The co-existence and co-transmission of neuropeptides and small molecule neurotransmitters in the same neuron is a fundamental aspect of almost all neurons across various species. However, the differences regarding their in vivo spatiotemporal dynamics and underlying molecular regulation remain poorly understood. Here, we developed a GPCR-activation-based (GRAB) sensor for detecting short neuropeptide F (sNPF) with high sensitivity and spatiotemporal resolution. Furthermore, we explore the differences of in vivo dynamics and molecular regulation between sNPF and acetylcholine (ACh) from the same neurons. Interestingly, the release of sNPF and ACh shows different spatiotemporal dynamics. Notably, we found that distinct synaptotagmins (Syt) are involved in these two processes, as Syt7 and Sytα for sNPF release, while Syt1 for ACh release. Thus, this new GRAB sensor provides a powerful tool for studying neuropeptide release and providing new insights into the distinct release dynamics and molecular regulation between neuropeptides and small molecule neurotransmitters.

Oncogenic mutations can destabilize signaling proteins, resulting in increased or unregulated activity. Thus, there is considerable interest in mapping the relationship between mutations and the stability of signaling proteins, to better understand the consequences of oncogenic mutations and potentially inform the development of new therapeutics. Here, we develop a tool to study protein-kinase stability in live mammalian cells and the effects of the HSP90 chaperone system on the stability of these kinases. We determine the expression levels of protein kinases by monitoring the fluorescence of fluorescent proteins fused to those kinases, normalized to that of co-expressed reference fluorescent proteins. We used this tool to study the dependence of Src- and Raf-family kinases on the HSP90 system. We demonstrate that this sensor reports on destabilization induced by oncogenic mutations in these kinases. We also show that Src-homology 2 and Src-homology 3 domains, which are required for autoinhibition of Src-family kinases, stabilize these kinase domains in the cell. Our expression-calibrated sensor enables the facile characterization of the effects of mutations and small-molecule drugs on protein-kinase stability.

Recently, fluorescent sensors have gained considerable attention due to their high sensitivity, low cost and noninvasiveness. Among the different materials that can be used for this purpose, carbon dots (CDs) represent valuable candidates for applications in sensing. These, indeed, are easily synthesized, show high quantum yield and are highly biocompatible. However, it was pointed out that the photoluminescence properties of these nanomaterials are strictly dependent on the synthetic and purification methods adopted. The presence of halloysite nanotubes (HNTs), a natural, low cost and biocompatible clay mineral, has been found to be efficient in obtaining small and highly monodispersed CDs without long and tedious purification techniques. Herein, we report the comparison of synthetic pathways for obtaining halloysite-N-doped CDs (HNTs-NCDs) that could be used in biological sensing. One was based on the synthesis of N-doped CDs by a bottom-up approach on HNTs\' surface by a MW pyrolysis process; the other one was based on the post-modification of pristine N-doped CDs with halloysite derivatives. The evaluation of the best synthetic route was performed by different physico-chemical techniques. It was found that the bottom-up approach led to the formation of N-doped CDs with different functional groups onto the HNTs\' surface. This evidence was also translated in the different fluorescence quantum yields and the existence of several functional groups in the obtained materials was investigated by potentiometric titrations. Furthermore, the ability of the synthesized nanomaterials as sensors for Fe3+ ions detection was assessed by spectroscopic measurements, and the cellular uptake was verified by confocal/fluorescence microscopies as well.

Microbes synthesize and secrete siderophores, that bind and solubilize precipitated or otherwise unavailable iron in their microenvironments. Gram (-) bacterial TonB-dependent outer membrane receptors capture the resulting ferric siderophores to begin the uptake process. From their similarity to fepA, the structural gene for the Escherichia coli ferric enterobactin (FeEnt) receptor, we identified four homologous genes in the human and animal ESKAPE pathogen Klebsiella pneumoniae (strain Kp52.145). One locus encodes IroN (locus 0027 on plasmid pII), and three other loci encode other FepA orthologs/paralogs (chromosomal loci 1658, 2380, and 4984). Based on the crystal structure of E. coli FepA (1FEP), we modeled the tertiary structures of the K. pneumoniae FepA homologs and genetically engineered individual Cys substitutions in their predicted surface loops. We subjected bacteria expressing the Cys mutant proteins to modification with extrinsic fluorescein maleimide (FM) and used the resulting fluorescently labeled cells to spectroscopically monitor the binding and transport of catecholate ferric siderophores by the four different receptors. The FM-modified FepA homologs were nanosensors that defined the ferric catecholate uptake pathways in pathogenic strains of K. pneumoniae. In Kp52.145, loci 1658 and 4984 encoded receptors that primarily recognized and transported FeEnt; locus 0027 produced a receptor that principally bound and transported FeEnt and glucosylated FeEnt (FeGEnt); locus 2380 encoded a protein that bound ferric catecholate compounds but did not detectably transport them. The sensors also characterized the uptake of iron complexes, including FeGEnt, by the hypervirulent, hypermucoviscous K. pneumoniae strain hvKp1.
Both commensal and pathogenic bacteria produce small organic chelators, called siderophores, that avidly bind iron and increase its bioavailability. Klebsiella pneumoniae variably produces four siderophores that antagonize host iron sequestration: enterobactin, glucosylated enterobactin (also termed salmochelin), aerobactin, and yersiniabactin, which promote colonization of different host tissues. Abundant evidence links bacterial iron acquisition to virulence and infectious diseases. The data we report explain the recognition and transport of ferric catecholates and other siderophores, which are crucial to iron acquisition by K. pneumoniae.

