Journal of Fluorescence

, Volume 22, Issue 3, pp 971–992 | Cite as

Fluorescence and Electrochemical Recognition of Nucleosides and DNA by A Novel Luminescent Bioprobe Eu(lll) -TNB

  • Hassan A. Azab
  • E. M. Mogahed
  • F. K. Awad
  • R. M. Abd El Aal
  • Rasha M. Kamel
Original Paper


The luminescence arising from lanthanide cations offers several advantages over organic fluorescent molecules: sharp, distinctive emission bands allow for easy resolution between multiple lanthanide signals; long emission lifetimes (μs –ms) make them excellent candidates for time-resolved measurements; and high resistance to photo bleaching allow for long or repeated experiments. A method is presented for determination of nucleosides using the effect of enhancement of fluorescence of the easily accessible europium(III)-TNB in presence of different nucleosides. The latter coordinates to Eu(III) -TNB and enhances its luminescence intensity as a result of the displacement of water from the inner coordination sphere of the central metal. A similar method for the determination of DNA based on the quenching of Eu(III)-TNB has been established. The interaction of Eu(III)-4,4,4 trifluoro-1-(2-naphthyl)1,3-butanedione (TNB) complex with nucleosides (NS) (guanosine, adenosine, cytidine , inosine) and DNA has been studied using normal and time-resolved luminescence techniques. Binding constants were determined at 293 K, 298 K, 303 K, 308 K and 313 K by using Benesi-Hildebrand equation. A thermodynamic analysis showed that the reaction is spontaneous with ΔG being negative. The enthalpy ΔH and the entropy ΔS of reactions were all determined. The formation of binary and ternary complexes of Eu (III) with nucleosides and TNB has been studied potentiometrically at (25.0 ± 0.1) °C and ionic strength I = 0.1−3 (KNO3) . The formation of the 1:1 binary and 1:1:1 ternary complexes are inferred from the corresponding titration curves. Initial estimates of the formation constants of the resulting species and the protonation constants of the different ligands used have been refined with the HYPERQUAD computer program. Electrochemical investigations for the systems under investigations have been carried out using cyclic voltammetry (CV), differential pulse polarography (DPP), and square wave voltammetry (SWV) on a glassy carbon electrode in I = 0.1 mol/L p-toluenesulfonate as supporting electrolyte.


Europium-TNB Adenosine Guanosine Cytidine Inosine DNA Time-resolved luminescence Voltammetry Glassy carbon electrode 


  1. 1.
    Jiao K, Wang QX, Sun W, Jian FF (2005) Synthesis, characterization and DNA-binding properties of a new cobalt(II) complex: Co(bbt)2Cl2. J Inorg Biochem 99:1369–1375PubMedCrossRefGoogle Scholar
  2. 2.
    Sigman DS, Graham DR, Aurora VD, Stern AM (1979) Oxygen-dependent cleavage of DNA by the 1,10-phenanthroline. cuprous complex. Inhibition of Escherichia coli DNA polymerase I. J Biol Chem 254:12269–12272PubMedGoogle Scholar
  3. 3.
    Yang ZS, Wang YL (2004) Electrochemically induced DNA cleavage by copper-bipyridyl complex. Electrochem Commun 6:158–163CrossRefGoogle Scholar
  4. 4.
    Cater MT, Bard AJ (1987) Voltammetric studies of the interaction of tris(1,10-phenanthroline)cobalt(III) with DNA. J Am Chem Soc 109:7528–7530CrossRefGoogle Scholar
  5. 5.
    Kawanishi S, Inoue S, Sano S (1986) Mechanism of DNA cleavage induced by sodium chromate(VI) in the presence of hydrogen peroxide. J Biol Chem 261:5952–5958PubMedGoogle Scholar
  6. 6.
    Chen QY, Li DH (1999) Interaction of a novel red-region fluorescent probe. Nile Blue, with DNA and its application to nucleic acids assay. Analyst 124:901–906PubMedCrossRefGoogle Scholar
  7. 7.
    Li YF, Huang CZ, Huang XH (2001) Determination of DNA by its enhancement effect of resonance light scattering by azur A. Anal Chim Acta 429:311–319CrossRefGoogle Scholar
  8. 8.
    Wang J, Xu D, Kawde AN, Polsky R (2001) Metal nanoparticle-based electrochemical stripping potentiometric detection of DNA Hybridization. Anal Chem 73:5576–5581PubMedCrossRefGoogle Scholar
  9. 9.
    Wang H, Li WR, Lu Y, Fu NN, Zhang HS (2005) Vibrational frequencies and infrared intensities of the hydrogen-bonded complexes of nitrous acid with ethers: ab initio and DFT studies. Spectrochim Acta A 61:2103–2107CrossRefGoogle Scholar
  10. 10.
    Zhang S, Zhong H, Ding C (2008) Chronocoulometric detection of DNA hybridization using reporter DNA probe modified with Au nanoparticles based on porous gold leaf electrode. Anal Chem 80:7206–7212PubMedCrossRefGoogle Scholar
  11. 11.
    Zou QC, Yan QJ, Song GW, Zhang SL, Wu LM (2007) Detection of DNA using cationic polyhedral oligomeric silsesquioxane nanoparticles as the probe by resonance light technique. Biosens Bioelectron 22:1461–1465PubMedCrossRefGoogle Scholar
  12. 12.
    Niu S, Singh G, Saraf RF (2007) Label-less fluorescence-based method to detect hybridization with applications to DNA micro-array. Biosens Bioelectron 23:714–720PubMedCrossRefGoogle Scholar
  13. 13.
    Fernandez MR, Gonzalez MJV, Garcia MED (1997) Room-temperature phosphorescent palladium–porphine probe for DNA determination. Anal Chem 69:2406–2410CrossRefGoogle Scholar
  14. 14.
    Churchwell MI, Beland FA, Doerge DR (2002) Quantification of multiple DNA adducts formed through oxidative stress using liquid chromatography and electrospray tandem mass spectrometry. Chem Res Toxicol 15:1295–1301PubMedCrossRefGoogle Scholar
  15. 15.
    Hason S, Pivonkova H, Vetter V, Fojta M (2008) Label-free sequence-specific DNA sensing using copper-enhanced anodic stripping of purine bases at boron-doped diamond electrodes. Anal Chem 80:2391–2399PubMedCrossRefGoogle Scholar
  16. 16.
    Benedict JD, Forsham PH, Stetten D (1949) The metabolism of uric acid in the normal and gouty human studied with the aid of isotopic uric acid. J Biol Chem 181:183PubMedGoogle Scholar
  17. 17.
    Waslien CI, Calloway DH, Margen S (1968) Uric acid production of men fed graded amounts of egg protein and yeast nucleic acid. Am J Med 21:892Google Scholar
  18. 18.
    Griebsch A, Zollner N (1974) Effect of ribomononucleotides given orally on uric acid production in man. Adv Exp Med Biol 41:443PubMedGoogle Scholar
  19. 19.
    Garrel DR, Verdy M, PetitClerc C, Martin C, Bruke D, Hamet P (1991) Lowering of HDL2-cholesterol and lipoprotein A-I particle levels by increasing the ratio of polyunsaturated to saturated fatty acids. Am J Clin Nutr 53:665PubMedGoogle Scholar
  20. 20.
    Loffler W, Grobner W, Medina R, Zollner N (1982) Influence of dietary purines on pool size, turnover, and excretion of uric acid during balance conditions. Isotope studies using 15N-uric acid. Res Exp Med (Berl) 181:113CrossRefGoogle Scholar
  21. 21.
    Yu TS, Berfe L, Gutman AB (1962) Renal function in gout. II. Efect of uric acid loading on renal excretion of uric acid. Am J Med 33:829PubMedCrossRefGoogle Scholar
  22. 22.
    Choi HK, Liu S, Curhan G (2005) Intake of purine-rich foods, protein, and dairy products and relationship to serum levels of uric acid: the Third National Health and Nutrition Examination Survey. Arthritis Rheum 52:283PubMedCrossRefGoogle Scholar
  23. 23.
    Harris MD, Siegel LB, Alloway JA (1999) Gout and hyperuricemia. Am Fam Physician 59:925PubMedGoogle Scholar
  24. 24.
    Gibson T, Rodgers AV, Simmonds HA, Court-Brown F, Todd E, Meilton V (1983) A controlled study of diet in patients with gout. Ann Rheum Dis 42:123PubMedCrossRefGoogle Scholar
  25. 25.
    Faller J, Fox IH (1982) Ethanol-induced hyperuricemia: evidence for increased urate production by activation of adenine nucleotide turnover. N Engl J Med 307:1598PubMedCrossRefGoogle Scholar
  26. 26.
    Cao G, Russel RM, Lischner N, Prior RL (1998) Serum antioxidant capacity is increased by consumption of strawberries, spinach, red wine or vitamin C in elderly women. J Nutr 128:2383PubMedGoogle Scholar
  27. 27.
    Yamaoka N, Kaneko K, Kudo Y, Aoki M, Yasuda M, Mawatari K, Yamada Y, Yamamoto T (2010) Analysis of purine in purine-rich cauliflower. Nucleos Nucleot Nucleic Acids 29:518CrossRefGoogle Scholar
  28. 28.
    Clifford AJ, Riumallo JA, Young VR, Scrimshaw NS (1976) Effect of oral purines on serum and urinary uric acid of normal. Hyperuricemic and gouty humans. J Nutr 106:428Google Scholar
  29. 29.
    QianbT CZ, Yang MS (2004) Determination of adenosine nucleotides in cultured cells by ion-pairing liquid chromatography-electrospray ionization mass spectrometry. Anal Biochem 325:77CrossRefGoogle Scholar
  30. 30.
    Cordell RL, Hill SJ, Ortori CA, Barrett DA (2008) Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry. J Chromatogr B 871:115CrossRefGoogle Scholar
  31. 31.
    Cai Z, Song F, Yang MS (2002) Capillary liquid chromatographic-high-resolution mass spectrometric analysis of ribonucleotides. J Chromatogr A 976:135PubMedCrossRefGoogle Scholar
  32. 32.
    Vela JE, Olson LY, Huang A, Fridland A, Ray AS (2007) Simultaneous quantitation of the nucleotide analog adefovir, its phosphorylated anabolites and 2′-deoxyadenosine triphosphate by ion-pairing LC/MS/MS. J Chromatogr B 848:335CrossRefGoogle Scholar
  33. 33.
    Carli D, Honorat M, Cohen S, Megherbi M, Vignal B, Dumontet C, Payen L, Guitton J (2009) Simultaneous quantification of 5-FU, 5-FUrd, 5-FdUrd, 5-FdUMP, dUMP and TMP in cultured cell models by LCMS/MS. J Chromatogr B 877:2937CrossRefGoogle Scholar
  34. 34.
    De Abreu RA, Van Baal JM, De Bruyn CH, Bakkeren JA, Schretlen ED (1982) Investigation of catecholamine metabolism using high-performance liquid chromatography. J Chromatogr 229:67PubMedCrossRefGoogle Scholar
  35. 35.
    Klawitter J, Schmitz V, Klawitter J, Leibfritz D, Christians U (2007) Development and validation of an assay for the quantification of 11 nucleotides using LC/LC–electrospray ionization–MS. Anal Biochem 365:230PubMedCrossRefGoogle Scholar
  36. 36.
    Gill BD, Indyk HE (2007) Determination of nucleotides and nucleosides in milks and pediatric formulas: a review. J AOAC Int 90:1354PubMedGoogle Scholar
  37. 37.
    Crauste C, Lefebvre I, Hovaneissian M, Puy JY, Roy B, Peyrottes S, Cohen S, Guitton J, Dumontet C, Perigaud C (2009) Validation and long-term evaluation of a modified on-line chiral analaytical method for therapeutic drug monitoring of (R, S)-methadone in clinical samples. J Chromatogr B 877:1417CrossRefGoogle Scholar
  38. 38.
    Elbanovski M, Makowska B (1996) The lanthanides as luminescent probes in investigations of biochemical systems J. Photochem Photobiol A 99:85CrossRefGoogle Scholar
  39. 39.
    Gudgin Dickson EF, Pollak A, Diamandis EP (1995) Time-resolved detection of lanthanide luminescence for ultrasensitive bioanalytical assays. J Photochem Photobiol B 27:3CrossRefGoogle Scholar
  40. 40.
    Lis S, Elbanowski M, Makowska B, Hnatejko Z (2002) Energy transfer in solution of lanthanide complexes. Photobiology A 150:233CrossRefGoogle Scholar
  41. 41.
    Azab HA, Anwar ZM, Ahmed RG (2010) Pyrimidine and purine mononucleotides recognition by trivalent lanthanide complexes with N-acetyl amino acids. J Chem Eng Data 55(1):459–475CrossRefGoogle Scholar
  42. 42.
    Azab HA, El-Korashy SA, Anwar ZM, Hussein BHM, Khairy GM (2010) Synthesis and fluorescence properties of Eu-anthracene-9-carboxylic acid towards N-acetyl amino acids and nucleotides in different solvents. Spectrochim Acta A Mol Biomol Spectrosc 75:21–27PubMedCrossRefGoogle Scholar
  43. 43.
    Azab HA, AboElNour KM, Sherif S (2007) Metal ion complexes containing di-, tripeptides and biologically important zwitterionic buffers. J Chem Eng Data 52:381–390CrossRefGoogle Scholar
  44. 44.
    Azab HA, El-Korashy SA, Anwar ZM, Hussein BHM, Khairy GM (2010) Eu(lll)-anthracene-9-carboxylic acid as a responsive luminescent bioprobe and its electroanalytical Interactions with N-acetyl amino acids, nucleotides and DNA. J Chem Eng Data 55:3130–3141CrossRefGoogle Scholar
  45. 45.
    Azab HA, Abd El-Gawad II, Kamel RM (2009) Ternary complexes formed by the fluorescent probe Eu (III)-9-anthracene carboxylic acid with pyrimidine and purine nucleobases. J Chem Eng Data 54:3069–3078CrossRefGoogle Scholar
  46. 46.
    Filip W, Mojmir S, Li AX, Azab HA, Bartha R, Hudson RHE (2007) A robust and convergent synthesis of dipeptides-DOTAM conjugates as chelators for lanthanide ions: new PARACEST MRI agents. Bioconjug Chem 18(5):1625–1636CrossRefGoogle Scholar
  47. 47.
    Orabi AS, Azab HA, ElDeghidy FS, Said H (2010) Ternary complexes of La(III), Ce(III), Pr(III) or Er(III) with adenosine 5’-mono,5’-di, and 5’-triphosphate as primary ligands and some biologically important zwitterionic buffers as secondary ligands. J Solution Chem 39:319–334CrossRefGoogle Scholar
  48. 48.
    Azab HA, Al-Deyab SS, Anwar ZM, Gharib RA (2011) Fluorescence and electrochemical probing of N-acetylamino acids, nucleotides and DNA by Eu(lll) -bathophenanthroline complex. J Chem Eng Data 56(4):833–849CrossRefGoogle Scholar
  49. 49.
    Azab HA, Al-Deyab SS, Anwar ZM, Kamel RM (2011) Potentiometric, electrochemical and fluorescence study of the coordination properties of the monomeric and dimeric complexes of Eu(lll) with nucleobases and PIPES. J Chem Eng Data 56:1960–1969CrossRefGoogle Scholar
  50. 50.
    Azab HA, Al-Deyab SS, Anwar ZM, Abd El-Gawad II, Kamel RM (2011) Comparison of the coordination tendency of amino acids, Nucleobases or mononucleotides towards the monomeric and dimeric lanthanide complexes with biologically important compounds. J Chem Eng Data 56:2613–2625CrossRefGoogle Scholar
  51. 51.
    Bates GR, Roy NR, Robinson AR (1964) Determination of pH: theory and practice. Wiley, New YorkGoogle Scholar
  52. 52.
    Gran G (1952) Determination of the equivalence point in potentiometric titration part II. Analyst 77:661CrossRefGoogle Scholar
  53. 53.
    May PM, Williams DR (1985) Computational methods for the determination of Formation Constants. In: Leggett DJ (ed). Plenum Press, New York, pp. 37-70.Google Scholar
  54. 54.
    Bjerrum J (1941) Metal amine complex formation in aqueous solution. Haase, CopenhagenGoogle Scholar
  55. 55.
    Irving H, Rossotti HS (1953) Methods for computing successive stability constants from experimental formation curves. J Chem Soc 3397.Google Scholar
  56. 56.
    De Stefano C, Princi P, Rigano C, Sammartano S (1987) Computer analysis of equilibrium data in solution. ESAB2M: An improved version of the ESAB program. Ann Chim (Rome) 77:643Google Scholar
  57. 57.
    Gans P, Vacca AJ (1996) Investigation of equilibria in solution. Determination of equilibrium constants with the HYPERQUAD suite of programs. Talanta 43:1739PubMedCrossRefGoogle Scholar
  58. 58.
    Kumar CV, Turner RS, Asunction EH (1993) Groove binding of a styrylcyanine dye to the DNA double helix: the salt effect. J Photochem Photobiol A Chem 74:231–238CrossRefGoogle Scholar
  59. 59.
    Son GS, Yeo JA, Kim JM, Kim SK, Moon HR, Nam W (1998) Base specific complex formation of norfloxacin with DNA. Biophys Chem 74:225–236PubMedCrossRefGoogle Scholar
  60. 60.
    Palu G, Valisena G, Ciarrocchi G, Gatto B, Palumbo M (1992) Quinolone binding to DNA is mediated by magnesium ions. Proc Natl Acad Sci USA 89:9671PubMedCrossRefGoogle Scholar
  61. 61.
    Ocaña JA, Barragán FJ, Callejon M (2004) Fluorescence and terbium sensitised luminescence determination of garenoxacin in human urine and serum. Talanta 63:691PubMedCrossRefGoogle Scholar
  62. 62.
    Teixeira LS, Grasso AN, Monteiro AM, Neto AMF, Vieira ND, Gidlund M, Courrol LC (2010) Enhancement on the europium emission band of europium chlortetracycline complex in the presence of LDL. Anal Biochem 400:19CrossRefGoogle Scholar
  63. 63.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Plenum Press, New York, pp. 87, 95, 237–249, 331.Google Scholar
  64. 64.
    Benesi HA, Hildebrand JH (1949) Spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons. J Am Chem Soc 71:2703CrossRefGoogle Scholar
  65. 65.
    Wei XF, Liu HZ (2000) The interaction between Triton X-100 and bovine serum albumin. Chin J Anal Chem 28:699Google Scholar
  66. 66.
    Kang J, Liu Y, Xie M, Li S, Jiang M, Wang Y (2004) Interactions of human serum albumin with chlorogenic acid and ferulic acid. Biochim Biophys Acta 1674:205PubMedCrossRefGoogle Scholar
  67. 67.
    Ross PD, Subranmanian S (1981) Thermodynamics of protein association reactions-forced contributing to stability. Biochemistry 20:3096PubMedCrossRefGoogle Scholar
  68. 68.
    Martin RB, Mariam YH (1979) Interactions between metal ions and nucleic bases, nucleosides, and nucleotides in solution. Met Ions Biol Syst 8:57Google Scholar
  69. 69.
    Martin RB (1985) Nucleoside sites for transition metal ion binding. Accounts Chem Res 18:32CrossRefGoogle Scholar
  70. 70.
    Patterson GS, Holm RH (1972) Effects of chelate ring substituents on the polarographic redox potentials of tris(ß-diketonato)ruthenium(II, III) complexes. Inorg Chem 11:2285CrossRefGoogle Scholar
  71. 71.
    Takeuchi T, Endo A, Shimizu K, Sato GP (1985) Electrochemical oxidation of tris(β-diketonato)-ruthenium(III) in acetonitrile solutions at platinum electrodes. J Electroanal Chem 185:185CrossRefGoogle Scholar
  72. 72.
    Collman JP (1965) Reactions of metal acetylacetonates. Angew Chem Int Ed Engl 4:132CrossRefGoogle Scholar
  73. 73.
    Brad AJ, Faulkner LR (1980) Electrochemical methods. Fundamentals and applications. Wiley, New York, 218Google Scholar
  74. 74.
    Oliveria-Brett AM, Piedade JAP, Silva LA, Diculescu VC (2004) Voltammetric determination of all DNA nucleotides. Anal Biochem 332:321CrossRefGoogle Scholar
  75. 75.
    Neidle S, Balasubramanian S (2006) Quadruplex nucleic acids. RSC Publishing, CambridgeCrossRefGoogle Scholar
  76. 76.
    Huppert JL (2008) Four-stranded nucleic acids: structure, function and targeting of G-quadruplexes. Chem Soc Rev 37:1375–1384PubMedCrossRefGoogle Scholar
  77. 77.
    Burge S, Parkinson GN, Hazel P, Todd AK, Neidle S (2006) Quadruplex DNA: sequence, topology and structure. Nucleic Acids Res 34:5402–5415PubMedCrossRefGoogle Scholar
  78. 78.
    Patel DJ, Phan AT, Kuryavyi V (2007) Human telomere, oncogenic promoter and 5’-UTR G-quadruplexes: diverse higher order DNA and RNA targets for cancer therapeutics. Nucleic Acids Res 35:7429–7455PubMedCrossRefGoogle Scholar
  79. 79.
    Parkinson GN, Lee MP, Neidle S (2002) Crystal structure of parallel quadruplexes from human telomeric DNA. Nature 417:876–880PubMedCrossRefGoogle Scholar
  80. 80.
    Haider S, Parkinson GN, Neidle S (2002) Crystal structure of the potassium form of an Oxytricha nova G-quadruplex. J Mol Biol 320:189–200PubMedCrossRefGoogle Scholar
  81. 81.
    Haider SM, Parkinson GN, Neidle S (2003) Structure of a G-quadruplex-ligand complex. J Mol Biol 326:117–125PubMedCrossRefGoogle Scholar
  82. 82.
    Phan AT, Kuryavyi V, Burge S, Neidle S, Patel DJ (2007) Structure of an unprecedented G-quadruplex scaffold in the human c-kit promoter. J Am Chem Soc 129:4386–4392PubMedCrossRefGoogle Scholar
  83. 83.
    Schaffitzel C, Berger I, Postberg J, Hanes J, Lipps HJ, Plückthun A (2001) In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc Natl Acad Sci USA 98:8572–8577PubMedCrossRefGoogle Scholar
  84. 84.
    Paeschke K, Simonsson T, Postberg J, Rhodes D, Lipps HJ (2005) Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo. Nat Struct Mol Biol 12:847–854PubMedCrossRefGoogle Scholar
  85. 85.
    Oganesian L, Bryan TM (2007) Physiological relevance of telomeric G-quadruplex formation: a potential drug target. Bioessays 29:155–165PubMedCrossRefGoogle Scholar
  86. 86.
    De Cian A, Grellier P, Mouray E, Depoix D, Bertrand H, Monchaud D, Teulade-Fichou MP, Mergny JL, Alberti P (2008) Plasmodium telomeric sequences: structure, stability and quadruplex targeting by small compounds. Chembiochem 9:2730–2739PubMedCrossRefGoogle Scholar
  87. 87.
    Balasubramanian S, Neidle S (2009) G-quadruplex nucleic acids as therapeutic targets. Curr Opin Chem Biol 13:345–353PubMedCrossRefGoogle Scholar
  88. 88.
    Baraldi PG, Tabrizi MA, Preti D, Fruttarolo F, Avitabile B, Bovero A, Pavani G, Carretero MCN, Romagnoli R (2003) DNA minor-groove binders. Design, synthesis, and biological evaluation of ligands structurally related to CC-1065, distamycin, andanthramycin. Pure Appl Chem 75:187–194CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Hassan A. Azab
    • 1
  • E. M. Mogahed
    • 2
  • F. K. Awad
    • 2
  • R. M. Abd El Aal
    • 2
  • Rasha M. Kamel
    • 2
  1. 1.Chemistry Department faculty of scienceSuez Canal UniversityIsmailiaEgypt
  2. 2.Chemistry Department, Faculty of ScienceSuez Canal UniversitySuezEgypt

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