Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Study on the adsorption of DNA on the layered double hydroxides (LDHs) Bin Li a,b, Pingxiao Wu a,b,c,⇑, Bo Ruan a, Paiyu Liu a, Nengwu Zhu a,b a
College of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, Guangzhou 510006, PR China c The Key Laboratory of Environmental Protection and Eco-Remediation of Guangdong Regular Higher Education Institutions, Guangzhou 510006, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Four kinds of layered double
hydroxides (LDHs) were synthesized to adsorb the DNA. We compared the adsorption between different LDHs and discovered the difference. Discussed the influence of various factors variables on adsorption process. Some models were used to discuss the adsorption mechanism between LDHs and DNA.
a r t i c l e
i n f o
Article history: Received 8 August 2013 Received in revised form 21 October 2013 Accepted 31 October 2013 Available online 7 November 2013 Keywords: Layered double hydroxides DNA Adsorption Mechanism
a b s t r a c t Four kinds of layered double hydroxides (LDHs) were prepared by chemical coprecipitation method and used as DNA adsorbents. Multiple characterization tools such as power X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and Standard electronic modules (SEM) were employed to characterize the LDHs. By examining the effect of initial concentration, solution pH, adsorption experiments were carried out to investigate the adsorption capacities of LDHs for DNA. The results revealed that the LDHs with Mg/Al = 3 had higher ability on adsorbing the DNA and were not affected by pH values. The LDHs exhibited excellent adsorption properties and completely adsorbed DNA within 2 h. The adsorption equilibrium data were fitted to the Langmuir and Freundlich models, showing that the Langmuir model which represented monolayer adsorption had better correlation with the adsorption linear equation. In addition, Circular dichroism (CD) spectrum, UV–vis spectorscopy and agarose gel electrophoresis revealed the integrity of DNA structure, suggesting that there had no damage on the DNA structure during the adsorption process. Ó 2013 Elsevier B.V. All rights reserved.
Introduction As genetic material, the existence of DNA takes an important part in the nature ecosystem. In soil environment, clay minerals, ⇑ Corresponding author at: College of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China. Tel.: +86 20 39380538; fax: +86 20 39383725. E-mail address: [email protected] (P. Wu). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.10.099
humic acids and soil adsorb the free DNA excreted by living cells or released by dying cells and protect them against the degradation of nuclease [1–5]. Then the organic–inorganic hybrids are formed and the absorbed DNA still has the ability to transform competent cells [6,7]. More and more researches pay close attention to the hybrids, which is an important part of the research field of modern soil microbial ecology. Because of the particular physical and chemical properties of clay minerals, understanding
388
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
the behavior of DNA being absorbed on the clay minerals has significant application on DNA bio-sensing and DNA purification. In recent years, soil biologists have been focus on the adsorption of DNA on surface-active particles. The species of soil particles, solution pH, ionic strength, the length and shape of DNA molecules and the concentration of Tris buffer are all significant influencing factors during adsorption. Based on the previous researches, the amount of DNA adsorbed on montmorillonite is higher than the DNA adsorbed on soil colloids and kaolinite, showing that the fine clays have better capability of DNA adsorption than coarse clays [8]. Natural allophanes adsorb markedly more DNA than synthetic allophone [9]. A marked decrease in the adsorption of DNA on mineral clays is observed with the increasing of pH from 2.0 to 9.0 [10,11]. The concentration of ionic in the solution affects the adsorption capability of the adsorbent. For instance, by raising ionic strength of the solution, the DNA retention on sand particles is increased because of the forming of the bridges between phosphate groups of DNA molecules and the negatively charged sites of sand [12–14]. Furthermore, there are also some investigations indicating that different metal ions play different roles during the adsorption. DNA bind strongly to mica surfaces in the existence of transition metals, while Ca2+ and Mg2+ can not play the same role [15]. Meanwhile, supercoiled plasmid DNA is not adsorbed well as linearized or open circular plasmid by sand and lower molecular mass DNA is adsorbed more on the particles than the higher molecular mass DNA [16]. Otherwise, the effect of Tris buffer on DNA adsorption depends on the kind of adsorbents. Such as, the natural allophane, kaolinite adsorb more DNA in the existence of Tris bufferl (pH = 7.0) instead of NaCl solution, just the opposite happens on the adsorption to montmorillonite [17]. Although numerous studies have been made on the adsorption of DNA on soil particles, it is rare to find the researches about the adsorption capacity of layered double hydroxide (LDH) for DNA, which is a type of double hydroxide, with a structure consisting of a positively charged brucite-like octahedral layer and interlayer anions [18]. As a soil particle, it can be easily synthesized by coprecipitation [22]. People always pay their attention on the intercalation between DNA and LDHs in the area of gene or drug delivery systems because of their unique structure and physicochemical properties [19–21]. In this paper, we are trying to investigate the binding characteristics of DNA by four different LDHs and study the mechanisms between them by all kinds of characterizations.
MgZnAl–CO3–LDH-2 ([Mg2+ + Zn2+]/Al3+ = 2) and MgZnAl–CO3– LDH-3 ([Mg2+ + Zn2+]/Al3+ = 3) were synthesized by using the same procedure. Characterization X-ray diffraction (XRD) measurements were performed on a Bruker D8-ADVANCE X-ray diffractometer, operated on Cu Ka radiation (k = 0.154 nm) at 40 kV and 40 mA, with step size of 0.02°, a rate of 17.7 s step1 and 2h range 2–70°. The infrared spectra (IR) of samples were collected by Bruker Vector 33 FTIR spectrometer using KBr pellets, in the range of 4000–400 cm1 with 4 cm1 resolution. Scanning electron microscopy (SEM) images were obtained by using a LEO 1530Vp microscopy at an accelerating voltage of 5.0 kV. The external surface areas (SSA) of LDHs were investigated by the method of N2 adsorption. Adsorption of DNA on LDHs LDH (50 mg) was mixed in a centrifuge tube with 10 ml 10 mM Tris buffer (pH = 7.0) containing DNA, the initial concentration of which ranged from 0 to 160 mg L1 for ternary LDHs and 0– 700 mg L1 for binary LDHs. Then continuously shook the suspension in an air-conditioned room at 25 ± 1 °C for 2 h. Thereafter, centrifuged the suspension at 10,000g for 10 min. The supernatant was analyzed by measuring the absorbance at k = 260 nm (A260), and the amount of DNA adsorbed at equilibrium by LDHs was calculated by the difference between the amount of DNA added and that remaining in the supernatant. The adsorptions were carried out three times. In addition, LDH-DNA complexes were obtained after reaction. In order to investigate the mechanism of adsorption, the adsorption data were analyzed using the different adsorption equations. In order to study how the adsorption capacities of LDHs for DNA influenced by the varying binding solution, the effect of adsorption time on DNA adsorption was studied by treating 50 mg LDH with 10 ml DNA solution. The working concentration of DNA was 200 mg L1. Then, continuously shook the mixture at 25 ± 1 °C for various time. The following operations were the same as what were mentioned above. The effect of solution pH was also investigated by adjusting the suspension pH in the range of 2.0–12.0.
