Fluorogenic Signaling Research Articles

A firm-push-to-open and light-push-to-lock strategy for a general chemical platform to develop activatable dual-modality NIR-II probes.

Activatable near-infrared (NIR) imaging in the NIR-II range is crucial for deep tissue bioanalyte tracking. However, designing such probes remains challenging due to the limited availability of general chemical strategies. Here, we introduced a foundational platform for activatable probes, using analyte-triggered smart modulation of the π-conjugation system of a NIR-II-emitting rhodamine hybrid. By tuning the nucleophilicity of the ortho-carboxy moiety, we achieved an electronic effect termed "firm-push-to-open and light-push-to-lock," which enables complete spirocyclization of the probe before sensing and allows for efficient zwitterion formation when the light-pushing aniline carbamate trigger is transformed into a firm-pushing aniline. This platform produces dual-modality NIR-II imaging probes with ~50-fold fluorogenic and activatable photoacoustic signals in live mice, surpassing reported probes with generally below 10-fold activatable signals. Demonstrating generality, we successfully designed probes for hydrogen peroxide (H2O2) and hydrogen sulfide (H2S). We envision a widespread adoption of the chemical platform for designing activatable NIR-II probes across diverse applications.

Deconvoluting nitric oxide-protein interactions with spatially resolved multiplex imaging.

Simultaneous imaging of nitric oxide (NO) and its proximal proteins should facilitate the deconvolution of NO-protein interactions. While immunostaining is a primary assay to localize proteins in non-genetically manipulated samples, NO imaging probes with immunostaining-compatible signals remain unexplored. Herein, probe NOP-1 was developed with an NO-triggered proximal protein labeling capacity and fluorogenic signals. The trick is to fuse the native chemical ligation of acyl benzotriazole with the protein-conjugation-induced fluorogenic response of Si-rhodamine fluorophore. NOP-1 predominantly existed in the non-fluorescent spirocyclic form. Yet, its acyl o-phenylenediamine moiety was readily activated by NO into acyl benzotriazole to conjugate proximal proteins, providing a fluorogenic response and translating the transient cellular NO signal into a permanent stain compatible with immunostaining. NOP-1 was utilized to investigate NO signaling in hypoglycemia-induced neurological injury, providing direct evidence of NO-induced apoptosis during hypoglycemia. Mechanistically, multiplex imaging revealed the overlap of cellular NOP-1 fluorescence with immunofluorescence for α-tubulin and NO2-Tyr. Importantly, α-tubulin was resolved from NOP-1 labeled proteins. These results suggest that NO played a role in hypoglycemia-induced apoptosis, at least in part, through nitrating α-tubulin. This study fills a crucial gap in current imaging probes, providing a valuable tool for unraveling the complexities of NO signaling in biological processes.

An Azomethine-H-Based Fluorogenic Sensor for Formic Acid.

Formic acid (FA) is an important C1-containing feedstock that serves as a masked source of dihydrogen gas (H2). To encourage the adoption of cleaner (noncarbonaceous) energy sources, FA detection and sensing is thus of considerable interest. Here, we examine the use of a commercially available dye, azomethine-H (Az-H), for FA sensing. Solution studies confirm that FA quenches both the absorbance and the luminescence properties of Az-H. FA was additionally found to attenuate a known Az-H (E)-to-(Z) conformational change, suggesting an Az-H/FA interaction, possibly through hydrogen bonding; this phenomenon was probed using 1H NMR spectroscopy. Moving toward a solid-state sensor, the Az-H probe was incorporated into a gelatin-based matrix. On exposure to FA, the luminescence of this system was found to increase in a FA-dependent manner, attributed to the formation of stable hydrogen-bonded structures, facilitating a (Z)-to-(E) isomerization via imine protonation, allowing for production of the more luminescent (E)-isomer. This fluorogenic signal was used as a FA sensor with an estimated detection limit of ca. 0.4 ppb FA vapor. This work constitutes an important step toward a highly sensitive FA sensor in both the solution and solid state, opening new space for the detection of organic acids in differing chemical environments.

