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Organisationseinheit der BAM
Functional nanomaterials (NM) of different size, shape, chemical composition, and surface chemistry are of increasing relevance for many key technologies of the twenty-first century. This includes polymer and silica or silica-coated nanoparticles (NP) with covalently bound surface groups, semiconductor quantum dots (QD), metal and metal oxide NP, and lanthanide-based NP with coordinatively or electrostatically bound ligands, as well as surface-coated nanostructures like micellar encapsulated NP. The surface chemistry can significantly affect the physicochemical properties of NM, their charge, their processability and performance, as well as their impact on human health and the environment. Thus, analytical methods for the characterization of NM surface chemistry regarding chemical identification, quantification, and accessibility of functional groups (FG) and surface ligands bearing such FG are of increasing importance for quality control of NM synthesis up to nanosafety. Here, we provide an overview of analytical methods for FG analysis and quantification with special emphasis on bioanalytically relevant FG broadly utilized for the covalent attachment of biomolecules like proteins, peptides, and oligonucleotides and address methodand material-related challenges and limitations. Analytical techniques reviewed include electrochemical titration methods, optical assays, nuclear magnetic resonance and vibrational spectroscopy, as well as X-ray based and thermal analysis methods, covering the last 5–10 years. Criteria for method classification and evaluation include the need for a signal-generating label, provision of either the total or derivatizable number of FG, need for expensive instrumentation, and suitability for process and production control during NM synthesis and functionalization.
Critical review of the determination of photoluminescence quantum yields of luminescent reporters
(2015)
A crucial variable for methodical performance evaluation and comparison of luminescent reporters is the photoluminescence quantum yield (Φ pl). This quantity, defined as the number of emitted photons per number of absorbed photons, is the direct measure of the efficiency of the conversion of absorbed photons into emitted light for small organic dyes, fluorescent proteins, metal–ligand complexes, metal clusters, polymeric nanoparticles, and semiconductor and up-conversion nanocrystals. Φ pl determines the sensitivity for the detection of a specific analyte from the chromophore perspective, together with its molar-absorption coefficient at the excitation wavelength. In this review we discuss different optical and photothermal methods for measuring Φ pl of transparent and scattering systems for the most common classes of luminescent reporters, and critically evaluate their potential and limitations. In addition, reporter-specific effects and sources of uncertainty are addressed. The ultimate objective is to provide users of fluorescence techniques with validated tools for the determination of Φ pl, including a series of Φ pl standards for the ultraviolet, visible, and near-infrared regions, and to enable better judgment of the reliability of literature data.
In order to assess the anisotropy-related uncertainties of relatively determined photoluminescence quantum yields (ΦPL) of molecular emitters and luminescent nanomaterials, we compared ΦPL values measured without and with polarizers using magic angle conditions and studied systematically the dependence of the detected emission intensity on the polarizer settings for samples of varying anisotropy. This includes a dispersion of a spherical quantum dot (QD) with an ideally isotropic emission, a solution of a common small organic dye in a fluid solvent as well as dispersions of elongated quantum dot rods (QDR) with an anisotropic luminescence and a small organic dye in a rigid polymeric matrix, as ideally anisotropic emitter. Our results show that for instruments lacking polarizers, anisotropy-related measurement uncertainties of relative photoluminescence quantum yields can amount to more than 40%, with the size of these systematic errors depending on the difference in emission anisotropy between the sample and the standard.
Many applications of nanometer- and micrometer-sized particles include their surface functionalization with linkers, sensor molecules, and analyte recognition moieties like (bio)ligands. This requires knowledge of the chemical nature and number of surface groups accessible for subsequent coupling reactions. Particularly attractive for the quantification of these groups are spectrophotometric and fluorometric assays, which can be read out with simple instrumentation. In this respect, we present here a novel Family of cleavable spectrophotometric and multimodal reporters for conjugatable amino and carboxyl surface groups on nano- and microparticles. This allows determination of particle-bound labels, unbound reporters in the supernatant, and reporters cleaved off from the particle surface, as well as the remaining thiol groups on particle, by spectrophotometry and inductively coupled optical emission spectrometry (32S ICP-OES). Comparison of the performance of these cleavable reporters with conductometry and conventional labels, utilizing changes in intensity or Color of absorption or emission, underlines the analytical potential of this versatile concept which elegantly circumvents Signal distortions by scattering and Encoding dyes and enables straightforward validation by method comparison.
