FRET-based Biosensor Research Articles

Novel FRET-Based Biosensors for Real-Time Monitoring of Estrogen Receptor Dimerization and Translocation Dynamics in Living Cells.

Estrogen receptors (ERs), comprising ER α and ER β, are crucial for regulating cell growth and differentiation via homo- and hetero-dimer formation. However, accurately detecting ER dimerization with precise spatiotemporal resolution remains a significant challenge. In this study, fluorescence resonance energy transfer-based biosensors to monitor ER dynamics in real-time, are developed and optimized.This approach involves comprehensive structural analysis, linker comparison, and the selection of optimal fluorescent protein pairs, resulting in three distinct biosensors capable of detecting all ER homo- and hetero-dimerizations within the nucleus. These biosensors are utilized to reveal interactions between ER α/β and calmodulin during dimer formation.Furthermore, by leveraging the ligand-binding domain (LBD) of ER β, ER ββ LBD biosensor is designed for real-time analysis of ER ββ homodimerization in the cytoplasm, enhancing the ability to screen ER dimerization-related drugs.Additionally, we developed a novel ER ββ translocation biosensor, which enables real-time observation of ER ββ translocation to the nucleus-a capability previously unavailable, is developed. This spatiotemporal analysis demonstrates the relevance of ER translocation in response to drug binding efficacy and extracellular matrix changes. Our biosensors offertransformative tools for studying ER dynamics, providing valuable insights for drug screening and the investigation of ER-related cellular processes.

Novel FRET-based Immunological Synapse Biosensor for the Prediction of Chimeric Antigen Receptor-T Cell Function.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer treatment. CARs are activated at the immunological synapse (IS) when their single-chain variable fragment (scFv) domain engages with an antigen, allowing them to directly eliminate cancer cells. Here, an innovative IS biosensor based on fluorescence resonance energy transfer (FRET) for the real-time assessment of CAR-IS architecture and signaling competence is presented. Using this biosensor, scFv variants for mesothelin-targeting CARs and identified as a novel scFv with enhanced CAR-T cell functionality despite its lower affinity than the original screened. The original CAR promoted internalization and trogocytosis, disrupting stable IS formation and impairing functionality are further observed. These findings emphasize the importance of enhancing IS quality rather than maximizing scFv affinity for superior CAR-T cell responses. Therefore, the FRET-based IS biosensor is a powerful tool for predicting CAR-T cell function, enabling the efficient engineering of next-generation CARs with enhanced antitumor potency.

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.

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.

Alteration of gene expression and protein solubility of the PI 5-phosphatase SHIP2 are correlated with Alzheimer’s disease pathology progression

A recent large genome-wide association study has identified EGFR (encoding the epidermal growth factor EGFR) as a new genetic risk factor for late-onset AD. SHIP2, encoded by INPPL1, is taking part in the signalling and interactome of several growth factor receptors, such as the EGFR. While INPPL1 has been identified as one of the most significant genes whose RNA expression correlates with cognitive decline, the potential alteration of SHIP2 expression and localization during the progression of AD remains largely unknown. Here we report that gene expression of both EGFR and INPPL1 was upregulated in AD brains. SHIP2 immunoreactivity was predominantly detected in plaque-associated astrocytes and dystrophic neurites and its increase was correlated with amyloid load in the brain of human AD and of 5xFAD transgenic mouse model of AD. While mRNA of INPPL1 was increased in AD, SHIP2 protein undergoes a significant solubility change being depleted from the soluble fraction of AD brain homogenates and co-enriched with EGFR in the insoluble fraction. Using FRET-based flow cytometry biosensor assay for tau-tau interaction, overexpression of SHIP2 significantly increased the FRET signal while siRNA-mediated downexpression of SHIP2 significantly decreased FRET signal. Genetic association analyses suggest that some variants in INPPL1 locus are associated with the level of CSF pTau. Our data support the hypothesis that SHIP2 is an intermediate key player of EGFR and AD pathology linking amyloid and tau pathologies in human AD.

