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Chemiluminescent probes based on 1,2-dioxetane scaffold are one of the most sensitive imaging modalities for detecting disease-related biomarkers and can obtain more accurate biological information in cells and in vivo. Due to the elimination of external light excitation, the background autofluorescence problem in fluorescence technology can be effectively avoided, providing ultrahigh sensitivity and signal-to-noise ratio for various applications. In this review, we highlight a comprehensive but concise overview of activatable 1,2-dioxetane-based chemiluminescent probes by reporting significant advances in accurate detection and bioimaging. The design principles and applications for reactive species, enzymes, and other disease-related biomarkers are systematically discussed and summarized. The challenges and potential prospects of chemiluminescent probes are also discussed to further promote the development of new chemiluminescence methods for biological analysis and diagnosis.

Organic phosphate biomolecules (OPBs) are indispensable components of eukaryotes and prokaryotes, such as acting as the fundamental components of cell membranes and important substrates for nucleic acids. They play pivotal roles in various biological processes, such as energy conservation, metabolism, and signal modulation. Due to the difficulty of detection caused by variety OPBs, investigation of their respective physiological effects in organisms has been restrained by the lack of efficient tools. Many small fluorescent probes have been employed for selective detection and monitoring of OPBs in vitro or in vivo due to the advantages of tailored properties, biodegradability and in situ high temporal and spatial resolution imaging. In this review, we summarize the recent advances in fluorescent probes for OPBs, such as nucleotides, NAD(P)H, FAD/FMN and PS. Importantly, we describe their identification mechanisms in detail and discuss the general strategies for these OPBs probe designs, which provide new insights and ideas for the future probe designs.

Thiophenols as high toxic environmental pollutants are poisonous for animals and aquatic organisms. Therefore, it is indispensable to monitor thiophenols in the environment. Herein, a novel near-infrared fluorescent probe was developed for the detection of thiophenols, which was easily prepared by one-step coupling of 2,4-dinitrobenzenesulfonyl chloride with Nile blue. The probe showed a significant near infrared (∼675 nm) fluorescence “turn-on” response to thiophenols with some good features including chromogenic reaction, high sensitivity and selectivity, fast response, near-infrared emission along with low detection limit (1.8 nM). The probe was employed to rapidly and visually determine thiophenols in several industrial wastewaters with good recoveries (90–110%). Moreover, this probe has been demonstrated good capability for imaging thiophenol in HeLa cells.

Microglia, the brain-resident macrophage, are involved in brain development and contribute to the progression of neural disorders. Despite the importance of microglia, imaging of live microglia at a cellular resolution has been limited to transgenic mice. Efforts have therefore been dedicated to developing new methods for microglia detection and imaging. Using a thorough structure-activity relationships study, we developed CDr20, a high-performance fluorogenic chemical probe that enables the visualization of microglia both in vitro and in vivo. Using a genome-scale CRISPR-Cas9 knockout screen, the UDP-glucuronosyltransferase Ugt1a7c was identified as the target of CDr20. The glucuronidation of CDr20 by Ugt1a7c in microglia produces fluorescence.

Sodium sulfite (Na2SO3) is widely used as a food additive, but the excessive residue of SO32− in food can cause irritative effects and damages to the human body. In this study, we reported a novel near-infrared (NIR) fluorescent probe (DCMQ) for visual detection of SO32− based on a chromogenic reaction. The probe can quantitatively determine SO32− with high specificity and sensitivity, fast response (

Hydrazine and bisulfite are poisonous to the environment and human health, hence it is necessary to monitor hydrazine and bisulfite in the environment and living cells. In this study, we reported a smart fluorescent probe (CMCA) for discriminative detection of hydrazine and bisulfite. The single fluorescent probe can respond to hydrazine and bisulfite with two different sets of colorimetric and ratiometric fluorescence signals together with high selectivity, fast response and low detection limit. The discriminative sensing behaviors of the probe for hydrazine and bisulfite were ascribed to different reaction mechanisms, which were confirmed by 1H NMR and HR-MS spectra. The probe has been successfully used to discriminatively determine hydrazine and bisulfite in industrial wastewater with good recoveries. Furthermore, the probe was employed to visualize hydrazine and bisulfite in live cells from different fluorescence channels with good performance. Therefore, this probe has great potential applications for discriminative detection of hydrazine and bisulfite in the environment and living cells.