Polyamidoamine (PAMAM) dendrimers were used to produce CdSe core/multi-shell fluorescent quantum dots (QDs) which are colloidally stable in aqueous solutions. The size, charge, and optical properties of QDs functionalized with the 4th (G4) and 5th (G5) generation of PAMAM were compared with amphiphilic polymer-covered QDs and used as criteria for the evaluation of the suitability of both water solubilization methods. As revealed by dynamic and electrophoretic light scattering (DLS and ELS), the hydrodynamic sizes of the QDs varied from 30 to 65 nm depending on QD type and dendrimer generation, with all QDs displaying highly positive surface charges, i.e., zeta potentials of around +50 mV in water. PAMAM functionalization yielded stable core/multi-shell QDs with photoluminescence quantum yields (Φ) of up to 45%. These dendrimer-covered QDs showed a smaller decrease in their Φ upon phase transfer compared with QDs made water soluble via encapsulation with amphiphilic brush polymer bearing polyoxyethylene/ polyoxypropylene chains.
This paper describes the production and characteristics of the nanoparticle test materials prepared for common use in the collaborative research project NanoChOp (Chemical and optical characterization of nanomaterials in biological systems), in casu suspensions of silica nanoparticles and CdSe/CdS/ZnS quantum dots (QDs). This paper is the first to illustrate how to assess whether nanoparticle test materials meet the requirements of a "reference material" (ISO Guide 30, 2015) or rather those of the recently defined category of "representative test material (RTM)" (ISO/TS 16195, 2013). The NanoChOp test materials were investigated with small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and centrifugal liquid sedimentation (CLS) to establish whether they complied with the required monomodal particle size distribution. The presence of impurities, aggregates, agglomerates, and viable microorganisms in the suspensions was investigated with DLS, CLS, optical and electron microscopy and via plating on nutrient agar. Suitability of surface functionalization was investigated with attenuated total reflection Fourier transform infrared spectrometry (ATR-FTIR) and via the capacity of the nanoparticles to be fluorescently labeled or to bind antibodies. Between-unit homogeneity and stability were investigated in terms of particle size and zeta potential. This paper shows that only based on the outcome of a detailed characterization process one can raise the status of a test material to RTM or reference material, and how this status depends on its intended use.
Effect of fluorescent staining on size measurements of polymeric nanoparticles using DLS and SAXS
(2015)
The influence of fluorescence on nanoparticle size measurements using dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) was investigated. For this purpose, two series of 100 nm-sized polymer nanoparticles stained with different concentrations of the fluorescent dyes DY555 and DY680 were prepared, absorbing/emitting at around 560 nm/590 nm and 695 nm/715 nm, respectively. SAXS measurements of these particle series and a corresponding blank control (without dye) revealed similar sizes of all particles within an uncertainty of 1 nm. DLS measurements carried out at three different laboratories using four different DLS instruments and two different laser wavelengths, i.e., 532 nm and 633 nm, revealed also no significant changes in size (intensity-weighted harmonic mean diameter, ZAverage) and size distribution (polydispersity index, PI) within and between the two dye-stained particle series and the blank sample. Nevertheless, a significant decrease of the detected correlation coefficients was observed with increasing dye concentration, due to the increased absorption of the incident light and thus, less coherent light scattering. This effect was wavelength dependent, i.e. only measurable for the dye-stained particles that absorb at the laser wavelength used for the DLS measurements.
