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608 KiB

Open AccessEditorial

Fluorescent Sensors for Biological Applications

by Hui-wang Ai

Sensors 2014, 14(9), 17829-17831; https://doi.org/10.3390/s140917829 - 25 Sep 2014

Cited by 13 | Viewed by 6773

Abstract

Fluorescence is one of the most important analytical methods used in biological studies. In the past decade or two, instrumentation in this field has greatly advanced, and now it is possible to detect single photons or fluorescent molecules [1,2], or break the Abbe [...] Read more.

Fluorescence is one of the most important analytical methods used in biological studies. In the past decade or two, instrumentation in this field has greatly advanced, and now it is possible to detect single photons or fluorescent molecules [1,2], or break the Abbe diffraction limit to distinguish two points spaced less than 50 nm apart [3]. Concurrently, the development of improved fluorescent probes, which can be coupled with state-of-the-art instruments, has been equally important. This special issue on “fluorescent biosensors” in Sensors reports recent results from eight research groups in the field of sensor development. It includes three review articles, and six research articles reporting original results. [...] Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

Research

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722 KiB

Open AccessArticle

Coumarin-Based Fluorescent Probes for Dual Recognition of Copper(II) and Iron(III) Ions and Their Application in Bio-Imaging

by Olimpo García-Beltrán, Bruce K. Cassels, Claudio Pérez, Natalia Mena, Marco T. Núñez, Natalia P. Martínez, Paulina Pavez and Margarita E. Aliaga

Sensors 2014, 14(1), 1358-1371; https://doi.org/10.3390/s140101358 - 13 Jan 2014

Cited by 82 | Viewed by 11534

Abstract

Two new coumarin-based “turn-off” fluorescent probes, (E)-3-((3,4-dihydroxybenzylidene)amino)-7-hydroxy-2H-chromen-2-one (BS1) and (E)-3-((2,4-dihydroxybenzylidene)amino)-7-hydroxy-2H-chromen-2-one (BS2), were synthesized and their detection of copper(II) and iron(III) ions was studied. Results show that both compounds are highly selective [...] Read more.

Two new coumarin-based “turn-off” fluorescent probes, (E)-3-((3,4-dihydroxybenzylidene)amino)-7-hydroxy-2H-chromen-2-one (BS1) and (E)-3-((2,4-dihydroxybenzylidene)amino)-7-hydroxy-2H-chromen-2-one (BS2), were synthesized and their detection of copper(II) and iron(III) ions was studied. Results show that both compounds are highly selective for Cu²⁺ and Fe³⁺ ions over other metal ions. However, BS2 is detected directly, while detection of BS1 involves a hydrolysis reaction to regenerate 3-amino-7-hydroxycoumarin (3) and 3,4-dihydroxybenzaldehyde, of which 3 is able to react with copper(II) or iron(III) ions. The interaction between the tested compounds and copper or iron ions is associated with a large fluorescence decrease, showing detection limits of ca. 10⁻⁵ M. Preliminary studies employing epifluorescence microscopy demonstrate that Cu²⁺ and Fe³⁺ ions can be imaged in human neuroblastoma SH-SY5Y cells treated with the tested probes. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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351 KiB

Open AccessArticle

Optimization of ERK Activity Biosensors for both Ratiometric and Lifetime FRET Measurements

by Pauline Vandame, Corentin Spriet, Franck Riquet, Dave Trinel, Katia Cailliau-Maggio and Jean-François Bodart

Sensors 2014, 14(1), 1140-1154; https://doi.org/10.3390/s140101140 - 10 Jan 2014

Cited by 22 | Viewed by 9335

Abstract

Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose [...] Read more.

Among biosensors, genetically-encoded FRET-based biosensors are widely used to localize and measure enzymatic activities. Kinases activities are of particular interest as their spatiotemporal regulation has become crucial for the deep understanding of cell fate decisions. This is especially the case for ERK, whose activity is a key node in signal transduction pathways and can direct the cell into various processes. There is a constant need for better tools to analyze kinases in vivo, and to detect even the slightest variations of their activities. Here we report the optimization of the previous ERK activity reporters, EKAR and EKAREV. Those tools are constituted by two fluorophores adapted for FRET experiments, which are flanking a specific substrate of ERK, and a domain able to recognize and bind this substrate when phosphorylated. The latter phosphorylation allows a conformational change of the biosensor and thus a FRET signal. We improved those biosensors with modifications of: (i) fluorophores and (ii) linkers between substrate and binding domain, resulting in new versions that exhibit broader dynamic ranges upon EGF stimulation when FRET experiments are carried out by fluorescence lifetime and ratiometric measurements. Herein, we characterize those new biosensors and discuss their observed differences that depend on their fluorescence properties. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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1081 KiB

