Fluorescent probes for bioimaging and biosensing

Single-molecule fluorescent probes can be used for nanoreporting, localization, and now multiplexing, but understanding their stochastic fluctuations in emission intensity is crucial for accurate signal interpretation. Here, the authors elucidate the blinking dynamics of rhodamine, BODIPY, and antraquinone dyes, demonstrate that multiplexing performance improves with photophysical differences, and suggest guidelines for the selection and design of organic fluorophores for single-molecule multiplexing.

Cathepsin D (CatD) is an aspartic acid protease involved in the immune response of macrophages to bacterial infection, and the development of pH insensitive CatD probes is vital in order to detect its activity across multiple cellular components. Here, the authors develop a water soluble and pH insensitive FRET-based fluorescent probe for the sensitive detection of CatD.

Fluorescent labeling of proteins by fluorophore Prodan is a useful tool to measure protein conformational changes via color switching, however, structure-based rules that predict where to emplace fluorophores that elicit robust fluorescent responses are challenging to establish. Here, the authors investigate mutated glucose-binding proteins labeled with Prodan derivatives, and uncover a mechanism for Prodan ligand-mediated color switching that enables structure-based biosensor design using specific steric interactions between fluorophore and protein.

Norepinephrine (NE) is a key neurotransmitter in the central nervous system of organisms however specifically tracking the transient NE dynamics with high spatiotemporal resolution in living systems remains a great challenge. Herein, the authors developed a small molecular fluorescent probe that can precisely anchor on neuronal cytomembranes and specifically respond to NE on a 100-ms timescale.

Specific detection of cellular superoxide is challenging. Here, the authors designed 1,2,4,5-tetrazine based fluorogenic probes for specific and sensitive imaging of superoxide, and applied them in high throughput screening of modulators of oxidative stress.

Studying the specific roles of macrophage subsets has been hampered by a lack of subset-specific probes. Here the authors report an M1 selective fluorescent probe named CDr17, and demonstrate the suitability of this probe for tracking M1 macrophages in vivo.

The main protease (M^pro) of SARS-CoV-2 is an important target for COVID-19 therapy. Here, a pair of genetically encoded BRET-based sensors for detecting M^pro activity are generated by sandwiching N-terminal autocleavage sites in between the mNeonGreen and NanoLuc proteins.

Fluorogenic detection of H₂O₂ in cells is established, but equivalent tools to monitor its cellular targets remain in their infancy. Here authors develop fluorogenic probes for detecting cysteine sulfenic acid, a redox modification inextricably linked to H₂O₂ signalling and oxidative stress.

Small molecule NIR-II fluorophores are of interest for a range of applications but can suffer from chemical and photostability issues. Here, the authors report on the development of an acceptor molecule with improved stability in alkaline conditions expanding the range of possible applications.

Expanding the responsive dyes repertoire is currently a developing field in biorthogonal chemistry. In this article, the authors develop fluorophores that turn on their near-infrared fluorescence upon biorthogonal reaction based on a “torsion-induced disaggregation” approach, allowing for sensitive in vivo imaging of tumors.

Super-resolution microscopy is a powerful tool for cellular studies but requires bright and stable fluorescent probes. Here, the authors report on a strategy to introduce quinoxaline motifs to conventional probes to make them brighter, more photostable, larger Stokes shift, and demonstrate the probes for biosensing applications.

Using metal coordination to assemble carbon nanodots (CND) into clusters can enhance their photophysical properties for applications in sensing and biomedicine. Here, carboxylate groups on the surface of carbon nanodots serve as ligands for the coordination of manganese ions, enabling the assembly of optically and magnetically active CND clusters in a one-step microwave-assisted synthesis in water.

To achieve high-contrast in fluorescence imaging of deep tissues is challenging. Here, the authors develop NIR-II fluorescent small molecules with high brightness and emission extending to 1900 nm, enabling in vivo imaging of deep tissues with enhanced signal-to-background ratios.

Monitoring of the hypertension drug felodipine is important to avoid unwanted side effects from high concentrations, but its sensitive detection remains challenging. Here, combining the advantages of ratiometric luminescence probes and near-infrared lanthanide luminescence sensing, multi-emission near-infrared magnetic core-shell nanoparticles based on lanthanide metal–organic frameworks are synthesized using a layer-by-layer method and shown to have excellent temperature and felodipine detection abilities.

Afterglow luminescence is promising for non-background molecular imaging in vivo. Here the authors report a ratiometric afterglow luminescent nanoplatform to generate activatable afterglow probes for quantification of specific analytes including NO.

Mechanically interlocked supramolecular molecules show potential as imaging probes for biomedical applications. Here, the authors developed synthetic routes based on multicomponent reactions to access rotaxane-based bimodal imaging agents for nuclear and optical detection using the cucurbit[6]uril CB[6]-mediated azide-alkyne click reaction.

Sensing small biomolecules in biofluids using host-guest chemosensors remains challenging, in part due to the impact of interfering components. Here, the authors develop a dual-macrocyclic rotaxane for tryptophan detection which can function in biofluids such as human serum and urine.