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4616 KiB

Open AccessArticle

A Toolbox of Genetically Encoded FRET-Based Biosensors for Rapid l-Lysine Analysis

by Victoria Steffen, Julia Otten, Susann Engelmann, Andreas Radek, Michael Limberg, Bernd W. Koenig, Stephan Noack, Wolfgang Wiechert and Martina Pohl

Sensors 2016, 16(10), 1604; https://doi.org/10.3390/s16101604 - 28 Sep 2016

Cited by 30 | Viewed by 8215

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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Figure 1

Figure 1
Schematic comparison between the sensor with the native (a) and the circular permutated (b) l-lysine-l-arginine-l-ornithine binding protein (LAO-BP) with ECFP (blue), Citrine (yellow), bound l-lysine (black) and the N- and C-terminus connecting linker (dark green). The blue and yellow spheres at the N- and C-terminus, respectively, indicate the attached fluorescent proteins. Full article ">Figure 2
Binding isotherms of the two sensors constructed with the native (nLAO-BP, black) and the circular permutated (cpLAO-BP, red) LAO binding protein. Also indicated are the readout parameters R0, Rsat, Kd (corresponding to the affinity), and ΔR (as a measure for the sensor sensitivity). Full article ">Figure 3
Toolbox of nine different Förster Resonance Energy Transfer (FRET)-based biosensor constructs based on the cpLAO-BP (green) from E. coli, using ECFP (blue) as a FRET donor, and Citrine (yellow) as a FRET acceptor. The linker sequences between the fluorescent proteins and the binding protein are shown in magenta. The spring symbolizes a flexible linker (F: (GGS)4), the block stands for a rigid linker (R: KLYPYDVPDYA), 0: no linker. The abbreviations for the nine different sensor constructs used in the text are shown next to their respective pictograms. Full article ">Figure 4
Binding isotherms of the nine different sensor toolbox variants. Next to the curves, the pictograms of the sensor constructs are shown. In all sections, the construct without additional linkers is shown in black. (a), the sensor variants without an N-terminal linker construct are presented; (b) the sensor variants with an N-terminal flexible linker construct are shown; (c) the sensor variants with an N-terminal rigid linker are shown; (d) the Kd-values for l-lysine of all constructs are listed. Full article ">Figure 5
Comparison of binding isotherms of the sensor prototype (00) in 20 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (black), in culture supernatant, and in fresh CGXII-medium, respectively. The curve in 20 mM MOPS buffer, pH 7.3 (black) was recorded in 96-well plates in a microtiter plate spectrofluorimeter (M-200, Tecan, Männedorf, Switzerland). 90 µL sensor solution (in 20 mM MOPS buffer, pH 7.3, sensor concentration OD515 nm = 0.2 equals 0.18 mg/mL) and 10 µL of the respective l-lysine stock solution (10×) were used to achieve the given l-lysine concentration by a 1:10 dilution. The calibration in fresh CGXII-medium with 10 g/L glucose [<a href="#B47-sensors-16-01604" class="html-bibr">47</a>] and culture supernatant of a C. glutamicum wildtype culture [<a href="#B38-sensors-16-01604" class="html-bibr">38</a>] in the stationary phase were recorded in Flowerplates® directly in the BioLector® cultivation device. 900 µL medium (pH 7, green) or culture supernatant (pH 7.5, red) and 100 µL sensor solution (in 20 mM MOPS buffer, pH 7.3, sensor concentration OD515 nm = 0.4 equals 0.36 mg/mL) were used. Full article ">Figure 6
Workflow for cultivation of l-lysine producing C. glutamicum with in-plate calibration: Overview of the cultivation and sampling process including the online and offline analytics. The cells were cultivated in one halve of the wells of a Flowerplate® while the other halve was reserved for calibration standards. Online analytics were performed during the entire cultivation time with optodes on the bottom of the well (measurement of pH and pO2) and with measurement of scattered light for observation of the biomass formation. The cultivation of l-lysine producing C. glutamicum DM1933-cells was performed in 24 identically inoculated wells of a Flowerplate®. At each sampling point, one row of the plate was sampled and subsequently the biosensor solution (in 20 mM MOPS buffer, pH 7.3, sensor concentration OD515 nm = 0.4 equals 0.36 mg/mL) was added. In the offline analytics l-lysine concentrations were determined with HPLC and the colorimetric Ninhydrin assay. Full article ">

2890 KiB

Open AccessArticle

Characterization of the ER-Targeted Low Affinity Ca²⁺ Probe D4ER

by Elisa Greotti, Andrea Wong, Tullio Pozzan, Diana Pendin and Paola Pizzo

Sensors 2016, 16(9), 1419; https://doi.org/10.3390/s16091419 - 2 Sep 2016

Cited by 32 | Viewed by 8811

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 (K_d) 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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6143 KiB

Open AccessArticle

Imaging of Metabolic Status in 3D Cultures with an Improved AMPK FRET Biosensor for FLIM

by George Chennell, Robin J. W. Willows, Sean C. Warren, David Carling, Paul M. W. French, Chris Dunsby and Alessandro Sardini

