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DNA adsorption onto calcium aluminate and silicate glass surfaces
Colloids and Surfaces B: Biointerfaces 117 (2014) 538–544
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DNA adsorption onto calcium aluminate and silicate glass surfaces Krista Carlson a,b,∗ , Lisa Flick c,1 , Matthew Hall b,2 a
Alfred University, 2 Pine St., Alfred 14802, NY, USA University of Utah, 201 Presidents Cir, Salt Lake City 84112, UT, USA c Monroe Community College, 1000 East Henrietta Rd, Rochester 14623, NY, USA b
a r t i c l e
i n f o
Article history: Received 10 April 2013 Received in revised form 4 November 2013 Accepted 10 November 2013 Available online 19 November 2013 Keywords: DNA adsorption Nucleic acid isolation Calcium aluminate glass Silicate glass
a b s t r a c t A common technique for small-scale isolation of genomic DNA is via adsorption of the DNA molecules onto a silica scaffold. In this work, the isolation capacities of calcium aluminate based glasses were compared against a commercially available silica scaffold. Silica scaffolds exhibit a negative surface at the physiological pH values used during DNA isolation (pH 5–9), while the calcium aluminate glass microspheres exhibit a positive surface charge. Isolation data demonstrates that the positively charged surface enhanced DNA adsorption over the negatively charged surface. DNA was eluted from the calcium aluminate surface by shifting the pH of the solution to above its IEP at pH 8. Iron additions to the calcium aluminate glass improved the chemical durability without compromising the surface charge. Morphology of the glass substrate was also found to affect DNA isolation; 43–106 m diameter soda lime silicate microspheres adsorbed a greater quantity of genomic DNA than silica fibers with an average diameter of ∼2 m. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The isolation of genomic DNA from cells is important in a wide range of applications ranging from genetic research to the identification of a crime suspect or a physically unidentifiable victim [1,2]. Commonly used rapid isolation methods often involve chemicals that can lead to DNA degradation and are toxic to both humans and the environment [2–5]. One of the most safe and effective small-scale isolation techniques involves the creation of electrostatic interactions between the negatively charged DNA phosphate backbone and a silica scaffold [6–15]. Isolation is typically performed within the physiological range between pH 5–9 [16]. As both the DNA and the silica substrate exhibit a negative surface charge within this range, the addition of agents that shield this negative charge are essential for electrostatic interactions to occur. Common methods to reduce electrostatic repulsion include varying the pH of the buffer solution and the electrolyte type and concentration [11–13]. Chaotropic agents are used in commercially available, smallscale silica-membrane DNA extraction technologies, such as the DNeasy kit available from Qiagen [17]. In this isolation technique,
∗ Corresponding author. Tel.: +1607 871 2486; fax: +1607 871 2354. E-mail addresses: [email protected] (K. Carlson), lfl[email protected] (L. Flick), [email protected] (M. Hall). 1 Tel.: 585 292 2721. 2 Tel.: 607 871 2486. 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.014
the chaotropic agent guanidine hydrochloride (GuHCl) is added to the initial buffer solution to enhance entrapment of the genomic DNA strands within the silica fibers. At the physiological pH of the buffer solution, GuHCl is protonated and encourages DNA adsorption to the negatively charged silicate surface through hydrogen bonds formed by its protonated amine groups [18]. GuHCl also disrupts water molecules associated with the DNA backbone, that could potentially hinder the ability of GuHCl to interact with positively charged surfaces [19]. However, problems arise when residual GuHCl remains after purification, interfering with downstream applications such as polymerase chain reaction (PCR) [11]. Cations of inorganic salts are commonly used instead of chaotropic agents to shield and/or bridge negatively charged species [11–15]. Monovalent cations (i.e. Na+ and K+ ) tend to shield and stabilize the negatively charged phosphate groups through the formation of weak ionic or electrostatic bonds, as they do not directly interact with the DNA backbone. Divalent cations, however, form both indirect and/or direct covalent bonds with either the negatively charged DNA backbone or silanol groups present on the surface of the silica scaffold. Nguyen et al. [12] reported the low ionic strength divalent cations (1 mM Ca2+ ) provided greater attachment efficiencies between pH 6–8 than high ionic strength solutions containing monovalent cations (300 mM NaCl); however, adsorption in both cases was irreversible. Irreversibility in 1 mM Ca2+ was attributed to specific binding between the phosphate backbone and the silica surface, appearing as a rigid plasmid adsorption layer. While no explanation was provided for the adsorption irreversibility in 300 mM NaCl, it is possible
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that the compaction of the electrical double layer in such a high ionic strength solution led to specific adsorption of the plasmid DNA. An alternative method to modifying the buffer solution is to manipulate the charge of the underlying scaffold material [19,20]. Jiang et al. examined the adsorption and elution efficiency of Fe3+ modified silica particles for salmon sperm DNA extraction [20]. The addition of these positive charges led to increased adsorption efficiency at pH 4, close to the pH 3.5 isoelectric point (IEP) of the modified scaffold. However, to get similar efficiency at pH 7, it was necessary to increase the NaCl concentration from 100 mM to 400 mM. An optimal scaffold material would exhibit a positive charge during adsorption and a negative charge during elution, within the physiological pH range where the DNA chain is the most stable [16]. Aluminum oxide membranes (AOM) are an example of a scaffold material that exhibits a positive surface charge until pH 9 [21]. These nanostructure surfaces can easily be electrochemically formed with pore sizes between 10–200 nm. However, limitations to these membranes stem from their small pore size, brittle nature, and potentially costly and difficult scalability. The novelty of the work presented here lies in the creation of glass scaffold materials with both inherently positive and negative surface charge within the physiological pH range. The surface charge of an oxide in an aqueous solution is dependent upon the pH and ions present in solution, as well as the electronegativity of the oxide cations and their coordination in the material [22–24]. Calcium aluminate based glasses were studied as potential scaffold materials as they exhibit a positively charged surface until pH 8 [25]. Experimental results indicate that the natural electrostatic attraction between the DNA and the scaffold led to increased adsorption capacity. Genomic DNA was eluted from the glass during short incubation times in a buffer solution with a pH value >8. Glasses were examined instead of their crystalline counterparts due to their flexibility in both chemical composition and form [26,27].
2. Materials and methods 2.1. Scaffold material preparation and characterization Calcium aluminate glasses were formed in the lab, and soda lime silicate (SLS) glass was obtained from Corning petri dishes. The melt time for the calcium aluminate glass containing iron (CAFe) was longer than the pure calcium aluminate (CA) glass as it was necessary for complete homogenization of the melt. Microspheres were formed by dropping glass frit into an oxygen–propane flame at 1900 ◦ C via a vibrating spatula. The flame was kept under oxidizing conditions to prevent the formation of a carbon coating. The irregularly shaped particles melted and became spherical as they traveled through the flame, eventually landing in a collection chamber. The unannealed microspheres were stored in sealed plastic containers in ambient atmosphere. Images of the scaffold were collected using an FEI environmental scanning electron microscope (ESEM), and the surface areas were obtained using a Micromeritics Tristar gas adsorption analyzer. A PerkinElmer inductively coupled plasma-optical emission spectrometer (ICP-OES) was used to determine the composition of the calcium aluminate glass microspheres. ICP-OES samples were prepared by placing 80 l of the centrifuged solution into a mixture of 1 ml de-ionized water and a drop of concentrated nitric acid to inhibit any potential precipitation of the dissolved species. The solutions were analyzed for all of the cations present in the various calcium aluminate compositions. The surface charge of the scaffold were measured using an Anton Paar electrokinetic analyzer and are reported elsewhere [25].
