Fluorescence Resonance Energy Transfer Pair Research Articles

Accurate and highly sensitive detection of Alzheimer's disease-related extracellular vesicles via förster resonance energy transfer

Alzheimer's disease (AD) is the most common neurodegenerative disease in the world and poses a huge challenge to global healthcare. Early and accurate detection of amyloid-β (1-42) (Aβ42), a key biomarker of AD, is crucial for effective diagnosis and intervention of AD. Specific or overexpressed proteins on extracellular vesicles (EVs) describe a close correlation with the occurrence and development of diseases. EVs are a very promising non-invasive biomarker for the diagnosis of AD and other diseases. As a sensitive, simple and rapid analytical method, fluorescence resonance energy transfer (FRET) has been widely applied in the detection of EVs. Herein, we developed a dual labelling strategy for simultaneously detecting EV membrane proteins of Aβ42 and CD63 based on FRET pair consisting of Au nanoclusters (AuNCs) and polydopamine nanospheres (PDANSs). The constructed nanoprobe, termed EVMPFAP assay, could specifically measure the Aβ42 and CD63 on EVs with excellent sensitivity, high specificity and satisfactory accuracy. The limit of detection of EVMPFAP assay was 1.4 × 103 particles mL−1 and the linear range was from 104 to 108 particles mL−1. EVMPFAP assay was successfully used to analyze plasma EVs to distinguish AD and healthy mice. We expect that EVMPFAP assay can be routinely applied for early diagnosis and development-monitoring of AD, thus facilitating the fight against AD.

Sensing of Gunshot Residue components from real sample using Fluorescence Resonance Energy Transfer

The present work represents a Fluorescence Resonance Energy Transfer (FRET) based sensing method for detecting Gunshot Residue (GSR) components. Two laser dyes Acf and RhB have been used as donor and acceptor respectively in the FRET pair. The real sample was collected after test firing in a forensic science laboratory. On the other hand, a standard GSR solution has been prepared in the laboratory. For the preparation of standard GSR solutions, we used the water solutions of the salts BaCl2, SbCl3, and Pb(NO3)2. The FRET efficiency was measured between Acf and RhB to sense the presence of GSR components (Pb+2, Ba+2, and Sb+3) in both real sample and standard solution by mixing the salts in aqueous solution. It has been observed that the FRET efficiency systematically decreases in the presence of GSR components. To amplify the FRET efficiency of the dye pair, inorganic clay dispersion (laponite) was used. The enhancement in FRET efficiency represents a better sensitivity of the proposed sensor. The current sensor is useful for the quantification of concentrations of the GSR components in a real sample.

Single-Molecule FRET-Based Multiplexed Detection.

Single-molecule multiplexed detection is a high-promise toolkit for the expanding field ofbiosensing and molecular diagnostics. Among many single-molecule techniques available today for biomarker sensing including fluorescence, force, electrochemical, spectroscopic, barcoding, and other techniques, fluorescence-based approaches are arguably the most widely used methods due to their high sensitivity, selectivity, and readily available fluorophore-labeling schemes for a wide variety of biomolecules. However, multiplexed imaging using fluorescence techniques has proven to be challenging due to the sophisticated labeling schemes often requiring multiple FRET (fluorescence resonance energy transfer) pairs and/or excitation sources, which lead to overlapping signals and complicate data analysis. Here, we describe a single-molecule FRET method that enables multiplexed analysis while still using only one FRET pair, and thus the described approach is a significant step forward from conventional FRET methods.

Carbon Dot‐Based Fluorescence Resonance Energy Transfer (FRET) Systems for Biomedical, Sensing, and Imaging Applications

AbstractCarbon dots (CDs) emerge as a potential group of photo‐luminescent nano‐materials due to their excellent optical, electrical, and chemical properties, as well as their competence in a wide range of environmental applications. CDs have unique and appealing properties such as excellent stability, low toxicity, water solubility, and derivability. When coupled with CDs, fluorescence resonance energy transfer (FRET) results in the development of highly sensitive ratiometric fluorescence sensor probes with potential applications in bio‐imaging, metal sensing, membrane dynamics, and environmental sensing. In this review, the progress and recent developments in CDs based FRET systems utilized for various environmental applications are conferred. An in‐depth description is provided regarding the numerous donor/acceptor systems which when integrated with CDs generate efficient FRET systems. The review enables researchers to identify and develop specific systems which can be utilized to generate a FRET pair with potential physico–chemical properties that aid the development of the same for various applications.

Single-Molecule FRET X.

Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon that has been repurposed as a biophysical tool to measure nanometer distances. With FRET by DNA eXchange, or FRET X, many points of interest (POIs) in a single object can be probed, overcoming a major limitation of conventional single-molecule FRET. In FRET X, short fluorescently labeled DNA imager strands specifically and transiently bind their complementary docking strands on a target molecule, such that at most a single FRET pair is formed at each point in time and multiple POIs on a single molecule can be readily probed. Here, we describe the sample preparation, image acquisition, and data analysis for structural analysis of DNA nanostructures with FRET X.

Multifunctional Nanoprobe Based on Fluorescence Resonance Energy Transfer for Furin Detection and Drug Delivery.

Triple-negative breast cancer is particularly difficult to treat because of its high degree of malignancy and poor prognosis. A fluorescence resonance energy transfer (FRET) nanoplatform plays a very important role in disease diagnosis and treatment due to its unique detection performance. Combining the properties of agglomeration-induced emission fluorophore and FRET pair, a FRET nanoprobe (HMSN/DOX/RVRR/PAMAM/TPE) induced by specific cleavage was designed. First, hollow mesoporous silica nanoparticles (HMSNs) were used as drug carriers to load doxorubicin (DOX). HMSN nanopores were coated with the RVRR peptide. Then, polyamylamine/phenylethane (PAMAM/TPE) was combined in the outermost layer. When Furin cut off the RVRR peptide, DOX was released and adhered to PAMAM/TPE. Finally, the TPE/DOX FRET pair was constituted. The overexpression of Furin in the triple-negative breast cancer cell line (MDA-MB-468 cell) can be quantitatively detected by FRET signal generation, so as to monitor cell physiology. In conclusion, the HMSN/DOX/RVRR/PAMAM/TPE nanoprobes were designed to provide a new idea for the quantitative detection of Furin and drug delivery, which is conducive to the early diagnosis and treatment of triple-negative breast cancer.

Engineering highly efficient NIR-II FRET platform for Background-Free homogeneous detection of SARS-CoV-2 neutralizing antibodies in whole blood

Förster or fluorescence resonance energy transfer (FRET) enables to probe biomolecular interactions, thus playing a vital role in bioassays. However, conventional FRET platforms suffer from limited sensitivity due to the low FRET efficiency and poor anti-interference of existing FRET pairs. Here we report a NIR-II (1000–1700 nm) FRET platform with extremely high FRET efficiency and exceptional anti-interference capability. This NIR-II FRET platform is established based on a pair of lanthanides downshifting nanoparticles (DSNPs) by employing Nd3+ doped DSNPs as an energy donor and Yb3+ doped DSNPs as an energy acceptor. The maximum FRET efficiency of this well-engineered NIR-II FRET platform reaches up to 92.2%, which is much higher than most commonly used ones. Owing to the all-NIR advantage (λex = 808 nm, λem = 1064 nm), this highly efficient NIR-II FRET platform exhibits extraordinary anti-interference in whole blood, and thus enabling background-free homogeneous detection of SARS-CoV-2 neutralizing antibodies in clinical whole blood sample with high sensitivity (limit of detection = 0.5 μg/mL) and specificity. This work opens up new opportunities for realizing highly sensitive detection of various biomarkers in biological samples with severe background interference.

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.

DNA Origami Curvature Sensors for Nanoparticle and Vesicle Size Determination with Single-Molecule FRET Readout.

