TY - JOUR A1 - Felbeck, Tom A1 - Behnke, Thomas A1 - Hoffmann, Katrin A1 - Grabolle, Markus A1 - Lezhnina, M.M. A1 - Kynast, U.H. A1 - Resch-Genger, Ute T1 - Nile-red-nanoclay hybrids: Red emissive optical probes for use in aqueous dispersion N2 - Water-dispersible and (bio)functionalizable nanoclays have a considerable potential as inexpensive carriers for organic molecules like drugs and fluorophores. Aiming at simple design strategies for red-emissive optical probes for the life sciences from commercial precursors with minimum synthetic effort, we systematically studied the dye loading behavior and stability of differently functionalized laponites. Here, we present a comprehensive study of the absorption and emission properties of the red emissive hydrophobic and neutral dye Nile Red, a well-known polarity probe, which is almost insoluble and nonemissive in water. Adsorption of this probe onto disk-shaped nanoclays was studied in aqueous dispersion as function of dye concentration, in the absence and presence of the cationic surfactant cetyltrimethylammonium bromide (CTAB) assisting dye loading, and as a function of pH. This laponite loading strategy yields strongly fluorescent nanoclay suspensions with a fluorescence quantum yield of 0.34 at low dye loading concentration. The dye concentration-, CTAB-, and pH-dependent absorption, fluorescence emission, and fluorescence excitation spectra of the Nile-Red6#8211;nanoclay suspensions suggest the formation of several Nile Red species including emissive Nile Red monomers facing a polar environment, nonemissive H-type dimers, and protonated Nile Red molecules that are also nonfluorescent. Formation of all nonemissive Nile Red species could be suppressed by modification of the laponite with CTAB. This underlines the great potential of properly modified and functionalized laponite nanodisks as platform for optical probes with drug delivery capacities, for example, for tumor and therapy imaging. Moreover, comparison of the Nile Red dimer absorption spectra with absorption spectra of previously studied Nile Red aggregates in dendrimer systems and micelles and other literature systems reveals a considerable dependence of the dimer absorption band on microenvironment polarity which has not yet been reported so far for H-type dye aggregates. KW - Nile Red KW - Dye KW - Laponite KW - Nanoclay KW - Photoluminescence KW - Fluorescence KW - Polarity probe KW - Aggregate KW - Dimer PY - 2013 DO - https://doi.org/10.1021/la402165q SN - 0743-7463 SN - 1520-5827 VL - 29 IS - 36 SP - 11489 EP - 11497 PB - American Chemical Society CY - Washington, DC AN - OPUS4-29017 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Behnke, Thomas A1 - Laux, Eva-Maria A1 - Hoffmann, Katrin A1 - Peters, S. A1 - Haueisen, J. A1 - Klemm, M. A1 - Resch-Genger, Ute T1 - Simple approaches to fluorescence lifetime standards using dye-quencher pairs N2 - Photoluminescence techniques are amongst the most widely used tools in the material and life sciences, with new and exciting applications continuously emerging. Advantages include their comparative ease of use, unique sensitivity, non-invasive character, and potential for multiplexing, remote sensing, and miniaturization. Increasing applications of fluorescence techniques in the life sciences and emerging medical applications of fluorescence microscopic techniques including 1P and 2P fluorescence microscopy combined with fluorescence lifetime imaging (FLIM) in e.g. in vivo eye diagnostics boosted the demand for robust, easy-to-use, and reliable fluorescence standards to ensure the reliability and comparability of fluorescence data. This includes fluorescence standards for the consideration of instrument-specific spectral and intensity distortions of measured signals and instrument performance validation, fluorescence intensity standards for the quantification of measured intensities and for signal referencing, and lately, also fluorescence lifetime standards. T2 - BMT 2012 - 46. DGBMT Jahrestagung CY - Jena, Germany DA - 16.09.2012 PY - 2012 DO - https://doi.org/10.1515/bmt-2012-4499 SN - 0013-5585 SN - 1862-278X VL - 57 IS - Suppl. 