ساخت زیست حسگر الکتروشیمیایی بر مبنی DNA فاقد نشانگر و اصلاح شده با نانو مواد جهت شناسایی ویروس موزاییک انجیر
نیلوفر رجبی
1
(گروه گیاهپزشکی، دانشکده علوم کشاورزی و صنایع غذایی، واحد علوم و تحقیقات دانشگاه آزاد اسلامی، تهران. صندوق پستی: 775- 14515 – کدپستی: 147789385)
محمد رضا صفرنژاد
2
(بخش تحقیقات ویروس شناسی، موسسه تحقیقات گیاه پزشکی کشور، سازمان تحقیقات آموزش و ترویج کشاورزی)
فرشاد رخشنده رو
3
(گیاه پزشکی، دانشکده علوم کشاورزی و صنایع غذایی، واحد علوم و تحقیقات دانشگاه اسلامی، تهران)
مسعود شمس بخش
4
(استاد گروه بیماری شناسی گیاهی، دانشکده کشاورزی، دانشگاه تربیت مدرس)
حجت الله ربانی
5
(گروه مهندسی آنتی ژن و آنتی بادی مرکز تحقیقات آنتی بادی مونوکلونال پژوهشکده ابن سینا، دانشگاه شهید بهشتی، تهران، ایران)
کلید واژه: ویروس موزاییک انجیر, زیست‌حسگر الکترو شیمیایی, بهینه¬سازی, کالیبراسیون,
چکیده مقاله :
امروزه زیست¬حسگر¬های الکترو¬شیمیایی به¬دلیل حساسیت و دقت بالا در ردیابی اسید¬های نوکلئیک هدف مورد توجه بسیار قرار گرفته¬اند. از طرفی به دلیل اهمیت اقتصادی درختان انجیر، تشخیص سریع و دقیق بیماری¬های آن از جمله بیماری موزائیک انجیر، Fig Mosaic Disease (FMD)، به¬عنوان یکی از مهم¬ترین و مخرب¬ترین بیماری¬های ویروسی درختان انجیر، نقش مؤثری در مدیریت این بیماری ایفا می¬کند. در این مطالعه، نانو¬زیست¬حسگر الکترو-شیمیایی مبتنی بر DNA و فاقد نشانگر مولکولی برای شناسایی کیفی و کمی ویروس FMV در نمونه‌های آلوده بهینه¬سازی شد. در این راستا، جهت بهینه¬سازی و بهبود عملکرد زیست‌حسگر طراحی شده، پارامتر¬های غلظت کاوشگر DNA برای تشکیل تک‌لایه‌های خود انباشته و زمان لازم جهت انجام فرآیند هیبریداسیون در محدوده زیستی با بیشترین تأثیر در زیست‌حسگر DNA، مورد بررسی قرار گرفت. با توجه به داده‌های منحنی کالیبراسیون، اولین محدوده خطی 3- میکرومولار تا 3 میکرومولار با معادله خط y = 4.81x+34.58 و ضریب تشخیص (9967/0=R2) به-دست آمد. زیست¬حسگر بهینه¬سازی شده در این پژوهش جهت تشخیص ماده ژنتیکی FMV دارای پایداری،گزینش‌پذیری بالا و همچنین کاربری آسان و سریع بوده که نوید یک روش تشخیصی با کارایی و دقت بالا جهت شناسایی این ویروس را می¬دهد.
Today, electrochemical biosensors are highly regarded due to their high sensitivity and accuracy in tracking target nucleic acids. On the other hand, due to the economic importance of fig trees, rapid and accurate diagnosis of its diseases, including Fig Mosaic Disease (FMD), which is one of the most important and destructive viral diseases of fig trees plays an effective role in the management and control of this disease. In this study, a novel label free electrochemical DNA-based biosensor was optimized to qualitatively and quantitatively identify the presence of FMV in the infected samples. In this regard, in order to optimize and improve the performance of the designed biosensor, the concentration parameters of the DNA probe to form self-assembled monolayers and the time required to perform the hybridization process in the biological range with the greatest impact on the DNA biosensor were investigated. According to the data of the calibration curve, the first linear range at -3 μM to 3 μM was obtained with the equation of the line y= 4.81x+34.58 and the detection coefficient (R2= 0.9967). In this research, the omtimized biosensor to detect a stable, high selective, easy and fast FMV-DNA, promises a diagnostic method with high efficiency and accuracy.
Adam, V., Huska, D., Hubalek, J. and Kizek, R. 2010. Easy to use and rapid isolation and detection of a viral nucleic acid by using paramagnetic microparticles and carbon nanotubes-based screen-printed electrodes. Microfluidics and Nanofluidics 8(3): 329- 339.
Alimoradian, M., Rakhshandehroo, F. and Shamsbakhsh, M. 2016. Prevalence and phylogenetic analysis of fig mosaic virus and fig badnavirus-1 in Iran. Journal of Plant Protection Research 56(2): 122-128.
Alsaheli, Z., Abdallah, A., Incerti, O., Shalaby, A., Youssef, S., Digiaro, M. and Elbeaino, T. 2021. Development of singleplex and multiplex real-time (Taqman®) RT-PCR assays for the detection of viruses associated with fig mosaic disease. Journal of Virological Methods 293: 114145.
Amiri, M., Bezaatpour, A., Jafari, H., Boukherroub, R. and Szunerits, S. 2018. Electrochemical methodologies for the detection of pathogens. ACS sensors 3(6): 1069-1086.
Authier, L., Grossiord, C., Brossier, P. and Limoges, B. 2001. Gold nanoparticle-based quantitative electrochemical detection of amplified human cytomegalovirus DNA using disposable microband electrodes. Analytical Chemistry 73(18): 4450-4456.
Brazaca, L.C., Dos Santos, P.L., de Oliveira, P.R., Rocha, D.P., Stefano, J.S., Kalinke, C., Munoz, R.A.A., Bonacin, J.A., Janegitz, B.C. and Carrilho, E. 2021. Biosensing strategies for the electrochemical detection of viruses and viral diseases–a review. Analytica Chimica Acta 1159: 338384.
Caglayan, K., Elci, E., Serce, C.U., Kaya, K., Gazel, M. and Medina, V. 2012. Detection of Fig mosaic virus in Virulifereous eriophyide mite Aceria ficus. Journal of Plant Pathology 94(3): 629-634.
Cesewski, E. and Johnson, B.N. 2020. Electrochemical biosensors for pathogen detection. Biosensors and Bioelectronics 159: 112214.
Dai Tran, L., Nguyen, B.H., Van Hieu, N., Tran, H.V., Le Nguyen, H. and Nguyen, P.X. 2011. Electrochemical detection of short HIV sequences on chitosan/Fe3O4 nanoparticle based screen printed electrodes. Materials Science and Engineering: C 31(2): 477-485.
Dalkılıccedil, Z., Mestav, H.O. and Kocataş, H. 2011. Genetic diversity of male fig (Ficus carica caprificus L.) genotypes with random amplified polymorphic DNA (RAPD) markers. African Journal of Biotechnology 10(4): 519-526.
Du, M., Yang, T., Li, X. and Jiao, K. 2012. Fabrication of DNA/graphene/polyaniline nanocomplex for label-free voltammetric detection of DNA hybridization. Talanta 88: 439-444.
Duffy, G. and Moore, E. 2017. Electrochemical immunosensors for food analysis: A review of recent developments. Analytical Letters 50(1): 1-32.
Elbeaino, T., Digiaro, M., Alabdullah, A., De Stradis, A., Minafra, A., Mielke, N., Castellano, M.A. and Martelli, G.P. 2009a. A multipartite single-stranded negative-sense RNA virus is the putative agent of fig mosaic disease. Journal of General Virology 90(5): 1281-1288.
Elbeaino, T., Digiaro, M. and Martelli, G.P. 2009b. Complete nucleotide sequence of four RNA segments of fig mosaic virus. Archives of Virology 154(11): 1719-1727.