The fluorescence quantum yield (QY) of CdSe dot/CdS rod (DR) nanoparticle ensembles is dependent on the Shell growth and excitation wavelength. We analyze the origin of this dependency by comparing the optical properties of DR ensembles to the results obtained in single-particle experiments. On the Ensemble level, we find that the QY of DRs with shell lengths shorter than 40 nm exhibits no dependence on the excitation wavelength, whereas for DRs with shell lengths longer than 50 nm, the QY significantly decreases for excitation above the CdS band gap. Upon excitation in the CdSe core, the ensemble QY, the fluorescence wavelength, and the fluorescence blinking behavior of individual particles are only dependent on the radial CdS shell thickness and not on the CDs shell length. If the photogenerated excitons can reach the CdSe core region, the fluorescence properties will be dependent only on the surface passivation in close vicinity to the CdSe core. The change in QY upon excitation above the band gap of CdS for longer DRs cannot be explained by nonradiative particles because the ratio of emitting DRs is found to be independent of the DR length. We propose a model after which the decrease in QY for longer CdS shells is due to an increasing fraction of nonradiative exciton recombination within the elongated shell. This is supported by an effective-mass-approximation-based calculation, which suggests an optimum length of DRs of about 40 nm, to combine the benefit of high CdS absorption cross section with a high fluorescence QY.
The large number of nanomaterial-based applications emerging in the materials and life sciences and the foreseeable increasing use of these materials require methods that evaluate and characterize the toxic potential of these nanomaterials to keep safety risks to people and environment as low as possible. As nanomaterial toxicity is influenced by a variety of parameters like size, shape, chemical composition, and surface chemistry, high throughput screening (HTS) platforms are recommended for assessing cytotoxicity. Such platforms are not yet available for genotoxicity testing. Here, we present first results obtained for application-relevant nanomaterials using an automatable genotoxicity platform that relies on the quantification of the phosphorylated histone H2AX (γ-H2AX) for detecting DNA double strand breaks (DSBs) and the automated microscope system AKLIDES® for measuring integral fluorescence intensities at different excitation wavelengths. This platform is used to test the genotoxic potential of 30 nm-sized citrate-stabilized gold nanoparticles (Au-NPs) as well as micellar encapsulated iron oxide nanoparticles (FeOx-NPs) and different cadmium (Cd)-based semiconductor quantum dots (QDs), thereby also searching for positive and negative controls as reference materials. In addition, the influence of the QD shell composition on the genotoxic potential of these Cd-based QDs was studied, using CdSe cores as well as CdSe/CdS core/shell and CdSe/CdS/ZnS core/shell/shell QDs. Our results clearly revealed the genotoxicity of the Au-NPs and its absence in the FeOx-NPs. The genotoxicity of the Cd-QDs correlates with the shielding of their Cd-containing core, with the core/shell/shell architecture preventing genotoxicity risks. The fact that none of these nanomaterials showed cytotoxicity at the chosen particle concentrations in a conventional cell viability assay underlines the importance of genotoxicity studies to assess the hazardous potential of nanomaterials.
Controlling thickness and tightness of surface passivation shells is crucial for many applications of core–shell nanoparticles (NP). Usually, to determine shell thickness, core and core/shell particle are measured individually requiring the availability of both nanoobjects. This is often not fulfilled for functional nanomaterials such as many photoluminescent semiconductor quantum dots (QD) used for bioimaging, solid state lighting, and display technologies as the core does not show the applicationrelevant functionality like a high photoluminescence (PL) quantum yield, calling for a whole nanoobject approach. By combining high-resolution transmission electron microscopy (HR-TEM) and X-ray photoelectron spectroscopy (XPS), a novel whole nanoobject approach is developed representatively for an ultrabright oleic acid-stabilized, thick shell CdSe/CdS QD with a PL quantum yield close to unity. The size of this spectroscopically assessed QD, is in the range of the information depth of usual laboratory XPS. Information on particle size and monodispersity were validated with dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) and compared to data derived from optical measurements. In addition to demonstrating the potential of this novel whole nanoobject approach for determining architectures of small nanoparticles, the presented results also highlight challenges faced by different sizing and structural analysis methods and method-inherent uncertainties.