Experimental Desorption of DNA on LDHs Chemicals Salmon sperm DNA whose purity ration assessed by A260nm/ A280m was higher than 1.8 was purchased from Sigma Chemical Co., Louis, MO. Magnesium nitrate hexahydrate (Mg(NO3)6H2O), aluminum nitrate nonahydrate (Al(NO3)9H2O), zinc nitrate hexahydrate (Zn(NO3)6H2O), sodium hydroxide pellets (NaOH), urea (CON2H4) were brought from Huaxin Company Guangzhou, Guangdong Province, China. All chemicals were analytical grade. Synthesis The LDHs were prepared by coprecipitation [23]. 0.04 mol of Mg(NO3)6H2O, 0.02 mol of Al(NO3)9H2O (the molar ratio of Mg2+/Al3+ was fixed at 2) and 0.35 mol of urea were dissolved in 250 ml deionized water under vigorous stirring. The resulting suspension was stirred for 48 h at 100 °C in the equipment of refluxing. After the suspension was cooled to room temperature, it was washed several times by deionized water and air-dried. It was signed as MgAl–CO3–LDH-2. MgAl–CO3–LDH-3 (Mg2+/Al3+ = 3),
In the desorption experiment, added 10 ml DNA (200 mg L1) to 50 mg powered LDH and shook the reaction system at 25 ± 1 °C for 2 h and centrifuged it at 10,000g for 10 min. Washed the residue by 1 ml of Tris buffer until there was no DNA in the supernatant. Spectral analysis The X-ray diffraction of LDH-DNA complexes were made by Xray diffractometer to find if there was any DNA inserting into the interlayer of LDH during the adsorption and to observe the structure of LDH after adsorption. The FTIR spectrum for MgAl–CO3– LDH-3 which had the highest capacity of DNA adsorption and LDH-DNA were performed on the FTIR spectroscopy to identify whether the ion exchanging occurred and the complex was formed. CD spectra of native DNA and DNA desorbed from LDH by Tris buffer were characterized by J-810 spectropolarimeter and each measurement was scanned three times. Furthermore, UV–vis spectra and agarose gel electrophoresis were employed to demonstrate the integrity of the released DNA.
389
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Results and discussions Characterization of LDH Fig. 1 showed the SEM images of MgAl–CO3–LDH-3 and MgZnAl–CO3–LDH-3. As shown in Fig. 1, the particles of MgAl–CO3– LDH-3 presented regular hexagon shape, with diameters from 0.5 to 2.0 lm. Moreover, it also had the structure of laminate with uniform shape, which was the typical structure of LDH. Furthermore, the dispersion of MgAl–CO3–LDH-3 was good as shown in the picture. By contrast, though MgZnAl–CO3–LDH-3 had the structure of laminate, it had poor dispersion and the lamellar intersected with each other, which looked like the petal of a rose. We suppose the reason is that incomplete recrystallization happened during the manufacturing operations. Adsorption analysis Effect of concentration of DNA on adsorption As shown in Fig. 2, the amount of DNA adsorbed on LDHs increased with the higher concentration of DNA, until reached the point of maximum amounts of adsorption. And there were no significant changes in the isothermal adsorption curves after the absorption reached equilibrium. The amount of DNA adsorbed on binary LDHs was far greater than ternary LDHs. In addition, MgAl–CO3–LDH-3 had higher adsorption capacity than that of MgAl–CO3–LDH-2 and the same situation occurred on the ternary LDHs, which can be attributed to the specific surface areas of LDHs particles (Table 1). That is, the larger the specific surface area, the greater the adsorption capacity. Langmuir (1) and Freundlich (2) models were used to analyze the adsorption data. Langmuir and Freundlich equations can be described as follows:
Fig. 2. Equilibrium adsorption of DNA on LDHs.
Table 1 Selected properties of LDHs studied. Samples
SSA (m2/g)
pH (H2O)
d003 (nm)
MgZnAl–CO3–LDH-2 MgZnAl–CO3–LDH-3 MgAl–CO3–LDH-2 MgAl–CO3–LDH-3
37.61 7.49 59.25 45.04
6.36 6.15 7.26 6.76
0.757 0.751 0.756 0.755
Q ¼ qm C e =ð1=b þ C e Þ
ð1Þ
Q ¼ K f C 1=n e
ð2Þ
where Q represents the amount of adsorbed DNA, qm is the maximum amount of DNA adsorbed, b and Ce are adsorption coefficient and the equilibrium concentration of DNA, respectively. Kf and n are both adsorption coefficients [24]. As shown in Table 2, both isotherm models were suitable for the DNA adsorption by LDHs. The Langmuir model fitted better, indicating that the mode of the DNA adsorbed on the LDHs was monolayer adsorption. Furthermore, the regression equations reflected the maximum amount of DNA adsorption intuitively. The qm of MgAl–CO3–LDH-3 was 81.01 mg/g, which was superior to MgZnAl–CO3–LDH-3 obviously.
Fig. 1. SEM images of MgAl–CO3–LDH-3 and MgZnAl–CO3–LDH-3.
Effect of contact time and kinetics study These four kinds of LDHs exhibited excellent adsorption capacity, making a good performance to reach equilibrium within 2 h and nearly no additional uptake occurred during the rest reaction time (Fig. 3). The adsorption rates of LDHs for DNA were high.
390
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Table 2 Regression equation of isotherm models and correlation coefficients. Adsorbent
Isothermal adsorption model
Equation
R2
MgAl–CO3–LDH-2
Langmuir
0.994
MgAl–CO3–LDH-3
Freundlich Langmuir
y = 77.55x/ (0.138 + x) y = 55.299(x0.069) y = 81.013x/ (0.97 + x) y = 51.065(x0.091) y = 10.017x/ (1.186 + x) y = 5.938(x0.12) y = 13.660x/ (1.073 + x) y = 8.819(x0.103)
MgZnAl–CO3– LDH-2 MgZnAl–CO3– LDH-3
Freundlich Langmuir Freundlich Langmuir Freundlich
0.964 0.999 0.937 0.959 0.859 0.993 0.951
Therefore, the contact time of 2 h was adopted by equilibrium adsorption isotherm. In order to further understand the mechanism of the adsorption process, many rate models were adopted to interpret the experimental data, such as the Pseudo-first-order (3), Pseudo-second-order (4) and Elovich (5) models [25]. The equations of the models were represented as follows:
lnðqe qt Þ ¼ ln qe k1 t
ð3Þ
t 1 1 ¼ þ t qt k2 q2e qe
ð4Þ
qt ¼
1 1 lnðabÞ þ ln t b b
As shown in Table 3, the coefficients of determination (R2) of Pseudo-second-order equation for LDHs were all very high, revealing that the equilibrium data corresponded to Pseudo-second-order best. In addition, the qe of the Pseudo-second-order equation were consistent with the experimental qe values. Therefore, the chemical adsorption was the rate limiting step of the adsorption process. Through the k2 comparison of the four kinds of LDHs, the adsorption rate followed the order: MgAl–CO3–LDH-3 > MgZnAl–CO3–LDH-3 > MgZnAl–CO3–LDH-2 > MgAl–CO3–LDH-2, the adsorption rate of MgAl–CO3–LDH-3 was also the best outside of the adsorption capacity, simultaneously.
