技术领域technical field
本发明涉及细胞富集仪器，具体涉及一种包括微流控芯片的循环肿瘤细胞检测仪器。The invention relates to a cell enrichment instrument, in particular to a circulating tumor cell detection instrument including a microfluidic chip.
背景技术Background technique
现代医学研究表明由原始病灶渗入外周血液循环的肿瘤细胞即循环肿瘤细胞，是造成癌症转移的“种子”。因此，循环肿瘤细胞具有重要的临床诊断和科学研究价值，主要体现于：(1)检测血液中循环肿瘤细胞比造影等传统诊断技术具有更高的精度，可为癌症的早期诊断和预后评估提供新方法手段，被称为液体活检技术。(2)带有与原始肿瘤相同的遗传信息，可为癌症转移机制的研究和抗癌药物的筛选提供重要标本。但由于血液中循环肿瘤细胞的数量异常稀少，这对有效分离和检测稀有循环肿瘤细胞提出了严峻的挑战。Modern medical research has shown that tumor cells that infiltrate from the original lesion into the peripheral blood circulation, that is, circulating tumor cells, are the "seeds" that cause cancer metastasis. Therefore, circulating tumor cells have important clinical diagnosis and scientific research value, which are mainly reflected in: (1) The detection of circulating tumor cells in blood has higher accuracy than traditional diagnostic techniques such as imaging, which can provide a basis for early diagnosis and prognosis evaluation of cancer; The new approach is called liquid biopsy technology. (2) With the same genetic information as the original tumor, it can provide important samples for the study of cancer metastasis mechanism and the screening of anticancer drugs. However, due to the extremely rare number of circulating tumor cells in blood, it poses a serious challenge to effectively isolate and detect rare circulating tumor cells.
目前市场上的循环肿瘤细胞检测仪器CellSearch系统，该仪器通过免疫捕获和荧光枚举来统计7.5毫升癌症病人血液中稀有循环肿瘤细胞的个数，以此来预测患者的生存期。除了该设备之外，越来越多的循环肿瘤细胞检测原型样机被研究报告，如Cynvenio的磁标记检测系统、基于白细胞反向标记的CTC-iChip平台等。但上述仪器主要借助工序复杂的免疫标记方法，存在(1)需要借助昂贵的免疫生化试剂，大幅提高了检测成本高，标记工序同时限制了单位时间的处理通量；(2)特异性强，使得仪器只适用于特定几种表达特异性标志物如上皮粘附因子的癌症病种；(3)经标记后的细胞将丧失活性，无法用于后续定量生物学研究。Currently on the market, the CellSearch system, an instrument for detecting circulating tumor cells, uses immunocapture and fluorescence enumeration to count the number of rare circulating tumor cells in 7.5 milliliters of cancer patient blood, so as to predict the patient's survival period. In addition to this device, more and more circulating tumor cell detection prototypes have been researched and reported, such as Cynvenio's magnetic label detection system, CTC-iChip platform based on white blood cell reverse labeling, etc. However, the above-mentioned instruments mainly rely on the immunolabeling method with complicated procedures, and there are (1) the need to use expensive immunobiochemical reagents, which greatly increases the cost of detection, and the labeling process also limits the processing throughput per unit time; (2) strong specificity, This makes the instrument only suitable for certain types of cancers that express specific markers such as epithelial adhesion factor; (3) The marked cells will lose their activity and cannot be used for subsequent quantitative biological research.
发明内容Contents of the invention
发明目的：本发明提供一种包括微流控芯片的循环肿瘤细胞检测仪器，解决了现有仪器需要标记细胞，处理通量小，检测成本高，适用病种少的问题。Purpose of the invention: The present invention provides a circulating tumor cell detection instrument including a microfluidic chip, which solves the problems that existing instruments need to label cells, have low processing throughput, high detection cost, and few applicable diseases.
技术方案：本发明所述的一种微流控芯片，包括从上而下堆叠的惯性分选模块和电阻抗检测模块，所述惯性分选模块上设置有若干个微流道分选结构，所述电阻抗检测模块上设置有串联的正弦预聚焦惯性流道和微电极传感流道，所述微电极传感流道上连接有检测微电极，所述惯性分选模块和电阻抗检测模块周边对应的位置上均设置有定位圆孔。Technical solution: A microfluidic chip according to the present invention includes an inertial sorting module and an electrical impedance detection module stacked from top to bottom, the inertial sorting module is provided with several microchannel sorting structures, The electrical impedance detection module is provided with a sinusoidal pre-focusing inertial channel and a microelectrode sensing channel connected in series, the microelectrode sensing channel is connected with a detection microelectrode, the inertial sorting module and the electrical impedance detection module Positioning circular holes are arranged at corresponding positions on the periphery.
