Förster Resonance Energy Transfer Efficiency Research Articles

Boosting FRET Efficiency of Chromophores with Aggregation-Caused Quenching by a Crystallization-Induced Precise Co-assembly Strategy.

Förster resonance energy transfer (FRET) plays a critical role in organic optoelectronic materials. However, developing facile and effective strategies to achieve high-efficiency energy harvesting of chromophores with aggregation-caused quenching (ACQ) remains an appealing yet challenging task, that has not yet been explored. Herein, a subtly strategy, crystallization-induced precise co-assembly (CIPCA) involving a molecular "lightening agent," to effectively improve FRET efficiency of ACQ chromophores is developed. Bis(phenylethynyl)anthracene (BPA) and bis(phenylethynyl)naphthacene (BPN) with significant ACQ effect are chosen as representative FRET donor and acceptor, respectively, and weakly-fluorescent octafluoronaphthalene (OFN) acted as the "lightening agent." Thanks to precise co-assembly with OFN, the PLQY of solid BPA is enhanced by 107%, and the BPN powder can be unprecedentedly lighted. More importantly, through such powerful CIPCA, the monotonous and weak emission for BPA@BPN can be remarkably regulated to colorful and much brighter ones with FRET efficiency improvement of as high as 180-270%. An in-depth understanding of FRET regulation is elucidated through a precise correlation of the supramolecular structures and properties. Such achievements allow to successfully fabricate distinct multi-stimuli-responsive fluorescent patterns and highly-emissive colorful flowers with high flexibility. This research provides an efficient strategy to improve the FRET efficiency of ACQ pairs.

Glyoxal-methyl-ethylene sulfonic acid fixative enhances the fixation of cytoskeletal structures for Förster resonance energy transfer measurements.

Förster resonance energy transfer (FRET) serves as a tool for measuring protein-protein interactions using various sensor molecules. The tension sensor module relies on FRET technology. In our study, this module was inserted within the actinin molecule to measure the surface tension of the cells. Given that the decay curve of FRET efficiency correlates with surface tension increase, precise and accurate efficiency measurement becomes crucial. Among the methods of FRET measurements, FRET efficiency remains the most accurate if sample fixation is successful. However, when cells were fixed with 4% paraformaldehyde (PFA), the actinin-FRET sensor diffused across the cytoplasm; this prompted us to explore fixation method enhancements. Glyoxal fixative has been reported to improve cytoskeletal morphologies compared to PFA. However, it was not known whether glyoxal fits FRET measurements. Glyoxal necessitates an acetic acid solution for fixation; however, acidic conditions could compromise fluorescence stability. We observed that the pH working range of glyoxal fixative aligns closely with MES (methyl-ethylene sulfonic acid) Good's buffer. Initially, we switched the acidic solution for MES buffer and optimized the fixation procedure for in vitro and in vivo FRET imaging. By comparing FRET measurements on hydrogels with known stiffness to tumor nodules in mouse lung, we estimated in vivo stiffness. The estimated stiffness of cancerous tissue was harder than the reported stiffness of smooth muscle. This discovery shed lights on how cancer cells perceive environmental stiffness during metastasis.

Development of mKate3/HaloTag7 (JFX650) and CFP/YFP Dual-Fluorescence (or Förster) Resonance Energy Transfer Pairs for Visualizing Dual-Molecular Activity.

Although several imaging strategies for dual fluorescence (or Förster) resonance energy transfer (FRET) biosensors have been reported, their implementation is challenging because of the limited performance of fluorescent proteins and the spectral overlap of FRET biosensors. These processes often require additional data calibration to eliminate artifacts. Many CFP/YFP FRET biosensors have been developed. In this study, we introduced the mKate3/HT7(JFX650) FRET pair, which effectively formed two pairs of FRET pairs for dual-FRET imaging when combined with the CFP/YFP FRET pair. The FRET donor mKate3 exhibited higher brightness than its predecessor mKate. The FRET acceptor, HT7(JFX650), is a HaloTag7 protein covalently conjugated with a far-red JFX650-THL ligand. The pair comprising mKate3 and HT7(JFX650) represents an excellent FRET dyad, exhibiting a high FRET efficiency ratio. To use the FRET pair for dual FRET biosensor imaging, we constructed PKA and K+ biosensors based on the mKate3/HT7(JFX650) FRET pair. These biosensors can be used along with CFP/YFP biosensors to simultaneously detect the responses of intracellular PKA/Src, PKA/Ca2+, and K+/Ca2+ under different stimuli. The findings revealed that dual FRET biosensors, which are based on the combination of CFP/YFP and mKate3/HT7 (JFX650), exhibit adequate compatibility and can be used to visualize multiple molecular activities in a live cell.

