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Superparamagnetic Nanoparticle-Polystyrene Bead Conjugates as Pathogen Capture Mimics: A Parametric Study of Factors Affecting Capture Efficiency and Specificity Arnold J. Kell,† Kanchana Somaskandan,† Gale Stewart,‡ Michel G. Bergeron,‡ and Benoit Simard*,† Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex DriVe, Ottawa, Ontario, Canada K1A 0R6, and Centre de Recherche en Infectiologie, Centre Hospitalier UniVersitaire de Que´ bec, PaVillon CHUL, UniVersite´ LaVal, Que´ bec City, Que´ bec, Canada G1V 4G2 ReceiVed September 18, 2007 There is currently significant interest in the miniaturization of disease detection platforms. As detection platforms decrease in size there is a need for the development of sample preparation protocols by which cells or biomarkers of interest can be concentrated from large volumes down to volumes more amenable to analysis within microfluidic devices. To address this issue, we present a series of magnetic confinement assays for polystyrene (PS) beads mediated through their covalent modification with a series of superparamagnetic nanoparticles, where the PS beads have many properties similar to bacteria, but are not pathogenic. The magnetic confinement of the PS beads is investigated as a function of (1) the overall nanoparticle size, (2) the loading of superparamagnetic content within the nanoparticle matrix, and (3) the viscosity and volume of the dispersion medium. We demonstrate that the time required for the magnetic capture of the PS beads by the superparamagnetic nanoparticles (1) decreases as the loading of superparamagnetic material into the nanoparticles increases and (2) increases as the viscosity and volume of the dispersion medium are increased. However, limitations in the magnetic confinement efficiency for the PS beads labeled with nanoparticles comprised of low loadings of superparamagnetic material can be overcome through the use of magnetic columns. These magnetic columns provide a practical and fast mode of sample preparation that should facilitate the magnetic concentration of cells and biomarkers from large volumes to volumes more amenable to incorporation into a microfluidic-based analysis system, where they can be analyzed/detected.
Introduction Biodiagnostics has risen to the forefront of focus for many research groups throughout the world, where it is believed that the early diagnosis of infectious diseases can potentially save thousands of lives each year.1 In seeking ways to decrease the time required to detect pathogenic infections, there is a lot of interest in utilizing microfluidic platforms. Within these platforms, it has been shown that harmful bacteria cells can be ruptured to liberate their genomic DNA,2 and the bacteria can be subsequently detected and identified based on this genomic DNA fingerprint within a microarray.3 However, if µ-TAS platforms are to reach their full potential with respect to rapidly detecting and identifying general bacterial infections, there are many challenges that must be addressed with respect to sample preparation. For example, a clinical blood sample that is to be tested for the presence of a bacteria infection will generally be as much as 10-20 mL in volume, whereas the capacity of most microfluidic devices is, at most, 100-200 µL. As such, it is critical that sample preparation methods be developed that can concentrate or harvest the bacteria cells of interest from clinical samples into volumes more suitable for analysis within a microfluidic device for analysis (i.e., from 10 to 20 mL to 100-200 µL). Functionalized superparamagnetic nanoparticles have already shown utility in the magnetic capture * Corresponding author. Email:
[email protected]. † National Research Council of Canada. ‡ Universite ´ Laval. (1) Picard, F. J.; Bergeron, M. G. Drug DiscoVery Today 2002, 7, 1092-1101. (2) Kido, H.; Micic, M.; Smith, D.; Zoval, J.; Norton, J.; Madou, M. Colloids Surf., B 2007, 58, 44-51. (3) Peytavi, R.; Raymond, F. R.; Gagne, D.; Picard, F. J.; Jia, G.; Zoval, J.; Madou, M.; Boissinot, K.; Boissinot, M.; Bissonnette, L.; Ouellette, M.; Bergeron, M. G. Clin. Chem. 2005, 51, (10) 1836-1844.
