Fluorescence Resonance Energy Transfer Biosensor Research Articles

A graphene oxide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of botulinum neurotoxin A (BoNT/A) enzymatic activity

Botulinum neurotoxins (BoNTs) are among the most potent toxic bacterial proteins for humans, which make them potential agents for bioterrorism. Therefore, an ultrasensitive detection of BoNTs and their active states is in great need as field-deployable systems for anti-terrorism applications. We report the construction of a novel graphene oxide (GO)-peptide based fluorescence resonance energy transfer (FRET) biosensor for ultrasensitive detection of the BoNT serotype A light chain (BoNT-LcA) protease activity. A green fluorescence protein (GFP) modified SNAP-25 peptide substrate (SNAP-25-GFP) was optimally designed and synthesized with the centralized recognition/cleavage sites. This FRET platform was constructed by covalent immobilization of peptide substrate on GO with BSA passivation which have advantages of low non-specific adsorption and high stability in protein abundant solution. BoNT-LcA can specifically cleave SNAP-25-GFP substrate covalently immobilized on GO to release the fragment with GFP. Based on fluorescence signal recovery measurement, the target BoNT-LcA was detected sensitively and selectively with the linear detection range from 1fg/mL to 1pg/mL. The limit of detection (LOD) for BoNT-LcA is around 1fg/mL.

A fluorescence resonance energy transfer (FRET) biosensor based on graphene quantum dots (GQDs) and gold nanoparticles (AuNPs) for the detection of mecA gene sequence of Staphylococcus aureus

In this work, a novel fluorescence resonance energy transfer (FRET) biosensor based on graphene quantum dots (GQDs) and gold nanoparticles (AuNPs) pairs was developed for Staphylococcus aureus specific gene sequence detection. This FRET biosensor platform was realized by immobilization of capture probes on GQDs and conjugation of reporter probes on AuNPs. Target oligos then co-hybridized with capture probes and reporter probes to form a sandwich structure which brought GQDs and AuNPs to close proximity to trigger FRET effect. The fluorescence signals before and after addition of targets were measured and the fluorescence quenching efficiency could reach around 87% with 100 nM target oligo. The limit of detection (LOD) of this FRET biosensor was around 1 nM for S.aureus gene detection. Experiments with both single-base mismatched oligos and double-base mismatched oligos demonstrated the good sequence selectivity of this FRET biosensor.

The use of a stably expressed FRET biosensor for determining the potency of cancer drugs.

Many cancer drugs are intended to kill cancer cells by inducing apoptosis. However, the potency assays used for measuring the bioactivity of these products are generally cell viability assays which do not distinguish between cell death and growth inhibition. Here we describe a cell-based fluorescence resonance energy transfer (FRET) biosensor designed to measure the bioactivity of apoptosis inducing cancer drugs. The biosensor contains cyan fluorescent protein (CFP) linked via caspase 3 and caspase 8 specific cleavage recognition sequences to yellow fluorescent protein (YFP). Upon caspase activation, as in the case of apoptosis induction, the linker is cleaved abolishing the cellular FRET signal. This assay closely reflects the mechanism of action of cancer drugs, in killing cancer cells and therefore can function as a potency test for different cancer drugs. We rigorously demonstrate this through characterization of a class of proteins targeting the death receptors. The one-step assay appears to be superior to other apoptosis-based assays because of its simplicity, convenience, and robustness.

A novel upconversion, fluorescence resonance energy transfer biosensor (FRET) for sensitive detection of lead ions in human serum.

There has been great progress in the development of fluorescence biosensors based on quantum dots (QDs) for the detection of lead ions. However, most methods are detecting lead ions in aqueous solution rather than in human serum due to the influence of protein autofluorescence in serum excited by visible light. Thus, we developed a novel fluorescence resonance energy transfer (FRET) biosensor by choosing the upconversion NaYF4:Yb(3+)/Tm(3+) nanoparticles as the energy donor and the CdTe QDs as the energy acceptor for lead ion detection. It is the first near infrared (NIR)-excited fluorescent probe for determination of lead ions in serum that is capable of overcoming self-luminescence from serum excitation with visible light. The sensor also shows high selectivity, a low detection limit (80 nm) and good linear Stern-Volmer characteristics (R = 0.996), both in the buffer and serum. This biosensor has great potential for versatile applications in lead ion detection in biological and analytical fields.