Multiple intermediates in the detergent-induced fusion of lipid vesicles

Detergent-induced vesicle interactions, critical for applications including virus inactivation, varies according to the detergent type and membrane composition, but the underlying mechanistic details remain underexplored. Here, we use a lipid mixing assay based on Förster resonance energy transfer (FRET), and single-vesicle characterization approaches to identify that sub-micron-sized vesicles are induced to fuse by the non-ionic detergent Triton-X-100. We demonstrate that the process is a multi-step mechanism, characterized by discrete values of FRET efficiency between membrane-embedded fluorophores, and involves permeabilization, vesicle docking, hemi-fusion and full lipid mixing at sub-solubilizing detergent concentrations. We also dissect the kinetics of vesicle fusion to surface-tethered vesicles using a label-free quartz-crystal microbalance with dissipation monitoring approach, opening a platform for biotechnology applications. The presented strategies provide mechanistic insight into the dynamics of vesicle fusion and have implications for applications including drug delivery and sensor development where transport and manipulation of encapsulated cargo is essential.

A Stochastic FRET Study on the Core Dimension of Polystyrene-block-Poly(Polyethylene Glycol Monomethyl Ether Acrylate) Micelles.

Polystyrene-b-poly(polyethylene glycol monomethyl ether acrylate) (PSt-b-PPEGA) copolymers featuring pyrene and perylene as the Förster resonance energy transfer (FRET) donor (denoted as D-BCP) and acceptor (denoted as A-BCP), respectively, were synthesized via the reversible addition and fragmentation chain transfer (RAFT) polymerization. These copolymers were then used to form DA-mixed micelles via a dialysis method. The micelles consisted of D-BCP (mole fraction fD = 0.04), A-BCP (fA = 0.04), and label-free PSt-b-PPEGA (fN = 0.92). The decrease in fluorescence intensity of pyrene in the micelles confirmed the occurrence of FRET, with an observed efficiency of 0.32. A Monte Carlo approach was employed to estimate the expected FRET efficiency, assuming the random fractional distribution of D-BCP and A-BCP, along with the random spatial distribution of pyrene and perylene within the micellar core. The observed FRET efficiency suggested a core radius (Rc) of 0.95R0, where R0 was the Förster critical distance. With R0 calculated to be 3.2 nm based on Förster theory, Rc was determined to be approximately 3.0 nm, aligning closely with the dried-out core radius estimated from aggregation number and polystyrene density. This stochastic FRET methodology was further applied to investigate the swelling behavior of the polymer micelles in a mixture of N,N-dimethylformamide (DMF) and water. Dynamic light scattering analysis revealed minimal change in core dimension below 60 vol % DMF content. However, FRET analysis provided a deeper insight, showing an increase in core radius with DMF content up to 20 vol %, followed by saturation up to 50 vol %, offering a more comprehensive understanding of the micelle swelling behavior.

Calibration of FRET-based biosensors using multiplexed biosensor barcoding.

Förster resonance energy transfer (FRET) between fluorescent proteins (FPs) is widely used in the design of genetically encoded fluorescent biosensors, which are powerful tools for monitoring the dynamics of biochemical activities in live cells. FRET ratio, defined as the ratio between acceptor and donor signals, is often used as a proxy for the actual FRET efficiency, which must be corrected for signal crosstalk using donor-only and acceptor-only samples. However, the FRET ratio is highly sensitive to imaging conditions, making direct comparisons across different experiments and over time challenging. Inspired by a method for multiplexed biosensor imaging using barcoded cells, we reasoned that calibration standards with fixed FRET efficiency can be introduced into a subset of cells for normalization of biosensor signals. Our theoretical analysis indicated that the FRET ratio of high-FRET species relative to non-FRET species slightly decreases at high excitation intensity, suggesting the need for calibration using both high and low FRET standards. To test these predictions, we created FRET donor-acceptor pairs locked in "FRET-ON" and "FRET-OFF" conformations and introduced them into a subset of barcoded cells. Our results confirmed the theoretical predictions and showed that the calibrated FRET ratio is independent of imaging settings. We also provided a strategy for calculating the FRET efficiency. Together, our study presents a simple strategy for calibrated and highly multiplexed imaging of FRET biosensors, facilitating reliable comparisons across experiments and supporting long-term imaging applications.