ð5Þ
where qe (mg/g) and qt (mg/g) are the amounts of DNA adsorbed on the LDHs at equilibrium and at time t (min); k1 and k2 are the adsorption rate constants of Pseudo-first-order equation and Pseudo-second-order equation; a (mg g1 min1) and b (g mg1) are the initial adsorption rate and the chemical adsorption parameter, respectively.
Effect of pH on adsorption As an important factor affecting adsorption experiment, the change of solution pH had an enormous impact on the DNA adsorption. The change of pH value leaded to the fluctuation of the material surface charge, so the surface charge had significance on adsorption process in some sense. Regardless of the changes of the solution pH, the capacities of DNA adsorption of binary LDHs were still stronger than ternary LDHs (Fig. 4). The maximum adsorption of DNA on all LDHs occurred at around pH 2.0, which probably caused by the precipitation of DNA on the surface of LDHs [26]. The adsorption capacities decreased dramatically with the increasing solution pH on ternary LDHs, while it almost had no influence on the adsorption capacity of binary LDHs. In addition, the capacity of DNA adsorption by ternary LDHs rose at around pH 5.0 and decreased again at around pH 8.0. The surface charges of LDHs and DNA were probably the main reasons for the different adsorption abilities. It reduced gradually with the elevation of pH and leaded to the weakness of coulombian force between LDHs and DNA [27], which was consist with the adsorption curve. The isoeletric point of DNA is around pH 5.0 [4], leading to the increasing of DNA adsorption by ternary LDHs around pH 5.0. Furthermore, the abilities of DNA adsorption by binary LDHs were not affected by the change of solution pH. It seems that there were some other forces between LDHs and DNA besides of coulombian force, so that the coulombian force was not the dominant element in the process of adsorption between binary LDHs and DNA. The adsorption processes is simulated in Fig. 5. XRD analysis
Fig. 3. Effect of contact time on the adsorption of DNA by LDHs.
Fig. 6 showed the XRD patterns of LDHs and LDH-DNA composites. The characteristic diffraction peaks of LDH-DNA still appeared after adsorption, revealing that the process of DNA adsorption did not do damage to the interlayer structure of LDHs. They all had a good crystallinity and there were no other impurities in the samples [28]. The new peaks appeared in 15–20° might be caused by the coordinate bond which formed after DNA was adsorbed on the surface of LDHs. XRD is an effective measure for detecting the intercalation of LDH structure. The basal spacing of LDHs was around 0.7 nm before adsorption and tiny changes appeared on it after adsorption. The basal spacing of LDHs would be more than 2.0 nm if the intercalation happened because of the width of
Table 3 The Pseudo-second-order parameters for DNA adsorption on LDHs. Samples
MgZnAl–CO3–LDH-2 MgZnAl–CO3–LDH-3 MgAl–CO3–LDH-2 MgAl–CO3–LDH-3
qe
(experiment)
8.858 13.152 39.774 39.913
(mg/g)
Pseudo-second-order K2 (g mg1 min1)
qe (mg/g)
R2
0.502 1.550 0.364 2.220
8.648 12.720 39.806 39.730
0.983 0.987 0.999 0.999
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Fig. 4. Effect of solution pH on the adsorption of DNA by LDHs.
391
Fig. 7. XRD patterns of MgAl–CO3–LDH-3 at different pH.
Fig. 5. Schematic of equilibrium interfacial structure for DNA on LDH surface: top view of the initial configuration for DNA adsorption on LDH.
Fig. 8. FTIR spectra of MgAl–CO3–LDH-3 and MgAl–CO3–LDH-3-DNA.
tiny changes. So we can came to the conclusion that the damage of the structure of LDHs can be ignored during the experiment. FTIR analysis
Fig. 6. XRD patterns of LDHs and LDH-DNA.
DNA which was 2.0 nm [29]. The changes of the basal spacing on LDHs before and after adsorption may due to the loss of H2O molecules inside or on the surface of LDHs. It can be concluded that DNA does not intercalated into the interlayer of LDHs but just be adsorbed on the surface of them. Considering the effect of pH on the structure of LDHs, XRD (MgAl–CO3–LDH-3) were accordingly done to inspect if the LDHs were dissolved, and the result was showed in Fig. 7. The peaks of LDHs reflection were all around 0.75 nm and became a little weaker with the increasing of pH. Meanwhile, the intensity of the peaks increased in initial stage and then declined. The basic structure of MgAl–CO3–LDH-3 was still in good shape, though there were some
Fig. 8 showed the FTIR spectra of MgAl–CO3–LDH-3-DNA composition and the spectra of MgAl–CO3–LDH-3 which had a higher ability on DNA adsorption. The broad bands observed at 3445 cm1 and 3448 cm1 were attributed to hydroxyl (O–H) stretching vibrations, which were the same as the bands of 1643 cm1 and 1650 cm1 [30]. The adsorption band at 1356 cm1 and 1357 cm1 were assigned to CO2 3 . The appearances of other peaks were on account of the basic structure of LDH. Compared with the unmodified LDH, a new band centered at 1228 cm1 standing for the vibrational modes of PO3 4 , which was part of the basic structure of DNA after adsorption [31,32]. These results demonstrated that there were some DNA molecules being adsorbed on the LDH after adsorption experiment, which was in accordance with the XRD patterns. Moreover, comparing the two curves, the characteristic absorption peaks of LDH after adsorption were almost the same as before, which turned out that ion exchange reaction did not happen during adsorption. CD analysis Fig. 9 showed the CD spectra of native DNA and DNA desorbed from LDHs. As a method to monitor the conformational transition
392
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Fig. 9. CD spectra of native DNA and DNA desorbed by LDHs: (a) native DNA, (b) DNA desorbed by MgAl–CO3–LDH-2, (c) DNA desorbed by MgAl–CO3–LDH-3, (d) DNA desorbed by MgZnAl–CO3–LDH-2, (e) DNA desorbed by MgZnAl–CO3–LDH-3.
Fig. 11. Electrophoresis analysis for the native DNA and DNA desorbed from LDHs: Lane 1, marker; Lane 2, native DNA; Lane 3, DNA desorbed by MgAl–CO3–LDH-2; Lane 4, DNA desorbed by MgAl–CO3–LDH-3; Lane 5, DNA desorbed by MgZnAl– CO3–LDH-2; Lane 6, DNA desorbed by MgZnAl–CO3–LDH-3.
the length of native DNA was nearly 1000 bp. Compared to the native DNA in lane 2, the migration behaviors of the desorbed DNA were almost the same as native DNA, besides of the brightness of the strips, which due to the concentration of DNA. As a measurement to demonstrate the length of DNA, the results clearly showed that the process of adsorption did not affect the length of DNA, which revealed that the desorbed DNA still had the biological activity. Conclusion
Fig. 10. UV–vis spectra of native DNA and DNA desorbed by LDHs: (a) native DNA, (b) DNA desorbed by MgZnAl–CO3–LDH-3, (c) DNA desorbed by MgAl–CO3–LDH-2, (d) DNA desorbed by MgZnAl–CO3–LDH-2, (e) DNA desorbed by MgAl–CO3–LDH-3.
