A NANOWIRE-INTEGRATED MICROFLUIDIC DEVICE FOR HYDRODYNAMIC TRAPPING AND ANCHORING OF BACTERIAL CELLS Donguk Kwon1, Jung Kim1, Soochan Chung1, and Inkyu Park1 1 Korea Advanced Institute of Science and Technology (KAIST), Daejeon, KOREA
ABSTRACT
DESIGN AND TRAPPING MECHANISM
In this work, we proposed a novel method for facile hydrodynamic trapping and anchoring of bacterial cells using nanowire array with fishnet-like structure in microfluidic channel. Vertically well-aligned ZnO nanowires were directly synthesized onto side walls of microslit structures by hydrothermal method to form mesh-like cage structures. We found that the mesh-like cages were effective in trapping and anchoring of Escherichia coli cells as model bacteria. In addition, we observed two anchoring modes; impaling and wedging, by electron microscopy and they resulted in irreversible and reversible damage to the anchored cells, respectively. We expected that the suggested bacterial cell trapping method can be used as a simple cell-manipulating platform for advanced microfluidic system.
In Figure 1(a), the design of the microfluidic device is shown. Cell solution entered into the four parallel chambers that are identical with each other. The empty region is present before the cage arrays to make cells spread. The cage arrays were arranged in a series of zigzags as shown in Figure 1(c). Each cage has slit structures with width of 3~4 μm. A photograph of the device is shown in Figure 1(b).
INTRODUCTION Trapping or retaining of biological cells at intended position is a key operation to develop an advanced microfluidic system for studying their biochemical reaction and behavior against external environment [1]. Trapping of biological targets in microfluidic device can provide controllable experimental environment for post-processes (e.g. monitoring [2], culturing [3], pairing [4], and cytotoxicity screening [5]). The trapping function also can be used as a means of detecting and analyzing platform itself [6]. Especially, in the case of bacterial cells, trapping the cells has some experimental difficulty due to the relatively small size of sub-micrometer dimension and mobility of the cells. Nevertheless, there have been many researches about the trapping of bacterial cells in microfluidic systems such as optical tweezing [7], dielectrophoretic trapping [8], magnetic trapping [9], and acoustic trapping [10]. However, these methods require expensive equipment or complicated setups to apply the trapping methods. Some researchers employed a hydrodynamic trapping method as an alternative, but this approach still has complexity in the flow control [11]. In this work, we developed nanowire-integrated microfluidic devices for facile hydrodynamic trapping and anchoring of bacterial cells and demonstrated that mesh-like cages formed by integrating ZnO nanowires are effective in trapping and anchoring of Escherichia coli (E. coli) as a model bacterium. Our method does not require expensive and complicate experimental instruments and delicate system control. It was shown that living bacteria were trapped both in the bare microslit structures and the nanowire-integrated structures with different efficiencies. We also observed two anchoring modes, impaling and wedging, resulted in irreversible damage or reversible deformation of the cell wall, respectively.
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Figure 1: Design of the device and trapping mechanism. (a) Schematic illustration of the microfluidic device from top view. (b) A photograph of the microfluidic device. (c) Detailed view of cage array in a series of zigzags. (d) A schematic diagram of the mesh-like cage formed by integration of ZnO nanowires. E. coli cells are trapped at the cage while they travel along the microchannel. A schematic diagram for trapping mechanism is described in Figure 1(d). ZnO nanowire array was employed to arrange dense nanostructures with shape of fishnet. Robust ZnO nanowires synthesized by hydrothermal method had high-aspect-ratio with diameter of 40~80 nm and length of
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and height of ~10 μm (Figure 3(a)). After hydrothermal synthesis of ZnO nanowire array, mesh-like cage structure was formed (Figure 3(b)). The length of ZnO nanowires was ~2 μm, so the slit could be filled with the nanowire array.
few micrometers [12]. The ZnO nanowires were also well-aligned vertically on the substrate where they grew on. Moreover, the nanowire array was dense enough to form a fishnet for trapping of sub-micrometer sized bacterial cells. Therefore, we integrated ZnO nanowire array directly onto the microslit structure to form a mesh-like cage as shown in Figure 1. When E. coli cells meet the mesh-like cage during travel along the microchannel, they will be trapped at the cage.
DEVICE FABRICATION Figure 2 shows the fabrication process of the device. First, chrome (Cr) etch mask was patterned on silicon substrate by photolithography and e-beam evaporation (Figure 2(a), (b)). Microslit structures were fabricated by deep reactive ion etching (DRIE) with the Cr etch mask (Figure 2(c)), which was removed from the substrate after DRIE (Figure 2(d)). Next, ZnO seed layer was deposited by sputtering directly onto the microslit structures (Figure 2(e)). Then, ZnO nanowires were synthesized hydrothermally in precursor solution at 95 C for 2 hours (Figure 2(f)). After synthesis of ZnO nanowires, PDMS cover was capped by stamp-and-stick method to ensure the compact capping and visibility (Figure 2(g)) [13].
Figure 3: A group of SEM images for detailed view of microslit structures on silicon substrate. (a) SEM images of bare microslit structures before integration of ZnO nanowires. (b) SEM images of mesh-like cages after integration of ZnO nanowires into microslit structures.
