基于MEMS技术的细胞破碎微流控芯片系统研究
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摘要
细胞破碎的目的是对初级样品实施细胞破碎处理以获得其中的内容物,例如蛋白质、脱氧核糖核酸(Deoxyribonucleic acid,简称DNA)、核糖核酸(RiboNucleic Acid,简称RNA),这对大多数细胞内物质的反应和检测进行研究的微流控芯片是必不可少的。近几年来电场法促使细胞破碎逐渐受到研究者们的关注,关于电子细胞破碎微流控芯片研究的报导数量也在逐渐上升。采用电场的方法进行细胞破碎是因为电场破碎法相对于传统的机械法、化学法、生物法等简化了样品提纯的过程、而且不会使蛋白质发生变性等,并且因为该方式采用了微型化,能较容易和其他芯片进行集成。
本文基于微机电系统(Micro-Electro-Mechanical Systems,简称MEMS)加工技术,设计并制备了一种运用较低的脉冲电压来实现细胞破碎的微流控芯片系统。该微流控芯片系统的主要任务是实现微流控系统的样品预处理,为生物、化学的后续的研究和分析奠定基础。
本论文中细胞破碎微流控芯片的制作是运用了微机电系统加工技术中的剥离工艺,采用标准光刻胶处理方法,以玻璃为基底,在玻璃上制作了Ti/Pt微电极。将外部的施加低脉冲电压施加在6对微电极上,使微电极产生细胞破碎所需的高场强。同时,本论文还设计了专用的细胞破碎信号发生器,该信号发生器是以C8051F020单片机为核心的硬件电路组成的。该信号发生器能实现对芯片施加外部电压信号,该电压信号可实现脉冲宽度、脉冲周期、脉冲电压幅度的调节。设计了细胞破碎效果检测系统,运用电荷耦合器件(Charge Coupled Device,简称CCD)将采集到的细胞图片传输到计算机,利用图像处理技术对细胞进行计数,可以计算出细胞破碎率。分别运用八邻域边界跟踪法和梯度Hough变换法对预处理后的细胞图像进行计数,结果表明利用八邻域边界跟踪标号进行细胞计数,对没有粘连细胞图像计数效果较好；但是当细胞图像中存在粘连情况时,对计数结果影响较大。本文提出了利用梯度Hough变换法实现显微细胞自动计数。梯度Hough变换在标准Hough变换基础上提高了对细胞计数的速度,并且结果显示其计数的准确率较八邻域边界跟踪法高。分别采用BHK21(幼仓鼠肾细胞),C2C12(小鼠成肌细胞)对芯片进行了细胞破碎实验检测。在倒置显微镜下在线观察BHK21、C2C12细胞破碎的过程,实验结果表明低脉冲电压形成的电场使这两种细胞都完全破碎了,并且对从破碎物进行了DNA质量检测。
由于该系统制作了电极距离为微米级的微小电极,3V的低脉冲电压就实现了细胞破碎。实验表明,低电压脉冲信号的宽度不同,其细胞破碎所需的电压和破碎率关联性。细胞破碎率和低电压脉冲信号的宽度、电压幅度和细胞悬浮液的电导率均有关系。通过实现发现干净的聚甲基丙烯酸甲酯(polymethyl methacrylate,简称PMMA)与芯片的玻璃衬底间形成了半封闭沟道,该沟道可以较好的防止施加低脉冲电压时产生气泡,实验表明产生的气泡与细胞悬浮液的电导率有关联。
本论文基于MEMS技术设计的细胞破碎微流控芯片系统,实现了在低电压脉冲信号的作用下使细胞以较高的破碎率进行了破碎,并且实现了在线检测细胞破碎率。该系统具有操作简单、所需破碎的脉冲电压低、制作成本低、微型化等优势。
The purpose of cell lysis is to break primary samples and to obtain inner contents (such as protein, DNA, RNA). Microfluidic chip is indispensable for the majority of studies on cellular reaction and detection. In recent years, electronic ways of cell lysis have been paid more attention by researchers and the number of relative reports is rising gradually. Compared with chemical ways and pyrogenation, electronic ways greatly simplify the purifying steps and don't change the protein properties. And its micromation can be conveniently integrated into other biochip samples.
Based on Micro-Electro-Mechanical Systems (abbreviation MEMS), a new microfluidic chips system for cell lysis under low voltage is designed and made in this thesis. It is used for the pretreatment in microfluidic system and this sets the base for later chemical and biological research and analysis. In this thesis the new system is made with the standard light-sensitive lacquer method of stripping technology and Ti/Pt microelectrodes on glass substrate. High field intensity can be obtained by pulsed voltage exerted by 6 pairs of microelectrodes. Meantime impulse signal generators especially for cell lysis is designed. It is composed of C8051F020 singlechip computer and applied circuit and imitated output circuit. This generator can justify impulse width, impulse cycle and impulse voltage extent. The new microfluidic chips system also included a cell lysis detecting effect system.
With charge coupled device, the detecting system transports the collected cell images to computers and count cells. Cell lysis rate can be calculated through image processing technology. With 8-connected boundary track and grads hough transform to count cells, the result shows that 8-connected boundary track is better and the effect is also better for non-conglutination cell images. But it matters greatly if there exists conglutination in images. This thesis put forward calculating cells with grads hough transform automatically. Grads hough transform is faster in calculating based on standard grads hough transform. The results shows that this method is more accurate than 8-connected boundary track in calculating. BHK21 and C2C12 are respectively used in the cell lysis experiments to test the chip. They are observed through inverted microscope. The observing result shows that impulse electrical field made these two kinds of cells completely broken and extracted DNA from broken cells successfully.
Since the distance between electrodes are measured by micron, the voltage needed for cell lysis declines to 3v. The experiment also shows that there is subtle difference between the needed voltage and cell lysis rate under the effect of different impulse signals. Cell lysis rate is relative with impulse width, impulse voltage and electrical conductivity of cell suspending liquid. Researches show that the semi-closed passage between clean polymethyl methacrylate and chips can effectively prevent the production of bubbles. The production of bubbles is also relative with electrical conductivity of cell suspending liquid.
In brief, this cell lysis microfluidic chips can break cells under a low voltage at a high breaking rate. It can be conveniently integrated into microfluidic systems or chip experiments for the pretreatment of samples. This system also has advantages, such as low impulse voltage, low cost and micromation etc.
本文基于微机电系统(Micro-Electro-Mechanical Systems,简称MEMS)加工技术,设计并制备了一种运用较低的脉冲电压来实现细胞破碎的微流控芯片系统。该微流控芯片系统的主要任务是实现微流控系统的样品预处理,为生物、化学的后续的研究和分析奠定基础。
本论文中细胞破碎微流控芯片的制作是运用了微机电系统加工技术中的剥离工艺,采用标准光刻胶处理方法,以玻璃为基底,在玻璃上制作了Ti/Pt微电极。将外部的施加低脉冲电压施加在6对微电极上,使微电极产生细胞破碎所需的高场强。同时,本论文还设计了专用的细胞破碎信号发生器,该信号发生器是以C8051F020单片机为核心的硬件电路组成的。该信号发生器能实现对芯片施加外部电压信号,该电压信号可实现脉冲宽度、脉冲周期、脉冲电压幅度的调节。设计了细胞破碎效果检测系统,运用电荷耦合器件(Charge Coupled Device,简称CCD)将采集到的细胞图片传输到计算机,利用图像处理技术对细胞进行计数,可以计算出细胞破碎率。分别运用八邻域边界跟踪法和梯度Hough变换法对预处理后的细胞图像进行计数,结果表明利用八邻域边界跟踪标号进行细胞计数,对没有粘连细胞图像计数效果较好；但是当细胞图像中存在粘连情况时,对计数结果影响较大。本文提出了利用梯度Hough变换法实现显微细胞自动计数。梯度Hough变换在标准Hough变换基础上提高了对细胞计数的速度,并且结果显示其计数的准确率较八邻域边界跟踪法高。分别采用BHK21(幼仓鼠肾细胞),C2C12(小鼠成肌细胞)对芯片进行了细胞破碎实验检测。在倒置显微镜下在线观察BHK21、C2C12细胞破碎的过程,实验结果表明低脉冲电压形成的电场使这两种细胞都完全破碎了,并且对从破碎物进行了DNA质量检测。