Mapping the distribution of tension across paxillin upon shear stress with FRET-based biosensor

Paxillin communicates with multiple signalling molecules in focal adhesions (FAs) and participates in the intracellular force transmission upon shear stress. Thus, paxillin is likely to contribute to establishing the shear stress induced-cell polarity. However, it is still unclear whether the tension across FAs proteins can direct the polarity establishments by providing spatial features, due to a lack of efficient manners. This work proposes a visualization approach containing a DNA-encoded biosensor and fluorescent image processing algorithm to collect the spatiotemporal features of tension across paxillin. The results indicate that the tension across paxillin shows polarity between the upstream and downstream zones of the cell along the direction of shear stress, which was mediated by the membrane fluidity and integrity of the cytoskeleton. It demonstrates that the spatial information from the upper surface of cells upon shear stress can be transmitted to the interior of FAs on the basal layer by the architecture consisting of plasma membrane and cytoskeleton. Paxillin is a potential participant in activating cell polarity by providing a spatial mechanical guide to related signaling molecules upon shear stress.Graphical

Unravelling molecular dynamics in living cells: Fluorescent protein biosensors for cell biology.

Genetically encoded, fluorescent protein (FP)-based Förster resonance energy transfer (FRET) biosensors are microscopy imaging tools tailored for the precise monitoring and detection of molecular dynamics within subcellular microenvironments. They are characterised by their ability to provide an outstanding combination of spatial and temporal resolutions in live-cell microscopy. In this review, we begin by tracing back on the historical development of genetically encoded FP labelling for detection in live cells, which lead us to the development of early biosensors and finally to the engineering of single-chain FRET-based biosensors that have become the state-of-the-art today. Ultimately, this review delves into the fundamental principles of FRET and the design strategies underpinning FRET-based biosensors, discusses their diverse applications and addresses the distinct challenges associated with their implementation. We place particular emphasis on single-chain FRET biosensors for the Rho family of guanosine triphosphate hydrolases (GTPases), pointing to their historical role in driving our understanding of the molecular dynamics of this important class of signalling proteins and revealing the intricate relationships and regulatory mechanisms that comprise Rho GTPase biology in living cells.

A FRET based ultrasensitive fluorescent aptasensor for 6′-sialyllactose detection

As a kind of human milk oligosaccharide, 6′-sialyllactose (6′-SL) plays an important role in promoting infant brain development and improving infant immunity. The content of 6′-SL in infant formula milk powder is thus one of the important nutritional indexes. Since the lacking of efficient and rapid detection methods for 6′-SL, it is of great significance to develop specific recognition elements and establish fast and sensitive detection methods for 6′-SL. Herein, using 6′-SL specific aptamer as the recognition element, catalytic hairpin assembly as the signal amplification technology and quantum dots as the signal label, a fluorescence biosensor based on fluorescence resonance energy transfer (FRET) was constructed for ultra-sensitive detection of 6′-SL. The detection limit of this FRET-based fluorescent biosensor is 0.3 nM, and it has some outstanding characteristics such as high signal-to-noise ratio, low time-consuming, simplicity and high efficiency in the actual sample detection. Therefore, it has broad application prospect in 6′-SL detection.

NaYF4:Yb/Ho upconversion nanoprobe incorporated gold nanoparticle (AuNP) based FRET immunosensor for the "turn-on" detection of cardiac troponin I.

Cardiac troponin I (cTnI) is a significant biomarker for acute heart attack. Hence, fast, economical, easy and real time monitoring of cardiac troponin I (cTnI) is of great importance in diagnosis and prognosis of heart failure in the healthcare domain. In this work, an immunoassay based on NaYF4:Yb/Ho based photon-upconversion nanoparticle (UCNP) with narrow emission peaks at 540 nm and 655 nm respectively, is synthesized. Then, it is encapsulated with amino functionalized silica using 3-aminopropyltriethoxysilane (APTES) to form APTES@SiO2-NaYF4:Yb/Ho UCNPs. When AuNPs is added to this system, the fluorescence is quenched by the electrostatic interaction with APTES@SiO2-NaYF4:Yb/Ho UCNPs, thereby exhibiting a FRET-based biosensor. When the cTnI antigen is introduced into the developed probe, an antibody-antigen complex is formed on the surface of the UCNPs resulting in fluorescence recovery. The developed sensor shows a linear response towards cTnI in the range from 0.1693 ng mL-1 to 1.9 ng mL-1 with a low limit of detection (LOD) of 5.5 × 10-2 ng mL-1. The probe exhibits adequate selectivity and sensitivity when compared with coexisting cardiac biomarkers, biomolecules and in real human serum samples.