Freezing Conformers for Gas-Phase Förster Resonance Energy Transfer.

Various techniques are available to illuminate geometric structures of molecular ions in gas phase, such as Förster Resonance Energy Transfer (FRET) informing on distances between two dyes covalently attached to a molecule. Typically, cationic rhodamines, which absorb and emit visible light, are used for labeling. Extensive work has revealed that the transition energy of a rhodamine is intricately linked to its nearby microenvironment, with nearby charges causing Stark-shifted emission. This occurs because the inter-dye Coulomb interaction is weaker in the excited state (S1) than in the ground state (S0) due to the increase in polarizability upon excitation. Therefore, absorption and emission spectra, along with FRET efficiencies, provide insights into structural motifs. At room temperature, multiple conformers often co-exist, leading to overlapping absorption bands among different conformers and broad spectra. To study specific conformers, it is necessary to isolate them, for example, using ion-mobility spectrometry. Another approach is to reduce temperature, which results in spectral narrowing and distinct absorption bands, allowing for the selection of specific conformers through selective excitation. Here, we describe the instrumentation used for cryogenically cold FRET experiments and discuss recent results for small model systems, as well as future directions for a technique still in its infancy.

Extremely Sensitive Genetically Encoded Temperature Indicator Enabling Measurement at the Organelle Level.

Intracellular temperature is a fundamental parameter in biochemical reactions. Genetically encoded fluorescent temperature indicators (GETIs) have been developed to visualize intracellular thermogenesis; however, the temperature sensitivity or localization capability in specific organelles should have been further improved to clearly capture when and where intracellular temperature changes at the subcellular level occur. Here, we developed a new GETI, gMELT, composed of donor and acceptor subunits, in which cyan and yellow fluorescent proteins, respectively, as a Förster resonance energy transfer (FRET) pair were fused with temperature-sensitive domains. The donor and acceptor subunits associated and dissociated in response to temperature changes, altering the FRET efficiency. Consequently, gMELT functioned as a fluorescence ratiometric indicator. Untagged gMELT was expressed in the cytoplasm, whereas versions fused with specific localization signals were targeted to the endoplasmic reticulum (ER) or mitochondria. All gMELT variations enabled more sensitive temperature measurements in cellular compartments than those in previous GETIs. The gMELTs, tagged with ER or mitochondrial targeting sequences, were used to detect thermogenesis in organelles stimulated chemically, a method previously known to induce thermogenesis. The observed temperature changes were comparable to previous reports, assuming that the fluorescence readout changes were exclusively due to temperature variations. Furthermore, we demonstrated how macromolecular crowding influences gMELT fluorescence given that this factor can subtly affect the fluorescence readout. Investigating thermogenesis with gMELT, accounting for factors such as macromolecular crowding, will enhance our understanding of intracellular thermogenesis phenomena.

Comparison of vinculin tension in cellular monolayers and three-dimensional multicellular aggregates.

Confocal frequency-domain fluorescence lifetime and Förster resonance energy transfer (FRET) microscopy of Chinese hamster ovary (CHO-K1) cells expressing the vinculin tension sensor (VinTS) is used to compare vinculin tension in three-dimensional (3D) multicellular aggregates and 2D cellular monolayers. In both 2D and 3D cultures, the FRET efficiency of VinTS is 5-6% lower than that of VinTL (p < 0.05), a tail-less control which cannot bind actin or paxillin. The difference between VinTS and VinTL FRET efficiency can be mitigated by treatment with the Rho-associated kinase inhibitor Y-27632, demonstrating that VinTS is under tension in both 2D and 3D cultures. However, there is an overall decrease in FRET efficiency of both VinTS and VinTL in the 3D multicellular aggregates compared with the 2D monolayers. Expression of VinTS in 2D and 3D cultures exhibits puncta consistent with cellular adhesions. While paxillin is present at the sites of VinTS expression in the 2D monolayers, it is generally absent from VinTS puncta in the 3D aggregates. The results suggest that VinTS experiences a modified environment in 3D aggregates compared with 2D monolayers and provide a basis for further investigation of molecular tension sensors in 3D tissue models.

Fluorogenic Rhodamine-Based Chemigenetic Biosensor for Monitoring Cellular NADPH Dynamics.