A series of highly photostable, lysosome-targeted BODIPYs with tunable fluorescence from green to the near-infrared region was developed. The probes were found to show an accurate lysosome-targeting ability with high selectivity and high photostability under continuous excitation irradiation. The results show that these lysosome-targeted BODIPYs offer a simple and convenient tool for fluorescence imaging of lysosomes, which will be valuable for understanding the activity and function of lysosomes in related cells.

Summary Elastic fibers are present in a variety of tissues and are responsible for their resilience. Until now, no optical contrast agent in the near-infrared (NIR) wavelength range of 700–900 nm has been reported for the imaging of elastic fibers. Here, we report the discovery of a NIR zwitterionic elastin probe ElaNIR (elastin NIR) through fluorescent-image-based screening. The probe was successfully applied for in vitro , ex vivo , and in vivo imaging by various imaging modalities. Age-related elastin differences shown by in vivo fluorescent and photoacoustic imaging indicated that ElaNIR can be a potentially convenient tool for uncovering changes of elastin in live models.

Drug-induced acute kidney injury (AKI), caused by renal drug metabolism, has been regarded as a main problem in clinical pharmacology and practice. However, due to the lack of effective biomarkers and noninvasive real-time tools, the early diagnosis of drug-induced AKI is still a crucial challenge. The superoxide anion (O2˙-), the preliminary reactive oxidative species, is closely related to drug-induced AKI. In this paper, we reported two new mitochondria-targeted fluorescent probes for investigating AKI via mapping the fluctuation of O2˙- with high sensitivity and selectivity by the combination of rational design and a probe-screening approach. Small-molecule fluorescent probes (Naph-O2˙- and NIR-O2˙- ) with high accuracy and excellent selectivity were successfully applied to detect endogenously produced O2˙- in living cells and tissues by dual-model confocal imaging, and to trap the fluctuation of the O2˙- level during the drug-induced nephrotoxicity. Moreover, probe NIR-O2˙- was also used to elucidate the protective effects of l-carnitine (LC) against drug-induced nephrotoxicity for the first time. Therefore, these probes may be potential chemical tools for exploring the roles of O2˙- in complex nephrotoxicity disease systems.

A new strategy that integrates the targeting group and response moiety together for the preparation of mitochondrial probe was developed. Bioimaging studies have shown that for the first time, the newly designed probe HDFL-Cys can first accumulate in mitochondria and then react with the analyte., Mitochondria are vital organelles that not only produce cellular energy but also participate in many biological processes. Recently, various fluorescent probes have been developed for mitochondrial imaging. However, due to the lack of suitable dyes or strategies, it is difficult for most reported mitochondrial targeting probes to prove whether the analytes they detected are from mitochondria. In addition, positive charge on mitochondrial probes can seriously affect the mitochondrial environment. To address these issues, we herein put forward a novel strategy for probe design based on a smart NIR dye (HDFL) for mitochondrial targeting detection. Compared to general mitochondrial targeting probes that are modified with a target site and a reaction site, the new strategy is to combine the two sites together for a mitochondrial probe that would provide accurate detection of analytes in mitochondria without interference. As a proof of concept, we synthesized a mitochondrial-targetable probe HDFL-Cys for cysteine. Bioimaging studies have shown that the new type of probe HDFL-Cys can first accumulate in mitochondria and then react with the analyte (cysteine) accompanied by the departure of the targeting group (lipophilic cation moieties). Thus, it can specifically detect the analyte in mitochondria without interference from extra-mitochondrial analytes. We anticipate that the new strategy based on the novel NIR dye HDFL may be a potential platform for developing desirable ratiometric fluorescent probes for mitochondrial imaging.

