ELECTROCHEMICAL DNA BIOSENSORS pot

PRESENTATION OVERVIEWz Electrochemical DNA Hybridization Sensing Strategies z Inosine based hybridization detection by using carbon paste electrode CPE z Detection of NOS-terminator, a

ELECTROCHEMICAL DNA

BIOSENSORS

Prof Dr Mehmet OZSOZ

Dept of Analytical Chemistry,

Faculty of Pharmacy, Ege University

35100 Bornova /Izmir, TURKEY

ozsozs@pharmacy.ege.edu.tr http://pharmacy.ege.edu.tr/~ozsozs

Imagination is more important than knowledge.

Albert Einstein

z If a man emties his purse into his head,

no man can take it away from him An

investment in knowledge always pays the

best interest.

Benjamin Franklin

PRESENTATION OVERVIEW

z Electrochemical DNA Hybridization Sensing

Strategies

z Inosine based hybridization detection by using

carbon paste electrode (CPE)

z Detection of NOS-terminator, a genetic

element present in GMOs

z Carbon Nanotubes

z Optical Biosensors

z If a man emties his purse into his head,

no man can take it away from him An

investment in knowledge always pays the

best interest.

Benjamin Franklin

DNA biosensor scheme

Basic principle of

a glucose biosensor

GOX β-D-glucose + O 2 + H 2 O Gluconolactone + H 2 O 2

Transducer Analytical signal

Electrochemical DNA Hybridization

Sensing Strategies

1.Label based

a) Hybridization indicators

– metal complexes

– organic dye molecules

– anticancer agents etc.

b) Labelled probe

- Metal label (Au or Ag-nanoparticles,)

- oligonucleotide containing -SH, -NH2, groups.

2 Label free

– Electrochemical signals of DNA purine bases

guanine, (Inosine), adenine

Label-free (guanine) sensing strategie

How is the probe DNA

HYBRIDIZATION INDICATORS

Ethidium Bromide

PNA vs DNA

Label free detection of Mis-matched (MM)

sequences by using PNA capture probes

Inosine is an electro-inactive analogue of guanine,

which can also bind to cytosine by forming two hydrogen bonds.

YES / NO SYSTEM for hybridization detection

No signal is observed from inosine modified probe.

After hybridization, a signal is derived from the guanine

bases in the target.

Oxidation signal of DNA bases

Guanine, Adenine Inosine, Adenine

Covalent immobilization of

Oligonucleotide onto graphite

perpendicular to the electrode

surface

modified Au nanoparticles upon binding to target DNA is a

well-described event.

Elghanian, R.; Storhoff, J J.; Mucic, R C.; Letsinger, R L.; Mirkin, C A "Selective

Colorimetric Detection of Polynucleotides Based on the Distance-Dependent Optical

Properties of Gold Nanoparticles," Science, 1997, 277, 1078-1080.

Color shift is only observed from the hybridization with the

target DNA

Au Electrode Study Procedure

Electrode system

DNA-Chip technology

Chip Design

What is Factor V Leiden

Mutation?

position of the human genome.

Experimental Procedure

z When hybridization was occured between probe and target

on CPE surface, a guanine oxidation signal at ~+1.00 V

was appeared The YES / NO system was established for

the electrochemical detection of allele – specific mutation

on Factor V.

THE DETECTION OF “ALLEL

SPECIFIC” GENE

Disposable genosensor, a new tool for the detection of

NOS-terminator, a genetic element present in GMOs

B Meric et al / Food Control 15 (2004) 621–626

z Genetically modified organisms (GMOs) are referred to as living organisms of vegetable or animal origin in which the artificial

introduction of foreign genes is able to confer novel

characteristics not

found in the wild type

The EU Council regulation on traceability and labeling for GMO

- A threshold of 0.9% is set for labeling, below which labeling of

GMOs is not required.

z PCR methods, based on exponential

amplification of a specific GMO DNA fragment

, are highly sensitive, but require sophisticated

instruments for post-amplification steps, such

as gel electrophoresis and fluorescent

imaging.

- Alternatively an electrochemical genosensor is

presented here for rapid analysis of GMO

related PCR products.

Since GMOs and their derivatives are widely used

in food industry, the analytical assays are needed

to provide the detection and the quanti.cation of

GMOs in food matrices

z Nos terminator is present in nearly all

GMOs due to its important in gene

regulation

z DNA extraction and PCR was performed

z From the amplification of the NOS-terminator

region,fragments of 180 bp (base pairs) were

obtained

Probe 5`-AAT GAT TAA TTG GGG GAC TCT AAT C-3’

Target 5’-GAT TAG AGT CCC CCA ATT AAT CAT T-3’

Carbon Nanotubes(CNT)

z Multi walled carbon nanotubes (MWNTs)

were used as nanowires which combined

DNA molecules to a carbon paste

electrode(CPE)

z Unique electronic and mechanical

properties and chemical stability

z CNT accelerate the electron transfer

Thymine probe: 5’-Amino-TTT TTT-3’

Adenine probe: 5’-Amino-AAA AAA-3’

Inosine substituted thymine tagged probe:

