Small-Molecule-Based Fluorescent Sensors for Selective Detection of Reactive Oxygen Species in Biological Systems.

Reactive oxygen species (ROS) encompass a collection of intricately linked chemical entities characterized by individually distinct physicochemical properties and biological reactivities. Although excessive ROS generation is well known to underpin disease development, it has become increasingly evident that ROS also play central roles in redox regulation and normal physiology. A major challenge in uncovering the relevant biological mechanisms and deconvoluting the apparently paradoxical roles of distinct ROS in human health and disease lies in the selective and sensitive detection of these transient species in the complex biological milieu. Small-molecule-based fluorescent sensors enable molecular imaging of ROS with great spatial and temporal resolution and have thus been appreciated as excellent tools for aiding discoveries in modern redox biology. We review a selection of state-of-the-art sensors with demonstrated utility in biological systems. By providing a systematic overview based on underlying chemical sensing mechanisms, we wish to highlight the strengths and weaknesses in prior sensor works and propose some guiding principles for the development of future probes.

iATPSnFR2: A high-dynamic-range fluorescent sensor for monitoring intracellular ATP.

We developed a significantly improved genetically encoded quantitative adenosine triphosphate (ATP) sensor to provide real-time dynamics of ATP levels in subcellular compartments. iATPSnFR2 is a variant of iATPSnFR1, a previously developed sensor that has circularly permuted superfolder green fluorescent protein (GFP) inserted between the ATP-binding helices of the ε-subunit of a bacterial F0-F1 ATPase. Optimizing the linkers joining the two domains resulted in a ~fivefold to sixfold improvement in the dynamic range compared to the previous-generation sensor, with excellent discrimination against other analytes, and affinity variants varying from 4 µM to 500 µM. A chimeric version of this sensor fused to either the HaloTag protein or a suitable spectrally separated fluorescent protein provides an optional ratiometric readout allowing comparisons of ATP across cellular regions. Subcellular targeting the sensor to nerve terminals reveals previously uncharacterized single-synapse metabolic signatures, while targeting to the mitochondrial matrix allowed direct quantitative probing of oxidative phosphorylation dynamics.

Direct measurement of dynamic attractant gradients reveals breakdown of the Patlak-Keller-Segel chemotaxis model.

Chemotactic bacteria not only navigate chemical gradients, but also shape their environments by consuming and secreting attractants. Investigating how these processes influence the dynamics of bacterial populations has been challenging because of a lack of experimental methods for measuring spatial profiles of chemoattractants in real time. Here, we use a fluorescent sensor for aspartate to directly measure bacterially generated chemoattractant gradients during collective migration. Our measurements show that the standard Patlak-Keller-Segel model for collective chemotactic bacterial migration breaks down at high cell densities. To address this, we propose modifications to the model that consider the impact of cell density on bacterial chemotaxis and attractant consumption. With these changes, the model explains our experimental data across all cell densities, offering insight into chemotactic dynamics. Our findings highlight the significance of considering cell density effects on bacterial behavior, and the potential for fluorescent metabolite sensors to shed light on the complex emergent dynamics of bacterial communities.

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

A bright red fluorescent genetically encoded sensor for lactate imaging.

Lactate plays a crucial role in energy metabolism and greatly impacts protein activities, exerting diverse physiological and pathological effects. Therefore, convenient lactate assays for tracking spatiotemporal dynamics in living cells are desirable. In this paper, we engineered and optimized a red fluorescent protein sensor for l-lactate named FiLa-Red. This indicator exhibited a maximal fluorescence change of 730 % and an apparent dissociation constant (Kd) of approximately 460 µM. By utilizing FiLa-Red and other sensors, we monitored energy metabolism in a multiplex manner by simultaneously tracking lactate and NAD+/NADH abundance in the cytoplasm, nucleus, and mitochondria. The FiLa-Red sensor is expected to be a useful tool for performing metabolic analysis in vitro, in living cells and in vivo.

A Highly Sensitive and Selective Fluorescent Sensor for Folic Acid Detection Based on D-penicillamine Stabilized Ag/Cu Alloy Nanoclusters.

