CN103131693A - Separation method of rare cells in blood - Google Patents
Separation method of rare cells in blood Download PDFInfo
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- CN103131693A CN103131693A CN2013100586530A CN201310058653A CN103131693A CN 103131693 A CN103131693 A CN 103131693A CN 2013100586530 A CN2013100586530 A CN 2013100586530A CN 201310058653 A CN201310058653 A CN 201310058653A CN 103131693 A CN103131693 A CN 103131693A
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Abstract
The invention discloses a separation method of rare cells in blood. According to the invention, a microchip is turned such that the microchip is in three different states. In the first state, a PBS buffering solution discharges air in the microchip. In the second state, a blood sample flows through the microchip, and detection is carried out. A microfluidic channel is positioned below a magnet. With the magnet and the magnetic field, and with a principle that the magnetic force of the rare cells is opposite to the gravity of the rare cells, the rare cells are separated from the blood sample. In the third state, the microchip is in a vertical state, such that the separation efficiency of rare cells and red blood cells is improved. When detection is finished, the microchip is in a vertical state, such that the glass slide with tumor cells can be fetched from the microchip, and subsequent analysis can be carried out. With the three states, the separation of the rare cells is achieved.
Description
Technical field
The present invention relates to a kind of blood testing, especially relate to the separation method of rare cell in a kind of blood.
Background technology
Blood rare cell separometer is the diagnostic tool of future generation that detects rare cell in the blood relevant to major disease, and rare cell comprises cell with specified protein mark of circulating tumor cell (CTC), pernicious stem cell and the pathology of circulation in blood sample etc.Relevant studies confirm that, the circulating tumor cell quantity that occurs in patient's blood sample and patient's early diagnosis and survival rate have very strong dependency.Therefore, be the key that improves tumor disease early discovery rate and personalized treatment to the rare cell determination and analysis of patient's blood sample.
The applying date that is all the applicant's application is on October 15th, 2012, the patent No. is that 201210389753.7 Chinese invention patent application file discloses " separometer of rare cell in a kind of blood ", its structure is as follows: comprise storer and pump, microchip, microchip comprises the reeded thin slice of tool and slide glass, and thin slice is arranged on and forms a microfluidic channel on slide glass; This microfluidic channel is connected between this storer and this pump, allows this blood sample from the memory stream to the pump;
A plurality of magnet are positioned at bottom this slide glass, form the Distribution of Magnetic Field of gradient along the length of slide glass, and slide glass is used for gathering the rare cell that microchip is caught.Also comprise the full wind-up for the direction of this microfluidic channel of rotation.Under the driving of full wind-up, this microfluidic channel in the vertical position, level attitude and symmetrical upturned position replace, and can make cellular segregation more effective.This patent document does not disclose the concrete rotating manner of microchip and the peculiar effect of bringing in detail.
Summary of the invention
Technical problem to be solved by this invention is to provide the separation method of rare cell in the better blood of a kind of separating effect.
The present invention solves the problems of the technologies described above the technical scheme that adopts: 1) prepare two storeies, a suction pump, a syringe pump and a microchip, microchip comprises the reeded thin slice of tool and slide glass, be provided with fluid inlet and liquid outlet on thin slice, thin slice is arranged on and forms a microfluidic channel on slide glass; Magnet is positioned at bottom this slide glass, and forms the Distribution of Magnetic Field of gradient along the length of slide glass, and slide glass is used for gathering the rare cell that microchip is caught;
2) first first memory is communicated with the fluid inlet of microchip by the first pipe connecting, suction pump is communicated with the liquid outlet of microchip by the second pipe connecting; Syringe pump is connected with first memory;
Utilize syringe pump to inject the PBS damping fluid to first memory, set the center of rotation of microchip, this center of rotation is positioned on the first pipe connecting, this center of rotation is near microchip, so that shorten the first required pipe connecting length as far as possible, rotating microchip makes microchip become 20-40 degree angle with horizontal plane, magnet is positioned at the top of slide glass, the liquid outlet of microchip is lower than the fluid inlet of microchip, utilize suction pump that the PBS damping fluid in first memory is drawn in microchip, with the Bas Discharged in microchip; This method can be clean with the air exhausting in microfluidic channel, avoids air to affect subsequent separation process to the interference of blood sample.
3) second memory that will deposit blood replaces first memory, rotating microchip makes microchip become horizontality, the fluid inlet of microchip or liquid outlet are down, then second memory is done and is rocked action, anti-Hemostatic Oral Liquid precipitation utilizes syringe pump that the blood in second memory is injected in microchip, then the static 1-5 of microchip hour, slowly through the microfluidic channel in microchip, slide glass gathers the rare cell that microchip is caught to blood under the effect of suction pump;
4) when the blood in microchip is rare, inject a small amount of PBS damping fluid with syringe pump in second memory, second memory is done and is rocked action, prevent PBS damping fluid precipitation, open suction pump, PBS damping fluid in second memory flows in microchip, and this process is cleaned microchip, repeated washing 2-4 time; Other blood molecules on slide glass are removed away convenient follow-up analytical work from slide glass.
