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Special Issue "FRET Biosensors"

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A special issue of Sensors (ISSN 1424-8220). This special issue belongs to the section "Biosensors".

Deadline for manuscript submissions: closed (30 June 2016)

Special Issue Editors

Guest Editor
Prof. Dr. Niko Hildebrandt

NanoBioPhotonics, Institut d’Electronique Fondamentale, Université Paris-Sud 91405 Orsay Cedex, France
Website | E-Mail
Interests: FRET; spectroscopy; imaging; diagnostics; immunoassays; multiplexing; biosensing; lanthanides; quantum dots; time-gating
Guest Editor
Dr. Igor Medintz

Laboratory for Biosensors and Biomaterials, Center for Biomolecular Science & Engineering, United States Naval Research Laboratory, Washington, D.C. USA
Website | E-Mail
Fax: +1 202 7679594
Interests: nanoparticle-biological interface, energy transfer, FRET, biosensing, enzymatic catalysis at a nanoparticle interface, nanoparticle-based cellular imaging
Guest Editor
Prof. Dr. Russ Algar

Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC Canada V6T 1Z1, Canada
Website | E-Mail
Phone: 604-822-2464
Interests: fluorescence; resonance energy transfer; assays, imaging; biosensing; point-of-care diagnostics; nanoparticles; enzymes; nucleic acids; surface chemistry

Special Issue Information

Dear Colleagues,

FRET or Förster resonance energy transfer is a versatile and sensitive tool for qualitative and quantitative analysis of biological interactions and processes. The access to a wide range of fluorescent materials, in conjunction with improved, easy-to-use, and yet very sophisticated microscopes and spectrometers, have made FRET a very prominent technique for biosensing. Fluorophores that are utilized in FRET now encompass organic dyes, fluorescent proteins, semiconductor quantum dots, metal chelates, various noble metal and other nanoparticles, intrinsically fluorescent amino acids, biological cofactors, and polymers, to name but a few members of this growing library. Hand-in-hand with materials development is the growing availability of numerous reactive and bioorthogonal chemistries to specifically attach such fluorophores to all types of biological molecules, ranging from proteins to DNA. The unique ability of FRET to probe nanoscale inter- and intramolecular separation distances, has also led to a rapidly growing field of structural FRET studies of biomolecules and biological complexes.

We invite manuscripts for this forthcoming Special Issue that describe all aspects pertinent to FRET-based biosensing and bioimaging. Both reviews and original research articles will be published. Reviews should provide an up-to-date and critical overview of the current state of the art in a particular application, such as diagnostics and protein–protein interactions, or a particular technique such as single-molecule FRET or FRET spectroscopic rulers. Original research papers that describe the utilization of FRET in biosensing, or new concepts and fundamental studies with potential relevance to biosensing, are also of interest. If you have a preliminary idea or suggestion you would like to discuss beforehand, please feel free to contact us. We look forward to and welcome your participation in this Special Issue.

Prof. Dr. Niko Hildebrandt
Dr. Igor Medintz
Prof. Dr. Russ Algar
Guest Editors

Submission

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. Papers will be published continuously (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are refereed through a peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Sensors is an international peer-reviewed Open Access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs).


Keywords

  • FRET
  • fluorescence
  • luminescence
  • nanotechnology
  • nanomaterials
  • fluorescent probes
  • spectroscopy
  • imaging
  • bioanalysis
  • diagnostics

Published Papers (22 papers)

