TY - JOUR A1 - Roloff, Alexander A1 - Nirmalananthan-Budau, Nithiya A1 - Rühle, Bastian A1 - Borcherding, H. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Quantification of Aldehydes on Polymeric Microbead Surfaces via Catch and Release of Reporter Chromophores N2 - Aldehyde moieties on 2D-supports or microand nanoparticles can function as anchor groups for the attachment of biomolecules or as reversible binding sites for proteins on cell surfaces. The use of aldehyde-based materials in bioanalytical and medical settings calls for reliable methods to detect and quantify this functionality. We report here on a versatile concept to quantify the accessible aldehyde moieties on particle surfaces through the specific binding and subsequent release of small reporter molecules such as fluorescent dyes and nonfluorescent chromophores utilizing acylhydrazone formation as a reversible covalent labeling strategy. This is representatively demonstrated for a set of polymer microparticles with different aldehyde labeling densities. Excess reporter molecules can be easily removed by washing, eliminating inaccuracies caused by unspecific adsorption to hydrophobic surfaces. Cleavage of hydrazones at acidic pH assisted by a carbonyl trap releases the fluorescent reporters rapidly and quasi-quantitatively and allows for their fluorometric detection at low concentration. Importantly, this strategy separates the signal-generating molecules from the bead surface. This circumvents common issues associated with light scattering and signal distortions that are caused by binding-induced changes in reporter fluorescence as well as quenching dye− dye interactions on crowded particle surfaces. In addition, we demonstrate that the release of a nonfluorescent chromophore via disulfide cleavage and subsequent quantification by absorption spectroscopy gives comparable results, verifying that both assays are capable of rapid and sensitive quantification of aldehydes on microbead surfaces. These strategies enable a quantitative comparison of bead batches with different functionalization densities, and a qualitative prediction of their coupling efficiencies in bioconjugations, as demonstrated in reductive amination reactions with Streptavidin. KW - Fluorescent label KW - Surface group quantification KW - Polymer particle KW - Cleavable linker KW - Catch and release assay PY - 2019 U6 - https://doi.org/10.1021/acs.analchem.8b05515 SN - 0003-2700 SN - 1520-6882 VL - 91 IS - 14 SP - 8827 EP - 8834 PB - ACS Publications AN - OPUS4-48284 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kage, Daniel A1 - Hoffmann, Katrin A1 - Borcherding, H. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Lifetime encoding in flow cytometry for bead‑based sensing of biomolecular interaction N2 - To demonstrate the potential of time-resolved flow cytometry (FCM) for bioanalysis, clinical diagnostics, and optically encoded bead-based assays, we performed a proof-of-principle study to detect biomolecular interactions utilizing fluorescence lifetime (LT)-encoded micron-sized polymer beads bearing target-specific bioligands and a recently developed prototype lifetime flow cytometer (LT-FCM setup). This instrument is equipped with a single excitation light source and different fluorescence detectors, one operated in the photon-counting mode for time-resolved measurements of fluorescence decays and three detectors for conventional intensity measurements in different spectral windows. First, discrimination of bead-bound biomolecules was demonstrated in the time domain exemplarily for two targets, Streptavidin (SAv) and the tumor marker human chorionic gonadotropin (HCG). In a second step, the determination of biomolecule concentration levels was addressed representatively for the inflammation-related biomarker tumor necrosis factor (TNF-α) utilizing fluorescence intensity measurements in a second channel of the LT-FCM instrument. Our results underline the applicability of LT-FCM in the time domain for measurements of biomolecular interactions in suspension assays. In the future, the combination of spectral and LT encoding and multiplexing and the expansion of the time scale from the lower nanosecond range to the longer nanosecond and the microsecond region is expected to provide many distinguishable codes. This enables an increasing degree of multiplexing which could be attractive for high throughput screening applications. KW - Fluorescence KW - Sensor KW - Assay KW - Protein KW - Multiplexing KW - Flow cytometry KW - Barcoding KW - Lifetime KW - Dye KW - Bead KW - Bead-based assay KW - Method KW - Quantification PY - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-516007 VL - 10 IS - 1 SP - 19477 PB - Nature AN - OPUS4-51600 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Kage, Daniel A1 - Fischer, Linn A1 - Hoffmann, Katrin A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Close spectroscopic look at dye-stained polymer microbeads N2 - Dye-stained micrometer-sized polymer beads are important tools in the life sciences with applications in biomedical, biochemical, and clinical research. Here, bead-based assays are increasingly used, for example, in DNA sequencing and the detection of autoimmune diseases or pathogenic microorganisms. Moreover, stained beads are employed as calibration tools for fluorescence microscopy and flow cytometry methods with increasing complexity. To address the requirements concerning the relevant fluorescence features, the spectroscopic properties of representative polymer beads with diameters ranging from about 1 to 10 μm stained with varying concentrations of rhodamine 6G were systematically assessed. The observed dependence of the spectral properties, fluorescence decay kinetics, and fluorescence quantum yields on bead size and dye loading concentration is attributed to different fluorescence characteristics of fluorophores located in the particle core and near-surface dye molecules. Supported by the fluorescence anisotropy measurements, the origin of the observed alteration of fluorescence features is ascribed to a combination of excitation energy transfer and polarity-related effects that are especially pronounced at the interface of the bead and the surrounding medium. The results of our studies underline the need to carefully control and optimize all Parameters that can affect the fluorescence properties of the dye-stained beads. KW - Fluorophore KW - Polymer particles KW - Photophysics KW - Life sciences KW - Standards PY - 2018 U6 - https://doi.org/10.1021/acs.jpcc.8b02546 SN - 1932-7447 VL - 122 IS - 24 SP - 12782 EP - 12791 PB - ACS Publications CY - Washington, DC AN - OPUS4-45453 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -