Logic-signal-based multiplex detection of MiRNAs with high tension hybridization and multiple signal amplification.

MicroRNAs (miRNAs) are crucial regulators of gene expression. The abnormal expression of miRNA is often closely related to many diseases. However, the accurate clinical profiling of miRNA expression remains a great challenge due to the high similarity and wide variety of miRNA sequence structures. Here, we report a highly specific and sensitive multiplex miRNA detection scheme with high tension hybridization and dual signal amplification based on the recyclable autocatalytic DNAzyme and a light harvesting conjugated polymer. Multiple signals can be read out simultaneously by single excitation through the efficient multiple fluorescence resonance energy transfer (FRET) between the conjugated polymer and different small molecule dyes. In addition, different types of logic gates can also be operated by observing the emission intensities of the labeling dyes with miRNAs as inputs, thus giving rise to a new way for the specific detection of certain miRNAs according to the logic signals. Furthermore, we successfully applied the strategy for multiple miRNA detection in cell lysates and the results agree well with those of qRT-PCR. Thus, we believe that this platform holds great potential for miRNA detection in biological samples.

3D Semiconducting Polymer/Graphene Networks: Toward Sensitive Photocathodic Enzymatic Bioanalysis.

This work reports the development of three-dimensional (3D) semiconducting polymer/graphene (SP/G) networks toward sensitive photocathodic enzymatic bioanalysis. Specifically, the porous 3D graphene was first synthesized via the hydrothermal and freeze-dry processes and then mixed with semiconducting polymer to obtain the designed hierarchical structure with unique porosity and large surface area. Afterward, the as-prepared hybrid was immobilized onto the indium tin oxide (ITO) for further characterizations. Exemplified by sarcosine oxidase (SOx) as a model biocatalyst, an innovative 3D SP/G-based photocathodic bioanalysis capable of sensitive and specific sarcosine detection was achieved. The suppression of cathodic photocurrent was observed in the as-developed photocathodic enzymatic biosystem due to the competition of oxygen consumption between the enzyme-biocatalyst process and O2-dependent photocathodic electrode. This work not only presented a unique protocol for 3D SP/G-based photocathodic enzymatic bioanalysis but also provided a new horizon for the design, development, and utilization of numerous 3D platforms in the broad field of general photoelectrochemical (PEC) bioanalysis.

Polymer Dots for Photoelectrochemical Bioanalysis.

Different from the most extensively used inorganic quantum dots (Qdots) for the current state-of-the-art photoelectrochemical (PEC) bioanalysis, this work reports the first demonstration of polymer dots (Pdots) for novel PEC bioanalysis. The semiconducting Pdots were prepared via the reprecipitation method and then immobilized onto the transparent indium tin oxide glass electrode for PEC biodetection of the model molecule l-cysteine. The experimental results revealed that the as-fabricated Pdots exhibited excellent and interesting PEC activity and good analytical performance of rapid response, high stability, wide linear range, and excellent selectivity. In particular, the PEC sensor could easily discriminate l-cysteine from reduced l-glutathione (l-GSH). This work manifested the great promise of Pdots in the field of PEC bioanalysis, and it is believed that our work could inspire the development of numerous functional Pdots with unique properties for innovative PEC bioanalytical purposes in the future.

Photomodified fluoranthene exerts more harmful effects as compared to intact fluoranthene by inhibiting growth and photosynthetic processes in wheat.

Polycyclic aromatic hydrocarbons (PAHs) are toxic to plants. Exposure of PAHs to sunlight can result in the formation of photomodified PAHs, which are generally more toxic than the parent compounds. In the present study, biochemical and physiological effects of intact fluoranthene (FLT) and photomodified FLT (P.FLT) in wheat were investigated. Our results demonstrated that the treatment of FLT and P.FLT, inhibited germination, seedling growth and pigment content. However, inhibition observed on primary photochemistry and photosystem II heterogeneity was more prominent in P.FLT treated plants. Both FLT and P.FLT affected the levels of antioxidant enzymes such as POD, SOD, CAT and GR to similar extent. Greater inhibitory effects of P.FLT than FLT may be ascribed to its effects on growth parameters and photochemistry rather than on the activities of various antioxidant enzymes.