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2.2. DNA adsorption and isolation from cells A 10 mg/ml calf thymus DNA solution from Invitrogen was diluted to a 0.01 mg/ml concentration in a 30 mM (0.35 wt.%) Trizma hydrochloride (Tris) buffer solution from Sigma (pH 7.4). The DNA concentration was based on adsorption isotherms examining DNA concentrations between 0.005–0.1 mg/ml. A 0.01 mg/ml concentration was used as it allowed for the most complete and reproducible elution values. The average size of the DNA strands was <2000 bp. Diluted solutions were stored at –20 ◦ C until use, when a desired amount was extracted and allowed to equilibrate at 23 ◦ C before adsorption experiments were performed. The glass microspheres were weighed out to 0.02 ± 0.0003 g and placed in centrifuge tubes (Pall Life Science) containing filters with a pore size of 0.45 m. The CA scaffold was rinsed with 100 l of the 30 mM Tris buffer for 15 min and then centrifuged at 6000 × g for 2 min. The remaining scaffold were not rinsed or rinsed for 1 min, followed by the same centrifuging procedure. The scaffolds were incubated with 100 l of the 0.01 mg/ml DNA solution for 1 min and then centrifuged at 6000 × g for 2 min. DNA elution was performed by incubating the microspheres in 100 l of the Qiagen AE elution buffer (10 mM Tris, 0.5 mM EDTA, pH 9) for 1 min. No agitation was performed during rinsing or elution. DNA isolation from mouse leukaemic monocyte macrophage cells (RAW264.7 cell line, American type tissue culture collection) was performed using the protocol and buffer solutions provided by Qiagen [17]. The initial wash buffers contained the chaotropic agent guanidine hydrochloride. The microsphere scaffold was kept at 0.02 g as this size allowed for consistent and complete removal of the cell debris and surfactant-containing lysis solution. 2.3. DNA quantification and characterization The concentration of DNA adsorbed onto the microspheres was quantified using a nanodrop spectrometer (Thermo Scientific). Quantification of the concentration of DNA was calculated using the Beer–Lambert law: C=
(A × ε) l
(1)
where C is the concentration of nucleic acids in solution (ng/l), A is the measured absorbance (AU), l is the path length of the measured solution (cm), and ε is the extinction coefficient of DNA (50 ng cm l−1 ). Absorption spectra were collected from 200 to 330 nm to examine the UV absorption band of the DNA base pairs at 260 nm, with salt absorption visible at 230 nm and any insoluble light scattering components visible at 320 nm [17]. A circular dichroism (CD) spectrophotometer (AVIV) was used to examine the conformation of 1 mg/ml calf thymus DNA in 30 mM Tris before and elution from the calcium aluminate and SLS microspheres. Solutions containing a mixture of leached ions from the CA scaffold and the DNA were also measured. Calf thymus DNA is in B-form and exhibits a CD spectrum with adsorption minima and maxima of similar magnitudes at ∼248 and ∼278 nm, respectively. Molar circular dichroism (ε) was reported from 200 to 330 nm using a silica cylindrical cell with a 0.1 mm path length. 3. Results and discussion 3.1. Scaffold characterization Compositions of the microspheres are listed in Table 1. The soda lime silicate (SLS) glass composition was estimated based on the common application of its frit source [26,27]. Energy dispersive spectroscopy (EDS) showed these fibers to be primarily composed of silica with trace amounts of sodium, aluminum, and potassium.
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Table 1 Compositions of the glass microspheres. All ICP-OES determined compositions have an error of <0.1 mol%. Name
CA CAFe SLSa a
Composition (mol%)
Melt time (h)
Na2 O
K2 O
CaO
Al2 O3
Fe2 O3
SiO2
0.3 5.2 12–13
– 1.4 ≤0.2
59.0 28.0 11–12
40.7 55.0 ≤0.8
– 10.4 ≤0.02
– – 74–75
0.5 1.5 NA
Composition estimated from [26,27], could also include trace amounts of MgO and SiO2 .
Table 2 Microsphere and Qiagen scaffold properties. Sample
Microsphere/fiber diameter (m)
SA (cm2 )
IEPb (pH)
CA 43–106 m CAFe 14–43 m CAFe 43–106 m SLS 43–106 m Qiagen filter
67 ± 36 28 ± 14 65 ± 44 61 ± 44 ∼2
1.1 ± 0.8 2.6 + 0.3 1.1 ± 0.9 1.2 ± 0.9 280 ± 31
∼9a 8.1 8.1 3.3 2.9
a b
Zeta potentialb (mV) pH 7
pH 8
pH 9
NA 18 18 −23 −24
NA <1 <1 −25 −24
NA −18 −18 −26 −24
Estimated from [25]. Measurements performed in 10 mM KCl, error within ±2 mV [25].