Selective chromogenic and fluorogenic signalling of Hg2+ ions using a benzothiazole-quinolinyl acrylate conjugate and its applications in the environmental water samples and living cells

We have developed a new chromogenic and fluorogenic chemosensor (L) based on benzothiazole conjugated with quinolinyl acrylate for the detection of Hg2+ ions with excellent selectivity and sensitivity. In the presence of Hg2+, the fluorescence of L was quenched from light blue to colorless. The L could work in the wide pH range of 5.0–8.0 without considerable interference from other tested ions. UV–Visible and fluorometric investigations revealed that the limit of detection (LOD) for Hg2+ was 1.0 μM (202 ppb) and 11.8 nM (2.38 ppb), respectively. Further, the applicability of probe L in the determination of Hg2+ in various water samples and biological samples were tested. Human Bone Osteosarcoma cell lines (HOS) were utilized for this study and satisfying results were obtained. These cell line results also further support the ‘on-off’ properties of probe L. Based on the colorimetric and fluorescent results we have designed a Boolean logic gate for futuristic applications.

Fluorescence Sensing of the Panhandle Structure of the Influenza A Virus RNA Promoter by Thiazole Orange Base Surrogate-Carrying Peptide Nucleic Acid Conjugated with Small Molecule

We have developed a new class of triplex-forming peptide nucleic acid (PNA)-based fluorogenic probes for sensing of the panhandle structure of the influenza A virus (IAV) RNA promoter region. Here, a small molecule (DPQ) capable of selectively binding to the internal loop structure was conjugated with triplex-forming forced intercalation of the thiazole orange (tFIT) probe with natural PNA nucleobases. The resulting conjugate, tFIT-DPQ, showed a significant light-up response (83-fold) upon strong (Kd = 107 nM) and structure-selective binding to the IAV RNA promoter region under physiological conditions (pH 7.0, 100 mM NaCl). We demonstrated the conjugation of these two units through the suitable spacer was key to show useful binding and fluorogenic signaling functions. tFIT-DPQ facilitated the sensitive and selective detection of IAV RNA based on its binding to the promoter region. Furthermore, we found that tFIT-DPQ could work as a sensitive indicator for screening of test compounds targeting the IAV RNA promoter region in the fluorescence indicator displacement assay.

A corrole-based fluorescent probe for detection of sulfur ion and its application in living cells

A dual signaling probe DPC-O-NBD formed through the conjugation of 5,15-bis(phenyl)-10-(4-hydroxylphenyl)-corrole (DPC-OH) with 4-chloro-7-nitrobe-nzo-2-oxa-1,3-diazole (NBD-Cl) is explored to detect sulfur ion (S2−). The key design principle is based on the nucleophilicity of S2−, which can be used to cleave the bridging ether bond in the probe to obtain both fluorogenic and chromogenic signaling behaviors. Importantly, the probe DPC-O-NBD not only showed a high sensitivity (LOD: 48 nM) and selectivity over other interfering species, but also exhibited a rapid response (∼18 s) towards S2−. Furthermore, DPC-O-NBD can also be used to detect S2− in both water and live cells, providing a potentially powerful approach for probing S2− chemistry in biological systems.

Discovery of a Broad-Spectrum Fluorogenic Agonist for Strigolactone Receptors through a Computational Approach.

Strigolactones (SLs) are plant hormones that play various roles in plant physiology, including provoking the germination of parasitic weeds Orobanche and Striga. A family of α/β-hydrolases have been proposed to be the SL receptor proteins. Effective assays for measuring the activity of SL receptors could promote the development of SL-related biology and chemistry. In this study, we developed a new approach called pharmacophore-linked probe virtual screening (PPVS). Its application yielded an effective "off-on" probe named Xilatone Red (XLR). This probe showed a broad spectrum and excellent sensitivity toward SL receptors, including ShD14 (Striga D14), for which the detection limit was determined to be in the micromolar range, outperforming that of the commercial fluorogenic agonist Yoshimulactone Green (YLG). Upon hydrolysis by SL receptors, XLR provided fluorogenic and colorimetric signaling responses. Furthermore, XLR could induce germination of Phelipanche aegyptiaca seeds and prevent Arabidopsis max4-1 branching defects at micromolar concentrations. Our molecular simulations revealed the essential factors in the molecular perception of XLR. We anticipate that this study can prompt the discovery of high-performance SL agonists/antagonists to combat parasitic weeds.