Dual-Targeting into the Mitochondria of Cancer Cells for Ratiometric Investigation of the Dynamic Fluctuation of Sulfur Dioxide and Formaldehyde with Two-Photon Integrated Semiconducting Polymer Dots.

Mitochondrial sulfur dioxide (SO2) and formaldehyde (FA) in cancer cells serve as important signal molecules in mediating multiple physiological and pathological activities. Accurate monitoring of the dynamic fluctuation of SO2 and FA in the mitochondria of cancer cells is important for insight into their relationships and functions in cancer, understanding cancer mechanism, and the role of mitochondrial homeostasis in cancer invasion and metastasis. Herein, a novel integrated two-photon semiconducting polymer dot (BF@Pdots) with dual-targeting (cancer cells and mitochondrial) and dual-emission in green and red regions, which is rationally designed through a four-step engineering strategy by using two newly synthesized functionalized polymers PFNA and FD-PSMA as precursors, has been developed for accurate tracking of the dynamic variation of SO2 and FA in the mitochondria of cancer cells. The sensing mechanism is on the basis of the fluorescence resonance energy transfer (FRET) process in BF@Pdots tuned by the reversible Michael addition reaction between the sensing-groups and SO2 (or FA). The integrated BF@Pdots nanoprobes display excellent performances in the accurate detection of the dynamic fluctuation of SO2 and FA such as precise positioning in the mitochondria of cancer cells, self-calibrating ratiometric, two-photon emission with long wavelength excitation, and fast reversible response. The BF@Pdots nanoprobes are also applied to the ratiometric detection of the dynamic fluctuation of exogenous and endogenous SO2 and FA in the mitochondria of cancer cells for the first time with satisfactory results. Taken together, this work will provide an attractive way to develop versatile integrated Pdots-based fluorescent probes through flexible molecular engineering for applications in accurate imaging of biomolecules in living systems.

Innovative Thin-Layer Chromatography/Fluorescence Detection Approach for Sensitive and Specific Determination of Ledipasvir in Rats' Feces and Pharmaceutical Dosage Form.

An innovative thin-layer chromatography method coupled with the fluorescence detection was developed for a specific estimation of ledipasvir. The separation was achieved on plates of silica gel 60 F254 using ethylacetate: hexane: acetonitrile: triethylamine; (6: 3.5: 1.5: 0.5, $\mathrm{v}/\mathrm{v}/\mathrm{v}/\mathrm{v}$) as a mobile phase system. The method involved the exposure of the developed thin-layer chromatography plate of ledipasvir to strong ultraviolet irradiation, resulting in a great enhancement in the fluorescence properties of ledipasvir. The irradiated plates were scanned after the excitation at 315 $\mathrm{nm}$. The method provided a sufficient separation of ledipasvir from sofosbuvir with ${R}_F$values of 0.28 and 0.36 for ledipasvir and sofosbuvir, respectively. The developed procedures were validated based on guidelines from the International Conference on Harmonization and Food and Drug Administration guidelines. The calibration curve was linear over the range of 5-50 $\mathrm{ng}/\mathrm{band}$. The excellent analytical features of the proposed method allow to the specific determination of ledipasvir in pharmaceutical tablets without interference from sofosbuvir or excipients. As the main elimination route for ledipasvir is via the fecal excretion (86%), the method was applied for the estimation of ledipasvir in fecal specimens with adequate recovery. In addition, the proposed method was applied for testing the content uniformity of ledipasvir in the pharmaceutical tablets.

A nucleic acid-specific fluorescent probe for nucleolus imaging in living cells.

Nucleus imaging is of great importance for understanding cellular processes of genetic expression, proliferation and growth, etc. Although many nucleic-acid selective dyes for nucleus staining are available, few of them meet multiple standards. Herein, we report a cationic fluorescence dye FTI that possesses visible light excitation (436 nm), orange emission (571 nm) and a large Stokes shift (~135 nm) for nucleic-acid staining. FTI displays an obvious and sensitive fluorescent response to DNA in vitro with a 6.4-fold quantum yield increasing. Co-staining and nucleic acid digest experiments in live cells demonstrate that FTI exhibits an unexpected selectivity for the nucleolus of the cells due to the stronger affinity to RNA than DNA. Because of good photostability and low cytotoxicity, FTI can accomplish a promising stain for DNA recognition in vitro and nucleolus-specific imaging in cancer cells.