5) rotate microchip and make microchip be in plumbness, the fluid inlet of microchip is convenient to erythrocyte and flows out microchip under action of gravity higher than liquid outlet, improves the rare cell separation efficiency; After detecting end, microchip is in plumbness, is convenient to take out from microchip with the slide glass of tumour cell, carries out subsequent analysis.
Compared with prior art, advantage of the present invention is that the upset microchip makes microchip be in three different states, first state: the PBS damping fluid avoids air to affect subsequent separation process to the interference of blood sample the Bas Discharged in microchip.Second state: allow the blood sample microchip of flowing through, microfluidic channel is positioned at the below of magnet, utilize this moment magnet that the principle of the opposite direction of the magnetic force of rare cell and rare cell gravity is separated rare cell from blood sample, the 3rd state: microchip is in plumbness, be convenient to erythrocyte and flow out microchip under action of gravity, more be conducive to the user to the fetching of slide glass, three states have been completed the separation to rare cell.
Description of drawings
Fig. 1 is the first view of the present invention;
Fig. 2 is the second view of the present invention;
Fig. 3 is third state schematic diagram of the present invention.
Embodiment
Embodiment is described in further detail the present invention below in conjunction with accompanying drawing.
The separation method of rare cell in a kind of blood, concrete steps are as follows:
1) prepare two storeies, a suction pump 1, a syringe pump and a microchip 2, microchip 2 comprises the reeded thin slice of tool and slide glass, is provided with fluid inlet 21 and liquid outlet 22 on thin slice, and thin slice is arranged on and forms a microfluidic channel on slide glass; Magnet is positioned at bottom this slide glass, and forms the Distribution of Magnetic Field of gradient along the length of slide glass, and slide glass is used for gathering the rare cell that microchip is caught;
2) first first memory is communicated with the fluid inlet 21 of microchip 2 by the first pipe connecting 4, suction pump 1 is communicated with the liquid outlet 22 of microchip by the second pipe connecting 6; Syringe pump is connected with first memory;
Utilize syringe pump to inject the PBS damping fluid to first memory, set the center of rotation 5 of microchip, this center of rotation 5 is positioned on the first pipe connecting 4, this center of rotation 5 is near microchip, rotate microchip and make microchip 2 become 20-40 degree angle with horizontal plane, magnet is positioned at the top of slide glass, and the liquid outlet 22 of microchip 2 is lower than the fluid inlet 21 of microchip 2, utilize suction pump 1 that the PBS damping fluid in first memory is drawn in microchip 2, with the Bas Discharged in microchip 2;
3) second memory 3 that will deposit blood replaces first memory, utilize syringe pump to the interior injection of second memory 3 blood to be detected, rotate microchip 2 and make 2 one-tenth horizontalitys of microchip, the fluid inlet 21 of microchip 2 or liquid outlet 22 are down, then second memory 3 is done and is rocked action, anti-Hemostatic Oral Liquid precipitation, after second memory 3 stops rocking, utilize syringe pump that the blood in second memory 3 is injected in microchip 2, then the static 1-5 of microchip 2 hour, blood is slow in the microfluidic channel in microchip 2 under the effect of suction pump 1, slide glass gathers the rare cell that microchip is caught,
4) when the blood in microchip 2 is rare, with syringe pump to a small amount of PBS damping fluid of the interior injection of second memory 3, second memory 3 is done and is rocked action, prevent PBS damping fluid precipitation, open suction pump 1, PBS damping fluid in second memory 3 flows in microchip 2, and this process is cleaned microchip 2, repeated washing 2-4 time;
5) rotate microchip 2 and make microchip 2 be in plumbness, the liquid outlet of microchip 2 or fluid inlet are towards a side, and the fluid inlet 21 of microchip is convenient to erythrocyte and flows out microchip under action of gravity higher than liquid outlet 22, improve the rare cell separation efficiency.After detecting end, microchip is in plumbness, is convenient to take out from microchip 2 with the slide glass of tumour cell, carries out subsequent analysis.