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Research

Jump to: Review, Other

Open AccessArticle A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis
Sensors 2016, 16(10), 1604; doi:10.3390/s16101604
Received: 2 August 2016 / Revised: 15 September 2016 / Accepted: 20 September 2016 / Published: 28 September 2016
PDF Full-text (4616 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective
[...] Read more.
Background: The fast development of microbial production strains for basic and fine chemicals is increasingly carried out in small scale cultivation systems to allow for higher throughput. Such parallelized systems create a need for new rapid online detection systems to quantify the respective target compound. In this regard, biosensors, especially genetically encoded Förster resonance energy transfer (FRET)-based biosensors, offer tremendous opportunities. As a proof-of-concept, we have created a toolbox of FRET-based biosensors for the ratiometric determination of l-lysine in fermentation broth. Methods: The sensor toolbox was constructed based on a sensor that consists of an optimized central lysine-/arginine-/ornithine-binding protein (LAO-BP) flanked by two fluorescent proteins (enhanced cyan fluorescent protein (ECFP), Citrine). Further sensor variants with altered affinity and sensitivity were obtained by circular permutation of the binding protein as well as the introduction of flexible and rigid linkers between the fluorescent proteins and the LAO-BP, respectively. Results: The sensor prototype was applied to monitor the extracellular l-lysine concentration of the l-lysine producing Corynebacterium glutamicum (C. glutamicum) strain DM1933 in a BioLector® microscale cultivation device. The results matched well with data obtained by HPLC analysis and the Ninhydrin assay, demonstrating the high potential of FRET-based biosensors for high-throughput microbial bioprocess optimization. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessArticle Characterization of the ER-Targeted Low Affinity Ca2+ Probe D4ER
Sensors 2016, 16(9), 1419; doi:10.3390/s16091419
Received: 5 July 2016 / Revised: 26 August 2016 / Accepted: 30 August 2016 / Published: 2 September 2016
PDF Full-text (2890 KB) | HTML Full-text | XML Full-text
Abstract
Calcium ion (Ca2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+]) within
[...] Read more.
Calcium ion (Ca2+) is a ubiquitous intracellular messenger and changes in its concentration impact on nearly every aspect of cell life. Endoplasmic reticulum (ER) represents the major intracellular Ca2+ store and the free Ca2+ concentration ([Ca2+]) within its lumen ([Ca2+]ER) can reach levels higher than 1 mM. Several genetically-encoded ER-targeted Ca2+ sensors have been developed over the last years. However, most of them are non-ratiometric and, thus, their signal is difficult to calibrate in live cells and is affected by shifts in the focal plane and artifactual movements of the sample. On the other hand, existing ratiometric Ca2+ probes are plagued by different drawbacks, such as a double dissociation constant (Kd) for Ca2+, low dynamic range, and an affinity for the cation that is too high for the levels of [Ca2+] in the ER lumen. Here, we report the characterization of a recently generated ER-targeted, Förster resonance energy transfer (FRET)-based, Cameleon probe, named D4ER, characterized by suitable Ca2+ affinity and dynamic range for monitoring [Ca2+] variations within the ER. As an example, resting [Ca2+]ER have been evaluated in a known paradigm of altered ER Ca2+ homeostasis, i.e., in cells expressing a mutated form of the familial Alzheimer’s Disease-linked protein Presenilin 2 (PS2). The lower Ca2+ affinity of the D4ER probe, compared to that of the previously generated D1ER, allowed the detection of a conspicuous, more clear-cut, reduction in ER Ca2+ content in cells expressing mutated PS2, compared to controls. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessArticle Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM
Sensors 2016, 16(8), 1312; doi:10.3390/s16081312
Received: 20 June 2016 / Revised: 10 August 2016 / Accepted: 12 August 2016 / Published: 19 August 2016
PDF Full-text (6143 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2
[...] Read more.
We describe an approach to non-invasively map spatiotemporal biochemical and physiological changes in 3D cell culture using Forster Resonance Energy Transfer (FRET) biosensors expressed in tumour spheroids. In particular, we present an improved Adenosine Monophosphate (AMP) Activated Protein Kinase (AMPK) FRET biosensor, mTurquoise2 AMPK Activity Reporter (T2AMPKAR), for fluorescence lifetime imaging (FLIM) readouts that we have evaluated in 2D and 3D cultures. Our results in 2D cell culture indicate that replacing the FRET donor, enhanced Cyan Fluorescent Protein (ECFP), in the original FRET biosensor, AMPK activity reporter (AMPKAR), with mTurquoise2 (mTq2FP), increases the dynamic range of the response to activation of AMPK, as demonstrated using the direct AMPK activator, 991. We demonstrated 3D FLIM of this T2AMPKAR FRET biosensor expressed in tumour spheroids using two-photon excitation. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessArticle Anchoring of FRET Sensors—A Requirement for Spatiotemporal Resolution
Sensors 2016, 16(5), 703; doi:10.3390/s16050703
Received: 8 April 2016 / Revised: 4 May 2016 / Accepted: 11 May 2016 / Published: 16 May 2016
PDF Full-text (3516 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within
[...] Read more.
