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2010年12月31日 星期五

生物感測晶片 (Biosensor DEP Chip)

Introduction
There is considerable and growing interest in electrical detection method and the remarkable capability of positioning and registration of cell with single-cell resolution. Recently, Iliescu et al. demonstrated a capillary-based microfludic chip with coplanar electrodes for detection of dead and live yeast cells by electrical detection [3]. However, the impedance measured by coplanar electrodes without positioning and registration function, it is difficult to measure the impedance of single cell due to the misplaced or overlapping phenomena occurred. A. Han and A. B. Frazier [4] presented a multi-layer and polymer-based microchip for positioning single cell in a cavity by pressure difference and analysis of each cell for a period of time, however, the fabrication process was too complicate and the electrical signal only could be extracted from low frequency range. In this paper, we proposed a DEP chip consisted of multilayer electrodes and microcavities array for trapping cells and further electrical measurement under single-cell level. The viability of two kinds of cell lines, NB4 and HL-60 can be clearly identified, and the effects of microcavity on impedance measurement ?also will be discussed by numerical simulation and experimental data.
Simulation
The purpose of numerical simulation is to obtain an optimal depth of microcavity for trapping cells efficiently by DEP force and explicit difference of impedance as electric detection for dead and live cells. Hence, there are three kinds of depth for SU-8 microcavity, 5, 10 and 15μm, were established in 2D model and analyzed by the CFD-ACE+ software and COMSOL (FEMLab) software for electric field and total current density, respectively. In the Fig. 1(a), the contour of electric field, E, showed the highest density occurred near the edge of middle electrode and the lowest one resulted in the bottom of microcavity, thus, cells could be moved into microcavity by negative DEP force. Three cases of various depths of microcavity are shown in the Fig. 1(b), the deeper depth of microcavity constructed higher electric field density. However, the DEP force depends on the instead of E. From Fig. 2, the place happened largest value of was the same as E, although the for deeper microcavity was higher but the difference between 10μm and 15μm was little. Consequently, different depth of microcavity has no significant influence on DEP trapping of particles. In addition, the contour of in the Fig. 2(a) showed the intensity variation was small as increasing the distance away from SU-8 surface, hence, it is necessary to calculate the effective height in the flow chamber that DEP force can affect the particles and place particles into the microcavities. In the Fig. 2(c), the cross-sectional profiles of for several distances away from the middle electrode upon the SU-8 surface were plotted. The intensity of dramatically descended as the distance away the middle electrode increased, for instance, as the height in the chamber was 28μm, the intensity of went to zero. Thus, if the suspension particle flows beyond 28μm height in the flow chamber, it would be difficult to trap particles by DEP force. For electric detection simulation, As the results for 10μm depth microcavity shown in the Fig. 3, the contour of total current density for with or without particle in the microcavity exhibits the highest density occurred at the edges of two impedance electrodes, and most part of current passed through the medium if there is no particle in the microcavity, on the other hand, the current flowed through the particle as the microcavity occupied by a dielectric particle. Therefore, the resulting impedances can be expected significantly different for the cases with or without particle.

Fig. 1. (a) The contour of electric field for 10μm depth SU-8 microcavity, the highest density of electric field is near middle electrode upon the SU-8 surface, and the lowest value occurred at the bottom of microcavity (b) the density of electric field along the surface of SU-8 for the different depths, 5, 10 and 15μm, respectively.
Fig. 2. (a) The gradient of electric field intensity, for 10μm depth SU-8 microcavity, the strongest DEP force happened near the top of SU-8 microcavity and move particle into microcavity by negative DEP (b) along the surface of SU-8 for different microcavity depths 5, 10 and 15μm, respectively. (C) The various profiles of? for different distances away from the middle electrode surface, such as 0, 2, 4 6, 10 and 28μm.
Fig. 3. The total current density as impedance measurement by applied? voltage is 0.2V on a pair of bottom electrodes, the frequency is 100kHz, and? the depth of microcavity is 10μm; (a) without particle in the SU-8 cavity; (b) the particle fixed in the SU-8 cavity.
Fabrication and Experimental SetupThe multilayer electrodes DEP chip is consisted of three parts, shown in the Fig. 4(k). The bottom electrodes for impedance measurement was patterned firstly shown as the Fig. 4(a) to (b). The middle part consisted of cavity-type 3D microstructures array and middle electrode for DEP trapping, which were made of thick photoresist layer, SU-8, and metal layer, Au, respectively, shown in Fig. 4(c) to (h). There are three kinds of depths for SU-8 microcavity, 5, 10 and 15μm. The diameter and spacing of microcavity array were both designed in the same scale with cells about 16μm. Each block of microcavity array consisted 10x10 array. The total area of SU-8 microcavity array was 5 mm square and the total number of cavities was 1500 in one chip. Thus, we can simultaneously trap 1500 cells in one experiment. The last part was a rectangular flow chamber with W*L*H = 7mm*50mm* 100μm, formed by double sided tape attached on the ITO glass, shown in the Fig. 4(i). The advantages of using double sided tape are not only easy to patterning but also for good quality of bonding between upper ITO glass and SU-8 layer shown in Fig. 4(j). 
Fig. 4. The microfabrication processes of multilayer electrodes DEP chip for single-cell level impedance measurement.
In addition, the edge of SU-8 microcavity is smooth enough to prevent any harmfulness as trapping cells into cavities shown in the Fig. 5(a). The layout of four pairs of impedance electrodes is shown in the Fig. 5(b). Due to the high efficient trapping capability, we thus can ensure that cells can be trapped upon these four pairs of impedance electrodes for further detection. The finished multilayer electrodes DEP chip is shown in Figure 5(c).
Fig. 6. (a) SEM pictures of SU-8 cayitys array, the diameter and spacing are both 16 μm and the depth is 10μm; (b) The layout of four pairs of impedance electrodes; (c) The picture of multilayer electrodes DEP chip.
The experimental setup was shown in Fig. 7. The two main purposes in this paper, basically, one is to trap cells in microcavity array by DEP force, another is to measure the impedance variation while cells were trapped. Here, we utilized a syringe pump (KDS-210, KD Scientific) to control the flow rate. The AC signal was generated by the function generator (33220A, Agilent) for DEP trapping. Forimpedance measurement, one precession LCR meter (Wayne Kerr-4620A, NEW Boston Street Woburn, MA) was operated by LabView programming with frequency ranging from 1 KHz to 3Hz and 0.2Vrms. In addition, a digital CCD also was mounted on OPTIMA biological microscope for monitoring the DEP force acting on the cells and capturing the in-situ image for post image processing.
Fig. 7. Experimental setup for DEP trap cell and impedance measurement.
Capability of Trapping Cells
The optical micrographs show the capability of trapping HL-60 cell. (a)-(c) The HL-60 cell subjected to negative DEP effect and would be attracted at the region of lower intensity of electric field by applied AC signal with 10VPP and 10KHz; (d)-(f) the cells began move toward to the maximum regions of electrical field intensity by positive DEP force as applied an AC signal with 10 Vpp and 500 KHz.
Fig. 8. The optical micrographs show the capability of trapping HL-60 cell.
Impedance Measurement Results
Fig. 9 and 10, represent the magnitude and phase of impedance spectrum from different DEP chips with 5μm and 10μm SU-8 layer, respectively. The experimental results for 5μm and 10μm depth microcavity were illustrated in the Fig. 9 and 10, respectively. All the impedance results for each condition showed the capacitance behavior, the impedance magnitude decreased when frequency increased. For the 5μm-depth microcavity chip, the impedance difference between only sucrose and live HL-60 cells immersed in sucrose solution was 60 KΩ at 10 KHz, meanwhile, the impedance difference between live and dead HL-60 cell was 80 KΩ. Hence, we can easily identify the microcavity whether with or without cells and the viability of cells based on the impedance measurement method. For the 10μm-depth microcavity chip, the impedance difference between only sucrose and live HL-60 cell immersed in sucrose solution was 120 KΩ at 10 KHz, which is twice larger than the value of 5μm-depth microcavity chip, thus, the thicker microcavity chip has higher sensitivity of cell sensing, which also agrees with simulation results. Besides the HL-60 cells, we utilized another cell line, NB4, for the purpose to differentiate different kinds of cells on the chip. As the results in the Fig. 10, the impedance difference between live HL60 cells and live NB4 cells at 10 KHz was about 190KΩ, therefore, we can use this chip as the key technology for cell separation.
Fig. 9. The results of impedance measurement for the 5μm-depth microcavity under four conditions: (1) air, (2) only DI water without cells, (3) only RO water without cells, (4) only sucrose solution without cells, (5) HL60 live cell immersed in sucrose solution, (6) HL60 dead cell immersed in sucrose solution, all conditions were applied 0.2V and the frequency range is 1K to 3MHz. (a) impedance magnitude (ohm); (b) phase (degree).
Fig. 10. The results of impedance measurement for the 10μm-depth microcavity under five conditions: (1) air, (2) only DI water without cells, (3) only RO water without cells, (4) only sucrose solution without cells, (5) HL60 live cell immersed in sucrose solution, (6) NB4 live cell immersed in sucrose solution,(7) NB4 dead cell immersed in sucrose solution, all conditions were applied 0.2V and the frequency range is 1K to 3M Hz. (a) impedance magnitude (ohm); (b) phase (degree).
Conclusions
We have designed and fabricated a DEP chip with multilayer electrodes and microcavity array for impedance measurement of single cell. The depth effects on impedance difference were analyzed by finite element method and verified by experimental results. This microchip not only provides an efficient way to immobilization cells in the microcavity for a long period of time without applying DEP force but also easily identifies the live and dead cells based on impedance measurement.

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