TY - JOUR A1 - Eisold, Ursula A1 - Kupstat, Annette A1 - Klier, Dennis Tobias A1 - Primus, Philipp-A. A1 - Pschenitza, Michael A1 - Niessner, Reinhard A1 - Knopp, Dietmar A1 - Kumke, Michael Uwe T1 - Probing the physicochemical interactions of 3-hydroxy-benzo[a]pyrene with different monoclonal and recombinant antibodies by use of fluorescence line-narrowing spectroscopy JF - Analytical & bioanalytical chemistry N2 - Characterization of interactions between antigens and antibodies is of utmost importance both for fundamental understanding of the binding and for development of advanced clinical diagnostics. Here, fluorescence line-narrowing (FLN) spectroscopy was used to study physicochemical interactions between 3-hydroxybenzo[a]pyrene (3OH-BaP, as antigen) and a variety of solvent matrices (as model systems) or anti-polycyclic aromatic hydrocarbon antibodies (anti-PAH). We focused the studies on the specific physicochemical interactions between 3OH-BaP and different, previously obtained, monoclonal and recombinant anti-PAH antibodies. Control experiments performed with non-binding monoclonal antibodies and bovine serum albumin (BSA) indicated that nonspecific interactions did not affect the FLN spectrum of 3OH-BaP. The spectral positions and relative intensities of the bands in the FLN spectra are highly dependent on the molecular environment of the 3OH-BaP. The FLN bands correlate with different vibrational modes of 3OH-BaP which are affected by interactions with the molecular environment (pi-pi interactions, H-bonding, or van-der-Waals forces). Although the analyte (3OH-BaP) was the same for all the antibodies investigated, different binding interactions could be identified from the FLN spectra on the basis of structural flexibility and conformational multiplicity of the antibodies' paratopes. KW - FLNS KW - Antibody KW - Paratope KW - Hapten KW - Polycyclic aromatic hydrocarbons Y1 - 2014 U6 - https://doi.org/10.1007/s00216-013-7584-8 SN - 1618-2642 SN - 1618-2650 VL - 406 IS - 14 SP - 3387 EP - 3394 PB - Springer CY - Heidelberg ER - TY - JOUR A1 - Kupstat, Annette A1 - Kumke, Michael Uwe A1 - Hildebrandt, Niko T1 - Toward sensitive, quantitative point-of-care testing (POCT) of protein markers miniaturization of a homogeneous time-resolved fluoroimmunoassay for prostate-specific antigen detection JF - The analyst : the analytical journal of the Royal Society of Chemistry N2 - Point-of-care testing (POCT) systems which allow for a sensitive, quantitative detection of protein markers are extremely useful for the early detection and therapy progress monitoring of cancer. However, currently commercially available POCT devices are mainly limited to the qualitative detection of protein markers. In this study we demonstrate the successive miniaturization of a sensitive and fast assay for the quantitative detection of prostate-specific antigen (PSA) using a well established and clinically approved homogeneous time-resolved fluoroimmunoassay technology (TRACE (R)) on a commercial plate-reader system (KRYPTOR (R)). Regarding the initial requirements for the development of POCT devices we applied a 30-fold assay volume reduction (150 mu L to 5 mu L) to achieve a reasonable lab-on-a-chip volume and a 24-fold and 120-fold excitation pulse energy reduction to achieve reasonable pulse energies for low-cost miniature excitation sources. Due to highly efficient optimization of key POCT parameters our miniaturized PSA assay achieved a 30% increased sensitivity and a 2-fold improved limit of detection compared to the standard plate-reader method. Our results demonstrate the successful implementation of key parameters for a significant miniaturization and for cost reduction in the clinically approved KRYPTOR (R) platform for protein detection. The technological alterations required are easy-to-implement and can be immediately adapted for more than 30 diagnostic protein markers already available for the KRYPTOR (R) platform. These features strongly recommend our assay format to be utilized in innovative, sensitive, quantitative POCT of protein markers. Y1 - 2011 U6 - https://doi.org/10.1039/c0an00684j SN - 0003-2654 VL - 136 IS - 5 SP - 1029 EP - 1035 PB - Royal Society of Chemistry CY - Cambridge ER - TY - JOUR A1 - Kupstat, Annette A1 - Ritschel, Thomas A1 - Kumke, Michael Uwe T1 - Oxazine Dye-Conjugated DNA Oligonucleotides Forster Resonance Energy Transfer in View of Molecular Dye-DNA Interactions JF - Bioconjugate chemistry N2 - In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5'-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive Forster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye-DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye-DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680-dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor-acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large Forster distance of similar to 7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins. Y1 - 2011 U6 - https://doi.org/10.1021/bc200379y SN - 1043-1802 VL - 22 IS - 12 SP - 2546 EP - 2557 PB - American Chemical Society CY - Washington ER - TY - THES A1 - Kupstat, Annette T1 - Förster resonance energy transfer (FRET)-based detection of molecular binding interactions - from fundamental research to application in life science Y1 - 2012 CY - Potsdam ER -