Protective effects of antioxidants on micronuclei induced by irradiated 9-fluorenone/N,N-dimethyl-p-toluidine in CHO cells.

9-Fluorenone (9F), the aromatic photosensitizer, is widely used as an initiator in visible-light (VL) cured resin systems. There is growing concern that 9F may produce genetic damage by inducing mutation. In this study, 9F in the presence or absence of reducing agent N,N-dimethyl-p-toluidine (DMT) with or without VL irradiation was analyzed for the induction of chromosomal aberrations indicated by micronuclei (MN) induced in CHO cells. Our data demonstrated that a dose-related increase in the frequency of MN and prolonged cell cycles in 9F with or without DMT in the presence or absence of VL irradiation (p < 0.05). The rank orders with respect to genotoxicity and cytotoxicity were found to be as follows: 9F/DMT +VL > 9F/DMT = 9F + VL > 9F. To determine whether oxidative stress could modulate MN induced by 9F/DMT with or without VL irradiation in CHO cells, cells were pretreated with N-acetyl-L-cysteine (NAC), ascorbic acid, and alpha-tocopherol. The pretreatment with antioxidants could diminish not only the prolonged cell cycle but also the decreased frequency of MN which is induced by 9F with or without DMT in the presence or absence of VL irradiation in CHO cells (p < 0.05). Our findings provide the evidences for the induction of MN by 9F in the presence or absence of DMT with or without VL irradiation in CHO cells, indicating clastogenic activity of 9F/DMT in vitro. These antioxidants act as the antagonists against the genotoxicity and cytotoxicity of 9F/DMT. Thus, leaching photoinitiator and reducing agent might be contributing the sources of oxidative stress.

Effects of visible light-irradiated camphorquinone and 9-fluorenone on murine oral mucosa.

The purpose of this study was to evaluate the histopathological effects of camphorquinone (CQ) and 9-fluorenone (9F) with or without visible light (VL) irradiation on the oral mucous membranes of mice. VL irradiation resulted in a higher degree of tissue damage after CQ or 9F application, particularly the latter. Necrosis and apoptosis were responsible for the tissue damage after application of either agent in the presence of VL irradiation.

Fluorene oxidation by solar-driven photo-Fenton process: toward mild pH conditions.

Polycyclic aromatic hydrocarbons (PAHs) are on the list of priority pollutants to be eliminated from the environment due to their carcinogenic and mutagenic action, chemical stability, and resistance to biodegradation. The aim of this study was to evaluate the degradation of fluorene, a well-known PAH, in aqueous solutions (0.03 and 0.08 mg L-1), by means of a solar-driven conventional (PF) and modified photo-Fenton mediated by ferrioxalate complexes (PFF). Photolysis was also employed for comparison purposes. PF reaction was evaluated at different pH values (2.8, 3.5, and 4.0) and iron concentrations (2, 5, 10, and 20 mg L-1). On the other hand, PFF studies were conducted at mild pH conditions (4.0, 5.0, and 6.0) and iron content of 2 mg L-1, keeping initial iron/oxalate molar ratio at 1:3. In both PF and PFF, the initial hydrogen peroxide/iron molar ratio was maintained at 5. In the presence of methanol as cosolvent for fluorene dissolution, the PF reaction was hampered and no consumption of H2O2 was observed during the reaction carried out at constant pH (2.8). This led to low degradation rates, similar to those achieved by photolysis. Under the same pH but using acetonitrile as cosolvent for fluorene dissolution, fluorene degradation was found to be proportional to the iron content used in the PF experiments. On the other hand, at an invariable iron concentration of 5 mg Fe2+ L-1, the increase in pH was accompanied by a decrease in the molar fraction of the most photoactive iron complex (FeOH2+) and ferric hydroxides precipitation, leading to a reduction in the fluorene degradation rate. With regard to the PFF tests, similar fluorene degradation performance was achieved at pH 4 and 5, while at pH 6 iron precipitation became relevant and the degradation rate was slightly slower. PFF has shown to be more efficient than the PF under the same pH (4) and iron concentration (2 mg L-1). Moreover, even at near neutral pH (6), fluorine degradation was shown to be feasible by using ferrioxalate complexes.