A multi-color labelling technique for visualizing multiple intracellular apparatuses in their native environment using small fluorescent probes remains challenging. This approach requires both orthogonal and biocompatible coupling reactions in heterogeneous biological systems with minimum fluorescence background noise. Here, we present a palette of BODIPY probes containing azide and cyclooctyne moieties for copper-free click chemistry in living cells. The probes, referred to as ‘tame probes’, are highly permeable and specific in nature, leaving no background noise in cells. Such probes, which are rationally designed through optimized lipophilicity, water solubility and charged van der Waals surface area, allow us to demonstrate rapid and efficient concurrent multi-labelling of intracellular target components. We show that these probes are capable of not only labelling organelles and engineered proteins, but also showing the intracellular glycoconjugates’ dynamics, through the use of metabolic oligosaccharide engineering technology in various cell types. The results demonstrated in this study thus provide flexibility for multi-spectral labelling strategies in native systems in a high spatiotemporal manner.

The need for detecting and labelling environmentally and biologically important analytes has driven considerable research efforts in developing fluorescent probes. During the sensing process, molecular motions (i.e., molecular rotations or vibrations) of a flexible fluorescent probe can be significantly altered by its embedding micro-environment or analyte, thereby leading to substantial changes in readout signals. Motion-induced change in emission (MICE) can be utilized as an effective sensing mechanism. However, in comparison to the well-understood sensing mechanisms, such as photo-induced electron transfer (PET), intramolecular charge transfer (ICT), aggregation-induced emission (AIE) and disaggregation-induced emission (DIE), MICE has not been systematically discussed to date. In this tutorial review, we will summarize the concept and mechanisms of MICE for developing single-molecular fluorescent probes, present unique advantages of MICE based sensors, demonstrate their various applications, and discuss technical challenges in this field. We expect that this review will promote a deeper understanding of MICE and facilitate the development of novel MICE based probes.

Hydrogen sulfide (H2S) has been found to be an important biological regulator that plays important roles in many physiological and pathological processes. Near-infrared (NIR) fluorescent probes capable of selectively detecting H2S in vivo will be useful tools to understand its mechanisms in biological processes. Herein, we reported an easily synthesized and stimuli-responsive NIR fluorescent probe (QCy7-HS) for selective evaluation of endogenous H2S in the living cells and mice. In response to cellular H2S stimulus, QCy7-HS is converted to QCy7 and shows a unique off-on near-infrared fluorescence signal change. The results of selectivity and kinetic studies indicated that our probe has high H2S binding capacity. Therefore, this probe was used for the fluorescence detection of H2S in cells. Moreover, the probe was applied for study exogenous and endogenous H2S in live mice, indicating that the new probe can be used as an efficient tool on H2S related events in living animals.

The novel ratiometric fluorescent probe HPQRB with an ESIPT effect based on Michael addition for highly sensitive and fast detection of sulfite in living HepG2 cells is reported. HPQRB can be easily synthesized by a two‐step condensation reaction. HPQRB has a large emission shift (Δλ=116 nm), which is beneficial for fluorescence imaging research, and its sulfite‐responsive site is based on a rhodamine‐like structure with the emission peak at 566 nm, which decreases with increasing sulfite concentration. and its HPQ structure always has an ESIPT effect throughout the reaction process, keeping the emission peak at 450 nm as a self‐reference. In particular, HPQRB has high selectivity for sulfite and responds quickly (within 30 s) with a low detection limit (44 nM). Furthermore, HPQRB has been successfully used for fluorescence imaging of sulfite in HepG2 cells, demonstrating the superior ability to detect sulfite under physiological conditions.

