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.
Trang 1Contents lists available atScienceDirect
Carbohydrate Polymers journal homepage:www.elsevier.com/locate/carbpol
Juan Carlos Medina-Llamasc, José Jarib Alcaraz-Espinozad, Celso Pinto de Meloa,d,⁎
a Pós-Graduação em Ciência de Materiais, Universidade Federal de Pernambuco, 50670-901 Recife, PE, Brazil
b 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
A R T I C L E I N F O
Keywords:
Maghemite
Chitosan
Polyaniline
Magnetic hybrid nanocomposite
DNA extraction
Human blood
A B S T R A C T
We examined the properties of the nanocompositeγ-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 theγ-Fe2O3@Chi@Pani na-nocomposite is a promising adsorbent for use in protocols for purification of DNA from complex samples
1 Introduction
The elucidation of DNA function in organisms represented an
un-precedent 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
knowl-edge, 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 needsfirst 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–li-quid 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; Medina-Llamas, Chávez-Guajardo, Andrade, Alves, & de Melo, 2014) These MNPs typically consist of a superparamagnetic nucleus (usually Fe3O4
orγ-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 ma-terials 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
https://doi.org/10.1016/j.carbpol.2018.05.034
Received 21 December 2017; Received in revised form 9 May 2018; Accepted 11 May 2018
⁎ 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).
Available online 22 May 2018
0144-8617/ © 2018 Elsevier Ltd All rights reserved
T
Trang 2this, 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
alter-native 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
re-levant examples of exploiting electrostatic complexes between cationic
polymer NPs and DNA can be found not only in thefield of gene
de-livery, 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
ad-sorption 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
cel-lulose, is composed of randomly distributed N-acetyl glucosamine and
D-glucosamine and can be obtained from the deacetylation of chitin
(Foster, Ho, Hook, Basuki, & Marçal, 2015) In its structure, Chi
pre-sents three potential reactive sites (two hydroxyl groups per glycosidic
unit and a primary amine) that are susceptible to a subsequent
mod-ification 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
lim-itations 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),
re-sulting in a material that preserves the intrinsic properties of Chi while
presenting an enhanced functionality for DNA adsorption and
trans-fection
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
bio-molecules, 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
proto-nation 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-Llamas, Andrade, & de Melo, 2015) We have exploited this latter me-chanism in a previous work, to create two solid phase adsorption ma-terials 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 resultingγ-Fe2O3@Chi@Pani composite by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and by measurements of their magnetic response
2 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), so-dium hydroxide (NaOH) and methanol (CH3OH) were obtained from Química Moderna (Brazil), while aniline (C6H5NH2), sodium phosphate monobasic (NaH2PO4), bibasic sodium phosphate (Na2HPO4) were ac-quired 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
X-100 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-pre-cipitation 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 min The re-sulting solution was dripped into a 250 mL round-bottom flask con-taining 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 1 h Afterwards, we slowly added 15 mL of an aqueous solution of NH4OH 28% (v/v) to theflask, and stirred for 30 min Fi-nally, 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 afinal brown powder was obtained
2.2.2 Synthesis of theγ-Fe2O3@Chi@Pani magnetic nanocomposite The polymerization of aniline on the surface of the Fe3O4@Chi MNPs was performed according to the established procedure ( Medina-Llamas 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-bottomflask, which was stirred for 15 min Subsequently, 20 mL of an aqueous so-lution of HCl (0.1 M) containing 18 mmol of APS was added The polymerization reaction was allowed to proceed for 24 h at 5 °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 char-acterized by Fourier Transform Infrared Spectroscopy (FTIR), Scanning
