ELECTROCHEMICAL DNA BIOSENORS

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ELECTROCHEMICAL DNA BIOSENORS

• Prof. Mehmet OZSOZ
• Ege University, Faculty of Pharmacy, Dept. of
  Analytical Chemistry,
• 35100 Bornova / IZMIR
• ozsozs_at_pharm.ege.edu.tr

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SUMMARY

• Whats 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

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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.

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• 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.

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DNA biosensor scheme
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Basic principle of a glucose biosensor

• GOX
  
• ?-D-glucose O2 H2O
  Gluconolactone H2O2
•  
•  
  Transducer
•  
  Analytical signal

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PNA vs. DNA
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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

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Examples for commonly used indicators in DNA
biosensors
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Inosine is an electro-inactive analogue of
guanine, which can also bind to cytosine by
forming two hydrogen bonds.
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Electrode system
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DNA-Chip technology
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Oxidation signal of DNA bases

• Guanine, Adenine Inosine,
  Adenine

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• 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.

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The Factor V Leiden mutation,

• designated as 1691 G gt 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.

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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

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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.

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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).

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YES / NO SYSTEM for hybridization detectionNo
signal is observed from inosine modified probe.
After hybridization, a signal is derived from
the guanine bases in the target.
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Experimental

1. CPE Activation 1.7V 60 sec. in 0.05M phosphate
   buffer solution (PBS).
2. Inosine-labelled probe immobilization 0.5V
   300s. in acetate buffer solution (ABS).
3. Washing step with ABS.
4. 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.
5. Washing step 1 SDS buffer 3s and then
   immediately dipped into blank Tris-HCl buffer
   solution(TBS).
6. Measurement The oxidation signal of guanine was
   measured by using differential pulse voltammetry
   (DPV) in blank ABS by scanning from-0.80-1.40V.

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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.

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Figure 1A Figure 1B
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Figure 2A Figure 2B
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THE ALLEL SPECIFIC DETECTION of MUTATION
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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

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DNA-Directed Attachment of Carbon Nanotubes for
EnhancedLabel-Free Electrochemical Detection of
DNA Hybridization
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Covalent immobilization of Oligonucleotide onto
graphite
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Part II

Electrochemical Genosensor based on colloidal
gold nanoparticles
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Gold nanoparticleshave 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. 

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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.
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.
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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.
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• 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.

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• 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.

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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

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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.

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Figure I (synt. oligonucleotides)
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Figure II (Synt. PCR)
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Figure III (PCR real sample)
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• WT probe with WT target at PGE
• R. S. D. 7.64 (n5).
• MT probe with the MT target at PGE
• R. S. D. 7.42 (n5).
• The detection limits, (S/N3)
• 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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TiO2 nanoparticles Studies
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0.1M TiO2 signal by using DPV
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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.
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Future work

• For this study,
• hybridization detection (after finding TiO2
  nanoparticles attractivity on ss or ds DNA) by
  using CPE and PGE.

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Electrochemical Coding of Single-NucleotidePolym
orphisms By Monobase-Modified Gold Nanoparticles
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Part III

• Electrochemical Genosensor for the Discrimination
  of HSV (Herpes Simplex Virus)
• Type I and II

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Herpes Simplex Virus

• Type I PCR Product
• 5TCAACTTCGACTGGCCCTTCTTGCTGGCCAAGCTGACGGACATTTACA
  AGGTCCCCCTGGAGACGGGTACGGCCGCATGAACGGCCGGGGCGTGTTTC
  GCGTGTGGGACATAGGCCAGAGCCACTTCCAGAAGCGCAGCAAGATAAAG
  GTGAACGGCATGGTGAGCATCGACATGTACGG 3
• Type II PCR Product
• 5TCAACTTCGACTGGCCCTTCGTCCTGACCAAGCTGACGGAGATCTACA
  AGGTCCCGCTCGAGACGGGTACGGGCGCATGAACGGCCGGGGTGTGTTCC
  GCGTGTGGGACATAGGCCAGAGCCACTTCCAGAAGCGCAGCAAGATAAAG
  GTGAACGGCATGGTGAACATCGACATGTACGG 3

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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.

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.
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HSV Tip1 ve Tip2 varyasyonlarinin ayiriminda PCR
ürünü örneklerinden prob dizi seçimiHSV Tip1
64208 ve 64386 nükleotidleri arasindaki bölgeye
ait PCR ürünü5 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 3HSV Tip2 64669 ve 64847
nükleotidleri arasindaki bölgeye ait PCR
Ürünü5 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
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FIGURE I

• Meldola Blue signal obtained from, hybridization
  between, A Probe TypeII and synthetic targetII,
  BProbe TypeII and synthetic targetI, C Probe
  TypeII only.

Meldola Blue signal obtained from, hybridization
between, A Probe TypeI and synthetic targetI,
BProbe TypeI and synthetic targetII, C Probe
TypeI only.
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HSV Çalismalarina Ait Bulgularin
Degerlendirilmesi

• Diferansiyel puls voltametri teknigi ile Prob
  derisiminin hibridizasyondan sonra alinan MDB
  yanita etkisi. A)probun karsiligi olan hedef ile
  hibridizasyonu, B) mutasyon içeren hedef dizi ile
  hibridizasyonu sonucu PGE yüzeyinde olusan MDB
  sinyalleri.

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En uygun hibridizasyon zamani saptandi

• Diferansiyel puls voltametri teknigi ile MDB
  indikatörlügünde hibridizasyon zamaninin
  incelenmesi
• A)probun karsiligi olan hedef ile hibridizasyonu,
  B) mutasyon içeren hedef dizi ile hibridizasyonu
  sonucu PGE yüzeyinde olusan MDB sinyalleri.

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PCR ürünü örnekler için kullanilacak en uygun
seyrelme orani saptandi.

• PCR ürünü örneklerinin seyrelme oraninin
  incelenmesi. A) probun karsiligi olan hedef ile
  hibridizasyonu, B) mutasyon içeren hedef dizi ile
  hibridizasyonu sonucu alinan elektrokimyasal
  sinyaller

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Gerçek hasta örnekleriyle yapilan çalismalarda
HSV Tip1 ve Tip2nin ayirimi net olarak gözlendi.
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FIGURE 2 Real Samples
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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.

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• 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.

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• 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

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THANK YOU