FRET biosensors have become a routine tool for investigating mechanisms and components of cell signaling. Strategies for improving them for particular applications are continuously sought. One important aspect to consider when designing FRET probes is the dynamic distribution and propagation of signals within living cells. We have addressed this issue by directly comparing an anchored (taFS) to a non-anchored (naFS) cleavable FRET sensor. We chose a microtubule-associated protein tau as an anchor, as microtubules are abundant throughout the cytosol of cells. We show that tau-anchored FRET sensors are concentrated at the cytoskeleton and enriched in the neurite-like processes of cells, providing high intensity of the total signal. In addition, anchoring limits the diffusion of the sensor, enabling spatiotemporally resolved monitoring of subcellular variations in enzyme activity. Thus, anchoring is an important aspect to consider when designing FRET sensors for deeper understanding of cell signaling. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessArticle Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy
Sensors 2016, 16(2), 258; doi:10.3390/s16020258
Received: 16 October 2015 / Revised: 17 January 2016 / Accepted: 16 February 2016 / Published: 19 February 2016
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Abstract
The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced
[...] Read more.
The gold nanoparticle (GNP) aggregation growth induced by deoxyribonucleic acid (DNA) is studied by laser scanning confocal and environmental scanning electron microscopies. As in the investigated case the direct light scattering analysis is not suitable, we observe the behavior of the fluorescence produced by a dye and we detect the aggregation by the shift and the broadening of the fluorescence peak. Results of laser scanning confocal microscopy images and the fluorescence emission spectra from lambda scan mode suggest, in fact, that the intruding of the hydrophobic moiety of the probe within the cationic surfactants bilayer film coating GNPs results in a Förster resonance energy transfer. The environmental scanning electron microscopy images show that DNA molecules act as template to assemble GNPs into three-dimensional structures which are reminiscent of the DNA helix. This study is useful to design better nanobiotechnological devices using GNPs and DNA. Full article
(This article belongs to the Special Issue FRET Biosensors)
Open AccessArticle Evaluating Quantum Dot Performance in Homogeneous FRET Immunoassays for Prostate Specific Antigen
Sensors 2016, 16(2), 197; doi:10.3390/s16020197
Received: 30 November 2015 / Revised: 11 January 2016 / Accepted: 2 February 2016 / Published: 4 February 2016
Cited by 6 | PDF Full-text (1975 KB) | HTML Full-text | XML Full-text
Abstract
The integration of semiconductor quantum dots (QDs) into homogeneous Förster resonance energy transfer (FRET) immunoassay kits for clinical diagnostics can provide significant advantages concerning multiplexing and sensitivity. Here we present a facile and functional QD-antibody conjugation method using three commercially available QDs with
[...] Read more.
The integration of semiconductor quantum dots (QDs) into homogeneous Förster resonance energy transfer (FRET) immunoassay kits for clinical diagnostics can provide significant advantages concerning multiplexing and sensitivity. Here we present a facile and functional QD-antibody conjugation method using three commercially available QDs with different photoluminescence (PL) maxima (605 nm, 655 nm, and 705 nm). The QD-antibody conjugates were successfully applied for FRET immunoassays against prostate specific antigen (PSA) in 50 µL serum samples using Lumi4-Tb (Tb) antibody conjugates as FRET donors and time-gated PL detection on a KRYPTOR clinical plate reader. Förster distance and Tb donor background PL were directly related to the analytical sensitivity for PSA, ...which resulted in the lowest limits of detection for Tb-QD705 (2 ng/mL), followed by Tb-QD655 (4 ng/mL), and Tb-QD605 (23 ng/mL). Duplexed PSA detection using the Tb-QD655 and Tb-QD705 FRET-pairs demonstrated the multiplexing ability of our immunoassays. Our results show that FRET based on QD acceptors is suitable for multiplexed and sensitive biomarker detection in clinical diagnostics. Full article
(This article belongs to the Special Issue FRET Biosensors)
Open AccessArticle Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly
Sensors 2015, 15(12), 30457-30468; doi:10.3390/s151229810
Received: 8 October 2015 / Revised: 23 November 2015 / Accepted: 26 November 2015 / Published: 4 December 2015
Cited by 1 | PDF Full-text (1707 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time
[...] Read more.
Understanding how to controllably modulate the efficiency of energy transfer in Förster resonance energy transfer (FRET)-based assemblies is critical to their implementation as sensing modalities. This is particularly true for sensing assemblies that are to be used as the basis for real time intracellular sensing of intracellular processes and events. We use a quantum dot (QD) donor -mCherry acceptor platform that is engineered to self-assemble in situ wherein the protein acceptor is expressed via transient transfection and the QD donor is microinjected into the cell. QD-protein assembly is driven by metal-affinity interactions where a terminal polyhistidine tag on the protein binds to the QD surface. Using this system, we show the ability to modulate the efficiency of the donor–acceptor energy transfer process by controllably altering either the ligand coating on the QD surface or the precise location where the QD-protein assembly process occurs. Intracellularly, a short, zwitterionic ligand mediates more efficient FRET relative to longer ligand species that are based on the solubilizing polymer, poly(ethylene glycol). We further show that a greater FRET efficiency is achieved when the QD-protein assembly occurs free in the cytosol compared to when the mCherry acceptor is expressed tethered to the inner leaflet of the plasma membrane. In the latter case, the lower FRET efficiency is likely attributable to a lower expression level of the mCherry acceptor at the membrane combined with steric hindrance. Our work points to some of the design considerations that one must be mindful of when developing FRET-based sensing schemes for use in intracellular sensing. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessArticle FRET-Based Nanobiosensors for Imaging Intracellular Ca2+ and H+ Microdomains
Sensors 2015, 15(9), 24662-24680; doi:10.3390/s150924662
Received: 24 July 2015 / Revised: 14 September 2015 / Accepted: 16 September 2015 / Published: 23 September 2015
Cited by 1 | PDF Full-text (1439 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here,
[...] Read more.
Semiconductor nanocrystals (NCs) or quantum dots (QDs) are luminous point emitters increasingly being used to tag and track biomolecules in biological/biomedical imaging. However, their intracellular use as highlighters of single-molecule localization and nanobiosensors reporting ion microdomains changes has remained a major challenge. Here, we report the design, generation and validation of FRET-based nanobiosensors for detection of intracellular Ca2+ and H+ transients. Our sensors combine a commercially available CANdot®565QD as an energy donor with, as an acceptor, our custom-synthesized red-emitting Ca2+ or H+ probes. These ‘Rubies’ are based on an extended rhodamine as a fluorophore and a phenol or BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid) for H+ or Ca2+ sensing, respectively, and additionally bear a linker arm for conjugation. QDs were stably functionalized using the same SH/maleimide crosslink chemistry for all desired reactants. Mixing ion sensor and cell-penetrating peptides (that facilitate cytoplasmic delivery) at the desired stoichiometric ratio produced controlled multi-conjugated assemblies. Multiple acceptors on the same central donor allow up-concentrating the ion sensor on the QD surface to concentrations higher than those that could be achieved in free solution, increasing FRET efficiency and improving the signal. We validate these nanosensors for the detection of intracellular Ca2+ and pH transients using live-cell fluorescence imaging. Full article
(This article belongs to the Special Issue FRET Biosensors)
Open AccessArticle Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity
Sensors 2015, 15(8), 17977-17989; doi:10.3390/s150817977
Received: 4 June 2015 / Revised: 18 July 2015 / Accepted: 21 July 2015 / Published: 23 July 2015
Cited by 3 | PDF Full-text (4592 KB) | HTML Full-text | XML Full-text
Abstract
We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated
[...] Read more.
We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity. Full article
(This article belongs to the Special Issue FRET Biosensors)
Open AccessArticle Generation of Red-Shifted Cameleons for Imaging Ca2+ Dynamics of the Endoplasmic Reticulum
Sensors 2015, 15(6), 13052-13068; doi:10.3390/s150613052
Received: 30 March 2015 / Revised: 21 May 2015 / Accepted: 26 May 2015 / Published: 4 June 2015
Cited by 4 | PDF Full-text (7022 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin
[...] Read more.
Cameleons are sophisticated genetically encoded fluorescent probes that allow quantifying cellular Ca2+ signals. The probes are based on Förster resonance energy transfer (FRET) between terminally located fluorescent proteins (FPs), which move together upon binding of Ca2+ to the central calmodulin myosin light chain kinase M13 domain. Most of the available cameleons consist of cyan and yellow FPs (CFP and YFP) as the FRET pair. However, red-shifted versions with green and orange or red FPs (GFP, OFP, RFP) have some advantages such as less phototoxicity and minimal spectral overlay with autofluorescence of cells and fura-2, a prominent chemical Ca2+ indicator. While GFP/OFP- or GFP/RFP-based cameleons have been successfully used to study cytosolic and mitochondrial Ca2+ signals, red-shifted cameleons to visualize Ca2+ dynamics of the endoplasmic reticulum (ER) have not been developed so far. In this study, we generated and tested several ER targeted red-shifted cameleons. Our results show that GFP/OFP-based cameleons due to miss-targeting and their high Ca2+ binding affinity are inappropriate to record ER Ca2+ signals. However, ER targeted GFP/RFP-based probes were suitable to sense ER Ca2+ in a reliable manner. With this study we increased the palette of cameleons for visualizing Ca2+ dynamics within the main intracellular Ca2+ store. Full article
(This article belongs to the Special Issue FRET Biosensors)
Open AccessArticle MMP-2/9-Specific Activatable Lifetime Imaging Agent
Sensors 2015, 15(5), 11076-11091; doi:10.3390/s150511076
Received: 2 April 2015 / Revised: 1 May 2015 / Accepted: 6 May 2015 / Published: 12 May 2015
Cited by 2 | PDF Full-text (4972 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable
[...] Read more.
Optical (molecular) imaging can benefit from a combination of the high signal-to-background ratio of activatable fluorescence imaging with the high specificity of luminescence lifetime imaging. To allow for this combination, both imaging techniques were integrated in a single imaging agent, a so-called activatable lifetime imaging agent. Important in the design of this imaging agent is the use of two luminophores that are tethered by a specific peptide with a hairpin-motive that ensured close proximity of the two while also having a specific amino acid sequence available for enzymatic cleavage by tumor-related MMP-2/9. Ir(ppy)3 and Cy5 were used because in close proximity the emission intensities of both luminophores were quenched and the influence of Cy5 shortens the Ir(ppy)3 luminescence lifetime from 98 ns to 30 ns. Upon cleavage in vitro, both effects are undone, yielding an increase in Ir(ppy)3 and Cy5 luminescence and a restoration of Ir(ppy)3 luminescence lifetime to 94 ns. As a reference for the luminescence activation, a similar imaging agent with the more common Cy3-Cy5 fluorophore pair was used. Our findings underline that the combination of enzymatic signal activation with lifetime imaging is possible and that it provides a promising method in the design of future disease specific imaging agents. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessArticle The Use of the LanthaScreen TR-FRET CAR Coactivator Assay in the Characterization of Constitutive Androstane Receptor (CAR) Inverse Agonists
Sensors 2015, 15(4), 9265-9276; doi:10.3390/s150409265
Received: 27 February 2015 / Revised: 3 April 2015 / Accepted: 14 April 2015 / Published: 21 April 2015
