单细胞分析的新方法和肾上腺素细胞传感器
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摘要
第一章对单细胞分析技术及其在生命科学研究上的应用性进行了综述。分别对毛细管电泳检测的理论和技术,2003年之后毛细管电泳检测方法在分析单细胞上的应用,微流控芯片在细胞培养、细胞操作和细胞内组分测定等方面的应用,影像学单细胞分析方法(包括荧光显微术、共聚焦激光扫描显微术、全内反射荧光显微术)在单细胞分析上的应用进行了简单介绍及综述。
第二章中我们建立了未见报道的微流控芯片电泳/柱端安培法检测单个细胞中化学组分的方法。在这个方法中,我们在双T型微流控芯片上实现了单个细胞的电动进样、转移和破膜,并使用安置在双T型微流控芯片的末端电化学检测器对单个细胞中的物质进行了检测。通过在微流控芯片的多通道体系中调整电压,完成了单个原生质体的电压控制进样,并通过施加一个220 V/cm的直流电压将细胞在芯片内溶膜。细胞溶膜后,细胞中要检测的物质在微流控芯片的分离通道中进行电泳分离,并在末端由电化学方法检测。用这个方法测定了单个小麦(Cha9)愈伤组织细胞中的抗坏血酸。
第三章中我们设计了利用荧光共振能量转移(FRET)原理检测CEA mRNA含量的方法。在这个方法中,将两种荧光染料标记的核酸探针与目标mRNA杂交,杂交后两种荧光染料的距离满足产生FRET的条件,通过检测核酸杂交后FRET的强度,可以测定目标mRNA的含量。我们用这种方法测定了MGC 803胃癌细胞提取液中CEA mRNA的含量。
第四章中我们将第三章所研究的FRET测定mRNA的原理应用到测定单个细胞内mRNA的含量。在至今所有报道的定量测定单细胞中化学组分分析的论文,细胞都是溶膜后再进行测定。在这种情况下,检测过程中细胞都已死亡。在绝大多数单细胞中化学组分定量分析的论文中,细胞是一个一个分析的,分析速度较慢,从细胞取样到测到信号通常需要十几分钟到几十分钟,即分析通量很低。在本章中我们研究成功了一种高通量定量测定活细胞中mRNA的方法。在这个方法中,首先用毛地黄皂苷将细胞膜蚀孔,使荧光核酸探针能够自由扩散进入细胞并同细胞内的mRNA杂交,荧光探针与mRNA杂交后在激光的照射下产生FRET。使用高灵敏CCD同时获取大量单个MGC 803细胞的FRET荧光图像,
In chapter one of this thesis, the techniques of single-cell analysis were reviewed briefly. These techniques were capillary electrophoresis (CE) over the past two years, microfluidic chip including the cell-culture, cell manipulation and detection of trance material in single cells and image analysis such as fluorescence microscopy, laser scanning confocal microscopy (LSCM) and total internal reflection fluorescence microscopy (TIRFM).In chapter two, an electrochemical method with a microfluidic device was developed for analysis of single cells. In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intercellular species were integrated in a microfluidic chip with a double-T injector coupled with an end-channel amperometric detector. A single cell was loaded at the double-T injector on the microfluidic chip by using electrical field. Then, the docked cell was lysed by a direct current electric field of 220 V/cm. The analyte of interest inside the cell was electrokinetically transported to the detection end of separation channel and was electrochemically detected. External standardization was used to quantify the analyte of interest in individual cells. Ascorbic acid (AA) in single wheat callus cells was chosen as the model compound. AA could be directly detected at a carbon fiber disk bundle electrode. The selectivity of electrochemical detection made the electropherogram simple. The technique described here could, in principle, be applied to a variety of electroactive species within single cells.In chapter three, determination of mRNA was carried out by fluorescence resonance energy transfer (FRET). In this method, two fluorescence nucleic acid probes were hybridized with target mRNA and the FRET between the two probes was detected. The concentration of the target mRNA could be determined through measuring the fluorescence intensity of FRET. The method investigated here was applied to determine mRNA in the extracts of MGC 803 cells.In chapter four, high-throughput single-cell analysis of mRNA was developed based on FRET. In this method, cells were first perforated with digitonin to improve
permeability. In this case, the probes could diffuse into cells easily. After the probes were hybridizui with target mRXT \, the FRET image was taken. The fluorescence intensity of FRET of individual cells was obtained using software MetaMorph. Based on the fluorescence intensity, the mRNA amount in single cells could be acquired. Since the FRET images of thirty cells could be taken simultaneously, the analysis throughput was high. This method was applied to determine CEA mRNA in single MGC 803 cells.In chapter five, a method for determination of glucose in individual cells was developed based on double enzymes reaction. In this method, a single cell was loaded in a capillary and then a solution containing 10 mmol/L (SDS), glucose oxidase (GOD), horseradish peroxidase (HRP) and 10-Acetyl-3,7-dihydroxy- phenoxazine (ADHP) was introduced into the capillary. After the cell was lysed by SDS, oxygen was reduced to H2O2 by GOD in the presence of glucose released from the lysed cell. At the same time, ADHP was converted to fluorescent resorufin by HRP. The amount of glucose in the cell could be obtained through detecting the fluorescence intensity of resorufin. This method was used to determine glucose amount in single MGC 803 cells.In chapter six, we investigated the cell sensor of epinephrine based on the cell signaling transduction. The principle is: After epinephrine combined with adrenergic receptor on the cell surface, the stimulated G protein within cells causes the signaling transduction to generate glucose. The generated glucose is released from the cells into the solution. The glucose in the solution is then detected using the method described in chapter five. The amount of epinephrine can be measured through determining glucose concentration. Since the glucose concentration is higher than the epinephrine concentration, implying amplification of the signal. It was found that the signal was amplified 38 times. This cell sensor was used to determine epinephrine.
