Magnetic extraction and purification of DNA from whole human blood using a γ-Fe2O3@Chitosan@Polyaniline hybrid nanocomposite

We examined the properties of the nanocomposite γ-Fe2O3@Chi@Pani as an adsorbent of deoxyribonucleic acid (DNA). As a model system, we used an aqueous solution of salmon sperm DNA, whose decreasing concentration was followed by monitoring the 260 nm UV–vis absorption.

Carbohydrate Polymers 197 (2018) 100–108 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Magnetic extraction and purification of DNA from whole human blood using a γ-Fe2O3@Chitosan@Polyaniline hybrid nanocomposite T Bruna Gomes Maciela, Romário Justino da Silvaa, Alicia Elizabeth Chávez-Guajardob, ⁎ Juan Carlos Medina-Llamasc, José Jarib Alcaraz-Espinozad, Celso Pinto de Meloa,d, a Pús-Graduaỗóo em Ciờncia de Materiais, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil Unidad Académica de Ciencias de la Tierra, Universidad Autónoma de Zacatecas, 98058 Zacatecas, Zac, Mexico c Centro de Estudios Científicos y Tecnológicos No 18, Instituto Politécnico Nacional, 98160 Zacatecas, Zac, Mexico d Departamento de Física, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil b A R T I C LE I N FO A B S T R A C T Keywords: Maghemite Chitosan Polyaniline Magnetic hybrid nanocomposite DNA extraction Human blood We examined the properties of the nanocomposite γ-Fe2O3@Chi@Pani as an adsorbent of deoxyribonucleic acid (DNA) As a model system, we used an aqueous solution of salmon sperm DNA, whose decreasing concentration was followed by monitoring the 260 nm UV–vis absorption After adjusting the data collected to a Langmuir isotherm curve, we estimated the adsorption capacity (qe) of the nanocomposite as 49.5 mg/g We also observed that the kinetic model of the DNA capture presents a mixed character, with both chemical mechanisms and intraparticle diffusion processes involved When the MNC was used to extract the DNA from complex samples (human blood), a capture rate of 80 ng/μL was achieved, with the collected fraction exhibiting good quality, as evaluated by PCR analysis and electrophoresis assays These results suggest that the γ-Fe2O3@Chi@Pani nanocomposite is a promising adsorbent for use in protocols for purification of DNA from complex samples Introduction The elucidation of DNA function in organisms represented an unprecedent revolution in science, with special impact on the methods used for studying living beings Nowadays, isolating, analyzing, and manipulating the DNA are essential steps in different areas of knowledge, such as i) the development of new biotechnological processes (Baeshen et al., 2014; Demain & Adrio, 2007) and new drugs (Debouck & Goodfellow, 1999), ii) finding the origin of diseases (Kruglyak, 1999), iii) making diagnostics (Drmanac et al., 1998), iv) broadening the scope of anthropological studies (Fernández et al., 2014), and v) solving legal issues with the help of DNA forensic research (Gill, Jeffreys, & Werrett, 1985) Nonetheless, to achieve success in those endeavors one needs first to extract and purify the DNA of interest, a challenging step that continues to be laborious and time-consuming (Tan & Yiap, 2009) In spite of the vast amount of procedures already available for DNA purification, as a general rule the current liquid–liquid and solid phase extraction methods protocols still face important drawbacks to be overcome In pursuing a more efficient extraction of DNA, several alternatives based on novel solid extraction matrices have been developed in the last decades Among these methods, those that employ nanoparticles (NPs) to adsorb the nucleic acid molecules ⁎ deserve special mention, since the characteristic high surface to volume ratio of the NPs favors the achievement of high yields in the capture of the targeted molecules (Martin & Mitchell, 1998; Yeung & Hsing, 2006) Extraction methods based on the use of magnetic nanoparticles (MNPs) appear as specially convenient for their simplicity, since in this case the fraction of interest can be easily separated from the reaction medium by use of a small magnet, thus avoiding the necessity of further laborious separation procedures (Lu, Salabas, & Schüth, 2007; MedinaLlamas, Chávez-Guajardo, Andrade, Alves, & de Melo, 2014) These MNPs typically consist of a superparamagnetic nucleus (usually Fe3O4 or γ-Fe2O3) enveloped by a corona of a DNA affinity ligand (Wierucka & Biziuk, 2014) Examining the recent literature, it is possible to identify two main alternative methods for coating the MNPs, either by enveloping the magnetic core using silica (Li, Zhang, & Gu, 2012; Sheng et al., 2016; Torney, Trewyn, Lin, & Wang, 2007) or by employing polymeric materials for this (Kievit et al., 2009; Medina-Llamas et al., 2014; Samal et al., 2012) Silica MNPs possess the advantage of exhibiting high porosity, and the charge distribution over their enormous superficial area can be adjusted to better capture DNA by changes of pH or by the controlled presence of chaotrophic agents (Li et al., 2012) In spite of Corresponding author at: Departamento de Física, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil E-mail address: celso@df.ufpe.br (C.P de Melo) https://doi.org/10.1016/j.carbpol.2018.05.034 Received 21 December 2017; Received in revised form May 2018; Accepted 11 May 2018 Available