Cited by 5 | PDF Full-text (940 KB) | HTML Full-text | XML Full-text
Abstract
The constitutive androstane receptor (CAR) is a critical nuclear receptor in the gene regulation of xenobiotic and endobiotic metabolism. The LanthaScreenTM TR-FRET CAR coactivator assay provides a simple and reliable method to analyze the affinity of a ligand to the human CAR ligand-binding
[...] Read more.
The constitutive androstane receptor (CAR) is a critical nuclear receptor in the gene regulation of xenobiotic and endobiotic metabolism. The LanthaScreenTM TR-FRET CAR coactivator assay provides a simple and reliable method to analyze the affinity of a ligand to the human CAR ligand-binding domain (LBD) with no need to use cellular models. This in silico assay thus enables the study of direct CAR ligands and the ability to distinguish them from the indirect CAR activators that affect the receptor via the cell signaling-dependent phosphorylation of CAR in cells. For the current paper we characterized the pharmacodynamic interactions of three known CAR inverse agonists/antagonists—PK11195, clotrimazole and androstenol—with the prototype agonist CITCO (6-(4-chlorophenyl)imidazo[2,1-b][1,3] thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) using the TR-FRET LanthaScreenTM assay. We have confirmed that all three compounds are inverse agonists of human CAR, with IC50 0.51, 0.005, and 0.35 μM, respectively. All the compounds also antagonize the CITCO-mediated activation of CAR, but only clotrimazole was capable to completely reverse the effect of CITCO in the tested concentrations. Thus this method allows identifying not only agonists, but also antagonists and inverse agonists for human CAR as well as to investigate the nature of the pharmacodynamic interactions of CAR ligands. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessArticle A Homogenous Fluorescence Quenching Based Assay for Specific and Sensitive Detection of Influenza Virus A Hemagglutinin Antigen
Sensors 2015, 15(4), 8852-8865; doi:10.3390/s150408852
Received: 22 January 2015 / Revised: 2 April 2015 / Accepted: 9 April 2015 / Published: 15 April 2015
Cited by 3 | PDF Full-text (1733 KB) | HTML Full-text | XML Full-text | Supplementary Files
Abstract
Influenza pandemics cause millions of deaths worldwide. Effective surveillance is required to prevent their spread and facilitate the development of appropriate vaccines. In this study, we report the fabrication of a homogenous fluorescence-quenching-based assay for specific and sensitive detection of influenza virus surface
[...] Read more.
Influenza pandemics cause millions of deaths worldwide. Effective surveillance is required to prevent their spread and facilitate the development of appropriate vaccines. In this study, we report the fabrication of a homogenous fluorescence-quenching-based assay for specific and sensitive detection of influenza virus surface antigen hemagglutinins (HAs). The core of the assay is composed of two nanoprobes namely the glycan-conjugated highly luminescent quantum dots (Gly-QDs), and the HA-specific antibody-modified gold nanoparticle (Ab-Au NPs). When exposed to strain-specific HA, a binding event between the HA and the two nanoprobes takes place, resulting in the formation of a sandwich complex which subsequently brings the two nanoprobes closer together. This causes a decrease in QDs fluorescence intensity due to a non-radiative energy transfer from QDs to Au NPs. A resulting correlation between the targets HA concentrations and fluorescence changes can be observed. Furthermore, by utilizing the specific interaction between HA and glycan with sialic acid residues, the assay is able to distinguish HAs originated from viral subtypes H1 (human) and H5 (avian). The detection limits in solution are found to be low nanomolar and picomolar level for sensing H1-HA and H5-HA, respectively. Slight increase in assay sensitivity was found in terms of detection limit while exposing the assay in the HA spiked in human sera solution. We believe that the developed assay could serve as a feasible and sensitive diagnostic tool for influenza virus detection and discrimination, with further improvement on the architectures. Full article
(This article belongs to the Special Issue FRET Biosensors)
Open AccessArticle Genotyping Single Nucleotide Polymorphisms Using Different Molecular Beacon Multiplexed within a Suspended Core Optical Fiber
Sensors 2014, 14(8), 14488-14499; doi:10.3390/s140814488
Received: 25 June 2014 / Revised: 1 August 2014 / Accepted: 5 August 2014 / Published: 8 August 2014
Cited by 3 | PDF Full-text (1373 KB) | HTML Full-text | XML Full-text
Abstract
We report a novel approach to genotyping single nucleotide polymorphisms (SNPs) using molecular beacons in conjunction with a suspended core optical fiber (SCF). Target DNA sequences corresponding to the wild- or mutant-type have been accurately recognized by immobilizing two different molecular beacons on
[...] Read more.
We report a novel approach to genotyping single nucleotide polymorphisms (SNPs) using molecular beacons in conjunction with a suspended core optical fiber (SCF). Target DNA sequences corresponding to the wild- or mutant-type have been accurately recognized by immobilizing two different molecular beacons on the core of a SCF. The two molecular beacons differ by one base in the loop-probe and utilize different fluorescent indicators. Single-color fluorescence enhancement was obtained when the immobilized SCFs were filled with a solution containing either wild-type or mutant-type sequence (homozygous sample), while filling the immobilized SCF with solution containing both wild- and mutant-type sequences resulted in dual-color fluorescence enhancement, indicating a heterozygous sample. The genotyping was realized amplification-free and with ultra low-volume for the required DNA solution (nano-liter). This is, to our knowledge, the first genotyping device based on the combination of optical fiber and molecular beacons. Full article
(This article belongs to the Special Issue FRET Biosensors)