Trang 3Electron Microscopy (SEM) and magnetic measurements FTIR spectra
of the samples of interest were obtained in the 4000–400 cm−1range
by using pressed KBr pellets in an IRTracer-100 spectrophotometer
(Shimadzu, Japan) SEM micrographs were obtained using a FEG-SEM
MIRA 3 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
de-termined by use of a Nanodrop 2000C spectrophotometer (Thermo
Scientific, USA) The integrity and quality of extracted DNA was
ver-ified 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
2.2.4 DNA adsorption experiments in a model system
The DNA adsorption experiments were performed as follows First,
we placed 4 mg ofγ-Fe2O3@Chi@Pani MNC and 2.5 mL of a glycine/
HCl pH 3.6 buffer solution in a 5 mL glass vial, and mixed them with aid
of a vortex for 5 s Subsequently, we added 2.5 mL of sperm salmon
DNA solution into the vial that was kept under stirring for 10 min 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 cleanflask A 2 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
ab-sorption peak of DNA (Fig 1)
In these studies, we exposed different amounts of the
γ-Fe2O3@Chi@Pani MNC (2, 3, 4 and 5 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
= ⎛
⎝
⎠
Adsorption C C
where C0is the initial concentration and Cfthefinal 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 500μL of a glycine/
HCl buffer solution (pH 3.6) in the glass vial that contained the MNC
and mixed the contents using in a vortex for 30 s Then, we magneti-cally confined the MNC and removed the supernatant Subsequently,
we added 5 mL of a buffer solution at pH 8.0 (Tris/HCl) to the glass vial, which was then stirred for 10 min in an orbital shaker at 230 rpm Finally, the MNC was once more magnetically confined, and the su-pernatant collected and analyzed by UV–vis
2.2.6 Extraction of DNA from whole human blood samples For the DNA extraction, we followed the procedure: in a 2 mL mi-crotube, we added 5μL of proteinase K, 100 μL of whole human blood and 500μL 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 5 min during this period of time After this, we added 1.2 mL of glycine/HCl/NaCl buffer solution (pH 3.3) and 4 mg of MNC
to the microtube, and allowed the DNA-MNC interaction to occur during 10 min Then, the MNC was magnetically confined and the su-pernatant discarded, and we then added 900μL of a solution prepared using 1.7 mL of ethanol for each 1 mL of a potassium acetate-Tris/HCl-EDTA 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 5 min for al-lowing the ethanol evaporation, and we added 30μL of an elution so-lution (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 su-pernatant 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 2.5μL of dNTPs 10X (0.2 mM); 2.5μL of PCR 10 X-MgCl2(200 mM Tris-HCl, pH 8.4 and 500 mM de KCl); 0.75μL of MgCl2(1.5 mM); 0.2μL of Taq; 15.6μL 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 1.5μL of the extracted DNA, totaling 25μL for each well, except for the negative control, where 1.5μL of nuclease free water was added The reagents used in this procedure were purchased from Invitrogen Life Technolo-gies
The amplifications were performed in a StepOnePlus thermocycler Initially, the sample was subjected to a temperature of 50 °C for 2 min, and afterwards the temperature was raised to 95 °C for 3 min to allow
Fig 1 Scheme of the experiment used to estimate the capacity of DNA adsorption by MNC
Trang 4denaturation 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 thefinal extension at 4 °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
pur-ification kit from Qiagen (Germany) After purpur-ification, the quality of
the obtained amplifications was examined by performing an agarose gel
electrophoresis, with use of a diamond dye (Promega, USA)
3 Results and discussion
3.1 Characterization of the Fe3O4@Chi MNPs and theγ-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 3(I)) In the iron oxide/chitosan/polyaniline spectrum (curve a), one can observe peaks at 636 cm−1and 577 cm−1that are due to Fe-O vibrations of the maghemite (γ-Fe2O3) iron oxide phase ( Medina-Llamas et al., 2014) In the iron oxide/chitosan spectrum (curve b), only one peak in 621 cm−1was found, which we associated to the Fe-O vibrations of the magnetite (Fe3O4), in an indication of the Fe3O4to γ-Fe2O3transformation 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−1and 2850 cm−1that may be attributed to the symmetric and asymmetric stretching of the eCH2e groups, respectively The peaks at 1564 cm−1and 1482 cm−1 are due to the C]C stretching of the quinoid and benzenoid rings, re-spectively, while the peak at 796 cm−1is attributed to CeH out of the plane deformation in the benzenoid ring The peaks at 1294 cm−1and