1 SP - 613 PB - De Gruyter CY - Berlin AN - OPUS4-27682 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - NIR-Fluorescent Polystyrene Nanoparticles: Design, Optical Characterization and Bioanalytical Applications T2 - Abteilungsseminar BfR CY - Berlin, Germany DA - 2012-06-15 PY - 2012 AN - OPUS4-26022 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Moser, Marko A1 - Nirmalananthan-Budau, Nithiya A1 - Behnke, Thomas A1 - Geißler, Daniel A1 - Resch-Genger, Ute T1 - Multimodal cleavable reporters versus conventional labels for optical quantification of accessible amino and carboxy groups on nano- and microparticles N2 - 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. KW - Polymer particles KW - Cleavable reporter KW - Optical quantification KW - Validation PY - 2018 DO - https://doi.org/10.1021/acs.analchem.8b00666 SN - 1520-6882 SN - 0003-2700 N1 - Geburtsname von Nirmalananthan-Budau, Nithiya: Nirmalananthan, N. - Birth name of Nirmalananthan-Budau, Nithiya: Nirmalananthan, N. VL - 90 IS - 9 SP - 5887 EP - 5895 PB - ACS Publ. CY - Washington, DC AN - OPUS4-44830 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - Bioimaging with rationally designed NIR-flourescent nanosensors T2 - NAMDIATREAM Winter School on Bioimaging, sensing and therapeutic applications of nanomaterials CY - Villars-sur-Ollon, Switzerland DA - 2013-03-03 PY - 2013 AN - OPUS4-27808 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - NIR-Emissive O2-Nanosensors for Biomarker Imaging T2 - Biosensor Symposium CY - Wildau, Germany DA - 2013-03-10 PY - 2013 AN - OPUS4-27810 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Quevedo, Pablo A1 - Behnke, Thomas A1 - Resch-Genger, Ute T1 - Streptavidin conjugation and quantification—a method evaluation for nanoparticles N2 - Aiming at the development of validated protocols for protein conjugation of nanomaterials and the determination of protein labeling densities, we systematically assessed the conjugation of the model protein streptavidin (SAv) to 100-, 500-, and 1000-nm-sized polystyrene and silica nanoparticles and dye-encoded polymer particles with two established conjugation chemistries, based upon achievable coupling efficiencies and labeling densities. Bioconjugation reactions compared included EDC/sulfo NHS ester chemistry for direct binding of the SAv to carboxyl groups at the particle surface and maleimide-thiol chemistry in conjunction with heterobifunctional PEG linkers and aminated nanoparticles (NPs). Quantification of the total and functional amounts of SAv on these nanomaterials and unreacted SAv in solution was performed with the BCA assay and the biotin–FITC (BF) titration, relying on different signal generation principles, which are thus prone to different interferences. Our results revealed a clear influence of the conjugation chemistry on the amount of NP crosslinking, yet under optimized reaction conditions, EDC/sulfo NHS ester chemistry and the attachment via heterobifunctional PEG linkers led to comparably efficient SAv coupling and good labeling densities. Particle size can obviously affect protein labeling densities and particularly protein functionality, especially for larger particles. For unstained nanoparticles, direct bioconjugation seems to be the most efficient strategy, whereas for dyeencoded nanoparticles, PEG linkers are to be favored for the prevention of dye–protein interactions which can affect protein functionality specifically in the case of direct SAv binding. Moreover, an influence of particle size on achievable protein labeling densities and protein functionality could be demonstrated. KW - Protein quantification KW - BCA assay KW - Biotin–FITC titration KW - Polystyrene nanoparticles PY - 2016 DO - https://doi.org/10.1007/s00216-016-9510-3 SN - 1618-2642 SN - 1618-2650 VL - 408 IS - 15 SP - 4133 EP - 4149 PB - Springer AN - OPUS4-36618 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - NIR-floureszente Mikro- und Nanopartikel für die Bioanalytik T2 - Zukunft Made in Adlershof CY - Berlin, Germany DA - 2009-09-08 PY - 2009 AN - OPUS4-20642 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - FLIM and Optical Imaging with NIR-Fluorescent Lifetime-Encoded Nanoparticles of well Known Surface Chemistry T2 - Anakon 2015 CY - Graz, Austria DA - 2015-03-23 PY - 2015 AN - OPUS4-33695 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - Targeted NIR-emissive nanosensors for bioimaging T2 - 7th Senftenberger Innovationsforum Multiparameteranalytik CY - Senftenberg, Germany DA - 2013-04-18 PY - 2013 AN - OPUS4-28468 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Behnke, Thomas A1 - Würth, Christian A1 - Laux, Eva-Maria A1 - Hoffmann, Katrin A1 - Resch-Genger, Ute T1 - Simple strategies towards bright polymer particles via one-step staining procedures N2 - In order to develop simple and versatile procedures for the preparation of red emissive particles, various one-step swelling procedures for the loading of fluorophores into nanometer- and micrometer-sized polystyrene particles were systematically assessed. Parameters studied for model dyes from common dye classes include the composition of the swelling medium, dye charge and polarity, dye concentration, and particle surface chemistry. The dye loading procedures were compared based upon the efficiency of dye incorporation, fluorescence intensity, and colloidal stability of the resulting particles as well as the absence of dye leaking as determined