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Luminescence. 2014 Aug;29(S1):6-55

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Abstract
O0001 On the efficiency of the peroxyoxalate system using a simple experimental and theoretical approach Felipe A. Augusto, Carolina P. Frias, Wilhelm J. Baader Instituto de Química, São Paulo, SP, Brazil The peroxyoxalate system is the most efficient intermolecular chemiluminescent reaction with chemiluminescence quantum yields reaching up to three orders of magnitude higher than other similar systems.(1) It consists in the reaction of an oxalic ester with hydrogen peroxide, typically catalyzed by a base, forming a high-energy intermediate which interacts with a compound called activator, ACT.(2,3) This interaction leads to the ACT in its singlet excited state in a mechanism that involves a charge/electron transfer initially from the ACT to the peroxide and then back to the ACT.(2,3) Several important details have been experimentally determined about the mechanism, like the cyclization rate constant and the direct interaction between the high-energy intermediate and the ACT.(4,5) However the identity of the high-energy intermediate is still a matter of discussion, with only a few possible intermediate structures being properly studied or definitely discarded.(6) The study and characterization of the peroxyoxalate reaction with a relatively small ACT, like naphthalene, could allow a detailed theoretical study of the chemiexcitation step, contrarily to the case of the compounds normally used as ACTs which would involve prohibitive computational costs. Therefore, the reaction of bis(2,4-dinitrophenyl) oxalate (DNPO) with hydrogen peroxide catalyzed by imidazole (IMI-H) was studied using naphthalene as ACT. The observed rate constant (kobs ) showed linear dependence with the [H2 O2 ] (Fig. , kH2O2 = 23 ± 1 L mol(-1) s(-1) ) and [IMI-H] (Fig. , kIMI-H = 202 ± 7 L mol(-1) s(-1) ) and no dependence with [DNPO] or [ACT] (data not shown). [Figure: see text] The study of the peroxyoxalate system, employing naphthalene as electronically simple ACT, indicates the existence of a linear relationship between the kobs and the H2 O2 as well as IMI-H concentration, in general agreement with the mechanism proposed for the initial steps of the transformation. This fact indicates a normal behavior of the system in the conditions utilized. Therefore, the peroxyoxalate reaction with naphthalene as ACT will now be subject to theoretical studies in order to elucidate the exact mechanism of the chemiexcitation step, with the intention to understand the reason for the extremely high efficiency of the system even so it involves, apparently, intermolecular electron transfer steps. References 1. Augusto FA, Souza GA, Souza Junior SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. 2. Ciscato LFML, Augusto FA, Weiss D, Bartoloni FH, Albrecht S, Brandl H, Zimmermann T, Baader WJ. ARKIVOC 2012;2012:391. 3. Bartoloni FH, Bastos EL, Ciscato LFML, Peixoto MMdeM, Santos APF, Santos CS, Oliveira S, Augusto FA, Pagano APE, Baader W. J. Quim. Nova 2011;34:544. 4. Da Silva SM, Casallanovo F, Oyamaguchi KH, Ciscato LFML, Stevani CV, Baader WJ. Luminescence 2002;17:313. 5. Ciscato LFML, Bartoloni FH, Bastos EL, Baader WJ. J. Org. Chem. 2009;74:8974. 6. Stevani CV, Campos IPA, Baader WJ. J. Chem. Soc., Perkin Trans. 2 1996;1645. O0002 Kinetic studies on the sodium salicylate catalyzed peroxyoxalate reaction Glalci A. Souza, Wilhelm J. Baader Instituto de Química da Universidade de São Paulo, São Paulo-SP, Brazil The peroxyoxalate reaction is the only chemiluminescence reaction which appears to involve the intermolecular Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism in its chemiexcitation step that possess highest emission quantum yields of up to 60%.(1-3) Detailed kinetic studies on this highly efficient CL system has been performed mainly using imidazole as base catalyst and the mechanistic elucidation of the complete reaction has shown that this compound is acting also as nucleophilic catalyst.(4-5) However, it has been shown also that the base and nucleophilic catalyst imidazole leads to a decrease in the CL emission quantum yield, apparently due to its interaction with the high-energy intermediate.(4-6) In order to further elucidate the mechanism of the peroxyoxalate system, the kinetics of the reaction were studied with sodium salicylate as base catalyst. The CL emission obtained in the reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) (0.1 mM) with hydrogen peroxide, catalyzed by sodium salicylate, in the presence of 9,10-diphenylanthracene (DPA) (0.2 mM) as activator, in ethyl acetate at 25 °C, was measured in different conditions. When the reaction was performed varying the sodium salicylate concentration, the rate constants corresponding to the emission decay increased with the base concentration, showing a saturation curve like behavior. The decay rate constant also increased with increasing hydrogen peroxide concentrations and at low H2 O2 concentrations the rate constants show a linear dependence on the hydrogen peroxide concentration allowing the determination of a bimolecular rate constant (kbim ). These rate constants showed to depend also on the sodium salicylate concentration; kbim  = (0.56 ± 0.06) 10(-3) ; (1.6 ± 0.2) 10(-3) and (2.04 ± 0.06) 10(-3)  L mol(-1)  s(-1) , for sodium salicylate concentrations of 0.3, 1.0 and 5.0 mmol L(-1) , respectively. Upon variation at higher hydrogen peroxide concentrations the rate constants showed saturation curves, indicating a change in the rate-limiting step. The rate constants corresponding to the initial rise in the emission intensity have been shown independent of the hydrogen peroxide concentration showing that this reagent does not participate in the reaction step measured in this part of the kinetic curve. Similarly to the behavior observed with imidazole, the CL emission quantum yield showed to decrease with an increase in the sodium salicylate concentration. The maximum quantum yields obtained with sodium salicylate were s (max)  = (1.24 ± 0.06) 10(-3) E mol(-1) when the sodium salicylate concentration was 0.5 mmol L(-1) . These data indicate that, like imidazole, also sodium salicylate appears to interaction with the high-energy intermediate in the peroxyoxalate reaction, which diminishes the CL emission quantum yields. Financial support; FAPESP, Capes, CNPq. 1. Stevani CV, Silva SM, Baader WJ. Eur. J. Org. Chem. 2000;4037. 2. Augusto FA, Souza GA, Souza Jr., SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013, 89, 1299. 3. Ciscato LFML, Augusto FA, Weiss D, Bartoloni FH, Bastos EL, Albrecht S, Brandl H, Zimmermann T, Baader, WJ. Arkivoc;2012 (iii), 391. 4. Silva SM, Casallanovo F, Oyamaguchi KH, Ciscato LFLM, Stevani CV, Baader WJ, Luminescence 2002;17:313. 5. Stevani CV, Lima DF, Toscano VG, Baader WJ, J. Chem. Soc. Perk. Trans. 2 1996;989. 6. Ciscato LFML., Bartoloni FH, Bastos EL., Baader WJ, J. Org. Chem. 2009;74:8974. O0003 How can quantitative bioluminescence and in-situ fluorescence of firefly oxyluciferin in luciferase be compared with theoretical calculations? Hidefumi Akiyama(a) , Yu Wang(a,b) , Miyabi Hiyama(a) , Toshimitsu Mochizuki(a) , Kanako Terakado(c) , Toru Nakatsu(c) (a) University of Tokyo, Kashiwa, Chiba 2778581, Japan (b) Institute of Genetics and Developmental Biology, Beijing 100101, Japan (c) Kyoto University, Sakyo-ku, Kyoto 6068501, China To investigate color-determination mechanisms from physicist points of view, we study quantitative spectra of firefly bioluminescence, and the in-situ absorption and fluorescence spectra of oxyluciferin contained in luciferase in a consumed reaction mixture, and intend to compare them with quantum-chemistry theoretical calculations. We have so far measured quantitative in-vitro firefly bioluminescence spectra influenced by pH, kinds of bivalent metal ions, temperatures, and mutant luciferase using our total-photon-flux spectrometer with our new light sta
ndards. We found that all the spectra were systematically and quantitatively decomposed into one environment-sensitive and two environment -insensitive Gaussian peaks, and that no intensity conversion between yellow-green and red emissions but mere intensity variation of the pH-sensitive green peak at 2.2 eV causes the changes in apparent emission colors [1,2]. Therefore, answers for the color-determination problem need not only the assignment of the peaks, but also the explanation of the intensity change of the green peak. [Figure: see text] We next measured in-situ absorption and fluorescence characteristics of oxyluciferin, still combined with luciferase in a consumed reaction mixture [3]. The in-situ absorption spectra indicated that neutral oxyluciferin was dominant, which was in weak pH-dependent equilibrium with oxyluciferin mono-anions. The neutral oxyluciferin was more dominant in luciferase environments than in bare water environments. The in-situ fluorescence spectra shown in Fig.  clarified that the neutral oxyluciferin is a blue emitter and the oxyluciferin mono-anion is a green emitter (Fig. ). Even in red-mutant luciferase environment, the in-situ fluorescence has shown strong blue and green fluorescent emissions (Fig. ). In short, the spectra of in-situ fluorescence of oxyluciferin in luciferase and those of bioluminescence have shown significant discrepancy. [Figure: see text] Therefore, the above-mentioned discrepancy between bioluminescence and in-situ fluorescence cast an important question, how they can be consistently compared with quantum-chemistry theoretical calculations, which are recently published in a large numbers. The above results may suggest that enzyme microenvironment affects the transition state in bioluminescence chemical reaction, but not the oxyluciferin states after the reaction. Physicists need to discuss this issue with biologists and chemists in the bioluminescence research community. 1. Ando Y, Niwa K, Yamada N, Irie T, Enomoto T, Kubota H, Ohmiya Y, Akiyama H. Nature Photonics 2008;2:44-47. 2. Wang Y, Akiyama H, Terakado K, Nakatsu T. Scientific Reports 2013;3:2490. 3. Wang Y, Hayamizu Y, Akiyama H. J. Phys. Chem. B 2014;118:2070-2076. O0004 Determination of the total phenolic / antioxidant content in honey samples using formaldehyde / potassium permanganate chemiluminiscence system in a novel microfluidics device Butheina A. M. Al Haddabi, Haider A. J. Al Lawati, FakhrEldin O. Suliman, Gouri B. Varma Sultan Qaboos University, Al-Khod, Oman Microfluidc device has been explored as a tool for the estimation of the total phenolic content/ antioxidant content in honey using acidic potassium permanganate chemiluminescence (KMnO4 -CL) detection system. Selected phenolic antioxidants including quercetin, catechin, gallic acid, caffeic acid and ferulic acid elicited analytically useful CL with detection limits ranging between 2.38 nmol L(-1) for galic acid and 33.9 nmol L(-1) O-coumaric acid for only 2 μL injection volume. The parameters that affect the CL signal intensity of each antioxidant were carefully optimized. . It was observed that formaldehyde can enhance the CL signal intensity of phenolic compounds up to 27 times (Fig. ). Additionally, it was observed that the chip volume and geometry both can play an important role in enhancing the CL signal intensity in this system. The CL signal intensity was enhanced five times when a spiral – flow split chip (SF) geometry was used, compared to the simple spiral chip (S) geometry commonly used (Fig. ). Other parameters were also optimized, including pH and concentration of reagents used and the flow rates. The effect of solvents and surfactants on CL signal intensities was also studied. The method was applied on Omani honey samples. Nine different honey samples resulted in total phenolic / antioxidant level range between 40 and 772 mg Kg(-1) with respect to gallic acid. Folin Coicalteu reagent (FCR) resulted in a good correlation with the developed method which was found to be a selective, rapid and sensitive method to estimate total phenolic / antioxidant level in a good agreement with reported results for honey samples. [Figure: see text] [Figure: see text] References 1. Costin JW, Barnett NW, Lewis SW, McGillivery DJ. Analytica Chimica Acta 2003;499:47-56. 2. Alvarez-Suarez JM, Gonzalez-Paramas AM, Santos-Buelga C, Battino M. J. Agric. Food Chem. 2010;58:9817-9824. O0005 Electrochemiluminescence sensor based on tris(2,2’bipyridyl)ruthenium(II)/poly(AHNSA) for chlorpheniramine maleate analysis Mohammed M. Alhinaai, Emad A. Khudaish Sultan Qaboos University, AlKhude, Muscat, Oman Since the discovery of impressive luminance property of tris(2,2’bipyridyl)ruthenium(II), [Ru(bpy)3 ](2+) , it becomes one of intensive used reagent for chemiluminescence and electrochemiluminescence (ECL) analysis with a wide range of coreactants. Immobilizing of the expensive Ru(II)-complex on the electrode surface began early 1980s using Nafion as an exchanger polymer [1] is still a hot and continuous topic by many research groups [2]. The main objective of the present work was to develop a solid-state ECL sensor based on immobilizing [Ru(bpy)3 ](2+) on a conducting polymer for chlorpheniramine maleate (CPM) determination. The sensor was fabricated by composite electropolymerization of 2.0 mM [Ru(bpy)3 ](2+) and 1.5 mM of 4-amino-3-hydroxy-naphthalene sulfonic acid (AHNSA) in acidic medium via potentiodynamic repetitive cycles between -0.8 and +2.0 V at 0.1 V s(-1) . The fabrication parameters were optimized carefully in order to produce a stable film and obtain an intense ECL signal. Figure  shows the electrochemical characterization of the composite surface film where a redox peak of [Ru(bpy)3 ](2/3+) are well defined at 1180 mV (anodic) and 980 mV (cathodic), respectively. Impedance spectroscopy analysis showed that the charge transfer resistance of the composite film is greatly lowered by doping Ru(II)-complex on the moiety of the PAHNSA which suggests that Ru(II)-complex is acting as a charge transfer center [3]. [Figure: see text] This sensor exhibited excellent ECL behaviour toward CPM analysis as shown in Fig.  with a good stability and reproducibility. The standard deviation of 16 measurements in flow stream was 2.35%. It also has a very good lifetime when stored in 5 °C for two weeks where the recovery measurement approached 94%. The sensor was also applied to estimate CPM in pharmaceutical preparations. The linear dynamic range was from 0.1 to 32 µg/mL (R(2)  = 0.9956). The detection limit was 23 µg/L and the recovery of real sample analysis was from 102.0% to 98.50% for syrup and tablets respectively. [Figure: see text] Interference studies showed no effect of common compounds and the acceptable molar concentration ratios of foreign species to CPM were higher than1000-fold for Na(+) , K(+) , NO3 (-) , SO3 (2-) , 100-fold for Mg(2+) , Al(3+) , NH4 (+) , Cl(-) , lactose, sucrose, and glucose, and 10-fold for Fe(3+) and Co(2+) . The analytical parameters such as pH, flow rate, buffer concentration were systematically tested. In conclusion, a novel composite polymeric film was fabricated using simple electrochemical method and applied for determination of CPM in real samples. The sensor showed a good stability and sensitivity regardless the matrix of the pharmaceutical preparation. References 1. Rubinstein I, Bard A. J. J. Am. Chem. SOC. 1980;102;6641-6842. 2. Su M, Wei W, Liu S. Analytica Chimica Acta 2011;704:16-32. 3. Zhang B, Shi S, Shi W, Sun Z, Kong X, Wei M, Duan X. Electrochimica Acta 2012;67:133-139. O0006 Experimental Evidence of the Occurrence of an Intermolecular Electron Transfer in the Catalyzed Decomposition of spiro-Alkyl-1,2-Dioxetanones Fernando Heering Bartoloni(2,1) , Marcelo Almeida de Oliveira(1) , Luiz Francisco Monteiro Leite Ciscato(2,1) , Felipe Alberto Augusto(1) , Erick Leite Bastos(1) , Wilhelm Josef Baader(1) (1) Departamento de Qu&iacu
