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Anaplasma Phagocytophilum’un Tayinine Yönelik Elektrokimyasal DNA Sensörü Geliştirilmesi

Yıl 2017, , 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

Kaynakça

  • [1]. Wang J.,Rivas G., Cai X., Palecek E., Nielsen P., Shiraishi H., Dontha N., Luo D., Parrado C.,Chicharro M., Farias P.A.M., Valera F.S., Grant D.H., Ozsoz M., Flair M.N., DNA electrochemical biosensors for environmental monitoring. A review, Analytica Chimica Acta,1997, 347 (1-2), 1.
  • [2]. Wang J., Towards genoelectronics: Electrochemical biosensing of DNA hybridization, Chem. Eur., J., 1999, 5 (6), 1681.
  • [3]. Wang J.,Rivas G., Luo D., Cai X., Valera F.S., Dontha N., DNA-modified electrode for the detection of aromatic amines, Anal. Chem.,1996, 68, 4365.
  • [4]. Özkan D., Erdem A., Kara P., Kerman K., Gooding J.J., Nielsen P.E., Özsöz M., Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes,Anal. Chim. Acta, 2002, 462 (1), 39.
  • [5]. Fojta M., Havran L., Fulneckova J., Kubicaova T., Adsorptive transfer stripping AC voltammetry DNA complexes with intercalators, Electroanal., 2000, 12, 926.
  • [6]. Wang J., Kawde A.N., Erdem A., Salasar M., Magnetic beads based label free electrochemical detection of DNA hybridisation, Analyst, 2001, 120, 2020.
  • [7]. Meriç B., Kerman K., Özkan D., Kara P., Özsöz M., Indicator-free DNA biosensor based on adenine and guanine signals, Electroanal., 2002, 14(18), 1245.
  • [8]. Lumbey-Woodyear T., Campbell C.N., Freeman E. Freeman Georgiou G. Heller A., Rapid Amperometric verification of PCR amplification of DNA, Anal. Chem.,1999, 71, 535.
  • [9]. Kara P., Kerman K., Özkan D., Meriç B., Erdem A., Özkan Z., Özsöz M., Electrochemical genosensor for the detection of interaction between methylene blue and DNA, Electrochem. Commun., 2002, 4, 705.
  • [10]. Arıksoysal D., Karadeniz H., Erdem A., Şengönül A., Sayıner A.A., Özsöz M., Label-free electrochemical hybridization genosensor for the detection of hepatitis B virus genotype on the development of lamivudine resistance, Anal. Chem., 2005, 77 (15), 4908.
  • [11]. Brabec V., Koudelka J., Oxidation of DNA at carbon electrodes the effect of the quality of the DNA sample, J. Electroanal. Chem., 1980, 116, 793.
  • [12]. Fojta M., Doffkova R., Palecek E., Determination of traces of RNA in submicrogram amounts of single -or double- stranded DNAs by means of nucleic acid-modified electrodes, Electroanal., 1996, 8(5), 420.
  • [13]. Erdem A, Meriç B., Kerman K., Dalbastı T., Özsöz M., Detection of interaction between metal complex indicator and DNA by using electrochemical biosensor, Electroanal., 1999, 11, 1372.
  • [14]. Jelen, F., Erdem A., Palecek, E., Cyclic voltammetry of echinomycin and its interaction with double-stranded and single-stranded DNA adsorbed at the electrode, Bioelectrochemistry, 2002, 55, 165.
  • [15]. Pividori M.I., Merkoçi A., Alegret S., Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods, Biosensors and Bioelectronics, 2000, 15, 291.
  • [16]. Wang A.H.J. Interactions between anti tumor drugs and DNA, NucleicAcidsand Molecular Biology, 1987, Vol.1, 52, (Ed; Eckstein, F.,Lilley, D.M.J.,)Springer-Verlag Berlin Heidelberg-Germany.
  • [17]. Özkan D., Karadeniz H., Erdem A., Mascini M., Özsöz M., Electrochemical genosensor for Mitomycin C- DNA interaction based on guanine signal, J. Pharmaceutical and Biomedical Analysis, 2004, 35, 905.
  • [18]. Dumitrache M.O., Paştiu A.I., Kalmár Z., Mircean V., Sándor A.D., Gherman C.M., Peştean C., Mihalca A.D., Cozma V., Northern white-breasted hedgehogs Erinaceus roumanicus as hosts for ticks infected with Borrelia burgdorferisensu lato and Anaplasma phagocytophilum in Romania, TicksTick-borneDis., 2013, 4 (3) , 214.
  • [19]. Woldehiwet Z., The natural history of Anaplasma phagocytophilum, Vet. Parasitology, 2010, 167, 108.
  • [20]. Stuen S., Torsteinbo W.O., Bergstrom K., Bardsen K., Superinfection occurs in Anaplasma phagocytophilum infected sheep irrespective of infection phase and protection status, Acta Vet. Scand., 2009, 5, 1.
  • [21]. Grøva L., Olesen I., Steinshamn H., Stuen S., Prevalence of Anaplasma phagocytophilum infection and effect on lamb growth, Acta Vet. Scand., 2011, 53, 30.
  • [22]. Stuen S., Anaplasma phagocytophilum – the most widespread tick-borne infection in animals in Europe, Vet. Res. Commun., 2007, 31 (1),79.
  • [23]. Kathrin H.,Rainer O., Henning F., Stefan O. B., Dieter H., Peter K., Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany, Int. J. Med. Microbiol., 2004, 293(37), 86.
  • [24]. Petrovec M, Sixl W, Marth E, Bushati N, Wust G, Domestic animals as indicatiors of Anaplasma species infections in Northern Albania, Annuals of the New York Academy of Sciences, 2003, 990, 112.
  • [25]. Rymaszewska A., Divergence within the marker region of the gro ESL operon in Anaplasma phagocytophilum. European Journal of Clinical Microbiology and Infectious Diseases, 2008, 27, 1025.
  • [26]. Smrdel K.S., Seret M., Duh D., Knap N., Avsic-Zupanc T. , Anaplasma phagocytophilum in ticks in Slovenia, ParasitesandVectors, 2010, 3, 210.
  • [27]. Wang J., Cai X., Rivas G., Shiraishi H. Stripping potentiometric transduction of DNA hybridization processes, Anal. Chim. Acta, 1996, 326, 141.
  • [28]. Palecek E., Fojta M., Differantial pulse voltammetric determination of RNA at the picomole level in the presence of DNA and nucleic acid component, Anal. Chem., 1994, 66, 1566.
  • [29]. Meriç B., Kerman K., Özkan D., Kara P., Erensoy S., Akarca U.S., Macsini M., Özsöz M., ). Electrochemical DNA biosensor for the detection of TT and Hepatitis B virus from PCR amplified real samples by using methylene blue,Talanta, 2002, 56 (5, 939.
  • [30]. Özkan D., Erdem A., Kara P., Kerman K., Gooding J.J., Nielsen P. E., Özsöz M., Electrochemical detection of hybridization using peptide nucleic acids and methylen blue on self-assembled alkanethiol monolayer modified gold electrodes, Electrochem. Commun., 2002, 4, 796.
  • [31]. Erdem A., Özsöz M., Voltammetry of the anticancer drug mitoxantrone and DNA, Turk. J. Chem, 2001, 25, 469.
  • [32]. Erdem A., Sayar F., Karadeniz H., Guven G., Ozsoz M., Piskin E., Development of Streptavidin Carrying Magnetic Nanoparticles and Their Applications in Electrochemical Nucleic Acid Sensor Systems, Electroanal,, 2007, 19, 798.

Development of Electrochemical DNA Sensor for The Determination of Anaplasma Phagocytophilum

Yıl 2017, , 748 - 758, 08.12.2017

Adil Elik , Gültekin Gökçe Çağdaş Ceylan

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

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

  • [1]. Wang J.,Rivas G., Cai X., Palecek E., Nielsen P., Shiraishi H., Dontha N., Luo D., Parrado C.,Chicharro M., Farias P.A.M., Valera F.S., Grant D.H., Ozsoz M., Flair M.N., DNA electrochemical biosensors for environmental monitoring. A review, Analytica Chimica Acta,1997, 347 (1-2), 1.
  • [2]. Wang J., Towards genoelectronics: Electrochemical biosensing of DNA hybridization, Chem. Eur., J., 1999, 5 (6), 1681.
  • [3]. Wang J.,Rivas G., Luo D., Cai X., Valera F.S., Dontha N., DNA-modified electrode for the detection of aromatic amines, Anal. Chem.,1996, 68, 4365.
  • [4]. Özkan D., Erdem A., Kara P., Kerman K., Gooding J.J., Nielsen P.E., Özsöz M., Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes,Anal. Chim. Acta, 2002, 462 (1), 39.
  • [5]. Fojta M., Havran L., Fulneckova J., Kubicaova T., Adsorptive transfer stripping AC voltammetry DNA complexes with intercalators, Electroanal., 2000, 12, 926.
  • [6]. Wang J., Kawde A.N., Erdem A., Salasar M., Magnetic beads based label free electrochemical detection of DNA hybridisation, Analyst, 2001, 120, 2020.
  • [7]. Meriç B., Kerman K., Özkan D., Kara P., Özsöz M., Indicator-free DNA biosensor based on adenine and guanine signals, Electroanal., 2002, 14(18), 1245.
  • [8]. Lumbey-Woodyear T., Campbell C.N., Freeman E. Freeman Georgiou G. Heller A., Rapid Amperometric verification of PCR amplification of DNA, Anal. Chem.,1999, 71, 535.
  • [9]. Kara P., Kerman K., Özkan D., Meriç B., Erdem A., Özkan Z., Özsöz M., Electrochemical genosensor for the detection of interaction between methylene blue and DNA, Electrochem. Commun., 2002, 4, 705.
  • [10]. Arıksoysal D., Karadeniz H., Erdem A., Şengönül A., Sayıner A.A., Özsöz M., Label-free electrochemical hybridization genosensor for the detection of hepatitis B virus genotype on the development of lamivudine resistance, Anal. Chem., 2005, 77 (15), 4908.
  • [11]. Brabec V., Koudelka J., Oxidation of DNA at carbon electrodes the effect of the quality of the DNA sample, J. Electroanal. Chem., 1980, 116, 793.
  • [12]. Fojta M., Doffkova R., Palecek E., Determination of traces of RNA in submicrogram amounts of single -or double- stranded DNAs by means of nucleic acid-modified electrodes, Electroanal., 1996, 8(5), 420.
  • [13]. Erdem A, Meriç B., Kerman K., Dalbastı T., Özsöz M., Detection of interaction between metal complex indicator and DNA by using electrochemical biosensor, Electroanal., 1999, 11, 1372.
  • [14]. Jelen, F., Erdem A., Palecek, E., Cyclic voltammetry of echinomycin and its interaction with double-stranded and single-stranded DNA adsorbed at the electrode, Bioelectrochemistry, 2002, 55, 165.
  • [15]. Pividori M.I., Merkoçi A., Alegret S., Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods, Biosensors and Bioelectronics, 2000, 15, 291.
  • [16]. Wang A.H.J. Interactions between anti tumor drugs and DNA, NucleicAcidsand Molecular Biology, 1987, Vol.1, 52, (Ed; Eckstein, F.,Lilley, D.M.J.,)Springer-Verlag Berlin Heidelberg-Germany.
  • [17]. Özkan D., Karadeniz H., Erdem A., Mascini M., Özsöz M., Electrochemical genosensor for Mitomycin C- DNA interaction based on guanine signal, J. Pharmaceutical and Biomedical Analysis, 2004, 35, 905.
