摘要
神经电生理研究,历来是神经科学研究的主要内容。但是,目前神经元电信号的记录,仍主要依赖于以膜片钳为代表的细胞膜微电极穿刺或钳制技术。该类方法因其对细胞的穿刺损害作用,难以实现长时程测量,同时也难以实现对神经元网络的多位点同时测量。有鉴于此,研究者们陆续采用微机械加工技术开展了基于细胞传感器(cell-based biosensor)的微电极阵列(microelectrode array,MEA)和场效应管(field effect transistor,FET)阵列等神经芯片(neurochip)技术的研究,用以实现神经元胞外电位的记录。作为一种体外检测的新型细胞芯片技术,其实质就是在MEA或FET阵列传感器芯片表面培养神经元,使神经元通过一层薄的电解液同芯片的电极或栅极相耦合,构成可以实现控制电路和神经系统双向通讯的生物芯片,从而对细胞的电生理特性进行传感测量。该技术以其可对多个细胞同时进行长期、无损检测的特点,已在药物筛选、环境检测等生物医学领域得到了初步的应用。同时,该芯片技术同样适用于神经系统在体研究,从而在脑的高级功能、神经修复、以及人工器官等研究领域展示出了诱人的前景。
本论文首先从器件设计和细胞培养两个关键技术入手,建立了小鼠胚胎干细胞(embryonic stem cells)来源的基于光寻址电位传感器(light addressable potentiometric sensor,LAPS)的新型神经芯片技术,并在药物筛选等研究的基础上,从离体与在体两个方面,将该技术应用到了生物嗅觉的研究之中,为神经芯片技术拓展了新的应用领域。本论文的主要内容和贡献如下:
1.利用LAPS的光寻址特性,实现了神经芯片的细胞跟踪定位检测。MEA和FET阵列芯片都有一个共同的局限,即由于检测位点均为芯片表面固定的电极或栅极,被测细胞需要培养生长在这些特定位点,所以给细胞培养技术提出了很高的要求。LAPS芯片则是基于微机械加工技术的另一类新型细胞传感器,细胞电位的改变可通过检测LAPS的光生电流得以测量。利用其光源的可寻址特性,方便对芯片表面随机培养细胞的跟踪定位,从而克服了上述阵列传感器固定检测位点几何特性的限制。
Neuronal electrophysiology is one of the main research fields of the neuroscience. However, recording methods of the electronical signals still depend on the microelectrode or the patch clamp sticking to the membrane of the cells. It is difficult for those techniques to realize long-term monitoring, especially, the multi channels synchronous recording of neuronal networks, due to their unavoidable damage to cells. Therefore, using microelectromechanical systems, researchers have developed neurochips of microelectrode array (MEA) or field effect transistor (FET) array, based on cell-based biosensor, to record the extracellular potentials of neurons. Being an in vitro recording system, neurochip means a technique of culturing neurons on the surface of MEA or FET arrays, where cells can couple with electrodes or gates of FET through a thin layer of electrolyte. The novel biochip allows the communication between chips and cells, as well as monitoring the electrical activities of neurons in vitro in a long-term and non-invasive way. With these merits, neurochip have been applied primarily to biotnedical studies such as drugs screening and environment detection. Furthermore, neurochip allows for investigating the high function of the brain, neuronal prostheses and the reconstruction of damaged sense organs, when used in vivo to the neural system.To settle the key techniques of device design and celluar culture, the paper illustrated a novel neurochip based on light addressable potentiometric sensor (LAPS), using excitable cells cultured from embryonic stem cells, which can be successfully applied to drugs screening. Moreover, the neurochip has also been employed to the research of the biological olfaction in vivo as well as in vitro, which is a new application of the neurochip research. The major contents and contributions of this thesis are given as the following aspects.1. Based on light addressing characters of LAPS, the neurocip realized cell tracking and detection. Both MEA and FET array are restricted to measuring the
extracelluar potential only at a limited number of active measuring sites (the tip of each individual microelectrode and the gate-electrode). It is difficult to culture cells just on those sites. LAPS is another commonly used semiconductor chip. When cells produce potential changes, which can be recorded by measuring the photocurrent generated from LAPS. By scanning the light-pointer along LAPS, cells at any desired position can be recorded, hence it can overcome the limitation of other neurochips mentioned before.2. A novel cell source for neurochip was provided, by inducing mouse embryonic stem cells in vitro differentiation culture. Neuronal cell is the bridge of the neurochip and neural system, which can sense the different physical and chemical stimulations of the neural system. Therefore, culture of the neuronal cells is very important to the neurochip. We induced mouse embryonic stem cells in vitro to differentiate into spontaneity beating cardiomyocytes and excited neurons respectively. Using extracellular potentials of the cells recorded by LAPS, a primary application in drugs screening of the stem cell chip was discussed.3. A bionic olfactory neurochip was designed based on a hybrid system of LAPS and olfactory neurons, which is sensitive to odors. Mammalian olfactory system can distinguish thousands of odors even in a minimal concentration level. In order to realize the biomimetic design of electronic nose on the principle of olfactory system, the chip was designed based on the electrophysiology detection of the olfactory cells. Through the simulative response under stimulations of the environment odorants, the results show that the bioelectronic nose of this biomimetic neurochip is sensitive to odorous changes. The technique widened the design idea of the traditional artificial nose.4. Dopamine in rat olfaction monitored in vivo, using a microprobe with microelectrode array. Based on the technique of microelectromechanical systems, a probe chip with 8 channels electrode arrays was fabricated. The sensor area of each electrode is 16 urn* 120 um or 0.3 urn* 120 um. With the very low cross talking noise of each channel, the probe could even detect Dopamine in 50 nM. Catecholamines were also monitored in vivo with the probe settling in rat nasal mucus. Using the
animal model of trigeminal stimulation by CO2, the results demonstrate the chip could monitor the time course of neurotransmitter substances by multiple sensor sites.
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