te;mica Fundamental do Instituto de Química da Universidade de São Paulo, Sao Paulo. SP, Brazil, (2) Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo Andre, SP, Brazil Several chemical and biochemical reactions have light as a co-product and some of them can show high quantum efficiencies, include firefly bioluminescence, the peroxyoxalate system, and the induced decomposition of 1,2-dioxetanes.(1) Cyclic peroxides have been frequently described as high-energy intermediates in the chemical formation of products in the electronic excited state because their decomposition fulfill both energetic and geometric criteria required for chemiexcitation. Nevertheless, the thermal decomposition of 1,2-dioxetanes and 1,2-dioxetanones results in inefficient chemiluminescence emission due to the preferential formation of products in the non-emissive triplet-excited state (ΦS  < 10(-4) E mol(-1) vs. ΦT  > 0.1 E mol(-1) ). ).(2,3) However, it has been reported that fluorescent polycyclic aromatic hydrocarbons with low oxidation potentials (referred to as activators, ACT) catalyze the decomposition of 3,3-dimethyl-1,2-dioxetanone (1), resulting in noticeable increase in light emission intensity, and high singlet chemiexcitation quantum yields (ΦS  = 0.1 E mol(-1) ).(4) Contrarily, our group found recently that the ΦS for the catalyzed decomposition of 1,2-dioxetanones, the model intermediate in firefly bioluminescence, were overestimated in several orders of magnitude.(5) Consequently, the validity of this system as a model for excited states formation might be questioned. Therefore, we report here our results of a kinetic study on the catalyzed decomposition of the spiro-substituted 1,2-dioxetanone derivatives, spiro-adamantyl-1,2-dioxetanone (2) and spiro-cyclopentyl-1,2-dioxetanone (3) by several activators and confirmed the occurrence of an intermolecular electron or charge transfer in this transformation. The 1,2-dioxetanone derivatives 2 and 3 were prepared, purified and handled as described elsewhere;(6) kinetic assays and data treatment were performed as detailed before.(5) The kobs values for the decomposition of 2 and 3, determined in toluene in the absence and in the presence of different ACTs, do not depend on the nature and concentration of the ACT (kobs (2, 50 °C) = (6 ± 1) 10(-3)  s(-1) and kobs (3, 25 °C) = (9 ± 3) × 10(-4)  s(-1) ). Therefore, the bimolecular rate constant (kCAT ) cannot be determined directly; however, the kCAT /kD ratios and the chemiexcitation quantum yields at the infinite ACT concentration (ΦS (∞) ) can be calculated for each ACT from the double reciprocal plots of the singlet quantum yields (ΦS ) versus the ACT concentrations.(5) The kCAT /kD values show linear free-energy relation with the ACT’s oxidation potential, indicating the importance of an intermolecular electron transfer from the ACT to the peroxide in the chemiexcitation step. The low efficiency in excited states formation in the catalyzed decomposition of these cyclic peroxides are rationalized by steric interactions between the activator and the bulky alkyl substituents on the peroxidic ring, thereby lowering the charge-transfer complex formation constant between the peroxide and the activator. Financial support; FAPESP, Capes, CNPq. 1. Augusto FA, Souza GA, Souza Junior SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. 2. Adam W, Baader WJ. J. Am. Chem. Soc. 1985;107:416. 3. Adam W, Baader WJ. Angew. Chem. Int. Ed. Engl. 1984;23:166. 4. Schuster GB, Schmidt SP. Adv. Phys. Org. Chem. 1982;18:187. 5. de Oliveira MA, Bartoloni FH., Augusto FA, Ciscato LFML, Bastos EL, Baader WJ. J. Org. Chem. 2012;77;10537. 6. Bartoloni FH, de Oliveira MA, Augusto FA, Ciscato LFML, Bastos EL, Baader JW. J. Braz. Chem. Soc. 2012;23:2093. O0007 Chemiluminescent detection of Nitric Oxide Martina Bancirova Palacký University, Olomouc, Czech Republic The chemistry of nitric oxide inside humans and other mammals is perhaps the most interesting aspect of this simple molecule’s behaviour. NO is involved in controlling blood pressure; transmitting nerve signals and a variety of other signalling processes. When tissues in the body become inflamed for long periods of time, the concentration of nitric oxide within them increases and this can be used to diagnose disease. But also NO secreted by activated cells appears to be a complex “cocktail” of substances (see Fig. .)(1) [Figure: see text] So is necessary to take in account the presence of ROS during the determination of nitric oxide. One of the chemiluminescent detections is based upon the chemiluminescence reaction between NO and the luminol (5-amino-2,3-dihydro-1,4-phthalazinedione)-H2 O2 system. The luminol-H2 O2 system is specifically reactive to NO, so that other nitrogen-containing compounds (organic nitrite, organic nitrate, and thio-nitroso compounds do not interfere. (2) The light emission of luminol as detection of bloodstains is a complex process based on hydrogen peroxide decomposition catalyzed by haemoglobin. Three common known methods of bloodstains; detection according to Grodsky, Weber and by Bluestar® Forensic reagent(3), are based on the luminol chemiluminescence (complex process based on hydrogen peroxide decomposition catalyzed by haemoglobin). The Bluestar® Forensic Magnum was chosen because of its declared stability as a “new” detection system for nitric oxide. The different dilutions (up to three orders) of the Bluestar® Forensic Magnum were used. The experiments were done by using luminometer (type FB 12, Berthold Detection Systems, Germany) in the total volume of 1 mL. Sodium azide was used as a specific quencher of singlet oxygen to prove its presence (see Fig. .) [Figure: see text] 1. Kroncke KD, Fehsel K, Kolb-Bachofen V. Nitric Oxide; Cytotoxicity versus Cytoprotection-How, Why, When, and Where?, NITRIC OXIDE; Biology and Chemistry 1997;1:107-120. 2. Kikuchi K, Nagano T, Hayakawa H, Hirata Y, Hirobe M. Detection of Nitric Oxide Production from a Perfused Organ by a Luminol-H202 System. Analytical Chemistry 1993;65:1794-1799. 3. Blum LJ, Esperança P, Rocquefelte S. A new high-performance reagent and procedure for latent bloodstain detection based on luminol chemiluminescence. Canadian Society of Forensic Science Journal. 2006;39:81-100. Financial support from the Czech Science Foundation, project 301/11/0767. O0008 An In-depth study on Blue electrochemiluminescent Iridium(III) complexes Gregory Barbante(a) , Egan Doeven(a) , Paul Francis(a) , Timothy Connell(c) , Paul Donnelly(c) , Conor Hogan(b) , David Wilson(b) (a) Deakin University, Geelong, Victoria, Australia (b) La Trobe University, Melbourne, Victoria, Australia (c) Melbourne University, Melbourne, Victoria, Australia Electrogenerated chemiluminescence (ECL) is a form of luminescence produced by high-energy reactions between electrogenerated precursors,([1,2]) in which the electronically excited states responsible for the emission of light can be generated through the annihilation between oxidised and reduced forms of the same species, or by using a sacrificial co-reactant. The application of a co-reactant ECL as a highly sensitive mode of detection has been predominantly based on the use of tris(2,2′-bipyridine)ruthenium(II) ([Ru(bpy)3 ](2+) ), and related polyimine-ruthenium(II) complexes, with characteristic orange/red emissions (max ca. 590-700 nm).([1,2]) Over the last decade, however, numerous researchers have begun to explore chemiluminescence and ECL reactions with cyclometalated iridium(III) complexes exhibiting a wide range of electrochemical properties and emission maxima that can be tuned through subtle changes in the structure of one or more ligands.([3]) These complexes have created new possibilities for multiplexed ECL detection systems.([4]) However, in contrast to the vast range of orange/red-emitting metal c
omplex electrochemiluminophores,([5,6]) relatively few blue emitters are available, and the most effective design of blue-emitting complexes for ECL detection is yet to be fully elucidated. We have therefore used electrochemical, spectroscopic and computational techniques to explore a series of blue-emitting iridium(III) complexes (see Fig. ) that exhibit various potentially attractive structural attributes for conventional and multiplexed ECL detection. Theoretical and experimental studies reveal the most effective strategies for the design of blue-shifted iridium(III) complexes for efficient electrogenerated chemiluminescence. Stabilisation of the HOMO while only moderately stabilising the LUMO increases the energy gap, thus ensuring favourable thermodynamics and kinetics for the reaction leading to the excited state. Of the iridium(III) complexes examined, [Ir(df-ppy)2 (ptb)](+) was most attractive as a blue-emitter for ECL detection, featuring a large hypsochromic shift (max = 454 and 484 nm), superior co-reactant ECL intensity than the archetypal homoleptic green and blue emitters; [Ir(ppy)3 ] and [Ir(df-ppy)3 ] (by over 16-fold and threefold, respectively), and greater solubility in polar solvents. We have therefore used electrochemical, spectroscopic and computational techniques to explore a series of blue-emitting iridium(III) complexes (see Fig. ) that exhibit various potentially attractive structural attributes for conventional and multiplexed ECL detection. Theoretical and experimental studies reveal the most effective strategies for the design of blue-shifted iridium(III) complexes for efficient electrogenerated chemiluminescence. Stabilisation of the HOMO while only moderately stabilising the LUMO increases the energy gap, thus ensuring favourable thermodynamics and kinetics for the reaction leading to the excited state. Of the iridium(III) complexes examined, [Ir(df-ppy)2 (ptb)](+) was most attractive as a blue-emitter for ECL detection, featuring a large hypsochromic shift (max = 454 and 484 nm), superior co-reactant ECL intensity than the archetypal homoleptic green and blue emitters; [Ir(ppy)3 ] and [Ir(df-ppy)3 ] (by over 16-fold and threefold, respectively), and greater solubility in polar solvents. [Figure: see text] 1. Bard AJ. Electrogenerated Chemiluminescence. Marcel Dekker; New York, 2004 2. Forster RJ, Keyes TE. Neuromethods 2013;80:347-367 3. Zanarini S, Felici M, Valenti G, Marcaccio M, Prodi L, Bonacchi S, Contreras-Carballada P, Williams RM, Feiters MC, Nolte RJM, De Cola L, Paolucci F. Chem. Eur. J. 2011;17:4640-4647 4. Doeven EH, Zammit EM, Barbante GJ, Hogan CF, Barnett NW, Francis PS. Angew. Chem. 2012;124:4430-4433 5. Barbante GJ, Hogan CF, Wilson DJD, Lewcenko NA, Pfeffer FM, Barnett NW, Francis PS. Analyst 2011;136:1329-1338 6. Gorman BA, Francis PS, Barnett NW. Analyst 2006;131:616-639 O0009 Chaetopterus variopedatus tissue autofluorescence spectral characteristics Anna Belousova(a) , Fyodor Kondrashov(b) , Maria Plyuscheva(b) (a) Moscow State University, Biological Faculty, Invertebrate Zoology Department, Moscow, Russia (b) Centre de Regulació Genòmica (CRG), Barcelona, Spain Chaetopterus variopedatus is a sedentary marine polychaete that lives in a parchment U-shaped tube. It has a body with three distinct regions, each region contains different morphologically developed segments. [3] The polychaete is famous for being capable of emitting light with its epithelium and producing blue luminous mucous. Studies on anatomy and morphology of phenomenon of its epithelial bioluminescence have pointed out some special areas of intensity, such as notopodial structures of middle and posterior regions. [2] Referring to the chemical approach, previous research on the chemical compounds of Chaetopterus bioluminescent system have proved that the photoprotein takes part in the reaction. [1] As for the whole biochemical pathway of the Chaetopterus bioluminescence – it is still a question to solve. Photoproteins that are involved in the luminescence reaction are known for becoming fluorescent after the reaction of luminescence takes place. [1] Therefore, the distribution of the products of the bioluminescence reaction can be studied using the confocal light microscopy methods. The autofluorescence itself is usually relatively stable either continious, or intensive. The distribution of fluorescence of different wavelengths was observed with a confocal microscope on several cross-sections of the worm’s body and on epithelium of various parts of the body. The specimens of the worms tissue exhibit fluorescence of some ranges of wavelengths – excited by lasers from 405 to 633 nm. Confocal observations have shown that the UV excitation of a tissue results most efficiently in the strong fast-bleaching fluorescence in far-red (630 nm) spectrum area. Purified far-red autofluorescent component which is supposed to be a part of bioluminescence reaction [4] is stored in the vesicles which can be visualised with the confocal microscopy. Though having a strong intensity, the far-red fluorescence is bleaching very fast which makes it more challenging to observe. References 1. Shimomura O. Bioluminescence; chemical principles and methods. – World Scientific, 2012. 2. Anctil M. The epithelial luminescent system of Chaetopterus variopedatus //Canadian Journal of Zoology. – Т. 57. – №. 6. – С, 1979;1290-1310. 3. Harvey EN. Bioluminescence. – Academic Press, 1952. 4. Branchini BR, et al. Chemical analysis of the luminous slime secreted by the marine worm Chaetopterus (Annelida, Polychaeta) //Photochemistry and photobiology. – Т. 90. – №.1. – С, 2014;247-251. [Figure: see text] [Figure: see text] O0010 Construction of lux operon of the ancestor of bioluminescent bacteria and proposal of evolutionary hypothetical theories on the origin and propagation of luminescent bacterial species Ramesh CH, Mohanraju R Pondicherry University, Pondicherry, India Using the processes, “Horizontal gene transfer (HGT) or Chromosomal exchange mechanism (CEM) or Acquisition, Plasmid DNA exchange, genetical processes like Interchromosomal rearrangements (ICR), and geological processes” we assembled lux genes together and constructed the luminescent bacterial ancestor that might have not yet been isolated or extincted in the biological evolution during the geological processes. The construction of lux operon of this luminescent ancestor was carried out with different lux genes such as the regulatory and structural genes, and with other genes which are flanking towards upstream and downstream of the lux operon of different luminescent bacterial species. The lux operon of this ancestor was constructed based on the approximate base pairs of different lux genes. The approximate order of lux operon is as follows luxZYLOPUMNQRSTICDABFEGH-ribEBHA. Where rib genes of rib operon are linked to downstream of the lux operon. Hypothetically it is possible to construct this luminescent bacteria ancestor, while we expect possibilities of finding of this luminescent ancestor. The processes “Insertion, Deletion, Horizontal gene transfer (HGT) or Chromosomal exchange mechanism (CEM) and Interchromosomal rearrangements (ICR), geological processes and Plasmid DNA exchange (PDE) might have given origin for modern luminescent bacteria. These processes provides base for this ancestor construction and supports for the presence or possibility in constructing the ancestor of luminescent bacteria. We also propose new strong hypothetical theories which speak about the existence of luminescent ancestor and origin of modern luminescent bacterial species. O0011 Ecological functions of shark luminescence Julien Claes, Jérôme Mallefet Laboratoire de Biologie Marine, Earth and Life Institute, Université catholique de Louvain, Louvain-la-Neuve, Belgium Introduction; Sharks from the Etmopteridae and Dalatiidae families are among the most enigmatic bioluminescent organisms. Although they encompass about 12% of current shark diversity, with
