Thin Films, Nano-Particles and 2D Materials
Arrays of NdFeB Clusters in PDMS Background Used to Levitate T47D Human Cells
2023 年 64 巻 9 号 p. 2143-2146
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2023 年 64 巻 9 号 p. 2143-2146
Sorting and trapping cells play an important role in fundamental cellular and biology research that enables the study of single-cell behaviors, which are different in comparison with a cluster of cells. Contactless handling techniques using different optical, mechanical, or magnetic phenomena have been studied for single-cell trapping. Among them, the diamagnetic force created by magnetic structures on cells is significant and stable both in time and in space. In this work, arrays of hard magnetic clusters in the PDMS background (hereafter called magnetic structure) were successfully fabricated using the magnetic imprinting method. The magnetic structure shows a proper magnetic property and the possibility to sort and trap T47D single cells via the diamagnetic levitation phenomenon at defined positions, which are both experimentally observed and theoretically calculated. The obtained results show the promise of developing a simple way to separate directly living cells.
Fig. 4 T47D cells levitate on the magnetic structure covered by a Si piece (a), dependence of the theoretical total force in the z-axis affecting a cell which is at the center of the gap between 4 squared-shape NdFeB clusters on the height from the surface of the magnetic structure (b).
Cell sorting and trapping are critical operations for many applications in pharmaceutical and fundamental biology. Arrangements of cells are advantageous for characterizing small quantities of cells among a wide population.1) Moreover, cell arrays allow monitoring of the behavior of individual cells for various durations spanning from minutes to days.2) Other applications of living cell arrays include but are not limited to diagnosing, identifying the role of cells in diseases, studying communication between cells, drug screening, and cell therapy.3,4)
Recently, contactless handling techniques have been developed for arraying and trapping cells by using the force which induced the optical effect,5) the mechanical effect,6) the dielectric, or the diamagnetic effects.7–9) A diamagnetic force created by magnetic structures is significant and stable both in time and space. Furthermore, the magnetic structures supply the magnetic field source without energy dissipation and heating. For these reasons, several techniques have been developed to fabricate magnetic structures such as the electroplating method,10) the self-assembly method,11) the topographically patterned method,12) the ink-jet printing,13) and the magnetic imprinting.14) The last one is a simple and low-cost method to fabricate rapidly the arrays of magnetic clusters without using high-tech fabrication systems and vacuum techniques.
In this study, the array of NdFeB clusters, embedded in the PDMS background, was successfully fabricated using the magnetic imprinting method. Each square-shape cluster has an average surface area of 50 × 50 µm2, a thickness of 8 µm, and an adjacent distance of 50 µm, respectively. The magnetic structure generates the calculated maximum intensity of magnetic induction in the z-axis (Bz), and the calculated highest gradient of Bz in the z-axis (dBz/dz) at 10 µm above its surface of 1.0 × 10−2 T, and 2.0 × 104 T/m, respectively. As the result, the magnetic structure showed a possibility to directly sort and trap breast cancer cells (T47D) at 16 µm above its surface, which is, to the best of our knowledge, the first trial for this kind of cells.
NdFeB micro-sized particles with original conditions as average diameter of 6 µm, weight density of 7.5 g/cm3, remnant magnetization (MR) of 45 emu/g ∼ 0.42 T, and coercive force (HC) of 2 kG ∼ 0.2 T were used.9) PDMS SYLGARD 184 Dow Corning was used as obtained. And, T47D cells were provided by Sigma Aldrich.
2.2 Fabrication processTo fabricate the magnetic structure, a master structure containing arrays of NdFeB micro-magnets with a lateral dimension of 50 × 50 µm2 and a thickness of 5 µm, deposited by the sputtering method on a patterned Si substrate was used.15) The fabrication process of the magnetic structure is illustrated in the Fig. 1.
Diagram of fabrication process of magnetic structure by magnetic imprint method.
Firstly, an etched Si piece with a thickness approximately of 10 µm was placed on the master structure to make a flat background. Next, the NdFeB particles was put on the Si surface. Thanks to the magnetic force induced by the master structure, the NdFeB particles moved to the high magnetic field strength regions, i.e., at the edges and surface of the micro-magnets in the master structure, and then formed nearly squared-shape clusters. Spatial distribution of the clusters on the Si surface is consistent to the master structure configuration.
The mixture of base and curing agent with ratio 10:1 PDMS was mixed well before baked at 70°C for 1 hour to form elastic layer and followed gradually poured on the as-produced clusters. Then, the PDMS layer was peeled off to obtain a PDMS background with the NdFeB clusters (Fig. 2(a)). Average dimension of these clusters was 8 µm for the depth, 50 × 50 µm2 for the surface area, and the adjacent distance between the clusters was about 50 µm (Fig. 2(b)). Finally, the magnetic structures were perpendicularly magnetized by an 2 T external magnetic to obtain a remanent magnetization with out-of-plane orientation.
Top-view image (a), cross-sectioned image (b) of the magnetic structure.
The X-ray diffraction (XRD) measurement was conducted to confirm the material composition. The XRD pattern of magnetic structured NdFeB material is depicted in Fig. 2(c), which indicates the peaks at 2θ ≈ 20.04°, 31.96°, 36.36°, 38.10°, 39.44°, 44.39°, 46.83°, 60.52° and 78.15° corresponding to lattice planes of (004), (310), (320), (105), (115), (006), (116), (008) and (00 10), respectively. The peak positions show a good agreement with previous reports16,17) that reveal the detailed composition of the NdFeB material to be Nd2Fe14B with tetragonal crystal structure system and space group of P42/mnmm, implying good stoichiometry. The unit cell structure of the NdFeB material is shown in Fig. 2(d) with lattice parameters of a = b = 8.81 Å, c = 12.21 Å, α = β = γ = 90°, and the unit cell volume of 946.31 Å3.
Scanning Hall probe measurement was used for measuring the component of magnetic induction intensity in the z-axis (Bz) at different heights above the surface of the magnetic structure, then gradient of Bz in the plane (dBz/dy) and gradient of Bz in the direction perpendicular to the plane (dBz/dz) were calculated. The Fig. 3(a) represents the data of the Bz of the magnetic structure which was obtained for an active area of 1 × 1 mm2 at 10 µm height above its surface. This result shows that the amplitude of the Bz componential magnetic induction produced by the NdFeB clusters is well consistent to the configuration and order of the clusters. However, correlative positions have a negligible inhomogeneity between which may relate to small variation of the width and thickness of the clusters, or distance between the clusters. Detailed plots of Bz, dBz/dy, and dBz/dz along a line parallel to the surface of the magnetic structure at the height of 10 µm (the white dash line in Fig. 2(a)) in Figs. 3(b), 3(c), and 3(d), respectively, are proper to the distribution of magnetic stray field generated from the clusters in the magnetic structure, and to the configuration of the magnetic structure, as well as agree with the magnetic space as in Fig. 3(a). In particularly, the maximum values of Bz, dBz/dy, and dBz/dz are obtained at positions around edges of the clusters, and are approximately of 1.0 × 10−2 T, 3.2 × 103 T/m, and 2.0 × 104 T/m, respectively.
3D image illustrates intensity of Bz magnetic induction at a plane above the surface of the magnetic structure of 10 µm (a), plot of Bz (b), plot of dBz/dy (c), and plot of dBz/dz (d) along the white dash line in Fig. 2(a).
By using the obtained magnetic structure, the breast cancer cells (T47D) were sorted and positioned relying on the diamagnetic levitation phenomenon. The T47D cell can be considered simply as a sphere with a diameter around 14 µm, and a weight of 5 × 10−8 g.11) The solution of T47D cells had been prepared with concentration of 105 cell/ml and the magnetic susceptibility of −7.7 × 10−8. The magnetic structure was covered by 10 µm-thick Si piece for protecting the surface of the magnetic structure and creating a flat, high-contrast plane before starting experiments.
The distribution of T47D cells on the surface of the magnetic structure was observed via an optical microscopy (Fig. 4(a)). Clearly to see that cells were suspended in the same plane, and stabilized at their own positions. The periodic arrangement of cells for both x and y directions in the plane was observed. The distance of adjacent cells was approximately 100 µm which equaled with the distance between adjacent void spaces (as illustrated as green line in Fig. 2(a)). These positions of cells have been shown the high agreement to the theory of the diamagnetic levitation phenomenon. This means that at these positions, the vertical magnetic force affected to cells to be counterbalanced the gravity force and the buoyant force. Consequently, the applied total force on cells becomes zero.
T47D cells levitate on the magnetic structure covered by a Si piece (a), dependence of the theoretical total force in the z-axis affecting a cell which is at the center of the gap between 4 squared-shape NdFeB clusters on the height from the surface of the magnetic structure (b).
The levitation height of T47D cells was also measured by varying the focusing plane of the optical microscope on the surface of cells and the substrate (i.e., Si layer). Indeed, the displacement of the focusing plane revealed that the surface of the Si and the stable position of cells were not in the same plane. Based on this displacement, the distance between cells and the surface of the Si layer was estimated by 6 µm. It meant that the levitation height from cell surface to the surface of the magnetic structure was around 16 µm, which is close to the theoretical value (the intersection points of the curve with the x-axis in Fig. 4(b)).
We have successfully fabricated the magnetic structure which consisted of arrays of squared-shape NdFeB clusters in PDMS background by the magnetic imprinting method. The fabrication design of the magnetic structure is controlled by the master structure, the magnetic powder. Depending on the size, the magnetic properties of cells, and the specific application, suitable master structures and magnetic powders will be selected for the fabrication so that we can obtain magnetic structures which generate proper magnetic field strength and gradient in the space. The good stoichiometric material belongs to the tetragonal crystal structure system and space group of P42/mnmm. The structure possessed the proper magnetic property and the possibility to sort and trap T47D cells via the diamagnetic levitation phenomenon which are both experimentally observed and theoretically calculated. The obtained results show the promising for development simple way to direct separation of living cells.
This research was funded by VNU Asia Research Center (ARC) from grants source by CHEY Institute for Advanced Studies, code CA.22.05A.
