Fluorescence Polarization: Recent Bioanalytical Applications, Pitfalls, and Future Trends

Goulko, Alevtina A.; Zhao, Qiang; Guthrie, Jeffrey; Zou, Hanfa; Le, X. Chris

doi:10.1007/4243_2008_021

Cited by 12 publications

(9 citation statements)

References 63 publications

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“…Using polarized excitation and emission filters, the instrument measures fluorescence perpendicular to the excitation plane (the ‘P-channel’) and fluorescence that is parallel to the excitation plane (the ‘S-channel’), and then it calculates FP in millipolarization units (mP) as follows: mP = [(S – P * G) / (S + P * G)] * 1000. The ‘G-factor’ is measured by the instrument as a correction for any bias toward the P channel (16). Polarization values of each ALEXA647-labeled ASO in 1× PBS at 2 nM concentration were subtracted from each measurement.…”

Section: Methodsmentioning

confidence: 99%

Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay

Gaus

Gupta

Chappell

et al. 2018

Nucleic Acids Research

111

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Interactions of chemically modified nucleic acid therapeutics with plasma proteins play an important role in facilitating distribution from the injection site to peripheral tissues by reducing renal clearance. Despite the importance of these interactions, analytical methods that can characterize binding constants with individual plasma proteins in a reliable and high throughput manner are not easily available. We developed a fluorescence polarization (FP) based assay and measured binding constants for the 25 most abundant human plasma proteins with phosphorothioate (PS) modified antisense oligonucleotides (ASOs). We evaluated the influence of sequence, sugar modifications, and PS content on ASO interactions with several abundant human plasma proteins and determined the effect of salt and pH on these interactions. PS ASOs were found to associate predominantly with albumin and histidine-rich glycoprotein (HRG) in mouse and human plasma by size-exclusion chromatography. In contrast, PS ASOs associate predominantly with HRG in monkey plasma because of higher concentrations of this protein in monkeys. Finally, plasma proteins capable of binding PS ASOs in human plasma were confirmed by employing affinity chromatography and proteomics. Our results indicate distinct differences in contributions from the PS backbone, nucleobase composition and oligonucleotide flexibility to protein binding.

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Section: Methodsmentioning

confidence: 99%

Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay

Gaus

Gupta

Chappell

et al. 2018

Nucleic Acids Research

111

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“…Many of these techniques rely on fundamental photophysical principles including Förster resonance energy transfer (FRET) [1], fluorescence polarization (FP) [2], and fluorescence correlation spectroscopy (FCS) [3]. These methods also draw from a diverse pool of fluorescent dyes exhibiting unique and varied photophysical properties [4].…”

Section: Introductionmentioning

confidence: 99%

Monitoring protein interactions and dynamics with solvatochromic fluorophores

Loving

Sainlos

Imperiali

2010

Trends in Biotechnology

268

196

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Solvatochromic fluorophores possess emission properties that are sensitive to the nature of the local microenvironment. These dyes have been exploited in applications ranging from the study of protein structural dynamics to the detection of protein-binding interactions. While the solvatochromic indole fluorophore of tryptophan has been utilized extensively for in vitro studies to advance our understanding of basic protein biochemistry, the emergence of new extrinsic synthetic dyes with improved properties in conjunction with recent developments in site-selective methods to incorporate these chemical tools into proteins now open the way for studies in more complex systems. Herein we discuss recent technological advancements and their application in the design of powerful reporters, which serve critical roles in modern cell biology and assay development.

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“…The analyte in the sample could displace the tracer from the complex, resulting in the change in the polarization value, which is related to the concentration of analyte . Single-reagent FPIA (SR-FPIA) is a one-step immunoassay, so the advantage in detection speed makes it more suitable for screening determination in a large number of samples . Throughout the past decades, FPIA has been used for the measurement of various small molecule analytes, such as toxins, − pesticides, , and veterinary drugs. − …”

Section: Introductionmentioning

confidence: 99%

“…15 Single-reagent FPIA (SR-FPIA) is a one-step immunoassay, so the advantage in detection speed makes it more suitable for screening determination in a large number of samples. 16 Throughout the past decades, FPIA has been used for the measurement of various small molecule analytes, such as toxins, 17−19 pesticides, 20,21 and veterinary drugs. 22−24 In this work, we describe the development of a sensitive onestep FPIA based on a class-specific mAb for the simultaneous determination of multiple FQ antibiotics in food samples.…”

Section: ■ Introductionmentioning

confidence: 99%

Simultaneous Determination of Multiple (Fluoro)quinolone Antibiotics in Food Samples by a One-Step Fluorescence Polarization Immunoassay

Wang

Eremin

et al. 2013

J. Agric. Food Chem.

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This paper describes a rapid one-step fluorescence polarization immunoassay (FPIA) for the simultaneous determination of multiple (fluoro)quinolone antibiotics (FQs) in food samples. Several fluorescent tracers were synthesized and evaluated in the FPIA method based on a broad-specificity of monoclonal antibodies toward FQs. The heterogeneous tracer, SAR-5-FAM, was considered as the optimal choice to prepare the immunocomplex single reagent, which allows a rapid and sensitive displacement reaction by addition of analytes. Optimized single-reagent FPIA exhibited broad cross-reactivities in the range of 7.8-172.2% with 16 FQs tested and was capable of determining most FQs at the level of maximum residue limits. Recoveries for spiked milk and chicken muscle samples were from 77.8 to 116%, with relative standard deviation lower than 17.4%. Therefore, this method could be applicable in routine screening analysis of multiple FQ residues in food samples.

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Fluorescence Polarization: Recent Bioanalytical Applications, Pitfalls, and Future Trends

Cited by 12 publications

References 63 publications

Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay

Characterization of the interactions of chemically-modified therapeutic nucleic acids with plasma proteins using a fluorescence polarization assay

Monitoring protein interactions and dynamics with solvatochromic fluorophores

Simultaneous Determination of Multiple (Fluoro)quinolone Antibiotics in Food Samples by a One-Step Fluorescence Polarization Immunoassay

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