by absorption and fluorescence spectroscopy, flow cytometry, and measurements of zeta potentials. In addition, for the first time, the influence of the amount of incorporated dye on the absolute fluorescence quantum yield and brightness of the fluorescent particles was investigated for selected chromophores in differently sized particles using a custom-made calibrated integrating sphere setup. Our results demonstrate the general suitability of these one-step loading procedures for efficient particle staining with neutral, zwitterionic, and charged fluorophores like oxazines, coumarines, squaraines, xanthenes, and cyanines emitting in the visible and near infrared. Dye polarity was identified as a suitable tool to estimate the loading efficiency of fluorophores into these polymer particles. KW - Fluorescence KW - Polystyrene KW - Particles KW - Encapsulation KW - Quantum yield KW - Zeta potential KW - Method KW - Label KW - Particle KW - Polymer KW - Absolute fluorescence quantum yield KW - Fluorophore KW - Dye content KW - Surface groups KW - Size KW - Brightness PY - 2012 DO - https://doi.org/10.1016/j.dyepig.2012.01.021 SN - 0143-7208 SN - 1873-3743 VL - 94 IS - 2 SP - 247 EP - 257 PB - Elsevier Ltd. CY - Kidlington AN - OPUS4-25481 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - Spectroscopic quantification of surface groups on micro- and nanoparticles N2 - Nanometer- and micrometer-sized particles are of increasing importance for a wide range of applications in the material and life sciences. This includes carriers for, e.g., drugs as well as dye molecules for use as multichromophoric reporters for signal enhancement in optical assays, platforms for DNA sequencing, and the fabrication of nanosensors and targeted probes for bioimaging studies. Application-relevant properties of such particles include their size (and size distribution), shape, colloidal stability, biocompatibility, and ease of subsequent functionalization, e.g., with linkers, sensor molecules, and targeting ligands. The latter requires knowledge of the number of groups effectively accessible for subsequent coupling reactions and hence, selective and sensitive methods of analysis, which can be ideally 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. For product control, robust, reliable and fast methods performable with inexpensive equipment are prefered. In this respect, we studied a variety of conventional labels for optical readout, utilizing changes in intensity and/or color of absorption and/or emission. In addition, we developed 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, they enable straightforward validation by method comparison with elemental analysis, ICP-OES or ICP-MS. Here, we present representative examples of newly synthesized cleavable labels and their application for the quantification of amino, thiol and carboxy surface groups on different nanomaterials and compare these results with measurements using conventional optical labels. T2 - 253rd ACS National Meeting CY - San Francisco, CA, USA DA - 02.04.2017 KW - Nanoparticles KW - Microparticles KW - Surface groups KW - Quantification KW - Cleavable linker PY - 2017 N1 - Geburtsname von Nirmalananthan-Budau, Nithiya: Nirmalananthan, N. - Birth name of Nirmalananthan-Budau, Nithiya: Nirmalananthan, N. AN - OPUS4-40004 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hoffmann, Katrin A1 - Behnke, Thomas A1 - Grabolle, Markus A1 - Resch-Genger, Ute T1 - Nanoparticle-encapsulated vis- and NIR-emissive fluorophores with different fluorescence decay kinetics for lifetime multiplexing N2 - Bioanalytical, clinical, and security applications increasingly require simple, efficient, and versatile strategies to measure an ever increasing number of analytes or events in parallel in a broad variety of detection formats as well as in conjunction with chromatographic separation techniques or flow cytometry. An attractive alternative to common optical multiplexing and encoding methods utilizing spectral multiplexing/color encoding and intensity encoding is lifetime multiplexing, which relies on the discrimination between different fluorescent reporters based on their fluorescence decay kinetics. Here, we propose a platform of surface-functionalizable polymeric nanoparticles stained with fluorophores differing in their fluorescence lifetimes as a new multiplexing and encoding approach. Proof-of-concept measurements with different sets of lifetime-encoded polystyrene nanoparticles are presented, obtained via staining of preformed particles with visible (vis)- and near-infrared (NIR)-emissive organic dyes, which display very similar absorption and emission spectra to enable excitation and detection at the same wavelengths, yet sufficiently different fluorescence decay kinetics in suspension, thereby