Fluorescence Resonance Energy Transfer Sensor Research Articles

A Comprehensive Exploration of Caspase Detection Methods: From Classical Approaches to Cutting-Edge Innovations.

The activation of caspases is a crucial event and an indicator of programmed cell death, also known as apoptosis. These enzymes play a central role in cancer biology and are considered one promising target for current and future advancements in therapeutic interventions. Traditional methods of measuring caspase activity such as antibody-based methods provide fundamental insights into their biological functions, and are considered essential tools in the fields of cell and cancer biology, pharmacology and toxicology, and drug discovery. However, traditional methods, though extensively used, are now recognized as having various shortcomings. In addition, these methods fall short of providing solutions to and matching the needs of the rapid and expansive progress achieved in studying caspases. For these reasons, there has been a continuous improvement in detection methods for caspases and the network of pathways involved in their activation and downstream signaling. Over the past decade, newer methods based on cutting-edge state-of-the-art technologies have been introduced to the biomedical community. These methods enable both the temporal and spatial monitoring of the activity of caspases and their downstream substrates, and with enhanced accuracy and precision. These include fluorescent-labeled inhibitors (FLIs) for live imaging, single-cell live imaging, fluorescence resonance energy transfer (FRET) sensors, and activatable multifunctional probes for in vivo imaging. Recently, the recruitment of mass spectrometry (MS) techniques in the investigation of these enzymes expanded the repertoire of tools available for the identification and quantification of caspase substrates, cleavage products, and post-translational modifications in addition to unveiling the complex regulatory networks implicated. Collectively, these methods are enabling researchers to unravel much of the complex cellular processes involved in apoptosis, and are helping generate a clearer and comprehensive understanding of caspase-mediated proteolysis during apoptosis. Herein, we provide a comprehensive review of various assays and detection methods as they have evolved over the years, so to encourage further exploration of these enzymes, which should have direct implications for the advancement of therapeutics for cancer and other diseases.

In Situ Quantitative Imaging of Plasma Membrane Stiffness in Live Cells Using a Genetically Encoded FRET Sensor.

Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.

An ultrasensitive smartphone-assisted bicolor-ratiometric fluorescence sensing platform based on a “noise purifier” for point-of-care testing of pathogenic bacteria in food

Non-specific binding in fluorescence resonance energy transfer (FRET) remains a challenge in foodborne pathogen detection, resulting in interference of high background signals. Herein, we innovatively reported a dual-mode FRET sensor based on a “noise purifier” for the ultrasensitive quantification of Escherichia coli O157:H7 in food. An efficient FRET system was constructed with polymyxin B-modified nitrogen-sulfur co-doped graphene quantum dots (N, S-GQDs@PMB) as donors and aptamer-modified yellow carbon dots (Y-CDs@Apt) as acceptors. Magnetic multi-walled carbon nanotubes (Fe@MWCNTs) were employed as a “noise purifier” to reduce the interference of the fluorescence background. Under the background purification mode, the sensitivity of the dual-mode signals of the FRET sensor has increased by an order of magnitude. Additionally, smartphone-assisted colorimetric analysis enabled point-of-care detection of E. coli O157:H7 in real samples. The developed sensing platform based on a “noise purifier” provides a promising method for ultrasensitive on-site testing of trace pathogenic bacteria in various foodstuffs.

Local autocrine plasticity signaling in single dendritic spines by insulin-like growth factors.

The insulin superfamily of peptides is essential for homeostasis as well as neuronal plasticity, learning, and memory. Here, we show that insulin-like growth factors 1 and 2 (IGF1 and IGF2) are differentially expressed in hippocampal neurons and released in an activity-dependent manner. Using a new fluorescence resonance energy transfer sensor for IGF1 receptor (IGF1R) with two-photon fluorescence lifetime imaging, we find that the release of IGF1 triggers rapid local autocrine IGF1R activation on the same spine and more than several micrometers along the stimulated dendrite, regulating the plasticity of the activated spine in CA1 pyramidal neurons. In CA3 neurons, IGF2, instead of IGF1, is responsible for IGF1R autocrine activation and synaptic plasticity. Thus, our study demonstrates the cell type-specific roles of IGF1 and IGF2 in hippocampal plasticity and a plasticity mechanism mediated by the synthesis and autocrine signaling of IGF peptides in pyramidal neurons.

