pubs.acs.org/NanoLett
Fifteen-Piconewton Force Detection from Neural Growth Cones Using Nanowire Arrays Waldemar Ha¨llstro¨m,† Monica Lexholm,† Dmitry B. Suyatin,† Greger Hammarin,† Dan Hessman,† Lars Samuelson,† Lars Montelius,† Martin Kanje,‡ and Christelle N. Prinz*,† †
Division of Solid State Physics and ‡ Department of Cell and Organism Biology, Lund University, Sweden ABSTRACT We used epitaxially grown monodisperse nanowire arrays to measure cellular forces with a spatial resolution of 1 µm. Nerve cells were cultured on the array and cellular forces were calculated from the displacement of the nanowire tips. The measurements were done in situ on live cells using confocal microscopy. Forces down to 15 pN were measured on neural growth cones, showing that this method can be used to study the fine details of growth-cone dynamics. KEYWORDS Mechanosensing, cellular force, picoNewton, nanowire, neuron
C
ellular mechanotransduction is a growing field of interest with recent studies showing that external and internal forces can alter cellular signaling and function.1,2 There are many ways to measure cellular forces in vitro. Optical tweezers and the micropipet method allow picoNewton forces to be measured.3–5 While being very sensitive, these techniques allow force measurements to be made simultaneously on a few points only on a cell. The elastic substrate method6 allows probing of cellular forces on an ensemble of cells. This method, as well as its derived method based on elastomer micropillar arrays, allows forces from nanoNewtons up to hundreds of nanoNewtons to be measured.7–9 Simultaneously the spatial resolution of these methods is limited by the size of the pillars/markers to 2 µm at best. Subcellular structures such as filipodia and lamelipodia show highly dynamic behavior as they probe their environment. Resolving the growth cone lamelipodia dynamics requires high spatial and temporal resolution. Nanowires with their high aspect ratio, high packing density, and small diameter have a great potential for detecting small forces with high spatial resolution. Biological cells can grow on silicon micro- and nanowire surfaces.10,11 We have shown that neurons can grow on top of a free-standing gallium phosphide (GaP) nanowire mat.12 Here we report a method using GaP nanowire arrays to detect cellular forces down to 15 pN. The forces are measured by quantifying the deflection of the nanowire tips in situ using confocal microscopy. The nanowires are fluorescently labeled and the confocal plane is set to the tip of the nanowires for deflection measurements. The tip displacement is translated to force using the linear elasticity theory. We demonstrate nanoNewton force detection with 80 nm diameter nanowire arrays on nerve fibers and 15 pN force detection from an
isolated neural growth cone with 40 nm diameter nanowire arrays with a spatial resolution of 1 µm. This method provides a good basis for live-cell force measurements and in vitro study of growth cone path finding and neural regeneration. GaP nanowire arrays were defined using electron-beamlithography (EBL) patterned gold seeds and grown epitaxially from GaP (111)B substrates. Arrays with 40 and 80 nm diameter nanowires were made. For the 40 nm diameter nanowire arrays, the EBL resist ZEP20A7 was spun at 9000 rpm on the substrate for 60 s. The sample was then baked on a hot plate at 180 °C for 2 min. A hexagonal array of single pixel dots was defined in the resist using an EBL system (Raith 150) working at 20 kV with single-pixel dose of 5 fAs. The distance between the dots was set to 1 µm depending on the sample. The samples were developed in o-xylene for 5 min before being rinsed in isopropanol (IPA) and blown dry with nitrogen. Ten nanometers of thermally evaporated gold were deposited on the surface before liftoff in Remover 1165 at 60 °C. The samples were then rinsed in deionized (DI) water and blown dry with nitrogen. For the 80 nm diameter nanowire arrays, the EBL resist PMMA950A5 was spun at 5000 rpm for 30 s followed by a 15 min bake on a hot plate at 160 °C. The EBL exposure was performed at 20 kV with a single pixel dose of 22 fAs. The samples were developed in a 1:3 mixture of methyl isobutyl cathone/IPA for 1 min, rinsed in IPA, and dried in nitrogen. A 20 nm gold layer was deposited by thermal evaporation and was followed by a lift-off in acetone at 60 °C. The samples were then rinsed in IPA and dried in nitrogen. All samples were stored in a nitrogen-filled glovebox until the nanowire growth. The nanowires were grown by metal-organic vapor-phase epitaxy (MOVPE) (AIX200/4, Aixtron AG) from the EBLdefined gold particles. In order to remove surface oxide and alloy the Au particles with the substrate, the samples were annealed at 470 °C for 10 min in hydrogen and phosphine