Cell engineering method using fluorogenic oligonucleotide signaling probes and flow cytometry.

ObjectiveChromovert® Technology is presented as a new cell engineering technology to detect and purify living cells based on gene expression.MethodsThe technology utilizes fluorogenic oligonucleotide signaling probes and flow cytometry to detect and isolate individual living cells expressing one or more transfected or endogenously-expressed genes.ResultsResults for production of cell lines expressing a diversity of ion channel and membrane proteins are presented, including heteromultimeric epithelial sodium channel (αβγ-ENaC), sodium voltage-gated ion channel 1.7 (NaV1.7-αβ1β2), four unique γ-aminobutyric acid A (GABAA) receptor ion channel subunit combinations α1β3γ2s, α2β3γ2s, α3β3γ2s and α5β3γ2s, cystic fibrosis conductance regulator (CFTR), CFTR-Δ508 and two G-protein coupled receptors (GPCRs) without reliance on leader sequences and/or chaperones. In addition, three novel plasmid-encoded sequences used to introduce 3′ untranslated RNA sequence tags in mRNA expression products and differentially-detectable fluorogenic probes directed to each are described. The tags and corresponding fluorogenic signaling probes streamline the process by enabling the multiplexed detection and isolation of cells expressing one or more genes without the need for gene-specific probes.ConclusionsChromovert technology is provided as a research tool for use to enrich and isolate cells engineered to express one or more desired genes.

Single-atom switching as a general approach to designing colorimetric and fluorogenic probes for mercury ions

By performing a single-atom replacement within common fluorophores, we have developed a facile and general strategy to prepare a broad-spectrum class of colorimetric and fluorogenic probes for the selective detection of mercury ions in aqueous environments. Thionation of carbonyl groups from existing fluorophore cores results in a great reduction of fluorescence. In the presence of mercury ions, the resulting thiocaged probes are efficiently desulfurized to their oxo derivatives, leading to pronounced changes in chromogenic and fluorogenic signals. Because these probes exhibit high selectivity, excellent sensitivity, good membrane-permeability, and rapid responses towards mercury ions, they are suitable for visualization of mercury in both aqueous and intracellular environments.

A Photoalkylative Fluorogenic Probe of Guttiferone A for Live Cell Imaging and Proteome Labeling in Plasmodium falciparum.

Guttiferone A (GA) 1, a polycyclic polyprenylated acylphloroglucinol (PPAP) isolated from the plant Symphonia globulifera (Clusiaceae), constitutes a novel hit in antimalarial drug discovery. PPAPs do not possess identified biochemical targets in malarial parasites up to now. Towards this aim, we designed and evaluated a natural product-derived photoactivatable probe AZC-GA 5, embedding a photoalkylative fluorogenic motif of the 7-azidocoumarin (AZC) type, devoted to studying the affinity proteins interacting with GA in Plasmodium falciparum. Probe 5 manifested a number of positive functional and biological features, such as (i) inhibitory activity in vitro against P. falciparum blood-stages that was superimposable to that of GA 1, dose–response photoalkylative fluorogenic properties (ii) in model conditions using bovine serum albumin (BSA) as an affinity protein surrogate, (iii) in live P. falciparum-infected erythrocytes, and (iv) in fresh P. falciparum cell lysate. Fluorogenic signals by photoactivated AZC-GA 5 in biological settings were markedly abolished in the presence of excess GA 1 as a competitor, indicating significant pharmacological specificity of the designed molecular probe relative to the native PPAP. These results open the way to identify the detected plasmodial proteins as putative drug targets for the natural product 1 by means of proteomic analysis.