minimizing instrumentation costs. Data analysis was performed with a linear combination approach in the lifetime domain. Our results and first cell experiments with these reporter sets underline the suitability of our multiplexing strategy for the discrimination between and the quantification of different labels. This simple and versatile concept can be extended to all types of fluorophores, thereby expanding the accessible time scale, and can be used, e.g., for the design of labels and targeted probes for fluorescence assays and molecular imaging, cellular imaging studies, and barcoding applications, also in conjunction with spectral and intensity encoding. KW - Fluorescent label KW - Multiplexing KW - Optical KW - encoding KW - Lifetime multiplexing KW - Fluorescence lifetime imaging FLIM KW - Nanoparticles PY - 2014 DO - https://doi.org/10.1007/s00216-013-7597-3 SN - 1618-2642 SN - 1618-2650 VL - 406 IS - 14 SP - 3315 EP - 3322 PB - Springer CY - Berlin AN - OPUS4-30077 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - Fluoreszente Polymer-Nanopartikel für die Bioanalytik T2 - Nano- und Mikropartikel für die biomedizinische Analytik CY - Potsdam-Golm DA - 2015-10-29 PY - 2015 AN - OPUS4-34723 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schneider, Ralf A1 - Weigert, Florian A1 - Lesnyak, V. A1 - Leubner, S. A1 - Lorenz, T. A1 - Behnke, Thomas A1 - Dubavik, A. A1 - Joswig, J.-O. A1 - Resch-Genger, Ute A1 - Gaponik, N. A1 - Eychmüller, A. ED - Resch-Genger, Ute ED - Schneider, Ralf T1 - pH and concentration dependence of the optical properties of thiol-capped CdTe nanocrystals in water and D2O N2 - The optical properties of semiconductor nanocrystals (SC NCs) are largely controlled by their size and surface chemistry, i.e., the chemical composition and thickness of inorganic passivation shells and the chemical nature and number of surface ligands as well as the strength of their bonds to surface atoms. The latter is particularly important for CdTe NCs, which – together with alloyed CdₓHg₁₋ₓTe – are the only SC NCs that can be prepared in water in high quality without the need for an additional inorganic passivation shell. Aiming at a better understanding of the role of stabilizing ligands for the control of the application-relevant fluorescence features of SC NCs, we assessed the influence of two of the most commonly used monodentate thiol ligands, thioglycolic acid (TGA) and mercaptopropionic acid (MPA), on the colloidal stability, photoluminescence (PL) quantum yield (QY), and PL decay behavior of a set of CdTe NC colloids. As an indirect measure for the strength of the coordinative bond of the ligands to SC NC surface atoms, the influence of the pH (pD) and the concentration on the PL properties of these colloids was examined in water and D₂O and compared to the results from previous dilution studies with a set of thiol-capped Cd₁₋ₓHgₓTe SC NCs in D₂O. As a prerequisite for these studies, the number of surface ligands was determined photometrically at different steps of purification after SC NC synthesis with Ellman’s test. Our results demonstrate ligand control of the pH-dependent PL of these SC NCs, with MPA-stabilized CdTe NCs being less prone to luminescence quenching than TGA-capped ones. For both types of CdTe colloids, ligand desorption is more pronounced in H₂O compared to D₂O, underlining also the role of hydrogen bonding and solvent molecules. KW - Quantum dots KW - Fluorescence KW - Ligand analysis KW - Nano particles PY - 2016 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-371253 DO - https://doi.org/10.1039/c6cp03123d VL - 18 IS - 28 SP - 19083 EP - 19092 PB - RSC CY - Cambridge AN - OPUS4-37125 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Natte, Kishore A1 - Behnke, Thomas A1 - Orts Gil, Guillermo A1 - Würth, Christian A1 - Friedrich, Jörg Florian A1 - Österle, Werner A1 - Resch-Genger, Ute T1 - Synthesis and characterisation of highly fluorescent core-shell nanoparticles based on Alexa dyes N2 - Current and future developments in the emerging field of nanobiotechnology are closely linked to the rational design of novel fluorescent nanomaterials, e.g. for biosensing and imaging applications. Here, the synthesis of bright near infrared (NIR)-emissive nanoparticles based on the grafting of silica nanoparticles (SNPs) with 3-aminopropyl triethoxysilane (APTES) followed by covalent attachment of Alexa dyes and their subsequent shielding by an additional silica shell are presented. These nanoparticles were investigated by dynamic light scattering (DLS), transmission electron microscopy (TEM) and fluorescence spectroscopy. TEM studies revealed the monodispersity of the initially prepared and fluorophorelabelled silica particles and the subsequent formation of raspberry-like structures after addition