Förster Resonance Energy Transfer Acceptor Research Articles

Phage-induced “one-to-many” FRET sensor for highly sensitive detection of Escherichia coli O157:H7

As a foodborne pathogen capable of causing severe illnesses, early detection of Escherichia coli O157:H7 (E. coli O157:H7) is crucial for ensuring food safety. While Förster resonance energy transfer (FRET) is an efficient and precise detection technique, there remains a need for amplification strategies to detect low concentrations of E. coli O157:H7. In this study, we presented a phage (M13)-induced “one to many” FRET platform for sensitively detecting E. coli O157:H7. The aptamers, which specifically recognize E. coli O157:H7 were attached to magnetic beads as capture probes for separating E. coli O157:H7 from food samples. The peptide O157S, which specifically targets E. coli O157:H7, and streptavidin binding peptide (SBP), which binds to streptavidin (SA), were displayed on the P3 and P8 proteins of M13, respectively, to construct the O157S-M13K07-SBP phage as a detection probe for signal output. Due to the precise distance (≈3.2 nm) between two neighboring N-terminus of P8 protein, the SA-labeled FRET donor and acceptor can be fixed at the Förster distance on the surface of O157S-M13K07-SBP via the binding of SA and SBP, inducing FRET. Moreover, the P8 protein, with ≈2700 copies, enabled multiple FRET (≈605) occurrences, amplifying FRET in each E. coli O157:H7 recognition event. The O157S-M13K07-SBP-based FRET sensor can detect E. coli O157:H7 at concentration as low as 6 CFU/mL and demonstrates excellent performance in terms of selectivity, detection time (≈3 h), accuracy, precision, practical application, and storage stability. In summary, we have developed a powerful tool for detecting various targets in food safety, environmental monitoring, and medical diagnosis.

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.

Giant Light‐Harvesting in Dye‐Loaded Nanoparticles Enhanced by Blank Hydrophobic Salts

AbstractLight‐harvesting is a fundamental process in nature, which inspires researchers to develop artificial systems for photocatalysis, photovoltaics, and biosensing. A previously introduced light‐harvesting nanoantenna, based on polymeric nanoparticles (NPs) loaded with rhodamine dyes and bulky hydrophobic counterions, provides a record‐breaking antenna effect ≈1000. However, the high dye cooperativity of its thousands of encapsulated dyes causes energy losses by traces of self‐quenched dye aggregates. Here, it is found that these imperfections can be suppressed by blank hydrophobic salts (BHS) formed by the same bulky counterion (fluorinated tetraphenylborate) with an optically inactive cation, analogs of ionic liquids. The presence of BHS increases twofold the fluorescence quantum yields and fluorescence lifetimes of NPs and suppresses their fluorescence blinking. This study assumes that BHS provides an excess of bulky counterions that excludes traces of dye aggregates. As a result, an efficient Forster resonance energy transfer (FRET) is achieved from 40 000 dye donors to a single acceptor within a 70 nm particle, leading to the antenna effect of 4800, which is by far the highest value reported to date. Using this nanoantenna, a single‐molecule detection of the FRET acceptor is realized at low excitation power using an RGB camera of a smartphone.

Characterizing the Ligand Shell Morphology of PEG-Coated ZnO Nanocrystals Using FRET Spectroscopy.

Poly(ethylene glycol) (PEG) ligands can inhibit proteins and other biomolecules from adhering to underlying surfaces, making them excellent surface ligands for nanocrystal (NC)-based drug carriers. Quantifying the PEG ligand shell morphology is important because its structure determines the permeability of biomolecules through the shell to the NC surface. However, few in situ analytical tools can reveal whether the PEG ligands form either an impenetrable barrier or a porous coating surrounding the NC. Here, we present a Förster resonance energy transfer (FRET) spectroscopy-based approach that can assess the permeability of molecules through PEG-coated ZnO NCs. In this approach, ZnO NCs serve as FRET donors, and freely diffusing molecules in the bulk solution are FRET acceptors. We synthesized a series of variable chain length PEG-silane-coated ZnO NCs such that the longest chain length ligands far exceed the Förster radius (R0), where the energy transfer (EnT) efficiency is 50%. We quantified the EnT efficiency as a function of the ligand chain length using time-resolved photoluminescence lifetime (TRPL) spectroscopy within the framework of FRET theory. Unexpectedly, the longest PEG-silane ligand showed equivalent EnT efficiency as that of bare, hydroxyl-passivated ZnO NCs. These results indicate that the "rigid shell" model fails and the PEG ligand shell morphology is more likely porous or in a patchy "mushroom state", consistent with transmission electron microscopy data. While the spectroscopic measurements and data analysis procedures discussed herein cannot directly visualize the ligand shell morphology in real space, the in situ spectroscopy approach can provide researchers with valuable information regarding the permeability of species through the ligand shell under practical biological conditions.