Open AccessArticle

Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

by Rok Gaber, Andreja Majerle, Roman Jerala and Mojca Benčina

Sensors 2013, 13(12), 16330-16346; https://doi.org/10.3390/s131216330 - 28 Nov 2013

Cited by 8 | Viewed by 7338

Abstract

To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a [...] Read more.

To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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817 KiB

Open AccessArticle

Temporal and Spatial Properties of a Yeast Multi-Cellular Amplification System Based on Signal Molecule Diffusion

by Michael Jahn, Annett Mölle, Gerhard Rödel and Kai Ostermann

Sensors 2013, 13(11), 14511-14522; https://doi.org/10.3390/s131114511 - 25 Oct 2013

Cited by 5 | Viewed by 7212

Abstract

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One [...] Read more.

We report on the spatial and temporal signaling properties of a yeast pheromone-based cell communication and amplifier system. It utilizes the Saccharomyces cerevisiae mating response pathway and relies on diffusion of the pheromone α–factor as key signaling molecule between two cell types. One cell type represents the α–factor secreting sensor part and the other the reporter part emitting fluorescence upon activation. Although multi-cellular signaling systems promise higher specificity and modularity, the complex interaction of the cells makes prediction of sensor performance difficult. To test the maximum distance and response time between sensor and reporter cells, the two cell types were spatially separated in defined compartments of agarose hydrogel (5 ´ 5 mm) and reconnected by diffusion of the yeast pheromone. Different ratios of sensor to reporter cells were tested to evaluate the minimum amount of sensor cells required for signal transduction. Even the smallest ratio, one α–factor-secreting cell to twenty reporter cells, generated a distinct fluorescence signal. When using a 1:1 ratio, the secreted pheromone induced fluorescence in a distance of up to four millimeters after six hours. We conclude from both our experimental results and a mathematical diffusion model that in our approach: (1) the maximum dimension of separated compartments should not exceed five millimeters in gradient direction; and (2) the time-limiting step is not diffusion of the signaling molecule but production of the reporter protein. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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1888 KiB

Open AccessArticle

Mutational Analysis of a Red Fluorescent Protein-Based Calcium Ion Indicator

by Haley J. Carlson and Robert E. Campbell

Sensors 2013, 13(9), 11507-11521; https://doi.org/10.3390/s130911507 - 2 Sep 2013

Cited by 8 | Viewed by 9237

Abstract

As part of an ongoing effort to develop genetically encoded calcium ion (Ca²⁺) indicators we recently described a new variant, designated CH-GECO2.1, that is a genetic chimera of the red fluorescent protein (FP) mCherry, calmodulin (CaM), and a peptide that binds [...] Read more.

As part of an ongoing effort to develop genetically encoded calcium ion (Ca²⁺) indicators we recently described a new variant, designated CH-GECO2.1, that is a genetic chimera of the red fluorescent protein (FP) mCherry, calmodulin (CaM), and a peptide that binds to Ca²⁺-bound CaM. In contrast to the closely related Ca²⁺ indicator R-GECO1, CH-GECO2.1 is characterized by a much higher affinity for Ca²⁺ and a sensing mechanism that does not involve direct modulation of the chromophore pK_a. To probe the structural basis underlying the differences between CH-GECO2.1 and R-GECO1, and to gain a better understanding of the mechanism of CH-GECO2.1, we have constructed, purified, and characterized a large number of variants with strategic amino acid substitutions. This effort led us to identify Gln163 as the key residue involved in the conformational change that transduces the Ca²⁺ binding event into a change in the chromophore environment. In addition, we demonstrate that many of the substitutions that differentiate CH-GECO2.1 and R-GECO1 have little influence on both the K_d for Ca²⁺ and the sensing mechanism, and that the interdomain linkers and interfaces play important roles. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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430 KiB

Open AccessArticle

DNA Hairpins as Temperature Switches, Thermometers and Ionic Detectors

by Anette Thyssen Jonstrup, Jacob Fredsøe and Anni Hangaard Andersen

Sensors 2013, 13(5), 5937-5944; https://doi.org/10.3390/s130505937 - 10 May 2013

Cited by 48 | Viewed by 11177

Abstract

Temperature is of major importance in most branches of science and technology as well as in everyday life, and with the miniaturization of electronic devices and the increasing ability to make research into small-scale systems, a specific need for very small thermostats and [...] Read more.

