Analytische Chemie
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The surface chemistry / functionalization of nanomaterials and microparticles largely controls the stability of these materials as well as their solubility and subsequent biofunctionalization and their interactions with biological systems. Moreover, in the case of some nanomaterials like semiconductor quantum dots or lanthanide-based upconversion nanocrystals, the ligand shell strongly affects their optical properties, e.g., via passivation of surface states and traps that favor luminescence quenching or the protection of surface atoms from quenching water molecules. This renders analytical methods for the quantification of surface groups like functionalities very important. Targets of broad interest are here amino, carboxyl, alkine and maleimide groups used for common bioconjugation reactions and typical ligands like thiols and polyethylene glycol (PEG) molecules of varying length, used for the tuning of material hydrophilicity and biocompatibility, minimization of unspecific interactions, prevention of biofouling, and enhancement of blood circulation times as well as surface-bound biomolecules like streptavidin or other biomolecules relevant e.g., for diagnostic assays. Here, we focus on simple optical methods relying on standard laboratory instrumentation, validated by method comparison and/or mass balances and present examples for their use for the characterization of different types of nanomaterials and microparticles.
Luminescence-based detection methods, ranging from fluorescence spectroscopy for photophysical and mechanistic studies over sensing applications, chromatographic separation techniques and the microarray technology with fluorescence detection to fluorescence microscopy, flow cytometry, single molecule spectroscopy, and molecular imaging to integrating sphere spectroscopy, are among the most widely used methods in the life and material sciences. This is due to e.g., their unique sensitivity enabling the detection of single molecules, potential for multiplexing, ease of combination with spatial resolution, and suitability for remote sensing. Many of these advantages are closely linked to the choice of suitable molecular and nanoscale fluorescent reporters, typically required for signal generation. This includes organic dyes without and with sensor function, fluorophore-encoded polymeric and silica nanoparticles as well as nanocrystalline systems like semiconductor quantum dots and upconversion phosphors, emitting in the visible (vis), near-infrared (NIR), and IR (infrared). Current challenges present the environment sensitivity of most fluorophores, rendering fluorescence spectra, measured intensities/fluorescence quantum yields, and fluorescence decay kinetics matrix-dependent, and instrument-specific distortions of measured fluorescence signals that need to be considered for quantification and comparability of data, particularly fluorescence spectra.
Here, current applications of luminescence-based methods and different types of reporters will be presented. In this context, suitable spectroscopic tools for the characteri-zation of the optical properties of fluorescent reporters and fluorophore-encoded microparticles, analytical tools for the determination of the surface chemistry of different types of particles, and different multiplexing strategies will be discussed.
In recent years, elemental imaging of biological samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is gaining in importance. Recent improvements regarding spatial resolution (down to 1 µm) and washout time make LA-ICP-MS particularly interesting for single cell analysis.
Many current nanomaterials can serve as contrast agents in cellular or tissue imaging, drug delivery vehicles or therapeutics, whereas others can cause toxic effects. In order to evaluate nano-bio interactions, the number of nanoparticles (NPs) inside cells as well as their localisation within cellular substructures is of particular interest.
LA-ICP-MS was used to study the NP pathway from uptake, via intracellular processing up to cell division. Fibroblast cells were incubated with different metallic NPs under varying experimental conditions. For LA analysis the cells were fixed with formaldehyde and dried.
Our results show that LA-ICP-MS is able to localise NP aggregates within cellular substructures. The NPs accumulate in the perinuclear region in the course of intracellular processing, e.g. multivesicular fusion and endosomal maturation, but do not enter the nucleus [1, 2]. A strong dependence of NP uptake on concentration and incubation time was found. Additionally, the number of NPs internalized by individual cells was determined and variations within the cell population became visible.
A new laser ablation system providing a short washout time (50 ms) together with small spot sizes (< 4 µm) and high repetition rates allows high spatial resolution applications. First results of cell imaging will be shown.
The findings demonstrate the potential of LA-ICP-MS providing insight into NP uptake and intracellular distribution dependent on experimental parameters.
Immunoassays are an important field of in vitro diagnostics, as they allow for a fast and highly sensitive detection of many biologically and diagnostically relevant analytes such as proteins, hormones, and pharmaceuticals. Fluorescence immunoassays (FIA), where the antibodies and/or antigens are labeled with luminescent reporters, can be easily read out directly by measuring the intensity, decay time, or polarization of the emitted light. Moreover, FIA enable the simultaneous detection of different analytes within a single sample (multiplexing) and are particularly suited for point-of-care (POC) diagnostics and high throughput screening (HTS). The application of luminescent nanoparticles as reporters in FIA could further improve assay sensitivity, as several 100 to 1000 luminophores can be incorporated or attached to such nanoscale carriers, thereby amplifying their absorption and/or emission signals simply by increasing the number of dye molecules.