EXPERIMENTAL SETUP FOR TRAPPING OF BACTERIAL CELLS In Figure 4, the experimental setup for monitoring of trapping bacterial cells is shown. The setup simply consists of the nanowire-integrated device, fluorescent microscope and syringe pump. E. coli cells were genetically encoded with red fluorescent proteins (RFPs), so they express the red fluorescence with the fluorescent microscopy. E. coli cells in phosphate buffered saline (PBS) solution were simply forced to flow by syringe pump at the flow rate of 0.1 ml/hr. Total trapping period was 30 minutes. Figure 2: Fabrication process of the nanowire-integrated microfluidic device. First, microslit structures were patterned by photolithography and e-beam evaporation and fabricated by DRIE on silicon substrate. Next, ZnO nanowires were synthesized directly onto the side walls of microslit structures through hydrothermal method. Finally, PDMS cover is capped by stamp-and-stick method to ensure the compact capping. In Figure 3, scanning electron microscope (SEM) images of microslit structures with ZnO nanowires are shown. Microslit structures without nanowire array were constructed in an array on silicon substrate with the slit width of 3~4 μm
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Figure 4: Experimental setup for monitoring of trapping bacterial cells.
After same trapping time (0.5 hour), a couple of E. coli cells were trapped in each unit of bare microslit structures as shown in Figure 7(a). On the other hand, dozens of E. coli cells were trapped and anchored in each unit of nanowire-integrated microslit structures as shown in Figure 7(b).
HYDRODYNAMIC TRAPPING MODES To understand how E. coli cells are trapped at the structures, we made E. coli cells flow through trapping chambers with bare microslit array. Originally, the microstructure of silicon material is not visible because it does not emit fluorescence. By introducing fluorescein isothiocyanate (FITC) into trapping chambers before injection of the cell solution, the silhouette of microslit structure could be discriminated clearly from surrounding area. Consequently, E. coli cells also could be detected as green fluorescent signals because the cell wall was coated with FITC (Figure 5). Although most cells just passed through the microslit structures, some cells which had relatively thicker diameter could be trapped at the microslit structures. As shown in Figure 5, we observed two different trapping modes in bare microslit structures. First, bending occurs when a cell is trapped in transverse direction at the microslit (Figure 5(a)). Second, wedging happens when a cell burrows into microslit from the apical terminus of the cell in longitudinal direction (Figure 5(b)). The trapping process did not exert as much shear force to trapped cells as generating fracture of the cells.
Figure 6: (a) Nanowire-integrated cage structures before injection of the cell solution. For the images (b) – (f), yellow mark arrays are overlapped artificially for visual help. E. coli cells were forced to flow during (b) 0 min. (c) 1 min. (d) 5 min. (e) 10 min. (f) 20 min. All the images were taken at the same spot.
Figure 5: Fluorescent images of two different trapping modes of E. coli in bare microslit structures. (a) Bending (b) Wedging
TRAPPING OF BACTERIAL CELLS WITH NANOWIRE ARRAY Results of trapping E. coli cells using mesh-like cages integrated with nanowire array are shown in Figure 6. E. coli cell solution whose concentration of 4x105 cells/ml was injected into trapping chambers to monitor the trapping progression with time at the mesh-like cages. Mesh-like cages were arranged as shown in Figure 6(a), and all the images of Figure 6 were taken at the same spot. For Figure 6(b) – (f), yellow mark array is overlapped to indicate position of cages. The number of E. coli cells trapped at the cages were increased and determined with red fluorescence. Most of cages were filled with E. coli cells within 10 minutes. After 20 minutes, E. coli cells started to clog even the bypassing channel area. To examine the effectiveness of mesh-like shape of ZnO nanowires in cell trapping, we compared the bacterial cells trapping tendency in the bare microslit structures and nanowire-integrated microslit structures at the same flow rate.
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Figure 7: Fluorescent images of the trapping chambers after same trapping period of half an hour (a) Bare microslit structures (b) Nanowire-integrated microslit structures
HYDRODYNAMIC ANCHORING MODES After trapping of E. coli cells, the cells were dried with the compact capping of PDMS cover. Then, we checked SEM images of the cells, and we observed two different anchoring modes. The first anchoring mode, impalement of E. coli on ZnO nanowires, is shown in Figure 8(a). E. coli cells were pierced by sharp tip of ZnO nanowires, and irreversible
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damage to cell wall occurred. The second mode, wedging, is shown in Figure 8(b). In the wedging mode, E. coli cells were deformed only in the reversible manner and stuck in the mesh-like cages.
Figure 8: SEM images of two different anchoring modes of E. coli in nanowire-integrated microslit structures. (a) Impaling of E. coli on ZnO nanowires (b) Wedging into microslit structures integrated with ZnO nanowires
CONCLUSION Trapping of biological targets at intended position is important to develop an advanced microfluidic system. In this work, novel hydrodynamic trapping and anchoring method of bacterial cells that utilizes the fishnet-like shape of nanowire array has been introduced. It was demonstrated that implementation of mesh-like cages integrated with ZnO nanowire array is effective in hydrodynamic trapping and anchoring of E. coli cells. The proposed method is simpler than other trapping methods without complicated working principle, and has the potential for direct connection to many applications such as bacterial cell assay or inactivation.
ACKNOWLEDGEMENTS This work was supported by the Center for Integrated Smart Sensors funded by the Ministry of Education, Science and Technology as Global Frontier Project (CISS-2012M3A6A6054201). Donguk Kwon would like to thank Prof. Je-Kyun Park for providing Escherichia coli cells and laboratory facilities.
CONTACT *D. Kwon, tel: +82-42-350-5240; E-mail:
[email protected]
REFERENCES [1] J. Nilsson, M. Evander, B. Hammarström, and T. Laurell, “Review of Cell and Particle Trapping in Microfluidic Systems”, Anal. Chim. Acta., vol. 649, pp. 141-157, 2009.
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