4-1 Current drop caused from polystyrene beads 4-1-1 49 pores device
The experiment was manipulated in following conditions: applied voltage with 10 mV in 10 Hz, used the device with 49 pores, and injected the sample with concentration of 106 beads / ml at the rate of 20 µl / min. In addition, the status of the pores was recorded under optical microscope simultaneously. The measurement result is shown in Figure 4-1. The current kept in horizontal at the initial 14 seconds, dropped abruptly from 14th to 20th second, at last, the pores were saturated and the curve slope turned gently at about 20th second. Based on these difference phenomena, the curve was divided into three regions as shown in Figure 4-1.
In region Ⅰ, the polystyrene beads were not yet arrived to the micro-pores sieve(Figure 4-2), RP value was not change, so the current kept in constant. Figure 4-3 indicated that the micropores were blocked by polystyrene beads gradually from the 14th to 22nd second. Therefore, Rp and RD increased and the ionic current decreased.
This phenomenon conformed to that indicated in region Ⅱ of Figure 4-1. Therefore, a conclusion could be judged that the ionic current drop is the result of the micropores are blocked by polystyrene beads.
4-1-2 9 pore device experiment
In the same condition, but replaced 49 pores device by 9 pores device to repeat the experiment. As shown in Figure 4-5 and Figure 4-6, when every bead was trapped on the sieve, the ionic current decreased obviously and resulted in a step in the curve.
And if there were not any bead be trapped on, the current curve kept in constant. It proof again that the current drop was caused by polystyrene beads be trapped on.
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Furthermore, we can know the beads quantity by counting the amount of steps in Figure 4-5. Every step represents one bead. When the amount of pores increase the current variation caused by every individual beads will decrease. Like the current curve shown in Figure 4-1, the current variation caused by one bead blocking is so small that there are not obvious step observed but a smooth descending curve.
Saturation time
Figure 4-1. The ionic current curve in following measurement conditions : applied voltage with 10 mV in 10 Hz, used the device with 49 pores, injected the sample with concentration of 106 beads/ml at the rate of 20 µl/min.
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Figure 4-2. The OM image in region I of Figure 4-1.
Figure 4-3. The OM image in region II of Figure 4-1.
Figure 4-4. The OM image in region III of Figure 4-1.
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Figure 4-5. The ionic current curve in following measurement conditions: applied voltage with 10 mV in 10 Hz, used the device with 9 pores, injected the
sample with concentration of 104 beads/ml at the rate of 20 µl/min.
Figure 4-6. The OM image corresponds. Every figure corresponds to the time labbeld in Figure 4-5.
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4-2 Beads measurement results and discussion
The previous experiment results and the simulation electrical circuit model indicated that the micro-pore impedance variation cause from the pore be blocked by beads in low frequency would more obvious than in high frequency. So in following experiment, low frequency – 10 Hz was chosen and the applied voltage is 10 mV. All the beads were dissolved in 0.1 M KCl which has similar conductance with 1X PBS.
The beads were composed by polystyrene with 10 ± 0.63 μm in diameter. The sample solution was injected into the flow channel by syringe pump in the flow rate of 20 µl / min. Pure 0.1 M KCl without polystyrene beads was injected in the initial 10 seconds, the sample solution that contain different concentration of polystyrene beads was injected form the 10th second.
In the following experiments, we used difference pore number micropores sieve device to detect the beads concentration. The pore number ranged from 1 to 484, and we expected to distinguish the beads concentration from 102 beads/ml to 106 beads /ml.
4-2-1 Resolving of bead concentrations
Apply the value in Table 3-2 and Table 3-3, it could be estimated that the ionic current might decline to 84% or lower after the polystyrene solution had injected when using 484 pores device.
Figure 4-7 to Figure 4-11 are the results that applied 484 pores device to detect
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difference concentration samples. Once polystyrene beads arrive the device and block the micro-pore channels, the ionic current will start to decrease. The variations of the ionic current in 102 to 104 beads / ml are almost the same in the initial 100 seconds.
Because the pore blocked rate is too low and the variations are smaller than the error amount so it couldn’t be distinguished. But the variation could be distinguished between 104 to 106 beads /ml, as shown in Figure 4-12.
Apply the value in Table 3-2 and Table 3-3, it could be estimated that the ionic current might decline to 77% or lower after the polystyrene solution had injected when using 100 pores device.
Figure 4-13 to Figure 4-17 are the results that applied 100 pores device to detect difference concentration samples. Once polystyrene beads arrive the device and block the micro-pore channels, the ionic current will start to decrease. The variation of ionic current at 100th second could be distinguished from 102 to 106 beads /ml, as shown in Figure 4-18.
Apply the value in Table 3-2 and Table 3-3, it could be estimated that the ionic current might decline to 71% or lower after the polystyrene solution had injected when using 49 pores device. Figure 4-19 to Figure 4-23 are the results that applied 49 pores device to detect difference concentration samples. Once polystyrene beads arrive the device and block the micro-pore channels, the ionic current will start to decrease. The variation of ionic current at 100th second could be distinguished from
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102 to 106 beads /ml, as shown in Figure 4-24.
Apply the value in Table 3-2 and Table 3-3, it could be estimated that the ionic current might decline to 60% or lower after the polystyrene solution had injected when using 9 pores device.
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Table 4-1 The actual value of electrolyte and device resistance for 484 pores device.
Pore number Rel (k) RD (k) CM (F) RT (k)
484 10.171 2.502 0.199 × 10 -9 12.673
Current variation rate: RT / (RT + RD) = 84%
Figure 4-7. The current decline curve for 102 beads/ml sample by 484 pores device.
Figure 4-8 The current decline curve for 103 beads/ml sample by 484 pores device.
Figure 4-9 The current decline curve for 104 beads / ml sample by 484 pores device.
Figure 4-10 The current decline curve for 105 beads/ml sample by 484 pores device.
Figure 4-11 The current decline curve for 106 beads/ml sample by 484 pores device.
Figure 4-12 The statistics of different concentrations for 484 pores device.
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Table 4-2 The actual value of electrolyte and device resistance for 100 pores device.
Pore number Rel (k) RD (k) CM (F) RT (k)
100 12.139 5.058 0.692 × 10 -9 17.197
Current variation rate: RT / (RT + RD) = 77%
Figure 4-13 The current decline curve for 102 beads / ml sample by 100 pores device.
Figure 4-14. The current decline curve for 103 beads / ml sample by 100 pores device.
Figure 4-15. The current decline curve for 104 beads / ml sample by 100 pores device.
Figure 4-16 The current decline curve for 105 beads / ml sample by 100 pores device.
Figure 4-17. The current decline curve for 106 beads / ml sample by 100 pores device.
Figure 4-18. The statistics of different concentrations for 100 pores device.
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Table 4-3 The actual value of electrolyte and device resistance for 49 pores device.
Pore number Rel (k) RD (k) CM (F) RT (k)
49 11.628 8.015 0.320 × 10-9 19.643
Current variation rate: RT / (RT + RD) = 71%
Figure 4-19. The current decline curve for 102 beads /ml sample by 49 pores device.
Figure 4-20. The current decline curve for 103 beads /ml sample by 49 pores device.
Figure 4-21. The current decline curve for 104 beads/ml sample by 49 pores device.
Figure 4-22. The current decline curve for 105 beads / ml sample by 49 pores device.
Figure 4-23. The current decline curve for 106 beads / ml sample by 49 pores device.
Figure 4-24. The statistics of different concentrations for 49 pores device.
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Table 4-4 The actual value of electrolyte and device resistance for 9 pores device.
Pore number Rel (k) RD (k) CM (F) RT (k)
9 11.757 26.096 0.551 × 10-9 38.853
Current variation rate: RT / (RT + RD) = 60%
Figure 4-25. The current decline curve for 102 beads /ml sample by 9 pores device.
Figure 4-26. The current decline curve for 103 beads / ml sample by 9 pores device.
Figure 4-27. The current decline curve for 104 beads / ml sample by 9 pores device.
Figure 4-28. The current decline curve for 105 beads /ml sample by 9 pores device.
Figure 4-29. The current decline curve for 106 beads/ml sample by 9 pores device.
Figure 4-30. The statistics of different concentrations for 9 pores device.
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4-2-2 Discussion
From the calculation results, we could know that when the all the micropores are blocked (saturate), the ionic current will drop to 71% of initial value for 49 pores device and 77% for 100 pores device. And the saturation time will different with the concentrations. By analyzing the saturation time, the sample concentration could be known. In Figure 4-31 and Figure 4-32 displayed the saturation time for 49 and 100 pores device of different concentration. Both of the curve showed that the lower of the concentration the longer of the saturation time, based on this concept, the concentration could be distinguished from 102 to 106 beads /ml. In addition, the detection time for 49 pores device is about 300 seconds and 100 pores device is about 450 seconds. Using the device with fewer pores can speed up the detection rate.
Figure 4-31. The saturation time for 49-pore device of different concentration.
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Figure 4-32. The saturation time for 100-pore device of different concentration.
In order to accelerate the detection rate, the ionic current values in specific time were compiled. The ionic current values at 50 s, 100 s and 150s after starting the measurement are indicated as Figure 4-33 to Figure 4-35. At 100th and 150th second, it could distinguish the concentration from 102 to 105 beads/ml by 49 pores device and distinguish the concentration from 103 to 106 beads/ml by 100 pores device. The sample concentration could be known within 2 min by applying this statistical method.
Figure 4-33. The ionic current values at 50 s after the measurement started.
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Figure 4-34. The ionic current values at 100 s after the measurement started.
Figure 4-35. The ionic current values at 150 s after the measurement started.
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4-3 Concentration of HeLa cells
From the conclusion of the beads experiments, we know that 49 pores micro-sieve device is more sensitive and faster to detect the beads concentration in the range from 102 to 106 beads/ml. So we used it to demonstrate the cells experiment.
Before the measurement, the HeLa cells concentration would be confirmed by the hemocytometer. The sample with concentration lower than 104 cells / ml could not be confirmed by it, we could only dilute the higher concentration sample with 1X PBS to obtain.
From the ionic current curve shown in Figure 4-36, it is obvious that different concentration’s curve has different slope. By analyzing the ionic current value at specific time, the concentration could be known.
In addition, HeLa cells measurement results are compared with polystyrenes’.
The tendency of the former is coincident with the latter, as shown in Figure 4-37.
Figure 4-36. HeLa cells measurement result by using the 49 pores device.
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Figure 4-37. The comparison of polystyrene beads and HeLa cells measurement results by using the 49-pore device. The current ratio at 100th second after starting the measurement.
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