of DNA, the changes of the conformation of DNA before and after adsorption were presented accurately. The positive band at 276 nm and a negative band at 247 nm illustrated that the native DNA was a typical B-form [33,34]. Compared with the native DNA, the CD spectra of DNA desorbed from LDHs remained unchanged, revealing that the conformation of DNA did not change during the adsorption. UV–vis analysis The native DNA and released DNA were detected by UV–vis spectra. As shown in Fig. 10, the characteristic adsorption peak of native DNA was at 259 nm, which was caused by p ? p or n ? p transition of conjugated double bonds of purine and pyrimidine derivatives [35,36]. DNA released from LDHs was tested by UV–vis again, and it should be noted that the adsorption peaks of released DNA were all around 259 nm which were the same as the native DNA [37]. The results were in accordance with the results of CD spectra, proving that the DNA was still in good condition whether it was adsorbed on LDHs or not. Gel retardation analysis DNA was desorbed from LDHs after adsorption, as shown in lanes 3–6 (Fig. 11). The strip of native DNA (lane 2) indicated that
The findings of our observations revealed that as the adsorbents of DNA,binary LDHs have advantage over ternary LDHs and are prior to some other materials natural or man-made. The different results of the adsorption are mainly due to the surface charges of DNA and LDHs. Coulombic force takes an important part in the adsorption except for binary LDHs. The capacities of the binary LDHs on adsorbing the DNA are independent on the solution pH, revealing that there might be some other mechanisms in the adsorption between binary LDHs and DNA except for electrostatic force. XRD and FTIR results indicate that the process of intercalation do not happen during the adsorption but only for surface adsorption, on the other side, the conclusion that the structures of LDHs are not affected during adsorption is also confirmed through the results of XRD and FTIR. Meanwhile, the structure of DNA is not damaged during adsorption, which is proved by the results of CD, UV–vis spectra and electrophoresis. In summary, as a new adsorption material, it may be a potential drug carrier with wide application because of its excellent adsorption property and no biological toxicity. Acknowledgements This work was financially supported by the Research Fund for the Doctoral Program of Higher Education of China (No. 20100172110028), National Science Foundation of China (Grant Nos. 41273122, 41073058, 40973075). References [1] E. Gallori, M. Bazzicalupo, D.L. Canto, R. Fani, P. Nannipieri, C. Vettori, G. Stotzky, FEMS Microbiol FEMS Microbiol. Ecol. 15 (1994) 119–126. [2] S.A.E. Blum, M.G. Lorenz, W. Wackernagel, Syst. Appl. Microbiol. 20 (1997) 513–521. [3] C. Crecchio, G. Stotzky, Soil Biol. Biochem. 30 (1998) 1061–1067.
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393 [4] G. Stotzky, J. Environ. Qual. 29 (2000) 691–705. [5] C. Crecchio, P. Ruggiero, M. Curci, C. Colombo, G. Palumbo, G. Stotzky, Soil Sci. Soc. Am. J. 69 (2005) 834–841. [6] F. Widmer, R.J. Seidler, L.S. Watrud, Mol. Ecol. 5 (1996) 603–613. [7] E. Paget, M. Lebrun, G. Freyssinet, P. Simonet, Eur. J. Soil Biol. 34 (1998) 81–88. [8] P. Cai, Q. Huang, X.W. Zhang, H. Chen, Soil Biol. Biochem. 38 (2006) 471–476. [9] K. Saeki, M. Sakai, S.I. Wada, Appl. Clay. Sci. 50 (2010) 493–497. [10] C.A.I. Goring, W.V. Bartholomew, Soil Sci. 74 (1952) 149–164. [11] M.P. Greaves, M.J. Wilson, Soil Biol. Biochem. 1 (1969) 317–323. [12] G. Romanowski, M.G. Lorenz, W. Wackernagel, Appl. Environ. Microbiol. 57 (1991) 1057–1061. [13] M. Franchi, J.P. Ferris, E. Gallori, Orig. Life Evol. Biosph. 33 (2003) 1–16. [14] P. Cai, Q.Y. Huang, X.W. Zhang, Appl. Clay. Sci. 32 (2006) 147–152. [15] T.H. Nguyen, K.L. Chen, M. Elimelech, Biomacromol 11 (2010) 1225–1230. [16] G. Pietramellara, M. Franchi, E. Gallori, P. Nannipieri, Biol. Fert. Soils 33 (2001) 402–409. [17] K. Saeki, T. Kunito, M. Sakai, Microbes. Environ. 26 (2011) 88–91. [18] H. Nakayama, A. Hatakeyama, M. Tsuhako, Int. J. Pharm. 393 (2010) 105–112. [19] K. Ladewig, M. Niebert, Z.P. Xu, P.P. Gray, G.Q. Lu, Appl. Clay. Sci. 48 (2010) 280–289. [20] Y.Y. Wong, H.M. Cooper, K. Zhang, M. Chen, P. Bartlett, Z.P. Xu, J. Colloid Interface Sci. 369 (2012) 453–459. [21] M. Chen, H.M. Cooper, J.Z. Zhou, P.F. Bartlett, Z.P. Xu, J. Colloid Interface Sci. 390 (2013) 275–281.
[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37]
393
J.H. Choy, M. Park, J.M. Oh, NanoBio. Techonol. 4 (2008) 349–367. H. Toshiyuki, O. Hitoshi, Appl. Clay. Sci. 45 (2009) 123–132. H. Freundlich, Z. Phys. Chem. 57 (1907) 385–471. P.X. Wu, Q. Zhang, Y.P. Dai, N.W. Zhu, Z. Dang, P. Li, J.H. Wu, X.D. Wang, Geoderma 164 (2011) 215–219. K. Saeki, T. Kunito, M. Sakai, Biol. Fertil. Soils 46 (2010) 531–535. Z.P. Xu, Y.G. Jin, S.M. Liu, Z.P. Hao, G.Q. Lu, J. Colloid Interface Sci. 326 (2008) 522–529. L.N. Sun, C.W. Hu, Mater. Res. Bull. 46 (2010) 1922–1927. H. Nakayama, A. Hatakeyama, M. Tsuhako, Int. J. Pharm. 393 (2010) 104–111. I. Ghorbel-Abid, A. Jrad, K. Nahdi, M. Trabelsi-Ayadi, Desalination 246 (2009) 595–604. E. Taillandier, J. Liquier, J.A. Taboury, in: R.J.H. Clarc, R.E., Hester (Eds.), Wiley, Heyden, 1985, pp. 65. I.D. Galina, Y.G. Nina, B.K. Elena, P.P. Olena, Talanta 53 (2000) 233–246. X.G. Sun, E.H. Cao, X.Y. Zhang, D.G. Liu, C.L. Bai, Inorg. Chem. Commun. 5 (2002) 181–186. P. Cai, Q.Y. Huang, X.W. Zhang, Sci. Technol. 40 (2006) 2971–2976. V.B. Morris, S. Neethu, T.E. Abraham, C.K.S. Pillai, C.P. Sharma, J. Biomed. Mater. Res. 89 (2009) 282. G.F. Zuo, Y.Z. Wan, X.G. Meng, Q. Zhao, K.J. Ren, S.R. Jia, J.H. Wang, Mater. Chem. Phys. 126 (2011) 470–475. J.H. Choy, S.Y. Kwak, J.S. Park, Y.J. Jeong, J. Portier, J. Am. Chem. Soc. 121 (1999) 1399–1400.
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Study on the adsorption of DNA on the layered double hydroxides (LDHs) Bin Li a,b, Pingxiao Wu a,b,c,⇑, Bo Ruan a, Paiyu Liu a, Nengwu Zhu a,b a
College of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China The Key Lab of Pollution Control and Ecosystem Restoration in Industry Clusters, Ministry of Education, Guangzhou 510006, PR China c The Key Laboratory of Environmental Protection and Eco-Remediation of Guangdong Regular Higher Education Institutions, Guangzhou 510006, PR China b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Four kinds of layered double
hydroxides (LDHs) were synthesized to adsorb the DNA. We compared the adsorption between different LDHs and discovered the difference. Discussed the influence of various factors variables on adsorption process. Some models were used to discuss the adsorption mechanism between LDHs and DNA.
a r t i c l e
i n f o
Article history: Received 8 August 2013 Received in revised form 21 October 2013 Accepted 31 October 2013 Available online 7 November 2013 Keywords: Layered double hydroxides DNA Adsorption Mechanism
a b s t r a c t Four kinds of layered double hydroxides (LDHs) were prepared by chemical coprecipitation method and used as DNA adsorbents. Multiple characterization tools such as power X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and Standard electronic modules (SEM) were employed to characterize the LDHs. By examining the effect of initial concentration, solution pH, adsorption experiments were carried out to investigate the adsorption capacities of LDHs for DNA. The results revealed that the LDHs with Mg/Al = 3 had higher ability on adsorbing the DNA and were not affected by pH values. The LDHs exhibited excellent adsorption properties and completely adsorbed DNA within 2 h. The adsorption equilibrium data were fitted to the Langmuir and Freundlich models, showing that the Langmuir model which represented monolayer adsorption had better correlation with the adsorption linear equation. In addition, Circular dichroism (CD) spectrum, UV–vis spectorscopy and agarose gel electrophoresis revealed the integrity of DNA structure, suggesting that there had no damage on the DNA structure during the adsorption process. Ó 2013 Elsevier B.V. All rights reserved.
Introduction As genetic material, the existence of DNA takes an important part in the nature ecosystem. In soil environment, clay minerals, ⇑ Corresponding author at: College of Environment and Energy, South China University of Technology, Guangzhou 510006, PR China. Tel.: +86 20 39380538; fax: +86 20 39383725. E-mail address: [email protected] (P. Wu). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.10.099
humic acids and soil adsorb the free DNA excreted by living cells or released by dying cells and protect them against the degradation of nuclease [1–5]. Then the organic–inorganic hybrids are formed and the absorbed DNA still has the ability to transform competent cells [6,7]. More and more researches pay close attention to the hybrids, which is an important part of the research field of modern soil microbial ecology. Because of the particular physical and chemical properties of clay minerals, understanding
388
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
the behavior of DNA being absorbed on the clay minerals has significant application on DNA bio-sensing and DNA purification. In recent years, soil biologists have been focus on the adsorption of DNA on surface-active particles. The species of soil particles, solution pH, ionic strength, the length and shape of DNA molecules and the concentration of Tris buffer are all significant influencing factors during adsorption. Based on the previous researches, the amount of DNA adsorbed on montmorillonite is higher than the DNA adsorbed on soil colloids and kaolinite, showing that the fine clays have better capability of DNA adsorption than coarse clays [8]. Natural allophanes adsorb markedly more DNA than synthetic allophone [9]. A marked decrease in the adsorption of DNA on mineral clays is observed with the increasing of pH from 2.0 to 9.0 [10,11]. The concentration of ionic in the solution affects the adsorption capability of the adsorbent. For instance, by raising ionic strength of the solution, the DNA retention on sand particles is increased because of the forming of the bridges between phosphate groups of DNA molecules and the negatively charged sites of sand [12–14]. Furthermore, there are also some investigations indicating that different metal ions play different roles during the adsorption. DNA bind strongly to mica surfaces in the existence of transition metals, while Ca2+ and Mg2+ can not play the same role [15]. Meanwhile, supercoiled plasmid DNA is not adsorbed well as linearized or open circular plasmid by sand and lower molecular mass DNA is adsorbed more on the particles than the higher molecular mass DNA [16]. Otherwise, the effect of Tris buffer on DNA adsorption depends on the kind of adsorbents. Such as, the natural allophane, kaolinite adsorb more DNA in the existence of Tris bufferl (pH = 7.0) instead of NaCl solution, just the opposite happens on the adsorption to montmorillonite [17]. Although numerous studies have been made on the adsorption of DNA on soil particles, it is rare to find the researches about the adsorption capacity of layered double hydroxide (LDH) for DNA, which is a type of double hydroxide, with a structure consisting of a positively charged brucite-like octahedral layer and interlayer anions [18]. As a soil particle, it can be easily synthesized by coprecipitation [22]. People always pay their attention on the intercalation between DNA and LDHs in the area of gene or drug delivery systems because of their unique structure and physicochemical properties [19–21]. In this paper, we are trying to investigate the binding characteristics of DNA by four different LDHs and study the mechanisms between them by all kinds of characterizations.
MgZnAl–CO3–LDH-2 ([Mg2+ + Zn2+]/Al3+ = 2) and MgZnAl–CO3– LDH-3 ([Mg2+ + Zn2+]/Al3+ = 3) were synthesized by using the same procedure. Characterization X-ray diffraction (XRD) measurements were performed on a Bruker D8-ADVANCE X-ray diffractometer, operated on Cu Ka radiation (k = 0.154 nm) at 40 kV and 40 mA, with step size of 0.02°, a rate of 17.7 s step1 and 2h range 2–70°. The infrared spectra (IR) of samples were collected by Bruker Vector 33 FTIR spectrometer using KBr pellets, in the range of 4000–400 cm1 with 4 cm1 resolution. Scanning electron microscopy (SEM) images were obtained by using a LEO 1530Vp microscopy at an accelerating voltage of 5.0 kV. The external surface areas (SSA) of LDHs were investigated by the method of N2 adsorption. Adsorption of DNA on LDHs LDH (50 mg) was mixed in a centrifuge tube with 10 ml 10 mM Tris buffer (pH = 7.0) containing DNA, the initial concentration of which ranged from 0 to 160 mg L1 for ternary LDHs and 0– 700 mg L1 for binary LDHs. Then continuously shook the suspension in an air-conditioned room at 25 ± 1 °C for 2 h. Thereafter, centrifuged the suspension at 10,000g for 10 min. The supernatant was analyzed by measuring the absorbance at k = 260 nm (A260), and the amount of DNA adsorbed at equilibrium by LDHs was calculated by the difference between the amount of DNA added and that remaining in the supernatant. The adsorptions were carried out three times. In addition, LDH-DNA complexes were obtained after reaction. In order to investigate the mechanism of adsorption, the adsorption data were analyzed using the different adsorption equations. In order to study how the adsorption capacities of LDHs for DNA influenced by the varying binding solution, the effect of adsorption time on DNA adsorption was studied by treating 50 mg LDH with 10 ml DNA solution. The working concentration of DNA was 200 mg L1. Then, continuously shook the mixture at 25 ± 1 °C for various time. The following operations were the same as what were mentioned above. The effect of solution pH was also investigated by adjusting the suspension pH in the range of 2.0–12.0.
