自动化细胞注射中微操作与微注射技术及实验研究
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摘要
细胞注射技术在生物、医疗等领域中扮演越来越重要的角色,但其手工或半自动化操作方式,存在实验效率低、注射量控制难等不足。研究一种自动化程度高、微注射量精确可控的细胞注射技术已成为细胞工程和机械工程的重要课题。
微流体数字化技术是南京理工大学微系统研究室发明的、拥有自主知识产权的重要研究成果,该技术为建立与信息化、能量传输及固体运动数字化有同等意义的物质传输数字化开辟了道路,对微流体系统的研究和技术进步有重要意义。本论文以微流体数字化技术为起点,研究自动化细胞注射中微操作与微注射技术。从操作机理、实现方法与装置、验证实验等方面,研究了自动化细胞注射的系统结构和自动补给、位姿调节、数字化进退针、数字化微注射这四项关键技术,取得以下成果:
提出了“多操作手分立式”细胞注射系统结构,简化了单操作手的任务和操作方式,降低了图像处理、伺服控制等单元的性能要求,增强了微小工件位置和姿态调节能力,为研制自动化细胞注射系统奠定基础。
研制了微流体数字化技术驱动的细胞自动补给仪样机,适合于不同尺寸细胞的定点输送,具有自动计数、自动分离和实时可控等特征。
提出了基于流场流动和机械运动耦合控制的细胞位姿调节技术。以细胞培养液为控制介质,分析了细胞位姿调节技术操作机理;建立了被操作细胞工程化模型和淹没射流控制模型;以吸持针为末端执行器,发明了细胞位姿调节用单细胞微操作装置。实现了三维位置坐标和三自由度姿态信息的精确控制。
提出了前后端双支撑结构的冲击式微进给驱动原理,建立了该驱动原理的动力学模型,消除了传统惯性式微进给机构质心前后移动引起的横向摆动,进给步长和运动方向实时可调,运行稳定可靠,行程不受限制。
研制了具有数字化进退针和数字化微注射双重功能的微注射仪,测试了其进给和喷射性能。具有进给精度高、瞬时速度快、过程实时可控等特征,精度可达纳米量级,刺膜实验效率达149枚/h;实现了注射时间节拍化、微注射量规整化,且脉冲序列人为可控,微注射分辨率达飞升量级。
以小鼠卵母细胞为实验对象,进行了自动化细胞注射验证实验研究,实验效率达12枚/h。结果表明:基于细胞自动补给、位姿调节、数字化进退针和数字化微注射这四项特征操作的自动化细胞注射是可行的,“多操作手分立式”系统结构提高了细胞注射自动化程度。
In the domain of biology, medical treatment and so on, cell injection plays a more and more important role. But there are many deficiencies of manual or semi-automatic manipulation, including low experimental efficiency, difficulty of injection volume control and so on. It is an important subject of cell engineering and mechanical engineering to research a cell injection technology, with the characters of high automation and precise control of microinjection volume.
Invented and patented by micro-system lab of Nanjing University of Science and Technology, Digitalization of Micro-Fluids is the most important research production lately. With the equivalent of informationization, digital energy transmission and solid movement, the field of the digital matter transportation is exploited by Micro-Fluidic Digitalization technology, which is greatly significant for the development of microsystem research and technology. Base on the Digitalization of Micro-Fluids, the technologies of micro-manipulation and microinjection is presented for automatic cell injection in this paper. The technologies include cell auto-feeding, position and attitude adjusting, digital piercing and withdrawing micropipette and digital microinjection. Considering the aspects of manipulation mechanism, method and device, confirmed and applied experiment, and so on, a novel system structure and the pivotal technologies of automatic cell injection is presented as follow.
A novel cell injection system structure with divided manipulators is brought forward. The task and operating method of every single manipulator is simplified. The performance demand of components, including micro image test, servocontrol and so on, is reduce. The manipulating ability of micro part position and attitude is enhanced. The novel cell injection system structure lays a foundation for the automatic cell injection system.
By the drive of micro-fluidic digital technology, the prototype of cell auto-feeder is developed. With characteristics of auto-count, auto-separation and real time control, the prototype can transport appointed for different size cell.
Based on coupling control of mechanical motion and flow field, an automatable adjusting technology for cellular position and attitude is proposed. The manipulating mechanism of adjusting technology for cell position and attitude is primarily analyzed with the control of cell culture medium. The engineering model of manipulated cell and control model of submerged jet is founded. With end-actuator of micro-holding pipette, an individual cell manipulation system is developed for cell position and attitude adjusting. Precise control for 3D position and 3 D-O-F attitude is achieved.
With the structure character of front- and back- end double support, a novel impact micro motion drive principle is proposed. The principle dynamical model is established. Compared with traditional inertial micro motion principle, the transverse vibration, caused by center mass motion, is removed. Step length and motion direction can be adjusted real timely. Working stability is improved and motion travel is unrestricted.
A novel micro-injector is presented with function of digital piercing and withdrawing micropipette, digital microinjection. Confirmed by performance experiment of motion and injection, the micro-injector motion precision is high, coming to nanometer. Experimenatal efficiency of cell membrane pierces reachs 149 cells per hour. The instantaneous speed is rapid. The working process is real time controllable with characteristics of rhythmic injection time and standardized injection volume, while the drive pulse is controllable artificially. The amount of microinjection may reach an ultra high resolution of the order of femtoliters.
Taking mouse oocyte as experimental object, automatic cell injection experiment is carried with experimental efficency of 12 cells per hour. By experimental confirmation, the automatic cell injection scheme, which is based on manipulation of cell auto-feeding, position and attitude adjusting, digital piercing and withdrawing micropipette, digital microinjection, is feasible. The automation of cell injection is enhanced by system structure with divided manipulators.
