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Co-evaluation of interaction parameters of genomic and plasmid DNA for a new chromatographic medium
International Journal of Biological Macromolecules 141 (2019) 1183–1190
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Co-evaluation of interaction parameters of genomic and plasmid DNA for a new chromatographic medium Onal Burcu Önal a, Ömür Acet a, Raúl Sanz b, Sanz-Perez Eloy S. Sanz-Pérez c, Erdonmez Demet Erdönmez d, Mehmet Odabasßı a,* a
Aksaray University, Faculty of Arts and Science, Chemistry Department, Aksaray, Turkey Department of Chemical and Environmental Technology, ESCET, Universidad Rey Juan Carlos, C/ Tulipán s/n, 28933 Móstoles, Madrid, Spain Department of Chemical, Energy, and Mechanical Technology, ESCET, Universidad Rey Juan Carlos, C/ Tulipán s/n, 28933 Móstoles, Madrid, Spain d Aksaray University, Faculty of Arts and Science, Biology Department, Aksaray, Turkey b c
a r t i c l e
i n f o
Article history: Received 8 June 2019 Received in revised form 26 August 2019 Accepted 9 September 2019 Available online 11 September 2019 Keywords: Pore expanded SBA-15 Hybrid cryogels DNA adsorption
a b s t r a c t Preparation of new sorbents specific to DNA has a great significance in many biomedical fields. This study reports a new sorbent with high surface area and porosity to immobilize nucleic acids having both high molecular weight like genomic DNA (gDNA) for potential use in therapy of some immune system disease and low molecular weight like plasmid DNA (pDNA) for diagnosis, gene therapy and DNA vaccination. For this aim, silica-based pore-expanded SBA-15 nanoparticles with aminopropyl-trimethoxysilane (APTMS) for decoration of Fe+3 ions (PE SBA-15-APTMS/Fe+3) were synthesized to get high surface area for high adsorption, and embedded into cryogel column for obtaining interconnected pores to avoid diffusion limitation of DNA samples because of their viscosity features. SEM, XRD, BET, and FTIR techniques were used for characterization of samples. Synthesized hybrid column showed a superior adsorption capacity of 751.5 mg/g NP for gDNA at pH 6 with an initial concentration of 2.0 mg/mL. Hybrid column presented excellent performance for pDNA when evaluated with agarose gel electrophoresis. Ó 2019 Published by Elsevier B.V.
1. Introduction Deoxyribonucleic acid (DNA) is a biopolymer consisting of adenine (A), guanine (G), cytosine (C) and thymine (t) bases, ribose (a pentose monosaccharide) and phosphate groups, and responsible to carry genetic information. While, in some cases, DNA (especially double stranded DNA) is used for treatment of a few autoimmune illness [1], on the other hand, some disciplines like medicine and molecular biology need pure plasmid DNA (pDNA) for some utilizations such as manufacturing of high protein amount, cloning [2], gene therapy [3], DNA vaccination [4] and molecular diagnosis of diverse sickness [5]. The usage of therapeutic genes is generally via injection of naked or covered genes (such as covering with lipid molecules). Thus, significant amount of DNA is needed; but, it is noted that large-scale DNA purification has some difficulties [6]. Different techniques have been applied by researchers for purification and separation of DNA from cell lysate (i.e., solvent extraction, ultracentrifugation, etc.) [7]. However, these techniques
⇑ Corresponding author at: Aksaray University, Faculty of Arts and Science, Chemistry Department, Biochemistry Division, 68100 Aksaray, Turkey. https://doi.org/10.1016/j.ijbiomac.2019.09.068 0141-8130/Ó 2019 Published by Elsevier B.V.
mainly involve the use of extra contaminant such as ethidium bromide, phenol and CsCl besides some enzymes, which are not recommended for production of therapeutics [8]. These techniques have also low purification amount and high cost. Hereby, to overcome disadvantages of some methods mentioned above for purification of DNA, a number of chromatographic techniques have been developed such as size-exclusion chromatography (SEC), expanded bed anion-exchange chromatography (EB-AEC) [9], reversed-phase liquid chromatography (RPLC) [10], and hydrophobic interaction chromatography (HIC) [11]. Among these methods, while some of them have limited adsorption capacities, others need organic solvents, which are toxic for bioscience, in elution steps [12]. Development of new separation media for DNA is an important event in the field of biomedicine and biotechnology. Particles, which serve large surface area and high adsorption capacity, fulfil an important task like effective binding surfaces for adsorption of biomolecules. Nowadays, the applications of meso and nano-structured materials in bioseparation have been widely scaled up [13]. Mesoporous silica nanoparticles due to their outstanding features such as relatively cheapness, non-toxicity and biodegradability compared to those are utilized in the preparation are unique nominees in biomedical applications such as gene delivery, controlled release, biosensors, etc. [14–17].
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Supermacroporous cryogels having three-dimensional interconnected compatible system with great mass flow have good chemical and mechanical stability. Upgrading the binding capacities of cryogels takes more and more attention in biotechnology and separation science [18]. Supermacroporous cryogels constructed with micro and nano-sized particles by embedding process are newly promising medium, and have been used extensively in separation science [19–23]. Because particleembedded supermacroporous cryogels are bearing superiority features of two sorbents such as particles (i.e., large surface area for high ligand density) and cryogels (i.e., large pores, short diffusion path, low pressure drop, etc.) to overpower of high viscosity and lower diffusion coefficients of DNA mixture [24,25]. Here, silica-based pore-expanded SBA-15 nanoparticles [26,27] (PE SBA-15) were synthesized and functionalized with aminopropyl-trimethoxysilane (APTMS). Then, functionalized particles (PE SBA-15-APTMS) were decorated with Fe+3 ions and named as (PE SBA-15-APTMS/Fe+3). Fe+3-attached particles were used to get hybrid column with embedding process. Prepared hybrid column (PE SBA-15-APTMS/Fe+3 EHC) was evaluated with regard to adsorption characteristics and interaction parameters for genomic and plasmid DNA (gDNA and pDNA). 2. Experimental 2.1. Chemicals pMAD a kind of pDNA which was isolated from E. coli DH5a strains grown in Luria-Bertani (LB) medium including ampicillin (100 lg/mL) was provided by Aksaray University, Molecular Biology Laboratory (Aksaray, Turkey). All chemicals used were purchased from Sigma (St. Louis, MO, USA). 2.2. Methods 2.2.1. Synthesis of pore-expanded SBA-15 Pore-expanded SBA 15 (PE SBA-15) was synthesized by modifying the original procedure by Prof. Stucky for conventional SBA-15 [28]. Namely, 1,3,5-triisopropylbenzene (TIPB, 95%, Sigma-Aldrich) was used as a swelling agent and NH4F (99.99%, Sigma-Aldrich) as a solubility enhancer, following the procedure described by Cao et al. [29]. A P123:TIPB ratio of 2.4:1 was employed in order to preserve mesoscopic structure at the same time that pore diameter is increased. Also, a hydrolysis temperature of 17 °C instead of 40 °C was used. The surfactant was removed from the mesostructured support by ethanol-extraction for 24 h and the obtained material was named PE SBA-15. PE-SBA-15 was modified by grafting with aminopropyltrimethoxysilane (APTMS). 1 g silica was dispersed in 250 mL toluene and subsequently, 6.89 mmol APTMS/g silica was added. The resulting mixture was refluxed for 24 h. The solid obtained was filtered under vacuum, and the excess of APTMS was removed by washing with toluene. The material obtained was denoted PE SBA-15-APTMS. 2.2.2. Incorporation of Fe+3 ions to PE SBA-15-APTMS Incorporation of Fe+3 ions to PE SBA-15-APTMS was conducted in 150 ppm Fe+3 solution at pH 4.7 (adjusted with 0.01 M HCl) at room temperature. The attachment reaction was carried out for 1 h, and the concentration of Fe+3 ions in the media was monitored by inductively coupled plasma optical emission spectroscopy (ICPOES) with a Varian Vista AX apparatus. Fe+3 ions attached to PE SBA-15-APTMS was calculated by equation (Eq. (1)).
