Fluorescent Protein-Based Sensing and Detection

A special issue of Biosensors (ISSN 2079-6374). This special issue belongs to the section "Optical and Photonic Biosensors".

Deadline for manuscript submissions: closed (30 August 2023) | Viewed by 12558

Special Issue Editors


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Guest Editor
Molecular and Cell Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Douglas, QLD, 4811, Australia
Interests: biomarkers; immunotherapy; IgE antibody; drug resistance; drug discovery; melioidosis; mycobacterium tuberculosis; zinc; copper; tropomyosin; DNA binding; green fluorescent protein; biosensors

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Guest Editor
Molecular and Cell Biology, College of Public Health, Medical and Veterinary Sciences, James Cook University, Douglas, QLD, 4811, Australia
Interests: streptococcus pyogenes; DNA binding; copper; drug discovery; acute rheumatic fever; melioidosis; rheumatic heart disease; mycobacterium tuberculosis; biosensors; green fluorescent protein; zinc

Special Issue Information

Dear Colleagues,

In 2008, Osamu Shimomura, Martin Chalfie and Roger Tsien were awarded the Nobel Prize in Chemistry for “the discovery and development of the green fluorescent protein, GFP”. Indeed, since its cloning and characterization in the early 1990s, this fluorescent protein has become one of the most useful and versatile molecular tools in modern science. Osamu Shimomura characterized the chromophore of GFP and elucidated the mechanism of bioluminescence in Aequora victoria. Martin Chalfie expressed the protein in heterologous expression systems, and Roger Tsien developed a multitude of applications and mutants of fluorescent proteins. The possibilities and applications of GFP and its variants seem endless, with new disruptive technologies and assays emerging each year spanning across the fields of biology, chemistry, biophysics, and medicine.

This Special Issue showcases original research on a selection of new developments, including in vitro and in vivo applications of fluorescent protein-based technologies. Special focus is dedicated to fluorescent protein-based sensing and detection systems, as well as review articles on these topics.

Dr. Patrick Schaeffer
Dr. Alanna E. Sorenson
Guest Editors

Manuscript Submission Information

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Keywords

  • fluorescent protein
  • GFP
  • sensing
  • detection
  • diagnostics
  • proteomics
  • protein–protein interaction
  • protein–ligand interaction
  • metal binding
  • protein engineering

Published Papers (5 papers)