Direct phoxim sensing based on fluorescent metal-organic framework of Nu-1000 induced FRET

To control and on-site evaluate the contamination of organophosphorus pesticides, one direct sensing approach based on spontaneous fluorescence resonance energy transfer (FRET) was developed in this study. The luminescent metal-organic frameworks (LMOFs), a novel achievement of MOFs, could provide adjustable adsorption capacity and fluorescence index to scientific analysis was first employed to evaluate phoxim molecules in vegetables and fruits. Through the π-π recognition and FRET sensing between phoxim molecules and skeleton of Nu-1000 LMOFs probes, an ultra-low detection limit of 1.65 pg/L in the range of 5×10−7 to 5×10−3 mg/mL (R2=0.998) was achieved in the presence of phoxim. Such simple and accurate LMOFs- FRET-based sensing strategy could offer valuable reference for developing sensitive FRET sensors and/or on-site evaluation approaches in the areas of food quality control and food safety.

HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin.

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Genetically Encoded FRET-Based Nanosensor for Real-Time Monitoring of A549 Exosomes: Early Diagnosis of Cancer.

Exosomes contain a plethora of unique disease biomarkers involving cellular homeostasis, infection dissemination, cancer development, and cardiac diseases. Exosomes originating from cancer cells have promising biomarkers for the early detection and assessment of the therapeutic response to cancer. The exosomal epidermal growth factor receptor (EGFR) is a potential biomarker which is overexpressed in cancer; thus, the level of EGFR expression is investigated by so many methods in a liquid and solid biopsy. The optimal method for isolating pure exosomal EGFRs has not been well understood so far. Current approaches are complicated and time-consuming, therefore hampering their clinical applications. Here, we demonstrate the creation of an innovative fluorescence resonance energy transfer (FRET) sensor, named ExoSen (exosome sensor), which can be implemented to determine the concentration of exosomal EGFRs at in vitro as well as in vivo levels. In this study, a sensing element for A549 exosomes, mitogen-inducible gene 6 (MIG6), has been employed between the FRET pair ECFP and Venus. MIG6 binding to ExoSen induced a conformational change that can be monitored by a variation in the FRET ratio. Moreover, the developed sensor, expressed in bacterial, yeast, and HEK-293T cells, demonstrates an increased FRET ratio with the addition of A549 exosomes, which can quantify the A549 exosomes noninvasively. The ExoSen enables rapid detection of A549 exosomes with great sensitivity at a concentration of 3.5 × 109 particles/mL. ExoSen is stable to pH fluctuations and provides a highly accurate, real-time optical readout in cell-based experiments by using confocal microscopy.

Novel Xanomeline-Containing Bitopic Ligands of Muscarinic Acetylcholine Receptors: Design, Synthesis and FRET Investigation

In the last few years, fluorescence resonance energy transfer (FRET) receptor sensors have contributed to the understanding of GPCR ligand binding and functional activation. FRET sensors based on muscarinic acetylcholine receptors (mAChRs) have been employed to study dual-steric ligands, allowing for the detection of different kinetics and distinguishing between partial, full, and super agonism. Herein, we report the synthesis of the two series of bitopic ligands, 12-Cn and 13-Cn, and their pharmacological investigation at the M1, M2, M4, and M5 FRET-based receptor sensors. The hybrids were prepared by merging the pharmacophoric moieties of the M1/M4-preferring orthosteric agonist Xanomeline 10 and the M1-selective positive allosteric modulator 77-LH-28-1 (1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone) 11. The two pharmacophores were connected through alkylene chains of different lengths (C3, C5, C7, and C9). Analyzing the FRET responses, the tertiary amine compounds 12-C5, 12-C7, and 12-C9 evidenced a selective activation of M1 mAChRs, while the methyl tetrahydropyridinium salts 13-C5, 13-C7, and 13-C9 showed a degree of selectivity for M1 and M4 mAChRs. Moreover, whereas hybrids 12-Cn showed an almost linear response at the M1 subtype, hybrids 13-Cn evidenced a bell-shaped activation response. This different activation pattern suggests that the positive charge anchoring the compound 13-Cn to the orthosteric site ensues a degree of receptor activation depending on the linker length, which induces a graded conformational interference with the binding pocket closure. These bitopic derivatives represent novel pharmacological tools for a better understanding of ligand-receptor interactions at a molecular level.

A gene-encoded FRET fluorescent sensor designed for detecting asymmetric dimethylation levels in vitro and in living cells.