  • [18]. Dumitrache M.O., Paştiu A.I., Kalmár Z., Mircean V., Sándor A.D., Gherman C.M., Peştean C., Mihalca A.D., Cozma V., Northern white-breasted hedgehogs Erinaceus roumanicus as hosts for ticks infected with Borrelia burgdorferisensu lato and Anaplasma phagocytophilum in Romania, TicksTick-borneDis., 2013, 4 (3) , 214.
  • [19]. Woldehiwet Z., The natural history of Anaplasma phagocytophilum, Vet. Parasitology, 2010, 167, 108.
  • [20]. Stuen S., Torsteinbo W.O., Bergstrom K., Bardsen K., Superinfection occurs in Anaplasma phagocytophilum infected sheep irrespective of infection phase and protection status, Acta Vet. Scand., 2009, 5, 1.
  • [21]. Grøva L., Olesen I., Steinshamn H., Stuen S., Prevalence of Anaplasma phagocytophilum infection and effect on lamb growth, Acta Vet. Scand., 2011, 53, 30.
  • [22]. Stuen S., Anaplasma phagocytophilum – the most widespread tick-borne infection in animals in Europe, Vet. Res. Commun., 2007, 31 (1),79.
  • [23]. Kathrin H.,Rainer O., Henning F., Stefan O. B., Dieter H., Peter K., Pathogens and symbionts in ticks: prevalence of Anaplasma phagocytophilum (Ehrlichia sp.), Wolbachia sp., Rickettsia sp., and Babesia sp. in Southern Germany, Int. J. Med. Microbiol., 2004, 293(37), 86.
  • [24]. Petrovec M, Sixl W, Marth E, Bushati N, Wust G, Domestic animals as indicatiors of Anaplasma species infections in Northern Albania, Annuals of the New York Academy of Sciences, 2003, 990, 112.
  • [25]. Rymaszewska A., Divergence within the marker region of the gro ESL operon in Anaplasma phagocytophilum. European Journal of Clinical Microbiology and Infectious Diseases, 2008, 27, 1025.
  • [26]. Smrdel K.S., Seret M., Duh D., Knap N., Avsic-Zupanc T. , Anaplasma phagocytophilum in ticks in Slovenia, ParasitesandVectors, 2010, 3, 210.
  • [27]. Wang J., Cai X., Rivas G., Shiraishi H. Stripping potentiometric transduction of DNA hybridization processes, Anal. Chim. Acta, 1996, 326, 141.
  • [28]. Palecek E., Fojta M., Differantial pulse voltammetric determination of RNA at the picomole level in the presence of DNA and nucleic acid component, Anal. Chem., 1994, 66, 1566.
  • [29]. Meriç B., Kerman K., Özkan D., Kara P., Erensoy S., Akarca U.S., Macsini M., Özsöz M., ). Electrochemical DNA biosensor for the detection of TT and Hepatitis B virus from PCR amplified real samples by using methylene blue,Talanta, 2002, 56 (5, 939.
  • [30]. Özkan D., Erdem A., Kara P., Kerman K., Gooding J.J., Nielsen P. E., Özsöz M., Electrochemical detection of hybridization using peptide nucleic acids and methylen blue on self-assembled alkanethiol monolayer modified gold electrodes, Electrochem. Commun., 2002, 4, 796.
  • [31]. Erdem A., Özsöz M., Voltammetry of the anticancer drug mitoxantrone and DNA, Turk. J. Chem, 2001, 25, 469.
  • [32]. Erdem A., Sayar F., Karadeniz H., Guven G., Ozsoz M., Piskin E., Development of Streptavidin Carrying Magnetic Nanoparticles and Their Applications in Electrochemical Nucleic Acid Sensor Systems, Electroanal,, 2007, 19, 798.
Toplam 32 adet kaynakça vardır.

Ayrıntılar

Bölüm Natural Sciences
Yazarlar

Adil Elik

Gültekin Gökçe

Çağdaş Ceylan

Yayımlanma Tarihi 8 Aralık 2017
Gönderilme Tarihi 21 Nisan 2017
Kabul Tarihi 30 Mayıs 2017
Yayımlandığı Sayı Yıl 2017

Kaynak Göster

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