Label free selective detection of estriol using graphene oxide-based fluorescence sensor

Water-soluble and fluorescent Graphene oxide (GO) is biocompatible, easy, and economical to synthesize. Interestingly, GO is also capable of quenching fluorescence. On the basis of its fluorescence and quenching abilities, GO has been reported to serve as an energy acceptor in a fluorescence resonance energy transfer (FRET) sensor. GO-based FRET biosensors have been widely reported for sensing of proteins, nucleic acid, ATP (Adenosine triphosphate), etc. GO complexes with fluorescent dyes and enzymes have been used to sense metal ions. Graphene derivatives have been used for sensing endocrine-disrupting chemicals like bisphenols and chlorophenols with high sensitivity and good reproducibility. On this basis, a novel GO based fluorescent sensor has been successfully designed to detect estriol with remarkable selectivity and sensitivity. Estriol is one of the three estrogens in women and is considered to be medically important. Estriol content of maternal urine or plasma acts as an important screening marker for estimating foetal growth and development. In addition, estriol is also used as diagnostic marker for diseases like breast cancer, osteoporosis, neurodegenerative and cardiovascular diseases, insulin resistance, lupus erythematosus, endometriosis, etc. In this present study, we report for the first time a rapid, sensitive with detection limit of 1.3 nM, selective and highly biocompatible method for label free detection of estriol under physiological conditions using fluorescence assay.

Determination of Salmonella typhimurium by a Fluorescence Resonance Energy Transfer Biosensor Using Upconversion Nanoparticles as Labels

A Salmonella typhimurium (S. typhimurium) biosensor based on a fluorescence resonance energy transfer between upconversion and gold nanoparticles is reported. NaYF4:Yb,Er nanoparticles were synthesized and modified with a S. typhimurium target DNA complementary sequence to form the sensor. Gold nanoparticles were modified with a S. typhimurium target DNA complementary sequence to constitute the quenching probe. In the presence of S. typhimurium target DNA, gold and upconversion nanoparticles formed a sandwich complex, and the upconversion fluorescence resonance energy transfer occurred. Under the optimal conditions, the relative fluorescence was proportional to the concentration of S. typhimurium target DNA in the range of 0.001 pmol/L to 1 pmol/L with a limit of detection of 1 fM. S. typhimurium was detected from 30 cfu/mL to 5150 cfu/mL with a detection limit of 3 cfu/mL. The procedure was successfully applied to determine S. typhimurium in milk and validated by a traditional plate counting method. The developed upconversion fluorescence resonance energy transfer method is simple, fast, sensitive, specific, and incorporates nanomaterials in biosensor design.

Development of a FRET Biosensor with High Specificity for Akt

The serine/threonine kinase Akt plays a critical role in cell proliferation, survival, and tumorigenesis. As a central kinase in the phosphatidylinositol 3-kinase pathway, its activation mechanism at the plasma membrane has been well characterized. However, the subcellular Akt activity in living cells is still largely unknown. Fluorescence resonance energy transfer (FRET)-based biosensors have emerged as indispensable tools to visualize the subcellular activities of signaling molecules. In this study, we developed a highly specific FRET biosensor for Akt based on the Eevee backbone, called Eevee-iAkt. Using inhibitors targeting kinases upstream and downstream of Akt, we showed that Eevee-iAkt specifically monitors Akt activity in living cells. To visualize Akt activity at different subcellular compartments, we targeted Eevee-iAkt to raft and non-raft regions of the plasma membrane, mitochondria, and nucleus in HeLa and Cos7 cells. Interestingly, we revealed substantial differences in Akt activation between HeLa and Cos7 cells upon epidermal growth factor (EGF) stimulation: Akt was transiently activated in HeLa cells with comparable levels at the plasma membrane, cytosol, and mitochondria. In contrast, sustained and spatially localized Akt activation was observed in EGF-stimulated Cos7 cells. We found high Akt activity at the plasma membrane, low activity in the cytosol, and no detectable activity at the mitochondria and nucleus in Cos7 cells. The Eevee-iAkt biosensor was shown to be a valuable tool to study the functional relationship between subcellular Akt activation and its anti-apoptotic role in living cells.