over 50 described species, their luminescence is rarely observed [1]. Moreover, contrary to the situation encountered in other animals, their intrinsic light organs (photophores) are primarily controlled by hormones rather than by nerves [2,3] and form a diversity of patterns whose adaptive advantage has long remained obscure. This work aims to synthetize recent advances made in the field of shark luminescence ecology as well as to present novel experimental data in order to inspire future research. It involves various techniques such as in vivo luminescence measurements [via an optic fibre coupled to a luminometer (Berthold FB12)], spectrophotometry [with a mini-spectrometer (Hamamatsu Photonics C10083CA)], stereology and visual modelling. Results and discussion; In the last five years, we observed and characterized the spontaneous luminescence of one dalatiid and three etmopterid shark species. At 0.5 prepelvic length, their luminescence [downward emission; λmax at 457-488 nm; ventral intensity = 0.34-130.78 Mq s(-1)  mm(-2) (n = 31)] appears physically similar to the residual downwelling light present in their environment, supporting a function of camouflage by counterillumination [1,4]. However, digital photography revealed that etmopterid luminescence (i) was not homogeneous on the ventral side (Fig. A), a result confirmed by stereological analysis of photophore distribution; and (ii) was also present dorsally, as dim glows underlying several structures including fin spines, eyes and nostrils (Fig. B). This leads to a variable angular distribution pattern along the etmopterid body, as it can be seen in E. spinax, whose luminescence adopts caudally (at the level of clade-specific lateral photophore markings) a more lateral distribution well suited for intraspecific communication (Fig. ). Visual modelling demonstrated that spine-associated glow signal the presence of the defensive fin spines to predators at several meters and hence might be used for aposematism [5]. Finally, the association of photophores with photoreceptive tissues likely provides a reference for counterillumination while nostril luminescence might be used as a torch to improve prey detection. We suggest the luminescence versatility of etmopterid sharks to have powered their rapid radiation in the deep-sea. Acknowledgments Julien M. Claes and Jérôme Mallefet are respectively postdoctoral researcher and research fellow of the Fonds National de la Recherche Scientifique (FNRS, Belgium). This is a contribution to the Biodiversity Research Center (BDIV) and to the Centre Interuniversitaire de Biologie Marine (CIBIM). References 1. Claes JM, Nilsson DE, Straube N, Collin SP, Mallefet JM. Sci. Rep. 2014;4:4328 2. Claes JM, Mallefet J. J. Exp. Biol. 2009;212:3684-3692. 3. Claes JM, Ho HC, Mallefet J. J. Exp. Biol. 2011;215:1691-1699. 4. Claes JM, Aksnes DL, Mallefet J. J. Exp. Mar. Biol. Ecol. 2010;388:28-32. 5. Claes JM, Dean MN, Nilsson DE, Hart NS, Mallefet J. Sci. Rep. 2013;3:1308. [Figure: see text] [Figure: see text] O0012 Prolonging Light Emission in Enhanced Chemiluminescence (ECL) Leopoldo Della Ciana(a) , Michele Zucchelli(a) , Luca Covello(a) , Dario Foglietta(a) , Ivan Yu Sakharov(b) (a) Cyanagen, Bologna, Italy (b) Department of Chemistry of Lomonosov Moscow State University, Moscow, Russia The chemiluminescent oxidation of luminol catalyzed by peroxidase finds wide application in the detection and quantitation of antigens, haptens, and nucleic acids and, in particular blotting tests, i.e., Western (proteins), Southern (DNA), Northern (RNA) blots, and ELISA. Because peroxidases are poor catalysts in luminol oxidation, certain compounds known as enhancers are added to the substrate mixture to increase chemiluminescent (CL) intensity. Although a number of compounds were successfully used in the enhancement of peroxidase-induced CL [1,2], currently the most effective enhancer is 3-(10-phenothiazinyl)propane-1-sulfonate (SPTZ) [3]. This compound can increase CL induced by horseradish peroxidase (HRP) and soybean peroxidase (SbP), by an order of magnitude when compared to previously known enhancers, such as p-iodophenol, p-coumaric acid or p-iodophenylboronic acid [3]. Furthermore, it was shown that the introduction of some 4-dialkylaminopyridines such as 4-morpholinopyridine (MORP) a reaction mixture containing luminol, hydrogen peroxide, and SPTZ resulted in a further 10-fold increase of CL intensity. Because 4-dialkylaminopyridines enhanced CL only in the presence of a primary enhancer (SPTZ) and did not act as enhancers in the absence of SPTZ, these compounds were named ” secondary enhancers” . A recent study [4] suggests that ” for an implementation of its enhancing ability, 4-dialkylaminopyridines should get bound to a protein fragment of peroxidase located near the entrance in the canal of the active site, where adsorption of peroxidase substrates commonly occurs The existence of such a complex near the active site may help in binding SPTZ to the peroxidase due to the formation of some charge transfer and ionic bonds between 4-dialkylaminopyridines and SPTZ and, consequently, may improve the efficiency of the enzymatic oxidation of SPTZ with the formation of SPTZ(•+) , that, reacting with luminol, results in the increase of CL intensity” . No significant effect was observed when secondary enhancers were included in the formulation containing primary enhancers other than SPTZ. Apart from chemiluminescent signal intensity, another important feature of ECL systems is light output duration. A prolonged light emission is highly desirable, especially in the blotting techniques, where esposure parameters may need adjustement, without the need to repeat the experiment. In addition, to increase detection it may be useful to prolong exposures to many hours. Again, when considering formulations with only primary enhancers, SPTZ substrates are by far the best in terms of prolonged light emission. The addition of secondary enhancers such as MORP causes a gradual decrease in light output duration, reaching its minimum at the highest initial signal level. Thus, the aim of this study is the search for additives and/or reaction conditions which could prolong light output in these systems. In particular, we have extended our screening to chelators, free-radical scavengers, electron/energy transfer mediators. As a result, we discovered some formulations with significantly improved light duration. These favorable properties were also observed in model dot-blot assays and ELISA. 1. Thorpe GHG, Kricka LJ, Moseley SR, Whitehead TP. Clin. Chem. 1985;31:1335. 2. Kricka LJ, Cooper M, Ji X. Anal. Biochem. 1996;240:119-125. 3. Marzocchi E, Grilli S, Della Ciana L, Prodi L, Mirasoli M, Roda A. Anal. Biochem. 2008;377:189. 4. Yu I, Sakharov MM. Vdovenko Analytical Biochemistry 2013;434:12-14. O0013 Marine luciferases; are they really taxon-specific? A putative luciferase evolved by co-option in an echinoderm lineage Jérôme Delroisse(a) , Patrick Flammang(a) , Jérôme Mallefet(b) (a) Biology of Marine Organisms and Biomimetics, University of Mons, Mons, Belgium (b) Laboratory of Marine Biology, Catholic University of Louvain, Louvain-La-Neuve, Belgium The bioluminescence reaction can be generalized as the oxidation of a luciferin substrate catalysed by a luciferase enzyme [1]. Although some luciferins are shared by phylogenetically distant organisms, it is commonly admitted that luciferases are clade-specific [2]. The European brittle star Amphiura filiformis emits a blue light using a coelenterazine-luciferase system [3, 4]. However, brittle star luciferases (and echinoderm luciferases in general) have not been characterized so far. Using genomic and transcriptomic data, we highlighted the presence of several putative coelenterazine-specific luciferase sequences in A. filiformis. Sequence comparisons revealed that these enzymes are similar to the luciferase of the luminous sea pansy Renilla sp (up to 47% of identical amino acids, up to 69% of general similarity) despite the
large phylogenetic distance between these two species. Luciferase-like genes are also predicted in the purple sea urchin genome and surprisingly, mRNAs were also specifically identified in different transcriptomes from non-luminous echinoderms. Luciferase-like protein expression in non-luminous organisms raises the question of whether luciferin could be the limitative parameter of the bioluminescence reaction. A physiological approach, performed on tube feet of the common sea star (organs expressing luciferase-like mRNA) demonstrated that coelenterazine supplementation did not induce light emission in crude extracts from tube feet. Therefore, this luciferase-like enzyme must have a different function in sea stars, as it was previously suggested for the purple sea urchin [5]. Assuming Renilla luciferase derived from haloalkane dehalogenases [6], we can hypothesize that haloalkane dehalogenases were presumably independently co-opted in luciferases in both Renilla sp and A. filiformis. Using anti-Renilla luciferase antibody, immunodetections were performed on the arm of A. filiformis. Specific immunolabeling was observed in the stroma of the spines, organs that we previously described as the unique photogenic areas (Fig. ). Our results confirm the probable implication of an enzyme similar to Renilla luciferase in the bioluminescence of the brittle star A. filiformis. Two luminous systems using the same luciferin and homologous luciferases seem to have emerged in a convergent manner in two phylogenetically distant species. The similar way of life of these benthic suspension-feeding species could constitute a strong selective pressure for the emergence of bioluminescence. [Figure: see text] Acknowledgements Jérôme Delroisse, Patrick Flammang and Jérôme Mallefet are respectively research fellow, research director and research associate of F.R.S.-FNRS (Fonds de la Recherche Scientifique). Thanks to Olga Ortega-Martinez, Sam Dupont and Magnus Rosenblad (University of Gothenburg) for the access to Amphiura genomic database. Contribution to the “Centre interuniversitaire de la Biologie Marine”. Work supported in part by a FRFC Grant n° 2.4590.11. References 1. Henry JP, Michelson AM. Bioluminescence. Photochemistry and Photobiology 1978;27(6);855-858. 2. Haddock SH, Moline MA, Case JF. Bioluminescence in the sea. Marine Science 2010;2. 3. Shimomura O. Bioluminescence; chemical principles and methods. World Scientific Publishing Company, 2012. 4. Mallefet J, Parmentier B, Mulliez X, Shimomura O, Morsomme P. Characterisation of Amphiura filiformis luciferase (Ophiuroidea, Echinodermata). In Echinoderms in a Changing World – Johnson (ed) CRC Press The Netherlands. 293, 2013. 5. Fortova A, Sebestova E, Stepankova V, Koudelakova T, Palkova L, Damborsky J, Chaloupkova R. DspA from Strongylocentrotus purpuratus; The first biochemically characterized haloalkane dehalogenase of non-microbial origin. Biochimie 2013;95(11);2091-2096. 6. Loening AM, Fenn TD, Wu AM, Gambhir SS. Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output. Protein Engineering Design and Selection 2006;19(9);391-400. O0014 Ultrasensitive bioanalytical application using silica nanoparticles doped with new thermochemiluminescent 1,2-dioxetane derivatives Massimo Di Fusco(a) , Massimo Guardigli(b) , Mara Mirasoli(b) , Arianna Quintavalla(b) , Marco Lombardo(b) , Claudio Trombini(b) , Aldo Roda(b) (a) CIRI-MAM, Alma Mater Studiorum, University of Bologna, Bologna, Italy (b) Department of Chemistry “G. Ciamician”, Alma Mater Studiorum, University of Bologna, Bologna, Italy Thermochemiluminescence (TCL), i.e., the light emission originating from the thermolysis of a suitable molecule, was proposed in the late ’80s as a detection technique for immunoassays [1]. Being TCL emission simply triggered by heat, this technique would allow for reagentless luminescence-based detection, thus simplifying the microfluidic network in miniaturized analytical devices and biosensors. However, TCL detection was abandoned due to methodological problems, such as the high operating temperature (200-250 °C) and the poorer detectability in comparison with other labels. Despite these advantages, TCL detection remains very attractive because it potentially offers the same advantages of other chemiluminescent techniques. Recently, we tried to overcome the problems related to the reported TCL studies and in particular we described the synthesis of a library of TCL acridine-based 1,2-dioxetane derivatives (1-11 in Fig. ) proposed as new TCL labels [2,3]. Suitable structural modifications were introduced to decrease the emission triggering temperature down to 80-100 °C and to produce highly efficient fluorophores in the singlet excited state. [Figure: see text] In the first stage of the work we evaluated the photophysical properties of the acridanone derivatives and the TCL properties of 1,2-dioxetane derivatives using an ITO-coated glass slide as heating element placed directly in contact with a thermoelectrically cooled CCD sensor through a fiber optic taper. Such a lensless contact imaging configuration combined adequate spatial resolution and high light collection efficiency within a small size portable device. We showed that the 10-ethylacetate-9-acridanone derivative moieties produced in the singlet excited state were the main responsible for luminescence emission with fluorescence quantum yields (ϕF ) in the range 0.1-0.5. In addition, with the more efficient 1,2-dioxetane derivative 10 we obtained a limit of detection 17 times lower than 1. Herein, we described the encapsulation of these 1,2-dioxetane derivatives in silica nanoparticles (SiNPs), both alone or together with fluorescent energy acceptors, to obtain amplification of the TCL signal and their superficial modification with biotin for biosensing applications. The amino-functionalized SiNPs loaded with TCL compounds and fluorescent energy acceptor dipyridamole (DP) or 9,10-bis(phenylethynyl)anthracene (BPEA) thanks to the signal amplification due to the large number of 1,2-dioxetane molecules (about 104) in each SiNP and the increased emission efficiency due to the energy transfer to the fluorescent acceptor, could be revealed by TCL imaging with a detectability close to that of the CL enzyme label horseradish peroxidase [2]. In conclusion, the new TCL compounds showed emission triggering temperatures much lower (i.e., < 100 °C) than the compounds used in the past and higher emission yield. In addition, the entrapment of this compound in functionalized SiNPs, used as probes to amplify the TCL signal exploiting the strong biotin-avidin interaction, demonstrated its suitability for the development of TCL-based immune or nucleic acid biosensors. 1. Hummelen JC, Luider TM, Wynberg H. Complementary immunoassays. Chapter 14, Ed. W.P. Collins, 1988;191-208. 2. Roda A, Di Fusco M, Quintavalla A, Guardigli M, Mirasoli M, Lombardo M, Trombini Anal C. Chem. 2012;84:9913-9919. 3. Di Fusco M, Quintavalla A, Trombini C, Lombardo M, Roda A, Guardigli M, Mirasoli M. J. Org. Chem. 2013;78:11238-11246. O0015 Strategies towards Multi-colour Electrochemiluminescence Sensors Egan Doeven, Gregory Barbante, Emily Kerr, Paul Francis Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds, Victoria, Australia In recent times electrochemiluminescence (ECL) has emerged as an important analytical technique, its selectivity and sensitivity making it suitable for the detection of a wide range of compounds. ECL is exploited routinely in commercial applications for rapid, sensitive detection and quantification of biomarkers, food borne pathogens and biowarfare agents. New applications of ECL as a sensing technique continue to appear in a wide range of fields, with a range of novel ECL-active luminophores having diverse properties being developed. Generally a single luminophore is excited in an ECL experiment without wavelength discrimination, for exa
mple tris(2,2′-bipyridyl)ruthenium(II) ([Ru(bpy)3 ](2+) ) emits strong co-reactant ECL centred at 620 nm. This work explores the use of multiple, selectively excited ECL luminophores, as well as the recently discovered inhibition of Ir(ppy)3 ECL(1) (under certain conditions) in order to selectively detect three emitting species in a single solution.(2) The emitting species of interest have been developed to have complimentary photophysical and electrochemical properties, and can thus be selectively excited via application of different electrode potentials. Quantification of the emission from the luminophores is investigated using two different approaches. Simultaneously collecting electrochemical and spectral data using a potentiostat and typical wavelength-sensitive detector such as a CCD can be exploited to generate a 3D map of the emission vs. applied potential. Alternatively, the use of a low cost consumer-level digital camera and image analysis algorithms to isolate and quantify the contribution of each complex has been explored. Using this approach we demonstrate simultaneous detection of three emitting species at the low micro-molar level. This low cost multiplexed ECL detection system has potential applications in the emerging fields of mobile phone based telemedicine, as well as expanding the utility of current ECL based assays. 1. Doeven EH, Zammit EM, Barbante GJ, Francis PS, Barnett NW, Hogan CF. A potential-controlled switch on/off mechanism for selective excitation in mixed electrochemiluminescent systems. Chemical Science 2013;4(3);977-982. 2. Doeven EH, Barbante GJ, Kerr E, Hogan CF, Endler JA, Francis PS. Red-green-blue electrogenerated chemiluminescence utilizing a digital camera as detector. Analytical Chemistry 2014. DOI; 10.1021/ac404135f. O0017 Application of quinone as a selective chemiluminescent reagent for determination of biothiols in biological fluids Mohamed Elgawish(a,b) , Naoya Kishikawa(a) , Kaname Ohyama(a) , Naotaka Kuroda(a) (a) Graduate School of Biomedical Sciences, Course of Pharmaceutical Sciences, Nagasaki, Japan (b) Pharmaceutical Chemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt Background The physiological significance of low molecular weight thiols is well recognised with the levels of these compounds within biological fluids such as plasma and urine serving as valuable biomarkers in a number of clinical situations(1) . While there is a clear need to monitor these important analytes and indeed many procedures proffered, considerable scope remains for the development of fast protocols that require minimal sample pre-treatment. The biological important of quinones can be assign to their electrophilic and versatile oxidative properties, which are capable of promoting Michael-addition with cellular thiols, such as free glutathione, cysteine, and cysteine residues of proteins and electron transfer in living system through redox cycling of quinone/semiquinone/quinol triad system. Michael-addition-type probes have been actively developed in recent years and exploited for the design of chromo-and fluorogenic probes for thiol sensing(1) . In this context, we applied for the first time the Michael-addition reaction for chemiluminescence (CL) determination of biothiols. The principal of the proposed method relies on the application of quinone as Michael acceptor to react rapidly and specifically with biothiols. The liberated adducts retain the redox cycling capability of parent quinones to react with reductant, dithiothreitol (DTT), releasing reactive oxygen species (ROS) which can be measured by luminol-CL assay.(2) Materials and Methods Sample preparation One hundred microliters of human plasma, diluted with 500 mmol/L HEPES buffer, pH 8.5 to approximately 300 μL, was mixed with 10 μL of tris (2-carboxyethyl) phosphine (TCEP) solution (100 mmol/L in HEPES buffer, pH 8.5) and allowed to react at room temperature for 15 min. 10 μL of menadione (MQ) solution (100 mmol/L in acetonitrile (ACN)) was added and the sample was spin for 15 min at room temperature. Oasis HLB 1 cm(3) /30 mg cartridges were used to isolate the resulting adducts from each biological sample. The cartridges were conditioned with 0.5 mL of methanol and equilibrated with 0.5 mL of purified water. The samples were passed through individual cartridges, after which the cartridges were washed two times with 250 μL of purified water. The target analytes were eluted with 150 μL of 40% ACN, followed by 150 μL of neat ACN. Each mixture was vortex mixed, diluted ten times, and 20 μL was then inject ed into the HPLC-CL system (Fig. ) [Figure: see text] Results Four of the most important thiols in our body, cysteine (CYS), homocysteine (HCY), glutathione (GSH), and N-acetylcysteine (NAC), were selected under our investigation. MQ, the highly reactive and selective compound of studied quinones, overcame the problems of commonly utilized probe and reacted with thiol group specifically and rapidly at lowest possible temperature. All studied thiols reacted with MQ in 0.5 M HEPES buffer, pH 8.5, at room temperature. The reaction was carried out for 5 min at MQ to thiol molar ratio of about ten. The reaction was specific to aminothiol compared with other aminoacids which shown no reactivity. The calibration curves of MQ-thiol adducts showed excellent linearity over the range 0.0025-2 µmol/L with excellent r values and the detection limits at a signal-to-noise ratio of 3 were 0.0002-0.0008 µmol/L for all analytes. The derivatization of thiols was occurred before solid phase extraction technique to prevail the high polarity which makes their extraction from biological matrices very difficult. The proposed method could successfully quantify the studied thiols in human plasma samples with reasonable accuracy and precision. The protocol shown here clearly provides a sound footing from which further studies can be advanced to the measurement of other sulfhydryl thiols and matrices. References 1. Chen X, Zhou Y, Peng X, Yoon J et al Chem. Soc. Rev. 2010;39:2120. 2. Elgawish MS, Shimomai C, Kishikawa N, Ohyama K, Wada M, Kuroda N et al Chem. Res. Toxicol. 2013;26:1409. O0018 CASPT2//CASSCF Study Of the Ring-opening Mechanism of Dewar Dioxetane Pooria Farahani(a,b) , Marcus Lundberg(a) , Roland Lindh(a) , Daniel Roca-Sanjuán(b) (a) Uppsala University, Uppsala, Sweden (b) Universitat de València, Valencia, Spain Light emission from the heating of Dewar benzene was reported by McCapra.(1) Since the process was observed to be dependent on the presence of oxygen and most of the chemiluminescence reactions occur through an O-O cleavage,(1) the light observed was suggested to be produced after the ring opening of an intermediate structure, named Dewar dioxetane (see Fig. ).(2) The oxidation of Dewar benzene might lead to Dewar dioxetane and, after O-O and C-C cleavage,to the 2,4-hexadiendial product. The thermally activated decomposition mechanism of the Dewar dioxetane has been studied here by the multiconfigurational CASPT2//CASSCF approach,(3,4,5) and accurate reaction path strategies based on minimum energy path and intrinsic reaction coordinate computations. A two-steps biradical mechanism is determined for the process. It involves asynchronous O-O’ and C-C’ bond cleavage as in the related system 1,2-dioxetane.(6) Moreover, a radiationless decay path to the ground-state potential energy surface has been determined for the molecule along the manifold of the excited triplet state, while in the excited singlet state the system evolves toward an equilibrium structure that might be responsible of the light emission. This findings provide clues for rationalizing the observed light and point to a higher efficiency of fluorescence than phosphorescence. [Figure: see text] References 1. McCapra F. QUARTERLY Rev. 1966;20:485. 2. Koo J-Y, Schmidt S, Schuster G. Proc. Natl. Acad. Sci. USA 1978;75:30-33. 3. Roca-Sanju&a
acute;n D, Aquilante F, Lindh R. WIREs Comput. Mol. Sci. 2012;2:585-603. 4. Andersson P, Malmqvist P.-Å, Roos B.O. J. Chem. Phys. 1992;96:1218. 5. Andersson P, Malmqvist P.-Å, Roos B.O, Sadlej A, Wolinski K. J. Chem. Phys. 1992;94:5483. 6. Farahani P, Roca-Sanjuán D, Zapata F, Lindh R. J. Chem. Theory Comput. 2013;9:5404-5411. O0019 Bioluminescence of Obelin; identification of the light emitters using QM/MM models Shufeng Chen(a,b) , Isabelle Navizet(c,e) , Roland Lindh(d) , Yajun Liu(b) , Nicolas Ferré(a) (a) Aix-Marseille Université, Marseille, France (b) Beijing Normal University, Beijing, China (c) Université Paris-Est, Marne-la-Vallée, France (d) Uppsala University, Uppsala, Sweden (e) University of Witwatersrand, Johannesburg, South Africa The chemiluminescent compound coelenterazine is related to the bioluminescence of a wide range of marine organisms, eg the Obelia Longissima hydrozoan. While the corresponding photochemical reaction (an oxidative decarboxylation of oxo-coelenterazine in which the coelenteramide product is in an excited electronic state) is commonly used as a luminescent probe,(1) the details of its mechanism are still unknown. In particular, the chemical nature of the light emitters responsible for the multi-modal bioluminescence and fluorescence emission spectra is still matter of debate. Up to now, the neutral coelenteramide molecule and its phenolate anion (obtained through a proton transfer towards the close His22 residue, hence forming an ion-pair) are the two most serious candidates.(2) Using hybrid QM/MM calculations,(3) we confirm the implication of the neutral coelenteramide in its first excited state as the primary light emitter (the computed TDDFT/MM vertical emission is 339 nm). However our results demonstrate that the postulated ion-pair is not a stable light emitter. Actually, an electron transfers together with the proton to form a diradical state (Fig. ) and the corresponding system ultimately evolves towards a point of degeneracy between the ground and first excited states. Hence a non-radiative decay path is suggested to compete with the light emission process. [Figure: see text] Alternatively, the phenolate coelenteramide is found to be a light emitter, as long as His22 looses another proton at the same time its accepts the one coming from coelenteramide, hence keeping its electric neutrality (the computed TDDFT/MM vertical emission is about 500 nm, in excellent agreement with the experimental λ(max) ). Our calculations show that the final location of the proton is not of primary importance. Finally, using the unique modeling capabilities of QM/MM calculations, and comparing our results with previous computations of coelenteramide in gas phase or in a solvent(4) , we assess the different contributions responsible for the color of the emitted light. Besides the protonation state of the luminophore, the steric constraints induced to the tight cavity in which coelenteramide is bound is the most important factor, far more than the electrostatic interaction with the protein. References 1. Frank LA, Borisova VV, Markova SV, Malikova NP, Stepanyuk GA, Vysotski ES. Violet and Greenish Photoprotein Obelin Mutants for Reporter Applications in Dual-color Assay. Anal. Bioanal. Chem. 2008;391:2891-2896. 2. Belogurova NV, Kudryasheva NS, Alieva RR, Sizykh AG. Spectral Components of Bioluminescence of Aequorin and Obelin. J. Photochem. Photobiol., B 2008;92:117-122. 3. Chen S-F, Navizet I, Lindh R, Liu Y-J, Ferré N. Hybrid QM/MM Simulations of the Obelin Bioluminescence and Fluorescence Reveal an Unexpected Light Emitter. J. Phys. Chem. B 2014 (in press, dx.doi.org/10.1021/jp412198w). 4. Chen S-F, Navizet, I, Roca-Sanjuá n D, Lindh R, Liu Y-J, Ferré N. Chemiluminescence of Coelenterazine and Fluorescence of Coelenteramide; A Systematic Theoretical Study. J. Chem. Theory Comput. 2012, 8, 2796-2807. O0020 Identification of a fluorescent compound from the bioluminescent polychaete Tomopteris Warren Francis(a,b) , Meghan Powers(a,b) , Steve Haddock(a) (a) Monterey Bay Aquarium Research Institute, Moss Landing, CA, USA (b) University of California, Santa Cruz, Santa Cruz, CA, USA Tomopteris is a cosmopolitan genus of luminous polychaetes that release bright yellow particles from the parapodia when stimulated. Although the yellow bioluminescence of this genus has been the subject of a few investigations, the chemistry is essentially unstudied. All that is known is that the reaction does not involve any of the known molecules, like coelenterazine. The connection between fluorescence and bioluminescence has been a topic of great discussion. A brief report half a century ago described the yellow fluorescence of the parapodia with an identical similar spectrum to the bioluminescence, which suggested that it may be the luciferin or terminal light-emitter. Here we report the isolation of an abundant, fluorescent yellow-orange compound found in the luminous exudate and in the body of the animals. LCMS revealed the mass to be 270 m/z with a molecular formula of C15 H10 O5 , which ultimately was shown to be aloe-emodin, an anthraquinone previously found in various Aloe plant species. From known redox properties and chemiluminescence from other anthraquinones, we hypothesize that aloe-emodin is the oxyluciferin for Tomopteris bioluminescence. O0021 Chemiluminescent methods for explosives (TNT, TATP, HMTD) detection Stefano Girotti(a) , Elida Ferri(a) , Marcello D’Elia(b) , Mara Mirasoli(c) , Aldo Roda(c) , Luigi Ripani(d) , Giuseppe Peluso(d) , Roberta Risoluti(e) , Elisabetta Maiolini(a) , Francesco Saverio Romolo(f,g) (a) Dipartimento di Farmacia e Biotecnologie, Università di Bologna, Bologna,, Italy (b) Gabinetto Regionale di Polizia Scientifica per l’Emilia Romagna, Bologna, Italy (c) Dipartimento di Chimica, Università di Bologna, Bologna, Italy (d) Reparto Investigazioni Scientifiche (RIS) Carabinieri, Roma, Italy (e) Dipartimento di Chimica, Università “La Sapienza”, Roma, Italy (f) Institut de Police Scientifique, Université de Lausanne, Lausanne, Switzerland (g) Legal Medicine Section, Università “La Sapienza”, Roma, Jamaica The terroristic attacks performed in the last ten years have focused the attention on the protection and security of the citizen and the detection of various types of explosives is included in this purpose. Our work was finalised to develop chemiluminescent methods that permit the detection of TNT (2,4,6-trinitrotoluene), TATP (Triacetone triperoxide) and HMTD (Hexamethylene triperoxide diamine), displaying higher detectabilityand ease of use with respect to currently available methods (1-2). TNT is one of the most employed explosives in the 20th century and at the same time one possible well known environmental pollutant for its toxicity (3). For these reasons its detection could permit the prevention of terrorism acts (or the identification of the explosive used for these purposes) and for an early sign of environmental pollution. TATP (Triacetone triperoxide) and HMTD (Hexamethylene triperoxide diamine) are compounds extremely instable because they contain peroxide groups (4). Due to their simple synthesis, which requires, as reagents, compounds easily available at any supermarket, and that can be performed at home, they are frequently used in terrorist attacks. For TATP and HMTD we developed a chemiluminescent method that permits their indirect identification. Upon treatment with aci

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Abstract
P0001 Theoretical study of oxalic peracid derivatives; insights on the high-energy intermediate of the peroxyoxalate system Felipe A. Augusto, Noriberto A. Pradie, Antônio C. Borin, Erick L. Bastos, Wilhelm J. Baader Instituto de Química, São Paulo, SP, Brazil The peroxyoxalate reaction is used as an analytical tool for the detection of several analytes due to its low cost and high sensibility.(1,2) Our group has proposed a simplified mechanism for the reaction of bis(2,4,6-trichlorophenyl) oxalate (oxalic ester) with hydrogen peroxide in the presence of imidazole (base) and 9,10-diphenylanthracene (activator).(3,4) More recently it has been possible to directly observe the chemiexcitation step, obtaining for the first time kinetic data related to this step.(5) Although being known for more than half a century and having been extensively studied, the peroxyoxalate system still has particularities that are a matter of discussion.(1,2) Among them are the reason for its high efficiency when compared to similar systems and the identity of the high-energy intermediate (HEI).(1,2) Several HEI have been proposed along the years, but only a few of them based on experimental evidence and even less were effectively studied or discarded.(6) In the present work, several oxalic peracid derivatives have their geometries optimized and their energy calculated by theoretical means (B3LYP/6-31 + G(d)), then, for each peracid, a proposed reaction path is studied. This path is selected based on several structures proposed as HEI, like the 1,2-dioxetanedione and other cyclic peroxidic derivatives. Based on the results obtained for each step and for each peracid, it is possible to draw the energetic profile for these reactions. Using data from the third and fifth step Hammett plots were made which indicate the charge distribution during the course of the reaction (Fig. ). [Figure: see text] The results obtained in the study of specific reaction steps of the peroxyoxalate reaction for several oxalic peracid derivatives indicate that the proposed mechanism for this reaction is energetically viable.(3,4) The Hammett plots obtained with the calculated data indicate the involvement of a negative charge in the transition state, as also indicated by kinetic results from our group (unpublished work). References 1. Ciscato LFML, Augusto FA, Weiss D, Bartoloni FH, Albrecht S, Brandl H, Zimmermann T, Baader WJ. ARKIVOC 2012;2012:391. 2. Bartoloni FH, Bastos EL, Ciscato LFML, Peixoto MMdeM, Santos APE, Santos CS, Oliveira S, Augusto FA, Pagano APE, Baader WJ. Quim. Nova 2011;34:544. 3. Stevani CV, Lima DF, Toscano VG, Baader WJ. J. Chem. Soc., Perkin Trans. 2 1996;989. 4. Da Silva SM, Casallanovo F., Oyamaguchi KH, Ciscato LFML, Stevani CV, Baader WJ. Luminescence 2002;17:313. 5. Ciscato LFML, Bartoloni FH, Bastos EL, Baader WJ. J. Org. Chem. 2009;74:8974. 6. Augusto FA, Souza GA, Souza Junior SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. P0002 Investigation into the phosphate buffer role on the peroxyoxalate system in aqueous medium Glalci A. Souza, Monica M. M. Peixoto, Wilhelm J. Baader Instituto de Química da Universidade de São Paulo, São Paulo-SP, Brazil The peroxyoxalate reaction is known as the only chemiluminescence system which involves the intermolecular Chemically Initiated Electron Exchange Luminescence (CIEEL) mechanism that possesses proven high quantum yields (up to 60%).(1,2) The mechanism of this reaction has been intensively studied in non-aqueous medium, however, is still not yet completely understood.(3-6) Contrarily, no detailed mechanistic studies on this reaction were performed in aqueous medium, important for many analytical applications.(2) In this work we report the results of a kinetic study of the reaction of oxalic esters such as bis(2,4,6-trichlorophenyl) oxalate (TCPO), bis(4-methylphenyl) oxalate (BMePO) and bis(4-methoxyphenyl) oxalate (BMPO) with hydrogen peroxide and phosphate buffer as catalyst, using 2,5-diphenyloxazole as activator. The reaction was performed in a binary system using 1,2-dimethoxyethane as the organic phase and phosphate buffer as the aqueous phase in a proportion of 1;1 (v/v). The influence of the reagent concentration as well as the pH of the medium on the kinetic parameters and the chemiluminescence quantum yields has also been investigated. Kinetics studies with TCPO using different H2 O2 concentrations allow the determination of the rate constant for oxalic ester perhydrolysis (kper ), from the linear correlation between the hydrogen peroxide concentration and the observed rate constants (kobs ). The results show that TCPO is more reactive at pH 8 (kper  = 14.7 ± 0.7 L mol(-1) s(-1) ), followed by pH 7 (kper  = 6.2 ± 0.3 L mol(-1) s(-1) ) and less reactive at pH 6 (kper  = 0.82 ± 0.02 L mol(-1) s(-1) ). Additionally, kinetics studies performed with BMePO at different H2 O2 concentrations showed the same reactivity tendency over the pH range from 6 to 8 as with TCPO. However the kper values are slightly higher for BMePO (pH 8; kper  = 61 ± 3 L mol(-1) s(-1) ; pH 7; kper  = 20 ± 1 L mol(-1) s(-1) and pH 6; kper  = 9.9 ± 0.1 L mol(-1) s(-1) ). Finally, BMPO presented a very interesting reactivity pattern in aqueous medium. Contrarily to TCPO and BMePO, BMPO is considerably more reactive at pH 6 than at pH 8 and its reactivity at pH 7 (kper  = 34 ± 1 L mol(-1) s(-1) ) is comparable to BMePO. At pH 6 and 8, the reaction is so fast that is not possible to measure the rate constants at high hydrogen peroxide concentrations (> 15 mmol L(-1) ) in order to establish the linear correlation between the hydrogen peroxide concentration and kobs values. The highest rate constants possible to measured were kobs  = 1.23 ± 0.5 s(-1) at pH 6 for [H2 O2 ] = 10 mmol L(-1) and kobs  = 1.18 ± 0.06 s(-1) at pH 8 for [H2 O2 ] = 15 mmol L(-1) . These results indicate that for TCPO general base catalysis by phosphate is predominant, which is more efficient at pH 8. For BMePO at pH = 6 the general acid catalysis is also important as indicated by it much higher kper value as compared to TCPO. Finally, for BMPO acid catalysis is predominant as indicated by the higher kper values in more acid pH values for this oxalic ester. Financial support; FAPESP, Capes, CNPq. 1. Stevani CV, Silva SM, Baader WJ. Eur. J. Org. Chem. 2000;4037. 2. Augusto F. A., Souza G. A., Souza Jr. S. P., Khalid M., Baader W. J. Photochem. Photobiol. 2013;89:1299. 3. Stevani CV, Campos IPA, Baader WJ. J. Chem. Soc., Perkin Trans. 2 1996;1645. 4. Da Silva SM, Casallanovo F, Oyamaguchi KH, Ciscato LFML, Stevani CV, Baader WJ. Luminescence 2002;17:313. 5. Silva SM, Wagner K, Weiss D, Beckert R, Baader WJ. Luminescence 2002;17:362. 6. Ciscato LFML, Bartoloni F. H, Bastos E. L, Baader WJ. J. Org. Chem. 2009;74:8974. P0003 Lanthanide Complexes with N’-(2-hydroxybenzylidene)-3-methoxybenzohydrazide; Synthesis, Thermal Behaviour, Biological Activities and Luminescent Properties. Abdulaziz Ajlouni(a) , Ziyad Taha(a) , Waleed Al Momani(b) (a) Department of Applied Chemical Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan, Jordan (b) Department of Allied Medical Sciences, Al Balqa’ Applied University, Amman, Jordan Novel Ln(III) complexes with N’-(2-hydroxybenzylidene)-3-methoxybenzohydrazide (L) have been synthesized. The ligand and its complexes were characterized based on elemental analyses, molar conductance, IR, 1H and 13C-NMR, UV-vis., and TGA studies. The conductivity data show a 1;2 electrolytic nature with a general formula [LnL2(NO3)2]NO3. The IR spectra reveal the coordination of the ligand through the azomethine nitr
ogen and the hydroxyl O-atom in addition to the carbonyl oxygen to the lanthanide ion. The coordinated nitrate ions behave in a bidentate fashion. Under the excitation, the luminescence emission properties for Sm, Tb, Eu and Dy complexes are observed. These observations show that the ligand favor energy transfers to the emitting energy level of these lanthanide ions. Furthermore, the antimicrobial activities of all complexes were studied against different types of bacteria. It was observed from the results that most of the synthesized complexes of the tested series possessed good antibacterial activity against bacteria and the microbial activities of the complexes in most cases are higher than that of the corresponding ligand. P0004 Comparative study of apoptosome formation and ATP oscillation in apoptosis and differentiation processes using luciferase Shiva Akbari-Birgani(a) , Saman Hosseinkhani(a) , Hossein Baharvand(b) (a) Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran (b) Department of Stem Cells and Developmental Biology at the Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran A powerful and sensitive tool to monitor cytochrome c release and pursuing apoptosome formation based on split-luciferase complementary assay has been recently developed (1). This biosensor detects and report apoptosome formation based on Apaf-1 oligomerization (Fig. P0004;1). In this study the split luciferase biosensor is used to compare apoptosome formation in differentiation with apoptosis processes. ATP oscillation is also measured in both processes using luciferase based assay (2). The mESC line Royan B16, derived from the C57BL6 mouse strain (3) was cultured according to the standard protocol. Mouse Embryonic Stem Cells (mESCs) were differentiated to cardiac cell by static suspension culture and ascorbic acid as a cardiac differentiation inducer of mESCs. In parallel with differentiation, Doxorubicine, an apoptogenic chemotherapy drug, was used to induce apoptosis in another group of mECS. Sampling was performed in series time. To monitor the release of cytochrome c, western blotting was applied. To assess apoptosome formation during apoptosis and differentiation, mESCs were transfected with two vectors containing split luciferase genes connected to APAF1 gene; pcDNA-NLuc-APAF1 and pcDNA-CLuc-APAF1, and then apoptosis and differentiation were induced. Luciferase activity was measured at different times after induction. Cellular ATP content was also compared in these two processes using luciferase. Our evidences have revealed that cytochrome c accumulates in cytosol and apoptosome machinery forms during differentiation of mESCs as well as apoptosis. But they are delayed and reduced in differentiation relative to apoptosis. Cellular ATP measurements showed ATP oscillations go parallel with apoptosome formation during both processes. In total our results indicate that mitochondrial apoptotic pathway involves in cardiac differentiation of mESCs as well as apoptosis. However, timing and intensity of cytochrome c release, apoptosome formation and ATP increment is delayed in differentiation. [Figure: see text] 1. Torkzadeh-Mahani M, Ataei F, Nikkhah M, Hosseinkhani S. Design and development of a whole-cell luminescent biosensor for detection of early-stage of apoptosis. Biosens Bioelectron 2012;38:362-368 2. Mohammadi S, Nikkhah M, Nazari M, Hosseinkhani S. Design of a coupled bioluminescent assay for a recombinant pyruvate kinase from a thermophilic Geobacillus. Photochem Photobiol 2011;87:1338-1345 3. Hassani SN, Totonchi M, Farrokhi A, Taei A, Larijani MR, Gourabi H, Baharvand H. Simultaneous suppression of TGF-beta and ERK signaling contributes to the highly efficient and reproducible generation of mouse embryonic stem cells from previously considered refractory and non-permissive strains. Stem Cell Rev 2012;8:472-481 P0006 Effects of alcohols on the fluorescence of Ca(2+) -discharged photoprotein obelin Roza Alieva(a) , Nadezhda Belogurova(b) , Alena Petrova(a) , Nadezhda Kudryasheva(a,b) (a) Siberian Federal University, Krasnoyarsk, Russia (b) Institute of Biophysics SB RAS, Krasnoyarsk, Russia Photoprotein obelin is stable enzyme-substrate complex of polypeptide and 2-hydroperoxycoelenterazine, which is responsible for bioluminescence of the marine hydroid Obelia longissima [1]. The bioluminescent is triggered by calcium ions. Obelin is not fluorescent, but the product of the bioluminescent reaction, enzyme-bound coelenteramide, is a fluorescent protein called “Ca(2+) -discharged” obelin. Discharged obelin is stable and nontoxic and its spectra are variable, it can be applied as fluorescent biomarker to visualize biochemical processes in biological and medical investigations. Variation of the color of biomarkers is important for these applications. As we showed previously [2-4], fluorescence spectra of discharged obelin are not completely stable; they depend on Ca(2+) concentration, exposure to higher temperature, and excitation wavelength. The effects of ethanol on bioluminescence of mutant obelins were found in [5]. Availability of the active center of the obelins to exogenous ethanol molecules was suggested as a reason for bioluminescence spectra changes. Influence of alcohol molecules on light-induced fluorescence of Ca(2+) -discharged obelin was not studied yet. Here we examined the intensity and color of light-induced fluorescence of Ca(2+) -discharged photoprotein obelin in the presence of alcohols (ethanol and glycerol) which are widely used as biomedical agents. Light-induced fluorescence spectra of Ca(2+) -discharged obelin were measured at different concentrations of the alcohols at 350- and 280-nm photoexcitation (corresponding to polypeptide-bound coelenteramide and tryptophan absorption regions). Emission spectra at 280 nm excitation (tryptophan absorption region) included three peaks with 348, 504, and 657 nm maxima, corresponding to fluorescence of tryptophan, enzyme-bound coelenteramide, and hypothetical indole-coelenteramide exciplex, respectively. The latter was found in photoprotein emission spectra recently [4]. The spectra were deconvolved into Gaussian components – ultraviolet (tryptophan emission), blue-green (coelenteramide emission), and red (hypothetical indole-coelenteramide exciplex emission). The addition of alcohols increases covariantly the fluorescence intensities and contributions of ultraviolet (346 nm, tryptophan), violet (420 nm, protonated form of coelenteramide), and red (655 nm, exciplex) components and decreases fluorescence of blue-green (503 and 565 nm, partly deprotonated forms of coelenteramide) components of Ca(2+) -discharged obelin fluorescence. The effects are related to changes of the proton transfer efficiency in the fluorescent state of coelenteramide. Therefore, two peculiarities should be taken into consideration when applying the discharged obelin as a fluorescent biomarker; (1) variation of fluorescence color and intensity in the presence of alcohols, and (2) dependence of emission spectra on the excitation wavelength. Details of these peculiarities are reported in [6]. Acknowledgements This work partly supported by; the Program ‘Molecular and Cellular Biology’ of the Russian Academy of Sciences; the Grant Ministry of Education and Science RF 11.G34.31.0058; the Grant B-14 of Ministry of Education and Science RF assigned to Siberian Federal University. References 1. Vysotski ES, Markova SV, Frank LA. Molecular Biology. 2006;40:355-367. 2. Belogurova NV, Kudryasheva NS. J Photochem Photobiol B 2010;101:103-108. 3. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. LUMINESCENCE 2012;27:96. 4. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem 2013;405:3351-3358. 5. Belogurova NV. Kudryasheva NS, Alieva RR. J Mol Struct. 2009;924-926:148-152. 6. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem DOI; 10.1007/s00216-014-7685-z. P0007 Mitochondrial genome Organization and Phylogenetic analysis of bioluminescent Elateroidea Danilo Trabuco Amaral
