Fluorescence Turn-on Research Articles

Tuning Tetramethoxy-acridiniums for Fluorophores and Organic Photoredox Catalysis.

Tetramethoxy substituted alkyl-acridiniums are readily available by facile nucleophilic aromatic substitution on tris(dimethoxyphenyl)carbenium, but are non-fluorescent, presumably due to intramolecular photoinduced electron transfer quenching. In this work we introduce electron withdrawing groups by electrophilic aromatic substitution reactions, leading to fluorescence turn-on. The acridiniums are moderately fluorescent (ϕf = 20%) with long fluorescene lifetimes (τf = 9 ns). The positive excited state reduction potentials (E*red = +1.6 V) make the TMAcr+ excellent electron acceptors in the excited state, and efficient reductive photoredox catalysts able to oxidize a broad range of substrates.

PhotoclickandReleaseforSpatiotemporallyLocalized TheranosticsofSingleCells viaIn Situ Generation of 1,3-Diaryl-1H-benzo[f]indazole-4,9-dione.

Bioorthogonal click-release chemistry is a cutting-edge tool for exploringand manipulating biomolecule functionsin native biological systems. However, it is challenging to achieve the precise regulation or therapy of individual cells via click-release strategies driven by proximity and thermodynamics. Herein, we propose a novel photoclick-release approach based on a photo-induced cycloaddition between 4,4'-bis(N-arylsydnone) orC-bithienyl-diarylsydnone and 2-arylamino-naphthoquinone via irradiation with405 or 485 nm light. Itconstructs 1,3-diaryl-1H-benzo[f]indazole-4,9-dione (BIZON) as a pharmacophore while releases an arylamine for fluorescence turn-on probing. Both of the photoclick reagents were tailored byconnecting to the triphenyl phosphoniumdeliverymotif for enrichment in the mitochondria of live cells.This enablesan intracellular photoclick and release under the control of 405 or 485 nm light. We then discovered that the in situ photo-generated BIZON is capable of photosensitizing upon 485 or 520 nm light to produce singlet oxygen inside the mitochondria under aerobic conditions. Therefore, werealized wash-free fluorescence tracking and subsequent anti-cancer efficacy at single-cell resolution by using global illumination, which provides a foundation for wavelength-gated single-cell theranostics.

Esterase-responsive Self-immolative Prodrugs for the Sustained Delivery of the Anticancer Drug 5-Fluorouracil with Turn-on Fluorescence.

Stimuli-responsive prodrugs of anticancer drugs are advantageous for the selective delivery of drugs to cancer cells with minimized off-target side effects. In the present study, esterase-activatable fluorogenic prodrugs of the chemotherapeutic drug 5-fluorouracil (5-FU) have been rationally designed and synthesized using multi-step organic synthesis. While 5-FU was connected directly with the fluorophore via a C-N bond in the prodrug BJ-50, an additional self-immolative benzylic spacer with a carbonate linker was incorporated in the prodrug BJ-92. Although absorption and emission spectroscopic studies revealed the activation of both the prodrugs by porcine liver esterase (PLE), reverse-phase HPLC studies confirmed the inability of BJ-50 to release the active drug 5-FU. In contrast, a sustained release of 5-FU and Cou-OH was observed from BJ-92 in the presence of PLE. The endogenous esterase-mediated activation of the prodrug BJ-92 was validated by the turn-on fluorescence in HeLa cells and the anti-proliferative activities in A549, HeLa, and HEK-293 cells. Modulation of the expression of a few cancer marker proteins by BJ-92 and 5-FU was studied to evaluate their anticancer activities. As esterases are overexpressed in cancer cells, the prodrug in the present study would be helpful in selectively delivering 5-FU to cancer cells with reduced off-target side-effects.