Upconversion fluorescence resonance energy transfer biosensor for sensitive detection of human immunodeficiency virus antibodies in human serum

A facile one-step approach was proposed to prepare hydrophilic and peptide-functionalized upconversion nanoparticles (UCNPs), which were used in the design of a biosensor for the sensitive and selective determination of human immunodeficiency virus antibodies in human serum based on FRET from the UCNPs to the graphene oxide.

Stochastic ERK Activation Induced by Noise and Cell-to-Cell Propagation Regulates Cell Density-Dependent Proliferation

The extracellular signal-regulated kinase (ERK) plays a central role in the signaling cascades of cell growth. Here, we show that stochastic ERK activity pulses regulate cell proliferation rates in a cell density-dependent manner. A fluorescence resonance energy transfer (FRET) biosensor revealed that stochastic ERK activity pulses fired spontaneously or propagated from adjacent cells. Frequency, but not amplitude, of ERK activity pulses exhibited a bell-shaped response to the cell density and correlated with cell proliferation rates. Consistently, synthetic ERK activity pulses generated by a light-switchable CRaf protein accelerated cell proliferation. A mathematical model clarified that 80% and 20% of ERK activity pulses are generated by the noise and cell-to-cell propagation, respectively. Finally, RNA sequencing analysis of cells subjected to the synthetic ERK activity pulses suggested the involvement of serum responsive factor (SRF) transcription factors in the gene expression driven by the ERK activity pulses.

Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system

To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.

A graphene oxide-based FRET sensor for rapid and sensitive detection of matrix metalloproteinase 2 in human serum sample

Graphene oxide (GO) has been widely used to develop fluorescence resonance energy transfer (FRET) biosensors for tumor markers (e.g., matrix metalloproteinases, MMPs) due to its superior fluorescence quenching capacity and unique adsorption characteristics for biomolecules. In this study, fluorescein isothiocyanate-labeled peptide (Pep-FITC) was assembled onto the GO surface through covalent binding to construct a GO-Pep-FITC FRET sensor for sensitive, rapid, and accurate detection of MMP-2 in complex serum samples. Compared to similar GO-based FRET sensors fabricated through physical adsorption, the as prepared ones via covalent binding are significantly more stable under physiological conditions, enabling their detection of MMP-2 with high sensitivity (detection limit: 2.5ng/mL). More importantly, it allows for rapid MMP-2 detection (within 3h) even in complex biological samples with satisfactory accuracy and the relative standard deviation ≤7.03%. Our studies further suggest that such a platform developed here for sensitive, rapid, and accurate detection of biomarkers holds great promise for clinical diagnosis of protease-related diseases.

Fluorescence resonance energy transfer imaging of cell signaling from in vitro to in vivo: Basis of biosensor construction, live imaging, and image processing

The progress in imaging technology with fluorescent proteins has uncovered a wide range of biological processes in developmental biology. In particular, genetically-encoded biosensors based on the principle of fluorescence resonance energy transfer (FRET) have been used to visualize spatial and temporal dynamics of intracellular signaling in living cells. However, development of sensitive FRET biosensors and their application to developmental biology remain challenging tasks, which has prevented their widespread use in developmental biology. In this review, we first overview general procedures and tips of imaging with FRET biosensors. We then describe recent advances in FRET imaging - namely, the use of optimized backbones for intramolecular FRET biosensors and transposon-mediated gene transfer to generate stable cell lines and transgenic mice expressing FRET biosensors. Finally, we discuss future perspectives of FRET imaging in developmental biology.

Application of FRET Biosensors in Energy Metabolism

Genetically encoded optical biosensors become a tool of choice for quantitative studies on distribution and concentration changes of ions and metabolites in living cells. In systems biology, they are expected to provide multi-scale analysis in space and time for an advanced understanding of both normal and diseased physiological states. Here we develop and apply fluorescent biosensors based on fluorescence resonance energy transfer (FRET) that are able to monitor directly or indirectly the cellular energy state. Such genetically encoded FRET sensors allow quantitative analysis of changes in adenylate pools or activation of signaling pathways triggered by such changes. This should yield new insight into the spatiotemporal organization of cellular energy metabolism.