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Research

16 pages, 3699 KiB  
Article
Development of an Efficient FRET-Based Ratiometric Uranium Biosensor
by Sandrine Sauge-Merle, Morgane Recuerda, Maria Rosa Beccia, David Lemaire, Rym Cherif, Nicolas Bremond, Fabienne Merola, Yasmina Bousmah and Catherine Berthomieu
Biosensors 2023, 13(5), 561; https://doi.org/10.3390/bios13050561 - 19 May 2023
Viewed by 1437
Abstract
The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist [...] Read more.
The dispersion of uranium in the environment can pose a problem for the health of humans and other living organisms. It is therefore important to monitor the bioavailable and hence toxic fraction of uranium in the environment, but no efficient measurement methods exist for this. Our study aims to fill this gap by developing a genetically encoded FRET-based ratiometric uranium biosensor. This biosensor was constructed by grafting two fluorescent proteins to both ends of calmodulin, a protein that binds four calcium ions. By modifying the metal-binding sites and the fluorescent proteins, several versions of the biosensor were generated and characterized in vitro. The best combination results in a biosensor that is affine and selective for uranium compared to metals such as calcium or other environmental compounds (sodium, magnesium, chlorine). It has a good dynamic range and should be robust to environmental conditions. In addition, its detection limit is below the uranium limit concentration in drinking water defined by the World Health Organization. This genetically encoded biosensor is a promising tool to develop a uranium whole-cell biosensor. This would make it possible to monitor the bioavailable fraction of uranium in the environment, even in calcium-rich waters. Full article
(This article belongs to the Special Issue Fluorescent Protein-Based Sensing and Detection)
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11 pages, 1859 KiB  
Article
Real-Time Temperature Sensing Using a Ratiometric Dual Fluorescent Protein Biosensor
by Alanna E. Sorenson and Patrick M. Schaeffer
Biosensors 2023, 13(3), 338; https://doi.org/10.3390/bios13030338 - 3 Mar 2023
Cited by 2 | Viewed by 2174
Abstract
Accurate temperature control within biological and chemical reaction samples and instrument calibration are essential to the diagnostic, pharmaceutical and chemical industries. This is particularly challenging for microlitre-scale reactions typically used in real-time PCR applications and differential scanning fluorometry. Here, we describe the development [...] Read more.
Accurate temperature control within biological and chemical reaction samples and instrument calibration are essential to the diagnostic, pharmaceutical and chemical industries. This is particularly challenging for microlitre-scale reactions typically used in real-time PCR applications and differential scanning fluorometry. Here, we describe the development of a simple, inexpensive ratiometric dual fluorescent protein temperature biosensor (DFPTB). A combination of cycle three green fluorescent protein and a monomeric red fluorescent protein enabled the quantification of relative temperature changes and the identification of temperature discrepancies across a wide temperature range of 4–70 °C. The maximal sensitivity of 6.7% °C−1 and precision of 0.1 °C were achieved in a biologically relevant temperature range of 25–42 °C in standard phosphate-buffered saline conditions at a pH of 7.2. Good temperature sensitivity was achieved in a variety of biological buffers and pH ranging from 4.8 to 9.1. The DFPTB can be used in either purified or mixed bacteria-encapsulated formats, paving the way for in vitro and in vivo applications for topologically precise temperature measurements. Full article
(This article belongs to the Special Issue Fluorescent Protein-Based Sensing and Detection)
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31 pages, 4854 KiB  
Article
Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing
by Taylor D. Krueger, Longteng Tang and Chong Fang
Biosensors 2023, 13(2), 218; https://doi.org/10.3390/bios13020218 - 2 Feb 2023
Cited by 4 | Viewed by 2381
Abstract
Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca2+) concentrations in vivo with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these [...] Read more.
Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca2+) concentrations in vivo with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these ions. REX-GECO1 is a recently developed red-green emission and excitation ratiometric FP-based biosensor that achieves a high dynamic range due to differences in the chromophore response to light excitation with and without calcium ions. Using steady-state electronic measurements (UV/Visible absorption and emission), along with time-resolved spectroscopic techniques including femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS), the potential energy surfaces of these unique biosensors are unveiled with vivid details. The ground-state structural characterization of the Ca2+-free biosensor via FSRS reveals a more spacious protein pocket that allows the chromophore to efficiently twist and reach a dark state. In contrast, the more compressed cavity within the Ca2+-bound biosensor results in a more heterogeneous distribution of chromophore populations that results in multi-step excited state proton transfer (ESPT) pathways on the sub-140 fs, 600 fs, and 3 ps timescales. These results enable rational design strategies to enlarge the spectral separation between the protonated/deprotonated forms and the Stokes shift leading to a larger dynamic range and potentially higher fluorescence quantum yield, which should be broadly applicable to the calcium imaging and biosensor communities. Full article
(This article belongs to the Special Issue Fluorescent Protein-Based Sensing and Detection)
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14 pages, 8779 KiB  
Article
A Selective Fluorescent l-Lactate Biosensor Based on an l-Lactate-Specific Transcription Regulator and Förster Resonance Energy Transfer
by Xianzhi Xu, Rong Xu, Shuang Hou, Zhaoqi Kang, Chuanjuan Lü, Qian Wang, Wen Zhang, Xia Wang, Ping Xu, Chao Gao and Cuiqing Ma
Biosensors 2022, 12(12), 1111; https://doi.org/10.3390/bios12121111 - 1 Dec 2022
Cited by 8 | Viewed by 2068
Abstract
Selective detection of l-lactate levels in foods, clinical, and bacterial fermentation samples has drawn intensive attention. Many fluorescent biosensors based on non-stereoselective recognition elements have been developed for lactate detection. Herein, the allosteric transcription factor STLldR from Salmonella enterica serovar Typhimurium LT2 [...] Read more.
Selective detection of l-lactate levels in foods, clinical, and bacterial fermentation samples has drawn intensive attention. Many fluorescent biosensors based on non-stereoselective recognition elements have been developed for lactate detection. Herein, the allosteric transcription factor STLldR from Salmonella enterica serovar Typhimurium LT2 was identified to be stereo-selectively respond to l-lactate. Then, STLldR was combined with Förster resonance energy transfer (FRET) to construct a fluorescent l-lactate biosensor FILLac. FILLac was further optimized by truncating the N- and C-terminal amino acids of STLldR between cyan and yellow fluorescent proteins. The optimized biosensor FILLac10N0C exhibited a maximum emission ratio change (ΔRmax) of 33.47 ± 1.91%, an apparent dissociation constant (Kd) of 6.33 ± 0.79 μM, and a limit of detection of 0.68 μM. FILLac10N0C was applied in 96-well microplates to detect l-lactate in bacterial fermentation samples and commercial foods such as Jiaosu and yogurt. The quantitation results of FILLac10N0C exhibited good agreement with that of a commercial l-lactate biosensor SBA-40D bioanalyzer. Thus, the biosensor FILLac10N0C compatible with high-throughput detection may be a potential choice for quantitation of l-lactate in different biological samples. Full article
(This article belongs to the Special Issue Fluorescent Protein-Based Sensing and Detection)
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10 pages, 2622 KiB  
Article
Evaluation of Hydroxycarboxylic Acid Receptor 1 (HCAR1) as a Building Block for Genetically Encoded Extracellular Lactate Biosensors
by Joel Wellbourne-Wood, Marc Briquet, Maxime Alessandri, Francesca Binda, Maylis Touya and Jean-Yves Chatton
Biosensors 2022, 12(3), 143; https://doi.org/10.3390/bios12030143 - 25 Feb 2022
Cited by 4 | Viewed by 3072
Abstract
The status of lactate has evolved from being considered a waste product of cellular metabolism to a useful metabolic substrate and, more recently, to a signaling molecule. The fluctuations of lactate levels within biological tissues, in particular in the interstitial space, are crucial [...] Read more.
The status of lactate has evolved from being considered a waste product of cellular metabolism to a useful metabolic substrate and, more recently, to a signaling molecule. The fluctuations of lactate levels within biological tissues, in particular in the interstitial space, are crucial to assess with high spatial and temporal resolution, and this is best achieved using cellular imaging approaches. In this study, we evaluated the suitability of the lactate receptor, hydroxycarboxylic acid receptor 1 (HCAR1, formerly named GPR81), as a basis for the development of a genetically encoded fluorescent lactate biosensor. We used a biosensor strategy that was successfully applied to molecules such as dopamine, serotonin, and norepinephrine, based on their respective G-protein-coupled receptors. In this study, a set of intensiometric sensors was constructed and expressed in living cells. They showed selective expression at the plasma membrane and responded to physiological concentrations of lactate. However, these sensors lost the original ability of HCAR1 to selectively respond to lactate versus other related small carboxylic acid molecules. Therefore, while representing a promising building block for a lactate biosensor, HCAR1 was found to be sensitive to perturbations of its structure, affecting its ability to distinguish between related carboxylic molecules. Full article
(This article belongs to the Special Issue Fluorescent Protein-Based Sensing and Detection)
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