Experimental Desorption of DNA on LDHs Chemicals Salmon sperm DNA whose purity ration assessed by A260nm/ A280m was higher than 1.8 was purchased from Sigma Chemical Co., Louis, MO. Magnesium nitrate hexahydrate (Mg(NO3)6H2O), aluminum nitrate nonahydrate (Al(NO3)9H2O), zinc nitrate hexahydrate (Zn(NO3)6H2O), sodium hydroxide pellets (NaOH), urea (CON2H4) were brought from Huaxin Company Guangzhou, Guangdong Province, China. All chemicals were analytical grade. Synthesis The LDHs were prepared by coprecipitation [23]. 0.04 mol of Mg(NO3)6H2O, 0.02 mol of Al(NO3)9H2O (the molar ratio of Mg2+/Al3+ was fixed at 2) and 0.35 mol of urea were dissolved in 250 ml deionized water under vigorous stirring. The resulting suspension was stirred for 48 h at 100 °C in the equipment of refluxing. After the suspension was cooled to room temperature, it was washed several times by deionized water and air-dried. It was signed as MgAl–CO3–LDH-2. MgAl–CO3–LDH-3 (Mg2+/Al3+ = 3),
In the desorption experiment, added 10 ml DNA (200 mg L1) to 50 mg powered LDH and shook the reaction system at 25 ± 1 °C for 2 h and centrifuged it at 10,000g for 10 min. Washed the residue by 1 ml of Tris buffer until there was no DNA in the supernatant. Spectral analysis The X-ray diffraction of LDH-DNA complexes were made by Xray diffractometer to find if there was any DNA inserting into the interlayer of LDH during the adsorption and to observe the structure of LDH after adsorption. The FTIR spectrum for MgAl–CO3– LDH-3 which had the highest capacity of DNA adsorption and LDH-DNA were performed on the FTIR spectroscopy to identify whether the ion exchanging occurred and the complex was formed. CD spectra of native DNA and DNA desorbed from LDH by Tris buffer were characterized by J-810 spectropolarimeter and each measurement was scanned three times. Furthermore, UV–vis spectra and agarose gel electrophoresis were employed to demonstrate the integrity of the released DNA.
389
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Results and discussions Characterization of LDH Fig. 1 showed the SEM images of MgAl–CO3–LDH-3 and MgZnAl–CO3–LDH-3. As shown in Fig. 1, the particles of MgAl–CO3– LDH-3 presented regular hexagon shape, with diameters from 0.5 to 2.0 lm. Moreover, it also had the structure of laminate with uniform shape, which was the typical structure of LDH. Furthermore, the dispersion of MgAl–CO3–LDH-3 was good as shown in the picture. By contrast, though MgZnAl–CO3–LDH-3 had the structure of laminate, it had poor dispersion and the lamellar intersected with each other, which looked like the petal of a rose. We suppose the reason is that incomplete recrystallization happened during the manufacturing operations. Adsorption analysis Effect of concentration of DNA on adsorption As shown in Fig. 2, the amount of DNA adsorbed on LDHs increased with the higher concentration of DNA, until reached the point of maximum amounts of adsorption. And there were no significant changes in the isothermal adsorption curves after the absorption reached equilibrium. The amount of DNA adsorbed on binary LDHs was far greater than ternary LDHs. In addition, MgAl–CO3–LDH-3 had higher adsorption capacity than that of MgAl–CO3–LDH-2 and the same situation occurred on the ternary LDHs, which can be attributed to the specific surface areas of LDHs particles (Table 1). That is, the larger the specific surface area, the greater the adsorption capacity. Langmuir (1) and Freundlich (2) models were used to analyze the adsorption data. Langmuir and Freundlich equations can be described as follows:
Fig. 2. Equilibrium adsorption of DNA on LDHs.
Table 1 Selected properties of LDHs studied. Samples
SSA (m2/g)
pH (H2O)
d003 (nm)
MgZnAl–CO3–LDH-2 MgZnAl–CO3–LDH-3 MgAl–CO3–LDH-2 MgAl–CO3–LDH-3
37.61 7.49 59.25 45.04
6.36 6.15 7.26 6.76
0.757 0.751 0.756 0.755
Q ¼ qm C e =ð1=b þ C e Þ
ð1Þ
Q ¼ K f C 1=n e
ð2Þ
where Q represents the amount of adsorbed DNA, qm is the maximum amount of DNA adsorbed, b and Ce are adsorption coefficient and the equilibrium concentration of DNA, respectively. Kf and n are both adsorption coefficients [24]. As shown in Table 2, both isotherm models were suitable for the DNA adsorption by LDHs. The Langmuir model fitted better, indicating that the mode of the DNA adsorbed on the LDHs was monolayer adsorption. Furthermore, the regression equations reflected the maximum amount of DNA adsorption intuitively. The qm of MgAl–CO3–LDH-3 was 81.01 mg/g, which was superior to MgZnAl–CO3–LDH-3 obviously.
Fig. 1. SEM images of MgAl–CO3–LDH-3 and MgZnAl–CO3–LDH-3.
Effect of contact time and kinetics study These four kinds of LDHs exhibited excellent adsorption capacity, making a good performance to reach equilibrium within 2 h and nearly no additional uptake occurred during the rest reaction time (Fig. 3). The adsorption rates of LDHs for DNA were high.
390
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Table 2 Regression equation of isotherm models and correlation coefficients. Adsorbent
Isothermal adsorption model
Equation
R2
MgAl–CO3–LDH-2
Langmuir
0.994
MgAl–CO3–LDH-3
Freundlich Langmuir
y = 77.55x/ (0.138 + x) y = 55.299(x0.069) y = 81.013x/ (0.97 + x) y = 51.065(x0.091) y = 10.017x/ (1.186 + x) y = 5.938(x0.12) y = 13.660x/ (1.073 + x) y = 8.819(x0.103)
MgZnAl–CO3– LDH-2 MgZnAl–CO3– LDH-3
Freundlich Langmuir Freundlich Langmuir Freundlich
0.964 0.999 0.937 0.959 0.859 0.993 0.951
Therefore, the contact time of 2 h was adopted by equilibrium adsorption isotherm. In order to further understand the mechanism of the adsorption process, many rate models were adopted to interpret the experimental data, such as the Pseudo-first-order (3), Pseudo-second-order (4) and Elovich (5) models [25]. The equations of the models were represented as follows:
lnðqe qt Þ ¼ ln qe k1 t
ð3Þ
t 1 1 ¼ þ t qt k2 q2e qe
ð4Þ
qt ¼
1 1 lnðabÞ þ ln t b b
As shown in Table 3, the coefficients of determination (R2) of Pseudo-second-order equation for LDHs were all very high, revealing that the equilibrium data corresponded to Pseudo-second-order best. In addition, the qe of the Pseudo-second-order equation were consistent with the experimental qe values. Therefore, the chemical adsorption was the rate limiting step of the adsorption process. Through the k2 comparison of the four kinds of LDHs, the adsorption rate followed the order: MgAl–CO3–LDH-3 > MgZnAl–CO3–LDH-3 > MgZnAl–CO3–LDH-2 > MgAl–CO3–LDH-2, the adsorption rate of MgAl–CO3–LDH-3 was also the best outside of the adsorption capacity, simultaneously.
