WO2024109896A1 - Rapid temperature control device for cell culture based on microfluidic chip - Google Patents
Rapid temperature control device for cell culture based on microfluidic chip Download PDFInfo
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- 238000004113 cell culture Methods 0.000 title claims abstract description 11
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Abstract
The present invention provides a rapid temperature control device for cell culture based on a microfluidic chip, comprising a water bath incubator, a temperature control system, and a microfluidic chip and a temperature-sensing probe that are provided in the water bath incubator. The water bath incubator is arranged on a microscope stage or a motorized displacement stage of a time-lapse microscopy imaging system, a hollow window is provided at the bottom of an incubator body of the water bath incubator, a transparent window is provided at the top of the incubator body, and the centers of the transparent window and the hollow window are both coaxial with the optical axis of an objective lens of the time-lapse microscopy imaging system; a glass sheet substrate of the microfluidic chip is in close fit with the hollow window; a thermostat of the temperature control system is connected to the temperature-sensing probe so as to monitor the ambient temperature in the water bath incubator; the thermostat is used for heating the water in a water storage tank to a target temperature and maintaining a constant temperature, and the constant-temperature water in the water storage tank enters the water bath incubator through a water inlet pipe and then flows out of the water storage tank though a water outlet pipe, so that the constant-temperature water circulates inside the water bath incubator to achieve stable temperature control.
Description
本发明涉及生物技术领域，特别涉及一种基于微流控芯片细胞培养的快速控温装置。The invention relates to the field of biotechnology, and in particular to a rapid temperature control device based on microfluidic chip cell culture.
微流控芯片技术结合延时显微成像技术能够实现对单个细胞进行长时间的监测培养。微流控芯片用于在显微成像下培养和实时监测细胞或微生物生长时，通常需要给定适合生物生长的温度条件，对于不同的细胞或微生物，其生长所需要的条件不一定相同，其中包括但不限于温度条件，控制微流控芯片中及其周围环境温度的快速变化是该过程的关键。Microfluidic chip technology combined with time-lapse microscopy technology can achieve long-term monitoring and cultivation of single cells. When microfluidic chips are used to culture and monitor the growth of cells or microorganisms in real time under microscopic imaging, temperature conditions suitable for biological growth are usually required. Different cells or microorganisms may not necessarily have the same growth conditions, including but not limited to temperature conditions. Controlling the rapid changes in temperature in the microfluidic chip and its surrounding environment is the key to the process.
目前，用于控制微流控芯片培养环境温度的方式主要有三种。第一种为可集成在显微镜上的温控箱，通过加热空气形成对流的方式对显微镜载物台上的微流控芯片进行控温，可实现较大的温控范围；但是温度分布均匀性较差，较大空间的空气加热体积导致升降温很慢，无法实现快速且稳定的变温效果。第二种为可置于显微镜载物台上的基于金属加热的小型恒温培养箱，直接对放置其中的微流控芯片进行加热或者制冷；小型恒温培养箱可以控制微流控芯片整体实现较为快速的升温过程，但由于其没有散热装置，在降温过程中需要等待较长的时间，此外，微流控芯片处于培养箱内部的空气环境中，升温速度依然受限于空气较大的比热容。第三种为利用半导体制冷片直接放在微流控芯片下方，通过电流控制进行加热或制冷操作,也有一些研究机构在微流控芯片上直接印制热电半导体控温线路，实现对芯片上温度的控制；半导体制冷片通常需要放在微流控芯片下方，但是显微镜镜头的活动空间将受限制，因而无法实现高通量的实验数据采集对于半导体制冷片不直接接触微流控芯片的设计，不均匀的温度传导导致微流控芯片上不同位置的温度均一性较差；对于将半导体材料直接加工到微流控芯片上，则要求必须是特制硬质绝缘片材芯片基板，对热电材料要求高，而高倍显微镜通常使用厚度为0.17mm的玻璃作为芯片基底，该种半导体控温方式应用范围有限。此外，使用半导体制冷方式的微流控芯片通常与室温空气接触，因此微流控芯片不能够稳定地维持预期温度。At present, there are three main ways to control the temperature of the microfluidic chip culture environment. The first is a temperature control box that can be integrated on a microscope. It controls the temperature of the microfluidic chip on the microscope stage by heating the air to form convection, which can achieve a larger temperature control range; however, the temperature distribution uniformity is poor, and the air heating volume in a large space causes the temperature to rise and fall very slowly, and it is impossible to achieve a fast and stable temperature change effect. The second is a small constant temperature incubator based on metal heating that can be placed on the microscope stage, which directly heats or cools the microfluidic chip placed therein; the small constant temperature incubator can control the overall microfluidic chip to achieve a relatively fast heating process, but because it has no heat dissipation device, it takes a long time to wait during the cooling process. In addition, the microfluidic chip is in the air environment inside the incubator, and the heating rate is still limited by the large specific heat capacity of the air. The third method is to use semiconductor cooling chips placed directly under the microfluidic chip to perform heating or cooling operations through current control. Some research institutions also directly print thermoelectric semiconductor temperature control circuits on microfluidic chips to control the temperature on the chip. Semiconductor cooling chips usually need to be placed under the microfluidic chip, but the activity space of the microscope lens will be limited, so high-throughput experimental data acquisition cannot be achieved. For designs where the semiconductor cooling chip does not directly contact the microfluidic chip, uneven temperature conduction leads to poor temperature uniformity at different locations on the microfluidic chip. For direct processing of semiconductor materials onto microfluidic chips, it is required to be a special hard insulating sheet chip substrate, which has high requirements for thermoelectric materials. High-power microscopes usually use glass with a thickness of 0.17 mm as the chip substrate. This type of semiconductor temperature control method has a limited application range. In addition, microfluidic chips using semiconductor cooling methods are usually in contact with room temperature air, so microfluidic chips cannot stably maintain the expected temperature.