online 22 May 2018 0144-8617/ © 2018 Elsevier Ltd All rights reserved Carbohydrate Polymers 197 (2018) 100–108 B.G Maciel et al Llamas, Andrade, & de Melo, 2015) We have exploited this latter mechanism in a previous work, to create two solid phase adsorption materials for the retrieval of nucleic acids (Medina-Llamas et al., 2014) In the present investigation, we aim to engineer a MNP of Chi grafted with Pani (γ-Fe2O3@Chi@Pani) for the retrieval of DNA from blood samples with a purity degree high enough that it could be amplified by PCR We characterized the resulting γ-Fe2O3@Chi@Pani composite by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and by measurements of their magnetic response this, use of silica-based MNPs raises some concerns not only with regard to the total time required for the complete retrieval of the targeted molecules, but also with respect to the quality of the purified DNA, since denaturing DNA agents, such as guanidine hydrochloride, need to be used in the process Therefore, more recent efforts have been focused in facilitating the adsorption-desorption processes by modifying the surface chemistry of the silica MNPs, usually by the addition of silanol moieties containing functional (such as amino) groups or polymers that would directly respond to simple changes in the pH of the medium (Pandit, Nanayakkara, Cao, Raghavan, & White, 2015; Sheng et al., 2016) Use of natural or synthetic cationic polymers appears as an alternative method of coating the MNPs, in such a manner that the resulting polymer MNPs (P-MNPs) structure consists of an isolated particle or a cluster of superparamagnetic nanoparticles covered by a functional cationic polymer (Chen et al., 2013; Kievit et al., 2009; Medina-Llamas et al., 2014) Although this methodology has not been widely explored for the purpose of retrieving and purifying DNA molecules, some relevant examples of exploiting electrostatic complexes between cationic polymer NPs and DNA can be found not only in the field of gene delivery, but also in the area of sensor development (Cheong et al., 2009; Dias, Hussain, Marcos, & Roque, 2011; Kaushik, Solanki, Ansari, Malhotra, & Ahmad, 2009; Kaushik, Solanki, Ansari, Sumana et al., 2009) One aim in designing new NP-MNPs for DNA retrieval is to produce biocompatible functional systems that would allow the adsorption of a large amount of the target molecules (Nishiyama & Kataoka, 2006; Samal et al., 2012) At the same time, it is desirable to select cost-effective procedures based on sustainable and ecofriendly materials On that regard, Chitosan (Chi) appears as a very promising coating material This polymer, which is the second most abundant polysaccharide after cellulose, is composed of randomly distributed N-acetyl glucosamine and D-glucosamine and can be obtained from the deacetylation of chitin (Foster, Ho, Hook, Basuki, & Marỗal, 2015) In its structure, Chi presents three potential reactive sites (two hydroxyl groups per glycosidic unit and a primary amine) that are susceptible to a subsequent modification and capable of interacting with negatively charged species (Samal et al., 2012) Quaternization and grafting have been examined as manners of functionalizing the Chi structure so as to surpass limitations such as pH sensitivity and low porosity, normally associated to the natural form of this polysaccharide (Belalia, Grelier, Benaissa, & Coma, 2008; Jayakumar, Prabaharan, Reis, & Mano, 2005; Marcasuzaa, Reynaud, Ehrenfeld, Khoukh, & Desbrieres, 2010) The latter has been successfully implemented by use of cationic polymers as poly(lysine) (Ply) (Yu et al., 2007) or polyethylene imine (Wong et al., 2006), resulting in a material that preserves the intrinsic properties of Chi while presenting an enhanced functionality for DNA adsorption and transfection In recent times, use of intrinsically conducting polymers (CPs) in biomedical applications have attracted larger attention Besides their good stability, relatively simple chemical composition, and peculiar electrical characteristics, CPs offer the possibility of entrapping biomolecules, a singular feature that has made possible to use them for the tailoring of a new class of smart materials for a variety of applications, such as drug delivery, the development of biosensors and the design of scaffolds for tissue engineering, among others (Guimard, Gomez, & Schmidt, 2007) However, the lack of solubility and the usual brittle nature of pristine CPs limit the range of their possible applications, and a potential manner to overcome such disadvantages is to use CPs as a component in composite materials Amongst the CPs, polyaniline (Pani) stands out due to its high conductivity, rich redox properties, and the existence of a second doping mechanism corresponding to the protonation from base emeraldine to salt emeraldine; in this process, half of the nitrogen atoms in the polymer chain acquire a positive charge that will be neutralized by the interaction with the anion resulting from the dissociation of the acid (Alcaraz-Espinoza, Chávez-Guajardo, Medina- Materials and methods 2.1 Materials The reagents iron chloride II tetrahydrate (FeCl2·4H2O), salmon sperm DNA, sodium dodecyl sulfate (SDS), ammonium persulfate (APS) and chitosan (molecular weight 50,000–190,000 Da and deacetylation degree 75–85%) were purchased from Sigma-Aldrich Acetic acid (CH3COOH), hydrochloric acid (HCl), ethylenediamine tetraacetic acid (EDTA), sodium chloride (NaCl), ammonium hydroxide (NH4OH), sodium hydroxide (NaOH) and methanol (CH3OH) were obtained from Química Moderna (Brazil), while aniline (C6H5NH2), sodium phosphate monobasic (NaH2PO4), bibasic sodium phosphate (Na2HPO4) were acquired from Nuclear (Brazil) Agarose, blue/orange loading dye 6X, diamond nucleic acid dye, glycine, proteinase K, Tris-Acetate-EDTA 40X and tris (hydroxymethyl) aminomethane (Tris) were purchased from Promega Iron (III) chloride hexahydrate (FeCl3·6H2O), Triton X100 and potassium acetate (CH3COOK) were obtained from Dinâmica (Brazil), Vetec (Brazil) and Applichem (Germany), respectively All reagents were of analytical grade and used as received, except for aniline that was vacuum distilled before use In all experiments, we used deionized water obtained from a Synergy system (Millipore, USA) 2.2 Methods 2.2.1 Synthesis of Fe3O4@Chi nanoparticles The Fe3O4@Chi MNPs were obtained by the chemical co-precipitation method described before (Alibeigi & Vaezi, 2008; Jiang et al., 2015) The process can be detailed as follows: a 100 mL round-bottom flask, where we added 20.0 mL of deionized water, 2.08 g of FeCl3·6H2O and 0.800 g of FeCl2·4H2O, was vigorously stirred for 10 The resulting solution was dripped into a 250 mL round-bottom flask containing 0.25 g of chitosan dissolved in 100 mL of an aqueous solution of acetic acid 1% (v/v) The solution was stirred and heated at 1000 rpm and 40 °C for h Afterwards, we slowly added 15 mL of an aqueous solution of NH4OH 28% (v/v) to the flask, and stirred for 30 Finally, the product obtained was washed three times with a solution of NaOH (0.03 M) and then once with deionized water The resulting material was then dried in a vacuum oven (40 °C) for 36 h, when a final brown powder was obtained 2.2.2 Synthesis of the γ-Fe2O3@Chi@Pani magnetic nanocomposite The polymerization of aniline on the surface of the Fe3O4@Chi MNPs was performed according to the established procedure (MedinaLlamas et al., 2014) Initially, we added 100 mL of HCl 0.1 M, 7.3 mmol of SDS, 60 mg of MNPs and 1.5 mM of aniline in a round-bottom flask, which was stirred for 15 Subsequently, 20 mL of an aqueous solution of HCl (0.1 M) containing 18 mmol of APS was added The polymerization reaction was allowed to proceed for 24 h at °C, under constant stirring The resulting product was then subjected to several successive washings with methanol (CH3OH) and deionized water, and finally dried at 40 °C for 24 h 2.2.3 Characterization The Fe3O4@Chi MNPs and γ-Fe2O3@Chi@Pani MNCs were characterized by Fourier Transform Infrared Spectroscopy (FTIR), Scanning 101 Carbohydrate Polymers 197 (2018) 100–108 B.G Maciel et al and mixed the contents using in a vortex for 30 s Then, we magnetically confined the MNC and removed the supernatant Subsequently, we added mL of a buffer solution at pH 8.0 (Tris/HCl) to the glass vial, which was then stirred for 10 in an orbital shaker at 230 rpm Finally, the MNC was once more magnetically confined, and the supernatant collected and analyzed by UV–vis Electron Microscopy (SEM) and magnetic measurements FTIR spectra of the samples of interest were obtained in the 4000–400 cm−1 range by using pressed KBr pellets in an IRTracer-100 spectrophotometer (Shimadzu, Japan) SEM micrographs were obtained using a FEG-SEM MIRA LM (TESCAN, Czech Republic) Magnetization curves were acquired by the use of a EV7 vibrating sample magnetometer (MicroSense, USA) In the experiments of DNA retrieval from both aqueous solutions and whole human blood samples, the DNA concentration was determined by use of a Nanodrop 2000C spectrophotometer (Thermo Scientific, USA) The integrity and quality of extracted DNA was verified through agarose gel electrophorese and conventional PCR, using a K33-15H (Kasvi, Brazil) horizontal electrophoresis sub-cell system, a MB-16 (Maestrogen, USA) UV (302 nm) transilluminator and a StepOnePlus thermocycler (Applied Biosystems, EUA), respectively 2.2.6 Extraction of DNA from whole human blood samples For the DNA extraction, we followed the procedure: in a mL microtube, we added μL of proteinase K, 100 μL of whole human blood and 500 μL of a lysis solution (water/triton X-100 at 1%, pH 6.0) This microtube was incubated at 56 °C for 20 min, being placed in the vortex for 30 s at every during this period of time After this, we added 1.2 mL of glycine/HCl/NaCl buffer solution (pH 3.3) and mg of MNC to the microtube, and allowed the DNA-MNC interaction to occur during 10 Then, the MNC was magnetically confined and the supernatant discarded, and we then added 900 μL of a solution prepared using 1.7 mL of ethanol for each mL of a potassium acetate-Tris/HClEDTA solution Afterwards, the microtube was stirred for 30 s in the vortex, and the MNC was once again magnetically confined and the supernatant discarded, with this process being repeated twice After this washing procedure, the microtube was left open for for allowing the ethanol evaporation, and we added 30 μL of an elution solution (Na2HPO4/NaH2PO4, pH 7.6) The microtube now containing the elution solution and the MNC was then shaken for 15 s, and the system was left in incubation for a period of 