Maximizing the performance of protein-based fluorescent biosensors

Abstract

Fluorescent protein (FP)-based biosensors are genetically encoded tools that enable the imaging of biological processes in the context of cells, tissues, or live animals. Though widely used in biological research, practically all existing biosensors are far from ideal in terms of their performance, properties, and applicability for multiplexed imaging. These limitations have inspired researchers to explore an increasing number of innovative and creative ways to improve and maximize biosensor performance. Such strategies include new molecular biology methods to develop promising biosensor prototypes, high throughput microfluidics-based directed evolution screening strategies, and improved ways to perform multiplexed imaging. Yet another approach is to effectively replace components of biosensors with self-labeling proteins, such as HaloTag, that enable the biocompatible incorporation of synthetic fluorophores or other ligands in cells or tissues. This mini-review will summarize and highlight recent innovations and strategies for enhancing the performance of FP-based biosensors for multiplexed imaging to advance the frontiers of research.

Fluorescent Protein-Based Sensors for Detecting Essential Metal Ions across the Tree of Life

“…210,211 Applications of chemigenetic sensors have primarily focused on mammalian cell lines and primary cells, however, and efforts to expand their use to plants, bacteria, and other organisms where the synthetic component would need to be efficiently delivered have been limited. 212,213 In the realm of transition metals, there are many Zn 2+ and several Cu + and/or Cu 2+ sensors, utilizing a few different binding domains, but there have been limited efforts in optimizing the dynamic range of these indicators compared to the Ca 2+ family (Tables 1 and 2). Among the other most prominent transition metals, there is one sensor family introduced each for Mn, Co, Ni, and Mo, but none for Fe.…”

“…Here, alternative genetically encoded platforms have made more progress . For example, chemigenetic sensors for Na + have been reported, which combine a synthetic crown ether-based Na + -binding domain with a fluorophore (synthetic or protein) and a tag protein that can be used for cellular targeting. , Applications of chemigenetic sensors have primarily focused on mammalian cell lines and primary cells, however, and efforts to expand their use to plants, bacteria, and other organisms where the synthetic component would need to be efficiently delivered have been limited. , In the realm of transition metals, there are many Zn 2+ and several Cu + and/or Cu 2+ sensors, utilizing a few different binding domains, but there have been limited efforts in optimizing the dynamic range of these indicators compared to the Ca 2+ family (Tables and ). Among the other most prominent transition metals, there is one sensor family introduced each for Mn, Co, Ni, and Mo, but none for Fe. − The development of sensors for some of these metal ions must overcome their rankings in the Irving–Williams series, which orders the first-row divalent transition metals according to the stabilities of their metal–ligand complexes (Mn 2+ < Fe 2+ < Co 2+ < Ni 2+ < Cu 2+ > Zn 2+ ) .…”

“…Achievements with the GCaMP series of Ca 2+ sensors can serve as inspiration and guidance. Recent improvements in accelerating directed evolution and screening approaches can aid in developing and optimizing an array of protein-based metal ion sensors . For example, microfluidic cytometry methods can be used to screen and optically tag and sort single cells. ,, These approaches enable testing of a wider range of variables in more cells than traditional screening on plates or in wells.…”

Nonbinary fungal signals and calcium‐mediated transduction in plant immunity and symbiosis

“…The development of bioluminescence resonance energy transfer (BRET)‐based aequorin‐GFP reporters, together with the introduction of high‐sensitivity photon‐counting cameras, revived the use of aequorin as the only available GECI that can so far generate quantitative subcellular resolution mapping of [Ca 2+ ] over time (Grenzi et al ., 2021). Moreover, the constant development of novel Ca 2+ biosensors with maximised performance (Chai et al ., 2023) opens up new perspectives in plant symbiotic and immunity studies, by allowing the detection of systemic signalling events at the level of the entire plant during different types of plant–microbe interactions.…”

Advancing cell surface modification in mammalian cells with synthetic molecules

“…Halo-Tag protein (HTP) is an engineered protein derived from the bacterial haloalkane dehalogenase, which selectively reacts with alkanes containing a terminal chloride group (chloroalkanes) forming a covalent bond. 167,168 Similar protein recognition tags, such as SNAP tags 169 and ACP tags, 170 could also be utilized in cell surface modication, but they will not be extensively discussed in this section. A two-step approach was utilized in the HTP strategy: the expression of HTP on the cellular membrane is achieved via genetic engineering methods and further combined with cargoes containing the chloroalkane.…”