Upconversion Fluorescence Resonance Energy Transfer Biosensor with Aromatic Polymer Nanospheres as the Lable-Free Energy Acceptor

We report a new upconversion fluorescence resonance energy transfer (UC-FRET) biosensor using poly-m-phenylenediamine (PMPD) nanospheres as the energy acceptor in this paper. A single-stranded DNA (ssDNA) tagged with a sulfydryl group at the 5'-terminus was covalently linked to poly(ethylenimine) (PEI) functionalized upconversion phosphors (UCPs, the energy donor). Because of the π-rich electronic structure of PMPD, self-assembly of the donor and the acceptor was achieved through the π-π stacking interaction between ssDNA and PMPD. The fluorescence of the donor was quenched by the acceptor in a PMPD-concentration-dependent manner. A maximum quenching degree of 90% was acquired, which was among the highest levels of all previous reports. Upon the formation of double-stranded DNA (dsDNA) between the target DNA and the probe DNA, the energy acceptor was separated from the donor due to the weakened interaction between dsDNA and PMPD. The fluorescence of UCPs was accordingly restored, and a linear response was obtained with the target concentration ranging from 0.1 to 6.0 nM. The limit of detection was calculated as 0.036 nM, which was a highly competitive sensitivity. The sensor also showed high precision, pronounced specificity, and the applicability to complicated sample matrix (human serum). The UCPs-PMPD FRET sensing platform takes advantages of both the optical merits of the upconversion donors and the superquenching ability and good water-solubility of the aromatic polymer nanoparticles. This study will open the opportunity to develop a new class of UC-FRET biosensors.

Fluorescence resonance energy transfer (FRET)-based biosensors: visualizing cellular dynamics and bioenergetics

Förster (or fluorescence) resonance energy transfer (FRET) is a process involving the radiation-less transfer of energy from a "donor" fluorophore to an "acceptor" fluorophore. FRET technology enables the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as the monitoring of protein-protein interactions, protein-DNA interactions, and protein conformational changes. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time. Lately, applications of FRET-based biosensors range from basic biological to biomedical disciplines. Despite the diverse applications of FRET, FRET-based sensors still face many challenges. There is an increasing need for higher fluorescence resolution and improved specificity of FRET biosensors. Additionally, as more FRET-based technologies extend to medical diagnostics, the affordability of FRET reagents becomes a significant concern. Here, we will review current advances and limitations of FRET-based biosensor technology and discuss future FRET applications.

Advantages of substituting bioluminescence for fluorescence in a resonance energy transfer-based periplasmic binding protein biosensor

A genetically encoded maltose biosensor was constructed, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP2) at the N-terminus and a Renilla luciferase variant (RLuc2) at the C-terminus. This Bioluminescence resonance energy transfer2 (BRET2) system showed a 30% increase in the BRET ratio upon maltose binding, compared with a 10% increase with an equivalent fluorescence resonance energy transfer (FRET) biosensor. BRET2 provides a better matched Förster distance to the known separation of the N and C termini of MBP than FRET. The sensor responded to maltose and maltotriose and the response was completely abolished by introduction of a single point mutation in the BRET2 tagged MBP protein. The half maximal effective concentration (EC50) was 0.37μM for maltose and the response was linear over almost three log units ranging from 10nM to 3.16μM maltose for the BRET2 system compared to an EC50 of 2.3μM and a linear response ranging from 0.3μM to 21.1μM for the equivalent FRET-based biosensor. The biosensor’s estimate of maltose in beer matched that of a commercial enzyme-linked assay but was quicker and more precise, demonstrating its applicability to real-world samples. A similar BRET2-based transduction scheme approach would likely be applicable to other binding proteins that have a “venus-fly-trap” mechanism.

A Multi-Functional Imaging Approach to High-Content Protein Interaction Screening