Sensors 2016, 16(8), 1312; https://doi.org/10.3390/s16081312 - 19 Aug 2016

Cited by 13 | Viewed by 9087

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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Schematic representation of the structure of AMPK, its regulation and function. Energetic status is sensed by the gamma subunit. The catalytic alpha subunit functions as the effector of this protein complex by phosphorylating target proteins for metabolic regulation. Full article ">Figure 2
Confocal TCSPC FLIM of AMPKAR-NES and T2AMPKAR-NES. Top panel, exemplar intensity images and lifetime maps of T2AMPKAR-NES for both DMSO exposed (Left) and 25 µM 991 activated (Right) cells are shown; Middle left panel, exemplar fluorescence decay profile (blue circles) plotted with double exponential fit to data (blue line), IRF (red dashed line) and residuals (lower); Middle right panel, data shown are from three separate experiments. Fluorescence lifetimes for individual cells are shown in dot plot; Lower left panel, mean difference in biosensor mean weighted fluorescence lifetime (n = 3). Lifetimes are shown in picoseconds (shown in image). Scale bar = 20 µm. Full article ">Figure 3
Time course of activation of AMPK by 991. (Left) time course montage of confocal TCSPC FLIM maps of T2AMPKAR-NES weighted mean fluorescence lifetimes, an image was acquired each minute; (Right) average weighted mean T2-AMPKAR-NES donor fluorescence lifetime in response to addition of 50 µM 991. The asterisk indicates the point of compound addition. Lifetimes are shown in picoseconds (shown in image). Scale bar = 100 µm. Full article ">Figure 4
T2AMPKAR-NES dose response to 991. Upper panel: montage of confocal TCSPC FLIM maps of the weighted mean lifetime for the dose response; Lower left panel: plot of the mean weighted mean lifetime dose response for 991 (n = 6); Lower right panel: AMPK activity detected by automated Western blotting (WES) of AMPK α phosphothreonine-172, phospho-ACC and AMPK β. Lifetimes are shown in picoseconds (shown in image). Scale bar = 100 µm. Full article ">Figure 5
TPE-TCSPC FLIM in spheroids expressing T2AMPKAR-T391A-NES. Top left panel: the weighted mean fluorescence lifetime map for 2D cultures. Top right panel: the weighted mean fluorescence lifetime map for three spheroids at different depths (shown in panel); Left lower panel: exemplar fluorescence decay profile plotted (blue circles) with double exponential fitting (blue line), IRF (red dashed line), and residuals (lower); Lower right panel: plot of the mean weighted mean fluorescence lifetime versus depth. Lifetimes are shown in picoseconds (shown in image). Scale bar = 100 µm. Full article ">Figure 6
Titration of 991 in spheroids expressing T2AMPKAR-NES using TPE TCSPC FLIM. Left panel: FLIM map of weighted mean fluorescence lifetime with increasing concentrations of 991 (shown in panel). Upper right panel: plot of the mean weighted mean fluorescence lifetime dose response for 991. Comparison of spheroids expressing T2AMPKAR-T391A-NES is also shown; Lower right panel: exemplar fluorescence decay profile (blue circles) plotted with double exponential fitting (blue line), IRF (red dashed line) and residuals (lower). Lifetimes are shown in picoseconds (shown in image). Scale bar = 100 µm. Full article ">Figure 7
Titration of phenformin in spheroids expressing T2AMPKAR-NES using TPE TCSPC FLIM. Left Panel: montage of FLIM maps of the weighted mean fluorescence lifetimes as phenformin concentration is increased (shown in panel). Upper right panel: plot of the whole spheroid and core mean weighted mean lifetimes; Lower right panel: exemplar images of the whole spheroid and core region segment. Lifetimes are shown in picoseconds (shown in image). Scale bars = 100 µm. Full article ">

3516 KiB

Open AccessArticle

Anchoring of FRET Sensors—A Requirement for Spatiotemporal Resolution

by Elena V. Ivanova, Ricardo A. Figueroa, Tom Gatsinzi, Einar Hallberg and Kerstin Iverfeldt

Sensors 2016, 16(5), 703; https://doi.org/10.3390/s16050703 - 16 May 2016

Cited by 3 | Viewed by 5467

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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Graphical abstract

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Full article ">Figure 1
Anchoring enables monitoring of FRET sensors throughout the cell including thin processes. (A) Schematic illustration of an anchored (taFS-VEID) and a non-anchored (naFS-VEID) FRET sensors. Both sensors contain a tandem VEID sequence between ECFP and EYFP. naFS-VEID was generated by introducing a stop codon at the 5′-end of tau-encoding sequence within the taFS-VEID cDNA; (B) Representative images of differentiated SH-SY5Y cells overexpressing taFS-VEID (upper panel) and naFS-VEID (lower panel); (C) Representative images of SK-N-AS cells overexpressing taFS-VEID (upper panel) and naFS-VEID (lower pannel). Note the absence of signal from taFS-VEID, but not naFS-VEID, in the nucleus. The epifluorecence images were linearly adjusted to display 0.1% saturated pixels. Scale bar 10 µm. Full article ">Figure 2
Anchored FRET sensors for detection of active caspases at subcellular level. (A) Representative ratiometric (FRET/ECFP) time-lapse images of SK-N-AS cells transfected with taFS-VEID (upper panel) or naFS-VEID (lower panel) and treated with 1 µM staurosporine. Note the local differences in FRET within the cells expressing taFS-VEID. The early decline in FRET in the central parts of the taFS-VEID-expressing cells is likely reflecting liberation of ECFP from the anchorage and its resulting ability to diffuse. Scale bar 10 µm. The video montage of the time lapse images is available as <a href="#app1-sensors-16-00703" class="html-app">Supplementary materials</a>; (B) Average of temporally aligned ratio values of the fraction of pixels from each cell retaining the highest FRET (10th percentile) is plotted over time (n = 8 for taFS and n = 5 for naFS); (C) Apoptotic stimuli induce specific fragmentation of anchored (taFS) and non-anchored (naFS) FRET sensors. Human neuroblastoma SK-N-AS cells overexpressing either of the sensors were treated with 1 µM staurosporine (STS) for 3 h. Total cell lysates were analyzed by western blot with anti-GFP antibodies. Full article ">

1264 KiB

Open AccessArticle

Detection of Gold Nanoparticles Aggregation Growth Induced by Nucleic Acid through Laser Scanning Confocal Microscopy

by Ramla Gary, Giovani Carbone, Gia Petriashvili, Maria Penelope De Santo and Riccardo Barberi

Sensors 2016, 16(2), 258; https://doi.org/10.3390/s16020258 - 19 Feb 2016

Cited by 8 | Viewed by 6329

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)

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1975 KiB

Open AccessArticle

Evaluating Quantum Dot Performance in Homogeneous FRET Immunoassays for Prostate Specific Antigen

by Shashi Bhuckory, Olivier Lefebvre, Xue Qiu, Karl David Wegner and Niko Hildebrandt

Sensors 2016, 16(2), 197; https://doi.org/10.3390/s16020197 - 4 Feb 2016

Cited by 35 | Viewed by 7526

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)

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Figure 1
Optical characterizations of Tb-AB conjugates. (A) Absorption spectrum (dotted line) of the Tb-AB conjugate, of which the Tb peak at 340 nm and the AB peak at 280 nm were used to calculate the Tb per AB labeling ratio. The PL spectrum (solid line) was acquired upon excitation in the Tb absorption band (365 ± 2 nm); (B) Pulsed (Xe flash lamp at 100 Hz) excitation (360 ± 2 nm) was used to measure the PL decay at 490.0 ± 0.5 nm. The slightly bi-exponential decay curve (τ1 = 0.4 ± 0.3 ms, τ2 = 2.70 ± 0.02 ms) gave an amplitude-averaged decay time of τ(Tb) = 2.6 ± 0.2 ms. Full article ">Figure 2
Optical characterizations of QD-AB conjugates. (A) Absorption (dotted lines) and PL (solid lines) of QD605 (blue), QD655 (green) and QD705 (red). To show the spectral overlap between Tb emission and QD absorption and the differences in PL wavelengths also the Tb PL spectrum is shown (gray spectrum in the background). Förster distances were calculated as R0(QD605) = 8.8 ± 0.4 nm, R0(QD655) = 10.5 ± 0.5 nm, and R0(QD705) = 11.2 ± 0.6 nm; (B) Pulsed (405 nm diode laser at 1 MHz) excitation was used to measure the PL decay curves at the respective PL peaks of the QDs. The multi-exponential decay curves (same colors as in A) gave amplitude-averaged decay times of τ(QD605) = 6.3 ± 1.2 ns, τ(QD655) = 14.0 ± 0.9 ns, and τ(QD705) = 80.0 ± 3.0 ns. Full article ">Figure 3
PSA immunoassay calibration curves of the different Tb-QD FRET systems using QD605 (A); QD655 (B); and QD705 (C). Bottom abscissae give total PSA (TPSA) concentrations in nM, top abscissae give TPSA concentration in ng/mL. LODs were determined using the linear parts of the calibration curves (solid lines) and the standard deviation of the FRET-ratio at zero TPSA concentration (Equation (4)). Full article ">Figure 4
PSA immunoassay calibration curves for the detection of PSA in a duplexed detection format using the Tb-QD655 (green) and Tb-QD705 (red) FRET-ratios measured from the same samples (for each concentration) that contained both QD655 and QD705 AB conjugates. Full article ">Figure 5
F(ab) conjugation of QD605, QD655, and QD705 (for sizes and shapes cf. <a href="#sensors-16-00197-t001" class="html-table">Table 1</a>) was performed by transferring the amine-reactive QDs to maleimide-reactive QDs (mal-QD) using a sulfo-EMCS crosslinker (left). IgG was fragmented to F(ab) using a commercial fragmentation kit. The available disulfides on the F(ab) were reduced to sulfhydryls, which allowed a conjugation to the mal-QDs. Full article ">Figure 6
Principle of the Tb-to-QD FRET immunoassay against PSA. Both QD and Tb AB conjugates (left) are present in the assay solution at a constant concentration. The addition of PSA leads to the formation of [QD-AB]-PSA-[Tb-AB] complexes and a close proximity of Tb and QD, which results in FRET (middle). This assay leads to a typical immunoassay calibration curve, for which the FRET intensity increases (first linearly and then approaching a maximum value when the PSA concentration reaches the concentration of QD-ABs or Tb-ABs and a formation of further FRET- pairs is not possible) with increasing PSA concentration (right). Full article ">