Elbeaino, T., Digiaro, M., Mielke-Ehret, N., Muehlbach, H.P. and Martelli, G.P. 2018. ICTV virus taxonomy profile: Fimoviridae. Journal of General Virology 99(11): 1478-1479.
Erdem, A., Kerman, K., Meric, B., Akarca, U.S. and Ozsoz, M. 1999. DNA electrochemical biosensor for the detection of short DNA sequences related to the hepatitis B virus. Electroanalysis 11(8): 586-587.
Esfandyarpour, R., Esfandyarpour, H., Harris, J.S. and Davis, R.W. 2013. Simulation and fabrication of a new novel 3D injectable biosensor for high throughput genomics and proteomics in a lab-on-a-chip device. Nanotechnology 24(46): 465301.
Felix, F.S. and Angnes, L. 2018. Electrochemical immunosensors–a powerful tool for analytical applications. Biosensors and Bioelectronics 102: 470-478.
Firrao, G., Moretti, M., Rosquete, M.R., Gobbi, E. and Locci, R. 2005. Nanobiotransducer for detecting flavescence dorée phytoplasma. Journal of Plant Pathology 87: 101-107.
Fotouhi, L., Hashkavayi, A.B. and Heravi, M.M. 2013. Interaction of sulfadiazine with DNA on a MWCNT modified glassy carbon electrode: Determination of DNA. International Journal of Biological Macromolecules 53: 101-106.
Furst, A.L. and Francis, M.B. 2018. Impedance-based detection of bacteria. Chemical Reviews 119(1): 700-726.
Guliy, O., Zaitsev, B., Larionova, O. and Borodina, I. 2019. Virus detection methods and biosensor technologies. Biophysics 64(6): 890-897.
Haji-Hashemi, H., Habibi, M.M., Safarnejad, M.R., Norouzi, P. and Ganjali, M.R. 2018a. Label-free electrochemical immunosensor based on electrodeposited Prussian blue and gold nanoparticles for sensitive detection of citrus bacterial canker disease. Sensors and Actuators B: Chemical 275: 61-68.
Haji-Hashemi, H., Norouzi, P., Safarnejad, M.R., Larijani, B., Habibi, M.M., Raeisi, H. and Ganjali, M.R. 2018b. Sensitive electrochemical immunosensor for citrus bacterial canker disease detection using fast Fourier transformation square-wave voltammetry method. Journal of Electroanalytical Chemistry 820: 111-117.
Haji-Hashemi, H., Safarnejad, M.R., Norouzi, P., Ebrahimi, M., Shahmirzaie, M. and Ganjali, M.R. 2019. Simple and effective label free electrochemical immunosensor for Fig Mosaic Virus detection. Analytical Biochemistry 566: 102-106.
Idili, A., Amodio, A., Vidonis, M., Feinberg-Somerson, J., Castronovo, M. and Ricci, F. 2014. Folding-upon-binding and signal-on electrochemical DNA sensor with high affinity and specificity. Analytical Chemistry 86(18): 9013-9019.
Ishikawa, K., Maejima, K., Netsu, O., Fukuoka, M., Nijo, T., Hashimoto, M., Takata, D., Yamaji, Y. and Namba, S. 2015. Rapid detection of fig mosaic virus using reverse transcription loop-mediated isothermal amplification. Journal of General Plant Pathology 81(5): 382-389.
Jafari, S., Dehghani, M., Nasirizadeh, N., Baghersad, M.H. and Azimzadeh, M. 2019. Label-free electrochemical detection of Cloxacillin antibiotic in milk samples based on molecularly imprinted polymer and graphene oxide-gold nanocomposite. Measurement 145: 22-29.
Kerman, K., Saito, M., Morita, Y., Takamura, Y., Ozsoz, M. and Tamiya, E. 2004. Electrochemical coding of single-nucleotide polymorphisms by monobase-modified gold nanoparticles. Analytical Chemistry 76(7): 1877-1884.
Khan, M., Hasan, M., Hossain, S., Ahommed, M. and Daizy, M. 2020. Ultrasensitive detection of pathogenic viruses with electrochemical biosensor: State of the art. Biosensors and Bioelectronics 166: 112431.
