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 Förster 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 Förster distance of ~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.

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The spectroscopic properties of a novel intramolecular energy transfer probe (ET probe)--consisting of 3-hydroxybenzo[a]pyrene (3OH-BaP) as the donor covalently linked to sulforhodamine B (SRB) as the acceptor--for the detection of polycyclic aromatic hydrocarbons (PAH) antibody binding were characterized. Absorption and fluorescence spectra as well as fluorescence decay curves were recorded in methanol and aqueous solution, respectively. For comparison, the parent chromophores 3OH-BaP and SRB were investigated as well. In the case of the ET probe, a very strong fluorescence quenching of the BaP-moiety-related emission due to an efficient energy transfer (energy transfer efficiency of about 0.95 for methanol) to the SRB moiety was observed. Upon addition of the PAH antibody, the fluorescence intensity and anisotropy of the BaP moiety was drastically increased. On the other hand, the fluorescence anisotropy of the SRB moiety did not change. The anisotropy results clearly indicate the binding of the antibody. On the basis of these findings, we concluded the following model: the BaP moiety is incorporated in the antibody binding site, whereas the SRB moiety sticks out from the binding site, restricting the motion of the BaP moiety, but leaving the SRB moiety uninfluenced. More important, this structure results in a disruption of the intramolecular energy transfer. The antibody-induced disruption of the intramolecular energy transfer is envisaged as a detection scheme in a future homogeneous competitive fluorescence immunoassay (FIA). This may provide a novel general detection principle for the immunodetection of low-molecular analytes (haptens) in a homogeneous competitive FIA format.

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®) on a commercial plate-reader system (KRYPTOR®). Regarding the initial requirements for the development of POCT devices we applied a 30-fold assay volume reduction (150 µL to 5 µL) 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® 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® platform. These features strongly recommend our assay format to be utilized in innovative, sensitive, quantitative POCT of protein markers.