Although dye-loaded polymeric and silica nanoparticles have been increasingly used as reporters in immunoassays, achievable signal amplification factors related to the use of particle reporters are still difficult to predict and quantify, which also hampers the comparability of different nanoscale reporters. To overcome this challenge, we performed a systematic comparison of spectroscopically and analytically well characterized particle labels in a homogeneous sandwich immunoassay format for the detection of the common inflammation biomarker C-reactive protein (CRP). Hereby, we studied the influence of particle parameters like size, surface chemistry, and dye loading concentration for different dye classes, i.e. organic dyes and metal ligand complexes, varying in their signal-relevant spectroscopic properties (molar absorption coefficients, photoluminescence quantum yields, Stokes shifts, and emission decay times), for different detection schemes (direct read-out vs. dye extraction). The emitters applied were chosen to be commercially available for a reasonable price, to absorb between 400 nm and 450 nm, and to emit in the visible region, as these parameters are accessible with most established microplate readers. Based upon our findings, we highlight the advantages and limitations of nanoscale reporters with respect to the choice of suitable particles, encoding dyes, and detection strategies, and compare the achievable sensitivities and dynamic ranges for our CRP model immunoassay.
New Approaches for the Quantification of Functional Groups on Micro- and Nanoparticle Surfaces
(2017)
Nanometer- and micrometer-sized particles are increasingly used as tolls in (bio)analytics with typical applications being carriers for e.g., drugs or dye molecules for use as multichromophoric reporters for signal amplification in optical assays, platforms for DNA sequencing as well as nanosensors and targeted probes for bioimaging studies.
The application of such particles in the material and life sciences is closely linked to their size (and size distribution), shape, colloidal stability, biocompatibility, and ease of subsequent functionalization, e.g., with linkers, targeting ligands, and sensor molecules. The latter requires knowledge of the number of groups effectively accessible for subsequent coupling reactions and hence, selective and sensitive methods of analysis. Ideally, these methods are be robust, reliable, fast, performable with inexpensive equipment, and can be employed for the characterization of a broad variety of particle systems independent of their optical properties, i.e., scattering or the presence of encoding dyes.
In this respect, we studied a variety of conventional and newly developed labels for optical readout on self-made particles with varying surface group density, utilizing e.g., changes in intensity and/or color of absorption and/or emission. We focus here on the development of a platform of cleavable and multimodal labels for optical assays which consist of a cleavable linker and an optically active reporter moiety. In contrast to conventional reporters measured directly at the particle surface, which are prone to signal distortions by scattering and encoding dyes, these cleavable labels can be detected colorimetrically or fluorometrically both bound at the particle surface and after quantitative cleavage of the linker in the transparent supernatant. Moreover, for heteroatom-containing reporters, they enable straightforward validation by method comparison with elemental analysis, ICP-OES or ICP-MS.
Here, we present representative examples of our newly synthesized cleavable and multimodal labels and their application as reporters for the quantification of amino, thiol and carboxy surface groups on different nanomaterials and compare these results with measurements using conventional optical labels and results from measurements providing the total number of surface groups.
Polymer nanoparticles are of increasing importance for a wide range of applications in the material and life sciences. This includes their application as carriers for e.g., analyte-responsive ligands for DNA sequencing platforms, drugs as well as dye molecules for use as multichromophoric reporters for signal enhancement in optical assays or the fabrication of nanosensors and targeted probes in bioimaging studies.
Application-relevant properties of nanometer- and micrometer-sized particles (NP) include their size (and size distribution), colloidal stability, biocompatibility, and ease of subsequent functionalization, e.g., with linkers, sensor molecules, and targeting ligands. In this respect, the knowledge of the chemical nature, the total number of surface groups and the number of groups accessible for subsequent coupling reactions with differently sized optical labels or biomolecules is mandatory. This requires robust, reliable and validated methods, which can be employed for the characterization of a broad variety of particle systems independent of their optical properties, i.e., scattering or the presence of encoding dyes, and can be preferably performed specifically, sensitively, and fast with inexpensive equipment. Particularly attractive methods are here straightforward colorimetric, and fluorometric assays. In this respect, we studied a variety of conventional labels for optical readout, utilizing e.g., a change in intensity and/or color of absorption and/or emission. While in common assays, most reporters are measured directly at the particle surface, which can easily lead to signal distortions by scattering and encoding dyes, we focus on the development of cleavable and multimodal labels. These labels are detectable both bound at the particle surface and after cleavage of a linker unit in the supernatant with different analytical methods like fluorometry together with elemental analysis, ICP-OES or ICP-MS for straightforward method validation by method comparison. Here, we present our newly-synthesized cleavable labels and their application for photometric quantification of amino, thiol and carboxy surface groups on different types of nanomaterials and compare the results obtained from surface group analysis relying on conventional labels.