Amphiphilic pyrene-functionalized triazoliums for ATP recognition

Four pyrene functionalized triazolium salts with different lengths of alkyl tail from C4 to C16, i.e., PYTAZ-C4, PYTAZ-C8, PYTAZ-C12 and PYTAZ-C16, have been designed and synthesized for ATP recognition. It was revealed that the longer alkyl tail anchored receptors PYTAZ-C12 and PYTAZ-C16 exhibit low CMC values, and self-assemble nanoaggregation at low concentration in aqueous solution, which show a pyrene-based excimer emission at 496 nm. However, the shorter alkyl anchored receptors PYTAZ-C4 and PYTAZ-C8 give only the pyrene-based monomer emission at 380 nm at the test concentration of 10 μM in aqueous solution. Importantly, PYTAZ-C12 and PYTAZ-C16 exhibit a good fluorescence turn-on response toward polyphosphate anions, especially ATP, with the detection limits of 2.5 μM and 0.77 μM, respectively. Furthermore, probe PYTAZ-C16 was successfully used for fluorescence imaging of intracellular ATP in living cells.

4,4'-Sulfonyldianiline Derived Aggregation-Induced Emission Luminogen for the Detection of Ofloxacin.

The excessive use of antibiotic ofloxacin (Oflx) can cause serious detrimental effects to human health. Therefore, the utmost research priority is required to develop facile methods to detect Oflx. Herein, a V-shaped aggregation-induced emission (AIE) active Schiff base SDANA was introduced for the fluorescent turn-on detection of Oflx. The Schiff base SDANA was synthesized by condensing 4,4'-sulfonyldianiline with two equivalents of 2-hydroxy-1-naphthaldehyde. The nearly non-fluorescent SDANA in DMSO showed strong orange emission with the increase in HEPES buffer (H2O, 10 mM, pH 7.4) fractions in DMSO from 70 %-95 % due to the combined effects of AIE and ESIPT. The DLS and SEM analyses were performed to complement the formation of self-aggregates of SDANA. With the addition of Oflx, the fluorescence emission of AIE luminogen (AIEgen) SDANA (λem=575 nm, λex=400 nm) was blue-shifted and enhanced at 530 nm. The interactions of Oflx over the surface of SDANA aggregates disrupted the intramolecular charge transfer and aggregation morphology of SDANA, which gave a distinct fluorescence response to detect Oflx. The detection limit for Oflx was estimated as 0.81 μM, and the developed probe AIEgen SDANA was applied for the quantification of Oflx in human blood serum.

Solvent-Selective Fluorescence Sensing of Mg2+ and Al3+ Ions by Pincer-Type NNO Schiff Base Ligand: An Experimental and DFT Optimized Approach.

A newly developed dual-functional fluorescence sensing probe (phenylhydrazinyl pyridine) Schiff base (SB) has been designed with good selectivity for distinguishing Mg2+ and Al3+ metal ions in different solvent solutions. SB exhibits quick and visual turn-on fluorescence enhancement in response to Mg2+ and Al3+ detection. The addition of Mg2+ in ACN-HEPES buffer (1 : 1, v/v, pH 7.2) at (λmax=390 nm) and Al3+ in MeOH-HEPES buffer (1 : 1, v/v, pH 7.2) at (λmax=360 nm) resulted in significant enhancement of fluorescence, up to 7-9 times. These low detection limits of 7.1×10-6 M (7.1 μM) and 5.15×10-7 M (0.51 μM) for Mg2+ and Al3+, respectively, have been achieved by this solvent-controlled platform. Due to the sensing potential towards Mg2+, the probe was utilized as an imaging material for breast cancer cells. 1H-NMR studies were utilized to explore SB's sensing mechanism through turn-on fluorescence. Density functional theory (DFT) calculations were utilized to validate optimized SB and its intricate geometries, which govern the sensing mechanism in the solvent environment. Such a probe has extensive potential applications in bioimaging and the assessment of the quality of wastewater.

LIFs-Based Fluorescent Analysis of Silver Ions in Water Using AIEgen-Embedded PVA Hydrogel.

The detection and quantification analysis of silver ions is important to protect human health and improve the level of wastewater treatment. In this work, a polyvinyl alcohol (PVA) hydrogel-based sensing material based on an aggregation-induced emission strategy was developed and combined with a portable laser-induced fluorescence spectroscopy (LIFs) device for the direct detection and quantification of silver ions in water. Considering the requirements of environmental water analysis regarding portability, reliability, and convenience, this fluorescence turn-on strategy was optimized accordingly to ensure the accuracy and stability of the analysis in complex natural water environmental. This sensing method showed operational excellence and good sensitivity (down to 1 ppb) and may provide a stable and convenient strategy for the on-site, real-time analysis of environmental water samples.