1236 cm−1 can be assigned to the C−N stretching vibration of the benzenoid ring, whereas the peak at 1107 cm−1can be assigned to the aromatic CeH in plane bending The characteristic peaks for the chit-osan (curve c) appeared at 3370 cm−1(OeH and NeH stretching vi-brations), 2873 cm−1(also due CeH stretching vibrations), 1643 cm−1
Fig 2 Procedure for extracting DNA from human blood samples
Fig 3 (I) IR spectra ofγ-Fe2O3@Chi@Pani MNC (a), Fe3O4@Chi MNP (b) and Chitosan (c) and (II) Magnetization curves of Fe3O4@Chi MNPs (a) and for the
γ-Fe2O3@Chi@Pani MNC (b)
Trang 5(NeH bending vibrations) and 1070 cm−1 (CeOeC stretching
vibra-tions) (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,
re-spectively 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
do 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 InFig 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 ofFig 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
dis-tribution was from 23 nm to 31 nm, with an average diameter of
(27 ± 4) nm
3.2 DNA adsorption experiments
To assess the DNA adsorption capacity of theγ-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
iden-tical samples In these initial experiments, we observed that after
10 min of interaction the average adsorption of theγ-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)
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 1 kb, Promega, USA) (PROMEGA, 2017)
InFig 5, we show the results of the agarose gel electrophoresis ex-periment, 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 γ-Fe2O3@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 de-gradation
As expected, use of theγ-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
Fig 4 SEM image of Fe3O4@Chi (a) andγ-Fe2O3@Chi@Pani (b) samples (Histograms of the particle size distribution in inset)
Fig 5 Agarose gel electrophoresis for: standard DNA diluted in nuclease-free
H2O (a), after interaction with Fe3O4@Chi MNPs (b) andγ-Fe2O3@Chi@Pani MNC (c)
Trang 6the MNC was used.
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) InFig 6, one
can observe that the adsorption capacity increases when either the
in-teraction time or the amount of MNC used is increased When only 2 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, 4 and 5 mg) would lead to a progressive
increase in the amount of retrieved DNA (20%, 40% and 70%,
re-spectively− curves b, c and d) within the 145 min of interaction, but
with no indication that the saturation regime was already attained We
have then decided to adopt a fixed amount of 2 mg of the
γ-Fe2O3@Chi@Pani MNC in the subsequent analyzes to ensure that the
equilibrium concentration was reached in each case
3.2.2 Effect of initial DNA concentration
After assessing both the appropriate amount of theγ-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, afixed
amount of 2 mg of the MNC placed to interact with 5 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 min 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
ad-sorption 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)
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
function of the equilibrium concentration Ce, in the form
e
e
0
(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)
C
q bq
C q
1
e
e m
e
where b is the Langmuir constant and qmis the maximum amount of adsorbed molecules (mg/g) The values of b and qmcan be determined
by plotting the data as a Ce/qevs Cegraph 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)
n logC
log e log f 1 e
(4) where qeis the adsorption capacity, and Kfand n are constants that can
be determined from a logqevs logKfplot InTable 1we present the values of the parameters of the Langmuir and Freundlich models ob-tained after adjusting the data In Fig S2, we show the corresponding linearfittings 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 cal-culated by use of Eq.(2)
Fig 6 DNA adsorption as a function of the interaction time for an amount of
2 mg (a), 3 mg (b), 4 mg (c) and 5 mg (d) ofγ-Fe2O3@Chi@Pani MNC In each
case, 2.5 mL of a 100 ng/μL DNA solution was used
Fig 7 Percentage of adsorption and adsorption capacity at equilibrium as a function of initial DNA concentration
Table 1 Isotherm parameters for the Langmuir and Freundlich models applied to the DNA adsorption on theγ-Fe2O3@Chi@Pani MNC
Langmuir Freundlich
q m
(mg/g)
b (mg/L)
R2
K f (mg/g)
