Recent developments of genetically encoded optical sensors for cell biology

Optical sensors are powerful tools for live cell research as they permit to follow the location, concentration changes or activities of key cellular players such as lipids, ions and enzymes. Most of the current sensor probes are based on fluorescence which provides great spatial and temporal precision provided that high‐end microscopy is used and that the timescale of the event of interest fits the response time of the sensor. Many of the sensors developed in the past 20 years are genetically encoded. There is a diversity of designs leading to simple or sometimes complicated applications for the use in live cells. Genetically encoded sensors began to emerge after the discovery of fluorescent proteins, engineering of their improved optical properties and the manipulation of their structure through application of circular permutation. In this review, we will describe a variety of genetically encoded biosensor concepts, including those for intensiometric and ratiometric sensors based on single fluorescent proteins, Forster resonance energy transfer‐based sensors, sensors utilising bioluminescence, sensors using self‐labelling SNAP‐ and CLIP‐tags, and finally tetracysteine‐based sensors. We focus on the newer developments and discuss the current approaches and techniques for design and application. This will demonstrate the power of using optical sensors in cell biology and will help opening the field to more systematic applications in the future.     

An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins

Many fluorescent proteins have been created to act as genetically encoded biosensors. With these sensors, changes in fluorescence report on chemical states in living cells. Transition metal ions such as copper, nickel, and zinc are crucial in many physiological and pathophysiological pathways. Here, we engineered a spectral series of optimized transition metal ion-binding fluorescent proteins that respond to metals with large changes in fluorescence intensity. These proteins can act as metal biosensors or imaging probes whose fluorescence can be tuned by metals. Each protein is uniquely modulated by four different metals (Cu²⁺, Ni²⁺, Co²⁺, and Zn²⁺). Crystallography revealed the geometry and location of metal binding to the engineered sites. When attached to the extracellular terminal of a membrane protein VAMP2, dimeric pairs of the sensors could be used in cells as ratiometric probes for transition metal ions. Thus, these engineered fluorescent proteins act as sensitive transition metal ion-responsive genetically encoded probes that span the visible spectrum.     

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.     

New highly sensitive and selective catalytic DNA biosensors for metal ions

While remarkable progress has been made in developing sensors for metal ions such as Ca(II) and Zn(II), designing and synthesizing sensitive and selective metal ion sensors remains a significant challenge. Perhaps the biggest challenge is the design and synthesis of a sensor capable of specific and strong metal binding. Since our knowledge about the construction of metal-binding sites in general is limited, searching for sensors in a combinatorial way is of significant value. Therefore, we have been able to use a combinatorial method called in vitro selection to obtain catalytic DNA that can bind a metal ion of choice strongly and specifically. The metal ion selectivity of the catalytic DNA was further improved using a 'negative selection' strategy where catalytic DNA that are selective for competing metal ions are discarded in the in vitro selection processes. By labeling the resulting catalytic DNA with a fluorophore/quencher pair, we have made a new class of metal ion fluorescent sensors that are the first examples of catalytic DNA biosensors for metal ions. The sensors combine the high selectivity of catalytic DNA with the high sensitivity of fluorescent detection, and can be applied to the quantitative detection of metal ions over a wide concentration range and with high selectivity. The use of DNA sensors in detection and quantification of lead ions in environmental samples such as water from Lake Michigan has been demonstrated. DNA is stable, cost-effective, environmentally benign, and easily adaptable to optical fiber and microarray technology for device manufacture. Thus, the DNA sensors explained here hold great promise for on-site and real-time monitoring of metal ions in the fields of environmental monitoring, developmental biology, clinical toxicology, wastewater treatment, and industrial process monitoring.     

Ratiometric detection of Zn(II) using chelating fluorescent protein chimeras

Fluorescent indicators for the real-time imaging of small molecules or metal ions in living cells are invaluable tools for understanding their physiological function. Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) between fluorescent protein domains have important advantages over synthetic probes, but often suffer from a small ratiometric change. Here, we present a new design approach to obtain sensors with a large difference in emission ratio between the bound and unbound states. De novo Zn(II)-binding sites were introduced directly at the surface of both fluorescent domains of a chimera of enhanced cyan and yellow fluorescent protein, connected by a flexible peptide linker. The resulting sensor ZinCh displayed an almost fourfold change in fluorescence emission ratio upon binding of Zn(II). Besides a high affinity for Zn(II), the sensor was shown to be selective over other physiologically relevant metal ions. Its unique biphasic Zn(II)-binding behavior could be attributed to the presence of two distinct Zn(II)-binding sites and allowed ratiometric fluorescent detection of Zn(II) over a concentration range from 10 nM to 1 mM. Size-exclusion chromatography and fluorescence anisotropy were used to provide a detailed picture of the conformational changes associated with each Zn(II)-binding step. The high affinity for Zn(II) was mainly due to a high effective concentration of the fluorescent proteins and could be understood quantitatively by modeling the peptide linker between the fluorescent proteins as a random coil. The strategy of using chelating fluorescent protein chimeras to develop FRET sensor proteins with a high ratiometric change is expected to be more generally applicable, in particular for other metal ions and small molecules.     