Ratiometric biosensors employing Förster Resonance Energy Transfer (FRET) enable the real-time tracking of metabolite dynamics. Here, we introduce an approach for generating a FRET-based biosensor in which changes in apparent FRET efficiency rely on the analyte-controlled fluorogenicity of a rhodamine rather than the commonly used distance change between donor-acceptor fluorophores. Our fluorogenic, rhodamine-based, chemigenetic biosensor (FOCS) relies on a synthetic, protein-tethered FRET probe, in which the rhodamine acting as the FRET acceptor switches in an analyte-dependent manner from a dark to a fluorescent state. This allows ratiometric sensing of the analyte concentration. We use this approach to generate a chemigenetic biosensor for nicotinamide adenine dinucleotide phosphate (NADPH). FOCS-NADPH exhibits a rapid and reversible response toward NAPDH with a good dynamic range, selectivity, and pH insensitivity. FOCS-NADPH allows real-time monitoring of cytosolic NADPH fluctuations in live cells during oxidative stress or after drug exposure. We furthermore used FOCS-NADPH to investigate NADPH homeostasis regulation through the pentose phosphate pathway of glucose metabolism. FOCS-NADPH is a powerful tool for studying NADPH metabolism and serves as a blueprint for the development of future fluorescent biosensors.

CsPbBr3 Perovskite Quantum Dots Encapsulated by a Polymer Matrix for Ultrasensitive Dynamic Imaging of Intracellular MicroRNA.

Herein, CsPbBr3 perovskite quantum dots (CPB PQDs)@poly(methyl methacrylate) (PMMA) (CPB@PMMA) nanospheres were used as energy donors with high Förster resonance energy transfer (FRET) efficiency and exceptional biocompatibility for ultrasensitive dynamic imaging of tiny amounts of microRNAs in living cells. Impressively, compared with traditional homogeneous single QDs as energy donors, CPB@PMMA obtained by encapsulating numerous CPB PQDs into PMMA as energy donors could not only significantly increase the efficiency of FRET via improving the local concentration of CPB PQDs but also distinctly avoid the problem of cytotoxicity caused by divulged heavy metal ions entering living cells. Most importantly, in the presence of target miRNA-21, DNA dendrimer-like nanostructures labeled with 6-carboxy-tetramethylrhodamine (TAMRA) were generated by the exposed tether interhybridization of the Y-shape structure, which could wrap around the surface of CPB@PMMA nanospheres to remarkably bridge the distance of FRET and increase the opportunity for effective energy transfer, resulting in excellent precision and accuracy for ultrasensitive and dynamic imaging of miRNAs. As proof of concept, the proposed strategy exhibited ultrahigh sensitivity with a detection limit of 45.3 aM and distinctly distinguished drug-irritative miRNA concentration abnormalities with living cells. Hence, the proposed enzyme-free CPB@PMMA biosensor provides convincing evidence for supplying accurate information, which could be expected to be a powerful tool for bioanalysis, diagnosis, and prognosis of human diseases.

Improved Exciton Diffusion through Modulating Förster Resonance Energy Transfer for Efficient Organic Solar Cells

Förster resonance energy transfer (FRET) is commonly utilized in organic solar cells (OSCs) to enhance the power conversion efficiency (PCE) by promoting molecular interactions. The PCE is greatly affected by the number of photo‐generated excitons that effectively reach interfaces between the donor and acceptor materials of OSCs. However, the correlation between FRET and exciton diffusion has received limited attention. Therefore, it is crucial to understand and manipulate FRET process for investigating exciton dynamics in OSCs. Herein, BTA3 and its derivatives are chosen as the third components and energy donors to elucidate the intrinsic photophysical mechanism of FRET in OSCs by manipulating their efficiency. The results unambiguously demonstrate that the addition of guest F‐BTA3 exhibits the highest FRET efficiency, leading to a significant enhancement in the PCE of both PM6:Y6 and PM6:PY‐IT host systems. By correlating FRET process and exciton dynamics, this enhancement to remarkable increment in exciton diffusion length is attributed. Experimental results and theoretical simulations indicate high FRET efficiency arises from strong dipole–dipole interaction, and exhibits a positive correlation with exciton diffusion length. This study showcases the effective regulation of exciton diffusion in OSCs through modulation of FRET, offering a novel perspective for optimizing the performance of organic photovoltaic devices.