(a,b) , Yasuo Mitani(c) , Yoshihiro Ohmiya(d) , Vadim R. Viviani(a,b) (a) Graduate School of Biotechnology and Environmental Monitoring (UFSCar – Sorocaba), Sorocaba, Sao Paulo, Brazil (b) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Sao Carlos, Sao Paulo, Brazil (c) Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Japan (d) Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan The bioluminescence in the superfamily Elateroidea is observed within Lampyridae (fireflies), Phengodidae/Rhagophthalmidae (railroad worm) and Elateridae (click-beetles) families. Bioluminescence may have evolved independently in these families, since most of the Elateroidea families such as Eucnemidae, Throscidae, Drilidae, Cantharidae, Lycidae do not display luminescence. In this study, molecular phylogenetic analyses with mitochondrial genome were performed to answer this question. We sequenced the mitochondrial genomes of (Elateridae) Hapsodrilus ignifer, Pyrearinus termitilluminans, a non-luminescent Elateridae (Lampyridae), Bicellonycha lividipennis, (Phengodidae) Brasilocerus sp.2 and Phrixothrix hirtus using Long-PCR reactions and the primer walking methodology. Elateridae and Lampyridae species showed a typical Colepotera mitochondrial genome, however, in Phengodidae genomes several rearrangements were observed (Fig. 1). In Brasilocerus sp.2 genome, we did not observe the tRNAs for Trp, Gln and Ile, probably deleted in this species. In P. hirtus genome, the tRNA-Tyr was rearranged before the tRNA-Trp and we observed a duplicated region of tRNA-Leu/COX2 between NADH2 gene and the control region. The phylogenetic analyses using the Bayesian methods showed four clades within Elateroidea, 1. Eucnemidae; 2. Elateridae (with Drilidae) 3. Lampyridae and Lycidae and 4. Phengodidae, Rhagophthalmidae and Cantharidae, (Cantharoidea). As discussed by Viviani et al. (2009) and Amaral et al. (2013), the bioluminescence within Elateroidea may have originated at three different times from the similar ancestor luciferase-like enzymes which was pH-insensitive. Within this scenario, the Elateridae and Phengodidae/ Rhagophthalmidae luciferases evolved directly from the ancestral enzyme and the pH sensitivity may have evolved later in lampyrids or within Lampyridae/Lycidae clade. Financial Support; FAPESP and CNPq Reference 1. Amaral DT, Arnoldi FGC, Rosa S.P., Viviani VR. Molecular phylogeny of Neotropical bioluminescent beetles (Coleoptera; Elateroidea) in southern and central Brazil. Luminescence. Luminescence, DOI 10.1002/bio.2561. 2. Viviani VR, Prado RA, Arnoldi FCG, Abdalla FC. An ancestral luciferase in the Malpighian tubules of a non-bioluminescent beetle! Photochem Photobiol Sci 2009;8:57-61. P0008 Report of two new cases of luminous termite mounds inside Amazon forest; phylogenetic considerations Danilo Trabuco Amaral(a,b) , Vadim R. Viviani(a,b) (a) Graduate School of Biotechnology and Environmental Monitoring (UFSCar – Sorocaba), Sorocaba, Sao Paulo, Brazil (b) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Sao Carlos, Sao Paulo, Brazil The phenomenon of luminous termite mounds is known from Central Brazil cerrados (savannas) and is caused by infestation of larvae of the click beetle Pyrearinus termitilluminans. During warm spring nights, larvae expose their bright thorax, and attract flying insects that will serve as preys. The Pyrearinus genus comprises more than 40 species distributed in South America, most of them occurring in Brazil, including species inhabiting termite mounds. Although, there are also reports of luminous termite mounds inside the Amazon forest (Costa and Vanin, 2010), information about these cases are still missing. Here we report the observation of two new cases of luminous termite nests inside the Amazon forest; (I) one in transitional area between Cerrado and Amazon forest at the margins of Araguaia´s river in the northwestern region of Tocantins state, which is also caused by P. termitilluminans, and (II) the other occurring inside Amazon forest at the margins of Juruena river in the northwest region of Mato Grosso state, which is caused by a different species, Pyrearinus fragilis. Our molecular studies, using COI and NADH2 mitochondrial genes showed a close relationship between P. termittiluminans occurring in forest termite mounds in Caseara-TO and in Cerrado termite mounds of PNE-GO, despite the distance of 1,000 Km and the distinct habitats (cerrado and forest). Our data also show that P. termitilluminans and P. fragilis that inhabit termite mounds in different regions separated by 2 basin rivers, form a monophyletic group. These results indicate that niche selection for termite mounds may have occurred in a common ancestor, and this characteristic was maintained in the group. Financial support; FAPESP and CNPq Reference 1. Costa C, Vanin SA. Coleoptera Larval Fauna Associated with Termite Nests (Isoptera) with Emphasis on the Bioluminescent Termite Nests from Central Brazil. Psyche; A Journal of Entomology 2010;2010:1-13. P0009 Molecular Insights of luciferase evolution in Elateridae family Danilo Trabuco Amaral(a,b) , Vadim R. Viviani(a,b) (a) Graduate School of Biotechnology and Environmental Monitoring (UFSCar – Sorocaba), Sorocaba, Sao Paulo, Brazil (b) Graduate School of Evolutive Genetics and Molecular Biology, Federal University of São Carlos (UFSCar), Sao Carlos, Sao Paulo, Brazil Bioluminescence in beetles occurs in Elateroidea superfamily, within Lampyridae (fireflies), Phengodidae/Rhagophthalmidae (railroadworms/starworms) and Elateridae (click beetles) families. In Elateridae, more than 9,000 species were described, however, less than 300, display bioluminescence. Most of them are found in the Neotropical region with Brazil hosting the richest diversity, with species occurring in the Amazon, Atlantic rain forest and Cerrado (savanna). Adult click beetles usually display two lanterns emitting green bioluminescence in the dorsal region and a lantern emitting green to orange bioluminescence in ventral region. The luciferases from these lanterns were shown to be coded by paralogous genes. However, the ontogenic origin of these luciferases is not clear. Thus, to elucidate the evolution of the lanterns in Elateridae, we sequenced the partial cDNA luciferase sequences of Brazilian species and compared with the phylogeny of the group inferred from the mitochondrial genome. The reconstructed tree from luciferases sequences separated the species in two clades (I) South-american species, and (II) Central-american species. Biogeographic events, as discussed by Feder & Velez (2009), may have influenced the variation of bioluminescence colors in Elateridae. In our study, we did not observed the diversity of bioluminescent colors occasioned by vicariant events, as occur in Pyrophorus plagiophthalamus population, however the genetic signature found in our study with the luciferases of Pyrearinus, Fulgeochlzus and Ptesimopsia genera suggest the occurrence of some vicariant events resulting in two well-defined clades in the phylogeny. Our phylogenetic analyses of Elateridae grouped in the same clade the luciferases with similar bioluminescence spectra. The similarity among luciferase sequences displaying similar color, could be explained by intergenic recombination which increases the relationship among luciferases of different lanterns, as already reported luciferases of dorsal and ventral lanterns of Jamaican Pyrophorus plagiophtalamus (Feder & Velez, 2009). On the other hand, the luciferases of larval stage (Pyrearinus termitilluminans and Pyrearinus fragilis), were grouped at the same clade, between abdominal and dorsal luciferases of adult stage. The larval luciferases displayed a lower similarity with adult luciferases. These results showed a divergence between adult and larval luciferases, indicating that luminescence in adult and larvae are produced
by two different isoenzymes. Financial Support; FAPESP and CNPq Reference 1. Feder JL, Velez S. Intergenic exchange, geographic isolation, and the evolution of bioluminescent color for Pyrophorus click beetles, Evolution, 2009, 5, 1203-1216. P0010 Development of a highly sensitive and rapid chemiluminescent assay for hydrogen sulfide Hidetoshi Arakawa, Chiaki Nishijima Showa university, Tokyo, Japan Hydrogen sulfide (H2 S) is attracting attention as one of three endogenously generated gaseous signaling compounds, the others being carbon monoxide and nitric oxide. The hydrogen sulfide in live cells is generated by the following three enzymes; cystathionine β-synthase (CBS); cystathionine γ-lyase (CSE); and 3-mercaptopyruvate sulfurtransferase (3MST). These enzymes are involved in neurotransmitter regulation and vasodilatation. However, hydrogen sulfide, the odorous component of waste and sewage, is a toxic gas; therefore, a highly sensitive and specific method for monitoring H2 S is desired in order to protect human health and the environment. Hydrogen sulfide is generally measured by gas chromatography, but this method requires special equipment. Fluorescent probes for hydrogen sulfide have also been recently developed as a simpler method. In order to analyze hydrogen sulfide rapidly and sensitively, we have developed a novel method using lucigenin chemiluminescence in the presence of copper ion (II). Materials and method (1) Assay method; Lucigenin chemiluminescent solution (0.2 mL; 5 µmol/l copper chloride (II), 0.04 mg/mL lucigenin, 0.1 mg/mL TritonX-100) was added to Na2 S solution (20 µl) diluted with phosphate buffer (pH 11.7). Chemiluminecence intensity was measured using an Aloka luminescence reader (Aloka Co. Japan) (waiting time, 10 s; integration time, 10 s). (2) Electron spin resonance method (ESR) for the analysis of radicals; DMPO (5 µl) and lucigenin luminescence reagent (250 µl) was added to Na2 S (25 µl), and the radicals generated were measured by ESR. Superoxide dismutase (SOD) and catalase were used as scavenging enzyme. Results and discussion This is a novel chemiluminescence method based on the principle that light is emitted by metal ions and hydrogen sulfide in the presence of lucigenin. The effects of several metal ions (copper (II), copper (I), zinc, magnesium and aluminum) were studied. Intense luminescence was generated with copper (II). Analysis of reactive oxygen species; Reactive oxygen involved in this chemiluminescent reaction was analyzed using ESR by the addition of a scavenging enzyme, SOD. The emission disappeared upon the addition of SOD. In addition, the generated radical species were analyzed by ESR using the spin trapping agent, DMPO. An ESR signal was observed in the presence of lucigenin. Lucigenin was essential for the generation of reactive oxygen. With the addition of both catalase and SOD, this signal essentially disappeared. This result indicates that the radical species is a superoxide anion. The concept is depicted in Fig. P0010;1. Next the effects of pH, lucigenin, copper (II) and phosphate salt concentration were examined to determine the optimal conditions. The results are shown in (1) of the Materials and Method. Time course; When the time course of chemiluminescence intensity was examined following the addition of Lucigenin chemiluminescent solution, the maximum light emission intensity was obtained within a few seconds, and then the emission gradually decreased. Consequently, this method is capable of rapid measurement (within 10 seconds). Validation of sensitivity, specificity and precision; Under the above conditions, a standard curve of Na2 S shows 1 µmol / l (20 pmol / assay) ~ 10 mmol / l (20 nmol / assay), and reproducibility was from 1.5 to 11.7% (n = 7), with 6.0% as the mean. The specificity of the method was examined using cysteine and glutathione as SH compounds. When compared to sodium sulfide standards at the same concentration, the emission intensity was 2.9% and 4.1 %, respectively, for cysteine and glutathione. Further, by adding maleic imide to the luminescent reagent, the specificity was able to be improved. Thus, this method was found to show high specificity for Na2 S. On-site assay for H2 S The development of an on-site analytical method for hydrogen sulfide is required for environmental and hot spring water analyses. Therefore, hydrogen sulfide was measured using a portable (6 × 16 cm) Lumitester emitting instrument (PD-20, Kikkoman, Japan). The sensitivity of the PD-20 instrument is low compared with more commonly used highly sensitive luminescence instruments. Therefore, for performing on-site measurements, it is necessary to further enhance the emission intensity. Screening of enhancer; The effects of 17 surfactants to enhance chemiluminescence intensity were examined. 3-((3-Cholamidopropyl) dimethylammonio) -1- propanesulfonate (CHAPS) was selected. This zwitterionic detergent increased the emission intensity about tenfold. In the presence of CHAPS, it was possible to obtain a detection limit of Na2 S of 1 × 10(-5) mol /l using the PD-20 instrument. Currently, we are investigating the application of this methodology to biological samples and environmental studies. [Figure: see text] P0011 Chemiluminescent investigation of the antioxidant properties of halogen derivatives of salicylaldehyde benzoyl hydrazone in ROS generating systems Vera Hadjimitova, Nadya Avakumova, Trayko Traykov Department of Medical Physics and Biophysics, Medical University of Sofia, Sofia, 1431, Bulgaria Hydrazones are considered as an important class of organic compounds possessing various biological activities viz antimicrobial, antiviral, anti-inflammatory, anticancer etc. [1]. The aim of the present investigation was to determine in vitro the antioxidant and radical scavenging properties of two derivatives of the iron chelator salicylaldehyde benzoyl hydrazone (SBH) (5-bromosalicylaldehyde-4-hydroxybenzoylhydrazone- B1 and 5-bromosalicylaldehyde-isocotinoilhydrazones – B2 ) and to examine the influence of the molecular structure on their interaction with reactive oxygen species (O2 (─•) , HO(•) ) [2]. ROS were registered by two types of luminol-dependent chemiluminescence assay – luminol-dependent CL in a system of potassium superoxide (KO2 ) – produced O2 (─•) and luminol-dependent CL in a system of iron-dependent hydroxyl radical formation. Detailed description of the methods was published by Hadjimitova et al [3]. The antioxidant and radical scavenging properties of the tested substances were investigated in the concentration range of 3-100 µmol/l. The chemiluminescent response was determined by calculating the area under the obtained chemiluminescent light curve (integral chemiluminescence) for samples containing the tested hydrazones and the control samples. The ratio of CL in the presence and in the absence of the drug was termed CL scavenging index (CL-SI). Over the whole studied concentration range all of the three hydrazones showed slight effect on the luminol-dependent chemiluminescence in the system of non-enzymatically generated O2 (─.) (Fig. ). The decrease of CL-SI was significant only for the highest concentration of 100 µmol/l and its value was about 75%. B2 showed moderate radical scavenging properties which remained unchanged through the tested concentration range. The obtained results demonstrate that the bromine atom doesn’t possess electron-donating activity. In the system iron-dependent hydroxyl radical formation the tested components decreased the chemiluminescence response in a concentration dependent manner (Fig. P0011;2). In the studied concentration range B2 has the strongest antioxidant effect against the HO(•) . At concentration of 10μmol/l the observed effect of B2 is twofold bigger compared with the one of SBH. At the highest concentration B1 and B2 decreased equally the chemilumnescent response. CL-SI is 30% less than this of SBH measured in the same expe