为了对微流控芯片的样品流和其他辅助流体进行流量稳定和调节，所述惯性分选模块上方堆叠有流量调节稳定模块，其上设置多个被动流量调节阀和分流流道，使样品流稳定后通过分流流道均分输出。In order to stabilize and regulate the flow of the sample flow and other auxiliary fluids of the microfluidic chip, a flow regulation and stabilization module is stacked above the inertial sorting module, and a plurality of passive flow regulation valves and split flow channels are arranged on it to make the sample flow After stabilization, the output is evenly distributed through the split flow channel.
为了提高单位时间的处理通量，所述若干微流道分选结构并联或串联，微流道分选结构为螺旋流道结构。In order to increase the processing flux per unit time, the several micro-channel sorting structures are connected in parallel or in series, and the micro-channel sorting structure is a spiral channel structure.
为了差分减除无细胞的空白背景液体产生的信号，检测微电极为一对或两对，且每对均对称分布在微电极传感流道左右两侧或上下两侧。In order to differentially subtract the signal produced by the cell-free blank background liquid, one or two pairs of detection microelectrodes are used, and each pair is symmetrically distributed on the left and right sides or the upper and lower sides of the microelectrode sensing flow channel.
为了方便各模块对准安装，所述流量调节稳定模块、惯性分选模块和电阻抗检测模块周边对应的位置上均设置有定位圆孔，定位圆孔数量为四个。In order to facilitate the alignment and installation of each module, positioning round holes are provided at the corresponding positions around the flow regulation stabilization module, inertial sorting module and electrical impedance detection module, and the number of positioning round holes is four.
本发明所述的包括微流控芯片的循环肿瘤细胞检测仪器，还包括进样系统、液体收集装置、信号采集分析系统、核心控制器和显示控制界面，核心控制器与进样系统，信号采集分析系统和显示控制界面均电连接以控制运作，所述进样系统与所述微流控芯片样品入口连接根据核心控制器指令驱动样品导入芯片，所述液体收集装置与所述微流控芯片样品出口连接收集经富集得到的目标液和废液，所述信号采集分析系统与检测微电极连接根据核心控制器指令采集分析检测电极所在区域阻抗变化诱发的响应电信号，所述显示控制界面根据核心控制器指令显示数据结果。The circulating tumor cell detection instrument comprising a microfluidic chip according to the present invention also includes a sampling system, a liquid collection device, a signal acquisition and analysis system, a core controller and a display control interface, a core controller and a sampling system, and a signal acquisition system. The analysis system and the display control interface are electrically connected to control the operation. The sample introduction system is connected to the sample inlet of the microfluidic chip to drive the sample into the chip according to the instructions of the core controller. The liquid collection device is connected to the microfluidic chip. The sample outlet is connected to collect the target liquid and waste liquid obtained through enrichment. The signal collection and analysis system is connected to the detection microelectrode to collect and analyze the response electrical signal induced by the impedance change in the area where the detection electrode is located according to the instructions of the core controller. The display control interface Display the data results according to the instructions of the core controller.
为了通过气压推动样品和缓冲液导入微流控芯片，所述进样系统包括试样管、缓冲液瓶和隔膜泵，所述试验管和缓冲液瓶通过气密管路与隔膜泵连接，并通过液路硅胶管与微流控芯片样品入口连接，隔膜泵产生驱动气源驱使所述试样管和缓冲液瓶内的液体导入微流控芯片。In order to push the sample and the buffer solution into the microfluidic chip through air pressure, the sample introduction system includes a sample tube, a buffer solution bottle and a diaphragm pump, and the test tube and the buffer solution bottle are connected with the diaphragm pump through an airtight pipeline, and The liquid silicone tube is connected to the sample inlet of the microfluidic chip, and the diaphragm pump generates a driving gas source to drive the liquid in the sample tube and the buffer bottle into the microfluidic chip.
为了根据气压控制气路的通断，所述进样系统还包括电磁阀和压力传感器，电磁阀设置在气密管路上控制气路的通断，电磁阀和隔膜泵与压力传感器均电连接并根据压力传感器反馈的气压信息控制运作。In order to control the on-off of the gas path according to the air pressure, the sampling system also includes a solenoid valve and a pressure sensor. The solenoid valve is arranged on the airtight pipeline to control the on-off of the gas path. The operation is controlled according to the air pressure information fed back by the pressure sensor.