Arginine methylation is involved in many important biological processes. PRMT1 is a major arginine methyltransferase in mammalian cells and is highly conserved in eukaryotes. It catalyzes the methylation of various of substrates, including histones, and PRMT1 has been reported to be overexpressed in many cancers, indicating that it is a potential therapeutic target. No tool for efficient methylation level detection in living cells has been available to date. In this work, we designed and constructed a gene-encoded fluorescence resonance energy transfer (FRET) fluorescent sensor for detecting dimethylation levels in living cells and evaluated its functional efficiency both in vitro and in living cells. Both site-directed mutagenesis and PRMT1 inhibition experiments verified that the fluorescent sensor responded to changes in PRMT1 activity and to different PRMT1-induced methylation levels in vitro. Finally, we verified that this optimized methyl sensor responded sensitively to changes in methylation levels in living cells by overexpressing and inhibiting PRMT1, which makes it a useful tool for real-time imaging of arginine methylation. As a new tool for detecting arginine dimethylation levels in living cells, the designed FRET sensor is very important for posttranslational studies and may show a wide range of applications.

A FRET sensor for the real-time detection of long chain acyl-CoAs and synthetic ABHD5 ligands

Intracellular long-chain acyl-coenzyme As (LC-acyl-CoAs) are thought to be under tight spatial and temporal controls, yet the ability to image LC-acyl-CoAs in live cells is lacking. Here, we developed a fluorescence resonance energy transfer (FRET) sensor for LC-acyl-CoAs based on the allosterically regulated interaction between α/β hydrolase domain-containing 5 (ABHD5) and Perilipin 5. The genetically encoded sensor rapidly detects intracellular LC-acyl-CoAs generated from exogenous and endogenous fatty acids (FAs), as well as synthetic ABHD5 ligands. Stimulation of lipolysis in brown adipocytes elevated intracellular LC-acyl-CoAs in a cyclic fashion, which was eliminated by inhibiting PNPLA2 (ATGL), the major triglyceride lipase. Interestingly, inhibition of LC-acyl-CoA transport into mitochondria elevated intracellular LC-acyl-CoAs and dampened their cycling. Together, these observations reveal an intimate feedback control between LC-acyl-CoA generation from lipolysis and utilization in mitochondria. We anticipate that this sensor will be an important tool to dissect intracellular LC-acyl-CoA dynamics as well to discover novel synthetic ABHD5 ligands.

Rapid detection of pathogenic bacteria based on a universal dual-recognition FRET sensing system constructed with aptamer-quantum dots and lectin-gold nanoparticles

The threat to public health from bacterial infections has led to an urgent need to develop simpler, faster and more reliable bacterial detection methods. In this work, we developed a universal dual-recognition based sandwich fluorescence resonance energy transfer (FRET) sensor by using specific aptamer-modified quantum dots (Aptamer-QDs) as energy donor and lectin concanavalin A (Con A) modified gold nanoparticles (Con A-AuNPs) as energy acceptor to achieve rapid and sensitive detection of Escherichia coli (E. coli) within 0.5 h. In the presence of the target E. coli, the energy donor of Aptamer-QDs and acceptor of Con A-AuNPs were close to each other, causing changes of FRET signals. Based on the constructed FRET sensor, a linear detection range of from 102 cfu/mL to 2 × 108 cfu/mL with the detection limit of 45 cfu/mL for E. coli was achieved. Furthermore, the FRET sensor was applied to detect E. coli in the milk and orange juice with the detection limit of 300 cfu/mL and 200 cfu/mL, respectively and recovery rate from 83.1% to 112.5%. The strategy holds great promise in pathogenic bacteria detection due to its rapid and sensitivity.

Fabrication of poly (aspartic) acid functionalized graphene quantum dots based FRET sensor for selective and sensitive detection of MAGE-A11 antigen

In the present work, we investigated a label-free fluorescence resonance energy transfer (FRET) based poly (aspartic) acid (PAsP) functionalized graphene quantum dot (PAsP-GQDs) immunosensor for the selective and sensitive sensing of MAGE-A11 antigen.Synthesis of GQDs was accomplished with hydrothermal carbonization of Onion hull (ONI-GQDs). Synthesized PAsP-GQDs were characterized for fluorescence performance, functional compositions and morphological analysis. To form a typical FRET system, functionalized GQDs conjugated with anti-MAGE-A11 (antibody) using EDC/NHS chemistry served as an energy donor, while graphene nanosheets served as energy acceptors (quenchers). The fabricated PAsP-GQDs@M−A11−Ab immunosensor demonstrated high selectivity to MAGE-A11 with a broader detection range about of 0.05 ng mL−1 – 5 μg mL−1 (R2 = 0.9906) and a detection limit of about of 5.6 pg mL−1, with a rapid response time of 12 min. The performance of the developed immunosensor was established using real sample analysis, which showed an average recovery of 96.8 % with % RSD 0.71 indicating the high precision and reproducibility of the method. Furthermore, to implicate the post-functionalization modifications, cellular bioimaging potential and cytotoxicity studies were conducted as a comparative assessment. The present immunosensing strategies can be utilized as an analytical tool for detecting MAGE-A11 in various cancers.