In this work, a highly sensitive and selective method for detecting folic acid (FA) was developed using D-penicillamine (DPA) stabilized Ag/Cu alloy nanoclusters (DPA@Ag/Cu NCs). The yellow emission of DPA@Ag/Cu NCs was found to be quenched upon the addition of FA to the system. The fluorescence intensity quenching value demonstrated a linear relationship with FA concentrations ranging from 0.01 to 1200 µM, with a limit of detection (LOD) of 5.3 nM. Furthermore, the detection mechanism was investigated through various characterization analyses, including high resolution transmission electron microscopy, fluorescence spectra, ultraviolet-visible absorption spectra, and fluorescence lifetime. The results indicated that the fluorescence quenching induced by FA was a result of electron transfer from FA to the ligands of DPA@Ag/Cu NCs. The selectivity of the FA sensor was also evaluated, showing that common amino acids and inorganic ions had minimal impact on the detection of FA. Moreover, the standard addition method was successfully applied to detect FA in human serum, chewable tablets and FA tablets with promising results. The use of DPA@Ag/Cu NCs demonstrates significant potential for detecting FA in complex biological samples.

Lantern-type G-quadruplex fluorescent sensors for detecting divalent metal ions.

Three thioflavin T (ThT) derivatives, namely ThT/ethylenediaminetetraacetic acid conjugates (E1T, E2T, and E1T1P), were designed and synthesized as sensing components for divalent metal ion detection. Furthermore, these ThT derivatives were used to design lantern-type G-quadruplex (G4) fluorescent sensors. The fluorescence intensities of the ThT derivatives decreased by 1.2- to 5.6-folds in the presence of Ni2+ and Cu2+, respectively, regardless of the topology of the utilized G4. Conversely, when Mn2+ and Zn2+ coexisted in antiparallel G4, the fluorescence intensities of E2T increased to approximately 3.3- and 2.3-folds, respectively, depending on the concentration of the divalent metal ion, allowing for quantitative analyses. The Job plot analysis revealed that the binding ratio of G4 and E2T changed from 2:1 to 1:2 with the increasing concentration of the divalent metal ions. These results indicated that the basic principle of such a lantern-type G4 sensor can be applied to the detection of divalent metal ions and other types of targets, such as proteins, and small molecules via ThT derivatization.

o-phenylenediamine Derived Fluorescent Carbon Quantum dots for Detection of Hg(II) in Environmental Water.

With the expansion of human activities, the consequent influx of mercury (Hg) into the food chain and the environment is seriously threatening human life. Herein, nitrogen and sulfur co-doped fluorescent carbon quantum dots (yCQDs) were prepared via a hydrothermal method using o-phenylenediamine (OPD) and taurine as precursors. The morphological characteristics as well as spectral features of yCQDs indicated that the photoluminescence mechanism should be the molecular state fluorophores of 2, 3-diaminophenothiazine (oxOPD), which is the oxide of OPD. The as-synthesized yCQDs exhibited sensitive recognition of Hg2+. According to the investigation in combination of UV-Vis absorption spectra, time-resolved fluorescence spectra and quantum chemical calculations, the abundant functional groups on the surface of yCQDs allowed Hg2+ to bind with yCQDs through various interactions, and the formed complexes significantly inhibited the absorption of excitation light, resulting in the static fluorescence quenching of yCQDs. The proposed yCQDs was utilized for Hg2+ sensing with the limit of detection calculated to be 4.50 × 10- 8 M. Furthermore, the recognition ability of yCQDs for Hg2+ was estimated in tap water, lake water and bottled water, and the results indicated that yCQDs have potential applications in monitoring Hg2+.

Synthesis of P-Rich Carbon Quantum Dots for Sensitive Fluorescent Detection of 2-Methylimidazole.

Using o-phenylenediamine as carbon source and phytic acid as phosphorus source, two P-rich carbon quantum dots RCDs and BCDs were synthesized successfully by changing the reaction temperature and time of hydrothermal method. It was found that RCDs with red emission could realize sensitive detection of 2-methylimidazole, and 2-methylimidazole had no obvious quenching effect on BCDs with blue emission, which made RCDs a sensitive, quick and selective fluorescence sensor for 2-methylimidazole detection. Under the optimal experimental conditions, the fluorescence intensity of RCDs decreased with the increasing of 2-methylimidazole concentration. The detection of 2-methylimidazole concentration by the carbon quantum dots sensor showed a good linear relationship in the range of 5 ~ 110 µM, and the low detection limit was 0.61 µM (S/N = 3). The sensor is able to detect 2-methylimidazole in lake water, enabling the application of real samples. The results show that this work provides a simple fluorescence method to detect 2-methylimidazole in water.