rimental condition. From the obtained results it can be concluded that the investigated hydrazones posses well expressed antioxidant properties against hydroxyl radicals, compared with the demonstrated versus the superoxide radicals. References; 1. Kumar P, Narasimhan B. Hydrazides/hydrazones as antimicrobial and anticancer agents in the new millennium. Mini Rev Med Chem 2013 Jun;13(7);971-87. 2. Nikolova-Mladenova B, Halachev N, Iankova R, Momekov G, Ivanov D. Characterization and cytotoxicity of new salicilaldehyde benzoydrazone derivatives as potential antiproliferative agent. Journal Arzeimittelforschung/drug research 2011;12-F:714-718. 3. Hadjimitova V, Traykov T, Mileva M, Ribarov St. Effect of some psychotropic drugs on luminol-dependent chemiluminescence induced by O2 (─•) , HO(•) , HOCL. Z Naturforsch C 2002;57:1066-1071. [Figure: see text] One ml sample of PBS, pH 7.4, is containing 0.1 mM luminol and the drug in shown concentrations (in control sample drug was omitted). The CL was measured immediately after addition of 20 µl KO2 solution. Therefore, CL was measured using “flash assay” option of the MultiUse program, every 50 miliseconds. [Figure: see text] One ml sample of PBS, pH 7.4, is containing 0.1 mM luminol, 0.1 mM Fe (3+) , 0.1 mM EDTA, 0.1 mM ascorbate, 0.1 mM H2O2 and either of tested drugs at concentration between 3 and 100 μM, or buffer for the controls. The CL was measured using “flash assay” option of the MultiUse program, every 50 milliseconds. P0012 Inhibition of oxygen free radicals induced luminol-dependent chemiluminescence by 4-methoxy derivatives of salicylaldehyde benzoyl hydrazone Nadya Avakumova, Vera Hadjimitova, Trayko Traykov Department of medical Physics and Biophysics, Medical University of Sofia, Sofia, 1431, Bulgaria The fact that reactive oxygen species are implicated in various pathological complications has initiated the development of design strategies for novel synthetic antioxidants possessing optimized antioxidant activity and solubility and reduced potential toxic effects [1]. It has been reported that among the vast spectrum of pharmacological activities viz. antimicrobial, antimalarial, analgesic, anti-inflammatory, antitumoral some hydrazone derivatives possess also antioxidant properties [2]. In this investigation we studied the antioxidant activities and free radical scavenging capacity of newly synthesized 4-methoxy derivatives of salicylaldehyde benzoyl hydrazone (SBH) – 4-methoxy-salicylaldehyde benzoyl hydrazone (M1), 4-methoxy-salicylaldehyde-4-hydroxybenzoyl hydrazone (M2), 4-methoxy-salicylaldehyde-isocotinoyl hydrazone (M3) [3]. The purpose was to compare the results measured using a luminol-dependent chemiluminescence in the presence of in vitro generated O2 (─•) and HO(•) in order to determine the influence of the molecular structure of the hydrazones on the studied properties. The ratio (in percentage) between the chemiluminescent response in the presence and the absence of the tested hydrazone was termed chemiluminescent scavenging index (CL-SI) and reflects the antioxidant properties of the investigated compound. A detailed overview of the method is given in [4]. In both assays the three derivatives present themselves as antioxidant with strong influence of the concentration on the CL-SI values.The ability of the SBH derivatives to scavenge O2 (─•) was tested in a system with potassium superoxide (Fig. 61). The obtained results suggest that the replacement of ─ CH group (M1) with ─ COH group or N atom (M2 and M3 respectively) in the hydrazide ring increases the antioxidant effect. The measurements of the luminol-dependent chemiluminescence in the system of iron-dependent hydroxyl radical formation showed more than threefold decrease in the CL-SI index, at concentration 100 µmol/l (Fig. 62). On the bases of the results it was proposed that the studied 4-methoxy derivatives of SBH are scavengers of HO(•) and the different tested substitution patterns on the benzene ring have slight but not significant influence on the investigated property at the highest tested concentration. The observed activity could be well explained with the electron-releasing inductive effect of the methyl group which redistributes the electron density in the molecule and improves the direct antioxidant electron-donating effect. References; 1. Schulman HM, Hermes-Lima M, Wang EM, Ponka P. In vitro antioxidant properties of the iron chelator pyridoxal isonicotinoyl hydrazone and some of its analogs. Redox Rep. 1995;1:373-378. 2. Rollas S, Küçükgüzel SG. Biological Activities of Hydrazone Derivatives Molecules 2007;12(8);1910-1939 3. Mladenova B, Momekov G, Ivanov D. Synthesis and physicochemical characteristics of new salicilaledehyde benzoylhydrazone derivatives with high cytotoxic activity. Pharmacia 2011;LVIII(1-4);41-44. 4. Hadjimitova V, Traykov T, Mileva M, Ribarov St. Effect of some psychotropic drugs on luminol-dependent chemiluminescence induced by O2 (─•) , HO(•) , HOCL. Z Naturforsch C 2002;57:1066-1071. [Figure: see text] The solution in the sample cuvette comprises 1 ml phosphate buffer solution pH 7.4, containing 0.1 mM luminol and the hydrazone derivatives at concentration as indicated. In control sample the studied hydrazones were omitted. The CL was measured immediately after addition of 20 µl KO2 solution [Figure: see text] The solution in the sample cuvette comprises 1 ml phosphate buffer solution pH 7.4, containing 0.1 mM luminol, 0.1 mM Fe(3+) , 0.1 mM EDTA, 0.1 mM ascorbate, 1mM H2 O2 and the hydrazone derivatives at concentrations as indicated, or a buffer for the controls. P0013 Peroxyoxalate reaction in aqueous carbonate buffer media Fernando H. Bartoloni(a,b) , Ana Paula E. Pagano(a) , Felipe A. Augusto(a) , Wilhelm J. Baader(a) (a) Departamento de Química Fundamental, Instituto de Química, Sao Paulo, SP, Brazil (b) Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo Andre, SP, Brazil The peroxyoxalate system is commonly known as the reaction of an oxalic ester with hydrogen peroxide in the presence of a base and an activator.(1,2) In analytical chemistry it is very important as a detection tool, because of its low costs and high sensibility.(2) Although the behavior of the system is well known in organic media,(3-5) most of these applications are for the detection of analytes in aqueous media, in which competitive processes like ester hydrolysis can reduce the high chemiluminescence quantum yields normally obtained by this reaction.(2,6) Therefore, kinetic studies of this reaction in aqueous media are extremely important to understand mechanistic details like the relationship between ester hydrolysis and perhydrolysis and how this relation affects the chemiexcitation efficiency. In order to characterize the peroxyoxalate reaction in aqueous medium, the reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) with hydrogen peroxide and fluorescein (FLU) as activator was studied in aqueous carbonate buffer. The observed rate constants (kobs ) of the chemiluminescence emission intensity decay showed to increase with increasing medium pH; typical experiments were performed in pH = 10.4, where carbonate shows maximum buffering capacity. The kobs values showed no dependence on the buffer concentration in a wide range of hydrogen peroxide concentrations (Fig. 63, for [H2 O2 ] = 10 mmol L(-1) ), indicating the occurrence of specific base catalysis. Thus, the observed rate constants showed linear dependence on the [HO2 (-) ] (Fig. 1), with a bimolecular perhydrolysis rate constant of kper  = 5.9 10(3) L mol(-1) s(-1) ; the pseudo-first order hydrolysis rate constant in these condition is k0  = 1.2 s(-1) ; leading to a bimolecular hydrolysis rate constant by hydroxide ion of khyd  = 4.8 10(3) L mol(-1) s(-1) (Fig. 63). [Figure: see text] The present study indicates that the peroxyoxalate reaction
in aqueous carbonate buffer medium leads to reproducible kinetic curves and this system can therefore be utilized for analytical assays in aqueous environment. Although dark hydrolysis occurs in this system, the emission efficiency is high enough for possible applications. The rate constants are linearly dependent on the peroxide concentration, however, independent of the carbonate concentration indicating specific base catalysis. Financial support; FAPESP, Capes, CNPq. References 1. Bartoloni FH, Bastos EL, Ciscato LFML, Peixoto MMdeM, Santos APF, Santos CS, Oliveira S., Augusto FA, Pagano APE, Baader WJ. Quim. Nova 2011;34:544. 2. Ciscato LFML, Augusto FA, Weiss D, Bartoloni FH, Albrecht S, Brandl H, Zimmermann T, Baader WJ. ARKIVOC 2012;2012:391. 3. Da Silva SM, Casallanovo F, Oyamaguchi KH, Ciscato LFML, Stevani CV, Baader WJ. Luminescence 2002;17:313. 4. Ciscato LFML, Bartoloni FH, Bastos EL, Baader WJ. J. Org. Chem. 2009;74:8974. 5. Stevani CV, Campos IPA, Baader WJ. J. Chem. Soc., Perkin Trans. 2 1996;1645. 6. Augusto FA, Souza GA, Souza Junior SP, Khalid M, Baader WJ. Photochem. Photobiol. 2013;89:1299. P0014 Change of fluorescence spectra of discharged photoprotein obelin under variation of physico-chemical conditions Nadezhda Belogurova(a,b) , Nadezhda Kudryasheva(a,b) (a) Institute of Biophysics SB RAS, Krasnoyarsk, Russia (b) Siberian Federal University, Krasnoyarsk, Russia The assay of calcium-regulated photoproteins is highly sensitive, non- hazardous and their bioluminescence reactions triggered by calcium ions are rapid and simple. It makes photoproteins attractive for applications as a reporter protein [1]. Obelin isolated from hydroid Obelia longissima is one of the most studied among photoproteins. The product of the bioluminescent reaction of obelin (enzyme-bound chromophore, coelenteramide) is a fluorescent protein; It is called ‘discharged’ obelin. Because discharged obelin is stable and nontoxic it can be used as a fluorescent marker in biological and medical investigations, for example, in cytology, histology, and cryology. Fluorescence spectra of discharged obelin (and hence, emission color) are variable; they might depend on external physic-chemical conditions [2,3]. Therefore, it is important to study changes of fluorescent characteristics of the discharged obelin under variation of physico-chemical conditions – temperature and/or accompanying agents. Fluorescent spectra of discharged obelin were studied under exposure to 40°C (0 – 12.5 hours) [4] and addition of glycerin (С = 0.06 – 0.36 М), ethanol (С = 0.01 – 1.18 М) [5], dimethyl sulfoxide (С = 0.002 – 2.65 М), and polyethylene glycol (С = 1.7 10-5 – 1.7 10-2 М). We chose glycerin, ethanol, dimethyl sulfoxide and polyethylene glycol as accompanying agents extensively used in cytology, histology, and cryology. Increase of exposure time and concentration of the agents decreased intensity and changed color of discharged obelin fluorescence. The discharged obelin spectra obtained under different physico-chemical conditions were analyzed by deconvolving into spectral Gauss components. A red peak (λmax  = 660 nm) attributed to indole-coelenteramide exciplex was newly discovered in the discharged obelin emission induced by UV radiation. The expansion of the known group of colors (from violet to yellow) by adding the red color increases the potential of discharged obelin as a colored biomarker for monitoring of biochemical processes. Decay of the fluorescence intensity at exposure to 40°C was analyzed [6]. Permissible ranges of the accompanying agent concentrations were defined. Acknowledgements This work supported by the Pogramme “Molecular and Cellular Biology” of the Russian Academy of Sciences. References; 1. Markova SV, Vysotski ES, Lee J. In Bioluminescence and Chemiluminescence, Case JF, Harring PJ, Robison BH, Haddock SHD, Kricka LJ, Stanley PE, Eds. Singapore; World Scientific Printers (S) Pte Ltd 2000;11:115-118. 2. Belogurova NV, Kudryasheva NS. J Photochem Photobiol B 2010;101:103-108. 3. Belogurova NV, Kudryasheva NS. LUMINESCENCE 2012;27:100. 4. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. LUMINESCENCE 2012;27:96. 5. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem DOI; 10.1007/s00216-014-7685-z. 6. Alieva RR, Belogurova NV, Petrova AS, Kudryasheva NS. Anal Bioanal Chem 2013;405:3351-3358. P0015 Purification of a Novel Luciferase from Luminous Fish, Parapriacanthus ransonneti Manabu Bessho, Yuichi Oba Nagoya University, Nagoya, Japan The shallow water fish Parapriacanthus ransonneti uses cypridinid luciferin for its bioluminescence. Haneda and Johnson (1958) demonstrated the luciferin-luciferase cross-reaction between P. ransonneti and luminous ostracod Vargula hilgendorfii. Then, Johnson et al. (1961) showed that the structure of luciferin in both species was identical, and concluded that P. ransonneti emits blue light by using ingested luciferin from luminous ostracods. However, the identity of the luciferase in this unique luminous system remains unresolved. This study seeks to identify the luciferase from P. ransonneti. We performed purification of the luciferase from the thoracic luminous organs of 200 P. ransonneti specimens collected in Japan using anion exchange chromatography, gel filtration chromatography and SDS-PAGE. We successfully obtained nearly pure protein, whose relative luminescence activity was 28 times higher than that of crude extracts from the thoracic luminous organs. Peptide sequencing of the active fractions is in progress. P0016 Chitosan- induced Au/Ag alloy nanoparticles dispersed in ion liquid and its application in developing an ultrasensitive glucose chemiluminescence biosensor Mohammad Javad Chaichi, Seyedeh Olia Alijanpour University of Mazandaran, Babolsar, Mazandaran, Iran Introduction Biosensors have attracted much attention during recent times because of the potential applications of these devices in the clinical diagnostics, environmental monitoring, pharmaceuticals, and food processing industries due to their fast response and ease of operation [1,2]. A novel glucose biosensor based on the chemiluminescence (CL) detection of enzymatically generated H2 O2 was constructed by one covalent immobilization of glucose oxidase in CL cell. The chemiluminescence-based biosensors are developing due to their high sensitivity and excellent performance. Reagents immobilization onto proper substrates plays an important role in the development of the high-quality CL-based biosensors [3]. In this regard, the immobilization of enzyme is a key step for constructing an enzyme-based CL biosensor. Emergence and recent advance of nanoscience and nanotechnology open new opportunities for the application of nanoparticles in bioanalysis [4]. Imidazolium ion based ion liquids (IL) have recently been shown as promising compounds for preparation and stabilization of nanomaterial, and have found application in transition metal catalyzed CL reactions [5]. This paper describes the synthesis and characterization of chitosan-induced Au/Ag alloy NPs dispersed in IL, and use of them in the construction of a novel biosensor for determination of glucose in pharmaceuticals sample by CL reaction. Materials and methods Chemiluminescence measurements were carried out by a Sirius tube luminometer (Berthold detection system, Germany) with a photomultiplier tube detector in a light-tight globular bottom glass cell of 10 mm diameter. The chitosan- induced Au/Ag alloy NPs dispersed in ion liquid were synthesised base on microwave method [6]. The CL biosensor based on covalent immobilization of glucose oxidase and gold-silver alloy NPs dispersed in ion liquid on glutaraldehyde-functionalized glass cell was fabricated. Results and discussion The analytical performance of the proposed biosensor was examined under the optimum condition. The calibration graph of emission intensity (I) versus glucose concentration was linear in the range of 1.8 × 10 (-6) to 7. 5 ×&thin
