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Katarzyna NIEMIROWICZ – Department of Experimental Pharmacology, Medical University of Białystok; Agnieszka Z. WILCZEWSKA – Institute of Chemistry, University of Białystok; Halina CAR – Department of Experimental Pharmacology, Medical University of Bialystok, Poland Please cite as: CHEMIK 2013, 67, 10, 836–841 Introduction Molecular biology deals with the explanation of how the functioning of living organisms is subject to the characteristics of particles, and especially biopolymers such as nucleic acids and proteins. Along with the development and advancement of molecular biology techniques, it was possible to develop several methods of separation of macromolecules using different physicochemical phenomena [1, 2]. These methods are based mainly on the properties of macromolecules, such as surface charge, the presence of functional groups and the generation of hydrophobic interactions [3]. Nanotechnology is an interdisciplinary science and its robust growth is directly related to the advances in medical science. There are high hopes associated with the use of magnetic nanoparticles (MNP – Magnetic Nanoparticles) in molecular biology. These display a number of features that enable them to provide a basis for their use as separation tools. In addition to the possessed surface charge and related electro-kinetic properties, they are characterized by magnetic properties which facilitate the separation process, and thereby the puriication of the resulting conjugate to a constant magnetic nano-phase. Another important feature of the nanostructures is a large surface area which allows the immobilization of many biologically active molecules at the same time [4÷6]. MNP are used in many biomedical applications, including the controlled delivery of drugs (DDS – Drug Delivery Systems) [7], targeted therapy (TT – Target Therapy) [8] and at the level of diagnostic separation in the sorting of cells [9], in isolation and detection of bacteria [10, 11] and also as nano-biosensors to detect exogenous substances [12]. This paper presents the synthesis and application of magnetic nanoparticles functionalized by aminopropyltrimethoxysilane (APTMS) for the immobilization of DNA. The analysis of the conjugates of DNAnanoparticle was performed by a spectroscopy of diffuse relectance in infrared (DRIFTS – Diffuse Relectance Infrared Fourier Transform Spectroscopy). Material and methods The hydrated salts of iron chloride (II) and (III), 25% solution of an ammonium base, 3-aminopropyltrimethoxysilane (APTMS) were purchased from Sigma-Aldrich company, water was deionized with a Milli-Q system (Millipore), a yeast species of Saccharomyces cerevisiae was used for testing. A magnetic separation was carried out with neodymium magnet. After obtaining a ferroluid suspension, APTMS was added (0.5 ml). The reaction was conducted under argon in a slightly alkaline medium in the presence of ultrasound. The synthesized nanoparticles were analyzed by infrared spectroscopy (FTIR) Figure 1. Fig. 1. FT-IR spectra of aminosilane nanoparticles Abbreviations: [%T] – transmittance FT-IR (KBr, ν, cm-1): 3428 – stretching N–H bonds; 1115 – stretching C-O bonds; 1035 – stretching Si–O bonds; 693 – stretching Fe–O bonds Isolating DNA from Saccharomyces cerevisiae Sodium chloride (4 g) and sodium dodecyl sulfate (30 mg) were dissolved in deionized water (90 ml). Then, 20 g of baker’s yeast (Saccharomyces cerevisiae) were added. The mixture was incubated for 15 min at 60°C in a water bath, then cooled in an ice bath for 5 min, sonicated for 1 min and iltered. Then, 5 drops of pineapple juice were added to the iltrate and dispensed into test tubes (10 ml). In the inal stage, chilled ethanol (95%) was added to each tube. DNA was knocked off in the form of ine white strands [14]. Immobilization of DNA on the surface of MNP Magnetic nanoparticles functionalized with o-aminopropyl (30 mg) groups were suspended by sonication in deionized water (10 ml) and then added to the extract containing isolated nucleic acid (10 ml). The nanoparticles were separated from the solution