Evaluation of a biomarker (NO) dynamics in inflammatory zebrafish and periodontitis saliva samples via a fast-response and sensitive fluorescent probe.

Many pathological processes include nitric oxide (NO), a signaling transduction molecule. Tumors, cardiovascular, cerebrovascular, neurodegenerative, and other illnesses are linked to abnormal NO levels. Thus, evaluating NO levels in vitro and in vivo is crucial for studying chemical biology process of associated disorders. This work devised and manufactured a coumarin-based fluorescent probe ZPS-NO to detect nitric oxide (NO). The reaction between ZPS-NO and NO produced a highly selective and sensitive optical response that caused a powerful fluorescence "turn-on" effect with a ultra-low NO detection limit of 14.5 nM. Furthermore, the probe was applied to sense and image NO in living cells and inflammatory model of zebrafish, as well as to detect NO in periodontitis patients' saliva samples. We anticipate that probe ZPS-NO will serve as a practical and effective tool for assessing the interactions and evaluation of periodontitis development.

In vivo bioimaging and detection of endogenous hypochlorous acid in lysosome using a near-infrared fluorescent probe.

The phagocyte's lysosome is the primary site of hypochlorous acid (HOCl) synthesis, and HOCl can be used as a biomarker for osteoarthritis diagnosis and treatment evaluation. Accurate detection of HOCl with high sensitivity and selectivity is required to understand its activities in healthy bio-systems and diseases. By integrating acceptable design principles and dye screening methodologies, we proposed and developed a novel near-infrared fluorescent HOCl sensing probe (FNIR-HOCl). The FNIR-HOCl probe has a quick reaction rate, high sensitivity (LOD = 70 nM), and excellent selectivity toward HOCl over other metal ions and reactive oxygen species. It has been successfully implemented to detect endogenous HOCl produced by RAW264.7 cells, as well as in vivo imaging towards mice with osteoarthritis. As a result, the probe FNIR-HOCl is extremely promising as a biological tool for revealing the roles of HOCl in various physiological and pathological contexts.

A novel near-infrared fluorescent probe with hemicyanine scaffold for sensitive mitochondrial pH detection and mitophagy study.

Mitochondria, as an energy-producing powerhouse in live cells, is considered to be directly linked to cellular health. However, dysfunctional mitochondria and abnormal mitochondria pH would possibly activate mitophagy, cell apoptosis and intercellular acidification process. In this work, we synthesized a novel near infrared fluorescent probe (FNIR-pH) for measurement of mitochondrial pH based on the hemicyanine skeleton as a fluorophore. The FNIR-pH probe functioned as a mitochondrial pH substrate and exhibited quick and sensitive turn-on fluorescence responses to mitochondrial pH in basic solution due to the deprotonation of hydroxy group in the structure. From pH 3.0 to 10.0, the FNIR-pH exhibited almost 100-fold increase in fluorescence intensity at 766 nm wavelength. The FNIR-pH also displayed superior selectivity to various metal ions, excellent photostability, and low cytotoxicity, which facilitated further biological application. Owing to the proper pKa value of 7.2, the FNIR-pH paved the way for real-time monitoring of mitochondria pH changes in live cells and sensitive sensing of mitophagy. Moreover, the FNIR-pH probe was also implemented for fluorescent imaging of tumor-bearing mice to validate its potential application for in vivo imaging of bioanalytes and biomarkers.

Upconversion Optogenetic Engineered Bacteria System for Time-Resolved Imaging Diagnosis and Light-Controlled Cancer Therapy.

Engineering bacteria can achieve targeted and controllable cancer therapy using synthetic biology technology and the characteristics of tumor microenvironment. Besides, the accurate tumor diagnosis and visualization of the treatment process are also vital for bacterial therapy. In this paper, a light control engineered bacteria system based on upconversion nanoparticles (UCNP)-mediated time-resolved imaging (TRI) was constructed for colorectal cancer theranostic and therapy. UCNP with different luminous lifetimes were separately modified with the tumor targeting molecule (folic acid) or anaerobic bacteria (Nissle 1917, EcN) to realize the co-localization of tumor tissues, thus improving the diagnostic accuracy based on TRI. In addition, blue light was used to induce engineered bacteria (EcN-pDawn-φx174E/TRAIL) lysis and the release of tumor apoptosis-related inducing ligand (TRAIL), thus triggering tumor cell death. In vitro and in vivo results indicated that this system could achieve accurate tumor diagnosis and light-controlled cancer therapy. EcN-pDawn-φx174E/TRAIL with blue light irradiation could inhibit 53% of tumor growth in comparison to that without blue light irradiation (11.8%). We expect that this engineered bacteria system provides a new technology for intelligent bacterial therapy and the construction of cancer theranostics.