of a silica precursor.Measurements of absolute fluorescence Quantum yields of these scattering particle suspensions with an integrating sphere setup demonstrated the influence of dye labelling density-dependent fluorophore Aggregation on the signaling behaviour of such nanoparticles. KW - Silica KW - Alexa dyes KW - Fluorescent particles KW - Quantum yields KW - Nanoparticles KW - Protective shell KW - Nanobiotechnology PY - 2012 DO - https://doi.org/10.1007/s11051-011-0680-9 SN - 1388-0764 SN - 1572-896X VL - 14 IS - 2 SP - 680-1 - 680-10 PB - Kluwer CY - Dordrecht AN - OPUS4-25872 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Behnke, Thomas T1 - NIR-Fluorescent Particles as Internal Standards in Biosensors T2 - Doktorandentagung CY - Attendorn, Germany DA - 2009-02-15 PY - 2009 AN - OPUS4-19420 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Laux, Eva-Maria A1 - Behnke, Thomas A1 - Hoffmann, Katrin A1 - Resch-Genger, Ute T1 - Keeping particles brilliant - simple methods for the determination of the dye content of fluorophore-loaded polymeric particles N2 - One of the most active research areas in the life and material sciences is the design and synthesis of fluorescent nano- and micrometre sized particles for applications e.g. as labels, sensor systems, and platforms for fluorescence assays or barcoding materials. The reliable and reproducible fabrication of such particles as well as many applications require accurate, simple, and versatile procedures for the determination of the dye content per particle which affects e.g. the brightness of these materials and their surface charge and thus, colloidal stability. Here, four fast and inexpensive spectroscopic methods for the quantification of the fluorophore content of beads are presented and compared for nanometre- and micrometre sized polystyrene particles loaded or labeled with commercial fluorophores, differing in dye class, charge, and hydrophilicity. This included the determination of the amount of incorporated dye from absorption spectra of bead suspensions, via dissolving of the polymer matrix, via extraction of the polymer matrix, and from the supernatant of the swelling solution or reaction mixture. Method validation was performed with a sulfur-containing dye and elemental analysis. Based upon this method comparison and the accomplishable uncertainties, two reliable strategies for particle characterization and bead process control are identified that can be easily extended to other materials. PY - 2012 DO - https://doi.org/10.1039/c2ay05822g SN - 1759-9660 SN - 1759-9679 VL - 4 IS - 6 SP - 1759 EP - 1768 PB - RSC Publ. CY - Cambridge AN - OPUS4-26051 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Moser, Marko A1 - Behnke, Thomas A1 - Hamers-Allin, Carolina A1 - Klein-Hartwig, Karin A1 - Falkenhagen, Jana A1 - Resch-Genger, Ute T1 - Quantification of PEG-maleimide ligands and coupling efficiencies on nanoparticles with Ellman's reagent N2 - The surface modification of nanometer- and micrometer-sized particles and planar substrates with polyethylene glycol (PEG) ligands of varying length is a very common strategy to tune the hydrophilicity and biocompatibility of such materials, minimize unspecific interactions, improve biofunctionalization efficiencies, and enhance blood circulation times. Nevertheless, simple methods for the quantification of PEG ligands are comparatively rare. Here, we present a new concept for the quantification of PEG ligands for maleimide-functionalized PEG molecules and the determination of PEG coupling efficiencies, exploiting the quantitative reaction of maleimide with ʟ-cysteine, and the subsequent determination of the unreacted thiol with the photometric Ellman's test. This is shown for heterobifunctional PEG spacers of varying length and amino-functionalized polystyrene nanoparticles (PS NP) without and with differently charged encoding dyes. The reaction of ʟ-cysteine with the Ellman's reagent was monitored photometrically and with electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) to derive the reaction mechanism and to obtain the stoichiometry factor for ʟ-cysteine quantification. Mass balances and quantification of ʟ-cysteine via its sulfur concentration using elemental analysis and inductively coupled plasma mass spectrometry (ICP-MS) confirmed the accuracy and reliability of this approach that can be extended to other surface groups and ligands. KW - Photometric assay KW - Surface analysis KW - Nanoparticle KW - Polyethylene glycol KW - PEG quantification KW - Ellman's test PY - 2015 DO - https://doi.org/10.1021/acs.analchem.5b02173 SN - 0003-2700 SN - 1520-6882 VL - 87 IS - 18 SP - 9376 EP - 9383 PB - American Chemical Society CY - Washington, DC AN - OPUS4-34468 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -