The translation of discoveries on extracellular vesicle (EV) based cancer biomarkers to personalised precision oncology requires the development of robust, sensitive and specific assays that are amenable to adoption in the clinical laboratory. Whilst a variety of elegant approaches for EV liquid biopsy have been developed, most of them remain as research prototypes due to the requirement of a high level of microfabrication and/or sophisticated instruments. Hence, this study is set to develop a simple DNA aptamer-enabled and fluorescence polarisation-based homogenous assay that eliminates the need to separate unbound detection ligands from the bound species for EV detection. High specificity is achieved by immobilising EVs with one set of antibodies and subsequently detecting them with a DNA aptamer targeting a distinct EV biomarker. This two-pronged strategy ensures the removal of most, if not all, non-EV substances in the input biofluids, including soluble proteins, protein aggregates or non-vesicular particles, prior to quantifying biomarker-positive EVs. A limit of detection of 5.0 × 106 EVs/mL was achieved with a linear quantification range of 5.0 × 108 to 2.0 × 1010 EVs/mL. Facilitated by a multiple parametric analysis strategy, this aptamer-guided fluorescence polarisation assay was capable of distinguishing EVs from three different types of solid cancer cells based on quantitative differences in the levels of the same sets of biomarkers on EVs. Given the simplicity of the method and its ease of implementation in automated clinical biochemistry analysers, this assay could be exploited for future EV-based continuous and real-time monitoring of the emergence of new macro- or micro-metastasis, cancer progression as well as the response to treatment throughout different stages of cancer management in the clinic.

The adenosine A2A receptor (A2AR) has been identified as a therapeutic target for treating neurodegenerative diseases and cancer. In recent years, we have highlighted the 2-aminoquinazoline heterocycle as an promising scaffold for designing new A2AR antagonists, exemplified by 6-bromo-4-(furan-2-yl)quinazolin-2-amine 1 (Ki (hA2AR) = 20 nM). Here, we report the synthesis of new 2-aminoquinazoline derivatives with substitutions at the C6- and C7-positions, and the introduction of aminoalkyl chains containing tertiary amines at the C2-position to enhance antagonist activity and solubility properties. Compound 5m showed a high affinity for hA2AR with a Ki value of 5 nM and demonstrated antagonist activity with an IC50 of 6 µM in a cyclic AMP assay. Introducing aminopentylpiperidine and 4-[(piperidin-1-yl)methyl]aniline substituents maintained the binding affinities (9x, Ki = 21 nM; 10d, Ki = 15 nM) and functional antagonist activities (9x, IC50 = 9 µM; 10d, IC50 = 5 µM) of the synthesized compounds while improving solubility. This study provides insights into the future development of A2AR antagonists for therapeutic applications.

Brucellosis in animals is an infectious disease caused by bacteria of the genus Brucella. Known methods for diagnosing brucellosis face some challenges, due to the difficulties in isolating and standardizing the natural brucellosis antigen. In this work, we investigated the possibility of using the fluorescence polarization assay (FPA) with synthetic glycoconjugate biosensing tracers to detect antibodies against Brucella as a new methodology for diagnosing brucellosis. Based on the received results, the synthetic fluorescein-labeled trisaccharide tracer is most effective for Brucellosis detection. This tracer is structurally related to the immune determinant fragment of the Brucella LPS buildup of N-formyl-d-perosamine units, connected via α-(1→3)-linkage at the non-reducing end and α-(1→2)-linkage at the reducing end. The sensitivity and specificity in the case of the use of trisaccharide tracer 3b were 71% and 100% (Yuden\'s method) and 87% and 88% (Euclidean method), respectively, which is comparable with the diagnostic efficiency of traditionally used serological methods, such as the agglutination test (AT), complement fixation test (CFT), and Rose Bengal test (RBT). Given the known advantages of FPA (e.g., speed, compactness of the equipment, and standard reagents) and the increased specificity of the developed test system, it would be appropriate to consider its widespread use for the diagnosis of brucellosis in animals, including rapid testing in the field.

Transthyretin (TTR) and thyroxine-binding globulin (TBG) are two major thyroid hormone (TH) distributor proteins in human plasma, playing important roles in stabilizing the TH levels in plasma, delivery of TH to target tissues, and trans-barrier transport. Binding of xenobiotics to these distributor proteins can potentially affect all these three important roles of distributor proteins. Therefore, fast and cost-effective experimental methods are required for both TTR and TBG to screen both existing and new chemicals for their potential binding. In the present study, the TTR-binding assay was therefore simplified, optimized and pre-validated, while a new TBG-binding assay was developed based on fluorescence polarization as a readout. Seven model compounds (including positive and negative controls) were tested in the pre-validation study of the optimized TTR-binding assay and in the newly developed TBG-binding assay. The dissociation constants of the natural ligand (thyroxine, T4) and potential competitors were determined and compared between two distributor proteins, showing striking differences for perfluorooctanesulfonic acid (PFOS) and perfluorooctanoic acid (PFOA).

Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB package for object-based analysis of FPM data. By combining flexible image segmentation and novel object-based analyses with a high-throughput FPM processing pipeline, OOPS empowers researchers to simultaneously study molecular order and orientation in individual biological structures; conduct population assessments based on morphological features, intensity statistics, and FPM measurements; and create publication-quality visualizations, all within a user-friendly graphical interface. Here, we demonstrate the power and versatility of our approach by applying OOPS to punctate and filamentous structures.

The widely used Laurdan probe has two conformers, resulting in different optical properties when embedded in a lipid bilayer membrane, as demonstrated by our previous simulations. Up to now, the two conformers\' optical responses have, however, not been investigated when the temperature and the phase of the membrane change. Since Laurdan is known to be both a molecular rotor and a solvatochromic probe, it is subject to a profound interaction with both neighboring lipids and water molecules. In the current study, molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for a DPPC membrane at eight temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. It is seen that for Conf-I, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The current work might, therefore, be of importance for biomedical studies on diseases, which are associated with cell membrane transformations.

The characterization of interactions between autophagy modifiers (Atg8-family proteins) and their natural ligands (peptides and proteins) or small molecules is important for a detailed understanding of selective autophagy mechanisms and for the design of potential Atg8 inhibitors that affect the autophagy processes in cells. The fluorescence polarization (FP) assay is a rapid, cost-effective, and robust method that provides affinity and selectivity information for small molecules and peptide ligands targeting human Atg8 proteins.This chapter introduces the basic principles of FP assays. In addition, a case study on peptide interaction with human Atg8 proteins (LC3/GABARAPs) is described. Finally, data analysis and quality control of FP assays are discussed for the proper calculation of Ki values for the measured compounds.

Ampicillin (AMP) is a β-lactam antibiotic that can inhibit bacterial wall synthesis. The overuse and misuse of AMP makes it micropollutant that commonly found in food and various environmental media. In this work, a fluorescence polarization sensor was designed to sensitive detection of trace ampicillin based on click chemistry, using graphene oxide (GO) as a fluorescence polarization (FP) signal enhancement element. First, when ampicillin binds to its aptamer (apt), the adjacent alkyne and azide groups are separated, hindering the click-linking reaction. When Carboxyfluorescein (FAM) fluorophore-labeled probe (C-FAM) is added, its protruding 3-terminal FAM is recognized and cleaved by exonuclease I (EXO I), releasing fluorophores free that could not be adsorbed on GO, resulting in a lo0wer polarization signal. If there is no AMP in the system, aptamer probe is connected to its complementary chain ends by a click reaction. After C-FAM hybridizes with apt, the apt/P duplex is opened and the prominent single-stranded ends adsorb on the GO, leading a significantly enhanced FP signal. According to the relationship between the difference in FP values and the concentrations of AMP, the limit of detection of proposed method is as low as 80 pg/mL. This assay has a wide linear range plus excellent selectivity, and has been applied to detect AMP in milk and river water samples with satisfactory results, which demonstrates that the FP sensor has great potential for practical applications in food safety and environmental protection fields.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors.

Fluorescence polarization (Fpol) imaging of methylene blue (MB) is a promising quantitative approach to thyroid cancer detection. Clinical translation of MB Fpol technology requires reduction of the data analysis time that can be achieved via deep learning-based automated cell segmentation with a 2D U-Net convolutional neural network. The model was trained and tested using images of pathologically diverse human thyroid cells and evaluated by comparing the number of cells selected, segmented areas, and Fpol values obtained using automated (AU) and manual (MA) data processing methods. Overall, the model segmented 15.8% more cells than the human operator. Differences in AU and MA segmented cell areas varied between - 55.2 and + 31.0%, whereas differences in Fpol values varied from - 20.7 and + 10.7%. No statistically significant differences between AU and MA derived Fpol data were observed. The largest differences in Fpol values correlated with greatest discrepancies in AU versus MA segmented cell areas. Time required for auto-processing was reduced to 10 s versus one hour required for MA data processing. Implementation of the automated cell analysis makes quantitative fluorescence polarization-based diagnosis clinically feasible.