* To whom correspondence should be addressed. Phone: +46 46 222 4796. Fax: +46 46 222 3637. E-mail:
[email protected]. Received for review: 08/17/2009 Published on Web: 01/26/2010 © 2010 American Chemical Society
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(NGF), and 1% extra cellular matrix gel (ECM) were added onto the substrates. The cells were incubated for 24 h at 37 °C in the dark under carbogen atmosphere (95% oxygen, 5% carbon dioxide) before cellular force investigation. After 24 h, the culture and the medium were transferred in a glassbottom Petri-dish (Mattek Corporation) with the substrate resting upside down on pieces of 60 µm thick AlOx filters deposited at the edges of the Petri dish (for sample support without affecting the cells). The Petri dish was transferred to a cell culture incubator mounted on the stage of a inverted laser scanning confocal microscope (Zeiss LSM510). The stage incubator temperature was set to 37 °C, and the atmosphere inside the incubator was composed of 5% of carbon dioxide. The cells of interest were spotted using standard fluorescence microscopy with a 20× objective. The cells were then studied using confocal microscopy with a 63× oil immersion objective (N.A. 1.4). The fluorescence of the GFP and the nanowires was collected in two separate tracks using the 488 and 561 nm lasers, respectively, with a pinhole of one airy unit (corresponding to a 0.8 µm thick slice). The few cell processes that were not growing on top on the nanowires were not investigated. To minimize phototoxicity effects, the laser power was set to the minimum value at which a signal was detected. The focus was adjusted to the tip of the nanowires and time-lapse images were recorded every 1 to 10 s, depending on the sample. The images were analyzed using Image J software and a particletracking plugin.14 For each frame of the time series, the nanowire tip was located and linked between consecutive frames. The confocal planar drift was calculated from an ensemble of immobile nanowires that were not associated with any cells and subtracted from the images. The deflection of each nanowire tip was then analyzed and translated into force according to the linear elasticity theory for hexagonal cross sections15
FIGURE 1. Scanning electron microscopy image of a 80 nm diameter, 4 µm long nanowire array. The nanowires are spaced 1 µm apart. Scale bar 2 µm.
(H2 and PH3) atmosphere in the MOVPE reactor. The nanowire growth was initiated by supplying trimethylgallium (Ga(CH3)3) in addition to the phosphine at 470 °C. Precursor molar fractions were 10-5 and 10-2 for trimethylgallium and phosphine, respectively, in a hydrogen carrier gas flow of 6 L min-1. The growth was conducted under low pressure (10 kPa). Growth time, temperature and gas pressures all affect the wire length, which in this case was controlled to be between 2.5 and 5 µm depending on the sample. The nanowire diameter is determined by the gold particle size and was typically 40 nm (for ZEP resist) and 80 nm (for PMMA resist). The nanowire growth time was in the order of a few minutes. The resulting GaP nanowires were grown in the [111] B-direction, perfectly perpendicular to the surface with very low tapering and with exceptional homogeneity in the dimensions of the nanowires. Figure 1 shows a scanning electron microscope (SEM) image of a nanowire hexagonal array. The nanowires have a diameter of 80 nm and a length of 4 µm. The spacing between the nanowires is 1 µm. Before the cell culture step, the arrays were fluorescently labeled as follows. The substrates were immersed in a 10 µg mL-1 laminin solution in phosphate buffered saline (PBS) for 30 min at 37 °C. The samples were then extensively rinsed in PBS and incubated with 1/200 rabbit antilaminin IgG (Sigma) in PBS with 0.25% bovine serum albumin (BSA) and 0.25% Triton X100 for 2 h at room temperature. After extensive rinsing in PBS, the substrates were incubated with 1/200 antirabbit-Alexa Fluor 594 secondary antibody (Invitrogen) in PBS with 0.25% Triton X100 and BSA. Dissociated cells from mouse dorsal root ganglia (DRG) were cultured as described elsewhere.13 Genetically modified mice expressing the green fluorescent protein (GFP) were used (Okabe). The DRG cells were dissociated in a 0.25% collagenase solution, rinsed, and resuspended in RPMI 1640 cell culture medium at a concentration of 300 nerve cells µL-1 approximately. Each fluorescently labeled nanowire array substrate was coated with 100 µL of the cell suspension and put in the incubator at 37 °C. After 5 min, most cells had attached to the substrate and 2 mL of RPMI 1640 with 10% calf serum, 42 ng/mL of neural growth factor © 2010 American Chemical Society
F)
3EI 15√3ED4 ∆x ) ∆x L3 256L3
(1)
where E is the Young’s modulus of the nanowires, I is the second moment of inertia, D is the nanowire diameter, L is the length of the nanowire and ∆x is the displacement of the nanowire tip. We estimated the Young’s modulus of GaP nanowires from the measurement of the nanowire eigenfrequencies using a stroboscopic imaging technique.16 The sample, with nanowires still attached as they were grown, was glued on top of a piezo-electric crystal and placed inside a vacuum chamber. The nanowire oscillation was actuated by applying an AC voltage to the piezo-electric crystal. Snapshots of the moving nanowire were obtained using an ordinary optical microscope with stroboscopic illumination. For each frequency, the oscillation could be studied by sweeping the delay of the light pulses relative to the driving frequency. 783
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tables.18 The 0.68 factor is a corrective factor taking into account the measured tapering of the nanowires. This factor was obtained from finite element calculation of resonance frequencies for both tapered and nontapered nanowires. The average value of the Young’s modulus of the GaP nanowires was 150 GPa with a standard deviation of 32 GPa. In the Supporting Information, we show a movie of a Schwann cell growing on top of a 2.5 µm long, 80 nm diameter nanowire array with a spacing of 1 µm between the wires. It is very clear that the cell processes grab and bend the wires as the cell grows on the substrate. In this case, the forces involved are rather large (on the order of tens of nanoNewtons) and cannot be measured within the linear approximation. Figure 3 shows the results obtained from nerve cells growing on a 4 µm long, 80 nm diameter nanowire array with a spacing of 1 µm between the nanowires. The nanowire spring constant is 9.74 × 10-3 N m-1 (see eq 1). The measurements were done at a crossing between two groups of nerve fibers (growing horizontally and vertically within the white frame in Figure 3a). The acquisition was done at a rate of one image per second and lasted for 10 min. From the diameter of the fibers (around 3 µm), we believe it corresponds to a bundle of axons rather than a single nerve process. Figure 3a shows a confocal microscopy image of the nerve processes at the beginning (left) and at the end of the force measurement (right). The nerve fibers have grown from several micrometers toward the left of the image. Figure 3b-e represents the nanowires that are within the white frame in Figure 3a. Figures 3b-d represents the nanowire displacement vectors at time 0 s (b), 5 min (c), and 10 min (d). Three nanowires of interest have been highlighted in red, green, and blue. Note that new forces were generated on the substrate at the leading edge of the fiber as illustrated at 5 and 10 min for the nanowires highlighted in red and green. Figure 3e represents all the nanowire displacement vectors during the 10 min measurement time. It is interesting to note that for the nanowire highlighted in blue, the force magnitude remained the same but its direction has changed. During the 10 min acquisition time, the force direction on that particular wire changed by more than 45°, reflecting the dynamic nature of the nerve fibers; as the leading edge of the fiber is growing and is defining the growth direction, the whole fiber realigns itself and straightens itself (the small detours taken by the growth cone at an earlier stage are not preserved as the axons grow).13 Figure 4 represents the force magnitude for the nanowires highlighted in blue, red, and green in Figure 3 using the same color coding. The black trace represents the noise, which is smaller than 1 nN, measured on an immobile nanowire that was not associated with any cells. The bending of the green wire started after 2 min approximately. The force increased rapidly to 8 nN in one minute. Thereafter, the force magnitude fluctuated between 7 and 12 nN during the investigation. The force exerted on the red wire emerged
FIGURE 2. Eigenfrequencies measured on 13 nanowires using a stroboscopic imaging technique. Each nanowire had two different frequencies (represented by blue squares and red discs) in different directions.
Both amplitude and phase curves could be obtained by sweeping the driving frequency. All nanowires had two different eigenfrequencies in different directions. Theoretically, a hexagonal cross-section should have a symmetric area moment of inertia and therefore the eigenfrequency should be the same in all directions. However, a slight geometric asymmetry will result in the existence of two different eigenfrequencies. The eigenfrequency splitting was different both in size and angle for different nanowires, which implies that the individual properties of the nanowires are responsible for this phenomenom. Figure 2 represents the two measured values on an ensemble of 13 nanowires. The average nanowire diameter was 70 nm with a 80 nm diameter base and a 60 nm diameter tip. The length of the nanowire was 4.8 µm. The variation in the frequency values between nanowires is mainly due to differences in size between nanowires. It could be greatly reduced by measuring the dimensions of each specific nanowire that we have measured the eigenfrequency of. On the other hand, we cannot know the exact dimensions of each individual nanowire during the force measurements, and the spread in the frequencies values reflects the geometrical properties of the arrays. We deducted two values of the Young’s modulus, the first one from the average higher eigenfrequency and the second one from the average lower eigenfrequency according to17
E)
4π2FAL4(0.68f)2 Iβ4
(2)
where F is the density of GaP, A is the cross section area, f is the eigenfrequency, and β ) 1.8751 can be found in © 2010 American Chemical Society
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FIGURE 4. Force measured from the deflection of the nanowires highlighted in blue red and green in Figure 3. The black curve represents the noise (deflection of a nanowire that was not associated with any cells).
FIGURE 3. Axonal bundle growth on a 4 µm long, 80 nm diameter nanowire array. The nerve fibers of interest are within the white frame in panel a and have grown several micrometers during the 10 min acquisition time. Panels b-d represent the nanowire-tips displacement at 0 min (b), 5 min (c), and 10 min (d). Three nanowires have been highlighted in red, green, and blue for which the white dots correspond to the original (no force) position. Panel e shows all tip displacements measured during the 10 min acquisition time, showing changes in force direction. Scale bar 2 µm. Force scale bar (yellow): 10 nN.
FIGURE 5. Neural growth cone on a 5 µm long, 40 nm diameter nanowire array with a spacing of 1 µm between the wires. The colored circles designate the nanowires that have been used in the force measurements shown in Figure 6. Scale bar: 1 µm
spacing of 1 µm between the wires. The spring constant for such nanowires is 3.12 × 10-4 N m-1. The growth cone was sending and retracting filipodia and lamelipodia without progressing significantly on the substrate. The nanowires highlighted in red and green were located at the edge of the growth cone. The blue wire was located at a kink of the neural process. Such kinks can undergo large morphological changes as the neural process is reshaping to its final morphology.
at t ) 4 min, when the fibers have reached the nanowire. The force increased slowly over two minutes and stabilized around 6 nN. The force of the blue wire remained constant around 6 nN over the entire period. To detect smaller forces, we used nanowires with a lower spring constant, in other words longer nanowires with smaller diameters (see eq 1). Figure 5 shows a single neural growth cone growing on an array of 5 µm long, 40 nm diameter nanowires with a © 2010 American Chemical Society
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on the nanowire probably helped promoting cell-growth and adhesion. For the 5 µm long 40 nm diameter nanowires, the positional fluctuations are estimated to be on the order of 3 nm, assuming an harmonic oscillator. The uncertainty in the measured position of each nanowire can be estimated by22,23
σ)
(3)
where λ is the wavelength of the detected photons, NA is the numerical aperture of the objective, and n is the number of photons collected in the 0.8 µm thick optical slice. Assuming a 30% coverage of laminin on the nanowires, a 1% photon detection efficiency, and 25 fluorophores associated to each 900 kD laminin protein, the expected uncertainty in the determination of the position of each nanowire is around 5 nm. Together with the positional fluctuations, this can be translated to a force of 2 pN. The method we present here has a great potential. One of its advantages is that the forces are measured in situ, thereby avoiding artifacts due to cell drying.24,25 Another advantage is the high number of probes that such arrays can offer: the field of view with the 63x objective is 143 × 143 µm2, making it possible to probe more than 23000 points simultaneously when having a 1 µm spacing between the wires. The homogeneity in the nanowire sizes is a crucial component, without which there would be a high uncertainty associated with the force calculation. Finally, the method we present is adaptable to a wide variety of cells and do not require fluorescent cells as one can visualize the cells using laser reflection or phase contrast microscopy instead of fluorescence microscopy as we did. One of its disadvantages is the bleaching of the fluorophores that label the nanowires. This problem can be easily resolved by using quantum dots instead of standard fluorophores for coating the nanowires. The quantum dots should be carefully chosen to minimize blinking in order to avoid unnecessary degradation in positioning accuracy. In conclusion, we present a method for measuring cellular forces from 15 pN up to tens of nanoNewtons using monodisperse gallium phosphide nanowire arrays with a spatial resolution of 1 µm. The forces are calculated from the measurement of the nanowire-tip displacements using confocal microscopy in situ. The forces measured on a neural growth cone are comparable to the ones measured on a growth cone lamelipodia using optical tweezers. While being less sensitive than the optical tweezers method, our method can be used to probe more than 20 000 points simultaneously with a 15 pN force resolution and therefore holds great promise for studying growth cones dynamics and axon regeneration.
FIGURE 6. Growth cone forces measured with the four nanowires designated by the circles in Figure 5 (using the same color coding). The black curve corresponds to the noise and was obtained by measuring the deflection of a nanowire that was not in contact with the cell (white circle in Figure 5). The noise is below 15 pN and the forces measured ranged from 15 to 80 pN.
Figure 6 shows the forces detected on the four nanowires that are highlighted in Figure 5. The black curve corresponds to the noise (nanowire circled in white in Figure 5), which is smaller than 15 pN. The nanowires highlighted in green and red are subjected to forces varying between 15 and 35 pN. Since hardly any net growth was occurring, the forces are expected to be modest. However, the force exerted on the blue wire reached more than 70 pN indicating a larger reorganization in the cytoskeleton. The acquisition rate was 0.1 s-1. The maximum force increase rate was 40 pN in 10 s. The measured values are comparable with forces measured on DRG growth cone lamelipodia using optical tweezers4 where the force exerted by the lamelipodia was of 20 pN and above, and the maximum increase rate was 10 pN s-1. These forces are also in good agreement with data obtained on chick embryo heart fibroblast lamelipodia, where the lamelipodia pulling force exerted on a fishpole probe was measured between 10 and 50 pN.19 This demonstrates the ability of GaP nanowire arrays to measure very small forces exerted by neural growth cones at many points simultaneously, a technique that could complement optical tweezers measurements and yield new information on growth cone dynamics. It is well known that cells respond to the rigidity of the extracellular matrix or substrate they are growing on.2,20,21 In compliant substrates, the cell focal adhesions do not mature and in extreme cases, the cells do not attach and proliferate.1 In that context, it is important to stress that the cells had a normal shape and that they were able to attach and grow on the substrate despite the low rigidity of the substrate (3.12 × 10-4 N m-1). For comparison, the spring constant of PDMS pillars ranges from 2.7 10-3 to 1.6 N m-1 and for a flat surface of PDMS, it is estimated to be around 10 N m-1.9 In our case, the laminin © 2010 American Chemical Society
λ NA√n
Acknowledgment. This work was performed within the Nanometer Structure Consortium and the Department of 786
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Cell and Organism Biology at Lund University and was financed by the Knut and Alice Wallenberg Foundation, the Swedish Research Council (VR), and the Swedish Foundation for Strategic Research (SSF). The authors thank Peter Ekstro¨m, Rita Walle´n, Karl Rosenberg, and Inger Antonsson for their help.
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Note Added after ASAP Publication. Figure 4 was modified in the version of this paper published ASAP January 26, 2010; the corrected version published ASAP January 28, 2010.
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Supporting Information Available. A movie of a Schwann cell growing on top of a 2.5 µm long, 80 nm diameter nanowire array with 1 µm spacing, as well as a movie of the corresponding nanowire deflections. This material is available free of charge via the Internet at http://pubs.acs.org.
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