A dual-caged resorufin probe for rapid screening of infections resistant to lactam antibiotics.

The alarming increase of antimicrobial resistance urges rapid diagnosis and pathogen specific infection management. This work reports a rapid screening assay for pathogenic bacteria resistant to lactam antibiotics. We designed a fluorogenic N-cephalosporin caged 3,7-diesterphenoxazine probe CDA that requires sequential activations to become fluorescent resorufin. A series of studies with recombinant ß-lactamases and clinically prevalent pathogens including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens demonstrated that CDA possessed superior sensitivity in reporting the activity of ß-lactamases including cephalosporinases and carbapenemases. After a simple filtration, lactam-resistant bacteria in urine samples could be detected at 103 colony-forming units per milliliter within 2 hours.

Mitochondrial copper depletion suppresses triple-negative breast cancer in mice.

Depletion of mitochondrial copper, which shifts metabolism from respiration to glycolysis and reduces energy production, is known to be effective against cancer types that depend on oxidative phosphorylation. However, existing copper chelators are too toxic or ineffective for cancer treatment. Here we develop a safe, mitochondria-targeted, copper-depleting nanoparticle (CDN) and test it against triple-negative breast cancer (TNBC). We show that CDNs decrease oxygen consumption and oxidative phosphorylation, cause a metabolic switch to glycolysis and reduce ATP production in TNBC cells. This energy deficiency, together with compromised mitochondrial membrane potential and elevated oxidative stress, results in apoptosis. CDNs should be less toxic than existing copper chelators because they favorably deprive copper in the mitochondria in cancer cells instead of systemic depletion. Indeed, we demonstrate low toxicity of CDNs in healthy mice. In three mouse models of TNBC, CDN administration inhibits tumor growth and substantially improves survival. The efficacy and safety of CDNs suggest the potential clinical relevance of this approach.

Near-infrared fluorescent probes with BODIPY donors and rhodamine and merocyanine acceptors for ratiometric determination of lysosomal pH variance.

Three fluorescent probes have been developed by conjugating three different BODIPY donors to rhodamine and merocyanine acceptors for ratiometric determination of lysosomal pH variations. Probe A consists of a 1,3,5,7-tetramethyl-BODIPY donor and a near-infrared rhodamine acceptor bearing a lysosome-targeting morpholine residue. Probe B is composed of a 3,5-dimethyl-BODIPY donor and a near-infrared rhodamine acceptor modified with an o-phenylenediamine residue. Probe C contains a 3-styrene-functionalized BODIPY donor with longer wavelength emission and a near-infrared merocyanine acceptor containing a morpholine residue. Under neutral or basic pH conditions, the probes only show fluorescence from the BODIPY donors under BODIPY excitation because the rhodamine and merocyanine acceptors maintain closed spirolactam configurations. However, excitation at BODIPY absorption wavelengths concomitant with gradual pH decrease results in fluorescence decreases with the BODIPY donors and fluorescence increases from the rhodamine and merocyanine acceptors due to through-bond energy transfer from the donors to the acceptors. This is because the spirolactam ring opens under more acidic conditions and fluorescence of the acceptors results from significantly improved π-conjugation. These experimental results are substantiated with theoretical calculations on models of the different probes. The probes have all been used to determine lysosome pH variations in HeLa cells. Probe B was further utilized to successfully detect pH fluctuations in HeLa cells under oxidative stress and with treatment of NH4Cl and chloroquine.

Near-infrared fluorescent probes based on TBET and FRET rhodamine acceptors with different pK a values for sensitive ratiometric visualization of pH changes in live cells.

Three near-infrared ratiometric fluorescent probes (A-C) based on TBET and FRET near-infrared rhodamine acceptors with different pK a values were designed and synthesized to achieve sensitive ratiometric visualization of pH variations in lysosomes in visible and near-infrared channels. Tetraphenylethene (TPE) was bonded to near-infrared rhodamine dyes through short electrical π -conjugation linkers to prevent an aggregation-caused quenching (ACQ) effect and allow highly efficient energy transfer of up to 98.9% from TPE donors to rhodamine acceptors. Probes A-C respond to pH variation from 7.4 to 3.0 in both buffer solutions and live cells with significant decreases of donor fluorescence and concomitant extraordinary increases of rhodamine acceptor fluorescence because of highly efficient energy transfer. In addition, probe C is capable of determining pH fluctuations in live cells treated with chloroquine. The probes show good photostability, excellent cell membrane permeability, high selectivity to pH, and two well-resolved emission peaks to ensure accurately comparative and quantitative analyses of intracellular pH changes.

