CN107937257A - A kind of circulating tumor cell separating chips and its detection method - Google Patents
A kind of circulating tumor cell separating chips and its detection method Download PDFInfo
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- CN107937257A CN107937257A CN201711205076.8A CN201711205076A CN107937257A CN 107937257 A CN107937257 A CN 107937257A CN 201711205076 A CN201711205076 A CN 201711205076A CN 107937257 A CN107937257 A CN 107937257A
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Abstract
The problem of a kind of circulating tumor cell separating chips and its detection method belong to circulating tumor cell separation technology field, solve existing apparatus complexity, and transmission mechanism is more, and precision is not easy to ensure.When chip rotates, sample introduction liquid rapidly radially moves out, and is pumped by transition piece in filter chamber, and the cell more than cell size selection membrane aperture is captured, and the cell less than cell size selection membrane aperture is transferred to filter chamber's inflow waste compartment.The present invention is small, light-weight, is convenient for carrying, using the cell seperation film with liquid filling hole, there is clog-free, high sensitivity, selectivity, quick, the liquid that capillary is stored in the fluid ancillary chamber below film triggers, complete wetting is kept in whole filter process so that fluid flow needs minimum pressure differential and filter more uniformly to occur over the entire film；This method provides uniform, the supper-fast cell separation technology without blocking, and the 1kPa during its pressure value is filtered than stock size is much smaller.
Description
Technical field
The invention belongs to circulating tumor cell separation technology field, and in particular to a kind of circulating tumor cell separating chips and
Its detection method.
Background technology
One major obstacle of circulating tumor cell (CTC) research is that its whole blood quantity is extremely rare, every 106-107A blood
Contain 1-10 CTC in liquid cell, machinable blood volume is limited.It is rare due to CTC, develop and effective collected process
As the committed step for detecting and characterizing CTC, CTC is 1-10 cell in the frequency of occurrences of every 1 milliliter of blood.In this respect,
Microfluidic platforms, which have become, possible solution, because the fine structure of the device allows accurate fluid to control and go back
The environment of biocompatibility can be provided for cell.Although report so far flat using the various CTC detections of microflow control technique
Platform, but these platforms all have some main limitations.Up to the present, the isolation of the CTC based on micro-fluidic chip of proposition
Technology has been taken root in cancer cell biology characteristic (i.e. surface marker is expressed) or physical property (i.e. size, density, dielectricity
Can, etc.).
The CTC that peripheral blood is flowed into from primary and metastatic tumo(u)r is early detection invasive cancer and individualized cancer
Important biomolecule marker in treatment.Since the concentration of CTC is extremely low, thus the CTC for obtaining high recycling and high-purity be one very
Big challenge.In addition, CTC is very fragile, therefore suggest analyzed interior when 6-8 is small after patient takes a blood sample strongly.So urgently
Exploitation is needed to be widely deployed in quick in clinical setting, efficient and stable CTC isolation technics.In the past from peripheral blood
In the experiment of middle Selective Separation CTC, the most common detection method based on immunoaffinity, this method has of a relatively high spirit
Quick property and specificity, but detection time length (>When 1-4 is small).
By existing technology is investigated, Chinese patent 201520684448.X is disclosed and patent principle of the present invention
More similar cell isolation method.Although the device has certain cell separating capacity, device design is complex,
Transmission mechanism is excessive, and precision is not easy to ensure；And it is bulky, it is complicated, it is not portable, it is impossible to be used in hospital laboratory and
The other monitoring of bed, is unfavorable for promoting and applying.
The content of the invention
In order to solve the problems in the existing technology, the present invention provides a kind of circulating tumor cell separating chips and its
Detection method, the technology of CTC is separated using size, and the rate of recovery of CTC is improved using the photolithography thin film of regular aperture and shape
And purity, made by the liquid phase diffusion that before whole filter process and in whole process, the chamber under cell seperation film is full of
With can be filtered whole membrane area, and mitigate blockage problem significantly.
The technical proposal for solving the technical problem of the invention is as follows：
A kind of circulating tumor cell separating chips, including substrate and upper cover, substrate and the upper cover sealing；Substrate includes：
Loading chamber, transition piece, filter chamber and waste compartment；The region that the upper cover corresponds to loading chamber sets sample holes；Set in loading chamber
One catch, the catch corresponding position set transition piece；The transition piece structure is inclined-plane from low to high at catch；It is described
Filter chamber upper end is cell size selective membrane, and the space of lower end is big by passage and waste compartment unicom, the height of the transition piece
In the position equal to cell size selective membrane；In the centrally disposed centrifugal hole of the chip；Using separating chips when, by into
Loading chamber sample introduction is given in sample hole, chip is installed on centrifugation apparatus, when chip rotates, sample introduction liquid is rapidly radially to outward transport
It is dynamic, pumped by transition piece in filter chamber, the cell more than cell size selection membrane aperture is captured, and is selected less than cell size
The cell of membrane aperture is transferred to filter chamber and flows into waste compartment.
A kind of separated detection method of circulating tumor cell, this method comprises the following steps：
Step 1：By sample introduction liquid in the culture medium for being supplemented with hyclone and antibiotic/antifungal agent, and cultivating
In case and CO2Cultivated under environment；
Step 2：The surface of chip is passivated with bovine serum albumin(BSA) by loading chamber；
Step 3：After incubation, chip is put drive by centrifugation apparatus and is rotated with certain rotating speed, makes the tire ox in loading chamber
Serum solution is moved on in waste compartment；
Step 4：Lavation buffer solution adds to loading chamber by sample holes, keeps the rotating speed of step 3 chip, and lavation buffer solution leads to
Cell size selection film is crossed by filter chamber to waste compartment；
Step 5：Keep centrifugal device to work according to the rotating speed of step 3, sample introduction liquid is added to by sample holes
Loading chamber；Cell in filter chamber is separated, and then washs filter chamber with lavation buffer solution；
Step 6：After separation, cell size selective membrane is removed into installation with carrying out dyeing processing on glass slide, by glimmering
Light microscope is observed and records data.
The beneficial effects of the invention are as follows：Patent of the present invention is small, it is light-weight, be convenient for carrying, can detect not only cancer
Disease shifts a large amount of CTC of patient, but also never can detect CTC in the patient of the opposite early stage of cancer symptoms,
This demonstrate the possibility that potential CTC is used as early diagnosis mark.Using the cell seperation film with liquid filling hole, it has
Have clog-free, high sensitivity (95.9 ± 3.1% rate of recovery), selectivity (>2.5log consumes leucocyte), it is quick (>3mL/min)
The advantages that, the liquid that capillary is stored in the fluid ancillary chamber below film triggers, and in whole filter process
Keep complete wetting so that fluid flow needs minimum pressure differential and filter more uniformly to occur over the entire film；And
Using the independent laboratory chip system for having fluid auxiliary isolation technics, unmarked point of the vivo CT C from whole blood is realized
From without previous sample treatment.Numerical simulation and experiment show that this method provides uniform, the supper-fast cell without blocking
Isolation technics, the 1kPa during its pressure value is filtered than stock size are much smaller.
Brief description of the drawings
A kind of circulating tumor cell separating chips explosive view of Fig. 1 present invention.
A kind of circulating tumor cell separating chips backside structure figure of Fig. 2 present invention.
A kind of circulating tumor cell separating chips schematic diagram of Fig. 3 present invention.
1 in figure, sealed silicon rubber cushion, 2, cell size selective membrane, 3, upper cover, 4, upper cover shading patch, 5, strong water absorption function film, 6,
Substrate shading is pasted, and 7, substrate, 8, seal O-ring and 9, cell size selective membrane gland.
Embodiment
The present invention is described in further details with reference to the accompanying drawings and examples.
As shown in Figure 1 and shown in Fig. 2, a kind of circulating tumor cell separating chips, the chip includes：It is sealed silicon rubber cushion 1, thin
Born of the same parents' size selection film 2, upper cover 3, upper cover shading patch 4, strong water absorption function film 5, substrate shading patch 6, substrate 7, seal O-ring 8 and thin
Born of the same parents' size selection membrane pressure lid 9.The chip is made of three single filter elements, can handle three different blood samples at the same time,
Each unit includes sample loading chamber, transition piece and filter chamber, and three filter elements share a waste compartment；The upper cover 3 with
Substrate 7 is bonded by double-sided pressure-sensitive adhesive tape, to realize the close assembling of chip；Sample holes in the upper cover 3, for into
Sample and ventilation, send sample introduction liquid into loading chamber, and the loading chamber sets catch and the filtration members with catch position correspondence, from by
Seen at nearly catch, filtration members are ramp structure from low to high；The substrate 7, which creates, passage, and forms mainstream in substrate 7
Body room；Being created in the substrate 7 has exit passageway and is provided with the region for placing cell size selective membrane 9, by seal O-ring 8
Fastened with cell size selective membrane gland 9, sealed silicon rubber cushion 1, form filter chamber.The height of the transition piece is more than or equal to
The position of cell size selective membrane 9, and with cell size selective membrane 9 close to.Cell size selective membrane gland 9 is easily disassembled, will
It, which is installed on glass slide, is used for successive image analysis and characterization of molecules.It is provided with and adds on the cell size selective membrane gland 9
Sample hole, for adding coating buffer；The chip waste compartment is bonded by strong water absorption function film 5 by double-sided pressure-sensitive adhesive tape, by force
Power absorbing membrane 5 can absorb the waste liquid of a part；The upper cover shading patch 4 is with substrate shading patch 6 respectively by double-sided pressure-sensitive adhesive
Band sticks to upper cover 3 and substrate 7, sample introduction liquid is not disturbed be subject to external environment；Placement hole is provided among the chip, is used for
It is connected with centrifugal device.In this example, above-mentioned upper cover 3, substrate 7 and cell size selective membrane gland 9 are made of makrolon.
Said chip is installed in centrifugal device, as shown in figure 3, when chip rotates, positioned at the sample introduction liquid of loading chamber
Rapidly radially outwards pumped, by being arranged on the catch and filtration members of loading chamber, sample introduction liquid, which is lightly pushed into, to be located at
In the filter chamber of substrate 7, the cell of large-size is captured by cell size selective membrane 2, and reduced size cell passes through cell ruler
Very little selective membrane 2 is to be transferred in waste compartment.In whole filter process, in the unit that upper cover 3 is formed with substrate 7, cell
Filter chamber under size selection film 2 is filled with liquid.Wherein filtration members are provided with inclined plane, for preventing cell due to centrifugal force
Effect and be subject to excessive pressure to cause cell damage or rupture, the pressure drop Δ P across cell size selective membrane 2 is maintained at
To prevent cellular damage below threshold value, this is vital.Selected by using below equation to calculate across cell size
The pressure drop of film 2：
Wherein μ is the coefficient of dynamic viscosity, and L is the thickness of cell size selective membrane 2, and d is 2 hole of cell size selective membrane
Footpath, Q are the flows on cell size selective membrane 2, and N is the quantity in hole on cell size selective membrane 2.
A kind of separated detection method of circulating tumor cell, its detection method include the following steps：
Step 1：All cells of sample introduction liquid are trained in the RPMI for being supplemented with 5%FBS and 1% antibiotic/antifungal agent
Support in base, and in 37 DEG C of incubator is set as and 5%CO2Cultivate, tested for mark-on, cell fluorescent dye under environment
Preliminary making.
Step 2：Before cell size selection is carried out, the surface of chip is passivated with BSA, by 1%BSA solution by upper
Well in lid 3, adds in the unit being made of upper cover 3 and substrate 7, so that surface passivation, prevents cell from adsorbing.
Step 3：After being incubated 30 minutes, chip is driven by centrifugal device to be rotated with certain rotating speed, this loads sample
BSA solution in room is moved on in waste compartment, but the filter chamber below film remains full of liquid.
Step 4：1mL lavation buffer solutions are added into loading chamber by the well in upper cover 3, and chip keeps previous step
Rotating speed, lavation buffer solution arrives waste compartment by cell size selective membrane 2, and the BSA in 2 lower chamber of cell size selective membrane
The lavation buffer solution displacement that solution is newly added.After surface passivation, 3mL samples liquid is added into loading by the well in upper cover 3
Room, without any sample pretreatment step.
Step 5：The cell in loading chamber is separated by rotary chip, then by loading chamber 1mL washing buffers
Liquid washes twice, and keeps original working speed of centrifugal device to work after each liquid feeding.
Step 6：After separation, cell size selective membrane 2 is removed into installation with carrying out dyeing processing on glass slide, by
Fluorescence microscope is observed and records data.
In this example, the hole of EDS maps is non-homogeneous form and random point on cell size selective membrane 2 described in above-mentioned steps
Cloth.The substrate 7 is provided with screw hole with cell size selective membrane gland 9, it is fastened by screw.Chip described in above-mentioned steps
Since the lower section of cell size selective membrane 2 is full of liquid phase, and then the barometric gradient and upper chambers produced in the detection in hypostegal cavity room
Barometric gradient transitivity in room is good, causes relatively uniform pressure drop, so liquid flow is uniform when crossing cell size selection film 2
Flowing.