Particle size is an important characteristic of materials with a direct effect on their physicochemical features. Besides nanoparticles, particle size and surface curvature are particularly important in the world of lipids and cellular membranes as the cell membrane undergoes conformational changes in many biological processes which leads to diverging local curvature values. On account of that, it is important to develop cost-effective, rapid and sufficiently precise systems that can measure the surface curvature on the nanoscale that can be translated to size for spherical particles. As an alternative approach for particle characterization, we present flexible DNA nanodevices that can adapt to the curvature of the structure they are bound to. The curvature sensors use Fluorescence Resonance Energy Transfer (FRET) as the transduction mechanism on the single-molecule level. The curvature sensors consist of segmented DNA origami structures connected via flexible DNA linkers incorporating a FRET pair. The activity of the sensors was first demonstrated with defined binding to different DNA origami geometries used as templates. Then the DNA origami curvature sensors were applied to measure spherical silica beads having different size, and subsequently on lipid vesicles. With the designed sensors, we could reliably distinguish different sized nanoparticles within a size range of 50-300 nm as well as the bending angle range of 50-180°. This study helps with the development of more advanced modular-curvature sensing devices that are capable of determining the sizes of nanoparticles and biological complexes.

A toolkit of customised protein sensors for interrogating mechanical forces in the cell.

Mechanical forces play a role in a wide variety of cellular processes, such as activation of ion channels upon mechanical stimulation, force-bearing proteins in the muscle and extracellular matrix, and spindle tension in chromosome segregation. Specific proteins must, therefore, be capable of sensing mechanical signals and giving biological outputs. The understanding of mechanical stimuli at the molecular level has been limited to date, though, due to the lack of tools that can accurately measure low forces in vivo. Moreover, these force sensors must be customised for each specific use, to cover the appropriate force regime. Here we report a toolkit for measuring different ranges of forces in the cell, made possible by the striking spring-like properties of the tandem-repeat protein class and their exceptional amenability to rational design. We design a panel of Fluorescence Resonance Energy Transfer (FRET)-based tension sensors using consensus tetratricopeptide (CTPR) proteins as the mechanosensitive linkers. These are flanked by the fluorescent protein mCherry and contain an engineered a tetracysteine motif in the protein sequence, which then binds tightly to the fluorescein arsenical hairpin binder dye (FlAsH), resulting in a FRET pair. Optical tweezers experiments on the CTPR linkers show that the proteins respond to physiological forces in a highly distinctive manner. We dissect the mechanics of the proteins by mutationally creating and then characterising a series of variants with systematically modified properties, to build create a toolbox of customisable, calibrated force sensors. Our toolkit is capable of probing the varied mechanotransduction processes within the cell beyond what is currently possible.

Multiplexed smFRET Nucleic Acid Sensing Using DNA Nanotweezers

The multiplexed detection of disease biomarkers is part of an ongoing effort toward improving the quality of diagnostic testing, reducing the cost of analysis, and accelerating the treatment processes. Although significant efforts have been made to develop more sensitive and rapid multiplexed screening methods, such as microarrays and electrochemical sensors, their limitations include their intricate sensing designs and semi-quantitative detection capabilities. Alternatively, fluorescence resonance energy transfer (FRET)-based single-molecule counting offers great potential for both the sensitive and quantitative detection of various biomarkers. However, current FRET-based multiplexed sensing typically requires the use of multiple excitation sources and/or FRET pairs, which complicates labeling schemes and the post-analysis of data. We present a nanotweezer (NT)-based sensing strategy that employs a single FRET pair and is capable of detecting multiple targets. Using DNA mimics of miRNA biomarkers specific to triple-negative breast cancer (TNBC), we demonstrated that the developed sensors are sensitive down to the low picomolar range (≤10 pM) and can discriminate between targets with a single-base mismatch. These simple hybridization-based sensors hold great promise for the sensitive detection of a wider spectrum of nucleic acid biomarkers.

Review of 2D MnO2 Nanosheets as FRET-Based Nanodot Fluorescence Quenchers in Chemosensing Applications

