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Catalytic DNA-Based Biosensors for Effective Detection of Lead Ions

Catalytic DNA-Based Biosensors for Effective Detection of Lead Ions. September 28 , 2009 Bishnu Regmi Warner Research Group Louisiana State University Baton Rouge, LA.

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Catalytic DNA-Based Biosensors for Effective Detection of Lead Ions

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  1. Catalytic DNA-Based Biosensors for Effective Detection of Lead Ions September 28 , 2009 Bishnu Regmi Warner Research Group Louisiana State University Baton Rouge, LA

  2. Outline • Objective • Articles • Background • Current Analytical Methods • DNAzyme-Based Methods • Colorimetric Method • Fluorometric Method • Comparison • Critique • Acknowledgement

  3. Objective • To develop a highly sensitive, selective, and more practical method for routine analysis of lead content in environmental and biological materials.

  4. Objective • Articles • Background • Current Analytical Methods • DNAzyme-Based Methods • Colorimetric Method • Fluorometric Method • Comparison • Critique • Acknowledgement

  5. Articles Label-Free Colorimetric Detection of Lead Ions with a Nanomolar Detection Limit and Tunable Dynamic Range by using Gold Nanoparticles and DNAzyme Zidong Wang, Jung Heon Lee, and Yi Lu* Adv. Mater. 2008, 20, 3263-3267 Engineering a Unimolecular DNA-Catalytic Probe for Single Lead Ion Monitoring Hui Wang, Youngmi Kim, Haipeng Liu, Zhi Zhu, Suwussa Bamrungsap, and Weihong Tan* J. Am. Chem. Soc. 2009, 131, 8221-8226

  6. Objective • Articles • Background • Current Analytical Methods • DNAzyme-Based Methods • Colorimetric Method • Fluorometric Method • Comparison • Critique • Acknowledgement

  7. Introduction • No known biological or physiological role in humans or other animals • But it has been widely used by human beings since ancient times • It has been used: • Manufacture of automotive batteries • Lead sheets, pipes, solder, bullets, insecticide, ceramic glazes, paints • Tetraethyl lead, additive in gasoline to increase the octane rating http://www.epa.gov/ttn/atw/hlthef/lead.html#ref1 (accessed September 11, 2009)

  8. Potential Sources Lead Exposure • Air: Combustion of gasoline, solid waste, oil, and coal; emissions from iron and steel manufacture, lead smelters, and tobacco smoke • Food and soil • Flaking paint, paint chips and dust • Drinking water • Workplace http://www.epa.gov/ttn/atw/hlthef/lead.html#ref1 (accessed September 11, 2009) Lead and Your Health, National Institute of Environmental Health Sciences

  9. Molecular Mechanism of Lead Toxicity • Similar to the divalent ions Ca2+ and Zn2+, hence inhibits or mimics their action • Able to interact with proteins--amine, carboxyl and sulfhydryl groups: distortion of enzymes and structural proteins • Binding of lead to transporter inhibit or alter the ion transport across the membrane • Inhibition of delta aminolevulinic acid dehydratase and ferrochelatase of heme biosynthetic pathway Cornelis et al. Handbook of Elemental Speciation II - Species in the Environment, Food, Medicine and Occupational Health, Wiley,2005, 262-264 Warren et al. TIBS1998, 23, 217-221

  10. Warren et al. TIBS1998, 23, 217-221

  11. Clinical Manifestations of Lead Poisoning • Severe cramping abdominal pain • Encephalopathy • Hypertension • Constipation • Elevated excretion of heme biosynthetic intermediates • Premature birth and low birth weights http://www.atsdr.cdc.gov/csem/lead/pbphysiologic_effects2.html (accessed September 12, 2009) Warren et al. TIBS1998, 23, 217-221

  12. Toxicity Levels Before mid-1960s: toxic threshold for children 60 µg/dL (600 ppb) 1978 : 30 µg/dL (300 ppb) 1985: 25 µg/dL (250 ppb) 1991 : 10 µg/dL (100 ppb) Adverse effects even below 10 µg/dL ‘Action level’ in water = 15 ppb 10 µg/dL = 0.1 ppm =100 ppb = 483 nM http://www.cdc.gov/nceh/lead/policy/changeBLL.htm http://www.labmanager.com/articles.asp?ID=60

  13. Status of Lead Poisoning in Louisiana Source: http://www.cdc.gov/nceh/lead/data/State_Confirmed_byYear_1997_to_2006.xls

  14. Objective • Articles • Background • Current Analytical Methods • DNAzyme-Based Methods • Colorimetric Method • Fluorometric Method • Comparison • Critique • Acknowledgement

  15. Current Analytical Methods • Flame atomic absorption spectrometry (AAS) • Graphite furnace atomic absorption spectrometry (GFAAS) • Anodic stripping voltammetry (ASV) • Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) • Inductively coupled plasma mass spectrometry (ICP-MS)

  16. Advantages and Limitations of ICP-MS • Commercially available • Extremely sensitive (ppt) • Very selective • Rapid • Multi-element analysis • Wide dynamic range of 105 • Good accuracy and precision • Instrument very expensive • High running cost due to large argon consumption ( 17 L/min) • Not suitable for on-site and in situ analysis • Requires sample pretreatment and skilled operators • Relatively big volumes of samples Schutz et al. Occupational and Environmental Medicine, 1996;53:736-740 Agilent ICP-MS Journal March 2005 – Issue 22 http://web.uct.ac.za/depts/geolsci/facilities/icpms/lectures/lec2.html (accessed September 12, 2009) Li et al. Analytica Chimica Acta2000, 419, 65-72

  17. Objective • Articles • Background • Current Analytical Methods • DNAzyme-Based Methods • Colorimetric Method • Fluorometric Method • Comparison • Critique • Acknowledgement

  18. DNAzymes • Discovered in 1994 • Single stranded DNA molecules that catalyze diverse chemical and biological reactions • Most of them require metal ions for their activity • Not found in nature, but can be obtained by in vitro selection Conserved core for 17E: CCGAGCCGGTCGAAA rA Adenosine Ribonucleotide Liu J., Cao Z., Lu Y. Chem. Rev.2009, 109, 1948-1998 Breaker R. R. and Joyce F. G. Chem. Biol. 1994, 1, 223-229

  19. Liu J., Cao Z., Lu Y. Chem. Rev.2009, 109, 1948-1998

  20. Mechanism of Cleavage Brown et al. Biochemistry 2003, 42, 7152-7161

  21. Preparation of Lead-Specific DNAzyme Prepared by in vitro selection procedure rA Adenosine Ribonucleotide Breaker R. R. and Joyce F. G. Chem. Biol. 1995, 2, 655-660

  22. Basic Principle of the Label-Free Colorimetric Assay Wang Z., Lee J. H., Lu Y. Adv.Mater. 2008, 20, 3263-3267

  23. Lead-Induced Cleavage and Effect of EDTA Wang Z., Lee J. H., Lu Y. Adv.Mater. 2008, 20, 3263-3267

  24. Calibration Curve and Selectivity Detection limit: 3nM Dynamic range: 3 nM - 1 µM. Linear fitting range: 3 nM -100 nM Wang Z., Lee J. H., Lu Y. Adv.Mater. 2008, 20, 3263-3267

  25. Calibration Curve at pH 5.5 Dynamic range: 120 nM-20 µM Wang Z., Lee J. H., Lu Y. Adv.Mater. 2008, 20, 3263-3267

  26. Conclusions • Simple • Fast • Sensitive and Selective • Low detection limit: 3 nM • Tunable dynamic range • Suitable for on-site and real-time detection of lead ions • Can be extended to other metal ions

  27. Objective • Articles • Background • Current Analytical Methods • DNAzyme-Based Methods • Colorimetric Method • Fluorometric Method • Comparison • Critique • Acknowledgement

  28. Basic Principle of the Fluorometric Method Wang et al.J. Am. Chem. Soc. 2009, 131, 8221-8226

  29. The Sequences used in the Study D10 5′-/Dabcyl/-TATCTCTTCTCCGAGCCGGTCGAAATAGTGAG(T)10ACTCACTATrAGGAAGAGATA-/FAM/-3′ D7 5′-/Dabcyl/-ATCTTCCGAGCCGGTCGAAATAGTGAG-(T)10ACTCACTATrAGGAAGAT-/FAM/-3′ D5 5′-/Dabcyl/-ATTCCCCGAGCCGGTCGAAATAGTGAG-(T)10ACTCACTATrAGGAAT-/FAM/-3′ 6-Fluorescein (FAM) phosphoramidite 5′-4-(4-Dimethylaminophenylazo)benzoic acid (Dabcyl) phosphoramidite Wang et al. J. Am. Chem. Soc. 2009, 131, 8221-8226 http://www.sigmaaldrich.com/etc/medialib/docs/SAFC/General_Information/6-fam_flyer.Par.0001.File.dat/6-fam_flyer.pdf

  30. Fluorescence Signal in the Presence and Absence of Lead Ions Wang H. et al. J. Am. Chem. Soc. 2009, 131, 8221-8226

  31. Calibration Curve D10, 200 nM Detection limit: 3 nM Quantifiable detection range: 2 nM to 20 µM Quantifiable detection range 3 nM to 20 uM Wang et al. J. Am. Chem. Soc. 2009, 131, 8221-8226

  32. Selectivity Studies of the Sensor Wang et al. J. Am. Chem. Soc. 2009, 131, 8221-8226

  33. Single Lead Ion Reaction Kinetics Wang et al. J. Am. Chem. Soc. 2009, 131, 8221-8226

  34. Conclusions • Simple • Rapid • High sensitivity with a quantifiable detection range 3 nM to 20 µM • High selectivity: more than 80-fold over other divalent metal ions • Detection limit: 1600 times better than atomic spectroscopy • Single ion monitoring

  35. Objective • Articles • Background • Current Analytical Methods • DNAzyme-Based Methods • Colorimetric Method • Fluorometric Method • Comparison • Critique • Acknowledgement

  36. Comparison Schutz et al. Occupational and Environmental Medicine, 1996;53:736-740 Thomas Robert, Practical Guide to ICP-MS, Marcel Dekker Inc. 2004, p271 Li J. et al. Analytica Chimica Acta2000, 419, 65-72 Wolf R. E. Atomic Spectroscopy1997, 18, 169-174

  37. Objective • Articles • Background • Current Analytical Methods • DNAzyme-Based Methods • Colorimetric Method • Fluorometric Method • Comparison • Critique • Acknowledgement

  38. Critique • Simple • Fast • Cost-effective • Sensitive • Selective • Suitable for on-site analysis • Do not describe the analysis of real world samples • Large error bars, low precision • For fluorometric method, the signal seems to level off at ~2 µM not at 20 µM • Selectivity more than 80-fold, does not seem from the figure 4

  39. Acknowledgements Dr. Warner Monica Sylvain Warner Research Group

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