using an external magnetic ield. The moistened substance was decanted and the nanoparticles were transferred via Pasteur pipette to aluminum foil, left to dry and analyzed by diffuse relectance spectroscopy DRIFTS (Fig. 2). The synthesis of magnetic nanoparticles MNP For the synthesis of magnetic nanoparticles based on iron oxide, a modiication of a known Massart method was used [13]. The basis of the method is a co-precipitation of hydrated iron chloride (II) and iron (III) in a 1:2 molar ratio of the reactants in a basic medium, which contained a 25% aqueous solution of an ammonium base. The synthesis was carried out in aqueous conditions in an argon atmosphere in deionized water. During the synthesis, a sonication was used. The MNP surface modiication of APTMS nanostructures The nanoparticles obtained in the irst step were subjected to a magnetic separation using a magnet, and puriied by washing several times with ethanol and dried in an oven at 50°C. The cleaned MNP (500 mg) was re-suspended in ethanol as a result of sonication. nr 10/2013 • tom 67 Fig. 2. DRIFTS spectra of MNP-DNA conjugate. Abbreviations: KM –units of Kubelk-Munk = log inverse relectance (log1/R) versus wavenumber. Opis widma: FT-IR (DRIFTS, ν, cm-1): 3415 – stretching N-H bonds; 1660 – stretching C=O bonds; 1632 – bending N–H bonds; 1600 – stretching C=C of the imidazole ring ; 1225 – assymetric stretching PO2 bonds; 1178 – stretching C–O bonds; 1031 – stretching Si–O bonds; 643 – stretching Fe–O stretching • 839 science Magnetic Nanoparticles as separators of nucleic acids science Results and discussion The separation and puriication of DNA is a key stage for the most of the procedures used in molecular biology. The main purpose of this step is to obtain a stable biological material of high quality and purity, regardless of its origin. When choosing a suitable method of isolation one must take into account many aspects such as: the nature and origin of the material, the type of nucleic acid (DNA, RNA) and the inal purpose of the isolated acid [15]. Most isolated DNA is genomic or plasmid. This paper presents the use of synthesized magnetic nanoparticles for the immobilization of deoxyribonucleic acid (DNA) for MNP with amino groups on their surface. The co-precipitation reaction, and then polycondensation of 3-aminopropyltrimethoxysilane, resulted in obtaining magnetic nanoparticles with terminal amino groups. The synthesized nanoparticles were analyzed in the infrared spectral in KBr using a Nicolet Magna IR 550 Series II spectrophotometer. It was possible to identify intense signals at 1115 and 1035 cm-1, derived respectively from the vibrations of stretched C–O and Si–O bonds. In the spectrum there was a signal of vibration of the stretched N–H bonds at 3428 cm-1 and the signal at 693 cm-1 corresponding to the vibrations of the stretched Fe–O bonds. To isolate DNA, a precipitation method with the yeast suspension was used. Using sodium chloride and the SDS surfactant caused a breakdown of cell membranes, nuclear envelope, organelle membranes and other intracellular membranes. The incubation at 60°C accelerated the process of disintegration of biological membranes and the denaturation of DNA-ase, which are the enzymes naturally occurring in cells, able to digest DNA. In the next step, the use of sonication led to a mechanical destruction of cell walls and to the release of cell contents into the mixture. An addition of fresh pineapple juice containing proteolytic enzymes, consumed the proteins present in the iltered mixture. The concentrated ethanol resulted in the precipitation of DNA from the solution. The next stage was to perform the immobilization of DNA on the aminosilane magnetic nanoparticles (Fig. 3). An originally cloudy solution, under the inluence of an external magnetic ield, gained clariication which conirms that there was an attachment of DNA to MNP . The immobilization mechanism was based on a non-covalent interaction of the surface amino groups of nanoparticles with the functional groups of DNA. Most likely, a hydrogen bond was developed between the electronegative nitrogen atoms