Anaplasma Phagocytophilum’un Tayinine Yönelik Elektrokimyasal DNA Sensörü Geliştirilmesi

Yıl 2017, Cilt: 38 Sayı: 4, 748 - 758, 08.12.2017

Adil Elik Gültekin Gökçe Çağdaş Ceylan

https://doi.org/10.17776/csj.349331

Öz

Bu çalışma, oldukça geniş bir konakçı gurubunda
enfeksiyona yol açan bir patojen olan Anaplasma
Phagocytophilum dizi seçimli DNA hibridizasyonunun voltametrik yöntemle
tayin edildiği tek kullanımlık elektrokimyasal DNA biyosensörü geliştirilmesine
yöneliktir. Herhangi bir etiketlemenin yapılmadığı bu elektrokimyasal
çalışmanın esasını DNA hibridizasyonu sonrasında Guanin bazının yükseltgenme
sinyalinin ölçülmesi ile bir dubleks oluşumunun izlenmesi oluşturur. Söz konusu
biyosensör tasarımı inozinmodifiye (guanin içermeyen) probun, kalem grafit
elektrota (PGE) immobilize edilmesi ve diferansiyel puls voltametrisi (DPV) ile
guanin yükseltgenme sinyalinin ölçülerek, dubleks oluşumu tayinini içerir. Bu
çalışmanın ilk aşamasında, Anaplasma
phagocytophilum temsil eden prob dizisi, aktive edilmiş kalem grafit
elektrot (PGE) yüzeyine yaş adsorbsiyon yöntemi ile immobilize edilmiş ve daha
sonra prob ve hedefi arasındaki hibridizasyonun varlığı 1000 mV’da gözlenen
guanin yükseltgenme sinyali ile tespit edilmiştir.

Yapılan
optimizasyon çalışmasında prob derişimi 25 µg/mL, prob immobilize süresi 6
dakika; hibridizasyon için hedef derişimi 40 µg/mL ve hibridizasyon süresinin
10 dakika olduğu gözlenmiştir. Geliştirilen elektrokimyasal biyosensörün
spesifikliği, baz sırasından bir bazın yeri (MM) ve tüm bazların yerleri farklı
(NC) olan hedef diziler kullanılarak, test edilmiştir. Ferri/Ferro siyanür
redoks sistemi altında elektrokimyasal empedans yöntemi kullanılarak yapılan
empedimetrik ölçümler ile DNA hibridizasyonunun gerçekleştirildiği ayrıca teyit
edilmiştir. Tayin sınırını (DL) belirlemek için hedef derişimi 0,78 µM ile 3,90
µM arasında değişen hedef diziler kullanılmış ve tayin sınırı 0,244 µM olarak bulunmuştur.

Anahtar Kelimeler

Elektrokimyasal DNA Sensörü; Kalem Grafit Elektrot, Anaplasma Phagocytophilum, Diferansiyel PulsVoltametrisi

Development of Electrochemical DNA Sensor for The Determination of Anaplasma Phagocytophilum

Öz

This
study was intended to enhance the disposable electrochemical DNA biosensor
through which Anaplasma phagocytophilum,
a pathogen causing infection in a
considerably wide host group, sequence selective DNA hybridization was detected
with a voltametric method. Monitoring the formation of a duplex by
measuring the oxidation signal of guanine base after DNA hybridization forms
the basis of this study in which no labeling was done. The biosensor design
contained immobilizing inosine modified (guanine-free) probe to the pencil
graphite electrode (PGE) and detecting duplex formation by measuring guanine
oxidation signal with differential pulse voltammetry (DPV).

In the first stage of this study, probe sequence representing Anaplasma
phagocytophilum was immobilized to the activated surface of
pencil graphite electrode (PGE) by wet adsorbtion method, and then the hybridization between probe
and its target was confirmed with guanine oxidation signal observed at 1000 mV.
In the optimization study, it was observed that probe concentration was 25
µg/mL, immobilization time was 6 minutes; for the hybridization, target
concentration was 40 µg/mL and hybridization time was 10 minutes. Selectivity
of biochemical biosensor developed was tested by using mismatch and non complementary
target sequences. It was also confirmed that DNA hybridization was carried out
with impedimetric measurements by using electrochemical impedance method under
the ferri/ferro cyanide redox system. To estimate the detection limit (DL),
target sequences whose concentrations varies between 0, 78 µM and 3,90 µM were
used, and it was found that the detection limit was 0,244 µM.

Anahtar Kelimeler

Electrochemical DNA Sensor, Pencil Graphite Electrode, Anaplasma Phagocytophilum, Differential Pulse Voltammetry

Kaynakça

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