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Fluorescence-Based Protein Biosensors

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

Deadline for manuscript submissions: closed (10 October 2023) | Viewed by 16898

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Special Issue Editors

Prof. Dr. Stefano Bettati

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Guest Editor

1. Centro Interdipartimentale Biopharmanet-TEC, Università di Parma, Parco Area delle Scienze 27/A, 43124 Parma, Italy
2. Dipartimento di Medicina e Chirurgia, Università di Parma, Via Gramsci 14, 43126 Parma, Italy
Interests: PLP-dependent enzymes; hemoglobin; allostery; fluorescence; protein–protein interactions

Prof. Dr. Stefano Bruno

E-Mail Website
Guest Editor

Department of Food and Drug, Università degli Studi di Parma, 43125 Parma, Italy
Interests: heme proteins; PLP-dependent proteins; biopharmaceuticals; spectroscopy; fast kinetics

Prof. Dr. Luca Ronda

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Guest Editor

Department of Medicine and Surgery, Università degli Studi di Parma, 43125 Parma, Italy
Interests: heme proteins; PEGylated proteins; solvatochromic fluorophores; biosensors; enzymes immobilization

Dr. Marialaura Marchetti

E-Mail Website
Guest Editor

Interdepartmental Center Biopharmanet-Tec, Università degli Studi di Parma, 43124 Parma, Italy
Interests: protein biosensors; enzymes; protein modification; fluorescence; drug discovery

Special Issue Information

Dear Colleagues,

Since the second half of the twentieth century, fluorescence-based sensors have gained increasing importance as powerful and versatile analytical tools, with applications within life, health, and food sciences. The introduction of proteins in the development of fluorescence-based biosensors has provided several advantages because of their detection sensitivity and specificity, ease of modification, and mild application conditions. These unique features allow for considerable flexibility and have greatly expanded the use of biosensors to study complex phenomena with unprecedented resolution, from cellular events to enzymatic reactions in cells, food, and soil.

The advent of novel approaches, such as metal-enhanced fluorescence, bioorthogonal chemistry, and CRISPR/Cas9 technologies, has opened up an even more comprehensive range of potential applications. In this context, this upcoming Issue will focus on the versatility of fluorescent, protein-based biosensors, covering all fields and applications.

Authors are invited to contribute significant original research articles and short communications focused on the development or applications of fluorescence- and protein-based biosensors. Reviews discussing emergent technologies and advances in the field are also welcome.

Prof. Dr. Stefano Bettati
Prof. Dr. Stefano Bruno
Prof. Dr. Luca Ronda
Dr. Marialaura Marchetti
Guest Editors

Manuscript Submission Information

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Keywords

fluorescent protein biosensor
fluorescence
FRET
detection
imaging
enzyme
protein binding
recombinant protein
cellular events
metals
pollutants
bioorthogonal reaction

Published Papers (7 papers)

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Research

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13 pages, 1973 KiB

Open AccessArticle

Endosome and Lysosome Membrane Properties Functionally Link to γ-Secretase in Live/Intact Cells

by Mei C. Q. Houser, Shane P. C. Mitchell, Priyanka Sinha, Brianna Lundin, Oksana Berezovska and Masato Maesako

Sensors 2023, 23(5), 2651; https://0-doi-org.brum.beds.ac.uk/10.3390/s23052651 - 28 Feb 2023

Cited by 1 | Viewed by 1597

Abstract

Our unique multiplexed imaging assays employing FRET biosensors have previously detected that γ-secretase processes APP C99 primarily in late endosomes and lysosomes in live/intact neurons. Moreover we have shown that Aβ peptides are enriched in the same subcellular loci. Given that γ-secretase is integrated into the membrane bilayer and functionally links to lipid membrane properties in vitro, it is presumable that γ-secretase function correlates with endosome and lysosome membrane properties in live/intact cells. In the present study, we show using unique live-cell imaging and biochemical assays that the endo-lysosomal membrane in primary neurons is more disordered and, as a result, more permeable than in CHO cells. Interestingly, γ-secretase processivity is decreased in primary neurons, resulting in the predominant production of long Aβ42 instead of short Aβ38. In contrast, CHO cells favor Aβ38 over the Aβ42 generation. Our findings are consistent with the previous in vitro studies, demonstrating the functional interaction between lipid membrane properties and γ-secretase and provide further evidence that γ-secretase acts in late endosomes and lysosomes in live/intact cells. Full article

(This article belongs to the Special Issue Fluorescence-Based Protein Biosensors)

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10 pages, 2446 KiB

Open AccessArticle

A Bioluminescent Sensor for Rapid Detection of PPEP-1, a Clostridioides difficile Biomarker

by Kevin K. Ng, Zachary E. Reinert, Jeroen Corver, Danica Resurreccion, Paul J. Hensbergen and Jennifer A. Prescher

Sensors 2021, 21(22), 7485; https://0-doi-org.brum.beds.ac.uk/10.3390/s21227485 - 11 Nov 2021