1707 KiB

Open AccessArticle

Modulation of Intracellular Quantum Dot to Fluorescent Protein Förster Resonance Energy Transfer via Customized Ligands and Spatial Control of Donor–Acceptor Assembly

by Lauren D. Field, Scott A. Walper, Kimihiro Susumu, Eunkeu Oh, Igor L. Medintz and James B. Delehanty

Sensors 2015, 15(12), 30457-30468; https://doi.org/10.3390/s151229810 - 4 Dec 2015

Cited by 11 | Viewed by 6402

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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Graphical abstract

Graphical abstract
Full article ">Figure 1
Fluorescent materials and Förster resonance energy transfer (FRET) rationale used in this study. (A) Schematic of CdSe-ZnS core-shell quantum dots (QDs) and various capping ligands; (B) Absorption and emission spectra of the QD and mCherry (mC) donor–acceptor pair showing significant overlap of the QD emission and mCherry absorption, allowing for FRET-sensitized emission of mCherry; (C) Intracellular QD-mCherry assembly strategies. mCherry was expressed either free in the cytosol or as a fusion to the C-terminus of the transmembrane receptor, CD1b. Upon microinjection of QDs, a His6 motif on the C-terminus of the mCherry drove the intracellular assembly of mCherry to the QD surface either in the cytosol or at the cytofacial leaflet of the plasma membrane. Full article ">Figure 2
Assembly and FRET analysis of the QD-mCherry (mC) donor–acceptor pair. (A) The 545 nm-emitting QDs capped with CL4 or PEG600-NTA were assembled with increasing ratios of mCherry and separated on 1% agarose gels; (B) Emission spectra of CL4 capped 550 nm QDs showing sensitization of QD donor emission with increasing ratio of mCherry acceptor. Data have been corrected for direct excitation of mCherry; (C) Plot of QD-mCherry FRET efficiency as a function of increasing QD-mCherry ratio. Line is fit to Equation (2). Full article ">Figure 3
Cytosolic assembly of QDs and mCherry. COS-1 cells expressing His6-terminated cytosolic mCherry proteins were microinjected with QDs coated with CL4, PEG600-NTA and PEG750-OMe. Fields were specifically imaged where cells contained mCherry only (red arrow), QDs only (blue arrow) or both QD and mCherry (yellow arrow) to allow for the appropriate FRET imaging controls. Note the level of efficient FRET present in cells injected with the CL4 QDs, with slightly less efficient FRET observed in cells injected with PEG600-NTA QDs. PEG750-OMe QDs appeared to mediate the least efficient FRET with intracellular mCherry. Scale bar is 20 µm. Full article ">Figure 4
Assembly of QDs and mCherry at the cytofacial leaflet of the plasma membrane. COS-1 cells expressing mCherry proteins on the cytofacial leaflet of the plasma membrane (as a fusion to CD1b) were microinjected with QDs coated with CL4, PEG600-NTA and PEG750-OMe. Imaging was performed as described for <a href="#sensors-15-29810-f003" class="html-fig">Figure 3</a>. Note the lower level of mCherry expression due to its localization at the plasma membrane. The FRET signal for the CL4 QDs bound to membrane expressed mCherry was ~50% that observed for cytosolic mCherry. Scale bar is 20 μm. Full article ">

1439 KiB

Open AccessArticle

FRET-Based Nanobiosensors for Imaging Intracellular Ca²⁺ and H⁺ Microdomains

by Alsu I. Zamaleeva, Guillaume Despras, Camilla Luccardini, Mayeul Collot, Michel De Waard, Martin Oheim, Jean-Maurice Mallet and Anne Feltz

Sensors 2015, 15(9), 24662-24680; https://doi.org/10.3390/s150924662 - 23 Sep 2015

Cited by 14 | Viewed by 8717

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)

