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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.
Recent developments in Förster resonance energy transfer (FRET) diagnostics using quantum dots
(2016)
The exceptional photophysical properties and the nanometric dimensions of colloidal semiconductor quantum dots (QD) have strongly attracted the bioanalytical community over the last approximately 20 y. In particular, the integration of QDs in the analysis of biological components and interactions, and the related diagnostics using Förster resonance energy transfer (FRET), have allowed researchers to significantly improve and diversify fluorescence-based biosensing. In this TRENDS article, we review some recent developments in QD-FRET biosensing that have implemented this technology in electronic consumer products, multiplexed analysis, and detection without light excitation for diagnostic applications. In selected examples of smartphone-based imaging, single- and multistep FRET, steady-state and time-resolved spectroscopy, and bio/chemiluminescence detection of QDs used as both FRET donors and acceptors, we highlight the advantages of QD-based FRET biosensing for multiplexed and sensitive diagnostics.
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.
Polymeric nanoparticles (NP) are of increasing importance for a wide range of applications in the material and life sciences, including their use as drug carriers, fluorescent sensors and multimodal reporters in a large variety of bioassays and bioimaging studies. Application-relevant properties of NP include their size (and size distribution), shape, optical properties, and ease of subsequent functionalization, e.g. with linkers, sensor molecules, and bioligands. In this respect, knowledge of the chemical nature, the total number of surface groups and the number of groups accessible for subsequent coupling reactions is mandatory.1 Commercially available polystyrene NP often contain different additives like stabilizers, radical starters and crosslinkers, which can influence the quantification of surface functionalities. Moreover, they often have unknown surface group densities that may vary from batch to batch, which complicates or even hampers their reliable use in many (bio)applications. To circumvent these issues, we synthesized amino- and carboxy-functionalized, monodisperse 100 nm polystyrene NP with three different, well-defined surface group densities. Using a recently developed approach for the quantification of functional groups on nano- and microparticles with cleavable reporters,2 we quantified the assessible functional groups on the self-synthesised PS particles via optical spectroscopy (photometry) and inductively coupled optical emission spectrometry (32S ICP-OES). In addition, we developed a fluorimetric approach for the quantification of surface functional groups on nanoparticles based on the labelling with luminescent lanthanide complexes (LLC). In contrast to common organic dyes, LLC are not prone to photo¬luminescence quenching arising from reabsorption or dye aggregation, and thus, enable a reliable fluorometric quantification of the assessible functional groups on NP surfaces. Moreover, lanthanide tags can be detected with high specificity and sensitivity with analytical techniques such as XPS and ICP-MS, which allow for the multimodal validation of the fluorometric quantification approach.
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.