sp;10 (-3) M (insert in Fig. 1), and the detection limit was 4.5 × 10(-7) M (S/N = 3). The regression equation was I = 1 × 10(+6) C + 14983 (where C is the glucose concentration, M) with a correlation coefficient of 0.9975 (n = 10). A complete analysis, including sampling and washing, could be performed in less than 2 min with a relative standard deviation of less than 2% for 5.0 × 10(-4) M glucose (n = 12). All those results indicated that the performance of the CL biosensor was compatible with those of the known and well assessed glucose CL sensors. The results with the present method for serum samples agreed with those obtained by the laboratory method. [Figure: see text] References; 1. Ionescu RE, Cosnier S, Marks RS. Anal. Chem. 2006;78:6327-6331. 2. Li B, Lan D, Zhang Z. Anal. Biochem. 2008;374:64-70. 3. Zhang Z, Zhang S, Zhang X. Rev. Anal. Chim. Acta 2005;541:37-47. 4. Haghighi B, Bozorgzadeh S.. Microchem. J. 2010;95:192-197. 5. Zhou Y. Curr. Nanosci. 2005;1:35-42. 6. Fan C, Li W, Zhao S, Chen J, Li X. Mater. Lett. 2008;62:3518-3520. P0017 The Study of Electrobioluminescence of Microscolex phosphoreus that found in Iran Mohammad javad Chaichi, Moslem Mansour Lakouraj, Shahram Ghasemi, Afsaneh Nemati University of Mazandaran, Babolsar, Mazandaran, Iran Introduction A method of the electrical pulse stimulation of an bioluminescence animal or electrobioluminescence(EBL) generates luminescence during a potential sweep or potential step programming [1-2] when the potential of electrode reaches to negative potential. This method has been previously demonstrated by Santhanam [1-4]. In EBL of the earthworm by potential sweep or electrolysis of an earthworm as electrode in an inert electrolyte solution produces luminescence [3]. This luminescence is due to the inflow of electron into the electrode, where oxygen is reduced to peroxide [3]. In continuation of these studies, we investigated the effect of addition salts solution to electrolyte solution on EBL process. It is a first report of existing of bioluminescent earthworm in Iran. Experimental In this method we used from connection Berthold detection systems, Sirius-tube luminometer and Dropsens, bipotentiostat/galvanostat (μStat 400) to each other. Each of instruments was connected to individual computer via different interface. Bioluminescent earthworms, that collect in the north of Iran, M. phosphoreus were washed well with distilled water and the outer surface were dried between the fold of filter paper. For producing earthworm electrode a gold wire (0.3mm × 5cm) was inserted into 15 segment of the post-clitelium region. Earthworm electrode as working electrode together with two graphite (2mm × 5cm) as the counter and reference electrode were placed into a cell which was made of pyrex. As shown in Fig. 65 the electrobioluminescence cell was placed instead of the common cell into luminometer instrument and it was connected to a μStat 400 by a function generator. Emission of light was investigated by luminometer instrument. Electrochemical behavior was investigated by cyclic voltammetry. [Figure: see text] Result and discussion The cyclic voltammetry of the earthworm electrode as base state recorded in 8mL of 0.1M phosphate buffer (pH = 7.5) as electrolyte solution with repetitive five cycles of the potential from 0.5 to -1.0 V and potential sweep rate of 50 mVs. Emission of the light was occurred continuously in the sweep towards negative potentials and the intensity of the emission was higher at more values of negative potential. First step is oxygen reduction. This step initiates luminescence in about E = -0.56 V. Generally it tends to move toward negative values. The second step in the reduction of O2 occurs at a more negative potential due to H2 O2 . This step would occur at about E = -0.90 V and in this potential, the intensity of the emission is maximum, after this potential at the return cycling toward positive potential, the light emission would return to the base value. For investigation the effect of addition salts solution to electrolyte solution on EBL process, 10μL of 10(-6) M salts added into electrobioluminescence cell and the effect of each other of the salts investigated individually. The curve of the cyclic voltammograms in presence of salts solution almost similar behavior during input potential programming. For example as seen in Fig. 66 for about Fe(3+) ion, the other ions such as Cu(2+) , Ca(2+) , Fe(2+) and Zn(2+) have enhance effect on the electrobioluminescence output, but Fe(2+) ion had more effective than the others. Therefore we can determine the ions and hydrogen peroxide by the EBL procedure. [Figure: see text] References; 1. Limaye NM, Santhanam KSV. Bioelectrochem. Bioenerg 1990;24:249-256. 2. Limaye NM, Santhanam KSV. Bioelectrochem. Bioenerg 1988;19:9-19. 3. Limaye NM, Santhanam KSV. Bioelectrochem. Bioenerg 1986;15:341-351. 4. Ismail SA, Limaye NM, Santhanam KSV. Bioelectrochem. Bioenerg 1985;14:405-416. P0018 BRET(1) -assay using the FDSSμCell imaging plate reader; monitoring agonist-induced β-arrestin recruitment to a G protein-coupled receptor (GPCR) Frédéric Finana(a) , Jean Marc D’Angelo(b) , Luc De Vries(a) , Isabelle Rauly Lestienne(a) , Quynh-nhu Trinh-Xuan Kramer(b) , Romain Kramer(b) , Cyril Guerinot(a) (a) Centre de Recherche Pierre Fabre, Castres, France (b) Hamamatsu Photonics Europe, Massy, France The GPCRs represent the largest family of cell surface receptors and are the main target for drugs available on the pharmaceutical market. To prevent receptors from both acute and chronic overstimulation, GPCR activity is regulated by an intensively studied mechanism called desensitization or internalization. Following ligand exposure, arrestins interact with phosphorylated GPCRs, uncoupling them from their cognate G protein,blocking further activation and promoting endocytosis (2). Interaction between receptors and β-arrestins is a measurable functional event in the GPCR-mediated signaling cascade. The biophysical technique named Bioluminescence Resonance Energy Transfer (BRET) has been widely used to monitor and quantify agonist-promoted β-arrestin recruitment, including high throughput screenings. For the first time, Pierre Fabre Research Institute presents this BRET1 application on the FDSS/μCELL imaging plate reader (HAMAMATSU PHOTONICS) by monitoring the activity of the dopaminergic D2 receptor (short splice form), a prototypic and well characterized GPCR. P0019 Opsin-based extraocular photoreception in a luminous brittle star Jérôme Delroisse(a) , Esther Ullrich-Lüter(b) , Olga Ortega-Martinez(c) , Jérôme Mallefet(d) , Patrick Flammang(a) (a) Biology of Marine Organisms and Biomimetics, University of Mons, Mons, Belgium (b) Museum für Naturkunde, Berlin, Germany (c) Department of Biological and Environmental Science, University of Gothenburg, Kristineberg, Sweden (d) Laboratory of Marine Biology, Catholic University of Louvain, Louvain-La-Neuve, Belgium In the marine world, photoreception and bioluminescence constitute two opposite but intermingled phenomena related to light. Photoreception is a prerequisite for the bioluminescence perception and a luminous signal have to be perceived – by a prey, a predator or a conspecific – to be functionally efficient. Additionally photoreception could also be needed for the control of the bioluminescence process. In 2009, Tong et al. showed that the bioluminescent organ of a sepiolid squid possess light detection capability and use it to control the bacterial population of the photophore (and indirectly the light emission) [1]. More recently [2], molecular markers of photoreception – such as opsins – have also been identified in the photocyte of a ctenophore leading to the same conclusion of an intimate coupling between light perception and emission. Behavioral, morphological and molecul
ar studies have shown that at least some echinoderms species have developed photoreception capabilities [3, 4]. Because of the lack of distinguishable eyes, echinoderm photoreception is usually considered as diffuse and tegumentary which makes that group particularly enigmatic! With the publication of the purple sea-urchin genome, a new window was opened on the understanding of the sensory capabilities in the phylum of echinoderms. The luminous brittle star Amphiura filiformis is known to use photoreception to synchronize its suspension feeding activity [5] but data are lacking concerning the molecular actors involved in the light perception. Because brittle star luminescence is considered to be highly controlled [6], could extraocular photoreception be linked to the light emission in these particular organisms? As previously shown in non-related marine species, extraocular photoreception could be used to control the photogenesis in brittle stars. Using transcriptomic and genomic data, we reported a decade of new putative opsin genes in the luminous brittle star A. filiformis. For nine A. filiformis opsin candidates, the Schiff base needed for the chromophore linkage was identified and the bona fide opsin status was confirmed. A. filiformis genome indeed codes for a large diversity of opsins including rhabdomeric and ciliary opsins but also minor groups of opsins such as Go opsins, neuropsins and peropsins. Transcriptome analysis and immunodetections revealed opsin expression in various organs of the brittle star. Interestingly, ciliary opsins and luciferase-like were co-immunodetected in the spines of the brittle star also described as the only light emitting areas of the brittle star. As light emission and perception actors seem to be expressed in a common organ, we hypothesize a dual role for the brittle star spine in both light emission and reception. A possible linkage between the two light-mediated processes is proposed. The optical implication of the spine skeleton in light perception and/or light emission is also suggested. Acknowledgements Contribution to the “Centre interuniversitaire de la Biologie Marine” (Belgium). Work supported in part by a FRFC Grant n° 2.4590.11. References 1. Tong D, Rozas NS, Oakley TH, Mitchell J, Colley NJ, McFall-Ngai MJ. Evidence for light perception in a bioluminescent organ. Proceedings of the National Academy of Sciences 2009;106(24);9836-9841. 2. Schnitzler CE, Pang K, Powers ML, Reitzel AM, Ryan JF, Simmons D, et al.. Genomic organization, evolution, and expression of photoprotein and opsin genes in Mnemiopsis leidyi; a new view of ctenophore photocytes. BMC biology 2012;10(1);107. 3. Hendler G. An echinoderm’s eye view of photoreception and vision. In Echinoderms; Munchen; Proceedings of the 11th International Echinoderm Conference, 6-10 October 2003, Munich, Germany. Taylor & Francis, 2006;339. 4. Ullrich-Lüter EM, D’Aniello S, Arnone MI. C-opsin Expressing Photoreceptors in Echinoderms. Integrative and comparative biology 2013;53(1);27-38. 5. Delroisse J, Flammang P, Mallefet J. Between emission and perception; do luminous brittlestars perceive their own light? In Proceedings of the 17th international symposium on Bioluminescence and Chemiluminescence, 2012. 6. Dewael Y, Mallefet J.. Luminescence in ophiuroids (Echinodermata) does not share a common nervous control in all species. Journal of experimental biology 2002;205(6);799-806. P0020 Luciferase-based microfluidic bioassays Ivan Densiov(a) , Anton Yakimov(a) , Kirill Lukyanenko(a) , Peter Belobrov(a,b) (a) Sibarian Federal University, Krasnoyarsk, Russia (b) Institute of biophysics SB RAS, Krasnoyarsk, Russia Bioluminescence inhibition assay based on components of bioluminescence system of luminous bacteria is the perspective express method for detection of organic and inorganic pollutants in liquids [1]. The method is based on interaction of pollutants with bacterial luciferase and NADH;FMN-oxidoreductase form the bacterial luminescence system, that leads to quenching of light emission and changing of a shape of measured kinetic curves. To make the portable device for environmental monitoring based on this method the automation is necessary. The purpose of the work was to test the possibility of automation of such bienzyme bioassay process through the integration of all reagents in one microfluidic chip. Channelized surface of the microfluidic chip was formed on the plate of polymethyl methacrylate (PMMA) by direct cutting [2] with Roland MDX-20. Luciferase from a recombinant strain of Escherichia coli, NADH-FMN-oxidoreductase from Vibrio fischeri, NADH and aldehyde were immobilized in starch gel [1] and placed in the reactor chamber (Fig. 67). FMN for reaction activation was deposited in a special chamber by the process of drop drying. Hermetic sealing was carried out by sticking a second plate of PMMA on 3M 467MR adhesive. [Figure: see text] Sample proceeded through the input channel into a chamber with FMN, where it dissolved FMN, and then stirred with it in the serpentine mixer [3]. When the sample with FMN entering the chamber with immobilized reagents and bioluminescence reaction starting. The kinetics of bioluminescence reactions was recorded during 200 seconds by the GloMax 20/20 single tube luminometer (Promega, USA). The test measurements of bioluminescence were conducted after 6 months of storage of chips with immobilized reagents in room conditions (23˚C, 1 atm, 30% relative humidity, direct sunlight). Second check was conducted after another 6 months. It was shown that the proposed microfluidic chip allows automating the process of bioassays; mixing of the sample with bienzyme systems and substrates – all in a single chip with pre-defined proportions of substrates without attracting qualified staff. The automation of bioassays in microfluidic chip allows softens requirements for the storage of immobilized reagents, as evidenced by the performance of chips stored under normal room conditions. This is true when stored for six months. After one year of storage the luciferase activity drops to 5% level. It was found that the adhesive used for hermetic sealing is hydrophobic and additional equipment (pump) must be used to fill the capillaries with the liquid sample. It was found that the luminescence intensity depends on the shape of the chamber with FMN (Fig. 68). [Figure: see text] The process of the luciferase-based bioassay based on quenching measuring can be automated by the usage of microfluidic technology and immobilization methods. The proposed topology, although in need to be further developed, but still demonstrates the ability to adapt the luciferase bioassay for use in more complex devices for personal ecology. 1. Esimbekova E, Kondik A, Kratasyuk V. Bioluminescent enzymatic rapid assay of water integral toxicity. Environmental Monitoring and Assessment 2013;185(7);5909-5916. 2. Howell Jr PB, Golden JP, Hilliard LR, Erickson JS, Mott DR, Ligler FS. Two simple and rugged designs for creating microfluidic sheath flow. Lab Chip 2008;8:1097-1103. 3. Capretto L, Cheng W, Hill M, Zhang X. Micromixing Within Microfluidic Devices. Top Curr Chem 2011;304:27-68. P0021 Quantum dynamics simulations of model chemiluminescence systems Ignacio Fernández Galván, Hans Karlsson, Michael Stenrup, Roland Lindh Uppsala Universitet, Uppsala, Sweden A common mechanistic feature of chemiluminescent and bioluminescent reactions is the existence of an “entropic trap”, a region in the configuration space where the system

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