Determination of the attenuation factor in fluorene-based molecular wires.

Fluorene-based bridges exhibit a molecular wire-like behaviour in C(60)-wire-exTTF systems with a very low attenuation factor (beta = 0.09 A(-1)).

Aqueous photodegradation and toxicity of the polycyclic aromatic hydrocarbons fluorene, dibenzofuran, and dibenzothiophene.

Decay kinetics resulting from the application of UV and UV/H(2)O(2) to the polycyclic aromatic hydrocarbons (PAHs) fluorene, dibenzofuran and dibenzothiophene was studied. Batch experiments were conducted with both low-pressure monochromatic (253.7nm) and medium pressure polychromatic (200-300nm) UV sources alone or in the presence of up to 25mg/L hydrogen peroxide, in a quasi-collimated beam apparatus. Degradation of all three PAHs, by both UV and UV/H(2)O(2), exhibited pseudo-first-order reaction kinetics and low quantum yields ranging from 1.4x10(-3) to 1.8x10(-2)mol/E using both UV lamps. Toxicity testing using a bioluminesence inhibition bioassay was correlated to the decay in concentration of the PAHs as analyzed analytically using HPLC. Results demonstrated that treatment efficacy of oxidative PAH degradation measured by following the decay of the target compound is best complemented by also evaluating the toxicity of the treated water due to byproduct formation concerns.

Photocatalysis of fluorene adsorbed onto TiO2.

The adsorption of fluorene onto TiO2 has been investigated by conducting equilibrium and kinetic experiments. Adsorption isotherms have been evaluated at two different pHs in the range of temperatures 296-325 K. The type III isotherm shapes obtained were modelled by considering several expressions taken from the literature. Temperature exerted a positive influence in fluorene uptake. Addition of phosphates involved a negative effect when computing the final equilibrium fluorene removal. The kinetic experiments carried out at 296 K corroborated the competitiveness of phosphates to occupy the active sites on the titania surface. Nevertheless, equilibrium conditions are faster achieved at pH 2 than at pH 5. The photocatalysis of fluorene at different initial concentrations of the parent compound revealed a slight improvement of the process at pH 5 if compared to the results obtained at pH 2. A Langmuir-Hinselwood representation of the data confirms the previous statement. Catalyst load shows an optimum, concentration values of the photocatalyst above the optimum provoke a decrease in the fluorene abatement rate. Reutilisation of the catalyst indicates that fluorene is completely eliminated from the solid, i.e. it is suggested that fluorene and intermediates are surface oxidised.

Photoinduced intermolecular electron transfer process of fullerene (C60) and amine-substituted fluorenes studied by laser flash photolysis.

Photoinduced intermolecular electron transfer process of fullerene (C60) with 9,9-bis(4-triphenylamino)fluorene (BTAF) and 9,9-dimethoxyethyl-2-diphenylaminofluorene (DAF) in toluene and benzonitrile has been investigated by nanosecond laser photolysis technique in the visible/near-IR regions. By the selective excitation of C60 using 532 laser light, it has been proved that the electron transfer takes place from the ground states BTAF and DAF to the triplet excited state of C60 ((3)C60*) by observing the radical anion of C60 and radical cation of BTAF and DAF. It was observed that the electron transfer of BTAF/(3)C60* is more efficient than DAF/(3)C60* reflecting the effect of amine-substitutents of the fluorene moiety on the efficiency of the electron transfer process. On addition of a viologen dication (OV(2+)), the electron of the anion radical of C60 mediates to OV(2+) yielding the OV(+). These results proved that the photosensitized electron-transfer/electron-mediating processes have been confirmed by the transient absorption spectral method.

Photodegradation of polyfluorene and fluorene oligomers with alkyl and aromatic disubstitutions.

The stability of fluorene-based compounds and polymers, especially at the bridged C-9 position under photoirradiation and thermal treatment, has claimed wide attention. We report the electronic, vibrational, and MALDI-TOF mass spectral combined studies for the fluorene oligomers with alkyl and aromatic substitutions under UV-light irradiation. The low-energy emission and the formation of ketonic defects after degradation highly depend on the proportion of alkyl substitution. The oligomer with fully aromatic substitution shows good stability, but when the proportion of alkyl substitution increases, their photostability rapidly decreases. The mass spectra show not only the mass of the fluorenone-fluorene trimer but also another new degradation product with a large mass (pristine oligomer plus 14) from alkyl oxidation, which testify to the assistance of alkyl side chain during degradation. We propose that the degradation of fluorene is a radical chain process propagated by alkyl side chains, and then the different stability between alkyl and aromatic substitution can be well explained.