Review

Jump to: Research, Other

Open AccessReview A Guide to Fluorescent Protein FRET Pairs
Sensors 2016, 16(9), 1488; doi:10.3390/s16091488
Received: 21 July 2016 / Revised: 23 August 2016 / Accepted: 25 August 2016 / Published: 14 September 2016
Cited by 1 | PDF Full-text (1939 KB) | HTML Full-text | XML Full-text
Abstract
Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET
[...] Read more.
Förster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessReview Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes
Sensors 2016, 16(8), 1173; doi:10.3390/s16081173
Received: 19 June 2016 / Revised: 18 July 2016 / Accepted: 19 July 2016 / Published: 27 July 2016
Cited by 1 | PDF Full-text (1895 KB) | HTML Full-text | XML Full-text
Abstract
Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the
[...] Read more.
Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore, rapid, cost-effective and reliable detection of mutations is an object of extensive research. Today, Förster resonance energy transfer (FRET) probes are among the most often used tools for the detection of nucleic acids and in particular, for the detection of mutations. However, multiple parameters must be taken into account in order to create efficient FRET probes that are sensitive to nucleic acid mutations. In this review; we focus on the design principles for such probes and available computational methods that allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cellular heterogeneity to gaining new knowledge of nucleic acid structures directly in living cells. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Figure 1

