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Sensors (Basel, Switzerland) Dec 2020Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of... (Review)
Review
Fluorescence polarization holds considerable promise for bioanalytical systems because it allows the detection of selective interactions in real time and a choice of fluorophores, the detection of which the biosample matrix does not influence; thus, their choice simplifies and accelerates the preparation of samples. For decades, these possibilities were successfully applied in fluorescence polarization immunoassays based on differences in the polarization of fluorophore emissions excited by plane-polarized light, whether in a free state or as part of an immune complex. However, the results of recent studies demonstrate the efficacy of fluorescence polarization as a detected signal in many bioanalytical methods. This review summarizes and comparatively characterizes these developments. It considers the integration of fluorescence polarization with the use of alternative receptor molecules and various fluorophores; different schemes for the formation of detectable complexes and the amplification of the signals generated by them. New techniques for the detection of metal ions, nucleic acids, and enzymatic reactions based on fluorescence polarization are also considered.
Topics: Biological Assay; Fluorescence Polarization; Fluorescent Dyes; Metals; Nucleic Acids
PubMed: 33322750
DOI: 10.3390/s20247132 -
International Journal of Molecular... Aug 2022Fluorescence polarization (FP) has been applied in detecting chemicals and biomolecules for early-stage diagnosis, food safety analyses, and environmental monitoring.... (Review)
Review
Fluorescence polarization (FP) has been applied in detecting chemicals and biomolecules for early-stage diagnosis, food safety analyses, and environmental monitoring. Compared to organic dyes, inorganic nanomaterials such as quantum dots have special fluorescence properties that can enhance the photostability of FP-based biosensing. In addition, nanomaterials, such as metallic nanoparticles, can be used as signal amplifiers to increase fluorescence polarization. In this review paper, different types of nanomaterials used in in FP-based biosensors have been reviewed. The role of each type of nanomaterial, acting as a fluorescent element and/or the signal amplifier, has been discussed. In addition, the advantages of FP-based biosensing systems have been discussed and compared with other fluorescence-based techniques. The integration of nanomaterials and FP techniques allows biosensors to quickly detect analytes in a sensitive and cost-effective manner and positively impact a variety of different fields including early-stage diagnoses.
Topics: Biosensing Techniques; Fluorescence Polarization; Metal Nanoparticles; Nanostructures; Quantum Dots
PubMed: 35955779
DOI: 10.3390/ijms23158625 -
Advanced Drug Delivery Reviews 2019Non-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacology of small molecule drugs. Fluorescence... (Review)
Review
Non-invasive measurement of drug-target engagement can provide critical insights in the molecular pharmacology of small molecule drugs. Fluorescence polarization/fluorescence anisotropy measurements are commonly employed in protein/cell screening assays. However, the expansion of such measurements to the in vivo setting has proven difficult until recently. With the advent of high-resolution fluorescence anisotropy microscopy it is now possible to perform kinetic measurements of intracellular drug distribution and target engagement in commonly used mouse models. In this review we discuss the background, current advances and future perspectives in intravital fluorescence anisotropy measurements to derive pharmacokinetic and pharmacodynamic measurements in single cells and whole organs.
Topics: Animals; Drug Discovery; Fluorescence Polarization; High-Throughput Screening Assays; Humans
PubMed: 29410158
DOI: 10.1016/j.addr.2018.01.019 -
Methods in Molecular Biology (Clifton,... 2014Methods and protocols are described when using fluorescence metrology to determine the average nanoparticle (np) size in colloids in the range of 1-10 nm. The technique...
Methods and protocols are described when using fluorescence metrology to determine the average nanoparticle (np) size in colloids in the range of 1-10 nm. The technique is based on determining the rotational correlation time of the np from the decay of fluorescence anisotropy of a dye that is electrostatically or covalently attached to the np as it undergoes Brownian rotation. The np size is then calculated from the Stokes-Einstein equation. The exemplar of silica nps is presented, but the approach can also be applied to other types of nps.
Topics: Colloids; Fluorescence; Fluorescence Polarization; Nanoparticles; Particle Size; Silicon Dioxide
PubMed: 24108630
DOI: 10.1007/978-1-62703-649-8_11 -
Biosensors Nov 2022Combinations of sulfonamides (SAs) and antibacterial synergists (ASGs) are frequently used for treating infectious diseases and promoting growth for animals, which cause...
Combinations of sulfonamides (SAs) and antibacterial synergists (ASGs) are frequently used for treating infectious diseases and promoting growth for animals, which cause potential hazards to food safety and human health. To realize the simultaneous detection of SAs and ASGs in food, a homogeneous and high-throughput screening dual-wavelength fluorescence polarization immunoassay (DWFPIA) was developed. In this study, three SAs tracers and three ASGs tracers were synthesized by fluoresceins with different linkers and paired with their corresponding monoclonal antibodies (mAbs), respectively. To achieve a high sensitivity and broad specificity, the combination of tracers SADMPM-HDF with the longest linker paring mAb 10E6 for SAs and tracer HaptenA-DSCA paring mAb 9C9 for ASGs were chosen for the development of DWFPIA, achieving surprising IC values for 23 SAs below 100 μg L and 5 ASGs below 50 μg L. The accuracy of DWFPIA was applied in real milk samples by typical sulfamethazine (SMZ) and trimethoprim (TMP), with recoveries of 81.7-97.2% and 78.6-103.6%, and coefficient of variations (CVs) below 18.9%, which could be completed within 15 min, including sample pretreatment. We firstly developed a simultaneous screening DWFPIA, covering all of the SAs and ASGs used in clinic and providing a great application potential in food safety analysis.
Topics: Animals; Humans; Fluorescence Polarization Immunoassay; Milk; Sulfonamides; Sulfanilamide; Anti-Bacterial Agents; Antibodies, Monoclonal
PubMed: 36421171
DOI: 10.3390/bios12111053 -
Chemical Reviews May 2010
Review
Topics: Animals; Fluorescence Polarization; Fluorescent Dyes; Humans; Immunoassay; Molecular Imaging
PubMed: 20232898
DOI: 10.1021/cr900267p -
Cell Reports Methods Mar 2022Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of...
Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of BAX are commonly limited to methodologies measuring endpoint phenotypes and do not assess activation of monomeric BAX. Here, we present FLAMBE, a fluorescence polarization ligand assay for monitoring BAX early activation, that measures activation-induced release of a peptide probe in real time. Using complementary parallel and tandem biochemical techniques, we validate, corroborate, and apply FLAMBE to a contemporary repertoire of BAX modulators, characterizing their contributions within the early steps of BAX activation. Additionally, we use FLAMBE to reveal that historically "dead" BAX mutants remain responsive to activation as quasi-functional monomers. We also identify data metrics for comparative analyses and demonstrate that FLAMBE data align with downstream functional observations. Collectively, FLAMBE advances our understanding of BAX activation and fills a methodological void for studying BAX with broad applications in cell biology and therapeutic development. BAX activation studies are invaluable platforms for studying cellular and pharmacological modulators of apoptosis. The gold standard for studying BAX function relies on membrane permeabilization assays, which assess the pore-forming activity of oligomeric BAX. However, there are currently no rapid or kinetic assays to interrogate real-time activation of monomeric BAX in solution, thereby limiting any molecular insights that occur upstream of mitochondrial permeabilization. Furthermore, available methods to observe the activation of monomeric BAX suffer from low throughput and static observations. To address this methodological gap, we developed FLAMBE, a kinetic fluorescence polarization-based assay to measure monomeric BAX activation in solution via concomitant displacement of a labeled peptide. This approach maintains the benefits of rapid kinetic data generation in a low-cost microplate format without requiring specialized equipment or large quantities of protein. FLAMBE compliments available experimental strategies and expands the accessibility of investigators to monitor early steps within the BAX activation continuum.
Topics: bcl-2-Associated X Protein; Fluorescence Polarization; Ligands; Mitochondrial Membranes; Peptides; Humans
PubMed: 35419554
DOI: 10.1016/j.crmeth.2022.100174 -
Expert Opinion on Drug Discovery 2015Fluorescence anisotropy (FA) is one of the major established methods accepted by industry and regulatory agencies for understanding the mechanisms of drug action and... (Review)
Review
INTRODUCTION
Fluorescence anisotropy (FA) is one of the major established methods accepted by industry and regulatory agencies for understanding the mechanisms of drug action and selecting drug candidates utilizing a high-throughput format.
AREAS COVERED
This review covers the basics of FA and complementary methods, such as fluorescence lifetime anisotropy and their roles in the drug discovery process. The authors highlight the factors affecting FA readouts, fluorophore selection and instrumentation. Furthermore, the authors describe the recent development of a successful, commercially valuable FA assay for long QT syndrome drug toxicity to illustrate the role that FA can play in the early stages of drug discovery.
EXPERT OPINION
Despite the success in drug discovery, the FA-based technique experiences competitive pressure from other homogeneous assays. That being said, FA is an established yet rapidly developing technique, recognized by academic institutions, the pharmaceutical industry and regulatory agencies across the globe. The technical problems encountered in working with small molecules in homogeneous assays are largely solved, and new challenges come from more complex biological molecules and nanoparticles. With that, FA will remain one of the major work-horse techniques leading to precision (personalized) medicine.
Topics: Animals; Drug Discovery; Drug Evaluation, Preclinical; Drug-Related Side Effects and Adverse Reactions; Fluorescence Polarization; High-Throughput Screening Assays; Humans; Long QT Syndrome; Precision Medicine
PubMed: 26289575
DOI: 10.1517/17460441.2015.1075001 -
Biosensors & Bioelectronics Apr 2021Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold...
Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/μL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.
Topics: Biosensing Techniques; COVID-19; COVID-19 Nucleic Acid Testing; CRISPR-Cas Systems; Equipment Design; Fluorescence Polarization; Humans; Molecular Diagnostic Techniques; Nucleic Acid Amplification Techniques; Pandemics; Point-of-Care Systems; SARS-CoV-2; Signal Processing, Computer-Assisted; Signal-To-Noise Ratio
PubMed: 33540323
DOI: 10.1016/j.bios.2021.113049 -
Angewandte Chemie (International Ed. in... Nov 2022Target-directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated...
Target-directed dynamic combinatorial chemistry has emerged as a useful tool for hit identification, but has not been widely used, in part due to challenges associated with analyses involving complex mixtures. We describe an operationally simple alternative: in situ inhibitor synthesis and screening (ISISS), which links high-throughput bioorthogonal synthesis with screening for target binding by fluorescence. We exemplify the ISISS method by showing how coupling screening for target binding by fluorescence polarization with the reaction of acyl-hydrazides and aldehydes led to the efficient discovery of a potent and novel acylhydrazone-based inhibitor of human prolyl hydroxylase 2 (PHD2), a target for anemia treatment, with equivalent in vivo potency to an approved medicine.
Topics: Humans; Drug Discovery; Fluorescence Polarization; Hypoxia-Inducible Factor-Proline Dioxygenases
PubMed: 36112310
DOI: 10.1002/anie.202211510