A Novel Rhodamine B Derivative as a "Turn-on" Fluorescent Sensor for Cu2+ with High Selectivity and Sensitivity.

The number of "turn-on" fluorescent probes for Cu2+ is relatively limited, and interference from other metal cations presents a significant challenge for these sensors. In this study, we synthesized and characterized a rhodamine B-based sensor, designated as RBHP, using 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP) and rhodamine B hydrazide. Selectivity, sensitivity, solvent effects, water content, and pH of RBHP in relation to Cu²âº were conducted. RBHP exhibited an exceptionally low fluorescence background signal in acetonitrile and demonstrated a "turn-on" fluorescent response to Cu²âº. The PMBP-based acylhydrazone moiety and acetonitrile as the detection solvent are crucial for the selective detection. RBHP shows potential as a highly selective and sensitive fluorescent sensor for Cu2+.

Design and Application of Cu2+ Fluorescent Sensor Based on Carbazole Derivatives.

A novel fluorescent sensor for the detection of Cu2+ was developed based on carbazole derivatives. After the addition of Cu2+, the sensor exhibited obvious fluorescence quenching phenomenon, and the optical signal variation also enabled the sensor to quantitatively analyze Cu2+ due to the formation of a stable 1:1 metal-ligand complex in a short time. In addition, the sensor possessed chemical reversibility and pH stability. The cell imaging and zebra fish experiments also verified its application value in biological system.

Spectroscopic Investigation of Coumarin Based Novel Fluorescent TURN OFF Sensor for the Selective Detection of Fe3+: In-vitro Live Cell Imaging Application.

The novel TURN-OFF fluorescent sensors 4-(Benzo[1,3]dioxol-5-yloxymethyl)-7-hydroxy-chromen-2-one (4BHC) and 4-(6-Bromo-benzo[1,3]dioxol-5-yloxymethyl)-7-hydroxy-chromen-2-one (4BBHC) are designed and synthesized for the spectrofluorometric detection of the biologically important Fe3+ ions, which has sensitive and selective fluorescence quenching over other competitive metal ions. The effectiveness of the sensors and rapid response are validated through UV-Visible, and fluorescence spectral changes. These spectral changes could be due to the formation of coordination bond between ligand and metal ion. The binding stoichiometry of both sensors with Fe3+ ions is studied with the help of Job's plot, which gives a 1:2 coordination ratio; this is further confirmed through DFT, IR and NMR studies. The association constants of 4BHC and 4BBHC are calculated through Benesie-Hildebrand plots, and they are found to be 6 × 104 M-1 and 11.2 × 104 M-1 respectively. Following, LOD is calculated to define the range of sensitivity of the proposed sensors and is found to be 3.43 µM and 2.14 µM respectively. The chemical hardness parameter suggested that both sensors are soft molecules. In addition, low cytotoxicity levels of 4BHC and 4BBHC led to the demonstration of their efficacy in In-Vitro imaging of Fe3+ ions inside living cells, which ensures that these sensors are promising candidates for bioimaging.

Aggregation-induced Emission Active Naphthalimide Derived Schiff Base for Detecting Cu2+ and Its Applications.

Herein, an aggregation-induced emission (AIE) active Schiff base (NHS) was synthesized by condensing naphthalimide hydrazide with salicylaldehyde. The non-fluorescent solution of NHS in DMSO turned to emissive NHS upon increasing the HEPES fraction in DMSO from 70 to 95%. The UV-Vis absorption and DLS studies supported the self-aggregation of NHS that restricted the intramolecular rotation and activated the ESIPT process. The blue fluorescence of AIE luminogen NHS in DMSO:HEPES (5:95, v/v, pH = 7.4) was examined by adding different metal ions (Al3+, Ca2+, Cd2+, Co2+, Cu2+, Cr2+, Fe2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, Pb2+ and Zn2+). NHS showed a selective fluorescence switch-off response for Cu2+ due to the chelation enhancement quenching effect (CHEQ). The quenching of NHS by Cu2+ was explored by using density functional theory (DFT) and Stern-Volmer plot. The practical utility of NHS was examined by quantitative and qualitative analysis of Cu2+ in real water samples.

Novel An Efficient Fluorescent Probe Based on Calix[4]triazacrown-5 With Naphthalimide Group for Co2+, Cd2+ and Dopamine Detection.