of some pathogen cells,4-7 disease biomarkers,8,9 and DNA10-12 and are strong candidates to assist in the sample preparation/ concentration of bacteria for detection in microfluidic devices. As such, we are interested in exploring the ability of a series of silica-encapsulated superparamagnetic iron oxide nanoparticles to mediate the magnetic capture of polystyrene beads (PS beads). PS beads have many physical properties in common with bacteria, including density, size, and shape and should allow us to ascertain if these nanoparticles are good candidates for sample preparation assays for pathogen detection in microfluidic devices. PS beads are also advantageous as they allow us to covalently attach the superparamagnetic nanoparticles to label the PS bead rather than utilizing specific antigen-antibody interactions, as would be required if a real bacteria were employed. Covalent labeling allows us to neglect problems arising from differences in avidity and steric hindrance between the nanoparticles of different sizes and the surface of the bacteria cell. It also allows us to neglect differences in ligand surface coverage on the nanoparticle because even if one bond forms between the nanoparticle and the PS (4) Gu, H.; Xu, K.; Xu, C.; Xu, B. Chem. Commun. 2006, 941-949. (5) Ho, K.-C.; Tsai, P.-J.; Lin, Y.-S.; Chen, Y.-C. Anal. Chem. 2004, 76, 7162-7168. (6) Lin, Y.-S.; Tsai, P.-J.; Weng, M.-F.; Chen, Y.-C. Anal. Chem. 2005, 77, 1753-1760. (7) Yitzhaki, S.; Zahavy, E.; Oron, C.; Fisher, M.; Keysary, A. Anal. Chem. 2006, 78, 6670-6673. (8) Georganopoulou, D. G.; Chang, L.; Nam, J.-M.; Thaxton, C. S.; Mufson, E. J.; Klein, W. L.; Mirkin, C. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 22732276. (9) Lin, P.-C.; Chou, P.-H.; Chen, S.-H.; Liao, H.-K.; Wang, K.-Y.; Chen, Y.-J.; Lin, C.-C. Small 2006, 2, 485-489. (10) Ashtari, P.; He, X.; Wang, K.; Gong, P. Talanta 2005, 67, 548-554. (11) Bruce, I. J.; Sen, T. Langmuir 2005, 21, 7029-7035. (12) Stoeva, S. I.; Huo, F.; Lee, J.-S.; Mirkin, C. A. J. Am. Chem. Soc. 2005, 127, 15362-15363.
10.1021/la7037476 CCC: $40.75 © 2008 American Chemical Society Published on Web 02/22/2008
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bead the two should remain coupled indefinitely. This would not be the case for inherently weak antibody/antigen based interactions. Finally, because the PS beads we employ are fluorescent, the magnetic confinement can be monitored via fluorescence spectroscopy rather than relying on culturing and plate counting the bacteria or other specialized microbiology characterization methods. The labeling of the PS beads with the silica-encapsulated iron oxide nanoparticles imparts superparamagnetic character to the bacteria mimic. This will make the PS beads responsive to the presence of an external magnetic field and allow them to be magnetically confined or concentrated from solution. We are interested in using this investigation as a model study to determine how the magnetic confinement of the PS bead is affected by (1) the overall size of the nanoparticles covalently anchored to its surface, (2) the loading of superparamagnetic material in the nanoparticle matrices, and (3) the viscosity and volume of the dispersion medium (i.e., to mimic a confinement carried out in blood, where blood is ∼3 times more viscous than water). Ultimately, the results of this investigation will allow us to elucidate how the architecture and properties of a variety of nanoparticles affect their ability to participate in assays for the rapid and efficient preparation of small volume biological samples for introduction of substrates onto microfluidic platforms for analysis and identification.
Results and Discussion 1. Silica Encapsulation of Magnetic Nanoparticles and Subsequent Surface Modification. As previously reported, commercially available, water soluble ferrofluid nanoparticles (mixture of Fe2O3 and Fe3O4, FexOy, EMG 304 Ferrotec, USA) (1-NP) can be encapsulated in silica using the Sto¨ber sol-gel process to generate
[email protected] Though the supplier indicated that a mixture of iron oxide species are present in the FexOy ferrofluid, we have previously determined the nanoparticles to be comprised almost entirely of Fe2O3.15 The overall diameter of the resulting nanoparticles can be controlled by changing the concentration of tetraethoxyorthosilane (TEOS) employed in the synthesis.13,15 For example, the overall diameters of the nanoparticles can be increased from ∼45 nm (2-NP) to ∼73 nm (3-NP) by increasing the relative concentration of TEOS employed in the reaction by 4-fold (see Supporting Information). TEM images of 1-NP, 2-NP, and 3-NP are shown in Figure 1. We found that significantly decreasing the thickness of the silica shell results in the irreversible aggregation of the nanoparticles. This has been reported previously and is due, primarily, to the population of 1-NP seed nanoparticles larger than 20 nm in diameter. These large nanoparticles are known to exhibit ferromagnetic behavior, which facilitates nanoparticle aggregation. However, when the silica shell is at least 10 nm thick, the core-core interactions responsible for the aggregation are screened and the resulting nanoparticles remain readily dispersible following centrifugation or magnetic confinement.15,16 Following silica encapsulation, 2-NP and 3-NP can be functionalized with amine and carboxylic acid groups as highlighted in Scheme 1. Though simple, these two functional groups have tremendous utility, allowing for the covalent conjugation of essentially any biomolecule to the surface of these nanoparticles. Within the (13) Lu, Y.; Yin, Y.; Mayers, B. T.; Xia, Y. Nano Lett. 2002, 2, 183-186. (14) Ma, D.; Guan, J.; Normadin, F.; Denommee, S.; Enright, G.; Veres, T.; Simard, B. Chem. Mater. 2006, 18, 1920-1927. (15) Ma, D.; Veres, T.; Clime, L.; Normandin, F.; Guan, J.; Kingston, D.; Simard, B. J. Phys. Chem. C 2007, 111, 1999-2007. (16) Philipse, A. P.; van Bruggen, M. P. B.; Pathmamanoharan, C. Langmuir 1994, 10, 92-99.
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context of this investigation, the functional groups are valuable as they allow us to covalently anchor the nanoparticles to the surface of the PS beads.17 Both 2-NP and 3-NP can be modified with 3-aminopropylmethyldiethoxysilane (APDEMS) directly following silica encapsulation, generating 2(NH2)-NP and 3(NH2)-NP, respectively. These modifications were characterized via chemical modification with fluorescamine. Fluorescamine is a unique molecule that becomes luminescent only following reaction with a primary amine. The quantity of amine in a given sample of 2(NH2)-NP and 3(NH2)-NP can be measured by comparing the fluorescence intensity of the fluorescamine-modified NP in comparison to the fluorescence intensity of a model reaction with known concentrations of ethanolamine.18-21 The relative amine surface coverages for 2(NH2)-NP and 3(NH2)-NP are 3.5 and 2.3 amines/nm2, respectively. This data is presented in the Supporting Information. The amine-modified particles can be easily converted to carboxylic acid-modified nanoparticles, 2(COOH)-NP and 3(COOH)-NP, via reaction with a 1% (w/v) solution of succinic anhydride in anhydrous DMF.21,22 The extent of succinic anhydride modification for the nanoparticles was qualitatively verified by the lack of reactivity in the presence of fluorescamine, suggesting that all accessible amines were converted to carboxylic acid groups. Examination of the resulting amine- and carboxylic acid-modified nanoparticles by TEM indicates the surface modification does not change the diameter of the nanoparticles (Figure 2). 2. Effect of Nanoparticle Size on the Magnetic Confinement Time and Efficiency of Polystyrene Beads (Bacteria Mimics). In order to impart superparamagnetic character to the PS beads, the silica-encapsulated iron oxide nanoparticles were covalently coupled to them. Though the experiments highlighted within the manuscript focus exclusively on carboxylic acid-modified nanoparticles reacting with amine-terminated PS beads (PSNH2), similar results were obtained with amine-terminated nanoparticles and carboxylic acid-modified PS beads. These results are included in the Supporting Information. The general reaction scheme employed to label the PS beads with nanoparticles is depicted in Scheme 2. Specifically, amine-terminated fluorescent PS beads (PS-NH2, 1.1 µm diameter) were covalently modified with a large excess of 2(COOH)-NP or 3(COOH)NP, where the goal is to elucidate how the size of the nanoparticle affects the magnetic capture efficiency of the PS bead. Specific details of the synthesis are provided in the Supporting Information. Because PS-NH2 is fluorescent, its 2(COOH)-NP- and 3(COOH)-NP-mediated magnetic confinement can be monitored by fluorescence spectroscopy as demonstrated in Figure 3. For example, the fluorescence emission signal is strong for 2(COOH)-NP-modified PS-NH2 (Figure 3A(i)) and 3(COOH)NP-modified PS-NH2 (Figure 3B(i)) immediately following surface labeling, whereas the intensity decreases by >80% following magnetic confinement (panels A(ii) and B(ii) of Figure 3, respectively) with a rare earth magnet. Though both 2(COOH)-NP and 3(COOH)-NP are able to confine PS-NH2 efficiently, the time required for >80% confinement mediated by 3(COOH)-NP is significantly longer than the analogous (17) Hermanson, G. T. Bioconjugate Techniques; Academic Press: San Diego, 1996. (18) DeBernardo, S.; Weigele, M.; Toome, V.; Manhart, K.; Leimgruber, W.; Leimgruber, W.; Weigele, M. Bo¨hlen, P.; Stein, S.; Udenfriend, S. Arch. Biochem. Biophys. 1974, 163, 390-399. (19) Udenfriend, S.; Stein, S.; Bo¨hlen, P.; Dairman, W. Leimgruber, W.; Weigele, M. Science 1972, 178, 871-872. (20) Diaz-Quijada, G. A.; Wayner, D. D. M. Langmuir 2004, 20, 9607-9611. (21) Kell, A. J.; Simard, B. Chem. Commun. 2007, 1227-1229. (22) Mahalingam, V.; Onclin, S.; Peter, M.; Ravoo, B. J.; Huskens, J.; Reinhoudt, D. N. Langmuir 2004, 20, 11756-11762.
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Figure 1. The silica (SiO2) coating of 1-NP (10 nm) (A) through the Sto¨ber synthesis to generate 2-NP (45 nm) (B) and 3-NP (73 nm) (C).
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Scheme 1. The General Reaction Scheme for the Modification of the Silica-Encapsulated Nanoparticlesa
a
Amine-modified nanoparticles are generated from aminopropylmethyldiethoxysilane (APDEMS) (A). Carboxylic acid-modified nanoparticles are prepared in DMF solvent with 1% w/v succinic anhydride (B)
capture by 2(COOH)-NP as highlighted in the bar graph in Figure 3C (32 vs 14 min). Not surprisingly, the magnetic confinement efficiency of PS-NH2 also scales with the nanoparticle surface coverage as evidenced by TEM. When PS-NH2 is covalently modified with a dense layer of 2(COOH)-NP or 3(COOH)-NP (Figure 3Di and 3Ei, respectively), the confinement time is minimized. Conversely, when 2(COOH)-NP or 3(COOH)-NP nonspecifically binds to the surface of PS-NH2 in the absence of EDC and NHS, the coverage is very sparse (panels D(ii) and E(ii) of Figure 3) and the confinement is very inefficient at short timescales (28 ( 3% and 2 ( 1%, respectively). It should also be noted that the surface coverage of 2(COOH)-NP on PS-NH2 is also dependent on the concentration of NP employed in the covalent coupling reaction (see Supporting Information), where significantly lowering the concentration of nanoparticle results in a more sparse surface coverage and a slower magnetic confinement. To rationalize the difference in time required for the magnetic confinement of PS-NH2 by 2(COOH)-NP or 3(COOH)NP, there are several characteristics inherent to each of the nanoparticles that must be considered. First, it should be noted that the surface coverage of functional groups on 3(COOH)NP is slightly lower (∼70-75%) than that of 2(COOH)-NP (Supporting Information). Though fewer functional groups are present on the surface of 3(COOH)-NP, we do not feel that this small difference can account for the large difference in time required for the efficient confinement of PS-NH2, because a single bond between the nanoparticles and the PS bead will permanently bind the two together. A much more important factor to consider is the number of silica-encapsulated iron oxide nanoparticles that can be accommodated on the PS bead surface, because these nanoparticles are ultimately responsible for imparting the magnetic character to the PS beads. As highlighted earlier, 2(COOH)-NP is ∼45 nm and 3(COOH)-NP is ∼73 nm in diameter. The large difference in diameter results in significantly different nanoparticle coverages on the resulting labeled PS beads. For example, ∼2278 2(COOH)-NP can be accommodated onto the surface of PS-NH2, whereas only ∼934 3(COOH)-NP can be anchored to the same surface. As such, we propose that the slower confinement time is directly related to the fact that fewer 3(COOH)-NP can be accommodated on the surface of the PS bead. Because the magnetic confinement of the PS bead is mediated by the iron oxide nanoparticle encapsulated in the silica matrix, fewer nanoparticles anchored to the PS bead surface means fewer iron oxide nanoparticles can participate in the magnetic confinement. This results in longer
magnetic confinement times. It is also worth noting that the relative mass of superparamagnetic content per nanoparticle will be higher for 2(COOH)-NP than that for 3(COOH)-NP simply because the nanoparticles possess the same iron oxide core, but 3(COOH)-NP has a thicker silica shell. As such, the percent mass contribution of the superparamagnetic component will be inherently larger for 2(COOH)-NP-modified PS-NH2 also. This data is highlighted in Table 1 and in the Supporting Information and will be addressed in more detail later in the next section. Together these data suggest that surface-modified silicaencapsulated iron oxide nanoparticles can be useful for the magnetic concentration of PS beads from aqueous solution. By extension, provided that a strong interaction can be developed between the two, these silica-encapsulated nanoparticles should also be quite useful for the confinement of bacteria from solution. However, to minimize the time required for the magnetic confinement, the nanoparticles must be small in order to maximize both the mass contribution of the superparamagnetic component of the nanoparticles and the number of nanoparticles that can interact with the surface of a PS bead (or bacteria). The silicaencapsulated iron oxide nanoparticles utilized thus far generally contain, at most, three superparamagnetic nanoparticles encapsulated within the silica shell (Figures 1 and 2).13,15 We now shift our focus to determine how the magnetic confinement of PSNH2 by 2(COOH)-NP compares to that of commercially available nanoparticles with significantly higher loadings of superparamagnetic nanoparticles (high superparamagnetic content) within quite large polymeric nanoparticle matrices (significantly less nanoparticles can be accommodated on the PS bead surface). 3. Effect of Particle Architecture on Magnetic Capture Time for PS Beads. It has been proposed that smaller nanoparticles offer advantages to biomolecule labeling and targeting strategies, as they should allow for superior target coverage in comparison to larger particles.4,9 In contrast, there are also reports that suggest the magnetic confinement of bacteria is more efficient with larger nanoparticles.23,24 However, because very large nanoparticles are not colloidally stable, there can be problems associated with them coming into direct contact with their targets as they readily settle/precipitate due to gravitational forces easily overcoming Brownian motion.23,24 As such, we are (23) Tu, S.-I.; Uknalis, J.; Gore, M.; Irwin, P. J. Rapid Methods Autom. Microbiol. 2002, 10, 185-195. (24) Tu, S.-I.; Uknalis, J.; Gore, M.; Irwin, P.; Feder, I. J. Rapid Methods Autom. Microbiol. 2003, 11, 35-46.
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Figure 2. The TEM images of the nanoparticles prepared during this investigation. Nanoparticles 2-NP and 3-NP are modified with APDEMS to generate 2(NH2)-NP (A) and 3(NH2)-NP (C), respectively. These nanoparticles can be subsequently reacted with succinic anhydride to yield 2(COOH)-NP (B) and 3(COOH)-NP (D), respectively.
interested in determining if the covalent modification of the PS bead and the time required for the magnetic confinement of PS-NH2 by 2(COOH)-NP can compete with that of commercially available carboxylic acid-terminated ademtech (A(COOH), 200 nm)25 and Dynal nanoparticles (D(COOH), 1 µm).26 This is an interesting query because both A(COOH) and D(COOH) particles are much larger than the 2-NP-based nanoparticles, so only 156 and 12 of these particles can be accommodated on a 1.1 µm PS-NH2 bead, respectively as opposed to ∼2278 and ∼934 for 2(COOH)-NP and 3(COOH)NP, respectively. However, despite being larger, A(COOH) and D(COOH) are comprised of 70% and 37% (by weight) superparamagnetic material, whereas 2(COOH)-NP and 3(COOH)-NP are comprised of ∼2.8 and 0.7% superparamagnetic material, assuming one iron oxide nanoparticle per nanoparticle (Table 1). Following covalent modification of PS-NH2 with large excesses of A(COOH) or D(COOH) in a protocol analogous (25) http://www.ademtech.com. (26) http://www.invitrogen.com/dynal.
to Scheme 2, the magnetic confinement of the modified PSNH2 was again monitored by fluorescence spectroscopy. The reactions are detailed in the Supporting Information. Though the capture efficiency mediated by 2(COOH)-NP rivals that of the commercially available beads, the time required for >80% capture of the PS-NH2 mediated by A(COOH) (4 min) and D(COOH) (2 min) is significantly faster than that by 2(COOH)-NP (14 min) (Figure 4). However, in contrast to what was observed for 2(COOH)-NP, when A(COOH) and D(COOH) are simply allowed to nonspecifically bind to the surface of PS-NH2 in the absence of EDC and NHS, the confinement is also very fast (Figure 4A). When the A(COOH)-modified PS-NH2 beads are observed by TEM, there are clearly more A(COOH) on the surface of PS-NH2 when EDC and NHS are used to covalently couple the two (Figure 4B) than when the particles bind to PSNH2 through nonspecific absorption (Figure 4C). However, the decreased number of A(COOH) that do nonspecifically bind to the surface of the PS-NH2 is able to mediate its rapid and efficient magnetic capture. The TEM images of the D(COOH)-modified PS-NH2 are less definitive, because both particles are of similar
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Scheme 2. The General Reaction Schemes for the Preparation of NP-Polystyrene Bead Conjugates
size (∼1 µm), and it appears that a single D(COOH) can probably confine PS-NH2 whether or not the two are covalently bound or nonspecifically “stuck” to one another. As expected, significantly more 2(COOH)-NP- and 3(COOH)-NP-based nanoparticles bind to the surface of the PS-NH2 (superior labeling, as seen in the corresponding TEM images), but the huge number of magnetic nanoparticles encapsulated within the A(COOH) and D(COOH) nanoparticles facilitate a much faster confinement of the PS beads. To explain this data, one must consider the magnetic properties of the resulting 2(COOH)-NP-, 3(COOH)-NP-, A(COOH)-, and D(COOH)-modified PS-NH2. Because 2(COOH)-NP and 3(COOH)-NP have, at most, three superparamagnetic cores encapsulated in a silica shell and A(COOH) and D(COOH) are highly loaded with superparamagnetic nanoparticles, there will be large differences in mass contribution of superparamagnetic material with respect to the total mass of the modified PS-NH2 beads. For example, when even a single A(COOH) or D(COOH) nonspecifically binds to PS-NH2, the overall percent mass contribution of the superparamagnetic nanoparticles (the mass of the superparamagnetic content of the nanoparticles vs the mass of PS-NH2 and the polymer matrix encapsulating the superparamagnetic material) remains high (Table 1). In contrast, if a single 2(COOH)-NP or 3(COOH)-NP nonspecifically binds to the surface of PS-NH2, the overall percent mass contribution of the superparamagnetic content is extremely small (∼3.74 × 10-4%, assuming that a single 1-NP is encapsulated in the silica shell). Under these conditions, 2(COOH)-NP and 3(COOH)-NP will not magnetically confine the PS-NH2 rapidly. It is only when a large number of binding events occur so that the superparamagnetic mass contribution of the 2(COOH)-NP becomes high enough, the modified PS-NH2 can respond relatively quickly to the presence of a rare earth magnet. For example, if we assume a close-packed monolayer of 2(COOH)-NP surrounding PS-NH2, there would be ∼2278 binding events, and the magnetic content would be ∼0.65%. The percentage superparamagnetic content decreases to ∼0.23% when 3(COOH)-NP is on the PS bead surface, which is expressed as a slower magnetic confinement for the 3(COOH)-NP-
modified PS-NH2. Interestingly, in the absence of a silica shell, ∼43678 10 nm 1-NPs could be anchored to the surface of the PS bead, resulting in a magnetic mass contribution as high as 14%. Though the time required for the magnetic capture mediated by 2(COOH)-NP is slower than that mediated by A(COOH) and D(COOH), the selectivity of capture is better because there must be very high coverage (many selective interactions) for 2(COOH)-NP to confine the PS beads quickly and efficiently, whereas only a few nonspecific interactions lead to the efficient confinement of the PS bead with A(COOH) and D(COOH). These results suggest that smaller nanoparticles with less superparamagnetic content could prove to be important in situations where, for example, one species of bacteria in a mixture of several other species of bacteria must be isolated. Here, any nonspecific absorption to the unwanted species of bacteria would result in a very slow capture, whereas the specific targeting of the bacteria of interest would result in a more rapid magnetic confinement. The magnetic confinement experiments discussed thus far have been carried out in small volume (1 mL) aqueous solutions. However, it is of important to investigate how the viscosity and the total volume of the dispersion medium affect the 2-NP-mediated magnetic confinement of the PS beads in order to elucidate how well these nanoparticles can concentrate a bacteria mimic from a model clinical sample. 4. Effect of the Medium, Viscosity, and Overall Solution Volume on Magnetic Capture Time for PS Beads by 2(COOH)-NP. The rapid magnetic confinement of PS-NH2 by 2(COOH)-NP requires the conjugate to overcome a significant amount of drag and Brownian motion as it moves through the solution toward the rare earth magnet, and as the viscosity increases, the drag experienced by the large conjugate also increases. To explore how the viscosity of the dispersion medium affects the magnetic confinement time for 2(COOH)NP-modified PS-NH2, an aqueous solution of MES buffer was spiked with 30% by weight of glycerol, resulting in a solution with a viscosity of 2.5 Pa/s.27 This viscosity is higher than MES buffer itself (∼1.00 Pa‚s) and is actually quite close to that of (27) http://www.dow.com/glycerine/resources/table18.htm.
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Figure 3. A typical magnetic capture experiment where a fluorescence emission spectrum for 2(COOH)-NP-modified PS-NH2 and 3(COOH)-NP-modified PS-NH2 was acquired prior to (A(i) and B(i), respectively) and following magnetic confinement (A(ii) and B(ii), respectively). The time required for magnetic confinement for each of the nanoparticles is represented as a bar graph in (C). Representative TEM images of 2(COOH)-NP-modified PS-NH2 (D(i)), 3(COOH)-NP-modified PS-NH2 (E(i)), and control reactions where 2(COOH)NP (D(ii)) and 3(COOH)-NP (E(ii)) were allowed to nonspecifically bind to the surface of PS-NH2. Note that the coverage is significantly higher in (D(i)) and (E(i)) than in (D(ii)) and (E(ii)), respectively. Table 1. The Magnetic Content (%) for the Various Nanoparticle-PS Bead Conjugates
nanoparticle
mass of PS-NH2 (g)
mass of single nanoparticle (g)
mass of NP-PS bead conjugate (g)
no. of NPs in NP-PS bead conjugate
mass of magnetic content (g)
% mass magnetic content
2(COOH)-NP 2(COOH)-NP 3(COOH)-NP 3(COOH)-NP A(COOH) A(COOH) D(COOH) D(COOH) 1-NP 1-NP
7.35 × 10-13 7.35 × 10-13 7.35 × 10-13 7.35 × 10-13 7.35 × 10-13 7.35 × 10-13 7.35 × 10-13 7.35 × 10-13 7.35 × 10-13 7.35 × 10-13
9.71 × 10-17 9.71 × 10-17 4.09 × 10-16 4.09 × 10-16 7.50 × 10-15 7.50 × 10-15 8.33 × 10-13 8.33 × 10-13 2.74 × 10-18 2.74 × 10-18
∼7.35 × 10-13 9.56 × 10-13 ∼7.35 × 10-13 1.12 × 10-12 7.42 × 10-13 1.91 × 10-12 1.57 × 10-12 1.07 × 10-11 ∼7.35 × 10-13 8.55 × 10-13
1 2278 1 934 1 156 1 12 1 43 678
2.74 × 10-18 6.24 × 10-15 2.74 × 10-18 2.56 × 10-15 5.25 × 10-15 8.19 × 10-13 3.08 × 10-13 3.70 × 10-12 2.74 × 10-18 1.20 × 10-13
3.73 × 10-4 0.65 3.73 × 10-4 0.23 0.71 43 20 34 3.73 × 10-4 14
whole blood. The 2(COOH)-NP-modified PS-NH2 as prepared above was dispersed in 1 mL of the 30% glycerol/MES and
magnetically confined with a rare earth magnet as described above. As the viscosity is increased from 1.00 to 2.50 Pa‚s, the
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Figure 4. The effect of covalent modification of 2(COOH)-NP, A(COOH), and D(COOH) to PS-NH2 on the time required for magnetic confinement (9) in comparison to that when nonspecific absorption mediates the capture (0) (A). Note that nonspecific absorption yields confinement times identical to those for the covalently modified PS beads for A(COOH) and D(COOH), whereas there is a significant difference for 2(COOH)-NP. The TEM images corresponding to the labeling of PS-NH2 with A(COOH) in the presence (B) and absence (C) of EDC and NHS and corresponding to the labeling of PS-NH2 with D(COOH) in the presence (D) and absence (E) of EDC and NHS. Note that the smoother beads in (D) and (E) are the PS-NH2, whereas the rougher beads are D(COOH). Table 2. The Capture Efficiency of 2(COOH)-NP-modified PS-NH2 from a Variety of Dispersion Medium, Including (A) MES Buffer and (B) 30% Glycerol in MES Buffer mode of magnetic confinement
volume (mL) of dispersion (medium)
capture efficiency (time)
solvent volume utilized for recovery
rare earth magnet rare earth magnet rare earth magnet rare earth magnet magnetic Filter (µ-column) magnetic Filter (µ-column)
1 mL (A) 1 mL (B) 10 mL (A) 10 mL (B) 1 mL (B) 15 mL (B)
>80% (14 min) >80% (35 min) >80% (∼360 min) 360 min) >99% (95% (80% capture of the labeled PS-NH2 in the more viscous solution (Table 2). Though we expected the time required for efficient capture to increase as the viscosity of the dispersion medium was increased, we did not anticipate the time to increase 2-3 fold in the blood mimic solution. The magnetic capture of 2(COOH)-NP-modified PS-NH2 also slowed as the volume of the dispersion solvent was increased. Diluting 2(COOH)NP modified PS-NH2 by increasing the dispersion volume from
recovery (%) >80 >80
80-90 81
1 to 10 mL in MES buffer results in a 24-fold increase in the time required to magnetically confine >80% of the PS bead. When 2(COOH)-NP modified PS-NH2 is diluted similarly in 30% glycerol in MES buffer, 95% of the labeled PS beads (Panels B and E(ii) of Figure 5). It is noteworthy that the 2(COOH)-modified PS beads can be retained on the column with flow rates as high as 1.5 mL/minute. Following removal from the separator, the ferromagnetic column demagnetizes (Figure 5C) and the 2(COOH)-modified PS-NH2 can be eluted from the column with only 200 µL of fresh 30% glycerol/MES (Figure 5D) with >90% recovery. Because the flow rate of the solution through the column can be as high as 1.5 mL/minute, the total time required for magnetic confinement and recovery of 2(COOH)NP modified PS-NH2 is as little as 2 min, which is comparable to the times acquired with A(COOH) and D(COOH). A significant advantage of this procedure for magnetically confining NP-modified PS beads is that regardless of the volume of solution eluted through the magnetized column, the PS beads will never be farther than ∼20-30 µm from a ferromagnetic bead. By expanding this study to sample volumes of 10-15 mL, 2(COOH)-NP-modified PS-NH2 can be magnetically trapped (>95%) within the column and recovered (∼80%) with only
Figure 5. A pictorial representation of a magnetic column separation. When the magnetic µ-column is placed in the magnet (A), the ferromagnetic bead column becomes magnetized. As a solution of 2(COOH)-NP-modified PS-NH2, F, is introduced to the column, the magnetized PS bead is retained within the column and only the solvent is eluted (B). Upon removal from the magnet, the magnetic column is demagnetized (C) and F/ can be recovered with a wash of fresh solvent (D). In (E), the separation is shown as a series of fluorescence emission spectra, where (E(i)) is the spectra of the 2(COOH)-NP-modified PS-NH2 prior to its introduction onto the magnetized column (A), (E(ii)) is the solvent eluted from the column (B). The conjugates can be recovered from the column when the filter is removed from the magnet (D), and the recovery is ∼80% (E (iii)).
200 µL of fresh 30% glycerol/MES in only ∼15-18 min (,10% of the time required for the magnetic confinement with a rare earth magnet, Table 2). This represents a 50-75-fold preconcentration, with very little loss of the labeled PS-NH2 bead from the original sample. This process is also very practical as the isolated sample could be transferred directly from the magnetic column to a microfluidic device for analysis. Because many academic, commercial, and government researchers are currently focused on utilizing microfluidic platforms to detect and identify bacteria from blood and food samples, it is important to rapidly transition from large volumes to small volumes. These results suggest that a combination of superparamagnetic nanoparticles and magnetic columns are promising for such sample preparation applications, where bacteria could be easily concentrated into very small volumes prior to introduction onto the µ-fluidic device for analysis.
Conclusions (28) Whitesides, G. M.; Kazlauskas, R. J.; Josephson, L. Trends Biotechnol. 1983, 1, 144-148. (29) http://www.miltenyibiotec.com.
Put together, the results of this investigation highlight several important characteristics of the superparamagnetic nanoparticle-
3502 Langmuir, Vol. 24, No. 7, 2008
mediated concentration of PS beads from solution. In general, we were able to show that nanoparticles with higher loadings of superparamagnetic material are capable of capturing the PS beads from solution much more quickly than those with lower loadings (i.e., thinner silica shell faster than thicker silica shell and higher loading of superparamagnetic material in larger nanoparticles faster than lower loading of superparamagnetic material in smaller nanoparticles). However, because of the large superparamagnetic content of the highly loaded large nanoparticles, nonspecific absorption between nanoparticles and PS beads can lead to their efficient magnetic capture. This situation could be problematic in situations where a specific cell or bacteria is to be isolated from a mixture of several other bacterial species. Though slower, silica-encapsulated iron oxide nanoparticles provide attractive alternatives for specific capture under such circumstances, where the confinement is rapid only when a large number of specific interactions between the nanoparticles and the PS bead occur. Finally, the time required for the efficient magnetic confinement of PS beads by the small silica-encapsulated iron oxide nanoparticles can be significantly decreased through the use of
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magnetic columns, where the PS beads can be concentrated by >50-fold in less than 20 min. This is a powerful mode of sample preparation and provides a practical, proof-of-principle demonstration that silica-encapsulated iron oxide nanoparticles can mediate the magnetic concentration of bacteria mimics from large volumes to volumes more amenable to incorporation into a microfluidic device for analysis. We are currently exploring these nanoparticles for such an application. Acknowledgment. We thank Genome Canada and Genome Quebec for the generous financial support. We would also like to thank Catherine Bibby for acquiring the TEM images and Chantal Paquet, Maurice Boissinot, Ann Huletsky, and Regis Peytavi for useful discussions. Supporting Information Available: Experimental details and examples of calculation of the percentage mass of the superparamagnetic content of the various nanoparticles are presented in the Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. LA7037476