Effect of surface charge of gold nanoparticles on fluorescence amplification of polydiacetylene-based liposomes

Polydiacetylene (PDA)-based liposome have been attractive as sensory platforms owing to their applicability in simultaneous detection of colorimetric and fluorogenic signals. As PDA show low quantum yield, gold nanoparticles (GNPs) were used to amplify the fluorescence of the PDA by localized surface plasmon resonance. In this study, positively and negatively surface charged GNPs at different concentrations were complexed with negatively charged PDA liposome (GNP/PDA). Positively charged GNPs caused tight binding at the surface of negatively charged PDA liposome, became aggregated due to colloidal instability and thereby dramatically quenched the fluorescence of PDA. While negatively charged GNPs sparsely placed on the surface of the PDA liposome, at the optimized complexing condition, generated ∼10%of fluorescence amplification compared to non-complexed PDA liposome as observed by the photoluminescence spectra of the stable colloids. Under the optimum incubation conditions, GNP/PDA liposome that specifically binds with Pb2+ via its phenolic group, exhibited increased fluorescence intensity compared to the non-complexed PDA liposome-at the same concentration of target Pb2+.

Development of three ways molecular logic gate based on water soluble phenazine fluorescent 'selective ion' sensor.

New hydrophilic fluorescent selective ion sensor based on phenazine and phthalazine moieties, 1,1'-(phenazine-2,3-diyl)-bis(3-(1,4-dihydroxyphthalazin-6-yl)urea) (1), has been designed, synthesized and characterized. Interestingly, sensor 1 exhibits prominent "turn-on" and "turn-off" fluorogenic signaling at 580 nm towards Fe2+ & AcO- and Sr2+ & Cu2+, respectively. The fluorescence titration experiments shed light on the nature of the interaction between 1 and guest molecules (Fe2+, Sr2+, Cu2+ and AcO-), which divulge that 1 is flexible enough to orient itself according to the size of the guest molecule. Water mediated excited-state intramolecular proton transfer (ESIPT) and photo-induced electron transfer (PET) mechanisms are responsible for the dual behavior of 1, which binds with guest molecules in 1:1 stoichiometry. Based on the significant duplex fluorescence response of 1, a molecular logic gate keypad lock with sixteen "on" passwords for a storage system has been developed.

Membrane‐Targetable Probes for Hg2+ Detection in Live Cells and Paper‐Based Devices

AbstractTwo novel fluorogenic probes (HGMem‐2 and −3) for detecting Hg2+ were designed, synthesized and characterized. Probes based on a commercially available fluorophore (fluorescein) with high brightness were equipped with/without a photolabile group (2‐nitrobenzyl group), a membrane‐anchoring unit (cholesterol) and a Hg2+‐reactive moiety. Taking advantage of the hydrophobicity of the cholesterol group in the probe, a paper‐based device spotted with HGMem‐2 was developed as a Hg2+ paper indicators, and showed feasibility in complex cell lysate. Only upon UV photolysis, HGMem‐3 displayed more than 1900‐fold fluorescence enhancement with the addition of Hg2+. Furthermore, they exhibited pronounced Hg2+‐selective fluorogenic signalling behavior over other 45 analytes. Importantly, HGMem‐3 was utilized to specifically visualize Hg2+ near cell membrane in live HepG2 cells with high spatiotemporal resolution, avoiding the influence of reactive species outside cells successfully. Hence, our newly developed membrane‐anchored Hg2+ sensors provide useful tools for versatile applications in biological and environmental research.

Chelator Probe with Exceptionally High Stokes Shift for Selective Detection of OAc− with Red Emission: Application as a Biosensor

AbstractA novel chelator probe with flexible structural configuration has been synthesized as chemosensor for selective detection of biotic anion like OAc− by chromogenic as well as fluorogenic signal. The nitro substituted chelator probe has shown pale yellow to deep yellow coloration upon detection of OAc− ion. Significantly the emission of the chelator probe upon recognition of the anion has been turned ON in low energy region which has been explained through PET‐ESIPT‐ILCT‐C=N isomerisation based pathways. The chelator probe has shown exceptionally high stroke's shift. The host guest interaction has been confirmed by different sophisticated analytical tools such as UV‐Vis, fluorescence, 1H‐NMR and FT‐IR. Significantly the fluorescence turn on phenomena has been utilized to detect intracellular OAc− inside candida albicans to explore the applicability of the chelator probe as bio sensor.