为了控制液路的通断，所述液路硅胶管上设置有电磁铁截止机构控制液路的通断。In order to control the on-off of the liquid path, the silicone tube of the liquid path is provided with an electromagnet cut-off mechanism to control the on-off of the liquid path.
为了方便拆卸诶更换微流控芯片，所述微流控芯片通过插槽以可拆卸的方式安装在循环肿瘤细胞检测仪器上。In order to facilitate disassembly and replacement of the microfluidic chip, the microfluidic chip is detachably installed on the circulating tumor cell detection instrument through a slot.
有益效果：本发明不需要标记细胞，处理通量高，自动分离获得高纯度的活体循环肿瘤细胞，并可检测获得细胞的多个生物物理参数，测试过程简单，效率高，适用范围广。Beneficial effects: the present invention does not require labeled cells, has high processing throughput, can automatically separate and obtain high-purity circulating tumor cells in vivo, and can detect and obtain multiple biophysical parameters of the cells. The test process is simple, efficient, and widely applicable.
附图说明Description of drawings
图1是循环肿瘤细胞检测仪器整体结构示意图；Figure 1 is a schematic diagram of the overall structure of a circulating tumor cell detection instrument;
图2是仪器去除外壳和隔板后内部结构示意图；Figure 2 is a schematic diagram of the internal structure of the instrument after removing the shell and partition;
图3是微流控芯片的爆炸视图；Figure 3 is an exploded view of the microfluidic chip;
图4是流量调节稳定模块的详细平面结构图；Fig. 4 is a detailed plane structure diagram of the flow regulating and stabilizing module;
图5是惯性分选模块的平面结构示意图；Fig. 5 is the plane structure schematic diagram of inertial sorting module;
图6是电阻抗检测模块的平面结构示意图；Fig. 6 is a schematic plan view of the electrical impedance detection module;
图7是气路控制单元的结构示意图；Fig. 7 is a schematic structural view of the gas path control unit;
图8是液路控制单元的结构示意图；Fig. 8 is a schematic structural diagram of a liquid circuit control unit;
图9是信号采集分析系统的运行流程示意图；Fig. 9 is a schematic diagram of the operation flow of the signal acquisition and analysis system;
图10是气压及鞘液流量和样品流量的输出特性图；Fig. 10 is an output characteristic diagram of air pressure, sheath fluid flow rate and sample flow rate;
图11是掺杂癌细胞的初始血液样品、经仪器1次富集和2富集收集样品的采样显微镜照片；Fig. 11 is a sampling micrograph of the initial blood sample doped with cancer cells, the first enrichment and the second enrichment by the instrument;
图12是多次重复实验后获得的统计学血细胞去除率及癌细胞回收率数据；Figure 12 is the statistical blood cell removal rate and cancer cell recovery rate data obtained after repeated experiments;
图13是预聚焦正弦流道中不同尺寸粒子的聚焦效果统计学分布图；Figure 13 is a statistical distribution diagram of the focusing effect of particles of different sizes in the pre-focusing sinusoidal flow channel;
图14是不同尺寸粒子在直流模式下检测获得的电阻抗谱图；Figure 14 is the electrical impedance spectrum obtained by detecting particles of different sizes in DC mode;
图15是掺杂癌细胞血液经过检测区域后获得的电阻抗谱及分析统计散点图。Fig. 15 is an electrical impedance spectrum obtained after blood doped with cancer cells passes through the detection area and a scatter diagram of analysis statistics.
具体实施方式Detailed ways
下面结合附图对本发明作进一步说明。The present invention will be further described below in conjunction with accompanying drawing.
如图1-2所示，循环肿瘤细胞检测仪器，包括微流控芯片1，进样系统2、液体收集装置、信号采集分析系统3、核心控制器4和显示控制界面5。核心控制器4与进样系统2，信号采集分析系统3和显示控制界面5均电连接以控制运作，进样系统2与微流控芯片1样品入口连接根据核心控制器4指令驱动样品导入芯片，液体收集装置与微流控芯片1样品出口连接收集经富集得到的目标液和废液，液体收集装置包括目标样品管224和废液瓶226，信号采集分析系统3与检测微电极133连接根据核心控制器4指令采集分析检测电极所在区域阻抗的变化诱发的响应电信号，显示控制界面5根据核心控制器4指令显示数据结果。As shown in Figure 1-2, the circulating tumor cell detection instrument includes a microfluidic chip 1, a sampling system 2, a liquid collection device, a signal acquisition and analysis system 3, a core controller 4 and a display control interface 5. The core controller 4 is electrically connected to the sampling system 2, the signal acquisition and analysis system 3 and the display control interface 5 to control the operation, and the sampling system 2 is connected to the sample inlet of the microfluidic chip 1 to drive the sample into the chip according to the instructions of the core controller 4 The liquid collection device is connected to the sample outlet of the microfluidic chip 1 to collect the target liquid and waste liquid obtained through enrichment, the liquid collection device includes a target sample tube 224 and a waste liquid bottle 226, and the signal collection and analysis system 3 is connected to the detection microelectrode 133 According to the instruction of the core controller 4, the response electric signal induced by the change of the impedance of the area where the detection electrode is located is collected and analyzed, and the display control interface 5 displays the data result according to the instruction of the core controller 4.
