Capture and alignment of phi29 viral particles in sub-40 nanometer porous alumina membranes
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- Moon, J., Akin, D., Xuan, Y. et al. Biomed Microdevices (2009) 11: 135. doi:10.1007/s10544-008-9217-0
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Bacteriophage phi29 virus nanoparticles and its associated DNA packaging nanomotor can provide for novel possibilities towards the development of hybrid bio-nano structures. Towards the goal of interfacing the phi29 viruses and nanomotors with artificial micro and nanostructures, we fabricated nanoporous Anodic Aluminum Oxide (AAO) membranes with pore size of 70 nm and shrunk the pores to sub 40 nm diameter using atomic layer deposition (ALD) of Aluminum Oxide. We were able to capture and align particles in the anodized nanopores using two methods. Firstly, a functionalization and polishing process to chemically attach the particles in the inner surface of the pores was developed. Secondly, centrifugation of the particles was utilized to align them in the pores of the nanoporous membranes. In addition, when a mixture of empty capsids and packaged particles was centrifuged at specific speeds, it was found that the empty capsids deform and pass through 40 nm diameter pores whereas the particles packaged with DNA were mainly retained at the top surface of the nanoporous membranes. Fluorescence microscopy was used to verify the selective filtration of empty capsids through the nanoporous membranes.
Keywordsphi29Nanoporous membraneAtomic layer depositionAlignmentCapture
For the separation, collection, and filtration of viruses, membrane-based technologies have been developed as useful and efficient methods (Lakshmi and Martin 1997; van Reis and Zydney 2001; Xu et al. 2005; Yang et al. 2006; Lee et al. 2002). Potentially, membranes are well adapted for these applications because their pore size and surface chemistry can be adjusted to the size and surface chemistry of an organism of interest, and the specific capture (selective sensing) of this organism could be possible. Several types of membranes have been employed for virus capture and separation; however, many of these have not been effective (van Voorthuizen et al. 2001; Otaki et al. 1998; Urase et al. 1996). For example, in case of microfiltration membranes, the pore size is typically much larger than the size of the virus particles that are tens of nanometers in size (van Voorthuizen et al. 2001; Otaki et al. 1998). The smaller pore size ultramembranes have uneven distribution of pore size, allowing virus particles to permeate into a small number of abnormally large pores (Urase et al. 1996). Although chemically specific filtration using nanoporous membranes has been shown for small molecules (Jirage et al. 1999; Lee and Martin 2001; Fernandez-Lopez et al. 2001; Chun and Stroeve 2002), specific bio-organism immobilization, capture, and detection remain to be a great technical challenge.
2.1 Material and reagents
High-purity Al foil (99.999%, 0.2 in. thickness) was purchased from Research and PVD Materials. Trimethylaluminum (TMA) and 3-(trimethoxysilyl)propyl aldehyde (TMSPA) were purchased from Aldrich and used without further purification. Deionized water was obtained from an ultrafiltration system (Milli-Q, Millipore) with a measured resistivity above 18 MΩcm and passed through a 0.22 μm filter to remove particulate matter.
2.2 Fabrication of nanoporous AAO membrane
2.3 Atomic layer deposition
We used an ASM F-120 ALD system for shrinking the pore size of AAO membrane. ALD Al2O3 films were deposited by using alternating pulse of trimethylaluminum (TMA) [Al(CH3)3] and water (H2O) precursors. H2O vapor was used to oxidize chemisorbed TMA. N2 was used as a carrier gas to transport precursor vapor to the membrane surface and purge reaction gases from the reactor during each half-reaction. The pulse time of TMA, H2O and N2 were 0.8 s, 1.2 s and 2.0 s, respectively. For our purpose, the deposition process was run for 133 to 211 cycles at 2–5 Torr at 300°C and the growth rate was 0.9 Å per cycle for Al2O3.
2.4 Array of phi29 procapsid on the alumina membranes
3% 3-(trimethoxysilyl)propyl aldehyde (TMSPA) solution was prepared in ethanol/H2O (95%/5%) and membranes were immersed to clean the ALD/AAO membrane for 3 h. After washing the aldehyde-silanized ALD/AAO with ethanol several times, the membranes were dried in a stream of nitrogen gas and heated at 110°C for 20 min. The aldehyde-silanized alumina membrane was incubated in a solution of procapsid (without pRNA; 0.01 mg/mL in TMS buffer) for 15 h at room temperature. The membrane was then immersed into fresh TMS buffer for 30 min. at room temperature to remove any unattached procapsids.
2.5 Procedure for the DNA-packaging of phi29 in vitro
Briefly, 1 μg of RNA in 1 μL RNase free H2O was mixed with 10 μL of purified procapsids (0.4 mg/ml) in TMS (100 mM NaCl, 10 mM MgCl2, 50 mM Tris, pH 7.8) and then incubated for 60 min at room temperature. The pRNA-enriched procapsids were then mixed with 3 μL of ATP (10 mM in TMS), 6 μL of gp16 (0.5 mg/mL), and 1 μL of DNA-gp3 (200 ng/μL) and incubated 60 min at room temperature to complete the DNA-packaging reaction. We used these DNA-packaged phi29 particles for the experiments described in subsequent sections.
3 Results and discussion
In this paper, we report the use of anodized aluminum oxide (AAO) membranes with narrow pore size distribution and chemically modifiable surfaces for alignment and capture of phi29 particles. Highly ordered nanoporous AAO membranes were prepared by a two-step anodization process similar to described above and also previously (Moon and Wei 2005), resulting in structures as shown in Fig. 2(a) and (b).
The nanopores of these AAO films are still big compared to the phi29 nanomotors. Next, we decreased the pore sizes using atomic layer deposition (ALD) which significantly shrinks but does not fully seal the nanopores (Xiong et al. 2005; Miikkulainen et al. 2008). ALD is a promising technique to form ultrathin films uniformly over a large wafer and 3-D structures such as deep trenches and deep holes with atomic-scale thickness controllability. ALD consists of two half reactions: (1) self-limiting chemisorption of metal-organic or metal-halide precursor on the wafer surface and (2) reaction of the chemisorbed these species with oxygen-containing species such as water vapor, O2, O3, or metal alkoxide. Generally ALD-growth is carried out at a relative low temperature (ca. 300°C) because thermal decomposition of precursor at higher temperatures deteriorates the thickness controllability. After the ALD process was completed, field-emission scanning electron microscope (FE-SEM; Hitachi S-4800) analysis of nanoporous ALD/AAO membranes revealed pore diameters on the order of 39 nm, 25 nm, and 15 nm, respectively, with increasing times used for the deposition (Fig. 2(c)–(h)).
In order to attach the biomaterial particles to the pores only and not to the top surface, the aldehyde-silanized ALD/AAO membranes were polished for 10 s with 0.06 μm polishing cloth (Red-Final C; Allied High Tech Products) to remove the aldehyde-silanized layer from the face of the alumina surface. After polishing, the membrane was placed into a solution containing 0.01 mg/ml of procapsid dispersed in the TMS buffer for 4 h. The resulting FESEM images in Fig. 4(d, e) show that indeed the particles were mostly attached in the pores. The histogram in Fig. 4(f) represents the quantity of particles attached to the membranes. The unpolished membrane shows that 50% of the biomaterial particle binding occurred in the pores and 50% out of the pores. In contrast, the polished membrane shows particle binding to be localized at about 84% in the pores and about 16% out of the pores.
We suggest that the empty procapsid undergoes structural changes due to centrifugal force (1680 x g) during the centrifugation based filtering. The capsids have thin walls compared to their diameters, for example, the wall thickness of the phi29 capsid is ∼1.5 nm, whereas its linear dimensions are on the order of 40–50 nm (Tao et al. 1998). Moreover, the protein: protein interactions within the empty procapsids may not be fully stabilized. These factors might allow the empty procapsid the flexibility to deform sufficiently to traverse through a pore smaller than its own physical dimensions.
Many important and also related aspects of bacteriophage phi29 morphogenesis and mechanoelastic properties have not been fully investigated yet. One of these aspects is the influence of the enclosed viral DNA on the mechanical properties of the viral particle and its stability. The prohead of phi29 has 10 hexameric units in its cylindrical equatorial region, and 11 pentameric and 20 hexameric units comprise icosahedral end-caps with T = 3 quasi-symmetry (Tao et al. 1998). In a recent study, under identical conditions, the comparison of the spring constants of the empty capsid and the viron of minute virus of mice (MVM), known to have icosahedral (T = 1) symmetry, showed that the presence of the genomic DNA leads to an increase of the particle stiffness by 3%, 42%, and 140% when probed along fivefold, threefold, and twofold symmetry axes, respectively (Carrasco et al. 2006). The DNA molecule could reinforce the particle by coating the internal surface of the capsid, thus increasing the effective capsid wall thickness homogeneously. By structural analogy, we believe this phenomenon may also explain why DNA-packaged phi29 did not traverse the AAO membrane pores in our case; however, more detailed studies are needed to fully characterize this effect.
Our studies lay the ground work for interfacing viral nanoparticles with the nanoporous AAO membranes. We used atomic-layer deposition to precisely shrink the pore size of the nanoporous alumina membranes down to 15 nm. In addition, the alignment of viral particles on the pores was demonstrated either by chemical functionalization and polishing or by a centrifugation process. In particular, the nanoporous ALD/AAO membranes can also be used to separate fully DNA-packaged and unpackaged phi29 particles from a mixture by the centrifugation method. The centrifugation while using the nanoporous membranes can be used for the filtration, purification, and concentration of various viruses.
We acknowledge the funding from the National Institutes of Health through the NIH Roadmap for Medical Research (PN2 EY 018230 Nanomedicine Development Center), and NIH R21 EB007474 FE-SEM images were taken at the Birck Nanotechnology Center Microscopy Facility. We also thank Dr. D. Sherman for the TEM micrographs.