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Figure 1
FRET-based red-emitting ion sensors: (A) Principle. Coupling a green-emitting quantum dot (QD: here CANdot®565) donor to a red-emitting rhodamine-based ion sensor, the acceptor, produces an analyte-dependent FRET signal upon donor excitation at 405 nm. The custom red-emitting ion sensors used here are Ca2+ (alternatively H+) indicators, the emission of which is quenched by PET in absence of their ligand. Analyte binding results in a strong fluorescence peaking at 602 nm; (B–D) Chemical structure of the sensors: All sensors are built on an extended rhodamine moiety (blue). The two Ca2+ sensor families incorporate a BAPTA moiety (green), without (B) and with (C) an oxygen introduced on one of the aromatic ring of the BAPTA for the lower and higher affinity families: CaRuby1 (µM-mM range) and CaRuby2 (sub-µM range), respectively. Substitutions (Z1, Z2 in red with Z1=Cl, Z2=H for the chloride derivatives, and Z1=H, Z2=F for the fluorine derivatives, Z1=H, Z2=Me for CaRu1-Me) yield compounds with a finely tunable KD for Ca2+ binding. The pH sensor family (HRubies, (D)) is based on the addition of a phenol instead of a BAPTA. Note that all compounds bear an azido/alkyne side arm for click chemistry and the resulting potential for high-yield coupling reactions. The azide bearing linker is introduced in the bridge between the two aromatic rings of the BAPTA for the CaRubies1, and on the additional oxygen for the CaRubies2. HR-PiAC bears an alkyne moiety at the ortho position of the phenol through a piperazine carbamate link; (E–G) Spectral properties of retained donor/acceptor pairs (E) normalized absorbance and emission spectra (dashed and plain lines, respectively) of QD565 (green) and CaRu-Me (red). Since there is only a slight Ca2+ sensitivity of CaRu-Me absorbance when switching from EGTA- to 2 mM Ca2+-containing solution, the KDs of CaRu-Me and QDCaRu-Me were similar, as expected. Similar properties are expected with CaRu2-F and HR-PiAC since their absorbance is respectively Ca2+ and pH insensitive, as illustrated in panels (F) and (G), where blue and red traces are in absence and presence of the analyte, respectively. Full article ">Figure 2
Assembly of FRET-based ion sensors. CANdots have a CdSe core surrounded by a double layer of CdZn and ZnS. The QD TOP/TOPO (Step 1) passivating layer was replaced by a hydrophilic coating peptide made by mixing 50% cysteine-(SH function) and 50% lysine-(NH2 function) terminated peptides (Cys-peptide: Ac-CGSESGGSESG(FCC)3F-amide and Lys-peptide: NH2-KGSESGGSESG(FCC)3F-amide, respectively). Independently, an azide/alkyne-terminated ligand was bound to a clickable NH2-PEG to form a PEGylated dye (Step 2). Nanoparticles were then functionalized by adding the pegylated rhodamine-based sensor (red dots) (Step 3) using a SH/NH2 crosslinking reaction. Other NH2 terminated ligands can be added using the same crosslinking reaction and are included in stoichiometric ratio (not shown). Full article ">Figure 3
FRET between a QD565 donor and rhodamine-based ion-sensing acceptor fluorophores. FRET was measured as donor quenching upon QD excitation at 407 nm and acceptor sensitization, as a function of A/D ratio. Experiments were performed at an elevated analyte concentration (500 µM) to fully relieve the PET quenching of the dye. (A) Series of mixed spectra obtained by increasing the number of PEG5kDa -CaRuby2-F while keeping QD concentration constant (40 nM), in saturating Ca2+, 500 µM. The A/D ratio was determined by absorbance measurements at 407 and 581 nm, to evaluate QD565 and CaRuby concentrations, respectively; (B) Relative donor quenching (QD photoluminescence, red) and FRET efficiency (black) are reported after linear unmixing of donor and acceptor spectra, see Supplementary Material 6. Green data points shows acceptor sensitization (FCaRuby (exc. @ 350 nm) − FQD (exc. @ 350 nm)/(FCaRuby (exc. @ 535 nm)); (C,D) Similar experiments were carried with QD-HR-PiAC, with the pH adjusted to 4. In both cases, symbols ▲, ● show results from two independent runs. Full article ">Figure 4
(A) Fluorometric titration of FRET-based nanobiosensor assemblies: QD-PEG5kDa-CaRu2F (prepared with A/D ratio= 7.4, and using the Invitrogen Ca2+ buffer kit to adjust [Ca2+]); Spectra (left) were obtained when following Ca2+ concentrations were successively applied: 1, 17, 38, 65, 100, 150, 225, 351, 602 nM and 1.35 and 39 µM from bottom to top traces; (right) Resulting titration curves using direct excitation at 545 nm or FRET excitation upon QDs excitation at 407-nm; (B) QD-PEG5kDa -HR-PiAC (A/D ratio= 5.6), universal pH buffer, see Supplementary 3, p. 830 in [<a href="#B20-sensors-15-24662" class="html-bibr">20</a>]). Fluorescence curves are corrected for the pH sensitivity of the QDs fluorescence (see <a href="#sensors-15-24662-s001" class="html-supplementary-material">Figure S3</a> for details). Spectra shown on the left correspond to pH 6, 7.45, 7.65, 8.3, 8.9, 10.3, 11.45 and 11.9 from top to bottom, respectively. On the right: measured KDs are similar when carrying fluorometric titration either by direct excitation (at 545 nm) or by FRET excitation (at 407 nm). Full article ">Figure 5
Live-cell imaging of intracellular ion distributions. (A–C) Confocal images of BHK-21 cells after incubation with QD-HRu PiAC (A) co-stained by Lysotracker Green; (B) and a merged image of the two channels (C). Scale bar is 20 µm; (D–G) Intracellular calibration of pH sensors in a suspension of BHK-21 cells using flow cytometry. Typical histograms showing the internalization of the QD565/H-Ruby pH sensors according to the H-Ruby intensity (in grey, the cell without sensors, and in red, the cells having internalized pH sensors) upon direct (D) and by FRET excitation (F). Calibration curves of the intracellular pH sensors clamped at different pH with the ionophore nigericin were obtained by direct (E) and by FRET excitation (G) of H-Ruby. Intracellular pH as measured by the red histograms of fluorescence H-Ruby in (D,F) was evaluated (black dots) by extrapolation on the calibration curves (E,G), respectively; (H,I) Read-out of local Ca2+ transients upon glutamate application on BHK cells stably expressing the NR1 and NR2A subunits of the N-methyl-D-aspartate receptors (NMDARs). (H) Firstly internalized biosensors (QD-CaRuby-CPP in a ratio 1:10:10) were localized by superimposition of the bright-field and time-averaged TIRF image of cultured BHK cells detected in the green channel upon 405-nm evanescent-wave excitation of the QDs. (I) Repetitive stimulation evokes reversible Ca2+ transients. Superimposed traces obtained in the red channel following 568-nm excitation are responses of the Ca2+ sensor to two successive bath applications of NMDAR agonists at a saturating concentration, with a 15 min long continuous bath perfusion of control saline for recovery from desensitization in between. <a href="#sensors-15-24662-f005" class="html-fig">Figure 5</a>H,I is reprinted with permission from Zamaleeva et al., 2014 [<a href="#B8-sensors-15-24662" class="html-bibr">8</a>]. Copyright 2015 American Chemical Society. Full article ">Figure 6
Twisted-intramolecular charge transfer in CaRuby (at the left) and HRuby (at the right) dyes. Full article ">

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Open AccessArticle

Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity

by Butaek Lim, Ji-In Park, Kyung Jin Lee, Jin-Won Lee, Tae-Wuk Kim and Young-Pil Kim

Sensors 2015, 15(8), 17977-17989; https://doi.org/10.3390/s150817977 - 23 Jul 2015

Cited by 10 | Viewed by 8263

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)

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7022 KiB

Open AccessArticle

Generation of Red-Shifted Cameleons for Imaging Ca²⁺ Dynamics of the Endoplasmic Reticulum

by Markus Waldeck-Weiermair, Helmut Bischof, Sandra Blass, Andras T. Deak, Christiane Klec, Thomas Graier, Clara Roller, Rene Rost, Emrah Eroglu, Benjamin Gottschalk, Nicole A. Hofmann, Wolfgang F. Graier and Roland Malli

Sensors 2015, 15(6), 13052-13068; https://doi.org/10.3390/s150613052 - 4 Jun 2015

Cited by 30 | Viewed by 9838

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)