如图3所示，微流控芯片1为仪器的核心单元，从上而下堆叠的流量调节稳定模块11、惯性分选模块12和电阻抗检测模块13，微流控芯片1通过插槽227以可拆卸的方式安装在循环肿瘤细胞检测仪器上，微流控芯片1可为一体化结构，其材质可采用塑料、聚合物薄膜、橡胶、硅基半导体材料中的一种，其加工方法可为微注塑成型、激光结合热塑封、微细机加工及精密3D打印等加工手段。微流控芯片1装配于夹具14中，通过专用插槽直接插于仪器装置上，实现流体的注入和引出。微流控芯片1设置为一次性、可抛弃式使用，以避免交叉污染。样品流量调节稳定模块11为非必须模块，主要用于分别对注入微流控芯片1的样品流和其他辅助流体，辅助流体如空白鞘液或清洗液，进行流量稳定和调节。其可选结构如图4所示，模块上布置多个被动流量调节阀，具体中心区域布置一个被动流量调节阀111用于稳定注入样品流的流量，并通过分流流道113均分输出。上下布置8个相同的被动流量调节阀112用于稳定空白鞘液的流量。单体被动流量调节阀的流量输出特性可通过改变结构设计参数实现调控，流量调节稳定模块11的总体输出流量可通过改变并行的单体被动流量调节阀111、112数量获得。样品流量调节稳定模块11周边布置定位圆孔114实现与其他模块的对准装配。如图5所示，所述细胞高通量惯性分选模块12由多个微流道分选结构121并联或串联而成，以提高单位时间的处理通量，微流道分选结构121采用螺旋流道结构，亦可采用其他惯性分选流道结构，依据循环肿瘤细胞和血细胞之间的尺寸差异，利用惯性微流道结构中诱导产生的微流体惯性效应(惯性迁移和Dean二次流)使得不同尺寸细胞聚焦平衡至流道宽度方向的不同位置，最终从不同出口分离导出。该模块设计避免了采用免疫标记的方法，采用纯物理的尺寸差异实现循环肿瘤细胞和血细胞的非标记分选，可有效收集获取活体循环肿瘤细胞标本。所述细胞惯性分选模块12的处理通量可采用串联或并联的单体螺旋流道数量来控制。细胞高通量惯性分选模块12周边布置定位圆孔122实现与其他模块的对准装配。As shown in Figure 3, the microfluidic chip 1 is the core unit of the instrument, and the flow regulation and stabilization module 11, the inertial sorting module 12 and the electrical impedance detection module 13 are stacked from top to bottom, and the microfluidic chip 1 passes through the slot 227 Installed on a circulating tumor cell detection instrument in a detachable manner, the microfluidic chip 1 can be an integrated structure, and its material can be one of plastic, polymer film, rubber, and silicon-based semiconductor materials, and its processing method can be It is a processing method such as micro-injection molding, laser combined with thermoplastic sealing, micro-machining and precision 3D printing. The microfluidic chip 1 is assembled in the fixture 14, and is directly inserted into the instrument device through a special slot to realize the injection and extraction of fluid. The microfluidic chip 1 is set to be disposable and disposable, so as to avoid cross-contamination. The sample flow regulation and stabilization module 11 is a non-essential module, which is mainly used to stabilize and regulate the flow of the sample flow injected into the microfluidic chip 1 and other auxiliary fluids, such as blank sheath fluid or cleaning fluid. Its optional structure is shown in Figure 4. Multiple passive flow regulating valves are arranged on the module. Specifically, a passive flow regulating valve 111 is arranged in the central area to stabilize the flow rate of the injected sample flow, and the output is evenly divided through the split flow channel 113. Eight identical passive flow regulating valves 112 are arranged up and down to stabilize the flow of the blank sheath fluid. The flow output characteristics of the single passive flow regulating valve can be regulated by changing the structural design parameters, and the overall output flow of the flow regulating and stabilizing module 11 can be obtained by changing the number of parallel single passive flow regulating valves 111 and 112 . Positioning round holes 114 are arranged around the sample flow regulating and stabilizing module 11 to realize alignment and assembly with other modules. As shown in Figure 5, the cell high-throughput inertial sorting module 12 is formed by a plurality of microfluidic sorting structures 121 connected in parallel or in series to improve the processing throughput per unit time, and the microfluidic sorting structure 121 adopts The spiral channel structure can also adopt other inertial sorting channel structures. According to the size difference between circulating tumor cells and blood cells, the microfluidic inertia effect (inertial migration and Dean secondary flow) induced in the inertial microchannel structure can be used. ) makes the cells of different sizes focus and balance to different positions in the width direction of the flow channel, and finally separate and export from different outlets. The design of this module avoids the use of immunolabeling methods, and uses purely physical size differences to achieve non-marker sorting of circulating tumor cells and blood cells, which can effectively collect circulating tumor cell samples in vivo. The processing throughput of the cell inertial sorting module 12 can be controlled by the number of individual spiral channels connected in series or in parallel. Positioning circular holes 122 are arranged around the cell high-throughput inertial sorting module 12 to realize alignment and assembly with other modules.
