Catalytic DNA-Based Biosensors Catalytic DNA-Based Biosensors for Effective Detection of Lead for Effective Detection of Lead

IonsIons

September 28 , 2009

Bishnu Regmi

Warner Research Group Louisiana State University

Baton Rouge, LA

Outline

• Objective• Articles• Background• Current Analytical Methods• DNAzyme-Based Methods

Colorimetric Method Fluorometric Method

• Comparison• Critique• Acknowledgement

Objective

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

• Objective

• Articles

• Background

• Current Analytical Methods

• DNAzyme-Based Methods

Colorimetric Method

Fluorometric Method

• Comparison

• Critique

• Acknowledgement

Articles

Label-Free Colorimetric Detection of Lead Ions with a Nanomolar Detection Limit and Tunable Dynamic Range by using Gold Nanoparticles and DNAzymeZidong Wang, Jung Heon Lee, and Yi Lu*

Adv. Mater. 2008, 20, 3263-3267

Engineering a Unimolecular DNA-Catalytic Probe for Single Lead Ion MonitoringHui Wang, Youngmi Kim, Haipeng Liu, Zhi Zhu, Suwussa Bamrungsap, and Weihong Tan*

J. Am. Chem. Soc. 2009, 131, 8221-8226

•Objective

• Articles

• Background

• Current Analytical Methods

• DNAzyme-Based Methods

Colorimetric Method

Fluorometric Method

• Comparison

• Critique

• Acknowledgement

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)

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

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

o Binding of lead to transporter inhibit or alter the ion transport across the membrane

o 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, 262-264Warren et al. TIBS 1998, 23, 217-221

Warren et al. TIBS 1998, 23, 217-221

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. TIBS 1998, 23, 217-221

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

10 µg/dL = 0.1 ppm =100 ppb = 483 10 µg/dL = 0.1 ppm =100 ppb = 483 nMnM

http://www.labmanager.com/articles.asp?ID=60

‘Action level’ in water = 15 ppb

http://www.cdc.gov/nceh/lead/policy/changeBLL.htm

BTLL defined in different years

0

100

200

300

400

500

600

1965 1970 1975 1980 1985 1990 1995 2000 2005

Years

Blo

od to

xic

lead

leve

l (pp

b)

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

Status of Lead Poisoning in Louisiana

0.00%

0.50%

1.00%

1.50%

2.00%

2.50%

3.00%

3.50%

1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007

Year

Per

cen

tag

e o

f te

sted

ch

ildre

n w

ith

blo

od

le

ad le

vel a

bo

ve 1

0 u

g/d

L

•Objective

• Articles

• Background

• Current Analytical Methods

• DNAzyme-Based Methods

Colorimetric Method

Fluorometric Method

• Comparison

• Critique

• Acknowledgement

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)

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

Schutz et al. Occupational and Environmental Medicine, 1996;53:736-740Agilent ICP-MS Journal March 2005 – Issue 22http://web.uct.ac.za/depts/geolsci/facilities/icpms/lectures/lec2.html (accessed September 12, 2009)Li et al. Analytica Chimica Acta 2000, 419, 65-72

• 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

•Objective

• Articles

• Background

• Current Analytical Methods

• DNAzyme-Based Methods

Colorimetric Method

Fluorometric Method

• Comparison

• Critique

• Acknowledgement

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

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

Conserved core for 17E:CCGAGCCGGTCGAAA

rA

Adenosine Ribonucleotide

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

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

Mechanism of Cleavage

Preparation of Lead-Specific DNAzyme

Prepared by in vitro selection procedure

Breaker R. R. and Joyce F. G. Chem. Biol. 1995, 2, 655-660

rA

Adenosine Ribonucleotide

Basic Principle of the Label-Free Colorimetric Assay

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

Lead-Induced Cleavage and Effect of EDTA

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

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

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

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

• Objective

• Articles

• Background

• Current Analytical Methods

• DNAzyme-Based Methods

Colorimetric Method

Fluorometric Method

• Comparison

• Critique

• Acknowledgement

Basic Principle of the Fluorometric Method

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

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′

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

N N N

HN

OP

N

O

OCN

6-Fluorescein (FAM) phosphoramidite

5′-4-(4-Dimethylaminophenylazo)benzoic acid (Dabcyl) phosphoramidite

Fluorescence Signal in the Presence and Absence of Lead Ions

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

Quantifiable detection range 3 nM to 20 uM

Calibration Curve

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

D10, 200 nM

Detection limit: 3 nM

Quantifiable detection range: 2 nM to 20 µM

Selectivity Studies of the Sensor

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

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

Single Lead Ion Reaction Kinetics

• 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

Conclusions

• Objective

• Articles

• Background

• Current Analytical Methods

• DNAzyme-Based Methods

Colorimetric Method

Fluorometric Method

• Comparison

• Critique

• Acknowledgement

ICP-MS Fluorometric

Colorimetric

LOD ~ 0 .1 nM 3 nM 3 nM

Precision 1-5% NA NA

Accuracy

1-10% NA NA

Dynamic Range 105 103 102

Selectivity Selective Selective Selective

Simplicity Sophisticated Simple Simplest

Instrument Cost $ 150-200 K $ 40-50 K $ 40-50 K

Comparison

Schutz et al. Occupational and Environmental Medicine, 1996;53:736-740Thomas Robert, Practical Guide to ICP-MS, Marcel Dekker Inc. 2004, p271Li J. et al. Analytica Chimica Acta 2000, 419, 65-72Wolf R. E. Atomic Spectroscopy 1997, 18, 169-174

• Objective

• Articles

• Background

• Current Analytical Methods

• DNAzyme-Based Methods

Colorimetric Method

Fluorometric Method

• Comparison

• Critique

• Acknowledgement

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

Acknowledgements

Dr. Warner Dr. Warner Monica Sylvain Monica Sylvain Warner Research Group Warner Research Group