A novel naphthalimide-substituted calix[4]triazacrown-5 (Nap-Calix) at cone conformation was designed and synthesized to employ as a fluorescent probe, which enables the simultaneously detection of Co2+ and Cd2+ metal ions as well as dopamine (DA). 1H-NMR, 13C-NMR, ESI-MS and elemental analysis techniques were carried out to characterize its structure. Cation binding property of Nap-Calix against various metal ions such as Ba2+, Co2+, Ni2+, Pb2+, Zn2+, and Cd2+ exhibited that the sensor selectively binds to Co2+ and Cd2+ metal ions with a remarkable affinity. Introduction of Co2+ and Cd2+ metal ions to a solution of Nap-Calix in DMF/water (1:1, v/v) resulted with a new emission band at 370 nm when excited at 283 nm. In addition, the fluorescence sensing affinity of the probe Nap-Calix against a catecholamine neurotransmitter (dopamine) was investigated in a wide range of concentration of DA (0-0.1 mmol L-1) in 50% DMF/PBS (pH = 5.0). The fluorescence intensity of Nap-Calix, with excitation/emission peaks at 283/327 nm, is highly enhanced by DA. It was also observed that Nap-Calix exhibits excellent fluorescence behavior towards DA with a very low detection limit as 0.21 µmol L-1.

A Novel Fluorescent Sensor for Detecting Ag+ and Hg2+ ions: A Combination of Theoretical and Experimental Studies.

A new fluorescent sensor based on diethylaminosalicylaldehyde-thiosemicarbazide (DST) was studied using a combination of density functional theory calculations and experimental investigations. DST was able to detect the metal ions Ag+ and Hg2+ in the presence of various competing metal ions and anions, with detection limits of 0.45 and 0.34 µM, respectively. The DST sensor could operate in a fully aqueous environment and within a wide pH range from 5 to 9. Density functional theory studies supported the experimental findings in determining the stable structures of the DST sensor and the complexes between DST and the Ag+ and Hg2+ ions, as well as elucidating the fluorescence ON-OFF mechanism in the DST sensor and the complexes.

Benzothiazole-Quinoline-Based Fluorescent Probe for Fe3+ and its Applications in Environmental and Biological Samples.

Due to the its high abundance, iron ion contamination and toxicity is one of the most challenging issue for living beings. Although, iron is extremenly important for several body functions, excess amount of iron in the body can also be fatal. In last century, rapid industrialization, iron extraction and mismanagement of industrial waste disposal leads to iron contamination in water bodies. Therefore, versatile iron sensors needs to be develop which can be employed for detection in biological as well as real water samples. 8-hydroxyquinoline is well-known for its strong affinity towards transition metals including Fe3+. In this regard, we have synthesised benzothiazole-quinoline derived 1,2,3- triazole (4HBTHQTz), in which 4-(benzo[d]thiazol-2-yl)phenolic (4-HBT) group acts as a fluorophore. 4HBTHQTz showed high fluorescence and induced a selective decrease in fluorescence with Fe3+ at 380 nm (λex. = 320 nm). The detection limit of 4HBTHQTz with Fe3+ is calculated as 0.64 µM, which is lower than the WHO recommended limit in drinking water. 4HBTHQTz works over the 5-8 pH range and has shown promising results for quantitative detection of Fe3+ in water samples collected from tap, river and seawater. 4HBTHQTz can also detect the Fe3+ in biological samples which is confirmed by fluorescence cell imaging using L929 mouse fibroblast cells. Overall, 4HBTHQTz showed advantages such as high selectivity, quick detection, and good limit of detection (LOD) for Fe3+.

Facile and Sustainable Electrostatic Integration of Eosin Y Dye for the Assay of the Atomoxetine Drug Through the Enhancement of the RRS Phenomenon.