5’-T T T TTTCAA I AC CAC CAC TTC I AA ACC-3’

Target: 5’-GGT TTC GAA GTG GTG GTC TTG-3’

Single-base mismatch:

5’-GGT TTC GAA G G G GTG GTC TTG-3’

Label-Free Electrochemical Detection of DNA Hybridization

Electroanalysis DOI: 10.1002/elan.200303025

Electrochemical Coding of

Single-Nucleotide Polymorphisms By Monobase-Modified

Gold Nanoparticles

Anal Chem.2004, 76,1877-1884

Electrochemical Genosensor

for the Discrimination of HSV

(Herpes Simplex Virus)

Type I and II

Analytica Chimica Acta 518 (2004) 69–76

Herpes Simplex Virus;

HSV Type I infects the nervous

Variations of HSV Type 1 and Type 2 discriminations and probes selection :

HSV Type 1 (between 64208 and 64386 bases sequences)

5’ ATC AAC TTC GAC TGG CCC TTC * TTG CTG GCC AAG CTG

GAC ATG TAC GG 3’

HSV Type 2 (between 64669 and 64847 bases sequences)

5’ ATC AAC TTC GAC TGG CCC TTC * GTC CTG ACC AAG CTG

ATC GAC ATG TAC GG 3’

Real Samples-2

Label-Free Electrochemical Hybridization

Genosensor for the Detection of Hepatitis

B Virus Genotype on the Development of

Lamivudine Resistance

-Lamivudine is a pyrimidine nucleoside analogue that

inhibits viral replication by blocking viral reverse transcriptase

-Continuous lamivudine therapy may lead to selection of

resistant strains

Anal Chem.2005, 77,4908-4917

z Hepatitis B virus (HBV) causes acute and

chronic hepatitis, chirrosis and

hepatocelular carcinoma.

z Hepatitis B virus infection is the ninth

mainly reason of death on the world

According to the World Health Organization (WHO)

more than 400 million people are chronically infected

by Hepatitis B virus (HBV) worldwide

Long-term treatment is often needed and that is

associated with many problems such as,

z low cure rate

z development of lamivudine resistance

z Lamivudine-resistance is associated with nucleotide

substitutions that induce amino acid changes in codon 204 of

the polymerase gene.

z HBV strains revealed isoleucine (I) or valine (V) substitutions

instead of methionine in the tyrosine(Y), methionine(M),

aspartate(D) and aspartate(D) motif (YMDD motif)

z These changes are named YVDD or YIDD which are the most

general described mutations

PCR MAP of HBV

catcctgctg ctatgcctca tcttcttgtt ggttcttctg gattatcaag

gtatgttgcc cgtttgtcct ctaattccag gatcaacaac aaccagtacg

ggaccatgca aaacctgcac gactcctgct caaggcaact ctatgtttcc

ctcatgttgc tgtacaaaac ctacggatgg aaattgcacc tgtattccca

tcccatcgtc ctgggctttc gcaaaatacc tatgggagtg ggcctcagtc

cgtttctctt ggctcagttt actagtgcca tttgttcagt ggttcgtagg

z YMDD mutants are caused by a point mutation from A to

G at the 743nd position (YVDD) and from G to T at the

745rd position (YIDD) of the human genome.

5’ – AAT ACC ACA TCA TCC ATA TA – 3’

type-1 HBVgenome-YVDD)

5’ – AAT ACC ACA TCA TCC AcA TA – 3’

type-2 HBVgenome-YIDD)

5’ – AAT ACC ACA TCA TCa ATA TA – 3’

5’- TAT ATG GAT GAT GTG GTA TT-3’

z Mutant type-1 target sequence-2(MT-1T=YVDD):

5’- TAT gTG GAT GAT GTG GTA TT-3’

z Mutant type-2 target sequence-3(MT-2T=YIDD):

5’- TAT ATt GAT GAT GTG GTA TT-3’

z Non comlpementary sequences:

5’ – AAT ACC TGT ATT CCT CGC CTG TC –3’

Hybridization detection procedure

Cloned PCR products

z The guanine signal obtained from the wild type probe modified PGE after

hybridization with samples 1, 2 and 3 gave a R S D value of 9.4%, 7.6%

and 10.2%

R S D value of 10.4% and 11.8%

R S D value of 7.4 % and 6.0% The detection limits, estimated from S /

target at PGE

Development of an SPR-based biosensor for the detection of lipophilic

phycotoxins in shellfish residues

A European Commission-funded Specific Targeted Research Project

Food Quality and Safety

Food- CT- 2004- 514055

Biosensors and their

application to improving

food safety and quality

Biosensors and their

application to improving

food safety and quality

The Classic Binding Molecule:

The Antibody

The Classic Binding Molecule:

The Antibody

IgG MW ~180,000

SPR

Prof.Arzum ERDEM, Prof

Mehmet OZSOZ, PhD.Std Kagan KERMAN, PhD Std Pinar

KARA, PhD.Std Dilsat OZKAN.

PhD.Std.Burcu MERIC, PhD Std

Pinar KARA, Assoc Prof.Arzum ERDEM, PhD.Std Dilsat

OZKAN, PhD.Std Kagan KERMAN.