Microsphere and Qiagen fiber scaffold dimensions and surface areas are listed in Table 2. CA, CAFe, and SLS glass frits between 43–106 m produced microspheres with diameters within this range. Smaller spheres from frit ≤43 m could only be produced with the calcium aluminate compositions; SLS tended to form large agglomerates or hollow microspheres >150 m. CA microspheres in this size range were not formed due to their low chemical durability in the DNA isolation experiments. Scanning electron micrographs of the various scaffolds are shown in Figs. 1–3. The CAFe frit and microspheres, shown in Fig. 1, represent the typical appearance of the calcium aluminates and SLS before and after microsphere formation. While the majority of the spheres exhibited smooth, solid surfaces, a small fraction of the microspheres from the SLS and CAFe contained holes or patches of crystallization, respectively, shown in Fig. 2. The Qiagen filter, displayed in Fig. 3, was found to be composed of a spacious mat of fibers with an average diameter of ∼2 m. Previously reported [26] IEP and zeta potentials of the SLS and calcium aluminate based microspheres in a 10 mM KCl solution are listed in Table 2. The sign and magnitude of the charge that develop on an oxide material when it is placed in an aqueous solution are dependent upon the electronegativity of the cation in the material and will vary with changes in solution pH, electrolyte compounds and concentration [22–24]. Silicon cations, the primary component of the SLS and Qiagen scaffolds, are more electronegative than the aluminum and calcium cations that compose the bulk of the CAFe scaffold. Cations with greater electronegativities will produce scaffolds with acidic surfaces. According to the Bronsted acid–base theory, acidic oxides will require a greater amount of protons to neutralize the charged surface, leading to IEPs at lower pH values. The zeta potential data, shown in Table 2, was found to agree with this theory, as the SLS and Qiagen scaffolds displayed acidic IEPs at pH 3.3 and 2.9, respectively, while the CAFe scaffold exhibited a basic IEP at pH 8.1. The IEP of the CA glass could not be determined using the electrokinetic analyzer due to its high solubility in aqueous solutions. Based on previously reported IEP values for similar glass compositions, this value is estimated to be around pH 9 [25]. The IEPs of the calcium aluminate scaffolds in the adsorption and elution buffer solutions are expected to be similar to the reported values measured in 10 mM KCl. Changes in electrolyte type and ionic strength will alter the magnitude of the zeta potential; however, as long as the counterions (in this case, negatively charged
ions) are indifferent to the scaffold, significant changes in IEP values should not be observed [22]. Out of all of the agents in the buffering solution, the chelating agent ethylenediaminetetraacetic acid (EDTA) has the greatest potential to chemically interact with positive ions on the calcium aluminate microspheres and alter the IEP. However, IEP changes during adsorption are not a concern as this agent is typically reserved for elution and in storage buffers to limit DNA interactions with di- and tri-valent cations [28–30]. The high affinity for EDTA to form complexes with cations makes it particularly desirable in elution solutions to encourage DNA detachment from the scaffold. The addition of iron to the calcium aluminate glass was essential as calcium aluminate glasses exhibit very low chemical durability in aqueous solutions. Silica can also be used as a compositional stabilizer; however, the addition of the electronegative silicon cation at concentrations required for increased chemical durability would cause a significant decrease in IEP. The presence of iron in the CAFe composition provided increased chemical durability without compromising the positive surface charge at physiological pH values. 3.2. DNA adsorption and isolation The concentration of calf thymus DNA eluted from the scaffolds, as well as the concentration of DNA isolated from the RAW264.7 cells is presented in Table 3. A student’s t-test performed on these reported means indicated that there is a statistical difference, at the 95% confidence level, between the DNA adsorption capacities of the CAFe and silica based glasses. At this same confidence level, there was no difference between the CA microspheres and the SLS and CAFe 43–106 microspheres. However, the low chemical durability of the CA, which prevented its use in the isolation Table 3 Concentration of calf thymus DNA eluted from the microspheres/fibers and DNA isolated from RAW 264.7 leukaemic monocyte macrophase cells. Sample
Concentration of calf thymus DNA (×10−3 g/cm2 )
Concentration of isolated DNA (g/cm2 )
CA 43–106 mm CAFe 14–43 m CAFe 43–106 m SLS 43–106 m Qiagen filter
7.32 ± 5.20 10.41 ± 2.25 9.01 ± 2.28 6.64 ± 1.46 0.35 ± 0.14
NA 0.80 ± 0.58 4.46 ± 1.52 3.77 ± 1.03 0.06 ± 0.01
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Fig. 1. CAFe (a) frit forms (b) microspheres once dropped through an oxygen–propane flame at 1900 ◦ C.
Fig. 2. Defects present on some of the SLS and CAFe microspheres. Flawed SLS microspheres (a) exhibited pitting, while crystallization was present on CAFe microspheres (b).
of RAW264.7, makes it difficult to draw any conclusions about adsorption capacity. Therefore, further discussion about CA will be concerned mainly with chemical durability. In the absence of chaotropic agents, the negatively charged phosphate backbone of the DNA showed greater affinity for the calcium aluminates than the silicate based scaffolds. When normalized for surface area, both size ranges of the CAFe microspheres eluted significantly greater amounts of calf thymus DNA than the SLS microspheres or the Qiagen filters. Surface charge played less role during DNA isolation from the RAW264.7 cells, as chaotropic agents were present, allowing bridging between the two negatively charged entities. The higher adsorption capacity of the SLS microspheres during nucleic acid isolation from the cells is attributed to the chaotropic agents effectively shielding the negative charge on the silicate glass surfaces. This charge shielding enabled the negatively charged SLS microspheres to isolate a greater quantity of DNA than the 14–43 m CAFe microspheres.
Although the surface charge most likely extends only a few nanometers into the solution [31], the elution data suggest that the reversal in CAFe surface charge at pH 9 is strong enough to cause DNA detachment. The zeta potential shifted from 8 mV at 7.4 (pH of AE buffer), to −18 mV at pH 9 (pH of elution buffer), while the silicate based glasses ranged from ranged from −23 to −26 mV over this range. DNA adsorption onto the CAFe became irreversible using buffer solutions comparable to the AE buffer but with ≤pH 8. Phosphate buffers were examined based on work by Colman et al. [32] on the isolation of DNA using porous columns packed with hydroxyapatite (HA) powder. A concentrated phosphate buffer solution was needed to elute the tightly bound DNA, as it had a higher affinity for the HA surface than the EDTA. Elution buffers with varying concentrations of Tris, EDTA, and sodium dihydrogen phosphate (Na2 HPO4 ) were examined with undesirable results. Higher measured elution values were mainly the result calcium and aluminum ions leached from the microspheres. This
Fig. 3. Images of (a) fibers from a Qiagen filter mat and (b) a single fiber.
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with quartz microbalance with dissipation (QCM-D) in conjunction with dynamic light scattering (DLS) could provide greater insight into the structure of the adsorbed layer in the various electrolyte solutions [11,12]. 3.3. Chemical stability of scaffolds during isolation
Fig. 4. Concentration of calcium (Ca2+ ) and aluminum (Al3+ ) ions leached from the CA, CAFe, and SLS microspheres after different incubation periods in DI water.