Semiconductor nanocrystals with a spherical (QDs) core and a spherical or a rod-shaped Shell, u.a., so-called Quantum dot-Quantum rods (QDQRs) are increasingly used as fluorescent Reporters or optically active components in the life and material science, e.g., in solid state lightening including Plasma Displays. (1,2) Morever, there is an increasing interest in materials with emission >800 nm for bioanalysis, medical diagnostics, and safety barcodes. Prerequisites for the mechanistic understanding of nonradiativ decay channels needed for the rational design of improved nanomaterials and the comparison of material Performance are reliable fluorescence measurements and validated methods for the assessment of their surface chemistry. (3,4) The latter is of particular importance for nanocrystalline Emitters, where surface states and the accessibility of emissive states by quenchers largely control photoluminescence properties. (5) Here, we present results from systematic spectroscopic studies including absolutely measured photolumunescence Quantum yields of different vissible and NIR emisisve QD and QDQRs Systems of varying particle architecture size and surface chemistries in Dispersion and embedded in salt crystals. (6,7)
Due to their unique physical properties, particularly their electronic and luminescent properties, graphene quantum dots (GQDs) are expected to be suitable for a wide range of applications in bioimaging, electro-optical and photonic materials or energy harvesting among others.1 Tuning the surface chemistry provides an efficient approach to modulate the fluorescence and distinct electronic properties of GQDs.2 Nevertheless, the role of surface chemistry on the electronic structure of GQDs remains poorly understood. In this presentation, we will compare systematically the electronic and chemical structures of GQDs functionalized with carboxylic and aminated groups to those of non-functionalized GQDs, combining theoretical and experimental approaches, here various photon-based spectroscopies. First, the electronic structure of GQDs was characterized by soft X-ray absorption (XA) and X-ray emission (XE) spectroscopies, probing unoccupied and occupied electronic states, respectively, at the carbon K edge for the first time. The interpretation of the XA/XE spectra was done based on theoretical calculations. Then, the chemical structure of the GQDs was characterized in situ by ATR-FTIR in water, thereby accounting for the importance of the interface between GQDs and water believed to play a central role in the chemical reactivity and the optical properties. We previously demonstrated that monitoring the OH vibrations of water molecules during exposure to humid air was a powerful method to probe H-bonding environment around carbon nanomaterials.3 For GQDs, clear surface-dependent water adsorption profiles are observed and discussed. Finally, UV/Vis absorption and photoluminescence measurements were done to characterize the optical properties of these GQDs. Our results suggest that the surface chemistry of the GQDs affects significantly their electronic structure and optical properties. These findings will contribute to an improved understanding of the structure–activity relationship of GQDs and other carbon nanomaterials with surface modifications.
There is an increasing interest in optical reporters like semiconductor and lanthanide-based nanocrystals with emission > 800 nm and recently also > 1000 nm for bioanalysis, medical diagnostics, and safety barcodes. Mandatory for the comparison of different emitter classes and the rational design of the next generation of reporters for the short wavelength infrared (SWIR) region are reliable and quantitative photoluminescence measurements in this challenging wavelength region. This is of special relevance for nanocrystalline emitters like semiconductor quantum dots and rods as well as for upconversion and downconversion nanocrystals, where surface states and the accessibility of emissive states by quenchers largely control accomplishable quantum yields and hence, signal sizes and detection sensitivities from the reporter side. Such measurements are currently hampered by the lack of suitable methods and standards for instrument calibration and validation as well as by the lack of quantum yield standards with emission > 800 nm and especially > 1000 nm.
In this respect, we present the design of integrating sphere setups for absolute and excitation power densitydependent measurements of emission spectra and quantum yields in the wavelength region of 650 to 1650 nm
including calibration strategies and first candidates for potential fluorescence standards. Subsequently, the photoluminescence properties of different types of nanocrystals are presented and discussed including absolute photoluminescence measurements of upconversion and down conversion emission in different solvents.