Development of Electrochemical DNA Sensor for The Determination of Anaplasma Phagocytophilum

Adil Elik; Gültekin Gökçe; Çağdaş Ceylan

doi:10.17776/csj.349331

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Anaplasma Phagocytophilum’un Tayinine Yönelik Elektrokimyasal DNA Sensörü Geliştirilmesi

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

Abstract

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.

Keywords

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

References

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Details

Primary Language

English

Subjects

-

Journal Section

Research Article

Authors

Adil Elik

Gültekin Gökçe

Çağdaş Ceylan

Publication Date

December 8, 2017

Submission Date

April 21, 2017

Acceptance Date

May 30, 2017

Published in Issue

Year 2017 Volume: 38 Number: 4

DOI

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

IZ

https://izlik.org/JA82MX92NR

Cite

RIS / Bibtex

APA

Elik, A., Gökçe, G., & Ceylan, Ç. (2017). Development of Electrochemical DNA Sensor for The Determination of Anaplasma Phagocytophilum. Cumhuriyet Science Journal, 38(4), 748-758. https://doi.org/10.17776/csj.349331