Nanolif kaplı kalem grafit biyosensör yüzeyinde spesifik nükleik asit dizilerinin hibridizasyonunun elektrokimyasal olarak tespiti
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Date
2016-06-28
Authors
Tanik, Nilay Aladağ
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Publisher
Uludağ Üniversitesi
Abstract
Bu çalışmada, nanolif kaplı grafit biyosensör yüzeyinde spesifik nükleik asit dizilerinin hibridizasyonunun elektrokimyasal olarak tespiti amaçlanmıştır. Bu amaçla, Alzheimer, Parkinson, Bipolar Bozukluk gibi nörodejeneratif hastalıklarda sıklıkla gözlenen nörotrofik faktörler ailesinden BDNF geninde meydana gelen Val66Met tek nokta mutasyonunun ilk kez elektrokimyasal tayinine yönelik bir biyosensör geliştirilmiştir. Bu polimorfizminin tayini için genelde kullanılmakta olan yöntemler, RFLP, RT-PCR ve DNA sekanslama yöntemleridir. Ancak bunlar pahalı yöntemler olup aynı zamanda oldukça zaman alan örnek hazırlama süreçleri gerektirmektedir. Tez kapsamında yapılan ön çalışmalarda, guanine dayalı yani indikatörsüz bir ayırımın mümkün olduğu görülmüş ve yöntem olarak DNA'daki en elektroaktif ve kararlı yanıt veren baz olan guanin bazının yaklaşık +1,0 V civarında verdiği yükseltgenme sinyaline dayalı indikatörsüz hibridizasyon algılama yöntemi kullanılmıştır. Literatürde ilk defa PGE yüzeyi elektrospin yöntemi ile nanolifle kaplanmıştır. Nanolif kaplı elektrot yüzeyine prob tutturmadan önce CV uygulanması, yüzeyden alınan guanin sinyalinin CV uygulanmamış ve nanolif kaplı olmayan PGE yüzeyine göre 4 kat arttışına sebep olmuştur. Çalışmada optimum koşulların belirlenmesinde tek sarmal DNA'da gözlenen guaninin oksidasyon sinyalinin, dsDNA(hibrit) ve tek bazı eşleşmemiş DNA(MM)'dan daha fazla olması esas alınmıştır ve mümkün olan en iyi ayırımın sağlandığı koşullar en iyi tayin koşulları olarak belirlenmiştir. Buna göre en uygun prob ve hedef tamponu 5X SSC, ölçüm tamponu ABS, prob konsantrasyonu 10µg/ml, hedef konsantrasyonu 15µg/ml, prob tutturma ve hibridizasyon süresi 30dk, hibridizasyondan sonraki yıkama süresinin 20sn olduğu görülmüştür. Tek sarmal DNA da yani hibridizasyon meydana gelmemiş dizilerde (prob, NC ve MM dizilerinde) guaninler açık olduğu için yükseltgenme sinyali yüksek, hibridizasyondan sonra ise sitozinle aralarında oluşturdukları hidrojen bağı köprüleri nedeniyle yükseltgenmesi kısmen kapalı duruma geldiğinden tek sarmala göre daha düşük sinyal vermesi beklenmiştir. Sonuç olarak tek sarmal olan prob diziden alınan sinyal en yüksek, hibrit diziden alınan sinyal en düşük, NC ve MM dizilerinden alınan sinyalin de hibridizasyon meydana gelmediği için prob dizisine yakın bir değerde olduğu görülmüştür. Ayrıca bu sonuç, geliştirilen biyosensörün seçimli şekilde hedefine bağlandığını ve polimorfizm tayinin mümkün olduğunu göstermiştir. Yöntemimizin kolay uygulanabilir olması, pahalı ekipmanlara gerek duyulmaması, hızlı cevap veren bir sistem olması (yaklaşık 65dk'da) ve herhangi bir toksik veya radyoaktif ajanın kullanılmaması nedenleriyle klasik yöntemlere güçlü bir alternatif yöntem geliştirilmiştir. Bu prototip biyosensör ile sadece bu gibi polimorfizmlerin ve mutasyonların tespiti değil, çevre, gıda, tıbbi tanı ve klinik uygulamalarda da kullanılabilecek mikroçip teknolojisinin altyapısı oluşturulmuştur.