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Full article ">Figure 1
Evaluation of GFP/OFP cameleons upon ER targeting: (A) schematic composition of D1ERGO-Cam1 and D1ERGO-Cam2; and (B) representative confocal images for colocalization analysis. INS-1 cells were co-transfected with D1ER and either D1ERGO-Cam1 or D1ERGO-Cam2, respectively. Images of GFP/OFP-based cameleons were taken from the mKO emission at 570 nm (left panels); D1ER was monitored in its CFP emission at 480 nm (middle panels); and an overlay of images (right panels); (C) Average curves of FRET measurements in HeLa cells expressing either D1ER (cyan curve, n = 14), D1ERGO-Cam1 (dark grey curve, n = 10) or D1ERGO-Cam2 (green curve, n = 7) upon treatment with 100 µM histamine and 15 µM BHQ in the absence of extracellular Ca2+. SOCE was accomplished by the subsequent addition of 2 mM Ca2+. Full article ">Figure 2
Functional evaluation of red-shifted cameleons in the ER. Permeabilized HeLa cells transfected with individual red-shifted sensors targeted to the ER were recorded in an extracellular Ca2+-free solution and in a 2 mM Ca2+ containing environment, respectively. Representative curves showing FRET signals of each indicator excited at 420, 450, 460, 470, 480 or 490 nm and emissions were collected from either (A) D1ERGO-Cam1 (dark grey curve), D1ERGO-Cam2 (green curve), D1ERRG-Cam1 (pink curve), D1ERRG-Cam2 (purple curve), D1ERGR (light grey curve), D1ERTG (violet curve) or (B) D1ERRC (light green curve), D1ERCR (grey curve), D1ERGmR2 (black curve), D1ERmR2G (dark red curve), D1ERmR2C (blue curve) or D1ERCmR2 (red curve). Full article ">Figure 3
Column statistics of maximal ER Ca2+-release in HeLa cells transfected with individual GFP/RFP-based cameleons. Bars representing FRET change upon treatment with 100 µM histamine and 15 µM BHQ detected by D1ERRG-Cam1 (pink bar, n = 13), D1ERRG-Cam2 (purple bar, n = 10), D1ERGR (light grey bar, n = 4), D1ERTG (violet bar, n = 7), D1ERRC (light green bar, n = 10), D1ERCR (grey bar, n = 4), D1ERGmR2 (black bar, n = 11), D1ERmR2G (dark red bar, n = 6), D1ERmR2C (blue bar, n = 11) or D1ERCmR2 (red bar, n = 7). * P < 0.05 for D1ERCmR2 vs. all other indicators. Full article ">Figure 4
Determination of the Kd in permeabilized HeLa cells expressing D1ERCmR2. [Ca2+] was titrated to quantify the FRET ratio in percentage upon Ca2+-binding within D1ERCmR2 at 1 µM (n = 21), 3 µM (n = 21), 10 µM (n = 37), 30 µM (n = 37), 100 µM (n = 26), 300 µM (n = 43), 1 mM (n = 35), 2 mM (n = 17) and at 10 mM (n = 18). Full article ">Figure 5
Dual visualization of fura-2 and D1ERCmR2 in single individual HeLa cells. (A) Confocal images of D1ERCmR2 expressing and fura-2 loaded HeLa cells. Subcellular structures of D1ERCmR2 were either illuminated at 473 or 515 nm and emissions were recorded at 525 (Clover, upper left panel) or 570 nm (mRuby2, upper right panel), respectively. Fura-2 was excited at 405 and emitted light was imaged at 460 nm (lower left panel). Images overlaid (lower right panel); (B) Representative curves of cytosolic and ER [Ca2+] in a single HeLa cell treated with 100 µM Histamine and 15 µM BHQ in a nominal Ca2+ free buffer and subsequent readdition of 2 mM Ca2+; (C) Zoom into event of SOCE reveals a delayed ER Ca2+ refilling; (D) Spatiotemporal correlation of [Ca2+]Cyto and [Ca2+]ER during SOCE in percentage of the maximum increase shown in panel C. Full article ">Figure 6
Time course of [Ca2+]Cyto and [Ca2+]ER in a single HEK E2 cell visualizing oscillatory traces in 1 mM Ca2+. (A) Single emission curves of fura-2 either excited at 340 nm (black curve) or 380 nm (blue curve); (B) Donor and FRET acceptor fluorescences of D1ERCmR2 at 560 nm (red curve) or 510 nm (green curve), respectively; (C) Overlay of ratio curves from fura-2 (blue curve) or D1ERCmR2 (red curve). Full article ">

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Open AccessArticle

MMP-2/9-Specific Activatable Lifetime Imaging Agent

by Marcus T.M. Rood, Marcel Raspe, Jan Bart ten Hove, Kees Jalink, Aldrik H. Velders and Fijs W.B. Van Leeuwen

Sensors 2015, 15(5), 11076-11091; https://doi.org/10.3390/s150511076 - 12 May 2015

Cited by 7 | Viewed by 12974

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)₃and 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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Graphical abstract

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Full article ">Figure 1
Schematic overview of the difference between the use of an activatable luminescence imaging agent (left) and an activatable lifetime imaging agents (right), both based on a hairpin motive. It is important to note that in the case of lifetime activation not only the intensity increases, but also the lifetime. This gives two parameters to follow the activation reaction. Full article ">Figure 2
(A,B) Excitation-emission plots of peptides 2L (A) and 2D (B). The peaks labeled as “FRET” are the peaks that show acceptor emission (670 nm) with donor excitation (550 nm); (C) Luminescence decay traces at 600 nm of 3L, 3D, and reference compound Ir(ppy)3-COOH in water. All compounds were excited with a 372 nm laser at 2.5 MHz. Full article ">Figure 3
(A–D) Cleavage assay of 2L and 2D by MMP-expressing cells (A,B) Emission intensity from peptides 2L (red circles) and 2D (blue squares) of the donor peak (Cy3, excitation 525 nm, emission 566 nm) (A) and the FRET peak (Cy5, excitation 525 nm, emission 666 nm) (B) in time; (C) Variations in emission spectra (excitation 525 nm) of 2L in time from blue (first time point) to red (last time point) and (D) the donor/acceptor ratio; (E–H) Cleavage assay of 3L and 3D by MMP-expressing cells; (E,G) Emission intensity changes from peptides 3L (red circles) and 3D (blue squares) of the peaks at 590 nm (E) and 666 nm (G) in time; (F,H) Change in emission of these peaks of 3L in time from blue (first time point) to red (last time point) with excitation at 420 nm (F) or 625 nm (H). Arrows indicate change in time. Full article ">Figure 4
Confocal images of SKOV-3 cells after 24 h incubation with 2L (A–C) or 2D (D–F). (A,D) Cy3 channel in green; (B,E) Cy5 channel in red; (D,F) Overlay of differential interference contrast, nuclear stain (Hoechst 33342, blue), Cy3 (green), and Cy5 (red). Excitation and emission wavelengths are described in the Experimental Section. Full article ">Figure 5
Confocal images of SKOV-3 cells after 24 h incubation with 3L (A–C) or 3D (D–F). (A,D) Ir(ppy)3 channel in green; (B,E) Cy5 channel in red; (C,F) Overlay of both channels. Yellow means colocalization of red and green. Full article ">Figure 6
(A) Luminescence decay traces of a suspension of SKOV-3 cells after 24 h incubation with 3L (red) or 3D (blue); (B) FLIM image of cells incubated with 3L; (C) FLIM image of cells incubated with 3D. The scalebar on the left depicts τ, going from 0 ns (blue) to 150 ns (red). Full article ">Figure 7
Synthesis of the activatable imaging agents discussed in this research. (a) Cy5, PyBOP, DIPEA, DMF; (b) TFA, H2O, TIS; (c) Cy3-NHS, H2O/DMSO; (d) Ir(ppy)3-β-Ala-COOH, DCC, NHS, H2O/DMSO. The amino acid sequence for cleavage is highlighted in red and comprised of either l-amino acid or d-amino acids. All other amino acids used to generate these structures were d-amino acids. Full article ">

