James W. Foley

Selective, Highly Sensitive Fluorescent Probe for the Detection of Sulfur Dioxide Derivatives in Aqueous and Biological Environments

On the basis of a unique nucleophilic addition reaction, a novel water-soluble broadly emitting (500-700 nm) fluorescent Probe 1 was developed for the rapid detection of SO2 derivatives in aqueous media. The positively-charged benzopyrylium moiety in Probe 1 provides both excellent water solubility, making this probe applicable in 100% aqueous environments, and the ability to function as a fluorescence quencher of the coumarin moiety. Probe 1 generates a nearly instantaneous strong fluorescence signal in response to SO2 derivatives having an 8.3 nM detection limit for bisufite. The resulting Probe 1-sulfite adduct emits in the green/red spectral region (λ(max) = 585 nm) with a large Stokes shift (139 nm). The probe exhibits excellent selectivity toward SO2 derivatives over other potential interfering agents including reactive sulfur-containing species. Importantly, we demonstrate that Probe 1 can be used for the real-time sensing and bioimaging of SO2 derivatives in living cells.

Broadly Applicable Strategy for the Fluorescence Based Detection and Differentiation of Glutathione and Cysteine/Homocysteine: Demonstration in Vitro and in Vivo

Glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are small biomolecular thiols that are present in all cells and extracellular fluids of healthy mammals. It is well-known that each plays a separate, critically important role in human physiology and that abnormal levels of each are predictive of a variety of different disease states. Although a number of fluorescence-based methods have been developed that can detect biomolecules that contain sulfhydryl moieties, few are able to differentiate between GSH and Cys/Hcy. In this report, we demonstrate a broadly applicable approach for the design of fluorescent probes that can achieve this goal. The strategy we employ is to conjugate a fluorescence-quenching 7-nitro-2,1,3-benzoxadiazole (NBD) moiety to a selected fluorophore (Dye) through a sulfhydryl-labile ether linkage to afford nonfluorescent NBD-O-Dye. In the presence of GSH or Cys/Hcy, the ether bond is cleaved with the concomitant generation of both a nonfluorescent NBD-S-R derivative and a fluorescent dye having a characteristic intense emission band (B1). In the special case of Cys/Hcy, the NBD-S-Cys/Hcy cleavage product can undergo a further, rapid, intramolecular Smiles rearrangement to form a new, highly fluorescent NBD-N-Cys/Hcy compound (band B2); because of geometrical constraints, the GSH derived NBD-S-GSH derivative cannot undergo a Smiles rearrangement. Thus, the presence of a single B1 or double B1 + B2 signature can be used to detect and differentiate GSH from Cys/Hcy, respectively. We demonstrate the broad applicability of our approach by including in our studies members of the Flavone, Bodipy, and Coumarin dye families. Particularly, single excitation wavelength could be applied for the probe NBD-OF in the detection of GSH over Cys/Hcy in both aqueous solution and living cells.

A red fluorescent probe for thiols based on 3-hydroxyflavone and its application in living cell imaging

A novel red (λmaxem = 632 nm) fluorescent probe based on 3-hydroxyflavone was designed and synthesized for thiol recognition with high sensitivity and excellent selectivity. Application of the probe for selective detection of intracellular thiols has been successfully demonstrated.

Ratiometric Fluorescent Probe for Lysosomal pH Measurement and Imaging in Living Cells Using Single-Wavelength Excitation

A novel lysosome-targeting ratiometric fluorescent probe (CQ-Lyso) based on the chromenoquinoline chromorphore has been developed for the selective and sensitive detection of intracellular pH in living cells. In acidic media, the protonation of the quinoline ring of CQ-Lyso induces an enhanced intramolecular charge transfer (ICT) process, which results in large red-shifts in both the absorption (104 nm) and emission (53 nm) spectra which forms the basis of a new ratiometric fluorescence pH sensor. This probe efficiently stains lysosomes with high Pearsons colocalization coefficients using LysoTrackerDeep Red (0.97) and LysoTrackerBlue DND-22 (0.95) as references. Importantly, we show that CQ-Lyso quantitatively measures and images lysosomal pH values in a ratiometric manner using single-wavelength excitation.

Antimicrobial Photodynamic Efficacy of Side‐chain Functionalized Benzo[a]phenothiazinium Dyes

5‐(Ethylamino)‐9‐diethylaminobenzo[a]phenothiazinium chloride (EtNBS) is a photosensitizer (PS) with broad antimicrobial photodynamic activity. The objective of this study was to determine the antimicrobial photodynamic effect of side chain/end group modifications of EtNBS on two representative bacterial Gram‐type‐specific strains. Two EtNBS derivatives were synthesized, each functionalized with a different side‐chain end‐group, alcohol or carboxylic acid. In solution, both exhibited photochemical properties consistent with those of the EtNBS parent molecule. In vitro photodynamic therapy experiments revealed an initial Gram‐type‐specificity with two representative strains; both derivatives were phototoxic to Staphylococcus aureus 29213 but the carboxylic acid derivative was nontoxic to Escherichia coli 25922. This difference in photodynamic efficacy was not due to a difference in the binding of the two molecules to the bacteria as the amount of both derivatives bound by bacteria was identical. Interestingly, the carboxylic acid derivative produced no fluorescence emission when observed in cultures of E. coli via fluorescence microscopy. These early findings suggest that the addition of small functional groups could achieve Gram‐type‐specific phototoxicity through altering the photodynamic activity of PSs and deserve further exploration in a larger number of representative strains of each Gram type.