49.5 1.89 0.99 22.39 5.88 0.37
Trang 73.4 Kinetics of the adsorption process
One can obtain more information on how theγ-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 q( e q t) logq e k t (Pseudo first order),
2.303
1
(5)
= + t, h=k q (Pseudo−second order: ), or
t
q t k id( )t0.5 C (Morris Wener),
(7) where qeand qtare 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
ac-cording to the Eqs.(5)–(7), respectively (seeTable 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
intra-particle diffusion mechanisms
We can rationalize these results if we assume that a competition
exists between these two kinetic models, and that the processes of
ad-sorption and abad-sorption of the DNA molecules on the MNC occur
con-comitantly, 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
γ-Fe2O3@Chi@Pani MNC exhibits an increased capture rate of the target
molecule
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
sui-table for an effective elution of the captured molecules, in the
sub-sequent 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
percentage of the γ-Fe2O3@Chi@Pani MNC (as well as the time re-quired 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 min of interaction Teng et al (Teng, Li, Yan, Zhao, & Yang, 2009) estimated a capacity of 46 mg/g in
20 min when using 10 mg of magnetite nanoparticles coated with me-soporous 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 min Zhang et al (Zhang et al., 2012) used 1 mg of polystyrene-coated me-soporous silica nanoparticles, and obtained an adsorption capacity of 110.7 mg/g after 1440 min of interaction Sun et al (Sun et al., 2014) reported that employing 0.5 mg of M-MSN@TEOS in 120 min 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 collabora-tors (Hu et al., 2015) have used an iron phosphate@Polyethyleneimine MNC that showed both higher adsorption and larger desorption capa-city in shorter times than theγ-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
3.7 Extraction of DNA from whole human blood samples After having established the most suitable conditions for the ex-traction 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 poly-merase, 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 un-stable nucleic acid, RNA chains are not well preserved due to the al-kaline hydrolysis and the presence of alkali metals that favor their degradation (Oivanen, Kuusela, & Lönnberg, 1998), and one could ex-pect 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 com-posed by DNA molecules In fact, before establishing the best extraction protocol to be followed, we examined how the results would be affected when either 40μg or 200 μg of RNase was added in the lysing step As the results shown in Fig S5 reveal for both cases, no significant dif-ferences could be detected either in the UV–vis spectra of the final
Table 2
Kinetic parameters for DNA adsorption with nanocompositeγ-Fe2O3@Chi@Pani
Pseudo first order Pseudo second order Morris-Weber
K 1 (min−1) q e (mg/g) R 2 K 2 (g/mg.min) q e (mg/g) R 2 K id (min−1) R 2
2.3 × 10−3 43.65 0.88 1.5 × 10−3 20 0.94 1.31 0.92
Trang 8extracted material or in the real-time PCR amplification curves relative
to what is obtained when no RNase was introduced in the process
Hence, in thefinal 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
inter-action 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
re-moved cellular debris, and magnesium and calcium in excess by adding
Tris-HCl/EDTA and potassium acetate and ethanol Finally, the
cap-tured 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 InFig 8(I), it is possible to observe the presence of well-defined
bands, with no traces, confirming the purity and integrity of the
pur-ified 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 theγ-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
am-plification by PCR became possible
4 Conclusion
We synthesized magnetite/chitosan nanoparticles that were later
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 nano-compositeγ-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 usingγ-Fe2O3@Chi@Pani MNC for the DNA adsorption is available in the literature We have found that these magnetic nanocomposites exhibit an enhanced e ffi-ciency 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 sam-ples, 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 diag-nostics
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 ex-pertise and use of the instrumentation needed to characterize our ma-terials
Appendix A Supplementary data
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