In the past decade, a variety of MnO2 nanostructures have been used in chemosensing applications because of their unique redox properties, structural diversity, and broad absorption band, which makes them good energy acceptors. The most common strategy in Förster (or fluorescence) resonance energy transfer (FRET)-based chemosensing assays is to use low-dimensional fluorescent dots like carbon-based dots, metal dots, and even fluorescent dyes to form donor–acceptor pairs with 2D MnO2 nanosheets (NSs). This review is a collection of the current library on fluorescent nanodots and MnO2 NSs-based FRET conjugates as sensing platforms with a focus on highlighting the strategies involved in the detection of various analytes. Commonly, an analyte-triggered redox reaction results in the decomposition of MnO2 NSs, leading to the release of fluorescent nanodots in solution for the turn-on detection of various reducing species. We intend to elaborately discuss the systematic upgradation of detection strategies involving fluorescent dots@2D MnO2 NSs. Recently, a cascade of redox reactions was judiciously employed to target several bioanalytes involving a fluorescent dots@MnO2 NSs FRET conjugate in a selective and sensitive manner. These sensing assays are categorized by the use of different fluorescent nanodots [carbon dots (CDs) and graphene quantum dots (GQDs), polymer dots, metal dots, etc.] with 2D MnO2 NSs as FRET pairs in different sensing applications. Further, the analytes were categorized as reducing species, oxidants, enzymes, microbes, etc. The focus was paid to the sensing efficiency of the analytical tool. As a part of the study, a brief account of the synthesis of nanodots and MnO2 NSs, their properties, and the FRET mechanism is also included. In a few cases, the sensing mechanism follows an inner filter effect without a prerequisite of the distance between the fluorophore and quencher units in the sensing of analytes, which are also covered in this collection. The available literature indicates a bright future for these FRET-based contemporary sensing strategies as environmentally benign, cost-effective, label-free techniques, which are discussed in the end.

PyFREC 2.0: Software for excitation energy transfer modeling.

Excitation energy transfer is a ubiquitous process of fundamental importance for understanding natural phenomena, such as photosynthesis, as well as advancing technologies ranging from photovoltaics to development of photosensitizers and fluorescent probes used to explore molecular interactions inside living cells. The current version of PyFREC 2.0 is an advancement of the previously reported software (D. Kosenkov, J. Comput. Chem. 2016, 37, 1847-1854). The current update is primarily focused on providing a computational tool based on Förster theory for bridging a gap between theoretically calculated molecular properties (e.g., electronic couplings, orientation factors, etc.) and experimentally measured emission and absorption spectra of molecules. The software is aimed to facilitate deeper understanding of photochemical mechanisms of fluorescence resonance energy transfer (FRET) in donor-acceptor pairs. Specific updates of the software include implementations of overlap integrals between donor emission and acceptor absorption spectra of FRET pairs, estimation of Strickler-Berg fluorescence lifetimes, calculation of Förster radii, energy transfer efficiency, and radiation zones that, in particular, determine applicability of the Förster theory.

A ratiometric fluorescent probe based on FRET for selective monitoring of tungsten in living cells

Tungsten is considered a critical metal due to its unique properties and economic importance. Recent research has linked high tungsten concentrations to pulmonary diseases and increased cancer risk. Tungsten is on the list of critical metals at high risk maintained by the European Union. We have developed a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor for the first time to determine tungsten ion efflux mechanisms and measure its concentration and distribution within living cells in real-time. For the construction of the nanosensor, TupA (tungsten binding protein), a highly selective protein, was sandwiched between ECFP and Venus, a FRET pair. SENTUN (SENsor for TUNgsten) was the name given to the newly developed nanosensor. SENTUN was highly specific and selective for tungsten and stable at physiological pH levels. We also created three mutants: SENTUN-118D, SENTUN-118K, and SENTUN-178W, with SENTUN-118K exhibiting the highest affinity for tungsten and its comprehensive range detection. The novelty of this sensor is that it is genetically encoded and can be used for real-time measurement of tungsten levels in living cells.

Real time optical detection of gold in living cells through genetically-encoded probe.

To study the efflux of gold (Au) in living cells, a genetically encoded fluorescence resonance energy transfer (FRET)-based sensor has been developed. The gold-sensing domain GolB from Salmonella typhimurium has been fused to the N- and C-termini of the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus respectively. In living cells, this probe is highly selective and sensitive to gold and it can withstand changes in variable pH ranges. GolSeN-25, the most efficient sensor variant, binds gold with an affinity (Kd) of 0.3 × 10−6 M, covering gold concentrations of nM to μM, and can be used for non-invasive real-time in vivo gold measurement in living cells. A simple and sensitive FRET probe was designed for the detection of gold with high selectivity and can be applied to the analysis of real samples.