A versatile cellulose nanocrystal‑carbon dots architecture: Preparation and environmental/biological applications

In this work, a versatile cellulose nanocrystal‑carbon dots (CNC-CDs) architecture is prepared and applied in environmental remediation and biological sensing. The citric acid (CA) is employed to extract CNCs and in-situ synthesize blue-emissive CDs. The carboxyl-rich surface of CNC-CDs enables the chelation of Hg2+ and fluorescence quenching of CDs, realizing the detection and adsorption of Hg2+ with a film comprised of the architecture. The limit of detection (LOD) is measured as 12.8 nM with satisfying retention efficiency. The versatility of CNC-CDs is demonstrated by the fabrication of a fluorescence resonance energy transfer (FRET) sensor using m-tetrakis(4-sulfonatophenyl)porphine (TSPP) as FRET acceptor and Fe3+ receptor. Fluorescence of the sensor is responsive to Fe3+ in a ratiometric manner, which significantly contributes to the visualized and sensitive Fe3+ detection (6.9 nM). In combination with excellent biocompatibility, the FRET sensor is capable of imaging intracellular Fe3+.

Directed evolution of the genetically encoded zinc(II) FRET sensor ZapCY1

Zinc(II) ions (Zn2+) play an essential role in living systems, with their delicate concentration balance differing among the various intracellular organelles. The spatiotemporal distribution and homeostasis of Zn2+ can be monitored through photoluminescence imaging using zinc sensors. Among such biosensors, genetically encoded fluorescent sensor proteins are attractive tools owing to their subcellular localization advantage and high biocompatibility. However, the limited fluorescent properties of these proteins, such as their insufficient quantum yield and dynamic range, restrict their practical use. In this study, we developed an expression–screening–directed evolution system and used it to improve ZapCY1, a genetically encoded fluorescence resonance energy transfer (FRET) sensor. After four rounds of directed evolution, the FRET dynamic range of the modified sensor (designated ZapTV-EH) was increased by 1.5–1.7-fold. With its enhanced signal-to-noise ratio and ability to detect a wide Zn2+ concentration range, ZapTV-EH proves to be a better visualization tool for monitoring Zn2+ at the subcellular level. Combined with the simplified subcloning and expression steps and sufficient mutant libraries, this directed evolution system may provide a more simple and efficient way to develop and optimize genetically encoded FRET sensors through high-throughput screening.

Conformational fingerprinting of allosteric modulators in metabotropic glutamate receptor 2.

Activation of G protein-coupled receptors (GPCRs) is an allosteric process. It involves conformational coupling between the orthosteric ligand binding site and the G protein binding site. Factors that bind at non-cognate ligand binding sites to alter the allosteric activation process are classified as allosteric modulators and represent a promising class of therapeutics with distinct modes of binding and action. For many receptors, how modulation of signaling is represented at the structural level is unclear. Here, we developed fluorescence resonance energy transfer (FRET) sensors to quantify receptor modulation at each of the three structural domains of metabotropic glutamate receptor 2 (mGluR2). We identified the conformational fingerprint for several allosteric modulators in live cells. This approach enabled us to derive a receptor-centric representation of allosteric modulation and to correlate structural modulation to the standard signaling modulation metrics. Single-molecule FRET analysis revealed that a NAM (egative allosteric modulator) increases the occupancy of one of the intermediate states while a positive allosteric modulator increases the occupancy of the active state. Moreover, we found that the effect of allosteric modulators on the receptor dynamics is complex and depend on the orthosteric ligand. Collectively, our findings provide a structural mechanism of allosteric modulation in mGluR2 and suggest possible strategies for design of future modulators.

Fiber-based lactate recordings with fluorescence resonance energy transfer sensors by applying an magnetic resonance-informed correction of hemodynamic artifacts.

