Analytical Biochemistry 398 (2010) 120–122
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles Zhi Shan a, Qi Wu a, Xianxiang Wang a, Zhongwu Zhou a, Ken D. Oakes b, Xu Zhang b, Qianming Huang a, Wanshen Yang a,* a b
Faculty of Science, Sichuan Agricultural University, Yaan 625014, PR China Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
a r t i c l e
i n f o
Article history: Received 30 July 2009 Received in revised form 17 October 2009 Accepted 3 November 2009 Available online 10 November 2009
a b s t r a c t A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic DNA/protein complex after lysis can be achieved simply by magnetic separation. Furthermore, the yield and purity of pDNA extracted by MNPs are comparable to those obtained using organic solvents or commercial kits. This time- and costeffective protocol does not require centrifugation or precipitation steps and has the potential for automated DNA extraction, especially within miniaturized lab chip applications. Ó 2009 Elsevier Inc. All rights reserved.
Because plasmid DNA (pDNA)1 is routinely used as a genetic engineering vector, the development of a rapid, simple, and costeffective pDNA extraction method is of considerable advantage. Conventional pDNA extraction techniques (requiring centrifugation, precipitation, and chromatography separation) are not easily adapted to automated systems. In contrast, magnetic nanoparticle extraction methods demonstrate remarkable simplicity owing to the nature of the particles, which serve as a DNA adsorbent within biological matrices [1,2]. To date, an array of surface-modified magnetic nanoparticles (MNPs), both chemically and biologically synthesized [1–9], have been successfully used for pDNA purification. Various procedures have been developed using MNPs with carboxyl [2–4], hydroxyl [5,6], and amino functional groups [7–9]; however, the underlying mechanism is the same, namely, adsorbing pDNA from cleared lysate [4–9]. Most MNP procedures involve two centrifugation steps: the first to harvest cells from liquid culture and the second to pellet denatured genomic DNA/protein complexes after cell disruption and neutralization. These centrifugation procedures are both time- and labor-intensive; moreover, this step was not amenable to the miniaturization and automation required of high-throughput biological sample preparation [2]. Furthermore, the potential for shearing damage to biomacromolecules during extensive centrifugation is unavoidable.
* Corresponding author. Fax: +86 835 2886136. E-mail address:
[email protected] (W. Yang). 1 Abbreviations used: pDNA, plasmid DNA; MNP, magnetic nanoparticle; CCE, cell capture efficiency; OD, optical density; SDS, sodium dodecyl sulfate; EDTA, ethylenediaminetetraacetic acid; PEG, polyethylene glycol; UV, ultraviolet. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.11.006
The current study demonstrated a centrifugation-free procedure for pDNA extraction from Escherichia coli using carboxyl-modified MNPs. This approach used magnet-assisted separation to harvest both E. coli cells from a fermentation culture and denatured genomic DNA/protein complexes after lysis. The quality and quantity of magnetic particle-purified pDNA were confirmed by comparison against pDNA obtained using organic solvents and a commercially available purification kit. Carboxyl-modified superparamagnetic nanoparticles were used as a multifunctional bioadsorbent [10]. FeCl3 and FeSO4 (in a molar ratio of 1.65 in a 4-M NaOH solution) were used to prepare Fe3O4 MNPs [4]. The Fe3O4 MNPs were later separated and further coated by polymerization of monomer methacrylic acid using an emulsion polymerization approach [10]. The coated MNPs were washed five times with deionized water to remove residual methacrylic acid monomers and other impurities. Then MNPs were dispersed in deionized water under sonication for 10 min to form a stable magnetic nanofluid (17 mg/ml by dry weight) that can be stored for several months at room temperature. The cell capture efficiency (CCE) of MNPs was evaluated using E. coli JM109 containing pET 15b vector as a model organism. Cells were grown in Luria–Bertani medium (pH 7.0) containing 50 lg/ml ampicillin at 37 °C overnight. For optimization of cell capture, two experiments were performed. The first investigated the effect of pH on CCE. In this experiment, 10 ll of MNPs was added to 1.5 ml of differing E. coli concentrations (determined by optical density [OD] at 600 nm), with OD values ranging from 0.07 to 1.0 using cell-free culture supernatant as diluent. Each cell concentration was pH adjusted (using 1 M HCl) to achieve a range of pH values (1.18–7.0). Then Eppendorf tubes containing the cells (varying in
Notes & Tips / Anal. Biochem. 398 (2010) 120–122
pH and concentration) were placed in a magnetic field generated by a Promega magnetic separation stand for 10 min [4]. After immobilization of the cell/nanoparticle complexes, the supernatant was assessed for optical density at 600 nm. The second experiment addressed the influence of MNP concentration on CCE, where the E. coli cell concentration was held constant (OD600 of 1.0, 1.5 ml). In this experiment, the magnetic separation time was set at 3 min, and adjusted pH values again ranged between 1.18 and 7.0 while the volume of MNP fluid added to each cell aliquot ranged between 10 and 75 ll. The CCE was calculated in terms of the difference in optical density before and after separation: CCE (%) = 100(a b)/a, where a and b represent the OD600 readings before and after magnetic separation, respectively. The results indicated that capture of cells with MNPs was essentially a pH-dependent process (Fig. 1). Less than 10% of total E. coli cells were captured between pH 5.0 and 7.0. However, when the pH was below 5.0, CCE increased sharply, achieving upward of 90% capture between pH 2.3 and 3.2 (Fig. 1A). E. coli bacteria cell walls possess a negative charge under neutral conditions owing to abundant carboxyl, phosphoryl, and hydroxyl groups present in macromolecules of the cell wall [11–13]. When these functional groups are protonated with acid (or negative charges are neutralized with cationic polymers), the cells readily aggregate within
Fig. 1. Cell capture efficiency as a function of fermentation culture pH. (A) Cell densities (OD600) ranged between 0.07 and 1.0, whereas the separation time and MNP volume were 10 min and 10 ll, respectively. (B) MNP volumes ranged between 10 and 75 ll, whereas the separation time and cell density (OD600) were 3 min and 1.0, respectively.
121
the culture medium, as described previously [12–14]. Hence, the addition of HCl will strongly suppress dissociation of these ionic groups on the cell wall [12–14] and carboxyl groups on the MNP surface, resulting in colloidal instability and flocculation of both E. coli cells and MNPs. The resultant cell/MNP complexes present within low-pH environments contributed to the increased CCE. However, a slight CCE decrease was observed below pH 2.0 (Fig. 1A), possibly due to partial restabilization of E. coli cells as a result of amino group ionization on the cell surface. Concentrations of cells and MNPs also played an important role in CCE. For instance, at pH 3.8 the highest capture efficiency (96.3%) was observed at an OD600 of 1.0, whereas at lower cell densities (OD600 of 0.07) the CCE dropped to 62.1% (Fig. 1A). Furthermore, a slight increase in CCE was observed with higher MNP concentrations (Fig. 1B), suggesting coflocculation as the bacteria capture mechanism given that high concentrations of cells and/or MNPs increased CCE. The main advantage of using more MNPs (35–75 ll) for capture was a significant reduction in separation time. Only the lowest MNP addition (10 ll) failed to achieve capture efficiencies greater than 90% (within appropriate pH ranges of 2.0–3.2) with only 3 min of magnetic separation (Fig. 1B). For subsequent studies, optimized conditions (50 ll of MNPs, pH 3.2) were used for cell capture. Typically, more than 95% of E. coli cells could be recovered within 1.5 min from 1.5 ml of overnight culture with OD600 higher than 0.2. The separated cell/MNP complexes could then be easily lysed for pDNA extraction. The ease of lysis can be ascribed to the nonuniform cell aggregation exhibited by the MNPs (Fig. 2A), that, rather than uniformly covering the entire cell surface, leave E. coli membranes susceptible to