Unraveling Burst Selection Bias in Single-Molecule FRET of Species with Unequal Brightness and Diffusivity.

Single-molecule free diffusion experiments enable accurate quantification of coexisting species or states. However, unequal brightness and diffusivity introduce a burst selection bias and affect the interpretation of experimental results. We address this issue with a photon-by-photon maximum likelihood method, burstML, which explicitly considers burst selection criteria. BurstML accurately estimates parameters, including photon count rates, diffusion times, Förster resonance energy transfer (FRET) efficiencies, and population, even in cases where species are poorly distinguished in FRET efficiency histograms. We develop a quantitative theory that determines the fraction of photon bursts corresponding to each species and thus obtain accurate species populations from the measured burst fractions. In addition, we provide a simple approximate formula for burst fractions and establish the range of parameters where unequal brightness and diffusivity can significantly affect the results obtained by conventional methods. The performance of the burstML method is compared with that of a maximum likelihood method that assumes equal species brightness and diffusivity, as well as standard Gaussian fitting of FRET efficiency histograms, using both simulated and real single-molecule data for cold-shock protein, protein L, and protein G. The burstML method enhances the accuracy of parameter estimation in single-molecule fluorescence studies.

Disrupting EGFR-HER2 Transactivation by Pertuzumab in HER2-Positive Cancer: Quantitative Analysis Reveals EGFR Signal Input as Potential Predictor of Therapeutic Outcome.

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

Resonant energy transfer among naturally available bio materials for white light emission

In this work we have studied the fluorescence of natural dyes and generated nearly pure white light with chromaticity intensity (CIE) coordinate (0.35,0.35). The colour rendering index (CRI) and colour temperature corresponding to the CIE coordinate are calculated and these dyes are ideal for cool white light emission. It was observed that a broadband and simultaneous emission involving anthocyanin and polyphenol ellergic acid from jamun, curcumin and chlorophyll from spinach leaves played a vital role in obtaining a CIE index close to that of pure white light. The white light emission is due to the Förster resonance energy transfer (FRET) from curcumin to anthocyanin and ellergic acid to curcumin. Efficiency of FRET is calculated and different possibilities studied. For the polyphenol ellergic acid, curcumin FRET pair the spectral overlap integral and the efficiency are 3.29 × 10−24 m2, 99.97% and for the curcumin, anthocyanin pair, they are 4.03 × 10–24 m2, 76%, respectively.

Boosting Excited-State Energy Transfer by Anchoring Dipole Orientation in Binary Thermally Activated Delayed Fluorescence/J-Aggregate Assemblies.

Förster resonance energy transfer (FRET) has been widely applied in fluorescence imaging, sensing and so on, while developing useful strategy of boosting FRET efficiency becomes a key issue that limits the application. Except optimizing spectral properties, promoting orientation factor (κ2) has been well discussed but rarely utilized for boosting FRET. Herein, we constructed binary nano-assembling of two thermally activated delayed fluorescence (TADF) emitters (2CzPN and DMAC-DPS) with J-type aggregate of cyanine dye (C8S4) as doping films by taking advantage of their electrostatic interactions. Time-resolved spectroscopic measurements indicated that 2CzPN/Cy-J films exhibit an order of magnitude higher kFRET than DMAC-DPS/Cy-J films. Further quantitative analysing on kFRET and kDET indicated higher orientation factor (κ2) in 2CzPN/Cy-J films play a key role for achieving fast kFRET, which was subsequently confirmed by anisotropic measurements. Corresponding DFT/TDDFT calculation revealed strong "two-point" electrostatic anchoring in 2CzPN/Cy-J films that is responsible for highly orientated transitions. We provide a new strategy for boosting FRET in nano-assemblies, which might be inspired for designing FRET-based devices of sensing, imaging and information encryption.

Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

Aptamer-free upconversion nanoparticle/silk biosensor system for low-cost and highly sensitive detection of antibiotic residues