Fluorescent probes with high pKa values based on traditional, near-infrared rhodamine, and hemicyanine fluorophores for sensitive detection of lysosomal pH variations.

Sterically hindered fluorescent probes (A-C) have been developed by introducing 2-aminophenylboronic acid pinacol ester to a traditional, A, a near-infrared rhodamine dye, B, and a near-infrared hemicyanine dye, C, forming closed spirolactam ring structures. Probe A was non-fluorescent under basic pH conditions whereas probes B and C were moderately fluorescent with fluorescence quantum yields of 9% and 5% in pH 7.4 PBS buffer containing 1% ethanol, respectively. With all probes increasing acidity leads to significant increases in fluorescence at 580 nm, 644 and 744 nm for probes A, B and C with fluorescence quantum yields of 26%, 21% and 10% in pH 4.5 PBS buffer containing 1% ethanol, respectively. Probes A, B and C were calculated to have pKa values of 5.81, 5.45 and 6.97. The difference in fluorescence under basic conditions is ascribed to easier opening of the closed spirolactam ring configurations due to significant steric hindrance between the 2-aminophenylboronic acid pinacol ester residue and an adjacent H atom in the xanthene derivative moiety in probe B or C. The probes show fast, reversible, selective and sensitive fluorescence responses to pH changes, and are capable of sensing lysosomal pH variations in living cells.

A FRET-Based Near-Infrared Fluorescent Probe for Ratiometric Detection of Cysteine in Mitochondria.

We report a near-infrared fluorescent probe A for the ratiometric detection of cysteine based on FRET from a coumarin donor to a near-infrared rhodamine acceptor. Upon addition of cysteine, the coumarin fluorescence increased dramatically up to 18-fold and the fluorescence of the rhodamine acceptor decreased moderately by 45 % under excitation of the coumarin unit. Probe A has been used to detect cysteine concentration changes in live cells ratiometrically and to visualize fluctuations in cysteine concentrations induced by oxidation stress through treatment with hydrogen peroxide or lipopolysaccharide (LPS). Finally, probe A was successfully applied for the in vivo imaging of Drosophila melanogaster larvae to measure cysteine concentration changes.

Detecting Zn(II) Ions in Live Cells with Near-Infrared Fluorescent Probes.

Two near-infrared fluorescent probes (A and B) containing hemicyanine structures appended to dipicolylamine (DPA), and a dipicolylamine derivative where one pyridine was substituted with pyrazine, respectively, were synthesized and tested for the identification of Zn(II) ions in live cells. In both probes, an acetyl group is attached to the phenolic oxygen atom of the hemicyanine platform to decrease the probe fluorescence background. Probe A displays sensitive fluorescence responses and binds preferentially to Zn(II) ions over other metal ions such as Cd2+ ions with a low detection limit of 0.45 nM. In contrast, the emission spectra of probe B is not significantly affected if Zn(II) ions are added. Probe A possesses excellent membrane permeability and low cytotoxicity, allowing for sensitive imaging of both exogenously supplemented Zn(II) ions in live cells, and endogenously releases Zn(II) ions in cells after treatment of 2,2-dithiodipyridine.

Near-Infrared Hybrid Rhodol Dyes with Spiropyran Switches for Sensitive Ratiometric Sensing of pH Changes in Mitochondria and Drosophila melanogaster First-Instar Larvae.

Near-infrared hybrid rhodol dyes (probes A and B) for sensitive ratiometric visualization of pH changes were prepared by incorporating hemicyanine dyes into traditional rhodol dyes. This approach was based on π-conjugation changes involving a rhodol hydroxyl group as a spiropyran switch upon pH changes. Electronic spectra of probes A-2 and B-2 contain sharp absorption peaks at 535 nm and fluorescence peaks at 558 nm with similar π-conjugation and a closed spiropyran form at a basic pH of 10.2. However, acidic pH conditions break down the hemiaminal ether groups, leading to indolenium moieties and significantly extending the π-conjugation within the rhodol fluorophores, resulting in additional near-infrared emissions for probes A-1 and B-1. As a result, probes A and B exhibit gradual decreases of the absorption peaks at 535 nm and gradual increases in absorption peaks at 609 and 622 nm upon transition from basic to acidic pH, respectively. Both probes display ratiometric fluorescence sensing responses to pH downgrades from 10.2 to 3.6 with visible fluorescence decreases at 558 nm, as well as corresponding increases of the near-infrared fluorescence peaks at 688 and 698 nm, respectively. They exhibit fast, sensitive, and selective fluorescence responses with clearly defined ratiometric features to pH changes and show low cytotoxicity and excellent cell permeability. Our probes were successfully applied to ratiometrically detect pH changes in mitochondria, D. melanogaster first-instar larvae, and to visualize the mitophagy process caused by either cell nutrient starvation or drug treatment.