Specific identification and monitoring of senescent cells are essential for the in-depth understanding and regulation of senescence-related life processes and diseases. Fluorescent sensors providing real-time and in situ information with spatiotemporal resolution are unparalleled tools and have contributed greatly to this field. This review focuses on the recent progress in fluorescent sensors for molecularly targeted imaging and real-time tracking of cellular senescence. The molecular design, sensing mechanisms, and biological activities of the sensors are discussed. The sensors are categorized by the types of markers and targeting ligands. Accordingly, their molecular recognition and fluorescent performance towards senescence biomarkers are summarized. Finally, the perspective and challenges in this field are discussed, which are expected to assist future design of next-generation sensors for monitoring cellular senescence.

Molecularly imprinted polymers have received wide attention from various fields owing to their pre-designable, recognition ability, and practicality. However, the disadvantages of the traditional embedding method, which include a slow recognition rate, uneven site recognition, low binding capacity, and incomplete template molecule elution, limit the development of molecular imprinting technology. Surface molecular imprinting techniques have been developed to effectively solve these problems, and different materials are used as carriers in the synthesis of molecularly imprinted polymers. Metal-organic frameworks (MOFs) show great potential as carriers. Because of their high porosity and specific surface area, MOFs can provide a large number of active sites for molecular imprinting, which can improve their detection sensitivity. The variable metal centers and organic ligands of MOF materials can also lead to multiple structures and functions. Numerous types of MOF materials have been synthesized, and the properties of these materials can be tailored by adjusting their pore size and introducing functional groups. MOFs and molecular imprinting technology can be combined to take full advantage of the specific adsorption of molecular imprinting technology and the large specific surface area and multiple active sites of MOFs, thereby expanding the application range of the resulting materials. In this paper, five aspects of the concept of MOF functionalization are discussed: introduction of special ligands, regulation of metal central sites, formation of MOF complexes, derivatization of MOFs, and sacrificial MOFs. The applications of MOF-based molecularly imprinted materials in catalysis, sample pretreatment, drug carriers, fluorescence sensors, and electrochemical sensors are also reviewed. Finally, the existing problems and future development of MOF-based molecularly imprinted materials are discussed and prospected.

A concept of a microfluidic fluorescent chemical sensing system is presented and demonstrated as a sensor for measurement of dissolved oxygen in water. The system utilizes on-line mixing of a fluorescent reagent with the analyzed sample, while it measures the fluorescence decay time of the mixture. The system is built entirely out of silica capillaries and optical fibers, and allows for very low consumption of the reagent (of the order of mL/month) and the analyzed sample (of the order of L/month). The proposed system can, thus, be applied to continuous on-line measurements, while utilizing a broad variety of different and proven fluorescent reagents or dyes. The proposed system allows for the use of relatively high-excitation light powers, as the flow-through concept of the system reduces the probability of the appearance of bleaching, heating, or other unwanted effects on the fluorescent dye/reagent caused significantly by the excitation light. The high amplitudes of fluorescent optical signals captured by an optical fiber allow for low-noise and high-bandwidth optical signal detection, and, consequently, the possibility for utilization of reagents with nanosecond fluorescent lifetimes.

Here, we present a ditopic ion-pair sensor, B1, containing the BODIPY reporter unit in its structure, which is shown to be able-thanks to the presence of two heterogeneous binding domains-to interact with anions in an enhanced manner in the presence of cations. This enables it to interact with salts even in 99% aqueous solutions, making B1 a good candidate in terms of visual salt detection in the aquatic environment. Receptor B1\'s ability to extract and release salt was applied in the transport of potassium chloride through a bulk liquid membrane. Working with a concentration of B1 in the organic phase and with the presence of a specific salt in an aqueous solution, an inverted transport experiment was also demonstrated. By varying the type and the amount of the anions added to B1, we were able to develop diverse optical responses, including a unique four-step ON1-OFF-ON2-ON3 output.

Metal cations such as Zn2+, Al3+, Hg2+, Cd2+, Sn2+, Fe2+, Fe3+ and Cu2+ play important roles in biology, medicine, and the environment. However, when these are not maintained in proper concentration, they can be lethal to life. Therefore, selective sensing of metal cations is of great importance in understanding various metabolic processes, disease diagnosis, checking the purity of environmental samples, and detecting toxic analytes. Schiff base probes have been largely used in designing fluorescent sensors for sensing metal ions because of their easy processing, availability, fast response time, and low detection limit. Herein, an in-depth report on metal ions recognition by some Schiff base fluorescent sensors, their sensing mechanism, their practical applicability in cell imaging, building logic gates, and analysis of real-life samples has been presented. The metal ions having biological, industrial, and environmental significance are targeted. The compiled information is expected to prove beneficial in designing and synthesis of the related Schiff base fluorescent sensors.

The design of selective metal-binding sites is a challenge in both small-molecule and macromolecular chemistry. Selective recognition of manganese (II)-the first-row transition metal ion that tends to bind with the lowest affinity to ligands, as described by the Irving-Williams series-is particularly difficult. As a result, there is a dearth of chemical biology tools with which to study manganese physiology in live cells, which would advance understanding of photosynthesis, host-pathogen interactions, and neurobiology. Here we report the rational re-engineering of the lanthanide-binding protein, lanmodulin, into genetically encoded fluorescent sensors for MnII, MnLaMP1 and MnLaMP2. These sensors with effective Kd(MnII) of 29 and 7 µM, respectively, defy the Irving-Williams series to selectively detect MnII in vitro and in vivo. We apply both sensors to visualize kinetics of bacterial labile manganese pools. Biophysical studies indicate the importance of coordinated solvent and hydrophobic interactions in the sensors\' selectivity. Our results establish lanmodulin as a versatile scaffold for design of selective protein-based biosensors and chelators for metals beyond the f-block.