Lanthanide‐FRET Molecular Beacons for microRNA Biosensing, Logic Operations, and Physical Unclonable Functions

AbstractTime‐resolved or time‐gated (TG) biosensing and bioimaging with luminescent lanthanide probes and Förster resonance energy transfer (FRET) have significantly advanced bioanalytical chemistry. However, the development of lanthanide‐based molecular beacons (MBs) has been rather limited. Here, we designed DNA stem‐loop MB probes against two different microRNAs (miR‐21 and miR‐27b) using Tb and Eu FRET donors and quenching (BHQ2) and fluorescent (Cy3) FRET acceptors. Limits of detection down to 190 pM and duplexed miR‐21/miR‐27b quantification at low nanomolar concentrations with Tb‐BHQ2 and Eu‐BHQ2 TG‐FRET MBs demonstrated the versatility and high analytical performance of lanthanide‐based MBs. The particular donor‐acceptor distances in the Tb‐Cy3 MB resulted in inverted nucleic acid target concentration‐dependent TG PL intensities in short (e. g., 0 to 40 μs) and long (e. g., 0.1 to 2.1 ms) TG detection windows after pulsed excitation. We showed that this specific feature of our TG‐FRET MBs can be adapted to the design of molecular logic devices (NOR, OR, NAND, AND, XNOR, XOR, IMPLEMENT, and INHIBIT). Moreover, the almost unlimited choice of TG detection windows and the distinct spectral features of Tb and Cy3 over a broad visible spectral range could be exploited to devise biophotonic physical unclonable functions for highly secure authentication and identification. Our study manifests the versatility of lanthanides for advanced biophotonic applications.

Quantitative Measurement of Drug Release Dynamics within Targeted Organelles Using Förster Resonance Energy Transfer.

Measuring the release dynamics of drug molecules after their delivery to the target organelle is critical to improve therapeutic efficacy and reduce side effects. However, it remains challenging to quantitatively monitor subcellular drug release in real time. To address the knowledge gap, a novel gemini fluorescent surfactant capable of forming mitochondria-targeted and redox-responsive nanocarriers is designed. A quantitative Förster resonance energy transfer (FRET) platform is fabricated using this mitochondria-anchored fluorescent nanocarrier as a FRET donor and fluorescent drugs as a FRET acceptor. The FRET platform enables real-time measurement of drug release from organelle-targeted nanocarriers. Moreover, the obtained drug release dynamics can evaluate the duration of drug release at the subcellular level, which established a new quantitative method for organelle-targeted drug release. This quantitative FRET platform can compensate for the absent assessment of the targeted release performances of nanocarriers, offering in-depth understanding of the drug release behaviors at the subcellular targets.

Direct Measurement of Polymer-Chain-End-to-End Distances by Using RAFT Chain Transfer Agent as the FRET Acceptor.

Förster resonance energy transfer (FRET) is a powerful tool for measuring distances between two molecules (donor and acceptor) in close proximity (1-10 nm), which can be employed for determining polymer end-to-end distances (Ree). However, previous works for labeling FRET pairs on chain-ends often involve relatively complex steps for materials preparation, potentially limiting their broad use in synthetic polymer systems. In this work, we introduce an anthracene-functionalized chain-transfer agent for reversible addition-fragmentation chain-transfer (RAFT) polymerizations, which can directly yield polymers containing FRET donor and acceptor molecules on respective chain-ends. This approach enables the direct use of FRET for characterizing the averaged Ree of polymers. Building on this platform, we investigate the averaged Ree of polystyrene (PS) and poly(methyl methacrylate) (PMMA) in a good solvent as a function of their molecular weight. Notably, the FRET results show good agreement with simulation results obtained from all-atom molecular dynamics, confirming its measurement accuracy. Overall, this work provides a facile and broadly applicable platform to directly determine the Ree of low molecular weight polymers by using FRET-based methods.