Fluorescent Probes Based on π-Conjugation Modulation between Hemicyanine and Coumarin Moieties for Ratiometric Detection of pH Changes in Live Cells with Visible and Near-infrared Channels.

We report two ratiometric fluorescent probes based on π-conjugation modulation between coumarin and hemicyanine moieties for sensitive ratiometric detection of pH alterations in live cells by monitoring visible and near-infrared fluorescence changes. In a π-conjugation modulation strategy, a coumarin dye was conjugated to a near-infrared hemicyanine dye via a vinyl connection while lysosome-targeting morpholine ligand and o-phenylenediamine residue were introduced to the hemicyanine dye to form closed spirolactam ring structures in probes A and B, respectively. The probes show only visible fluorescence of the coumarin moiety under physiological and basic conditions because the hemicyanine moieties retain their closed spirolactam ring structures. However, decrease of pH to acidic condition causes spirolactam ring opening, and significantly enhances π-conjugation within the probes, thus generating new near-infrared fluorescence peaks of the hemicyanine at 755 nm and 740 nm for probes A and B, respectively. Moreover, the probes display ratiometric fluorescence response to pH with decreases of the coumarin fluorescence and increases of the hemicyanine fluorescence when pH changes from 7.4 to 2.5. The probes are fully capable of imaging pH changes in live cells with good ratiometric responses in visible and near-infrared channels, and effectively avoid fluorescence blind spots under neutral and basic pH conditions - an issue that typical intensity-based pH fluorescent probes run into. The probe design platform reported herein can be easily applied to prepare a variety of ratiometric fluorescent probes for detection of biological thiols, metal ions, reactive oxygen and nitrogen species by introducing appropriate functional groups to hemicyanine moiety.

New near-infrared rhodamine dyes with large Stokes shifts for sensitive sensing of intracellular pH changes and fluctuations.

New near-infrared rhodamine dyes with large Stokes shifts were developed and applied for sensitive detection of cellular pH changes and fluctuations by incorporating an additional amine group with fused rings into the rhodamine dyes to enhance the electron donating ability of amine groups and improve the spectroscopic properties of the dyes.

Ratiometric Near-Infrared Fluorescent Probes Based On Through-Bond Energy Transfer and π-Conjugation Modulation between Tetraphenylethene and Hemicyanine Moieties for Sensitive Detection of pH Changes in Live Cells.

In this paper, we present three ratiometric near-infrared fluorescent probes (A-C) for accurate, ratiometric detection of intracellular pH changes in live cells. Probe A consists of a tetraphenylethene (TPE) donor and near-infrared hemicyanine acceptor in a through-bond energy transfer (TBET) strategy, while probes B and C are composed of TPE and hemicyanine moieties through single and double sp2 carbon-carbon bond connections in a π-conjugation modulation strategy. The specific targeting of the probes to lysosomes in live cells was achieved by introducing morpholine residues to the hemicyanine moieties to form closed spirolactam ring structures. Probe A shows aggregation-induced emission (AIE) property at neutral or basic pH, while probes B and C lack AIE properties. At basic or neutral pH, the probes only show fluorescence of TPE moieties with closed spirolactam forms of hemicyanine moieties, and effectively avoid blind fluorescence imaging spots, an issue which typical intensity-based pH fluorescent probes encounter. Three probes show ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with TPE fluorescence decreases and hemicyanine fluorescence increases, because acidic pH makes the spirolactam rings open to enhance π-conjugation of hemicyanine moieties. However, probe A shows much more sensitive ratiometric fluorescence responses to pH changes from 7.0 to 3.0 with remarkable ratio increase of TPE fluorescence to hemicyanine fluorescence up to 238-fold than probes B and C because of its high efficiency of energy transfer from TPE donor to the hemicyanine acceptor in the TBET strategy. The probe offers dual Stokes shifts with a large pseudo-Stokes shift of 361 nm and well-defined dual emissions, and allows for colocalization of the imaging readouts of visible and near-infrared fluorescence channels to achieve more precisely double-checked ratiometric fluorescence imaging. These platforms could be employed to develop a variety of novel ratiometric fluorescent probes for accurate detection of different analytes in applications of chemical and biological sensing, imaging, and diagnostics by introducing appropriate sensing ligands to hemicyanine moieties to form on-off spirolactam switches.

