Fluorescence Quenching-Based Assays for Hydrolyzing Enzymes. Application of Time-Resolved Fluorometry in Assays for Caspase, Helicase, and Phosphatase

Abstract

We have developed assay technologies to measure hydrolyzing enzymes based on homogeneous time-resolved fluorescence quenching (TruPoint). High sensitivity was obtained using fluorescent europium chelates as labels, internally quenched by suitable quenchers and released upon enzymatic reaction. This approach allows robust and sensitive monitoring of low enzyme activities. The assay technology and the choice of donor-acceptor pairs were evaluated in three different enzymatic assays, a protease related to apoptosis, helicase involved in DNA unwinding, and phosphatase having an important role in cellular signaling cascades. All the assays produced an increasing signal, were sensitive, and had a good dynamic range. There were significant differences in optimized quenchers for each of the assays depending on the size, flexibility, and rigidity of the substrates. Also, clear differences in the energy-transfer reactions, their requirements for spectral overlapping, ionic interactions, and energy-transfer distances were found. Each of the enzymatic assays was briefly tested in a high-throughput screening environment by analyzing signal dynamics and statistical relevance as Z' factors.

Persistent luminescence nanoprobe for biosensing and lifetime imaging of cell apoptosis via time-resolved fluorescence resonance energy transfer

“…3d), which showed a detection range from 1.0 Â 10 À4 to 10 unit mL À1 with a detection limit of 2.4 Â 10 À5 unit mL À1 at 3s. This result was much better than those of an AueFe 2 O 3 nanoparticles-based fluorescent detection method (0.10e3.0 unit mL À1 ) and commercial europium(III) chelates-based TRF detection method (0.18e7.1 unit mL À1 ) [52,53]. For further comparison, the conventional steady-state fluorescence measurements were also conducted, which showed a detection limit of 3.3 Â 10 À2 unit mL À1 under identical conditions (Fig.…”

Enhancing helicase-dependent amplification by fusing the helicase with the DNA polymerase

“…One strand (44-mer) is labeled with fluorescent europium and the other one (26-mer) is labeled with its quencher. (Karvinen et al, 2004). In this assay, the presence of capture strand (26-mer), complementary to the quencher strand, prevents reannealing of the substrate strands.…”

Squeezing Fluorescent Dyes into Nanoscale Containers—The Supramolecular Approach to Radiative Decay Engineering

“…They were employed, among others, for the sensitive detection of DNA and proteins [78], and for a variety of enzyme assays [79][80][81]. Recently, a novel type of TRF assay based on DBO as a truly fluorescent (rather than phosphorescent) label has been introduced, thereby by-passing the need for rare earth metal ions [82].…”