Transcript ELECTROCHEMICAL GENOSENSOR BASED ON COLLOIDAL …

ELECTROCHEMICAL DNA
BIOSENORS
Prof. Mehmet OZSOZ
Ege University, Faculty of Pharmacy,
Dept. of Analytical Chemistry,
35100 Bornova / IZMIR
[email protected]
SUMMARY







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
Introduction

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 onsite monitoring schemes are required for
quick preventive action and early diagnosis.

Therefore, genosensors have recently been
the subject of extensive research activities.
DNA biosensor scheme
Basic principle of
a glucose biosensor
GOX
-D-glucose + O2 + H2O
Gluconolactone + H2O2
Transducer
Analytical signal
PNA vs. DNA
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
Examples for commonly used indicators in DNA biosensors
HYBRIDIZATION INDICATORS
Methylene blue
Ruthenium bipyridine
Cobalt
phenanthroline
Inosine is an electro-inactive analogue of guanine,
which can also bind to cytosine by forming two hydrogen bonds.
Electrode system
DNA-Chip technology
Oxidation signal of DNA bases
Guanine, 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.
The Factor V Leiden mutation,

designated as 1691 G > A or R506Q, 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.
Sequences
Wild-type (WT) capture probe :
5’ – AAT ACC TIT ATT CCT CIC CTI TC – 3’
Wild-type target :
5’ – GAC AGG CGA GGA ATA CAG GTA TT – 3’
Mutant (MT) capture probe :
5’ – AAT ACC TIT ATT CCT TIC CTI TC – 3’
Mutant target :
5’ – GAC AGG CAA GGA ATA CAG GTA TT – 3’
Part I

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.
In this study,

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).
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.
Experimental
1)
2)
3)
4)
5)
6)
CPE Activation : 1.7V 60 sec. in 0.05M phosphate
buffer solution (PBS).
Inosine-labelled probe immobilization : +0.5V 300s.
in acetate buffer solution (ABS).
Washing step with ABS.
Hybridization with the synthetic target or PCR
sample : Capt. probe modified CPE was inverted
and ~10µl of the target/ denatured PCR amplicon
(heating in a water bath at 950C for 6 min. and
subsequent freezing in ice bath for 2 min.) was
pipetted directly onto the capture probe.
Washing step %1 SDS buffer 3s and then
immediately dipped into blank Tris-HCl buffer
solution(TBS).
Measurement : The oxidation signal of guanine was
measured by using differential pulse voltammetry
(DPV) in blank ABS by scanning from-0.80-+1.40V.
Experimental Procedure

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.
Figure 1A
Figure 1B
Figure 2A
Figure 2B
THE ALLEL SPECIFIC DETECTION
of MUTATION
Carbon Nanotubes(CNT)

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
DNA-Directed Attachment of Carbon Nanotubes for Enhanced
Label-Free Electrochemical Detection of DNA Hybridization
Covalent immobilization of
Oligonucleotide onto graphite
Part II
Electrochemical Genosensor
based on colloidal gold
nanoparticles
Gold nanoparticles
have been an attractive material in
research for a long time …
Mirkin, C. A.; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J.
Nature 1996, 382, 607.
The visible color shift and aggregation of oligonucleotide
modified Au nanoparticles upon binding to target DNA is a welldescribed event.
Color shift is only
observed from the
hybridization with the
target DNA.
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.
Nanoelectrodes with nanoparticles
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.
Park, S.-J.; Taton, T. A.; Mirkin, C. A. "Array-Based Electrical Detection of DNA Using
Nanoparticle Probes," Science, 2002, 295, 1503-1506.
Our strategy depended on pure
electrochemistry of Au nanoparticles :
 When
hybridization occured between
complementary probes conjugated to Au
nanoparticles and target on pencil
graphite electrode (PGE) surface, Au
oxide wave at about  1.20 V appeared.

The changes 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 Nhydroxysulfosuccinimide (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
Synthetic PCR product:
5’ – CCT GCC CCA ATC CCT TTA TTA CCC CCT CCT TCA GAC ACC
TCT AAC CTC TTC TGG CTC AAA AAG AGA ATT GGG GGC TTA
GGG TCG GAA CCC AAG CTT AGA ACT TTA AGC AAC AAG ACC
ACC ACT TCG AAA CC –3’
Thiol-capped probe:
5’ – SH – C6H5 - GGT TTC GAA GTG GTG GTC TTG – 3’
Wild-type (WT) capture probe:
5’ – NH2 - AAT ACC TIT ATT CCT CIC CTI TC – 3’
Wild-type target:
5’ – GAC AGG CGA GGA ATA CAG GTA TT – 3’
Mutant (MT) capture probe:
5’ – NH2 - AAT ACC TIT ATT CCT TIC CTI TC – 3’
Mutant target:
5’ – GAC AGG CAA GGA ATA CAG GTA TT – 3’
Results

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.
Figure I (synt. oligonucleotides)
Figure II (Synt. PCR)
Figure III (PCR real sample)
WT probe with WT target at PGE
%R. S. D. = 7.64 % (n=5).
MT probe with the MT target at PGE
% R. S. D. = 7.42 % (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.
TiO2 nanoparticles Studies
0.1M TiO2 signal by using DPV
Behavior of TiO2-nanoparticles
modified CPE based on guanine signal
2500
Current (nA)
2000
1500
1000
500
0
CPE+Probe(P)
Ti-CPE+P
CPE+Hybrid(H)
Ti-CPE+H
Histomogram showed that, bare and TiO2 modofied carbon paste electrode(CPE)
behaviours, when the probe or hybrid immobilized onto the electrode surface.In the first
column, synthetic probe seguence modified (ssDNA) bare CPE, in the second column,
synthetic probe seguence modified (ssDNA) TiO2 contained CPE, In the third column,
synthetic hybrid modified (dsDNA) bare CPE, In the forth column, synthetic hybrid
modified (dsDNA) TiO2 contained CPE. Also the similar results obtained with pencil
graphite electrodes.
Future work