topic is discussed in greater detail in Section 3.3. Gel electrophoresis performed on the RAW264.7 cells also showed significant shearing in a buffer containing 30 mM Na2 HPO4 (unpublished data). Establishing how DNA tends to adsorb or become physically entrapped in the microsphere bed is important in determining why the larger microsphere sizes isolated a greater quantity of nucleic acids per surface area than the smaller CAFe microspheres or the Qiagen filter. The similarity in nucleic acid concentration between the 43–106 m CAFe and SLS microspheres suggests that, under the influence of a chaotropic agent, the major factor in nucleic acid isolation ability is substrate morphology and size. The Qiagen filter contained a mat composed of ∼2 m fibers which are much thinner than the genomic nucleic acid strands that can range from a couple microns to centimeters in length [1]. The high curvature of the fibers is unable to isolate strands ≤50,000 bp due to the shearing force generated during centrifuging [17]. The use of microspheres was anticipated to increase the adsorption ability via the reduction of strand repulsion through lower surface curvature and by their ability to physically entrap the larger strands in the tightly packed microsphere mat. Particles with a lower surface curvature might allow for more of the DNA backbone to adsorb, therefore, reducing the repulsive interaction volume of an unattached segment in solution. A lower interaction volume in solution could increase the possibility for DNA to adsorb in the surrounding region. Eluted DNA from glass filter mats and microspheres was separated by gel electrophoresis, and DNA fragments greater than 12,000 bp in length were observed in all samples. Based on the elution data, it appears that either physical entrapment or specific adsorption inhibited complete elution of the DNA. Further assessment of these surface
Silicate based glasses used for DNA isolation are pre-rinsed with acidic, aqueous solutions that remove mobile alkali or alkaline earth ions. Pre-rinsing the calcium aluminates with these solutions does not eliminate mobile ions, as the leached ions are the result of network dissolution due to aqueous attack. The calcium–aluminum network of the CAFe glass is highly susceptible to aqueous attack from dissociated water molecules [33,34]. As shown in Fig. 4, there is no protection for the calcium aluminate based glass surfaces against hydrogen ion attack in DI water. While both glasses leach ions, the CAFe has significantly less degradation as the iron stabilizes the glass network. The hydrated gel-layer formed during aqueous attack begins to morph into the hydrated calcium aluminate crystals after 30 min, as shown in Fig. 5a. Surface degradation was restricted during the adsorption/elution testing by the presence of organics and/or other compounds in solution which inhibit aqueous attack through hydrogen bonding with dissociated water molecules [33,34]. The retardation and/or inhibition of calcium aluminate hydration are highly dependent upon the number of groups an organic has available for hydrogen bonding and also its concentration in solution. Tris was thought to provide adequate resistance against aqueous attack due to the participation of its primary amine in hydrogen bonding. Fig. 6 demonstrates this ability as only 1.0 mM Ca2+ leached from the CA scaffold after 30 min in 30 mM Tris, significantly less than the 39.9 mM Ca2+ leached in DI water over the same time period. No calcium was found to have leached from CAFe or SLS microspheres, and no aluminum was detected in the buffer solutions incubated with any of the samples. An increase in leached ion concentration for the calcium aluminates after 60 min is due to the breakdown of the hydrated layer. As the protective hydrated layer detaches from the surface, a fresh calcium aluminate surface is exposed and attacked until a new hydrated layer is formed. Fig. 5b shows that the surface of the CA microspheres incubated in Tris for 30 min have some damage. Degradation is noticeable in the formation of a gel-layer where microspheres were in contact with one another. Buffers with a minimum of 30 mM Tris were found to be optimal for inhibiting degradation; however, microspheres also appeared chemically stable in the AE buffer (10 mM Tris, 0.5 mM EDTA) and in wash buffers containing the chaotropic agent GuHCl.
Fig. 5. Result of aqueous attack on CA microspheres. Hydrated calcium aluminate crystals (a) begin to form on the CA surface after 30 min in DI water. A 30 mM Tris buffer solution retards attack (b), exhibiting the majority of hydration layer degradation between the microspheres.
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Fig. 8. CD spectra of 1 mg/ml calf thymus DNA in 30 mM Tris with and without 2.5 mM Ca2+ leached from the CA microspheres.
Fig. 6. Concentration of calcium (Ca2+ ) leached from the CA, CAFe, and SLS microspheres after different incubation periods in 30 mM Tris. Aluminum ion concentrations are now shown as it remained undetectable even after a 24 h incubation period.
Tris-induced stabilization of the CAFe microspheres makes it possible to potentially use this material as a replacement for silica scaffolds. However, dissolved cations were found to alter the adsorbance values, sometimes hindering an accurate assessment of calf thymus DNA adsorption. Fig. 7 illustrates the effect of pre-rinse times on calf thymus DNA adsorption values. Without pre-rinsing both the SLS and CAFe scaffolds in ≥30 mM Tris for at least 1 min, measured UV absorbance values were higher than those measured for the original quantity of DNA in solution. The less chemically stable CA microspheres required a 15 min pre-rinse before accurate values could be obtained. This 15 min rinse time corresponds to the plateau in the leached calcium concentration shown in Fig. 5. As discussed earlier, the Tris molecule limits calcium aluminate degradation by preventing aqueous attack on the network structure. Once the Tris has stabilized the surface, leaching does not continue during the adsorption and elution process. CAFe and SLS microspheres did not leach detectable amounts of ions after a 1 min incubation with 30 mM Tris; therefore, pre-rinsing was unnecessary if only elution values were measured, as the adsorption phase acted as a pre-rinse.
No conformation change to the B-form DNA was visible after elution from any of the scaffolds. Incubating the calf thymus DNA with 2.5 mM Ca2+ , formed from incubating the CA microspheres in 30 mM Tris for 1 h, also exhibited no effect on conformation (Fig. 8). However, contamination due to broken pieces of hydrated scaffold, as well as lengthy pre-rinse times, makes the CAFe scaffold a more attractive option. 4. Conclusions Calf thymus DNA exhibited a greater affinity for the positively charged CAFe microspheres in the absence of chaotropic agents than for the negatively charged SLS and Qiagen scaffolds. CAFe 43–106 m also isolated a higher normalized concentration of RAW 264.7 cell DNA as the surface charge of the CAFe was still strong enough to electrostatically attract the DNA backbone. Shifting to basic pH values above the IEP of the material allowed DNA to detach. Scaffold morphology was also found to play a significant role in DNA isolation. In the presence of chaotropic agents, the 43–106 m SLS microspheres isolated more DNA than the 14–43 m CAFe microspheres. It is suggested that the chaotropic agents shielded the negative SLS surface enough to allow for the scaffold curvature to play a greater role in isolation. It was also demonstrated that the chemical attack on the calcium–aluminate network could be inhibited though the agents present in the buffer solutions. Through further compositional optimization, these materials have the potential to provide quick, effective, and low cost isolation of genomic DNA using buffer solutions within the physiological pH range. Acknowledgments The authors would like to acknowledge the National Science Foundation’s Graduate Research Fellowship Program for funding this research. The authors declare no competing interests. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.11.014. References
Fig. 7. Effect of different 30 mM Tris pre-rinse times on UV measured DNA calf thymus adsorption. CAFe and SLS microspheres provided accurate DNA adsorbance values after a 1 min pre-rinse. CA microspheres required a 15 min pre-rinse before leached calcium ions did not interfere with the UV absorbance measurements.
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[18] M. Garcia-Mira, J. Sanchez-Ruiz, PH corrections and protein ionization in water/guanidinium chloride, Biophys. J. 81 (2001) 3489–3502. [19] M. Winters, J. Richter, S. Sagar, A. Lee, R. Lander, Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities, Biotechnol. Prog. 19 (2003) 440–447. [20] S. Jiang, J. Zhuang, C. Wang, J. Li, W. Yang, Highly efficient adsorption of DNA on Fe3+ - iminodiacetic acid modified silica particles, Colloids Surf. A: Physiochem. Eng. Asp. 409 (2012) 143–148. [21] C. Ingham, J. Maat, W. deVos, Where bio meets nano: the many uses for nanoporous aluminum oxide in biotechnology, Biotechnol. Adv. 30 (2012) 1089–1099. [22] R. Hunter, Zeta Potential in Colloid Science: Principles and Applications, Academic Press, New York, 1981. [23] G. Parks, The isoelectric points of solid oxides, solid hydroxides, and aqueous hydroxo complex systems, Chem. Rev. 65 (1965) 177–198. [24] C. Noguera, Physics and Chemistry at Oxide Surfaces, Cambridge University Press, New York, 1996. [25] K. Carlson, M. Hall, Streaming potential measurements performed on silicate and calcium aluminate glass microspheres, Colloids Surf. A: Physicochem. Eng. Asp. 325 (2008) 101–106. [26] A. Varshneya, Fundamentals of Inorganic Glasses, Academic Press, New York, 1993. [27] J. Shelby, Introduction to Glass Science and Technology, Royal Society of Chemistry, Cambridge, 2005. [28] J. Grady, N. Chasteen, D. Harris, Radicals from good’s buffers, Anal. Biochem. 173 (1988) 111–115. [29] N. Good, S. Izawa, in: A. SanPietro (Ed.), Hydrogen Ion Buffers, 24, Academic Press, New York, 1972. [30] R. Durst, B. Staples, Tris/Tris•HCl. A standard buffer for use in the physiological pH range, Clin. Chem. 18 (1972) 206–208. [31] K. Mahanta, G. Mishra, M. Kansal, Estimation of electric double layer thickness from linearized and nonlinear solutions of Poisson–Boltzman equation for a single type of ions, Appl. Clay Sci. 59-60 (2012) 1–7. [32] A. Colman, M. Byers, S. Primrose, A. Lyons, Rapid purification of plasmid DNAs by hydroxyapatite chromatography, Biochemistry 76 (1978) 615–619. [33] F. Lea, The Chemistry of Cement and Concrete, Chemical Publishing Co., New York, 1971. [34] J. Young, Effect of organic compounds on the interconversions of calcium aluminate hydrates: hydration of tricalcium aluminate, J. Am. Ceram. Soc. 53 (1970) 65–69.
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Short communication
DNA adsorption onto calcium aluminate and silicate glass surfaces Krista Carlson a,b,∗ , Lisa Flick c,1 , Matthew Hall b,2 a
Alfred University, 2 Pine St., Alfred 14802, NY, USA University of Utah, 201 Presidents Cir, Salt Lake City 84112, UT, USA c Monroe Community College, 1000 East Henrietta Rd, Rochester 14623, NY, USA b
a r t i c l e
i n f o
Article history: Received 10 April 2013 Received in revised form 4 November 2013 Accepted 10 November 2013 Available online 19 November 2013 Keywords: DNA adsorption Nucleic acid isolation Calcium aluminate glass Silicate glass
a b s t r a c t A common technique for small-scale isolation of genomic DNA is via adsorption of the DNA molecules onto a silica scaffold. In this work, the isolation capacities of calcium aluminate based glasses were compared against a commercially available silica scaffold. Silica scaffolds exhibit a negative surface at the physiological pH values used during DNA isolation (pH 5–9), while the calcium aluminate glass microspheres exhibit a positive surface charge. Isolation data demonstrates that the positively charged surface enhanced DNA adsorption over the negatively charged surface. DNA was eluted from the calcium aluminate surface by shifting the pH of the solution to above its IEP at pH 8. Iron additions to the calcium aluminate glass improved the chemical durability without compromising the surface charge. Morphology of the glass substrate was also found to affect DNA isolation; 43–106 m diameter soda lime silicate microspheres adsorbed a greater quantity of genomic DNA than silica fibers with an average diameter of ∼2 m. © 2013 Elsevier B.V. All rights reserved.
1. Introduction The isolation of genomic DNA from cells is important in a wide range of applications ranging from genetic research to the identification of a crime suspect or a physically unidentifiable victim [1,2]. Commonly used rapid isolation methods often involve chemicals that can lead to DNA degradation and are toxic to both humans and the environment [2–5]. One of the most safe and effective small-scale isolation techniques involves the creation of electrostatic interactions between the negatively charged DNA phosphate backbone and a silica scaffold [6–15]. Isolation is typically performed within the physiological range between pH 5–9 [16]. As both the DNA and the silica substrate exhibit a negative surface charge within this range, the addition of agents that shield this negative charge are essential for electrostatic interactions to occur. Common methods to reduce electrostatic repulsion include varying the pH of the buffer solution and the electrolyte type and concentration [11–13]. Chaotropic agents are used in commercially available, smallscale silica-membrane DNA extraction technologies, such as the DNeasy kit available from Qiagen [17]. In this isolation technique,
∗ Corresponding author. Tel.: +1607 871 2486; fax: +1607 871 2354. E-mail addresses: [email protected] (K. Carlson), lfl[email protected] (L. Flick), [email protected] (M. Hall). 1 Tel.: 585 292 2721. 2 Tel.: 607 871 2486. 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.11.014
the chaotropic agent guanidine hydrochloride (GuHCl) is added to the initial buffer solution to enhance entrapment of the genomic DNA strands within the silica fibers. At the physiological pH of the buffer solution, GuHCl is protonated and encourages DNA adsorption to the negatively charged silicate surface through hydrogen bonds formed by its protonated amine groups [18]. GuHCl also disrupts water molecules associated with the DNA backbone, that could potentially hinder the ability of GuHCl to interact with positively charged surfaces [19]. However, problems arise when residual GuHCl remains after purification, interfering with downstream applications such as polymerase chain reaction (PCR) [11]. Cations of inorganic salts are commonly used instead of chaotropic agents to shield and/or bridge negatively charged species [11–15]. Monovalent cations (i.e. Na+ and K+ ) tend to shield and stabilize the negatively charged phosphate groups through the formation of weak ionic or electrostatic bonds, as they do not directly interact with the DNA backbone. Divalent cations, however, form both indirect and/or direct covalent bonds with either the negatively charged DNA backbone or silanol groups present on the surface of the silica scaffold. Nguyen et al. [12] reported the low ionic strength divalent cations (1 mM Ca2+ ) provided greater attachment efficiencies between pH 6–8 than high ionic strength solutions containing monovalent cations (300 mM NaCl); however, adsorption in both cases was irreversible. Irreversibility in 1 mM Ca2+ was attributed to specific binding between the phosphate backbone and the silica surface, appearing as a rigid plasmid adsorption layer. While no explanation was provided for the adsorption irreversibility in 300 mM NaCl, it is possible
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that the compaction of the electrical double layer in such a high ionic strength solution led to specific adsorption of the plasmid DNA. An alternative method to modifying the buffer solution is to manipulate the charge of the underlying scaffold material [19,20]. Jiang et al. examined the adsorption and elution efficiency of Fe3+ modified silica particles for salmon sperm DNA extraction [20]. The addition of these positive charges led to increased adsorption efficiency at pH 4, close to the pH 3.5 isoelectric point (IEP) of the modified scaffold. However, to get similar efficiency at pH 7, it was necessary to increase the NaCl concentration from 100 mM to 400 mM. An optimal scaffold material would exhibit a positive charge during adsorption and a negative charge during elution, within the physiological pH range where the DNA chain is the most stable [16]. Aluminum oxide membranes (AOM) are an example of a scaffold material that exhibits a positive surface charge until pH 9 [21]. These nanostructure surfaces can easily be electrochemically formed with pore sizes between 10–200 nm. However, limitations to these membranes stem from their small pore size, brittle nature, and potentially costly and difficult scalability. The novelty of the work presented here lies in the creation of glass scaffold materials with both inherently positive and negative surface charge within the physiological pH range. The surface charge of an oxide in an aqueous solution is dependent upon the pH and ions present in solution, as well as the electronegativity of the oxide cations and their coordination in the material [22–24]. Calcium aluminate based glasses were studied as potential scaffold materials as they exhibit a positively charged surface until pH 8 [25]. Experimental results indicate that the natural electrostatic attraction between the DNA and the scaffold led to increased adsorption capacity. Genomic DNA was eluted from the glass during short incubation times in a buffer solution with a pH value >8. Glasses were examined instead of their crystalline counterparts due to their flexibility in both chemical composition and form [26,27].