940 KiB

Open AccessArticle

The Use of the LanthaScreen TR-FRET CAR Coactivator Assay in the Characterization of Constitutive Androstane Receptor (CAR) Inverse Agonists

by Alejandro Carazo and Petr Pávek

Sensors 2015, 15(4), 9265-9276; https://doi.org/10.3390/s150409265 - 21 Apr 2015

Cited by 22 | Viewed by 9034

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 ?M, 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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Graphical abstract

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Principle of the TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) LanthaSceenTM CAR Coactivator Assay. Full article ">Figure 2
Optimization of the TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) LanthaSceenTM Assay. Our TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) experiments were slightly modified to study whether a protocol alteration influenced the response to the CAR agonist. In (A), the reaction mixture was composed in the following order: CITCO, the fluorescein-labeled anti-PGC1α coactivator, Tb-labeled GST antibody and CAR LBD; in (B), the reaction mixture was composed in the following order: CAR LBD, the fluorescein-labeled anti-PGC1α coactivator, Tb-labeled GST antibody and CITCO; and in (C), the standard protocol was followed (the order of CITCO, CAR LBD, the fluorescein-labeled anti-PGC1α coactivator and Tb-labeled GST antibody). The dotted line represents background nonspecific fluorescence in the absence of CAR LBD. Full article ">Figure 3
Effect of PK11195 on CAR LBD activity in the TR-FRET LanthaScreenTM CAR Coactivator Assay. PK11195 in a serial dilution was tested in inverse agonistic or antagonistic modes together with the prototype CAR agonist CITCO (1 μM concentration) using the TR-FRET assay. Data are presented as the relative activation to background activity (no CAR LBD in the reaction mixture, set to 0%) and to the effect of CITCO (1 μM) set as 100% activation. The dotted line represents the constitutive activity of CAR LBD (vehicle-treated samples). Data are presented as the means and S.D. from three independent experiments (n = 3). Dose response curves were fitted using a sigmoidal dose response equation with a variable slope employing the software GraphPad PRISM ver. 6.06. Full article ">Figure 4
Effect of androstenol on CAR LBD activity in the TR-FRET LanthaScreenTM CAR Coactivator Assay. Androstenol in a serial dilution was tested in inverse agonistic or antagonistic modes together with the prototype CAR agonist CITCO (1 μM concentration) using the TR-FRET assay. Data are presented as the relative activation to background activity (set to 0%) and to the effect of CITCO (1 μM) set as 100% activation. The dotted line represents constitutive activity of CAR LBD (vehicle-treated samples). Data are presented as the means and S.D. from three independent experiments (n = 3). Full article ">Figure 5
Effect of clotrimazole on CAR LBD activity in the TR-FRET LanthaScreenTM CAR Coactivator Assay. Clotrimazole in a serial dilution was tested in inverse agonistic or antagonistic modes together with the prototype CAR agonist CITCO (1 μM concentration) using the TR-FRET assay. Data are presented as the relative activation to background activity (set to 0%) and to the effect of CITCO (1 μM) set as 100% activation. The dotted line represents constitutive activity of CAR LBD (vehicle-treated samples). Data are presented as the means and S.D. from three independent experiments (n = 3). Full article ">

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Open AccessArticle

A Homogenous Fluorescence Quenching Based Assay for Specific and Sensitive Detection of Influenza Virus A Hemagglutinin Antigen

by Longyan Chen and Suresh Neethirajan

Sensors 2015, 15(4), 8852-8865; https://doi.org/10.3390/s150408852 - 15 Apr 2015

Cited by 20 | Viewed by 8805

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)

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Figure 1

Figure 1
Schematic illustration of the assay architecture for sensing avian influenza virus (AIV) surface antigen HA. Full article ">Figure 2
(a) Absorbance spectra of QDs with and without APBA conjugation; (b) FTIR spectra of bare carboxyl QDs, APBA-QDs and Glycan-QDs (6G). Full article ">Figure 3
(a) Fluorescent spectra of 6G-QDs/mAb-H1-Au NPs mixture with and without antigen H1N1-HA; (b) TEM image of 6G-QDs/mAb-H1-Au NPs mixture after incubation with H1N1- HA. Blue arrow indicates Ab-Au NPs which has higher electron density, while red arrow indicates Gly-QDs which has a relative lower electron density. Full article ">Figure 4
Cross-reactivity of two assays with human influenza virus H1N1 HA and avian influenza virus H5N1-HA. The mixtures, prepared with 6G-QDs/mAb-H1-Au NPs and with 3GQDs/mAb-H5-Au NPs, were tested using HAs derived from A/New Caledonia/20/1999 and A/Viet Nam/1194/2004 (120 nM), respectively. The control experiment was carried out using BSA (150 μM), (N = 3). Full article ">Figure 5
Detection of human influenza virus H1N1-HA and avian H5N1-HA by 6G-QDs/mAb- H1-Au NPs mixture and 3G-QDs/mAb-H5-Au NPs mixture, in PBS (a) and in human sera spiked PBS (b), respectively. (N = 3). Full article ">

1373 KiB

Open AccessArticle

Genotyping Single Nucleotide Polymorphisms Using Different Molecular Beacon Multiplexed within a Suspended Core Optical Fiber

by Linh Viet Nguyen, Sara Giannetti, Stephen Warren-Smith, Alan Cooper, Stefano Selleri, Annamaria Cucinotta and Tanya Monro

Sensors 2014, 14(8), 14488-14499; https://doi.org/10.3390/s140814488 - 8 Aug 2014

Cited by 9 | Viewed by 6094

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)