Wavelength tunable tetraphenylethene fluorophore dyads: Synthesis, aggregation-induced emission and Cl2 gas detection

We designed and synthesized three new tetraphenylethene (TPE) derivatives (CZ-TPE, DMA-TPE and TPA-TPE) modified with different electron rich group (carbazole, N, N-dimethylaniline and triphenylamine segments, respectively) by Knoevenagel condensation reaction. The chemical structures of these compounds are identified by NMR, MS and IR. The absorption and emission of these dyes were investigated by UV-Vis and fluorescence spectroscopy, respectively. The results shown three dyes exhibited evident aggregation-induced emission (AIE) or aggregation-induced emission enhancement (AIEE) properties with wavelength tunable emission. Their emission color can be tuned from green to orange in tetrahydrofuran/H2O solutions, from green to red in solid state or pure tetrahydrofuran solutions. In addition, the solution or film of TPA-TPE shown results in distinct fluorescence from “ON” to “Off” observation as well as color change from orange yellow to colorless by naked eyes when the addition of toxic Cl2 gas, which can be used as an excellent dual chromo- and fluorogenic signal changes that is well suited for monitoring Cl2 gas in solution, or solid film, conveniently. These results indicate TPA-TPE is a promising candidate chemosensor for the detection of Cl2 gas.

Dual signaling of hypochlorite in tap water by selective oxidation of phenylselenylated dichlorofluorescein

A novel hypochlorite-selective signaling probe based on the oxidative transformation of phenylselenylated 2′,7′-dichlorofluorescein 1 to its selenoxide analogue was investigated. In a pH 7.0 phosphate buffer system (containing 1% DMF as a solubilizer), the designed probe 1 exhibited pronounced colorimetric and fluorogenic hypochlorite signaling behavior over other representative oxidants such as hydrogen peroxide, tert-butyl hydroperoxide, perborate, and percarbonate as well as a range of environmentally relevant metal ions and anions. The signaling was due to the hypochlorite-assisted oxidation of the probe’s selenyl moiety to selenoxide. Hypochlorite signaling was unaffected by the presence of common metal ions and anions as a background. The hypochlorite-selective fluorescence signaling of probe 1 has a detection limit of 3.9×10−8M (0.002ppm). Finally, the determination of hypochlorite concentration in tap water using a smartphone as a stand-alone portable signaling detection and processing tool was successfully demonstrated.

Field-deployable, quantitative, rapid identification of active Ebola virus infection in unprocessed blood.

The West African Ebola virus outbreak underlined the importance of delivering mass diagnostic capability outside the clinical or primary care setting in effectively containing public health emergencies caused by infectious disease. Yet, to date, there is no solution for reliably deploying at the point of need the gold standard diagnostic method, real time quantitative reverse transcription polymerase chain reaction (RT-qPCR), in a laboratory infrastructure-free manner. In this proof of principle work, we demonstrate direct performance of RT-qPCR on fresh blood using far-red fluorophores to resolve fluorogenic signal inhibition and controlled, rapid freeze/thawing to achieve viral genome extraction in a single reaction chamber assay. The resulting process is entirely free of manual or automated sample pre-processing, requires no microfluidics or magnetic/mechanical sample handling and thus utilizes low cost consumables. This enables a fast, laboratory infrastructure-free, minimal risk and simple standard operating procedure suited to frontline, field use. Developing this novel approach on recombinant bacteriophage and recombinant human immunodeficiency virus (HIV; Lentivirus), we demonstrate clinical utility in symptomatic EBOV patient screening using live, infectious Filoviruses and surrogate patient samples. Moreover, we evidence assay co-linearity independent of viral particle structure that may enable viral load quantification through pre-calibration, with no loss of specificity across an 8 log-linear maximum dynamic range. The resulting quantitative rapid identification (QuRapID) molecular diagnostic platform, openly accessible for assay development, meets the requirements of resource-limited countries and provides a fast response solution for mass public health screening against emerging biosecurity threats.