Claims (1)
1. the separation method of rare cell in a blood is characterized in that concrete steps are as follows:
1) prepare two storeies, a suction pump, a syringe pump and a microchip, microchip comprises the reeded thin slice of tool and slide glass, is provided with fluid inlet and liquid outlet on thin slice, and thin slice is arranged on and forms a microfluidic channel on slide glass; Magnet is positioned at bottom this slide glass, and forms the Distribution of Magnetic Field of gradient along the length of slide glass, and slide glass is used for gathering the rare cell that microchip is caught;
2) first first memory is communicated with the fluid inlet of microchip by the first pipe connecting, suction pump is communicated with the liquid outlet of microchip by the second pipe connecting; Syringe pump is connected with first memory;
Utilize syringe pump to inject the PBS damping fluid to first memory, set the center of rotation of microchip, this center of rotation is positioned on the first pipe connecting, this center of rotation is near microchip, rotate microchip and make microchip become 20-40 degree angle with horizontal plane, magnet is positioned at the top of slide glass, and the liquid outlet of microchip is lower than the fluid inlet of microchip, utilize suction pump that the PBS damping fluid in first memory is drawn in microchip, with the Bas Discharged in microchip;
3) second memory that will deposit blood replaces first memory, rotating microchip makes microchip become horizontality, the fluid inlet of microchip or liquid outlet are down, then second memory is done and is rocked action, anti-Hemostatic Oral Liquid precipitation utilizes syringe pump that the blood in second memory is injected in microchip, then the static 1-5 of microchip hour, slowly through the microfluidic channel in microchip, slide glass gathers the rare cell that microchip is caught to blood under the effect of suction pump;
4) when the blood in microchip is rare, inject a small amount of PBS damping fluid with syringe pump in second memory, second memory is done and is rocked action, prevent PBS damping fluid precipitation, open suction pump, PBS damping fluid in second memory flows in microchip, and this process is cleaned microchip, repeated washing 2-4 time;
5) rotate microchip and make microchip be in plumbness, the fluid inlet of microchip is convenient to erythrocyte and flows out microchip under action of gravity higher than liquid outlet; After detecting end, will take out from microchip with the slide glass of tumour cell, carry out subsequent analysis.
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| CN201310058653.0A CN103131693B (en) | 2013-02-25 | 2013-02-25 | Separation method of rare cells in blood |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152345A (en) * | 2014-08-14 | 2014-11-19 | 江苏瑞明生物科技有限公司 | Microfluidic type single-cell separator |
| CN106969964A (en) * | 2017-02-23 | 2017-07-21 | 宁波美晶医疗技术有限公司 | The negative enrichment method and kit of rare cell in a kind of blood based on micro-fluidic and immune Magneto separate |
| CN110031622A (en) * | 2019-05-06 | 2019-07-19 | 中山大学 | A kind of minute yardstick magnetic enrichment detection device and detection method for immunoassay |
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| US4710472A (en) * | 1985-09-25 | 1987-12-01 | The United States Of America As Represented By The Secretary Of The Navy | Magnetic separation device |
| CN101142314A (en) * | 2004-03-03 | 2008-03-12 | 综合医院公司 | Magnetic device for isolation of cells and biomolecules in a microfluidic environment |
| CN102360010A (en) * | 2011-08-05 | 2012-02-22 | 上海交通大学 | Integrated microfluidic chip for capture of cancer cells in whole blood |
| CN102458665A (en) * | 2009-04-22 | 2012-05-16 | 临床基因组学股份有限公司 | Method and apparatus for isolating a target bioentity from a biological sample |
| CN102911864A (en) * | 2012-10-15 | 2013-02-06 | 宁波美晶医疗技术有限公司 | Separator for rare cells in blood |
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2013
- 2013-02-25 CN CN201310058653.0A patent/CN103131693B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US4710472A (en) * | 1985-09-25 | 1987-12-01 | The United States Of America As Represented By The Secretary Of The Navy | Magnetic separation device |
| CN101142314A (en) * | 2004-03-03 | 2008-03-12 | 综合医院公司 | Magnetic device for isolation of cells and biomolecules in a microfluidic environment |
| CN102458665A (en) * | 2009-04-22 | 2012-05-16 | 临床基因组学股份有限公司 | Method and apparatus for isolating a target bioentity from a biological sample |
| CN102360010A (en) * | 2011-08-05 | 2012-02-22 | 上海交通大学 | Integrated microfluidic chip for capture of cancer cells in whole blood |
| CN102911864A (en) * | 2012-10-15 | 2013-02-06 | 宁波美晶医疗技术有限公司 | Separator for rare cells in blood |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152345A (en) * | 2014-08-14 | 2014-11-19 | 江苏瑞明生物科技有限公司 | Microfluidic type single-cell separator |
| CN106969964A (en) * | 2017-02-23 | 2017-07-21 | 宁波美晶医疗技术有限公司 | The negative enrichment method and kit of rare cell in a kind of blood based on micro-fluidic and immune Magneto separate |
| CN106969964B (en) * | 2017-02-23 | 2020-02-18 | 宁波美晶医疗技术有限公司 | Negative phase enrichment method and kit for rare cells in blood based on micro-fluidic and immunomagnetic separation |
| CN110031622A (en) * | 2019-05-06 | 2019-07-19 | 中山大学 | A kind of minute yardstick magnetic enrichment detection device and detection method for immunoassay |
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| CN103131693B (en) | 2014-12-24 |
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