Single-residue mutation in protein kinase C toggles between cancer and neurodegeneration.

Conventional protein kinase C (cPKC) isozymes tune the signaling output of cells, with loss-of-function somatic mutations associated with cancer and gain-of-function germline mutations identified in neurodegeneration. PKC with impaired autoinhibition is removed from the cell by quality-control mechanisms to prevent the accumulation of aberrantly active enzyme. Here, we examine how a highly conserved residue in the C1A domain of cPKC isozymes permits quality-control degradation when mutated to histidine in cancer (PKCβ-R42H) and blocks down-regulation when mutated to proline in the neurodegenerative disease spinocerebellar ataxia (PKCγ-R41P). Using FRET-based biosensors, we determined that mutation of R42 to any residue, including lysine, resulted in reduced autoinhibition as indicated by higher basal activity and faster agonist-induced plasma membrane translocation. R42 is predicted to form a stabilizing salt bridge with E655 in the C-tail and mutation of E655, but not neighboring E657, also reduced autoinhibition. Western blot analysis revealed that whereas R42H had reduced stability, the R42P mutant was stable and insensitive to activator-induced ubiquitination and down-regulation, an effect previously observed by deletion of the entire C1A domain. Molecular dynamics (MD) simulations and analysis of stable regions of the domain using local spatial pattern (LSP) alignment suggested that P42 interacts with Q66 to impair mobility and conformation of one of the ligand-binding loops. Additional mutation of Q66 to the smaller asparagine (R42P/Q66N), to remove conformational constraints, restored degradation sensitivity. Our results unveil how disease-associated mutations of the same residue in the C1A domain can toggle between gain- or loss-of-function of PKC.

Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status.

Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b' and a' domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b' and a' domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

Specific detection of tau seeding activity in Alzheimer’s disease using rationally designed biosensor cells

BackgroundThe prion-like propagation of tau in neurodegenerative disorders implies that misfolded pathological tau can recruit the normal protein and template its aggregation. Here, we report the methods for the development of sensitive biosensor cell lines for the detection of tau seeding activity.ResultsWe performed the rational design of novel tau probes based on the current structural knowledge of pathological tau aggregates in Alzheimer’s disease. We generated Förster resonance energy transfer (FRET)-based biosensor stable cell lines and characterized their sensitivity, specificity, and overall ability to detect bioactive tau in human samples. As compared to the reference biosensor line, the optimized probe design resulted in an increased efficiency in the detection of tau seeding. The increased sensitivity allowed for the detection of lower amount of tau seeding competency in human brain samples, while preserving specificity for tau seeds found in Alzheimer’s disease.ConclusionsThis next generation of FRET-based biosensor cells is a novel tool to study tau seeding activity in Alzheimer’s disease human samples, especially in samples with low levels of seeding activity, which may help studying early tau-related pathological events.

Specific oligonucleotide-modified QDs as a powerful platform for SARS Cov-2 virus detection by FRET-based biosensors

FRET-based nano-bioconjugate sensors (nano-biosensors) could be examined in coronavirus disease (COVID-19) diagnosis. In this work, a nano-biosensor for SARS Cov-2 virus detection was established based on fluorescence resonance energy transfer (FRET) between oligonucleotide-modified CdTe/ZnS quantum dots (QDs) as a donor and Cy3 as an acceptor. UV–visible spectrophotometry, FT-IR spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to characterize the chemical and physical structures of the nano-biosensor. Under optimized conditions, the detection limit of complementary target DNA (ctDNA) was 2.19 × 10−9 µM. The linear equation and regression were y = 181.8x + 1 and R2 = 0.9762. In addition, capture DNA/QDs ratios were modified under different conditions. Finally, under optimal conditions, the feasibility of the current nano-biosensor evaluated for monitoring SARS Cov-2 in real samples.

Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Spatiotemporal Coordination of Rac1 and Cdc42 at the Whole Cell Level during Cell Ruffling.

Rho-GTPases are central regulators within a complex signaling network that controls cytoskeletal organization and cell movement. The network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, along with their numerous effectors that provide mutual regulation through feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling, using a simulation model that couples GTPase signaling with cell morphodynamics and captures the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of time-lapsed recordings of cell dynamics and GTPase activity. Our data-driven modeling approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data.