Fabrication of Helical Nanoribbon Polydiacetylene via Supramolecular Gelation: Circularly Polarized Luminescence and Novel Diagnostic Chiroptical Signals for Sensing.

Four kinds of commercially available diacetylene (DA) monomers with different chain length, diacetylene positions were fabricated into the organogels via mixing with a chaperone gelator, an amphiphilic l-histidine ester derivative LHC18 that can help the nongelator to form gels. Upon photo irradiation with a 254 nm UV light, the white gels underwent topochemical reaction and turned into red or blue gels, depending on the DA monomer structures. Through the gel formation, the molecular chirality of LHC18 can be transferred to the polydiacetylene (PDA) and helical nanoribbon structures were obtained. The blue gels showed a clear response to stimuli such as pH variation, heating, mechanical force and organic solvents, and turned into red gels. Interestingly, the blue gel showed strong supramolecular chirality, which could be turned off or changed into red phase CD signals. Such changes in chiroptical signals depended on the external heating and various organic solvents. In the case of heating, the blue gel changed into red one, which showed both strong CD signals and circularly polarized luminescence. In the case of organic solvents, although all the tested solvents made the blue gel to red, only some of them could keep the CD signals, thus providing additional sensing capacity of the PDA system. So far, the blue-to-red color change and the "fluorescence on" was widely used as colorimetric and fluorogenic diagnostic signals for PDA, here we showed an additional chiroptical diagnostic signal for a more precise sensing by using the helical PDA.

Multi-Amplified Sensing of MicroRNA by a Small DNA Fragment-Driven Enzymatic Cascade Reaction.

Combining technological developments such as nanomaterials, DNA nanotechnology, and functional enzymes has great potential to facilitate next generation high performance molecular diagnostic systems. In this work, we describe a microRNA (miRNA) detection assay that combines target recycling and isothermal amplification in an elegantly designed enzyme-mediated cascade reaction. Target recycling is driven by the action of duplex-specific nuclease (DSN), resulting in highly amplified translation of input miRNA to short output DNA fragments. These fragments act as highly specific initiators of rolling circle amplification (RCA), an isothermal reaction that outputs a large volume of polymeric DNAzymes per initiator, and finally a fluorogenic output signal. Based on careful electrophoretic analysis we observed that the DSN produces ca. 10 nt DNA fragments from DNA/miRNA duplexes, regardless of the length of DNA strands. Target recycling yielded ca. 5 orders of magnitude amplification through the DSN-assisted recycling system on magnetic particles, and the RCA yielded a further 2 orders of magnitude. The final assay exhibited a limit of detection of 1.8 fM of miRNA spiked into 20% human serum, and showed excellent selectivity for miR-21 versus single base-mismatched sequences and other cancer-related miRNAs. The developed assay was further employed to determine accurate amounts of miR-21 in total RNA samples extracted from human cancer cell lines and normal cells, confirming the applicability of the assay for direct and absolute quantification of mature specific miRNA in real biological samples.

Hg2+-selective fluorogenic signaling probe based on the hydrolysis of hydrazone

A simple Hg2+-selective fluorogenic signaling probe based on methoxynaphthalene hydrazone was investigated. Hydrazone 1 gave prominent fluorescent signals in response to Hg2+ ions in a 1:1 mixture of acetate buffer (pH 4.8) and DMSO. The fluorescence signaling was due to the Hg2+-selective hydrolysis of hydrazone to afford the strongly fluorescent methoxynaphthaldehyde. Hg2+-selective signaling of 1 was not affected by the presence of representative metal ions as the background. Hg2+-selective fluorescence signaling by 1 was possible with a detection limit of 6.0×10−6M. As a practical application, convenient detection of Hg2+ ions using a smartphone as a portable signaling processor was conducted.