2-1 Device fabrication process
In this research, we applied a convenient micro-fabrication process. These processes took place in National Chiao Tung University Nano Facility Center (NFC).
And the following equipment were used: Wet bench, Low pressure chemical vapor deposition system (LPCVD), Vacuum oven, Photoresist spinner, Optical microscope, Double side mask aligner and Dielectric material reactive ion etching system (RIE 200L). The fabrication process was described below.
2-1-1 Silicon wafer clean
In this experiment, double side polished (110) silicon wafers with 250 µm thickness were used. These wafers were cleaned in the following process:
H2SO4 : H2O2 = 3 : 1 in 75 ℃ for 10 min DI water rinse for 5 min
HF : H2O = 1 : 100 in room temperature until the surface is hydrophobic.
DI water rinse for 5 min
NH4OH : H2O2 : H2O = 1 : 4 : 20 in 75 ℃ for 10 min DI water rinse for 5 min
HCl : H2O2 : H2O = 1 : 1 : 6 in 75 ℃ for 10 min DI water rinse for 5 min
HF : H2O = 1 : 100 in room temperature until the surface is hydrophobic.
DI water rinse for 5 min
Because the wafer is too slight to stand high stress, after the clean process, we used nitride air gun to blow away the residual water instead using spin dryer. Then, baking the silicon wafer in 150 ℃ for 10 min to take out the residual moisture.
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2-1-2 Silicon nitride deposition
Low stress silicon nitride was deposited by Low Pressure Chemical Vapor Deposition (LPCVD) system. The wafers was put into the furnace, then, rising the furnace temperature to 825 ℃. After the temperature was rose to the assigned temperature, importing NH3 and SiH2Cl2 at the flow rate of 17 sccm and 85 sccm for 90 min to deposit 500 nm low stress nitride.
After this process, we can obtain a silicon wafer with 500 nm silicon nitride membrane at both sides. The structure is depicted in Figure 2-1.
Figure 2-1. The schematic of a silicon wafer with 500 nm silicon nitride membrane at both side.
2-1-3 Top side pattern
Figure 2-2. The top side mask pattern.
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In order to obtain a region of silicon nitride membrane in which length and width are both excess 200 µm on the bottom side after wet etching, we opened square windows with 600 µm in length on the top side as show in Figure 2-2.
First, coat HMDS to improve the adhesion between the silicon wafer and photoresist. After that, coat positive photoresist (FH 6400) by spinner. The first step rotation rate is 500 rpm for 10 sec; the second step rotation rate is 2500 rpm for 25 sec.
Then, soft bake at 95 ℃ for 90 sec. Expose the photoresist under light intensity 130 millijoule per square centimeter. Development by the developer (FHD-5) for 20 sec.
Hard bake at 120 ℃ for 90 sec.
After pattering the photoresist, the silicon nitride was etched by Dielectric Materials Reactive Ion Etching System (RIE 200L). We infused O2 and CF4 into the chamber at the flow rate of 80 sccm and 20 sccm as reactive gas. The reaction time is 400 sec to remove the 500 nm silicon nitride.
Last, soak the wafer in acetone 10 min to dissolve the photoresist. The wafers cannot be shaken by ultrasonic oscillator because the wafer would break during this process.
After this process, we can obtain a silicon nitride membrane with a square on it as depicted at Figure 2-3.
Figure 2-3. The schematic of the wafer after dry etching, there’s a square on silicon nitride membrane.
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2-1-4 Bottom side pattern
Figure 2-4. The bottom side mask pattern.
There were different quantities of the micro-pores designed on the bottom side mask. The pore size is 7 µm in diameter with 14 µm pitch as show in Figure 2-4.
Difference pore number were designed, they are 9, 49, 100, 484 individually as show in Figure 2-5.
Figure 2-5. Design of different pore numbers on the mask.
The following steps to define the bottom side silicon nitride pattern are similar with the top side pattern process. After this process, we can obtain a silicon nitride
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membrane with micropore array, and the pore sizes and pore quantities are the same with the mask. The cross sectional schematic was depicted at Figure 2-6.
Figure 2-6. The schematic of silicon nitride membrane with micropore array.
2-1-5 Wet etching
To etch the spare silicon between the membranes we dipped the silicon wafer into 30 wt% KOH solution at 80 ℃ for 5 hours. In order to let the surface be smoother, 4 % isopropyl alcohol (IPA) was added into the solution. This solution would anisotropy etch the silicon, the etch rate for (111) plane is remarkable slower than other planes. So there would be a trapezoid chamber in the silicon wafer after the etching process as illustrated in Figure 2-7.
Figure 2-7. The schematic of the micro-pores sieve.
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2-2 Device analysis
The micropore devices were examined by SEM and OM to confirm the consistence to the design. After that, pore diameter was analyzed by ImageJ and Si3N4
membrane thickness was characterized by N&K analyzer.