Relating daily solar ultraviolet radiation dose in salt marsh-associated estuarine systems to laboratory assessments of photoactivated polycyclic aromatic hydrocarbon toxicity.

Estuaries of the southeastern United States not only serve an important nursery function but also are common repositories of polycyclic aromatic hydrocarbons (PAHs) derived from upland activities. Thus, these habitats may be at risk for PAH phototoxicity. To better characterize this risk, a daily survey of ultraviolet-A (UV-A; 320-400 nm) irradiance was performed at Leadenwah Creek (Wadmalaw Island, SC, USA) on June 27 and August 1, 2003. In addition, laboratory assays were completed using two light exposure regimes: One that was typical of historical phototoxicity assessments (continuous light [C-UV]), and a more environmentally realistic regime (ER-UV). On both survey days, irradiance at a depth of 10 cm exhibited a pattern generally similar to that observed at the surface, whereas irradiance at the bottom of the creek was a function of both tidal height and time of day. Total UV-A dose at a 10-cm depth on June 27 and August 1, 2003 was 4.37 and 4.78 J/cm2, respectively. Attenuation coefficients on both days varied as a function of tidal height. In the laboratory, larval grass shrimp (Palaemonetes pugio) exposed to an ER-UV regime for these habitats (photoperiod, 12:12-h light:dark; total daily UV-A dose, 4.40 J/cm2) exhibited a 2.5-fold decrease in toxicity compared with those exposed to the C-UV regime (photoperiod, 24:0-h light:dark; total daily UV dose, 1.50 J/cm2), despite a threefold higher UV dose in the ER-UV regime. The lower potency under the ER-UV regime likely is attributable to the presence of a 12-h dark period allowing for recovery. The consequences of these results are discussed in the context of habitat-specific UV-A dose and its relevance to future laboratory assessments of PAH phototoxicity.

Photoactivated polycyclic aromatic hydrocarbon toxicity in medaka (Oryzias latipes) embryos: relevance to environmental risk in contaminated sites.

The hazard for photoactivated toxicity of polycyclic aromatic hydrocarbons (PAHs) has been clearly demonstrated; however, to our knowledge, the risk in contaminated systems has not been characterized. To address this question, a median lethal dose (LD50) for fluoranthene photoactivated toxicity in medaka (Orvzias latipes) embryos was determined experimentally and then compared with ultraviolet-A (UV-A; 320-400 nm) radiation exposures in a PAH-contaminated field site. The dose metric, J/cm2/ microg fluoranthene/g egg wet weight, provided the means to estimate risk as the depth where the LD50 level would be exceeded at realistic field PAH concentrations, based on estimates of UV-A exposure. The estimates were made using 30 years of solar radiation data for Duluth (MN, USA) and measurements of water-column UV-A transmittance in a PAH-contaminated field site. Medaka embryo failure was strongly related to tissue PAH concentration and UV-A exposure. The LD50 was estimated to be 12.64 J/cm2/ microg fluoranthene/g egg wet weight; the 95% confidence interval was 8.46 to 19.7 J/cm2/microg fluoranthene/g egg wet weight. Embryo failures were characterized by undifferentiated cell proliferation that occurred very early in development. No partial effects or embryo/larval malformations were observed. Estimates of the depth at which the LD50 would be exceeded in the contaminated field site ranged from 10.7 cm (clear-sky conditions and lowest attenuation) to 0.0 cm (cloudy conditions and highest attenuation). Similar calculations were done using water-column attenuation estimates from 12 sites across the Great Lakes (USA). For these, the depths at which the LD50 would be exceeded ranged from 0.00 to 271.6 cm under the conditions described above. These results suggest that PAH phototoxicity may be a risk factor in specific contaminated sites, and they provide a framework for assessing that risk.

Irradiation-induced adduct formation of RNA with carcinogenic arylamine derivatives.

