A novel Förster donor-acceptor dye pair for an immunoassay based on resonance energy transfer (RET) is characterized with respect to its photophysical properties. As donor and acceptor, we chose the long-wavelength excitable cyanine dyes Cy5 and Cy5.5, respectively. Due to the perfect spectral overlap, an exceptionally high R(0) value of 68.7 A is obtained in solution. For biochemical applications, antibodies (IgG) are labeled with Cy5, while a tracer for competitive binding is synthesized by labeling bovine serum albumin (BSA) with an analyte derivative and Cy5.5. Binding the dyes to proteins at a low dye/protein ratio increases the fluorescence lifetimes and quantum yields, leading to an enhanced R(0) value of 85.2 A. At higher dye/protein ratios, the formation of nonfluorescent dimeric species causes a decrease in the fluorescence lifetime and quantum yield due to RET from monomeric dyes to dimers within one protein molecule. The Förster distances could be calculated using the dimer absorption spectra to 83.9 and 83.6 A for Cy5 and Cy5.5, respectively. Upon binding of the Cy5-labeled IgG to the tracer, efficient quenching of Cy5 fluorescence is observed. Steady-state and time-resolved measurements reveal that approximately 50% of the quenching results in Förster-type RET, while the residual quenching effect is caused by static quenching processes. The applicability of this dye pair is demonstrated in a homogeneous competitive immunoassay for the pesticide simazine.
A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) a and fi binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flow-through fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.
The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 x 2 cm2 containing 25 x 25 wells, corresponding to approximately 15,000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 microg/L, or 1.11 x 10(-16) mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 microg/L, or 1.87 x 10(-13) mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 microg/L.
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