Near-infrared (NIR) fluorescence imaging technique is garnering increasing research attention due to various advantages. However, most NIR fluorescent probes still suffer from a false signals problem owing to their instability in real application. Especially in a pathological environment, many NIR probes can be easily destroyed due to the excessive generation of highly reactive species and causing a distorted false signal. Herein, we proposed an approach for developing a new stable NIR dye platform with an optically tunable group to eliminate false signals using the combination of dyes screening and rational design strategy. The conception is validated by the construction of two high-fidelity NIR fluorescent probes (NIR-LAP and NIR-ONOO–) sensing leucine aminopeptidase (LAP) and peroxynitrite (ONOO–), the markers of hepatotoxicity. These probes (NIR-LAP and NIR-ONOO–) were demonstrated to sensitively and accurately monitor LAP and ONOO– (detection limit: 80 mU/L for LAP and 90 nM for ONOO–), thereby allow...

Adenosine triphosphate (ATP), commonly produced in mitochondria, is required by almost all the living organisms; thus fluorescent probes for monitoring mitochondrial ATP levels fluctuation are essential and highly desired. Herein, we report a multisite-binding switchable fluorescent probe, ATP-Red 1, which selectively and rapidly responds to intracellular concentrations of ATP. Live-cell imaging indicated that ATP-Red 1 mainly localized to mitochondria with good biocompatibility and membrane penetration. In particular, with the help of ATP-Red 1, we successfully observed not only the decreased mitochondrial ATP levels in the presence of KCN and starvation state, but also the increased mitochondrial ATP levels in the early stage of cell apoptosis. These results indicate that ATP-Red 1 is a useful tool for investigating ATP-relevant biological processes.

The screening of highly toxic chemical vapor is critical for environmental monitoring and human health maintenance. Herein, we reported a chemosensor (AC-HPD) for rapid and specific detection of highly toxic oxalyl chloride vapor, which adopted anthracene carboxyimide as a fluorophore and 3-aminopropanol as the reaction site. Upon coupling of oxalyl chloride with NH and OH groups, AC-HPD exhibited remarkable colorimetric and ratiometric fluorescence dual-mode responses towards oxalyl chloride via the intramolecular charge transfer effect. AC-HPD could determine oxalyl chloride with features of rapid response, excellent specificity, low detection limit (95 nM), as well as obvious color and fluorescence change. Furthermore, AC-HPD-embedded test strip was successfully fabricated and used as a flexible sensing device for rapid, sensitive and visible monitoring oxalyl chloride vapor.

Sensitivity and detection limit are two vital factors that affect fluorophores-based sensing and imaging system. However, it remains a challenge to improve the sensitivity and detection limit of fluorophores, largely due to their limited response and photophysical properties. In this study, we report for the first time, a novel approach to enhance the sensitivity and detection limit of probes using silica nanoparticles, also known as silica nanoparticles-enhanced fluorescence (SiEF). SiEF can drastically improve the fluorescence intensities and detection limit of fluorophores. A SiEF-improved fluorescent sensor array for rapid and sensitive identification of different heavy metal ions is achieved, and a 3D spatial dispersion graph is obtained based on the SiEF-improved fluorescent sensor array, which provides a lower concentration dependent pattern than fluorophores alone, allowing qualitative, quantitative, and sensitive detection of heavy metal ions. Furthermore, with UV lamp irradiation of the sensor-metal ion mixtures, the output signals enable direct visual of heavy metal ions with low concentration. Thus, the SiEF approach provides a simple and practical strategy for fluorescent probes to improve their sensitivity and detection limit in analytes sensing.

Aggregation of amyloid β-peptide (Aβ) is implicated in the pathology of Alzheimer’s disease (AD), with the soluble, Aβ oligomeric species thought to be the critical pathological species. Identification and characterization of intermediate species formed during the aggregation process is crucial to the understanding of the mechanisms by which oligomeric species mediate neuronal toxicity and following disease progression. Probing these species proved to be extremely challenging, as evident by the lack of reliable sensors, due to their heterogeneous and transient nature. We describe here an oligomer-specific fluorescent chemical probe, BoDipy-Oligomer (BD-Oligo), developed through the use of the diversity-oriented fluorescent library approach (DOFLA) and high-content, imaging-based screening. This probe enables dynamic oligomer monitoring during fibrillogenesis in vitro and shows in vivo Aβ oligomers staining possibility in the AD mice model.

