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 What¶s a biosensor?
 Electrochemical DNA Hybridization Sensing
Strategies
 Inosine based hybridization detection by using
carbon paste electrode (CPE)
 Gold nanoparticles based detection of hybridization
by using disposable pencil graphite electrode (PGE)
 Detection of Factor V Leiden Mutation by using CPE
and PGE from real PCR samples.
 Carbon Nanotubes
 TiO2 nanoparticles
 à


 The detection of specific DNA sequences provides
the basis for detecting a wide variety of infectious
and inherited diseases.

 Traditional methods for DNA sequencing, based

on the coupling of electrophoretic separations and
radioisotopic detection, are labor intensive and
time consuming, and are thus not well suited for
routine and rapid medical analysis, particularly for
point-of-care tasks.
 Electrochemical hybridization biosensors
(genosensors) for the detection of DNA
sequences may greatly reduce the assay
time and simplify its protocol. Such fast on-
site monitoring schemes are required for
quick preventive action and early diagnosis.

 Therefore, genosensors have recently been

the subject of extensive research activities.
  

 Basic principle of
a glucose biosensor

GOX
°-D-glucose + O2 + H2O Gluconolactone + H2O2

Transducer
Analytical signal
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4.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
Examples for commonly used indicators in DNA biosensors

„
     
       

e th yle n e b lue
V u th e n ium b ip yrid in e

o b a lt
p h e n a n th roline
Inosine is an electro-inactive analogue of guanine,
which can also bind to cytosine by forming two hydrogen bonds.
|lectrode system
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£uanine, Adenine Inosine, Adenine
Electrochemical DNA biosensors were
described for the electrochemical DNA
detection procedure based on oxidation
signals of guanine and Au nanoparticles to
detect an inherited disease; Factor V Leiden
Mutation using polymerase chain reaction
(PCR) amplicons and synthetic
oligonucleotides.
?
 
   

 
 designated as 4
4 G > A or R50 , is the
major heritable risk factor for venous
thromboembolism.
 This mutation in the coagulation factor V
gene results in the resistance of Factor V to
inactivation by activated protein C (APC).
 If the coagulation Factor V cannot be
inactivated, blood coagulates in venums.
 uequences
Wild-type (WT) capture probe :
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utant ( T) capture probe :

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 An electrochemical DNA biosensor was described
for the detection of Factor V Leiden mutation and
the discrimination of mutation type using the
oxidation signal of guanine in connection with
DPV for the first time.
 There have not yet been any literature reports
about the detection of heterozygous or
homozygous mutations from PCR amplified
amplicons by using the guanine signal without any
modifications in the native bases or any external
labels.
 à 


 
 Inosine substituted synthetic
oligonucleotide capture probes related to the
wild ± type or mutant type amplicons were
used and these probes were hybridized with
their complementary DNA sequences
(target sequence or PCR amplicons) at
carbon paste electrode (CPE).
 V|u / NO uVuT| for hybridization detection
No signal is observed from inosine modified probe.
After hybridization, a signal is derived from the guanine
bases in the target.
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 When hybridization was occured between probe and target

on CPE surface, a guanine oxidation signal at ~+4.00 V
was appeared. The YES / NO system was established for
the electrochemical detection of allele ± specific mutation
on Factor V.
   
   
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 Multi walled carbon nanotubes (MWNTs)
were used as nanowires which combined
DNA molecules to a carbon paste
electrode(CPE)
 Unique electronic and mechanical
properties and chemical stability
 CNT accelerate the electron transfer
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 The visible color shift and aggregation of oligonucleotide
modified Au nanoparticles upon binding to target DNA is a well-
described event.

Color shift is only

observed from the
hybridization with the
target DNA.

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Hybridization forms a self-assembly of Au nanoparticles in the nanogap between two

nanoelectrodes. Silver precipitation on Au nanoparticles facilitates the electrical flow
from one electrode to the other.
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 When hybridization occured between

complementary probes conjugated to Au
nanoparticles and target on pencil
graphite electrode (PGE) surface, Au
oxide wave at about > 4.20 V appeared.
 Thechanges in this electrochemical signal
was used to detect hybridization.
Specific probes were immobilized onto the Au
nanoparticles in two different modes;

 a) Inosine substituted probes were covalently

attached from their amino groups at 5` end using
N-(3-dimethylamino)propyl)-N¶-
ethylcarbodiimide hydrochloride (EDC) and N-
hydroxysulfosuccinimide (NHS) as a coupling
agent onto a carboxylate terminated L-cysteine
self assembled monolayer (SAM) preformed on
the Au nanoparticles and

 b) Probes with a hexanethiol group at their 5¶

phosphate end formed a SAM on Au nanoparticles.
 The base sequences used
uynthetic
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 For the detection of hybridization between the

Factor V Leiden WT or MT capture probe
immobilized Au nanoparticles and target DNA, an
aliquot of the probe modified Au nanoparticles is
simply introduced onto the target immobilized
electrode.
 The appearance of the Au oxidation signal
confirmed the presence of the sought-after DNA
sequence.
 à
   
 
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 WT probe with WT target at PGE
%R. S. D. = 7.  % (n=5).
MT probe with the MT target at PGE
% R. S. D. = 7.2 % (n=5).
The detection limits, (S/N=3)
0.78 fmole/mL target with WT probe modified gold
nanoparticles
0.83 fmole/mL target with MT probe modified gold
nanoparticles.
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 For thisstudy,
hybridization detection (after finding TiO2
nanoparticles¶ attractivity on ss or ds DNA)
by using CPE and PGE.
 |lectrochemical oding of
uingle-Nucleotide

olymorphisms By onobase- odified
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In routine analysis; the

discrimination between Type I
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detection system.

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eldola Blue signal obtained from, eldola Blue signal obtained from,
hybridization between, A:
robe TypeI hybridization between, A:
robe TypeII and
and synthetic targetI, B:
robe TypeI and synthetic targetII, B:
robe TypeII and
synthetic targetII, :
robe TypeI only. synthetic targetI, :
robe TypeII only.
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 The competition in DNA genosensors is
about making them cheaper and easier to
use. In this presentation, the appearance of
the Au signal or a guanine signal or the
changes in Meldola Blue signal enable the
monitoring of hybridization at a carbon
electrode in a simple way at a short time.

 The success of PGE over existing carbon

electrodes, is its commercial availability.
The developed method also has a sufficient
detection limit for real-world analysis in
regard to diagnosis.

This procedure also eliminates the use of

toxic chemicals such as ethidium bromide,
which is commonly used in the gel
electrophoresis step of the reference methods
in mutation analysis.
 #4 (left to right) Assoc.
Prof.Arzum ERDEM, Prof.
Mehmet OZSOZ, PhD.Std. Kagan
KERMAN, Master Std. Pinar
KARA, PhD.Std. Dilsat OZKAN.
# 
(left to right)
PhD.Std.Burcu MERIC, Master
Std. Pinar KARA, Assoc.
Prof.Arzum ERDEM, PhD.Std.
Dilsat OZKAN, PhD.Std. Kagan
KERMAN.

#( (left
to right) Prof.
Mehmet
OZSOZ and ,
Master Std.
Hakan
KARADENIZ
?
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