如图6所示，所述电阻抗检测模块13由正弦预聚焦惯性流道131及微电极传感流道132串联而成，经分选之后的循环肿瘤细胞在正弦预聚焦惯性流道131中被重新聚焦排列成规则粒子列，并逐个通过检测区域，微电极传感流道132检测微电极133为一对或两对，检测模式为直流或交流，两对微电极133情况下，可差分减除背景(无细胞的空白背景液体)产生的信号。交流模式下低频时获取的是细胞的尺寸信息，高频时获取的是细胞的介电参数如细胞膜电容、细胞质电阻等，而细胞的个数由产生的电阻抗峰数量所获得。直流模式下获取的是细胞尺寸和数量信息。正弦预聚焦惯性流道131可以是对称或非对称形式，正弦波可为圆波或方波。微电极传感流道132中微电极133可为银/氯化银电极与高浓度电介质构成的液体电极，亦可为金属电极、铟锡氧化物薄膜电极。电阻抗检测模块13周边布置定位圆孔134实现与其他模块的对准装配。As shown in Figure 6, the electrical impedance detection module 13 is composed of a sinusoidal pre-focusing inertial flow channel 131 and a microelectrode sensing flow channel 132 in series, and the sorted circulating tumor cells are in the sinusoidal pre-focusing inertial flow channel 131 They are refocused and arranged into regular particle columns, and pass through the detection area one by one. The microelectrode sensing flow channel 132 detects one or two pairs of microelectrodes 133, and the detection mode is DC or AC. In the case of two pairs of microelectrodes 133, differential Signals due to background (empty background fluid without cells) were subtracted. In the AC mode, the size information of the cell is obtained at low frequency, and the dielectric parameters of the cell such as cell membrane capacitance and cytoplasmic resistance are obtained at high frequency, and the number of cells is obtained by the number of electrical impedance peaks generated. In the direct current mode, the cell size and quantity information is obtained. The sinusoidal pre-focusing inertial channel 131 can be in a symmetrical or asymmetrical form, and the sine wave can be a circular wave or a square wave. The microelectrode 133 in the microelectrode sensing channel 132 can be a liquid electrode composed of a silver/silver chloride electrode and a high-concentration dielectric, or a metal electrode or an indium tin oxide film electrode. Positioning round holes 134 are arranged around the electrical impedance detection module 13 to realize alignment and assembly with other modules.
如图7-8所示，进样系统包括试样管223、缓冲液瓶225、隔膜泵211、电磁阀212和压力传感器213，进样系统2由气路控制单元21和液路控制单元22控制，其中，气路控制单元由隔膜泵211、电磁阀212、压力传感器213及气密性管路214构成，隔膜泵211用于产生驱动气源；电磁阀212用于控制气路的通断；压力传感器213用于监测管路中气压，并反馈控制隔膜泵211及电磁阀212的运作，试验管223和缓冲液瓶225通过气密管路214与隔膜泵211连接，并通过液路硅胶管222与微流控芯片1样品入口连接，隔膜泵211产生驱动气源驱使试样管223和缓冲液瓶225内的液体导入微流控芯片1。电磁阀212设置在气密管路214上控制气路的通断，电磁阀212和隔膜泵211与压力传感器213均电连接并根据压力传感器213反馈的气压信息控制运作。液路控制单元22有电磁铁截止机构221和硅胶管222构成，通过气路提供的气压推动样品管224和缓冲液瓶225的液体进入硅胶管222，而液路的通断借助电磁铁加电后推动截止机构221来实现。经富集和检测后的活体循环肿瘤细胞流入目标样品管224收集，分离后的血细胞废液则进入废液瓶226收集。其中，在分选模式下，缓冲液和样品液被分别注入螺旋通道的两个入口，缓冲液起到辅助分选的作用。在清洗模式下，缓冲液起到清洗管路的作用。As shown in Figures 7-8, the sampling system includes a sample tube 223, a buffer bottle 225, a diaphragm pump 211, a solenoid valve 212, and a pressure sensor 213. The sampling system 2 consists of a gas circuit control unit 21 and a liquid circuit control unit 22. control, wherein the gas circuit control unit is composed of a diaphragm pump 211, a solenoid valve 212, a pressure sensor 213 and an airtight pipeline 214, the diaphragm pump 211 is used to generate the driving gas source; the solenoid valve 212 is used to control the on-off of the gas circuit ; The pressure sensor 213 is used to monitor the air pressure in the pipeline, and feeds back the operation of the diaphragm pump 211 and the solenoid valve 212. The test tube 223 and the buffer bottle 225 are connected with the diaphragm pump 211 through the airtight pipeline 214, and are connected to the diaphragm pump 211 through the liquid path silica gel. The tube 222 is connected to the sample inlet of the microfluidic chip 1 , and the diaphragm pump 211 generates a driving gas source to drive the liquid in the sample tube 223 and the buffer bottle 225 into the microfluidic chip 1 . The solenoid valve 212 is set on the airtight pipeline 214 to control the on-off of the air circuit. The solenoid valve 212 and the diaphragm pump 211 are electrically connected to the pressure sensor 213 and controlled according to the air pressure information fed back by the pressure sensor 213 . The liquid circuit control unit 22 is composed of an electromagnet cut-off mechanism 221 and a silicone tube 222. The air pressure provided by the air circuit pushes the liquid in the sample tube 224 and the buffer solution bottle 225 into the silicone tube 222, and the liquid circuit is switched on and off by means of an electromagnet. Push the cut-off mechanism 221 to realize. The enriched and detected circulating tumor cells in vivo flow into the target sample tube 224 for collection, and the separated blood cell waste liquid enters the waste liquid bottle 226 for collection. Wherein, in the sorting mode, the buffer solution and the sample solution are respectively injected into the two inlets of the spiral channel, and the buffer solution plays the role of auxiliary sorting. In the cleaning mode, the buffer plays the role of cleaning the pipeline.
如图9所示，信号采集分析系统3包含激励信号的发生、响应信号的获取与放大、信号的处理及分析等核心功能。与检测微电极连接，将产生的特定直流或交流激励信号经过转换后施加于检测微电极133上，当细胞经过检测微电极133时由于其空间占位效应，细胞将替换相应体积的背景溶液，从而引起两个检测微电极133间阻抗的变化。诱发的响应电信号经过转换和放大，并经降噪和提取后传输至计算机或仪器集成处理器软件系统，最终可分析获得细胞生物物理特性数据。As shown in FIG. 9 , the signal acquisition and analysis system 3 includes core functions such as generation of excitation signals, acquisition and amplification of response signals, and signal processing and analysis. It is connected with the detection microelectrode, and the generated specific DC or AC excitation signal is applied to the detection microelectrode 133 after being converted. When the cells pass through the detection microelectrode 133 due to its space occupation effect, the cells will replace the corresponding volume of background solution. As a result, the impedance between the two detection microelectrodes 133 changes. The evoked response electrical signal is converted and amplified, and then transmitted to the computer or instrument integrated processor software system after noise reduction and extraction, and finally can be analyzed to obtain cell biophysical characteristic data.
整个装置里，核心控制器4控制隔膜泵211、电磁阀212、压力传感器213、电磁铁截止机构221连接、信号采集分析系统3的运作，具备循环肿瘤细胞富集、浓缩、检测及管路清洗多种功能模式，分选模式即由血液样品中分离获得目标循环肿瘤细胞，去除大量存在的背景血细胞；浓缩模式用于将收集的目标循环肿瘤细胞样品中进一步去除空白液体，以提高循环肿瘤细胞的浓度；检测模式是在富集模式基础上进一步提取获得活体循环肿瘤细胞的多种生物物理特性数据。显示控制界面5用于实现仪器运作的控制、仪器问题的报警、功能模式切换及检测结果的输出显示。In the whole device, the core controller 4 controls the diaphragm pump 211, the solenoid valve 212, the pressure sensor 213, the connection of the solenoid cut-off mechanism 221, and the operation of the signal acquisition and analysis system 3, which has the functions of enrichment, concentration, detection and pipeline cleaning of circulating tumor cells A variety of functional modes, the sorting mode is to separate the target circulating tumor cells from the blood sample, and remove a large number of background blood cells; the concentration mode is used to further remove the blank liquid from the collected target circulating tumor cell samples to improve the circulating tumor cell The concentration; the detection mode is based on the enrichment mode to further extract and obtain various biophysical characteristics data of circulating tumor cells in vivo. The display control interface 5 is used to realize the control of the instrument operation, the alarm of the instrument problem, the switching of the function mode and the output display of the detection result.
