Objective To investigate the fluorescence resonance energy transfer (FRET) effect between dylight (DL) and AuNP (AuNP), and to construct a new fluorescence immunoassay for insulin in combination with the immunocompetition method. Methods Insulin antigen (Ag) and insulin antibody (Ab) were conjugated with DL and AuNP respectively to form DL-Ag conjugate and AUNp-AB conjugate. A novel fluorescence immunoassay for insulin was developed on the basis of FRET effect and the immune competition response between them. Then the performance of the method was evaluated and its application in actual samples was explored. Results The fluorescence immunoassay showed high sensitivity (0.015 ng/mL), short measurement time (4 min) and good specificity. It was successfully used in the measurement of serum insulin, and the recovery was between 96.9% and 121.1%. Conclusion FRET effect between AuNP and DL can be applied to develop a fluorescence immunoassay for the measurement of serum insulin.
Fluorescence Resonance Energy Transfer ; Insulin ; Immunoassay

The theory of fluorescence resonance energy transfer (FRET) and methods of fluorescence detection in fluorescent-quantitative polymerase chain reaction (FQ-PCR) are introduced in this article. Applications of FRET in fluorescence detection of PCR are emphatically discussed, and FRET research progress and future trends are pointed out too.
Fluorescence Resonance Energy Transfer ; methods ; trends ; Polymerase Chain Reaction ; methods

Animals ; Fluorescence ; Fluorescence Recovery After Photobleaching ; Fluorescence Resonance Energy Transfer ; Fluorometry ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry

Proteins are dynamic, fluctuating between multiple conformational states. Protein dynamics, spanning orders of magnitude in time and space, allow proteins to perform specific functions. Moreover, under certain conditions, proteins can morph into a different set of conformations. Thus, a complete understanding of protein structural dynamics can provide mechanistic insights into protein function. Here, we review the latest developments in methods used to determine protein ensemble structures and to characterize protein dynamics. Techniques including X-ray crystallography, cryogenic electron microscopy, and small angle scattering can provide structural information on specific conformational states or on the averaged shape of the protein, whereas techniques including nuclear magnetic resonance, fluorescence resonance energy transfer (FRET), and chemical cross-linking coupled with mass spectrometry provide information on the fluctuation of the distances between protein domains, residues, and atoms for the multiple conformational states of the protein. In particular, FRET measurements at the single-molecule level allow rapid resolution of protein conformational states, where information is otherwise obscured in bulk measurements. Taken together, the different techniques complement each other and their integrated use can offer a clear picture of protein structure and dynamics.
Fluorescence Resonance Energy Transfer ; Magnetic Resonance Spectroscopy ; Protein Conformation ; Proteins ; chemistry ; physiology

As a classic fluorescent detect technique, fluorescence resonance energy transfer (FRET) has been widely used in biological researches. Researchers have developed a series of fluorescence detect probes which were based on FRET. Caspase family plays an important role in apoptosis pathway, especially Caspase-8 which located, at the initial of death receptor mediated apoptosis pathway, whose its activation can trigger subsequent precaspases' activation and lead to apoptosis. So it is of great significance to detect the activation of Caspase-8 in apoptosis assay. In this study, a fluorescent probe based on FRET has been designed which can detect the activity change of Caspase-8 in cells. To identify the effectiveness and specificity of the probe, we measure the Caspase-8 activity under the Caspase-8 specifically activated apoptosis inducer RGD-TRAIL with the flow cytometry FRET detection platform. The results show that the probe can respond to the activity change of Caspase-8 in apoptotic cells, and the change can be quantified rapidly by flow cytometry. The study provides a more efficient and convenient detection method of Caspase-8 activity in living cells.
Apoptosis ; Caspase 8 ; metabolism ; Flow Cytometry ; Fluorescence Resonance Energy Transfer ; Fluorescent Dyes ; Humans

BACKGROUND: Ever since medical professionals have recognized the important role of ADAMTS-13 in the pathogenesis of TTP, several methods to diagnosis the activity of ADAMTS-13 in the plasma of TTP patients haves been developed. However these assays have not been widely used in practice because they are cumbersome and they require several days to complete. In this study we examine the new, rapid ADAMTS-13 activity assay that uses fluorescence resonance energy transfer and we compared it with the conventional assay to determine its diagnostic advantage. METHODS: Seven TTP patients were compared with 60 healthy controls. The plasma ADAMTS-13 activity was measured using the fluorescence-quenching substrate assay method. The results were compared with the results of performing multimer analysis of SDS-agarose gel electrophoresis. RESULTS: It took only 2 hour to complete the fluorescence-quenching substrate assay. The median ADAMTS-13 activity using the fluorescence-quenching substrate was 5.9% (range: 0~29.9%) for the patient group and 99.1% (range: 74.4~143.3%) for the healthy group, respectively. The median ADAMTS-13 activity using multimer analysis of SDS-agarose gel electrophoresis was 5.6% (range: 1.6~28.8%) for the patients group and 87.7% (range: 44.1~120.9%) for the healthy group, respectively. The ADAMTS-13 activities of the two assays were well correlated (correlation coefficient: 0.69). CONCLUSION: The quantification of ADAMTS-13 activity with using the fluorescence-quenching substrate is rapid and highly specific for the diagnosis of TTP and it is expected to be used widely in the diagnosis of TTP.
Diagnosis* ; Electrophoresis ; Fluorescence Resonance Energy Transfer ; Humans ; Plasma ; Purpura, Thrombotic Thrombocytopenic*