Q ¼ ðCi Cf ÞV=m
ð1Þ
Here, Ci and Cf represent initial and final Fe+3 concentrations, respectively (mg/mL); of the ions in the solution before and after incorporation, respectively (mg/mL); Q symbolizes attached Fe+3 amount onto the PE SBA-15-APTMS (mg/g); V is the solution capacity (mL) and m is amount of PE SBA-15-APTMS (g). After the attachment of Fe+3 ions, the sample obtained was named as PE SBA-15-APTMS/Fe+3. Fe+3 leakage studies from PE SBA-15-APTMS/Fe+3 were also conducted in various media with different pHs (4.0–7.0) containing 1.0 M NaCl. For this aim, PE SBA-15-APTMS/Fe+3 were stirred in the mentioned media at room temperature for 24 h. The Fe+3 concentration in the solution was calculated by ICP-OES. 2.2.3. Organization of PE SBA-15-APTMS/Fe+3 embedded column Here, 1.75 mL of 2-hydroxyethyl methacrylate (HEMA) and 40 mg of N,N0 -methylene-bis acrylamide (MBAm) used as main monomer and crosslinker, respectively were mixed with 2.2 mL of deionized water. Then, this mixture was blended with 45 mg of PE SBA-15-APTMS/Fe+3. Obtained mixture was poured into a plastic syringe with 0.8 cm i.d., and last volume was filled up to 5 mL with deionized water. After addition of 100 lL (10% (w/v) APS and 20 lL TEMED used as free radical producer and catalyst, respectively, the final mixture was incubated at 12 °C for 24 h. Later, syringe was taken to room temperature, and water was used several times for cleaning the column to remove contamination after thawing. The obtained column named as Fe+3 incorporated APTMS attached PE SBA-15 embedded hybrid column (PE SBA15-APTMS/Fe+3 EHC) was kept in buffer including 0.02% sodium azide at 4 °C until usage. 2.3. Characterization techniques 2.3.1. Physico-chemical characterization PE-SBA-15 large scale ordering was assessed by means of low angle X-ray diffractograms acquired in a powder Philips X’PertMPD diffractometer using a CuKa monochromatic radiation. Nitrogen adsorption-desorption isotherms at 77 K were carried out in a Micrometrics TRISTAR-3000 equipment. Surface area was obtained by fitting isotherm data points between 0.05 and 0.2 to the BET equation, pore diameter by using the B.J.H. model applied to cylindrical pores in the adsorption branch of the isotherms, and pore volume at a relative pressure of 0.97 [30]. The organic nitrogen loaded by grafting was measured in a Flash 2000 elemental analyzer equipped with a thermal conductivity detector. Scanning Electron Micrography (SEM) was used to observe the internal structure of the samples prepared. A Hitachi TM-1000 apparatus with a tungsten filament and a 15 kV accelerating voltage was used. Besides, semi-quantitative elemental analyses were performed with an EDX (OXFORD) detector.
2.4. Adsorption-desorption studies of DNA from aqueous solutions Evaluation of PE SBA-15-APTMS/Fe+3 EHC regarding to adsorption capacity for gDNA and pDNA was carried out in different media. For this aim, effects of concentration, pH, ionic strength, flow rate, and temperature were investigated. A peristaltic pump (ALITEA, Sweden) was used in experiments to send the solutions into the column. Amount of DNA in media before and after adsorption was red by UV spectrophotometer (Shimadzu, Tokyo, Japan, Model 1601) at 260 nm. All elution steps were carried out only in 20 min by applying 0.05 M phosphate buffer (pH 8) including 1 M NaCl at a flow rate of 1.0 mL/min. After all adsorption cycles, the cryogel column was cleaned up with water and equilibrated with operational buffer for the next process.
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2.5. Purification of E. coli plasmid DNA Two types of pDNA having two different molecular weight such as pMAD (9666 bp) and pUC18 (2686 bp) were grown in E.coli cells in different cultures to determine the efficiency of PE SBA-15APTMS/Fe+3 EHC. For purification process of pDNA samples, 1.5 mL of E.coli cells in LB (Laura Bertani) media was centrifuged. After removing of supernatants, the precipitates were resuspended in 250 lL GTE (Glucose/Tris/EDTA) solutions, and left at room temperature for 5 min. Then, cell lysis was done by adding 500 lL of sodium hydroxide and sodium dodecyl sulfate (NaOH/SDS) solutions. After lysis in alkaline conditions, obtained lysates were resuspended in 10 mL of acetate buffers at pH 6 for use in IMAC column. In order to determine the adsorption efficiency of PE SBA-15-APTMS/Fe+3 EHC for pDNA, 10 mL of obtained two kinds of pDNA having different molecular weights were passed through PE SBA-15-APTMS/Fe+3 EHC, separately. After washing stage with 10 mL of acetate buffer, elution of column was carried out by applying 0.05 M phosphate buffer (pH 8) including 1 M NaCl at a flow rate of 1.0 mL/min for 10 min. Obtained eluents were loaded on 0.8% of agarose gel and undergone to electrophoresis treatment at a constant electric field. The gel was run at 100 V for 30 min, and then obtained images were photographed. 3. Results and discussion 3.1. Characterization of PE SBA and PE SBA-15-APTMS X-ray powder diffraction spectra acquired for siliceous PE SBA15 and amine-containing PE SBA-15-APTMS are presented in Fig. 1. Siliceous PE SBA-15 showed the three characteristic peaks of p6mm bidimensional hexagonal structure. These peaks have been previously described for pore-expanded SBA [31], and are analogous to those reported for conventional SBA [32]. However, X-ray diffraction peaks for pore-expanded materials show a shift to lower angles due to the larger cell parameters (a0) of these materials (13.9 nm in this case) compared to conventional SBA supports (12.0 nm). Regarding the intensity of diffraction peaks, it is lower for PE SBA-15 than for conventional SBA materials and the high angle peaks corresponding to planes (1 1 0) and (2 0 0) were not as clear, likely due to a slight loss of the mesostructure during the pore-expansion process.
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The diffraction spectra of PE SBA-15-APTMS sample showed a similar pattern but with a lower intensity. This change is due to the grafting process, since it yielded a certain pore filling with organic moieties, thus decreasing X-ray diffraction inside the pores. Nevertheless, it can be observed that the hexagonal structure of PE SBA-15 was not modified after the grafting step. Fig. 2 shows the N2 adsorption-desorption isotherms obtained for PE-SBA-15 siliceous material and amine-containing PE SBA15-APTMS while Table 1 lists the main textural properties of these samples. Both isotherms can be categorized as type IV according to the IUPAC classification [30]. The steep increase in the amount of nitrogen adsorbed at a relative pressure around 0.80 can be ascribed to the presence of a narrow and wide mesoporosity. As seen in Table 1 summarizing PE SBA-15 textural properties, this sample showed a BET surface of 452 m2/g, a mean pore diameter of 11.7 nm and a total pore volume of 1.02 cm3/g. These values represent a much higher pore diameter for pore-expanded SBA-15 compared to the conventional silica (ca. 9 nm) though this increase was achieved at the expense of a reduction in the specific surface, which is typically between 500 and 700 m2/g for conventional SBA [28,33]. As a result of the grafting process and the subsequent loading of the porous structure with amines, the remaining volume inside PE SBA-15-APTMS was lower than the observed for PE SBA-15, thus adsorbing a smaller amount of nitrogen (see Fig. 2). Consequently, all textural properties were also lower than those corresponding to PE SBA-15 silica, though not reaching the saturation of the porous structure. The cryogels prepared with PE SBA-15-APTMS/Fe+3 were analysed by Scanning Electron Microscopy (SEM) with images being shown in Fig. 3. The layout of silica particles in the polymer matrix can be observed in Fig. 3a and b. As seen, the cryogel polymer is providing a structural support with silica particles being deposited on top of it. This arrangement is coherent with the independent synthesis of PE SBA-15-APTMS/Fe+3 and its subsequent deposition on the cryogel. Fig. 3c allows a closer study of PE SBA-15-APTMS/ Fe+3 material formed by particles of ca. 1 lm arranged in their characteristic chain-like array [29,34]. This fact supports the idea that silica particles were no altered by their immobilization inside the cryogel structure. Fe+3 ions attached to particles was determined to be 500 mg/g NP. Some studies were performed to see if there is any ion leakage, and no leakage was determined. This result confirmed that washing treatment was satisfying. 3.2. Adsorption studies for DNA to PE SBA-15-APTMS/Fe+3 EHC 3.2.1. Effect of pH It is important to identify the optimal circumstances for an adsorbent regarding adsorption parameters. Adsorption of DNA on IMAC based adsorbents is principally built up on chelation of metal ions with oxygen groups on phosphate structure of DNA. As seen in Fig. 4A, maximum adsorbed DNA was observed at pH 6.0 (0.05 M acetate buffer). Adsorption capacity of DNA decreased before and after pH 6.0. DNA has an isoelectric point of around pH 5 [35]. It is possible to interpret this situation as follows: After pH 6, Fe+3 ions on SBA-15 may be hydroxylated and therefore, a repulsion may take place between negatively charged DNA and adsorbent surface. On the other hand, at lower pHs than 6, because of protonation of DNA, a repulsion can occur between positively charged DNA and Fe+3 ions, as well [36]. A putative representation for this scenario is given in Fig. 5.
Fig. 1. X-ray powder diffraction spectra for PE-SBA and PE-SBA-AP.
3.2.2. Effect of medium DNA concentration DNA adsorption performance of PE SBA-15-APTMS/Fe+3 EHC was checked with different DNA concentration range of 0.25–
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Fig. 2. a) N2 adsorption-desorption isotherms and b) pore size distribution of siliceous corresponding to for PE-SBA and PE-SBA-AP.
Table 1 Textural properties of pore-expanded SBA-15 before and after being modified with APTMS. Sample
SBET (m2/g)
DP (nm)
VP (cm3/g)
d100 (nm)
a0 (nm)
e (nm)
PE SBA-15 PE SBA-15-APTMS
452 158
11.7 9.2
1.02 0.38
12.0 –
13.9 –
2.2 –
3.0 mg/mL and results were shown in Fig. 4B. Formation of hydrogen bonds, dehydration and electrostatic effects are main parameters playing role in DNA adsorption [37]. Usage of metal ions as ligand both reduces electrostatic repulsion between surface and DNA molecules and enhances the adsorption capacity. At the same time, binding of metal ions to DNA molecules supress the surface charge intensity of DNA which results in higher diffusion of DNA into surface pores and higher adsorption [38]. When compared to our previous studies [1,39], we gave priority to Fe+3 ions as ligand for DNA immobilization because of affinity of it for phosphate structure [40]. Adsorbed DNA molecules on PE SBA-15-APTMS/ Fe+3 EHC by Fe+3 ions has a remarkable degree (up to 751.5 mg/g NP). As seen, the amount of DNA coupled to cryogel nearly caught a plateau in a concentration of 2.0 mg/mL as a consequence of feeding of the binding area. It is noted that non-specific DNA adsorption on Fe+3 minus PE SBA-15-APTMS EHC was came true as 55.1 mg/g. There are plenty of reports in the literature being offered by considering the adsorption capacities. A few of them are given in Table 2. It is obviously seen here that when compared with adsorbent shape and surface functionalities, the result of our study is considerably high in the view of adsorption capacity. This result may be explained with embedded nanosized particles having extensive surface area and short diffusion path and low pressure drop of cryogel column. 3.2.3. Effect of flow rate Flow rate is another significant parameter which has influence on protein adsorption in column performance. Experiment conditions implemented in the range of 0.5 and 3.0 mL/min showed that efficient flow rate was 0.5 mL/min. By reducing the flow rate, contact time between protein and ligand spends long time. Thus, adsorption of DNA molecules to pore walls of cryogels assured high adsorption capacity. 3.2.4. Effect of ionic strength An adsorption process is mainly affected from ionic strength in media. To test this effect on DNA adsorption onto PE SBA-15APTMS/Fe+3 EHC, some experiments were accomplished with DNA solutions including NaCl in the range of 0–3.0 M (Fig. 6A). Based on results of ionic strength experiments, it can make inferences that increasing of ionic strength was concluded in low
adsorption. When salt ions are solved in an adsorption media, different kinds of ions are piled up onto surface with opposite charges, and prevent the adsorption of biomolecules. This case ends up with lower adsorption [18]. 3.2.5. Effect of temperature As for the temperature effect on DNA adsorption, this parameter was investigated between the temperatures of 5–35 °C and the highest DNA adsorption was observed at 25 °C. There are different types of interactions such as electrostatic, hydrophobic and/or coordination between metal ion and biomolecule in metal affinity chromatography, and dominant one depends on main functional groups (i.e. carboxyl, sulfhydryl, amine, etc.) interacting with metal ion, adjacent groups (i.e., aromatic rings) on biomolecule and metal ion. Determination of relative dominant interaction is not easy every time [40,41]. As seen in Fig. 6B, DNA adsorption increased up to 25 °C first, and then decreased. This situation may be explained by dominance of ionic interaction after 25 °C. 3.2.6. Column efficiency for pDNA samples from bacterial lysates In order to test the adsorption efficiencies of pDNA from E. coli lysate samples having different molecular weights to PE SBA-15APTMS/Fe+3 EHC, desorption solutions were run on agarose gel electrophoresis, and obtained lanes are given in Fig. 7. Here, lane 1 shows a marker having different molecular weights up to 12,000 bp. Lanes 2–4 and lanes 5–7 present the adsorption performance of pUC18 (2686 bp) and pMAD (9666 bp) plasmid samples from E. coli lysates, respectively. As seen especially from lanes 4 and 7 that adsorption and elution process of pDNA samples were carried out, successfully. According to these results, it can be said that prepared PE SBA-15-APTMS/Fe+3 EHC shows a satisfactory result for purification of pDNA samples from cell lysate. 3.2.7. Reusability studies We used the same column during all studies about 30 times without losing its adsorption performance to determine the reusability factors. For the desorption of DNA molecules from the PE SBA-15-APTMS/Fe+3 EHC, when 1 M NaCl solution was used, it wasn’t seen any significant desorption in the media. This situation may be explained with interaction strength between phosphate groups of DNA and Fe+3 ions used as ligand [40]. After using of pH 8 phosphate buffer including 1 M NaCl, approximately all of
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Fig. 4. pH effect on DNA adsorption onto PE-SBA-AP-Fe+3 EHC, amount of particle: 15 mg; DNA concentration: 1 mg/mL; flow rate: 1 mL/min; T: 25 °C (A); DNA concentration effect on adsorption onto PE-SBA-AP-Fe+3 EHC, amount of particle: 15 mg; pH 6.0; flow rate: 1 mL/min; T: 25 °C (B).
Ce Ce Kd ¼ þ Qe Qm Qm
ð3Þ
Here, Ce/Qe versus Ce are plotted. 1/Qm and Kd/Qm values can be obtained from the slope and intercept of the straight line, respectively.
ln Q e ¼
Fig. 3. Scanning electron micrographs of cryogel containing PE-SBA-AP-Fe+3.
the adsorbed DNA molecules were desorbed from PE SBA-15APTMS/Fe+3 EHC. At the end of about 30 cycles, a decreasing of only 2% was seen in adsorption capacity.
1 ln C e þ ln K F n
ð4Þ
As for Freundlich model, when lnQe versus lnCe are plotted, 1/n and Kf values are provided from the slope and intercept of the straight line, respectively. From two equations, while Ce (mg/mL) and Qe (mg/g) show adsorbed molecule concentration in equilibrium and amount of adsorbed molecules per unit weight of sorbent, Qm and Kf indicates the maximum adsorption capacity of adsorbent (mg/g) according to Langmuir and Freundlich, respectively. Also, Kd and n show dissociation constant and heterogeneity, respectively. The values calculated from Langmuir and Freundlich isotherm equations are presented in Table 3. As shown here, correlation coefficient (R2) of Langmuir equation is closer to 1. Therefore, it can be inferred from this result that adsorption of DNA molecules on PE SBA-15-APTMS/Fe+3 EHC actualized at monolayer and uniformly.
4. Conclusions 3.3. Investigation of adsorption models In order to characterize the binding kinetic parameters of DNA adsorption on PE SBA-15-APTMS/Fe+3 EHC, two adsorption models such as Langmuir [42] and Freundlich [43]] having formula shown in Eq. (3) and Eq. (4), respectively were reviewed.
According to our knowledge, such a study for the purification of plasmid and genomic DNA has been given comprehensively together in one paper for the first time. Here, aminopropyltrimethoxysilane immobilized silica-based pore-expanded SBA15 nanoparticles having large surface area were prepared. After
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Fig. 5. Putative showing of electron charge shift on IMAC sorbent by pH..
Table 2 Comparison of maximum DNA adsorption capacities of some sorbents in the literature. Sorbent
Adsorption capacity (mg/g)
Ref.
Cibacron Blue 3GA Cu2+-attached magnetite nanoparticles Zirconia magnetic nanocomposite Silanized polymeric nanoparticles Fe+3-attached sporopollenin particles Poly(vinyl alcohol) nanofibrous membranes Ethylene-vinyl alcohol nanofibrous membranes with citric acid PE-SBA-AP-Fe+3 EHC column
32.5 mg/g 19.97 mg/g 53.5 mg/g 672.41 mg/g 109 mg/g 177 mg/g 284 mg/g
[45] [46] [47] [48] [49] [50] [51]
751.5 mg/g
In this study
Fig. 7. Agarose gel (0.8%) electrophoresis of pDNA from E. coli cells stained with ethidium bromide. Lane 1: Ladder (12,000 bp); lane 2: pUC18 plasmid (2686 bp) before adsorption; lane 3: pUC18 after adsorption; lane 4: pUC18 desorption; lane 5: pMAD plasmid (9666 bp) before adsorption; lane 6: pMAD after adsorption; lane 7 pMAD desorption..
Table 3 The parameters obtained from adsorption isotherms. Adsorption isotherm models Ce Qe
¼
Ce Qm
þ
Kd Qm
Langmuir model
lnQ e ¼ 1n lnC e þ lnK F Freundlich model
Fig. 6. Ionic strength effect on DNA adsorption onto PE-SBA-AP-Fe+3 EHC, amount of particle: 15 mg; pH 6.0; DNA concentration: 1 mg/mL; flow rate: 1 mL/min; T: 25 °C (A); effect of temperature on DNA adsorption onto PE-SBA-AP-Fe+3 EHC, amount of particle: 15 mg; pH 6.0 initial DNA concentration: 1 mg/mL; flow rate: 1 mL/min (B).
decorating with Fe+3 ions, they were embedded into cryogel column to test the efficiency of this column for DNA molecules having different molecular weight such as genomic and plasmid DNA samples. gDNA molecules having high molecular weight compared to
Adsorption parameters
Isotherm values
Qm Kd R2 Equation (Y=) Kf n R2 Equation (Y=)
769.2 (mg/g) 0.0692 (mg/mL) 0.999 0.0013x + 9.105 664.6 (mg/g) 4.46 0.9113 0.233x + 6.5202
pDNA passed through easily from IMAC cryogel column owning a supermacroporous texture, which prevents diffusion limitation especially for viscous solutions including molecules with high molecular weight [44]. Prepared column reached to a high adsorption level (751.5 mg/g NP for gDNA) because of embedded nanoparticles possessing high surface area. Prepared IMAC column was also tested for pDNA samples having different molecular weight from E. coli lysates. The specificity of this column to DNA molecules was demonstrated through pDNA samples, and results were given by agarose gel electrophoresis. In epitome, it can be made inferences from these results that prepared IMAC column
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(i.e. PE SBA-15-APTMS/Fe+3 EHC) may be used successfully for nucleic acid separation having both high and low molecular weight.