of NH2 groups and the hydrogen atoms of the functional groups of deoxyribonucleic acid. In addition, it can be assumed that the surface charges on the surface of the nanoparticles and of the nucleic acid played a role in the immobilization. Aminosilane nanoparticles contain a positive surface charge, while the nucleic acid has a negative charge. It is due to the presence of ortophosphoric acid residues in its structure. The obtained conjugate of MNP – DNA was analyzed by the infrared relection method (Fig. 2). It revealed the presence of signals characteristic to nucleic acids. These included signals at: 1660–1632 cm– 1 of C=O stretching vibrations and bending vibrations of NH, 1600 cm– 1 stretching vibration of C=C of the imidazole ring. The PO2 phosphate groups were identiied on the basis of the signals at 1225 cm– 1 characteristic of the asymmetric stretching vibrations and at 1096 cm– 1, corresponding to the symmetric stretching vibrations. In addition, some characteristic signals were observed for the nanoparticles at 1031 cm– 1 corresponding to the stretching vibrations of the Si –O and the stretching vibrations of Fe–O bonds at 643 cm-1. The described method allows to carry out a rapid and simple immobilization of DNA without the need for speciic surface complexes of the ligands such as streptavidin – biotin system [16, 17]. The technique based on the use of silica microspheres was developed by Chen et al [18]. The resulting microspheres were used as the adsorbent used in the extraction of DNA from plant material to the solid phase. There are many procedures describing the method of DNA isolation, which result in obtaining a RNA-free DNA, proteins and other compounds that may interfere in subsequent analyzes. Isolation tends to break the nucleic – protein complex (DNP) and to extract DNA in a possibly unaltered state, biologically active and chemically stable. Due to the size and sensitivity of the chromosomal DNA it is virtually impossible to conduct its isolation in an unaltered state as a part of the DNA undergoes a mechanical damage. The traditional methods of isolation are all multi-step and labor-intensive processes. They require the use of several reagents, as well as a high precision from the person carrying out the process. When performing an isolation, a lot of mistakes can be made, which may lead to the destruction of or damage to the structure of DNA, or result in an incomplete isolation and obtaining a material that is not suitable for further research. Summary and Conclusions As a result of the reaction of the co-precipitation of iron oxide, and then the poly-condensation of 3-aminopropyltrimethoxysilane, magnetic nanostructures were obtained of a core-shell type with amino groups on their surface. A large surface area of nanoparticles is also a large area of possible impacts of magnetic nanoparticles of the amino groups with functional groups contained in the chains of deoxyribonucleic acid, which in turn allows the adsorption of DNA from a solution. The application of the obtained nanoparticles provides an opportunity to use them as ixed magnetic sorbents used for separating nucleic acids (by adsorption) during their isolation from the solution. The proposed working method of isolating nucleic acids may provide a basis for setting new standards in diagnostic molecular biology. Acknowledgement This study was performed under the project “Studying, researching, commercializing – of the doctoral support program at UMB”, Measure 8.2.1 of the Human Capital Operational Programme, co-inanced by the European Union under the European Social Fund Programme Literature 1. 2. 3. 4. 5. Fig. 3. Immobilization of DNA on aminosilane nanoparticles. Abbreviations: MNP – magnetic nanoparticles; DNA – deoxyribonucleic acid; MNP-DNA – conjugate magnetic nanoparticles – deoxyribonucleic acid 840 • 6. Walkowiak B., Kochmańska V.: Elektroforeza przykłady zastosowań – Opracowanie zbiorowe. Amersham Biosciences 2002 Łódź, Warszawa, 2002, s. 6–7 Turner PC., McLennan AG., Bates AD, White MRH.: Biologia Molekularna – krótkie wykłady, PWN 2009 Warszawa, s 17–144 HYPERLINK https://atecentral.net/g19552 30.07.2013 Shokrollahi H. Structure, synthetic methods, magnetic properties and biomedical applications of ferroluids. Mater Sci Eng C Mater Biol Appl. 2013, 33, 2476–2487. 