A clinical role for Förster resonance energy transfer in molecular diagnostics of disease

Qiu, Xue; Hildebrandt, Niko

doi:10.1080/14737159.2019.1649144

Cited by 32 publications

(34 citation statements)

References 40 publications

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“…Continuous detection of enzyme activities (proteases, phosphatases, polymerase, kinases, telomerase, etc.) is an important parameter in medical diagnosis [27].…”

Section: Discussionmentioning

confidence: 99%

Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity

Incir

Kaplan

Bilgin

et al. 2021

Sakarya University Journal of Science

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Proteases are closely associated with many pathological conditions. Efficient detection of protease activity may be useful for diagnosis, prognosis, and the development of new therapeutic biomolecules. Fluorescent Resonance Energy Transfer (FRET) is defined as the non-radioactive energy transfer that occurs between two fluorophores. Fluorescent proteins are widely used in FRET biosensors because they can be genetically encoded and compatible with cells. Fluorescent Protein based FRET (FP-FRET) biosensors are used to monitor biological processes such as enzyme activity, intracellular ion concentration, conformational changes, protein-protein interactions. In this study, it was aimed to detect protease activity using an FP-FRET biosensor and TEV protease was chosen as a model enzyme. The plasmid encoding the mNeonGreen-TEV-mRuby3 fluorescent protein-based FRET biosensor was constructed. The gene of the designed FP-FRET biosensor was expressed in Escherichia coli DH5α cells using recombinant DNA techniques and purified using Ni-NTA affinity chromatography. As a result, the activity of the TEV protease enzyme was determined by emission measurements performed in the spectrofluorometer using the produced FP-FRET biosensor. The usability of the designed FP-FRET biosensor in the determination of protease enzyme activity was demonstrated.

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“…Continuous detection of enzyme activities (proteases, phosphatases, polymerase, kinases, telomerase, etc.) is an important parameter in medical diagnosis [27].…”

Section: Discussionmentioning

confidence: 99%

Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity

Incir

Kaplan

Bilgin

et al. 2021

Sakarya University Journal of Science

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“…Homogeneous immunoassays lacking the washing or separation steps are very appealing detection tools as they provide significant advantages regarding simplicity, rapidity, and less instrumentation requirement. As previously demonstrated by Pulli et al [18] and Arola et al [19], the fact that the two antibodies involved in the immunocomplex-based recognition of a low molecular weight analyte are inevitably brought into very close interaction provides an excellent basis for the utilization of the fluorescence/Förster resonance energy transfer (FRET) process for the signal generation in a homogeneous assay [20,21]. In the FRET process, energy is transferred from a light-excited donor fluorophore through a dipoledipole coupling interaction to an acceptor fluorophore, which then releases the energy as light at higher wavelength [20,22,23].…”

Section: Introductionmentioning

confidence: 95%

A 15-min non-competitive homogeneous assay for microcystin and nodularin based on time-resolved Förster resonance energy transfer (TR-FRET)

Akter

Lamminmäki

2021

Anal Bioanal Chem

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Simple and rapid methods are required for screening and analysis of water samples to detect cyanobacterial cyclic peptide hepatotoxins: microcystin/nodularin. Previously, we reported a highly sensitive non-competitive heterogeneous assay for microcystin/nodularin utilizing a generic anti-immunocomplex (anti-IC) single-chain fragment of antibody variable domains (scFv) isolated from a synthetic antibody library together with a generic adda ((2S,3S,4E,6E,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid)-specific monoclonal antibody (Mab) recognizing the common adda part of the microcystin/nodularin. Using the same antibody pair, here we report a homogeneous non-competitive assay for microcystin/nodularin based on TR-FRET (time-resolved Förster resonance energy transfer) measurement. The anti-IC scFv labeled with Alexa Fluor 680 and the Mab labeled with europium enabled the FRET process to occur in the presence of microcystin/nodularin. The TR-FRET signal is proportional to the toxin concentration in the sample. The rapid (15 min) homogeneous assay without requiring any washing step detected all the tested nine toxin variants (microcystin-LR, -dmLR, -RR, -dmRR, -YR, -LY, -LF -LW, and nodularin-R). Very good signal to blank ratio (~13) was achieved using microcystin-LR and the sample detection limit (blank+3SD of blank) for microcystin-LR was ~0.3 μg/L (~0.08 μg/L in 80-μL reaction well). The practical application of the TR-FRET assay was demonstrated with water samples spiked with microcystin-LR as well as with environmental water. The average recoveries of microcystin-LR from spiked water ranged from 65 to 123%. Good correlation (r2 = 0.73 to 0.99) with other methods (liquid chromatography-mass spectrometry and previously reported heterogeneous assay) was found when environmental samples were analyzed. The developed wash-free assay has the potential to play as a quick screening tool to detect microcystin/nodularin from water below the World Health Organization’s guideline limit (1 μg/L of microcystin-LR). Graphical abstract

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“…In biology, FRET constructs are routinely used to probe the conformation of proteins or nucleic acids, DNA hybridization, cellular membrane potential, and ligand-receptor or antibody-antigen interactions. [1][2][3][4] FRET relies on resonant dipole-dipole energy transfer (ET) between a photo-excited donor (D) and a groundstate acceptor (A) molecule. For efficient ET, the donors must lie in close proximity to acceptors and overlap spectrally, i.e., the donor emission must overlap with the acceptor absorption.…”

Section: Introductionmentioning

confidence: 99%

Enhancing FRET biosensing beyond 10 nm with photon avalanche nanoparticles

2020

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A clinical role for Förster resonance energy transfer in molecular diagnostics of disease

Cited by 32 publications

References 40 publications

Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity

Development of a Fluorescent Protein Based FRET Biosensor for Determination of Protease Activity

A 15-min non-competitive homogeneous assay for microcystin and nodularin based on time-resolved Förster resonance energy transfer (TR-FRET)

Enhancing FRET biosensing beyond 10 nm with photon avalanche nanoparticles

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