[23]
[24]
Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
[25]
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Co-evaluation of interaction parameters of genomic and plasmid DNA for a new chromatographic medium Onal Burcu Önal a, Ömür Acet a, Raúl Sanz b, Sanz-Perez Eloy S. Sanz-Pérez c, Erdonmez Demet Erdönmez d, Mehmet Odabasßı a,* a
Aksaray University, Faculty of Arts and Science, Chemistry Department, Aksaray, Turkey Department of Chemical and Environmental Technology, ESCET, Universidad Rey Juan Carlos, C/ Tulipán s/n, 28933 Móstoles, Madrid, Spain Department of Chemical, Energy, and Mechanical Technology, ESCET, Universidad Rey Juan Carlos, C/ Tulipán s/n, 28933 Móstoles, Madrid, Spain d Aksaray University, Faculty of Arts and Science, Biology Department, Aksaray, Turkey b c
a r t i c l e
i n f o
Article history: Received 8 June 2019 Received in revised form 26 August 2019 Accepted 9 September 2019 Available online 11 September 2019 Keywords: Pore expanded SBA-15 Hybrid cryogels DNA adsorption
a b s t r a c t Preparation of new sorbents specific to DNA has a great significance in many biomedical fields. This study reports a new sorbent with high surface area and porosity to immobilize nucleic acids having both high molecular weight like genomic DNA (gDNA) for potential use in therapy of some immune system disease and low molecular weight like plasmid DNA (pDNA) for diagnosis, gene therapy and DNA vaccination. For this aim, silica-based pore-expanded SBA-15 nanoparticles with aminopropyl-trimethoxysilane (APTMS) for decoration of Fe+3 ions (PE SBA-15-APTMS/Fe+3) were synthesized to get high surface area for high adsorption, and embedded into cryogel column for obtaining interconnected pores to avoid diffusion limitation of DNA samples because of their viscosity features. SEM, XRD, BET, and FTIR techniques were used for characterization of samples. Synthesized hybrid column showed a superior adsorption capacity of 751.5 mg/g NP for gDNA at pH 6 with an initial concentration of 2.0 mg/mL. Hybrid column presented excellent performance for pDNA when evaluated with agarose gel electrophoresis. Ó 2019 Published by Elsevier B.V.
1. Introduction Deoxyribonucleic acid (DNA) is a biopolymer consisting of adenine (A), guanine (G), cytosine (C) and thymine (t) bases, ribose (a pentose monosaccharide) and phosphate groups, and responsible to carry genetic information. While, in some cases, DNA (especially double stranded DNA) is used for treatment of a few autoimmune illness [1], on the other hand, some disciplines like medicine and molecular biology need pure plasmid DNA (pDNA) for some utilizations such as manufacturing of high protein amount, cloning [2], gene therapy [3], DNA vaccination [4] and molecular diagnosis of diverse sickness [5]. The usage of therapeutic genes is generally via injection of naked or covered genes (such as covering with lipid molecules). Thus, significant amount of DNA is needed; but, it is noted that large-scale DNA purification has some difficulties [6]. Different techniques have been applied by researchers for purification and separation of DNA from cell lysate (i.e., solvent extraction, ultracentrifugation, etc.) [7]. However, these techniques
⇑ Corresponding author at: Aksaray University, Faculty of Arts and Science, Chemistry Department, Biochemistry Division, 68100 Aksaray, Turkey. https://doi.org/10.1016/j.ijbiomac.2019.09.068 0141-8130/Ó 2019 Published by Elsevier B.V.
mainly involve the use of extra contaminant such as ethidium bromide, phenol and CsCl besides some enzymes, which are not recommended for production of therapeutics [8]. These techniques have also low purification amount and high cost. Hereby, to overcome disadvantages of some methods mentioned above for purification of DNA, a number of chromatographic techniques have been developed such as size-exclusion chromatography (SEC), expanded bed anion-exchange chromatography (EB-AEC) [9], reversed-phase liquid chromatography (RPLC) [10], and hydrophobic interaction chromatography (HIC) [11]. Among these methods, while some of them have limited adsorption capacities, others need organic solvents, which are toxic for bioscience, in elution steps [12]. Development of new separation media for DNA is an important event in the field of biomedicine and biotechnology. Particles, which serve large surface area and high adsorption capacity, fulfil an important task like effective binding surfaces for adsorption of biomolecules. Nowadays, the applications of meso and nano-structured materials in bioseparation have been widely scaled up [13]. Mesoporous silica nanoparticles due to their outstanding features such as relatively cheapness, non-toxicity and biodegradability compared to those are utilized in the preparation are unique nominees in biomedical applications such as gene delivery, controlled release, biosensors, etc. [14–17].
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Supermacroporous cryogels having three-dimensional interconnected compatible system with great mass flow have good chemical and mechanical stability. Upgrading the binding capacities of cryogels takes more and more attention in biotechnology and separation science [18]. Supermacroporous cryogels constructed with micro and nano-sized particles by embedding process are newly promising medium, and have been used extensively in separation science [19–23]. Because particleembedded supermacroporous cryogels are bearing superiority features of two sorbents such as particles (i.e., large surface area for high ligand density) and cryogels (i.e., large pores, short diffusion path, low pressure drop, etc.) to overpower of high viscosity and lower diffusion coefficients of DNA mixture [24,25]. Here, silica-based pore-expanded SBA-15 nanoparticles [26,27] (PE SBA-15) were synthesized and functionalized with aminopropyl-trimethoxysilane (APTMS). Then, functionalized particles (PE SBA-15-APTMS) were decorated with Fe+3 ions and named as (PE SBA-15-APTMS/Fe+3). Fe+3-attached particles were used to get hybrid column with embedding process. Prepared hybrid column (PE SBA-15-APTMS/Fe+3 EHC) was evaluated with regard to adsorption characteristics and interaction parameters for genomic and plasmid DNA (gDNA and pDNA). 2. Experimental 2.1. Chemicals pMAD a kind of pDNA which was isolated from E. coli DH5a strains grown in Luria-Bertani (LB) medium including ampicillin (100 lg/mL) was provided by Aksaray University, Molecular Biology Laboratory (Aksaray, Turkey). All chemicals used were purchased from Sigma (St. Louis, MO, USA). 2.2. Methods 2.2.1. Synthesis of pore-expanded SBA-15 Pore-expanded SBA 15 (PE SBA-15) was synthesized by modifying the original procedure by Prof. Stucky for conventional SBA-15 [28]. Namely, 1,3,5-triisopropylbenzene (TIPB, 95%, Sigma-Aldrich) was used as a swelling agent and NH4F (99.99%, Sigma-Aldrich) as a solubility enhancer, following the procedure described by Cao et al. [29]. A P123:TIPB ratio of 2.4:1 was employed in order to preserve mesoscopic structure at the same time that pore diameter is increased. Also, a hydrolysis temperature of 17 °C instead of 40 °C was used. The surfactant was removed from the mesostructured support by ethanol-extraction for 24 h and the obtained material was named PE SBA-15. PE-SBA-15 was modified by grafting with aminopropyltrimethoxysilane (APTMS). 1 g silica was dispersed in 250 mL toluene and subsequently, 6.89 mmol APTMS/g silica was added. The resulting mixture was refluxed for 24 h. The solid obtained was filtered under vacuum, and the excess of APTMS was removed by washing with toluene. The material obtained was denoted PE SBA-15-APTMS. 2.2.2. Incorporation of Fe+3 ions to PE SBA-15-APTMS Incorporation of Fe+3 ions to PE SBA-15-APTMS was conducted in 150 ppm Fe+3 solution at pH 4.7 (adjusted with 0.01 M HCl) at room temperature. The attachment reaction was carried out for 1 h, and the concentration of Fe+3 ions in the media was monitored by inductively coupled plasma optical emission spectroscopy (ICPOES) with a Varian Vista AX apparatus. Fe+3 ions attached to PE SBA-15-APTMS was calculated by equation (Eq. (1)).