Kuehn, B.M. 2016. Wearable biosensors studied for clinical monitoring and treatment. Jama 316(3): 255-257.
Lau, Y., Wu, H., Wee, E.J., Trau, M., Wang, Y. and Botella, J.R. 2017. Specific and sensitive isothermal electrochemical biosensor for plant pathogen DNA detection with colloidal gold nanoparticles as probes. Scientific Reports 7: 38896.
Liu, S.J., Nie, H.G., Jiang, J.H., Shen, G.L. and Yu, R.Q. 2009. Electrochemical sensor for mercury (II) based on conformational switch mediated by interstrand cooperative coordination. Analytical Chemistry 81(14): 5724-5730.
Liu, Y.T., Deng, J., Xiao, X.L., Ding, L., Yuan, Y.L., Li, H., Li, X.T., Yan, X.N. and Wang, L.L. 2011. Electrochemical sensor based on a poly (para-aminobenzoic acid) film modified glassy carbon electrode for the determination of melamine in milk. Electrochimica Acta 56(12): 4595-4602.
Lucarelli, F., Marrazza, G., Turner, A.P. and Mascini, M. 2004. Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors. Biosensors and Bioelectronics 19(6): 515-530.
Luo, X., Lee, T.M.H. and Hsing, I.M. 2008. Immobilization-free sequence-specific electrochemical detection of DNA using ferrocene-labeled peptide nucleic acid. Analytical Chemistry 80(19): 7341-7346.
Mazlan, N.F., Tan, L.L, Karim, N.H.A., Heng, L.Y., Jamaluddin, N.D., Yusof, N.YM., Quay, D.H.X. and Khalid, B. 2019. Acrylic-based genosensor utilizing metal salphen labeling approach for reflectometric dengue virus detection. Talanta 198: 358-370.
Mazloum-Ardakani, M., Hosseinzadeh, L. and Khoshroo, A. 2015. Label-free electrochemical immunosensor for detection of tumor necrosis factor α based on fullerene-functionalized carbon nanotubes/ionic liquid. Journal of Electroanalytical Chemistry 757: 58-64.
Mikkelsen, S.R. 1996. Electrochecmical biosensors for DNA sequence detection. Electroanalysis 8 (1): 15-19.
Mishra, G.K., Sharma, V. and Mishra, R.K. 2018. Electrochemical aptasensors for food and environmental safeguarding: A review. Biosensors 8(2): 28.
Movilli, J., Rozzi, A., Ricciardi, R., Corradini, R. and Huskens, J. 2018. Control of probe density at DNA biosensor surfaces using poly (l-lysine) with appended reactive groups. Bioconjugate Chemistry 29(12): 4110-4118.
Nguyen, M. and Haenni, A.L. 2003. Expression strategies of ambisense viruses. Virus Research 93(2): 141-150.
Palecek, E. and Fojta, M. 2005. Electrochemical DNA sensors. Pp. 127-192. In: Willner, I. and Katz, E. (eds.). Bioelectronics: From Theory to Applications. Wiley‐VCH Verlag GmbH & Co. KGaA.
Papadakis, G., Skandalis, N., Dimopoulou, A., Glynos, P. and Gizeli, E. 2015. Bacteria murmur: application of an acoustic biosensor for plant pathogen detection. PLoS One 10(7): e0132773.
Patel, R., Mitra, B., Vinchurkar, M., Adami, A., Patkar, R., Giacomozzi, F., Lorenzelli, L. and Baghini, M.S. 2022. A review of recent advances in plant-pathogen detection systems. Heliyon 8(12): e11855.
Patel, R., Vinchurkar, M., Shaikh, A.M., Patkar, R., Adami, A., Giacomozzi, F., Ramesh, R., Pramanick, B., Lorenzelli, L. and Baghini, M.S. 2023. Part I: Non-faradaic electrochemical impedance-based DNA biosensor for detecting phytopathogen-Ralstonia solanacearum. Bioelectrochemistry 150: 108370.
Pividori, M., Merkoci, A. and Alegret, S. 2000. Electrochemical genosensor design: immobilisation of oligonucleotides onto transducer surfaces and detection methods. Biosensors and Bioelectronics 15(5-6): 291-303.