Cited by 4 | Viewed by 2416

Abstract

Current assays for Clostridioides difficile in nonhospital settings are outsourced and time-intensive, resulting in both delayed diagnosis and quarantining of infected individuals. We designed a more rapid point-of-care assay featuring a “turn-on” bioluminescent readout of a C. difficile-specific protease, PPEP-1. NanoLuc, a bright and stable luciferase, was “caged” with a PPEP-1-responsive peptide tail that inhibited luminescence. Upon proteolytic cleavage, the peptide was released and NanoLuc activity was restored, providing a visible readout. The bioluminescent sensor detected PPEP-1 concentrations as low as 10 nM. Sensor uncaging was achieved within minutes, and signal was captured using a digital camera. Importantly, the sensor was also functional at ambient temperature and compatible with fecal material, suggesting that it can be readily deployed in a variety of settings. Full article

(This article belongs to the Special Issue Fluorescence-Based Protein Biosensors)

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11 pages, 14571 KiB

Open AccessArticle

Structural Analysis of a Genetically Encoded FRET Biosensor by SAXS and MD Simulations

by Ines Reinartz, Mona Sarter, Julia Otten, Henning Höfig, Martina Pohl, Alexander Schug, Andreas M. Stadler and Jörg Fitter

Sensors 2021, 21(12), 4144; https://0-doi-org.brum.beds.ac.uk/10.3390/s21124144 - 16 Jun 2021

Cited by 6 | Viewed by 2381

Abstract

Inspired by the modular architecture of natural signaling proteins, ligand binding proteins are equipped with two fluorescent proteins (FPs) in order to obtain Förster resonance energy transfer (FRET)-based biosensors. Here, we investigated a glucose sensor where the donor and acceptor FPs were attached to a glucose binding protein using a variety of different linker sequences. For three resulting sensor constructs the corresponding glucose induced conformational changes were measured by small angle X-ray scattering (SAXS) and compared to recently published single molecule FRET results (Höfig et al., ACS Sensors, 2018). For one construct which exhibits a high change in energy transfer and a large change of the radius of gyration upon ligand binding, we performed coarse-grained molecular dynamics simulations for the ligand-free and the ligand-bound state. Our analysis indicates that a carefully designed attachment of the donor FP is crucial for the proper transfer of the glucose induced conformational change of the glucose binding protein into a well pronounced FRET signal change as measured in this sensor construct. Since the other FP (acceptor) does not experience such a glucose induced alteration, it becomes apparent that only one of the FPs needs to have a well-adjusted attachment to the glucose binding protein. Full article

(This article belongs to the Special Issue Fluorescence-Based Protein Biosensors)

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12 pages, 2992 KiB

Open AccessArticle

A Compact Detection Platform Based on Gradient Guided-Mode Resonance for Colorimetric and Fluorescence Liquid Assay Detection

by Jing-Jhong Gao, Ching-Wei Chiu, Kuo-Hsing Wen and Cheng-Sheng Huang

Sensors 2021, 21(8), 2797; https://0-doi-org.brum.beds.ac.uk/10.3390/s21082797 - 15 Apr 2021

Cited by 2 | Viewed by 1889

Abstract

This paper presents a compact spectral detection system for common fluorescent and colorimetric assays. This system includes a gradient grating period guided-mode resonance (GGP-GMR) filter and charge-coupled device. In its current form, the GGP-GMR filter, which has a size of less than 2.5 mm, can achieve a spectral detection range of 500–700 nm. Through the direct measurement of the fluorescence emission, the proposed system was demonstrated to detect both the peak wavelength and its corresponding intensity. One fluorescent assay (albumin) and two colorimetric assays (albumin and creatinine) were performed to demonstrate the practical application of the proposed system for quantifying common liquid assays. The results of our system exhibited suitable agreement with those of a commercial spectrometer in terms of the assay sensitivity and limit of detection (LOD). With the proposed system, the fluorescent albumin, colorimetric albumin, and colorimetric creatinine assays achieved LODs of 40.99 and 398 and 25.49 mg/L, respectively. For a wide selection of biomolecules in point-of-care applications, the spectral detection range achieved by the GGP-GMR filter can be further extended and the simple and compact optical path configuration can be integrated with a lab-on-a-chip system. Full article

(This article belongs to the Special Issue Fluorescence-Based Protein Biosensors)

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16 pages, 5002 KiB

Open AccessArticle

Metal-Modified Montmorillonite as Plasmonic Microstructure for Direct Protein Detection

by Giorgia Giovannini, Denis Garoli, Patrick Rupper, Antonia Neels, René M. Rossi and Luciano F. Boesel

Sensors 2021, 21(8), 2655; https://0-doi-org.brum.beds.ac.uk/10.3390/s21082655 - 09 Apr 2021