A cyanine-based fluorescent cassette with aggregation-induced emission for sensitive detection of pH changes in live cells.

An aggregation-induced emission (AIE) cyanine-based fluorescent cassette with a large pseudo-Stokes shift was designed and prepared to sensitively image pH changes in live cells via through-bond energy transfer (TBET) from a tetraphenylethene (TPE) donor to a cyanine acceptor.

New Near-infrared Fluorescent Probes with Single-photon Anti-Stokes-shift Fluorescence for Sensitive Determination of pH Variances in Lysosomes with a Double-Checked Capability.

Two near-infrared luminescent probes with Stokes-shift and single-photon anti-Stokes-shift fluorescence properties for sensitive determination of pH variance in lysosomes have been synthesized. A morpholine residue in probe A which serves as a targeting group for lysosomes in viable cells was attached to the fluorophores via a spirolactam moiety while a mannose residue was ligated to probe B resulting in increased biocompatibility and solubility in water. Probes A and B contain closed spirolactam moieties, and show no Stokes-shift or anti-Stokes-shift fluorescence under neutral or alkali conditions. However, the probes incrementally react to pH variance from 7.22 to 2.76 with measurable increases in both Stokes-shift and anti-Stokes-shift fluorescence at 699 nm and 693 nm under 645 nm and 800 nm excitation, respectively. This acid-activated fluorescence is produced by the breaking of the probe spirolactam moiety, which greatly increased overall π-conjugation in the probes. These probes possess upconversion near-infrared fluorescence imaging advantages including minimum cellular photo-damage, tissue penetration, and minimum biological fluorescence background. They display excellent photostability with low dye photobleaching and show good biocompatibility. They are selective and capable of detecting pH variances in lysosomes at excitation with two different wavelengths, i.e., 645 and 800 nm.

A Redox Conjugated Polymer-Based All-Solid-State Reference Electrode.

This work reports the design, synthesis, and characterization of a novel redox-active conjugated polyaniline containing quinone moiety as a solid state reference electrode. The union of electro-active quinone with π-conjugated polyaniline was created by the first chemical synthesis of para-dimethoxybenzene-functionalized aniline as a monomer using a palladium-mediated coupling. The successful polymerization of the as-prepared monomer was accomplished without acid additives. Its post-polymerization modification with strong Lewis acid boron tribromide furnished unique poly (aniline quinone/hydroquinone) with desired properties for all-solid-state reference electrode (RE) applications. The electrochemical responses from the conjugated polyaniline backbone in this unique polymer have been "suppressed" by the quinone pendant. The resulting poly (aniline quinone) showed a quasi-reversible redox process from the redox behavior of the pendant quinone. The stable electrode potential of this poly (aniline quinone/hydroquinone) suggested that it was a single phase in which the amounts of totally reduced and totally oxidized species could be maintained at a constant in various solvents and electrolytes. Its electrochemical stability was excellent with 95% peak current retention after continuous cyclic voltammetric testing. The aniline and quinone moieties in poly (aniline quinone/hydroquinone) render it to have both hydrophilic and hydrophobic compatibility. It showed excellent behavior as a reference electrode in aqueous and non-aqueous media and can be used in both non-zero current and zero-current conditions, providing a stable potential with a maximum potential drift of ~4.7 mV over ten consecutive days.

Luminescent Probes for Sensitive Detection of pH Changes in Live Cells through Two Near-Infrared Luminescence Channels.

Two water-soluble near-infrared luminescent probes, which possess both conventional intense Stokes fluorescence and unique single-photon frequency upconversion luminescence (FUCL), were developed for sensitive and selective detection of pH changes in live cells. The water solubility and biocompatibility of these probes were achieved by introducing mannose residues through 2,2'-(ethylenedioxy)diethylamine tethered spacers to a near-infrared conventional fluorescence (CF) and FUCL organic fluorophore. At a pH higher than 7.4, the probes have ring-closed spirocyclic lactam structures, thus are colorless and nonfluorescent. Nevertheless, they sensitively respond to acidic pH values, with a drastic structural change to ring-opened spirocyclic lactam forms, which cause significant absorbance increases at 714 nm. Correspondingly, their near-infrared CF and FUCL intensities at 740 nm are also significantly enhanced when excited by 690 and 808 nm, respectively. The probes hold a variety of advantages such as high sensitivity, excellent reversibility and selectivity to pH over metal ions, low cellular autofluorescence background interference, good cell membrane permeability and photostability, as well as low cytotoxicity. Our results have successfully proven that these probes can visualize intracellular lysosomal pH changes in live cells by monitoring both near-infrared CF and FUCL changes.