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Schematic diagram of the final stage of the suspended core optical fiber (SCF) core functionalized with dual-type molecular beacons. Picture of the SCF cross section shown on the right side is a scanning electron microscope (SEM) image of the SCF used in this work. Full article ">
Experiment setup for fluorescence measurement of the dual-type molecular beacons (MBs) immobilized SCF filled with DNA solutions. SMF and MMF are abbreviations for single mode and multimode mode optical fiber, respectively. The green laser operating at 532 nm was used to excite HEX dyes (for wild-type sequence) and the red laser operating at 638 nm was used to excite Cy5 dyes (for mutant-type sequence). Full article ">
Comparison of fluorescence enhancement between dual-type MB immobilized SCF and control SCF upon filling with target DNA solution. The control SCF shows negligible fluorescence enhancement. Full article ">
Hybridization test of the dual-type MB immobilized SCF filled with solution containing either one type of DNA sequence, e.g., wild or mutant sequence (homozygous) or both type (heterozygous). Fluorescence enhancement clearly indicates (a,b) the homozygous type or (c) heterozygous type; (d) The averaged value over four measurements of spectra as shown in (a–c), crossing point of HEX and Cy5 fluorescence indicate heterozygousity and well separated values of HEX and Cy5 fluorescence represent homozygousity. Full article ">

Review

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1939 KiB

Open AccessReview

A Guide to Fluorescent Protein FRET Pairs

by Bryce T. Bajar, Emily S. Wang, Shu Zhang, Michael Z. Lin and Jun Chu

Sensors 2016, 16(9), 1488; https://doi.org/10.3390/s16091488 - 14 Sep 2016

Cited by 341 | Viewed by 40662

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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Figure 1
The principle of fluorescence resonance energy transfer (FRET). (A) Spectral overlap between mClover3 and mRuby3. The spectral overlap integrand (a product of fd(λ), εA and λ4 in the Equation (2)) is indicated by the black dashed line; (B) FRET efficiency (FRET E) versus distance. The FRET E varies with the sixth power of distance between donor and acceptor. The Förster radius (r0) is the distance at which 50% FRET occurs. Compared to ECFP-EYFP, mClover3-mRuby3 exhibits a larger FRET E change because of the larger r0 at which the given FRET biosensor operates; (C) Two types of FRET biosensors: intramolecular and intermolecular FRET biosensors. The sensing domains undergo conformational changes (intramolecular) or inter-domain interactions upon biochemical changes, leading to the change in FRET E; (D) The relationship between the intensity ratio of acceptor to donor (IA/ID) and FRET E. The ratio of peaks of the emission spectrum acquired by a sensitivity-normalized spectrum-scanning device is non-linearly related to the actual FRET E. However, it is important to note that ratios taken through filter cubes and cameras are not equivalent to ratios derived from a spectrum-scanning device, as filter cubes pass different amounts of light depending on the transmission spectra and cameras exhibit wavelength-dependent sensitivity. Full article ">Figure 2
Normalized excitation (or absorbance) and emission spectra of FPs of representative two-color FRET pairs. (A) mTurquoise2-mCitrine, a CFP-YFP pair; (B) mClover3-mRuby3, a GFP-RFP pair; (C) eqFP650-iRFP, an FFP-IFP pair; (D) mAmetrine-tdTomato, a LSS-FP based pair; (E) mEGFP-sREACh, a dark FP-based pair; (F) EYFP-rsTagRFP, an optical highlighter FP-based pair. Full article ">Figure 3
Normalized excitation (or absorbance) and emission spectra of FPs of representative four-color FRET pairs: (A) mTagRFP-sfGFP and mVenus-mKOκ pairs, two FRET pairs with two excitations; and (B) ECFP-cpVenus and LSSmOrange-mKate2 pairs, two FRET pairs with single excitation. Full article ">

1895 KiB

Open AccessReview

Revealing Nucleic Acid Mutations Using Förster Resonance Energy Transfer-Based Probes

by Nina P. L. Junager, Jacob Kongsted and Kira Astakhova

Sensors 2016, 16(8), 1173; https://doi.org/10.3390/s16081173 - 27 Jul 2016

Cited by 19 | Viewed by 9380

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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1504 KiB

Open AccessReview

The ?-Lactamase Assay: Harnessing a FRET Biosensor to Analyse Viral Fusion Mechanisms

by Daniel M. Jones and Sergi Padilla-Parra

Sensors 2016, 16(7), 950; https://doi.org/10.3390/s16070950 - 23 Jun 2016

Cited by 27 | Viewed by 10307

Abstract

The ?-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 ?-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)

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CCF2-AM is composed of a Hydroxycoumarin donor conjugated to a Fluorescein acceptor via a β-lactam ring. In the absence of β-lactamase, excitation at 409 nm promotes Förster resonance energy transfer (FRET) between the fluorescent donor and acceptor molecules, resulting in emission at 520 nm. Cleavage of the β-lactam ring by β-lactamase separates the two molecules, disrupting FRET and producing a fluorescence shift from 520 nm to 447 nm. Full article ">Figure 2
A representation of a HIV-1 particle is shown. β-lactamase can be packaged into nascent HIV-1 virions by fusing it to the accessory protein Vpr. For purposes of clarity, several HIV-1 proteins are not depicted. Full article ">Figure 3
(1) Fusion between the virus particle and target cell (either at the cell membrane or from within endosomes) liberates the encapsulated β-lactamase (2) The enzyme is then able to access the cytoplasmic CCF2-AM FRET substrate (3) CCF2-AM cleavage occurs and the fluorescence profile is altered, indicating fusion has occurred. Full article ">Figure 4
Methodology pipelines for the TA-BlaM and RT-BlaM assays. Virus particles are allowed to fuse before the addition of CCF2-AM in the TA-BlaM assay, and cells are typically fixed before analysis. The red box in the TA-BlaM flow diagram applies to fusion kinetic assays and is omitted under fusion endpoint assay conditions. In a RT-BlaM assay, target cells are loaded with CCF2-AM before being exposed to virus, meaning fusion can be monitored in live cells. The timing used for several steps (x,y and z) vary and are discussed in more detail in <a href="#sec4-sensors-16-00950" class="html-sec">Section 4</a> of the main text. MOI = multiplicity of infection. Full article ">Figure 5
To obtain kinetic measurements of fusion using a TA-BlaM assay (Left), fusion must be stopped at various time points using a known fusion inhibitor (red cross). The level of fusion that occurred up to the time of inhibition can then be quantified. In a RT-BlaM assay (Right), fusion can be measured in real time in the absence of fusion inhibitors. Full article ">

1550 KiB

Open AccessReview

Fluorescent Proteins as Genetically Encoded FRET Biosensors in Life Sciences

by Bernhard Hochreiter, Alan Pardo-Garcia and Johannes A. Schmid

Sensors 2015, 15(10), 26281-26314; https://doi.org/10.3390/s151026281 - 16 Oct 2015