Radiolysis of N2O-saturated solutions of transfer RNA (tRNA) and the arylacethydroxamic acids, N-hydroxy-N-2-acetylaminofluorene and N-hydroxy-N-4-acetylaminobiphenyl; their corresponding acetamides, 2-acetylaminofluorene and 4-acetylaminofluorene; or the O-glucuronide of N-hydroxy-N-2-acetylaminofluorene resulted in adduct formation of the nucleic acid with these carcinogenic arylamine derivatives. The yield of adducts on irradiation of the arylacethdroxamic acids with tRNA was greater than that for their corresponding acetamides or the O-glucuronide. The fluorenylacethydroxamic acid and acetamide were also more reactive than the biphenyl analogs. Adduct formation resulting from radiolysis of tRNA and the arylacethydroxamic acids or the O-glucuronide proceeded with retention of both the aromatic nucleus and the N-acetyl group. The yields of adducts were much greater for irradiated mixtures than for irradiation of either component alone followed by mixing. Evaluation of the data shows that initial modification of the tRNA or the carcinogen can lead to adduct formation. In the case of primary radical attack of the nucleic acid, it has been shown that short-lived reactive RNA intermediates are responsible for a major fraction of the observed yield of adducts in the irradiated mixtures. Comparative studies showed that irradiation under conditions that favor reaction of oxidizing radicals enhanced formation of the adducts. Oxygen was shown to protect RNA from irradiation-induced binding of the arylacethydroxamine acids due to competition of O2 with the carcinogen for the reactive RNA intermediates.

The effect of camphorquinone (CQ) and CQ-related photosensitizers on the generation of reactive oxygen species and the production of oxidative DNA damage.

Recent evidence suggests that following visible-light (VL) irradiation, CQ and the CQ-related photosensitizers benzil (BZ), benzophenone (BP), and 9-fluorenone (9-F) generate initiating radicals that may indiscriminately react with molecular oxygen forming reactive oxygen species (ROS). The purpose of this investigation was to determine whether VL-irradiated CQ, BZ, BP, and 9-F cause DNA damage due to the generation of ROS in vitro. ROS formation by CQ and CQ-related photosensitizers+/-dimethyl-p-toluidine (DMT) was investigated in a cell-free system with VL irradiation. DNA damage was determined using PhiX-174 RF I supercoiled double-stranded plasmid DNA and ROS quantified with 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO-9-AC), a fluorogenic ROS-sensitive probe. VL-irradiated CQ, BZ, BP, and 9-F (+/-DMT) produced significant DNA damage at 0.1, 0.5, and 1.0 mM and in a concentration-dependent manner (p<0.05). TEMPO-9-AC revealed that all investigated VL-irradiated photosensitizers produced significant amounts of ROS with BZ in the presence of DMT generating the most ROS after 30, 60, and 90 min. VL-irradiated CQ, BZ, BP, and 9-F +/-DMT continued to generate significant amounts of ROS 90 min after VL irradiation. As a result, future investigations should evaluate the effect of VL-irradiated photosensitizers in cells and possible protective effects provided by antioxidants.

Photochemical instability of 1-nitropyrene, 3-nitrofluoranthene, 1,8-dinitropyrene and their parent polycyclic aromatic hydrocarbons.

The environmental contaminants pyrene, 1-nitropyrene, 1,8-dinitropyrene, fluoranthene, and 3-nitrofluoranthene were exposed to light (greater than or equal to 310 nm) either in DMSO, or following coating onto silica. Under all conditions tested the pyrenyl were less stable than the fluoranthenyl compounds. During irradiation in DMSO or on silica, 1-nitropyrene had half-lives of 1.2 and 6 days, while those of 3-nitrofluoranthene were 12.5 and greater than 20 days, respectively. The photodecomposition of 1,8-dinitropyrene resembled that of 1-nitropyrene with half-lives of 0.7 and 5.7 days. A principle photodecomposition product of 1,8-dinitropyrene was identified as 1-nitropyren-8-ol. It was also found that when the nitroarenes were exposed to light, the loss of compound was associated with a concomitant loss of mutagenicity in Salmonella typhimurium strain TA98. The mechanism of nitrated polycyclic aromatic hydrocarbon decomposition and 1-nitropyren-8-ol formation, and the relevance to the atmospheric disposition of these compounds are discussed.