用本发明的检测仪检测时，首先仪器开机完成自检，所有功能初始化；在插槽227中插入集成可抛弃微流控芯片1，加载含待测血液的试样管223、样品收集管224，并灌装缓冲液至缓冲液瓶225。按下仪器启动键，亦可根据需求自定义选择其中的特定单体功能模式如分选、浓缩、检测及管路清洗。仪器自动运行，分离获得高纯度的活体循环肿瘤细胞样品，并可非标记检测获得多种生物物理信息数据。仪器进行管路清洗，丢弃集成可抛弃微流控芯片1。其中，如图9所示，信号采集分析系统具体流程是首先编程生成m序列数字信号，经D/A转换为模拟信号后施加到检测微电极133上，从微电极133上获得的响应电流先后经过差分放大器、低通过滤器转换后进行采集，对采集到的数字信号施加快速M变化得到系统的脉冲响应，进而施加快速傅里叶变换即可得到细胞的宽频阻抗信息。其中，快速M变化是实现系统响应信号和m序列激励信号相关计算的一种快速方法。图10为开发仪器的系统压力及鞘液和样品液输出流量特性图。由图可知，系统气压随着隔膜泵211的工作而呈现逐渐增加，当仪器关闭时压力降至零值。样品流量和鞘液流量在气压蓄力，电磁阀212和电磁铁截止机构221放开后，呈现瞬时的波动，随之由于样品流量调节稳定模块11的存在而迅速稳定至预设值。为减少流量波动对细胞分选和检测效果的影响，可将仪器运行区段，即仪器进行细胞分选和检测的时间区段进行略微延时。When using the detection instrument of the present invention to detect, firstly, the instrument is turned on to complete the self-inspection, and all functions are initialized; the integrated disposable microfluidic chip 1 is inserted into the slot 227, and the sample tube 223 and the sample collection tube 224 containing the blood to be tested are loaded. , and filling the buffer solution to the buffer solution bottle 225. Press the start button of the instrument, and you can also customize and select specific monomer function modes such as sorting, concentration, detection and pipeline cleaning according to your needs. The instrument operates automatically, separates and obtains high-purity circulating tumor cell samples in vivo, and can obtain various biophysical information data through non-labeled detection. The pipeline of the instrument is cleaned, and the integrated disposable microfluidic chip 1 is discarded. Among them, as shown in Figure 9, the specific process of the signal acquisition and analysis system is to program and generate m-sequence digital signals at first, convert them into analog signals by D/A and then apply them to the detection microelectrodes 133, and the response currents obtained from the microelectrodes 133 are successively After being converted by a differential amplifier and a low-pass filter, it is collected, and the collected digital signal is subjected to a fast M change to obtain the impulse response of the system, and then the fast Fourier transform is applied to obtain the broadband impedance information of the cell. Among them, the fast M change is a fast method to realize the correlation calculation between the system response signal and the m-sequence excitation signal. Figure 10 is a characteristic diagram of the system pressure and output flow rate of sheath liquid and sample liquid of the developed instrument. It can be seen from the figure that the system air pressure gradually increases with the operation of the diaphragm pump 211, and the pressure drops to zero when the instrument is turned off. The sample flow rate and the sheath fluid flow rate fluctuate instantaneously after the air pressure is charged and the solenoid valve 212 and the solenoid cut-off mechanism 221 are released, and then quickly stabilize to the preset value due to the existence of the sample flow rate adjustment and stabilization module 11 . In order to reduce the impact of flow fluctuations on cell sorting and detection effects, the instrument operation section, that is, the time period for the instrument to perform cell sorting and detection, can be slightly delayed.
采用本发明检测仪器对样品进行检测，具体样品为掺杂癌细胞的血样，为准确表征分选结果，在1毫升血液中掺入了较多的荧光标记乳腺癌细胞。对样品进行一次富集和两次富集后，用显微镜拍摄收集目标样品显微照片如图11所示，上列为明场模式，下列为荧光模式，(a)为初始样品液的取样显微照片，(b)为一次富集后的取样显微照片，(c)为两次富集后的取样显微照片，针对不同样品多次试验后得到获得的统计学结果图12所示结果表明发现完成一次富集后，血细胞的移除率已达到93％以上，而癌细胞捕获率约为85％。对上述产品液进行二次富集，进一步移除产品液中的血细胞后，发现血细胞移除率上升至99％以上，而癌细胞捕获率略下降至80％左右。为了获得正弦预聚焦惯性流道131出口区域，流道宽度50微米，流道高度25微米，流道长度4厘米，10微米、15微米及20微米合成粒子在50～200微升/分钟流量下的聚焦排列特性，通过高速摄像机拍摄粒子运动图片，并经过图像堆叠之后获得的粒子分布轨迹图如图13所示，由图可知，粒子在进入检测区域时呈现良好排列的粒子列，从而避免多个细胞/粒子同时经过检测区域而导致的假性错误检测结果。图14为直流模式下检测获得的10微米、15微米及20微米粒子电阻抗信号谱，单个粒子经过检测微电极133获得一个信号峰，统计信号峰的数量可准确获得经过粒子的个数。同时，信号峰的峰值与待检测的粒子/细胞尺寸之间呈现明显的线性对应关系，通过标定可实现细胞尺寸的准确检测。图15上部为裂解红细胞血液中掺杂乳腺癌细胞系(MCF-7)的电阻抗信号谱。下部为统计获得电阻抗信号峰峰值和峰宽散点图。由图可知，可通过电阻抗信号峰的峰值来有效鉴别白细胞和乳腺癌细胞。在交流模式下还可以获得其他多种生物物理特性数据。The detection instrument of the present invention is used to detect the samples, and the specific samples are blood samples doped with cancer cells. In order to accurately characterize the sorting results, more fluorescently labeled breast cancer cells are mixed into 1 ml of blood. After enriching the sample once and twice, the photomicrographs of the collected target samples were taken with a microscope, as shown in Figure 11. The upper column is the bright field mode, and the lower column is the fluorescence mode. (a) is the sampling display of the initial sample solution. Microphotograph, (b) is a sampling microphotograph after one enrichment, (c) is a sampling microphotograph after two enrichments, and the statistical results obtained after multiple experiments for different samples are shown in Figure 12. It shows that after one enrichment is completed, the removal rate of blood cells has reached more than 93%, while the capture rate of cancer cells is about 85%. After secondary enrichment of the above product solution and further removal of blood cells in the product solution, it was found that the removal rate of blood cells rose to over 99%, while the capture rate of cancer cells dropped slightly to about 80%. In order to obtain the outlet area of the sinusoidal pre-focusing inertial channel 131, the channel width is 50 microns, the channel height is 25 microns, the channel length is 4 cm, and the synthetic particles of 10 microns, 15 microns and 20 microns are under the flow rate of 50-200 microliters/minute Figure 13 shows the particle distribution trajectories obtained by taking particle motion pictures with a high-speed camera and stacking the images. It can be seen from the figure that the particles present a well-arranged particle column when they enter the detection area, thereby avoiding multiple False false detection results caused by cells/particles passing through the detection area at the same time. Figure 14 is the electrical impedance signal spectrum of 10 micron, 15 micron and 20 micron particles detected in DC mode. A single particle passes through the detection microelectrode 133 to obtain a signal peak, and the number of passing particles can be accurately obtained by counting the number of signal peaks. At the same time, there is an obvious linear correspondence between the peak value of the signal peak and the size of the particle/cell to be detected, and the accurate detection of the cell size can be realized through calibration. The upper part of Fig. 15 is the electrical impedance signal spectrum of a breast cancer cell line (MCF-7) doped in lysed red blood cells. The lower part is a scatter diagram of the peak-to-peak value and peak width of the electrical impedance signal obtained statistically. It can be seen from the figure that white blood cells and breast cancer cells can be effectively identified by the peak value of the electrical impedance signal peak. A variety of other biophysical property data can also be obtained in the AC mode.
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| CN201711414340.9ACN108226547A (en) | 2017-12-22 | 2017-12-22 | Circulating tumor cell detecting instrument including micro-fluidic chip |
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| CN201711414340.9ACN108226547A (en) | 2017-12-22 | 2017-12-22 | Circulating tumor cell detecting instrument including micro-fluidic chip |
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| RJ01 | Rejection of invention patent application after publication | Application publication date:20180629 | |
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