10 min, with care being taken to ensure a good contact between the MNC and the elution solution (Fig 2) Finally, the MNC was magnetically confined, when the supernatant was collected and analyzed by UV–vis To establish whether or not the DNA extracted from blood samples by use of the MNC was in fact of adequate quality for molecular biology protocols, the collected material was subjected to a PCR procedure for checking the amplification of a specific DNA sequence determined by the primer used To accomplish this, we implemented a master mix, using 2.5 μL of dNTPs 10X (0.2 mM); 2.5 μL of PCR 10 X-MgCl2 (200 mM Tris-HCl, pH 8.4 and 500 mM de KCl); 0.75 μL of MgCl2 (1.5 mM); 0.2 μL of Taq; 15.6 μL of free water, 1.0 μL of each primer (10 pmol) − forward CAT GTA CGT TGC TAT CCA GGC and reverse CTC CTT AAT GTC ACG CAC GAT (Mera, Heimfeld, & Faustman, 2014) Finally, we added 1.5 μL of the extracted DNA, totaling 25 μL for each well, except for the negative control, where 1.5 μL of nuclease free water was added The reagents used in this procedure were purchased from Invitrogen Life Technologies The amplifications were performed in a StepOnePlus thermocycler Initially, the sample was subjected to a temperature of 50 °C for min, and afterwards the temperature was raised to 95 °C for to allow 2.2.4 DNA adsorption experiments in a model system The DNA adsorption experiments were performed as follows First, we placed mg of γ-Fe2O3@Chi@Pani MNC and 2.5 mL of a glycine/ HCl pH 3.6 buffer solution in a mL glass vial, and mixed them with aid of a vortex for s Subsequently, we added 2.5 mL of sperm salmon DNA solution into the vial that was kept under stirring for 10 by use of a SHKE 2000 E-CLASS orbital shaker (Barnstead Lab-Line, USA), operating at 230 rpm After that, the MNC was magnetically confined and the supernatant stored in a clean flask A μL aliquot was used for the absorbance measurements in the Nanodrop For the quantification of the remaining DNA concentration in solution along the adsorption process, we determined the intensity of the characteristic 260 nm absorption peak of DNA (Fig 1) In these studies, we exposed different amounts of the γFe2O3@Chi@Pani MNC (2, 3, and mg) during distinct interaction times (5, 10, 15, 25, 35, 45, 65, 85, 105, 125 and 145 min) with the DNA samples We also evaluated the adsorption capacity of the MNC as a function of the DNA concentration (4.5; 8.5; 17.0; 33.5; 55; 118; and 173 ng/μL) in the solution The relationship C0 − Cf ⎞ Adsorption% = ⎛ x100 ⎝ C0 ⎠ ⎜ ⎟ (1) where C0 is the initial concentration and Cf the final concentration (i.e., after the interaction with the MNC) of DNA in solution (both in ng/μL), was used to determine the percentage of adsorbed DNA 2.2.5 DNA desorption experiments in model system After performing the adsorption experiments, we tested the DNA desorption capacity of the MNC For this, we placed 500 μL of a glycine/ HCl buffer solution (pH 3.6) in the glass vial that contained the MNC Fig Scheme of the experiment used to estimate the capacity of DNA adsorption by MNC 102 Carbohydrate Polymers 197 (2018) 100–108 B.G Maciel et al Fig Procedure for extracting DNA from human blood samples (Fig 3(I)) In the iron oxide/chitosan/polyaniline spectrum (curve a), one can observe peaks at 636 cm−1 and 577 cm−1 that are due to Fe-O vibrations of the maghemite (γ-Fe2O3) iron oxide phase (MedinaLlamas et al., 2014) In the iron oxide/chitosan spectrum (curve b), only one peak in 621 cm−1 was found, which we associated to the Fe-O vibrations of the magnetite (Fe3O4), in an indication of the Fe3O4 to γFe2O3 transformation that occurs after the Pani chains polymerize on the surface of the magnetic nanoparticles (Alibeigi & Vaezi, 2008) In curve (a), the characteristic peaks of the Pani and chitosan are present In the case of Pani, there are peaks at 2920 cm−1 and 2850 cm−1 that may be attributed to the symmetric and asymmetric stretching of the eCH2e groups, respectively The peaks at 1564 cm−1 and 1482 cm−1 are due to the C]C stretching of the quinoid and benzenoid rings, respectively, while the peak at 796 cm−1 is attributed to CeH out of the plane deformation in the benzenoid ring The peaks at 1294 cm−1 and 1236 cm−1 can be assigned to the C−N stretching vibration of the benzenoid ring, whereas the peak at 1107 cm−1 can be assigned to the aromatic CeH in plane bending The characteristic peaks for the chitosan (curve c) appeared at 3370 cm−1 (OeH and NeH stretching vibrations), 2873 cm−1 (also due CeH stretching vibrations), 1643 cm−1 denaturation of the double strand DNA This step of denaturation at 95 °C, which was maintained for 30 s, was followed by the annealing of the oligonucleotides at 50 °C for 45 s This process was terminated with the extension step at 72 °C for 30 s, and the final extension at °C for infinite time, a step necessary to prevent undesired amplicon reactions that may occur before the removal of the sample from the equipment The denaturation, annealing and extension procedures were repeated for 34 additional cycles For the purification of the amplicons, we used the QIAquick purification kit from Qiagen (Germany) After purification, the quality of the obtained amplifications was examined by performing an agarose gel electrophoresis, with use of a diamond dye (Promega, USA) Results and discussion 3.1 Characterization of the Fe3O4@Chi MNPs and the γ-Fe2O3@Chi@ Pani MNCs We performed FTIR analysis to examine the composition of the iron oxide/chitosan MNPs and iron oxide/chitosan/polyaniline MNCs Fig (I) IR spectra of γ-Fe2O3@Chi@Pani MNC (a), Fe3O4@Chi MNP (b) and Chitosan (c) and (II) Magnetization curves of Fe3O4@Chi MNPs (a) and for the γFe2O3@Chi@Pani MNC (b) 103 Carbohydrate Polymers 197 (2018) 100–108 B.G Maciel et al Fig SEM image of Fe3O4@Chi (a) and γ-Fe2O3@Chi@Pani (b) samples (Histograms of the particle size distribution in inset) (NeH bending vibrations) and 1070 cm−1 (CeOeC stretching vibrations) (Gregorio-Jauregui et al., 2012) By analyzing the magnetization curves of the samples (Fig 3(II)), we observed that at room temperature both the Fe3O4@Chi MNPs and the γ-Fe2O3@Chi@Pani MNC exhibit a superparamagnetic behavior with a saturation magnetization (Ms) of 42 emu/g and 22 emu/g, respectively We attribute this decrease in the Ms of the MNC to the presence of incorporated Pani, since the magnetization measurements take into account the total mass of the sample and the polymer chains not contribute to the magnetic response (Medina-Llamas et al., 2014) This is an additional result in agreement with the hypothesis that Pani is incorporated in the hybrid Fe3O4@Chi MNPs structure We used SEM to investigate the morphology and size distribution of the MNP and MNC In Fig 4, one can observe that both the Fe3O4@Chi MNPs (Fig 4a) and the γ-Fe2O3@Chi@Pani MNC (Fig 4b) exhibit a nearly spherical shape with a size distribution in the nanoscale range To estimate the particle size of the synthesized materials, we used the ImageJ software to measure the diameter of 100 particles in several SEM micrographs After plotting the particle diameters as histograms (see inset of Fig 4a and b), we found that the Fe3O4@Chi MNPs have a size distribution in the 17–31 nm range, with an average diameter of (23 ± 6) nm, while for the γ-Fe2O3@Chi@Pani MNC the size distribution was from 23 nm to 31 nm, with an average diameter of (27 ± 4) nm An important question to be addressed is whether the use of either the MNP or the MNC would cause any level of degradation of the captured DNA To examine this, we repeated the adsorption experiment but this time using as target a standard commercial kit with fragments of known size (DNA Ladder of kb, Promega, USA) (PROMEGA, 2017) In Fig 5, we show the results of the agarose gel electrophoresis experiment, where one can see the standard DNA diluted in nuclease-free H2O before (lane (a)) and after interaction with the Fe3O4@Chi MNPs (lane (b)), whose bands present a lower intensity, and with the γFe2O3@Chi@Pani MNC lane (c), whose bands are more intense These results confirm that the material collected by use of either the MNPs or the MNCs is of good quality with no perceptible evidence of DNA degradation As expected, use of the γ-Fe2O3@Chi@Pani MNC resulted in a larger degree of adsorption than when the Fe3O4@Chi MNP was adopted as adsorbent Therefore, in the subsequent adsorption experiments only 3.2 DNA adsorption experiments To assess the DNA adsorption capacity of the γ-Fe2O3@Chi@Pani MNC, we first investigated a model system, which was prepared by dissolving strands of salmon sperm DNA in an aqueous solution at an approximate concentration of 60 ng/μL For comparison purposes, we also tested the adsorption capacity of the Fe3O4@Chi MNPs for identical samples In these initial experiments, we observed that after 10 of interaction the average adsorption of the γ-Fe2O3@Chi@Pani MNC was of the order of 61%, which corresponds to a capture of DNA of approximately 36.2 ng/μL On the other hand, when using the MNPs in a similar procedure the relative amount of captured material was smaller (16.8 ng/μL), corresponding to an average adsorption of only 29% (see Fig S1 in the Supplementary Material) Fig Agarose gel electrophoresis for: standard DNA diluted in nuclease-free H2O (a), after interaction with Fe3O4@Chi MNPs (b) and γ-Fe2O3@Chi@Pani MNC (c) 104 Carbohydrate Polymers 197 (2018) 100–108 B.G Maciel et al Fig Percentage of adsorption and adsorption capacity at equilibrium as a function of initial DNA concentration Fig DNA adsorption as a function of the interaction time for an amount of mg (a), mg (b), mg (c) and mg (d) of γ-Fe2O3@Chi@Pani MNC In each case, 2.5 mL of a 100 ng/μL DNA solution was used function of the equilibrium concentration Ce, in the form qe = the MNC was used (C0 − Ce ) V m (2) where m is the mass of the adsorbent (in g) and V is the volume (L) of the solution, at a given temperature The adsorption isotherms can provide relevant information about the distribution of the molecules between the solution (liquid phase) and the adsorbent (solid phase), once one adjusts the experimental data to either the Langmuir or the Freundlich models (Javadian et al., 2014) An isotherm obeys the Langmuir model when the adsorption occurs with the formation of a monolayer of the adsorbed material on a homogeneous surface, according to the relation (Faust & Aly, 2013) 3.2.1 Interaction time and optimal MNC amount Different amounts (2–5 mg) of the γ-Fe2O3@Chi@Pani MNC were allowed to interact with a DNA solution of concentration equal to 100 ng/μL, during different time intervals (0 a 145 min) In Fig 6, one can observe that the adsorption capacity increases when either the interaction time or the amount of MNC used is increased When only mg of MNC was used, a saturation of the adsorption was observed within the allotted time, with a low amount of captured material (curve a) Use of higher amounts of MNC (3, and mg) would lead to a progressive increase in the amount of retrieved DNA (20%, 40% and 70%, respectively − curves b, c and d) within the 145 of interaction, but with no indication that the saturation regime was already attained We have then decided to adopt a fixed amount of mg of the γFe2O3@Chi@Pani MNC in the subsequent analyzes to ensure that the equilibrium concentration was reached in each case Ce C = + e qe bqm qm (3) where b is the Langmuir constant and qm is the maximum amount of adsorbed molecules (mg/g) The values of b and qm can be determined by plotting the data as a Ce/qe vs Ce graph On the other hand, the Freundlich model, which is more suitable for the description of an adsorption process that occurs with the formation of multilayers on a heterogeneous surface, obeys the relation (Ho, Porter, & McKay, 2002) 3.2.2 Effect of initial DNA concentration After assessing both the appropriate amount of the γ-Fe2O3@Chi@ Pani MNC to be used and the most adequate interaction time for a good adsorption rate of the dissolved DNA, we investigated the effect of the initial DNA concentration upon the adsorption process For this, a fixed amount of mg of the MNC placed to interact with mL from solutions of different initial concentrations of DNA (4.5; 8.5; 17.0; 33.5; 55; 118; and 173 ng/μL), with the mixtures being stirred during 60 in an orbital shaker In Fig 7, one can observe that at low concentrations (4.5; 8.5; 17.0 ng/μL) the amount of MNC used was able to completely adsorb the dissolved DNA One can also notice that, as the DNA concentration increases, the percentage of adsorbed material decreases and the adsorption capacity increases For instance, for the concentrations of 33.5 ng/μL and 118 ng/μL the corresponding adsorption percentage was of the order of 54% and 17%, while the adsorption capacity was 45 mg and 49.5 mg DNA/g MNC, respectively Thus, although the percentage of adsorption is decreased, the mass adsorption capacity increases, due to the increase in the corresponding driving force (Wasewar, 2010) log qe = logKf + logCe n (4) where qe is the adsorption capacity, and Kf and n are constants that can be determined from a logqe vs logKf plot In Table we present the values of the parameters of the Langmuir and Freundlich models obtained after adjusting the data In Fig S2, we show the corresponding linear fittings One can note that, as indicated by the values of the coefficient of determination R2, the DNA adsorption follows a Langmuir process; in fact, the experimental value for the adsorption capacity determined according to this model (49.5 mg/g) agrees with that calculated by use of Eq (2) Table Isotherm parameters for the Langmuir and Freundlich models applied to the DNA adsorption on the γ-Fe2O3@Chi@Pani MNC Langmuir 3.3 Adsorption isotherms To analyze the characteristics of the adsorption process, we first examined how the adsorption capacity at equilibrium (qe) varies as a 105 qm (mg/g) b (mg/L) 49.5 1.89 Freundlich Kf(mg/g) R2 0.99 22.39 n R2 5.88 0.37 Carbohydrate Polymers 197 (2018) 100–108 B.G Maciel et al percentage of the γ-Fe2O3@Chi@Pani MNC (as well as the time required to complete each of these steps) to those of other adsorbents described in the literature – see Table S1 The γ-Fe2O3@Chi@Pani MNC has an adsorption capacity of 49.5 mg/g, which is reached after 60 of interaction Teng et al (Teng, Li, Yan, Zhao, & Yang, 2009) estimated a capacity of 46 mg/g in 20 when using 10 mg of magnetite nanoparticles coated with mesoporous silica, and to promote the DNA desorption they used a Tris/ EDTA solution at pH 8.0, which was heated at 60 °C during 20 Zhang et al (Zhang et al., 2012) used mg of polystyrene-coated mesoporous silica nanoparticles, and obtained an adsorption capacity of 110.7 mg/g after 1440 of interaction Sun et al (Sun et al., 2014) reported that employing 0.5 mg of M-MSN@TEOS in 120 adsorbed 10.6 mg/g However, note that in the latter work guanidine thiocyanate was used This reagent is not only toxic, but also absorbs at wavelengths near 260 nm; hence, additional purification steps need to be performed to eliminate its presence from the medium Finally, Hu and collaborators (Hu et al., 2015) have used an iron phosphate@Polyethyleneimine MNC that showed both higher adsorption and larger desorption capacity in shorter times than the γ-Fe2O3@Chi@Pani MNC; however, in this process it was necessary to adopt centrifugation as an additional step Compared to these adsorbents, the MNC described in this work presents the competitive advantage of demanding a simple procedure for the magnetic separation of the captured DNA, while not requiring the use of toxic reagents, such as guanidine thiocyanate 3.4 Kinetics of the adsorption process One can obtain more information on how the γ-Fe2O3@Chi@Pani MNC interacts with the DNA strands by studying the kinetics of the adsorption process For this, we adjusted the experimental data to the pseudo first order, pseudo second order and Morris-Weber kinetic models (Haerifar & Azizian, 