For this study,
hybridization detection (after finding TiO2
nanoparticles’ attractivity on ss or ds DNA)
by using CPE and PGE.
Electrochemical Coding of
Single-Nucleotide
Polymorphisms By Monobase-Modified
Gold Nanoparticles
Part III
Electrochemical Genosensor
for the Discrimination of HSV
(Herpes Simplex Virus)
Type I and II
Herpes Simplex Virus;
Type I PCR Product
5’TCAACTTCGACTGGCCCTTCTTGCTGGCCAAGCTGACG
GACATTTACAAGGTCCCCCTGGAGACGGGTACGGCCGCA
TGAACGGCCGGGGCGTGTTTCGCGTGTGGGACATAGGCC
AGAGCCACTTCCAGAAGCGCAGCAAGATAAAGGTGAACG
GCATGGTGAGCATCGACATGTACGG 3’
Type II PCR Product
5’TCAACTTCGACTGGCCCTTCGTCCTGACCAAGCTGACG
GAGATCTACAAGGTCCCGCTCGAGACGGGTACGGGCGCA
TGAACGGCCGGGGTGTGTTCCGCGTGTGGGACATAGGCC
AGAGCCACTTCCAGAAGCGCAGCAAGATAAAGGTGAACG
GCATGGTGAACATCGACATGTACGG 3’
HSV Type I infects the nervous
system,
however HSV Type II infects the
genital system.
In routine analysis; the
discrimination between Type I
and II is done by sequence
detection system.
.
HSV Tip1 ve Tip2 varyasyonlarının ayırımında
PCR ürünü örneklerinden prob dizi seçimi:
HSV Tip1 64208 ve 64386 nükleotidleri arasındaki bölgeye ait
PCR ürünü:
5’ ATC AAC TTC GAC TGG CCC TTC TTG CTG GCC AAG CTG ACG
GAC A TT TAC AAG GTC CCC CTG GAC GGG TAC GGC CGC ATG AAC
GGC CGG GGC GTG TTT CGC GTG TGG GAC ATA GGC CAG AGC CAC TTC
CAG AAG CGC AGC AAG ATA AAG GTG AAC GGC ATG GTG AGC ATC
GAC ATG TAC GG 3’
HSV Tip2 64669 ve 64847 nükleotidleri arasındaki bölgeye ait
PCR Ürünü:
5’ ATC AAC TTC GAC TGG CCC TTC GTC CTG ACC AAG CTG ACG
GAG A TC TAC AAG GTC CCG CTC GAC GGG TAC GGG CGC ATG AAC
GGC CGG GGT GTG TTC CGC GTG TGG GAC ATC GGC CAG AGC CAC TTC
CAG AAG CGC AGC AAG ATA AAG GTG AAC GGC ATG GTG AAC ATC GAC
ATG TAC GG 3’
FIGURE I:
Meldola Blue signal obtained from,
hybridization between, A: Probe TypeI
and synthetic targetI, B:Probe TypeI and
synthetic targetII, C: Probe TypeI only.
Meldola Blue signal obtained from,
hybridization between, A: Probe TypeII and
synthetic targetII, B:Probe TypeII and
synthetic targetI, C: Probe TypeII only.
HSV Çalışmalarına Ait Bulguların
Değerlendirilmesi:

Diferansiyel puls voltametri tekniği ile Prob derişiminin hibridizasyondan sonra alınan
MDB yanıta etkisi. A)probun karşılığı olan hedef ile hibridizasyonu, B) mutasyon
içeren hedef dizi ile hibridizasyonu sonucu PGE yüzeyinde oluşan MDB sinyalleri.
En uygun hibridizasyon zamanı saptandı
Diferansiyel puls voltametri tekniği ile MDB indikatörlüğünde hibridizasyon
zamanının incelenmesi;
A)probun karşılığı olan hedef ile hibridizasyonu, B) mutasyon içeren hedef dizi
ile hibridizasyonu sonucu PGE yüzeyinde oluşan MDB sinyalleri.

PCR ürünü örnekler için kullanılacak en
uygun seyrelme oranı saptandı.

PCR ürünü örneklerinin seyrelme oranının incelenmesi. A) probun
karşılığı olan hedef ile hibridizasyonu, B) mutasyon içeren hedef dizi
ile hibridizasyonu sonucu alınan elektrokimyasal sinyaller
Gerçek hasta örnekleriyle yapılan
çalışmalarda HSV Tip1 ve Tip2’nin ayırımı
net olarak gözlendi.
FIGURE 2: Real Samples
Conclusion

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.
Photo-1: (left to right) Assoc.
Prof.Arzum ERDEM, Prof.
Mehmet OZSOZ, PhD.Std. Kagan
KERMAN, Master Std. Pinar
KARA, PhD.Std. Dilsat OZKAN.
Photo-2: (left to right)
PhD.Std.Burcu MERIC, Master
Std. Pinar KARA, Assoc.
Prof.Arzum ERDEM, PhD.Std.
Dilsat OZKAN, PhD.Std. Kagan
KERMAN.
Photo-3: (left
to right) Prof.
Mehmet
OZSOZ and ,
Master Std.
Hakan
KARADENIZ
THANK YOU