【作者】 张玲；

【导师】 于军；

【作者基本信息】 华中科技大学 ， 微电子学与固体电子学， 2008， 硕士

【摘要】 胞解芯片的目的是对初级样品实施破胞处理以获得其中的内容物(蛋白质、DNA、RNA),这对大多数对细胞内物质的反应和检测进行研究的生物芯片是必不可少的。近几年来电子法胞解逐渐受到研究者们的关注,关于电子胞解芯片研究的报导数量也在逐渐上升。采用电场的方法进行胞解是因为电子胞解法相对于传统的化学胞解法、热解法等大大简化了样品提纯的步骤、不会造成蛋白质变性等,而且因为它的微型化能方便地集成到其他生物芯片中去提供样品处理过程。在本文里设计并制作了一种采用低电压进行细胞破碎的电子胞解芯片。此胞解芯片被设计用于微流控系统的样品前处理部分以进行系统中的后续研究和分析。它也可用来给一般的诊断和生物实验制备样品。文中胞解芯片的制备采用了剥离工艺的标准光刻胶处理方法,在玻璃基底上制备了Ti/Pt微电极。通过向6对微电极对施加脉冲电压得到胞解所需的高场强(kV/cm)。同时还设计了胞解专用的脉冲信号发生器。它由AT89S52单片机及其应用电路、模拟输出电路组成,能发出波形规整的直流脉冲。分别采用BHK21(幼仓鼠肾细胞),C2C12(小鼠成肌细胞)对芯片进行了胞解实验测试。在倒置显微镜下观察到了BHK21,C2C12细胞的胞解,实验观察结果表明脉冲电场使得两种细胞都完全裂解了。因为制备了间距为微米级的电极,胞解所需电压被降至3V,胞解所需场强约为0.6kV/cm。实验表明,在不同宽度的脉冲信号作用下,胞解所需的电压和胞解率有微妙的变化。胞解率与脉冲宽度、电压和细胞悬浮液的电导率都有关。研究发现干净的PMMA与玻璃基底间形成的半封闭通道可以有效地防止施加脉冲电压时气泡的产生,气泡的产生也与细胞悬浮液的电导率有关。总之,此胞解芯片可以在低的直流脉冲电压下使细胞以较高的胞解率破解。它可以方便地被集成到微流控系统或芯片实验室中去进行样品前处理。更多还原

【Abstract】 Cell lysis is an essential step in biochips which based-on the research of intracellular materials reaction and analysis for extracting intracellular materials such as DNA, RNA, and protein. Electrical cell lysis had been demonstrated in a number of reports in recent years and interest in electrical cell lysis had increased. We adopted the means of using electric field lysing because it can greatly simplify purification steps for preparation of biological samples and avoid denaturing of protein compared with conventional chemical methods and can be integrated into other bio-chip conveniently because its micromation.A low-voltage electrical cell lysis chip was designed and fabricated. It was designed to be a part of microfluidics systems where cell lysing was needed to for further analysis. It also can be used for sample preparation in general diagnosis and bio-experiments. Ti/Pt microelectrode was fabricated based on glass by standard photoresist processing of lift-off processing. The cell lysis chip acquire the high electric field strength(kV/cm)for cell lysis by applying an pulsed voltage to six pairs of micro-gap electrodes. A pulse generator was designed also. It was made with a AT89S52 microcontroller application circuit and an analogue circuit of output. It can generate regular DC pulses. BHK21, C2C12 cells are tested with the micro-fabricated chip. The lysis of BHK21, C2C12 cells were observed by a inverted microscope. The experimental observation suggests that BHK21, C2C12 cells were completely disintegrated by the pulsed electric field. In addition, the voltage required for lysing was reduced to only 3V by making the electrode gap on the order of microns and the intensity of the electric field needed is 0.6kV/cm.The experimental observation suggests that the voltage for cell lysis and the lysis rate had changed when applied different duration of pulses. Lysis rate was influenced by the voltage and duration of the pulse and the conductance of the cell suspension. We found the half-close micro-channels formed by clean PMMA with glass was useful for preventing bubbling when applied pulsed electric field.In conclusion, the present cell lysis chip is capable of cell lysis at a low DC pulsed voltage with high lysis rate; thus, it is suitable for a sample pretreatment component of a microfluidics or lab-on-a-chip.更多还原