Bioorthogonal cyclopropenones for investigating RNA structure.

RNA sequences encode secondary and tertiary structures that impact protein production and other cellular processes. Misfolded RNAs can also potentiate disease, but the complete picture is lacking. To establish more comprehensive and accurate RNA structure-function relationships, new methods are needed to interrogate RNA and trap native conformations in cellular environments. Existing tools primarily rely on electrophiles that are constitutively "on" or triggered by UV light, often resulting in high background reactivity. We developed an alternative, chemically triggered approach to crosslink RNAs using bioorthogonal cyclopropenones (CpOs). These reagents selectively react with phosphines to provide ketenes-electrophiles that can trap neighboring nucleophiles to forge covalent crosslinks. As proof-of-concept, we synthesized a panel of CpOs and appended them to thiazole orange (TO-1). The TO-1 conjugates bound selectively to a model RNA aptamer (Mango) with nanomolar affinity, confirmed by fluorescence turn-on. After phosphine administration, covalent crosslinks were formed between the CpO probes and RNA. The degree of crosslinking was both time and dose-dependent. We further applied the chemically triggered tools to model RNAs in biologically relevant conditions. Collectively, this work expands the toolkit of probes for studying RNA and its native conformations.

Redox Oxygen Species-Responsive Nanotheranostics with Dual-Channel Fluorescent Turn-On for Early Diagnosis and Targeted Therapy of Alzheimer's Disease.

Current diagnosis and treatment strategies mainly focus on the pathologies of the mid-to-late stage of AD (Alzheimer's disease), with clinical outcomes that are far from ideal. Herein, we developed the ROS (reactive oxygen species)-responsive brain neuronal targeting nanotheranostic platforms that possess the dual-channel fluorescent "turn-on" properties and release drugs in AD neurons in response to ROS, thereby simultaneously facilitating the diagnosis and therapy of early AD. Through the modification of acetylcholine receptor targeting RVG29 peptide, the nanotheranostics penetrated BBB and accumulated into diseased neurons in an intact form, consequently maximizing the diagnostic and therapeutic performance. The anti-oxidative drug baicalein conjugated onto the surface of nanotheranostics via ROS-cleavable boronate ester linkage rapidly released for ROS scavenging, while the encapsulated fluorophores turned on their fluorescence for AD diagnosis upon microenvironment stimuli. This nanotheranostic strategy exhibited highly sensitivity with a ROS detection limit of up to 100µm and accurately early detection of ROS in 3×Tg AD mice at 6 months of age in vivo. In addition, it could also rescue memory defects, scavenge oxidative stress, attenuate neuroinflammation and enhance neuroprotective effect in 3×Tg AD mice. This work opens up a promising and smart strategy for early diagnosis and therapy in neurodegenerative disease.

Selective Recognition of the Dimeric NG16 Parallel G-Quadruplex Structure Using Synthetic Turn-On Red Fluorescent Protein Chromophore.

Red fluorescent protein (RFP)-based fluorescent probes that can selectively interact with specific nucleic acids are of great importance for therapeutic and bioimaging applications. Herein, we have reported the synthesis of RFP chromophores for selective recognition of G-quadruplex nucleic acids in vitro and ex vivo. We identified DFHBI-DM as a fluorescent turn-on probe that binds to the dimeric NG16 parallel quadruplex with superior selectivity and sensitivity over various parallel, antiparallel, and hybrid topologies. The binding of DFHBI-DM to NG16 exhibited excellent photophysical properties, including high binding affinity, large Stokes shift, high photostability, and quantum yield. The MD simulation study supports the 1:1 binding stoichiometry. It confirms the planar conformation of DFHBI-DM, which makes strong binding interactions with a flat quartet of NG16 compared to other antiparallel and hybrid topologies. The cell imaging and MTT assays revealed that DFHBI-DM is a biocompatible and efficient fluorescent probe for intracellular imaging of NG16. Overall, these results demonstrated that DFHBI-DM could be an effective fluorescent G4-stabilizing agent for the dimeric NG16 parallel quadruplex, and it could be a promising candidate for further exploration of bioimaging and therapeutic applications.

