Evaluation of Bioresist patterning resolution
As Bioresist is photopatternable, we can create any arbitrary 2D shape using photolithography. We evaluated the patterning resolution of Bioresist by changing the line and space ratio of the pattern. In this evaluation, the thickness of Bioresist was fixed at 4 μm for manipulation of Synechocystis sp. PCC 6803, which is a kind of cyanobacteria with a diameter of a few micrometers. Figure 3 shows examples of the fabricated patterns where the widths of the target patterns were 5, 10, 20, 50 and 100 μm, respectively. From Figure 3, we can see that the patterning resolution depends on the line and space ratio of the design, and that a larger line and space ratio gives higher resolution. For example, in the cases where the line and space ratios were 1:1, 1:2, and 1:3, the resolutions of the line patterns were 10, 10, and 5 μm, respectively. In the case where the line and space ratio was 1:2 and line width is 5 μm, unexposed resist remains on the pattern as a dissolved remainder. The reason the results depend on the design is one of the features of Bioresist. Bioresist patterns swell during development in the photofabrication process because the development process requires isopropyl alcohol (IPA) as an etchant, which causes expansion of Bioresist in the photo fabrication process.
Evaluation of temperature dependent expansion of Bioresist
We evaluated the expansion rate of Bioresist patterns by changing the temperature of the environment. The expansion rate is defined as shown in Equation 1
(1)
where D
0
and D
t
are the diameter of patterned gel and the diameter of swollen gel at each temperature t respectively. The environmental temperature was controlled by a thermo plate (TOKAI HIT. Co. Ltd., TP-CH110R) which is placed under the microfluidic chip. The photo of the shape of gel pattern was taken by the vision sensor attached to the microscope. We waited 10 minutes to confirm stabilization of each temperature for experiment. We measured the shape of patterned gel during temperature increasing phase, and then decreasing phase. The expansion rate is defined as the ratio between the swollen diameter at each temperature condition and the patterned diameter of the fabricated pillar. Figure 4 shows how Bioresist patterns change. The pattern diameter and the height are 20 μm and 4 μm, respectively. From Figures 4(b) through (c), we can see that the shape of Bioresist is swollen at the low temperature condition, and that the shape shrinks with high temperature. Figure 5 shows the relationship between the expansion rate and temperature. From Figure 5, we can see that Bioresist has a phase transition, and the transition temperature is approximately 30°C. Moreover, the hysteresis between the heating and cooling phases is small. The measured expansion rates were 1.1 through 1.7 with respect to temperature.
Gel-valve evaluation
We designed the gel-valve based on the results for patterning resolution and expansion rate. In this case, the width of the gel-valve structure and the gap between gels are designed as 20 μm and 10 μm, respectively, as shown in Figure 6(a). Figures 6(b) and (c) show the fabricated valve and a scanning electron microscope (SEM) image of the gel-valve, respectively. The gel-valve is normally closed at room temperature (less than 30°C), and opens when electrical current is applied to the microheater at the bottom of the patterned Bioresist. Figure 7 shows how the gel-valve moves by switching the heating on and off; the closing and opening movements are controlled by changing the electrical current to the microheater.
Figure 8 shows the relationship between the pressures in microchannel when the syringe pump was actuated. The pressure in the microchannel was measured using a pressure sensor (Copal electronics, PA-830) at the inlet of the channel. From 0 through 32 seconds the measured pressure increased, and then decreased with respect to time. This result corresponded to leakage that occurred due to the detachment of the PDMS cover from the glass substrate. From Figure 8, we confirmed that the leakage pressure of the microchannel using the gel-valves was 199.5 kPa.
Frequency characteristics of the gel-valve were evaluated as shown in Figure 9. Amplitude and frequency of the input current to the microheater were controlled by a function generator (NF Corporation, WF1974). The input signals were square waves; the currents were 33.2, 38.2, and 43.7 mA respectively; and the frequency was changed from 0.1 to 10 Hz. As a general tendency, the displacement of phase lag decreases with respect to the increase in frequency. When the applied current was 33.2 mA, the gel-valve did not shrink faster than 1 Hz because the valve was not heated enough owing to the low current. On the other hand, when the applied current was 43.7 mA, the valve was well-actuated up to 5 Hz, but it did not swell enough at 10 Hz because the gel was not cooled enough at high frequency. From these results, we confirm that the amplitude of the applied current has a certain optimum value owing to the heating and cooling balance of the total system, and we set the current as 38.2 mA for the following experiments. In this case, we can actuate the gel-valve at 5 Hz. In our experiment in 3.3 through 3.4, we fixed pH of the surrounding condition of the gel-valve using DI water. Since the expansion motion of the gel-actuator depends on its size and pH of the surrounding condition, we will investigate these effects on the response time with the desired culture condition of target cells in future [27],[28].
Application: microvalve and cell sorter
The gel-valve was evaluated as an on-chip flow control component. In this experiment, Synechocystis sp. PCC 6803, which is a kind of cyanobacteria with a diameter of a few micrometers, was used as a tracer. Figure 10(a) shows sequential photos of the movement of a Synechocystis bacterium flowing in the channel. The transportation of Synechocystis was controlled and stop-and-go motion was observed with respect to the closing and opening of the gel-valve; we could also observe Synechocystis through the valve (Additional file 1). Moreover, we demonstrated cell sorting by the combination of gel-valves shown in Figure 10(b). Two gel-valves were placed downstream of the branch of the microchannel. The flow was switched by changing the applied current to each valve, and the cells were sorted at the branch. From these results, we confirmed that the gel-valve works successfully, and that we can control the transportation of cells changing only the applied electrical current to the microheater.