Sulfur dioxide (SO2) can be endogenously produced by enzymes in mitochondria during oxidation of H2S or sulphur-containing amino acids, and plays important roles in several physiological processes. However, the design and synthesis of fluorescent probes which can detect mitochondrial SO2 and its derivatives in living cells still remain unresolved. Herein, we report the preparation of a lipophilic cationic dye 1 (Mito-Ratio-SO2), which targets the mitochondria in living cells and is sensitive to the presence of SO2 derivatives. The ratiometric probe Mito-Ratio-SO2 displays a 170 nm blue-shift in emission with two well-resolved emission bands upon addition of sulfite. Mechanistic studies indicate that three probe-SO2 adducts coexist after reaction, as supported by liquid chromatography and density function theory investigations. Importantly, the ratiometric probe is highly selective for sulfite over other bio-species including H2S. Fluorescence co-localization studies indicate that the probe localizes solely in the mitochondria of HeLa cells. Last but not least, fluorescent imaging of HeLa cells successfully demonstrates the detection of intrinsically generated intracellular SO2 derivatives in living cells.

In this paper, we report a new strategy for constructing a dye library with large Stokes shifts. By coupling a dark donor with BODIPY acceptors of tunable high quantum yield, a novel dark resonance energy transfer (DRET)-based library, named BNM, has been synthesized. Upon excitation of the dark donor (BDN) at 490 nm, the absorbed energy is transferred to the acceptor (BDM) with high efficiency, which was tunable in a broad range from 557 nm to 716 nm, with a high quantum yield of up to 0.8. It is noteworthy to mention that the majority of the non-radiative energy loss of the donor was converted into the acceptor's fluorescence output with a minimum leak of donor emission. Fluorescence imaging tested in live cells showed that the BNM compounds are cell-permeable and can also be employed for live-cell imaging. This is a new library which can be excited through a dark donor allowing for strong fluorescence emission in a wide range of wavelengths. Thus, the BNM library is well suited for high-throughput screening or multiplex experiments in biological applications by using a single laser excitation source.

Neuronal accumulation of tau aggregates is a pathological hallmark in multiple neurodegenerative disorders, collectively called tauopathies. A tau aggregation sensor that can monitor abnormal tau aggregation in neurons would facilitate the study of tau aggregation processes and the discovery of tau aggregation blockers. Here, we describe a BODIPY-fluorescence sensor (BD-tau) that selectively responds to pathological tau aggregates in live cells.

A new strategy for constructing large Stokes shift dyes by coupling a low quantum yield (less than 1%) BODIPY donor (BDN) with tunable high quantum yield BODIPY acceptors (BDM) has been explored to synthesize a set of novel Dark Resonance Energy Transfer (DRET) dyes, named BNM. The low quantum yield of the donor is ascribed to the intramolecular rotation of the phenyl rings, which has been proven by controlling the viscosity and temperature of the solvent. However, upon excitation of BNM compounds at the donor absorption wavelength, tunable emissions from 560 nm to 617 nm were obtained, with a high quantum yield of up to 0.75. Ultrafast dynamic studies demonstrated that the absorbed energy was transferred to the acceptor (BDM) with a high energy transfer rate, before being quenched by non-radiative intramolecular rotations. Using a dark donor makes it possible to avoid fluorescence leaks from donor emission. This is a new set of RET dyes that can be excited by a low quantum yield donor to emit a tunable wide range of high fluorescence emission.