Development of an Efficient FRET-Based Ratiometric Uranium Biosensor.

The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist for this. Our study aims to fill this gap by developing a genetically encoded FRET-based ratiometric uranium biosensor. This biosensor was constructed by grafting two fluorescent proteins to both ends of calmodulin, a protein that binds four calcium ions. By modifying the metal-binding sites and the fluorescent proteins, several versions of the biosensor were generated and characterized in vitro. The best combination results in a biosensor that is affine and selective for uranium compared to metals such as calcium or other environmental compounds (sodium, magnesium, chlorine). It has a good dynamic range and should be robust to environmental conditions. In addition, its detection limit is below the uranium limit concentration in drinking water defined by the World Health Organization. This genetically encoded biosensor is a promising tool to develop a uranium whole-cell biosensor. This would make it possible to monitor the bioavailable fraction of uranium in the environment, even in calcium-rich waters.

PO-02-154 CHRONIC SYMPATHETIC REMODELING IN HEART FAILURE IMPACTS WHOLE-HEART CAMP SIGNALING AND ARRHYTHMOGENESIS

Elevated sympathetic tone and b-AR desensitization are characteristics associated with heart failure (HF) and are contributors to the initiation of arrhythmia. Studies have shown that altered b-AR responsiveness in HF leads to altered compartmentalization and imparied sub-cellular cAMP signaling. Yet, if cAMP signaling is heterogeneously remodeled throughout the intact heart, and how this may contribute to arrhythmia is unknown. To assess whole-heart cAMP signaling and functional responses to adrenergic activation in HF. HF was induced via transverse aortic constriction in cardiac specific CAMPER reporter mice, expressing a FRET-based cAMP biosensor. HF was confirmed by echocardiography and hearts were Langendorff-perfused for simultaneous cAMP and Ca2+ imaging on an integrated whole-heart optical mapping FRET imaging system. Sham mice underwent the same procedure but without constriction of the aorta. Baseline cAMP levels, assessed as the FRET ratio, were uniform throughout the ventricles of male and female Sham hearts. Conversely, in HF, baseline levels of cAMP were higher in the apex vs. the base, particularly in female hearts. Low-dose b1-AR stimulation (100nM NE) led to greater cAMP response in male HF hearts vs. Sham, this response was uniform across the male heart. In female HF hearts, there was a trend toward a greater cAMP response in basal, but not apical regions. At high dose b1-AR stimulation (500nM NE), male and female HF hearts had reduced cAMP responses compared to Sham, mainly in the female apex, where dose-dependent responses were lost. Similar apex-base differences were evident following b1 and b2-AR stimulation (100-500nM Epi), with lower cAMP responses in the female apex in HF. Uniform cAMP responses in male and female Sham hearts were accompanied by uniform shortening of the Ca2+ transient duration (CaTD) in response to low dose b1-AR stimulation. Following HF, baseline CaTD was shorter than in Sham hearts and the response to b-AR stimulation was reduced. Moreover, CaTD responses were no longer spatially uniform, where apical CaTD shortened beyond that of basal regions in HF. At high dose b1-AR stimulation, ventricular arrhythmia was observed in 8/10 HF hearts, vs 0/4 Sham hearts. Using whole-heart cAMP and Ca2+ imaging, we have shown cAMP activity and responses became spatially heterogeneous in HF, mainly in female hearts. Heterogeneity in cAMP activity was associated with heterogenous Ca2+ responses and ventricular arrhythmia.

PO-455617-7 CHRONIC SYMPATHETIC REMODELING IN HEART FAILURE IMPACTS WHOLE-HEART CAMP SIGNALING AND ARRHYTHMOGENESIS.