2013; Largergren, 1898; Plazinski & Rudzinski, 2009) Each one of these models can be expressed in its linearized form according to log (qe − qt ) = log qe − t qt = h + qe t , k1 t 2.303 (Pseudo − first order), (5) h = k2 qe2 (Pseudo − second order: ), or (6) qt = kid (t )0.5 + C (Morris − Wener), (7) where qe and qt are the amounts (in mg) of DNA adsorbed per mass unit (g) of the MNC, at equilibrium and at time t, respectively, while k1 (min−1), k2 (g/mg.min) and kid(min−1) are the pseudo-first-order, pseudo-second-order and intraparticle diffusion adsorption kinetic rate constants, respectively, t (min) is the time and C is an intraparticle diffusion constant The constants of these models were calculated from the intercept and the slope of the plots, which were constructed according to the Eqs (5)–(7), respectively (see Table 2) As suggested by the comparison of the corresponding coefficients and by the fittings shown in Fig S3, the process is best described (R2 = 0.94) by a chemical process (i.e., the pseudo-second order model), with a possible contribution (R2 = 0.92) of Morris-Weber intraparticle diffusion mechanisms We can rationalize these results if we assume that a competition exists between these two kinetic models, and that the processes of adsorption and absorption of the DNA molecules on the MNC occur concomitantly, as schematically represented in Fig S4 According to this view, the DNA strands initially interact with the polyaniline chains through the electrostatic attraction between the cationic charges of the amino groups and the negatively charged DNA phosphate groups (Fig S4-1) Afterwards, the DNA migrates through the interstices of the Pani coating, reaching the more internal chitosan sites (which also have amine groups) (Fig S4-2) As a result of this dual process, the γFe2O3@Chi@Pani MNC exhibits an increased capture rate of the target molecule 3.7 Extraction of DNA from whole human blood samples After having established the most suitable conditions for the extraction of DNA strands dissolved in an aqueous medium, we proceed to examine the performance of the MNC for the capture of DNA present in human blood samples, a system of much greater complexity Blood is a tissue composed of plasma and serum (liquid), with solid components consisting of red cells (erythrocytes), white blood cells (leukocytes) and platelets (thrombocytes), whose main function is to transport oxygen and nutrients throughout the body (Butler, 2009) Complex samples such as blood have components called inhibitors – such as hemoglobin, immunoglobulin G, and lactoferrin (Kermekchiev, Kirilova, Vail, & Barnes, 2009) – that prevent the action of DNA polymerase, thus making unfeasible the direct application of whole blood in PCR amplification procedures Taking this into account, we considered the blood composition in the development of a MNC based protocol for the capture of DNA with a satisfactory purity for molecular biology applications As a more unstable nucleic acid, RNA chains are not well preserved due to the alkaline hydrolysis and the presence of alkali metals that favor their degradation (Oivanen, Kuusela, & Lönnberg, 1998), and one could expect that the resulting RNA fragments should compete for adsorption on the active sites of the MNC However, in human blood the overall amount of RNA is much lesser than that of DNA (Chomczynski et al., 2016), so that at the end the extracted material will be mostly composed by DNA molecules In fact, before establishing the best extraction protocol to be followed, we examined how the results would be affected when either 40 μg or 200 μg of RNase was added in the lysing step As the results shown in Fig S5 reveal for both cases, no significant differences could be detected either in the UV–vis spectra of the final 3.5 DNA desorption experiments It is known (Cao, Easley, Ferrance, & Landers, 2006; Matsumoto, Yako, Minagawa, & Tanioka, 2007) that the adsorption of DNA strands occurs more effectively at lower pH values, with a significant decrease being observed at pH > 6.5 Since the latter pH range is the most suitable for an effective elution of the captured molecules, in the subsequent desorption studies for the model system we used a pH 8.0 Tris/ HCl buffer solution Our results indicated a desorption rate of (66.0 ± 0.97) % for the MNC 3.6 γ-Fe2O3@Chi@Pani MNC as an adsorbent: comparison to other materials We compared the adsorption capacity and the desorption Table Kinetic parameters for DNA adsorption with nanocomposite γ-Fe2O3@Chi@Pani Pseudo first order Pseudo second order Morris-Weber K1 (min−1) qe (mg/g) R2 K2 (g/mg.min) qe (mg/g) R2 Kid (min−1) R2 2.3 × 10−3 43.65 0.88 1.5 × 10−3 20 0.94 1.31 0.92 106 Carbohydrate Polymers 197 (2018) 100–108 B.G Maciel et al Fig (I) Images of agarose gel electrophoresis of the DNA extracted from human blood using Fe3O4@Chi MNPs (a) and γ-Fe2O3@Chi@Pani MNC (b) and (II) electrophoresis experiment using reference DNA standard (a), PCR amplified DNA extracted from human blood by use of MNPs (b), negative sample (c) and PCR amplified DNA extracted from human blood by use of MNC (d) coated with polyaniline by means of emulsion polymerization of the aniline monomers During the polymerization in an aqueous medium, a transformation of the magnetite nanoparticles to the maghemite phase