Fluorescent probes have emerged as an essential tool in the molecular recognition events in biological systems; however, due to the complex structures of certain biomolecules, it remains a challenge to design small-molecule fluorescent probes with high sensitivity and selectivity. Inspired by the enzyme-catalyzed reaction between biomolecule and probe, we present a novel combination-reaction two-step sensing strategy to improve sensitivity and selectivity. Based on this strategy, we successfully prepared a turn-on fluorescent reduced nicotinamide adenine dinucleotide (NADH) probe, in which boronic acid was introduced to bind with NADH and subsequently accelerate the sensing process. This probe shows remarkably improved sensitivity (detection limit: 0.084 μM) and selectivity to NADH in the absence of any enzymes. In order to improve the practicality, the boronic acid was further modified to change the measurement conditions from alkalescent (pH 9.5) to physiological environment (pH 7.4). Utilizing these probes, we not only accurately quantified the NADH weight in a health care product but also evaluated intracellular NADH levels in live cell imaging. Thus, these bio-inspired fluorescent probes offer excellent tools for elucidating the roles of NADH in biological systems as well as a practical strategy to develop future sensitive and selective probes for complicated biomolecules.

Intracellular viscosity is a fundamental physical parameter that indicates the functioning of cells. In this work, we developed a simple boron-dipyrromethene (BODIPY)-based probe, BTV, for cellular mitochondria viscosity imaging by coupling a simple BODIPY rotor with a mitochondria-targeting unit. The BTV exhibited a significant fluorescence intensity enhancement of more than 100-fold as the solvent viscosity increased. Also, the probe showed a direct linear relationship between the fluorescence lifetime and the media viscosity, which makes it possible to trace the change of the medium viscosity. Furthermore, it was demonstrated that BTV could achieve practical applicability in the monitoring of mitochondrial viscosity changes in live cells through fluorescence lifetime imaging microscopy (FLIM).

Fluorescence labelling of an intracellular biomolecule in native living cells is a powerful strategy to achieve in-depth understanding of the biomolecule's roles and functions. Besides being nontoxic and specific, desirable labelling probes should be highly cell permeable without nonspecific interactions with other cellular components to warrant high signal-to-noise ratio. While it is critical, rational design for such probes is tricky. Here we report the first predictive model for cell permeable background-free probe development through optimized lipophilicity, water solubility and charged van der Waals surface area. The model was developed by utilizing high-throughput screening in combination with cheminformatics. We demonstrate its reliability by developing CO-1 and AzG-1, a cyclooctyne- and azide-containing BODIPY probe, respectively, which specifically label intracellular target organelles and engineered proteins with minimum background. The results provide an efficient strategy for development of background-free probes, referred to as ‘tame' probes, and novel tools for live cell intracellular imaging., The success of a fluorescent dye as a molecular probe to monitor the intracellular activity of biomolecules depends on its physicochemical characteristics. Here, the authors use a predictive model to identify key features that allow them to design cell permeable, background-free fluorescent probes.

A series of structurally novel anion receptors , , and in which a ferrocene unit and a fluorescent moiety are linked to two imidazolium rings have been designed and prepared from 1,1'-bis(imidazolylmethyl)ferrocene. Their crystal structures revealed that these receptors are capable of incorporating anions such as PF(6)(-) and Br(-). Consequently, the anion binding studies were carried out using various techniques including electrochemistry (CV and OSWV), fluorescence, UV-vis, and (1)H NMR spectroscopy. All the receptors showed a special electrochemical response to the F(-) anion with a remarkable cathodic shift of more than 260 mV and displayed a unique selectivity for F(-) and AcO(-) anions with fluorescence enhancement over various other anions of present interest (Cl(-), Br(-), I(-), HSO(4)(-), H(2)PO(4)(-)). In addition, for receptor , obvious absorption changes were observed when the H(2)PO(4)(-) anion was added while other anions (F(-), Cl(-), Br(-), I(-), AcO(-), HSO(4)(-)) showed only a minor influence on the UV-vis spectra. (1)H NMR titrations demonstrated that receptors and can bind anions through (C-H)(+)X(-) hydrogen bonds and showed strong affinity and high selectivity for the AcO(-) anion in acetonitrile.