第二章中我们建立了未见报道的微流控芯片电泳/柱端安培法检测单个细胞中化学组分的方法。在这个方法中,我们在双T型微流控芯片上实现了单个细胞的电动进样、转移和破膜,并使用安置在双T型微流控芯片的末端电化学检测器对单个细胞中的物质进行了检测。通过在微流控芯片的多通道体系中调整电压,完成了单个原生质体的电压控制进样,并通过施加一个220 V/cm的直流电压将细胞在芯片内溶膜。细胞溶膜后,细胞中要检测的物质在微流控芯片的分离通道中进行电泳分离,并在末端由电化学方法检测。用这个方法测定了单个小麦(Cha9)愈伤组织细胞中的抗坏血酸。
第三章中我们设计了利用荧光共振能量转移(FRET)原理检测CEA mRNA含量的方法。在这个方法中,将两种荧光染料标记的核酸探针与目标mRNA杂交,杂交后两种荧光染料的距离满足产生FRET的条件,通过检测核酸杂交后FRET的强度,可以测定目标mRNA的含量。我们用这种方法测定了MGC 803胃癌细胞提取液中CEA mRNA的含量。
第四章中我们将第三章所研究的FRET测定mRNA的原理应用到测定单个细胞内mRNA的含量。在至今所有报道的定量测定单细胞中化学组分分析的论文,细胞都是溶膜后再进行测定。在这种情况下,检测过程中细胞都已死亡。在绝大多数单细胞中化学组分定量分析的论文中,细胞是一个一个分析的,分析速度较慢,从细胞取样到测到信号通常需要十几分钟到几十分钟,即分析通量很低。在本章中我们研究成功了一种高通量定量测定活细胞中mRNA的方法。在这个方法中,首先用毛地黄皂苷将细胞膜蚀孔,使荧光核酸探针能够自由扩散进入细胞并同细胞内的mRNA杂交,荧光探针与mRNA杂交后在激光的照射下产生FRET。使用高灵敏CCD同时获取大量单个MGC 803细胞的FRET荧光图像,
In chapter one of this thesis, the techniques of single-cell analysis were reviewed briefly. These techniques were capillary electrophoresis (CE) over the past two years, microfluidic chip including the cell-culture, cell manipulation and detection of trance material in single cells and image analysis such as fluorescence microscopy, laser scanning confocal microscopy (LSCM) and total internal reflection fluorescence microscopy (TIRFM).In chapter two, an electrochemical method with a microfluidic device was developed for analysis of single cells. In this method, cell injection, loading and cell lysis, and electrokinetic transportation and detection of intercellular species were integrated in a microfluidic chip with a double-T injector coupled with an end-channel amperometric detector. A single cell was loaded at the double-T injector on the microfluidic chip by using electrical field. Then, the docked cell was lysed by a direct current electric field of 220 V/cm. The analyte of interest inside the cell was electrokinetically transported to the detection end of separation channel and was electrochemically detected. External standardization was used to quantify the analyte of interest in individual cells. Ascorbic acid (AA) in single wheat callus cells was chosen as the model compound. AA could be directly detected at a carbon fiber disk bundle electrode. The selectivity of electrochemical detection made the electropherogram simple. The technique described here could, in principle, be applied to a variety of electroactive species within single cells.In chapter three, determination of mRNA was carried out by fluorescence resonance energy transfer (FRET). In this method, two fluorescence nucleic acid probes were hybridized with target mRNA and the FRET between the two probes was detected. The concentration of the target mRNA could be determined through measuring the fluorescence intensity of FRET. The method investigated here was applied to determine mRNA in the extracts of MGC 803 cells.In chapter four, high-throughput single-cell analysis of mRNA was developed based on FRET. In this method, cells were first perforated with digitonin to improve
permeability. In this case, the probes could diffuse into cells easily. After the probes were hybridizui with target mRXT \, the FRET image was taken. The fluorescence intensity of FRET of individual cells was obtained using software MetaMorph. Based on the fluorescence intensity, the mRNA amount in single cells could be acquired. Since the FRET images of thirty cells could be taken simultaneously, the analysis throughput was high. This method was applied to determine CEA mRNA in single MGC 803 cells.In chapter five, a method for determination of glucose in individual cells was developed based on double enzymes reaction. In this method, a single cell was loaded in a capillary and then a solution containing 10 mmol/L (SDS), glucose oxidase (GOD), horseradish peroxidase (HRP) and 10-Acetyl-3,7-dihydroxy- phenoxazine (ADHP) was introduced into the capillary. After the cell was lysed by SDS, oxygen was reduced to H2O2 by GOD in the presence of glucose released from the lysed cell. At the same time, ADHP was converted to fluorescent resorufin by HRP. The amount of glucose in the cell could be obtained through detecting the fluorescence intensity of resorufin. This method was used to determine glucose amount in single MGC 803 cells.In chapter six, we investigated the cell sensor of epinephrine based on the cell signaling transduction. The principle is: After epinephrine combined with adrenergic receptor on the cell surface, the stimulated G protein within cells causes the signaling transduction to generate glucose. The generated glucose is released from the cells into the solution. The glucose in the solution is then detected using the method described in chapter five. The amount of epinephrine can be measured through determining glucose concentration. Since the glucose concentration is higher than the epinephrine concentration, implying amplification of the signal. It was found that the signal was amplified 38 times. This cell sensor was used to determine epinephrine.
引文
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