微流体数字化技术是南京理工大学微系统研究室发明的、拥有自主知识产权的重要研究成果,该技术为建立与信息化、能量传输及固体运动数字化有同等意义的物质传输数字化开辟了道路,对微流体系统的研究和技术进步有重要意义。本论文以微流体数字化技术为起点,研究自动化细胞注射中微操作与微注射技术。从操作机理、实现方法与装置、验证实验等方面,研究了自动化细胞注射的系统结构和自动补给、位姿调节、数字化进退针、数字化微注射这四项关键技术,取得以下成果:
提出了“多操作手分立式”细胞注射系统结构,简化了单操作手的任务和操作方式,降低了图像处理、伺服控制等单元的性能要求,增强了微小工件位置和姿态调节能力,为研制自动化细胞注射系统奠定基础。
研制了微流体数字化技术驱动的细胞自动补给仪样机,适合于不同尺寸细胞的定点输送,具有自动计数、自动分离和实时可控等特征。
提出了基于流场流动和机械运动耦合控制的细胞位姿调节技术。以细胞培养液为控制介质,分析了细胞位姿调节技术操作机理;建立了被操作细胞工程化模型和淹没射流控制模型;以吸持针为末端执行器,发明了细胞位姿调节用单细胞微操作装置。实现了三维位置坐标和三自由度姿态信息的精确控制。
提出了前后端双支撑结构的冲击式微进给驱动原理,建立了该驱动原理的动力学模型,消除了传统惯性式微进给机构质心前后移动引起的横向摆动,进给步长和运动方向实时可调,运行稳定可靠,行程不受限制。
研制了具有数字化进退针和数字化微注射双重功能的微注射仪,测试了其进给和喷射性能。具有进给精度高、瞬时速度快、过程实时可控等特征,精度可达纳米量级,刺膜实验效率达149枚/h;实现了注射时间节拍化、微注射量规整化,且脉冲序列人为可控,微注射分辨率达飞升量级。
以小鼠卵母细胞为实验对象,进行了自动化细胞注射验证实验研究,实验效率达12枚/h。结果表明:基于细胞自动补给、位姿调节、数字化进退针和数字化微注射这四项特征操作的自动化细胞注射是可行的,“多操作手分立式”系统结构提高了细胞注射自动化程度。
In the domain of biology, medical treatment and so on, cell injection plays a more and more important role. But there are many deficiencies of manual or semi-automatic manipulation, including low experimental efficiency, difficulty of injection volume control and so on. It is an important subject of cell engineering and mechanical engineering to research a cell injection technology, with the characters of high automation and precise control of microinjection volume.
Invented and patented by micro-system lab of Nanjing University of Science and Technology, Digitalization of Micro-Fluids is the most important research production lately. With the equivalent of informationization, digital energy transmission and solid movement, the field of the digital matter transportation is exploited by Micro-Fluidic Digitalization technology, which is greatly significant for the development of microsystem research and technology. Base on the Digitalization of Micro-Fluids, the technologies of micro-manipulation and microinjection is presented for automatic cell injection in this paper. The technologies include cell auto-feeding, position and attitude adjusting, digital piercing and withdrawing micropipette and digital microinjection. Considering the aspects of manipulation mechanism, method and device, confirmed and applied experiment, and so on, a novel system structure and the pivotal technologies of automatic cell injection is presented as follow.
A novel cell injection system structure with divided manipulators is brought forward. The task and operating method of every single manipulator is simplified. The performance demand of components, including micro image test, servocontrol and so on, is reduce. The manipulating ability of micro part position and attitude is enhanced. The novel cell injection system structure lays a foundation for the automatic cell injection system.
By the drive of micro-fluidic digital technology, the prototype of cell auto-feeder is developed. With characteristics of auto-count, auto-separation and real time control, the prototype can transport appointed for different size cell.
Based on coupling control of mechanical motion and flow field, an automatable adjusting technology for cellular position and attitude is proposed. The manipulating mechanism of adjusting technology for cell position and attitude is primarily analyzed with the control of cell culture medium. The engineering model of manipulated cell and control model of submerged jet is founded. With end-actuator of micro-holding pipette, an individual cell manipulation system is developed for cell position and attitude adjusting. Precise control for 3D position and 3 D-O-F attitude is achieved.
With the structure character of front- and back- end double support, a novel impact micro motion drive principle is proposed. The principle dynamical model is established. Compared with traditional inertial micro motion principle, the transverse vibration, caused by center mass motion, is removed. Step length and motion direction can be adjusted real timely. Working stability is improved and motion travel is unrestricted.
A novel micro-injector is presented with function of digital piercing and withdrawing micropipette, digital microinjection. Confirmed by performance experiment of motion and injection, the micro-injector motion precision is high, coming to nanometer. Experimenatal efficiency of cell membrane pierces reachs 149 cells per hour. The instantaneous speed is rapid. The working process is real time controllable with characteristics of rhythmic injection time and standardized injection volume, while the drive pulse is controllable artificially. The amount of microinjection may reach an ultra high resolution of the order of femtoliters.
Taking mouse oocyte as experimental object, automatic cell injection experiment is carried with experimental efficency of 12 cells per hour. By experimental confirmation, the automatic cell injection scheme, which is based on manipulation of cell auto-feeding, position and attitude adjusting, digital piercing and withdrawing micropipette, digital microinjection, is feasible. The automation of cell injection is enhanced by system structure with divided manipulators.
引文
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