Small Molecule Quinoxaline Fluorescent Probe for AcO- Real-Time Detection in Vivo.

Fluorescent probes to detect biologically important acetate ion (AcO-) are essential for regulating substance metabolism, alleviating inflammatory symptoms, reducing cancer incidence, and diagnosing early diseases. However, the relatively small charge-to-atomic radius ratio in AcO- and its triangular spatial structure pose challenges in recognition and often lead to interference from other anions in detection methods. Herein, we introduce a quinoxaline fluorescent probe, o-(4-(2-(3-oxo-3,4 -dihydroqui-noxalin-2-yl)vinyl)phenyl) dimethylaminothiophene ester (QPDMT), specifically design and synthetic for the accurate detection of AcO-. This probe leverages molecular nucleophilicity and electron transfer to undergo a reaction that releases the fluorophore upon cleavage of the thioformyl ether bond, exhibiting a turn-on fluorescence response at 530nm. QPDMT exhibits an impressively low detection limit of 30 nM, a rapid response time of 20min, a robust linear response in the 1-9 µM range and excellent fluorescence quantum yield, 0.32. Importantly, this probe demonstrates low cytotoxicity, making it an ideal candidate for endogenous AcO- detection in living cells and organisms.

Fluorescence Turn-ON Displacement Assays with Cucurbit[7]uril-Thiophenylpyridinium Complexes as Host-Dye Reporter Pairs.

The N-methyl-4-thiophenylpyridinium cation (ThioPy) is a high affinity (Kd ca. 5 nM), fast-exchanging fluorescent probe for cucurbit[7]uril (CB7). The CB7/ThioPy complex shows a unique fluorescence turn-ON response upon displacement by an analyte in sensing application. This enabled the development of a real-time fluorescence assay with the MRFA peptide for the protease thermolysin, which is also suitable for the cancer biomarker cathepsin B. Moreover, liposome encapsulation of CB7/ThioPy in large unilamellar vesicles (LUVs) provided mechanistic insight into intravesicular dye displacement reactions.

Double detection of mycotoxins based on aptamer induced Fe3O4@TiO2@Ag Core − Shell nanoparticles “turn on” fluorescence resonance energy transfer

Multiple and sensitive mycotoxin detection is an essential early-warning mechanism for safeguarding human health, and preserving the environment. We synthesized a turn-on Fluorescence Resonance Energy Transfer (FRET) aptamer sensor based on the unique fluorescence quenching and substrate recognition characteristics of Ag NTs (energy receptors) and aptamer modified Fe3O4@TiO2 NP (energy donor) to detect multiple toxins using a single diagnostic approach. The addition of aflatoxin B1 (AFB1) and ochratoxin A (OTA) resulted in a change in fluorescence intensity at 510 and 650 nm, which can be employed for simultaneous recognition with detection limits of 0.94 ng·mL−1 (R2 = 0.997) and 0.54 ng·mL−1 (R2 = 0.995). The aptasensors have been successfully applied for the determination of AFB1 and OTA in grain and oil samples with high recovery rates. The approach provides novel possibilities for the development of sensitive and selective aptasensors with potential applications in aptamer-recognized multifunctional biosensing.

Terminal deoxynucleotidyl transferase-mediated CRISPR sensing platform for simple and point-of-care detection of cobalt pollution

