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 - Hennig, Andreas A1 - Hoffmann, Angelika A1 - Borcherding, H. A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Quantification of surface functional groups on polymer microspheres by supramolecular host-guest interactions N2 - We introduce a method to determine the number of accessible functional groups on a polymer microsphere surface based on the interaction between the macrocyclic host cucurbit[7]uril (CB7) and a guest reacted to the microsphere surface. After centrifugation, CB7 in the supernatant is quantified by addition of a fluorescent dye. The difference between added and detected CB7 affords the number of accessible surface functional groups. KW - Cucurbituril KW - Acridine orange KW - Fluorescence KW - Polymer surface KW - Surface modification KW - Quantification PY - 2011 U6 - https://doi.org/10.1039/c1cc11692d SN - 0022-4936 SN - 0009-241x SN - 1359-7345 SN - 1364-548x VL - 47 IS - 27 SP - 7842 EP - 7844 PB - Royal Society of Chemistry CY - Cambridge AN - OPUS4-24034 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Hennig, Andreas A1 - Borcherding, H. A1 - Jäger, Christian A1 - Hatami, Soheil A1 - Würth, Christian A1 - Hoffmann, Angelika A1 - Hoffmann, Katrin A1 - Thiele, T. A1 - Schedler, U. A1 - Resch-Genger, Ute T1 - Scope and limitations of surface functional quantification methods: exploratory study with poly(acrylic acid)-grafted micro- and nanoparticles N2 - The amount of grafted poly(acrylic acid) on poly(methyl methacrylate) micro- and nanoparticles was quantified by conductometry, 13C solid-state NMR, fluorophore labeling, a supramolecular assay based on high-affinity binding of cucurbit[7]uril, and two colorimetric assays based on toluidine blue and nickel complexation by pyrocatechol violet. The methods were thoroughly validated and compared with respect to reproducibility, sensitivity, and ease of use. The results demonstrate that only a small but constant fraction of the surface functional groups is accessible to covalent surface derivatization independently of the total number of surface functional groups, and different contributing factors are discussed that determine the number of probe molecules which can be bound to the polymer surface. The fluorophore labeling approach was modified to exclude artifacts due to fluorescence quenching, but absolute quantum yield measurements still indicate a major uncertainty in routine fluorescence-based surface group quantifications, which is directly relevant for biochemical assays and medical diagnostics. Comparison with results from protein labeling with streptavidin suggests a porous network of poly(acrylic acid) chains on the particle surface, which allows diffusion of small molecules (cutoff between 1.6 and 6.5 nm) into the network. KW - Polymers KW - Surface groups KW - Quantification KW - Fluorescence PY - 2012 U6 - https://doi.org/10.1021/ja302649g SN - 0002-7863 SN - 1520-5126 VL - 134 IS - 19 SP - 8268 EP - 8276 PB - American Chemical Society CY - Washington, DC AN - OPUS4-26002 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Brunner, Claudia A1 - Hoffmann, Katrin A1 - Thiele, T. A1 - Schedler, U. A1 - Jehle, H. A1 - Resch-Genger, Ute T1 - Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control N2 - Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a 'green' dye and a 'red' dye in a polymer matrix. These dyes present 'Cy3' and 'Cy5' analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests. KW - New reference material KW - Microarray KW - Fluorescence KW - Standard KW - Calibration slide PY - 2015 U6 - https://doi.org/10.1007/s00216-014-8450-z SN - 1618-2642 SN - 1618-2650 VL - 407 IS - 11 SP - 3181 EP - 3191 PB - Springer CY - Berlin AN - OPUS4-32580 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -