pubs.acs.org/Langmuir © 2010 American Chemical Society
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Adhesion Force Studies of Nanofibers and Nanoparticles Malcolm Xing,†,^,# Wen Zhong,*,‡, Xiuling Xu,‡ and Douglas Thomson§ Department of Mechanical Engineering, ‡Department of Textile Sciences, §Department of Electrical Engineering, Department of Medical Microbiology, and ^Department of Biochemistry and Medical Genetics, University of Manitoba, and #Manitoba Institute of Child Health, Winnipeg, MB R3T 2N2, Canada )
†
Received January 29, 2010. Revised Manuscript Received June 1, 2010 Surface adhesion between nanofibers and nanoparticles has attracted attention for potential biomedical applications, but the measurement has not been reported. Adhesion forces were measured using a polystyrene (PS) nanoparticle attached to an atomic force microscopy (AFM) tip/probe. Electrospun PS nanofibers of different diameters were tapped with the probe to study the effect of fiber diameters on adhesion force. Both AFM experiments and numerical models suggest that the adhesion force increases with increased fiber diameters. Numerical models further demonstrated that local deformation of the fiber surface, including the flattening of surface asperities and the nanofiber wrapping around the particle during contact, may have a significant impact on the adhesion force. The adhesion forces are in the order of 100 nN, much smaller than the adhesion forces of the gecko foot hair, but much larger than that of the receptor-ligand pair, antibody-antigen pair, and single-stranded DNA from a substrate. Adhesion forces of nanofibers with roughness were predicted by numerical analysis. This study is expected to provide approaches and information useful in the design of nanomedicine and scaffold based on nanofibers for tissue engineering and regenerative medicine.
1. Introduction Because of its involvement in a wide variety of applications, adhesion, an important aspect of nanofibers and nanoparticles, attracts extensive attention. Regular-patterned nanofibers maximize the adhesion force of gecko toes to allow rapid and stable attachment of pads to surfaces.1,2 Thus, the design and development of directionally sensitive dry adhesives based on polymer nanofibrils has been inspired.3 Drug-loaded nanoparticle delivery is one of the most promising methods of improving the efficiency of drug delivery and of inducing the differentiation of stem cells. When therapeutic molecules are entrapped within nanoparticles, they can be protected from proteinase and DNAase degradation. The knowledge of the effects of adhesion forces in nanostructures is valuable to the development of biomimicking nanomaterials and will help to (1) improve the drug loading efficiency of nanoparticles as a result of the clarification of the interface between nanoparticles and drugs, (2) increase gene transfection efficiency of nanoparticles as a result of the clarification of the interface between gene-loaded nanoparticles and cells, (3) enhance the cell adhesion to nanofiber scaffolds that mimic extra cellular matrix by understanding the cells/nanofibers interface, and (4) augment the long-term release capability of the drug supplemented scaffold applied in tissue engineering and regenerative medicine. Adhesion is a prerequisite for a drug to enter nanoparticles, for nanoparticles to pack genes, for nanoparticles to be uptaken by cells, and for cells *Corresponding author. Tel: 1-204-474-9913, Fax: 1-204-474-7593, E-mail:
[email protected]. (1) Tian, Y.; Pesika, N.; Zeng, H. B.; Rosenberg, K.; Zhao, B. X.; McGuiggan, P.; Autumn, K.; Israelachvili, J. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 19320. (2) Peattie, A. M.; Full, R. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 18595. (3) Jeong, H. E.; Lee, J. K.; Kim, H. N.; Moon, S. H.; Suh, K. Y. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5639. (4) Koh, H. S.; Yong, T.; Chan, C. K.; Ramakrishna, S. Biomaterials 2008, 29, 3574. (5) Jia, J.; Duan, Y. Y.; Yu, J.; Lu, J. W. J. Biomed. Mater. Res. A 2008, 86, 364. (6) Grafahrend, D.; Calvet, J. L.; Salber, J.; Dalton, P. D.; Moeller, M.; Klee, D. J. Mater. Sci.-Mater. Med. 2008, 19, 1479. (7) Prabhakaran, M. P.; Venugopal, J.; Chan, C. K.; Ramakrishna, S. Nanotechnology 2008, 19, 455102.
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to survive and proliferate in scaffolds. As a result, great efforts have been made to improve the adhesion of cells to nanofibers.4-7 For fibers of small scale, the technical applications of surface adhesion forces and interactions between nanofibers and dust, small particles, bacteria, targeted biomolecules, ligands, drug particles, or cells is of great importance.8 Quantitative measurement of the adhesion forces between submicrometer particles and nanofibrous materials has become essential in different fields of research. By gluing a silica sphere with a radius of 3.5 μm to the end of an atomic force microscopy (AFM) cantilever, a direct force measurement between a single particle and a planar surface can be determined.9 Drelich et al.10 used AFM techniques to determine the surface tension of polymer materials from pull-off force measurements. The measurement involves contact of a spherical particle probe (radius R) with a planar sample. The correlation between the pull-off force (F) and the surface tension of the material (γS) is F ¼ 2cπRγS
ð1Þ
where c is a constant: in the DMT model, c = 2, and in the JKR model, c = 1.5. Kaushik et al.11 studied the dynamics of microparticle detachment from surfaces using the AFM force-displacement curves obtained when the particles were repeatedly attached and detached from the surface. A similar process was conducted to determine the adhesion between polymer surfaces according to the force required for the AFM tip to completely remove the polymer layer from the substrate.12 Up to now, there has been little work concerning adhesion between nanoparticles and nanofibers. However, such adhesion (8) Pan, N.; Zhong, W. Fluid transport phenomena in fibrous materials; Textile Institute: Woodhead Publishing: Cambridge, England, 2006; p 93. (9) Ducker, W. A.; Senden, T. J.; Pashley, R. M. Nature 1991, 353, 239. (10) Drelich, J.; Tormoen, G. W.; Beach, E. R. J. Colloid Interface Sci. 2004, 280, 484. (11) Kaushik, A.; Srinivasa, A. R.; Phares, D. J. Particulate Sci. Technol. 2007, 25, 387. (12) Dvir, H.; Jopp, J.; Gottlieb, M. J. Colloid Interface Sci. 2006, 304, 58.
Published on Web 06/16/2010
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Figure 1. SEM images of PS nanofibers electrospun from 25% (A), 30% (B), and 35% (C) PS/DMF solutions, respectively.
Figure 2. SEM images of the PS particle-attached cantilever (A) and PS particle used in pull-off force measurement (B).
has a considerable influence on the performance of nanofibers in a variety of applications, such as the design of nanomedicine, nanocomposites, drug-supplemented scaffolds for tissue engineering and regenerative medicine, filters, and protective devices. Greater research efforts are necessary to gain a better understanding of the interfacial interactions between submicrometer particles and nanofibrous materials. The present study was initiated to measure the adhesion forces between nanofibers and a colloidal polystyrene (PS) AFM probe. Variously sized electrospun PS nanofibers were used to determine experimentally and numerically the relationship between fiber diameter and the adhesion force. The Classical JKR model and the finite element method (FEM) were used to simulate the interaction between nanofiber and nanoparticle.
2. Materials and Methods 2.1. Preparation of Polystyrene Nanofibers. Three measured amounts of polystyrene (PS) pellets (Mw 190 000 Da, from Scientific Polymer Products, Ontario, NY) were dissolved in N,N0 dimethylformamide (DMF) (analytical grade, Fisher) by stirring them overnight at 50 C to obtain clear solutions with concentrations of 25%, 30%, and 35% by weight, respectively. PS pellets and DMF were used as received. The PS nanofibers (Figure 1) were prepared by electrospinning the PS/DMF solutions. Samples of electrospun PS nanofibers were vacuum-coated with a layer of gold prior to examination with a scanning electron microscope (SEM, Cambridge Stereoscan 120, accelerating voltage 20 kV) to determine their surface morphology and size. 2.2. Force Measurement. Adhesion force measurement was obtained from an atomic force microscope (Dimensions 3100 SPM, Digital Instruments Veeco, Santa Barbara, CA) and a cantilever to which a 1 μm diameter PS particle (mean diameter of 1.05 ( 0.01 μm) was attached (supplied by Novascan Technologies). Spring constant of the cantilever used in this study was 11810 DOI: 10.1021/la100443d
0.58 N/m (provided by the manufacturer). Young’s modulus and Poisson’s ratio of the PS particle (provided by the manufacturer) were 3-3.5 GPa and 0.34, respectively. SEM images of a PS particle-attached cantilever are shown in Figure 2. Figure 2A shows an overhead view of the cantilever, and Figure 2B shows that the PS particle has a uniform dimension and a smooth surface. Force curves were obtained as a plot of the deflection signal of the cantilever against the vertical displacement of the base of the cantilever (scanner displacement). The cantilever deflection can be converted into force according to Hooke’s Law. To ensure that the PS particle had a centric contact with the PS nanofiber during measurements, the colloid probe started at a distance from the selected nanofiber, touching first the surface of the substrate (a cover glass) on which nanofibers were deposited (position a). The probe then stepped vertically toward the nanofiber, making a measurement at each stop. At a certain point, the probe had a centric contact with the PS nanofiber (position b), then moved to position c having noncentric contact with the fiber. Finally, the colloid probe moved across the nanofiber and touched the substrate surface again on the other side of the fiber (position d). The track of the colloid probe in the force measurement process is illustrated in Figure 3. The adhesion between the nanoparticle and the substrate is large at the beginning (position a). As the nanoparticle approaches the nanofiber noncentrically, the adhesion drops, rises a little at the point of centric contact between nanoparticle and nanofiber (position b), drops again at the noncentric contact points (position c), and finally rises dramatically when the nanoparticle touches the substrate again (position d). The adhesive force vs distance curve along this track is asymmetric, allowing a determination of the adhesion force between the nanoparticle and the nanofiber at the point of centric contact. For the purpose of comparison, adhesion measurements were obtained also on the PS films (casted by a spin-coater) through the PS particle-attached cantilever. Langmuir 2010, 26(14), 11809–11814
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Figure 3. Adhesive force vs distance when the probe moves vertically across the nanofiber (R = 240 nm). Table 1. Adhesion Forces between the PS Particle and PS Nanofibers of Different Diameters
#
average radius (nm)
adhesive force (nN)
rms roughness (Rq, nm)
FJKR (nN)
FE simulated adhesive force FFE (nN)
1 2 3 film
520 320 240 N/A
217 ( 15 178.2 ( 21.6 159.5 ( 12.7 384.9 ( 9.3
34 54 28 8
145.2 111.3 91.0 282.6
234 198 181 420
The root mean squared (rms) roughness of a nanofiber was also determined by AFM in this study, as listed in Table 1. The rms roughness (Rq) is defined as sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi n 1X Rq ¼ Rti n i¼1
ð3Þ
where Rt is the maximum height of the profile defined as the differences between the maximum peak height and the maximum valley depth. 2.3. Numerical Analysis. 2.3.1. Model Loads. The finite element method was used to simulate the interaction between a nanoparticle and a nanofiber. To mimic the experiments through numerical analysis, the process was divided into two steps: (1) the nanoparticle approaches and contacts the nanofiber, and (2) the nanoparticle detaches from the fiber. The Lennard-Jones potential (L-J) p(d) will be considered as the function loading in the finite element model at the interfaces.13 When the nanoparticle approaches the nanofiber (before contact) and when the nanoparticle detaches from the nanofiber 8Δγ pðdÞ ¼ 3e
" 3 # e 9 e d d
ð4Þ
where p is the Lennard-Jones potential, d the distance between the two surfaces, Δγ the adhesion work, and e the equilibrium distance at zero contact. Here, Δγ is set to 10 mJ/m2 and e to 0.5 nm.13,14 To simulate the adhesion force or pull-off force, cable elements that function only under tension force were designed and used in the two contacting surfaces. Each cable element was set with a (13) Greenwood, J. A. Proc. R. Soc. London, Ser. A: Math. Phys. Eng. Sci. 1997, 453, 1277. (14) Gao, H. J.; Yao, H. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 7851.
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Figure 4. FEM model of a cantilever with a nanoparticle probe in contact with a nanofiber. maximum stress of 20 MPa.14 During the pulling-off process, the axial stresses of the cable element were deactivated in the computation, which mimicked the separation of two contact bodies with molecular interaction forces. 2.3.2. Models. Generally, nanofibers are assumed to be in cylindrical in shape and nanoparticles spherical. ANSYS (version 10.0, Pittsburgh, PA) was used to simulate the interactions between a nanoparticle and a nanofiber or the interactions between a nanoparticle and a planar film. The element type solid 185 was used (Figure 4). To simulate the pull-off process, the “death and birth” algorithm of the cable element was activated to mimic the failure when the element had reached its limit; the failed element was given a zero stiffness matrix. Two processes were simulated: (1) the probe attaches to the fiber, simulated by giving a displacement loading (negative loading indicating compression) to the cantilever or the nanoparticle, where the initial gap between fiber and particle was 0.35 nm; (2) the probe detaches from the fiber, simulated by giving the same displacement loading as in the first process (positive loading indicating a pulling force). Thirty substeps were set to complete the loading processes. The reaction forces (Frc) at the bottom line of the nanofiber or the bottom surface of the nanofilm were calculated from the solution of the substeps. The maximum adhesion force or pull-off force (Fad) was obtained in accordance with the following equilibrium condition X
Frc ¼
X
Fad
ð5Þ
2.3.3. Roughness Model. Roughness is an important parameter in the study of adhesion between two surfaces. A roughness model (Figure 5) was developed to investigate the effects of roughness on adhesion forces. The spherical probe (R3 = 500 nm) was assumed to have a smooth surface. The fiber was assumed to have a regularly indented circular cross section. The radius R1 represents DOI: 10.1021/la100443d
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Figure 5. Roughness model.
Figure 6. Typical force curve: PS particle approaching, then retracting from a PS nanofiber of radius 240 nm. The abscissca is the Z-piezo traveling distance. the radius at a peak; R2 represents the radius of the fiber at the full depth of the valley. The difference between R1 and R2 gives the maximum height of the profile Rf = R1 - R2. L1 and L2 denote the radians (in degrees) of the regular peaks and indent lengths along the circumference. The roughness width of the surface (RW) can be calculated by averaging the two sectors: RW = πR1(L1 þ L2)/ 360. By varying R2, L1, and L2, fiber surfaces having different roughness could be modeled. The adhesion force between a nanoparticle and a nanofiber in the roughness model was also tested under different displacement loadings to investigate the flattening effect: Contact loading could be such that the surface asperities are squashed flat.
3. Results and Discussion 3.1. Adhesion Forces between Nanofibers and Colloid Particle. Figure 6 shows a typical force curve provided by the AFM, when the PS particle (radius 0.5 μm) probe approaches and then retracts from the studied object, a fiber or a spin-coated PS film. The probe starts at a distance from the studied surface and descends to approach the surface of the film. The slight deflection in the force curve indicates that the attractive forces near the surface pull the tip down. The cantilever bends upward when the probe descends further to press into the surface. Upon retraction from the surface, the probe ascends until the forces are in equilibrium with the surface forces, and the cantilever relaxes downward. When the probe continues retraction and ascends further, the cantilever bends downward as surface attraction holds onto the probe. The probe finally breaks free, and the cantilever rebounds sharply upward. The rest of the force curve shows that probe continues to ascend and leave the surface of the film. The start of the retraction curve shifts slightly from the end 11812 DOI: 10.1021/la100443d
of the approach curve (highest force applied on approach). This may be caused by a slight rotation/displacement of the nanofiber when the nanoparticle probe starts to retract from the nanofiber. Experimental determination of the adhesive force between the probe and a nanofiber is more complicated because it is difficult to ensure that the probe is making a centric contact with the fiber. In our design, the probe steps vertically across the fiber, making contact with the fiber at small intervals, under the assumption that one contact will be nearest to the centric contact. Figure 3 also gives a sample curve of the adhesive forces vs distances when the probe moves vertically across a nanofiber with a radius of 240 nm and illustrates the different positions where the probe touches or moves away from the fiber. The adhesive force vs distance curve is asymmetric: the adhesive force between the probe and substrate (i.e., the cover glass) is quite large; as the probe approaches the fiber, there is a drop in the adhesive force (noncentric contact between the probe and the fiber), followed by a small peak (centric contact) and another drop (noncentric contact). The adhesive force rises when the probe leaves the fiber and contacts the glass substrate again. In this way, three single nanofibers with different diameters were tested. The adhesive force between a probe (1 μm in diameter) and nanofibers with different diameters at their centric contact was determined from the curves of the adhesive force vs distance and is listed in Table 1. The average radii of these nanofibers, as determined by AFM scanning, are also listed in Table 1. In the experiments, the Z-piezo traveling distances from the touching point (when approaching) to the retraction starting point were kept identical for all curves used for analysis. This portion of Z-piezo traveling distance corresponds to the displacement loading that was applied on the nanoparticle in the FEM simulations. For the purpose of comparison, the adhesive force between the probe and nanofibers was calculated according to the traditional JKR methods, which is the suitable model for soft elastic solids10 FJKR ¼
3 πRWA 2
ð6Þ
Here, R is the radius of the probe in contact with a planar film; R = RFRP/(RF þ RP) is the contact between the probe (radius RP) and a fiber (radius RF); WA is the energy of adhesion between two polystyrene surfaces. WA equals 2γSV (solid-vapor surface energy of polystyrene, typically 30 mJ/m2).15 The results of the calculated FJKR are listed in Table 1. A displacement loading of 0.6 μm was given to the cantilever. A FEM simulated force curve (including both approaching and retraction tracks) is shown in Figure 7. The adhesion force estimated from the simulated force curve agrees with the adhesion force derived from the experimental force curve in Figure 6. The adhesion forces estimated using JKR and finite element models (listed in Table 1) show a tendency similar to that obtained from the AFM experiments; that is, the adhesion forces increased as the fiber diameter increased. The increase in fiber diameter leads to an increase in contact area between the nanofiber and the nanoparticle, resulting in an increase of adhesion force. The planar film can be regarded as a fiber with an infinite radius presenting the maximum adhesive force. It should be noted that the abscissa in Figures 6 and 7 reflects the Z-piezo traveling distance and the displacement of the cantilever base that is attached to the piezo. It is not the actual displacement or deformation of the nanoparticle probe. It has been (15) Hodges, C. S.; Cleaver, J. A. S.; Ghadiri, M.; Jones, R.; Pollock, H. M. Langmuir 2002, 18, 5741.
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Article Table 2. Adhesion Forces Estimated by the Roughness Model (nN) with 5 nm Displacement Loading L1 (degree) R2 (nm)
7.5
15
220 200
158 127
172 145
Table 3. Adhesion Forces Estimated by the Roughness Model (nN) with 7 nm Displacement Loading L1 (degree)
Figure 7. Simulated force curve showing force vs displacement loading of the nanoparticle on the nanofiber: PS particle approaching then retracting from a PS nanofiber of radius 240 nm.
Figure 8. Adhesion force and contact radius vs displacement loadings, estimated by FEM.
estimated by FEM that the maximum deformation of the particle is about 5 nm with a 0.6 μm Z-piezo traveling distance (see Supporting Information Figure S1). Another factor that may affect the contact area between the nanoparticle and nanofiber is the displacement loading. The contact area further impacts the adhesion force. This effect can be investigated by FEM. The adhesion forces between a nanoparticle (r = 500 μm) and a nanofiber (r = 520 nm) under different displacement loadings (applied on the nanoparticle) were estimated using FEM and are shown in Figure 8. The simulation results demonstrate that an increase in the displacement loading leads to increases in the contact radius and significant rises in adhesion forces. Large contact areas may result from the wrapping of the nanofiber around the nanoparticles when the nanoparticle presses into the nanofiber, significantly increasing the adhesion force between them. The JKR model underestimates the adhesion forces, as shown in Table 1, perhaps because of the assumption of a circular contact area between the two bodies and smooth surfaces. In fact, the area of contact between a sphere and a cylinder is not always circular in shape. The adhesion forces estimated from the FE numerical analysis agreed more closely with the experimental results, although the estimates are somewhat higher than the results from the AFM experiments. This may be the result of (1) again, the smooth surface assumption, and (2) the fact that an average elastic modulus (3.25 GPa) was used in the computational model. In this work, the results from both the experiments and the models indicate that the adhesion force between a nanoparticle Langmuir 2010, 26(14), 11809–11814
R2 (nm)
7.5
15
220 200
186 143
202 178
and a nanofiber is on the order of 100 nN: much larger than that between individual ligand-receptor pairs (∼102 pN),16,17 antibodyantigen (∼102 pN),18 single-stranded DNA-graphite substrate (∼101 pN), and between different nanoparticles (∼10 nN).19 To our knowledge, the numeric order of adhesion forces between nanofibers is still unknown, but it may be larger than 102 nN based on the calculation from a single pulling of gecko foot hair (104 nN).20 3.3. Roughness Model. The roughness model demonstrates the estimated adhesion forces with varying roughness parameters: R2 (220 and 200 nm), and L1 (7.5 and 15), to represent the maximum heights of the profile and roughness width respectively. The fiber radius R1 was kept constant at 240 nm. Since the roughness was assumed to be regularly patterned for the purposes of this model, the rms roughness of the fiber surface is equal to the maximum heights of the profile: Rf = R1 - R2. The adhesion forces were predicted through the roughness model, as shown in Tables 2 and 3. Results in Table 2 suggest that roughness is vitally related to the adhesion between the two surfaces: about 20 nm difference in rms roughness (ΔR2 = 220 - 200 = 20 nm) or roughness width (ΔL1 π R1= 7.5 3.14 240/360 = 16 nm) may lead to a variance of around 101 nN in adhesion forces. This magnitude of adhesion force is larger than those of receptorligand, DNA peeling, and antibody-antigen adhesions. Because the roughness of nanofibers can so profoundly affect a biological process, it must never be neglected during the design of nanopartices and nanofibers used in tissue engineering and nanomedicine. Surface roughness and adhesion forces may also be affected by the “flattening effect” of the force applied to the surface of the fiber. In some cases, the force may be such that the surface asperities are squashed flat. An FEM study was performed to represent such conditions. Table 3 reports the FEM estimated adhesion forces between the nanoparticle and the “rough” nanofiber under different displacement loadings. The adhesion force increases when the displacement loading rises from 5 to 7 nm (applied on the nanoparticle); the “flattening” of the surface has a significant effect on the adhesion forces. It should be noted that the asperities in our model are long linear areas, so compression is only lateral; therefore, any increases in the adhesion force can only come from lateral smoothing of the surface. In other words, the contact area increase from real asperities may be still larger. (16) Florin, E. L.; Moy, V. T.; Gaub, H. E. Science 1994, 264, 415. (17) Moy, V. T.; Florin, E. L.; Gaub, H. E. Science 1994, 266, 257. (18) Willemsen, O. H.; Snel, M. M.; van der Werf, K. O.; de Grooth, B. G.; Greve, J.; Hinterdorfer, P.; Gruber, H. J.; Schindler, H.; van Kooyk, Y.; Figdor, C. G. Biophys. J. 1998, 75, 2220. (19) Xu, L. P.; Pradhan, S.; Chen, S. Langmuir 2007, 23, 8544. (20) Autumn, K.; Liang, Y. A.; Hsieh, S. T.; Zesch, W.; Chan, W. P.; Kenny, T. W.; Fearing, R.; Full, R. J. Nature 2000, 405, 681.
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4. Conclusion We initiated work to measure the adhesion forces between nanoparticles and nanofibers using AFM. PS nanoparticleattached AFM probes were used to study PS nanofibers of different sizes. To ensure a centric contact of nanoparticle with the nanofiber during each measurement, the colloid probe was allowed to make multiple measurements in a vertical track across the fiber. The adhesion forces measured along this track showed asymmetric changes. As a result, the centric contact point and the adhesion force at this point could be determined. JKR and FEM models were used to estimate the adhesion forces. Both experiments and models showed that adhesion forces rose with increased fiber diameters. The JKR model underestimated the adhesion forces, while results from FEM aligned more closely with the experimental results. Both the experiments and models suggest that the adhesion force between a nanofiber and a
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nanoparticle is on the order of 100 nN, which is within the range of relating some important bioadhesion phenomena. The roughness model further suggests that surface roughness is so critical a parameter in the determination of adhesion forces that it can never be neglected in biomedical applications. The flattening of surface asperities and the wrapping of the nanofiber around the nanoparticle both significantly increase the contact area and result in an increased adhesion force. Acknowledgment. Our work is supported by the NSERC (Natural Sciences and Engineering Research Council of Canada) Discovery Grant, NSERC RTI (Research Tools and Instrument) Grant and Manitoba Medical Service Foundation (MMSF). Supporting Information Available: Additional information as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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