Optimizing Upconversion Nanoparticles for FRET Biosensing.

Upconversion nanoparticles (UCNPs) are some of the most promising nanomaterials for bioanalytical and biomedical applications. One important challenge to be still solved is how UCNPs can be optimally implemented into Förster resonance energy transfer (FRET) biosensing and bioimaging for highly sensitive, wash-free, multiplexed, accurate, and precise quantitative analysis of biomolecules and biomolecular interactions. The many possible UCNP architectures composed of a core and multiple shells doped with different lanthanoid ions at different ratios, the interaction with FRET acceptors at different possible distances and orientations via biomolecular interaction, and the many and long-lasting energy transfer pathways from the initial UCNP excitation to the final FRET process and acceptor emission make the experimental determination of the ideal UCNP-FRET configuration for optimal analytical performance a real challenge. To overcome this issue, we have developed a fully analytical model that requires only a few experimental configurations to determine the ideal UCNP-FRET system within a few minutes. We verified our model via experiments using nine different Nd-, Yb-, and Er-doped core-shell-shell UCNP architectures within a prototypical DNA hybridization assay using Cy3.5 as an acceptor dye. Using the selected experimental input, the model determined the optimal UCNP out of all theoretically possible combinatorial configurations. An extreme economy of time, effort, and material was accompanied by a significant sensitivity increase, which demonstrated the powerful feat of combining a few selected experiments with sophisticated but rapid modeling to accomplish an ideal FRET biosensor.

Detecting RNA–Protein Interactions With EGFP‐Cy3 FRET by Acceptor Photobleaching

Förster Resonance Energy Transfer (FRET) is a great tool for cell biologists to investigate molecular interactions in live specimens. FRET is a distance-dependent phenomenon which can detect molecular interactions at distances between 1-10 nm. Several FRET approaches are reported in the literature for live and fixed cells to study protein-protein interactions; this protocol provides details of acceptor photobleaching as a FRET method to study RNA-Protein interactions. Cy3-labeled RNA foci (FRET acceptors) are photobleached at the intra-cellular site of interest (the nuclei) and the intensity of the EGFP-tagged proteins (FRET donors) at that same site are measured pre- and post- photobleaching. In principle, FRET is detected if the intensity of EGFP increases after photobleaching of Cy3. This protocol describes necessary steps and appropriate controls to conduct FRET measurements by the acceptor photobleaching method. Successful applications of this protocol will provide data to support the conclusion that EGFP-labeled proteins directly interact with Cy3-labeled RNA at the site of photobleaching. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: FRET in fixed cells Alternate Protocol: FRET in live cells.

Biosensor Optimization Using a Förster Resonance Energy Transfer Pair Based on mScarlet Red Fluorescent Protein and an mScarlet-Derived Green Fluorescent Protein.

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, and less autofluorescence. Here, we describe the development of an mScarlet-derived green fluorescent protein (designated as mWatermelon) and its use as a FRET donor to the red fluorescent protein mScarlet-I as a FRET acceptor. We tested the functionality of this FRET pair by engineering biosensors for the detection of protease activity, Ca2+, and K+. Furthermore, we described a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I.

Steric Barrier‐Steered Reaction Sites in Micellar Catalysis

AbstractIn the pursuit of an advanced micellar catalysis system, the reaction sites in micelles determine its catalytic performances. However, the intrinsic polarity gradient of micelles anchors the reactants in the fixed regions, leading to an unmet challenge to steer the reaction sites to the optimal ones. To overcome this limitation, a Förster resonance energy transfer (FRET) spectroscopic ruler is established to position the true sites of reactants in micellar catalysis by using tetraphenylethylene (TPE) in the aggregation‐induced emission (AIE) micelles as the FRET donor and Nile red as the FRET acceptor. Subsequently, the customizable TPE steric barriers in the AIE micelles are apt to mediate the reactants into the polar micellar surface, the semipolar palisade layer, or the nonpolar internal layer, achieving the corresponding catalytic efficiencies. Additionally, the universality of steric barrier‐steered reaction sites in micellar catalysis is further demonstrated by the simple incorporation of small nonpolar molecules into the commercially available surfactants. To the authors’ knowledge, this is the first report on the design of steric barriers to steer the reaction sites in micellar catalysis. These findings open up attractive perspectives for the exploration of new classes of efficient micellar catalysis systems in a variety of reactions.