The suggested study follows specific protocols to guarantee that the atomoxetine drug analysis approach is environmentally friendly and sustainable. A number of recently created methods were used as prospective evidence for environmental sustainability and applicability, which is an essential point to emphasize. The current study introduces a new and very unique technology using ultrasensitive spectrofluorimetry to identify the atomoxetine (AXT) medication. A one-step, one-pot, direct spectrofluorimetric technique was used in this study's methodology, which was determined to be effective and environmentally sustainable for the drug's evaluation and validation. When AXT and EY reagent were mixed in an acidic setting, a highly resonant Rayleigh scattering product was promptly generated. The application of fluorimetric analysis was built on a novel theory that the augmentation in dye response subsequent to the addition of AXT was directly correlated to the resultant complex's molecular mass, as determined at a wavelength of 365 nm. The complexation progression caused a considerable rise in the molecular mass, from 292.82 (AXT+) to 983.68 (AXT-EY) g mol-1. The quantum yield for the coupled product was measured. The linearity was determined to be between 30 and 1500 ng mL-1, while the sensitivity values were found to be between 9.2 and 28.1 ng mL-1. Analyzing AXT-EY complexes allowed us to determine the best values for all of the system's adjustable parameters. The system showed adherence to International Council for Harmonization (ICH) criteria without difficulties. The recommended procedure was then evaluated for environmental friendliness using many current environmental safety metrics. Fortunately, the current analytical technique is also recognized as a white one by the WAC standards, which integrate functional and ecological features using the Green/Red/Blue (RGB 12) design. A novel instrument called BAGI was used to assess the feasibility of the proposed approach in the field of analytical chemistry.

Hazardous 2,4,6-Trinitrophenol (TNP) Detection in Water by Amine and Azine Functionalized Metal-Organic Framework.

A Zn(II)-based metal-organic framework (MOF) decorated with amine and azine functionalities, TMU-17-NH2 (formulated as [Zn(H2ata)(L)].2DMF; L = 1,4-bis(4-pyridyl)-2,3-diaza-2,3-butadiene and H2ata = 2-aminoterephthalic acid) has been successfully synthesized via a solvothermal method. According to crystallographic studies, the synthesized TMU-17-NH2 has three dimensional cuboidal structure with the pore surface decorated with free amine (-NH2) and azine (= N-N =) functional groups. The photoluminescence investigations proved that the synthesized MOF can be effectively utilized for selective detection of 2,4,6-trinitrophenol (TNP) in water with an apparent turn-off quenching response. Its limits of detection (LOD) for TNP was 9.4 ppb and competitive nitro explosive testing confirmed its higher selectivity towards TNP (over other nitro explosives). Calculations based on density functional theory (DFT) and spectrum overlap were utilized to evaluate the sensing mechanisms. This MOF-based fluorescence sensing technique for TNP had a high sensitivity (Ksv = 3.26 × 104 M-1).

Chemical sensors detect and resolve proteome aggregation in peripheral neuropathy cell model induced by chemotherapeutic agents.

As a consequence of somatosensory nervous system injury or disease, neuropathic pain is commonly associated with chemotherapies, known as chemotherapy-induced peripheral neuropathy (CIPN). However, the mechanisms underlying CIPN-induced proteome aggregation in neuronal cells remain elusive due to limited detection tools. Herein, we present series sensors for fluorescence imaging (AggStain) and proteomics analysis (AggLink) to visualize and capture aggregated proteome in CIPN neuronal cell model. The environment-sensitive AggStain imaging sensor selectively binds and detects protein aggregation with 12.3 fold fluorescence enhancement. Further, the covalent AggLink proteomic sensor captures cellular aggregated proteins and profiles their composition via LC-MS/MS analysis. This integrative sensor platform reveals the presence of proteome aggregation in CIPN cell model and highlights its potential for broader applications in assessing proteome stability under various cellular stress conditions.

A highly sensitive fluorescence sensor for tobacco mosaic virus RNA based on DSN cycle and ARGET ATRP double signal amplification.

Early and sensitive detection of tobacco mosaic virus (TMV) is of great significance for improving crop yield and protecting germplasm resources. Herein, we constructed a novel fluorescence sensor to detect TMV RNA (tRNA) through double strand specific nuclease (DSN) cycle and activator regenerative electron transfer atom transfer radical polymerization (ARGET ATRP) dual signal amplification strategy. The hairpin DNA complementarily paired with tRNA was used as a recognition unit to specifically capture tRNA. By the double-stranded DNA hydrolyzed with DSN, tRNA is released to open more hairpin DNA, and more complementary DNA (cDNA) is bound to the surface of the magnetic beads (MBs) to achieve the first amplification. After binding with the initiator, the cDNA employed ARGET ATRP to attach more fluorescent signal molecules to the surface of MBs, thus achieving the second signal amplification. Under the optimal experimental conditions, the logarithm of fluorescence intensity versus tRNA concentration showed a good linear relationship in the range of 0.01-100 pM, with a detection limit of 1.03 fM. The limit of detection (LOD) was calculated according to LOD = 3 N/S. Besides, the sensor showed good reproducibility and stability, which present provided new method for early and highly sensitive detection for plant viruses.