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1 Nanoscale lateral displacement arrays for the separation of exosomes and colloids down to 20 nm Benjamin H. Wunsch, Joshua T. Smith, Stacey M. Gifford, Chao Wang, Markus Brink, Robert Bruce, Robert H. Austin, Gustavo Stolovitzky, and Yann Astier SUPPLEMENTAL INFORMATION 1. Array structures Nanofluidic chips were fabricated on 200 mm wafers (Supplementary Fig. 1a) to enable high-quality fluorescence imaging of nanoscale polymer beads and bio-colloids when coupled with a custom fluidic jig as described elsewhere. 1 Optical contact lithography followed by a combination of electron beam (ebeam) and deep-ultra violet (DUV) lithography schemes were utilized consecutively to define microchannel and nanofluidic pillar array features (Supplementary Fig. 1b), respectively, in an SiO 2 hard mask on bulk silicon substrates. Following hard mask definition, all features were transferred into silicon in tandem using a reactive-ion etch (RIE). Two dimensional Fast Fourier transform (FFT) analysis of the arrays (Supplementary Fig. 1c) confirmed a maximum angle, θ max, of 5.8 very close to the design value of 5.7. θ max = 5.7 was chosen as it corresponds to a relatively small row shift fraction δ = 0.1 leading to a well-defined row repeat N = 10, where θ max = tan -1 (δ) and N = 1/ δ. 2 Having a precise N simplifies design criteria while a small δ increases the efficiency of a particular gap size in sorting the smallest possible particle, since the critical diameter D c to sort a particle in the parabolic flow model is given by D c = 2αGδ with α being a scaling factor depending on the flow profile and G is the distance between pillars. This sorting efficiency becomes crucial to induce sorting of very small entities such as proteins, particularly as gaps are scaled in the tens of nanometers regime, pushing the limits of fabrication and ability to effectively wet these features. Supplementary Figure 1d shows the cross-sectional SEM image of a G = 134 nm pillar array. Gap sizes for each array tested were determined by measuring 3 randomly chosen sets of 5 adjacent gaps across the ~35 µm width of the pillar arrays (an average of 15 gaps per array), including 1 set chosen near the array center and the other 2 sets nearer to each edge of the array. Gap variation from pillar top to bottom was minor but not negligible so measurements were taken at pillar mid-height as indicated by the dotted line in Supplementary Figure 1d. Thermal oxidation after pillar etching on parallel-processed wafers permitted controllable means of tuning of the gap size to effectively narrow the gap to a desired width based on the results from an SEM cross-section of a send-ahead wafer for each array fabricated. NATURE NANOTECHNOLOGY 1
2 Supplementary Figure 1. Fabricated fluidic chips containing sorting arrays with maximum angle θmax = 5.7º. (a) Fluidic chips printed on a 200mm wafer using mixed DUV and electron beam lithography. (b) Optical image showing microfluidic channels joined by nanochannel features, including pillar-sorting arrays. (c) 2D FFT confirming successful patterning of the design angle θ t (d) Scanning electron microscope image of a sorting array with pillar pitch λ = 400nm and G = 134nm. Gap scaling and uniformity is demonstrated through RIE optimization and thermal oxidation of the Si posts, permitting well-controlled gap widths. 2 NATURE NANOTECHNOLOGY
3 SUPPLEMENTARY INFORMATION 2. Fluorescent bead size distribution Supplementary Figure 2. Fluorescent polystyrene beads. (a-c) Example SEM images of D P = 110 nm (a), 50 nm (b) and 20 nm (c) beads coated with a layer of evaporated Ti / Au (1 nm / 10 nm). Scale bars represent 200 nm. (d) Histograms of bead diameters measured from SEM images. (e) Properties of bead samples used in nanodld experiments. a: Mean diameter ± standard deviation. NATURE NANOTECHNOLOGY 3
4 3. Analysis of nanodld fluorescence microscopy images for particle behavior 3.1 Analysis of fluorescent polystyrene bead displacement: Fluorescence image videos of the array outlet are analyzed using custom software to determine the migration angle of the displaced particle flux. In the current array configuration, the flux of particles across the array is displaced towards the collection wall, forming a fluorescent, triangular pattern (white triangle on Supplementary Fig. 3a). A depletion region, where particles have been displaced out, appears on the opposite side of the array from the collection wall (dark triangle in Supplementary Fig. 3a). The extent of this depletion region at the outlet of the array determines the lateral displacement, W, of the particle flux. Determination of the lateral displacement is complicated by the fact that the edge of the particle flux, seen in the cross-section of fluorescence intensity across the array outlet, has a continuous form with no sharp cut-off. We estimate the edge of the particle flux, and thus W, using the inflection point of the fluorescence intensity (Supplementary Fig. 3b). This assumes that the fluorescent intensity distribution corresponds to the particle density distribution. Inlet Flow Collection Wall Triangle (Particle Fluorescence) Displacement L θ Outlet W Nanopillar Array Normalized Count (A.U.) Lateral Position (µm) 1st Derivative Normalized Count (A.U.) Normalized Count (A.U.) Lateral Position (µm) st Derivative Normalized Count (A.U.) Supplementary Figure 3: Determination of lateral displacement from particle fluorescence images. (a) Schematic of nano-dld array showing particle flux entering from left (inlet) to right (outlet). Particles are displaced upwards towards the collection wall of the array, forming a fluorescent triangle pattern (wedge), from which the migration angle, θ, and lateral displace, W, can be measured. The lateral displacement is taken at the outlet of the array. In the schematic, the particles are completely displacing (bumping mode) so θ = θmax = 5.7º, the maximum angle of the arrays used in this work. (b,c) Plots of fluorescent line profiles taken at the outlets of arrays for (b) complete displacement and (c) partial displacement modes. The red lines show the 1 st derivative of the fluorescent line profiles after smoothing, whose maximum corresponds to the inflection point (black dot). The lateral displacement, W, is taken as the distance between the bottom wall of the array (opposite the collection wall) and the inflection 4 NATURE NANOTECHNOLOGY
5 SUPPLEMENTARY INFORMATION point. Using the length of the array, L, and the lateral displacement, W, the migration angle can be calculated from tan(θ) = W/L. The blue line in (c) is the 1 st derivative taken before a 50-point smooth of the data (red line). The migration angle, θ, is defined as tan(θ) = W L -1, where L is the length of the array. For a completely displaced particle sample, all particles end up at the collection wall at the end of the array, and θ = θ max = 5.7, the maximum angle of the array. For no displacement, the particle flux covers the entire outlet, and θ = 0. The displacement efficiency is defined as: = = tan ~ = tan 100 To compare the effectiveness of sorting particles for a given D P and G, a figure of merit, FOM, is defined as the ratio between the lateral displacement of the particles, and the distance needed to fully displace the particles across the array: = tan = ηtan From the definition of the figure of merit, the full-displacement length can be defined as: = The parameter L C is the length of an array required to fully bump a particle to the collection wall, assuming the particle starts at the far wall of the device channel. NATURE NANOTECHNOLOGY 5
6 0.6 Bump Mode P Scaling Ratio, D P G Zigzag Mode Row Shift Fraction, εδ Supplementary Figure 4. Polystyrene fluorescent bead displacement as a function of particle diameter, D P, compared to critical diameter needed for displacement in a parabolic flow according to the lane model of DLD. Bead values are given for a given row shift fraction, δ = 0.10, and scaling ratio D PG -1. Value shading represents the percentage maximum angle, P. The black line is the calculated critical diameter scaling ratio, D CG -1 ~1.16 δ According to the lane model of DLD, beads with scaling ratios below the critical line should exhibit zigzag mode, P = 0%, and not displace within the array, while those above should show complete displacement, P = 100%. 6 NATURE NANOTECHNOLOGY
7 SUPPLEMENTARY INFORMATION Supplementary Figure 5. (a) (Left) Optical microscope image, 20x magnification, of a typical full-width injection nanodld device, showing the overall configuration of the array. (Center) SEM images of inlet and outlet regions bordering the nanodld array. (Right) Fluorescence microscopy images of fluorescent polystyrene beads flowing into the inlet region (top row) and exiting the array outlet region (bottom row), corresponding to those shown in the SEM images. The lateral displacement modes for zigzag, partial, and bumping are shown for D p = 20 nm / G = 214 nm, D p = 50 nm / G = 134 nm, and D p = 110 nm / G = 235 nm, respectively. The migration angle, θ, indicates the lateral displacement of the particle flux in the array. (b) Percentage maximum angle of fluorescent polystyrene beads displaced in nano-dld arrays as a function of the scaling ratio between particle diameter and gap size. Nominal bead diameters are 110 nm (red squares), 50 nm (cyan circles) and 20 nm (blue triangles). Error bars represent the standard deviation of at least three independent experiments. The line at D pg -1 = 0.37 represents the theoretical critical diameter DC, calculated according to the DLD lane model in parabolic fluid flow, at which beads are expected to be in bumping mode. P = 100% corresponds to complete displacement of beads (bumping mode), P = 0% corresponds to no displacement (zigzag mode), and 0% < P < 100% indicates partial displacement mode. NATURE NANOTECHNOLOGY 7
8 Supplementary Table 1. Performance parameters for nanodld displacement of fluorescence polystyrene beads. Particle Diameter, D P (nm) Array Gap Size, G (nm) Scaling Ratio, D P /G Percent Maximum Angle, P (%) Displacement Efficiency, η Figure of Merit, FOM Full Displacement Length, L C (mm) ± % ± % ± % ± % ± % ± % Analysis of Exosome Displacement: For exosomes, single-particles trajectories are recorded in fluorescence microscope images, instead of a flux density, as in the case of fluorescent polystyrene beads. We obtain a distribution of single-particle events instead of a continuous distribution determined by the average fluorescence density. In general, flowing particles form a streak or trace in a given video frame. For each particle observed, the location of the head of the particle s trace is manually marked per frame of video. The collection of x,y-coordinate pairs taken from the combined number of frames (typically 200) defines the trajectory of the particle within the image frame of the video. From the collected θ of all the single-particle trajectories, a histogram of the distribution of particles based on their deflection can be generated. This distribution gives the spread of particle positions after traveling 70 µm from the inlet. From the histogram the migration angle, θ, can be obtained as tan(θ) = W / 70. Supplementary Figure 6 shows the main steps in the data analysis. 8 NATURE NANOTECHNOLOGY
9 SUPPLEMENTARY INFORMATION Supplementary Figure 6. Exosome particle analysis in nanodld array. (a) Composite fluorescent microscopy image of a typical exosome particle trajectory in a G = 235 nm array. Scale bar is 10 µm. For each frame of the trajectory, the position of the particle s trace is recorded. The combined coordinates for each trajectory can be offset to form a unified starting position plot, (b), which shows the spread in the exosome particle ensemble. The black line at lateral position W = 0 µm is the ideal zigzag mode trajectory, and at W = 8.1 µm the ideal bump mode trajectory. The dashed line at W = 4.8 µm is the ideal trajectory at the mean ending lateral displacement, W. The displacement of each trajectory can be calculated to generate the histogram in (c) which gives the distribution of exosome particles in the lateral direction of the nanodld after traveling 70 µm. NATURE NANOTECHNOLOGY 9
10 4. Electron Microscopy of Exosomes For scanning electron microscopy, SEM, sample preparation was carried out by first applying 1 droplet of exosome solution on a glass slide and letting it dry in a sterile environment. A 2 nm conductive metal layer was sputtered onto the sample using a Hummer Sputter System (Anatech Ltd.) with a Au:Pd, 60:40 target. Imaging was performed with a Zeiss Leo 1560 Scanning Electron Microscope at 5k ev. Cryo-electron microscopy was contracted out to the Electron Microscopy Resource Laboratory in the Perelman School of Medicine, University of Pennsylvania. Sample preparation and imaging was carried out by Dr. Dewight Williams. For sample preparation, 10 µl of as-obtained human urine derived exosomes were diluted to 25 µl using PBS. Supplementary Figure 7 shows a typical EM image of the as-obtained exosomes. Supplementary Figure 7. Cryo-electron microscopy image of as-obtained human urine derived exosomes. 10 NATURE NANOTECHNOLOGY
11 SUPPLEMENTARY INFORMATION 5. Modeling of Exosome Displacement Distribution The simulated exosome displacement distribution was generated to compare with the measured displacement histogram. As the simulated distribution makes use of the exosome size distribution, this model allows us to determine if the measured exosome particle size distribution taken from SEM is consistent with the displacement dynamics which depends of particle size. The model is based on the same principles used by Heller and Bruss. 4 The displacement histogram gives the lateral displacement measured for each exosome trajectory after travelling a distance L = 70 µm from the array inlet, at v ob = 253 µm s -1 (the average speed of the exosomes). It is important to note that the measured displacement histogram is based on offsetting all particle trajectories to a single starting origin, W = 0. Lateral displacement can cause the particle distribution to shift to positive x, while diffusion will spread the distribution in both positive and negative x. For modeling, the particle sizes were taken from the binning of the SEM data, with D p = nm, with D p = 10 nm. For each particle size, the scaled distribution was modeled with a Gaussian function. The model accounts for diffusion by computing the Stokes-Einstein bulk diffusivity for each particle size. = 3 With T = absolute temperature, k = Boltzmann s constant, and µ is the shear viscosity of water ( kg cm -1 s -1 ) The full-width at half max, w, of the Gaussian was determined from the diffusion over the given time, τ, calculated from the distance and speed of travel. = = The position of the mean of the Gaussian, x 0, was determined from the parameterized percentage maximum angle, P, data taken for the polystyrene beads in a G = 235 nm nanodld array. The lateral displacement, W, is calculated from the parameterized P. =Δ = Δ tan With θ max = 5.7⁰. The distribution F i for the ith particle size was calculated at each lateral displacement position, W = [-15,15] µm, and the distributions summed to form the final displacement distribution, F. NATURE NANOTECHNOLOGY 11
12 Δ = = Δ With A i the normalized frequency of the ith particle size as measured from SEM. Supplementary Figure 8 shows a plot of final displacement distribution, F, and individual Gaussians, F i, showing the major contribution of the distribution density comes from particles with D p = nm. Normalized Particle Count nm 50 nm Summed Curve 70 nm 80 nm 90 nm Exosome Displacement, ΔW (μm) Supplementary Figure 8. Modeled exosome displacement distribution. Plot of the total summed curve (dashed red line) and individual Gaussian distributions (black line, red shading), normalized by the summed curve integrated area. The main Gaussians contributing to the model distribution are labeled with the binned exosome size, D p, showing that the majority of the model curve comes from contributions of particles between nm. 12 NATURE NANOTECHNOLOGY
13 SUPPLEMENTARY INFORMATION 6. Fractionation of Exosomes Supplementary Figure 9. Hypothesis testing of the null hypothesis that the before and after separation particle size distributions were the same, against the alternative hypothesis that the after separation would be shifted to smaller values. A Student s t-test yielded a P-value< , and a Kolmogorov Smirnov test resulted in a P- value of This supports the rejection of the null hypothesis in favor of the alternative that exosome particle size distributions after separation are enriched in smaller urine derived exosomes compared to the before separation samples. 7. Particle Volume Fraction Supplementary Table 2. Volume fractions of fluorescence polystyrene beads. Nominal Experimental Volume Particles per Array Unit Cell Particle Size, Concentration Fraction D P (nm) (particles ml -1 ) (%) nanodld Gap Size, G (nm) For all devices, the pillar height was 400 nm except for G = 42 nm, in which case it was 200 nm. NATURE NANOTECHNOLOGY 13
14 8. Estimation of Particle Induced Distortion of Flow In cases when the particle concentration is high (particle-laden flows), it is clear that the effects of other particles on a single particle can change that particle trajectory. But this can happen even in very dilute particle suspensions. Indeed, even the presence of a single finite size particle modifies the boundary condition of the fluid system, which now has to verify the non-slip boundary condition at the surface of the particle. The motion of a particle in a fluid flow is described by the Maxey-Riley equation: L1 L2 L3 R1 R2 R3 R4 R5 R6 Where m p and m f denote the mass of the particle and the mass of the liquid displaced by the particle respectively, a is the radius of the particle (assumed to be a sphere), and µ and are the dynamic and kinematic viscosities, respectively. g i denotes the i th component of the gravitational acceleration vector and u i denotes the j th component of the undisturbed velocity field which is a function of position and time. The vector Y(t) denotes the spatial coordinates of the particle center of mass at time t. W i is the i th component of the difference between the particle center-of-mass velocity v i, and the undisturbed velocity field (i.e., the velocity field without a particle) such that W i = v i(t) u i(y(t), t). The term for the time derivative of a fluid element: is shorthand = + We will call U 0 the typical unperturbed fluid velocity scale. Likewise, W 0 is taken to be a representative velocity scale for W i. L will denote the characteristic length scale in the unperturbed flow. The equation above is an approximation valid when the particle Reynolds number R p=aw 0/ and the shear Reynolds number R s=re a 2 /L 2 are small, where Re= LU 0/ is the unperturbed flow Reynolds number. 14 NATURE NANOTECHNOLOGY
15 SUPPLEMENTARY INFORMATION In our experiments U 0 is m/sec (we ll take the later as an upper bound when we calculate the Reynolds numbers), and L is half the gap size. We will take U 0 to be an upper bound for W 0. The diameter of the particle ranges from 20 nm to 100nm, and in what follows we will take L = m and a = m (notice that this is the smallest particle radius and gap size tested). Using the numbers described above, and the kinematic viscosity of water to be 10-6 m 2 s-1 we have that for our system: = < = = = = = Therefore, the conditions that R p<<1 and R s<<1 are verified and we can proceed with the Maxey-Riley formulation. We will assume that the density of the particles is the same as the density of the solvent (nearly true for polystyrene particles and exosomes). Dividing the Maxey Riley equation by 6πaµU 0 we can estimate the relative order of importance of each term. We get from each of the terms of the equation: 1 ~ 2 ~ 3 ~ 0 4 ~ 3 ~ 5 ~ 1 ~ 6 ~ 2 ~ NATURE NANOTECHNOLOGY 15
16 It can be observed that except for the term resulting from Faxén s correction (R4), all the other terms are a function of the shear Reynolds number, and smaller than (That is, were it not for Faxén s correction, which accounts for the curvature of the flow field, the velocity of the particle would differ from the velocity of the fluid by < 0.3%). As expected, Faxén s correction would become less important as the size of the particle decreases for a fixed gap size. Neglecting the smaller terms, the simplified Maxely-Riley form is given by: = ~ 1 6 Where v is the difference in the fluid velocities between the particle laden and undistributed flows, D p is the particle diameter, and G is the gap size of the pillar array. As the scaling factor approaches 0.5 (close to the empirically observed threshold for bumping), v ~ 4% of the undisturbed velocity. This suggests the particle induced disturbance of the local fluid flow is small, and that the effect of this perturbation on the nanodld operation is minimal. We recognized that the simplicity of a dimensional analysis argument, while allowing an estimate of the magnitude of the particles effect on the fluid flow, does not take into account the full complexity of the nanodld system. The fluid passing through the pillar gaps leads to regions of compact streamlines, in which small disturbances of the flow may have greater impact on determining the partitioning of particles between the bump and zigzag mode. In addition, small disturbances in the flow may be amplified by the fact that a single particle encounters thousands of pillar gaps in its trajectory across the device, and therefore the small effects may become cumulatively larger. Some effects are neglected in the Maxey-Riley formulation. The sphere is assumed to be far from the walls, and therefore particle-boundary interactions are excluded. The effect of this interaction is an added drag and lift force, as well as the potential for lateral displacement of fluid. The Stokes drag, taken in the formula above to be the one corresponding to a uniform flow far from boundaries, should be modified to take into account the additional drag created by such boundaries. 5 This can be considered to be an increase in the viscosity and therefore would result in a still smaller Reynolds numbers which does not modify our basic conclusion. The lift force, perpendicular to the local flow direction, can be estimated to have an effect smaller than the shear Reynolds number and therefore it is of the same order of the corrections discussed lines above. 6 Particle-induced lateral transport comes from an asymmetry in the flow around the particle which leads to a net displacement of fluid to the side of the particle as it traverses along a boundary. 7 The effect scales with a 3 as well as the particle Reynolds 16 NATURE NANOTECHNOLOGY
17 SUPPLEMENTARY INFORMATION number, and thus is expected to be negligibly small in the current nanodld system. References 1. Wang, C. et al. Hydrodynamics of Diamond-Shaped Gradient Nanopillar Arrays for Effective DNA Translocation into Nanochannels. ACS Nano 9, (2015). 2. Huang, L. R., Cox, E. C., Austin, R. H. & Sturm, J. C. Continuous particle separation through deterministic lateral displacement. Science (80-. ). 304, (2004). 3. Inglis, D. W., Davis, J. A., Austin, R. H. & Sturm, J. C. Critical particle size for fractionation by deterministic lateral displacement. Lab Chip 6, (2006). 4. Heller, M. & Bruus, H. A theoretical analysis of the resolution due to diffusion and size dispersion of particles in deterministic lateral displacement devices. J. Micromechanics Microengineering 18, (2008). 5. Leach, J. et al. Comparison of Faxén s correction for a microsphere translating or rotating near a surface. Phys. Rev. E 79, (2009). 6. Liu, C., Xue, C., Sun, J. & Hu, G. A generalized formula for inertial lift on a sphere in microchannels. Lab Chip 16, (2016). 7. Amini, H., Sollier, E., Weaver, W. M. & Di Carlo, D. Intrinsic particle-induced lateral transport in microchannels. PNAS 109, (2012). NATURE NANOTECHNOLOGY 17
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