2. Materials and methods 2.1. Scaffold material preparation and characterization Calcium aluminate glasses were formed in the lab, and soda lime silicate (SLS) glass was obtained from Corning petri dishes. The melt time for the calcium aluminate glass containing iron (CAFe) was longer than the pure calcium aluminate (CA) glass as it was necessary for complete homogenization of the melt. Microspheres were formed by dropping glass frit into an oxygen–propane flame at 1900 ◦ C via a vibrating spatula. The flame was kept under oxidizing conditions to prevent the formation of a carbon coating. The irregularly shaped particles melted and became spherical as they traveled through the flame, eventually landing in a collection chamber. The unannealed microspheres were stored in sealed plastic containers in ambient atmosphere. Images of the scaffold were collected using an FEI environmental scanning electron microscope (ESEM), and the surface areas were obtained using a Micromeritics Tristar gas adsorption analyzer. A PerkinElmer inductively coupled plasma-optical emission spectrometer (ICP-OES) was used to determine the composition of the calcium aluminate glass microspheres. ICP-OES samples were prepared by placing 80 l of the centrifuged solution into a mixture of 1 ml de-ionized water and a drop of concentrated nitric acid to inhibit any potential precipitation of the dissolved species. The solutions were analyzed for all of the cations present in the various calcium aluminate compositions. The surface charge of the scaffold were measured using an Anton Paar electrokinetic analyzer and are reported elsewhere [25].
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2.2. DNA adsorption and isolation from cells A 10 mg/ml calf thymus DNA solution from Invitrogen was diluted to a 0.01 mg/ml concentration in a 30 mM (0.35 wt.%) Trizma hydrochloride (Tris) buffer solution from Sigma (pH 7.4). The DNA concentration was based on adsorption isotherms examining DNA concentrations between 0.005–0.1 mg/ml. A 0.01 mg/ml concentration was used as it allowed for the most complete and reproducible elution values. The average size of the DNA strands was <2000 bp. Diluted solutions were stored at –20 ◦ C until use, when a desired amount was extracted and allowed to equilibrate at 23 ◦ C before adsorption experiments were performed. The glass microspheres were weighed out to 0.02 ± 0.0003 g and placed in centrifuge tubes (Pall Life Science) containing filters with a pore size of 0.45 m. The CA scaffold was rinsed with 100 l of the 30 mM Tris buffer for 15 min and then centrifuged at 6000 × g for 2 min. The remaining scaffold were not rinsed or rinsed for 1 min, followed by the same centrifuging procedure. The scaffolds were incubated with 100 l of the 0.01 mg/ml DNA solution for 1 min and then centrifuged at 6000 × g for 2 min. DNA elution was performed by incubating the microspheres in 100 l of the Qiagen AE elution buffer (10 mM Tris, 0.5 mM EDTA, pH 9) for 1 min. No agitation was performed during rinsing or elution. DNA isolation from mouse leukaemic monocyte macrophage cells (RAW264.7 cell line, American type tissue culture collection) was performed using the protocol and buffer solutions provided by Qiagen [17]. The initial wash buffers contained the chaotropic agent guanidine hydrochloride. The microsphere scaffold was kept at 0.02 g as this size allowed for consistent and complete removal of the cell debris and surfactant-containing lysis solution. 2.3. DNA quantification and characterization The concentration of DNA adsorbed onto the microspheres was quantified using a nanodrop spectrometer (Thermo Scientific). Quantification of the concentration of DNA was calculated using the Beer–Lambert law: C=
(A × ε) l
(1)
where C is the concentration of nucleic acids in solution (ng/l), A is the measured absorbance (AU), l is the path length of the measured solution (cm), and ε is the extinction coefficient of DNA (50 ng cm l−1 ). Absorption spectra were collected from 200 to 330 nm to examine the UV absorption band of the DNA base pairs at 260 nm, with salt absorption visible at 230 nm and any insoluble light scattering components visible at 320 nm [17]. A circular dichroism (CD) spectrophotometer (AVIV) was used to examine the conformation of 1 mg/ml calf thymus DNA in 30 mM Tris before and elution from the calcium aluminate and SLS microspheres. Solutions containing a mixture of leached ions from the CA scaffold and the DNA were also measured. Calf thymus DNA is in B-form and exhibits a CD spectrum with adsorption minima and maxima of similar magnitudes at ∼248 and ∼278 nm, respectively. Molar circular dichroism (ε) was reported from 200 to 330 nm using a silica cylindrical cell with a 0.1 mm path length. 3. Results and discussion 3.1. Scaffold characterization Compositions of the microspheres are listed in Table 1. The soda lime silicate (SLS) glass composition was estimated based on the common application of its frit source [26,27]. Energy dispersive spectroscopy (EDS) showed these fibers to be primarily composed of silica with trace amounts of sodium, aluminum, and potassium.
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Table 1 Compositions of the glass microspheres. All ICP-OES determined compositions have an error of <0.1 mol%. Name
CA CAFe SLSa a
Composition (mol%)
Melt time (h)
Na2 O
K2 O
CaO
Al2 O3
Fe2 O3
SiO2
0.3 5.2 12–13
– 1.4 ≤0.2
59.0 28.0 11–12
40.7 55.0 ≤0.8
– 10.4 ≤0.02
– – 74–75
0.5 1.5 NA
Composition estimated from [26,27], could also include trace amounts of MgO and SiO2 .
Table 2 Microsphere and Qiagen scaffold properties. Sample
Microsphere/fiber diameter (m)
SA (cm2 )
IEPb (pH)
CA 43–106 m CAFe 14–43 m CAFe 43–106 m SLS 43–106 m Qiagen filter
67 ± 36 28 ± 14 65 ± 44 61 ± 44 ∼2
1.1 ± 0.8 2.6 + 0.3 1.1 ± 0.9 1.2 ± 0.9 280 ± 31
∼9a 8.1 8.1 3.3 2.9
a b
Zeta potentialb (mV) pH 7
pH 8
pH 9
NA 18 18 −23 −24
NA <1 <1 −25 −24
NA −18 −18 −26 −24
Estimated from [25]. Measurements performed in 10 mM KCl, error within ±2 mV [25].