由于该系统制作了电极距离为微米级的微小电极,3V的低脉冲电压就实现了细胞破碎。实验表明,低电压脉冲信号的宽度不同,其细胞破碎所需的电压和破碎率关联性。细胞破碎率和低电压脉冲信号的宽度、电压幅度和细胞悬浮液的电导率均有关系。通过实现发现干净的聚甲基丙烯酸甲酯(polymethyl methacrylate,简称PMMA)与芯片的玻璃衬底间形成了半封闭沟道,该沟道可以较好的防止施加低脉冲电压时产生气泡,实验表明产生的气泡与细胞悬浮液的电导率有关联。
本论文基于MEMS技术设计的细胞破碎微流控芯片系统,实现了在低电压脉冲信号的作用下使细胞以较高的破碎率进行了破碎,并且实现了在线检测细胞破碎率。该系统具有操作简单、所需破碎的脉冲电压低、制作成本低、微型化等优势。
The purpose of cell lysis is to break primary samples and to obtain inner contents (such as protein, DNA, RNA). Microfluidic chip is indispensable for the majority of studies on cellular reaction and detection. In recent years, electronic ways of cell lysis have been paid more attention by researchers and the number of relative reports is rising gradually. Compared with chemical ways and pyrogenation, electronic ways greatly simplify the purifying steps and don't change the protein properties. And its micromation can be conveniently integrated into other biochip samples.
Based on Micro-Electro-Mechanical Systems (abbreviation MEMS), a new microfluidic chips system for cell lysis under low voltage is designed and made in this thesis. It is used for the pretreatment in microfluidic system and this sets the base for later chemical and biological research and analysis. In this thesis the new system is made with the standard light-sensitive lacquer method of stripping technology and Ti/Pt microelectrodes on glass substrate. High field intensity can be obtained by pulsed voltage exerted by 6 pairs of microelectrodes. Meantime impulse signal generators especially for cell lysis is designed. It is composed of C8051F020 singlechip computer and applied circuit and imitated output circuit. This generator can justify impulse width, impulse cycle and impulse voltage extent. The new microfluidic chips system also included a cell lysis detecting effect system.
With charge coupled device, the detecting system transports the collected cell images to computers and count cells. Cell lysis rate can be calculated through image processing technology. With 8-connected boundary track and grads hough transform to count cells, the result shows that 8-connected boundary track is better and the effect is also better for non-conglutination cell images. But it matters greatly if there exists conglutination in images. This thesis put forward calculating cells with grads hough transform automatically. Grads hough transform is faster in calculating based on standard grads hough transform. The results shows that this method is more accurate than 8-connected boundary track in calculating. BHK21 and C2C12 are respectively used in the cell lysis experiments to test the chip. They are observed through inverted microscope. The observing result shows that impulse electrical field made these two kinds of cells completely broken and extracted DNA from broken cells successfully.
Since the distance between electrodes are measured by micron, the voltage needed for cell lysis declines to 3v. The experiment also shows that there is subtle difference between the needed voltage and cell lysis rate under the effect of different impulse signals. Cell lysis rate is relative with impulse width, impulse voltage and electrical conductivity of cell suspending liquid. Researches show that the semi-closed passage between clean polymethyl methacrylate and chips can effectively prevent the production of bubbles. The production of bubbles is also relative with electrical conductivity of cell suspending liquid.
In brief, this cell lysis microfluidic chips can break cells under a low voltage at a high breaking rate. It can be conveniently integrated into microfluidic systems or chip experiments for the pretreatment of samples. This system also has advantages, such as low impulse voltage, low cost and micromation etc.
引文
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