Effect of bis-triazoles on a ribose-based fluorescent sensor

We synthesized two ribosyl-based fluorescent sensors. Both sensors have an anthracene as the fluorophore, but they differ in the recognition site for metal ions. One (3) has two ribosyl esters, and the other (6) has two triazole groups linked to two ribosyl esters. Among the metal ions examined in MeOH, compound 3 displayed a large chelation-enhanced fluorescence (CHEF) effect with Hg(2+) and Cu(2+) ions, and compound 6 displayed a large chelation-quenched fluorescence (CHQF) effect with Cu(2+) and Ni(2+) ions. The results demonstrated that the absence (sensor 3) and presence (sensor 6) of an incorporated bis-triazole group in a ribosyl-based fluorescent sensor conferred different preferences and distinct binding modes for metal ions.     

Design and development of genetically encoded fluorescent sensors to monitor intracellular chemical and physical parameters

Over the past decades many researchers have made major contributions towards the development of genetically encoded (GE) fluorescent sensors derived from fluorescent proteins. GE sensors are now used to study biological phenomena by facilitating the measurement of biochemical behaviors at various scales, ranging from single molecules to single cells or even whole animals. Here, we review the historical development of GE fluorescent sensors and report on their current status. We specifically focus on the development strategies of the GE sensors used for measuring pH, ion concentrations (e.g., chloride and calcium), redox indicators, membrane potential, temperature, pressure, and molecular crowding. We demonstrate that these fluroescent protein-based sensors have a shared history of concepts and development strategies, and we highlight the most original concepts used to date. We believe that the understanding and application of these various concepts will pave the road for the development of future GE sensors and lead to new breakthroughs in bioimaging.     

Designs,applications, and limitations of genetically encoded fluorescent sensors to explore plant biology

The understanding of signaling and metabolic processes in multicellular organisms requires knowledge of the spatial dynamics of small molecules and the activities of enzymes, transporters, and other proteins in vivo, as well as biophysical parameters inside cells and across tissues. The cellular distribution of receptors, ligands, and activation state must be integrated with information about the cellular distribution of metabolites in relation to metabolic fluxes and signaling dynamics in order to achieve the promise of in vivo biochemistry. Genetically encoded sensors are engineered fluorescent proteins that have been developed for a wide range of small molecules, such as ions and metabolites, or to report biophysical processes, such as transmembrane voltage or tension. First steps have been taken to monitor the activity of transporters in vivo. Advancements in imaging technologies and specimen handling and stimulation have enabled researchers in plant sciences to implement sensor technologies in intact plants. Here, we provide a brief history of the development of genetically encoded sensors and an overview of the types of sensors available for quantifying and visualizing ion and metabolite distribution and dynamics. We further discuss the pros and cons of specific sensor designs, imaging systems, and sample manipulations, provide advice on the choice of technology, and give an outlook into future developments.

Different types of genetically encoded sensors in plants can be used to quantify and visualize ion and metabolite distributions and dynamics.     

Recent progress of TP/NIR fluorescent probes for metal ions

Metal ions are of significance in various pathological and physiological processes. As such, it is crucial to monitor their levels in organisms. Two-photon (TP) and near-infrared (NIR) fluorescence imaging has been utilized to monitor metal ions because of minimal background interference, deeper tissue depth penetration, lower tissue self-absorption, and reduced photodamage. In this review, we briefly summarize recent progress from 2020 to 2022 of TP/NIR organic fluorescent probes and inorganic sensors in the detection of metal ions. Additionally, we present an outlook for the development of TP/NIR probes for bio-imaging, diagnosis of diseases, imaging-guided therapy, and activatable phototherapy.     

Simultaneous detection of ca(2+) and diacylglycerol signaling in living cells

Phospholipase C produces two second messengers - diacylglycerol (DAG), which remains in the membrane, and inositol triphosphate (IP(3)), which triggers the release of calcium ions (Ca(2+)) from intracellular stores. Genetically encoded sensors based on a single circularly permuted fluorescent protein (FP) are robust tools for studying intracellular Ca(2+) dynamics. We have developed a robust sensor for DAG based on a circularly permuted green FP that can be co-imaged with the red fluorescent Ca(2+) sensor R-GECO for simultaneous measurement of both second messengers.     

Genetically encoded fluorescent sensors of membrane potential

Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.     