occurs, resulting in the formation of maghemite/chitosan/polyaniline hybrid nanocomposites We examined the properties of the nanocomposite γ-Fe2O3@Chi@Pani as an adsorbent of DNA, using a model system consisting of an aqueous solution of salmon sperm DNA To the best of our knowledge, no previous report of using γ-Fe2O3@Chi@Pani MNC for the DNA adsorption is available in the literature We have found that these magnetic nanocomposites exhibit an enhanced efficiency when compared to that of other adsorbents discussed in the literature, and we present arguments suggesting that this observed larger degree of capture is associated to a dual adsorption/diffusion character of the interaction between the DNA and the nanoparticles When we applied these MNCs to extract DNA from human blood samples, the quality and purity of the collected material had the adequate levels for use in PCR amplification experiments We believe that these hybrid nanocomposites represent a promising low cost and convenient material that could be easily incorporated in purification protocols for different applications in molecular biology and in the area of diagnostics extracted material or in the real-time PCR amplification curves relative to what is obtained when no RNase was introduced in the process Hence, in the final protocol we first added proteinase K to the whole blood sample to induce the proteins present to degrade, and then a lysis solution (water-triton X-100) is used to disrupt the cellular membrane Then, we added a glycine/HCl/NaCl solution for promoting the interaction between the MNC and the DNA of the lysed sample After that, the DNA-MNC complex with could be separated with the help of a magnet and the supernatant discarded In a subsequent step, we removed cellular debris, and magnesium and calcium in excess by adding Tris-HCl/EDTA and potassium acetate and ethanol Finally, the captured DNA fraction was eluted by applying a Na2HPO4/NaH2PO4, with the objective of deprotonating the Pani chains and so interrupting the interaction By using UV–vis absorption spectroscopy to assess the amount of DNA extracted, we determined an average DNA adsorption capacity of (54.2 ± 0.08) ng/μL for the Fe3O4@Chi MNPs and of (80 ± 0.14) ng/ μL for the γ-Fe2O3@Chi@Pani MNC and a value for the 260/280 ratio of 1.86 and 1.71, respectively In all UV–vis spectra, the peak at 260 nm (which corresponds to DNA absorption) is well-defined Iron oxide presents an absorption in the range from 250 to 400 nm, chitosan around 300 nm and Pani at 323 and 636 nm In Fig S6, we show the (200–800) nm spectrum of the DNA from blood sample of the MNC, confirming the absence of spurious absorption bands We must stress, however, that the UV–vis absorption results can only be taken as a convenient indicator of the quality of the sample (OGT, 2011); hence, we carried out an agarose gel electrophoresis to assess the integrity and purity of the DNA extracted from the blood sample In Fig 8(I), it is possible to observe the presence of well-defined bands, with no traces, confirming the purity and integrity of the purified nucleic acid fraction To a further confirmation of this, we submitted the extracted DNA sample to a PCR amplification with the β-actin primers and used the resulting material in an agarose gel electrophoresis experiment (Fig 8(II)), comparing the observed bands to those of a reference standard (ID 4501885a1) The presence of a significant band associated to fragments corresponding to 250 base pairs demonstrated that the DNA fraction extracted by use of the γ-Fe2O3@Chi@Pani MNC has the adequate quality use in molecular biology procedures In fact, since no inhibitors were present in the DNA extracted using the MNC, its amplification by PCR became possible Acknowledgment This work was supported by the Brazilian agencies FACEPE, CNPq, CAPES, Brazilian Ministry of Health BGM, RJS and JJAE are grateful for CAPES, CNPq and FACEPE scholarships, respectively Juan Medina and Alicia Chávez are grateful to the Mexican agency CONACyT The authors would like to thank Dr Alexandre Ricalde Rodrigues, Dr Valdir Balbino, the technical staff of the Department of Fundamental Chemistry and the of Department of Physics of the UFPE for their expertise and use of the instrumentation needed to characterize our materials Appendix A Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.carbpol.2018.05.034 References Alcaraz-Espinoza, J J., Chávez-Guajardo, A E., Medina-Llamas, J C., Andrade, C A S., & de Melo, C P (2015) Hierarchical composite polyaniline–(electrospun polystyrene) fibers applied to heavy metal remediation ACS Applied Materials & Interfaces, 7(13), 7231–7240 Alibeigi, 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Extraction of DNA from whole human blood samples For the DNA extraction, we followed the procedure: in a mL microtube, we added μL of proteinase K, 100 μL of whole human blood and 500 μL of a. .. and (II) electrophoresis experiment using reference DNA standard (a) , PCR amplified DNA extracted from human blood by use of MNPs (b), negative sample (c) and PCR amplified DNA extracted from human. .. B.G Maciel et al Fig Percentage of adsorption and adsorption capacity at equilibrium as a function of initial DNA concentration Fig DNA adsorption as a function of the interaction time for an amount