Rajabi, N., Safarnejad, M.R., Rakhshandehroo, F., Shamsbakhsh, M. and Rabbani, H. 2022. Developing of specific monoclonal recombinant antibody fused to alkaline phosphatase (AP) for one-step detection of fig mosaic virus. 3 Biotech 12(4): 88.
Rana, K., Mittal, J., Narang, J., Mishra, A. and Pudake, R.N. 2021. Graphene based electrochemical DNA biosensor for detection of False Smut of Rice (Ustilaginoidea virens). The Plant Pathology Journal 37(3): 291-298.
Rasheed, P.A. and Sandhyarani, N. 2017. Carbon nanostructures as immobilization platform for DNA: a review on current progress in electrochemical DNA sensors. Biosensors and Bioelectronics 97: 226-237.
Rastogi, M. and Singh, S.K. 2019. Advances in molecular diagnostic approaches for biothreat agents. Pp: 281-310. In: Singh, S.K. and Kuhn, J.H. (eds). Defense Against Biological Attacks. Springer.
Saylan, Y., Erdem, O., Ünal, S. and Denizli, A. 2019. An alternative medical diagnosis method: biosensors for virus detection. Biosensors 9(2): 65.
Sedlackova, E., Bytesnikova, Z., Birgusova, E., Svec, P., Ashrafi, A.M., Estrela, P. and Richtera, L. 2020. Label-free DNA biosensor using modified reduced graphene oxide platform as a DNA methylation assay. Materials 13(21): 4936.
Shahmirzaie, M., Safarnejad, M.R., Rakhshandehroo, F., Safarpour, H., Rabbani, H., Zamanizadeh, H.R. and Elbeaino, T. 2019. Production of a polyclonal antiserum against recombinant nucleocapsid protein and its application for the detection of fig mosaic virus. Journal of Virological Methods 265: 22-25.
Silvestrini, M., Fruk, L., Moretto, L.M. and Ugo, P. 2015. Detection of DNA hybridization by methylene blue electrochemistry at activated nanoelectrode ensembles. Journal of Nanoscience and Nanotechnology 15(5): 3437-3442.
Sun, X., He, P., Liu, S., Ye, J. and Fang, Y. 1998. Immobilization of single-stranded deoxyribonucleic acid on gold electrode with self-assembled aminoethanethiol monolayer for DNA electrochemical sensor applications. Talanta 47(2): 487-495.
Tian, B., Gao, F., Fock, J., Dufva, M. and Hansen, M.F. 2020. Homogeneous circle-to-circle amplification for real-time optomagnetic detection of SARS-CoV-2 RdRp coding sequence. Biosensors and Bioelectronics 165: 112356.
Topkaya, S.N., Azimzadeh, M. and Ozsoz, M. 2016. Electrochemical biosensors for cancer biomarkers detection: Recent advances and challenges. Electroanalysis 28(7): 1402-1419.
Uliana, C.V., Tognolli, J.O. and Yamanaka, H. 2011. Application of factorial design experiments to the development of a disposable amperometric DNA biosensor. Electroanalysis 23(11): 2607-2615.
Walia, J.J., Salem, N.M. and Falk, B.W. 2009. Partial sequence and survey analysis identify a multipartite, negative-sense RNA virus associated with fig mosaic. Plant Disease 93(1): 4-10.
Walpita, P. and Flick, R. 2005. Reverse genetics of negative-stranded RNA viruses: a global perspective. FEMS Microbiology Letters 244(1): 9-18.
Wu, G., Ji, H., Hansen, K., Thundat, T., Datar, R., Cote, R., Hagan, M.F., Chakraborty, A.K. and Majumdar, A. 2001. Origin of nanomechanical cantilever motion generated from biomolecular interactions. Proceedings of the National Academy of Sciences 98(4): 1560-1564.
Yu, H.L.L., Maslova, A. and Hsing, I.M. 2017. Rational design of electrochemical DNA biosensors for Point of Care applications. ChemElectroChem 4(4): 795-805.