In this study, specifıc nucleic acid sequences hybridization on the nanofiber coated pencil graphite biosensor surface is intended to detect by electrochemically. For this purpose, biosensor has been developed at the first time for the electrochemical detection of Val66Met single point mutations occurred in the BDNF gene which is frequently observed neurodegenerative diseases such as Alzheimer, Parkinson, and bipolar disorder. For determining this polymorphism, these methods are generally used: RFLP, RT-PCR, and DNA sequencing. But, these methods are expensive and they also requires expertise and quite time-consuming sample preparation processes. It was found that to be possible the detection of hybridization with label-free methods which are based on a guanine signal in the preliminary studies of these thesis. In this method, the oxidation signal of most electroactive and stable DNA's base, guanine, approximately at about +1.0V is used. It is the first time that PGE surface is coated with nanofibers with electrospinning method in the literature. Application of CV to the nanofiber coated electrode surfaces before the probe attachment has led to a 4-fold increase in oxidation signal of guanine when compared with the untreated and uncoated surface of PGE. For the determining of optimum operation conditions, it was based on that the oxidation signal of guanin signal at the ssDNA is more than the signal at the dsDNA and MM DNA. The conditions which give the best possible distinction between the ssDNA, dsDNA and MM DNA were determined as the optimum conditions of assay. Accordingly, optimum probe, target, and measurement buffer were found 5X SSC, 5X SSC and ABS, respectively, probe and target concentration were found 10μg/ml and 15μg/ml, probe immobilization and hybridization time were found 30min, and washing time after the hybridization was found 20s. ssDNA is the meaning of sequences which are not occur hybridization (probe, NC, and MM) had higher oxidation signal of guanin. Because, when the hybridization occurs, there is a decrease in electrochemical signals due to the interaction of free guanines of the probe with complementary cytosine bases present in the target sequence. This is because of the guanine bases are closed with the cytosine bases in the target sequences. Consequently, signal received from the probe was high, signal received from the hybrid was low, since the signals received from the NC and MM sequences, in which hybridization not occur, were found to be close to the value of the probe signal. Moreover, these results showed that the developed biosensor selectively connect to the target and showed that it is possible to determination of polymorphisms. Our method is easy to apply, no need to expensive equipment, fast response system (about 65min), and no using of any toxic or radioactive agents. For these reasons, it is powerful alternative to classical methods. This prototype biosensor can be used not only the detection of polymorphisms and mutations and also can be used environment, food, medical diagnostics and in clinical applications to create the infrastructure of the microchip technology.
In this study, specifıc nucleic acid sequences hybridization on the nanofiber coated pencil graphite biosensor surface is intended to detect by electrochemically. For this purpose, biosensor has been developed at the first time for the electrochemical detection of Val66Met single point mutations occurred in the BDNF gene which is frequently observed neurodegenerative diseases such as Alzheimer, Parkinson, and bipolar disorder. For determining this polymorphism, these methods are generally used: RFLP, RT-PCR, and DNA sequencing. But, these methods are expensive and they also requires expertise and quite time-consuming sample preparation processes. It was found that to be possible the detection of hybridization with label-free methods which are based on a guanine signal in the preliminary studies of these thesis. In this method, the oxidation signal of most electroactive and stable DNA's base, guanine, approximately at about +1.0V is used. It is the first time that PGE surface is coated with nanofibers with electrospinning method in the literature. Application of CV to the nanofiber coated electrode surfaces before the probe attachment has led to a 4-fold increase in oxidation signal of guanine when compared with the untreated and uncoated surface of PGE. For the determining of optimum operation conditions, it was based on that the oxidation signal of guanin signal at the ssDNA is more than the signal at the dsDNA and MM DNA. The conditions which give the best possible distinction between the ssDNA, dsDNA and MM DNA were determined as the optimum conditions of assay. Accordingly, optimum probe, target, and measurement buffer were found 5X SSC, 5X SSC and ABS, respectively, probe and target concentration were found 10μg/ml and 15μg/ml, probe immobilization and hybridization time were found 30min, and washing time after the hybridization was found 20s. ssDNA is the meaning of sequences which are not occur hybridization (probe, NC, and MM) had higher oxidation signal of guanin. Because, when the hybridization occurs, there is a decrease in electrochemical signals due to the interaction of free guanines of the probe with complementary cytosine bases present in the target sequence. This is because of the guanine bases are closed with the cytosine bases in the target sequences. Consequently, signal received from the probe was high, signal received from the hybrid was low, since the signals received from the NC and MM sequences, in which hybridization not occur, were found to be close to the value of the probe signal. Moreover, these results showed that the developed biosensor selectively connect to the target and showed that it is possible to determination of polymorphisms. Our method is easy to apply, no need to expensive equipment, fast response system (about 65min), and no using of any toxic or radioactive agents. For these reasons, it is powerful alternative to classical methods. This prototype biosensor can be used not only the detection of polymorphisms and mutations and also can be used environment, food, medical diagnostics and in clinical applications to create the infrastructure of the microchip technology.
Description
Keywords
Elektrokimyasal DNA biyosensör, Nanolif, Elektrospin, Elektrochemical DNA biosensor, SNP, Nanofiber, Elektrospinning
Citation
Tanik, N. A. (2016). Nanolif kaplı kalem grafit biyosensör yüzeyinde spesifik nükleik asit dizilerinin hibridizasyonunun elektrokimyasal olarak tespiti. Yayınlanmamış doktora tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.