Designs and Applications of Multi-stimuli Responsive FRET Processes in AIEgen-Functionalized and Bi-fluorophoric Supramolecular Materials

Materials capable of displaying strong ratiometric fluorescence with Förster resonance energy transfer (FRET) processes have attracted much research interest because of various chemosensor and biomedical applications. This review highlights several popular strategies in designing FRET-OFF/ON mechanisms of ratiometric fluorescence systems. In particular, the developments of organic and polymeric FRET materials featuring aggregation-induced emission-based luminogens (AIEgens), supramolecular assemblies, photochromic molecular switches and surfactant-induced AIE/FRET mechanisms are presented. AIEgens have been frequently employed as FRET donor and/or acceptor fluorophores to obtain enhanced ratiometric fluorescences in solution and solid states. Since AIE effects and FRET processes rely on controllable distances between fluorophores, many interesting fluorescent properties can be designed by regulating aggregation states in polymers and supramolecular systems. Photo-switchable fluorophores, such as spiropyran and diarylethene, provide drastic changes in fluorescence spectra upon photo-induced isomerizations, leading to photo-switching mechanisms to activate/deactivate FRET processes. Supramolecular assemblies offer versatile platforms to regulate responsive FRET processes effectively. In rotaxane structures, the donor-acceptor distance and FRET efficiency can be tuned by acid/base-controlled shuttling of the macrocycle component. The tunable supramolecular interactions are strongly influenced by external factors (such as pH values, temperatures, analytes, surfactants, UV-visible lights, etc.), which induce the assembly and disassembly of host-guest systems and thus their FRET-ON/FRET-OFF behavior. In addition, the changes in donor or acceptor fluorescence profiles upon detections of analytes can also sufficiently alter the FRET behavior and result in different ratiometric fluorescence outputs. The strategies and examples provided in this review offer the insights and toolkits for future FRET-based material developments.

Universal Concept for Bright, Organic, Solid-State Emitters─Doping of Small-Molecule Ionic Isolation Lattices with FRET Acceptors.

Brightly fluorescent solid-state materials are highly desirable for bioimaging, optoelectronic applications, and energy harvesting. However, the close contact between π-systems most often leads to quenching. Recently, we developed small-molecule ionic isolation lattices (SMILES) that efficiently isolate fluorophores while ensuring very high densities of the dyes. Nevertheless, efficient Förster resonance energy transfer (FRET) energy migration in such dense systems is inevitable. While attractive for energy harvesting applications, FRET also significantly compromises quantum yields of fluorescent solids by funneling the excitation energy to dark trap states. Here, we investigate the underlying property of FRET and exploit it to our favor by intentionally introducing fluorescent dopants into SMILES materials, acting as FRET acceptors with favorable photophysical properties. This doping is shown to outcompete energy migration to dark trap states while also ruling out reabsorption effects in dense SMILES materials, resulting in universal fluorescent solid-state materials (thin films, powders, and crystals) with superior properties. These include emission quantum yields reaching as high as 50-65%, programmable fluorescence lifetimes with mono-exponential decay, and independent selection of absorption and emission maxima. The volume normalized brightness of these FRET-based SMILES now reach values up to 32,200 M-1 cm-1 nm-3 and can deliver freely tunable spectroscopic properties for the fabrication of super-bright advanced optical materials. It is found that SMILES prohibit PET quenching between donor and acceptor dyes that is observed for non-SMILES mixtures of the same dyes. This allows a very broad selection of donor and acceptor dyes for use in FRET SMILES.

Small-Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications.

For fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state non-radiatively, and are promising alternatives to fluorescent FRET acceptors. Small-molecule (dark) quenchers have been widely used as acceptors in FRET-based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small-molecule quenchers that are used in FRET-based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophore-quencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

Small‐Molecule Quenchers for Förster Resonance Energy Transfer: Structure, Mechanism, and Applications

AbstractFor fluorogenic probes, Förster resonance energy transfer (FRET) is one of the most widely used mechanisms to realize the detection of biochemical activities. Dark quenchers relax from the excited state to the ground state non‐radiatively, and are promising alternatives to fluorescent FRET acceptors. Small‐molecule (dark) quenchers have been widely used as acceptors in FRET‐based probes to monitor various physiological processes with minimum background signal. Herein, we summarize the relevant advances of small‐molecule quenchers that are used in FRET‐based probes. This Review is intended to provide suggestions regarding the rationale design and selection of appropriate fluorophore–quencher FRET pairs, which are fully compatible with challenging analytical applications in various biological systems. Finally, an outlook of the future biomedical applications and developments of this field is presented.

Multiple Docking of Fluorescent Dyes to Fibrillar Insulin

The co-localization of the donor and acceptor fluorophores capable of transferring the energy by the Fӧrster mechanism, on the molecular scaffold of amyloid fibrils offers new opportunities not only for refinement of the amyloid detection and structural analysis, but also for designing photonic nanodevices on their basis. The assembly of these systems involves the non-covalent dye-protein interactions which can hardly be characterized in terms of a precise dye location within the fibril structure that is required for fabricating the FRET-based light harvesting systems or photonic nanowires. In view of this, the dye-fibril binding process deserves a detailed in silico study. In the previous molecular docking studies of the FRET donors and acceptors interacting with the insulin model fibrils we considered only one ligand during the simulation procedure. However, the real situation is much more complicated, when the multiple ligands can compete for the same binding site, a direct complexation between the dyes on the fibril scaffold can take place, the spatial distribution of the bound fluorophores can be unfavorable for the energy transfer, etc. In addition, the mutual orientation of the donor and acceptor molecules essentially contribute to the efficiency of the Förster resonance energy transfer (FRET) in the investigated systems. The present study was undertaken to gain molecular docking insight into the binding of the donor (Thioflavin T) and acceptor (Congo Red or a phosphonium dye TDV) fluorophores to the insulin amyloid fibrils using the multiple docking approach. The employed PatcDock and SwissDock webserves provided evidence for the preferable association of all dyes with the fibril grooves. The protein-ligand interaction profiler (PLIP) was employed for analyzing the (InsF + ThT +CR) and (InsF + ThT + TDV) systems. The revealed binding modes and the types of the dye-fibril interactions may be of importance for a more detailed analysis of the FRET process in amyloid systems and may serve as a background for further in silico studies of the cascade FRET on the amyloid fibril scaffold.

Construction of Self-Assembling Lipopeptide-Based Benign Nanovesicles to Prevent Amyloid Fibril Formation and Reduce Cytotoxicity of GxxxGxxxGxxxG Motif.

Alzheimer's disease, a progressive severe neurodegenerative disorder, has been until now incurable, in spite of serious efforts worldwide. We have designed self-assembled myristoyl-KPGPK lipopeptide-based biocompatible nanovesicles, which can inhibit amyloid fibrillation made by the transmembrane GxxxGxxxGxxxG motif of Aβ-protein and human myelin protein zero as well as reduce their neurotoxicity. Various spectroscopic and microscopic investigations illuminate that the lipopeptide-based nanovesicles dramatically inhibit random coil-to-β-sheet transformation of Aβ25-37 and human myelin protein zero protein precursor, which is the prerequisite of GxxxGxxxGxxxG motif-mediated fibril formation. Förster resonance energy transfer (FRET) assay using synthesized Cy-3 (FRET donor) and Cy-5 (FRET acceptor)-conjugated Aβ25-37 also exhibits that nanovesicles strongly inhibit the fibril formation of Aβ25-37. The mouse neuro-2a neuroblastoma cell line is used, which revealed the GxxxGxxxGxxxG-mediated cytotoxicity. However, the neurotoxicity has been diminished by co-incubating the GxxxGxxxGxxxG motif with the nanovesicles.

Molecular Transport within Polymer Brushes: A FRET View at Aqueous Interfaces.