Open AccessReview The β-Lactamase Assay: Harnessing a FRET Biosensor to Analyse Viral Fusion Mechanisms
Sensors 2016, 16(7), 950; doi:10.3390/s16070950
Received: 5 May 2016 / Revised: 8 June 2016 / Accepted: 20 June 2016 / Published: 23 June 2016
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Abstract
The β-lactamase (BlaM) assay was first revealed in 1998 and was demonstrated to be a robust Förster resonance energy transfer (FRET)-based reporter system that was compatible with a range of commonly-used cell lines. Today, the BlaM assay is available commercially as a kit
[...] Read more.
The β-lactamase (BlaM) assay was first revealed in 1998 and was demonstrated to be a robust Förster resonance energy transfer (FRET)-based reporter system that was compatible with a range of commonly-used cell lines. Today, the BlaM assay is available commercially as a kit and can be utilised readily and inexpensively for an array of experimental procedures that require a fluorescence-based readout. One frequent application of the BlaM assay is the measurement of viral fusion—the moment at which the genetic material harboured within virus particles is released into the cytosol following successful entry. The flexibility of the system permits evaluation of not only total fusion levels, but also the kinetics of fusion. However, significant variation exists in the scientific literature regarding the methodology by which the assay is applied to viral fusion analysis, making comparison between results difficult. In this review we draw attention to the disparity of these methodologies and examine the advantages and disadvantages of each approach. Successful strategies shown to render viruses compatible with BlaM-based analyses are also discussed. Full article
(This article belongs to the Special Issue FRET Biosensors)
Open AccessReview Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences
Sensors 2015, 15(10), 26281-26314; doi:10.3390/s151026281
Received: 20 June 2015 / Accepted: 8 October 2015 / Published: 16 October 2015
Cited by 9 | PDF Full-text (1550 KB) | HTML Full-text | XML Full-text
Abstract
Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore,
[...] Read more.
Fluorescence- or Förster resonance energy transfer (FRET) is a measurable physical energy transfer phenomenon between appropriate chromophores, when they are in sufficient proximity, usually within 10 nm. This feature has made them incredibly useful tools for many biomedical studies on molecular interactions. Furthermore, this principle is increasingly exploited for the design of biosensors, where two chromophores are linked with a sensory domain controlling their distance and thus the degree of FRET. The versatility of these FRET-biosensors made it possible to assess a vast amount of biological variables in a fast and standardized manner, allowing not only high-throughput studies but also sub-cellular measurements of biological processes. In this review, we aim at giving an overview over the recent advances in genetically encoded, fluorescent-protein based FRET-biosensors, as these represent the largest and most vividly growing group of FRET-based sensors. For easy understanding, we are grouping them into four categories, depending on their molecular mechanism. These are based on: (a) cleavage; (b) conformational-change; (c) mechanical force and (d) changes in the micro-environment. We also address the many issues and considerations that come with the development of FRET-based biosensors, as well as the possibilities that are available to measure them. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessReview Biosensing with Förster Resonance Energy Transfer Coupling between Fluorophores and Nanocarbon Allotropes
Sensors 2015, 15(6), 14766-14787; doi:10.3390/s150614766
Received: 19 March 2015 / Revised: 1 June 2015 / Accepted: 5 June 2015 / Published: 23 June 2015
Cited by 7 | PDF Full-text (2728 KB) | HTML Full-text | XML Full-text
Abstract
Nanocarbon allotropes (NCAs), including zero-dimensional carbon dots (CDs), one-dimensional carbon nanotubes (CNTs) and two-dimensional graphene, exhibit exceptional material properties, such as unique electrical/thermal conductivity, biocompatibility and high quenching efficiency, that make them well suited for both electrical/electrochemical and optical sensors/biosensors alike. In particular,
[...] Read more.
Nanocarbon allotropes (NCAs), including zero-dimensional carbon dots (CDs), one-dimensional carbon nanotubes (CNTs) and two-dimensional graphene, exhibit exceptional material properties, such as unique electrical/thermal conductivity, biocompatibility and high quenching efficiency, that make them well suited for both electrical/electrochemical and optical sensors/biosensors alike. In particular, these material properties have been exploited to significantly enhance the transduction of biorecognition events in fluorescence-based biosensing involving Förster resonant energy transfer (FRET). This review analyzes current advances in sensors and biosensors that utilize graphene, CNTs or CDs as the platform in optical sensors and biosensors. Widely utilized synthesis/fabrication techniques, intrinsic material properties and current research examples of such nanocarbon, FRET-based sensors/biosensors are illustrated. The future outlook and challenges for the research field are also detailed. Full article
(This article belongs to the Special Issue FRET Biosensors)
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Open AccessReview Förster Resonance Energy Transfer between Quantum Dot Donors and Quantum Dot Acceptors
Sensors 2015, 15(6), 13288-13325; doi:10.3390/s150613288
Received: 22 April 2015 / Revised: 29 May 2015 / Accepted: 1 June 2015 / Published: 5 June 2015
Cited by 17 | PDF Full-text (8289 KB) | HTML Full-text | XML Full-text
Abstract
Förster (or fluorescence) resonance energy transfer amongst semiconductor quantum dots (QDs) is reviewed, with particular interest in biosensing applications. The unique optical properties of QDs provide certain advantages and also specific challenges with regards to sensor design, compared to other FRET systems. The
[...] Read more.
Förster (or fluorescence) resonance energy transfer amongst semiconductor quantum dots (QDs) is reviewed, with particular interest in biosensing applications. The unique optical properties of QDs provide certain advantages and also specific challenges with regards to sensor design, compared to other FRET systems. The brightness and photostability of QDs make them attractive for highly sensitive sensing and long-term, repetitive imaging applications, respectively, but the overlapping donor and acceptor excitation signals that arise when QDs serve as both the donor and acceptor lead to high background signals from direct excitation of the acceptor. The fundamentals of FRET within a nominally homogeneous QD population as well as energy transfer between two distinct colors of QDs are discussed. Examples of successful sensors are highlighted, as is cascading FRET, which can be used for solar harvesting. Full article
(This article belongs to the Special Issue FRET Biosensors)
Figures