The detection of antibiotics is crucial for safeguarding the environment, ensuring food safety, and promoting human health. However, developing a rapid, convenient, low-cost, and sensitive method for antibiotic detection presents significant challenges. Herein, an aptamer-free biosensor was successfully constructed using upconversion nanoparticles (UCNPs) coated with silk fibroin (SF), based on Förster resonance energy transfer (FRET) and the charge-transfer effect, for detecting roxithromycin (RXM). A synergistic FRET efficiency was achieved by utilizing alizarin red and RXM complexes as energy acceptors, with UCNP as the energy donor, and immobilizing an ultrathin SF protein corona within 10 nm. The biosensor detects RXM in deionized water with high sensitivity primarily through monolayer adsorption, with a detection range of 1.0 nM–141.6 nM and a detection limit as low as 0.68 nM. The performance of this biosensor was compared with the ultra-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method for detecting antibiotics in river water separately and a strong correlation between the two methods was observed. The biosensor exhibited long-term stability in aqueous solutions (up to 60 d) with no attenuation of fluorescence intensity. Furthermore, the biosensor's applicability extended to the highly sensitive detection of other antibiotics, such as azithromycin. This study introduces a low-cost, eco-friendly, and highly sensitive method for antibiotic detection, with broad potential for future applications in environmental, healthcare, and food-related fields.

FRETpredict: a Python package for FRET efficiency predictions using rotamer libraries

Förster resonance energy transfer (FRET) is a widely-used and versatile technique for the structural characterization of biomolecules. Here, we introduce FRETpredict, an easy-to-use Python software to predict FRET efficiencies from ensembles of protein conformations. FRETpredict uses a rotamer library approach to describe the FRET probes covalently bound to the protein. The software efficiently and flexibly operates on large conformational ensembles such as those generated by molecular dynamics simulations to facilitate the validation or refinement of molecular models and the interpretation of experimental data. We provide access to rotamer libraries for many commonly used dyes and linkers and describe a general methodology to generate new rotamer libraries for FRET probes. We demonstrate the performance and accuracy of the software for different types of systems: a rigid peptide (polyproline 11), an intrinsically disordered protein (ACTR), and three folded proteins (HiSiaP, SBD2, and MalE). FRETpredict is open source (GPLv3) and is available at github.com/KULL-Centre/FRETpredict and as a Python PyPI package at pypi.org/project/FRETpredict.

Specific FRET Probes Sensitive to Chitosan-Based Polymeric Micelles Formation, Drug-Loading, and Fine Structural Features.

Förster resonance energy transfer (FRET) probes are a promising tool for studying numerous biochemical processes. In this paper, we show the application of the FRET phenomenon to observe the micelle formation from surfactants, micelles self-assembling from chitosan grafted with fatty acid (oleic-OA, or lipoic-LA), cross-linking of SH groups in the micelle's core, and inclusion and release of the model drug cargo from the micelles. Using the carbodiimide approach, amphiphilic chitosan-based polymers with (1) SH groups, (2) crosslinked with S-S between polymer chains, and (3) without SH and S-S groups were synthesized, followed by characterization by FTIR and NMR spectroscopy. Two pairs of fluorophores were investigated: 4-methylumbelliferon-trimethylammoniocinnamate-rhodamine (MUTMAC-R6G) and fluorescein isothiocyanate-rhodamine (FITC-R6G). While FITC-R6G has been described before as an FRET-producing pair, for MUTMAC-R6G, this has not been described. R6G, in addition to being an acceptor fluorophore, also serves as a model cytostatic drug in drug-release experiments. As one could expect, in aqueous solution, FRET effect was poor, but when exposed to the micelles, both MUTMAC-R6G and FITC-R6G yielded a pronounced FRET effect. Most likely, the formation of micelles is accompanied by the forced convergence of fluorophores in the hydrophobic micelle core by a donor-to-acceptor distance (r) significantly closer than in the aqueous buffer solution, which was reflected in the increase in the FRET efficiency (E). Therefore, r(E) could be used as analytical signal of the micelle formation, including critical micelle concentration (CMC) and critical pre-micelle concentration (CPMC), yielding values in good agreement with the literature for similar systems. We found that the r-function provides analytically valuable information about the nature and mechanism of micelle formation. S-S crosslinking between polymer chains makes the micelle more compact and stable in the normal physiological conditions, but loosens in the glutathione-rich tumor microenvironment, which is considered as an efficient approach in targeted drug delivery. Indeed, we found that R6G, as a model cytostatic agent, is released from micelles with initial rate of 5%/h in a normal tissue microenvironment, but in a tumor microenvironment model (10 mM glutathione), the release of R6G from S-S stitched polymeric micelles increased up to 24%/h. Drug-loading capacity differed substantially: from 75-80% for nonstitched polymeric micelles to ~90% for S-S stitched micelles. Therefore, appropriate FRET probes can provide comprehensive information about the micellar system, thus helping to fine-tune the drug delivery system.