Organic and inorganic nanoparticles (NPs) are increasingly used as drug carriers, fluorescent sensors, and multimodal labels in the life and material sciences. These applications require knowledge of the chemical nature, total number of surface groups, and the number of groups accessible for subsequent coupling of e.g., antifouling ligands, targeting bioligands, or sensor molecules. To establish the concept of catch-and-release assays, cleavable probes were rationally designed from a quantitatively cleavable disulfide moiety and the optically detectable reporter 2-thiopyridone (2-TP). For quantifying surface groups on nanomaterials, first, a set of monodisperse carboxy-and amino-functionalized, 100 nm-sized polymer and silica NPs with different surface group densities was synthesized. Subsequently, the accessible functional groups (FGs) were quantified via optical spectroscopy of the cleaved off reporter after its release in solution. Method validation was done with inductively coupled plasma optical emission spectroscopy (ICP-OES) utilizing the sulfur atom of the cleavable probe. This comparison underlined the reliability and versatility of our probes, which can be used for surface group quantification on all types of transparent, scattering, absorbing and/or fluorescent particles. The correlation between the total and accessible number of FGs quantified by conductometric titration, qNMR, and with our cleavable probes, together with the comparison to results of conjugation studies with differently sized biomolecules reveal the potential of catch-and-release reporters for surface analysis. Our findings also underline the importance of quantifying particularly the accessible amount of FGs for many applications of NPs in the life sciences.
The syntheses, structures, and photophysical properties of two new zinc(II) complexes bearing the tridentate N,N′-dimethyl-N,N′-dipyridin-2-ylpyridine-2,6-diamine (ddpd) ligand are presented. Structural investigations through single-crystal X-ray diffractometry, NMR spectroscopy, and density functional theory calculations revealed a diverse coordination behavior that depends on the counterion. Spectroscopic (UV/Vis and emission spectroscopy) and theoretical techniques (DFT and time-dependent DFT calculations) were employed to explore the photophysical properties of the complexes.
The excitation wavelength (lexc) dependence of the photoluminescence (PL) quantum yield (FPL) and decay behavior (tPL) of a series of CdSe/CdS quantum dot/Quantum rods (QDQRs), consisting of the same spherical CdSe core and rod-shaped CdS shells, with aspect ratios ranging from 2 to 20 was characterized. lexc between 400–565 nm were chosen to cover the first excitonic absorption band of the CdSe core material, the onset of absorption of the CDs shell, and the region of predominant shell absorption. A strong lexc dependence of relative and absolutely measured FPL and tPL was found particularly for the longer QDQRs with higher aspect ratios. This is attributed to combined contributions from a length-dependent shell-to-core exciton localization efficiency, an increasing number of defect states within the shell for the longest QDQRs, and probably also the presence of absorbing, yet non-emitting shell material. Although the FPL values of the QDQRs decrease at shorter wavelength, the extremely high extinction coefficients introduced by the shell outweigh this effect, leading to significantly higher brightness values at wavelengths below the absorption onset of the CdS Shell compared with direct excitation of the CdSe cores. Moreover, our results present also an interesting example for the comparability of absolutely measured FPL using an integrating sphere setup and FPL values measured relative to common FPL standards, and underline the Need for a correction for particle scattering for QDQRs with high aspect ratios.
Heterogeneous sandwich immunoassays are widely used for biomarker detection in bioanalysis and medical diagnostics. The high analyte sensitivity of the current “gold standard” enzyme-linked immunosorbent assay (ELISA) originates from the signal-generating enzymatic amplification step, yielding a high number of optically detectable reporter molecules. For future point-of-care testing (POCT) and point-of-need applications, there is an increasing interest in more simple detection strategies that circumvent time-consuming and temperature-dependent enzymatic reactions. A common concept to aim for detection limits comparable to those of enzymatic amplification reactions is the usage of polymer nanoparticles (NP) stained with a large number of chromophores. We explored different simple NP-based signal amplification strategies for heterogeneous sandwich immunoassays
that rely on an extraction-triggered release step of different types of optically detectable reporters. Therefore, streptavidinfunctionalized polystyrene particles (PSP) are utilized as carriers for (i) the fluorescent dye coumarin 153 (C153) and (ii) hemin (hem) molecules catalyzing the luminol reaction enabling chemiluminescence (CL) detection. Additionally, (iii) NP labeling with hemin-based microperoxidase MP11 was assessed. For each amplification approach, the PSP was first systematically optimized regarding size, loading concentration, and surface chemistry. Then, for an immunoassay for the inflammation marker C- eactive protein (CRP), the analyte sensitivity achievable with optimized PSP
ystems was compared with the established ELISA concept for photometric and CL detection. Careful optimization led to a limit of detection (LOD) of 0.1 ng/mL for MP11-labeled PSP and CL detection, performing similarly well to a photometric ELISA (0.13 ng/mL), which demonstrates the huge potential of our novel assay concept.