystals and target antibodies was investigated using Dot Blot experiments and LA-ICP-MS.
Luminescent nanocrystals like core/shell semiconductor quantum dots and lanthanide doped nanophosphors as well as gold nanoclusters with emission in the visible (vis) and particularly in the near infrared (NIR) and short wavelength infrared (SWIR) region have been increasingly used as reporters in the life sciences and for bioimaging studies in the last years. This has led to sophisticated core-shell particle architectures of different chemical composition utilizing semiconductor quantum dots and lanthanide-based nanocrystals and initiated the design of gold nanoclusters with different ligands. In addition, this led to an increasing number of quantitative spectroscopic studies focusing on the key performance parameter photoluminescence quantum yield to identify optimum particle structures. In the following, an overview of different classes of nanocrystalline emitters and their photophysics is provided and examples for the absolute characterization of the photoluminescence properties of these different vis/NIR/SWIR emitters are shown including excitation power density-dependent studies on the ensemble and single particle level. Also, the impact of such measurements on a profound mechanistic understanding of the underlying nonradiative deactivation pathways is highlighted as required for reporter design.
Surface functionalization of nanomaterials is nowadays at the core of many applications of functional materials in the life and material sciences. Examples range from membranes and microarrays over bead-based assays, and next generation sequencing to nanometer-sized optical reporters, nanosensors, and magnetic and optical contrast agents. Typical function-nalization steps include silanization and grafting reactions with reactive monomers to introduce functional groups like amino or carboxylic acid groups or the attachment of ligands like polyethylene glycol (PEG) molecules and biomolecules. [1-3] This enables to tune e.g., dis-persibility, hydrophilicity and biocompatibility, minimize unspecific interactions, improve biofunctionalization efficiencies, and enhance blood circulation times and allows for the use of nanomaterials as reporters in assays or the design of targeted probes for bioimaging.
At the core of all functionalization strategies are reliable and validated methods for surface group and ligand quantification that can be preferably performed with routine laboratory instrumentation, require only small amounts of substances, and are suitable for many different types of nanomaterials. [3] There is meanwhile a considerable need to make these methods traceable. We present here versatile and simple concepts for the quantification of common functional groups, ligands, and biomolecules on different types of organic and inorganic nanomaterials, using conventional and newly developed cleavable and multimodal reporters, that can be detected with optical spectroscopy. [4-7] These reporters are chosen to enable method validation with the aid of method comparisons and mass balances. Also, strategies how to make these simple assays traceable to SI units using quantitative nuclear resonance spectroscopy (qNMR) and X-ray photoelectron spectroscopy (XPS) are derived.
Accurate and quantitative photoluminescence measurements are mandatory for the comparison of different emitter classes and the rational design of the next generation of molecular and nanoscale reporters as well as for most applications relying on their luminescence features in the life and material sciences and nanobiophotonics. In the following, procedures for the determination of the spectroscopic key parameter photoluminescence quantum yield, i.e., the number of emitted per absorbed photons, in the UV/vis/NIR/SWIR are presented including pitfalls and achievable uncertainties and material-specific effects related to certain emitter classes are addressed. Special emphasis is dedicated to luminescent nanocrystals.
Inorganic nanocrystals with linear and nonlinear luminescence in the ultraviolet, visible, near infrared and shortwave infrared like semiconductor quantum dots and spectrally shifting lanthanide-based nanophosphors have meanwhile found applications in the life and material sciences ranging from optical reporters for bioimaging and sensing over security barcodes to solid state lighting and photovoltaics. These nanomaterials commonly have increasingly sophisticated core/shell particle architectures with shells of different chemical composition and thickness to minimize radiationless deactivation at the particle surface that is usually the main energy loss mechanism [1]. For lanthanide-based spectral shifters, particularly for very small nanoparticles, also surface coatings are needed which protect near-surface lanthanide ions from luminescence quenching by high energy vibrators like O-H groups and prevent the disintegration of these nanoparticles under high dilution conditions. [2,3,4]. The identification of optimum particle structures requires quantitative spectroscopic studies focusing on the key performance parameter photoluminescence quantum yield [5,6], ideally flanked by single particle studies to assess spectroscopic inhomogeneities on a particle-to-particle level for typical preparation methods [7], Moreover, in the case of upconversion nanoparticles with a multi-photonic and hence, excitation power density (P)-dependent luminescence, quantitative luminescence studies over a broad P range are required to identify particle architectures that are best suited for applications in fluorescence assays up to fluorescence microscopy. Here, we present methods to quantify the photoluminescence of these different types of emitters in the vis/NIR/SWIR and as function of Pand demonstrate the importance of such measurements for a profound mechanistic understanding of the nonradiative deactivation pathways in semiconductor and upconversion nanocrystals of different size and particle architecture in different environments.
