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Catalytic DNA-Based Biosensors for Effective Detection of Lead Ions

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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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slide1

Catalytic DNA-Based Biosensors for Effective Detection of Lead Ions

September 28 , 2009

Bishnu Regmi

Warner Research Group Louisiana State University Baton Rouge, LA

slide2

Outline

  • Objective
  • Articles
  • Background
  • Current Analytical Methods
  • DNAzyme-Based Methods
    • Colorimetric Method
    • Fluorometric Method
  • Comparison
  • Critique
  • Acknowledgement
slide3

Objective

  • To develop a highly sensitive, selective, and more practical method for routine analysis of lead content in environmental and biological materials.
slide4

Objective

  • Articles
  • Background
  • Current Analytical Methods
  • DNAzyme-Based Methods
    • Colorimetric Method
    • Fluorometric Method
  • Comparison
  • Critique
  • Acknowledgement
slide5

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

slide6

Objective

  • Articles
  • Background
  • Current Analytical Methods
  • DNAzyme-Based Methods
    • Colorimetric Method
    • Fluorometric Method
  • Comparison
  • Critique
  • Acknowledgement
slide7

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)

slide8

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

slide9

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

slide11

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

slide12

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

slide13

Status of Lead Poisoning in Louisiana

Source:

http://www.cdc.gov/nceh/lead/data/State_Confirmed_byYear_1997_to_2006.xls

slide14

Objective

  • Articles
  • Background
  • Current Analytical Methods
  • DNAzyme-Based Methods
    • Colorimetric Method
    • Fluorometric Method
  • Comparison
  • Critique
  • Acknowledgement
slide15

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)
slide16

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

slide17

Objective

  • Articles
  • Background
  • Current Analytical Methods
  • DNAzyme-Based Methods
    • Colorimetric Method
    • Fluorometric Method
  • Comparison
  • Critique
  • Acknowledgement
slide18

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

slide20

Mechanism of Cleavage

Brown et al. Biochemistry 2003, 42, 7152-7161

slide21

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

slide22

Basic Principle of the Label-Free Colorimetric Assay

Wang Z., Lee J. H., Lu Y. Adv.Mater. 2008, 20, 3263-3267

slide23

Lead-Induced Cleavage and Effect of EDTA

Wang Z., Lee J. H., Lu Y. Adv.Mater. 2008, 20, 3263-3267

slide24

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

slide25

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

slide26

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
slide27

Objective

  • Articles
  • Background
  • Current Analytical Methods
  • DNAzyme-Based Methods
    • Colorimetric Method
    • Fluorometric Method
  • Comparison
  • Critique
  • Acknowledgement
slide28

Basic Principle of the Fluorometric Method

Wang et al.J. Am. Chem. Soc. 2009, 131, 8221-8226

slide29

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

slide30

Fluorescence Signal in the Presence and Absence of Lead Ions

Wang H. et al. J. Am. Chem. Soc. 2009, 131, 8221-8226

slide31

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

slide32

Selectivity Studies of the Sensor

Wang et al. J. Am. Chem. Soc. 2009, 131, 8221-8226

slide33

Single Lead Ion Reaction Kinetics

Wang et al. J. Am. Chem. Soc. 2009, 131, 8221-8226

slide34

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
slide35

Objective

  • Articles
  • Background
  • Current Analytical Methods
  • DNAzyme-Based Methods
    • Colorimetric Method
    • Fluorometric Method
  • Comparison
  • Critique
  • Acknowledgement
slide36

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

slide37

Objective

  • Articles
  • Background
  • Current Analytical Methods
  • DNAzyme-Based Methods
    • Colorimetric Method
    • Fluorometric Method
  • Comparison
  • Critique
  • Acknowledgement
slide38

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
slide39

Acknowledgements

Dr. Warner Monica Sylvain Warner Research Group