Elevated sympathetic tone and b-AR desensitization are characteristics associated with heart failure (HF) and are contributors to the initiation of arrhythmia. Studies have shown that altered b-AR responsiveness in HF leads to altered compartmentalization and imparied sub-cellular cAMP signaling. Yet, if cAMP signaling is heterogeneously remodeled throughout the intact heart, and how this may contribute to arrhythmia is unknown. To assess whole-heart cAMP signaling and functional responses to adrenergic activation in HF. HF was induced via transverse aortic constriction in cardiac specific CAMPER reporter mice, expressing a FRET-based cAMP biosensor. HF was confirmed by echocardiography and hearts were Langendorff-perfused for simultaneous cAMP and Ca2+ imaging on an integrated whole-heart optical mapping FRET imaging system. Sham mice underwent the same procedure but without constriction of the aorta. Baseline cAMP levels, assessed as the FRET ratio, were uniform throughout the ventricles of male and female Sham hearts. Conversely, in HF, baseline levels of cAMP were higher in the apex vs. the base, particularly in female hearts. Low-dose b1-AR stimulation (100nM NE) led to greater cAMP response in male HF hearts vs. Sham, this response was uniform across the male heart. In female HF hearts, there was a trend toward a greater cAMP response in basal, but not apical regions. At high dose b1-AR stimulation (500nM NE), male and female HF hearts had reduced cAMP responses compared to Sham, mainly in the female apex, where dose-dependent responses were lost. Similar apex-base differences were evident following b1 and b2-AR stimulation (100-500nM Epi), with lower cAMP responses in the female apex in HF. Uniform cAMP responses in male and female Sham hearts were accompanied by uniform shortening of the Ca2+ transient duration (CaTD) in response to low dose b1-AR stimulation. Following HF, baseline CaTD was shorter than in Sham hearts and the response to b-AR stimulation was reduced. Moreover, CaTD responses were no longer spatially uniform, where apical CaTD shortened beyond that of basal regions in HF. At high dose b1-AR stimulation, ventricular arrhythmia was observed in 8/10 HF hearts, vs 0/4 Sham hearts. Using whole-heart cAMP and Ca2+ imaging, we have shown cAMP activity and responses became spatially heterogeneous in HF, mainly in female hearts. Heterogeneity in cAMP activity was associated with heterogenous Ca2+ responses and ventricular arrhythmia.

FRET Based Biosensor: Principle Applications Recent Advances and Challenges.

Förster resonance energy transfer (FRET)-based biosensors are being fabricated for specific detection of biomolecules or changes in the microenvironment. FRET is a non-radiative transfer of energy from an excited donor fluorophore molecule to a nearby acceptor fluorophore molecule. In a FRET-based biosensor, the donor and acceptor molecules are typically fluorescent proteins or fluorescent nanomaterials such as quantum dots (QDs) or small molecules that are engineered to be in close proximity to each other. When the biomolecule of interest is present, it can cause a change in the distance between the donor and acceptor, leading to a change in the efficiency of FRET and a corresponding change in the fluorescence intensity of the acceptor. This change in fluorescence can be used to detect and quantify the biomolecule of interest. FRET-based biosensors have a wide range of applications, including in the fields of biochemistry, cell biology, and drug discovery. This review article provides a substantial approach on the FRET-based biosensor, principle, applications such as point-of-need diagnosis, wearable, single molecular FRET (smFRET), hard water, ions, pH, tissue-based sensors, immunosensors, and aptasensor. Recent advances such as artificial intelligence (AI) and Internet of Things (IoT) are used for this type of sensor and challenges.

Nimesulide, a COX-2 inhibitor, sensitizes pancreatic cancer cells to TRAIL-induced apoptosis by promoting DR5 clustering †

ABSTRACT Nimesulide is a nonsteroidal anti-inflammatory drug and a COX-2 inhibitor with antitumor and antiproliferative activities that induces apoptosis in oral, esophagus, breast, and pancreatic cancer cells. Despite being removed from the market due to hepatotoxicity, nimesulide is still an important research tool being used to develop new anticancer drugs. Multiple studies have been done to modify the nimesulide skeleton to develop more potent anticancer agents and related compounds are promising scaffolds for future development. As such, establishing a mechanism of action for nimesulide remains an important part of realizing its potential. Here, we show that nimesulide enhances TRAIL-induced apoptosis in resistant pancreatic cancer cells by promoting clustering of DR5 in the plasma membrane. In this way, nimesulide acts like a related compound, DuP-697, which sensitizes TRAIL-resistant colon cancer cells in a similar manner. Our approach applies a time-resolved FRET-based biosensor that monitors DR5 clustering and conformational states in the plasma membrane. We show that this tool can be used for future high-throughput screens to identify novel, nontoxic small molecule scaffolds to overcome TRAIL resistance in cancer cells.