The excessive use of cobalt in various chemical industries and arbitrary discharge of industrial wastewater have led to increased cobalt pollution in soil and water resources, increasing the risk of human exposure to high concentrations of cobalt and necessitating an urgent need for on-site monitoring platform for cobalt pollution. In this study, the terminal deoxynucleotidyl transferase (TdT)-CRISPR platform has been developed. In this platform, cobalt as a cofactor of TdT, can significantly improve the tailing efficiency of TdT-mediated extension. Therefore, when cobalt is present, the detection probe can be extended with poly(T) tails through the TdT-mediated extension, which can be subsequently served as the DNA activator for Cas12a, leading to the cleavage of fluorescence reporter molecules and triggering turn-on fluorescence signals. Consequently, this dual amplification sensing strategy of TdT-CRISPR platform demonstrated exceptional sensitivity (0.83 nM) and high specificity for cobalt over other ions. Furthermore, the method was successfully employed for the detection of cobalt in tap water and river samples. CRISPR-lateral flow assays (CRISPR-LFAs) were evaluated in this study for the simple and point-of-care detection of cobalt pollution. The assays are capable of detecting cobalt concentrations as low as 50 nM, which is significantly lower than the environmental standards of 16.9 μM, through strip analysis with the naked eye. These results commonly suggest that the TdT-CRISPR platform holds significant promise for monitoring cobalt pollution, providing a robust and sensitive solution for on-site detection and contributing to the mitigation of cobalt contamination risks in environmental matrices.

Seeing and Cleaving: Turn-Off Fluorophore Uncaging and Its Application in Hydrogel Photopatterning and Traceable Neurotransmitter Photocages.

The advancements in targeted drug release and experimental neuroscience have amplified the scientific interest in photolabile protecting groups (PPGs) and photouncaging. The growing need for the detection of uncaging events has led to the development of reporters with fluorescence turn-on upon uncaging. In contrast, fluorescent tags with turn-off properties have been drastically underexplored, although there are applications where they would be sought after. In this work, a rhodamine-based fluorescent tag is developed with signal turn-off following photouncaging. One-photon photolysis experiments reveal a ready loss of red fluorescence signal upon UV (365 nm) irradiation, while no significant change is observed in control experiments in the absence of PPG or with irradiation around the absorption maximum of the fluorophore (595 nm). The two-photon photolysis of the turn-off fluorescent tag is explored in hydrogel photolithography experiments. The hydrogel-bound tag enables the power-, dwell time-, and wavelength-dependent construction of intricate patterns and gradients. Finally, a prominent caged neurotransmitter (MNI-Glu) is modified with the fluorescent tag, resulting in the glutamate precursor named as GlutaTrace with fluorescence traceability and turn-off upon photouncaging. GlutaTrace is successfully applied for the visualization of glutamate precursor distribution following capillary microinjection and for the selective excitation of neurons in a mouse brain model.

Robust Natural Light-Absorbable and -Degradable AIE Photosensitizers for Fluorescence Labeling and Efficient Photodynamic Eradication of Algal Pollutants.

Current water pollution caused by the excessive proliferation of harmful algae urges green methods that can efficiently utilize natural light to treat algal pollution. Herein, a series of aggregation-induced emission (AIE) photosensitizers that can efficiently harness sunshine were synthesized for the environmentally friendly and biocompatible treatment of algal pollution. By tuning the number of thiophene units and the electron conjugation degree, the photosensitizers' absorptions were broadened to cover the whole visible light range. The positive charges guided photosensitizers to aggregate on algal cell surfaces, resulting in a turn-on fluorescence signal and robust reactive oxygen species generation under sunshine, thereby achieving fluorescence labeling and photodynamic eradication of algae. The eradication outcomes demonstrated that the AIE photosensitizers significantly outperformed the commercial algaecide ALG. At 20 ppm photosensitizers, 90.4% and 94.2% killing rates were achieved for C. reinhardtii and C. vulgaris, respectively, 2.8- and 3.6-fold higher than those from the same concentration of ALG. Excellent performances in inhibiting algae growth were also verified with efficiency superior to that of ALG. Importantly, the photosensitizers can self-degrade into biocompatible fragments under irradiation to avoid secondary pollution. The developed photosensitizers that possess sunshine convertibility and degradability provide an efficient tool for algal treatment, showing broad research and application prospects.

A near-infrared fluorescent probe with a large Stokes shift for the detection and imaging of biothiols in vitro and in vivo.