2-2-1 SEM and OM image
Figure 2-8(a) is optical microscope (OM) image of the device. From the image, it could be know that the top side pattern and bottom side pattern had been aligned together; the micro-pores array is located at the center of the silicon fillister dug in the wet etching process. To inspect more detail of the device, it was examined under scanning electron microscope (SEM). It could be confirmed that there are micro-pores array channels located on the silicon nitride membrane. The SEM image is shown in Figure 2-8 (b). possessed in National Nano Device Laboratories (NDL).
The measurement points are composed by a 3x3 array, the array center is the
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center of the wafer and the interval between points is 3 centimeter as shown in Figure 2-9. The average thickness is 505 ± 26.5 nm according to the statistical result.
Figure 2-9. The positions of silicon nitride thickness measurement points.
2-2-3 Pore diameter analysis
Since the pore diameter would be little inaccuracy in the process of exposure, develop and dry etching, it should be reconfirmed. ImageJ was applied to analyze the diameter the result shows that the average pore diameter is 6.68 ± 0.39 μm.
Figure 2-10. The pore diameter was analyzed by ImageJ.
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2-3 Cell culture process
HeLa cells were used in this research to demonstrate the cell counting. Cells were cultivated in the medium containing 90% DMEM medium and 10% newborn calf serum. We displaced the medium to trypsin and put the dish into incubator for 2 min to separate the cells from the dish. Then, add the medium had been mention previous into the dish and draw out all the solution to centrifugal tube. Put the centrifugal tube into centrifuge; adjust the rotation rate at 1500 rpm for 3 min; draw out the solution and keep the HeLa cells in the tube. At last, add 1X PBS into the tube and adjust the cell concentration. Before measuring the concentration by micropores sieve, we applied hemocytometer to estimate the cell concentrations.
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2-4 Polystyrene beads sample preparing process
In order to carry out the research more convenient, we used polystyrene beads instead of cells to demonstrate the experiment. Nonionic white polystyrene latex was purchased from InvitrogenTM. The size of the polystyrene beads is 10 ± 0.63 µm in diameter. Polystyrene beads dissolved in DI water and the concentration is 7.4 × 107 beads/ml.
The polystyrene beads solution was diluted with various volume ratios of 0.1M KCl. We diluted the polystyrene beads into 1.5 × 102, 1.5 × 103, 1.5 × 104, 1.5 × 105 and 1.5 × 106/ml in concentration. And use hemocytometer to re-confirm the concentration. But from the image of hemocytometer, we could only recognize the concentration from 106 to 104 / ml. So the other concentrations were diluted from the sample with a know concentration.
Figure 2-11. The image of hemocytometer for 1.5 × 106 beads/ml solution.
Figure 2-12. The image of hemocytometer for 1.5 × 105 beads/ml solution.
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Figure 2-13. The image of hemocytometer for 1.5 × 104 beads/ml solution.
Figure 2-14. The image of hemocytometer for 1.5 × 103 beads/ml solution.
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2-5 Acrylic block design
There was a T-shaped channel inside the acrylic block as showed in Figure 2-16.
And the dimensions are shown in Figure 2-15. One side of the channel was the inlet that let the sample solution flow in, anther side was used to fix the Ag/AgCl reference electrodes and the other was aligned with the micropores array to let the sample flow through the chip.
Figure 2-15. The lateral view of the acrylic block.
Figure 2-16. The illustration of the acrylic fixture for micropore sieve device.
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2-6 Microfluidic Fixture Materials：
Acrylic block mention in chapter 2-5 × 2
O-ring with internal diameter 3.6 mm and external diameter 6.2 mm × 2 O-ring with internal diameter 1.2 mm and external diameter 3.2 mm × 2 Ag/AgCl reference electrode × 2
Teflon tube in 10 centimeter × 2 Micro-sieve device × 1
Syringe × 1
Screws and nuts × 2
PDMS with thickness 0.8 mm and a hole with diameter 1 mm × 2 Difference concentrations beads solution × 500 µl
The micro-sieve was clipped between two acrylic blocks which had a 1 mm hole aligned with the micro-pores array. To avoid the solution leaking from cracks, we put the PDMS between the acrylic blocks and the micro-sieve device and plugged the O-rings in the channel terminal. The Ag/AgCl reference electrodes were inserted into the acrylic blocks and put an O-ring around it to avoid solution leaking. The sample solution was loading in a syringe which connecting to a Teflon tube and the tube was inserted into the inlet channel. And the set up would be fixed by screws and nuts. The sketch was depicted in Figure 2-17.
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Figure 2-17. The photograph of the microfluidic channel including the arcrylic fixture.
The sample solution was injected into the channel and passed through the pores by the syringe pump in a constant flow rate. Ag/AgCl reference electrodes were inserted into the flow channel and connected to the lock-in amplifier (SR-850) to measure the ion-current.
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2-7 Set-up of Measurement equipment
The voltage was applied via a lock-in amplifier (SR850) by connecting to the reference electrodes. The data acquisition was fulfilled via a DAQ card and a computer.
Figure 2-18. The illustration of the microfluidic channel and measurement system.
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