现有兼容延时显微成像***的微流控芯片快速控温装置无法实现温度的快速变化及稳定控温。Existing microfluidic chip rapid temperature control devices compatible with time-lapse microscopy imaging systems cannot achieve rapid temperature changes and stable temperature control.
本发明提供一种基于微流控芯片细胞培养的快速控温装置，所述快速控温装置与延时显微成像***兼容，其包括：水浴培养箱，其设置在延时显微成像***的显微镜载物台或电动位移台上方；所述水浴培养箱包括箱体以及设置在所述箱体上的进水口和出水口，所述箱体的底部设置有镂空视窗，所述箱体的顶部设置有透明视窗，所述镂空视窗和所述透明视窗的中心均与延时显微成像***的物镜光轴同轴；微流控芯片，其设置在所述水浴培养箱的所述镂空视窗上，所述微流控芯片的玻璃片基底与所述镂空视窗紧密配合；温感探头，其设置所述水浴培养箱内并靠近所述微流控芯片或设置在所述微流控芯片上；控温***，其包括蓄水池、恒温器、连接所述进水口的进水管道和连接所述出水口的出水管道；其中，所述恒温器与所述温感探头连接，以用于监测所述水浴培养箱内的环境温度；所述恒温器用于使所述蓄水池内的水达到目的温度并保持恒温，所述蓄水池内的恒温水通过所述进水管道进入所述水浴培养箱并通过所述出水管道再流出所述蓄水池，进而实现以恒温水在所述水浴培养箱内循环流动以稳定控温。The present invention provides a rapid temperature control device based on microfluidic chip cell culture, the rapid temperature control device is compatible with a time-lapse microscopic imaging system, and comprises: a water bath incubator, which is arranged above a microscope stage or an electric displacement stage of the time-lapse microscopic imaging system; the water bath incubator comprises a box body and a water inlet and a water outlet arranged on the box body, a hollow window is arranged at the bottom of the box body, a transparent window is arranged at the top of the box body, and the centers of the hollow window and the transparent window are both coaxial with the objective lens optical axis of the time-lapse microscopic imaging system; a microfluidic chip is arranged on the hollow window of the water bath incubator, and a glass sheet substrate of the microfluidic chip is connected to the hollow window. The windows are tightly fitted; a temperature sensing probe is arranged in the water bath incubator and close to the microfluidic chip or on the microfluidic chip; a temperature control system includes a water reservoir, a thermostat, a water inlet pipe connected to the water inlet, and a water outlet pipe connected to the water outlet; wherein the thermostat is connected to the temperature sensing probe to monitor the ambient temperature in the water bath incubator; the thermostat is used to make the water in the water reservoir reach the target temperature and maintain a constant temperature, the constant temperature water in the water reservoir enters the water bath incubator through the water inlet pipe and flows out of the water reservoir through the water outlet pipe, thereby realizing the circulation of constant temperature water in the water bath incubator to stabilize the temperature control.
可选地，所述箱体包括底座和设置在所述底座上的顶盖，所述镂空视窗设置在所述底座上，所述透明视窗设置在所述顶盖上。Optionally, the box body includes a base and a top cover arranged on the base, the hollow window is arranged on the base, and the transparent window is arranged on the top cover.
可选地，所述镂空视窗的设计满足高倍物镜对所述微流控芯片的最大面积成像。Optionally, the hollow window is designed to allow a high-power objective lens to image the maximum area of the microfluidic chip.
可选地，该快速控温装置还包括设置在所述水浴培养箱内与所述底座可拆卸连接的芯片压片，所述芯片压片包括用于压紧所述微流控芯片的弹簧针或橡皮圈。Optionally, the rapid temperature control device further comprises a chip pressing sheet disposed in the water bath incubator and detachably connected to the base, wherein the chip pressing sheet comprises a spring pin or a rubber ring for pressing the microfluidic chip.
可选地，所述芯片压片通过螺丝与所述底座连接。Optionally, the chip pressing piece is connected to the base via screws.
可选地，所述芯片压片的周侧设置有隔水橡皮圈，所述底座的上表面周侧开设有橡皮圈槽，所述顶盖的下表面设置有与所述橡皮圈槽配合的隔水橡皮圈。Optionally, a waterproof rubber ring is provided on the peripheral side of the chip pressing sheet, a rubber ring groove is opened on the peripheral side of the upper surface of the base, and a waterproof rubber ring matching with the rubber ring groove is provided on the lower surface of the top cover.
可选地，所述底座和所述顶盖通过螺丝连接。Optionally, the base and the top cover are connected by screws.
可选地，所述箱体上还设置有芯片管路进口和芯片管路出口。Optionally, the box body is also provided with a chip pipeline inlet and a chip pipeline outlet.
可选地，所述芯片管路进口和所述芯片管路出口设置在所述顶盖上。Optionally, the chip pipeline inlet and the chip pipeline outlet are arranged on the top cover.
可选地，所述控温***还包括蠕动泵，所述进水口管道连接所述蠕动泵以便于所述蓄水池内的恒温水传输至所述水浴培养箱的内部。Optionally, the temperature control system further comprises a peristaltic pump, and the water inlet pipe is connected to the peristaltic pump so as to facilitate the transmission of the constant temperature water in the water reservoir to the interior of the water bath incubator.
本发明的有益效果为：The beneficial effects of the present invention are:
本发明提供一种基于微流控芯片细胞培养的快速控温装置，快速控温装置与延时显微成像***兼容，其包括水浴培养箱、控温***以及设置在水浴培养箱内的微流控芯片和温感探头。其中，水浴培养箱设置在延时显微成像***的显微镜载物台或电动位移台上方，水浴培养箱的箱体底部设置有镂空视窗，箱体顶部设置有透明视窗，透明视窗和镂空视窗的中心均与延时显微成像***的物镜光轴同轴；微流控芯片设置在镂空视窗上，其玻璃片基底与所述镂空视窗紧密配合；控温***的恒温器与温感探头连接，以用于监测水浴培养箱内的环境温度；恒温器用于加热蓄水池内的水达到目的温度并保持恒温，蓄水池内的恒温水通过进水管道进入水浴培养箱并通过出水管道再流出蓄水池，进而实现恒温水在水浴培养箱内循环流动以稳定控温。细胞培养液通过芯片管路进口进入微流控芯片中并通过芯片管路出口流出，用于提供持续培养细胞所需的营养环境。The present invention provides a rapid temperature control device for cell culture based on a microfluidic chip, which is compatible with a time-lapse microscopy imaging system, and includes a water bath incubator, a temperature control system, and a microfluidic chip and a temperature sensing probe arranged in the water bath incubator. The water bath incubator is arranged above a microscope stage or an electric displacement stage of the time-lapse microscopy imaging system, a hollow window is arranged at the bottom of the water bath incubator, and a transparent window is arranged at the top of the water bath incubator, and the centers of the transparent window and the hollow window are coaxial with the optical axis of the objective lens of the time-lapse microscopy imaging system; the microfluidic chip is arranged on the hollow window, and its glass sheet base is closely matched with the hollow window; the thermostat of the temperature control system is connected to the temperature sensing probe to monitor the ambient temperature in the water bath incubator; the thermostat is used to heat the water in the water reservoir to reach the target temperature and maintain a constant temperature, and the constant temperature water in the water reservoir enters the water bath incubator through a water inlet pipe and flows out of the water reservoir through a water outlet pipe, thereby realizing the circulation of constant temperature water in the water bath incubator to stabilize the temperature control. The cell culture fluid enters the microfluidic chip through the chip pipeline inlet and flows out through the chip pipeline outlet to provide the nutritional environment required for continuous cell culture.
图1是本申请提供的一种基于微流控芯片细胞培养的快速控温装置的一实施例的结构示意图。FIG1 is a schematic structural diagram of an embodiment of a rapid temperature control device for cell culture based on a microfluidic chip provided in the present application.
图2是图1提供的实施例中的水浴培养箱的分解结构示意图。FIG. 2 is a schematic diagram of the exploded structure of the water bath incubator in the embodiment provided in FIG. 1 .
附图标记说明如下：The following are the descriptions of the reference numerals:
10、水浴培养箱；11、底座；111、镂空视窗；112、橡皮圈槽；113、螺纹孔；12、温感探头；13、芯片压片；131、弹簧针；14、顶盖；141、透明视窗；142、透明板；143、隔水橡皮圈；144、进水口；145、出水口；146、芯片管路出口；147、芯片管路进口；20、控温***；21、蓄水池；22、恒温器；23、蠕动泵；24、第一管路；25、第二管路；26、进水管道；27、出水管道；A、显微镜载物台；B、物镜；C、培养液管；D、废液管。10. Water bath incubator; 11. Base; 111. Hollow window; 112. Rubber ring groove; 113. Threaded hole; 12. Temperature sensor; 13. Chip pressing sheet; 131. Spring pin; 14. Top cover; 141. Transparent window; 142. Transparent plate; 143. Water-proof rubber ring; 144. Water inlet; 145. Water outlet; 146. Chip pipeline outlet; 147. Chip pipeline inlet; 20. Temperature control system; 21. Water reservoir; 22. Thermostat; 23. Peristaltic pump; 24. First pipeline; 25. Second pipeline; 26. Water inlet pipeline; 27. Water outlet pipeline; A. Microscope stage; B. Objective lens; C. Culture medium tube; D. Waste liquid tube.
体现本发明特征与优点的典型实施方式将在以下的说明中详细叙述。应理解的是本发明能够在不同的实施方式上具有各种的变化，其皆不脱离本发明的范围，且其中的说明及图示在本质上是当作说明之用，而非用以限制本发明。Typical embodiments that embody the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various changes in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations therein are essentially used for illustration purposes rather than for limiting the present invention.
在本申请的描述中，需要理解的是，术语“中心”、“纵向”、“横向”、“长度”、“宽度”、“厚度”、“上”、“下”、“前”、“后”、“左”、“右”、“竖直”、“水平”、“顶”、“底”、“内”、“外”、“顺时针”、“逆时针”等指示的方位或位置关系为基于附图所示的方位或位置关系，仅是为了便于描述本申请和简化描述，而不是指示或暗示所指的装置或元件必须具有特定的方位、以特定的方位构造和操作，因此不能理解为对本申请的限制。此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个所述特征。在本申请的描述中，“多个”的含义是两个或两个以上，除非另有明确具体的限定。In the description of the present application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.
为了进一步说明本发明的原理和结构，现结合附图对本发明的优选实施例进行详细说明。In order to further illustrate the principle and structure of the present invention, preferred embodiments of the present invention are now described in detail with reference to the accompanying drawings.
请参阅图1和图2，本申请提供一种基于微流控芯片细胞培养的快速控温装置，该快速控温装置与延时显微成像***兼容，其包括水浴培养箱10、控温***20以及设置在水浴培养箱内的微流控芯片和温感探头12，温感探头12置于微流控芯片附近或微流控芯片上，用于实时监测温度。Please refer to Figures 1 and 2. The present application provides a rapid temperature control device for cell culture based on a microfluidic chip. The rapid temperature control device is compatible with a time-lapse microscopy imaging system. It includes a water bath incubator 10, a temperature control system 20, and a microfluidic chip and a temperature sensing probe 12 arranged in the water bath incubator. The temperature sensing probe 12 is placed near or on the microfluidic chip for real-time temperature monitoring.
请参阅图2，水浴培养箱10设置在延时显微成像***的显微镜载物台A或电动位移台上方，其包括箱体以及设置在箱体上的进水口144和出水口145，箱体的底部设置有镂空视窗111，箱体的顶部设置有透明视窗141，透明视窗141和镂空视窗111的中心均与延时显微成像***的物镜B光轴同轴，通过在箱体的顶部和底部均开视窗的设计实现光照需求。Please refer to Figure 2. The water bath incubator 10 is arranged above the microscope stage A or the electric translation stage of the time-lapse microscopy imaging system. It includes a box body and a water inlet 144 and a water outlet 145 arranged on the box body. A hollow window 111 is arranged at the bottom of the box body, and a transparent window 141 is arranged at the top of the box body. The centers of the transparent window 141 and the hollow window 111 are coaxial with the optical axis of the objective lens B of the time-lapse microscopy imaging system. The lighting requirements are achieved by opening windows at the top and bottom of the box body.
具体地在本实施例中，箱体包括底座11和设置在底座11上的顶盖14，底座11与顶盖14形成一容置腔室。Specifically, in this embodiment, the box body includes a base 11 and a top cover 14 disposed on the base 11 , and the base 11 and the top cover 14 form a containing chamber.
底座11为凹槽结构，并与显微镜载物台A或电动位移台兼容，用于延时显微成像实验与应用。凹槽结构的底座11可以被水溶液填充，以浸没设置在水浴培养箱10内部的微流控芯片。底座11的中部设置有用于支撑微流控芯片的凹槽结构的镂空视窗111，微流控芯片的玻璃片基底与镂空视窗111紧密配合。优选地，镂空视窗111的设计满足高倍物镜对微流控芯片的最大面积成像。可以理解地是，底座11的尺寸和形状可根据应用平台调整设计，实现与不同种类和型号的载物台或电动位移台相匹配。The base 11 is a groove structure and is compatible with the microscope stage A or the electric translation stage for time-lapse microscopic imaging experiments and applications. The base 11 with a groove structure can be filled with an aqueous solution to immerse the microfluidic chip disposed inside the water bath incubator 10. A hollow window 111 with a groove structure for supporting the microfluidic chip is provided in the middle of the base 11, and the glass sheet substrate of the microfluidic chip is tightly matched with the hollow window 111. Preferably, the design of the hollow window 111 satisfies the maximum area imaging of the microfluidic chip by a high-power objective lens. It can be understood that the size and shape of the base 11 can be adjusted and designed according to the application platform to match with different types and models of stages or electric translation stages.
进一步地，在本实施例中还包括设置在水浴培养箱10内与底座11可拆卸连接的芯片压片13。芯片压片13通过螺丝与底座11连接，芯片压片13上设置有用于压紧微流控芯片玻璃基底的弹簧针131，微流控芯片的玻璃片基底与镂空视窗111适配，芯片压片13上的弹簧针131均匀分布在镂空视窗111的正上方，也即微流控芯片的玻璃片基底的正上方，通过弹簧针131的挤压，微流控芯片的玻璃基底与底座11紧密的固定在一起。可以理解地是，由于芯片压片131和底座11可拆卸连接，可以方便地更换不同种类和形状的微流控芯片。在满足压紧微流控芯片的玻璃片基地的前提下，另一种可替代弹簧针131的结构为橡皮圈结构。Furthermore, in the present embodiment, a chip pressing plate 13 is also included, which is arranged in the water bath incubator 10 and is detachably connected to the base 11. The chip pressing plate 13 is connected to the base 11 by screws, and a spring pin 131 for pressing the glass substrate of the microfluidic chip is provided on the chip pressing plate 13. The glass substrate of the microfluidic chip is adapted to the hollow window 111. The spring pins 131 on the chip pressing plate 13 are evenly distributed directly above the hollow window 111, that is, directly above the glass substrate of the microfluidic chip. Through the squeezing of the spring pins 131, the glass substrate of the microfluidic chip is tightly fixed to the base 11. It can be understood that since the chip pressing plate 131 and the base 11 are detachably connected, microfluidic chips of different types and shapes can be easily replaced. On the premise of satisfying the glass substrate of the microfluidic chip, another structure that can replace the spring pin 131 is a rubber ring structure.
顶盖14为与底座11对应的凸槽结构，其中部设置有透明视窗141，透明视窗141内固定固定有透明板142。顶盖14的透明视窗141和底座11的镂空视窗111的中心均与延时显微成像***的物镜B的光轴同轴。在将该快速控温装置集成到延时显微成像***时，透明视窗141能够使落射光投射照射到水浴培养箱10内的微流控芯片上，透明视窗141足够大，避免了对落射光的遮挡；由于高倍成像时需使用浸油物镜，物镜B与样品距离仅几百微米，中间有物镜镜油填充，物镜B需要足够的活动空间来保证大范围的采样，镂空视窗111的设计最大限度保留微流控芯片的成像空间。The top cover 14 is a convex groove structure corresponding to the base 11, and a transparent window 141 is provided in the middle thereof, and a transparent plate 142 is fixed in the transparent window 141. The centers of the transparent window 141 of the top cover 14 and the hollow window 111 of the base 11 are coaxial with the optical axis of the objective lens B of the time-lapse microscopy imaging system. When the rapid temperature control device is integrated into the time-lapse microscopy imaging system, the transparent window 141 can project incident light onto the microfluidic chip in the water bath incubator 10. The transparent window 141 is large enough to avoid blocking the incident light. Since an oil immersion objective lens is required for high-magnification imaging, the distance between the objective lens B and the sample is only a few hundred microns, and the objective lens oil is filled in the middle. The objective lens B needs sufficient space to ensure a large range of sampling. The design of the hollow window 111 retains the imaging space of the microfluidic chip to the maximum extent.
顶盖14的一侧开设有进水口144、出水口145、芯片管路进口147和芯片管路出口146。连接培养液管C的第一管路24从顶盖14的芯片管路进口147进入水浴培养箱10内部后于微流控芯片连接，微流控芯与第二管路25连接，第二管路25从顶盖14的芯片管路出口146穿出水浴培养箱10并连接废液管D，如此可提供持续培养细胞所需的营养环境。可以理解地是，进水口144、出水口145、芯片管路进口146和芯片管路出口147还可以根据实际需要设置在顶盖14的其他位置。A water inlet 144, a water outlet 145, a chip pipeline inlet 147 and a chip pipeline outlet 146 are provided on one side of the top cover 14. The first pipeline 24 connected to the culture liquid tube C enters the water bath incubator 10 from the chip pipeline inlet 147 of the top cover 14 and is connected to the microfluidic chip. The microfluidic core is connected to the second pipeline 25. The second pipeline 25 passes through the water bath incubator 10 from the chip pipeline outlet 146 of the top cover 14 and is connected to the waste liquid pipe D, so as to provide the nutritional environment required for continuous cell culture. It can be understood that the water inlet 144, the water outlet 145, the chip pipeline inlet 146 and the chip pipeline outlet 147 can also be set at other positions of the top cover 14 according to actual needs.
优选地，可根据需求将第一管路24和第二管路25置于水浴培养箱10内的非视窗空间位置，芯片管路进口147和芯片管路出口146的外侧用管道刃环和分体接头密封固定在顶盖14上。Preferably, the first pipeline 24 and the second pipeline 25 can be placed in a non-window space position in the water bath incubator 10 as required, and the outer sides of the chip pipeline inlet 147 and the chip pipeline outlet 146 are sealed and fixed to the top cover 14 with pipeline blade rings and split joints.
顶盖14与底座11的四周设置的相互对应的螺纹孔113，可以使得二者通过螺丝连接。进一步地，底座11的上表面周侧开设有橡皮圈槽112，顶盖14的下表面设置有与橡皮圈槽112配合的隔水橡皮圈143。通过底座11和顶盖14的螺接实现挤压隔水橡皮圈143以对二者连接处进行隔水，底座11和顶盖14组合成具有蓄水功能的水浴培养箱10，对浸没其中的微流控芯片进行快速控温。The top cover 14 and the base 11 are provided with corresponding threaded holes 113 around them, so that the two can be connected by screws. Furthermore, a rubber ring groove 112 is provided on the upper surface of the base 11, and a waterproof rubber ring 143 that cooperates with the rubber ring groove 112 is provided on the lower surface of the top cover 14. The waterproof rubber ring 143 is squeezed by screwing the base 11 and the top cover 14 to waterproof the connection between the two. The base 11 and the top cover 14 are combined into a water bath incubator 10 with a water storage function, which can quickly control the temperature of the microfluidic chip immersed therein.
请再参阅图1，控温***20，其包括蓄水池21、恒温器22、蠕动泵23、连接进水口144的进水管道26和连接出水口145的出水管道27，进水管道26和出水管道27共同放置于蓄水池21中，进水管道26与蠕动泵23连接以便于蓄水池21内的恒温水传输至水浴培养箱10以使得水充满水浴培养箱10的内部，恒温器22用于使蓄水池21内的水达到目的温度并保持恒温，蓄水池21内的恒温水通过进水管道26进入水浴培养箱10并通过出水管道27再流出蓄水池21，进而实现以恒温水在水浴培养箱10内循环流动以稳定控温；恒温器22与温感探头12连接，以用于监测水浴培养箱10内的环境温度，若需要快速改变微流控芯片的环境温度，只通过调整设置恒温器22的目标温度，将蓄水池21中的水快速置换为目标温度的水进行循环即可，实现将微流控芯片的监测温度达到目标温度。微流控芯片在水浴环境中的温度扰动极小，温度切换准确、稳定，可以实现快速变温，在3min内完成温度切换并维持稳定，温度范围可达50℃。Please refer to FIG. 1 again. The temperature control system 20 includes a water reservoir 21, a thermostat 22, a peristaltic pump 23, a water inlet pipe 26 connected to the water inlet 144, and a water outlet pipe 27 connected to the water outlet 145. The water inlet pipe 26 and the water outlet pipe 27 are placed together in the water reservoir 21. The water inlet pipe 26 is connected to the peristaltic pump 23 so that the constant temperature water in the water reservoir 21 is transferred to the water bath incubator 10 so that the water fills the inside of the water bath incubator 10. The thermostat 22 is used to make the water in the water reservoir 21 reach the target temperature and maintain the constant temperature. The constant temperature water enters the water bath incubator 10 through the water inlet pipe 26 and flows out of the water reservoir 21 through the water outlet pipe 27, thereby realizing the circulation of the constant temperature water in the water bath incubator 10 to stabilize the temperature control; the thermostat 22 is connected to the temperature sensing probe 12 to monitor the ambient temperature in the water bath incubator 10. If the ambient temperature of the microfluidic chip needs to be changed quickly, the target temperature of the thermostat 22 can be adjusted and set to quickly replace the water in the water reservoir 21 with water of the target temperature for circulation, so that the monitoring temperature of the microfluidic chip reaches the target temperature. The temperature disturbance of the microfluidic chip in the water bath environment is extremely small, the temperature switching is accurate and stable, and rapid temperature change can be achieved. The temperature switching is completed within 3 minutes and maintained stable, and the temperature range can reach 50°C.
可以理解地是，水浴培养箱10的芯片管路进口147、芯片管路出口146、进水口144和出水口145的数量和位置，在满足快速变温的前提下可任意设计更改。It is understandable that the number and position of the chip pipeline inlet 147, the chip pipeline outlet 146, the water inlet 144 and the water outlet 145 of the water bath incubator 10 can be arbitrarily designed and changed under the premise of satisfying rapid temperature change.
本申请提供一种基于微流控芯片细胞培养的快速控温装置，快速控温装置与延时显微成像***兼容，其包括水浴培养箱10、控温***20以及设置在水浴培养箱10内的微流控芯片和温感探头12。其中，水浴培养箱10设置在延时显微成像***的显微镜载物台A或电动位移台上方，水浴培养箱10的箱体底部设置有镂空视窗111，箱体顶部设置有透明视窗141，透明视窗141和镂空视窗111的中心均与延时显微成像***的物镜B光轴同轴；微流控芯片设置在镂空视窗111上，其玻璃片基底与镂空视窗111紧密配合；控温***20的恒温器22与温感探头12连接，以用于监测水浴培养箱10内的环境温度；恒温器22用于使蓄水池21内的水达到目的温度并保持恒温，蓄水池21内的恒温水通过进水管道26进入水浴培养箱10并通过出水管道27再流出蓄水池21，进而实现恒温水在水浴培养箱10内循环流动以稳定控温。该快速控温装置已经实现，并已经经过长期实验的验证证明其可行性和有效性，使用该快速控温装置对大肠杆菌细胞在37℃条件下培养6小时后3min内切换到10℃条件下培养并得到延时显微成像实验结果，所获得的实验数据均可使用图像分析软件进行分析和统计。The present application provides a rapid temperature control device for cell culture based on a microfluidic chip. The rapid temperature control device is compatible with a time-lapse microscopy imaging system and includes a water bath incubator 10, a temperature control system 20, and a microfluidic chip and a temperature sensing probe 12 arranged in the water bath incubator 10. Among them, the water bath incubator 10 is arranged above the microscope stage A or the electric translation stage of the time-lapse microscopy imaging system, a hollow window 111 is arranged at the bottom of the water bath incubator 10, and a transparent window 141 is arranged at the top of the water bath incubator 10, and the centers of the transparent window 141 and the hollow window 111 are coaxial with the optical axis of the objective lens B of the time-lapse microscopy imaging system; the microfluidic chip is arranged on the hollow window 111, and its glass sheet base is tightly matched with the hollow window 111; the thermostat 22 of the temperature control system 20 is connected to the temperature sensing probe 12 to monitor the ambient temperature in the water bath incubator 10; the thermostat 22 is used to make the water in the water reservoir 21 reach the target temperature and maintain a constant temperature, the constant temperature water in the water reservoir 21 enters the water bath incubator 10 through the water inlet pipe 26 and flows out of the water reservoir 21 through the water outlet pipe 27, thereby realizing the circulation of constant temperature water in the water bath incubator 10 to stabilize the temperature control. This rapid temperature control device has been realized and its feasibility and effectiveness have been verified by long-term experiments. The rapid temperature control device was used to culture Escherichia coli cells at 37°C for 6 hours and then switched to 10°C within 3 minutes to obtain time-lapse microscopy experimental results. The experimental data obtained can be analyzed and counted using image analysis software.
虽然已参照几个典型实施方式描述了本发明，但应当理解，所用的术语是说明和示例性、而非限制性的术语。由于本发明能够以多种形式具体实施而不脱离发明的精神或实质，所以应当理解，上述实施方式不限于任何前述的细节，而应在随附权利要求所限定的精神和范围内广泛地解释，因此落入权利要求或其等效范围内的全部变化和改型都应为随附权利要求所涵盖。Although the present invention has been described with reference to several typical embodiments, it should be understood that the terms used are illustrative and exemplary, rather than restrictive. Since the present invention can be embodied in a variety of forms without departing from the spirit or essence of the invention, it should be understood that the above embodiments are not limited to any of the foregoing details, but should be interpreted broadly within the spirit and scope defined by the appended claims, so all changes and modifications falling within the scope of the claims or their equivalents should be covered by the appended claims.
Claims (10)
- 一种基于微流控芯片细胞培养的快速控温装置，所述快速控温装置与延时显微成像***兼容，其特征在于，包括：A rapid temperature control device for cell culture based on a microfluidic chip, wherein the rapid temperature control device is compatible with a time-lapse microscopic imaging system, and is characterized in that it comprises:水浴培养箱，其设置在延时显微成像***的显微镜载物台或电动位移台上方；所述水浴培养箱包括箱体以及设置在所述箱体上的进水口和出水口，所述箱体的底部设置有镂空视窗，所述箱体的顶部设置有透明视窗，所述镂空视窗和所述透明视窗的中心均与延时显微成像***的物镜光轴同轴；A water bath incubator, which is arranged above a microscope stage or an electric translation stage of a time-lapse microscopy imaging system; the water bath incubator comprises a box body and a water inlet and a water outlet arranged on the box body, a hollow window is arranged at the bottom of the box body, and a transparent window is arranged at the top of the box body, and the centers of the hollow window and the transparent window are both coaxial with the objective lens optical axis of the time-lapse microscopy imaging system;微流控芯片，其设置在所述水浴培养箱的所述镂空视窗上，所述微流控芯片的玻璃片基底与所述镂空视窗紧密配合；A microfluidic chip, which is arranged on the hollow window of the water bath incubator, and the glass substrate of the microfluidic chip is closely matched with the hollow window;温感探头，其设置所述水浴培养箱内并靠近所述微流控芯片或设置在所述微流控芯片上；A temperature sensing probe is arranged in the water bath incubator and close to the microfluidic chip or arranged on the microfluidic chip;控温***，其包括蓄水池、恒温器、连接所述进水口的进水管道和连接所述出水口的出水管道；A temperature control system, comprising a water reservoir, a thermostat, a water inlet pipe connected to the water inlet, and a water outlet pipe connected to the water outlet;其中，所述恒温器与所述温感探头连接，以用于监测所述水浴培养箱内的环境温度；所述恒温器用于使所述蓄水池内的水达到目的温度并保持恒温，所述蓄水池内的恒温水通过所述进水管道进入所述水浴培养箱并通过所述出水管道再流出所述蓄水池，进而实现以恒温水在所述水浴培养箱内循环流动以稳定控温。Among them, the thermostat is connected to the temperature sensing probe to monitor the ambient temperature in the water bath incubator; the thermostat is used to make the water in the water reservoir reach the target temperature and maintain a constant temperature, and the constant temperature water in the water reservoir enters the water bath incubator through the water inlet pipe and flows out of the water reservoir through the water outlet pipe, thereby realizing the circulation of constant temperature water in the water bath incubator to stabilize the temperature control.
- 根据权利要求1所述的快速控温装置，其特征在于，所述箱体包括底座和设置在所述底座上的顶盖，所述镂空视窗设置在所述底座上，所述透明视窗设置在所述顶盖上。The rapid temperature control device according to claim 1 is characterized in that the box body includes a base and a top cover arranged on the base, the hollow window is arranged on the base, and the transparent window is arranged on the top cover.
- 根据权利要求2所述的快速控温装置，其特征在于，所述镂空视窗的设计满足高倍物镜对所述微流控芯片的最大面积成像。The rapid temperature control device according to claim 2 is characterized in that the design of the hollow window satisfies the maximum area imaging of the microfluidic chip by a high-power objective lens.
- 根据权利要求2所述的快速控温装置，其特征在于，还包括设置在所述水浴培养箱内与所述底座可拆卸连接的芯片压片，所述芯片压片包括用于压紧所述微流控芯片的弹簧针或橡皮圈。The rapid temperature control device according to claim 2 is characterized in that it also includes a chip pressing sheet disposed in the water bath incubator and detachably connected to the base, the chip pressing sheet including a spring pin or a rubber ring for pressing the microfluidic chip.
- 根据权利要求4所述的快速控温装置，其特征在于，所述芯片压片通过螺丝与所述底座连接。The rapid temperature control device according to claim 4 is characterized in that the chip pressing sheet is connected to the base by screws.
- 根据权利要求2所述的快速控温装置，其特征在于，所述芯片压片的周侧设置有隔水橡皮圈，所述底座的上表面周侧开设有橡皮圈槽，所述顶盖的下表面设置有与所述橡皮圈槽配合的隔水橡皮圈。The rapid temperature control device according to claim 2 is characterized in that a waterproof rubber ring is provided on the peripheral side of the chip pressing sheet, a rubber ring groove is opened on the peripheral side of the upper surface of the base, and a waterproof rubber ring matching the rubber ring groove is provided on the lower surface of the top cover.
- 根据权利要求6所述的快速控温装置，其特征在于，所述底座和所述顶盖通过螺丝连接。The rapid temperature control device according to claim 6 is characterized in that the base and the top cover are connected by screws.
- 根据权利要求1所述的快速控温装置，其特征在于，所述箱体上还设置有芯片管路进口和芯片管路出口。The rapid temperature control device according to claim 1 is characterized in that a chip pipeline inlet and a chip pipeline outlet are also provided on the box body.
- 根据权利要求8所述的快速控温装置，其特征在于，所述芯片管路进口和所述芯片管路出口设置在所述顶盖上。The rapid temperature control device according to claim 8 is characterized in that the chip pipeline inlet and the chip pipeline outlet are arranged on the top cover.
- 根据权利要求1所述的快速控温装置，其特征在于，所述控温***还包括蠕动泵，所述进水口管道连接所述蠕动泵以便于所述蓄水池内的恒温水传输至所述水浴培养箱的内部。The rapid temperature control device according to claim 1 is characterized in that the temperature control system also includes a peristaltic pump, and the water inlet pipe is connected to the peristaltic pump to facilitate the transmission of the constant temperature water in the water reservoir to the interior of the water bath incubator.
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2022
- 2022-11-24 CN CN202211479951.2A patent/CN115747044A/en active Pending
-
2023
- 2023-11-23 WO PCT/CN2023/133765 patent/WO2024109896A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CN115747044A (en) | 2023-03-07 |
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