In this study, a new near-infrared (NIR) fluorescent turn-on probe featuring a large Stokes shift (198nm) was developed for the detection of biothiols. The probe was based on a dicyanoisophorone derivative serving as the fluorophore and a 2,4-dinitrobenzenesulfonyl (DNBS) group functioning as both a recognition site and a fluorescence quencher. In the absence of biothiols, the fluorescence of the probe was low due to the photoinduced electron transfer (PET) effect between the fluorophore and DNBS. Upon the presence of biothiols, the DNBS group underwent a nucleophilic aromatic substitution reaction with the sulfhydryl group of biothiols, leading to the release of the fluorophore and a notable emission peak at 668nm. This developed probe exhibited exceptional selectivity and sensitivity to biothiols in solution, with an impressive detection limit of 28nM for cysteine (Cys), 22nM for homocysteine (Hcy), and 24nM for glutathione (GSH). Furthermore, the probe demonstrated its applicability by successfully visualizing both endogenous and exogenous biothiols in living systems.

Highly selective peptide-based fluorescent turn-on probe with large Stokes shift for the detection of Cd(II) and its applications in bio-imaging and smartphone-assisted recognition

Cadmium ions (Cd2+) is a ubiquitous environmental pollutant that endangers all forms of life, and it has become critical to develop simple and highly selective detection methods for monitoring Cd2+. Herein, a new fluorescent turn-on probe DPGC with dansyl fluorophore labelled tripeptide (ProGlyCys-NH2) was successfully synthesized using solid-phase peptide synthesis technology with Fmoc chemistry. Interestingly, DPGC showed high specificity and fluorescence “turn on” response towards Cd2+ in 100% aqueous solution. In addition, the limit of detection (LOD) for Cd2+ recognition by DPGC was calculated to be 12.4 nM. The pH test showed that DPGC had a wide detection range of 7–11 for Cd2+, and the response time was also less than 30 s. Notably, the DPGC-Cd2+ complex forms a 2:1 binding stoichiometry was determined using fluorescence titration, Job's plot, ESI-HRMS and DFT calculations. Additionally, the binding of DPGC with Cd2+ was found fluorescence reversible on addition of EDTA. Meanwhile, DPGC has excellent membrane permeability and low cytotoxicity, and was successfully employed for detecting Cd2+in living cells and zebrafish. More importantly, the colour change of Cd2+ solution with different concentrations under 365 nm UV light was detected by a smartphone App to achieve semi-quantitative visual analysis of Cd2+ ions.

A Fluorescence Enhancement Sensor Based on Silver Nanoclusters Protected by Rich-G-DNA for ATP Detection.

In this study, a turn-on fluorescence sensor for the detection of adenosine 5'-triphosphate (ATP) was developed and tested using ATP-DNA2-Ag NCs. The results showed that the fluorescence of ATP-DNA2-Ag NCs was significantly enhanced with the addition of ATP. The fluorescence enhancement was a result of the specific binding activity of the ATP aptamer and ATP, which caused G-rich sequences to approach the dark DNA-Ag NCs, owing to the alteration in ATP aptamer conformation. The proposed sensor demonstrated a good linear range of 18-42 mM and a limit of detection (LOD) of 2.8 μM. The sensor's features include sensitivity, selectivity, and simple operation. In addition, the proposed sensor successfully measured ATP in 100-fold diluted fetal bovine serum.

A Dicyanoisophorone-based Fluorescent Turn-on Probe for Rapid Detecting Thiophenol in Aqueous Medium and Living Cell Imaging

A novel colorimetric and fluorescent thiophenol probe based on dicyanoisophorone has been successfully achieved, which has low-cost, easy operation, high selectivity, sensitivity and stability. The chemosensor shows a large Stokes shift and approximately 170 nm when excited at 510 nm. ISO-DiNO2 could be utilized as “Turn-on” and a naked-eyes chemosensor to detect PhSH, which is accompanied by a distinct color shift from red to dark purple with strong red fluorescence at 365 nm UV-light. Its limit of detection was determined to be 1.15 μM. More importantly, ISO-DiNO2 can react instantaneously (<10 s) with PhS−. In addition, ISO-DiNO2 has been utilized in test paper strips, water sample together with imaging of PhS− in living Raw264.7 cells, demonstrating that ISO-DiNO2 has excellent and promising applications.