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Halina Car, Katarzyna Niemirowicz, Izabela Święcicka, Agnieszka Z. Wilczewska. Zgłoszenie patentowe P.401362,: Uniwersytet Medyczny w Białymstoku i Uniwersytet w Białymstoku 11. Tang Y, Li Z, He N, Zhang L, Ma C, Li X, Li C, Wang Z, Deng Y, He L. Preparation of functional magnetic nanoparticles mediated with PEG-4000 and application in Pseudomonas aeruginosa rapid detection. J Biomed Nanotechnol. 2013, 9, 312–317. 12. Luo F, Zheng L, Chen S, Cai Q, Lin Z, Qiu B, Chen G. An aptamer-based luorescence biosensor for multiplex detection using unmodiied gold nanoparticles. Chem Commun (Camb). 2012 May 22. [Epub ahead of print] 13. Massart R. Preparation of aqueous magnetic liquids in alkaline and acidic media. IEEE Transactions on Magnetics. 1981, 17, 1247–1248 14. HYPERLINK“http://lesson.org.pl/iles/lessons/pl/biol/izolacja_DNA_z_cebuli.pdf” 30.07.2013 15. HYPERLINK “http://pl.shvoong.com/exact-sciences/biology/1909253-izolacja-dna-hodowlanych-kom%C3%B3rek-eukariotycznych/ “ 30.07.2013 16. Huang SC, Stump MD, Weiss R, Caldwell KD. Binding of biotinylated DNA to streptavidin-coated polystyrene latex: effects of chain length and particle size. Anal Biochem. 1996, 237, 115–122. 17. Largy E, Hamon F, Teulade-Fichou MP. A streptavidin paramagnetic-particle based competition assay for the evaluation of the optical selectivity of quadruplex nucleic acid luorescent probes. Methods. 2012, 57, 129–137. 18. Chen F., Shi R., Xue Y., Chen L.,Wan QH.: Templated synthesis of monodisperse mesoporous maghemite/silica microspheres for magnetic separation of genomic DNA, Journal of Magnetism and Magnetic Materials, 2010, 322, 2439–2445. Katarzyna NEMIROWICZ – M.Sc., is a graduate (chemistry) of the Faculty of Biology and Chemistry, University of Bialystok (2011) and Laboratory Medicine at the Medical University of Bialystok (2012). At present she is the second year PhD student in the Department of Experimental Pharmacology, Medical University of Bialystok, Faculty of Medicine. Since February 2013 she has been a principal investigator the grant Prelude. She is also the beneiciary of a program “I study, investigate, commercialize” – UMB doctoral support program funded by the European Union under the European Social Fund as well as the scholar of Polpharma Science Foundation Scholarship. Research interests: organic synthesis, nanotechnology and targeted therapy. She is the author and co-author of ive research papers, 13 papers and communications runs at national and international conferences, as well as a patent application. e-mail: [email protected], phone: +85 748 55 54 Agnieszka Z. WILCZEWSKA – Ph.D, Doctoral thesis defended at the Faculty of Chemistry of the University of Warsaw in 1998. Currently, she works at the Faculty of Biology and Chemistry, University of Bialystok, Department of Chemistry of Natural Products. Scientiic interest: vinyl polymers, MADIX polymerization and nanotechnology. e-mail: [email protected] Halina CAR – M.D., Ph.D., is a graduate of the Faculty of Medicine, Medical University of Bialystok (1987). She obtained the degree of Doctor of Medicine in 1990, and defended her habilitation thesis in 2007. In the years 1987÷2010 she worked in the Department of Pharmacology, Medical University of Bialystok. For the last three years has been a head of the Department of Experimental Pharmacology, Medical University of Bialystok. She works as Voivodshipl Consultant in Clinical Pharmacology. Research interests: learning and memory, neurodegeneration and possibilities of their therapy, tumor processes in the brain, targeted therapy. She is the author and co-author of 58 scientiic articles published in the international medical journals and 80 papers and communications presented at national and international conferences, as well as a patent application e-mail: [email protected], [email protected], phone:+85 748 55 54 nr 10/2013 • tom 67 • 841