ð5Þ
where qe (mg/g) and qt (mg/g) are the amounts of DNA adsorbed on the LDHs at equilibrium and at time t (min); k1 and k2 are the adsorption rate constants of Pseudo-first-order equation and Pseudo-second-order equation; a (mg g1 min1) and b (g mg1) are the initial adsorption rate and the chemical adsorption parameter, respectively.
Effect of pH on adsorption As an important factor affecting adsorption experiment, the change of solution pH had an enormous impact on the DNA adsorption. The change of pH value leaded to the fluctuation of the material surface charge, so the surface charge had significance on adsorption process in some sense. Regardless of the changes of the solution pH, the capacities of DNA adsorption of binary LDHs were still stronger than ternary LDHs (Fig. 4). The maximum adsorption of DNA on all LDHs occurred at around pH 2.0, which probably caused by the precipitation of DNA on the surface of LDHs [26]. The adsorption capacities decreased dramatically with the increasing solution pH on ternary LDHs, while it almost had no influence on the adsorption capacity of binary LDHs. In addition, the capacity of DNA adsorption by ternary LDHs rose at around pH 5.0 and decreased again at around pH 8.0. The surface charges of LDHs and DNA were probably the main reasons for the different adsorption abilities. It reduced gradually with the elevation of pH and leaded to the weakness of coulombian force between LDHs and DNA [27], which was consist with the adsorption curve. The isoeletric point of DNA is around pH 5.0 [4], leading to the increasing of DNA adsorption by ternary LDHs around pH 5.0. Furthermore, the abilities of DNA adsorption by binary LDHs were not affected by the change of solution pH. It seems that there were some other forces between LDHs and DNA besides of coulombian force, so that the coulombian force was not the dominant element in the process of adsorption between binary LDHs and DNA. The adsorption processes is simulated in Fig. 5. XRD analysis
Fig. 3. Effect of contact time on the adsorption of DNA by LDHs.
Fig. 6 showed the XRD patterns of LDHs and LDH-DNA composites. The characteristic diffraction peaks of LDH-DNA still appeared after adsorption, revealing that the process of DNA adsorption did not do damage to the interlayer structure of LDHs. They all had a good crystallinity and there were no other impurities in the samples [28]. The new peaks appeared in 15–20° might be caused by the coordinate bond which formed after DNA was adsorbed on the surface of LDHs. XRD is an effective measure for detecting the intercalation of LDH structure. The basal spacing of LDHs was around 0.7 nm before adsorption and tiny changes appeared on it after adsorption. The basal spacing of LDHs would be more than 2.0 nm if the intercalation happened because of the width of
Table 3 The Pseudo-second-order parameters for DNA adsorption on LDHs. Samples
MgZnAl–CO3–LDH-2 MgZnAl–CO3–LDH-3 MgAl–CO3–LDH-2 MgAl–CO3–LDH-3
qe
(experiment)
8.858 13.152 39.774 39.913
(mg/g)
Pseudo-second-order K2 (g mg1 min1)
qe (mg/g)
R2
0.502 1.550 0.364 2.220
8.648 12.720 39.806 39.730
0.983 0.987 0.999 0.999
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Fig. 4. Effect of solution pH on the adsorption of DNA by LDHs.
391
Fig. 7. XRD patterns of MgAl–CO3–LDH-3 at different pH.
Fig. 5. Schematic of equilibrium interfacial structure for DNA on LDH surface: top view of the initial configuration for DNA adsorption on LDH.
Fig. 8. FTIR spectra of MgAl–CO3–LDH-3 and MgAl–CO3–LDH-3-DNA.
tiny changes. So we can came to the conclusion that the damage of the structure of LDHs can be ignored during the experiment. FTIR analysis
Fig. 6. XRD patterns of LDHs and LDH-DNA.
DNA which was 2.0 nm [29]. The changes of the basal spacing on LDHs before and after adsorption may due to the loss of H2O molecules inside or on the surface of LDHs. It can be concluded that DNA does not intercalated into the interlayer of LDHs but just be adsorbed on the surface of them. Considering the effect of pH on the structure of LDHs, XRD (MgAl–CO3–LDH-3) were accordingly done to inspect if the LDHs were dissolved, and the result was showed in Fig. 7. The peaks of LDHs reflection were all around 0.75 nm and became a little weaker with the increasing of pH. Meanwhile, the intensity of the peaks increased in initial stage and then declined. The basic structure of MgAl–CO3–LDH-3 was still in good shape, though there were some
Fig. 8 showed the FTIR spectra of MgAl–CO3–LDH-3-DNA composition and the spectra of MgAl–CO3–LDH-3 which had a higher ability on DNA adsorption. The broad bands observed at 3445 cm1 and 3448 cm1 were attributed to hydroxyl (O–H) stretching vibrations, which were the same as the bands of 1643 cm1 and 1650 cm1 [30]. The adsorption band at 1356 cm1 and 1357 cm1 were assigned to CO2 3 . The appearances of other peaks were on account of the basic structure of LDH. Compared with the unmodified LDH, a new band centered at 1228 cm1 standing for the vibrational modes of PO3 4 , which was part of the basic structure of DNA after adsorption [31,32]. These results demonstrated that there were some DNA molecules being adsorbed on the LDH after adsorption experiment, which was in accordance with the XRD patterns. Moreover, comparing the two curves, the characteristic absorption peaks of LDH after adsorption were almost the same as before, which turned out that ion exchange reaction did not happen during adsorption. CD analysis Fig. 9 showed the CD spectra of native DNA and DNA desorbed from LDHs. As a method to monitor the conformational transition
392
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393
Fig. 9. CD spectra of native DNA and DNA desorbed by LDHs: (a) native DNA, (b) DNA desorbed by MgAl–CO3–LDH-2, (c) DNA desorbed by MgAl–CO3–LDH-3, (d) DNA desorbed by MgZnAl–CO3–LDH-2, (e) DNA desorbed by MgZnAl–CO3–LDH-3.
Fig. 11. Electrophoresis analysis for the native DNA and DNA desorbed from LDHs: Lane 1, marker; Lane 2, native DNA; Lane 3, DNA desorbed by MgAl–CO3–LDH-2; Lane 4, DNA desorbed by MgAl–CO3–LDH-3; Lane 5, DNA desorbed by MgZnAl– CO3–LDH-2; Lane 6, DNA desorbed by MgZnAl–CO3–LDH-3.
the length of native DNA was nearly 1000 bp. Compared to the native DNA in lane 2, the migration behaviors of the desorbed DNA were almost the same as native DNA, besides of the brightness of the strips, which due to the concentration of DNA. As a measurement to demonstrate the length of DNA, the results clearly showed that the process of adsorption did not affect the length of DNA, which revealed that the desorbed DNA still had the biological activity. Conclusion
Fig. 10. UV–vis spectra of native DNA and DNA desorbed by LDHs: (a) native DNA, (b) DNA desorbed by MgZnAl–CO3–LDH-3, (c) DNA desorbed by MgAl–CO3–LDH-2, (d) DNA desorbed by MgZnAl–CO3–LDH-2, (e) DNA desorbed by MgAl–CO3–LDH-3.