Cited by 15 | Viewed by 2552

Abstract

Thanks to its negative surface charge and high swelling behavior, montmorillonite (MMT) has been widely used to design hybrid materials for applications in metal ion adsorption, drug delivery, or antibacterial substrates. The changes in photophysical and photochemical properties observed when fluorophores interact with MMT make these hybrid materials attractive for designing novel optical sensors. Sensor technology is making huge strides forward, achieving high sensitivity and selectivity, but the fabrication of the sensing platform is often time-consuming and requires expensive chemicals and facilities. Here, we synthesized metal-modified MMT particles suitable for the bio-sensing of self-fluorescent biomolecules. The fluorescent enhancement achieved by combining clay minerals and plasmonic effect was exploited to improve the sensitivity of the fluorescence-based detection mechanism. As proof of concept, we showed that the signal of fluorescein isothiocyanate can be harvested by a factor of 60 using silver-modified MMT, while bovine serum albumin was successfully detected at 1.9 µg/mL. Furthermore, we demonstrated the versatility of the proposed hybrid materials by exploiting their plasmonic properties to develop liquid label-free detection systems. Our results on the signal enhancement achieved using metal-modified MMT will allow the development of highly sensitive, easily fabricated, and cost-efficient fluorescent- and plasmonic-based detection methods for biomolecules. Full article

(This article belongs to the Special Issue Fluorescence-Based Protein Biosensors)

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Review

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21 pages, 2512 KiB

Open AccessReview

Genetically Encoded Biosensors for the Fluorescence Detection of O₂ and Reactive O₂ Species

by Marialaura Marchetti, Luca Ronda, Monica Cozzi, Stefano Bettati and Stefano Bruno

Sensors 2023, 23(20), 8517; https://0-doi-org.brum.beds.ac.uk/10.3390/s23208517 - 17 Oct 2023

Cited by 2 | Viewed by 1008

Abstract

The intracellular concentrations of oxygen and reactive oxygen species (ROS) in living cells represent critical information for investigating physiological and pathological conditions. Real-time measurement often relies on genetically encoded proteins that are responsive to fluctuations in either oxygen or ROS concentrations. The direct binding or chemical reactions that occur in their presence either directly alter the fluorescence properties of the binding protein or alter the fluorescence properties of fusion partners, mostly consisting of variants of the green fluorescent protein. Oxygen sensing takes advantage of several mechanisms, including (i) the oxygen-dependent hydroxylation of a domain of the hypoxia-inducible factor-1, which, in turn, promotes its cellular degradation along with fluorescent fusion partners; (ii) the naturally oxygen-dependent maturation of the fluorophore of green fluorescent protein variants; and (iii) direct oxygen binding by proteins, including heme proteins, expressed in fusion with fluorescent partners, resulting in changes in fluorescence due to conformational alterations or fluorescence resonance energy transfer. ROS encompass a group of highly reactive chemicals that can interconvert through various chemical reactions within biological systems, posing challenges for their selective detection through genetically encoded sensors. However, their general reactivity, and particularly that of the relatively stable oxygen peroxide, can be exploited for ROS sensing through different mechanisms, including (i) the ROS-induced formation of disulfide bonds in engineered fluorescent proteins or fusion partners of fluorescent proteins, ultimately leading to fluorescence changes; and (ii) conformational changes of naturally occurring ROS-sensing domains, affecting the fluorescence properties of fusion partners. In this review, we will offer an overview of these genetically encoded biosensors. Full article

(This article belongs to the Special Issue Fluorescence-Based Protein Biosensors)

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36 pages, 4373 KiB

Open AccessReview

Multiphoton Laser Fabrication of Hybrid Photo-Activable Biomaterials

by Margaux Bouzin, Amirbahador Zeynali, Mario Marini, Laura Sironi, Riccardo Scodellaro, Laura D’Alfonso, Maddalena Collini and Giuseppe Chirico

Sensors 2021, 21(17), 5891; https://0-doi-org.brum.beds.ac.uk/10.3390/s21175891 - 01 Sep 2021

Cited by 11 | Viewed by 3696

Abstract

The possibility to shape stimulus-responsive optical polymers, especially hydrogels, by means of laser 3D printing and ablation is fostering a new concept of “smart” micro-devices that can be used for imaging, thermal stimulation, energy transducing and sensing. The composition of these polymeric blends is an essential parameter to tune their properties as actuators and/or sensing platforms and to determine the elasto-mechanical characteristics of the printed hydrogel. In light of the increasing demand for micro-devices for nanomedicine and personalized medicine, interest is growing in the combination of composite and hybrid photo-responsive materials and digital micro-/nano-manufacturing. Existing works have exploited multiphoton laser photo-polymerization to obtain fine 3D microstructures in hydrogels in an additive manufacturing approach or exploited laser ablation of preformed hydrogels to carve 3D cavities. Less often, the two approaches have been combined and active nanomaterials have been embedded in the microstructures. The aim of this review is to give a short overview of the most recent and prominent results in the field of multiphoton laser direct writing of biocompatible hydrogels that embed active nanomaterials not interfering with the writing process and endowing the biocompatible microstructures with physically or chemically activable features such as photothermal activity, chemical swelling and chemical sensing. Full article

(This article belongs to the Special Issue Fluorescence-Based Protein Biosensors)

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