Functional imaging can provide a level of quantification that is not possible in what might be termed traditional high-content screening. This is due to the fact that the current state-of-the-art high-content screening systems take the approach of scaling-up single cell assays, and are therefore based on essentially pictorial measures as assay indicators. Such phenotypic analyses have become extremely sophisticated, advancing screening enormously, but this approach can still be somewhat subjective. We describe the development, and validation, of a prototype high-content screening platform that combines steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM). This functional approach allows objective, quantitative screening of small molecule libraries in protein-protein interaction assays. We discuss the development of the instrumentation, the process by which information on fluorescence resonance energy transfer (FRET) can be extracted from wide-field, acceptor fluorescence anisotropy imaging and cross-checking of this modality using lifetime imaging by time-correlated single-photon counting. Imaging of cells expressing protein constructs where eGFP and mRFP1 are linked with amino-acid chains of various lengths (7, 19 and 32 amino acids) shows the two methodologies to be highly correlated. We validate our approach using a small-scale inhibitor screen of a Cdc42 FRET biosensor probe expressed in epidermoid cancer cells (A431) in a 96 microwell-plate format. We also show that acceptor fluorescence anisotropy can be used to measure variations in hetero-FRET in protein-protein interactions. We demonstrate this using a screen of inhibitors of internalization of the transmembrane receptor, CXCR4. These assays enable us to demonstrate all the capabilities of the instrument, image processing and analytical techniques that have been developed. Direct correlation between acceptor anisotropy and donor FLIM is observed for FRET assays, providing an opportunity to rapidly screen proteins, interacting on the nano-meter scale, using wide-field imaging.

Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

Endothelial nitric oxide synthase (eNOS)-mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1-mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca(2+) ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor L-N(G)-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca(2+)-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1-Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition.

Myosin-IXA Regulates Collective Epithelial Cell Migration by Targeting RhoGAP Activity to Cell-Cell Junctions

Epithelial tissues undergo extensive collective movements during morphogenesis, repair, and renewal. Collective epithelial cell migration requires the intercellular coordination of cell-cell adhesions and the establishment of anterior-posterior polarity, while maintaining apical-basal polarity, but how this is achieved at the molecular level is not well understood. Using an RNA interference-based screen to identify Rho family GTPase regulators required for the collective migration of human bronchial epithelial cells, we identified myosin-IXA (gene name: Myo9a). Depletion of myosin-IXA, a RhoGAP and actin motor protein, in collectively migrating cells led to altered organization of the actin cytoskeleton and tension-dependent disruption of cell-cell adhesions, followed by an inability to form new adhesions resulting in cell scattering. Closer examination revealed that myosin-IXA is required during the formation of junction-associated actin bundles soon after cell-cell contact. Structure-function analysis of myosin-IXA revealed that the motor domain is necessary and sufficient for binding to actin filaments, whereas expression of the RhoGAP domain partially rescued the cell scattering phenotype induced by myosin-IXA depletion. Finally, a fluorescence resonance energy transfer biosensor revealed a significant increase in Rho activity at nascent cell-cell contacts in myosin-IXA depleted cells compared to controls. We propose that myosin-IXA locally regulates Rho and the assembly of thin actin bundles associated with nascent cell-cell adhesions and that this is required to sustain the collective migration of epithelial cells.

FRET imaging and statistical signal processing reveal positive and negative feedback loops regulating the morphology of randomly migrating HT-1080 cells.

Cell migration plays an important role in many physiological processes. Rho GTPases (Rac1, Cdc42, RhoA) and phosphatidylinositols have been extensively studied in directional cell migration. However, it remains unclear how Rho GTPases and phosphatidylinositols regulate random cell migration in space and time. We have attempted to address this issue using fluorescence resonance energy transfer (FRET) imaging and statistical signal processing. First, we acquired time-lapse images of random migration of HT-1080 fibrosarcoma cells expressing FRET biosensors of Rho GTPases and phosphatidyl inositols. We developed an image-processing algorithm to extract FRET values and velocities at the leading edge of migrating cells. Auto- and cross-correlation analysis suggested the involvement of feedback regulations among Rac1, phosphatidyl inositols and membrane protrusions. To verify the feedback regulations, we employed an acute inhibition of the signaling pathway with pharmaceutical inhibitors. The inhibition of actin polymerization decreased Rac1 activity, indicating the presence of positive feedback from actin polymerization to Rac1. Furthermore, treatment with PI3-kinase inhibitor induced an adaptation of Rac1 activity, i.e. a transient reduction of Rac1 activity followed by recovery to the basal level. In silico modeling that reproduced the adaptation predicted the existence of a negative feedback loop from Rac1 to actin polymerization. Finally, we identified MLCK as the probable controlling factor in the negative feedback. These findings quantitatively demonstrate positive and negative feedback loops that involve actin, Rac1 and MLCK, and account for the ordered patterns of membrane dynamics observed in randomly migrating cells.