Microsphere and Qiagen fiber scaffold dimensions and surface areas are listed in Table 2. CA, CAFe, and SLS glass frits between 43–106 m produced microspheres with diameters within this range. Smaller spheres from frit ≤43 m could only be produced with the calcium aluminate compositions; SLS tended to form large agglomerates or hollow microspheres >150 m. CA microspheres in this size range were not formed due to their low chemical durability in the DNA isolation experiments. Scanning electron micrographs of the various scaffolds are shown in Figs. 1–3. The CAFe frit and microspheres, shown in Fig. 1, represent the typical appearance of the calcium aluminates and SLS before and after microsphere formation. While the majority of the spheres exhibited smooth, solid surfaces, a small fraction of the microspheres from the SLS and CAFe contained holes or patches of crystallization, respectively, shown in Fig. 2. The Qiagen filter, displayed in Fig. 3, was found to be composed of a spacious mat of fibers with an average diameter of ∼2 m. Previously reported [26] IEP and zeta potentials of the SLS and calcium aluminate based microspheres in a 10 mM KCl solution are listed in Table 2. The sign and magnitude of the charge that develop on an oxide material when it is placed in an aqueous solution are dependent upon the electronegativity of the cation in the material and will vary with changes in solution pH, electrolyte compounds and concentration [22–24]. Silicon cations, the primary component of the SLS and Qiagen scaffolds, are more electronegative than the aluminum and calcium cations that compose the bulk of the CAFe scaffold. Cations with greater electronegativities will produce scaffolds with acidic surfaces. According to the Bronsted acid–base theory, acidic oxides will require a greater amount of protons to neutralize the charged surface, leading to IEPs at lower pH values. The zeta potential data, shown in Table 2, was found to agree with this theory, as the SLS and Qiagen scaffolds displayed acidic IEPs at pH 3.3 and 2.9, respectively, while the CAFe scaffold exhibited a basic IEP at pH 8.1. The IEP of the CA glass could not be determined using the electrokinetic analyzer due to its high solubility in aqueous solutions. Based on previously reported IEP values for similar glass compositions, this value is estimated to be around pH 9 [25]. The IEPs of the calcium aluminate scaffolds in the adsorption and elution buffer solutions are expected to be similar to the reported values measured in 10 mM KCl. Changes in electrolyte type and ionic strength will alter the magnitude of the zeta potential; however, as long as the counterions (in this case, negatively charged
ions) are indifferent to the scaffold, significant changes in IEP values should not be observed [22]. Out of all of the agents in the buffering solution, the chelating agent ethylenediaminetetraacetic acid (EDTA) has the greatest potential to chemically interact with positive ions on the calcium aluminate microspheres and alter the IEP. However, IEP changes during adsorption are not a concern as this agent is typically reserved for elution and in storage buffers to limit DNA interactions with di- and tri-valent cations [28–30]. The high affinity for EDTA to form complexes with cations makes it particularly desirable in elution solutions to encourage DNA detachment from the scaffold. The addition of iron to the calcium aluminate glass was essential as calcium aluminate glasses exhibit very low chemical durability in aqueous solutions. Silica can also be used as a compositional stabilizer; however, the addition of the electronegative silicon cation at concentrations required for increased chemical durability would cause a significant decrease in IEP. The presence of iron in the CAFe composition provided increased chemical durability without compromising the positive surface charge at physiological pH values. 3.2. DNA adsorption and isolation The concentration of calf thymus DNA eluted from the scaffolds, as well as the concentration of DNA isolated from the RAW264.7 cells is presented in Table 3. A student’s t-test performed on these reported means indicated that there is a statistical difference, at the 95% confidence level, between the DNA adsorption capacities of the CAFe and silica based glasses. At this same confidence level, there was no difference between the CA microspheres and the SLS and CAFe 43–106 microspheres. However, the low chemical durability of the CA, which prevented its use in the isolation Table 3 Concentration of calf thymus DNA eluted from the microspheres/fibers and DNA isolated from RAW 264.7 leukaemic monocyte macrophase cells. Sample
Concentration of calf thymus DNA (×10−3 g/cm2 )
Concentration of isolated DNA (g/cm2 )
CA 43–106 mm CAFe 14–43 m CAFe 43–106 m SLS 43–106 m Qiagen filter
7.32 ± 5.20 10.41 ± 2.25 9.01 ± 2.28 6.64 ± 1.46 0.35 ± 0.14
NA 0.80 ± 0.58 4.46 ± 1.52 3.77 ± 1.03 0.06 ± 0.01
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Fig. 1. CAFe (a) frit forms (b) microspheres once dropped through an oxygen–propane flame at 1900 ◦ C.
Fig. 2. Defects present on some of the SLS and CAFe microspheres. Flawed SLS microspheres (a) exhibited pitting, while crystallization was present on CAFe microspheres (b).
of RAW264.7, makes it difficult to draw any conclusions about adsorption capacity. Therefore, further discussion about CA will be concerned mainly with chemical durability. In the absence of chaotropic agents, the negatively charged phosphate backbone of the DNA showed greater affinity for the calcium aluminates than the silicate based scaffolds. When normalized for surface area, both size ranges of the CAFe microspheres eluted significantly greater amounts of calf thymus DNA than the SLS microspheres or the Qiagen filters. Surface charge played less role during DNA isolation from the RAW264.7 cells, as chaotropic agents were present, allowing bridging between the two negatively charged entities. The higher adsorption capacity of the SLS microspheres during nucleic acid isolation from the cells is attributed to the chaotropic agents effectively shielding the negative charge on the silicate glass surfaces. This charge shielding enabled the negatively charged SLS microspheres to isolate a greater quantity of DNA than the 14–43 m CAFe microspheres.
Although the surface charge most likely extends only a few nanometers into the solution [31], the elution data suggest that the reversal in CAFe surface charge at pH 9 is strong enough to cause DNA detachment. The zeta potential shifted from 8 mV at 7.4 (pH of AE buffer), to −18 mV at pH 9 (pH of elution buffer), while the silicate based glasses ranged from ranged from −23 to −26 mV over this range. DNA adsorption onto the CAFe became irreversible using buffer solutions comparable to the AE buffer but with ≤pH 8. Phosphate buffers were examined based on work by Colman et al. [32] on the isolation of DNA using porous columns packed with hydroxyapatite (HA) powder. A concentrated phosphate buffer solution was needed to elute the tightly bound DNA, as it had a higher affinity for the HA surface than the EDTA. Elution buffers with varying concentrations of Tris, EDTA, and sodium dihydrogen phosphate (Na2 HPO4 ) were examined with undesirable results. Higher measured elution values were mainly the result calcium and aluminum ions leached from the microspheres. This
Fig. 3. Images of (a) fibers from a Qiagen filter mat and (b) a single fiber.
