Ratiometric Near-Infrared Fluorescent Probes Based On Through-Bond Energy Transfer and ?-Conjugation Modulation between Tetraphenylethene and Hemicyanine Moieties for Sensitive Detection of pH Changes in Live Cells.
ABSTRACT: 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 ?-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 ?-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.
Project description:We report two ratiometric fluorescent probes based on ?-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 ?-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 ?-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.
Project description: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 ? -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.
Project description: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.
Project description: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 ?-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.
Project description: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 ?-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.
Project description: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 ?-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 ?-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 ?-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.
Project description:Two-photon excitation (TPE) probe-based fluorescence imaging has become one of the most attractive diagnostic techniques to investigate biomolecules and biological events in live cells and tissues. At the current stage most of the TPE-based sensing is reflected by fluorescence intensity changes. Nevertheless the mere altering of intensity could be facilely affected by ambient conditions. On the other hand, TPE probes based on an intramolecular charge transfer (ICT) strategy could solve this problem to some extent with a morphology change-induced emission shift. However their applications are yet constrained due to the inherent limitation of ICT, e.g. the high degree of overlap of two emissions bands and shifts of the TPE maxima. To achieve the desired TPE-based sensing and to circumvent the problems stated above, we adapted a Förster resonance energy transfer (FRET) strategy to develop small molecule ratiometric TPE fluorescent probes. Our FRET-based ratiometric TPE fluorescent probe displays a remarkable emission shift (up to 125 nm) with two well-resolved emission bands. Hence the ratio of these two emission bands could enable the measurement of fluorescence changes more accurately, thus further improving imaging in live cells and deep tissues. To the best of our knowledge, the current reported probe has the largest emission shift among all the small molecule ratiometric TPE fluorescent probes while the maximum TPE wavelength remains unchanged. This work has provided a FRET approach to fabricate novel small molecule ratiometric TPE fluorescent probes that improve imaging in deep tissues.
Project description:We report five fluorescent probes based on coumarin-hybridized fluorescent dyes with spirolactam ring structures (A-E) to detect pH changes in live cell by monitoring visible and near-infrared fluorescence changes. Under physiological or basic conditions, the fluorescent probes A, B, C, D and E preserve their spirolactam ring-closed forms and only display fluorescent peaks in the visible region corresponding to coumarin moieties at 497, 483, 498, 497 and 482 nm, respectively. However, at acidic pH, the rings of the spirolactam forms of the fluorescent probes A, B, C, D and E open up, generating new near-infrared fluorescence peaks at 711, 696, 707, 715, and 697 nm, respectively, through significantly extended ?-conjugation to coumarin moieties of the fluorophores. The fluorescent probes B and E can be applied to visualize pH changes by monitoring visible as well as near-infrared fluorescence changes. This helps avoid fluorescence imaging blind spots at neutral or basic pH, which typical pH fluorescent probes encounter. The probes exhibit high sensitivity to pH changes, excellent photostability, low auto-fluorescence background and good cell membrane permeability.
Project description:A fluorescence resonance energy transfer (FRET)-based near-infrared fluorescent probe (B?) for double-checked sensitive detection of intracellular pH changes has been synthesized by binding a near-infrared rhodamine donor to a near-infrared cyanine acceptor through robust C-N bonds via a nucleophilic substitution reaction. To demonstrate the double-checked advantages of probe B?, a near-infrared probe (A) was also prepared by modification of a near-infrared rhodamine dye with ethylenediamine to produce a closed spirolactam residue. Under basic conditions, probe B? shows only weak fluorescence from the cyanine acceptor while probe A displays nonfluorescence due to retention of the closed spirolactam form of the rhodamine moiety. Upon decrease in solution pH level, probe B? exhibits a gradual fluorescence increase from rhodamine and cyanine constituents at 623 nm and 743 nm respectively, whereas probe A displays fluorescence increase at 623 nm on the rhodamine moiety as acidic conditions leads to the rupture of the probe spirolactam rings. Probes A and B? have successfully been used to monitor intracellular pH alternations and possess pKa values of 5.15 and 7.80, respectively.
Project description:Hydrogen peroxide (H2O2) plays a key role in the progression of human illnesses, such as autoimmune and auto-inflammatory diseases, infectious diseases, diabetes, and cancer, etc. In this work, we have discribed a novel probe, TPE-TLE, which remarkably displayed AIE property and ratiometric fluorescence emission profiles in the presence of H2O2. This ratiometric fluorescent probe with AIE property exhibits outstanding features such as the well-resolved emission peaks, high sensitivity, high selectivity, low cytotoxicity, and good cell-membrane permeability. These excellent attributes enable us to demonstrate the ratiometric imaging of endogenously produced H2O2 in macrophages and cancer cells based on the novel ratiometric probe with AIE property for the first time. By comparing two kinds of cells, it is firstly found that cancer cells should contain much more endogenous H2O2 than macrophages. We expect that TPE-TLE will be useful fluorescent platform for the development of a variety of ratiometric fluorescent probes with AIE property to achieve unique biological applications.