Open AccessReview QD-Based FRET Probes at a Glance
Sensors 2015, 15(6), 13028-13051; doi:10.3390/s150613028
Received: 8 April 2015 / Revised: 20 May 2015 / Accepted: 22 May 2015 / Published: 4 June 2015
Cited by 13 | PDF Full-text (3238 KB) | HTML Full-text | XML Full-text
Abstract
The unique optoelectronic properties of quantum dots (QDs) give them significant advantages over traditional organic dyes, not only as fluorescent labels for bioimaging, but also as emissive sensing probes. QD sensors that function via manipulation of fluorescent resonance energy transfer (FRET) are of
[...] Read more.
The unique optoelectronic properties of quantum dots (QDs) give them significant advantages over traditional organic dyes, not only as fluorescent labels for bioimaging, but also as emissive sensing probes. QD sensors that function via manipulation of fluorescent resonance energy transfer (FRET) are of special interest due to the multiple response mechanisms that may be utilized, which in turn imparts enhanced flexibility in their design. They may also function as ratiometric, or “color-changing” probes. In this review, we describe the fundamentals of FRET and provide examples of QD-FRET sensors as grouped by their response mechanisms such as link cleavage and structural rearrangement. An overview of early works, recent advances, and various models of QD-FRET sensors for the measurement of pH and oxygen, as well as the presence of metal ions and proteins such as enzymes, are also provided. Full article
(This article belongs to the Special Issue FRET Biosensors)

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Open AccessAddendum Addendum: Hochreiter, B.; Pardo-Garcia, A.; Schmid, J.A. Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences. Sensors 2015, 15, 26281–26314
Sensors 2015, 15(11), 29182; doi:10.3390/s151129182
Received: 9 November 2015 / Accepted: 10 November 2015 / Published: 18 November 2015
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Abstract The authors wish to add an Acknowledgments section to their paper published in Sensors [1], doi:10.3390/s151026281, website: http://www.mdpi.com/1424-8220/15/10/26281. [...] Full article
(This article belongs to the Special Issue FRET Biosensors)

Planned Papers

The below list represents only planned manuscripts. Some of these manuscripts have not been received by the Editorial Office yet. Papers submitted to MDPI journals are subject to peer-review.

Title: Towards cellular imaging of Ca2+ and H+ microdomains
Type:
Ariticle
Author:
Alsu I. Zamaleeva, 1,2,3,*, Despras Guillaume, 4,5,6, Camilla Luccardini, 1,2,3, Mayeul Collot, 4,5,6, Martin Oheim 7,8, Michel De Waard 9,10, Jean-Maurice Mallet 4,5,6, Anne Feltz 1,2,3,*
1
Ecole Normale Supérieure, Institut de Biologie de l'ENS (IBENS), Paris F-75005, France
2
INSERM U1024, Paris F-75005, France
3
CNRS UMR 8197, Paris F-75005, France
4
UPMC Université́ Paris 06, Ecole Normale Supérieure (ENS), Paris, F-75005 France
5
CNRS UMR 7203, Paris F-75005, France
6
Laboratory of Biomolecules (LBM), Paris F-75005, France
7
Brain Physiology Laboratory, Université Paris Descartes, PRES Sorbonne Paris Cité, Paris F-75006, France
8
CNRS UMR 8118, Paris F-75006, France
9
Inserm U836, Grenoble Neuroscience Institute, Research Group 3, LabEx Ion Channel Science and Therapeutics, BP170, 38042 Grenoble Cedex 09, France
10
Université Joseph Fourier, Grenoble, France
Abstract:
Semiconductor nanocrystals (NCs) or quantum dots (Qdots) are strong, point-like emitters that are increasingly used for biological and medical imaging. Here, we report on FRET-based Ca2+ and H+ nanobiosensors that are used for the intracellular detection of local cationic transients. For this purpose, we selected the commercially available CANdot®565QD as the donor and customized red-emitting rhodamine-based cationic indicators as the acceptor. The QDs were stably functionalized using the same SH/maleimide (NH2) cross-link chemistry that is utilized for all wanted reactants, which were mixed at the desired final stoichiometry; an ionic sensor and cell penetrating peptides were utilized to favor internalization. Our work validates the use of these constructs for the detection of intracellular Ca2+ and pH transients in living cells.
Keywords:
Quantum dot biosensors, Nanoparticle surface chemistry, FRET-based Ca2+ and H+ probes, Red emitting indicator, Intracellular Ca2+ and H+ fluorometry, Cell penetrating peptide.

Type of Paper: Review
Tentative Title: Revealing Nucleic Acid Mutations Using FRET Probes
Authors: Nina P. L. Junager, Jacob Kongsted and Kira Astakhova
Affiliations: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark
Abstract: Nucleic acid mutations are of tremendous importance in modern clinical work, biotechnology and in fundamental studies of nucleic acids. Therefore a rapid, cost effective, and reliable detection of mutations is a subject of extensive research.
Today, FRET probes are among the most frequently used tools for this task. However, multiple parameters must be taken into account in order to create efficient FRET probes which are sensitive to nucleic acid mutations.
In this review, we focus on the design principles of such probes and available computational methods which allow for their rational design. Applications of advanced, rationally designed FRET probes range from new insights into cancer heterogeneity at the single-cell level to gaining new knowledge on nucleic acid structures directly in living cells.


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