Q ¼ ðCi Cf ÞV=m
ð1Þ
Here, Ci and Cf represent initial and final Fe+3 concentrations, respectively (mg/mL); of the ions in the solution before and after incorporation, respectively (mg/mL); Q symbolizes attached Fe+3 amount onto the PE SBA-15-APTMS (mg/g); V is the solution capacity (mL) and m is amount of PE SBA-15-APTMS (g). After the attachment of Fe+3 ions, the sample obtained was named as PE SBA-15-APTMS/Fe+3. Fe+3 leakage studies from PE SBA-15-APTMS/Fe+3 were also conducted in various media with different pHs (4.0–7.0) containing 1.0 M NaCl. For this aim, PE SBA-15-APTMS/Fe+3 were stirred in the mentioned media at room temperature for 24 h. The Fe+3 concentration in the solution was calculated by ICP-OES. 2.2.3. Organization of PE SBA-15-APTMS/Fe+3 embedded column Here, 1.75 mL of 2-hydroxyethyl methacrylate (HEMA) and 40 mg of N,N0 -methylene-bis acrylamide (MBAm) used as main monomer and crosslinker, respectively were mixed with 2.2 mL of deionized water. Then, this mixture was blended with 45 mg of PE SBA-15-APTMS/Fe+3. Obtained mixture was poured into a plastic syringe with 0.8 cm i.d., and last volume was filled up to 5 mL with deionized water. After addition of 100 lL (10% (w/v) APS and 20 lL TEMED used as free radical producer and catalyst, respectively, the final mixture was incubated at 12 °C for 24 h. Later, syringe was taken to room temperature, and water was used several times for cleaning the column to remove contamination after thawing. The obtained column named as Fe+3 incorporated APTMS attached PE SBA-15 embedded hybrid column (PE SBA15-APTMS/Fe+3 EHC) was kept in buffer including 0.02% sodium azide at 4 °C until usage. 2.3. Characterization techniques 2.3.1. Physico-chemical characterization PE-SBA-15 large scale ordering was assessed by means of low angle X-ray diffractograms acquired in a powder Philips X’PertMPD diffractometer using a CuKa monochromatic radiation. Nitrogen adsorption-desorption isotherms at 77 K were carried out in a Micrometrics TRISTAR-3000 equipment. Surface area was obtained by fitting isotherm data points between 0.05 and 0.2 to the BET equation, pore diameter by using the B.J.H. model applied to cylindrical pores in the adsorption branch of the isotherms, and pore volume at a relative pressure of 0.97 [30]. The organic nitrogen loaded by grafting was measured in a Flash 2000 elemental analyzer equipped with a thermal conductivity detector. Scanning Electron Micrography (SEM) was used to observe the internal structure of the samples prepared. A Hitachi TM-1000 apparatus with a tungsten filament and a 15 kV accelerating voltage was used. Besides, semi-quantitative elemental analyses were performed with an EDX (OXFORD) detector.
2.4. Adsorption-desorption studies of DNA from aqueous solutions Evaluation of PE SBA-15-APTMS/Fe+3 EHC regarding to adsorption capacity for gDNA and pDNA was carried out in different media. For this aim, effects of concentration, pH, ionic strength, flow rate, and temperature were investigated. A peristaltic pump (ALITEA, Sweden) was used in experiments to send the solutions into the column. Amount of DNA in media before and after adsorption was red by UV spectrophotometer (Shimadzu, Tokyo, Japan, Model 1601) at 260 nm. All elution steps were carried out only in 20 min by applying 0.05 M phosphate buffer (pH 8) including 1 M NaCl at a flow rate of 1.0 mL/min. After all adsorption cycles, the cryogel column was cleaned up with water and equilibrated with operational buffer for the next process.
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2.5. Purification of E. coli plasmid DNA Two types of pDNA having two different molecular weight such as pMAD (9666 bp) and pUC18 (2686 bp) were grown in E.coli cells in different cultures to determine the efficiency of PE SBA-15APTMS/Fe+3 EHC. For purification process of pDNA samples, 1.5 mL of E.coli cells in LB (Laura Bertani) media was centrifuged. After removing of supernatants, the precipitates were resuspended in 250 lL GTE (Glucose/Tris/EDTA) solutions, and left at room temperature for 5 min. Then, cell lysis was done by adding 500 lL of sodium hydroxide and sodium dodecyl sulfate (NaOH/SDS) solutions. After lysis in alkaline conditions, obtained lysates were resuspended in 10 mL of acetate buffers at pH 6 for use in IMAC column. In order to determine the adsorption efficiency of PE SBA-15-APTMS/Fe+3 EHC for pDNA, 10 mL of obtained two kinds of pDNA having different molecular weights were passed through PE SBA-15-APTMS/Fe+3 EHC, separately. After washing stage with 10 mL of acetate buffer, elution of column was carried out by applying 0.05 M phosphate buffer (pH 8) including 1 M NaCl at a flow rate of 1.0 mL/min for 10 min. Obtained eluents were loaded on 0.8% of agarose gel and undergone to electrophoresis treatment at a constant electric field. The gel was run at 100 V for 30 min, and then obtained images were photographed. 3. Results and discussion 3.1. Characterization of PE SBA and PE SBA-15-APTMS X-ray powder diffraction spectra acquired for siliceous PE SBA15 and amine-containing PE SBA-15-APTMS are presented in Fig. 1. Siliceous PE SBA-15 showed the three characteristic peaks of p6mm bidimensional hexagonal structure. These peaks have been previously described for pore-expanded SBA [31], and are analogous to those reported for conventional SBA [32]. However, X-ray diffraction peaks for pore-expanded materials show a shift to lower angles due to the larger cell parameters (a0) of these materials (13.9 nm in this case) compared to conventional SBA supports (12.0 nm). Regarding the intensity of diffraction peaks, it is lower for PE SBA-15 than for conventional SBA materials and the high angle peaks corresponding to planes (1 1 0) and (2 0 0) were not as clear, likely due to a slight loss of the mesostructure during the pore-expansion process.