Molecular permeability through polymer brush chains is implicated in surface lubrication, wettability, and solute capture and release. Probing molecular transport through polymer brushes can reveal information on the polymer nanostructure, with a permeability that is dependent on chain conformation and grafting density. Herein, we introduce a brush system to study the molecular transport of fluorophores from an aqueous droplet into the external “dry” polymer brush with the vapour phase above. The brushes consist of a random copolymer of N-isopropylacrylamide and a Förster resonance energy transfer (FRET) donor-labelled monomer, forming ultrathin brush architectures of about 35 nm in solvated height. Aqueous droplets containing a separate FRET acceptor are placed onto the surfaces, with FRET monitored spatially around the 3-phase contact line. FRET is used to monitor the transport from the droplet to the outside brush, and the changing internal distributions with time as the droplets prepare to recede. This reveals information on the dynamics and distances involved in the molecular transport of the FRET acceptor towards and away from the droplet contact line, which are strongly dependent on the relative humidity of the system. We anticipate our system to be extremely useful for studying lubrication dynamics and surface droplet wettability processes.

Multiplexed Biosensing and Bioimaging Using Lanthanide-Based Time-Gated Förster Resonance Energy Transfer.

The necessity to scrutinize more and more biological molecules and interactions both in solution and on the cellular level has led to an increasing demand for sensitive and specific multiplexed diagnostic analysis. Photoluminescence (PL) detection is ideally suited for multiplexed biosensing and bioimaging because it is rapid and sensitive and there is an almost unlimited choice of fluorophores that provide a large versatility of photophysical properties, including PL intensities, spectra, and lifetimes.The most frequently used technique to detect multiple parameters from a single sample is spectral (or color) multiplexing with different fluorophores, such as organic dyes, fluorescent proteins, quantum dots, or lanthanide nanoparticles and complexes. In conventional PL biosensing approaches, each fluorophore requires a distinct detection channel and excitation wavelength. This drawback can be overcome by Förster resonance energy transfer (FRET) from lanthanide donors to other fluorophore acceptors. The lanthanides' multiple and spectrally narrow emission bands over a broad spectral range can overlap with several different acceptors at once, thereby allowing FRET from one donor to multiple acceptors. The lanthanides' extremely long PL lifetimes provide two important features. First, time-gated (TG) detection allows for efficient suppression of background fluorescence from the biological environment or directly excited acceptors. Second, temporal multiplexing, for which the PL lifetimes are adjusted by the interaction with the FRET acceptor, can be used to determine specific biomolecules and/or their conformation via distinct PL decays. The high signal-to-background ratios, reproducible and precise ratiometric and homogeneous (washing-free) sensing formats, and higher-order multiplexing capabilities of lanthanide-based TG-FRET have resulted in significant advances in the analysis of biomolecular recognition. Applications range from fundamental analysis of biomolecular interactions and conformations to high-throughput and point-of-care in vitro diagnostics and DNA sequencing to advanced optical encoding, using both liquid and solid samples and in situ, in vitro, and in vivo detection with high sensitivity and selectivity.In this Account, we discuss recent advances in lanthanide-based TG-FRET for the development and application of advanced immunoassays, nucleic acid sensing, and fluorescence imaging. In addition to the different spectral and temporal multiplexing approaches, we highlight the importance of the careful design and combination of different biological, organic, and inorganic molecules and nanomaterials for an adjustable FRET donor-acceptor distance that determines the ultimate performance of the diagnostic assays and conformational sensors in their physiological environment. We conclude by sharing our vision on how progress in the development of new sensing concepts, material combinations, and instrumentation can further advance TG-FRET multiplexing and accelerate its translation into routine clinical practice and the investigation of challenging biological systems.

Assessment of cAMP-PKA Signaling in Candida glabrata by FRET-Based Biosensors.

Fluorescence or Förster resonance energy transfer (FRET)-based biosensors are used to monitor activity through molecular pathways inside the cell. Binding of secondary metabolites or enzyme-guided modification of protein targets can be assessed by quantifying the rate of energy transfer between two adequate fluorophores. The AKAR3 sensor contains a protein kinase A (PKA) phosphorylation site, which upon phosphorylation interacts with a ligand domain, bringing together FRET donor and acceptor fluorophores and allowing FRET. The EPAC2 sensor contains a cyclic adenosine monophosphate (cAMP)-binding domain. Upon binding of cAMP, donor and acceptor molecules are separated, thereby lowering energy transfer. Since the cAMP-PKA pathway is of great importance for regulation of growth, survival, and virulence in Candida species, monitoring the activity of this pathway in a time- and space-resolved manner allows for detailed investigation of potential drug targets. In this chapter, we describe how these FRET-based biosensors can be used in a practical setup.