A colorimetric and ratiometric fluorescent probe for Cu2+ with a large red shift and its imaging in living cells

The ester derived from picolinic acid and 4-hydroxynaphthalimide has been developed as a colorimetric and ratiometric fluorescent probe for the detection of Cu2+. Upon exposure to Cu2+ in a buffered solution, the probe displays a large red shift both in its absorption (110 nm) and emission (143 nm) spectra. This novel probe has a high sensitivity and an excellent selectivity for Cu2+ ions over competing metal ions (K+, Na+, Zn2+, Ni2+, Mn2+, Mg2+, Ca2+, Hg2+, Cd2+, Pb2+, Fe2+, Co2+, Ba2+, Ag+ and Fe3+). Most importantly, this new picolinate probe can successfully detect Cu2+ in living cells.

Real-time fluorescence monitoring of phenothiazinium photosensitizers and their anti-mycobacterial photodynamic activity against Mycobacterium bovis BCG in in vitro and in vivo models of localized infection

An objective was to explore the photodynamic activity of two cationic photosensitizers (PS) (benzo[a]phenothiazinium chloride and benzo[a]phenoselenazinium chloride) against Mycobacterium bovis BCG both in vitro and in a murine model of BCG-granuloma. The hypothesis being tested in this study was that cationic molecules could best interact with the negatively charged membrane of BCG as a model for mycobacterial infection. Cells in culture were incubated with various concentrations of PS and subsequently illuminated using a 635 nm diode laser. Dark- and light-induced killing profiles were generated as a function of fluence and dye concentration. In vivo, local injection of the PS into subcutaneous Mycobacterium-induced granuloma sites in murine model was followed by red light illumination of the same area. A special microscope was fabricated for real-time in vivo fluorescent microscopy to monitor EtNBS delivery to subcutaneous murine granulomata. Both PS demonstrated good in vitro antimycobacterial photodynamic activity with varying degrees of toxicity under dark conditions. Real time in vivo monitoring of benzophenothiazine chloride in the mouse model indicated that this fluorescent photosensitizer was delivered rapidly to the subcutaneous granuloma site. In vivo, photosensitizer specific dark- and photo-toxicities depended on the structure, concentration of the photosensitizer and the light dose utilized. Cationic phenothiazine photosensitizers are promising candidates for use in anti-mycobacterial PDT for localized diseases such as cutaneous and pulmonary granulomata.

7-Azabicyclo[2.2.1]heptane as a Unique and Effective Dialkylamino Auxochrome Moiety: Demonstration in a Fluorescent Rhodamine Dye

A new type of a highly fluorescent sulforhodamine dye, 221SR, was designed and synthesized using 7-azabicylco[2.2.1]heptyl moieties as the electron donating auxochrome groups. Using the prototypical dye tetramethylsulforhodamine, TMSR, as a benchmark, we show this new dye has higher fluorescent quantum yields, Phi, (Phi = 0.95 vs Phi = 0.65 at 20 degrees C), emission efficiencies that are invariant in the 20--> 60 degrees C temperature range (Phi = 0.95 vs Phi = 0.38 at 60 degrees C), and fluorescence lifetimes that increase with a rise in temperature (20 --> 60 degrees C) as compared to a decrease for the benchmark dye (3.8 --> 3.9 vs 2.8 --> 1.7 ns). Importantly, photostability studies found the azabicyclic rhodamine to be many times more stable than its tetramethyl analogue. To the best our knowledge this is the first report of the use of an apex-N-substituted azabicycloalkane as an electron donor group in any class of donor-acceptor dye. Thus, the concept of using an apex-substitutes bicyclic amine as a donor moiety constitutes a new paradigm for simultaneously inducing remarkable beneficial effects on both emission efficiency and photostability in a donor-acceptor fluorophore.

5,9-Diaminodibenzo[a,j]phenoxazinium Chloride: A Rediscovered Efficient Long Wavelength Fluorescent Dye

We have evaluated the chemical, photophysical and photostability properties of 5,9-diaminodibenzo[a,j]phenoxazinium chloride, 3, and its bis-5,9-ethylamino analogue, 4, with the goal of determining if they have characteristics that are compatible with the requirements of a useful fluorescent probe. In order to gauge the potential utility of these fluorophores in biological and non-biological applications, these data were compared to those obtained for Oxazine 118, 1, and Cresyl Violet, 2, two well known fluorescent dyes that differ in molecular structure from the title dye 3 by having two or one fewer benzo moieties fused to a generic oxazine ring structure, respectively. The findings of this investigation show that 3, as well as bis-ethylamino analogue, 4, have fluorescent lifetimes, quantum yields and photostabilities that compare favorably with the lower order benchmark fluorophores 1 and 2. Moreover, both dibenzo dyes have the highly desirable properties of absorbing and emitting further in the red and far red /near infrared spectral region, respectively, than do their less conjugated analogues. Taken together, these results suggest that 3 constitutes an archetype upon which a new class of long wavelength fluororescent reporters might be based.

FIB machining of occulting masks for imaging of extrasolar planets

We are developing the ability for Focused Ion Beam (FIB) machining of occulting masks for use in coronagraphs. These masks will be used as soft-edged Lyot stops to suppress light from stars and allow direct imaging of extrasolar planets. The FIB approach is attractive because it has the potential for higher precision than mechanical machining and for larger volumes than electron-beam lithography. The mask fabrication process is trifold: 1) a transparent material-currently, poly(methyl methacrylate) (PMMA)-is doped with dyes; 2) the mask shape is FIB milled into the material; and 3) the mask is coated with another layer of index-matching transparent absorber. Using a Zeiss NVision 40 FIB system, we have fabricated conical-shaped masks of various slopes in dye-doped PMMA. Inherent in this process is the advantage of control of the features through programming the ion beam track. We have also optically characterized these masks as well as the dye-doped absorbing material. We have found that the dye-doped PMMA has a very high absorbance, >1 OD.