At the core of luminescence color and lifetime Tuning of rare earth doped upconverting nanoparticles (UCNPs), is the understanding of the Impact of the particle architecture for commonly used sensitizer (S) and activator (A) Ions. In this respect, a series of core@Shell NaYF4 UCNPs doped with Yb3+ and Ho3+ ions are presented here, where the same dopant concentrations are distributed in different particle architectures following the scheme: YbHo core and YbHo@..., ...@YbHo, Yb@Ho, Ho@Yb, YbHo@Yb, and Yb@YbHo core-Shell NPs. As refealed by quantitative steady-state and time-resolved luminescence studies, the relative spatial Distribution of the A and S ions in the UCNPs and their protection from surface quenching has a critical Impact on ther luminescence characteristics. Although the increased amount of Yb3+ Ions boosts UCNP Performance by amplifying the Absorption, the Yb3+ ions can also efficiently dissipate the energy stored in the material through energy Migration to the surface, thereby reducing the Overall energy Transfer Efficiency to the activator ions. The results provide yet another proof that UC Phosphor chemistry combined with materials Engineering through intentional core@shell structures may help to fine-tune the luminescence Features of UCNPs for their specific future applications in biosensing, bioimaging, photovoltaics, and Display technologies.
We investigated the correlation between the thickness of the ZnS shell of CdSeZnS quantum dots (QDs), the stability of the particles, and the fluorescence quantum yield. As a measure for stability, a new shell quality test was developed. This test is based on the reaction of the QDs with photochemically formed thiophenol radicals and communicates an imperfect ZnS shell by a rapid and complete loss of fluorescence. The quantum yield increases from less than 5% for unshelled CdSe up to 50%, with an increase in ZnS shell thickness up to 0.60.8 nm. At the same time, the particles become significantly more stable, as revealed by the shell test.
In der klinischen Diagnostik werden für zytometrische Messverfahren bereits eine Reihe von Reagenzien eingesetzt zur Markierung von Antikörper eingesetzt, um die Detektion von Biomarkern mittels Fluoreszenz- oder Flugzeitmassenspektrometrie zu ermöglichen.
Seit kurzem ist auch eine Imaging Mass Cytometry Kombination direkt erhältlich, wodurch der Nachweis von Biomarkern in Gewebeschnitten erreicht werden kann. Dazu wird eine Kopplung von Laser Ablation und induktiv gekoppeltem Plasma Massenspektrometrie eingesetzt, wobei ähnlich der Massenzytometrie, zuvor Antikörper mit Metallen markiert, und im Anschluss mit dem Gewebeschnitt inkubiert werden. Durch die hohe Ortsauflösung können die Biomarker lokalisiert, und zukünftig vielleicht auch quantifiziert werden.
Insbesondere Lanthanide eignen sich als Markierungsmetalle, da sie einen niedrigen Untergrund und chemisch ähnliches Verhalten zueinander aufweisen. Allein durch diese Elemente können bereits etwa 15 Parameter unterschieden werden, was durch isotopenreine Standards weiter gesteigert werden kann.
Vom Markierungsgrad abhängig werden unterschiedlich viele Metalle am Antikörper gebunden, und beeinflussen so die Sichtbarkeit im ICP-MS. Nanopartikel könnten daher eine deutliche Steigerung der Sensitivität bewirken. GdVO4 Nanokristalle scheinen bisher sehr vielversprechend und bieten neben multiparametrischen Anwendungen auch Multimodalität.
Die Synthese der Nanokristalle zeigte hohe Homogenität und Reproduzierbarkeit in Partikelgröße in der Zusammensetzung. Ein erstes Experiment mit einer Zellkultur konnte bereits die effiziente Markierung der Zellen unter Beweis stellen, wobei durch hohe Signalstärke auch subzelluläre Auflösung in der LA-ICP-MS erreicht werden konnte.