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with quartz microbalance with dissipation (QCM-D) in conjunction with dynamic light scattering (DLS) could provide greater insight into the structure of the adsorbed layer in the various electrolyte solutions [11,12]. 3.3. Chemical stability of scaffolds during isolation
Fig. 4. Concentration of calcium (Ca2+ ) and aluminum (Al3+ ) ions leached from the CA, CAFe, and SLS microspheres after different incubation periods in DI water.
topic is discussed in greater detail in Section 3.3. Gel electrophoresis performed on the RAW264.7 cells also showed significant shearing in a buffer containing 30 mM Na2 HPO4 (unpublished data). Establishing how DNA tends to adsorb or become physically entrapped in the microsphere bed is important in determining why the larger microsphere sizes isolated a greater quantity of nucleic acids per surface area than the smaller CAFe microspheres or the Qiagen filter. The similarity in nucleic acid concentration between the 43–106 m CAFe and SLS microspheres suggests that, under the influence of a chaotropic agent, the major factor in nucleic acid isolation ability is substrate morphology and size. The Qiagen filter contained a mat composed of ∼2 m fibers which are much thinner than the genomic nucleic acid strands that can range from a couple microns to centimeters in length [1]. The high curvature of the fibers is unable to isolate strands ≤50,000 bp due to the shearing force generated during centrifuging [17]. The use of microspheres was anticipated to increase the adsorption ability via the reduction of strand repulsion through lower surface curvature and by their ability to physically entrap the larger strands in the tightly packed microsphere mat. Particles with a lower surface curvature might allow for more of the DNA backbone to adsorb, therefore, reducing the repulsive interaction volume of an unattached segment in solution. A lower interaction volume in solution could increase the possibility for DNA to adsorb in the surrounding region. Eluted DNA from glass filter mats and microspheres was separated by gel electrophoresis, and DNA fragments greater than 12,000 bp in length were observed in all samples. Based on the elution data, it appears that either physical entrapment or specific adsorption inhibited complete elution of the DNA. Further assessment of these surface
Silicate based glasses used for DNA isolation are pre-rinsed with acidic, aqueous solutions that remove mobile alkali or alkaline earth ions. Pre-rinsing the calcium aluminates with these solutions does not eliminate mobile ions, as the leached ions are the result of network dissolution due to aqueous attack. The calcium–aluminum network of the CAFe glass is highly susceptible to aqueous attack from dissociated water molecules [33,34]. As shown in Fig. 4, there is no protection for the calcium aluminate based glass surfaces against hydrogen ion attack in DI water. While both glasses leach ions, the CAFe has significantly less degradation as the iron stabilizes the glass network. The hydrated gel-layer formed during aqueous attack begins to morph into the hydrated calcium aluminate crystals after 30 min, as shown in Fig. 5a. Surface degradation was restricted during the adsorption/elution testing by the presence of organics and/or other compounds in solution which inhibit aqueous attack through hydrogen bonding with dissociated water molecules [33,34]. The retardation and/or inhibition of calcium aluminate hydration are highly dependent upon the number of groups an organic has available for hydrogen bonding and also its concentration in solution. Tris was thought to provide adequate resistance against aqueous attack due to the participation of its primary amine in hydrogen bonding. Fig. 6 demonstrates this ability as only 1.0 mM Ca2+ leached from the CA scaffold after 30 min in 30 mM Tris, significantly less than the 39.9 mM Ca2+ leached in DI water over the same time period. No calcium was found to have leached from CAFe or SLS microspheres, and no aluminum was detected in the buffer solutions incubated with any of the samples. An increase in leached ion concentration for the calcium aluminates after 60 min is due to the breakdown of the hydrated layer. As the protective hydrated layer detaches from the surface, a fresh calcium aluminate surface is exposed and attacked until a new hydrated layer is formed. Fig. 5b shows that the surface of the CA microspheres incubated in Tris for 30 min have some damage. Degradation is noticeable in the formation of a gel-layer where microspheres were in contact with one another. Buffers with a minimum of 30 mM Tris were found to be optimal for inhibiting degradation; however, microspheres also appeared chemically stable in the AE buffer (10 mM Tris, 0.5 mM EDTA) and in wash buffers containing the chaotropic agent GuHCl.
Fig. 5. Result of aqueous attack on CA microspheres. Hydrated calcium aluminate crystals (a) begin to form on the CA surface after 30 min in DI water. A 30 mM Tris buffer solution retards attack (b), exhibiting the majority of hydration layer degradation between the microspheres.
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Fig. 8. CD spectra of 1 mg/ml calf thymus DNA in 30 mM Tris with and without 2.5 mM Ca2+ leached from the CA microspheres.
Fig. 6. Concentration of calcium (Ca2+ ) leached from the CA, CAFe, and SLS microspheres after different incubation periods in 30 mM Tris. Aluminum ion concentrations are now shown as it remained undetectable even after a 24 h incubation period.
Tris-induced stabilization of the CAFe microspheres makes it possible to potentially use this material as a replacement for silica scaffolds. However, dissolved cations were found to alter the adsorbance values, sometimes hindering an accurate assessment of calf thymus DNA adsorption. Fig. 7 illustrates the effect of pre-rinse times on calf thymus DNA adsorption values. Without pre-rinsing both the SLS and CAFe scaffolds in ≥30 mM Tris for at least 1 min, measured UV absorbance values were higher than those measured for the original quantity of DNA in solution. The less chemically stable CA microspheres required a 15 min pre-rinse before accurate values could be obtained. This 15 min rinse time corresponds to the plateau in the leached calcium concentration shown in Fig. 5. As discussed earlier, the Tris molecule limits calcium aluminate degradation by preventing aqueous attack on the network structure. Once the Tris has stabilized the surface, leaching does not continue during the adsorption and elution process. CAFe and SLS microspheres did not leach detectable amounts of ions after a 1 min incubation with 30 mM Tris; therefore, pre-rinsing was unnecessary if only elution values were measured, as the adsorption phase acted as a pre-rinse.
No conformation change to the B-form DNA was visible after elution from any of the scaffolds. Incubating the calf thymus DNA with 2.5 mM Ca2+ , formed from incubating the CA microspheres in 30 mM Tris for 1 h, also exhibited no effect on conformation (Fig. 8). However, contamination due to broken pieces of hydrated scaffold, as well as lengthy pre-rinse times, makes the CAFe scaffold a more attractive option. 4. Conclusions Calf thymus DNA exhibited a greater affinity for the positively charged CAFe microspheres in the absence of chaotropic agents than for the negatively charged SLS and Qiagen scaffolds. CAFe 43–106 m also isolated a higher normalized concentration of RAW 264.7 cell DNA as the surface charge of the CAFe was still strong enough to electrostatically attract the DNA backbone. Shifting to basic pH values above the IEP of the material allowed DNA to detach. Scaffold morphology was also found to play a significant role in DNA isolation. In the presence of chaotropic agents, the 43–106 m SLS microspheres isolated more DNA than the 14–43 m CAFe microspheres. It is suggested that the chaotropic agents shielded the negative SLS surface enough to allow for the scaffold curvature to play a greater role in isolation. It was also demonstrated that the chemical attack on the calcium–aluminate network could be inhibited though the agents present in the buffer solutions. Through further compositional optimization, these materials have the potential to provide quick, effective, and low cost isolation of genomic DNA using buffer solutions within the physiological pH range. Acknowledgments The authors would like to acknowledge the National Science Foundation’s Graduate Research Fellowship Program for funding this research. The authors declare no competing interests. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2013.11.014. References
Fig. 7. Effect of different 30 mM Tris pre-rinse times on UV measured DNA calf thymus adsorption. CAFe and SLS microspheres provided accurate DNA adsorbance values after a 1 min pre-rinse. CA microspheres required a 15 min pre-rinse before leached calcium ions did not interfere with the UV absorbance measurements.
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