Temperature is of major importance in most branches of science and technology as well as in everyday life, and with the miniaturization of electronic devices and the increasing ability to make research into small-scale systems, a specific need for very small thermostats and thermometers has been created. Here we describe how DNA molecules can be used as nanoscale sensors to meet these requirements. We illustrate how the hybridization kinetics between bases in DNA molecules combined with conformational changes of the DNA backbone can be exploited in the construction of simple but versatile temperature switches and thermometers, which can be built into electronic systems. DNA based sensors are at the same time applicable as ion detectors to monitor the chemical environment of a specific system. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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Review

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219 KiB

Open AccessReview

Applications of Delayed Fluorescence from Photosystem II

by Ya Guo and Jinglu Tan

Sensors 2013, 13(12), 17332-17345; https://doi.org/10.3390/s131217332 - 16 Dec 2013

Cited by 20 | Viewed by 6640

Abstract

While photosystem II (PSII) of plants utilizes light for photosynthesis, part of the absorbed energy may be reverted back and dissipated as long-term fluorescence (delayed fluorescence or DF). Because the generation of DF is coupled with the processes of forward photosynthetic activities, DF [...] Read more.

While photosystem II (PSII) of plants utilizes light for photosynthesis, part of the absorbed energy may be reverted back and dissipated as long-term fluorescence (delayed fluorescence or DF). Because the generation of DF is coupled with the processes of forward photosynthetic activities, DF contains the information about plant physiological states and plant-environment interactions. This makes DF a potentially powerful biosensing mechanism to measure plant photosynthetic activities and environmental conditions. While DF has attracted the interest of many researchers, some aspects of it are still unknown because of the complexity of photosynthetic system. In order to provide a holistic picture about the usefulness of DF, it is meaningful to summarize the research on DF applications. In this short review, available literature on applications of DF from PSII is summarized. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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857 KiB

Open AccessReview

Illumination of the Spatial Order of Intracellular pH by Genetically Encoded pH-Sensitive Sensors

by Mojca Benčina

Sensors 2013, 13(12), 16736-16758; https://doi.org/10.3390/s131216736 - 5 Dec 2013

Cited by 101 | Viewed by 13430

Abstract

Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated [...] Read more.

Fluorescent proteins have been extensively used for engineering genetically encoded sensors that can monitor levels of ions, enzyme activities, redox potential, and metabolites. Certain fluorescent proteins possess specific pH-dependent spectroscopic features, and thus can be used as indicators of intracellular pH. Moreover, concatenated pH-sensitive proteins with target proteins pin the pH sensors to a definite location within the cell, compartment, or tissue. This study provides an overview of the continually expanding family of pH-sensitive fluorescent proteins that have become essential tools for studies of pH homeostasis and cell physiology. We describe and discuss the design of intensity-based and ratiometric pH sensors, their spectral properties and pH-dependency, as well as their performance. Finally, we illustrate some examples of the applications of pH sensors targeted at different subcellular compartments. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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593 KiB

Open AccessReview

Genetically Encoded Fluorescent Redox Probes

by Wei Ren and Hui-Wang Ai

Sensors 2013, 13(11), 15422-15433; https://doi.org/10.3390/s131115422 - 11 Nov 2013

Cited by 26 | Viewed by 8658

Abstract

Redox processes are involved in almost every cell of the body as a consequence of aerobic life. In the past decades, redox biology has been increasingly recognized as one of the key themes in cell signaling. The progress has been accelerated by development [...] Read more.

Redox processes are involved in almost every cell of the body as a consequence of aerobic life. In the past decades, redox biology has been increasingly recognized as one of the key themes in cell signaling. The progress has been accelerated by development of fluorescent probes that can monitor redox conditions and dynamics in cells and cell compartments. This short paper focuses on fluorescent redox probes that are genetically encoded, and discusses their properties, molecular mechanism, advantages and pitfalls. Our recent work on reaction-based encoded probes that are responsive to particular redox signaling molecules is also reviewed. Future challenges and directions are also commented. Full article

(This article belongs to the Special Issue Fluorescent Biosensors)

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