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The diffraction spectra of PE SBA-15-APTMS sample showed a similar pattern but with a lower intensity. This change is due to the grafting process, since it yielded a certain pore filling with organic moieties, thus decreasing X-ray diffraction inside the pores. Nevertheless, it can be observed that the hexagonal structure of PE SBA-15 was not modified after the grafting step. Fig. 2 shows the N2 adsorption-desorption isotherms obtained for PE-SBA-15 siliceous material and amine-containing PE SBA15-APTMS while Table 1 lists the main textural properties of these samples. Both isotherms can be categorized as type IV according to the IUPAC classification [30]. The steep increase in the amount of nitrogen adsorbed at a relative pressure around 0.80 can be ascribed to the presence of a narrow and wide mesoporosity. As seen in Table 1 summarizing PE SBA-15 textural properties, this sample showed a BET surface of 452 m2/g, a mean pore diameter of 11.7 nm and a total pore volume of 1.02 cm3/g. These values represent a much higher pore diameter for pore-expanded SBA-15 compared to the conventional silica (ca. 9 nm) though this increase was achieved at the expense of a reduction in the specific surface, which is typically between 500 and 700 m2/g for conventional SBA [28,33]. As a result of the grafting process and the subsequent loading of the porous structure with amines, the remaining volume inside PE SBA-15-APTMS was lower than the observed for PE SBA-15, thus adsorbing a smaller amount of nitrogen (see Fig. 2). Consequently, all textural properties were also lower than those corresponding to PE SBA-15 silica, though not reaching the saturation of the porous structure. The cryogels prepared with PE SBA-15-APTMS/Fe+3 were analysed by Scanning Electron Microscopy (SEM) with images being shown in Fig. 3. The layout of silica particles in the polymer matrix can be observed in Fig. 3a and b. As seen, the cryogel polymer is providing a structural support with silica particles being deposited on top of it. This arrangement is coherent with the independent synthesis of PE SBA-15-APTMS/Fe+3 and its subsequent deposition on the cryogel. Fig. 3c allows a closer study of PE SBA-15-APTMS/ Fe+3 material formed by particles of ca. 1 lm arranged in their characteristic chain-like array [29,34]. This fact supports the idea that silica particles were no altered by their immobilization inside the cryogel structure. Fe+3 ions attached to particles was determined to be 500 mg/g NP. Some studies were performed to see if there is any ion leakage, and no leakage was determined. This result confirmed that washing treatment was satisfying. 3.2. Adsorption studies for DNA to PE SBA-15-APTMS/Fe+3 EHC 3.2.1. Effect of pH It is important to identify the optimal circumstances for an adsorbent regarding adsorption parameters. Adsorption of DNA on IMAC based adsorbents is principally built up on chelation of metal ions with oxygen groups on phosphate structure of DNA. As seen in Fig. 4A, maximum adsorbed DNA was observed at pH 6.0 (0.05 M acetate buffer). Adsorption capacity of DNA decreased before and after pH 6.0. DNA has an isoelectric point of around pH 5 [35]. It is possible to interpret this situation as follows: After pH 6, Fe+3 ions on SBA-15 may be hydroxylated and therefore, a repulsion may take place between negatively charged DNA and adsorbent surface. On the other hand, at lower pHs than 6, because of protonation of DNA, a repulsion can occur between positively charged DNA and Fe+3 ions, as well [36]. A putative representation for this scenario is given in Fig. 5.
Fig. 1. X-ray powder diffraction spectra for PE-SBA and PE-SBA-AP.
3.2.2. Effect of medium DNA concentration DNA adsorption performance of PE SBA-15-APTMS/Fe+3 EHC was checked with different DNA concentration range of 0.25–
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Fig. 2. a) N2 adsorption-desorption isotherms and b) pore size distribution of siliceous corresponding to for PE-SBA and PE-SBA-AP.
Table 1 Textural properties of pore-expanded SBA-15 before and after being modified with APTMS. Sample
SBET (m2/g)
DP (nm)
VP (cm3/g)
d100 (nm)
a0 (nm)
e (nm)
PE SBA-15 PE SBA-15-APTMS
452 158
11.7 9.2
1.02 0.38
12.0 –
13.9 –
2.2 –
3.0 mg/mL and results were shown in Fig. 4B. Formation of hydrogen bonds, dehydration and electrostatic effects are main parameters playing role in DNA adsorption [37]. Usage of metal ions as ligand both reduces electrostatic repulsion between surface and DNA molecules and enhances the adsorption capacity. At the same time, binding of metal ions to DNA molecules supress the surface charge intensity of DNA which results in higher diffusion of DNA into surface pores and higher adsorption [38]. When compared to our previous studies [1,39], we gave priority to Fe+3 ions as ligand for DNA immobilization because of affinity of it for phosphate structure [40]. Adsorbed DNA molecules on PE SBA-15-APTMS/ Fe+3 EHC by Fe+3 ions has a remarkable degree (up to 751.5 mg/g NP). As seen, the amount of DNA coupled to cryogel nearly caught a plateau in a concentration of 2.0 mg/mL as a consequence of feeding of the binding area. It is noted that non-specific DNA adsorption on Fe+3 minus PE SBA-15-APTMS EHC was came true as 55.1 mg/g. There are plenty of reports in the literature being offered by considering the adsorption capacities. A few of them are given in Table 2. It is obviously seen here that when compared with adsorbent shape and surface functionalities, the result of our study is considerably high in the view of adsorption capacity. This result may be explained with embedded nanosized particles having extensive surface area and short diffusion path and low pressure drop of cryogel column. 3.2.3. Effect of flow rate Flow rate is another significant parameter which has influence on protein adsorption in column performance. Experiment conditions implemented in the range of 0.5 and 3.0 mL/min showed that efficient flow rate was 0.5 mL/min. By reducing the flow rate, contact time between protein and ligand spends long time. Thus, adsorption of DNA molecules to pore walls of cryogels assured high adsorption capacity. 3.2.4. Effect of ionic strength An adsorption process is mainly affected from ionic strength in media. To test this effect on DNA adsorption onto PE SBA-15APTMS/Fe+3 EHC, some experiments were accomplished with DNA solutions including NaCl in the range of 0–3.0 M (Fig. 6A). Based on results of ionic strength experiments, it can make inferences that increasing of ionic strength was concluded in low
adsorption. When salt ions are solved in an adsorption media, different kinds of ions are piled up onto surface with opposite charges, and prevent the adsorption of biomolecules. This case ends up with lower adsorption [18]. 3.2.5. Effect of temperature As for the temperature effect on DNA adsorption, this parameter was investigated between the temperatures of 5–35 °C and the highest DNA adsorption was observed at 25 °C. There are different types of interactions such as electrostatic, hydrophobic and/or coordination between metal ion and biomolecule in metal affinity chromatography, and dominant one depends on main functional groups (i.e. carboxyl, sulfhydryl, amine, etc.) interacting with metal ion, adjacent groups (i.e., aromatic rings) on biomolecule and metal ion. Determination of relative dominant interaction is not easy every time [40,41]. As seen in Fig. 6B, DNA adsorption increased up to 25 °C first, and then decreased. This situation may be explained by dominance of ionic interaction after 25 °C. 3.2.6. Column efficiency for pDNA samples from bacterial lysates In order to test the adsorption efficiencies of pDNA from E. coli lysate samples having different molecular weights to PE SBA-15APTMS/Fe+3 EHC, desorption solutions were run on agarose gel electrophoresis, and obtained lanes are given in Fig. 7. Here, lane 1 shows a marker having different molecular weights up to 12,000 bp. Lanes 2–4 and lanes 5–7 present the adsorption performance of pUC18 (2686 bp) and pMAD (9666 bp) plasmid samples from E. coli lysates, respectively. As seen especially from lanes 4 and 7 that adsorption and elution process of pDNA samples were carried out, successfully. According to these results, it can be said that prepared PE SBA-15-APTMS/Fe+3 EHC shows a satisfactory result for purification of pDNA samples from cell lysate. 3.2.7. Reusability studies We used the same column during all studies about 30 times without losing its adsorption performance to determine the reusability factors. For the desorption of DNA molecules from the PE SBA-15-APTMS/Fe+3 EHC, when 1 M NaCl solution was used, it wasn’t seen any significant desorption in the media. This situation may be explained with interaction strength between phosphate groups of DNA and Fe+3 ions used as ligand [40]. After using of pH 8 phosphate buffer including 1 M NaCl, approximately all of
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Fig. 4. pH effect on DNA adsorption onto PE-SBA-AP-Fe+3 EHC, amount of particle: 15 mg; DNA concentration: 1 mg/mL; flow rate: 1 mL/min; T: 25 °C (A); DNA concentration effect on adsorption onto PE-SBA-AP-Fe+3 EHC, amount of particle: 15 mg; pH 6.0; flow rate: 1 mL/min; T: 25 °C (B).
