在体生物电子鼻气味检测及嗅觉感知机理研究

发布时间：2018-01-18 09:25

本文关键词：在体生物电子鼻气味检测及嗅觉感知机理研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：生物嗅觉系统具有敏锐的气味感知能力,被认为是最高效的感测系统之一。随着社会文明的推进,气体检测技术用于提高人类生存质量,保障人类健康,已被广泛应用于食品安全、缉毒防爆、疾病诊断、环境监测等领域。基于仿生电子鼻和离体生物电子鼻的研究经验,本文提出一种新型的在体生物电子鼻系统:以哺乳动物的嗅上皮作为初级气味感受器,气味信息在嗅觉系统中修饰处理后,利用多通道微电极阵列提取嗅觉系统中多个神经元信号,通过多维度模式识别算法,对神经信号进行解码,提取出气味相关信息,从而实现气味检测。本文首先从使用寿命、重复性、特异性、灵敏度等方面对其气味检测性能进行分析;同时,通过提取分析嗅皮层响应信号和记录特定荧光标记的嗅小球区域响应信号两种方法,探索了进一步提高生物电子鼻检测特异性的方法;同时,本文讨论了生物电子鼻系统在食品新鲜度检测、爆炸物TNT检测领域的应用潜力。此外,从基础研究角度,本文研究了清醒状态和睡眠状态下动物的嗅觉感知差异形成机理,即嗅觉门控机理。结合光遗传学、动物行为学、在体电生理记录等方法,分析比较了不同状态下嗅觉系统的LFP和峰电位响应信号,研究结果显示嗅觉门控现象的产生依赖于脑区间神经元活动的同步性,即局部嗅觉区域和跨脑区间神经元的同步发放引起嗅觉感知,而非神经元的绝对活动强度。本文主要的创新性研究工作为:1.深入研究了在体生物电子鼻对痕量气味的检测性能和识别能力使用多通道植入式微电极提取清醒大鼠嗅球并分析神经元对气味刺激的响应,实现了在体生物电子鼻对单分子气味和混合气味的高灵敏度检测。实验结果表明由于保证了神经元的在体环境和嗅觉系统完整性,相比于离体生物电子鼻,该电子鼻使用寿命可达3周;采用PCA方法对大鼠僧帽/丛状细胞群的响应信号进行分类,不仅可以有效实现不同官能团单分子气体的区分,也可以区分相似气味的单分子和混合(自然)气体。研究进一步分析了该电子鼻对不同新鲜度的食品挥发物气体的响应方式,结果表明不同存储时间的食品气味诱发不同的峰电位响应活动;此外,该电子鼻对食品中常见气味香芹酮的最低检测浓度可达到10-10M。研究验证了该电子鼻检测单分子和混合气味的可行性,也初步证明了其在痕量气味、食品检测等领域的应用前景。2.提出了基于大鼠侧嗅束响应记录的生物电子鼻,提高了气味检测的性能嗅球中的僧帽/丛状细胞对气味信息整合编码后,其轴突整合成侧嗅束将信息传递到嗅觉皮层,因此,相比于局部嗅球的僧帽/丛状细胞,侧嗅束中携带了更完整的气味信息。为了进一步提高在体生物电子鼻气味检测特异性,我们将多通道电极植入区域从嗅球转向侧嗅束。由于刺激诱发的僧帽/丛状细胞活动经过颗粒细胞等中间神经元的侧抑制作用修饰后,其输出活动携带了完整的气味信息,研究结果表明从侧嗅束记录到的神经元峰电位自发频率低,而气味诱发的发放频率高,使得气味响应信噪比增加,提高气味响应灵敏度。因此,通过检测大鼠侧嗅束响应信号,提高了在体生物电子鼻的气味检测特异性。3.提出了基于转基因小鼠的生物电子鼻,实现了爆炸物TNT的定量检测使用在特定嗅感觉神经元表达绿色荧光蛋白的M72-IRES-tauGFP转基因小鼠,通过光学成像方法对M72嗅小球精确定位,将MEA探针植入到M72嗅小球区域,在不同动物之间实现特定嗅小球的响应记录,结果显示M72嗅小球对含有苯环的物质具有高敏感度,检测特异性高,进一步使用TNT刺激,发现其对TNT的响应也具有高敏感度,同时,其对TNT的检测下限小于10-6M。因此,该电子鼻不仅能实现苯环物质的特异性检测,在爆炸物TNT检测领域也具有应用价值。4.探索研究了哺乳动物在清醒和睡眠状态下的气味感知能力差异的机理以M72-ChR2-YFP和OMP-ChR2-GFP转基因小鼠为研究模型,使用蓝光激活嗅感觉神经元,同时记录多个嗅觉区域在清醒和睡眠状态下的LFP和峰电位响应信号。实验结果表明在睡眠状态下,嗅觉系统的LFP响应幅值和峰电位响应频率均不小于清醒状态,证明了嗅觉感知形成不依赖于神经群活动强度和单个神经元发放频率;进一步研究发现,在清醒状态下,刺激嗅感觉神经元使得不同嗅觉区域的Y波(30-80 Hz)能量增加,而睡眠状态下γ波能量不发生变化,由于γ振荡反映了神经元活动的同步性,表明了嗅觉感知形成依赖于神经元间的同步性。研究结果表明,嗅觉门控受到神经元活动同步性影响,清醒状态下响应神经元活动同步性高于睡眠状态。
[Abstract]:The biological olfactory system has a sense of smell sharp, is considered to be one of the most efficient sensing system. Along with the social civilization progress, gas detection technology used to improve the quality of human life, the protection of human health, has been widely used in food safety, drug explosion, disease diagnosis, environmental monitoring and other fields. The bionic electronic nose and from the research experience of organisms based on electronic nose, the paper puts forward a novel in vivo biological electronic nose system: the mammalian olfactory epithelium as primary odorant receptors, odor information modification in the olfactory system, using multi electrode array signal extraction of multiple neurons in olfactory system, through multi dimension pattern recognition algorithm, the decoding of neural signals to extract relevant information so as to realize the air smell, odor detection. This paper from the service life, repeatability, specificity, sensitivity and other aspects Analyze the odor detection performance; at the same time, through the extraction and analysis of response signals of two methods of glomerulus area entorhinal cortex response signal and record the specific fluorescent marker, explore the methods to further improve the biological specificity of detection by electronic nose; at the same time, this paper discusses the biological electronic nose system in food freshness detection, the potential application field of explosion TNT detection. In addition, from the perspective of basic research, this paper studies the olfactory perception differences awake and sleep state of animal and the formation mechanism of olfactory gating mechanism. Combined with the light of genetics, animal behavior, in vivo electrophysiological recording methods, analysis and comparison of the LFP and the peak potential of the olfactory system response under different conditions the research result shows that the synchronization signal of olfactory gating phenomenon depends on brain neuron activity, namely synchronous firing local regional and cross olfactory neurons in brain regions By olfactory perception, rather than absolute intensity of neurons. The main contributions of this thesis are as follows: 1. in-depth study of the in vivo biological electronic nose detection performance and the ability to identify trace odor using multi-channel implantable microelectrode extraction of olfactory bulb in conscious rats and analyze the response of neural element odors, realized in the body biological electronic nose to smell and odor molecules mixed high sensitivity detection. Experimental results show that with the guarantee of neurons in the olfactory system and environmental integrity, compared to the in vitro biological electronic nose, the electronic nose service life of up to 3 weeks; using the method of PCA response signal on rat mitral / tufted cells group classification, distinguish not only can effectively achieve the different functional groups of single molecule gas, can also distinguish between single molecules and mixed similar odor (NATURAL) gas. Further analysis of the electric nose The response to food volatile gas of different freshness. The results show that the peak potential of different storage time and different food odor evoked response activities; in addition, the electronic nose to smell the lowest detectable concentration of common carvone in food can reach 10-10M. study to verify the feasibility of the electronic nose detection of single molecules and mixed smell, too preliminary proved its trace odor, the application prospect of.2. in food detection of rat lateral olfactory tract response of electronic nose based on biological records, improve the performance of olfactory odor detection in mitral / plexiform cells of odor information integration after its integration into the axon encoding, olfactory tract side information transfer to the olfactory cortex, therefore, compared to the local olfactory bulb mitral / tufted cells, lateral olfactory tract with more complete odor information. In order to further improve the in vivo detection of specific electronic nose, We will be multi-channel electrode implantation area from the olfactory bulb to lateral olfactory tract. Due to stimulation of the mitral / plexiform cell activity through intermediate granule cell neurons side inhibition after modification, the output activities carry complete odor information, the results show that the lateral olfactory tract on neuronal spikes recorded spontaneous low frequency and high frequency distribution odor evoked response, the smell of the SNR increases, improve the odor response sensitivity. Therefore, through the detection of rat lateral olfactory tract response signal, improve the in vivo biological electronic nose odor detection of specific.3. of biological electronic nose based on transgenic mice, the M72-IRES-tauGFP transgenic mice quantitative detection of TNT explosives used in the specific olfactory sensory neurons expressing green fluorescent protein, glomeruli and precise localization of M72 by the optical imaging method, the MEA probe into olfactory M72 Small area response records achieve specific glomeruli in different animal. The results showed that M72 sensitivity to glomeruli containing benzene substances with high specificity, detection, further use of TNT stimulation, the response to TNT has a high sensitivity, at the same time, the TNT detection limit of less than 10-6M. so the electronic nose can not only realize the specific detection of benzene substances, in the field of TNT explosive detection also has the application value of.4. explores the odor perception ability difference during wakefulness and sleep state of the mammalian mechanism based on M72-ChR2-YFP and OMP-ChR2-GFP transgenic mice model for the study, the use of blue light activated olfactory sensory neurons, while recording a plurality of smell regional response LFP and peak potential during wakefulness and sleep state signal. Experimental results show that in the sleep state, the olfactory system LFP response response amplitude and peak potential The frequency is not less than the waking state, proved that olfactory perception does not depend on the formation of neural group activity intensity and single neuron firing frequency; the further study found that in the awake state, stimulation of olfactory sensory neurons makes different olfactory region Y wave (30-80 Hz) energy increases, and sleep state of gamma wave energy does not change, because gamma oscillation reflects the synchronization of neuronal activity, show that the synchronization of olfactory perception depends on the formation between neurons. The results show that the sense of smell is affected by synchronous gating of neuronal activity, conscious response to synchronous neuronal activity than sleep.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q43;TP216

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