Claims (10)
1. a kind of circulating tumor cell separating chips, including substrate and upper cover, substrate and the upper cover sealing；It is characterized in that,
Substrate includes：Loading chamber, transition piece, filter chamber and waste compartment；The region that the upper cover corresponds to loading chamber sets sample holes；Loading
Indoor one catch of setting, the catch corresponding position set transition piece；The transition piece structure is from low to high at catch
Inclined-plane；The filter chamber upper end is cell size selective membrane, and the space of lower end passes through passage and waste compartment unicom, the transition piece
Height be more than or equal to cell size selective membrane position；In the centrally disposed centrifugal hole of the chip；Using separating chips
When, loading chamber sample introduction is given by sample holes, chip is installed on centrifugation apparatus, when chip rotates, the quick footpath of sample introduction liquid
To moving out, pumped by transition piece in filter chamber, the cell more than cell size selection membrane aperture is captured, less than cell
The cell of size selection membrane aperture is transferred to filter chamber and flows into waste compartment.
2. a kind of circulating tumor cell separating chips according to claim 1, it is characterised in that the substrate and upper cover are led to
Double-sided pressure-sensitive adhesive tape is crossed to be bonded.
3. a kind of circulating tumor cell separating chips according to claim 1, it is characterised in that the filter chamber and substrate
It is flexibly connected, the filter chamber back side, which is set gradually, to be sealed and be loaded by seal O-ring and cell size selective membrane gland.
A kind of 4. circulating tumor cell separating chips according to claim 1, it is characterised in that the cell size selection
Random distribution has the hole of non-homogeneous form on film, and cell size selective membrane is detachable structure.
5. a kind of circulating tumor cell separating chips according to claim 4, it is characterised in that when processing is containing fragile thin
During the sample introduction liquid of born of the same parents, the pressure drop Δ P across cell size selective membrane is maintained at below threshold value and prevents cellular damage, by using
Below equation calculates the pressure drop across cell size selective membrane：
Wherein μ is the coefficient of dynamic viscosity, and L is the thickness of cell size selective membrane, and d is the aperture of cell size selective membrane, and Q is
The flow of cell size selective membrane, N are the quantity of cell size selection fenestra.
6. a kind of circulating tumor cell separating chips according to claim 1, it is characterised in that outside the substrate and upper cover
All set shading to paste, a strong water absorption function film is set in the upper cover.
A kind of 7. detection method of circulating tumor cell separating chips described in based on claim 1-6, it is characterised in that the party
Method includes the following steps：
Step 1：By sample introduction liquid in the culture medium for being supplemented with hyclone and antibiotic/antifungal agent, and in the incubator
And CO2Cultivated under environment；
Step 2：The surface of chip is passivated with bovine serum albumin(BSA) by loading chamber；
Step 3：After incubation, chip is put drive by centrifugation apparatus and is rotated with certain rotating speed, makes the hyclone in loading chamber
Solution is moved on in waste compartment；
Step 4：Lavation buffer solution adds to loading chamber by sample holes, keeps the rotating speed of step 3 chip, lavation buffer solution passes through thin
Born of the same parents' size selection film is by filter chamber to waste compartment；
Step 5：Keep centrifugal device to work according to the rotating speed of step 3, sample introduction liquid is added into loading by sample holes
Room；Cell in filter chamber is separated, and then washs loading chamber with lavation buffer solution；
Step 6：After separation, cell size selective membrane is removed into installation with carrying out dyeing processing on glass slide, is shown by fluorescence
Micro mirror is observed and records data.
8. detection method according to claim 7, it is characterised in that in the step 1, all cells are being supplemented with
In the culture medium of 1% antibiotic of 5%FBS hyclones/antifungal agent, and in 37 DEG C of incubator is set as and 5%CO2Ring
Cultivate, tested for mark-on, cell fluorescent dye preliminary making under border.
9. detection method according to claim 7, it is characterised in that in the step 3, carrying out cell size selection
Before, the surface of chip is passivated with bovine serum albumin(BSA), 1% bovine serum albumin solution is added into sample by chip upper cover
Well.
10. detection method according to claim 7, it is characterised in that in the step 3, keep filter chamber to be full of liquid
Body.
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Cited By (3)
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| CN109917123A (en) * | 2019-04-19 | 2019-06-21 | 广州安诺科技股份有限公司 | A kind of residual detection device of agriculture based on DELFIA and detection method |
| CN110982665A (en) * | 2019-11-22 | 2020-04-10 | 上海理工大学 | Multi-channel sample introduction device and method for sorting and detecting circulating tumor cells |
| CN111073798A (en) * | 2020-03-04 | 2020-04-28 | 山东第一医科大学(山东省医学科学院) | Centrifugal circulation tumor cell step-by-step separation device |
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