Shining light on signaling and metabolic networks by genetically encoded biosensors

Fluorescent labels have revolutionized cell biology. Signaling intermediates and metabolites can be measured in real time with subcellular spatial resolution. Most of these sensors are based on fluorescent proteins, and many report fluorescence resonance energy transfer. Because the biosensors are genetically encoded, a toolbox for addressing cell biological questions at the systems level is now available. Fluorescent biosensors are able to determine the localization of proteins and their dynamics, to reveal the cellular and subcellular localization of the respective interactions and activities, and to provide complementary data on the steady state levels of ions, metabolites, and signaling intermediates with high temporal and spatial resolution. They represent the basis for cell-based high-throughput assays that are necessary for a systems perspective on plant cell function.     

Frex and FrexH: Indicators of metabolic states in living cells

Reduced nicotinamide adenine dinucleotide (NADH) and its oxidized form play central roles in energy and redox metabolisms. For many years, researchers have relied on the weak NADH endogenous fluorescence signal to determine the NADH level in living cells. We recently reported a series of genetically encoded fluorescent sensors highly specific for NADH. These sensors allow real-time, quantitative measurement of this significant molecule in different subcellular compartments. In this study, we provide a more detailed discussion of the benefits and limitations of these genetically encoded fluorescent sensors. These sensors are utilized in most laboratories without the need for sophisticated instruments because of their superior sensitivity and specificity. They are also viable alternatives to existing techniques for measuring the endogenous fluorescence of intracellular NAD(P)H.     

Photochemical tools for studying metal ion signaling and homeostasis

Metal ions have well-established catalytic and structural roles in proteins. Much of the knowledge acquired about metalloenzymes has been derived using spectroscopic techniques and X-ray crystallography, but these methodologies are less effective for studying metal ions that are not tightly bound to biomacromolecules. In order to prevent deleterious chemistry, cells tightly regulate the uptake, distribution, and intracellular concentrations of metal ions. Investigation into these homeostasis mechanisms has necessitated the development of alternative ways to study metal ions. Photochemical tools such as small molecule and protein-based fluorescent sensors as well as photocaged complexes have provided insight into the homeostasis and signaling mechanisms of Ca(2+), Zn(2+), and Cu(+), but a comprehensive picture of metal ions in biology will require additional development of these techniques, which are reviewed in this Current Topics article.     

Facile synthesis of anthracene-appended amino acids as highly selective and sensitive fluorescent Fe3+ ion sensors

We designed new fluorescent chemical sensors for Fe³⁺ ion detection, by conjugating amino acids as receptors into an anthracene fluorophore. The conjugates were synthesized in solid phase by Fmoc-chemistry. Fluorescence sensors containing Asp (1) and Glu (2) both had exclusive selectivity for Fe³⁺ in 100% aqueous solution and in a mixed organic–aqueous solvent system. Other metal ions did not interfere with the detection ability of the sensors for Fe³⁺. The sensors detect Fe³⁺ ions via a chelation-enhanced fluorescent quenching effect. The binding affinity, reversible monitoring, and pH sensitivity of the sensors were investigated. In addition, detection of fluoride ion among halide ions was done by a chemosensing ensemble method with 1–Fe³⁺ and 2–Fe³⁺ complexes.     

Impact of labile metal nanoparticles on cellular homeostasis. Current developments in imaging,synthesis and applications

BackgroundThe use of nanomaterials is constantly increasing in electronics, cosmetics, food additives, and is emerging in advanced biomedical applications such as theranostics, bio-imaging and therapeutics. However their safety raises concerns and requires appropriate methods to analyze their fate in vivo.Scope of reviewIn this review, we describe the current knowledge about the toxicity of labile metal (ZnO, CuO and Ag) nanoparticles (NPs) both at the organism and cellular levels, and describe the pathways that are triggered to maintain cellular homeostasis. We also describe advanced elemental imaging approaches to analyze intracellular NP fate. Finally, we open the discussion by presenting recent developments in terms of synthesis and applications of Ag and CuO NPs.Major conclusionsLabile metal nanoparticles (MeNPs) release metal ions that trigger a cellular response involving biomolecules binding to the ions followed by regulation of the redox balance. In addition, specific mechanisms are set up by the cell in response to physiological ions such as Cu(I) and Zn(II). Among all types of NPs, labile MeNPs induce the strongest inflammatory responses which are most probably due to the combined effects of the NPs and of its released ions. Interestingly, recent developments in imaging technologies enable the intracellular visualization of both the NPs and their ions and promise new insights into nanoparticle fate and toxicity.General significanceThe exponential use of nanotechnologies associated with the difficulties of assessing their impact on health and the environment has prompted scientists to develop novel methodologies to characterize these nanoobjects in a biological context.     

Nanowire sensors for multiplexed detection of biomolecules

Nanowire-based detection strategies provide promising new routes to bioanalysis that could one day revolutionize the healthcare industry. This review covers recent developments in nanowire sensors for multiplexed detection of biomolecules such as nucleic acids and proteins. We focus on encoded nanowire suspension arrays and semiconductor nanowire-based field-effect transistors. Nanowire assembly and integration with microchip technology is emphasized as a key step toward the ultimate goal of multiplexed detection at the point of care using portable, low power, electronic biosensor chips.