Cited by 145 | Viewed by 23278

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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Full article ">Figure 1
The basic principles of fluorescence and FRET. (A) Jablonski diagram, explaining the effect of fluorescence. Absorption of a photon by the fluorophore raises an electron to an excited energy state. Within this state, the electron drops to the ground level via radiationless vibrational relaxation. The excited state is very instable, leading to a relaxation back to the ground state within a few nanoseconds. The energy quantum of this difference is emitted via a photon of a specific wavelength, which is respectively longer than the absorbed wavelength, i.e., contains less energy; (B) Jablonski Diagram explaining the effect of FRET. Here, the energy that is released from the relaxation of the donor is taken up by a suitable acceptor in close proximity, leading to the excitation of one of its electrons, and further to the emission of a photon by the acceptor rather than the donor; (C) Correlation between the FRET efficiency and the distance between the fluorophores. The Förster radius R0 is the distance, where 50% of energy is transferred via FRET. Full article ">Figure 2
Structure and spectrum of fluorescent proteins. (A) The three-dimensional structure of Green fluorescent Protein (GFP) as it occurs naturally in the jellyfish species Aequorea Victoria [<a href="#B5-sensors-15-26281" class="html-bibr">5</a>]; (B,C) The spectrum of the fluorescent protein FRET-pair; (B) Cerulean (a cyan FP) and Venus (a yellow FP); and (C) GFP and RFP, also showing the overlap between donor emission and acceptor excitation, which is an important factor for the usability of a FRET-pair. Full article ">Figure 3
Effects of FRET on the spectroscopic properties. (A) FRET produces a change in the emissions intensities of the donor (EmD) and the acceptor (EmA), clearly measurable as a change in ratio; (B) FRET has a characteristic effect on the fluorescent lifetime τ, the delay between excitation and emission. Full article ">Figure 4
The basic measuring principle of fluorescence lifiteme in (A) time-domain, where the time between the exciting light pulse, and emission detection is measured in multiple single measurements to obtain a specific photon count curve; and (B) frequency domain, in which the delay in the oscillation of the electromagnetic wave is determined. Full article ">Figure 5
The different types of fluorescent-protein based FRET-biosensors. FRET biosensors indicate a biological change through a change in the FRET signal caused by an alteration of the donor-acceptor distance by (A) cleavage of a linker domain; (B) a conformational change due to internal alterations or (C) a conformational change due to the effect of an external mechanical force. Furthermore, the FRET signal might also be altered (D) by a change of the fluorescence properties triggered by a change of the surrounding environment of the fluorophores. The given schemes are only basic examples and many biosensors include alterations to these basic principles, e.g., for conformational change based sensors, the process can be inverted with a high FRET signal at resting state, and the decrease of said signal after posttranslational modification, or the fluorophore pair of a biosensor could be integrated between sensor domains in the middle of a construct. Full article ">

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Open AccessReview

Biosensing with Förster Resonance Energy Transfer Coupling between Fluorophores and Nanocarbon Allotropes

by Shaowei Ding, Allison A. Cargill, Suprem R. Das, Igor L. Medintz and Jonathan C. Claussen

Sensors 2015, 15(6), 14766-14787; https://doi.org/10.3390/s150614766 - 23 Jun 2015

Cited by 30 | Viewed by 9652

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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(a) The fluorescence spectra of NS-co-doped fluorescent carbon nanodots (NSCDs) as treated with different concentrations of methotrexate (MTX) ranging from 0–50.0 µM; the intensity decreases as the concentration of MTX increases and FRET is inhibited. The inset shows photographs that correspond to the increasing concentrations of MTX; (b) The linear relationship between fluorescence and MTX concentration. Reproduced with permission from Wang et al. [<a href="#B42-sensors-15-14766" class="html-bibr">42</a>]. Copyright 2015 Biosensors and Bioelectronics, Elsevier. PL, photoluminescence. Full article ">Figure 2
(a) Scheme showing the FRET process from upconverting phosphors (UCPs) to CNPs; (b) interference testing shows that the fluorescent intensity increases dramatically in the presence of thrombin. Reproduced with permission from Wang, Bao, Liu and Pang [<a href="#B84-sensors-15-14766" class="html-bibr">84</a>]. Copyright 2011 American Chemical Society. Full article ">Figure 3
Microscopic images of the NBD nanotubes upon the addition of the fluorescence acceptor dye QSY7, which quenches NBD via FRET. Reprinted with permission from Kameta et al. [<a href="#B85-sensors-15-14766" class="html-bibr">85</a>]. Copyright 2007 American Chemical Society. Full article ">Figure 4
FRET causes the aptamer to bind to graphene, thus quenching the fluorescence of an attached dye. The fluorescence is recovered when quadruplex-thrombin is formed, as it has a weak affinity to graphene, thus removing the dyes from the graphene. Reproduced with permission from Chang et al. [<a href="#B88-sensors-15-14766" class="html-bibr">88</a>]. Copyright 2010 American Chemical Society. Full article ">Figure 5
Intensity at donor emission (max 520 nm) of DNA-D-NT. (a) Förster resonance energy transfer (FRET) is clearly occurring, as indicated by the two alternating peaks. This, in turn, indicates DNA hybridization on nanotube surfaces. Emission of the donor on DNA-NT decreases with additions of attachment to the acceptor, thus the alternating peaks. The addition of complement conjugated with acceptor (cDNA-A) actually increases the donor emission at higher concentrations; (b) No FRET between donor and acceptor appears, indicating that there is no hybridization occurring, and the donor fluorescence remains significantly lower in the presence of cDNA-A than without it. Reproduced with permission from Jeng et al. [<a href="#B57-sensors-15-14766" class="html-bibr">57</a>]. Copyright 2006, American Chemical Society. Full article ">Figure 6
(a) Scheme showing DNA analysis using three probes (P5, P6, P7) in the presence of a blue T5 target; (b–d) fluorescence spectra for multicolor detection showing corresponding wavelengths when the probe is in the presence of different targets: (b) blue T5 at 494/526 nm/nm; (c) red T6 643/666 nm/nm; and (d) orange T7 587/609 nm/nm. Reproduced with permission from He et al. [<a href="#B90-sensors-15-14766" class="html-bibr">90</a>]. Copyright 2010 Advanced Functional Materials, John Wiley and Sons. Full article ">

8289 KiB

Open AccessReview

Förster Resonance Energy Transfer between Quantum Dot Donors and Quantum Dot Acceptors

by Kenny F. Chou and Allison M. Dennis

Sensors 2015, 15(6), 13288-13325; https://doi.org/10.3390/s150613288 - 5 Jun 2015

Cited by 237 | Viewed by 23802

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)

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3238 KiB

Open AccessReview

QD-Based FRET Probes at a Glance

by Armen Shamirian, Aashima Ghai and Preston T. Snee

Sensors 2015, 15(6), 13028-13051; https://doi.org/10.3390/s150613028 - 4 Jun 2015

Cited by 57 | Viewed by 12585

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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599 KiB

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

by Bernhard Hochreiter, Alan Pardo-Garcia and Johannes A. Schmid

Sensors 2015, 15(11), 29182; https://doi.org/10.3390/s151129182 - 18 Nov 2015

Viewed by 3552

Abstract

The authors wish to add an Acknowledgments section to their paper published in Sensors [1], doi:10.3390/s151026281, website: https://www.mdpi.com/1424-8220/15/10/26281. [...] Full article

(This article belongs to the Special Issue FRET Biosensors)

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