Ce Ce Kd ¼ þ Qe Qm Qm
ð3Þ
Here, Ce/Qe versus Ce are plotted. 1/Qm and Kd/Qm values can be obtained from the slope and intercept of the straight line, respectively.
ln Q e ¼
Fig. 3. Scanning electron micrographs of cryogel containing PE-SBA-AP-Fe+3.
the adsorbed DNA molecules were desorbed from PE SBA-15APTMS/Fe+3 EHC. At the end of about 30 cycles, a decreasing of only 2% was seen in adsorption capacity.
1 ln C e þ ln K F n
ð4Þ
As for Freundlich model, when lnQe versus lnCe are plotted, 1/n and Kf values are provided from the slope and intercept of the straight line, respectively. From two equations, while Ce (mg/mL) and Qe (mg/g) show adsorbed molecule concentration in equilibrium and amount of adsorbed molecules per unit weight of sorbent, Qm and Kf indicates the maximum adsorption capacity of adsorbent (mg/g) according to Langmuir and Freundlich, respectively. Also, Kd and n show dissociation constant and heterogeneity, respectively. The values calculated from Langmuir and Freundlich isotherm equations are presented in Table 3. As shown here, correlation coefficient (R2) of Langmuir equation is closer to 1. Therefore, it can be inferred from this result that adsorption of DNA molecules on PE SBA-15-APTMS/Fe+3 EHC actualized at monolayer and uniformly.
4. Conclusions 3.3. Investigation of adsorption models In order to characterize the binding kinetic parameters of DNA adsorption on PE SBA-15-APTMS/Fe+3 EHC, two adsorption models such as Langmuir [42] and Freundlich [43]] having formula shown in Eq. (3) and Eq. (4), respectively were reviewed.
According to our knowledge, such a study for the purification of plasmid and genomic DNA has been given comprehensively together in one paper for the first time. Here, aminopropyltrimethoxysilane immobilized silica-based pore-expanded SBA15 nanoparticles having large surface area were prepared. After
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Fig. 5. Putative showing of electron charge shift on IMAC sorbent by pH..
Table 2 Comparison of maximum DNA adsorption capacities of some sorbents in the literature. Sorbent
Adsorption capacity (mg/g)
Ref.
Cibacron Blue 3GA Cu2+-attached magnetite nanoparticles Zirconia magnetic nanocomposite Silanized polymeric nanoparticles Fe+3-attached sporopollenin particles Poly(vinyl alcohol) nanofibrous membranes Ethylene-vinyl alcohol nanofibrous membranes with citric acid PE-SBA-AP-Fe+3 EHC column
32.5 mg/g 19.97 mg/g 53.5 mg/g 672.41 mg/g 109 mg/g 177 mg/g 284 mg/g
[45] [46] [47] [48] [49] [50] [51]
751.5 mg/g
In this study
Fig. 7. Agarose gel (0.8%) electrophoresis of pDNA from E. coli cells stained with ethidium bromide. Lane 1: Ladder (12,000 bp); lane 2: pUC18 plasmid (2686 bp) before adsorption; lane 3: pUC18 after adsorption; lane 4: pUC18 desorption; lane 5: pMAD plasmid (9666 bp) before adsorption; lane 6: pMAD after adsorption; lane 7 pMAD desorption..
Table 3 The parameters obtained from adsorption isotherms. Adsorption isotherm models Ce Qe
¼
Ce Qm
þ
Kd Qm
Langmuir model
lnQ e ¼ 1n lnC e þ lnK F Freundlich model
Fig. 6. Ionic strength effect on DNA adsorption onto PE-SBA-AP-Fe+3 EHC, amount of particle: 15 mg; pH 6.0; DNA concentration: 1 mg/mL; flow rate: 1 mL/min; T: 25 °C (A); effect of temperature on DNA adsorption onto PE-SBA-AP-Fe+3 EHC, amount of particle: 15 mg; pH 6.0 initial DNA concentration: 1 mg/mL; flow rate: 1 mL/min (B).
decorating with Fe+3 ions, they were embedded into cryogel column to test the efficiency of this column for DNA molecules having different molecular weight such as genomic and plasmid DNA samples. gDNA molecules having high molecular weight compared to
Adsorption parameters
Isotherm values
Qm Kd R2 Equation (Y=) Kf n R2 Equation (Y=)
769.2 (mg/g) 0.0692 (mg/mL) 0.999 0.0013x + 9.105 664.6 (mg/g) 4.46 0.9113 0.233x + 6.5202
pDNA passed through easily from IMAC cryogel column owning a supermacroporous texture, which prevents diffusion limitation especially for viscous solutions including molecules with high molecular weight [44]. Prepared column reached to a high adsorption level (751.5 mg/g NP for gDNA) because of embedded nanoparticles possessing high surface area. Prepared IMAC column was also tested for pDNA samples having different molecular weight from E. coli lysates. The specificity of this column to DNA molecules was demonstrated through pDNA samples, and results were given by agarose gel electrophoresis. In epitome, it can be made inferences from these results that prepared IMAC column
B. Önal et al. / International Journal of Biological Macromolecules 141 (2019) 1183–1190
(i.e. PE SBA-15-APTMS/Fe+3 EHC) may be used successfully for nucleic acid separation having both high and low molecular weight.
[23]
[24]
Declaration of competing interest On behalf of all authors, the corresponding author states that there is no conflict of interest.
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