of DNA, the changes of the conformation of DNA before and after adsorption were presented accurately. The positive band at 276 nm and a negative band at 247 nm illustrated that the native DNA was a typical B-form [33,34]. Compared with the native DNA, the CD spectra of DNA desorbed from LDHs remained unchanged, revealing that the conformation of DNA did not change during the adsorption. UV–vis analysis The native DNA and released DNA were detected by UV–vis spectra. As shown in Fig. 10, the characteristic adsorption peak of native DNA was at 259 nm, which was caused by p ? p or n ? p transition of conjugated double bonds of purine and pyrimidine derivatives [35,36]. DNA released from LDHs was tested by UV–vis again, and it should be noted that the adsorption peaks of released DNA were all around 259 nm which were the same as the native DNA [37]. The results were in accordance with the results of CD spectra, proving that the DNA was still in good condition whether it was adsorbed on LDHs or not. Gel retardation analysis DNA was desorbed from LDHs after adsorption, as shown in lanes 3–6 (Fig. 11). The strip of native DNA (lane 2) indicated that
The findings of our observations revealed that as the adsorbents of DNA,binary LDHs have advantage over ternary LDHs and are prior to some other materials natural or man-made. The different results of the adsorption are mainly due to the surface charges of DNA and LDHs. Coulombic force takes an important part in the adsorption except for binary LDHs. The capacities of the binary LDHs on adsorbing the DNA are independent on the solution pH, revealing that there might be some other mechanisms in the adsorption between binary LDHs and DNA except for electrostatic force. XRD and FTIR results indicate that the process of intercalation do not happen during the adsorption but only for surface adsorption, on the other side, the conclusion that the structures of LDHs are not affected during adsorption is also confirmed through the results of XRD and FTIR. Meanwhile, the structure of DNA is not damaged during adsorption, which is proved by the results of CD, UV–vis spectra and electrophoresis. In summary, as a new adsorption material, it may be a potential drug carrier with wide application because of its excellent adsorption property and no biological toxicity. Acknowledgements This work was financially supported by the Research Fund for the Doctoral Program of Higher Education of China (No. 20100172110028), National Science Foundation of China (Grant Nos. 41273122, 41073058, 40973075). References [1] E. Gallori, M. Bazzicalupo, D.L. Canto, R. Fani, P. Nannipieri, C. Vettori, G. Stotzky, FEMS Microbiol FEMS Microbiol. Ecol. 15 (1994) 119–126. [2] S.A.E. Blum, M.G. Lorenz, W. Wackernagel, Syst. Appl. Microbiol. 20 (1997) 513–521. [3] C. Crecchio, G. Stotzky, Soil Biol. Biochem. 30 (1998) 1061–1067.
B. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 121 (2014) 387–393 [4] G. Stotzky, J. Environ. Qual. 29 (2000) 691–705. [5] C. Crecchio, P. Ruggiero, M. Curci, C. Colombo, G. Palumbo, G. Stotzky, Soil Sci. Soc. Am. J. 69 (2005) 834–841. [6] F. Widmer, R.J. Seidler, L.S. Watrud, Mol. Ecol. 5 (1996) 603–613. [7] E. Paget, M. Lebrun, G. Freyssinet, P. Simonet, Eur. J. Soil Biol. 34 (1998) 81–88. [8] P. Cai, Q. Huang, X.W. Zhang, H. Chen, Soil Biol. Biochem. 38 (2006) 471–476. [9] K. Saeki, M. Sakai, S.I. Wada, Appl. Clay. Sci. 50 (2010) 493–497. [10] C.A.I. Goring, W.V. Bartholomew, Soil Sci. 74 (1952) 149–164. [11] M.P. Greaves, M.J. Wilson, Soil Biol. Biochem. 1 (1969) 317–323. [12] G. Romanowski, M.G. Lorenz, W. Wackernagel, Appl. Environ. Microbiol. 57 (1991) 1057–1061. [13] M. Franchi, J.P. Ferris, E. Gallori, Orig. Life Evol. Biosph. 33 (2003) 1–16. [14] P. Cai, Q.Y. Huang, X.W. Zhang, Appl. Clay. Sci. 32 (2006) 147–152. [15] T.H. Nguyen, K.L. Chen, M. Elimelech, Biomacromol 11 (2010) 1225–1230. [16] G. Pietramellara, M. Franchi, E. Gallori, P. Nannipieri, Biol. Fert. Soils 33 (2001) 402–409. [17] K. Saeki, T. Kunito, M. Sakai, Microbes. Environ. 26 (2011) 88–91. [18] H. Nakayama, A. Hatakeyama, M. Tsuhako, Int. J. Pharm. 393 (2010) 105–112. [19] K. Ladewig, M. Niebert, Z.P. Xu, P.P. Gray, G.Q. Lu, Appl. Clay. Sci. 48 (2010) 280–289. [20] Y.Y. Wong, H.M. Cooper, K. Zhang, M. Chen, P. Bartlett, Z.P. Xu, J. Colloid Interface Sci. 369 (2012) 453–459. [21] M. Chen, H.M. Cooper, J.Z. Zhou, P.F. Bartlett, Z.P. Xu, J. Colloid Interface Sci. 390 (2013) 275–281.
[22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37]
393
J.H. Choy, M. Park, J.M. Oh, NanoBio. Techonol. 4 (2008) 349–367. H. Toshiyuki, O. Hitoshi, Appl. Clay. Sci. 45 (2009) 123–132. H. Freundlich, Z. Phys. Chem. 57 (1907) 385–471. P.X. Wu, Q. Zhang, Y.P. Dai, N.W. Zhu, Z. Dang, P. Li, J.H. Wu, X.D. Wang, Geoderma 164 (2011) 215–219. K. Saeki, T. Kunito, M. Sakai, Biol. Fertil. Soils 46 (2010) 531–535. Z.P. Xu, Y.G. Jin, S.M. Liu, Z.P. Hao, G.Q. Lu, J. Colloid Interface Sci. 326 (2008) 522–529. L.N. Sun, C.W. Hu, Mater. Res. Bull. 46 (2010) 1922–1927. H. Nakayama, A. Hatakeyama, M. Tsuhako, Int. J. Pharm. 393 (2010) 104–111. I. Ghorbel-Abid, A. Jrad, K. Nahdi, M. Trabelsi-Ayadi, Desalination 246 (2009) 595–604. E. Taillandier, J. Liquier, J.A. Taboury, in: R.J.H. Clarc, R.E., Hester (Eds.), Wiley, Heyden, 1985, pp. 65. I.D. Galina, Y.G. Nina, B.K. Elena, P.P. Olena, Talanta 53 (2000) 233–246. X.G. Sun, E.H. Cao, X.Y. Zhang, D.G. Liu, C.L. Bai, Inorg. Chem. Commun. 5 (2002) 181–186. P. Cai, Q.Y. Huang, X.W. Zhang, Sci. Technol. 40 (2006) 2971–2976. V.B. Morris, S. Neethu, T.E. Abraham, C.K.S. Pillai, C.P. Sharma, J. Biomed. Mater. Res. 89 (2009) 282. G.F. Zuo, Y.Z. Wan, X.G. Meng, Q. Zhao, K.J. Ren, S.R. Jia, J.H. Wang, Mater. Chem. Phys. 126 (2011) 470–475. J.H. Choy, S.Y. Kwak, J.S. Park, Y.J. Jeong, J. Portier, J. Am. Chem. Soc. 121 (1999) 1399–1400.