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.

A FRET pair for quantitative and superresolution imaging of amyloid fibril formation

The presence of neuritic plaques and amyloid fibrils arising from the misfolding of certain proteins is the principal molecular indicator of neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Methodologies for studying the early stages of amyloid aggregation are rapidly arising to provide a better understanding of the mechanism of fibrillization and cytotoxicity and to identify potential targets for diagnosis and therapy. The method presented here involves the simultaneous use of two different fluorophores, a quinolimide derivative and Nile Blue A. These are capable of interacting with and reporting on the formation of preamyloid aggregates and fibrils of apoferritin through fluorescence resonance energy transfer (FRET), which occurs between them, thus maximizing the contrast in detection and quantitative information of such amyloid species by using multidimensional fluorescence lifetime imaging microscopy (FLIM).

Rational engineering of ratiometric calcium sensors with bright green and red fluorescent proteins

Ratiometric genetically encoded calcium indicators (GECIs) record neural activity with high brightness while mitigating motion-induced artifacts. Recently developed ratiometric GECIs primarily employ cyan and yellow-fluorescent fluorescence resonance energy transfer pairs, and thus fall short in some applications that require deep tissue penetration and resistance to photobleaching. We engineered a set of green-red ratiometric calcium sensors that fused two fluorescent proteins and calcium sensing domain within an alternate configuration. The best performing elements of this palette of sensors, Twitch-GR and Twitch-NR, inherited the superior photophysical properties of their constituent fluorescent proteins. These properties enabled our sensors to outperform existing ratiometric calcium sensors in brightness and photobleaching metrics. In turn, the shot-noise limited signal fidelity of our sensors when reporting action potentials in cultured neurons and in the awake behaving mice was higher than the fidelity of existing sensors. Our sensor enabled a regime of imaging that simultaneously captured neural structure and function down to the deep layers of the mouse cortex.

Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies.

The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET (fluorescence resonance energy transfer) couple. Fluorescence correlation spectroscopy and photon-burst detection were employed to examine FRET pair labeled DENV-PRs freely diffusing in solution. The measurements were performed with two double mutants and with two dye couples. The data provide strong evidence that an equilibrium of at least two conformations of DENV-PR exists in solution. The competitive inhibitor stabilizes the closed state. Because the open and closed conformations appear to coexist in solution, our results support the picture of a conformational selection rather than that of an induced fit mechanism with respect to the inhibitor-induced formation of the closed state.

MASH-FRET: A Simplified Approach for Single-Molecule Multiplexing Using FRET.

Multiplexed detection has been a big motivation in biomarker analysis as it not only saves cost and labor but also improves the reliability of diagnosis. Among the many approaches for multiplexed detection, fluorescence resonance energy transfer (FRET)-based multiplexing is gaining popularity particularly due to its low background and quantitative nature. Although several FRET-based approaches have been developed for multiplexing, they require either multiple FRET pairs in combination with multiple excitation sources or complicated algorithms to accurately assign signals for individual FRET pairs. Therefore, the need for multiple FRET pairs and multiple excitation sources not only complicates the experimental design but also increases the cost and labor. In this regard, multiplexed sensing by tuning the interdye distance of a single FRET pair could be an ideal solution if identification of multiple FRET efficiencies in a single imaging is possible. Here, implementing a program called MASH-FRET, we evaluated the rigor and capability of this program in identifying seemingly overlapped FRET populations obtained from a multiplexed detection experiment using a single FRET pair. Through MASH-FRET-enabled bootstrap-based analysis of FRET data (also called BOBA-FRET), we demonstrated that the resolution and statistical confidence of the poorly resolved or even unresolved FRET populations can be readily determined. Using simulated FRET data, we further demonstrated that the program can easily identify FRET populations separated by ∼0.1 in mean FRET values, indicating an upper limit of ∼9-fold multiplexing without the need for complicated labeling schemes and multiexcitation sources. Therefore, this paper presents a data analysis approach on an existing platform that has a great potential to simplify the technological needs for multiplexing and to broaden the scope of FRET-based single-molecule analyses.