.Significance: Fluorescence resonance energy transfer (FRET) sensors offer enormous benefits when studying neurophysiology through confocal microscopy. Yet, their use for fiber-based in vivo recordings is hampered by massive confounding effects and has therefore been scarcely reported.Aim: We aim to investigate whether in vivo fiber-based lactate recordings in the rodent brain are feasible with FRET sensors and implement a correction algorithm for the predominant hemodynamic artifact.Approach: We performed fiber-based FRET recordings of lactate (Laconic) and calcium (Twitch-2B) simultaneously with functional MRI and pharmacological MRI. MR-derived parameters were applied to correct hemodynamic artifacts. Results of FRET measurements were validated by local field potential, magnetic resonance spectroscopy, and blood analysis.Results: Hemodynamic artifacts dominated fiber-based in vivo FRET measurements with both Laconic and Twitch-2B. Our MR-based correction algorithm enabled to remove the artifacts and detect lactate and calcium changes during sensory stimulation or intravenous lactate injections.Conclusions: In vivo fiber-based lactate recordings are feasible using FRET-based sensors. However, signal corrections are required. MR-derived hemodynamic parameters can successfully be applied for artifact correction.

Single-Molecule Sensor for High-Confidence Detection of miRNA.

MicroRNAs (miRNAs) play a crucial role in regulating gene expression and have been linked to many diseases. Therefore, sensitive and accurate detection of disease-linked miRNAs is vital to the emerging revolution in early diagnosis of diseases. While the detection of miRNAs is a challenge due to their intrinsic properties such as small size, high sequence similarity among miRNAs and low abundance in biological fluids, the majority of miRNA-detection strategies involve either target/signal amplification or involve complex sensing designs. In this study, we have developed and tested a DNA-based fluorescence resonance energy transfer (FRET) sensor that enables ultrasensitive detection of a miRNA biomarker (miRNA-342-3p) expressed by triple-negative breast cancer (TNBC) cells. The sensor shows a relatively low FRET state in the absence of a target but it undergoes continuous FRET transitions between low- and high-FRET states in the presence of the target. The sensor is highly specific, has a detection limit down to low femtomolar (fM) without having to amplify the target, and has a large dynamic range (3 orders of magnitude) extending to 300 000 fM. Using this strategy, we demonstrated that the sensor allows detection of miRNA-342-3p in the miRNA-extracts from cancer cell lines and TNBC patient-derived xenografts. Given the simple-to-design hybridization-based detection, the sensing platform developed here can be used to detect a wide range of miRNAs enabling early diagnosis and screening of other genetic disorders.

Measurement of Macromolecular Crowding in Rhodobacter sphaeroides under Different Growth Conditions.

ABSTRACTThe bacterial cytoplasm is a very crowded environment, and changes in crowding are thought to have an impact on cellular processes including protein folding, molecular diffusion and complex formation. Previous studies on the effects of crowding have generally compared cellular activity after imposition of stress. In response to different light intensities, in unstressed conditions, Rhodobacter sphaeroides changes the number of 50-nm intracytoplasmic membrane (ICM) vesicles, with the number varying from a few to over a thousand per cell. In this work, the effects of crowding induced by ICM vesicles in photoheterotrophic R. sphaeroides were investigated using a fluorescence resonance energy transfer (FRET) sensor and photoactivated localization microscopy (PALM). In low light grown cells where the cytoplasm has large numbers of ICM vesicles, the FRET probe adopts a more condensed conformation, resulting in higher FRET ratio readouts compared to high light cells with fewer ICM vesicles. The apparent diffusion coefficients of different sized proteins, PAmCherry, PAmCherry-CheY6, and L1-PAmCherry, measured via PALM showed that diffusion of protein molecules >27 kDa decreased as the number of ICM vesicles increased. In low light R. sphaeroides where the crowding level is high, protein molecules were found to diffuse more slowly than in aerobic and high light cells. This suggests that some physiological activities might show different kinetics in bacterial species whose intracellular membrane organization can change with growth conditions.

FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs.

With the development of fluorescent proteins (FPs) and advanced optical microscopy techniques, Förster or fluorescence resonance energy transfer (FRET) has become a powerful tool for real-time noninvasive visualization of a variety of biological processes, including kinase activities, with high spatiotemporal resolution in living cells and organisms. FRET can be detected in appropriately configured microscopes as changes in fluorescence intensity, lifetime, and anisotropy. Here, we describe the preparation of samples expressing FP-based FRET sensors for RhoA kinase, intensity- and lifetime-based FRET imaging, and postimaging data analysis.

(Invited) Nanostructured Fluorescence Sensors Toward Point-of-Care Testing

The mechanism of fluorescence resonance energy transfer (FRET) was studied after inorganic nanomaterials have replaced organic dyes as energy donors and acceptors. Quantum dots and graphene oxide have been used for construction of FRET sensors. Next the fluorophores are incorporated to paper-based lateral flow assays toward point-of-care testing. The fluorescence sensors have sued for detection of heavy metal ions and protein biomarkers.