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.
Biosensing Techniques ; trends ; Fluorescence Resonance Energy Transfer ; Luminescent Proteins ; metabolism ; Research ; trends

Polymer micelles formed by self-assembly of amphiphilic polymers are widely used in drug delivery, gene delivery and biosensors, due to their special hydrophobic core/hydrophilic shell structure and nanoscale. However, the structural stability of polymer micelles can be affected strongly by environmental factors, such as temperature, pH, shear force in the blood and interaction with non-target cells, leading to degradations and drug leakage as drug carriers. Therefore, researches on the structural integrity and in vivo distribution of micelle-based carriers are very important for evaluating their therapeutic effect and clinical feasibility. At present, fluorescence resonance energy transfer (FRET) technology has been widely used in real-time monitoring of aggregation, dissociation and distribution of polymer micelles ( in vitro and in vivo). In this review, the polymer micelles, characteristics of FRET technology, structure and properties of the FRET-polymer micelles are briefly introduced. Then, methods and mechanism for combinations of several commonly used fluorescent probes into polymer micelles structures, and progresses on the stability and distribution studies of FRET-polymer micelles ( in vitro and in vivo) as drug carriers are reviewed, and current challenges of FRET technology and future directions are discussed.
Micelles ; Drug Carriers/chemistry* ; Polymers/chemistry* ; Fluorescence Resonance Energy Transfer ; Polyethylene Glycols/chemistry*

Mechanical force has essential effects on cellular behaviors such as proliferation, migration and differentiation, and the mechanism of mechanotransduction is still one of the hot spots in mechanobiology study. Traditional methods could not provide accurate evaluation of the protein activation signal upon mechanical stress application. The development of fluorescence protein technology greatly promoted the understanding of mechanotransduction. In particular, genetically-encoded biosensors based on fluorescence resonance energy transfer (FRET) technique has achieved a real-time dynamic observation of living cell signal protein activity, which provides a powerful tool for the in-depth study of biomechanics. In this paper, we provide a summary on recent progress of FRET application in biomechanics. Firstly we introduce the FRET technology, and then we summarize three methods to integrate the mechanical stimulation with the FRET imaging system on cell experiments. After that, the important progress of biomechanical research on signal pathway made by FRET technology, such as cytoskeleton, Rho family, calcium and cellular physical stress visualization, are also discussed. Finally, we point out the bottleneck of the future development in FRET technology, and also make the prospect of the application of FRET in mechanotransduction. In summary, FRET technology provides a powerful tool for the studies of mechanotransduction, which will advance our systematic understanding on the molecular mechanisms about how cells respond to mechanical stimulation.
Biomechanical Phenomena ; Biosensing Techniques ; Fluorescence Resonance Energy Transfer ; Humans ; Luminescent Proteins ; chemistry ; Mechanotransduction, Cellular ; Microscopy, Fluorescence ; Signal Transduction ; Stress, Mechanical

PURPOSE: The in vitro study suggested that proline to serine polymorphism in codon 475 (C1423T) of the A Disintegrin and Metalloprotease with ThromboSpondin type 1 repeats-13 (ADAMTS-13) gene is related to reduced activity of ADAMTS- 13. In this study, the frequency of the Pro475Ser polymorphism in Koreans was studied and plasma ADAMTS-13 activity was measured to find out whether this polymorphism contributes to decreased ADAMTS-13 activity in Koreans. PATIENTS AND METHODS: The frequency of the C1423T allele of the ADAMTS13 gene was studied along with measuring plasma ADAMTS-13 activity in 250 healthy Korean individuals. RESULTS: The allele frequency of C1423T polymorphism was 4%, and the median activity of CT type was 107 (69-143)%, which was lower than in controls with the CC genotype [118 (48-197)%, (p=0.021)]. CONCLUSION: Therefore, the Pro475Ser polymorphism seems to be popular in the Korean population, and attenuates ADAMTS-13 plasma activity.
ADAM Proteins/blood/*genetics/metabolism ; Asian Continental Ancestry Group ; Fluorescence Resonance Energy Transfer ; Gene Frequency ; Genotype ; Humans ; *Polymorphism, Genetic