The Application of Fluorescence Lifetime Readouts in High-Throughput Screening

Abstract

Measurement of fluorescence lifetime is a well-established technique, which has recently been introduced into the portfolio of assay formats used in high-throughput screening (HTS). This investigation establishes appropriate conditions for using lifetime measurements to reduce the impact of compound interference effects during large-scale HTS of corporate screening files. Experimental data on mixtures of standard fluorophores and interfering compounds (from 5 HTS campaigns) have been combined with a theoretical model to identify the minimum data quality required, defined by the photon count in the peak channel, for discrimination of biological activity. Single-component fluorophore lifetimes can be recovered with an error of 1%, with a peak photon count of 10 2 , but the same accuracy with a 2-component decay requires a peak photon count of 10 3 . When a 3rd component is introduced, the minimum peak count increases to 10 4 . The influence of scattered light on lifetime determination was investigated using an emulsion (diameters 25-675 nm). The measured decays of interfering compounds, identified as autofluorescent, show that the vast majority have a very short lifetime that can readily be resolved from the reporter fluorophore, using appropriate data-fitting methods. ( analytical methods has given benefits of increased sensitivity and speed along with reduced reaction volume and environmental impact, especially in the application of highthroughput screening (HTS) of large corporate compound collections.1 However, compared to radiometric assay formats, fluorescence methods introduce increased opportunities for compound interference effects.2 The fraction of compounds in an HTS campaign that interfere with bioassays in a deleterious manner may be significant compared to those having genuine biological activity leading to a greater burden on compound logistics in hit follow-up.3 More important, however, a significant proportion of reproducible false positives can cause difficulties in the analysis of the screen output, with the worst-case scenario being when a compound with genuine biological activity is overlooked in favor of interfering compounds. 4 Several modes of action have been described for interfering compounds, which are a consequence of their underlying physicochemical properties. Weakly or insoluble compounds can interfere with optical detection by generating light-scattering aggregates. More subtly, aggregates themselves can act as adsorption centers and sequestrate biomolecules in a manner that mimics a true inhibition-type response. 5,6 Other optical effects include light absorption due to colored compounds, quenching by a compound of the fluorescent emission of a reporter fluorophore, and compounds having intrinsic fluorescence over the spectral range of the reporter fluorophore.2,7 To attempt to address these deficiencies, the HTS community has advanced assay and detection technology, with approaches such as time-resolved fluorescence resonant energy transfer, now available.8 Although some success ...

SERS active Ag nanoparticles in mesoporous silicon: detection of organic molecules and peptide–antibody assays

“…The biological assay on the latter substrate did not present any particular Raman feature except than a broad band around 1650 cm –1 because of the O–H bending mode of water. In the case of peptide immobilized on Ag NPs, the SERS spectrum showed weak bands at 1453–1430 cm –1 and 1624 cm –1 ascribed respectively to CH 2 scissor/N–H deformation and Amide I mode; a strong resonance can be noticed at 1763 cm –1 , which probably derives from nonhydrogen bonded amide C = O groups …”

“…Spectra were collected on the samples rinsed and again dipped in ddH 2 O. The immobilized peptides on the Ag surface captured antibodies from solution yielding characteristic SERS features at 1148 cm –1 (NH 3 deformation), 1272 cm –1 (Amide III) and 1506 cm –1 (Amide II). The first two are peculiar of the antibody–peptide complex, while the third is a mode of the stand‐alone antibody, as can be argued by the Raman spectra of the same antibody spotted on the silvered p‐Si substrate without immobilized peptides.…”

Spectroscopy on the wing: Naturally inspired SERS substrates for biochemical analysis

“…Among these, fluorescence has been the most widely used readout method for industrial application, frequently in enzyme-linked immunosorbent assays (ELISA) [4], since high sensitivity is critical for the immunoassay detection. However, fluorescence-based readouts rely upon an extraneous fluorescent label that can alter binding interaction and can be unreliable when assaying in the presence of autofluorescent or scattering compounds [5].…”

A Fluorescence Lifetime–Based Binding Assay to Characterize Kinase Inhibitors

“…24 For example, in a recent study, Moger and colleagues showed that approximately 100 times more photons counts (which would require a 100-fold increase in read time per well) were required to accurately fit data from a 3-component decay mixture relative to that of a 2component decay mixture while maintaining equivalent data accuracy. 25 In an HTS setting using a probe that possesses complex, multiexponential decays in both the free and the bound states, it is impractical to accurately fit such curves. However, in our experience, fitting the data to a single exponential decay equation is sufficient for both the screening and characterization of binding interactions such as those presented here.…”