sodium dodecyl sulfate (SDS) and alkaline solution. Cell lysis and pDNA purification details were as follows. Once the cell/MNP complexes were firmly immobilized on the tube wall, the culture supernatant was discarded. Captured cell/MNP complexes were resuspended in 100 ll of solution I (25 mM Tris [pH 8.0], 10 mM ethylenediaminetetraacetic acid [EDTA], and 400 lg/ ml RNase A), followed by the addition of 200 ll of solution II (0.2 M NaOH and 1% [w/v] SDS). The resultant mixture was incubated on ice for 3 min after gentle mixing. Genomic DNA, proteins, and other cell debris were precipitated with the addition of 150 ll of solution III (3 M potassium acetate, pH 5.5). The precipitate with trapping MNPs inside was separated magnetically. The cleared alkaline lysate supernatant was transferred to a new 1.5-ml Eppendorf tube for pDNA extraction. A 1/10 volume of MNPs was added to the tube and well mixed with the supernatant by five pipetting cycles, followed by mixing with an equal volume of binding buffer (15% PEG [polyethylene glycol] 8000 and 2.5 M NaCl) [4]. The MNPs were immobilized, and the supernatant was removed using a pipet. The pellet was rinsed with 750 ll of cold 70% ethanol. After removal and evaporation of the ethanol, the pDNA was eluted in 50 ll of TE buffer (10 mM Tris [pH 8.0] and 1 mM EDTA) at room temperature for 0.5 min. The MNPs were then immobilized with the supernatant transferred to a DNase/RNase-free Eppendorf tube. To compare the MNP technique against existing methods, pDNA extraction was also performed using organic solvents and a commercial kit (Qiagen, USA) according to the molecular cloning laboratory manual [15] and the manufacturer’s instructions, respectively. The yield and purity of pDNA were analyzed by ultraviolet (UV) spectroscopy and agarose gel electrophoresis. The bands were visualized under UV light by GoldView staining (SBS Genetech, China) using the Gel Doc XR System (Bio-Rad, USA). In an evaluation of extraction methods (performed in triplicate), comparable pDNA was extracted by MNPs (8.57 ± 0.13 lg) from 1.5 ml of overnight culture as was extracted with organic solvents (10.36 ± 0.11 lg) and by the commercial kit (9.32 ± 0.05 lg), as illustrated in Fig. 2B. The pDNA purity, as estimated by the
122
Notes & Tips / Anal. Biochem. 398 (2010) 120–122
(Fig. 2B) with BglII and EcoRI (Sangon, China), indicating the feasibility of the purified DNA for downstream applications. Because the recovery of cells, the removal of floc after neutralization, and the purification of pDNA all were accomplished with rapid magnetic separation rather than centrifugal processes, the entire procedure required less than 20 min, whereas the comparable established methods took at least 30 min. Furthermore, the current method required less handling and no hazardous reagents such as phenol and chloroform. The setup costs associated with the current method are much less than those of centrifugation-dependent methods. In addition, the E. coli cells can be captured by carboxylated MNPs rather than expensive immunomagnetic particles. In summary, multifunctional MNPs proved to be a time- and cost-effective pDNA preparation technique independent of centrifugation and hazardous organic solvents. Not only are the pH-sensitive magnetic nanoparticles well suited for routine laboratory use, but also the simplicity of this approach indicates their potential for automated pDNA purification. Acknowledgments This work was supported by a research grant from Sichuan Agricultural University. The authors thank Yi Zhou for transmission electron microscope (TEM) imaging and helpful discussions. References
Fig. 2. (A) Transmission electron microscope (TEM) image of E. coli cells captured with MNPs. The scale bar represents 250 nm. (B) Agarose gel electrophoresis of pDNA digested with two restriction endonucleases (BglII and EcoRI). Lanes 1, 2, and 3: pDNA extracted with standard organic solvents, a commercial Qiagen kit, and MNPs, respectively; lanes 4, 5, and 6: restriction digestion of pDNA extracted with standard organic solvents, the Qiagen kit, and MNPs, respectively; lane M: DNA molecular weight markers (fragment sizes 0.5, 1.0, 2.0, 3.5, 5.5, and 7 kb).
OD260/OD280 ratio, was approximately 1.8, indicating that the extracted DNA was pure with negligible protein contamination. The compatibility of the pDNA isolated by the current method was demonstrated by a successful double restriction digestion
[1] Z.M. Saiyed, C. Bochiwal, H. Gorasia, S.D. Telang, C.N. Ramchand, Application of magnetic particles (Fe3O4) for isolation of genomic DNA from mammalian cells, Anal. Biochem. 356 (2006) 306–308. [2] X. Xie, X. Nie, B. Yu, X. Zhang, Rapid enrichment of leucocytes and genomic DNA from blood based on bifunctional core–shell magnetic nanoparticles, J. Magn. Magn. Mater. 311 (2007) 416–420. [3] T.R. Sarkar, J. Irudayaraj, Carboxyl-coated magnetic nanoparticles for mRNA isolation and extraction of supercoiled plasmid DNA, Anal. Biochem. 379 (2008) 130–132. [4] Z. Shan, W. Yang, X. Zhang, Q. Huang, H. Ye, Preparation and characterization of carboxyl-group functionalized superparamagnetic nanoparticles and the potential for bio-applications, J. Braz. Chem. Soc. 18 (2007) 1329–1335. [5] M.J. Davies, J.I. Taylor, N. Sachsinger, L.J. Bruce, Isolation of plasmid DNA using magnetite as a solid phase adsorbent, Anal. Biochem. 262 (1998) 92–94. [6] C.L. Chiang, C.S. Sung, C.Y. Chen, Application of silica-magnetite nanocomposites to the isolation of ultrapure plasmid DNA from bacterial cells, J. Magn. Magn. Mater. 305 (2006) 483–490. [7] C.L. Chiang, C.S. Sung, T.F. Wu, C.Y. Chen, C.Y. Hsu, Application of superparamagnetic nanoparticles in purification of plasmid DNA from bacterial cells, J. Chromatogr. B 822 (2005) 54–60. [8] X. He, H. Huo, K. Wang, W. Tan, P. Gong, J. Ge, Plasmid DNA isolation using amino-silica coated magnetic nanoparticles (ASMNPs), Talanta 73 (2007) 764– 769. [9] B. Yoza, A. Arakaki, T. Matsunaga, DNA extraction using bacterial magnetic particles modified with hyperbranched polyamidoamine dendrimer, J. Biotechnol. 101 (2003) 219–228. [10] S. Yu, G.M. Chow, Carboxyl group (–CO2H) functionalized ferrimagnetic iron oxide nanoparticles for potential bio-applications, J. Mater. Chem. 14 (2004) 2781–2786. [11] M. Torimura, S. Ito, K. Kano, T. Ikeda, Y. Esaka, T. Ueda, Surface characterization and on-line activity measurements of microorganisms by capillary zone electrophoresis, J. Chromatogr. B 721 (1999) 31–37. [12] S. Barany, A. Szepesszentgyörgyi, Flocculation of cellular suspensions by polyelectrolytes, Adv. Colloid Interface Sci. 111 (2004) 117–129. [13] K.E. Eboigbodin, J.J. Ojeda, C.A. Biggs, Investigating the surface properties of Escherichia coli under glucose controlled conditions and its effect on aggregation, Langmuir 23 (2007) 6691–6697. [14] S.P. Strand, M.S. Vandvik, K.M. Vrum, K. Stgaard, Screening of chitosans and conditions for bacterial flocculation, Biomacromolecules 2 (2001) 126–133. [15] J. Sambrook, D.W. Russell, Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.
