本研究利用微機電系統製程技術製作微小電極，以方波脈衝於電極上產生足以電穿孔裂解細胞之電場，並應用於紅血球裂解，釋出糖化血色素研究。研究策略為設計平面式裂解晶片包含方形、尖端與齒梳型三種不同電極形狀，電極間距50 μm施加不同頻率(100 Hz、250 Hz、500 Hz、1000 Hz、2000 Hz)、不同電壓(10~40 V)之訊號，探討紅血球受影響之裂解效率，由實驗得知方形電極裂解效率優於齒梳型優於尖端型設計，隨著電壓上升而效率提高，於電壓40 V、頻率500 Hz有最高裂解效率66.7%。本研究以有限元素法分析軟體來模擬微電極中之電場強度分布，由模擬分析結果得知，平面式裂解晶片因尖端放電效果能產生足夠強度裂解電場，並設計了平行板裂解晶片，有效提升電場範圍避免電場不均與高度限制問題，效率較平面式裂解晶片佳，在電場強度25000 V/m其裂解效率達到95%，透過化學改質與雙面膠流道技術製作出微流體裂解晶片，藉由提高親水性與毛細現象，使檢體不須借助外力逕自流向偵測端，由紫外光-可見光吸收光譜儀檢測破壞細胞膜提升之血紅素濃度，血紅素波長吸收值1.06，相當接近完全破裂控制組之吸收值1.07，證明裂解晶片實驗後之血液能有效釋出血紅素。

SUMMARY

This thesis presents an irreversible electroporation system for red blood cell lysis. The microchip device and three types of microelectrodes were designed and fabricated using MEMS and microfluidic technology. After integrating two different electrodes, we simulated and analyzed the suitable electric field for cell lysis by a finite element software, ANSYS, and found that when the parallel plate electrodes was at 5V, a notable lysis rate 95% was achieved in 10 seconds. Therefore, the proposed system has the advantage of less lysis time and higher lysis rate.

Key words: cell lysis, electroporation, MEMS, glycated hemoglobin, biomedical sensor, microfluidic chip

INTRODUCTION

As the increased the number of patients with diabetes, market of blood glucose testing grows 10.7% every year. For example, glycosylated hemoglobin (HbA1c), the combination of HbA with glucose, is used for detecting diabetes or controlling blood glucose level. But, because hemoglobin and HbA1c are present in the red blood cells (RBC), cell lysis is required before detection. A common way to lyse cells is to use a lysis buffer, but the procedure is complicated and time-consuming. If the electric field intensity exceeds a critical value, the cell membrane becomes permeable to release the subcellular materials. In our study, we lysed RBCs by electroporation, which has the advantages of easy power supply, simple operation, and less chemical reactions.

MATERIALS AND METHODS

The electrodes was made of indium tin oxide, which has high electrical conductivity. Our three chips include a planar electrode chip, a parallel plate electrode chip and a microfluidic chip. The planar electrode chip was packaged by double-sided tape (30 μm in thickness), and the reaction region was 10 ×12 mm. Inside the planar electrode chip were 5000 micro electrodes categorized into three types: square, saw-tooth, and comb. The gap between the electrodes was 50 μm, and each electrodes generated a corresponding electric field. Then, the parallel plate electrode chip was also packaged by double-sided tape (200 μm in thickness), and the reaction region was 5 ×5 mm. In the microfluidic chips, two types of electrodes were integrated by surface modification and capillarity, and an electric field was generated between the upper electrode and the lower electrode. So that the cell lysis could be easily observed and measured.
ANSYS 13.0 software was used to analyze the model of the chips and the distribution and intensity of the electric field to ensure its sufficient intensity. Function generator and high voltage amplifier were used to generate square pulses signal to lyse the RBCs. Oxygenated hemoglobin and deoxygenated hemoglobin have similar peak values, so we could judge the degree of hemolysis. When hemolysis occurred, the RBCs released hemoglobin, and the absorption spectra of hemoglobin was obtained at wavelength 420 nm.

RESULTS AND DISCUSSION

The results reveal that sufficient electric field intensity was generated, and the lysis rate of the planar electrode chip are shown in Table 1. Because of point discharge, the lysis rate of square and comb electrodes are more than that of saw-tooth electrodes. A maximum lysis efficiency of 66.7% was measured in square electrodes at 500 Hz and 40 V.
Then, the parallel plate electrode chip was free from uneven distribution of the electric field and the height restrictions, and the cell lysis is presented in Figure 1, in which the cells maintained shape before lysis and broke down after lysis. Inputting 5 voltage to parallel plate electrode chip led to the lysis rate 95% in whole blood within 10 seconds. Thus, this chip has the advantages of less operating time, lower energy consumption, and smaller volumes of specimen (5 μl). The lysis rate is shown in Figure 2.
After measuring the degree of hemolysis by UV-Vis, we observed that the absorbance in specimen after lysis was pretty close to complete hemolysis. The result reveals that this experiment can effectively release hemoglobin from the RBCs. Also, we verified the effect of surface modification on the microfluidic chip by contact angle detection. Compared with the unmodified chip, the contact angle of the modified chip reduced from 74° to 33°, and the effect of modification lasted for four weeks. With the rise of hydrophilicity, the microfluidic chips did not need external force because the specimen was carried to the detection region by surface modification and capillarity.

CONCLUSION

In this thesis, we proved that the simulation of electric field by ANSYS was feasible and reliable for chip design and fabricated miniaturization of lysis chip. By MEMS and microfluidic technology, the chip has the advantages of high lysis and hemolysis rate, small volume of specimen and short operating time. Therefore, the proposed system provides a new way for cell lysis and disease detection.

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